Reduction of MDSCs with all-trans retinoic acid improves CAR therapy efficacy for sarcomas

PubMed Central

Long, Adrienne H.; Highfill, Steven L.; Cui, Yongzhi; Smith, Jillian P.; Walker, Alec J.; Ramakrishna, Sneha; El-Etriby, Rana; Galli, Susana; Tsokos, Maria G.; Orentas, Rimas J.; Mackall, Crystal L.

2016-01-01

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CARs) have shown impressive activity against B cell malignancies, and preliminary results suggest that T cells expressing a first generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18/18 (100%) of osteosarcomas, 2/15 (13%) of rhabdomyosarcomas, and 7/35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2+ sarcoma and neuroblastoma cell lines in vitro. However in xenograft models, GD2-CAR T cells had no antitumor effect against GD2+ sarcoma, despite effectively controlling GD2+ neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSCs) that inhibited human CAR T-cell responses in vitro. Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. PMID:27549124

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination

PubMed Central

Ham, Jungoh; Costa, Carlotta; Sano, Renata; Lochmann, Timothy L.; Sennott, Erin M.; Patel, Neha U.; Dastur, Anahita; Gomez-Caraballo, Maria; Krytska, Kateryna; Hata, Aaron N.; Floros, Konstantinos V.; Hughes, Mark T.; Jakubik, Charles T.; Heisey, Daniel A.R.; Ferrell, Justin T.; Bristol, Molly L.; March, Ryan J.; Yates, Craig; Hicks, Mark A.; Nakajima, Wataru; Gowda, Madhu; Windle, Brad E.; Dozmorov, Mikhail G.; Garnett, Mathew J.; McDermott, Ultan; Harada, Hisashi; Taylor, Shirley M.; Morgan, Iain M.; Benes, Cyril H.; Engelman, Jeffrey A.; Mossé, Yael P.; Faber, Anthony C.

2016-01-01

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression. PMID:26859456

Predictive Modeling of Neuroblastoma Growth Dynamics in Xenograft Model After Bevacizumab Anti-VEGF Therapy.

PubMed

He, Yixuan; Kodali, Anita; Wallace, Dorothy I

2018-06-14

Neuroblastoma is the leading cause of cancer death in young children. Although treatment for neuroblastoma has improved, the 5-year survival rate of patients still remains less than half. Recent studies have indicated that bevacizumab, an anti-VEGF drug used in treatment of several other cancer types, may be effective for treating neuroblastoma as well. However, its effect on neuroblastoma has not been well characterized. While traditional experiments are costly and time-consuming, mathematical models are capable of simulating complex systems quickly and inexpensively. In this study, we present a model of vascular tumor growth of neuroblastoma IMR-32 that is complex enough to replicate experimental data across a range of tumor cell properties measured in a suite of in vitro and in vivo experiments. The model provides quantitative insight into tumor vasculature, predicting a linear relationship between vasculature and tumor volume. The tumor growth model was coupled with known pharmacokinetics and pharmacodynamics of the VEGF blocker bevacizumab to study its effect on neuroblastoma growth dynamics. The results of our model suggest that total administered bevacizumab concentration per week, as opposed to dosage regimen, is the major determining factor in tumor suppression. Our model also establishes an exponentially decreasing relationship between administered bevacizumab concentration and tumor growth rate.

Heterogeneous distribution of alectinib in neuroblastoma xenografts revealed by matrix-assisted laser desorption ionization mass spectrometry imaging: a pilot study.

PubMed

Ryu, Shoraku; Hayashi, Mitsuhiro; Aikawa, Hiroaki; Okamoto, Isamu; Fujiwara, Yasuhiro; Hamada, Akinobu

2018-01-01

The penetration of the anaplastic lymphoma kinase (ALK) inhibitor alectinib in neuroblastomas and the relationship between alectinib and ALK expression are unknown. The aim of this study was to perform a quantitative investigation of the inter- and intra-tumoural distribution of alectinib in different neuroblastoma xenograft models using matrix-assisted laser desorption ionization MS imaging (MALDI-MSI). The distribution of alectinib in NB1 (ALK amplification) and SK-N-FI (ALK wild-type) xenograft tissues was analysed using MALDI-MSI. The abundance of alectinib in tumours and intra-tumoural areas was quantified using ion signal intensities from MALDI-MSI after normalization by correlation with LC-MS/MS. The distribution of alectinib was heterogeneous in neuroblastomas. The penetration of alectinib was not significantly different between ALK amplification and ALK wide-type tissues using both LC-MS/MS concentrations and MSI intensities. Normalization with an internal standard increased the quantitative property of MSI by adjusting for the ion suppression effect. The distribution of alectinib in different intra-tumoural areas can alternatively be quantified from MS images by correlation with LC-MS/MS. The penetration of alectinib into tumour tissues may not be homogenous or influenced by ALK expression in the early period after single-dose administration. MALDI-MSI may prove to be a valuable pharmaceutical method for elucidating the mechanism of action of drugs by clarifying their microscopic distribution in heterogeneous tissues. © 2017 The British Pharmacological Society.

Mutation-Independent Activation of the Anaplastic Lymphoma Kinase in Neuroblastoma.

PubMed

Regairaz, Marie; Munier, Fabienne; Sartelet, Hervé; Castaing, Marine; Marty, Virginie; Renauleaud, Céline; Doux, Camille; Delbé, Jean; Courty, José; Fabre, Monique; Ohta, Shigeru; Vielh, Philippe; Michiels, Stefan; Valteau-Couanet, Dominique; Vassal, Gilles

2016-02-01

Activating mutations of anaplastic lymphoma kinase (ALK) have been identified as important players in neuroblastoma development. Our goal was to evaluate the significance of overall ALK activation in neuroblastoma. Expression of phosphorylated ALK, ALK, and its putative ligands, pleiotrophin and midkine, was screened in 289 neuroblastomas and 56 paired normal tissues. ALK was expressed in 99% of tumors and phosphorylated in 48% of cases. Pleiotrophin and midkine were expressed in 58% and 79% of tumors, respectively. ALK activation was significantly higher in tumors than in paired normal tissues, together with ALK and midkine expression. ALK activation was largely independent of mutations and correlated with midkine expression in tumors. ALK activation in tumors was associated with favorable features, including a younger age at diagnosis, hyperdiploidy, and detection by mass screening. Antitumor activity of the ALK inhibitor TAE684 was evaluated in wild-type or mutated ALK neuroblastoma cell lines and xenografts. TAE684 was cytotoxic in vitro in all cell lines, especially those harboring an ALK mutation. TAE684 efficiently inhibited ALK phosphorylation in vivo in both F1174I and R1275Q xenografts but demonstrated antitumor activity only against the R1275Q xenograft. In conclusion, ALK activation occurs frequently during neuroblastoma oncogenesis, mainly through mutation-independent mechanisms. However, ALK activation is not associated with a poor outcome and is not always a driver of cell proliferation and/or survival in neuroblastoma. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN-amplified neuroblastoma.

PubMed

Boboila, Shuobo; Lopez, Gonzalo; Yu, Jiyang; Banerjee, Debarshi; Kadenhe-Chiweshe, Angela; Connolly, Eileen P; Kandel, Jessica J; Rajbhandari, Presha; Silva, Jose M; Califano, Andrea; Yamashiro, Darrell J

2018-06-07

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo.

PubMed

Murray, Jayne; Valli, Emanuele; Yu, Denise M T; Truong, Alan M; Gifford, Andrew J; Eden, Georgina L; Gamble, Laura D; Hanssen, Kimberley M; Flemming, Claudia L; Tan, Alvin; Tivnan, Amanda; Allan, Sophie; Saletta, Federica; Cheung, Leanna; Ruhle, Michelle; Schuetz, John D; Henderson, Michelle J; Byrne, Jennifer A; Norris, Murray D; Haber, Michelle; Fletcher, Jamie I

2017-09-01

The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

PEST-containing nuclear protein mediates the proliferation, migration, and invasion of human neuroblastoma cells through MAPK and PI3K/AKT/mTOR signaling pathways.

PubMed

Wu, Dong-Dong; Gao, Ying-Ran; Li, Tao; Wang, Da-Yong; Lu, Dan; Liu, Shi-Yu; Hong, Ya; Ning, Hui-Bin; Liu, Jun-Ping; Shang, Jia; Shi, Jun-Feng; Wei, Jian-She; Ji, Xin-Ying

2018-05-02

PEST-containing nuclear protein (PCNP), a novel nuclear protein, is involved in cell proliferation and tumorigenesis. However, the precise mechanism of action of PCNP in the process of tumor growth has not yet been fully elucidated. ShRNA knockdown and overexpression of PCNP were performed in human neuroblastoma cells. Tumorigenic and metastatic effects of PCNP were examined by tumor growth, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo. PCNP over-expression decreased the proliferation, migration, and invasion of human neuroblastoma cells and down-regulation of PCNP showed reverse effects. PCNP over-expression increased protein expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved poly adenosine diphosphate-ribose polymerase, as well as ratios of B-cell lymphoma-2 (Bcl-2)-associated X protein/Bcl-2 and Bcl-2-associated death promoter/B-cell lymphoma-extra large in human neuroblastoma cells, however PCNP knockdown exhibited reverse trends. PCNP over-expression increased phosphorylations of extracellular signal-regulated protein kinase 1/2, p38, c-Jun N-terminal kinase, as well as decreased phosphorylations of phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), nevertheless PCNP knockdown exhibited opposite effects. Furthermore, PCNP over-expression significantly reduced the growth of human neuroblastoma xenograft tumors by down-regulating angiogenesis, whereas PCNP knockdown markedly promoted the growth of human neuroblastoma xenograft tumors through up-regulation of angiogenesis. PCNP mediates the proliferation, migration, and invasion of human neuroblastoma cells through mitogen-activated protein kinase and PI3K/AKT/mTOR signaling pathways, implying that PCNP is a therapeutic target for patients with neuroblastoma.

Targeting tachykinin receptors in neuroblastoma.

PubMed

Henssen, Anton G; Odersky, Andrea; Szymansky, Annabell; Seiler, Marleen; Althoff, Kristina; Beckers, Anneleen; Speleman, Frank; Schäfers, Simon; De Preter, Katleen; Astrahanseff, Kathy; Struck, Joachim; Schramm, Alexander; Eggert, Angelika; Bergmann, Andreas; Schulte, Johannes H

2017-01-03

Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma.

Polo-like kinase 1 is a therapeutic target in high-risk neuroblastoma.

PubMed

Ackermann, Sandra; Goeser, Felix; Schulte, Johannes H; Schramm, Alexander; Ehemann, Volker; Hero, Barbara; Eggert, Angelika; Berthold, Frank; Fischer, Matthias

2011-02-15

High-risk neuroblastoma remains a therapeutic challenge for pediatric oncologists. The Polo-like kinase 1 (PLK1) is highly expressed in many human cancers and is a target of the novel small-molecule inhibitor BI 2536, which has shown promising anticancer activity in adult malignancies. Here, we investigated the effect of BI 2536 on neuroblastoma cells in vitro and in vivo to explore PLK1 as a potential target in high-risk neuroblastoma therapy. PLK1 transcript levels were analyzed by microarrays in 476 primary neuroblastoma specimens, and correlation with prognostic markers and patient outcome was examined. To explore the effect of PLK1 inhibition on neuroblastoma cells, 7 cell lines were treated with BI 2536 and changes in growth properties were determined. Furthermore, nude mice with IMR-32 and SK-N-AS xenografts were treated with BI 2536. PLK1 is highly expressed in unfavorable neuroblastoma and in neuroblastoma cell lines. Expression of PLK1 is associated with unfavorable prognostic markers such as stage 4, age >18 months, MYCN amplification, unfavorable gene expression-based classification, and adverse patient outcome (P < 0.001 each). On treatment with nanomolar doses of BI 2536, all neuroblastoma cell lines analyzed showed significantly reduced proliferation, cell cycle arrest, and cell death. Moreover, BI 2536 abrogated growth of neuroblastoma xenografts in nude mice. Elevated PLK1 expression is significantly associated with high-risk neuroblastoma and unfavorable patient outcome. Inhibition of PLK1 using BI 2536 exhibits strong antitumor activity on human neuroblastoma cells in vitro and in vivo, opening encouraging new perspectives for the treatment of high-risk neuroblastoma. ©2010 AACR.

The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

PubMed

Guan, J; Tucker, E R; Wan, H; Chand, D; Danielson, L S; Ruuth, K; El Wakil, A; Witek, B; Jamin, Y; Umapathy, G; Robinson, S P; Johnson, T W; Smeal, T; Martinsson, T; Chesler, L; Palmer, R H; Hallberg, B

2016-09-01

The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients. © 2016. Published by The Company of Biologists Ltd.

MicroRNA-542-5p as a Novel Tumor Suppressor in Neuroblastoma

PubMed Central

Bray, Isabella; Tivnan, Amanda; Bryan, Kenneth; Foley, Niamh H; Watters, Karen M; Tracey, Lorraine; Davidoff, Andrew M; Stallings, Raymond L

2011-01-01

Several studies have implicated the dysregulation of microRNAs in neuroblastoma pathogenesis, an often fatal paediatric cancer arising from precursor cells of the sympathetic nervous system. Our group and others have demonstrated that lower expression of miR-542-5p is highly associated with poor patient survival, indicating a potential tumor suppressive function. Here, we demonstrate that ectopic over-expression of this miRNA decreases the invasive potential of neuroblastoma cell lines in vitro, along with primary tumor growth and metastases in an orthotopic mouse xenograft model, providing the first functional evidence for the involvement of miR-542-5p as a tumor suppressor in any type of cancer. PMID:21310526

High-Dose, Single-Fraction Irradiation Rapidly Reduces Tumor Vasculature and Perfusion in a Xenograft Model of Neuroblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jani, Ashish; Shaikh, Fauzia; Barton, Sunjay

Purpose: To characterize the effects of high-dose radiation therapy (HDRT) on neuroblastoma tumor vasculature, including the endothelial cell (EC)–pericyte interaction as a potential target for combined treatment with antiangiogenic agents. Methods and Materials: The vascular effects of radiation therapy were examined in a xenograft model of high-risk neuroblastoma. In vivo 3-dimensional contrast-enhanced ultrasonography (3D-CEUS) imaging and immunohistochemistry (IHC) were performed. Results: HDRT significantly reduced tumor blood volume 6 hours after irradiation compared with the lower doses used in conventionally fractionated radiation. There was a 63% decrease in tumor blood volume after 12-Gy radiation compared with a 24% decrease after 2 Gy. Analysis ofmore » tumor vasculature by lectin angiography showed a significant loss of small vessel ends at 6 hours. IHC revealed a significant loss of ECs at 6 and 72 hours after HDRT, with an accompanying loss of immature and mature pericytes at 72 hours. Conclusions: HDRT affects tumor vasculature in a manner not observed at lower doses. The main observation was an early reduction in tumor perfusion resulting from a reduction of small vessel ends with a corresponding loss of endothelial cells and pericytes.« less

Novel multi-targeted ErbB family inhibitor afatinib blocks EGF-induced signaling and induces apoptosis in neuroblastoma.

PubMed

Mao, Xinfang; Chen, Zhenghu; Zhao, Yanling; Yu, Yang; Guan, Shan; Woodfield, Sarah E; Vasudevan, Sanjeev A; Tao, Ling; Pang, Jonathan C; Lu, Jiaxiong; Zhang, Huiyuan; Zhang, Fuchun; Yang, Jianhua

2017-01-03

Neuroblastoma is the most common extracranial solid tumor in children. The ErbB family of proteins is a group of receptor tyrosine kinases that promote the progression of various malignant cancers including neuroblastoma. Thus, targeting them with small molecule inhibitors is a promising strategy for neuroblastoma therapy. In this study, we investigated the anti-tumor effect of afatinib, an irreversible inhibitor of members of the ErbB family, on neuroblastoma. We found that afatinib suppressed the proliferation and colony formation ability of neuroblastoma cell lines in a dose-dependent manner. Afatinib also induced apoptosis and blocked EGF-induced activation of PI3K/AKT/mTOR signaling in all neuroblastoma cell lines tested. In addition, afatinib enhanced doxorubicin-induced cytotoxicity in neuroblastoma cells, including the chemoresistant LA-N-6 cell line. Finally, afatinib exhibited antitumor efficacy in vivo by inducing apoptosis in an orthotopic xenograft neuroblastoma mouse model. Taken together, these results show that afatinib inhibits neuroblastoma growth both in vitro and in vivo by suppressing EGFR-mediated PI3K/AKT/mTOR signaling. Our study supports the idea that EGFR is a potential therapeutic target in neuroblastoma. And targeting ErbB family protein kinases with small molecule inhibitors like afatinib alone or in combination with doxorubicin is a viable option for treating neuroblastoma.

Polyamine pathway inhibition as a novel therapeutic approach to treating neuroblastoma

PubMed Central

Gamble, Laura D.; Hogarty, Michael D.; Liu, Xueyuan; Ziegler, David S.; Marshall, Glenn; Norris, Murray D.; Haber, Michelle

2012-01-01

Polyamines are highly regulated essential cations that are elevated in rapidly proliferating tissues, including diverse cancers. Expression analyses in neuroblastomas suggest that up-regulation of polyamine pro-synthetic enzymes and down-regulation of catabolic enzymes is associated with poor prognosis. Polyamine sufficiency may be required for MYCN oncogenicity in MYCN amplified neuroblastoma, and targeting polyamine homeostasis may therefore provide an attractive therapeutic approach. ODC1, an oncogenic MYCN target, is rate-limiting for polyamine synthesis, and is overexpressed in many cancers including neuroblastoma. Inhibition of ODC1 by difluoromethylornithine (DFMO) decreased tumor penetrance in TH-MYCN mice treated pre-emptively, and extended survival and synergized with chemotherapy in treating established tumors in both TH-MYCN and xenograft models. Efforts to augment DFMO activity, or otherwise maximally reduce polyamine levels, are focused on antagonizing polyamine uptake or augmenting polyamine export or catabolism. Since polyamine inhibition appears to be clinically well tolerated, these approaches, particularly when combined with chemotherapy, have great potential for improving neuroblastoma outcome in both MYCN amplified and non-MYCN amplified neuroblastomas. PMID:23181218

Didymin: an orally active citrus flavonoid for targeting neuroblastoma

PubMed Central

Singhal, Sharad S.; Singhal, Sulabh; Singhal, Preeti; Singhal, Jyotsana; Horne, David; Awasthi, Sanjay

2017-01-01

Neuroblastoma, a rapidly growing yet treatment responsive cancer, is the third most common cancer of children and the most common solid tumor in infants. Unfortunately, neuroblastoma that has lost p53 function often has a highly treatment-resistant phenotype leading to tragic outcomes. In the context of neuroblastoma, the functions of p53 and MYCN (which is amplified in ~25% of neuroblastomas) are integrally linked because they are mutually transcriptionally regulated, and because they together regulate the catalytic activity of RNA polymerases. Didymin is a citrus-derived natural compound that kills p53 wild-type as well as drug-resistant p53-mutant neuroblastoma cells in culture. In addition, orally administered didymin causes regression of neuroblastoma xenografts in mouse models, without toxicity to non-malignant cells, neural tissues, or neural stem cells. RKIP is a Raf-inhibitory protein that regulates MYCN activation, is transcriptionally upregulated by didymin, and appears to play a key role in the anti-neuroblastoma actions of didymin. In this review, we discuss how didymin overcomes drug-resistance in p53-mutant neuroblastoma through RKIP-mediated inhibition of MYCN and its effects on GRK2, PKCs, Let-7 micro-RNA, and clathrin-dependent endocytosis by Raf-dependent and -independent mechanisms. In addition, we will discuss studies supporting potential clinical impact and translation of didymin as a low cost, safe, and effective oral agent that could change the current treatment paradigm for refractory neuroblastoma. PMID:28187004

Didymin: an orally active citrus flavonoid for targeting neuroblastoma.

PubMed

Singhal, Sharad S; Singhal, Sulabh; Singhal, Preeti; Singhal, Jyotsana; Horne, David; Awasthi, Sanjay

2017-04-25

Neuroblastoma, a rapidly growing yet treatment responsive cancer, is the third most common cancer of children and the most common solid tumor in infants. Unfortunately, neuroblastoma that has lost p53 function often has a highly treatment-resistant phenotype leading to tragic outcomes. In the context of neuroblastoma, the functions of p53 and MYCN (which is amplified in ~25% of neuroblastomas) are integrally linked because they are mutually transcriptionally regulated, and because they together regulate the catalytic activity of RNA polymerases. Didymin is a citrus-derived natural compound that kills p53 wild-type as well as drug-resistant p53-mutant neuroblastoma cells in culture. In addition, orally administered didymin causes regression of neuroblastoma xenografts in mouse models, without toxicity to non-malignant cells, neural tissues, or neural stem cells. RKIP is a Raf-inhibitory protein that regulates MYCN activation, is transcriptionally upregulated by didymin, and appears to play a key role in the anti-neuroblastoma actions of didymin. In this review, we discuss how didymin overcomes drug-resistance in p53-mutant neuroblastoma through RKIP-mediated inhibition of MYCN and its effects on GRK2, PKCs, Let-7 micro-RNA, and clathrin-dependent endocytosis by Raf-dependent and -independent mechanisms. In addition, we will discuss studies supporting potential clinical impact and translation of didymin as a low cost, safe, and effective oral agent that could change the current treatment paradigm for refractory neuroblastoma.

Rho-associated kinase is a therapeutic target in neuroblastoma.

PubMed

Dyberg, Cecilia; Fransson, Susanne; Andonova, Teodora; Sveinbjörnsson, Baldur; Lännerholm-Palm, Jessika; Olsen, Thale K; Forsberg, David; Herlenius, Eric; Martinsson, Tommy; Brodin, Bertha; Kogner, Per; Johnsen, John Inge; Wickström, Malin

2017-08-08

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN -driven neuroblastoma growth in TH- MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

A ketogenic diet supplemented with medium-chain triglycerides enhances the anti-tumor and anti-angiogenic efficacy of chemotherapy on neuroblastoma xenografts in a CD1-nu mouse model.

PubMed

Aminzadeh-Gohari, Sepideh; Feichtinger, René Günther; Vidali, Silvia; Locker, Felix; Rutherford, Tricia; O'Donnel, Maura; Stöger-Kleiber, Andrea; Mayr, Johannes Adalbert; Sperl, Wolfgang; Kofler, Barbara

2017-09-12

Neuroblastoma (NB) is a pediatric malignancy characterized by a marked reduction in aerobic energy metabolism. Recent preclinical data indicate that targeting this metabolic phenotype by a ketogenic diet (KD), especially in combination with calorie restriction, slows tumor growth and enhances metronomic cyclophosphamide (CP) therapy of NB xenografts. Because calorie restriction would be contraindicated in most cancer patients, the aim of the present study was to optimize the KD such that the tumors are sensitized to CP without the need of calorie restriction. In a NB xenograft model, metronomic CP was combined with KDs of different triglyceride compositions and fed to CD1-nu mice ad libitum . Metronomic CP in combination with a KD containing 8-carbon medium-chain triglycerides exerted a robust anti-tumor effect, suppressing growth and causing a significant reduction of tumor blood-vessel density and intratumoral hemorrhage, accompanied by activation of AMP-activated protein kinase in NB cells. Furthermore, the KDs caused a significant reduction in the serum levels of essential amino acids, but increased those of serine, glutamine and glycine. Our data suggest that targeting energy metabolism by a modified KD may be considered as part of a multimodal treatment regimen to improve the efficacy of classic anti-NB therapy.

The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

PubMed

Eliassen, Liv Tone; Berge, Gerd; Leknessund, Arild; Wikman, Mari; Lindin, Inger; Løkke, Cecilie; Ponthan, Frida; Johnsen, John Inge; Sveinbjørnsson, Baldur; Kogner, Per; Flaegstad, Trond; Rekdal, Øystein

2006-08-01

Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN-amplified and non-MYCN-amplified cell lines. Non-transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno- and fluorescence-labeled LfcinB revealed that the peptide co-localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase-6, -7 and -9 followed by cell death. However, neither addition of the pan-caspase inhibitor, zVAD-fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH-SY-5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic- and the mitochondria membrane. Copyright (c) 2006 Wiley-Liss, Inc.

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model.

PubMed

Lu, Jiaxiong; Guan, Shan; Zhao, Yanling; Yu, Yang; Woodfield, Sarah E; Zhang, Huiyuan; Yang, Kristine L; Bieerkehazhi, Shayahati; Qi, Lin; Li, Xiaonan; Gu, Jerry; Xu, Xin; Jin, Jingling; Muscal, Jodi A; Yang, Tianshu; Xu, Guo-Tong; Yang, Jianhua

2017-08-01

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of specific activity on neuroblastoma uptake of I-123-meta-iodobenzylguanidine in nude mice xenografted with SK-N-SH cells.

PubMed

Farahati, J; Coenen, H; Dutschka, K; Stuben, G; Knuhmann, K; Budach, W; Kremens, B; Reiners, C

1997-01-01

The effect of specific activity of meta[I-123]iodobenzylguanidine ([I-123]MIBG) on neuroblastoma uptake was studied in a nude mouse model (NMRI nu/nu) xenografted subcutaneously with SK-N-SH cells. Groups of eight animals received [I-123]MIBG intravenously with a specific activity of greater than or equal to 260 GBq/mu mol (no-carrier-added), 3.7 GBq/mu mol, 37 MBq/mu mol, and 0.37 MBq/mu mol, respectively. All animals in the group injected with 0.37 MBq/mu mol died immediately after the injection. Al 4 and 24 h, there was no significant effect of specific activity on tumor uptake of [I-123]MIBG in the different groups. The uptake of non-tumor tissue was in general lower with 37 MBq/mu mol compared to higher specific activities. The differences in blood, heart, liver, spleen and lungs were statistically significant at 24 h, whereas at 4 h significant differences were only present in the heart, liver and lungs. The results suggest that for the treatment of children with neuroblastoma a lower specific activity of radioiodinated MIBG may minimize the radiation exposure to non-tumor tissue but not to the tumor. Higher mass of MIBG >0.5 mu mol/g, however, is considered as lethal dose in our nude mice model and corresponding doses may cause toxic side effects in human.

Reactivating TP53 signaling by the novel MDM2 inhibitor DS-3032b as a therapeutic option for high-risk neuroblastoma

PubMed Central

Arnhold, Viktor; Schmelz, Karin; Proba, Jutta; Winkler, Annika; Wünschel, Jasmin; Toedling, Joern; Deubzer, Hedwig E.; Künkele, Annette; Eggert, Angelika; Schulte, Johannes H.; Hundsdoerfer, Patrick

2018-01-01

Fewer than 50% of patients with high-risk neuroblastoma survive five years after diagnosis with current treatment protocols. Molecular targeted therapies are expected to improve survival. Although MDM2 has been validated as a promising target in preclinical models, no MDM2 inhibitors have yet entered clinical trials for neuroblastoma patients. Toxic side effects, poor bioavailability and low efficacy of the available MDM2 inhibitors that have entered phase I/II trials drive the development of novel MDM2 inhibitors with an improved risk-benefit profile. We investigated the effect of the novel MDM2 small molecular inhibitor, DS-3032b, on viability, proliferation, senescence, migration, cell cycle arrest and apoptosis in a panel of six neuroblastoma cell lines with different TP53 and MYCN genetic backgrounds, and assessed efficacy in a murine subcutaneous model for high-risk neuroblastoma. Re-analysis of existing expression data from 476 primary neuroblastomas showed that high-level MDM2 expression correlated with poor patient survival. DS-3032b treatment enhanced TP53 target gene expression and induced G1 cell cycle arrest, senescence and apoptosis. CRISPR-mediated MDM2 knockout in neuroblastoma cells mimicked DS-3032b treatment. TP53 signaling was selectively activated by DS-3032b in neuroblastoma cells with wildtype TP53, regardless of the presence of MYCN amplification, but was significantly reduced by TP53 mutations or expression of a dominant-negative TP53 mutant. Oral DS-3032b administration inhibited xenograft tumor growth and prolonged mouse survival. Our in vitro and in vivo data demonstrate that DS-3032b reactivates TP53 signaling even in the presence of MYCN amplification in neuroblastoma cells, to reduce proliferative capacity and cause cytotoxicity. PMID:29416773

Rho-associated kinase is a therapeutic target in neuroblastoma

PubMed Central

Dyberg, Cecilia; Fransson, Susanne; Andonova, Teodora; Sveinbjörnsson, Baldur; Lännerholm-Palm, Jessika; Olsen, Thale K.; Martinsson, Tommy; Brodin, Bertha; Kogner, Per; Johnsen, John Inge

2017-01-01

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma. PMID:28739902

Dextran-Catechin: An anticancer chemically-modified natural compound targeting copper that attenuates neuroblastoma growth

PubMed Central

Vittorio, Orazio; Brandl, Miriam; Cirillo, Giuseppe; Kimpton, Kathleen; Hinde, Elizabeth; Gaus, Katharina; Yee, Eugene; Kumar, Naresh; Duong, Hien; Fleming, Claudia; Haber, Michelle; Norris, Murray; Boyer, Cyrille; Kavallaris, Maria

2016-01-01

Neuroblastoma is frequently diagnosed at advanced stage disease and treatment includes high dose chemotherapy and surgery. Despite the use of aggressive therapy survival rates are poor and children that survive their disease experience long term side effects from their treatment, highlighting the need for effective and less toxic therapies. Catechin is a natural polyphenol with anti-cancer properties and limited side effects, however its mechanism of action is unknown. Here we report that Dextran-Catechin, a conjugated form of catechin that increases serum stability, is preferentially and markedly active against neuroblastoma cells having high levels of intracellular copper, without affecting non-malignant cells. Copper transporter 1 (CTR1) is the main transporter of copper in mammalian cells and it is upregulated in neuroblastoma. Functional studies showed that depletion of CTR1 expression reduced intracellular copper levels and led to a decrease in neuroblastoma cell sensitivity to Dextran-Catechin, implicating copper in the activity of this compound. Mechanistically, Dextran-Catechin was found to react with copper, inducing oxidative stress and decreasing glutathione levels, an intracellular antioxidant and regulator of copper homeostasis. In vivo, Dextran-Catechin significantly attenuated tumour growth in human xenograft and syngeneic models of neuroblastoma. Thus, Dextran-Catechin targets copper, inhibits tumour growth, and may be valuable in the treatment of aggressive neuroblastoma and other cancers dependent on copper for their growth. PMID:27374085

Artemisinin reduces cell proliferation and induces apoptosis in neuroblastoma.

PubMed

Zhu, Shunqin; Liu, Wanhong; Ke, Xiaoxue; Li, Jifu; Hu, Renjian; Cui, Hongjuan; Song, Guanbin

2014-09-01

Artemisinin, a natural product from the Chinese medicinal plant, Artemisia annua L., is commonly used in the treatment of malaria, and has recently been reported to have potent anticancer activity in various types of human tumors. Yet, the effect of artemisinin on neuroblastoma is still unclear. In the present study, we aimed to investigate the effects of artemisinin on neuroblastoma cells. We observed that artemisinin significantly inhibited cell growth and proliferation, and caused cell cycle arrest in the G1 phase in neuroblastoma cell lines. Annexin V-FITC/PI staining assay revealed that artemisinin markedly induced apoptosis. Soft agar assays revealed that artemisinin suppressed the ability of clonogenic formation of neuroblastoma cells and a xenograft study in NOD/SCID mice showed that artemisinin inhibited tumor growth and development in vivo. Therefore, our results suggest that the Chinese medicine artemisinin could serve as a novel potential therapeutic agent in the treatment of neuroblastoma.

Meta-[{sup 211}At]astatobenzylguanidine (MABG): In vivo evaluation in an athymic mouse human neuroblastoma xenograft model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vaidyanathan, G.; Friedman, H.S.; Keir, S.T.

1996-05-01

Because of the short range and high linear energy transfer of {sup 211}At {alpha}-particles, the MIBG analogue MABG might be useful for the therapy of micrometastatic neuroblastoma and previous in vitro studies have demonstrated that under single-cell conditions, the cytotoxicity of MABG is > 1000 times higher than [{sup 131}I]MIBG. A paired label protocol was used to compare the tissue distribution of MABG and [{sup 131}I]MIBG in athymic mice bearing subcutaneous SK-N-SH human neuroblastoma xenografts from 1-24 hr after injection. In tumor, significantly higher (p < 0.05) uptake was observed for MABG (3.8 {plus_minus} 0.8%ID/g vs 3.1 {plus_minus} 0.7%ID/g atmore » 8 hr). Pretreatment with desipramine reduced tumor uptake of MABG by 43%, suggesting that accumulation was related to the uptake-1 mechanism. Significantly higher uptake of MABG also was observed in normal tissue targets. For example, at 8 hr, heart uptake of MABG was 6.0 {plus_minus} 0.9 % ID/g compared with 4.5 {plus_minus} 0.8%ID/g for [{sup 131}I]MIBG. Two strategies were investigated to increase the tumor-to-hear uptake ratio. Pretreatment of mice with unlabeled MIBG (4 mg/kg) increased MABG tumor uptake by 1.5-fold while reducing uptake in several normal tissues including heart. The vesicular uptake blocker tetrabenazine (TBZ; 20 mg/kg), reduced MABG hear uptake by 30% of control values with not significant decrease in tumor levels. We conclude that MABG deserves further evaluation as a potential agent for the treatment of neuroblastoma, particularly in combination with strategies to minimize radiation dose to normal target tissues.« less

Targeted inhibition of histone H3K27 demethylation is effective in high-risk neuroblastoma.

PubMed

Lochmann, Timothy L; Powell, Krista M; Ham, Jungoh; Floros, Konstantinos V; Heisey, Daniel A R; Kurupi, Richard I J; Calbert, Marissa L; Ghotra, Maninderjit S; Greninger, Patricia; Dozmorov, Mikhail; Gowda, Madhu; Souers, Andrew J; Reynolds, C Patrick; Benes, Cyril H; Faber, Anthony C

2018-05-16

High-risk neuroblastoma is often distinguished by amplification of MYCN and loss of differentiation potential. We performed high-throughput drug screening of epigenetic-targeted therapies across a large and diverse tumor cell line panel and uncovered the hypersensitivity of neuroblastoma cells to GSK-J4, a small-molecule dual inhibitor of lysine 27 of histone 3 (H3K27) demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and histone demethylase Jumonji D3 (JMJD3). Mechanistically, GSK-J4 induced neuroblastoma differentiation and endoplasmic reticulum (ER) stress, with accompanying up-regulation of p53 up-regulated modulator of apoptosis (PUMA) and induction of cell death. Retinoic acid (RA)-resistant neuroblastoma cells were sensitive to GSK-J4. In addition, GSK-J4 was effective at blocking the growth of chemorefractory and patient-derived xenograft models of high-risk neuroblastoma in vivo. Furthermore, GSK-J4 and RA combination increased differentiation and ER stress over GSK-J4 effects and limited the growth of neuroblastomas resistant to either drug alone. In MYCN -amplified neuroblastoma, PUMA induction by GSK-J4 sensitized tumors to the B cell lymphoma 2 (BCL-2) inhibitor venetoclax, demonstrating that epigenetic-targeted therapies and BCL-2 homology domain 3 mimetics can be rationally combined to treat this high-risk subset of neuroblastoma. Therefore, H3K27 demethylation inhibition is a promising therapeutic target to treat high-risk neuroblastoma, and H3K27 demethylation can be part of rational combination therapies to induce robust antineuroblastoma activity. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Cyclin-Dependent Kinase Inhibitor AT7519 as a Potential Drug for MYCN-Dependent Neuroblastoma.

PubMed

Dolman, M Emmy M; Poon, Evon; Ebus, Marli E; den Hartog, Ilona J M; van Noesel, Carel J M; Jamin, Yann; Hallsworth, Albert; Robinson, Simon P; Petrie, Kevin; Sparidans, Rolf W; Kok, Robbert J; Versteeg, Rogier; Caron, Huib N; Chesler, Louis; Molenaar, Jan J

2015-11-15

MYCN-dependent neuroblastomas have low cure rates with current multimodal treatment regimens and novel therapeutic drugs are therefore urgently needed. In previous preclinical studies, we have shown that targeted inhibition of cyclin-dependent kinase 2 (CDK2) resulted in specific killing of MYCN-amplified neuroblastoma cells. This study describes the in vivo preclinical evaluation of the CDK inhibitor AT7519. Preclinical drug testing was performed using a panel of MYCN-amplified and MYCN single copy neuroblastoma cell lines and different MYCN-dependent mouse models of neuroblastoma. AT7519 killed MYCN-amplified neuroblastoma cell lines more potently than MYCN single copy cell lines with a median LC50 value of 1.7 compared to 8.1 μmol/L (P = 0.0053) and a significantly stronger induction of apoptosis. Preclinical studies in female NMRI homozygous (nu/nu) mice with neuroblastoma patient-derived MYCN-amplified AMC711T xenografts revealed dose-dependent growth inhibition, which correlated with intratumoral AT7519 levels. CDK2 target inhibition by AT7519 was confirmed by significant reductions in levels of phosphorylated retinoblastoma (p-Rb) and nucleophosmin (p-NPM). AT7519 treatment of Th-MYCN transgenic mice resulted in improved survival and clinically significant tumor regression (average tumor size reduction of 86% at day 7 after treatment initiation). The improved efficacy of AT7519 observed in Th-MYCN mice correlated with higher tumor exposure to the drug. This study strongly suggests that AT7519 is a promising drug for the treatment of high-risk neuroblastoma patients with MYCN amplification. ©2015 American Association for Cancer Research.

Effect of Citrus bergamia juice on human neuroblastoma cells in vitro and in metastatic xenograft models.

PubMed

Navarra, M; Ursino, M R; Ferlazzo, N; Russo, M; Schumacher, U; Valentiner, U

2014-06-01

Neuroblastoma is the most common extracranial pediatric solid tumor with poor prognosis in children with disseminated stage of disease. A number of studies show that molecules largely distributed in commonly consumed fruits and vegetables may have anti-tumor activity. In this study we evaluate the effect of Citrus bergamia (bergamot) juice (BJ) in vitro and in a spontaneous metastatic neuroblastoma SCID mouse model. Qualitative and quantitative characterizations of BJ flavonoid fractions were performed by RP-HPLC/PDA/MS. We show that BJ significantly affects SK-N-SH and LAN-1 cell proliferation in vitro, but fails to reduce primary tumor weight in vivo. Moreover, BJ reduced cell adhesiveness and invasion of LAN-1 and SK-N-SH cells in vitro and the number of pulmonary metastases under consideration of the number of tumor cells in the blood in mice inoculated with LAN-1 cells in vivo. These effects without any apparent sign of systemic toxicity confirm the potential clinical interest of BJ and lay the basis for further investigation in cancer. Copyright © 2014. Published by Elsevier B.V.

Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma.

PubMed

Maser, Tyler; Rich, Maria; Hayes, David; Zhao, Ping; Nagulapally, Abhinav B; Bond, Jeffrey; Saulnier Sholler, Giselle

2017-06-01

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates dopamine and other catecholamines by O-methylation. Tolcapone, a drug commonly used in the treatment of Parkinson's disease, is a potent inhibitor of COMT and previous studies indicate that Tolcapone increases the bioavailability of dopamine in cells. In this study, we demonstrate that Tolcapone kills neuroblastoma (NB) cells in preclinical models by inhibition of COMT. Treating four established NB cells lines (SMS-KCNR, SH-SY5Y, BE(2)-C, CHLA-90) and two primary NB cell lines with Tolcapone for 48 h decreased cell viability in a dose-dependent manner, with IncuCyte imaging and Western blotting indicating that cell death was due to caspase-3-mediated apoptosis. Tolcapone also increased ROS while simultaneously decreasing ATP-per-cell in NB cells. Additionally, COMT was inhibited by siRNA in NB cells and showed similar increases in apoptotic markers compared to Tolcapone. In vivo xenograft models displayed inhibition of tumor growth and a significant decrease in time-to-event in mice treated with Tolcapone compared to untreated mice. These results indicate that Tolcapone is cytotoxic to neuroblastoma cells and invite further studies into Tolcapone as a promising novel therapy for the treatment of neuroblastoma. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism

PubMed Central

Mandriota, Stefano J.; Valentijn, Linda J.; Lesne, Laurence; Betts, David R.; Marino, Denis; Boudal-Khoshbeen, Mary; London, Wendy B.; Rougemont, Anne-Laure; Attiyeh, Edward F.; Maris, John M.; Hogarty, Michael D.; Koster, Jan; Molenaar, Jan J.; Versteeg, Rogier

2015-01-01

Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma. PMID:26053094

PCI-24781 (abexinostat), a novel histone deacetylase inhibitor, induces reactive oxygen species-dependent apoptosis and is synergistic with bortezomib in neuroblastoma

PubMed Central

Sholler, Giselle Saulnier; Currier, Erika A.; Dutta, Akshita; Slavik, Marni A.; Illenye, Sharon A.; Mendonca, Maria Cecilia F.; Dragon, Julie; Roberts, Stephen S.; Bond, Jeffrey P.

2014-01-01

In this study, we investigated the cytotoxic effects of a broad-spectrum histone deacetylase (HDAC) inhibitor, PCI-24781, alone and in combination with the proteasome inhibitor bortezomib in neuroblastoma cell lines. The combination was shown to induce synergistic cytotoxity involving the formation of reactive oxygen species. The cleavage of caspase-3 and PARP, as determined by western blotting, indicated that cell death was primarily due to apoptosis. Xenograft mouse models indicated increased survival among animals treated with this combination. The Notch signaling pathway and MYCN gene expression were quantified by reverse transcription-polymerase chain reaction (PCR) in cells treated with PCI-24781 and bortezomib, alone and in combination. Notch pathway expression increased in response to an HDAC inhibitor. NFKB1 and MYCN were both significantly down regulated. Our results suggest that PCI-24781 and bortezomib are synergistic in neuroblastoma cell lines and may be a new therapeutic strategy for this disease. PMID:25520806

9-ING-41, a small-molecule glycogen synthase kinase-3 inhibitor, is active in neuroblastoma.

PubMed

Ugolkov, Andrey V; Bondarenko, Gennadiy I; Dubrovskyi, Oleksii; Berbegall, Ana P; Navarro, Samuel; Noguera, Rosa; O'Halloran, Thomas V; Hendrix, Mary J; Giles, Francis J; Mazar, Andrew P

2018-05-25

Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3β (GSK-3β) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3β expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3β inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.

Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma.

PubMed

Hassannia, Behrouz; Wiernicki, Bartosz; Ingold, Irina; Qu, Feng; Van Herck, Simon; Tyurina, Yulia Y; Bayır, Hülya; Abhari, Behnaz A; Angeli, Jose Pedro Friedmann; Choi, Sze Men; Meul, Eline; Heyninck, Karen; Declerck, Ken; Chirumamilla, Chandra Sekhar; Lahtela-Kakkonen, Maija; Van Camp, Guy; Krysko, Dmitri V; Ekert, Paul G; Fulda, Simone; De Geest, Bruno G; Conrad, Marcus; Kagan, Valerian E; Berghe, Wim Vanden; Vandenabeele, Peter; Berghe, Tom Vanden

2018-06-25

High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor-like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.

mda-7/IL-24 induces cell death in neuroblastoma through a novel mechanism involving AIF and ATM

PubMed Central

Bhoopathi, Praveen; Lee, Nathaniel; Pradhan, Anjan K.; Shen, Xue-Ning; Das, Swadesh K.; Sarkar, Devanand; Emdad, Luni; Fisher, Paul B.

2016-01-01

Advanced stages of neuroblastoma, the most common extracranial malignant solid tumor of the central nervous system in infants and children, are refractive to therapy. Ectopic expression of melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) promotes broad-spectrum antitumor activity in vitro, in vivo in pre-clinical animal models and in a Phase I clinical trial in patients with advanced cancers, without harming normal cells. mda-7/IL-24 exerts cancer-specific toxicity (apoptosis or toxic autophagy) by promoting ER stress and modulating multiple signal transduction pathways regulating cancer cell growth, invasion, metastasis, survival and angiogenesis. To enhance cancer-selective expression and targeted anti-cancer activity of mda-7/IL-24 we created a tropism-modified Cancer Terminator Virus (Ad.5/3-CTV), which selectively replicates in cancer cells producing robust expression of mda-7/IL-24. We now show that Ad.5/3-CTV induces profound neuroblastoma anti-proliferative activity and apoptosis in a caspase 3/9-independent manner both in vitro and in vivo in a tumor xenograft model. Ad.5/3-CTV promotes these effects through a unique pathway involving apoptosis inducing factor (AIF) translocation into the nucleus. Inhibiting AIF rescued neuroblastoma cells from Ad.5/3-CTV-induced cell death, whereas pan-caspase inhibition failed to promote survival. Ad.5/3-CTV infection of neuroblastoma cells increased ATM phosphorylation instigating nuclear translocation and increased γ–H2AX, triggering nuclear translocation and intensified expression of AIF. These results were validated further using two ATM small molecule inhibitors that attenuated PARP cleavage by inhibiting γ–H2AX, which in turn inhibited AIF changes in Ad.5/3-CTV-infected neuroblastoma cells. Taken together, we elucidate a novel pathway for mda-7/IL-24-induced caspase-independent apoptosis in neuroblastoma cells mediated through modulation of AIF, ATM and γ–H2AX. PMID:27197168

Anti-cancer effect of oncolytic adenovirus-armed shRNA targeting MYCN gene on doxorubicin-resistant neuroblastoma cells.

PubMed

Li, Yuan; Zhuo, Baobiao; Yin, Yiyu; Han, Tao; Li, Shixian; Li, Zhengwei; Wang, Jian

2017-09-09

Chemotherapy is one of the few effective choices for patients with neuroblastoma. However, the development of muti-drug resistance (MDR) to chemotherapy is a major obstacle to the effective treatment of advanced or recurrent neuroblastoma. The muti-drug resistance-associated protein (MRP), which encodes a transmembrane glycoprotein, is a key regulator of MDR. The expression of MRP is a close correlation with MYCN oncogene in neuroblastoma. We have recently shown ZD55-shMYCN (oncolytic virus armed with shRNA against MYCN) can down-regulate MYCN to inhibit tumor cells proliferation and induce apoptosis in neuroblastoma. Here we further report ZD55-shMYCN re-sensitized doxorubicin-resistant cells to doxorubicin (as shown by reduced proliferation, increased apoptosis, and inhibited cell migration), and reduced the in vivo growth rate of neuroblastoma xenografts by down-regulation of MRP expression. Sequential therapy with doxorubicin did not affect the replication of ZD55-shMYCN in doxorubicin-resistant neuroblastoma cells, but decreased the expression of Bcl-2, Bcl-X L , MMP-1. Thus, this synergistic effect of ZD55-shMYCN in combination with doxorubicin provides a novel therapy strategy for doxorubicin-resistant neuroblastoma, and is a promising approach for further clinical development. Copyright © 2017 Elsevier Inc. All rights reserved.

Dual targeting of MDM2 and BCL2 as a therapeutic strategy in neuroblastoma.

PubMed

Van Goethem, Alan; Yigit, Nurten; Moreno-Smith, Myrthala; Vasudevan, Sanjeev A; Barbieri, Eveline; Speleman, Frank; Shohet, Jason; Vandesompele, Jo; Van Maerken, Tom

2017-08-22

Wild-type p53 tumor suppressor activity in neuroblastoma tumors is hampered by increased MDM2 activity, making selective MDM2 antagonists an attractive therapeutic strategy for this childhood malignancy. Since monotherapy in cancer is generally not providing long-lasting clinical responses, we here aimed to identify small molecule drugs that synergize with idasanutlin (RG7388). To this purpose we evaluated 15 targeted drugs in combination with idasanutlin in three p53 wild type neuroblastoma cell lines and identified the BCL2 inhibitor venetoclax (ABT-199) as a promising interaction partner. The venetoclax/idasanutlin combination was consistently found to be highly synergistic in a diverse panel of neuroblastoma cell lines, including cells with high MCL1 expression levels. A more pronounced induction of apoptosis was found to underlie the synergistic interaction, as evidenced by caspase-3/7 and cleaved PARP measurements. Mice carrying orthotopic xenografts of neuroblastoma cells treated with both idasanutlin and venetoclax had drastically lower tumor weights than mice treated with either treatment alone. In conclusion, these data strongly support the further evaluation of dual BCL2/MDM2 targeting as a therapeutic strategy in neuroblastoma.

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Neuroblastoma

PubMed Central

Gillory, Lauren A.; Megison, Michael L.; Stewart, Jerry E.; Mroczek-Musulman, Elizabeth; Nabers, Hugh C.; Waters, Alicia M.; Kelly, Virginia; Coleman, Jennifer M.; Markert, James M.; Gillespie, G. Yancey; Friedman, Gregory K.; Beierle, Elizabeth A.

2013-01-01

Despite intensive research efforts and therapeutic advances over the last few decades, the pediatric neural crest tumor, neuroblastoma, continues to be responsible for over 15% of pediatric cancer deaths. Novel therapeutic options are needed for this tumor. Recently, investigators have shown that mice with syngeneic murine gliomas treated with an engineered, neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ134.5 gene, enabling replication in tumor cells but precluding infection of normal neural cells. We hypothesized that M002 would also be effective in the neural crest tumor, neuroblastoma. We showed that M002 infected, replicated, and decreased survival in neuroblastoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly decreased tumor growth, and that this effect was augmented with the addition of ionizing radiation. Importantly, survival could be increased by subsequent doses of radiation without re-dosing of the virus. Finally, these studies showed that the primary entry protein for oHSV, CD111 was expressed by numerous neuroblastoma cell lines and was also present in human neuroblastoma specimens. We concluded that M002 effectively targeted neuroblastoma and that this oHSV may have potential for use in children with unresponsive or relapsed neuroblastoma. PMID:24130898

Targeted drug delivery using genetically engineered diatom biosilica.

PubMed

Delalat, Bahman; Sheppard, Vonda C; Rasi Ghaemi, Soraya; Rao, Shasha; Prestidge, Clive A; McPhee, Gordon; Rogers, Mary-Louise; Donoghue, Jacqueline F; Pillay, Vinochani; Johns, Terrance G; Kröger, Nils; Voelcker, Nicolas H

2015-11-10

The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.

Calculated and TLD-based absorbed dose estimates for I-131-labeled 3F8 monoclonal antibody in a human neuroblastoma xenograft nude mouse model.

PubMed

Ugur, O; Scott, A M; Kostakoglu, L; Hui, T E; Masterson, M E; Febo, R; Sgouros, G; Rosa, E; Mehta, B M; Fisher, D R

1995-01-01

Preclinical evaluation of the therapeutic potential of radiolabeled antibodies is commonly performed in a xenografted nude mouse model. To assess therapeutic efficacy it is important to estimate the absorbed dose to the tumor and normal tissues of the nude mouse. The current study was designed to accurately measure radiation does to human neuroblastoma xenografts and normal organs in nude mice treated with I-131-labeled 3F8 monoclonal antibody (MoAb) against disialoganglioside GD2 antigen. Absorbed dose estimates were obtained using two different approaches: (1) measurement with teflon-imbedded CaSO4:Dy mini-thermoluminescent dosimeters (TLDs) and (2) calculations using mouse S-factors. The calculated total dose to tumor one week after i.v. injection of the 50 microCi I-131-3F8 MoAb was 604 cGy. The corresponding decay corrected and not corrected TLD measurements were 109 +/- 9 and 48.7 +/- 3.4 cGy respectively. The calculated to TLD-derived dose ratios for tumor ranged from 6.1 at 24 h to 5.5 at 1 week. The light output fading rate was found to depend upon the tissue type within which the TLDs were implanted. The decay rate in tumor, muscle, subcutaneous tissue and in vitro, were 9.5, 5.0, 3.7 and 0.67% per day, respectively. We have demonstrated that the type of tissue in which the TLD was implanted strongly influenced the in vivo decay of light output. Even with decay correction, a significant discrepancy was observed between MIRD-based calculated and CaSO4:Dy mini-TLD measured absorbed doses. Batch dependence, pH of the tumor or other variables associated with TLDs which are not as yet well known may account for this discrepancy.

Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

PubMed

Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

2017-05-01

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients. Published [2016]. This article is a U.S. Government work and is in the public domain in the USA.

Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma.

PubMed

Kocak, H; Ackermann, S; Hero, B; Kahlert, Y; Oberthuer, A; Juraeva, D; Roels, F; Theissen, J; Westermann, F; Deubzer, H; Ehemann, V; Brors, B; Odenthal, M; Berthold, F; Fischer, M

2013-04-11

Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression.

High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition

PubMed Central

Bate-Eya, Laurel T.; den Hartog, Ilona J.M.; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.; Dolman, M. Emmy M.

2016-01-01

The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression. PMID:27056887

Targeted Doxorubicin-Loaded Bacterially Derived Nano-Cells for the Treatment of Neuroblastoma.

PubMed

Sagnella, Sharon M; Trieu, Jennifer; Brahmbhatt, Himanshu; MacDiarmid, Jennifer A; MacMillan, Alex; Whan, Renee M; Fife, Christopher M; McCarroll, Joshua A; Gifford, Andrew J; Ziegler, David S; Kavallaris, Maria

2018-05-01

Advanced stage neuroblastoma is an aggressive disease with limited treatment options for patients with drug-resistant tumors. Targeted delivery of chemotherapy for pediatric cancers offers promise to improve treatment efficacy and reduce toxicity associated with systemic chemotherapy. The EnGeneIC Dream Vector (EDV TM ) is a nanocell, which can package chemotherapeutic drugs and target tumors via attachment of bispecific proteins to the surface of the nanocell. Phase I trials in adults with refractory tumors have shown an acceptable safety profile. Herein we investigated the activity of EGFR-targeted and doxorubicin-loaded EDV TM ( EGFR EDV TM Dox ) for the treatment of neuroblastoma. Two independent neuroblastoma cell lines with variable expression of EGFR protein [SK-N-BE(2), high; SH-SY-5Y, low] were used. EGFR EDV TM Dox induced apoptosis in these cells compared to control, doxorubicin, or non-doxorubicin loaded EGFR EDV TM In three-dimensional tumor spheroids, imaging and fluorescence life-time microscopy revealed that EGFR EDV TM Dox had a marked enhancement of doxorubicin penetration compared to doxorubicin alone, and improved penetration compared to non-EGFR-targeted EDV TM Dox , with enhanced spheroid penetration leading to increased apoptosis. In two independent orthotopic human neuroblastoma xenograft models, short-term studies (28 days) of tumor-bearing mice led to a significant decrease in tumor size in EGFR EDV TM Dox -treated animals compared to control, doxorubicin, or non-EGFR EDV TM Dox There was increased TUNEL staining of tumors at day 28 compared to control, doxorubicin, or non-EGFR EDV TM Dox Moreover, overall survival was increased in neuroblastoma mice treated with EGFR EDV TM Dox ( P < 0007) compared to control. Drug-loaded bispecific-antibody targeted EDVs TM offer a highly promising approach for the treatment of aggressive pediatric malignancies such as neuroblastoma. Mol Cancer Ther; 17(5); 1012-23. ©2018 AACR . ©2018 American Association for Cancer Research.

The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines.

PubMed

Kolbinger, Fiona R; Koeneke, Emily; Ridinger, Johannes; Heimburg, Tino; Müller, Michael; Bayer, Theresa; Sippl, Wolfgang; Jung, Manfred; Gunkel, Nikolas; Miller, Aubry K; Westermann, Frank; Witt, Olaf; Oehme, Ina

2018-06-09

High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma.

PubMed

Lodrini, Marco; Sprüssel, Annika; Astrahantseff, Kathy; Tiburtius, Daniela; Konschak, Robert; Lode, Holger N; Fischer, Matthias; Keilholz, Ulrich; Eggert, Angelika; Deubzer, Hedwig E

2017-10-17

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients.

Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma

PubMed Central

Lodrini, Marco; Sprüssel, Annika; Astrahantseff, Kathy; Tiburtius, Daniela; Konschak, Robert; Lode, Holger N.; Fischer, Matthias; Keilholz, Ulrich; Eggert, Angelika; Deubzer, Hedwig E.

2017-01-01

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients. PMID:29156716

Somatostatin in neuroblastoma and ganglioneuroma.

PubMed

Kogner, P; Borgström, P; Bjellerup, P; Schilling, F H; Refai, E; Jonsson, C; Dominici, C; Wassberg, E; Bihl, H; Jacobsson, H; Theodorsson, E; Hassan, M

1997-10-01

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. Somatostatin may inhibit neuroblastoma growth and induce apoptosis in vitro and was therefore investigated. Using a radioimmunoassay, we found that all ganglioneuromas contained high somatostatin concentrations (> 16 pmol/g), significantly higher than neuroblastomas (n = 117, median 2.8 pmol/g), healthy adrenals, Wilms' tumours, phaeochromocytomas and other neuroendocrine tumours (P < 0.001). Neuroblastomas contained more somatostatin than control tumours (P < 0.001-0.05). Neuroblastomas amplified for the MYCN oncogene contained less somatostatin than non-amplified tumours (1.2 pmol/g versus 4.0 pmol/g, respectively; P = 0.026). In a clinically unfavourable neuroblastoma subset (age > 12 months, stage 3 or 4) 16 children with high concentrations of somatostatin in primary tumours had a better prognosis than 23 with low somatostatin (46.7% versus 0% survival at 5 years, P < 0.005). Scintigraphy using 111In-pentetreotide identified tumours expressing high-affinity somatostatin receptors in vivo. However, no significant correlation was found between somatostatin receptor expression and peptide content in 15 tumours. Similarly, human SH-SY5Y neuroblastoma xenografts grown in nude rats showed low somatostatin concentrations, but were positive for somatostatin receptor scintigraphy. Treatment of these rats with the somatostatin analogue octreotide seemed to upregulate in vivo receptor expression of somatostatin and vasoactive intestinal peptide more effectively than 13-cis retinoic acid. In conclusion, somatostatin in neuroblastoma is associated with differentiation to benign ganglioneuromas in vivo and favourable outcome in advanced tumours. Furthermore, somatostatin receptor scintigraphy may identify tumours with high-affinity receptors in children that might benefit from targeted therapy using synthetic somatostatin analogues.

Dual targeting of wild-type and mutant p53 by small molecule RITA results in the inhibition of N-Myc and key survival oncogenes and kills neuroblastoma cells in vivo and in vitro.

PubMed

Burmakin, Mikhail; Shi, Yao; Hedström, Elisabeth; Kogner, Per; Selivanova, Galina

2013-09-15

Restoration of the p53 function in tumors is a promising therapeutic strategy due to the high potential of p53 as tumor suppressor and the fact that established tumors depend on p53 inactivation for their survival. Here, we addressed the question whether small molecule RITA can reactivate p53 in neuroblastoma and suppress the growth of neuroblastoma cells in vitro and in vivo. The ability of RITA to inhibit growth and to induce apoptosis was shown in seven neuroblastoma cell lines. Mechanistic studies were carried out to determine the p53 dependence and the molecular mechanism of RITA-induced apoptosis in neuroblastoma, using cell viability assays, RNAi silencing, co-immunoprecipitation, qPCR, and Western blotting analysis. In vivo experiments were conducted to study the effect of RITA on human neuroblastoma xenografts in mice. RITA induced p53-dependent apoptosis in a set of seven neuroblastoma cell lines, carrying wild-type or mutant p53; it activated p53 and triggered the expression of proapoptotic p53 target genes. Importantly, p53 activated by RITA inhibited several key oncogenes that are high-priority targets for pharmacologic anticancer strategies in neuroblastoma, including N-Myc, Aurora kinase, Mcl-1, Bcl-2, Wip-1, MDM2, and MDMX. Moreover, RITA had a strong antitumor effect in vivo. Reactivation of wild-type and mutant p53 resulting in the induction of proapoptotic factors along with ablation of key oncogenes by compounds such as RITA may be a highly effective strategy to treat neuroblastoma. ©2013 AACR.

High-Affinity GD2-Specific CAR T Cells Induce Fatal Encephalitis in a Preclinical Neuroblastoma Model

PubMed Central

Richman, Sarah A.; Nunez-Cruz, Selene; Moghimi, Babak; Li, Lucy Z.; Gershenson, Zachary T.; Mourelatos, Zissimos; Barrett, David M.; Grupp, Stephan A.; Milone, Michael C.

2018-01-01

The GD2 ganglioside, which is abundant on the surface of neuroblastoma cells, is targeted by an FDA-approved therapeutic monoclonal antibody and is an attractive tumor-associated antigen for cellular immunotherapy. Chimeric antigen receptor (CAR)–modified T cells can have potent antitumor activity in B-cell malignancies, and trials to harness this cytolytic activity toward GD2 in neuroblastoma are under way. In an effort to enhance the antitumor activity of CAR T cells that target GD2, we generated variant CAR constructs predicted to improve the stability and the affinity of the GD2-binding, 14G2a-based, single-chain variable fragment (scFv) of the CAR and compared their properties in vivo. We included the E101K mutation of GD2 scFv (GD2-E101K) that has enhanced antitumor activity against a GD2+ human neuroblastoma xenograft in vivo. However, this enhanced antitumor efficacy in vivo was concomitantly associated with lethal central nervous system (CNS) toxicity comprised of extensive CAR T-cell infiltration and proliferation within the brain and neuronal destruction. The encephalitis was localized to the cerebellum and basal regions of the brain that display low amounts of GD2. Our results highlight the challenges associated with target antigens that exhibit shared expression on critical normal tissues. Despite the success of GD2-specific antibody therapies in the treatment of neuroblastoma, the fatal neurotoxicity of GD2-specific CAR T-cell therapy observed in our studies suggests that GD2 may be a difficult target antigen for CAR T-cell therapy without additional strategies that can control CAR T-cell function within the CNS. PMID:29180536

The Proteasome Inhibitor Bortezomib Enhances ATRA-Induced Differentiation of Neuroblastoma Cells via the JNK Mitogen-Activated Protein Kinase Pathway

PubMed Central

Luo, Peihua; Lin, Meili; Li, Lin; Yang, Bo; He, Qiaojun

2011-01-01

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib. PMID:22087283

Antagonizing Bcl-2 Family Members Sensitizes Neuroblastoma and Ewing’s Sarcoma to an Inhibitor of Glutamine Metabolism

PubMed Central

Olsen, Rachelle R.; Mary-Sinclair, Michelle N.; Yin, Zhirong; Freeman, Kevin W.

2015-01-01

Neuroblastomas (NBL) and Ewing’s sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated metabolism of cancer as a therapeutic strategy, this approach has not been fully examined in NBL and EWS. In this study, we first tested a panel of metabolic inhibitors and identified the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) as the most potent chemotherapeutic across all NBL and EWS cell lines tested. Myc, a master regulator of metabolism, is commonly overexpressed in both of these pediatric malignancies and recent studies have established that Myc causes cancer cells to become “addicted” to glutamine. We found DON strongly inhibited tumor growth of multiple tumor lines in mouse xenograft models. In vitro, inhibition of caspases partially reversed the effects of DON in high Myc expressing cell lines, but not in low Myc expressing lines. We further showed that induction of apoptosis by DON in Myc-overexpressing cancers is via the pro-apoptotic factor Bax. To relieve inhibition of Bax, we tested DON in combination with the Bcl-2 family antagonist navitoclax (ABT-263). In vitro, this combination caused an increase in DON activity across the entire panel of cell lines tested, with synergistic effects in two of the N-Myc amplified neuroblastoma cell lines. Our study supports targeting glutamine metabolism to treat Myc overexpressing cancers, such as NBL and EWS, particularly in combination with Bcl-2 family antagonists. PMID:25615615

Anti-tumor effect of AZD8055 against neuroblastoma cells in vitro and in vivo.

PubMed

Xu, Dong-Qing; Toyoda, Hidemi; Yuan, Xiao-Jun; Qi, Lei; Chelakkot, Vipin Shankar; Morimoto, Mari; Hanaki, Ryo; Kihira, Kentarou; Hori, Hiroki; Komada, Yoshihiro; Hirayama, Masahiro

2018-04-15

Neuroblastoma (NB) is one of the most common solid tumors in children. High-risk NB remains lethal in about 50% of patients despite comprehensive and intensive treatments. Activation of PI3K/Akt/mTOR signaling pathway correlates with oncogenesis, poor prognosis and chemotherapy resistance in NB. Due to its central role in growth and metabolism, mTOR seems to be an important factor in NB, making it a possible target for NB. In this study, we investigated the effect of AZD8055, a potent dual mTORC1-mTORC2 inhibitor, in NB cell lines. Our data showed that mTOR signaling was extensively activated in NB cells. The activity of mTOR and downstream molecules were down-regulated in AZD8055-treated NB cells. Significantly, AZD8055 effectively inhibited cell growth and induced cell cycle arrest, autophagy and apoptosis in NB cells. Moreover, AZD8055 significantly reduced tumor growth in mice xenograft model without apparent toxicity. Taken together, our results highlight the potential of mTOR as a promising target for NB treatment. Therefore, AZD8055 may be further investigated for treatment in clinical trials for high risk NB. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of Critical Domains within the Tumor Suppressor CASZ1 Required for Transcriptional Regulation and Growth Suppression

PubMed Central

Virden, Ryan A.

2012-01-01

CASZ1 is a zinc finger (ZF) transcription factor that is critical for controlling the normal differentiation of subtypes of neural and cardiac muscle cells. In neuroblastoma tumors, loss of CASZ1 is associated with poor prognosis and restoration of CASZ1 function suppresses neuroblastoma tumorigenicity. However, the key domains by which CASZ1 transcription controls developmental processes and neuroblastoma tumorigenicity have yet to be elucidated. In this study, we show that loss of any one of ZF1 to ZF4 resulted in a 58 to 79% loss in transcriptional activity, as measured by induction of tyrosine hydroxylase promoter-luciferase activity, compared to that of wild-type (WT) CASZ1b. Mutation of ZF5 or deletion of the C-terminal sequence of amino acids (aa) 728 to 1166 (a truncation of 38% of the protein) does not significantly alter transcriptional function. A series of N-terminal truncations reveals a critical transcriptional activation domain at aa 31 to 185 and a nuclear localization signal at aa 23 to 29. Soft agar colony formation assays and xenograft studies show that WT CASZ1b is more active in suppressing neuroblastoma growth than CASZ1b with a ZF4 mutation or a deletion of aa 31 to 185. This study identifies key domains needed for CASZ1b to regulate gene transcription. Furthermore, we establish a link between loss of CASZ1b transcriptional activity and attenuation of CASZ1b-mediated inhibition of neuroblastoma growth and tumorigenicity. PMID:22331471

miR-137 regulates the constitutive androstane receptor and modulates doxorubicin sensitivity in parental and doxorubicin-resistant neuroblastoma cells

PubMed Central

Takwi, Apana A; Wang, Yue-Ming; Wu, Jing; Michaelis, Martin; Cinatl, Jindrich; Chen, Taosheng

2013-01-01

Chemotherapy is the most common treatment for cancer. However, multidrug resistance (MDR) remains a major obstacle to effective chemotherapy, limiting the efficacy of both conventional chemotherapeutic and novel biologic agents. The constitutive androstane receptor (CAR), a xenosensor, is a key regulator of MDR. It functions in xenobiotic detoxification by regulating the expression of phase I drug metabolizing enzymes and ATP-binding cassette (ABC) transporters, whose overexpression in cancers and whose role in drug resistance make them potential therapeutic targets for reducing MDR. MicroRNAs (miRNAs) are endogenous negative regulators of gene expression and have been implicated in most cellular processes, including drug resistance. Here we report the inversely related expression of miR-137 and CAR in parental and doxorubicin-resistant neuroblastoma cells, wherein miR-137 is down-regulated in resistant cells. miR-137 over-expression resulted in down-regulation of CAR protein and mRNA (via mRNA degradation); it sensitized doxorubicin-resistant cells to doxorubicin (as shown by reduced proliferation, increased apoptosis, and increased G2-phase cell cycle arrest) and reduced the in vivo growth rate of neuroblastoma xenografts. We observed similar results in cellular models of hepatocellular and colon cancers, indicating that the doxorubicin-sensitizing effect of miR-137 is not tumor type-specific. Finally, we show for the first time a negative feedback loop whereby miR-137 down-regulates CAR expression and CAR down-regulates miR-137 expression. Hypermethylation of the miR-137 promoter and negative regulation of miR-137 by CAR contribute in part to reduced miR-137 expression and increased CAR and MDR1 expression in doxorubicin-resistant neuroblastoma cells. These findings demonstrate that miR-137 is a crucial regulator of cancer response to doxorubicin treatment, and they identify miR-137 as a highly promising target to reduce CAR-driven doxorubicin resistance. PMID:23934188

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma.

PubMed

Fisher, T; Golan, H; Schiby, G; PriChen, S; Smoum, R; Moshe, I; Peshes-Yaloz, N; Castiel, A; Waldman, D; Gallily, R; Mechoulam, R; Toren, A

2016-03-01

Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma

PubMed Central

Fisher, T.; Golan, H.; Schiby, G.; PriChen, S.; Smoum, R.; Moshe, I.; Peshes-Yaloz, N.; Castiel, A.; Waldman, D.; Gallily, R.; Mechoulam, R.; Toren, A.

2016-01-01

Background Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. Methods We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ9-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Results Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Conclusions Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl. PMID:27022310

Improved outcome of 131I-mIBG treatment through combination with external beam radiotherapy in the SK-N-SH mouse model of neuroblastoma.

PubMed

Corroyer-Dulmont, Aurélien; Falzone, Nadia; Kersemans, Veerle; Thompson, James; Allen, Danny P; Able, Sarah; Kartsonaki, Christiana; Malcolm, Javian; Kinchesh, Paul; Hill, Mark A; Vojnovic, Boris; Smart, Sean C; Gaze, Mark N; Vallis, Katherine A

2017-09-01

To assess the efficacy of different schedules for combining external beam radiotherapy (EBRT) with molecular radiotherapy (MRT) using 131 I-mIBG in the management of neuroblastoma. BALB/c nu/nu mice bearing SK-N-SH neuroblastoma xenografts were assigned to five treatment groups: 131 I-mIBG 24h after EBRT, EBRT 6days after 131 I-mIBG, EBRT alone, 131 I-mIBG alone and control (untreated). A total of 56 mice were assigned to 3 studies. Study 1: Vessel permeability was evaluated using dynamic contrast-enhanced (DCE)-MRI (n=3). Study 2: Tumour uptake of 131 I-mIBG in excised lesions was evaluated by γ-counting and autoradiography (n=28). Study 3: Tumour volume was assessed by longitudinal MR imaging and survival was analysed (n=25). Tumour dosimetry was performed using Monte Carlo simulations of absorbed fractions with the radiation transport code PENELOPE. Given alone, both 131 I-mIBG and EBRT resulted in a seven-day delay in tumour regrowth. Following EBRT, vessel permeability was evaluated by DCE-MRI and showed an increase at 24h post irradiation that correlated with an increase in 131 I-mIBG tumour uptake, absorbed dose and overall survival in the case of combined treatment. Similarly, EBRT administered seven days after MRT to coincide with tumour regrowth, significantly decreased the tumour volume and increased overall survival. This study demonstrates that combining EBRT and MRT has an enhanced therapeutic effect and emphasizes the importance of treatment scheduling according to pathophysiological criteria such as tumour vessel permeability and tumour growth kinetics. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma.

PubMed

Sime, Wondossen; Niu, Qiankun; Abassi, Yasmin; Masoumi, Katarzyna Chmielarska; Zarrizi, Reihaneh; Køhler, Julie Bonne; Kjellström, Sven; Lasorsa, Vito Alessandro; Capasso, Mario; Fu, Haian; Massoumi, Ramin

2018-04-24

BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells.

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway.

PubMed

Thress, Kenneth; Macintyre, Terry; Wang, Haiyun; Whitston, Dave; Liu, Zhong-Ying; Hoffmann, Ethan; Wang, Tao; Brown, Jeffrey L; Webster, Kevin; Omer, Charles; Zage, Peter E; Zeng, Lizhi; Zweidler-McKay, Patrick A

2009-07-01

Tropomyosin-related kinases (TrkA, TrkB, and TrkC) are receptor tyrosine kinases that, along with their ligands, the neurotrophins, are involved in neuronal cell growth, development, and survival. The Trk-neurotrophin pathway may also play a role in tumorigenesis through oncogenic fusions, mutations, and autocrine signaling, prompting the development of novel Trk inhibitors as agents for cancer therapy. This report describes the identification of AZ-23, a novel, potent, and selective Trk kinase inhibitor. In vitro studies with AZ-23 showed improved selectivity over previous compounds and inhibition of Trk kinase activity in cells at low nanomolar concentrations. AZ-23 showed in vivo TrkA kinase inhibition and efficacy in mice following oral administration in a TrkA-driven allograft model and significant tumor growth inhibition in a Trk-expressing xenograft model of neuroblastoma. AZ-23 represents a potent and selective Trk kinase inhibitor from a novel series with the potential for use as a treatment for cancer.

68Ga-DOTATOC and FDG PET Imaging of Preclinical Neuroblastoma Models.

PubMed

Provost, Claire; Prignon, Aurélie; Cazes, Alex; Combaret, Valérie; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Montravers, Françoise; Talbot, Jean-Noël

2016-09-01

Somatostatine receptors subtype 2 (SSTR2) are regarded as a potential target in neuroblastoma (NB) for imaging and promising therapeutic approaches. The purpose of this study was to evaluate and compare the SSTR2 status by (68)Ga-[tetraxetan-D-Phe1, Tyr3]-octreotide ((68)Ga-DOTATOC) positron-emission tomography (PET) and the tumour metabolic activity by (18)F-fluorodeoxyglucose (FDG) PET in different experimental models of NB. Three cell lines of human NB with different levels of expression of SSTR2 were grafted into nude mice. Animals were imaged with FDG and (68)Ga-DOTATOC and the maximum standardized uptake value (SUVmax) was determined to quantify tracer uptake. Ex vivo biodistribution of (68)Ga-DOTATOC and immunohistochemical analysis of NB xenografts were performed. Compared with FDG, the SUVmax of (68)Ga-DOTATOC uptake by the tumour was lower but the ratio to background was higher; there was a strong positive correlation between SUVmax values observed with the two tracers (r(2)=0.65). Sorting the cell lines according to uptake of FDG or (68)Ga-DOTATOC, injected activity per gram of tissue, Ki67 index or expression of SSTR2 assessed visually led to the same classification. (68)Ga-DOTATOC allows preclinical imaging of NB according to the intensity of the expression of SSTR2. In contrast with what has been reported for neuroendocrine tumours, in this NB model, the (68)Ga-DOTATOC uptake was positively correlated with FDG uptake and with Ki67 index, usual markers of tumour aggressiveness. If confirmed in humans, this result would favour a theranostic application of (68)Ga-DOTATOC in NB, even in advanced stages. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN

DTIC Science & Technology

2014-09-01

AWARD NUMBER: W81XWH-13-1-0220 TITLE: A Genetically Engineered Mouse Model of Neuroblastoma ...CONTRACT NUMBER A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN 5b. GRANT NUMBER W81XWH-13-1-0220 5c...common ALK mutations in neuroblastoma , F1174L and R1275Q. We have determined that in tumors cells expressing mutated ALK, different downstream

Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

PubMed

Babae, Negar; Bourajjaj, Meriem; Liu, Yijia; Van Beijnum, Judy R; Cerisoli, Francesco; Scaria, Puthupparampil V; Verheul, Mark; Van Berkel, Maaike P; Pieters, Ebel H E; Van Haastert, Rick J; Yousefi, Afrouz; Mastrobattista, Enrico; Storm, Gert; Berezikov, Eugene; Cuppen, Edwin; Woodle, Martin; Schaapveld, Roel Q J; Prevost, Gregoire P; Griffioen, Arjan W; Van Noort, Paula I; Schiffelers, Raymond M

2014-08-30

Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

Network Modeling of microRNA-mRNA Interactions in Neuroblastoma Tumorigenesis Identifies miR-204 as a Direct Inhibitor of MYCN.

PubMed

Ooi, Chi Yan; Carter, Daniel R; Liu, Bing; Mayoh, Chelsea; Beckers, Anneleen; Lalwani, Amit; Nagy, Zsuzsanna; De Brouwer, Sara; Decaesteker, Bieke; Hung, Tzong-Tyng; Norris, Murray D; Haber, Michelle; Liu, Tao; De Preter, Katleen; Speleman, Frank; Cheung, Belamy B; Marshall, Glenn M

2018-06-15

Neuroblastoma is a pediatric cancer of the sympathetic nervous system where MYCN amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN +/+ mice to age-matched wild-type controls, we devised a predicted miRNA-mRNA interaction network. Among the miRNA-mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with MYCN -amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation in vitro and tumorigenesis in vivo Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis. Significance: Network modeling of miRNA-mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. Cancer Res; 78(12); 3122-34. ©2018 AACR . ©2018 American Association for Cancer Research.

Predicting neuroblastoma using developmental signals and a logic-based model.

PubMed

Kasemeier-Kulesa, Jennifer C; Schnell, Santiago; Woolley, Thomas; Spengler, Jennifer A; Morrison, Jason A; McKinney, Mary C; Pushel, Irina; Wolfe, Lauren A; Kulesa, Paul M

2018-07-01

Genomic information from human patient samples of pediatric neuroblastoma cancers and known outcomes have led to specific gene lists put forward as high risk for disease progression. However, the reliance on gene expression correlations rather than mechanistic insight has shown limited potential and suggests a critical need for molecular network models that better predict neuroblastoma progression. In this study, we construct and simulate a molecular network of developmental genes and downstream signals in a 6-gene input logic model that predicts a favorable/unfavorable outcome based on the outcome of the four cell states including cell differentiation, proliferation, apoptosis, and angiogenesis. We simulate the mis-expression of the tyrosine receptor kinases, trkA and trkB, two prognostic indicators of neuroblastoma, and find differences in the number and probability distribution of steady state outcomes. We validate the mechanistic model assumptions using RNAseq of the SHSY5Y human neuroblastoma cell line to define the input states and confirm the predicted outcome with antibody staining. Lastly, we apply input gene signatures from 77 published human patient samples and show that our model makes more accurate disease outcome predictions for early stage disease than any current neuroblastoma gene list. These findings highlight the predictive strength of a logic-based model based on developmental genes and offer a better understanding of the molecular network interactions during neuroblastoma disease progression. Copyright © 2018. Published by Elsevier B.V.

Neuroblastoma, a Paradigm for Big Data Science in Pediatric Oncology

PubMed Central

Salazar, Brittany M.; Balczewski, Emily A.; Ung, Choong Yong; Zhu, Shizhen

2016-01-01

Pediatric cancers rarely exhibit recurrent mutational events when compared to most adult cancers. This poses a challenge in understanding how cancers initiate, progress, and metastasize in early childhood. Also, due to limited detected driver mutations, it is difficult to benchmark key genes for drug development. In this review, we use neuroblastoma, a pediatric solid tumor of neural crest origin, as a paradigm for exploring “big data” applications in pediatric oncology. Computational strategies derived from big data science–network- and machine learning-based modeling and drug repositioning—hold the promise of shedding new light on the molecular mechanisms driving neuroblastoma pathogenesis and identifying potential therapeutics to combat this devastating disease. These strategies integrate robust data input, from genomic and transcriptomic studies, clinical data, and in vivo and in vitro experimental models specific to neuroblastoma and other types of cancers that closely mimic its biological characteristics. We discuss contexts in which “big data” and computational approaches, especially network-based modeling, may advance neuroblastoma research, describe currently available data and resources, and propose future models of strategic data collection and analyses for neuroblastoma and other related diseases. PMID:28035989

Neuroblastoma, a Paradigm for Big Data Science in Pediatric Oncology.

PubMed

Salazar, Brittany M; Balczewski, Emily A; Ung, Choong Yong; Zhu, Shizhen

2016-12-27

Pediatric cancers rarely exhibit recurrent mutational events when compared to most adult cancers. This poses a challenge in understanding how cancers initiate, progress, and metastasize in early childhood. Also, due to limited detected driver mutations, it is difficult to benchmark key genes for drug development. In this review, we use neuroblastoma, a pediatric solid tumor of neural crest origin, as a paradigm for exploring "big data" applications in pediatric oncology. Computational strategies derived from big data science-network- and machine learning-based modeling and drug repositioning-hold the promise of shedding new light on the molecular mechanisms driving neuroblastoma pathogenesis and identifying potential therapeutics to combat this devastating disease. These strategies integrate robust data input, from genomic and transcriptomic studies, clinical data, and in vivo and in vitro experimental models specific to neuroblastoma and other types of cancers that closely mimic its biological characteristics. We discuss contexts in which "big data" and computational approaches, especially network-based modeling, may advance neuroblastoma research, describe currently available data and resources, and propose future models of strategic data collection and analyses for neuroblastoma and other related diseases.

Mouse neuroblastoma cell based model and the effect of epileptic events on calcium oscillations and neural spikes

NASA Astrophysics Data System (ADS)

Kim, Suhwan; Baek, Juyeong; Jung, Unsang; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

2013-05-01

Recently, Mouse neuroblastoma cells are considered as an attractive model for the study of human neurological and prion diseases, and intensively used as a model system in different areas. Among those areas, differentiation of neuro2a (N2A) cells, receptor mediated ion current, and glutamate induced physiological response are actively investigated. The reason for the interest to mouse neuroblastoma N2A cells is that they have a fast growing rate than other cells in neural origin with a few another advantages. This study evaluated the calcium oscillations and neural spikes recording of mouse neuroblastoma N2A cells in an epileptic condition. Based on our observation of neural spikes in mouse N2A cell with our proposed imaging modality, we report that mouse neuroblastoma N2A cells can be an important model related to epileptic activity studies. It is concluded that the mouse neuroblastoma N2A cells produce the epileptic spikes in vitro in the same way as produced by the neurons or the astrocytes. This evidence advocates the increased and strong level of neurotransmitters release by enhancement in free calcium using the 4-aminopyridine which causes the mouse neuroblastoma N2A cells to produce the epileptic spikes and calcium oscillation.

Inhibition of serine/threonine phosphatase PP2A enhances cancer chemotherapy by blocking DNA damage induced defense mechanisms.

PubMed

Lu, Jie; Kovach, John S; Johnson, Francis; Chiang, Jeffrey; Hodes, Richard; Lonser, Russell; Zhuang, Zhengping

2009-07-14

A variety of mechanisms maintain the integrity of the genome in the face of cell stress. Cancer cell response to chemotherapeutic and radiation-induced DNA damage is mediated by multiple defense mechanisms including polo-like kinase 1 (Plk-1), protein kinase B (Akt-1), and/or p53 pathways leading to either apoptosis or cell cycle arrest. Subsequently, a subpopulation of arrested viable cancer cells may remain and recur despite aggressive and repetitive therapy. Here, we show that modulation (activation of Akt-1 and Plk-1 and repression of p53) of these pathways simultaneously results in paradoxical enhancement of the effectiveness of cytotoxic chemotherapy. We demonstrate that a small molecule inhibitor, LB-1.2, of protein phosphatase 2A (PP2A) activates Plk-1 and Akt-1 and decreases p53 abundance in tumor cells. Combined with temozolomide (TMZ; a DNA-methylating chemotherapeutic drug), LB-1.2 causes complete regression of glioblastoma multiforme (GBM) xenografts without recurrence in 50% of animals (up to 28 weeks) and complete inhibition of growth of neuroblastoma (NB) xenografts. Treatment with either drug alone results in only short-term inhibition/regression with all xenografts resuming rapid growth. Combined with another widely used anticancer drug, Doxorubicin (DOX, a DNA intercalating agent), LB-1.2 also causes marked GBM xenograft regression, whereas DOX alone only slows growth. Inhibition of PP2A by LB-1.2 blocks cell-cycle arrest and increases progression of cell cycle in the presence of TMZ or DOX. Pharmacologic inhibition of PP2A may be a general method for enhancing the effectiveness of cancer treatments that damage DNA or disrupt components of cell replication.

Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC

PubMed Central

Hadjidaniel, Michael D.; Muthugounder, Sakunthala; Hung, Long T.; Sheard, Michael A.; Shirinbak, Soheila; Chan, Randall Y.; Nakata, Rie; Borriello, Lucia; Malvar, Jemily; Kennedy, Rebekah J.; Iwakura, Hiroshi; Akamizu, Takashi; Sposto, Richard; Shimada, Hiroyuki; DeClerck, Yves A.; Asgharzadeh, Shahab

2017-01-01

Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack MYCN amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking MYCN amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the MYC oncogene. Analysis of human neuroblastoma tumor specimens revealed that MYC up-regulation correlates with markers of TAM infiltration. In an IL6ko neuroblastoma model, the absence of IL-6 protein had no effect on tumor development and prevented neither STAT3 activation nor MYC up-regulation. In contrast, inhibition of JAK-STAT activation using AZD1480 or the clinically admissible inhibitor ruxolitinib significantly reduced TAM-mediated growth of neuroblastomas implanted subcutaneously in NOD scid gamma mice. Our results point to a unique mechanism in which TAMs promote tumor cells that lack amplification of an oncogene common to the malignancy by up-regulating transcriptional expression of a distinct oncogene from the same gene family, and underscore the role of IL-6-independent activation of STAT3 in this mechanism. Amplification of MYCN or constitutive up-regulation of MYC protein is observed in approximately half of high-risk tumors; our findings indicate a novel role of TAMs as inducers of MYC expression in neuroblastomas lacking independent oncogene activation. PMID:29207662

Transcriptional Profiling Reveals a Common Metabolic Program for Tumorigenicity in High-Risk Human Neuroblastoma and Mouse Neuroblastoma Sphere-Forming Cells

PubMed Central

Liu, Mengling; Xia, Yingfeng; Ding, Jane; Ye, Bingwei; Zhao, Erhu; Choi, Jeong-Hyeon; Alptekin, Ahmet; Yan, Chunhong; Dong, Zheng; Huang, Shuang; Yang, Liqun; Cui, Hongjuan; Zha, Yunhong; Ding, Han-Fei

2017-01-01

Summary High-risk neuroblastoma remains one of the deadliest childhood cancers. Identification of metabolic pathways that drive or maintain high-risk neuroblastoma may open new avenues of therapeutic interventions. Here we report the isolation and propagation of neuroblastoma sphere-forming cells with self-renewal and differentiation potential from tumors of TH-MYCN mice, an animal model of high-risk neuroblastoma with MYCN amplification. Transcriptional profiling reveals that mouse neuroblastoma sphere-forming cells acquire a metabolic program characterized by transcriptional activation of the cholesterol and serine-glycine synthesis pathways, primarily as a result of increased expression of sterol regulatory element-binding factors and Atf4, respectively. This metabolic reprogramming is recapitulated in high-risk human neuroblastomas and is prognostic for poor clinical outcome. Genetic and pharmacological inhibition of the metabolic program markedly decreases the growth and tumorigenicity of both mouse neuroblastoma sphere-forming cells and human neuroblastoma cell lines. These findings suggest a therapeutic strategy for targeting the metabolic program of high-risk neuroblastoma. PMID:27705805

Transcriptional Profiling Reveals a Common Metabolic Program in High-Risk Human Neuroblastoma and Mouse Neuroblastoma Sphere-Forming Cells.

PubMed

Liu, Mengling; Xia, Yingfeng; Ding, Jane; Ye, Bingwei; Zhao, Erhu; Choi, Jeong-Hyeon; Alptekin, Ahmet; Yan, Chunhong; Dong, Zheng; Huang, Shuang; Yang, Liqun; Cui, Hongjuan; Zha, Yunhong; Ding, Han-Fei

2016-10-04

High-risk neuroblastoma remains one of the deadliest childhood cancers. Identification of metabolic pathways that drive or maintain high-risk neuroblastoma may open new avenues of therapeutic interventions. Here, we report the isolation and propagation of neuroblastoma sphere-forming cells with self-renewal and differentiation potential from tumors of the TH-MYCN mouse, an animal model of high-risk neuroblastoma with MYCN amplification. Transcriptional profiling reveals that mouse neuroblastoma sphere-forming cells acquire a metabolic program characterized by transcriptional activation of the cholesterol and serine-glycine synthesis pathways, primarily as a result of increased expression of sterol regulatory element binding factors and Atf4, respectively. This metabolic reprogramming is recapitulated in high-risk human neuroblastomas and is prognostic for poor clinical outcome. Genetic and pharmacological inhibition of the metabolic program markedly decreases the growth and tumorigenicity of both mouse neuroblastoma sphere-forming cells and human neuroblastoma cell lines. These findings suggest a therapeutic strategy for targeting the metabolic program of high-risk neuroblastoma. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Mouse neuroblastoma cell-based model and the effect of epileptic events on calcium oscillations and neural spikes

NASA Astrophysics Data System (ADS)

Kim, Suhwan; Jung, Unsang; Baek, Juyoung; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

2013-01-01

Recently, mouse neuroblastoma cells have been considered as an attractive model for the study of human neurological and prion diseases, and they have been intensively used as a model system in different areas. For example, the differentiation of neuro2a (N2A) cells, receptor-mediated ion current, and glutamate-induced physiological responses have been actively investigated with these cells. These mouse neuroblastoma N2A cells are of interest because they grow faster than other cells of neural origin and have a number of other advantages. The calcium oscillations and neural spikes of mouse neuroblastoma N2A cells in epileptic conditions are evaluated. Based on our observations of neural spikes in these cells with our proposed imaging modality, we reported that they can be an important model in epileptic activity studies. We concluded that mouse neuroblastoma N2A cells produce epileptic spikes in vitro in the same way as those produced by neurons or astrocytes. This evidence suggests that increased levels of neurotransmitter release due to the enhancement of free calcium from 4-aminopyridine causes the mouse neuroblastoma N2A cells to produce epileptic spikes and calcium oscillations.

Origin and initiation mechanisms of neuroblastoma.

PubMed

Tsubota, Shoma; Kadomatsu, Kenji

2018-05-01

Neuroblastoma is an embryonal malignancy that affects normal development of the adrenal medulla and paravertebral sympathetic ganglia in early childhood. Extensive studies have revealed the molecular characteristics of human neuroblastomas, including abnormalities at genome, epigenome and transcriptome levels. However, neuroblastoma initiation mechanisms and even its origin are long-standing mysteries. In this review article, we summarize the current knowledge about normal development of putative neuroblastoma sources, namely sympathoadrenal lineage of neural crest cells and Schwann cell precursors that were recently identified as the source of adrenal chromaffin cells. A plausible origin of enigmatic stage 4S neuroblastoma is also discussed. With regard to the initiation mechanisms, we review genetic abnormalities in neuroblastomas and their possible association to initiation mechanisms. We also summarize evidences of neuroblastoma initiation observed in genetically engineered animal models, in which epigenetic alterations were involved, including transcriptomic upregulation by N-Myc and downregulation by polycomb repressive complex 2. Finally, several in vitro experimental methods are proposed that hopefully will accelerate our comprehension of neuroblastoma initiation. Thus, this review summarizes the state-of-the-art knowledge about the mechanisms of neuroblastoma initiation, which is critical for developing new strategies to cure children with neuroblastoma.

The mutational landscape of MYCN, Lin28b and ALK F1174L driven murine neuroblastoma mimics human disease.

PubMed

De Wilde, Bram; Beckers, Anneleen; Lindner, Sven; Kristina, Althoff; De Preter, Katleen; Depuydt, Pauline; Mestdagh, Pieter; Sante, Tom; Lefever, Steve; Hertwig, Falk; Peng, Zhiyu; Shi, Le-Ming; Lee, Sangkyun; Vandermarliere, Elien; Martens, Lennart; Menten, Björn; Schramm, Alexander; Fischer, Matthias; Schulte, Johannes; Vandesompele, Jo; Speleman, Frank

2018-02-02

Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems ( ALK, Th- MYCN, Dbh- MYCN and Lin28b ). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.

Zebrafish as a model to study neuroblastoma development.

PubMed

Casey, Mattie J; Stewart, Rodney A

2018-05-01

Neuroblastoma is a pediatric solid tumor arising from embryonic neural crest progenitor cells that normally generate the peripheral sympathetic nervous system. As such, the location of neuroblastoma tumors is correlated with the distribution of major post-ganglionic clusters throughout the sympathetic chain, with the highest incidence in the adrenal medulla or lumbar sympathetic ganglia (~65%). Neuroblastoma is an enigmatic tumor that can spontaneously regress with minimal treatment or become highly metastatic and develop resistance to aggressive treatments, including radiation and high-dose chemotherapy. Age of diagnosis, stage of disease and cellular and genetic features often predict whether the tumor will regress or advance to metastatic disease. Recent efforts using molecular and genomic technologies have allowed more accurate stratification of patients into low-, intermediate- and high-risk categories, thereby allowing for minimal intervention in low-risk patients and providing potential new therapeutic targets, such as the ALK receptor tyrosine kinase, for high-risk or relapsed patients. Despite these advances, the overall survival of high-risk neuroblastoma patients is still less than 50%. Furthermore, next-generation sequencing has revealed that almost two-thirds of neuroblastoma tumors do not contain obvious pathogenic mutations, suggesting that epigenetic mechanisms and/or a perturbed cellular microenvironment may heavily influence neuroblastoma development. Understanding the mechanisms that drive neuroblastoma, therefore, will likely require a combination of genomic, developmental and cancer biology approaches in whole animal systems. In this review, we discuss the contributions of zebrafish research to our understanding of neuroblastoma pathogenesis as well as the potential for this model system to accelerate the identification of more effective therapies for high-risk neuroblastoma patients in the future.

In Vivo magnetic resonance imaging of xenografted tumors using FTH1 reporter gene expression controlled by a tet-on switch.

PubMed

He, Xiaoya; Cai, Jinhua; Li, Hao; Liu, Bo; Qin, Yong; Zhong, Yi; Wang, Longlun; Liao, Yifan

2016-11-29

As a promising magnetic resonance imaging (MRI) reporter, ferritin has been used to track cells in vivo; however, its continuous overexpression can be cytotoxic, which restricts its application. In this study, we aimed to develop a switch to turn this genetic reporter "on" or "off" while monitoring cell grafts via MRI. To accomplish this, we genetically modified the ferritin heavy chain (FTH1) with a Tet-On switch and assessed the expression of FTH1 in transduced neuroblastoma cells (SK-N-SH) in vitro and in xenografted tumors in vivo. We found that FTH1 expression induced by doxycycline (Dox) in SK-N-SH-FTH1 cells depended on treatment dose and duration. We successfully detected T2-weighted MRI contrast in cell grafts after switching "on" the reporter gene using Dox, and this contrast disappeared when we switched it "off". The genetic reporter FTH1 can thus be switched "on" or "off" throughout longitudinal monitoring of cell grafts, limiting expression to when MRI contrast is needed. The controllable imaging system we have developed minimizes risks from constitutive reporter gene overexpression and facilitates tumor cell monitoring in vitro and in vivo.

Colony stimulating factor 1 receptor blockade improves the efficacy of chemotherapy against human neuroblastoma in the absence of T lymphocytes.

PubMed

Webb, Matthew W; Sun, Jianping; Sheard, Michael A; Liu, Wei-Yao; Wu, Hong-Wei; Jackson, Jeremy R; Malvar, Jemily; Sposto, Richard; Daniel, Dylan; Seeger, Robert C

2018-04-17

Tumor-associated macrophages can promote growth of cancers. In neuroblastoma, tumor-associated macrophages have greater frequency in metastatic versus loco-regional tumors, and higher expression of genes associated with macrophages helps to predict poor prognosis in the 60% of high-risk patients who have MYCN-non-amplified disease. The contribution of cytotoxic T-lymphocytes to anti-neuroblastoma immune responses may be limited by low MHC class I expression and low exonic mutation frequency. Therefore, we modelled human neuroblastoma in T-cell deficient mice to examine whether depletion of monocytes/macrophages from the neuroblastoma microenvironment by blockade of CSF-1R can improve the response to chemotherapy. In vitro, CSF-1 was released by neuroblastoma cells, and topotecan increased this release. In vivo, neuroblastomas formed by subcutaneous co-injection of human neuroblastoma cells and human monocytes into immunodeficient NOD/SCID mice had fewer human CD14 + and CD163 + cells and mouse F4/80 + cells after CSF-1R blockade. In subcutaneous or intra-renal models in immunodeficient NSG or NOD/SCID mice, CSF-1R blockade alone did not affect tumor growth or mouse survival. However, when combined with cyclophosphamide plus topotecan, the CSF-1R inhibitor BLZ945, either without or with anti-human and anti-mouse CSF-1 mAbs, inhibited neuroblastoma growth and synergistically improved mouse survival. These findings indicate that depletion of tumor-associated macrophages from neuroblastomas can be associated with increased chemotherapeutic efficacy without requiring a contribution from T-lymphocytes, suggesting the possibility that combination of CSF-1R blockade with chemotherapy might be effective in patients who have limited anti-tumor T-cell responses. © 2018 UICC.

Silencing Intersectin 1 Slows Orthotopic Neuroblastoma Growth in Mice.

PubMed

Harris, Jamie; Herrero-Garcia, Erika; Russo, Angela; Kajdacsy-Balla, Andre; O'Bryan, John P; Chiu, Bill

2017-11-01

Neuroblastoma accounts for 15% of all pediatric cancer deaths. Intersectin 1 (ITSN1), a scaffold protein involved in phosphoinositide 3-kinase (PI3K) signaling, regulates neuroblastoma cells independent of MYCN status. We hypothesize that by silencing ITSN1 in neuroblastoma cells, tumor growth will be decreased in an orthotopic mouse tumor model. SK-N-AS neuroblastoma cells transfected with empty vector (pSR), vectors expressing scrambled shRNA (pSCR), or shRNAs targeting ITSN1 (sh#1 and sh#2) were used to create orthotopic neuroblastoma tumors in mice. Volume was monitored weekly with ultrasound. End-point was tumor volume >1000 mm. Tumor cell lysates were analyzed with anti-ITSN1 antibody by Western blot. Orthotopic tumors were created in all cell lines. Twenty-five days post injection, pSR tumor size was 917.6±247.7 mm, pSCR was 1180±159.9 mm, sh#1 was 526.3±212.8 mm, and sh#2 was 589.2±74.91 mm. sh#1-tumors and sh#2-tumors were smaller than pSCR (P=0.02), no difference between sh#1 and sh#2. Survival was superior in sh#2-tumors (P=0.02), trended towards improved survival in sh#1-tumors (P=0.09), compared with pSCR-tumors, no difference in pSR tumors. Western blot showed decreased ITSN1 expression in sh#1 and sh#2 compared with pSR and pSCR. Silencing ITSN1 in neuroblastoma cells led to decreased tumor growth in an orthotopic mouse model. Orthotopic animal models can provide insight into the role of ITSN1 pathways in neuroblastoma tumorigenesis.

TRPM7 maintains progenitor-like features of neuroblastoma cells: implications for metastasis formation

PubMed Central

Middelbeek, Jeroen; Kamermans, Alwin; Kuipers, Arthur J.; Hoogerbrugge, Peter M.; Jalink, Kees; van Leeuwen, Frank N.

2015-01-01

Neuroblastoma is an embryonal tumor derived from poorly differentiated neural crest cells. Current research is aimed at identifying the molecular mechanisms that maintain the progenitor state of neuroblastoma cells and to develop novel therapeutic strategies that induce neuroblastoma cell differentiation. Mechanisms controlling neural crest development are typically dysregulated during neuroblastoma progression, and provide an appealing starting point for drug target discovery. Transcriptional programs involved in neural crest development act as a context dependent gene regulatory network. In addition to BMP, Wnt and Notch signaling, activation of developmental gene expression programs depends on the physical characteristics of the tissue microenvironment. TRPM7, a mechanically regulated TRP channel with kinase activity, was previously found essential for embryogenesis and the maintenance of undifferentiated neural crest progenitors. Hence, we hypothesized that TRPM7 may preserve progenitor-like, metastatic features of neuroblastoma cells. Using multiple neuroblastoma cell models, we demonstrate that TRPM7 expression closely associates with the migratory and metastatic properties of neuroblastoma cells in vitro and in vivo. Moreover, microarray-based expression profiling on control and TRPM7 shRNA transduced neuroblastoma cells indicates that TRPM7 controls a developmental transcriptional program involving the transcription factor SNAI2. Overall, our data indicate that TRPM7 contributes to neuroblastoma progression by maintaining progenitor-like features. PMID:25797249

Advances in the translational genomics of neuroblastoma

PubMed Central

Bosse, Kristopher R.; Maris, John M.

2015-01-01

Neuroblastoma is an embryonal malignancy that commonly affects young children and is remarkably heterogenous in its malignant potential. Recently, the genetic basis of neuroblastoma has come into focus, which has catalyzed not only a more comprehensive understanding of neuroblastoma tumorigenesis, but has also revealed novel oncogenic vulnerabilities that are being leveraged therapeutically. Neuroblastoma is a model pediatric solid tumor in its use of recurrent genomic alterations, such as high-level MYCN amplification, for risk stratification. Given the relative paucity of recurrent activating somatic point mutations or gene fusions in primary neuroblastoma tumors studied at initial diagnosis, innovative treatment approaches beyond small molecules targeting mutated or dysregulated kinases will be required moving forward to achieve noticeable improvements in overall patient survival. However, the clonally acquired, oncogenic aberrations in relapsed neuroblastomas are currently being defined and may offer an opportunity to improve patient outcomes with molecularly targeted therapy directed towards aberrantly regulated pathways in relapsed disease. This review will summarize the current state of knowledge of neuroblastoma genetics and genomics, highlighting the improved prognostication and potential therapeutic opportunities that have arisen from recent advances in understanding germline predisposition, recurrent segmental chromosomal alterations, somatic point mutations and translocations, and clonal evolution in relapsed neuroblastoma. PMID:26539795

Mutations in PIK3CA are infrequent in neuroblastoma

PubMed Central

Dam, Vincent; Morgan, Brian T; Mazanek, Pavel; Hogarty, Michael D

2006-01-01

Background Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Methods Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. Results We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. Conclusion These data suggest that activating mutations in the Ras/Raf-MAPK/PI3K signaling cascades occur infrequently in neuroblastoma. Further, despite compelling evidence for MYC and RAS cooperation in vitro and in vivo to promote tumourigenesis, activation of RAS signal transduction does not constitute a preferred secondary pathway in neuroblastomas with MYCN deregulation in either human tumors or murine models. PMID:16822308

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition.

PubMed

Henssen, Anton; Althoff, Kristina; Odersky, Andrea; Beckers, Anneleen; Koche, Richard; Speleman, Frank; Schäfers, Simon; Bell, Emma; Nortmeyer, Maike; Westermann, Frank; De Preter, Katleen; Florin, Alexandra; Heukamp, Lukas; Spruessel, Annika; Astrahanseff, Kathy; Lindner, Sven; Sadowski, Natalie; Schramm, Alexander; Astorgues-Xerri, Lucile; Riveiro, Maria E; Eggert, Angelika; Cvitkovic, Esteban; Schulte, Johannes H

2016-05-15

Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470-81. ©2015 AACR. ©2015 American Association for Cancer Research.

Advances in emerging drugs for the treatment of neuroblastoma.

PubMed

Berlanga, Pablo; Cañete, Adela; Castel, Victoria

2017-03-01

Neuroblastoma is the most common solid extracranial tumor of childhood. Outcome for children with high-risk neuroblastoma remains suboptimal. More than half of children diagnosed with high-risk neuroblastoma either do not respond to conventional therapies or relapse after treatment with dismal prognosis. Areas covered: This paper presents a short review of the state of the art in the current treatment of high-risk neuroblastoma. An updated review of new targeted therapies in this group of patients is also presented. Expert opinion: In order to improve prognosis for high-risk patients there is an urgent need to better understand spatial and temporal heterogeneity and obtain new predictive preclinical models in neuroblastoma. Combination strategies with conventional chemotherapy and/or other targeted therapies may overcome current ALK inhibitors resistance. Improvement of international and transatlantic cooperation to speed clinical trials accrual is needed.

Activated ALK Collaborates with MYCN in Neuroblastoma Pathogenesis

PubMed Central

Zhu, Shizhen; Lee, Jeong-Soo; Guo, Feng; Shin, Jimann; Perez-Atayde, Antonio R.; Kutok, Jeffery L.; Rodig, Scott J.; Neuberg, Donna S.; Helman, Daniel; Feng, Hui; Stewart, Rodney A.; Wang, Wenchao; George, Rani E.; Kanki, John P.; Look, A. Thomas

2012-01-01

SUMMARY Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analogue following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma. PMID:22439933

An efficient and robust MRI-guided radiotherapy planning approach for targeting abdominal organs and tumours in the mouse

PubMed Central

Bird, Luke; Tullis, Iain D. C.; Newman, Robert G.; Corroyer-Dulmont, Aurelien; Falzone, Nadia; Azad, Abul; Vallis, Katherine A.; Sansom, Owen J.; Muschel, Ruth J.; Vojnovic, Borivoj; Hill, Mark A.; Fokas, Emmanouil; Smart, Sean C.

2017-01-01

Introduction Preclinical CT-guided radiotherapy platforms are increasingly used but the CT images are characterized by poor soft tissue contrast. The aim of this study was to develop a robust and accurate method of MRI-guided radiotherapy (MR-IGRT) delivery to abdominal targets in the mouse. Methods A multimodality cradle was developed for providing subject immobilisation and its performance was evaluated. Whilst CT was still used for dose calculations, target identification was based on MRI. Each step of the radiotherapy planning procedure was validated initially in vitro using BANG gel dosimeters. Subsequently, MR-IGRT of normal adrenal glands with a size-matched collimated beam was performed. Additionally, the SK-N-SH neuroblastoma xenograft model and the transgenic KPC model of pancreatic ductal adenocarcinoma were used to demonstrate the applicability of our methods for the accurate delivery of radiation to CT-invisible abdominal tumours. Results The BANG gel phantoms demonstrated a targeting efficiency error of 0.56 ± 0.18 mm. The in vivo stability tests of body motion during MR-IGRT and the associated cradle transfer showed that the residual body movements are within this MR-IGRT targeting error. Accurate MR-IGRT of the normal adrenal glands with a size-matched collimated beam was confirmed by γH2AX staining. Regression in tumour volume was observed almost immediately post MR-IGRT in the neuroblastoma model, further demonstrating accuracy of x-ray delivery. Finally, MR-IGRT in the KPC model facilitated precise contouring and comparison of different treatment plans and radiotherapy dose distributions not only to the intra-abdominal tumour but also to the organs at risk. Conclusion This is, to our knowledge, the first study to demonstrate preclinical MR-IGRT in intra-abdominal organs. The proposed MR-IGRT method presents a state-of-the-art solution to enabling robust, accurate and efficient targeting of extracranial organs in the mouse and can operate with a sufficiently high throughput to allow fractionated treatments to be given. PMID:28453537

A Primary Xenograft Model of Small Cell Lung Cancer Reveals Irreversible Changes in Gene Expression Imposed by Culture In-Vitro

PubMed Central

Daniel, Vincent C.; Marchionni, Luigi; Hierman, Jared S.; Rhodes, Jonathan T.; Devereux, Wendy L.; Rudin, Charles M.; Yung, Rex; Parmigani, Giovanni; Dorsch, Marion; Peacock, Craig D.; Watkins, D. Neil

2009-01-01

Traditional approaches to the preclinical investigation of cancer therapies rely on the use of established cell lines maintained in serum-based growth media. This is particularly true of small cell lung cancer (SCLC), where surgically resected tissue is rarely available. Recent attention has focused on the need for better models that preserve the integrity of cancer stem cell populations, as well as three-dimensional tumor-stromal interactions. Here we describe a primary xenograft model of SCLC in which endobronchial tumor specimens obtained from chemo-naive patients are serially propagated in vivo in immunodeficient mice. In parallel, cell lines grown in conventional tissue culture conditions were derived from each xenograft line, passaged for 6 months, and then re-implanted to generate secondary xenografts. Using the Affymetrix platform, we analyzed gene expression in primary xenograft, xenograft-derived cell line, and secondary xenograft, and compared these data to similar analyses of unrelated primary SCLC samples and laboratory models. When compared to normal lung, primary tumors, xenografts and cell lines displayed a gene expression signature specific for SCLC. Comparison of gene expression within the xenograft model identified a group of tumor-specific genes expressed in primary SCLC and xenografts that was lost during the transition to tissue culture, and that was not regained when the tumors were re-established as secondary xenografts. Such changes in gene expression may be a common feature of many cancer cell culture systems, with functional implications for the use of such models for preclinical drug development. PMID:19351829

mTOR ATP-competitive inhibitor INK128 inhibits neuroblastoma growth via blocking mTORC signaling

PubMed Central

Zhang, Huiyuan; Dou, Jun; Yu, Yang; Zhao, Yanling; Fan, Yihui; Cheng, Jin; Xu, Xin; Liu, Wei; Guan, Shan; Chen, Zhenghu; shi, Yan; Patel, Roma; Vasudevan, Sanjeev A; Zage, Peter E; Zhang, Hong; Nuchtern, Jed G; Kim, Eugene S; Fu, Songbin; Yang, Jianhua

2015-01-01

High-risk neuroblastoma often develops resistance to high-dose chemotherapy. The mTOR signaling cascade is frequently deregulated in human cancers and targeting mTOR signaling sensitizes many cancer types to chemotherapy. Here, using a panel of neuroblastoma cell lines, we found that the mTOR inhibitor INK128 showed inhibitory effects on both anchorage-dependent and independent growth of neuroblastoma cells and significantly enhanced the cytotoxic effects of doxorubicin (Dox) on these cell lines. Treatment of neuroblastoma cells with INK128 blocked the activation of downstream mTOR signaling and enhanced Dox-induced apoptosis. Moreover, INK128 was able to overcome the established chemoresistance in the LA-N-6 cell line. Using an orthotopic neuroblastoma mouse model, we found that INK128 significantly inhibited tumor growth in vivo. In conclusion, we have shown that INK128-mediated mTOR inhibition possessed substantial antitumor activity and could significantly increase the sensitivity of neuroblastoma cells to Doxorubicin therapy. Taken together, our results indicate that using INK128 can provide additional efficacy to current chemotherapeutic regimens and represent a new paradigm in restoring drug sensitivity in neuroblastoma. PMID:25425103

The iron chelator Dp44mT suppresses osteosarcoma’s proliferation, invasion and migration: in vitro and in vivo

PubMed Central

Li, Pengcheng; Zheng, Xun; Shou, Kangquan; Niu, Yahui; Jian, Chao; Zhao, Yong; Yi, Wanrong; Hu, Xiang; Yu, Aixi

2016-01-01

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells, including neuroblastoma, neuroepithelioma and prostate cancer. However, whether Dp44mT has anticancer effects in osteosarcoma is still unknown. Here, we investigated the antitumor action of Dp44mT in osteosarcoma and its underlying mechanisms. A human osteosarcoma 143B cell line in vitro and 143B xenograft in nude mice in vivo were utilized, the anticancer effects of Dp44mT were examined through methods of MTT assay, transwell, wound healing assay, flow cytometry, western blot, immunohistochemistry and H&E staining. We showed that Dp44mT inhibits cell proliferation, invasion and migration in vitro. In addition, flow cytometry further illustrated that Dp44mT suppression of 143B cell proliferation, invasion and migration were partially due to induction of cell apoptosis, cell cycle arrest in S phase and ROS production. Also in vitro and in vivo, the expression levels of Bcl2, Bax, Caspase3, Caspase9, LC3-II, β-catenin and its downstream targets such as C-myc and Cyclin D1 demonstrated that cell apoptosis and autophagy, as well as Wnt/β-catenin pathway were involved in Dp44mT induced osteosarcoma suppression. The Dp44mT inhibition of osteosarcoma was further verified via animal models. The findings indicated that in vivo Dp44mT showed a significant reduction in the 143B xenograft tumor growth and metastasis. In conclusion, our data demonstrated that Dp44mT has effective anticancer capability in osteosarcoma and that may represent a promising treatment strategy for osteosarcoma. PMID:28078009

Detection of neuroendocrine tumors using promoter-specific secreted Gaussia luciferase.

PubMed

Tseng, Alan Wei-Shun; Akerstrom, Victoria; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

2016-01-01

Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors.

Increasing Antiproliferative Properties of Endocannabinoids in N1E-115 Neuroblastoma Cells through Inhibition of Their Metabolism

PubMed Central

Hamtiaux, Laurie; Hansoulle, Laurie; Dauguet, Nicolas; Muccioli, Giulio G.; Gallez, Bernard; Lambert, Didier M.

2011-01-01

The antitumoral properties of endocannabinoids received a particular attention these last few years. Indeed, these endogenous molecules have been reported to exert cytostatic, apoptotic and antiangiogenic effects in different tumor cell lines and tumor xenografts. Therefore, we investigated the cytotoxicity of three N-acylethanolamines – N-arachidonoylethanolamine (anandamide, AEA), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA) - which were all able to time- and dose-dependently reduce the viability of murine N1E-115 neuroblastoma cells. Moreover, several inhibitors of FAAH and NAAA, whose presence was confirmed by RT-PCR in the cell line, induced cell cytotoxicity and favored the decrease in cell viability caused by N-acylethanolamines. The most cytotoxic treatment was achieved by the co-incubation of AEA with the selective FAAH inhibitor URB597, which drastically reduced cell viability partly by inhibiting AEA hydrolysis and consequently increasing AEA levels. This combination of molecules synergistically decreased cell proliferation without inducing cell apoptosis or necrosis. We found that these effects are independent of cannabinoid, TRPV1, PPARα, PPARγ or GPR55 receptors activation but seem to occur through a lipid raft-dependent mechanism. These findings further highlight the interest of targeting the endocannabinoid system to treat cancer. More particularly, this emphasizes the great potential benefit of designing novel anti-cancerous therapies based on the association of endocannabinoids and inhibitors of their hydrolysis. PMID:22046372

Studies of the Tumor Microenvironment in Pathogenesis of Neuroblastoma

DTIC Science & Technology

2013-07-01

NOTES 14. ABSTRACT The NBL -Tag neuroblastoma tumors were assessed for presence of macrophages and their role in promoting tumor growth...Anti-IL6 antibody therapy on a co-culture of macrophages and NBL -Tag tumor cell line (NBT2) did not decrease the tumor- promoting effects of ‘trained...characterization of a recently described 100% penetrant transgenic murine neuroblastoma model ( NBL -Tag) established lack of MYCN amplification using

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma

PubMed Central

Fabian, Johannes; Opitz, Desirée; Althoff, Kristina; Lodrini, Marco; Hero, Barbara; Volland, Ruth; Beckers, Anneleen; de Preter, Katleen; Decock, Anneleen; Patil, Nitin; Abba, Mohammed; Kopp-Schneider, Annette; Astrahantseff, Kathy; Wünschel, Jasmin; Pfeil, Sebastian; Ercu, Maria; Künkele, Annette; Hu, Jamie; Thole, Theresa; Schweizer, Leonille; Mechtersheimer, Gunhild; Carter, Daniel; Cheung, Belamy B.; Popanda, Odilia; von Deimling, Andreas; Koster, Jan; Versteeg, Rogier; Schwab, Manfred; Marshall, Glenn M.; Speleman, Frank; Erb, Ulrike; Zoeller, Margot; Allgayer, Heike; Simon, Thorsten; Fischer, Matthias; Kulozik, Andreas E.; Eggert, Angelika; Witt, Olaf; Schulte, Johannes H.; Deubzer, Hedwig E.

2016-01-01

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma. PMID:27572323

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma.

PubMed

Fabian, Johannes; Opitz, Desirée; Althoff, Kristina; Lodrini, Marco; Hero, Barbara; Volland, Ruth; Beckers, Anneleen; de Preter, Katleen; Decock, Anneleen; Patil, Nitin; Abba, Mohammed; Kopp-Schneider, Annette; Astrahantseff, Kathy; Wünschel, Jasmin; Pfeil, Sebastian; Ercu, Maria; Künkele, Annette; Hu, Jamie; Thole, Theresa; Schweizer, Leonille; Mechtersheimer, Gunhild; Carter, Daniel; Cheung, Belamy B; Popanda, Odilia; von Deimling, Andreas; Koster, Jan; Versteeg, Rogier; Schwab, Manfred; Marshall, Glenn M; Speleman, Frank; Erb, Ulrike; Zoeller, Margot; Allgayer, Heike; Simon, Thorsten; Fischer, Matthias; Kulozik, Andreas E; Eggert, Angelika; Witt, Olaf; Schulte, Johannes H; Deubzer, Hedwig E

2016-10-11

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.

Sphingosine-1-Phosphate Receptor-1 Promotes Environment-Mediated and Acquired Chemoresistance.

PubMed

Lifshitz, Veronica; Priceman, Saul J; Li, Wenzhao; Cherryholmes, Gregory; Lee, Heehyoung; Makovski-Silverstein, Adar; Borriello, Lucia; DeClerck, Yves A; Yu, Hua

2017-11-01

Drug resistance is a major barrier for the development of effective and durable cancer therapies. Overcoming this challenge requires further defining the cellular and molecular mechanisms underlying drug resistance, both acquired and environment-mediated drug resistance (EMDR). Here, using neuroblastoma (NB), a childhood cancer with high incidence of recurrence due to resistance to chemotherapy, as a model we show that human bone marrow-mesenchymal stromal cells induce tumor expression of sphingosine-1-phosphate receptor-1 (S1PR1), leading to their resistance to chemotherapy. Targeting S1PR1 by shRNA markedly enhances etoposide-induced apoptosis in NB cells and abrogates EMDR, while overexpression of S1PR1 significantly protects NB cells from multidrug-induced apoptosis via activating JAK-STAT3 signaling. Elevated S1PR1 expression and STAT3 activation are also observed in human NB cells with acquired resistance to etoposide. We show in vitro and in human NB xenograft models that treatment with FTY720, an FDA-approved drug and antagonist of S1PR1, dramatically sensitizes drug-resistant cells to etoposide. In summary, we identify S1PR1 as a critical target for reducing both EMDR and acquired chemoresistance in NB. Mol Cancer Ther; 16(11); 2516-27. ©2017 AACR . ©2017 American Association for Cancer Research.

Association of high microvessel αvβ3 and low PTEN with poor outcome in stage 3 neuroblastoma: rationale for using first in class dual PI3K/BRD4 inhibitor, SF1126

PubMed Central

Singh, Alok R.; Joshi, Shweta; Vega, Francisco M.; Guo, Pinzheng; Xu, Jingying; Groshen, Susan; Ye, Wei; Millard, Melissa; Campan, Mihaela; Morales, Guillermo; Garlich, Joseph R.; Laird, Peter W.; Seeger, Robert C.; Shimada, Hiroyuki

2017-01-01

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma. PMID:28881723

Transmembrane adaptor protein PAG1 is a novel tumor suppressor in neuroblastoma

PubMed Central

Agarwal, Saurabh; Ghosh, Rajib; Chen, Zaowen; Lakoma, Anna; Gunaratne, Preethi H.; Kim, Eugene S.; Shohet, Jason M.

2016-01-01

(NB) is the most common extracranial pediatric solid tumor with high mortality rates. The tyrosine kinase c-Src has been known to play an important role in differentiation of NB cells, but the mechanism of c-Src regulation has not been defined. Here, we characterize PAG1 (Cbp, Csk binding protein), a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in NB. Clinical cohort analysis demonstrate that low expression of PAG1 is a significant prognostic factor for high stage disease, increased relapse, and worse overall survival for children with NB. PAG1 knockdown in NB cells promotes proliferation and anchorage-independent colony formation with increased activation of AKT and ERK downstream of c-Src, while PAG1 overexpression significantly rescues these effects. In vivo, PAG1 overexpression significantly inhibits NB tumorigenicity in an orthotopic xenograft model. Our results establish PAG1 as a potent tumor suppressor in NB by inhibiting c-Src and downstream effector pathways. Thus, reactivation of PAG1 and inhibition of c-Src kinase activity represents an important novel therapeutic approach for high-risk NB. PMID:26993602

Evidence of chromaffin oxygen sensing in neuroblastoma.

PubMed

Hedborg, F; Franklin, G; Norrman, J; Grimelius, L; Wassberg, E; Hero, B; Schilling, F; Berthold, F; Harms, D; Sandstedt, B

2001-01-01

With the aid of IGF2 and VEGF in situ hybridization; tyrosine hydroxylase, chromogranin A, and Ki67 immunohistochemistry; and TUNEL staining applied to a large series of clinical neuroblastomas and to an animal model, we show here that stroma-poor neuroblastomas show evidence of chromaffin differentiation similar to that of type 1 small intensely fluorescent (SIF) cells and that this occurs in a vascular-dependent fashion, indicating a role for local tumor hypoxia in the differentiation process.

NRF2 Mediates Neuroblastoma Proliferation and Resistance to Retinoic Acid Cytotoxicity in a Model of In Vitro Neuronal Differentiation.

PubMed

de Miranda Ramos, Vitor; Zanotto-Filho, Alfeu; de Bittencourt Pasquali, Matheus Augusto; Klafke, Karina; Gasparotto, Juciano; Dunkley, Peter; Gelain, Daniel Pens; Moreira, José Cláudio Fonseca

2016-11-01

Retinoic acid (RA) morphogenetic properties have been used in different kinds of therapies, from neurodegenerative disorders to some types of cancer such as promyelocytic leukemia and neuroblastoma. However, most of the pathways responsible for RA effects remain unknown. To investigate such pathways, we used a RA-induced differentiation model in the human neuroblastoma cells, SH-SY5Y. Our data showed that n-acetyl-cysteine (NAC) reduced cells' proliferation rate and increased cells' sensitivity to RA toxicity. Simultaneously, NAC pre-incubation attenuated nuclear factor erythroid 2-like factor 2 (NRF2) activation by RA. None of these effects were obtained with Trolox ® as antioxidant, suggesting a cysteine signalization by RA. NRF2 knockdown increased cell sensibility to RA after 96 h of treatment and diminished neuroblastoma proliferation rate. Conversely, NRF2 overexpression limited RA anti-proliferative effects and increased cell proliferation. In addition, a rapid and non-genomic activation of the ERK 1/2 and PI3K/AKT pathways revealed to be equally required to promote NRF2 activation and necessary for RA-induced differentiation. Together, we provide data correlating NRF2 activity with neuroblastoma proliferation and resistance to RA treatments; thus, this pathway could be a potential target to optimize neuroblastoma chemotherapeutic response as well as in vitro neuronal differentiation protocols.

Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse.

PubMed

Chen, Lindi; Humphreys, Angharad; Turnbull, Lisa; Bellini, Angela; Schleiermacher, Gudrun; Salwen, Helen; Cohn, Susan L; Bown, Nick; Tweddle, Deborah A

2016-12-27

Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ~85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression.

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma

PubMed Central

Li, Nan; Fu, Haiying; Hewitt, Stephen M.; Dimitrov, Dimiter S.

2017-01-01

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma. PMID:28739923

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

PubMed

Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

2017-08-08

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

The pre-rRNA processing factor DEF is rate limiting for the pathogenesis of MYCN-driven neuroblastoma.

PubMed

Tao, T; Sondalle, S B; Shi, H; Zhu, S; Perez-Atayde, A R; Peng, J; Baserga, S J; Look, A T

2017-07-06

The nucleolar factor, digestive organ expansion factor (DEF), has a key role in ribosome biogenesis, functioning in pre-ribosomal RNA (pre-rRNA) processing as a component of the small ribosomal subunit (SSU) processome. Here we show that the peripheral sympathetic nervous system (PSNS) is very underdeveloped in def-deficient zebrafish, and that def haploinsufficiency significantly decreases disease penetrance and tumor growth rate in a MYCN-driven transgenic zebrafish model of neuroblastoma that arises in the PSNS. Consistent with these findings, DEF is highly expressed in human neuroblastoma, and its depletion in human neuroblastoma cell lines induces apoptosis. Interestingly, overexpression of MYCN in zebrafish and in human neuroblastoma cells results in the appearance of intermediate pre-rRNAs species that reflect the processing of pre-rRNAs through Pathway 2, a pathway that processes pre-rRNAs in a different temporal order than the more often used Pathway 1. Our results indicate that DEF and possibly other components of the SSU processome provide a novel site of vulnerability in neuroblastoma cells that could be exploited for targeted therapy.

MYCN Promotes the Expansion of Phox2B-Positive Neuronal Progenitors to Drive Neuroblastoma Development

PubMed Central

Alam, Goleeta; Cui, Hongjuan; Shi, Huilin; Yang, Liqun; Ding, Jane; Mao, Ling; Maltese, William A.; Ding, Han-Fei

2009-01-01

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B+ neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin+ cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B+ neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development. PMID:19608868

TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival.

PubMed

Chavali, P L; Saini, R K R; Zhai, Q; Vizlin-Hodzic, D; Venkatabalasubramanian, S; Hayashi, A; Johansson, E; Zeng, Z-j; Mohlin, S; Påhlman, S; Hansford, L; Kaplan, D R; Funa, K

2014-10-30

Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan-Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival

PubMed Central

Chavali, P L; Saini, R K R; Zhai, Q; Vizlin-Hodzic, D; Venkatabalasubramanian, S; Hayashi, A; Johansson, E; Zeng, Z-j; Mohlin, S; Påhlman, S; Hansford, L; Kaplan, D R; Funa, K

2014-01-01

Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan–Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties. PMID:25356871

EGFR gene overexpression retained in an invasive xenograft model by solid orthotopic transplantation of human glioblastoma multiforme into nude mice.

PubMed

Yi, Diao; Hua, Tian Xin; Lin, Huang Yan

2011-03-01

Orthotopic xenograft animal model from human glioblastoma multiforme (GBM) cell lines often do not recapitulate an extremely important aspect of invasive growth and epidermal growth factor receptor (EGFR) gene overexpression of human GBM. We developed an orthotopic xenograft model by solid transplantation of human GBM into the brain of nude mouse. The orthotopic xenografts sharing the same histopathological features with their original human GBMs were highly invasive and retained the overexpression of EGFR gene. The murine orthotopic GBM models constitute a valuable in vivo system for preclinical studies to test novel therapies for human GBM.

Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.

PubMed

Carlson, Brett L; Pokorny, Jenny L; Schroeder, Mark A; Sarkaria, Jann N

2011-03-01

Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short-term explant cultures or intracranial xenografts. This unit describes detailed procedures for establishment, maintenance, and utilization of a primary GBM xenograft panel for the purpose of using them as tumor models for basic or translational studies.

Treatment of Neuroblastoma with an Engineered “Obligate” Anaerobic Salmonella typhimurium Strain YB1

PubMed Central

Ning, Bo-Tao; Yu, Bin; Chan, Shing; Chan, Jian-liang; Huang, Jian-Dong; Chan, Godfrey Chi-Fung

2017-01-01

Purpose Neuroblastoma is an embryonic solid tumor derived from the progenitors of the sympathetic nervous system. More than half of the patients developed metastatic disease at the time of initial diagnosis and had poor outcome with current therapeutic approaches. In recent years, some obligate and facultative anaerobic bacteria were reported to target the hypoxic and necrotic region of solid tumor models and caused tumor regression. We recently successfully constructed an “obligate” anaerobic Salmonella strain YB1 that was applied in breast cancer nude mice model by us. Here, we report the application of YB1 in neuroblastoma treatment. Methods The anti-cancer effect and side-effects of YB1 was examined in both in vitro and in vivo experiment. Previous established orthotopic neuroblastoma SCID/beige murine model using SK-NLP/luciferase cell line was adopted. Results In vitro, YB1 induced apoptosis for up to 31.4% of the neuroblastoma cells under anaerobic condition, three times more than that under aerobic condition (10.9%). The expression of both Toll like Receptor 4 and 5 (TLR4 and TLR5) in cancer cells were significantly up-regulated (p<0.05, p<0.01 respectively) after the treatment of YB1 under anaerobic condition. In mouse model, YB1 preferentially accumulated inside the core of the tumors, rather than in normal tissues as our previous reported. This is suggestive of the hypoxic nature of tumor core. Tumor growth was significantly retarded in YB1 treatment group (n=6, P<0.01). Furthermore, there was no long-term organ damage noted in all the organs examined including heart, lung, liver, spleen and brain in the YB1 treated mice. Conclusion The genetic modified Salmonella strain YB1 is a promising anti-tumor strategy against the tumor bulk for neuroblastoma. Future study can be extended to other common cancer types to verify the relative efficacy on different neoplastic cells. PMID:28775780

Novel target for high-risk neuroblastoma identified in pre-clinical research | Center for Cancer Research

Cancer.gov

Pre-clinical research by investigators at the Center for Cancer Research and their colleagues have identified a number of novel epigenetic targets for high-risk neuroblastoma and validated a promising new targeted inhibitor in pre-clinical models.  Read more...

Translational development of difluoromethylornithine (DFMO) for the treatment of neuroblastoma

PubMed Central

Bassiri, Hamid; Benavides, Adriana; Haber, Michelle; Gilmour, Susan K.; Norris, Murray D.

2015-01-01

Neuroblastoma is a childhood tumor in which MYC oncogenes are commonly activated to drive tumor progression. Survival for children with high-risk neuroblastoma remains poor despite treatment that incorporates high-dose chemotherapy, stem cell support, surgery, radiation therapy and immunotherapy. More effective and less toxic treatments are sought and one approach under clinical development involves re-purposing the anti-protozoan drug difluoromethylornithine (DFMO; Eflornithine) as a neuroblastoma therapeutic. DFMO is an irreversible inhibitor of ornithine decarboxylase (Odc), a MYC target gene, bona fide oncogene, and the rate-limiting enzyme in polyamine synthesis. DFMO is approved for the treatment of Trypanosoma brucei gambiense encephalitis (“African sleeping sickness”) since polyamines are essential for the proliferation of these protozoa. However, polyamines are also critical for mammalian cell proliferation and the finding that MYC coordinately regulates all aspects of polyamine metabolism suggests polyamines may be required to support cancer promotion by MYC. Pre-emptive blockade of polyamine synthesis is sufficient to block tumor initiation in an otherwise fully penetrant transgenic mouse model of neuroblastoma driven by MYCN, underscoring the necessity of polyamines in this process. Moreover, polyamine depletion regimens exert potent anti-tumor activity in pre-clinical models of established neuroblastoma as well, in combination with numerous chemotherapeutic agents and even in tumors with unfavorable genetic features such as MYCN, ALK or TP53 mutation. This has led to the testing of DFMO in clinical trials for children with neuroblastoma. Current trial designs include testing lower dose DFMO alone (2,000 mg/m2/day) starting at the completion of standard therapy, or higher doses combined with chemotherapy (up to 9,000 mg/m2/day) for patients with relapsed disease that has progressed. In this review we will discuss important considerations for the future design of DFMO-based clinical trials for neuroblastoma, focusing on the need to better define the principal mechanisms of anti-tumor activity for polyamine depletion regimens. Putative DFMO activities that are both cancer cell intrinsic (targeting the principal oncogenic driver, MYC) and cancer cell extrinsic (altering the tumor microenvironment to support anti-tumor immunity) will be discussed. Understanding the mechanisms of DFMO activity are critical in determining how it might be best leveraged in upcoming clinical trials. This mechanistic approach also provides a platform by which iterative pre-clinical testing using translational tumor models may complement our clinical approaches. PMID:26835380

Optical Imaging and Gene Therapy with Neuroblastoma-Targeting Polymeric Nanoparticles for Potential Theranostic Applications.

PubMed

Lee, Jangwook; Jeong, Eun Ju; Lee, Yeon Kyung; Kim, Kwangmeyung; Kwon, Ick Chan; Lee, Kuen Yong

2016-03-02

Recently, targeted delivery systems based on functionalized polymeric nanoparticles have attracted a great deal of attention in cancer diagnosis and therapy. Specifically, as neuroblastoma occurs in infancy and childhood, targeted delivery may be critical to reduce the side effects that can occur with conventional approaches, as well as to achieve precise diagnosis and efficient therapy. Thus, biocompatible poly(d,l-lactide-co-glycolide) (PLG) nanoparticles containing an imaging probe and therapeutic gene are prepared, followed by modification with rabies virus glycoprotein (RVG) peptide for neuroblastoma-targeting delivery. RVG peptide is a well-known neuronal targeting ligand and is chemically conjugated to PLG nanoparticles without changing their size or shape. RVG-modified nanoparticles are effective in specifically targeting neuroblastoma both in vitro and in vivo. RVG-modified nanoparticles loaded with a fluorescent probe are useful to detect the tumor site in a neuroblastoma-bearing mouse model, and those encapsulating a therapeutic gene cocktail (siMyc, siBcl-2, and siVEGF) significantly suppressed tumor growth in the mouse model. This approach to designing and tailoring of polymeric nanoparticles for targeted delivery may be useful in the development of multimodality systems for theranostic approaches. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Restricted growth of attenuated poliovirus strains in cultured cells of a human neuroblastoma.

PubMed

Agol, V I; Drozdov, S G; Ivannikova, T A; Kolesnikova, M S; Korolev, M B; Tolskaya, E A

1989-09-01

Cultured cells of a human neuroblastoma, SK-N-MC, were found to be highly resistant to Sabin attenuated poliovirus types 1 and 2 strains; no appreciable cytopathic effect was observed, and the total harvest was generally in the order of 1 PFU per cell or less. On the other hand, related neurovirulent strains of these antigenic types produced a relatively good (2 orders of magnitude higher) yield in a markedly protracted infectious cycle. The limited growth of the attenuated virus in the neuroblastoma cells appeared to be confined to a minor cell subpopulation. Experiments with intratypic (type 1) poliovirus recombinants suggested that the major genetic determinants limiting reproduction of the attenuated polioviruses in the neuroblastoma cells are located in the 5' half of the viral RNA, although the 3' half also appears to contribute somewhat to this phenotype. The possibility that neuroblastoma cells may represent an in vitro model for studying poliovirus neurovirulence is briefly discussed.

A three-gene expression signature model for risk stratification of patients with neuroblastoma.

PubMed

Garcia, Idoia; Mayol, Gemma; Ríos, José; Domenech, Gema; Cheung, Nai-Kong V; Oberthuer, André; Fischer, Matthias; Maris, John M; Brodeur, Garrett M; Hero, Barbara; Rodríguez, Eva; Suñol, Mariona; Galvan, Patricia; de Torres, Carmen; Mora, Jaume; Lavarino, Cinzia

2012-04-01

Neuroblastoma is an embryonal tumor with contrasting clinical courses. Despite elaborate stratification strategies, precise clinical risk assessment still remains a challenge. The purpose of this study was to develop a PCR-based predictor model to improve clinical risk assessment of patients with neuroblastoma. The model was developed using real-time PCR gene expression data from 96 samples and tested on separate expression data sets obtained from real-time PCR and microarray studies comprising 362 patients. On the basis of our prior study of differentially expressed genes in favorable and unfavorable neuroblastoma subgroups, we identified three genes, CHD5, PAFAH1B1, and NME1, strongly associated with patient outcome. The expression pattern of these genes was used to develop a PCR-based single-score predictor model. The model discriminated patients into two groups with significantly different clinical outcome [set 1: 5-year overall survival (OS): 0.93 ± 0.03 vs. 0.53 ± 0.06, 5-year event-free survival (EFS): 0.85 ± 0.04 vs. 0.042 ± 0.06, both P < 0.001; set 2 OS: 0.97 ± 0.02 vs. 0.61 ± 0.1, P = 0.005, EFS: 0.91 ± 0.8 vs. 0.56 ± 0.1, P = 0.005; and set 3 OS: 0.99 ± 0.01 vs. 0.56 ± 0.06, EFS: 0.96 ± 0.02 vs. 0.43 ± 0.05, both P < 0.001]. Multivariate analysis showed that the model was an independent marker for survival (P < 0.001, for all). In comparison with accepted risk stratification systems, the model robustly classified patients in the total cohort and in different clinically relevant risk subgroups. We propose for the first time in neuroblastoma, a technically simple PCR-based predictor model that could help refine current risk stratification systems. ©2012 AACR.

A Three-Gene Expression Signature Model for Risk Stratification of Patients with Neuroblastoma

PubMed Central

Garcia, Idoia; Mayol, Gemma; Ríos, José; Domenech, Gema; Cheung, Nai-Kong V.; Oberthuer, André; Fischer, Matthias; Maris, John M.; Brodeur, Garrett M.; Hero, Barbara; Rodríguez, Eva; Suñol, Mariona; Galvan, Patricia; de Torres, Carmen; Mora, Jaume; Lavarino, Cinzia

2014-01-01

Purpose Neuroblastoma is an embryonal tumor with contrasting clinical courses. Despite elaborate stratification strategies, precise clinical risk assessment still remains a challenge. The purpose of this study was to develop a PCR-based predictor model to improve clinical risk assessment of patients with neuroblastoma. Experimental Design The model was developed using real-time PCR gene expression data from 96 samples and tested on separate expression data sets obtained from real-time PCR and microarray studies comprising 362 patients. Results On the basis of our prior study of differentially expressed genes in favorable and unfavorable neuroblastoma subgroups, we identified three genes, CHD5, PAFAH1B1, and NME1, strongly associated with patient outcome. The expression pattern of these genes was used to develop a PCR-based single-score predictor model. The model discriminated patients into two groups with significantly different clinical outcome [set 1: 5-year overall survival (OS): 0.93 ± 0.03 vs. 0.53 ± 0.06, 5-year event-free survival (EFS): 0.85 ± 0.04 vs. 0.042 ± 0.06, both P < 0.001; set 2 OS: 0.97 ± 0.02 vs. 0.61 ± 0.1, P = 0.005, EFS: 0.91 ± 0.8 vs. 0.56 ± 0.1, P = 0.005; and set 3 OS: 0.99 ± 0.01 vs. 0.56 ± 0.06, EFS: 0.96 ± 0.02 vs. 0.43 ± 0.05, both P < 0.001]. Multivariate analysis showed that the model was an independent marker for survival (P < 0.001, for all). In comparison with accepted risk stratification systems, the model robustly classified patients in the total cohort and in different clinically relevant risk subgroups. Conclusion We propose for the first time in neuroblastoma, a technically simple PCR-based predictor model that could help refine current risk stratification systems. PMID:22328561

Accelerating drug development for neuroblastoma - New Drug Development Strategy: an Innovative Therapies for Children with Cancer, European Network for Cancer Research in Children and Adolescents and International Society of Paediatric Oncology Europe Neuroblastoma project.

PubMed

Moreno, Lucas; Caron, Hubert; Geoerger, Birgit; Eggert, Angelika; Schleiermacher, Gudrun; Brock, Penelope; Valteau-Couanet, Dominique; Chesler, Louis; Schulte, Johannes H; De Preter, Katleen; Molenaar, Jan; Schramm, Alexander; Eilers, Martin; Van Maerken, Tom; Johnsen, John Inge; Garrett, Michelle; George, Sally L; Tweddle, Deborah A; Kogner, Per; Berthold, Frank; Koster, Jan; Barone, Giuseppe; Tucker, Elizabeth R; Marshall, Lynley; Herold, Ralf; Sterba, Jaroslav; Norga, Koen; Vassal, Gilles; Pearson, Andrew Dj

2017-08-01

Neuroblastoma, the commonest paediatric extra-cranial tumour, remains a leading cause of death from cancer in children. There is an urgent need to develop new drugs to improve cure rates and reduce long-term toxicity and to incorporate molecularly targeted therapies into treatment. Many potential drugs are becoming available, but have to be prioritised for clinical trials due to the relatively small numbers of patients. Areas covered: The current drug development model has been slow, associated with significant attrition, and few new drugs have been developed for neuroblastoma. The Neuroblastoma New Drug Development Strategy (NDDS) has: 1) established a group with expertise in drug development; 2) prioritised targets and drugs according to tumour biology (target expression, dependency, pre-clinical data; potential combinations; biomarkers), identifying as priority targets ALK, MEK, CDK4/6, MDM2, MYCN (druggable by BET bromodomain, aurora kinase, mTORC1/2) BIRC5 and checkpoint kinase 1; 3) promoted clinical trials with target-prioritised drugs. Drugs showing activity can be rapidly transitioned via parallel randomised trials into front-line studies. Expert opinion: The Neuroblastoma NDDS is based on the premise that optimal drug development is reliant on knowledge of tumour biology and prioritisation. This approach will accelerate neuroblastoma drug development and other poor prognosis childhood malignancies.

Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells.

PubMed

Shum, Thomas; Omer, Bilal; Tashiro, Haruko; Kruse, Robert L; Wagner, Dimitrios L; Parikh, Kathan; Yi, Zhongzhen; Sauer, Tim; Liu, Daofeng; Parihar, Robin; Castillo, Paul; Liu, Hao; Brenner, Malcolm K; Metelitsa, Leonid S; Gottschalk, Stephen; Rooney, Cliona M

2017-11-01

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer. Significance: The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. Cancer Discov; 7(11); 1238-47. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201 . ©2017 American Association for Cancer Research.

What's New in Neuroblastoma Research and Treatment?

MedlinePlus

... New in Neuroblastoma Research? Neuroblastoma About Neuroblastoma What’s New in Neuroblastoma Research? Important research into neuroblastoma is ... group . Other gene changes might help researchers find new treatments that work on certain types of neuroblastoma ...

Novel Dedifferentiated Liposarcoma Xenograft Models Reveal PTEN Down-Regulation as a Malignant Signature and Response to PI3K Pathway Inhibition

PubMed Central

Smith, Kathleen B.; Tran, Linh M.; Tam, Brenna M.; Shurell, Elizabeth M.; Li, Yunfeng; Braas, Daniel; Tap, William D.; Christofk, Heather R.; Dry, Sarah M.; Eilber, Fritz C.; Wu, Hong

2014-01-01

Liposarcoma is a type of soft tissue sarcoma that exhibits poor survival and a high recurrence rate. Treatment is generally limited to surgery and radiation, which emphasizes the need for better understanding of this disease. Because very few in vivo and in vitro models can reproducibly recapitulate the human disease, we generated several xenograft models from surgically resected human dedifferentiated liposarcoma. All xenografts recapitulated morphological and gene expression characteristics of the patient tumors after continuous in vivo passages. Importantly, xenograftability was directly correlated with disease-specific survival of liposarcoma patients. Thus, the ability for the tumor of a patient to engraft may help identify those patients who will benefit from more aggressive treatment regimens. Gene expression analyses highlighted the association between xenograftability and a unique gene expression signature, including down-regulated PTEN tumor-suppressor gene expression and a progenitor-like phenotype. When treated with the PI3K/AKT/mTOR pathway inhibitor rapamycin alone or in combination with the multikinase inhibitor sorafenib, all xenografts responded with increased lipid content and a more differentiated gene expression profile. These human xenograft models may facilitate liposarcoma research and accelerate the generation of readily translatable preclinical data that could ultimately influence patient care. PMID:23416162

Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ.

PubMed

Joo, Kyeung Min; Kim, Jinkuk; Jin, Juyoun; Kim, Misuk; Seol, Ho Jun; Muradov, Johongir; Yang, Heekyoung; Choi, Yoon-La; Park, Woong-Yang; Kong, Doo-Sik; Lee, Jung-Il; Ko, Young-Hyeh; Woo, Hyun Goo; Lee, Jeongwu; Kim, Sunghoon; Nam, Do-Hyun

2013-01-31

Frequent discrepancies between preclinical and clinical results of anticancer agents demand a reliable translational platform that can precisely recapitulate the biology of human cancers. Another critical unmet need is the ability to predict therapeutic responses for individual patients. Toward this goal, we have established a library of orthotopic glioblastoma (GBM) xenograft models using surgical samples of GBM patients. These patient-specific GBM xenograft tumors recapitulate histopathological properties and maintain genomic characteristics of parental GBMs in situ. Furthermore, in vivo irradiation, chemotherapy, and targeted therapy of these xenograft tumors mimic the treatment response of parental GBMs. We also found that establishment of orthotopic xenograft models portends poor prognosis of GBM patients and identified the gene signatures and pathways signatures associated with the clinical aggressiveness of GBMs. Together, the patient-specific orthotopic GBM xenograft library represent the preclinically and clinically valuable "patient tumor's phenocopy" that represents molecular and functional heterogeneity of GBMs. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

The metastatic microenvironment: lung-derived factors control the viability of neuroblastoma lung metastasis.

PubMed

Maman, Shelly; Edry-Botzer, Liat; Sagi-Assif, Orit; Meshel, Tsipi; Yuan, Weirong; Lu, Wuyuan; Witz, Isaac P

2013-11-15

Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations-lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung-derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro-apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung-derived factors affected various additional progression-linked cellular characteristics of neuroblastoma cells, such as the expression of stem-cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease. Copyright © 2013 UICC.

Therapeutic effects of autologous lymphocytes activated with trastuzumab for xenograft mouse models of human breast cancer.

PubMed

Nakagawa, Shinichiro; Matsuoka, Yusuke; Ichihara, Hideaki; Yoshida, Hitoji; Yoshida, Kenshi; Ueoka, Ryuichi

2013-01-01

Trastuzumab (TTZ) is molecular targeted drug used for metastatic breast cancer patients overexpressing human epidermal growth factor receptor 2 (HER2). Therapeutic effects of lymphocytes activated with TTZ (TTZ-LAK) using xenograft mouse models of human breast cancer (MDA-MB-453) cells were examined in vivo. Remarkable reduction of tumor volume in a xenograft mouse models intravenously treated with TTZ-LAK cells after the subcutaneously inoculated of MDA-MB-453 cells was verified in vivo. The migration of TTZ-LAK cells in tumor of mouse models subcutaneously inoculated MDA-MB-453 cells was observed on the basis of histological analysis using immunostaining with CD-3. Induction of apoptosis in tumor of xenograft mice treated with TTZ-LAK cells was observed in micrographs using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method. It was noteworthy that the therapeutic effects of TTZ-LAK cells along with apoptosis were obtained for xenograft mouse models of human breast tumor in vivo.

Xenograft tumors derived from malignant pleural effusion of the patients with non-small-cell lung cancer as models to explore drug resistance.

PubMed

Xu, Yunhua; Zhang, Feifei; Pan, Xiaoqing; Wang, Guan; Zhu, Lei; Zhang, Jie; Wen, Danyi; Lu, Shun

2018-05-09

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) fusions show dramatic responses to specific tyrosine kinase inhibitors (TKIs); however, after 10-12 months, secondary mutations arise that confer resistance. We generated a murine xenograft model using patient-derived NSCLC cells isolated from the pleural fluid of two patients with NSCLC to investigate the mechanisms of resistance against the ALK- and EGFR-targeted TKIs crizotinib and osimertinib, respectively. Genotypes of patient biopsies and xenograft tumors were determined by whole exome sequencing (WES), and patients and xenograft-bearing mice received targeted treatment (crizotinib or osimertinib) accordingly. Xenograft mice were also treated for prolonged periods to identify whether the development of drug resistance and/or treatment responses were associated with tumor size. Finally, the pathology of patients biopsies and xenograft tumors were compared histologically. The histological characteristics and chemotherapy responses of xenograft tumors were similar to the actual patients. WES showed that the genotypes of the xenograft and patient tumors were similar (an echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) gene fusion (patient/xenograft: CTC15035 EML4-ALK ) and EGFR L858R and T790M mutations (patient/xenograft: CTC15063 EGFR L858R, T790M )). After continuous crizotinib or osimertinib treatment, WES data suggested that acquired ALK E1210K mutation conferred crizotinib resistance in the CTC15035 EML4-ALK xenograft, while decreased frequencies of EGFR L858R and T790M mutations plus the appearance of v-RAF murine sarcoma viral oncogene homolog B (BRAF) G7V mutations and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha (PIK3C2A) A86fs frame shift mutations led to osimertinib resistance in the CTC15063 EGFR L858R, T790M xenografts. We successfully developed a new method of generating drug resistance xenograft models from liquid biopsies using microfluidic technology, which might be a useful tool to investigate the mechanisms of drug resistance in NSCLC.

Next generation patient-derived prostate cancer xenograft models

PubMed Central

Lin, Dong; Xue, Hui; Wang, Yuwei; Wu, Rebecca; Watahiki, Akira; Dong, Xin; Cheng, Hongwei; Wyatt, Alexander W; Collins, Colin C; Gout, Peter W; Wang, Yuzhuo

2014-01-01

There is a critical need for more effective therapeutic approaches for prostate cancer. Research in this area, however, has been seriously hampered by a lack of clinically relevant, experimental in vivo models of the disease. This review particularly focuses on the development of prostate cancer xenograft models based on subrenal capsule grafting of patients’ tumor tissue into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This technique allows successful development of transplantable, patient-derived cancer tissue xenograft lines not only from aggressive metastatic, but also from localized prostate cancer tissues. The xenografts have been found to retain key biological properties of the original malignancies, including histopathological and molecular characteristics, tumor heterogeneity, response to androgen ablation and metastatic ability. As such, they are highly clinically relevant and provide valuable tools for studies of prostate cancer progression at cellular and molecular levels, drug screening for personalized cancer therapy and preclinical drug efficacy testing; especially when a panel of models is used to cover a broader spectrum of the disease. These xenograft models could therefore be viewed as next-generation models of prostate cancer. PMID:24589467

Genetically Engineered Cancer Models, But Not Xenografts, Faithfully Predict Anticancer Drug Exposure in Melanoma Tumors

PubMed Central

Combest, Austin J.; Roberts, Patrick J.; Dillon, Patrick M.; Sandison, Katie; Hanna, Suzan K.; Ross, Charlene; Habibi, Sohrab; Zamboni, Beth; Müller, Markus; Brunner, Martin; Sharpless, Norman E.

2012-01-01

Background. Rodent studies are a vital step in the development of novel anticancer therapeutics and are used in pharmacokinetic (PK), toxicology, and efficacy studies. Traditionally, anticancer drug development has relied on xenograft implantation of human cancer cell lines in immunocompromised mice for efficacy screening of a candidate compound. The usefulness of xenograft models for efficacy testing, however, has been questioned, whereas genetically engineered mouse models (GEMMs) and orthotopic syngeneic transplants (OSTs) may offer some advantages for efficacy assessment. A critical factor influencing the predictability of rodent tumor models is drug PKs, but a comprehensive comparison of plasma and tumor PK parameters among xenograft models, OSTs, GEMMs, and human patients has not been performed. Methods. In this work, we evaluated the plasma and tumor dispositions of an antimelanoma agent, carboplatin, in patients with cutaneous melanoma compared with four different murine melanoma models (one GEMM, one human cell line xenograft, and two OSTs). Results. Using microdialysis to sample carboplatin tumor disposition, we found that OSTs and xenografts were poor predictors of drug exposure in human tumors, whereas the GEMM model exhibited PK parameters similar to those seen in human tumors. Conclusions. The tumor PKs of carboplatin in a GEMM of melanoma more closely resembles the tumor disposition in patients with melanoma than transplanted tumor models. GEMMs show promise in becoming an improved prediction model for intratumoral PKs and response in patients with solid tumors. PMID:22993143

PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes

PubMed Central

Ferguson, Ross; Subramanian, Vasanta

2016-01-01

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease. PMID:27391595

PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes.

PubMed

Ferguson, Ross; Subramanian, Vasanta

2016-01-01

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease.

Novel dedifferentiated liposarcoma xenograft models reveal PTEN down-regulation as a malignant signature and response to PI3K pathway inhibition.

PubMed

Smith, Kathleen B; Tran, Linh M; Tam, Brenna M; Shurell, Elizabeth M; Li, Yunfeng; Braas, Daniel; Tap, William D; Christofk, Heather R; Dry, Sarah M; Eilber, Fritz C; Wu, Hong

2013-04-01

Liposarcoma is a type of soft tissue sarcoma that exhibits poor survival and a high recurrence rate. Treatment is generally limited to surgery and radiation, which emphasizes the need for better understanding of this disease. Because very few in vivo and in vitro models can reproducibly recapitulate the human disease, we generated several xenograft models from surgically resected human dedifferentiated liposarcoma. All xenografts recapitulated morphological and gene expression characteristics of the patient tumors after continuous in vivo passages. Importantly, xenograftability was directly correlated with disease-specific survival of liposarcoma patients. Thus, the ability for the tumor of a patient to engraft may help identify those patients who will benefit from more aggressive treatment regimens. Gene expression analyses highlighted the association between xenograftability and a unique gene expression signature, including down-regulated PTEN tumor-suppressor gene expression and a progenitor-like phenotype. When treated with the PI3K/AKT/mTOR pathway inhibitor rapamycin alone or in combination with the multikinase inhibitor sorafenib, all xenografts responded with increased lipid content and a more differentiated gene expression profile. These human xenograft models may facilitate liposarcoma research and accelerate the generation of readily translatable preclinical data that could ultimately influence patient care. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma

PubMed Central

Spel, Lotte; Boelens, Jaap-Jan; van der Steen, Dirk M.; Blokland, Nina J.G.; van Noesel, Max M.; Molenaar, Jan J.; Heemskerk, Mirjam H.M.

2015-01-01

Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20–40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neuroblastoma treatment, these immune escape mechanisms restrain clinical results. Therefore, we aimed to improve neuroblastoma immunogenicity to further the development of antigen-specific immunotherapy against neuroblastoma. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells which thereby readily sensitize neuroblastoma cells for recognition by CTLs. We show that oncoprotein PRAME serves as an immunodominant antigen for neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cells induce MHC I upregulation in neuroblastoma through contact-dependent secretion of IFNγ. Our results demonstrate remarkable plasticity in the peptide/MHC I surface expression of neuroblastoma cells, which is reversed when neuroblastoma cells experience innate immune attack by sensitized NK cells. These findings support the exploration of NK cells as adjuvant therapy to enforce neuroblastoma-specific CTL responses. PMID:26452036

Significance of clinical and biologic features in Stage 3 neuroblastoma: a report from the International Neuroblastoma Risk Group project.

PubMed

Meany, Holly J; London, Wendy B; Ambros, Peter F; Matthay, Katherine K; Monclair, Tom; Simon, Thorsten; Garaventa, Alberto; Berthold, Frank; Nakagawara, Akira; Cohn, Susan L; Pearson, Andrew D J; Park, Julie R

2014-11-01

International Neuroblastoma Staging System (INSS) Stage 3 neuroblastoma is a heterogeneous disease. Data from the International Neuroblastoma Risk Group (INRG) database were analyzed to define patient and tumor characteristics predictive of outcome. Of 8,800 patients in the INRG database, 1,483 with INSS Stage 3 neuroblastoma and complete follow-up data were analyzed. Secondary analysis was performed in 1,013 patients (68%) with MYCN-non-amplified (NA) tumors. Significant prognostic factors were identified via log-rank test comparisons of survival curves. Multivariable Cox proportional hazards regression model was used to identify factors independently predictive of event-free survival (EFS). Age at diagnosis (P < 0.0001), tumor MYCN status (P < 0.0001), and poorly differentiating/undifferentiated histology (P = 0.03) were independent predictors of EFS. Compared to other Stage 3 subgroups, outcome was inferior for patients ≥ 547 days with MYCN-NA neuroblastoma (P < 0.0001), and within this cohort, serum ferritin ≥ 96 ng/ml was associated with inferior EFS (P = 0.02). For patients <547 days of age with MYCN-NA tumors, serum ferritin levels were prognostic of overall survival (OS) (P = 0.04) and chromosome 11q aberration was prognostic of EFS (P = 0.03). Among patients with INSS Stage 3 neuroblastoma patients, age at diagnosis, MYCN status and histology predict outcome. Patients <547 days of age with MYCN-NA tumors that lack chromosome 11q aberrations or those with serum ferritin <96 ng/ml have excellent prognosis and should be considered for therapy reduction. Prospective clinical trials are needed to identify optimal therapy for those patients ≥ 547 days of age with undifferentiated histology or elevated serum ferritin. © 2014 Wiley Periodicals, Inc.

Clinical potentials of methylator phenotype in stage 4 high-risk neuroblastoma: an open challenge.

PubMed

Banelli, Barbara; Merlo, Domenico Franco; Allemanni, Giorgio; Forlani, Alessandra; Romani, Massimo

2013-01-01

Approximately 20% of stage 4 high-risk neuroblastoma patients are alive and disease-free 5 years after disease onset while the remaining experience rapid and fatal progression. Numerous findings underline the prognostic role of methylation of defined target genes in neuroblastoma without taking into account the clinical and biological heterogeneity of this disease. In this report we have investigated the methylation of the PCDHB cluster, the most informative member of the "Methylator Phenotype" in neuroblastoma, hypothesizing that if this epigenetic mark can predict overall and progression free survival in high-risk stage 4 neuroblastoma, it could be utilized to improve the risk stratification of the patients, alone or in conjunction with the previously identified methylation of the SFN gene (14.3.3sigma) that can accurately predict outcome in these patients. We have utilized univariate and multivariate models to compare the prognostic power of PCDHB methylation in terms of overall and progression free survival, quantitatively determined by pyrosequencing, with that of other markers utilized for the patients' stratification utilizing methylation thresholds calculated on neuroblastoma at stage 1-4 and only on stage 4, high-risk patients. Our results indicate that PCDHB accurately distinguishes between high- and intermediate/low risk stage 4 neuroblastoma in agreement with the established risk stratification criteria. However PCDHB cannot predict outcome in the subgroup of stage 4 patients at high-risk whereas methylation levels of SFN are suggestive of a "methylation gradient" associated with tumor aggressiveness as suggested by the finding of a higher threshold that defines a subset of patients with an extremely severe disease (OS <24 months). Because of the heterogeneity of neuroblastoma we believe that clinically relevant methylation markers should be selected and tested on homogeneous groups of patients rather than on patients at all stages.

Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity

PubMed Central

Klement, Giannoula; Baruchel, Sylvain; Rak, Janusz; Man, Shan; Clark, Katherine; Hicklin, Daniel J.; Bohlen, Peter; Kerbel, Robert S.

2000-01-01

Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 105:R15–R24 (2000). PMID:10772661

Altered expression of miRNAs and methylation of their promoters are correlated in neuroblastoma.

PubMed

Maugeri, Marco; Barbagallo, Davide; Barbagallo, Cristina; Banelli, Barbara; Di Mauro, Stefania; Purrello, Francesco; Magro, Gaetano; Ragusa, Marco; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

2016-12-13

Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5'-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5'-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma.

Altered expression of miRNAs and methylation of their promoters are correlated in neuroblastoma

PubMed Central

Di Mauro, Stefania; Purrello, Francesco; Magro, Gaetano; Ragusa, Marco; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

2016-01-01

Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5′-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5′-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma. PMID:27829219

Prussian blue nanoparticle-based photothermal therapy combined with checkpoint inhibition for photothermal immunotherapy of neuroblastoma.

PubMed

Cano-Mejia, Juliana; Burga, Rachel A; Sweeney, Elizabeth E; Fisher, John P; Bollard, Catherine M; Sandler, Anthony D; Cruz, Conrad Russell Y; Fernandes, Rohan

2017-02-01

We describe "photothermal immunotherapy," which combines Prussian blue nanoparticle (PBNP)-based photothermal therapy (PTT) with anti-CTLA-4 checkpoint inhibition for treating neuroblastoma, a common, hard-to-treat pediatric cancer. PBNPs exhibit pH-dependent stability, which makes them suitable for intratumorally-administered PTT. PBNP-based PTT is able to lower tumor burden and prime an immune response, specifically an increased infiltration of lymphocytes and T cells to the tumor area, which is complemented by the antitumor effects of anti-CTLA-4 immunotherapy, providing a more durable treatment against neuroblastoma in an animal model. We observe 55.5% survival in photothermal immunotherapy-treated mice at 100days compared to 12.5%, 0%, 0%, and 0% survival in mice receiving: anti-CTLA-4 alone, PBNPs alone, PTT alone, and no treatment, respectively. Additionally, long-term surviving, photothermal immunotherapy-treated mice exhibit protection against neuroblastoma rechallenge, suggesting the development of immunity against these tumors. Our findings suggest the potential of photothermal immunotherapy in improving treatments for neuroblastoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Human Neuroblastoma: From Basic Science to Clinical Debut of Cellular Oncogenes

NASA Astrophysics Data System (ADS)

Schwab, Manfred

Neuroblastoma is a childhood embryonic tumor of migrating neuroectodermal cells derived from the neural crest and destined for the adrenal medulla and the sympathetic nervous system. It very often has a rapidly progressive clinical course, and although many advances have been made in understanding the development of this tumor, improving the survival rates particularly in patients with metastatic tumor has been a frustrating experience. The mechanisms leading to neuroblastoma are largely unclear, but nonrandom chromosomal changes discovered early suggested the involvement of genetic alterations. Most prominent among these is the amplification of the oncogene MYCN, which identifies a group of patients who have a particularly dire prognosis. Amplified MYCN is used today as a prognostic marker on which therapy design is based to a large extent. An unusual aspect of neuroblastoma is the high rate at which tumors regress spontaneously, even in infants with extensive liver involvement and numerous subcutaneous nodules. Identifying the molecular and cellular basis of spontaneous regression could result in improved therapeutic approaches. Neuroblastoma is a model tumor with many fascinating aspects but has remained a challenge to the pediatric oncologist

Comprehensive Molecular Profiling of African-American Prostate Cancer to Inform on Prognosis and Disease Biology

DTIC Science & Technology

2016-10-01

prostate cancer through sequencing xenografts and tissue samples. Qualify novel drivers of AR- prostate cancer through in vitro models. Develop novel...ability of RNASEH2A to modulate radio-sensitivity in prostate cancer cell lines and xenograft models. 3: Investigate RNASEH2A as a marker of radio...lung cancer clinical management. List of the Specific Aims: Aim 1: To establish patient-derived xenografts (PDX) models of pre-neoplastic lesions

A Splice Variant of the Human Ion Channel TRPM2 Modulates Neuroblastoma Tumor Growth through Hypoxia-inducible Factor (HIF)-1/2α*

PubMed Central

Chen, Shu-jen; Hoffman, Nicholas E.; Shanmughapriya, Santhanam; Bao, Lei; Keefer, Kerry; Conrad, Kathleen; Merali, Salim; Takahashi, Yoshinori; Abraham, Thomas; Hirschler-Laszkiewicz, Iwona; Wang, JuFang; Zhang, Xue-Qian; Song, Jianliang; Barrero, Carlos; Shi, Yuguang; Kawasawa, Yuka Imamura; Bayerl, Michael; Sun, Tianyu; Barbour, Mustafa; Wang, Hong-Gang; Madesh, Muniswamy; Cheung, Joseph Y.; Miller, Barbara A.

2014-01-01

The calcium-permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full-length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of forkhead box transcription factor 3a (FOXO3a) and superoxide dismutase 2. Cells expressing the dominant negative short isoform (TRPM2-S) had reduced FOXO3a and superoxide dismutase 2 levels, reduced calcium influx in response to oxidative stress, and enhanced reactive oxygen species, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared with tumors expressing TRPM2-L. Expression of hypoxia-inducible factor (HIF)-1/2α was significantly reduced in TRPM2-S-expressing tumor cells as was expression of target proteins regulated by HIF-1/2α including those involved in glycolysis (lactate dehydrogenase A and enolase 2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (cytochrome oxidase 4.1/4.2 in complex IV). The reduction in HIF-1/2α was mediated through both significantly reduced HIF-1/2α mRNA levels and increased levels of von Hippel-Lindau E3 ligase in TRPM2-S-expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S-expressing cells after doxorubicin treatment was rescued by gain of HIF-1 or -2α function. These data suggest that TRPM2 activity is important for tumor growth and for cell viability and survival following doxorubicin treatment and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2α, mitochondrial function, and mitophagy. PMID:25391657

Orthotopic Patient-Derived Glioblastoma Xenografts in Mice.

PubMed

Xu, Zhongye; Kader, Michael; Sen, Rajeev; Placantonakis, Dimitris G

2018-01-01

Patient-derived xenografts (PDX) provide in vivo glioblastoma (GBM) models that recapitulate actual tumors. Orthotopic tumor xenografts within the mouse brain are obtained by injection of GBM stem-like cells derived from fresh surgical specimens. These xenografts reproduce GBM's histologic complexity and hallmark biological behaviors, such as brain invasion, angiogenesis, and resistance to therapy. This method has become essential for analyzing mechanisms of tumorigenesis and testing the therapeutic effect of candidate agents in the preclinical setting. Here, we describe a protocol for establishing orthotopic tumor xenografts in the mouse brain with human GBM cells.

Neuroblastoma

MedlinePlus

... Staying Safe Videos for Educators Search English Español Neuroblastoma KidsHealth / For Parents / Neuroblastoma What's in this article? ... infancy, the chance of recovery is good. About Neuroblastoma Neuroblastoma most commonly starts in the tissue of ...

The inhibition of 45A ncRNA expression reduces tumor formation, affecting tumor nodules compactness and metastatic potential in neuroblastoma cells

PubMed Central

Russo, Debora; Poggi, Alessandro; Villa, Federico; Brizzolara, Antonella; Canale, Claudio; Mescola, Andrea; Daga, Antonio; Russo, Claudio; Nizzari, Mario; Florio, Tullio; Menichini, Paola; Pagano, Aldo

2017-01-01

We recently reported the in vitro over-expression of 45A, a RNA polymerase III-transcribed non-coding (nc)RNA, that perturbs the intracellular content of FE65L1 affecting cell proliferation rate, short-term response to genotoxic stress, substrate adhesion capacity and, ultimately, increasing the tumorigenic potential of human neuroblastoma cells. In this work, to deeply explore the mechanism by which 45A ncRNA contributes to cancer development, we targeted in vitro and in vivo 45A levels by the stable overexpression of antisense 45A RNA. 45A downregulation leads to deep modifications of cytoskeleton organization, adhesion and migration of neuroblastoma cells. These effects are correlated with alterations in the expression of several genes including GTSE1 (G2 and S phase-expressed-1), a crucial regulator of tumor cell migration and metastatic potential. Interestingly, the downregulation of 45A ncRNA strongly affects the in vivo tumorigenic potential of SKNBE2 neuroblastoma cells, increasing tumor nodule compactness and reducing GTSE1 protein expression in a subcutaneous neuroblastoma mouse model. Moreover, intracardiac injection of neuroblastoma cells showed that downregulation of 45A ncRNA also influences tumor metastatic ability. In conclusion, our data highlight a key role of 45A ncRNA in cancer development and suggest that its modulation might represent a possible novel anticancer therapeutic approach. PMID:28029658

The inhibition of 45A ncRNA expression reduces tumor formation, affecting tumor nodules compactness and metastatic potential in neuroblastoma cells.

PubMed

Penna, Ilaria; Gigoni, Arianna; Costa, Delfina; Vella, Serena; Russo, Debora; Poggi, Alessandro; Villa, Federico; Brizzolara, Antonella; Canale, Claudio; Mescola, Andrea; Daga, Antonio; Russo, Claudio; Nizzari, Mario; Florio, Tullio; Menichini, Paola; Pagano, Aldo

2017-01-31

We recently reported the in vitro over-expression of 45A, a RNA polymerase III-transcribed non-coding (nc)RNA, that perturbs the intracellular content of FE65L1 affecting cell proliferation rate, short-term response to genotoxic stress, substrate adhesion capacity and, ultimately, increasing the tumorigenic potential of human neuroblastoma cells. In this work, to deeply explore the mechanism by which 45A ncRNA contributes to cancer development, we targeted in vitro and in vivo 45A levels by the stable overexpression of antisense 45A RNA.45A downregulation leads to deep modifications of cytoskeleton organization, adhesion and migration of neuroblastoma cells. These effects are correlated with alterations in the expression of several genes including GTSE1 (G2 and S phase-expressed-1), a crucial regulator of tumor cell migration and metastatic potential. Interestingly, the downregulation of 45A ncRNA strongly affects the in vivo tumorigenic potential of SKNBE2 neuroblastoma cells, increasing tumor nodule compactness and reducing GTSE1 protein expression in a subcutaneous neuroblastoma mouse model. Moreover, intracardiac injection of neuroblastoma cells showed that downregulation of 45A ncRNA also influences tumor metastatic ability. In conclusion, our data highlight a key role of 45A ncRNA in cancer development and suggest that its modulation might represent a possible novel anticancer therapeutic approach.

Inhibition of neuroblastoma cell proliferation with omega-3 fatty acids and treatment of a murine model of human neuroblastoma using a diet enriched with omega-3 fatty acids in combination with sunitinib.

PubMed

Barnés, Carmen M; Prox, Daniela; Christison-Lagay, Emily A; Le, Hau D; Short, Sarah; Cassiola, Flavia; Panigrahy, Dipak; Chaponis, Deviney; Butterfield, Catherine; Nehra, Deepika; Fallon, Erica M; Kieran, Mark; Folkman, Judah; Puder, Mark

2012-02-01

We investigated the use of dietary omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) in the treatment of neuroblastoma both as a sole agent and in combination with sunitinib, a broad-spectrum tyrosine kinase receptor inhibitor. Substitution of all dietary fat with menhaden oil (ω-3 PUFA rich) resulted in a 40-70% inhibition of tumor growth and a statistically significant difference in the levels of several PUFAs (18:2 ω-6, 20:4 ω-6, 22:4 ω-6, 20:5 ω-3) as compared with a control diet. Furthermore, tumors from animals on the ω-3 fatty acid (FA)-enriched diet had an elevated triene/tetraene ratio suggestive of a change in local eicosanoid metabolism in these tissues similar to that seen with essential fatty acid deficiency. The ω-3 FA-enriched diet also decreased tumor-associated inflammatory cells and induced mitochondrial changes suggestive of mitochondrial damage. Combination treatment with sunitinib resulted in further reduction in tumor proliferation and microvessel density. These findings suggest a potential role for ω-3 PUFAs in the combination treatment of neuroblastoma. We used a murine model of orthotopic and subcutaneous human neuroblastoma and diets that differ in the FA content to define the optimal dietary ω-3/omega-6 (ω-6) FA ratio required for the inhibition of these tumors.

Theranostic gas-generating nanoparticles for targeted ultrasound imaging and treatment of neuroblastoma.

PubMed

Lee, Jangwook; Min, Hyun-Su; You, Dong Gil; Kim, Kwangmeyung; Kwon, Ick Chan; Rhim, Taiyoun; Lee, Kuen Yong

2016-02-10

The development of safe and efficient diagnostic/therapeutic agents for treating cancer in clinics remains challenging due to the potential toxicity of conventional agents. Although the annual incidence of neuroblastoma is not that high, the disease mainly occurs in children, a population vulnerable to toxic contrast agents and therapeutics. We demonstrate here that cancer-targeting, gas-generating polymeric nanoparticles are useful as a theranostic tool for ultrasound (US) imaging and treating neuroblastoma. We encapsulated calcium carbonate using poly(d,l-lactide-co-glycolide) and created gas-generating polymer nanoparticles (GNPs). These nanoparticles release carbon dioxide bubbles under acidic conditions and enhance US signals. When GNPs are modified using rabies virus glycoprotein (RVG) peptide, a targeting moiety to neuroblastoma, RVG-GNPs effectively accumulate at the tumor site and substantially enhance US signals in a tumor-bearing mouse model. Intravenous administration of RVG-GNPs also reduces tumor growth in the mouse model without the use of conventional therapeutic agents. This approach to developing theranostic agents with disease-targeting ability may provide useful strategy for the detection and treatment of cancers, allowing safe and efficient clinical applications with fewer side effects than may occur with conventional agents. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of RNA-seq and microarray-based models for clinical endpoint prediction.

PubMed

Zhang, Wenqian; Yu, Ying; Hertwig, Falk; Thierry-Mieg, Jean; Zhang, Wenwei; Thierry-Mieg, Danielle; Wang, Jian; Furlanello, Cesare; Devanarayan, Viswanath; Cheng, Jie; Deng, Youping; Hero, Barbara; Hong, Huixiao; Jia, Meiwen; Li, Li; Lin, Simon M; Nikolsky, Yuri; Oberthuer, André; Qing, Tao; Su, Zhenqiang; Volland, Ruth; Wang, Charles; Wang, May D; Ai, Junmei; Albanese, Davide; Asgharzadeh, Shahab; Avigad, Smadar; Bao, Wenjun; Bessarabova, Marina; Brilliant, Murray H; Brors, Benedikt; Chierici, Marco; Chu, Tzu-Ming; Zhang, Jibin; Grundy, Richard G; He, Min Max; Hebbring, Scott; Kaufman, Howard L; Lababidi, Samir; Lancashire, Lee J; Li, Yan; Lu, Xin X; Luo, Heng; Ma, Xiwen; Ning, Baitang; Noguera, Rosa; Peifer, Martin; Phan, John H; Roels, Frederik; Rosswog, Carolina; Shao, Susan; Shen, Jie; Theissen, Jessica; Tonini, Gian Paolo; Vandesompele, Jo; Wu, Po-Yen; Xiao, Wenzhong; Xu, Joshua; Xu, Weihong; Xuan, Jiekun; Yang, Yong; Ye, Zhan; Dong, Zirui; Zhang, Ke K; Yin, Ye; Zhao, Chen; Zheng, Yuanting; Wolfinger, Russell D; Shi, Tieliu; Malkas, Linda H; Berthold, Frank; Wang, Jun; Tong, Weida; Shi, Leming; Peng, Zhiyu; Fischer, Matthias

2015-06-25

Gene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model. We generate gene expression profiles from 498 primary neuroblastomas using both RNA-seq and 44 k microarrays. Characterization of the neuroblastoma transcriptome by RNA-seq reveals that more than 48,000 genes and 200,000 transcripts are being expressed in this malignancy. We also find that RNA-seq provides much more detailed information on specific transcript expression patterns in clinico-genetic neuroblastoma subgroups than microarrays. To systematically compare the power of RNA-seq and microarray-based models in predicting clinical endpoints, we divide the cohort randomly into training and validation sets and develop 360 predictive models on six clinical endpoints of varying predictability. Evaluation of factors potentially affecting model performances reveals that prediction accuracies are most strongly influenced by the nature of the clinical endpoint, whereas technological platforms (RNA-seq vs. microarrays), RNA-seq data analysis pipelines, and feature levels (gene vs. transcript vs. exon-junction level) do not significantly affect performances of the models. We demonstrate that RNA-seq outperforms microarrays in determining the transcriptomic characteristics of cancer, while RNA-seq and microarray-based models perform similarly in clinical endpoint prediction. Our findings may be valuable to guide future studies on the development of gene expression-based predictive models and their implementation in clinical practice.

Establishing prostate cancer patient derived xenografts: lessons learned from older studies.

PubMed

Russell, Pamela J; Russell, Peter; Rudduck, Christina; Tse, Brian W C; Williams, Elizabeth D; Raghavan, Derek

2015-05-01

Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43-46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model. © 2015 The Authors. The Prostate published by Wiley Periodicals, Inc.

Establishing Prostate Cancer Patient Derived Xenografts: Lessons Learned From Older Studies

PubMed Central

Russell, Pamela J; Russell, Peter; Rudduck, Christina; Tse, Brian W-C; Williams, Elizabeth D; Raghavan, Derek

2015-01-01

Background Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. Methods We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Results Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43–46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Conclusions Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model. Prostate 75: 628–636, 2015. © The Authors. The Prostate published by Wiley Periodicals, Inc. PMID:25560784

ALK is a MYCN target gene and regulates cell migration and invasion in neuroblastoma

PubMed Central

Hasan, Md. Kamrul; Nafady, Asmaa; Takatori, Atsushi; Kishida, Satoshi; Ohira, Miki; Suenaga, Yusuke; Hossain, Shamim; Akter, Jesmin; Ogura, Atsushi; Nakamura, Yohko; Kadomatsu, Kenji; Nakagawara, Akira

2013-01-01

Human anaplastic lymphoma kinase (ALK) has been identified as an oncogene that is mutated or amplified in NBLs. To obtain a better understanding of the molecular events associated with ALK in the pathogenesis of NBL, it is necessary to clarify how ALK gene contributes to NBL progression. In the present study, we found that ALK expression was significantly high in NBL clinical samples with amplified MYCN (n = 126, P < 0.01) and in developing tumors of MYCN-transgenic mice. Indeed, promoter analysis revealed that ALK is a direct transcriptional target of MYCN. Overexpression and knockdown of ALK demonstrated its function in cell proliferation, migration and invasion. Moreover, treatment with an ALK inhibitor, TAE-684, efficiently suppressed such biological effects in MYCN amplified cells and tumor growth of the xenograft in mice. Our present findings explore the fundamental understanding of ALK in order to develop novel therapeutic tools by targeting ALK for aggressive NBL treatment. PMID:24356251

Artificial neural network classifier predicts neuroblastoma patients' outcome.

PubMed

Cangelosi, Davide; Pelassa, Simone; Morini, Martina; Conte, Massimo; Bosco, Maria Carla; Eva, Alessandra; Sementa, Angela Rita; Varesio, Luigi

2016-11-08

More than fifty percent of neuroblastoma (NB) patients with adverse prognosis do not benefit from treatment making the identification of new potential targets mandatory. Hypoxia is a condition of low oxygen tension, occurring in poorly vascularized tissues, which activates specific genes and contributes to the acquisition of the tumor aggressive phenotype. We defined a gene expression signature (NB-hypo), which measures the hypoxic status of the neuroblastoma tumor. We aimed at developing a classifier predicting neuroblastoma patients' outcome based on the assessment of the adverse effects of tumor hypoxia on the progression of the disease. Multi-layer perceptron (MLP) was trained on the expression values of the 62 probe sets constituting NB-hypo signature to develop a predictive model for neuroblastoma patients' outcome. We utilized the expression data of 100 tumors in a leave-one-out analysis to select and construct the classifier and the expression data of the remaining 82 tumors to test the classifier performance in an external dataset. We utilized the Gene set enrichment analysis (GSEA) to evaluate the enrichment of hypoxia related gene sets in patients predicted with "Poor" or "Good" outcome. We utilized the expression of the 62 probe sets of the NB-Hypo signature in 182 neuroblastoma tumors to develop a MLP classifier predicting patients' outcome (NB-hypo classifier). We trained and validated the classifier in a leave-one-out cross-validation analysis on 100 tumor gene expression profiles. We externally tested the resulting NB-hypo classifier on an independent 82 tumors' set. The NB-hypo classifier predicted the patients' outcome with the remarkable accuracy of 87 %. NB-hypo classifier prediction resulted in 2 % classification error when applied to clinically defined low-intermediate risk neuroblastoma patients. The prediction was 100 % accurate in assessing the death of five low/intermediated risk patients. GSEA of tumor gene expression profile demonstrated the hypoxic status of the tumor in patients with poor prognosis. We developed a robust classifier predicting neuroblastoma patients' outcome with a very low error rate and we provided independent evidence that the poor outcome patients had hypoxic tumors, supporting the potential of using hypoxia as target for neuroblastoma treatment.

Colorectal cancer patient-derived xenografted tumors maintain characteristic features of the original tumors.

PubMed

Cho, Yong Beom; Hong, Hye Kyung; Choi, Yoon-La; Oh, Ensel; Joo, Kyeung Min; Jin, Juyoun; Nam, Do-Hyun; Ko, Young-Hyeh; Lee, Woo Yong

2014-04-01

Despite significant improvements in colon cancer outcomes over the past few decades, preclinical development of more effective therapeutic strategies is still limited by the availability of clinically relevant animal models. To meet those clinical unmet needs, we generated a well-characterized in vivo preclinical platform for colorectal cancer using fresh surgical samples. Primary and metastatic colorectal tumor tissues (1-2 mm(3)) that originate from surgery were implanted into the subcutaneous space of nude mice and serially passaged in vivo. Mutation status, hematoxylin and eosin staining, short tandem repeat profiling, and array comparative genomic hybridization were used to validate the similarity of molecular characteristics between the patient tumors and tumors obtained from xenografts. From surgical specimens of 143 patients, 97 xenograft models were obtained in immunodeficient mice (establish rate = 67%). Thirty-nine xenograft models were serially expanded further in mice with a mean time to reach a size of 1000-1500 mm(3) of 90 ± 20 d. Histologic and immunohistochemical analyses revealed a high degree of pathologic similarity including histologic architecture and expression of CEA, CK7, and CD20 between the patient and xenograft tumors. Molecular analysis showed that genetic mutations, genomic alterations, and gene expression patterns of each patient tumor were also well conserved in the corresponding xenograft tumor. Xenograft animal models derived from fresh surgical sample maintained the key characteristic features of the original tumors, suggesting that this in vivo platform can be useful for preclinical development of novel therapeutic approaches to colorectal cancers. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterisation of the p53 pathway in cell lines established from TH-MYCN transgenic mouse tumours.

PubMed

Chen, Lindi; Esfandiari, Arman; Reaves, William; Vu, Annette; Hogarty, Michael D; Lunec, John; Tweddle, Deborah A

2018-03-01

Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors.

Modeling Leukemogenesis in the Zebrafish Using Genetic and Xenograft Models.

PubMed

Rajan, Vinothkumar; Dellaire, Graham; Berman, Jason N

2016-01-01

The zebrafish is a widely accepted model to study leukemia. The major advantage of studying leukemogenesis in zebrafish is attributed to its short life cycle and superior imaging capacity. This chapter highlights using transgenic- and xenograft-based models in zebrafish to study a specific leukemogenic mutation and analyze therapeutic responses in vivo.

microRNA-221 Enhances MYCN via Targeting Nemo-like Kinase and Functions as an Oncogene Related to Poor Prognosis in Neuroblastoma.

PubMed

He, Xiao-Yan; Tan, Zheng-Lan; Mou, Qin; Liu, Fang-Jie; Liu, Shan; Yu, Chao-Wen; Zhu, Jin; Lv, Lin-Ya; Zhang, Jun; Wang, Shan; Bao, Li-Ming; Peng, Bin; Zhao, Hui; Zou, Lin

2017-06-01

Purpose: MYCN is one of the most well-characterized genetic markers of neuroblastoma. However, the mechanisms as to how MYCN mediate neuroblastoma tumorigenesis are not fully clear. Increasing evidence has confirmed that the dysregulation of miRNAs is involved in MYCN-mediated neuroblastoma tumorigenesis, supporting their potential as therapeutic targets for neuroblastoma. Although miR-221 has been reported as one of the upregulated miRNAs, the interplay between miR-221 and MYCN-mediated neuroblastoma progression remains largely elusive. Experimental Design: The expression of miR-221 in the formalin-fixed, paraffin-embedded tissues from 31 confirmed patients with neuroblastoma was detected by locked nucleic acid- in situ hybridization and qRT-PCR. The correlation between miR-221 expression and clinical features in patients with neuroblastoma was assessed. The mechanisms as to how miR-221 regulate MYCN in neuroblastoma were addressed. The effect of miR-221 on cellular proliferation in neuroblastoma was determined both in vitro and in vivo Results: miR-221 was significantly upregulated in neuroblastoma tumor cells and tissues that overexpress MYCN, and high expression of miR-221 was positively associated with poor survival in patients with neuroblastoma. Nemo-like kinase (NLK) as a direct target of miR-221 in neuroblastoma was verified. In addition, overexpression of miR-221 decreased LEF1 phosphorylation but increased the expression of MYCN via targeting of NLK and further regulated cell cycle, particularly in S-phase, promoting the growth of neuroblastoma cells. Conclusions: This study provides a novel insight for miR-221 in the control of neuroblastoma cell proliferation and tumorigenesis, suggesting potentials of miR-221 as a prognosis marker and therapeutic target for patients with MYCN overexpressing neuroblastoma. Clin Cancer Res; 23(11); 2905-18. ©2016 AACR . ©2016 American Association for Cancer Research.

Xenograft model for therapeutic drug testing in recurrent respiratory papillomatosis.

PubMed

Ahn, Julie; Bishop, Justin A; Akpeng, Belinda; Pai, Sara I; Best, Simon R A

2015-02-01

Identifying effective treatment for papillomatosis is limited by a lack of animal models, and there is currently no preclinical model for testing potential therapeutic agents. We hypothesized that xenografting of papilloma may facilitate in vivo drug testing to identify novel treatment options. A biopsy of fresh tracheal papilloma was xenografted into a NOD-scid-IL2Rgamma(null) (NSG) mouse. The xenograft began growing after 5 weeks and was serially passaged over multiple generations. Each generation showed a consistent log-growth pattern, and in all xenografts, the presence of the human papillomavirus (HPV) genome was confirmed by polymerase chain reaction (PCR). Histopathologic analysis demonstrated that the squamous architecture of the original papilloma was maintained in each generation. In vivo drug testing with bevacizumab (5 mg/kg i.p. twice weekly for 3 weeks) showed a dramatic therapeutic response compared to saline control. We report here the first successful case of serial xenografting of a tracheal papilloma in vivo with a therapeutic response observed with drug testing. In severely immunocompromised mice, the HPV genome and squamous differentiation of the papilloma can be maintained for multiple generations. This is a feasible approach to identify therapeutic agents in the treatment of recurrent respiratory papillomatosis. © The Author(s) 2014.

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

PubMed

Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

2012-08-01

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

OSI-930: a novel selective inhibitor of Kit and kinase insert domain receptor tyrosine kinases with antitumor activity in mouse xenograft models.

PubMed

Garton, Andrew J; Crew, Andrew P A; Franklin, Maryland; Cooke, Andrew R; Wynne, Graham M; Castaldo, Linda; Kahler, Jennifer; Winski, Shannon L; Franks, April; Brown, Eric N; Bittner, Mark A; Keily, John F; Briner, Paul; Hidden, Chris; Srebernak, Mary C; Pirrit, Carrie; O'Connor, Matthew; Chan, Anna; Vulevic, Bojana; Henninger, Dwight; Hart, Karen; Sennello, Regina; Li, An-Hu; Zhang, Tao; Richardson, Frank; Emerson, David L; Castelhano, Arlindo L; Arnold, Lee D; Gibson, Neil W

2006-01-15

OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.

Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model.

PubMed

Chen, Shanshan; Li, Xuechun; Chen, Rongming; Yin, Mingang; Zheng, Qiuhong

2016-09-01

Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo , and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression.

Differential Splicing of Oncogenes and Tumor Suppressor Genes in African- and Caucasian-American Populations: Contributing Factor in Prostate Cancer Disparities

DTIC Science & Technology

2015-10-01

signaling protein as defined by in vitro assays and mouse xenograft studies, ii) is associated with worse prognosis in patients, and iii) is resistant to...available. Specific Aim 2. To characterize oncogenic differences of splice variant pairs in vivo using xenograft animal models. Task 1. Validate...idelalisib as defined by in vitro assays and mouse xenograft models. In contrast, the corresponding EA isoform (PI3Kδ-L) encodes a less aggressive isoform

Breast Cancer Therapy Using Antibody-Endostatin Fusion Proteins

DTIC Science & Technology

2008-04-01

animal tumor and/or human xenograft models. Task 9. Anti-tumor activity in human tumor xenografts (Months 18 -24) Anti-tumor efficacy in human...breast cancer SK-BR-3 xenografts in SCID mice. SK-BR-3 was implanted on the flank of SCID mice.9 The treatment was repeated (shown below in Fig. 18 ...untreated group (p=0.0141) (Fig. 18 ). KEY RESEARCH ACCOMPLISHMENTS o Treatment of established SK-BR-3 xenografts in SCID mice with the αHER2

Antitumor effect of bevacizumab in a xenograft model of canine hemangiopericytoma.

PubMed

Michishita, Masaki; Uto, Tatsuya; Nakazawa, Ryota; Yoshimura, Hisashi; Ogihara, Kikumi; Naya, Yuko; Tajima, Tsuyoshi; Azakami, Daigo; Kishikawa, Seigo; Arai, Toshiro; Takahashi, Kimimasa

2013-01-01

Canine hemangiopericytoma (CHP) is characterized by frequent local recurrence and increased invasiveness. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in tumors. The aim of the present study was to investigate the effect of a single dose of bevacizumab on a xenograft model of CHP. VEGF protein was secreted from cultured CHP cells and interacted with bevacizumab. Bevacizumab treatment suppressed tumor growth by inhibiting tumor angiogenesis, whereas no significant differences were observed in the proliferation index and apoptosis rates of treated and untreated mice. Thus, bevacizumab had antitumor effects in a xenograft model of CHP.

A protein in neuroblastoma could be a target of immunotoxins or immunotherapy | Center for Cancer Research

Cancer.gov

A cell surface protein, glycoprotein glypican-2 (GPC2), has been found to be an effective therapeutic target in cell cultures and mouse models that mimic childhood neuroblastoma.  The CCR scientists who made this discovery, reported July 24, 2017, in PNAS, have also produced immunotoxins and chimeric antigen receptor (CAR) T cells, a type of immunotherapy, that have shown

Tumor spheroid model for the biologically targeted radiotherapy of neuroblastoma micrometastases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, K.A.; Mairs, R.; Murray, T.

Neuroblastoma is a pediatric malignancy with a poor prognosis at least partly attributable to an early pattern of dissemination. New approaches to treatment of micrometastases include targeted radiotherapy using radiolabeled antibodies or molecules which are taken up preferentially by tumor cells. Multicellular tumor spheroids (MTS) resemble micrometastases during the avascular phase of their development. A human neuroblastoma cell line (NBl-G) was grown as MTS and incubated briefly with a radiolabeled monoclonal antibody ({sup 131}I-UJ13A) directed against neuroectodermal antigens. Spheroid response was evaluated in terms of regrowth delay or proportion sterilized. A dose-response relationship was demonstrated in terms of {sup 131}Imore » activity or duration of incubation. Control experiments using unlabeled UJ13A, radiolabeled nonspecific antibody (T2.10), radiolabeled human serum albumin, and radiolabeled sodium iodide showed these to be relatively ineffective compared to {sup 131}I-UJ13A. The cell line NBl-G grown as MTS has also been found to preferentially accumulate the radiolabeled catecholamine precursor molecule m-({sup 131}I)iodobenzylguanidine compared to cell lines derived from other tumor types. NBl-G cells grown as MTS provide a promising laboratory model for targeted radiotherapy of neuroblastoma micrometastases using radiolabeled antibodies or m-iodobenzylguanidine.« less

Novel synthetic monoketone transmute radiation-triggered NFκB-dependent TNFα cross-signaling feedback maintained NFκB and favors neuroblastoma regression.

PubMed

Aravindan, Sheeja; Natarajan, Mohan; Awasthi, Vibhudutta; Herman, Terence S; Aravindan, Natarajan

2013-01-01

Recently, we demonstrated that radiation (IR) instigates the occurrence of a NFκB-TNFα feedback cycle which sustains persistent NFκB activation in neuroblastoma (NB) cells and favors survival advantage and clonal expansion. Further, we reported that curcumin targets IR-induced survival signaling and NFκB dependent hTERT mediated clonal expansion in human NB cells. Herein, we investigated the efficacy of a novel synthetic monoketone, EF24, a curcumin analog in inhibiting persistent NFκB activation by disrupting the IR-induced NFκB-TNFα-NFκB feedback signaling in NB and subsequent mitigation of survival advantage and clonal expansion. EF24 profoundly suppressed the IR-induced NFκB-DNA binding activity/promoter activation and, maintained the NFκB repression by deterring NFκB-dependent TNFα transactivation/intercellular secretion in genetically varied human NB (SH-SY5Y, IMR-32, SK-PN-DW, MC-IXC and SK-N-MC) cell types. Further, EF24 completely suppressed IR-induced NFκB-TNFα cross-signaling dependent transactivation/translation of pro-survival IAP1, IAP2 and Survivin and subsequent cell survival. In corroboration, EF24 treatment maximally blocked IR-induced NFκB dependent hTERT transactivation/promoter activation, telomerase activation and consequent clonal expansion. EF24 displayed significant regulation of IR-induced feedback dependent NFκB and NFκB mediated survival signaling and complete regression of NB xenograft. Together, the results demonstrate for the first time that, novel synthetic monoketone EF24 potentiates radiotherapy and mitigates NB progression by selectively targeting IR-triggered NFκB-dependent TNFα-NFκB cross-signaling maintained NFκB mediated survival advantage and clonal expansion.

Novel Synthetic Monoketone Transmute Radiation-Triggered NFκB-Dependent TNFα Cross-Signaling Feedback Maintained NFκB and Favors Neuroblastoma Regression

PubMed Central

Aravindan, Sheeja; Natarajan, Mohan; Awasthi, Vibhudutta; Herman, Terence S.; Aravindan, Natarajan

2013-01-01

Recently, we demonstrated that radiation (IR) instigates the occurrence of a NFκB-TNFα feedback cycle which sustains persistent NFκB activation in neuroblastoma (NB) cells and favors survival advantage and clonal expansion. Further, we reported that curcumin targets IR-induced survival signaling and NFκB dependent hTERT mediated clonal expansion in human NB cells. Herein, we investigated the efficacy of a novel synthetic monoketone, EF24, a curcumin analog in inhibiting persistent NFκB activation by disrupting the IR-induced NFκB-TNFα-NFκB feedback signaling in NB and subsequent mitigation of survival advantage and clonal expansion. EF24 profoundly suppressed the IR-induced NFκB-DNA binding activity/promoter activation and, maintained the NFκB repression by deterring NFκB-dependent TNFα transactivation/intercellular secretion in genetically varied human NB (SH-SY5Y, IMR-32, SK–PN–DW, MC-IXC and SK–N-MC) cell types. Further, EF24 completely suppressed IR-induced NFκB-TNFα cross-signaling dependent transactivation/translation of pro-survival IAP1, IAP2 and Survivin and subsequent cell survival. In corroboration, EF24 treatment maximally blocked IR-induced NFκB dependent hTERT transactivation/promoter activation, telomerase activation and consequent clonal expansion. EF24 displayed significant regulation of IR-induced feedback dependent NFκB and NFκB mediated survival signaling and complete regression of NB xenograft. Together, the results demonstrate for the first time that, novel synthetic monoketone EF24 potentiates radiotherapy and mitigates NB progression by selectively targeting IR-triggered NFκB-dependent TNFα-NFκB cross-signaling maintained NFκB mediated survival advantage and clonal expansion. PMID:23967300

Neuroblastoma

MedlinePlus

Cancer - neuroblastoma ... Neuroblastoma can occur in many areas of the body. It develops from the tissues that form the ... pressure, digestion, and levels of certain hormones. Most neuroblastomas begin in the abdomen, in the adrenal gland, ...

Utility of a human-mouse xenograft model and in vivo near-infrared fluorescent imaging for studying wound healing.

PubMed

Shanmugam, Victoria K; Tassi, Elena; Schmidt, Marcel O; McNish, Sean; Baker, Stephen; Attinger, Christopher; Wang, Hong; Shara, Nawar; Wellstein, Anton

2015-12-01

To study the complex cellular interactions involved in wound healing, it is essential to have an animal model that adequately mimics the human wound microenvironment. Currently available murine models are limited because wound contraction introduces bias into wound surface area measurements. The purpose of this study was to demonstrate utility of a human-mouse xenograft model for studying human wound healing. Normal human skin was harvested from elective abdominoplasty surgery, xenografted onto athymic nude (nu/nu) mice, and allowed to engraft for 3 months. The graft was then wounded using a 2-mm punch biopsy. Wounds were harvested on sequential days to allow tissue-based markers of wound healing to be followed sequentially. On the day of wound harvest, mice were injected with XenoLight RediJect cyclooxygenase-2 (COX-2) probe and imaged according to package instructions. Immunohistochemistry confirms that this human-mouse xenograft model is effective for studying human wound healing in vivo. Additionally, in vivo fluorescent imaging for inducible COX-2 demonstrated upregulation from baseline to day 4 (P = 0·03) with return to baseline levels by day 10, paralleling the reepithelialisation of the wound. This human-mouse xenograft model, combined with in vivo fluorescent imaging provides a useful mechanism for studying molecular pathways of human wound healing. © 2013 The Authors. International Wound Journal © 2013 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

PubMed Central

Shipley, Mackenzie M.; Mangold, Colleen A.; Szpara, Moriah L.

2016-01-01

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease. PMID:26967710

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

PubMed

Shipley, Mackenzie M; Mangold, Colleen A; Szpara, Moriah L

2016-02-17

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

The current contribution of molecular factors to risk estimation in neuroblastoma patients.

PubMed

Berthold, F; Sahin, K; Hero, B; Christiansen, H; Gehring, M; Harms, D; Horz, S; Lampert, F; Schwab, M; Terpe, J

1997-10-01

The association of molecular characteristics with prognosis has been reported, but not their relationship with each other and their impact in the context of known clinical risk factors. In this study, data of 1249 consecutive intent-to-treat-neuroblastoma patients with more than 1 year follow-up were examined by multivariate analysis using loglinear and Cox proportional hazard regression models on a stage-related basis (stages 1-3: 600, 4S: 116, 4: 533). In a first step, risk factors were identified from 18 selected clinical variables, and risk groups defined. The second step investigated whether molecular characteristics (MYCN, LOH 1p, del 1p, CD44, N-ras, NGF-R, bcl-2, APO-1 (CD95)) contributed additional prognostic information to the model. The loglinear model demonstrated several interactions between clinical factors. By the Cox regression model, seven independent clinical risk factors were found for stages 1-3, seven for stage 4 and two for stage 4S. By subsequent introduction of all molecular variables, MYCN amplification only added significant prognostic information to the clinical factors in localised and stage 4 neuroblastoma. The models allowed the definition of risk groups for stages 1-3 patients by age (e beta = 5.09) and MYCN (e beta = 4.26), for stage 4 by MYCN (e beta = 2.78) and number of symptoms (e beta = 2.44) and for stage 4S by platelet count (e beta = 3.91) and general condition (e beta = 2.99). Molecular factors and in particular MYCN contribute significantly to risk estimation. In conjunction with clinical factors, they are powerful tools to define risk groups in neuroblastoma.

Regulation of neuroblastoma differentiation by forkhead transcription factors FOXO1/3/4 through the receptor tyrosine kinase PDGFRA

PubMed Central

Mei, Yang; Wang, Zhanxiang; Zhang, Lei; Zhang, Yiru; Li, Xiaoyu; Liu, Huihui; Ye, Jing; You, Han

2012-01-01

Neuroblastoma is a common childhood malignant tumor originated from the neural crest-derived sympathetic nervous system. A crucial early event in neuroblastoma pathogenesis is arrested differentiation of neuroblasts at various stages. Treatment of neuroblastoma with TPA and PDGF-BB leads to terminal differentiation of neuroblastoma cells. However, the signaling pathways that are involved in this process remain largely unknown. Here, we report that inhibition of endogenous FOXO proteins attenuated TPA/PDGF-BB mediated differentiation of neuroblastoma cells. Activated FOXO transcription factors acted on PDGFRA promoter to direct its basal mRNA expression as well as its induction upon serum deprivation. Depletion of endogenous PDGFRA in neuroblastoma cells significantly diminished neurite formation and extension under TPA/PDGF-BB treatment. Furthermore, ectopic expression of PDGFRA abolished the blockage of neuroblastoma differentiation by FOXOs inhibition. These findings define the FOXO–PDGFRA axis as crucial mechanistic components that govern TPA-induced neuroblastoma differentiation. PMID:22411791

Photothermal Therapy Generates a Thermal Window of Immunogenic Cell Death in Neuroblastoma.

PubMed

Sweeney, Elizabeth E; Cano-Mejia, Juliana; Fernandes, Rohan

2018-04-17

A thermal "window" of immunogenic cell death (ICD) elicited by nanoparticle-based photothermal therapy (PTT) in an animal model of neuroblastoma is described. In studies using Prussian blue nanoparticles to administer photothermal therapy (PBNP-PTT) to established localized tumors in the neuroblastoma model, it is observed that PBNP-PTT conforms to the "more is better" paradigm, wherein higher doses of PBNP-PTT generates higher cell/local heating and thereby more cell death, and consequently improved animal survival. However, in vitro analysis of the biochemical correlates of ICD (ATP, high-motility group box 1, and calreticulin) elicited by PBNP-PTT demonstrates that PBNP-PTT triggers a thermal window of ICD. ICD markers are highly expressed within an optimal temperature (thermal dose) window of PBNP-PTT (63.3-66.4 °C) as compared with higher (83.0-83.5 °C) and lower PBNP-PTT (50.7-52.7 °C) temperatures, which both yield lower expression. Subsequent vaccination studies in the neuroblastoma model confirm the in vitro findings, wherein PBNP-PTT administered within the optimal temperature window results in long-term survival (33.3% at 100 d) compared with PBNP-PTT administered within the higher (0%) and lower (20%) temperature ranges, and controls (0%). The findings demonstrate a tunable immune response to heat generated by PBNP-PTT, which should be critically engaged in the administration of PTT for maximizing its therapeutic benefits. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Maturation of the developing human fetal prostate in a rodent xenograft model

PubMed Central

Saffarini, Camelia M.; McDonnell, Elizabeth V.; Amin, Ali; Spade, Daniel J.; Huse, Susan M.; Kostadinov, Stefan; Hall, Susan J.; Boekelheide, Kim

2015-01-01

Background Prostate cancer is the most commonly diagnosed non-skin cancer in men. The etiology of prostate cancer is unknown, although both animal and epidemiologic data suggest that early life exposures to various toxicants, may impact DNA methylation status during development, playing an important role. Methods We have developed a xenograft model to characterize the growth and differentiation of human fetal prostate implants (gestational age 12-24 weeks) that can provide new data on the potential role of early life stressors on prostate cancer. The expression of key immunohistochemical markers responsible for prostate maturation was evaluated, including p63, cytokeratin 18, α-smooth muscle actin, vimentin, caldesmon, Ki-67, prostate specific antigen, estrogen receptor-α, and androgen receptor. Xenografts were separated into epithelial and stromal compartments using laser capture microdissection (LCM), and the DNA methylation status was assessed in >480,000 CpG sites throughout the genome. Results Xenografts demonstrated growth and maturation throughout the 200 days of post-implantation evaluation. DNA methylation profiles of laser capture micro-dissected tissue demonstrated tissue-specific markers clustered by their location in either the epithelium or stroma of human prostate tissue. Differential methylated promoter region CpG-associated gene analysis revealed significantly more stromal than epithelial DNA methylation in the 30 and 90-day xenografts. Functional classification analysis identified CpG-related gene clusters in methylated epithelial and stromal human xenografts. Conclusion This study of human fetal prostate tissue establishes a xenograft model that demonstrates dynamic growth and maturation, allowing for future mechanistic studies of the developmental origins of later life proliferative prostate disease. PMID:24038131

Pancreatic cancer ascites xenograft–an expeditious model mirroring advanced therapeutic resistant disease

PubMed Central

Schvimer, Michael; Atias, Dikla; Halperin, Sharon; Buzhor, Ella; Raitses-Gurevich, Maria; Cohen, Keren; Pri-Chen, Sara; Wilson, Julie; Denroche, Robert E.; Lungu, Ilinca; Bartlett, John M.S.; Mbabaali, Faridah; Yarden, Yosef; Nataraj, Nishanth Belugali; Gallinger, Steven; Berger, Raanan

2017-01-01

Pancreatic ductal adenocarcinoma has limited treatment options. There is an urgent need for developing appropriate pre-clinical models recapitulating metastatic disease, the most common clinical scenario at presentation. Ascites accumulation occurs in up to 20–30% of patients with pancreatic cancer; this milieu represents a highly cellular research resource of metastatic peritoneal spread. In this study, we utilized pancreatic ascites/pleural effusion cancer cells to establish patient derived xenografts. Ascites/pleural effusion-patient derived xenografts were established from twelve independent cases. Xenografts were serially passed in nude mice and tissue bio-specimen banking has been established. Histopathology of emergent tumors demonstrates poorly to moderately differentiated, glandular and mucin producing tumors, mirroring morphology of primary pancreatic cancer tumors. Whole genome sequencing of six patient derived xenografts samples demonstrates common mutations and structural variations similar to those reported in primary pancreatic cancer. Xenograft tumors were dissociated to single-cells and in-vitro drug sensitivity screen assays demonstrated chemo-resistance, correlating with patient clinical scenarios, thus serving as a platform for clinically relevant translational research. Therefore, establishment of this novel ascites/pleural effusion patient derived xenograft model, with extensive histopathology and genomic characterization, opens an opportunity for the study of advanced aggressive pancreatic cancer. Characterization of metastatic disease and mechanisms of resistance to therapeutics may lead to the development of novel drug combinations. PMID:28489577

A protein in neuroblastoma could be a target of immunotoxins or immunotherapy | Center for Cancer Research

Cancer.gov

A cell surface protein, glycoprotein glypican-2 (GPC2), has been found to be an effective therapeutic target in cell cultures and mouse models that mimic childhood neuroblastoma.  The CCR scientists who made this discovery, reported July 24, 2017, in PNAS, have also produced immunotoxins and chimeric antigen receptor (CAR) T cells, a type of immunotherapy, that have shown promise against this solid tumor. Read more...

A Real-Time Non-invasive Auto-bioluminescent Urinary Bladder Cancer Xenograft Model.

PubMed

John, Bincy Anu; Xu, Tingting; Ripp, Steven; Wang, Hwa-Chain Robert

2017-02-01

The study was to develop an auto-bioluminescent urinary bladder cancer (UBC) xenograft animal model for pre-clinical research. The study used a humanized, bacteria-originated lux reporter system consisting of six (luxCDABEfrp) genes to express components required for producing bioluminescent signals in human UBC J82, J82-Ras, and SW780 cells without exogenous substrates. Immune-deficient nude mice were inoculated with Lux-expressing UBC cells to develop auto-bioluminescent xenograft tumors that were monitored by imaging and physical examination. Lux-expressing auto-bioluminescent J82-Lux, J82-Ras-Lux, and SW780-Lux cell lines were established. Xenograft tumors derived from tumorigenic Lux-expressing auto-bioluminescent J82-Ras-Lux cells allowed a serial, non-invasive, real-time monitoring by imaging of tumor development prior to the presence of palpable tumors in animals. Using Lux-expressing auto-bioluminescent tumorigenic cells enabled us to monitor the entire course of xenograft tumor development through tumor cell implantation, adaptation, and growth to visible/palpable tumors in animals.

A family-based study of gene variants and maternal folate and choline in neuroblastoma: A Report from the Children’s Oncology Group

PubMed Central

Mazul, Angela L; Siega-Riz, Anna Maria; Weinberg, Clarice R; Engel, Stephanie M.; Zou, Fei; Carrier, Kathryn S.; Basta, Patricia V; Vaksman, Zalman; Maris, John M; Diskin, Sharon J; Maxen, Charlene; Naranjo, Arlene; Olshan, Andrew F

2016-01-01

Purpose Neuroblastoma is a childhood cancer of the sympathetic nervous system with embryonic origins. Previous epidemiologic studies suggest maternal vitamin supplementation during pregnancy reduces the risk of neuroblastoma. We hypothesized offspring and maternal genetic variants in folate-related and choline-related genes are associated with neuroblastoma and modify the effects of maternal intake of folate, choline and folic acid. Methods The Neuroblastoma Epidemiology in North America (NENA) study recruited 563 affected children and their parents through the Children’s Oncology Group’s Childhood Cancer Research Network. We used questionnaires to ascertain pre-pregnancy supplementation and estimate usual maternal dietary intake of folate, choline and folic acid. We genotyped 955 genetic variants related to folate or choline using DNA extracted from saliva samples and used a log-linear model to estimate both child and maternal risk ratios and stratum-specific risk ratios for gene-environment interactions. Results Overall, no maternal or offspring genotypic results met criteria for a false discovery rate (FDR) Q-value <0.2. Associations were also null for gene-environment interaction with pre-pregnancy vitamin supplementation, dietary folic acid and folate. FDR significant gene-choline interactions were found for offspring SNPs rs10489810 and rs9966612 located in MTHFD1L and TYMS, respectively, with maternal choline dietary intake dichotomized at the first quartile. Conclusion These results suggest that variants related to one-carbon metabolism are not strongly associated with neuroblastoma. Choline-related variants may play a role; however, the functional consequences of the interacting variants are unknown and require independent replication. PMID:27541142

A family-based study of gene variants and maternal folate and choline in neuroblastoma: a report from the Children's Oncology Group.

PubMed

Mazul, Angela L; Siega-Riz, Anna Maria; Weinberg, Clarice R; Engel, Stephanie M; Zou, Fei; Carrier, Kathryn S; Basta, Patricia V; Vaksman, Zalman; Maris, John M; Diskin, Sharon J; Maxen, Charlene; Naranjo, Arlene; Olshan, Andrew F

2016-10-01

Neuroblastoma is a childhood cancer of the sympathetic nervous system with embryonic origins. Previous epidemiologic studies suggest maternal vitamin supplementation during pregnancy reduces the risk of neuroblastoma. We hypothesized offspring and maternal genetic variants in folate-related and choline-related genes are associated with neuroblastoma and modify the effects of maternal intake of folate, choline, and folic acid. The Neuroblastoma Epidemiology in North America (NENA) study recruited 563 affected children and their parents through the Children's Oncology Group's Childhood Cancer Research Network. We used questionnaires to ascertain pre-pregnancy supplementation and estimate usual maternal dietary intake of folate, choline, and folic acid. We genotyped 955 genetic variants related to folate or choline using DNA extracted from saliva samples and used a log-linear model to estimate both child and maternal risk ratios and stratum-specific risk ratios for gene-environment interactions. Overall, no maternal or offspring genotypic results met criteria for a false discovery rate (FDR) Q-value <0.2. Associations were also null for gene-environment interaction with pre-pregnancy vitamin supplementation, dietary folic acid, and folate. FDR-significant gene-choline interactions were found for offspring SNPs rs10489810 and rs9966612 located in MTHFD1L and TYMS, respectively, with maternal choline dietary intake dichotomized at the first quartile. These results suggest that variants related to one-carbon metabolism are not strongly associated with neuroblastoma. Choline-related variants may play a role; however, the functional consequences of the interacting variants are unknown and require independent replication.

miR-335 and miR-363 regulation of neuroblastoma tumorigenesis and metastasis.

PubMed

Qiao, Jingbo; Lee, Sora; Paul, Pritha; Theiss, Lauren; Tiao, Joshua; Qiao, Lan; Kong, Andrew; Chung, Dai H

2013-08-01

microRNA (miRNA) functions broadly as post-transcriptional regulators of gene expression, and disproportionate miRNAs can result in dysregulation of oncogenes in cancer cells. We have previously shown that gastrin-releasing peptide receptor (GRP-R) signaling regulates tumorigenicity of neuroblastoma cells. Herein, we sought to characterize miRNA profile in GRP-R silenced neuroblastoma cells, and to determine the role of miRNAs on tumorigenicity and metastatic potential. Human neuroblastoma cell lines, BE(2)-C and SK-N-SH, were used for our study. Stably transfected GRP-R silenced cells were assessed for miRNA profiles. Cells were transfected with miR-335, miR-363, or miR-CON, a nontargeting control, and in vitro assays were performed. In vivo functions of miR-335 and miR-363 were also assessed in a spleen-liver metastasis murine model. GRP-R silencing significantly increased expression of miR-335 and miR-363 in BE(2)-C cells. Overexpression of miR-335 and miR-363 decreased tumorigenicity as measured by clonogenicity, anchorage-independent growth, and metastasis determined by cell invasion assay and liver metastasis in vivo. We report, for the first time, that GRP-R-mediated tumorigenicity and increased metastatic potential in neuroblastoma are regulated, in part, by miR-335 and miR-363. A better understanding of the anti-tumor functions of miRNAs could provide valuable insights to discerning molecular mechanisms responsible for neuroblastoma metastasis. Copyright © 2013 Mosby, Inc. All rights reserved.

Quantitative modeling of clinical, cellular, and extracellular matrix variables suggest prognostic indicators in cancer: a model in neuroblastoma.

PubMed

Tadeo, Irene; Piqueras, Marta; Montaner, David; Villamón, Eva; Berbegall, Ana P; Cañete, Adela; Navarro, Samuel; Noguera, Rosa

2014-02-01

Risk classification and treatment stratification for cancer patients is restricted by our incomplete picture of the complex and unknown interactions between the patient's organism and tumor tissues (transformed cells supported by tumor stroma). Moreover, all clinical factors and laboratory studies used to indicate treatment effectiveness and outcomes are by their nature a simplification of the biological system of cancer, and cannot yet incorporate all possible prognostic indicators. A multiparametric analysis on 184 tumor cylinders was performed. To highlight the benefit of integrating digitized medical imaging into this field, we present the results of computational studies carried out on quantitative measurements, taken from stromal and cancer cells and various extracellular matrix fibers interpenetrated by glycosaminoglycans, and eight current approaches to risk stratification systems in patients with primary and nonprimary neuroblastoma. New tumor tissue indicators from both fields, the cellular and the extracellular elements, emerge as reliable prognostic markers for risk stratification and could be used as molecular targets of specific therapies. The key to dealing with personalized therapy lies in the mathematical modeling. The use of bioinformatics in patient-tumor-microenvironment data management allows a predictive model in neuroblastoma.

ImmunoPET Imaging of Insulin-Like Growth Factor 1 Receptor in a Subcutaneous Mouse Model of Pancreatic Cancer

DTIC Science & Technology

2016-06-30

In addition, 89Zr-labeled nonspecific human IgG (89Zr-Df-IgG) displayed minimal uptake in IGF-1R positive MIA PaCa-2 tumor xenografts (3.63 ± 0.95...scanning using the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Human Pancreatic Adenocarcinoma Xenograft Mouse Model...biodistribution was performed to confirm the PET ROI data. The biodistribution of 89Zr-Df-1A2G11 was compared between the xenograft -bearing mice (Figure 6

β-Arrestin1 and Distinct CXCR4 Structures Are Required for Stromal Derived Factor-1 to Downregulate CXCR4 Cell-Surface Levels in Neuroblastoma

PubMed Central

Clift, Ian C.; Bamidele, Adebowale O.; Rodriguez-Ramirez, Christie; Kremer, Kimberly N.

2014-01-01

CXC chemokine receptor 4 (CXCR4) is a G protein–coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343–352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein–coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in β-arrestin1, we showed that β-arrestin1 is required for the CXCR4 343–352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both β-arrestin1 expression and the 343–352 region. Together, these results characterize CXCR4 structural domains and β-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. β-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis. PMID:24452472

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2

PubMed Central

Chen, Liying; Dharia, Neekesh V.; Ross, Linda; Iniguez, Amanda Balboni; Conway, Amy Saur; Wang, Emily Jue; Veschi, Veronica; Lam, Norris; Gustafson, W. Clay; Nasholm, Nicole; Vazquez, Francisca; Weir, Barbara A.; Ali, Levi D.; Pantel, Sasha; Jiang, Guozhi; Harrington, William F.; Lee, Yenarae; Goodale, Amy; Lubonja, Rakela; Krill-Burger, John M.; Meyers, Robin M.; Root, David E.; Bradner, James E.; Golub, Todd R.; Roberts, Charles W.M.; Hahn, William C.; Weiss, William A.; Thiele, Carol J.

2017-01-01

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma. PMID:29202477

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2.

PubMed

Chen, Liying; Alexe, Gabriela; Dharia, Neekesh V; Ross, Linda; Iniguez, Amanda Balboni; Conway, Amy Saur; Wang, Emily Jue; Veschi, Veronica; Lam, Norris; Qi, Jun; Gustafson, W Clay; Nasholm, Nicole; Vazquez, Francisca; Weir, Barbara A; Cowley, Glenn S; Ali, Levi D; Pantel, Sasha; Jiang, Guozhi; Harrington, William F; Lee, Yenarae; Goodale, Amy; Lubonja, Rakela; Krill-Burger, John M; Meyers, Robin M; Tsherniak, Aviad; Root, David E; Bradner, James E; Golub, Todd R; Roberts, Charles Wm; Hahn, William C; Weiss, William A; Thiele, Carol J; Stegmaier, Kimberly

2018-01-02

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

MIBG avidity correlates with clinical features, tumor biology, and outcomes in neuroblastoma: A report from the Children's Oncology Group.

PubMed

DuBois, Steven G; Mody, Rajen; Naranjo, Arlene; Van Ryn, Collin; Russ, Douglas; Oldridge, Derek; Kreissman, Susan; Baker, David L; Parisi, Marguerite; Shulkin, Barry L; Bai, Harrison; Diskin, Sharon J; Batra, Vandana; Maris, John M; Park, Julie R; Matthay, Katherine K; Yanik, Gregory

2017-11-01

Prior studies suggest that neuroblastomas that do not accumulate metaiodobenzylguanidine (MIBG) on diagnostic imaging (MIBG non-avid) may have more favorable features compared with MIBG avid tumors. We compared clinical features, biologic features, and clinical outcomes between patients with MIBG nonavid and MIBG avid neuroblastoma. Patients had metastatic high- or intermediate-risk neuroblastoma and were treated on Children's Oncology Group protocols A3973 or A3961. Comparisons of clinical and biologic features according to MIBG avidity were made with chi-squared or Fisher exact tests. Event-free (EFS) and overall (OS) survival compared using log-rank tests and modeled using Cox models. Thirty of 343 patients (8.7%) had MIBG nonavid disease. Patients with nonavid tumors were less likely to have adrenal primary tumors (34.5 vs. 57.2%; P = 0.019), bone metastases (36.7 vs. 61.7%; P = 0.008), or positive urine catecholamines (66.7 vs. 91.0%; P < 0.001) compared with patients with MIBG avid tumors. Nonavid tumors were more likely to be MYCN amplified (53.8 vs. 32.6%; P = 0.030) and had lower norepinephrine transporter expression. Patients with MIBG nonavid disease had a 5-year EFS of 50.0% compared with 38.7% for patients with MIBG avid disease (P = 0.028). On multivariate testing in high-risk patients, MIBG avidity was the sole adverse prognostic factor for EFS identified (hazard ratio 1.77; 95% confidence interval 1.04-2.99; P = 0.034). Patients with MIBG nonavid neuroblastoma have lower rates of adrenal primary tumors, bone metastasis, and catecholamine secretion. Despite being more likely to have MYCN-amplified tumors, these patients have superior outcomes compared with patients with MIBG avid disease. © 2017 Wiley Periodicals, Inc.

Molecular Classification Substitutes for the Prognostic Variables Stage, Age, and MYCN Status in Neuroblastoma Risk Assessment.

PubMed

Rosswog, Carolina; Schmidt, Rene; Oberthuer, André; Juraeva, Dilafruz; Brors, Benedikt; Engesser, Anne; Kahlert, Yvonne; Volland, Ruth; Bartenhagen, Christoph; Simon, Thorsten; Berthold, Frank; Hero, Barbara; Faldum, Andreas; Fischer, Matthias

2017-12-01

Current risk stratification systems for neuroblastoma patients consider clinical, histopathological, and genetic variables, and additional prognostic markers have been proposed in recent years. We here sought to select highly informative covariates in a multistep strategy based on consecutive Cox regression models, resulting in a risk score that integrates hazard ratios of prognostic variables. A cohort of 695 neuroblastoma patients was divided into a discovery set (n=75) for multigene predictor generation, a training set (n=411) for risk score development, and a validation set (n=209). Relevant prognostic variables were identified by stepwise multivariable L1-penalized least absolute shrinkage and selection operator (LASSO) Cox regression, followed by backward selection in multivariable Cox regression, and then integrated into a novel risk score. The variables stage, age, MYCN status, and two multigene predictors, NB-th24 and NB-th44, were selected as independent prognostic markers by LASSO Cox regression analysis. Following backward selection, only the multigene predictors were retained in the final model. Integration of these classifiers in a risk scoring system distinguished three patient subgroups that differed substantially in their outcome. The scoring system discriminated patients with diverging outcome in the validation cohort (5-year event-free survival, 84.9±3.4 vs 63.6±14.5 vs 31.0±5.4; P<.001), and its prognostic value was validated by multivariable analysis. We here propose a translational strategy for developing risk assessment systems based on hazard ratios of relevant prognostic variables. Our final neuroblastoma risk score comprised two multigene predictors only, supporting the notion that molecular properties of the tumor cells strongly impact clinical courses of neuroblastoma patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.

PubMed

Emdal, Kristina B; Pedersen, Anna-Kathrine; Bekker-Jensen, Dorte B; Tsafou, Kalliopi P; Horn, Heiko; Lindner, Sven; Schulte, Johannes H; Eggert, Angelika; Jensen, Lars J; Francavilla, Chiara; Olsen, Jesper V

2015-04-28

SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines. Our findings suggest that Cbl-b limits NGF-TrkA signaling to control the length of neurites. Copyright © 2015, American Association for the Advancement of Science.

Neuroblastoma treatment in the post-genomic era.

PubMed

Esposito, Maria Rosaria; Aveic, Sanja; Seydel, Anke; Tonini, Gian Paolo

2017-02-08

Neuroblastoma is an embryonic malignancy of early childhood originating from neural crest cells and showing heterogeneous biological, morphological, genetic and clinical characteristics. The correct stratification of neuroblastoma patients within risk groups (low, intermediate, high and ultra-high) is critical for the adequate treatment of the patients.High-throughput technologies in the Omics disciplines are leading to significant insights into the molecular pathogenesis of neuroblastoma. Nonetheless, further study of Omics data is necessary to better characterise neuroblastoma tumour biology. In the present review, we report an update of compounds that are used in preclinical tests and/or in Phase I-II trials for neuroblastoma. Furthermore, we recapitulate a number of compounds targeting proteins associated to neuroblastoma: MYCN (direct and indirect inhibitors) and downstream targets, Trk, ALK and its downstream signalling pathways. In particular, for the latter, given the frequency of ALK gene deregulation in neuroblastoma patients, we discuss on second-generation ALK inhibitors in preclinical or clinical phases developed for the treatment of neuroblastoma patients resistant to crizotinib.We summarise how Omics drive clinical trials for neuroblastoma treatment and how much the research of biological targets is useful for personalised medicine. Finally, we give an overview of the most recent druggable targets selected by Omics investigation and discuss how the Omics results can provide us additional advantages for overcoming tumour drug resistance.

Generation of human acute lymphoblastic leukemia xenografts for use in oncology drug discovery

PubMed Central

Holmfeldt, Linda

2015-01-01

The establishment of reproducible mouse models of acute lymphoblastic leukemia (ALL) is necessary to provide in vivo therapeutic models that recapitulate human ALL, and for amplification of limiting amounts of primary tumor material. A frequently used model is the primary xenograft model that utilizes immunocompromised mice and involves injection of primary patient tumor specimens into mice, and subsequent serial passaging of the tumors by retransplants of cells harvested from the mouse bone marrow and spleen. The tumors generated can then be used for genomic profiling, ex vivo compound testing, mechanistic studies and retransplantation. This unit describes detailed procedures for the establishment and maintenance of primary ALL xenograft panels for potential use in basic research or translational studies. PMID:25737157

Review: The Role of Radiolabeled DOTA-Conjugated Peptides for Imaging and Treatment of Childhood Neuroblastoma.

PubMed

Alexander, Natasha; Vali, Reza; Ahmadzadehfar, Hojjat; Shammas, Amer; Baruchel, Sylvain

2018-01-01

Childhood neuroblastoma is a heterogenous disease with varied clinical presentation and biology requiring different approaches to investigation and management. Metaiodobenzylguanidine (MIBG) is an essential component of metastatic staging for neuroblastoma and has been used as a treatment strategy for relapsed and refractory neuroblastoma. However, as 10% of children with neuroblastoma will have 123I-MIBG non-avid imaging and up to 60% with relapsed and refractory neuroblastoma will require further treatment with 131I-MIBG, alternative radioisotopes have been investigated for imaging and treatment. Neuroblastoma tumors express mostly somatostatin receptor- 2 (SSTR2) that can be targeted by somatostatin analogues including DOTA-conjugated peptides e.g. DOTATATE, DOTATOC. This review summarizes the rationale, utility and experience of DOTA-conjugated peptides in imaging and treatment of childhood neuroblastoma. Radiolabeled DOTA-peptides are used routinely in adults to image neuroendocrine tumors and have potential to be used to image and treat neuroblastoma. 68Ga-DOTATATE PET/CT has been shown to have better sensitivity, quicker clearance and administration times, reduced radiation exposure and limited toxicity compared to 123I-MIBG. Therapeutic studies of peptide receptor radionuclides e.g. 177Lu-DOTATATE in patients with relapsed neuroblastoma have used 68Ga- DOTATATE PET/CT to determine eligibility for therapy. Further studies would need to investigate appropriate indications, timings, scoring and clinical significance of radiolabeled DOTA-peptide conjugated PET/CT imaging in childhood neuroblastoma. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Targeting of PHOX2B expression allows the identification of drugs effective in counteracting neuroblastoma cell growth

PubMed Central

Zanni, Eleonora Di; Bianchi, Giovanna; Ravazzolo, Roberto; Raffaghello, Lizzia; Ceccherini, Isabella; Bachetti, Tiziana

2017-01-01

The pathogenic role of the PHOX2B gene in neuroblastoma is indicated by heterozygous mutations in neuroblastoma patients and by gene overexpression in both neuroblastoma cell lines and tumor samples. PHOX2B encodes a transcription factor which is crucial for the correct development and differentiation of sympathetic neurons. PHOX2B overexpression is considered a prognostic marker for neuroblastoma and it is also used by clinicians to monitor minimal residual disease. Furthermore, it has been observed that neuronal differentiation in neuroblastoma is dependent on down-regulation of PHOX2B expression, which confirms that PHOX2B expression may be considered a target in neuroblastoma. Here, PHOX2B promoter or 3′ untranslated region were used as molecular targets in an in vitro high-throughput approach that led to the identification of molecules able to decrease PHOX2B expression at transcriptional and likely even at post-transcriptional levels. Further functional investigations carried out on PHOX2B mRNA levels and biological consequences, such as neuroblastoma cell apoptosis and growth, showed that chloroquine and mycophenolate mofetil are most promising agents for neuroblastoma therapy based on down-regulation of PHOX2B expression. Finally, a strong correlation between the effect of drugs in terms of down-regulation of PHOX2B expression and of biological consequences in neuroblastoma cells confirms the role of PHOX2B as a potential molecular target in neuroblastoma. PMID:29069774

HIF2α reduces growth rate but promotes angiogenesis in a mouse model of neuroblastoma

PubMed Central

Favier, Judith; Lapointe, Stéphanie; Maliba, Ricardo; Sirois, Martin G

2007-01-01

Background HIF2α/EPAS1 is a hypoxia-inducible transcription factor involved in catecholamine homeostasis, vascular remodelling, physiological angiogenesis and adipogenesis. It is overexpressed in many cancerous tissues, but its exact role in tumour progression remains to be clarified. Methods In order to better establish its function in tumourigenesis and tumour angiogenesis, we have stably transfected mouse neuroblastoma N1E-115 cells with the native form of HIF2α or with its dominant negative mutant, HIF2α (1–485) and studied their phenotype in vitro and in vivo. Results In vitro studies reveal that HIF2α induces neuroblastoma cells hypertrophy and decreases their proliferation rate, while its inactivation by the HIF2α (1–485) mutant leads to a reduced cell size, associated with an accelerated proliferation. However, our in vivo experiments show that subcutaneous injection of cells overexpressing HIF2α into syngenic mice, leads to the formation of tumour nodules that grow slower than controls, but that are well structured and highly vascularized. In contrast, HIF2α (1–485)-expressing neuroblastomas grow fast, but are poorly vascularized and quickly tend to extended necrosis. Conclusion Together, our data reveal an unexpected combination between an antiproliferative and a pro-angiogenic function of HIF2α that actually seems to be favourable to the establishment of neuroblastomas in vivo. PMID:17655754

The Aurora kinase inhibitor CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo

PubMed Central

Faisal, Amir; Vaughan, Lynsey; Bavetsias, Vassilios; Sun, Chongbo; Atrash, Butrus; Avery, Sian; Jamin, Yann; Robinson, Simon P.; Workman, Paul; Blagg, Julian; Raynaud, Florence I.; Eccles, Suzanne A.; Chesler, Louis; Linardopoulos, Spiros

2015-01-01

The Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation and cytokinesis. Aurora A and B overexpression has also been associated with various human cancers and as such, they have been extensively studied as novel anti-mitotic drug targets. Here we characterise the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC50 values in both biochemical and cellular assays and exhibits anti-proliferative activity against a wide range of human solid tumour cell lines. CCT137690 efficiently inhibits histone H3 and TACC3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumour cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 (TK1) downregulation and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma (TH-MYCN) that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumour growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN amplified neuroblastoma. PMID:21885865

Intratumoral Delivery of Interferonγ-Secreting Mesenchymal Stromal Cells Repolarizes Tumor-Associated Macrophages and Suppresses Neuroblastoma Proliferation In Vivo.

PubMed

Relation, Theresa; Yi, Tai; Guess, Adam J; La Perle, Krista; Otsuru, Satoru; Hasgur, Suheyla; Dominici, Massimo; Breuer, Christopher; Horwitz, Edwin M

2018-06-01

Neuroblastoma, the most common extracranial solid tumor in childhood, remains a therapeutic challenge. However, one promising patient treatment strategy is the delivery of anti-tumor therapeutic agents via mesenchymal stromal cell (MSC) therapy. MSCs have been safely used to treat genetic bone diseases such as osteogenesis imperfecta, cardiovascular diseases, autoimmune diseases, and cancer. The pro-inflammatory cytokine interferon-gamma (IFNγ) has been shown to decrease tumor proliferation by altering the tumor microenvironment (TME). Despite this, clinical trials of systemic IFNγ therapy have failed due to the high blood concentration required and associated systemic toxicities. Here, we developed an intra-adrenal model of neuroblastoma, characterized by liver and lung metastases. We then engineered MSCs to deliver IFNγ directly to the TME. In vitro, these MSCs polarized murine macrophages to the M1 phenotype. In vivo, we attained a therapeutically active TME concentration of IFNγ without increased systemic concentration or toxicity. The TME-specific IFNγ reduced tumor growth rate and increased survival in two models of T cell deficient athymic nude mice. Absence of this benefit in NOD SCID gamma (NSG) immunodeficient mouse model indicates a mechanism dependent on the innate immune system. IL-17 and IL-23p19, both uniquely M1 polarization markers, transiently increased in the tumor interstitial fluid. Finally, the MSC vehicle did not promote tumor growth. These findings reveal that MSCs can deliver effective cytokine therapy directly to the tumor while avoiding systemic toxicity. This method transiently induces inflammatory M1 macrophage polarization, which reduces tumor burden in our novel neuroblastoma murine model. Stem Cells 2018;36:915-924. © AlphaMed Press 2018.

Genetic susceptibility to neuroblastoma

PubMed Central

Tolbert, Vanessa P.; Coggins, Grace E.; Maris, John M.

2017-01-01

Until recently, the genetic basis of neuroblastoma, a heterogeneous neoplasm arising from the developing sympathetic nervous system, remained undefined. The discovery of gain-of-function mutations in the ALK receptor tyrosine kinase gene as the major cause of familial neuroblastoma led to the discovery of identical somatic mutations and rapid advancement of ALK as a tractable therapeutic target. Inactivating mutations in a master regulator of neural crest development, PHOX2B, have also been identified in a subset of familial neuroblastomas. Other high penetrance susceptibility alleles likely exist, but together these heritable mutations account for less than 10% of neuroblastoma cases. A genome-wide association study of a large neuroblastoma cohort identified common and rare polymorphisms highly associated with the disease. Ongoing resequencing efforts aim to further define the genetic landscape of neuroblastoma. PMID:28458126

National Cancer Institute Pediatric Preclinical Testing Program: Model Description for In Vitro Cytotoxicity Testing

PubMed Central

Kang, Min H.; Smith, Malcolm A.; Morton, Christopher L.; Keshelava, Nino; Houghton, Peter J.; Reynolds, C. Patrick

2010-01-01

Background The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program (PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials. Procedures In vitro cytotoxicity testing employs a semi-automated fluorescence-based digital imaging cytotoxicity assay (DIMSCAN) that has a 4-log dynamic range of detection. Curve fitting of the fractional survival data of the cell lines in response to various concentrations of the agents was used to calculate relative IC50, absolute IC50, and Ymin values The panel of 23 pediatric cancer cell lines included leukemia (n=6), lymphoma (n=2), rhabdomyosarcoma (n=4), brain tumors (n=3), Ewing family of tumors (EFT, n=4), and neuroblastoma (n=4). The doubling times obtained using DIMSCAN were incorporated into data analyses to estimate the relationship between input cell numbers and final cell number. Results We report in vitro activity data for three drugs (vincristine, melphalan, and etoposide) that are commonly used for pediatric cancer and for the mTOR inhibitor rapamycin, an agent that is currently under preclinical investigation for cancer. To date, the PPTP has completed in vitro testing of 39 investigational and approved agents for single drug activity and two investigational agents in combination with various “standard” chemotherapy drugs. Conclusions This robust in vitro cytotoxicity testing system for pediatric cancers will enable comparisons to response data for novel agents obtained from xenograft studies and from clinical trials. PMID:20922763

Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells.

PubMed

Rust, Aleksander; Leese, Charlotte; Binz, Thomas; Davletov, Bazbek

2016-05-31

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells.

Neuroblastoma arginase activity creates an immunosuppressive microenvironment that impairs autologous and engineered immunity

PubMed Central

Mussai, Francis; Egan, Sharon; Hunter, Stuart; Webber, Hannah; Fisher, Jonathan; Wheat, Rachel; McConville, Carmel; Sbirkov, Yordan; Wheeler, Kate; Bendle, Gavin; Petrie, Kevin; Anderson, John; Chesler, Louis; De Santo, Carmela

2015-01-01

Neuroblastoma is the most common extra cranial solid tumour of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumour cells suppress T cell proliferation, through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34+ progenitor proliferation. Finally we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1 specific TCR and GD2-specific CAR engineered T cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for neuroblastoma patients. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumour and blood that leads to impaired immune surveillance and sub-optimal efficacy of immunotherapeutic approaches. PMID:26054597

Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.

PubMed

Boeva, Valentina; Louis-Brennetot, Caroline; Peltier, Agathe; Durand, Simon; Pierre-Eugène, Cécile; Raynal, Virginie; Etchevers, Heather C; Thomas, Sophie; Lermine, Alban; Daudigeos-Dubus, Estelle; Geoerger, Birgit; Orth, Martin F; Grünewald, Thomas G P; Diaz, Elise; Ducos, Bertrand; Surdez, Didier; Carcaboso, Angel M; Medvedeva, Irina; Deller, Thomas; Combaret, Valérie; Lapouble, Eve; Pierron, Gaelle; Grossetête-Lalami, Sandrine; Baulande, Sylvain; Schleiermacher, Gudrun; Barillot, Emmanuel; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

2017-09-01

Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

Somatic PTPN11 Mutation in a Child With Neuroblastoma and Protein Losing Enteropathy.

PubMed

Obasaju, Patience; Brondon, Jennifer; Mir, Sabina; Fordham, Lynn A; Lee, Sang; Blatt, Julie

2018-05-01

Neuroblastoma and protein losing enteropathy (PLE) are diagnoses commonly seen by oncologists and gastroenterologists, respectively. The concurrence of these 2 entities is rare, and not well explained. We describe the sixth case of PLE in a child with neuroblastoma, and the first for which genetic information is available. Tumor DNA had a mutation in the PTPN11 gene, which has been described in neuroblastoma, and in Noonan syndrome-a diagnosis in which neuroblastoma and PLE independently have been reported. Constitutional DNA was normal. Genetic studies in future patients will be needed to support the link between neuroblastoma and PLE.

Tobacco, alcohol and illicit drugs during pregnancy and risk of neuroblastoma: systematic review.

PubMed

Müller-Schulte, Eloise; Kurlemann, Gerhard; Harder, Anja

2017-11-21

To determine whether prenatal and perinatal maternal consumption of alcohol, tobacco and/or illicit drugs is associated with risk of neuroblastoma. Medline and Embase (both from inception to February 2017), and reference lists of included studies. To be eligible, a study had to be an original report including data on intake of alcohol, tobacco smoking and/or consumption of illicit drugs during pregnancy and risk of neuroblastoma in the child. From eligible studies, data study characteristics as well as effect measures and confounders were extracted. We assessed unadjusted and confounder-adjusted estimates, performed risk of bias analysis, constructed random-effects models and assessed heterogeneity. We identified 14 case-control studies (1987-2016) involving a total of 3114 children with neuroblastoma. Meta-analysis of unadjusted estimates showed an association between alcohol (OR 1.26; 95% CI 1.07 to 1.49), tobacco (OR 1.22; 95% CI 1.04 to 1.44) and illicit drug consumption during pregnancy and risk of neuroblastoma during childhood, with illicit drug consumption showing the strongest association (OR 3.26; 95% CI 1.36 to 7.86). However, adjusted estimates were highly heterogeneous. All studies were at high risk of bias. Smoking, alcohol or illicit drugs during pregnancy might play a role in the development of neuroblastoma. However, well-designed studies are needed to assess whether these exposures are causal and whether time period during pregnancy, dose or co-consumption of substances is critical. Registration number CRD42016036165. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis.

PubMed

Kollareddy, Madhu; Sherrard, Alice; Park, Ji Hyun; Szemes, Marianna; Gallacher, Kelli; Melegh, Zsombor; Oltean, Sebastian; Michaelis, Martin; Cinatl, Jindrich; Kaidi, Abderrahmane; Malik, Karim

2017-09-10

Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

A Phase I Study of Zoledronic Acid and Low Dose Cyclophosphamide in Recurrent/Refractory Neuroblastoma: A New Approaches to Neuroblastoma Therapy (NANT) Study

PubMed Central

Russell, Heidi V.; Groshen, Susan G.; Ara, Tasnim; DeClerck, Yves A.; Hawkins, Randy; Jackson, Hollie A.; Daldrup-Link, Heike E.; Marachelian, Araz; Skerjanec, Andrej; Park, Julie R.; Katzenstein, Howard; Matthay, Katherine K.; Blaney, Susan M.; Villablanca, Judith G.

2010-01-01

Background Zoledronic acid, a bisphosphonate, delays progression of bone metastases in adult malignancies. Bone is a common metastatic site of advanced neuroblastoma. We previously reported efficacy of zoledronic acid in a murine model of neuroblastoma bone invasion prompting this Phase I trial of zoledronic acid with cyclophosphamide in children with neuroblastoma and bone metastases. The primary objective was to determine recommended dosing of zoledronic acid for future trials. Procedure Escalating doses of intravenous zoledronic acid were given every 28 days with oral metronomic cyclophosphamide (25 mg/m2/day). Toxicity, response, zoledronic acid pharmacokinetics, bone turnover markers, serum IL-6, and sIL-6R were evaluated. Results Twenty-one patients, median age 7.5 (range 0.8 - 25.6) years were treated with 2 mg/m2 (n=4), 3 mg/m2 (n=3), or 4 mg/m2 (n=14) zoledronic acid. Fourteen patients were evaluable for dose escalation. A median of one (range 1-18) courses was given. Two dose limiting toxicities (Grade 3 hypophosphatemia) occurred at 4 mg/m2 zoledronic acid. Other Grade 3-4 toxicities included hypocalcemia (n=2), elevated transaminases (n=1), neutropenia (n=2), anemia (n=1), lymphopenia (n=1), and hypokalemia (n=1). Osteosclerosis contributed to fractures in one patient after 18 courses. Responses in evaluable patients included 1 partial response, 9 stable disease (median 4.5 courses, range 3-18), and 10 progressions. Zoledronic acid pharmacokinetics were similar to adults. Markers of osteoclast activity and serum IL-6 levels decreased with therapy. Conclusions Zoledronic acid with metronomic cyclophosphamide is well tolerated with clinical and biologic responses in recurrent/refractory neuroblastoma. The recommended dose of zoledronic acid is 4 mg/m2 every 28 days. PMID:21671363

Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma.

PubMed

Antonoff, Mara B; Chugh, Rohit; Skube, Steven J; Dudeja, Vikas; Borja-Cacho, Daniel; Clawson, Kimberly A; Vickers, Selwyn M; Saluja, Ashok K

2010-09-01

Our recent work demonstrated that treatment of neuroblastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma. Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis. Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis. Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death. Copyright 2010 Elsevier Inc. All rights reserved.

Targeting Metabolic Survival Pathways in Lung Cancer via Combination Therapy

DTIC Science & Technology

2014-06-01

B1, non-small cell lung cancer, glutamine metabolism, biguanides 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF...combination therapy (months 15-16) Task 5. In vivo testing of biguanide and glutamine metabolism inhibitors in xenograft models of LKB1-proficient and...combination therapies in xenograft mice (months 12-15) IACUC and ACURO approval have been granted for in vivo xenograft studies, which will commence in

Novel targeted therapy for neuroblastoma: silencing the MXD3 gene using siRNA.

PubMed

Duong, Connie; Yoshida, Sakiko; Chen, Cathy; Barisone, Gustavo; Diaz, Elva; Li, Yueju; Beckett, Laurel; Chung, Jong; Antony, Reuben; Nolta, Jan; Nitin, Nitin; Satake, Noriko

2017-09-01

BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide-common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

Overview and recent advances in the treatment of neuroblastoma.

PubMed

Whittle, Sarah B; Smith, Valeria; Doherty, Erin; Zhao, Sibo; McCarty, Scott; Zage, Peter E

2017-04-01

Children with neuroblastoma have widely divergent outcomes, ranging from cure in >90% of patients with low risk disease to <50% for those with high risk disease. Recent research has shed light on the biology of neuroblastoma, allowing for more accurate risk stratification and treatment reduction in many cases, although newer treatment strategies for children with high-risk and relapsed neuroblastoma are needed to improve outcomes. Areas covered: Neuroblastoma epidemiology, diagnosis, risk stratification, and recent advances in treatment of both newly diagnosed and relapsed neuroblastoma. Expert commentary: The identification of newer tumor targets and of novel cell-mediated immunotherapy agents may lead to novel therapeutic approaches, and clinical trials for regimens designed to target individual genetic aberrations in tumors are underway. A combination of therapeutic modalities will likely be required to improve survival and cure rates for patients with high-risk neuroblastoma.

Comparison of the Gene Expression Profiles of Human Hematopoietic Stem Cells between Humans and a Humanized Xenograft Model.

PubMed

Matsuzawa, Hideyuki; Matsushita, Hiromichi; Yahata, Takashi; Tanaka, Masayuki; Ando, Kiyoshi

2017-04-20

The aim of this study is to evaluate the feasibility of NOD/Shi-scid-IL2Rγ null (NOG) mice transplanted with human CD34 + /CD38 - /Lin -/low hematopoietic cells from cord blood (CB) as an experimental model of the gene expression in human hematopoiesis. We compared the gene expressions of human CD34 + /CD38 - /Lin -/low cells from human bone marrow (BM) and in xenograft models. The microarray data revealed that 25 KEGG pathways were extracted from the comparison of human CD34 + /CD38 - /Lin -/low HSCs between CB and BM, and that 17 of them--which were mostly related to cellular survival, RNA metabolism and lymphoid development--were shared with the xenograft model. When the probes that were commonly altered in CD34 + /CD38 - /Lin -/low cells from both human and xenograft BM were analyzed, most of them, including the genes related hypoxia, hematopoietic differentiation, epigenetic modification, translation initiation, and RNA degradation, were downregulated. These alterations of gene expression suggest a reduced differentiation capacity and likely include key alterations of gene expression for settlement of CB CD34 + /CD38 - /Lin -/low cells in BM. Our findings demonstrate that the xenograft model of human CB CD34 + /CD38 - /Lin -/low cells using NOG mice was useful, at least in part, for the evaluation of the gene expression profile of human hematopoietic stem cells.

Real-time investigation of cytochrome c release profiles in living neuronal cells undergoing amyloid beta oligomer-induced apoptosis

NASA Astrophysics Data System (ADS)

Lee, Jae Young; Park, Younggeun; Pun, San; Lee, Sung Sik; Lo, Joe F.; Lee, Luke P.

2015-06-01

Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid β oligomer (AβO)-induced apoptosis, as a model Alzheimer's disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy.Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid β oligomer (AβO)-induced apoptosis, as a model Alzheimer's disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02390d

Peginterferon Beta-1a Shows Antitumor Activity as a Single Agent and Enhances Efficacy of Standard of Care Cancer Therapeutics in Human Melanoma, Breast, Renal, and Colon Xenograft Models.

PubMed

Boccia, Antonio; Virata, Cyrus; Lindner, Daniel; English, Nicki; Pathan, Nuzhat; Brickelmaier, Margot; Hu, Xiao; Gardner, Jennifer L; Peng, Liaomin; Wang, Xinzhong; Zhang, Xiamei; Yang, Lu; Perron, Keli; Yco, Grace; Kelly, Rebecca; Gamez, James; Scripps, Thomas; Bennett, Donald; Joseph, Ingrid B; Baker, Darren P

2017-01-01

Because of its tumor-suppressive effect, interferon-based therapy has been used for the treatment of melanoma. However, limited data are available regarding the antitumor effects of pegylated interferons, either alone or in combination with approved anticancer drugs. We report that treatment of human WM-266-4 melanoma cells with peginterferon beta-1a induced apoptotic markers. Additionally, peginterferon beta-1a significantly inhibited the growth of human SK-MEL-1, A-375, and WM-266-4 melanoma xenografts established in immunocompromised mice. Peginterferon beta-1a regressed large, established WM-266-4 xenografts in nude mice. Treatment of SK-MEL-1 tumor-bearing mice with a combination of peginterferon beta-1a and the MEK inhibitor PD325901 ((R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide) significantly improved tumor growth inhibition compared with either agent alone. Examination of the antitumor activity of peginterferon beta-1a in combination with approved anticancer drugs in breast and renal carcinomas revealed improved antitumor activity in these preclinical xenograft models, as did the combination of peginterferon beta-1a and bevacizumab in a colon carcinoma xenograft model.

More than the genes, the tumor microenvironment in neuroblastoma

PubMed Central

Borriello, Lucia; Seeger, Robert C.; Asgharzadeh, Shahab; DeClerck, Yves A.

2017-01-01

Neuroblastoma is the second most common solid tumor in children. Since the seminal discovery of the role of amplification of the MYCN oncogene in the pathogenesis of neuroblastoma in the 1980s, much focus has been on the contribution of genetic alterations in the progression of this cancer. However it is now clear that not only genetic events play a role but that the tumor microenvironment (TME) substantially contributes to the biology of neuroblastoma. In this article, we present a comprehensive review of the literature on the contribution of the TME to the ten hallmarks of cancer in neuroblastoma and discuss the mechanisms of communication between neuroblastoma cells and the TME that underlie the influence of the TME on neuroblastoma progression. We end our review by discussing how the knowledge acquired over the last two decades in this field is now leading to new clinical trials targeting the TME. PMID:26597947

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.

PubMed

Bosse, Kristopher R; Raman, Pichai; Zhu, Zhongyu; Lane, Maria; Martinez, Daniel; Heitzeneder, Sabine; Rathi, Komal S; Kendsersky, Nathan M; Randall, Michael; Donovan, Laura; Morrissy, Sorana; Sussman, Robyn T; Zhelev, Doncho V; Feng, Yang; Wang, Yanping; Hwang, Jennifer; Lopez, Gonzalo; Harenza, Jo Lynne; Wei, Jun S; Pawel, Bruce; Bhatti, Tricia; Santi, Mariarita; Ganguly, Arupa; Khan, Javed; Marra, Marco A; Taylor, Michael D; Dimitrov, Dimiter S; Mackall, Crystal L; Maris, John M

2017-09-11

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target. Copyright © 2017 Elsevier Inc. All rights reserved.

More than the genes, the tumor microenvironment in neuroblastoma.

PubMed

Borriello, Lucia; Seeger, Robert C; Asgharzadeh, Shahab; DeClerck, Yves A

2016-09-28

Neuroblastoma is the second most common solid tumor in children. Since the seminal discovery of the role of amplification of the MYCN oncogene in the pathogenesis of neuroblastoma in the 1980s, much focus has been on the contribution of genetic alterations in the progression of this cancer. However it is now clear that not only genetic events play a role but that the tumor microenvironment (TME) substantially contributes to the biology of neuroblastoma. In this article, we present a comprehensive review of the literature on the contribution of the TME to the ten hallmarks of cancer in neuroblastoma and discuss the mechanisms of communication between neuroblastoma cells and the TME that underlie the influence of the TME on neuroblastoma progression. We end our review by discussing how the knowledge acquired over the last two decades in this field is now leading to new clinical trials targeting the TME. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Environment-Mediated Drug Resistance in Neuroblastoma

DTIC Science & Technology

2014-10-01

Neuroblastoma PRINCIPAL INVESTIGATOR: Yves A. DeClerck CONTRACTING ORGANIZATION... Neuroblastoma 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0571 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) DE CLERCK, YVES 5d. PROJECT...experiments have demonstrated that monocytes collaborate with MSC in inducing STAT3-dependent drug resistance in neuroblastoma . Further

Overcoming the Mechanism of Radioresistance in Neuroblastoma

DTIC Science & Technology

2014-06-01

of Radioresistance in Neuroblastoma PRINCIPAL INVESTIGATOR: Brian Marples PhD CONTRACTING ORGANIZATION: William Beaumont Hospital Inc...COVERED 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Overcoming the Mechanism of Radioresistance in Neuroblastoma 5b. GRANT NUMBER 5c. PROGRAM...for highly aggressive advanced-stage neuroblastoma remains poor despite a multidisciplinary approach involving aggressive surgery, chemotherapy and

Cell Proliferation in Neuroblastoma

PubMed Central

Stafman, Laura L.; Beierle, Elizabeth A.

2016-01-01

Neuroblastoma, the most common extracranial solid tumor of childhood, continues to carry a dismal prognosis for children diagnosed with advanced stage or relapsed disease. This review focuses upon factors responsible for cell proliferation in neuroblastoma including transcription factors, kinases, and regulators of the cell cycle. Novel therapeutic strategies directed toward these targets in neuroblastoma are discussed. PMID:26771642

YAP promotes tumorigenesis and cisplatin resistance in neuroblastoma.

PubMed

Yang, Chao; Tan, Juan; Zhu, Jun; Wang, Shan; Wei, Guanghui

2017-06-06

The transcriptional co-activator Yes-associated protein (YAP) is essential for Hippo pathway-driven tumorigenesis in various cancers. However, the expression and function of YAP in neuroblastoma remains elusive. Here, we show that YAP was highly expressed in Neuroblastoma (NB) and expression levels correlated with advanced tumor staging. Knockdown of YAP significantly impaired neuroblastoma proliferation, tumorigenesis, and invasion in vitro. Injection of the YAP inhibitor, Peptide 17, dramatically prevented neuroblastoma subcutaneous tumor growth by efficiently downregulating YAP expression in tumors. Additionally, less proliferative and more apoptotic cells were found in the Peptide 17 treatment group. Furthermore, YAP inhibition significantly inhibited cisplatin-resistant neuroblastoma proliferation, tumorigenesis, and invasion in vitro. The combination of Peptide 17 with low-dose cisplatin efficiently impaired cisplatin-resistant NB subcutaneous tumor growth, being as effective as high-dose cisplatin. Notably, the combination therapy caused lesser liver toxicity in mice compared to the high-dose cisplatin treatment group. Collectively, this work identifies YAP as a novel regulator of neuroblastoma proliferation, tumorigenesis, and invasion and indicates that YAP is a potential therapeutic target for cisplatin-resistant neuroblastoma.

Knockdown of zinc transporter ZIP8 expression inhibits neuroblastoma progression and metastasis in vitro.

PubMed

Mei, Zhengrong; Yan, Pengke; Wang, Ying; Liu, Shaozhi; He, Fang

2018-05-02

Neuroblastoma is one of the leading causes of cancer‑associated mortality worldwide, particularly in children, partially due to the absence of effective therapeutic targets and diagnostic biomarkers. Therefore, novel molecular targets are critical to the development of therapeutic approaches for neuroblastoma. In the present study, the functions of zinc transporter ZIP8 (Zip8), a member of the zinc transporting protein family, were investigated as novel molecular targets in neuroblastoma cancer cells. The proliferation rates of neuroblastoma cancer cells were significantly decreased when Zip8 was knocked down by lentiviral‑mediated RNA interference. Study of the molecular mechanism suggested that Zip8 modulated the expression of key genes involved in the nuclear factor‑κB signaling pathway. Furthermore, Zip8 depletion suppressed the migratory potential of neuroblastoma cancer cells by reducing the expression levels of matrix metalloproteinases. In conclusion, the results of the present study suggested that Zip8 was an important regulator of neuroblastoma cell proliferation and migration, indicating that Zip8 may be a potential anticancer therapeutic target and a promising diagnostic biomarker for human neuroblastoma.

Targeting Nuclear EGFR: Strategies for Improving Cetuximab Therapy in Lung Cancer

DTIC Science & Technology

2015-12-01

xenograft models to determine if, in vivo, SFK or AKT inhibition can block EGFR translocation to the nucleus leading to increased membrane expression...resistant NSCLC tumors using mouse xenografts . Mice bearing resistant tumors will be treated with 1) vehicle, 2) cetuximab, 3) dasatinib, 4) MK2206...phosphorylation of Y1101 and EGFR dimerization with HER3 leading to nuclear translocation. Key Research Accomplishments for Aim 2: • In vivo xenograft tumors

Achaete-Scute Homolog 1 Expression Controls Cellular Differentiation of Neuroblastoma

PubMed Central

Kasim, Mumtaz; Heß, Vicky; Scholz, Holger; Persson, Pontus B.; Fähling, Michael

2016-01-01

Neuroblastoma, the major cause of infant cancer deaths, results from fast proliferation of undifferentiated neuroblasts. Treatment of high-risk neuroblastoma includes differentiation with retinoic acid (RA); however, the resistance of many of these tumors to RA-induced differentiation poses a considerable challenge. Human achaete-scute homolog 1 (hASH1) is a proneural basic helix-loop-helix transcription factor essential for neurogenesis and is often upregulated in neuroblastoma. Here, we identified a novel function for hASH1 in regulating the differentiation phenotype of neuroblastoma cells. Global analysis of 986 human neuroblastoma datasets revealed a negative correlation between hASH1 and neuron differentiation that was independent of the N-myc (MYCN) oncogene. Using RA to induce neuron differentiation in two neuroblastoma cell lines displaying high and low levels of hASH1 expression, we confirmed the link between hASH1 expression and the differentiation defective phenotype, which was reversed by silencing hASH1 or by hypoxic preconditioning. We further show that hASH1 suppresses neuronal differentiation by inhibiting transcription at the RA receptor element. Collectively, our data indicate hASH1 to be key for understanding neuroblastoma resistance to differentiation therapy and pave the way for hASH1-targeted therapies for augmenting the response of neuroblastoma to differentiation therapy. PMID:28066180

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

PubMed

Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

2017-05-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

PubMed Central

Gaviglio, Angela L.; Knelson, Erik H.; Blobe, Gerard C.

2017-01-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. PMID:28174207

Genome-wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma.

PubMed

Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J; Almutairi, Bader; Etchevers, Heather C; McConville, Carmel; Malik, Karim T A; Brown, Keith W

2017-04-01

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome-wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest-expressing tumors had reduced relapse-free survival. Our functional studies showed that knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

PubMed Central

Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R.; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J.; Almutairi, Bader; Etchevers, Heather C.; McConville, Carmel; Malik, Karim T. A.

2016-01-01

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. PMID:27862318

Genetic susceptibility to neuroblastoma: current knowledge and future directions.

PubMed

Ritenour, Laura E; Randall, Michael P; Bosse, Kristopher R; Diskin, Sharon J

2018-05-01

Neuroblastoma, a malignancy of the developing peripheral nervous system that affects infants and young children, is a complex genetic disease. Over the past two decades, significant progress has been made toward understanding the genetic determinants that predispose to this often lethal childhood cancer. Approximately 1-2% of neuroblastomas are inherited in an autosomal dominant fashion and a combination of co-morbidity and linkage studies has led to the identification of germline mutations in PHOX2B and ALK as the major genetic contributors to this familial neuroblastoma subset. The genetic basis of "sporadic" neuroblastoma is being studied through a large genome-wide association study (GWAS). These efforts have led to the discovery of many common susceptibility alleles, each with modest effect size, associated with the development and progression of sporadic neuroblastoma. More recently, next-generation sequencing efforts have expanded the list of potential neuroblastoma-predisposing mutations to include rare germline variants with a predicted larger effect size. The evolving characterization of neuroblastoma's genetic basis has led to a deeper understanding of the molecular events driving tumorigenesis, more precise risk stratification and prognostics and novel therapeutic strategies. This review details the contemporary understanding of neuroblastoma's genetic predisposition, including recent advances and discusses ongoing efforts to address gaps in our knowledge regarding this malignancy's complex genetic underpinnings.

microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Sung, Derek; Li, Monica; Kosti, Adam; Lin, Gregory; Chen, Yidong; Pertsemlidis, Alexander; Hsiao, Tzu-Hung; Du, Liqin

2015-01-01

microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms—inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis. PMID:25760387

Modeling of Chronic Myeloid Leukemia: An Overview of In Vivo Murine and Human Xenograft Models

PubMed Central

Vellenga, Edo

2016-01-01

Over the past years, a wide variety of in vivo mouse models have been generated in order to unravel the molecular pathology of Chronic Myeloid Leukemia (CML) and to develop and improve therapeutic approaches. These models range from (conditional) transgenic models, knock-in models, and murine bone marrow retroviral transduction models followed by transplantation. With the advancement of immunodeficient xenograft models, it has become possible to use human stem/progenitor cells for in vivo studies as well as cells directly derived from CML patients. These models not only mimic CML but also have been instrumental in uncovering various fundamental mechanisms of CML disease progression and tyrosine kinase inhibitor (TKI) resistance. With the availability of iPSC technology, it has become feasible to derive, maintain, and expand CML subclones that are at least genetically identical to those in patients. The following review provides an overview of all murine as well as human xenograft models for CML established till date. PMID:27642303

SERPINE2 is a possible candidate promotor for lymph node metastasis in testicular cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagahara, Akira; Nakayama, Masashi; Oka, Daizo

2010-01-22

Testicular germ cell tumors (TGCTs) commonly metastasize to the lymph node or lung. However, it remains unclear which genes are associated with TGCT metastasis. The aim of this study was to identify gene(s) that promoted human TGCT metastasis. We intraperitoneally administered conditioned medium (CM) from JKT-1, a cell-line from a human testicular seminoma, or JKT-HM, a JKT-1 cell sub-line with high metastatic potential, into mice with JKT-1 xenografts. Administration of CM from JKT-HM significantly promoted lymph node metastasis. A cDNA microarray analysis showed that JKT-HM cells highly expressed the Serpine peptidase inhibitor, clade E, member 2 (SERPINE2), which encodes amore » secreted protein. Administration of CM from SERPINE2-silenced JKT-HM cells inhibited lymph node metastasis in the xenograft model, compared with administration of CM from JKT-HM cells. There was no significant difference in xenograft volume. Moreover, administration of CM from SERPINE2-over-expressing JKT-1 was likely to promote lymph node metastasis in the xenograft model. There was no difference in the in vitro proliferation or migration of JKT-1 cells cultured with CM from JKT-HM cells, compared to that with CM from JKT-1. There was no promotion of proliferation or lymphangiogenesis in the xenografts, as measured by Ki-67 and LYVE-1 immunohistochemistry, respectively. Although we could not clarify how SERPINE2 promoted lymph node metastasis, it may be a promoter in the development of lymph node metastasis in the human seminoma cells in a mouse xenograft model.« less

Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA.

PubMed

Costa, Delfina; Gigoni, Arianna; Würth, Roberto; Cancedda, Ranieri; Florio, Tullio; Pagano, Aldo

2014-01-01

Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential. Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in which differentiation is induced by retinoic acid treatment or stable overexpression of NDM29 non-coding RNA, both conditions characterized by a neuron-like differentiated phenotype. We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged. Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29. These data suggest the efficacy of metformin as neuroblastoma anticancer agent, and support the requirement of further studies on the possible role of the differentiation status on the antiproliferative effects of this drug.

Serum-circulating miRNAs predict neuroblastoma progression in mouse model of high-risk metastatic disease.

PubMed

Ramraj, Satish Kumar; Aravindan, Sheeja; Somasundaram, Dinesh Babu; Herman, Terence S; Natarajan, Mohan; Aravindan, Natarajan

2016-04-05

Circulating miRNAs have momentous clinical relevance as prognostic biomarkers and in the progression of solid tumors. Recognizing novel candidates of neuroblastoma-specific circulating miRNAs would allow us to identify potential prognostic biomarkers that could predict the switch from favorable to high-risk metastatic neuroblastoma (HR-NB). Utilizing mouse models of favorable and HR-NB and whole miRnome profiling, we identified high serum levels of 34 and low levels of 46 miRNAs in animals with HR-NB. Preferential sequence homology exclusion of mouse miRNAs identified 25 (11 increased; 14 decreased) human-specific prognostic marker candidates, of which, 21 were unique to HR-NB. miRNA QPCR validated miRnome profile. Target analysis defined the candidate miRNAs' signal transduction flow-through and demonstrated their converged roles in tumor progression. miRNA silencing studies verified the function of select miRNAs on the translation of at least 14 target proteins. Expressions of critical targets that correlate tumor progression in tissue of multifarious organs identify the orchestration of HR-NB. Significant (>10 fold) increase in serum levels of miR-381, miR-548h, and miR-580 identify them as potential prognostic markers for neuroblastoma progression. For the first time, we identified serum-circulating miRNAs that predict the switch from favorable to HR-NB and, further imply that these miRNAs could play a functional role in tumor progression.

Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms

PubMed Central

Monsma, David J; Cherba, David M; Eugster, Emily E; Dylewski, Dawna L; Davidson, Paula T; Peterson, Chelsea A; Borgman, Andrew S; Winn, Mary E; Dykema, Karl J; Webb, Craig P; MacKeigan, Jeffrey P; Duesbery, Nicholas S; Nickoloff, Brian J; Monks, Noel R

2015-01-01

Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAFV600E models showed acquired drug resistance, one BRAFV600E model had a complete and durable response, and a BRAFV600V model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations. PMID:26101714

Patient-derived xenograft in zebrafish embryos: a new platform for translational research in neuroendocrine tumors.

PubMed

Gaudenzi, Germano; Albertelli, Manuela; Dicitore, Alessandra; Würth, Roberto; Gatto, Federico; Barbieri, Federica; Cotelli, Franco; Florio, Tullio; Ferone, Diego; Persani, Luca; Vitale, Giovanni

2017-08-01

Preclinical research on neuroendocrine tumors usually involves immortalized cell lines and few animal models. In the present study we described an in vivo model based on patient-derived xenografts of neuroendocrine tumor cells in zebrafish (Danio rerio) embryos, allowing a rapid analysis of the angiogenic and invasive potential. Patient-derived neuroendocrine tumor cells were transplanted in 48 hours post-fertilization Tg(fli1a:EGFP) y1 zebrafish embryos that express enhanced green fluorescent protein in the entire vasculature. Neuroendocrine tumor cells, stained with CM-Dil, were injected into the subperidermal (perivitelline) space, close to the developing subintestinal venous plexus. A proper control group, represented by zebrafish injected with only D-PBS, was included in this study. Angiogenic and invasive potentials of each patient-derived xenograft were evaluated by both epifluorescence and confocal microscopes. Six out of eight neuroendocrine tumor samples were successfully transplanted in zebrafish embryos. Although the implanted tumor mass had a limited size (about 100 cells for embryos), patient-derived xenografts showed pro-angiogenic (5 cases) and invasive (6 cases) behaviors within 48 hours post injection. Patient-derived xenograft in zebrafish embryos appears to be a reliable in vivo preclinical model for neuroendocrine tumors, tumors with often limited cell availability. The rapidity of this procedure makes our model a promising platform to perform preclinical drug screening and opens a new scenario for personalized treatment in patients with neuroendocrine tumors.

184AA3: a xenograft model of ER+ breast adenocarcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hines, William C.; Kuhn, Irene; Thi, Kate

Despite the prevalence and significant morbidity resulting from estrogen receptor positive (ER +) breast adenocarcinomas, there are only a few models of this cancer subtype available for drug development and arguably none for studying etiology. Those models that do exist have questionable clinical relevance. Given our goal of developing luminal models, we focused on six cell lines derived by minimal mutagenesis from normal human breast cells, and asked if any could generate clinically relevant xenografts, which we then extensively characterized. Xenografts of one cell line, 184AA3, consistently formed ER + adenocarcinomas that had a high proliferative rate and other features consistentmore » with “luminal B” intrinsic subtype. Squamous and spindle cell/mesenchymal differentiation was absent, in stark contrast to other cell lines that we examined or others have reported. We explored intratumoral heterogeneity produced by 184AA3 by immunophenotyping xenograft tumors and cultured cells, and characterized marker expression by immunofluorescence and flow cytometry. A CD44 High  subpopulation was discovered, yet their tumor forming ability was far less than CD44 Low  cells. Single cell cloning revealed the phenotypic plasticity of 184AA3, consistent with the intratumoral heterogeneity observed in xenografts. Characterization of ER expression in cultures revealed ER protein and signaling is intact, yet when estrogen was depleted in culture, and in vivo, it did not impact cell or tumor growth, analogous to therapeutically resistant ER +  cancers. In conclusion, this model is appropriate for studies of the etiology of ovarian hormone independent adenocarcinomas, for identification of therapeutic targets, predictive testing, and drug development.« less

184AA3: a xenograft model of ER+ breast adenocarcinoma

DOE PAGES

Hines, William C.; Kuhn, Irene; Thi, Kate; ...

2015-12-12

Despite the prevalence and significant morbidity resulting from estrogen receptor positive (ER +) breast adenocarcinomas, there are only a few models of this cancer subtype available for drug development and arguably none for studying etiology. Those models that do exist have questionable clinical relevance. Given our goal of developing luminal models, we focused on six cell lines derived by minimal mutagenesis from normal human breast cells, and asked if any could generate clinically relevant xenografts, which we then extensively characterized. Xenografts of one cell line, 184AA3, consistently formed ER + adenocarcinomas that had a high proliferative rate and other features consistentmore » with “luminal B” intrinsic subtype. Squamous and spindle cell/mesenchymal differentiation was absent, in stark contrast to other cell lines that we examined or others have reported. We explored intratumoral heterogeneity produced by 184AA3 by immunophenotyping xenograft tumors and cultured cells, and characterized marker expression by immunofluorescence and flow cytometry. A CD44 High  subpopulation was discovered, yet their tumor forming ability was far less than CD44 Low  cells. Single cell cloning revealed the phenotypic plasticity of 184AA3, consistent with the intratumoral heterogeneity observed in xenografts. Characterization of ER expression in cultures revealed ER protein and signaling is intact, yet when estrogen was depleted in culture, and in vivo, it did not impact cell or tumor growth, analogous to therapeutically resistant ER +  cancers. In conclusion, this model is appropriate for studies of the etiology of ovarian hormone independent adenocarcinomas, for identification of therapeutic targets, predictive testing, and drug development.« less

Integrative genomics reveals hypoxia inducible genes that are associated with a poor prognosis in neuroblastoma patients.

PubMed

Applebaum, Mark A; Jha, Aashish R; Kao, Clara; Hernandez, Kyle M; DeWane, Gillian; Salwen, Helen R; Chlenski, Alexandre; Dobratic, Marija; Mariani, Christopher J; Godley, Lucy A; Prabhakar, Nanduri; White, Kevin; Stranger, Barbara E; Cohn, Susan L

2016-11-22

Neuroblastoma is notable for its broad spectrum of clinical behavior ranging from spontaneous regression to rapidly progressive disease. Hypoxia is well known to confer a more aggressive phenotype in neuroblastoma. We analyzed transcriptome data from diagnostic neuroblastoma tumors and hypoxic neuroblastoma cell lines to identify genes whose expression levels correlate with poor patient outcome and are involved in the hypoxia response. By integrating a diverse set of transcriptome datasets, including those from neuroblastoma patients and neuroblastoma derived cell lines, we identified nine genes (SLCO4A1, ENO1, HK2, PGK1, MTFP1, HILPDA, VKORC1, TPI1, and HIST1H1C) that are up-regulated in hypoxia and whose expression levels are correlated with poor patient outcome in three independent neuroblastoma cohorts. Analysis of 5-hydroxymethylcytosine and ENCODE data indicate that at least five of these nine genes have an increase in 5-hydroxymethylcytosine and a more open chromatin structure in hypoxia versus normoxia and are putative targets of hypoxia inducible factor (HIF) as they contain HIF binding sites in their regulatory regions. Four of these genes are key components of the glycolytic pathway and another three are directly involved in cellular metabolism. We experimentally validated our computational findings demonstrating that seven of the nine genes are significantly up-regulated in response to hypoxia in the four neuroblastoma cell lines tested. This compact and robustly validated group of genes, is associated with the hypoxia response in aggressive neuroblastoma and may represent a novel target for biomarker and therapeutic development.

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

PubMed

Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

2017-09-01

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Halofuginone suppresses growth of human uterine leiomyoma cells in a mouse xenograft model.

PubMed

Koohestani, Faezeh; Qiang, Wenan; MacNeill, Amy L; Druschitz, Stacy A; Serna, Vanida A; Adur, Malavika; Kurita, Takeshi; Nowak, Romana A

2016-07-01

Does halofuginone (HF) inhibit the growth of human uterine leiomyoma cells in a mouse xenograft model? HF suppresses the growth of human uterine leiomyoma cells in a mouse xenograft model through inhibiting cell proliferation and inducing apoptosis. Uterine leiomyomas are the most common benign tumors of the female reproductive tract. HF can suppress the growth of human uterine leiomyoma cells in vitro. The mouse xenograft model reflects the characteristics of human leiomyomas. Primary leiomyoma smooth muscle cells from eight patients were xenografted under the renal capsule of adult, ovariectomized NOD-scid IL2Rγ(null) mice (NSG). Mice were treated with two different doses of HF or vehicle for 4 weeks with six to eight mice per group. Mouse body weight measurements and immunohistochemical analysis of body organs were carried out to assess the safety of HF treatment. Xenografted tumors were measured and analyzed for cellular and molecular changes induced by HF. Ovarian steroid hormone receptors were evaluated for possible modulation by HF. Treatment of mice carrying human UL xenografts with HF at 0.25 or 0.50 mg/kg body weight for 4 weeks resulted in a 35-40% (P < 0.05) reduction in tumor volume. The HF-induced volume reduction was accompanied by increased apoptosis and decreased cell proliferation. In contrast, there was no significant change in the collagen content either at the transcript or protein level between UL xenografts in control and HF groups. HF treatment did not change the expression level of ovarian steroid hormone receptors. No adverse pathological effects were observed in other tissues from mice undergoing treatment at these doses. While this study did test the effects of HF on human leiomyoma cells in an in vivo model, HF was administered to mice whose tolerance and metabolism of the drug may differ from that in humans. Also, the longer term effects of HF treatment are yet unclear. The results of this study showing the effectiveness of HF in reducing UL tumor growth by interfering with the main cellular processes regulating cell proliferation and apoptosis are in agreement with previous studies on the effects of HF on other fibrotic diseases. HF can be considered as a candidate for reducing the size of leiomyomas, particularly prior to surgery. This project was funded by NIH PO1HD057877 and R01 HD064402. Authors report no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

HOXC9-Induced Differentiation in Neuroblastoma Development

DTIC Science & Technology

2014-10-01

Neuroblastoma Development PRINCIPAL INVESTIGATOR: Han-Fei Ding RECIPIENT: Georgia Health Sciences University Research Institute, Inc... Neuroblastoma  Development   5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0613 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...role in determining the differentiation states of neuroblastoma tumors, with higher levels of HOXC9 promoting differentiation. At the cellular level

Neuroblastoma-like schwannoma in a case of schwannomatosis: Report of a rare case

PubMed Central

Sulhyan, Kalpana R; Deshmukh, Bhakti D; Gosavi, Alka V; Ramteerthakar, Nayan A

2015-01-01

Schwannomatosis is a term used to describe patients with multiple nonvestibular schwannomas with no other stigmata of neurofibromatosis type-2 (NF2). Neuroblastoma-like schwannoma is a rare subtype of schwannoma, with histological features resembling a neuroblastoma. This case is probabaly the second case of very uncommon neuroblastoma-like schwannoma, in a patient of schwannomatosis. PMID:26715928

Neuroblastoma-like schwannoma in a case of schwannomatosis: Report of a rare case.

PubMed

Sulhyan, Kalpana R; Deshmukh, Bhakti D; Gosavi, Alka V; Ramteerthakar, Nayan A

2015-10-01

Schwannomatosis is a term used to describe patients with multiple nonvestibular schwannomas with no other stigmata of neurofibromatosis type-2 (NF2). Neuroblastoma-like schwannoma is a rare subtype of schwannoma, with histological features resembling a neuroblastoma. This case is probabaly the second case of very uncommon neuroblastoma-like schwannoma, in a patient of schwannomatosis.

Restoration of promyelocytic leukemia protein-nuclear bodies in neuroblastoma cells enhances retinoic acid responsiveness.

PubMed

Yu, Jiang Hong; Nakajima, Ayako; Nakajima, Hiroshi; Diller, Lisa R; Bloch, Kenneth D; Bloch, Donald B

2004-02-01

Neuroblastoma is the most common solid tumor of infancy and is believed to result from impaired differentiation of neuronal crest embryonal cells. The promyelocytic leukemia protein (PML)-nuclear body is a cellular structure that is disrupted during the pathogenesis of acute promyelocytic leukemia, a disease characterized by impaired myeloid cell differentiation. During the course of studies to examine the composition and function of PML-nuclear bodies, we observed that the human neuroblastoma cell line SH-SY5Y lacked these structures and that the absence of PML-nuclear bodies was a feature of N- and I-type, but not S-type, neuroblastoma cell lines. Induction of neuroblastoma cell differentiation with 5-bromo-2'deoxyuridine, all-trans-retinoic acid, or IFN-gamma induced PML-nuclear body formation. PML-nuclear bodies were not detected in tissue sections prepared from undifferentiated neuroblastomas but were present in neuroblasts in differentiating tumors. Expression of PML in neuroblastoma cells restored PML-nuclear bodies, enhanced responsiveness to all-trans-retinoic acid, and induced cellular differentiation. Pharmacological therapies that increase PML expression may prove to be important components of combined modalities for the treatment of neuroblastoma.

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

PubMed

Bettayeb, Karima; Tirado, Oscar M; Marionneau-Lambot, Séverine; Ferandin, Yoan; Lozach, Olivier; Morris, Jonathan C; Mateo-Lozano, Silvia; Drueckes, Peter; Schächtele, Christoph; Kubbutat, Michael H G; Liger, François; Marquet, Bernard; Joseph, Benoît; Echalier, Aude; Endicott, Jane A; Notario, Vicente; Meijer, Laurent

2007-09-01

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuatormore » (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.« less

Immobilization of concanavalin A receptors during differentiation of neuroblastoma cells.

PubMed

Fishman, M C; Dragsten, P R; Spector, I

1981-04-30

Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We have used the technique of fluorescence photobleaching recovery to study the lateral mobility of cell-surface constituents during the differentiation of neuroblastoma clone N1E-115 cells. The concanavalin A (Con A) binding sites appear as discrete patches distributed over the entire cell surface and exhibit lateral mobility in undifferentiated cells comparable with that of surface glycoproteins of other cells. After induction of differentiation, however, the vast majority of Con A binding sites become immobilized, and we present data which suggest that the mechanism of this immobilization may involve linkage to the internal actin network.

Nano-Bio-Mechanics of Neuroblastoma Cells Using AFM

NASA Astrophysics Data System (ADS)

Bastatas, Lyndon; Matthews, James; Kang, Min; Park, Soyeun

2011-10-01

We have conducted an in vitro study to determine the elastic moduli of neurobalstoma cell lines using atomic force microscopy. Using a panel of cell lines established from neuroblastoma patients at different stages of disease progress and treatment, we have investigated the differences in elastic moduli during a course of cancer progression and chemotherapy. The cells were grown on the hard substrates that are chemically functionalized to enhance adhesion. We have performed the AFM indentation experiments with different applied forces from the AFM probe. For the purpose of the comparison between cell lines, the indentations were performed only on cell centers. The obtained force-distance curves were analyzed using the Hertz model in order to extract the elastic moduli. We have found that the elastic moduli of human neuroblastoma cells significantly varied during the disease progression. We postulate that the observed difference might be affected by the treatment and chemotherapy.

Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

PubMed Central

Powers, John T; Tsanov, Kaloyan M; Pearson, Daniel S; Roels, Frederik; Spina, Catherine S; Ebright, Richard; Seligson, Marc; de Soysa, Yvanka; Cahan, Patrick; Theiβen, Jessica; Tu, Ho-Chou; Han, Areum; Kurek, Kyle C; LaPier, Grace S; Osborne, Jihan K; Ross, Samantha J; Cesana, Marcella; Collins, James J; Berthold, Frank; Daley, George Q

2016-01-01

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumor suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. However, here we show that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN mRNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN-amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma pathogenesis with broad implications for cancer pathogenesis. PMID:27383785

RSRC1 and CPZ gene polymorphisms with neuroblastoma susceptibility in Chinese children.

PubMed

Tang, Jue; Liu, Wei; Zhu, Jinhong; Zhang, Jiao; Wang, Feng-Hua; Liang, Jiang-Hua; Zeng, Jia-Hang; Wang, Hui; Xia, Huimin; He, Jing

2018-07-01

Two new neuroblastoma susceptibility loci at 3q25 (RSRC1 rs6441201 G > A) and 4p16 (CPZ rs3796725 T > C and rs3796727 A > G) were identified by a genome-wide association study (GWAS) involving Italians, African Americans and European Americans. In this case-control study with 393 neuroblastoma cases and 812 controls, we investigated the association between these three polymorphisms and neuroblastoma susceptibility in Chinese population. We found that participants harboring the RSRC1 rs6441201A allele were associated with an increased risk of neuroblastoma (AA vs. GG: adjusted OR = 1.55, 95% CI = 1.03-2.34, P = 0.036). No significant association between the CPZ polymorphisms (rs3796725 T > C and rs3796727A > G) and neuroblastoma susceptibility was observed. In conclusion, our results confirm that the RSRC1 rs6441201A allele is associated with neuroblastoma susceptibility in Chinese population. Copyright © 2018 Elsevier B.V. All rights reserved.

miR-129 inhibits tumor growth and potentiates chemosensitivity of neuroblastoma by targeting MYO10.

PubMed

Wang, Xiqian; Li, Jing; Xu, Xiao; Zheng, Jiachun; Li, Qingbo

2018-07-01

Although the treatment strategies for neuroblastoma (NB) develop rapidly, a considerable number of patients could not benefit from chemotherapy. Here, we revealed a miR-129-MYO10 axis that regulated neuroblastoma growth and chemosensitivity. Mechanistically, MYO10 was up-regulated in neuroblastoma tissues and associated with poor overall survival. While overexpression of MYO10 enhanced tumor growth, genetic inhibition of MYO10 led to growth-inhibitory and chemopotentiating effects in neuroblastoma. MYO10 was further identified as a target of miR-129. Our data showed that miR-129 down-regulated MYO10 expression and subsequently suppressed cell growth. Re-expression of MYO10 significantly rescued miR129-mediated proliferation repression and chemosensitivity. In conclusion, our results demonstrated that miR-129 inhibited neuroblastoma growth and potentiated chemosensitivity by targeting MYO10, which may represent promising targets and rational therapeutic options for neuroblastoma. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2.

PubMed

Ito, K; Miyamoto, R; Tani, H; Kurita, S; Kobayashi, M; Tamura, K; Bonkobara, M

2018-02-01

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Previously, the kinase inhibitor dasatinib was shown to have potent growth inhibitory activity against HS cells in vitro, possibly via targeting the EPHA2 receptor. Here, the in vivo effect of dasatinib in HS cells was investigated using a xenograft mouse model. Moreover, the expression status of EPHA2 was examined in six HS cell lines, ranging from insensitive to highly sensitive to dasatinib. In the HS xenograft mouse model, dasatinib significantly suppressed tumor growth, as illustrated by a decrease in mitotic and Ki67 indices and an increase in apoptotic index in tumor tissues. On Western blot analysis, EPHA2 was only weakly detected in all HS cell lines, regardless of sensitivity to dasatinib. Dasatinib likely results in the inhibition of xenograft tumor growth via a mechanism other than targeting EPHA2. The findings of this study suggest that dasatinib is a targeted therapy drug worthy of further exploration for the treatment of canine HS. © 2017 John Wiley & Sons Ltd.

Setting up a wide panel of patient-derived tumor xenografts of non-small cell lung cancer by improving the preanalytical steps.

PubMed

Ilie, Marius; Nunes, Manoel; Blot, Lydia; Hofman, Véronique; Long-Mira, Elodie; Butori, Catherine; Selva, Eric; Merino-Trigo, Ana; Vénissac, Nicolas; Mouroux, Jérôme; Vrignaud, Patricia; Hofman, Paul

2015-02-01

With the ongoing need to improve therapy for non-small cell lung cancer (NSCLC) there has been increasing interest in developing reliable preclinical models to test novel therapeutics. Patient-derived tumor xenografts (PDX) are considered to be interesting candidates. However, the establishment of such model systems requires highly specialized research facilities and introduces logistic challenges. We aimed to establish an extensive well-characterized panel of NSCLC xenograft models in the context of a long-distance research network after careful control of the preanalytical steps. One hundred fresh surgically resected NSCLC specimens were shipped in survival medium at room temperature from a hospital-integrated biobank to animal facilities. Within 24 h post-surgery, tumor fragments were subcutaneously xenografted into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical, aCGH and next-generation sequencing approaches. For this model system, the tumor take rate was 35%, with higher rates for squamous carcinoma (60%) than for adenocarcinoma (13%). Patients for whom PDX tumors were obtained had a significantly shorter disease-free survival (DFS) compared to patients for whom no PDX tumors (P = 0.039) were obtained. We established a large panel of PDX NSCLC models with a high frequency of mutations (29%) in EGFR, KRAS, NRAS, MEK1, BRAF, PTEN, and PI3KCA genes and with gene amplification (20%) of c-MET and FGFR1. This new patient-derived NSCLC xenograft collection, established regardless of the considerable time required and the distance between the clinic and the animal facilities, recapitulated the histopathology and molecular diversity of NSCLC and provides stable and reliable preclinical models for human lung cancer research. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

BATTLE: Biomarker-Based Approaches of Targeted Therapy for Lung Cancer Elimination

DTIC Science & Technology

2007-04-01

localization, which • The combination of erlotinib and Ad-dnIGF-1R synergistically inhibits the growth of tumors in xenograft mouse models . able outcomes...of erlotinib and Ad-dnIGF-1R synergistically inhibits the growth of tumors in xenograft mouse models . Specific Aim 2.3: To investigate the...biomarkers and adaptive randomization via hierarchical Bayes modeling . 2) To study the molecular mechanisms of response and resistance to targeted

Progesterone is essential for maintenance and growth of uterine leiomyoma.

PubMed

Ishikawa, Hiroshi; Ishi, Kazutomo; Serna, Vanida Ann; Kakazu, Rafael; Bulun, Serdar E; Kurita, Takeshi

2010-06-01

Uterine leiomyomata (ULs) represent the most common tumor in women and can cause abnormal uterine bleeding, large pelvic masses, and recurrent pregnancy loss. Although the dependency of UL growth on ovarian steroids is well established, the relative contributions of 17beta-estradiol and progesterone are yet to be clarified. Conventionally, estradiol has been considered the primary stimulus for UL growth, and studies with cell culture and animal models support this concept. In contrast, no research model has clearly demonstrated a requirement of progesterone in UL growth despite accumulating clinical evidence for the essential role of progesterone in this tumor. To elucidate the functions of ovarian steroids in UL, we established a xenograft model reflecting characteristics of these tumors by grafting human UL tissue beneath the renal capsule of immunodeficient mice. Leiomyoma xenografts increased in size in response to estradiol plus progesterone through cell proliferation and volume increase in cellular and extracellular components. The xenograft growth induced by estradiol plus progesterone was blocked by the antiprogestin RU486. Furthermore, the volume of established UL xenografts decreased significantly after progesterone withdrawal. Surprisingly, treatment with estradiol alone neither increased nor maintained the tumor size. Although not mitogenic by itself, estradiol induced expression of progesterone receptor and supported progesterone action on leiomyoma xenografts. Taken together, our findings define that volume maintenance and growth of human UL are progesterone dependent.

A Computational Framework for Design and Development of Novel Prostate Cancer Therapies

DTIC Science & Technology

2015-09-01

a ‘wound healing’ assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model. Cell Death and Disease (2014) 5...autophagy and apoptosis in prostate cancer cells, and inhibits prostate cancer xenograft tumor growth in vivo. To our knowledge, this is the first report...sensitizer, and blocking of autophagy by CQ promotes CTN06-induced cell death. CTN06 inhibits PC3 xenograft tumor growth in vivo. Given the in vitro

Regulating Prostate Cancer Sensitivity to Chemotherapy through Translational Control of CCAAT Enhancer Binding Proteins

DTIC Science & Technology

2015-08-01

ratio in LNCaP and PC3 cells and suppression of CEBPB sensitized these cells to bortezomib in vitro. PC3 xenografts deficient in CEBPB showed...resistant growth of PCa tumors in a mouse xenograft model. shNTV or shCEBPB LNCaP cells were subcutaneously engrafted into male NSG mice and when tumors...was monitored weekly by caliper measurement for 8-weeks (Fig. 3B). We observed significant suppression of CRPC growth in xenografts expressing shC

Targeting Therapy Resistant Tumor Vessels

DTIC Science & Technology

2008-08-01

No 6 C8161 s.c. xenografts No 5 K14-HPV16 skin cancer No 4 MDA-MB-435 orthotopic xenografts No 4 AGR TRAMP PIN lesions TRAMP PIN lesions Yes 18 TRAMP...CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c...Summary We developed three tumor models under this project: 4T1 mouse breast cancer and MDA-MB-435 human cancer xenograft tumors treated with anti

HOTAIR gene polymorphisms contribute to increased neuroblastoma susceptibility in Chinese children.

PubMed

Yang, Xu; He, Jing; Chang, Yitian; Luo, Annie; Luo, Ailing; Zhang, Jiao; Zhang, Ruizhong; Xia, Huimin; Xu, Ling

2018-06-15

Neuroblastoma is the most frequently diagnosed extracranial solid tumor in children. Previous studies have shown that single-nucleotide polymorphisms in some genes are associated with the risk of multiple cancers, including neuroblastoma. Although Hox transcript antisense intergenic RNA (HOTAIR) gene polymorphisms have been investigated in a variety of cancers, to the authors' knowledge the relationships between HOTAIR gene polymorphisms and neuroblastoma susceptibility have not been reported to date. The objective of the current study was to evaluate the correlation between HOTAIR gene polymorphisms and neuroblastoma risk in Chinese children. The authors genotyped 6 polymorphisms (rs920778 A>G, rs12826786 C>T, rs4759314 A>G, rs7958904 G>C, rs874945 C>T, and rs1899663 C>A) of the HOTAIR gene in 2 Chinese populations including 393 neuroblastoma cases and 812 healthy controls. The strength of the associations was evaluated using odds ratios and 95% confidence intervals. Further stratification analyses were conducted to explore the association between the HOTAIR gene polymorphisms rs12826786 C>T, rs874945 C>T, and rs1899663 C>A with neuroblastoma susceptibility in terms of age, sex, clinical stage of disease, and sites of origin. The authors found that the rs12826786 C>T (P =.013), rs874945 C>T (P =.020), and rs1899663 C>A (P =.029) polymorphisms were significantly associated with increased neuroblastoma risk. In stratification analyses, these associations were more predominant in females and among patients with tumor in the retroperitoneal region or mediastinum. The remaining 3 polymorphisms were not found to be related to neuroblastoma susceptibility. The results of the current study verified that HOTAIR gene polymorphisms are associated with increased neuroblastoma risk and suggest that HOTAIR gene polymorphisms might be a potential biomarker for neuroblastoma susceptibility. Cancer 2018;124:2599-606. © 2018 American Cancer Society. © 2018 American Cancer Society.

PD-L1 Is a Therapeutic Target of the Bromodomain Inhibitor JQ1 and, Combined with HLA Class I, a Promising Prognostic Biomarker in Neuroblastoma.

PubMed

Melaiu, Ombretta; Mina, Marco; Chierici, Marco; Boldrini, Renata; Jurman, Giuseppe; Romania, Paolo; D'Alicandro, Valerio; Benedetti, Maria C; Castellano, Aurora; Liu, Tao; Furlanello, Cesare; Locatelli, Franco; Fruci, Doriana

2017-08-01

Purpose: This study sought to evaluate the expression of programmed cell death-ligand-1 (PD-L1) and HLA class I on neuroblastoma cells and programmed cell death-1 (PD-1) and lymphocyte activation gene 3 (LAG3) on tumor-infiltrating lymphocytes to better define patient risk stratification and understand whether this tumor may benefit from therapies targeting immune checkpoint molecules. Experimental Design: In situ IHC staining for PD-L1, HLA class I, PD-1, and LAG3 was assessed in 77 neuroblastoma specimens, previously characterized for tumor-infiltrating T-cell density and correlated with clinical outcome. Surface expression of PD-L1 was evaluated by flow cytometry and IHC in neuroblastoma cell lines and tumors genetically and/or pharmacologically inhibited for MYC and MYCN. A dataset of 477 human primary neuroblastomas from GEO and ArrayExpress databases was explored for PD-L1, MYC, and MYCN correlation. Results: Multivariate Cox regression analysis demonstrated that the combination of PD-L1 and HLA class I tumor cell density is a prognostic biomarker for predicting overall survival in neuroblastoma patients ( P = 0.0448). MYC and MYCN control the expression of PD-L1 in neuroblastoma cells both in vitro and in vivo Consistently, abundance of PD-L1 transcript correlates with MYC expression in primary neuroblastoma. Conclusions: The combination of PD-L1 and HLA class I represents a novel prognostic biomarker for neuroblastoma. Pharmacologic inhibition of MYCN and MYC may be exploited to target PD-L1 and restore an efficient antitumor immunity in high-risk neuroblastoma. Clin Cancer Res; 23(15); 4462-72. ©2017 AACR . ©2017 American Association for Cancer Research.

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and 5-bromotetrandrine in xenograft nude-mice

PubMed Central

Chen, Baoan; Cheng, Jian; Wu, Yanan; Gao, Feng; Xu, Wenlin; Shen, Huilin; Ding, Jiahua; Gao, Chong; Sun, Qian; Sun, Xinchen; Cheng, Hongyan; Li, Guohong; Chen, Wenji; Chen, Ningna; Liu, Lijie; Li, Xiaomao; Wang, Xuemei

2009-01-01

In this paper we establish the xenograft leukemia model with stable multidrug resistance in nude mice and to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe3O4 (MNP-Fe3O4) combined with daunorubicin (DNR) in vivo. Two subclones of K562 and K562/A02 cells were inoculated subcutaneously into the back of athymic nude mice (1 × 107 cells/each) respectively to establish leukemia xenograft models. Drug-resistant and sensitive tumor-bearing nude mice were assigned randomly into five groups which were treated with normal saline; DNR; NP-Fe3O4 combined with DNR; 5-BrTet combined with DNR; 5-BrTet and MNP-Fe3O4 combined with DNR, respectively. The incidence of formation, growth characteristics, weight, and volume of tumors were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of Bcl-2, and BAX were detected by Western blot. Bcl-2, BAX, and caspase-3 genes were also detected. For K562/A02 cells xenograft tumors, 5-BrTet and MNP-Fe3O4 combined with DNR significantly suppressed growth of tumor. A histopathologic examination of tumors clearly showed necrosis of the tumors. Application of 5-BrTet and MNP-Fe3O4 inhibited the expression of Bcl-2 protein and upregulated the expression of BAX and caspase-3 proteins in K562/A02 cells xenograft tumor. It is concluded that 5-BrTet and MNP-Fe3O4 combined with DNR had a significant tumor-suppressing effect on a MDR leukemia cells xenograft model. PMID:19421372

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and 5-bromotetrandrine in xenograft nude-mice.

PubMed

Chen, Baoan; Cheng, Jian; Wu, Yanan; Gao, Feng; Xu, Wenlin; Shen, Huilin; Ding, Jiahua; Gao, Chong; Sun, Qian; Sun, Xinchen; Cheng, Hongyan; Li, Guohong; Chen, Wenji; Chen, Ningna; Liu, Lijie; Li, Xiaomao; Wang, Xuemei

2009-01-01

In this paper we establish the xenograft leukemia model with stable multidrug resistance in nude mice and to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe(3)O(4) (MNP-Fe(3)O(4)) combined with daunorubicin (DNR) in vivo. Two subclones of K562 and K562/A02 cells were inoculated subcutaneously into the back of athymic nude mice (1 x 10(7) cells/each) respectively to establish leukemia xenograft models. Drug-resistant and sensitive tumor-bearing nude mice were assigned randomly into five groups which were treated with normal saline; DNR; NP-Fe(3)O(4) combined with DNR; 5-BrTet combined with DNR; 5-BrTet and MNP-Fe(3)O(4) combined with DNR, respectively. The incidence of formation, growth characteristics, weight, and volume of tumors were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of Bcl-2, and BAX were detected by Western blot. Bcl-2, BAX, and caspase-3 genes were also detected. For K562/A02 cells xenograft tumors, 5-BrTet and MNP-Fe(3)O(4) combined with DNR significantly suppressed growth of tumor. A histopathologic examination of tumors clearly showed necrosis of the tumors. Application of 5-BrTet and MNP-Fe(3)O(4) inhibited the expression of Bcl-2 protein and upregulated the expression of BAX and caspase-3 proteins in K562/A02 cells xenograft tumor. It is concluded that 5-BrTet and MNP-Fe(3)O(4) combined with DNR had a significant tumor-suppressing effect on a MDR leukemia cells xenograft model.

Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model.

PubMed

Abe, Shinji; Kaneko, Mika Kato; Tsuchihashi, Yuki; Izumi, Toshihiro; Ogasawara, Satoshi; Okada, Naoto; Sato, Chiemi; Tobiume, Makoto; Otsuka, Kenji; Miyamoto, Licht; Tsuchiya, Koichiro; Kawazoe, Kazuyoshi; Kato, Yukinari; Nishioka, Yasuhiko

2016-09-01

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti-human podoplanin mAb, NZ-1, and a rat-human chimeric anti-human podoplanin antibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplanin-positive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ-8, and NZ-12, a novel rat-human chimeric anti-human podoplanin antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a(+) ) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human natural killer (NK) (CD56(+) ) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56(+) ) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK (CD56(+) ) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

A Model for Spheroid versus Monolayer Response of SK-N-SH Neuroblastoma Cells to Treatment with 15-Deoxy-PGJ 2

PubMed Central

Dunham, Ann; Chen, Paula X.; Chen, Michelle; Huynh, Milan; Rheingold, Evan; Prosper, Olivia

2016-01-01

Researchers have observed that response of tumor cells to treatment varies depending on whether the cells are grown in monolayer, as in vitro spheroids or in vivo. This study uses data from the literature on monolayer treatment of SK-N-SH neuroblastoma cells with 15-deoxy-PGJ 2 and couples it with data on growth rates for untreated SK-N-SH neuroblastoma cells grown as multicellular spheroids. A linear model is constructed for untreated and treated monolayer data sets, which is tuned to growth, death, and cell cycle data for the monolayer case for both control and treatment with 15-deoxy-PGJ 2. The monolayer model is extended to a five-dimensional nonlinear model of in vitro tumor spheroid growth and treatment that includes compartments of the cell cycle (G 1, S, G 2/M) as well as quiescent (Q) and necrotic (N) cells. Monolayer treatment data for 15-deoxy-PGJ 2 is used to derive a prediction of spheroid response under similar treatments. For short periods of treatment, spheroid response is less pronounced than monolayer response. The simulations suggest that the difference in response to treatment of monolayer versus spheroid cultures observed in laboratory studies is a natural consequence of tumor spheroid physiology rather than any special resistance to treatment. PMID:28044089

Progress towards personalized therapeutics: biologic- and risk-directed therapy for neuroblastoma.

PubMed

Gustafson, William Clay; Matthay, Katherine K

2011-10-01

Neuroblastoma, a tumor of the developing peripheral sympathetic nervous system, is the most common and deadly extracranial solid tumor of childhood. Risk-stratification and risk-adapted therapy play a large role in the modern treatment of neuroblastoma. Recently, through extensive international collaboration, new guidelines for risk stratification have emerged that will aid in international cooperative studies, as well as clarifying therapeutic options for patients. Current therapies for low- and intermediate-risk neuroblastoma have resulted in excellent prognoses for these risk strata, and current efforts are concentrated on chemotherapy reduction. By contrast, much more gradual progress has been made in improving survival for high-risk neuroblastoma patients, despite significant chemotherapy intensification. Current investigations focus on overcoming resistance by elucidating the molecular/genetic causes of neuroblastoma tumorigenesis and progression, with the aim of developing more effective biologically targeted therapies for this disease.

Transcriptional Inducers of Acetylcholinesterase Expression as Novel Antidotes for Protection Against Chemical Warfare Agents

DTIC Science & Technology

2005-10-01

neuroblastoma cell line , P19 and a human neuroblastoma cell line SH - SY5Y (data not shown). Effect of trichostatin A on...mouse neuroblastoma P19 cell line and a human neuroblastoma cell line SH - SY5Y . More experiments are needed to prove the potential of AChE expression in...treatment of nerve agent exposure. MATERIALS AND METHODS Neuronal cell lines and

Marrow Derived Antibody Library for the Treatment of Neuroblastoma

DTIC Science & Technology

2015-12-01

Award Number: W81XWH-12-1-0332 TITLE: Marrow-Derived Antibody Library for the Treatment of Neuroblastoma PRINCIPAL INVESTIGATOR: Giselle...Marrow-Derived Antibody Library for Treatment of Neuroblastoma 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...to Spectrum Health. 14. ABSTRACT Neuroblastoma (NB) is the most common solid tumor in children, which accounts for 15% of all pediatric cancer deaths

Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

DTIC Science & Technology

2015-10-01

Award Number: W81XWH-13-1-0241 TITLE: Identifying that Regulate Neuroblastoma Cell Differentiation PRINCIPAL INVESTIGATOR: Dr. Liqin Du...inducing miRNA, miR- 449a. We examined the differentiation-inducing function of miR-449a in multiple neuroblastoma cell lines. We have demonstrated that...miR-449a functions as an inducer of cell differentiation in neuroblastoma cell lines with distinct genetic backgrounds, including the MYCN

Targeting Notch pathway induces growth inhibition and differentiation of neuroblastoma cells.

PubMed

Ferrari-Toninelli, Giulia; Bonini, Sara Anna; Uberti, Daniela; Buizza, Laura; Bettinsoli, Paola; Poliani, Pietro Luigi; Facchetti, Fabio; Memo, Maurizio

2010-12-01

High-risk neuroblastoma is a severe pediatric tumor characterized by poor prognosis. Understanding the molecular mechanisms involved in tumor development and progression is strategic for the improvement of pharmacological therapies. Notch was recently proposed as a pharmacological target for the therapy of several cancers and is emerging as a new neuroblastoma-related molecular pathway. However, the precise role played by Notch in this cancer remains to be studied extensively. Here, we show that Notch activation by the Jagged1 ligand enhances the proliferation of neuroblastoma cells, and we propose the possible use of Notch-blocking γ-secretase inhibitors (GSIs) in neuroblastoma therapy. Two different GSIs, Compound E and DAPT, were tested alone or in combination with 13-cis retinoic acid (RA) on neuroblastoma cell lines. SH-SY5Y and IMR-32 cells were chosen as paradigms of lower and higher malignancy, respectively. Used alone, GSIs induced complete cell growth arrest, promoted neuronal differentiation, and significantly reduced cell motility. The combination of GSIs and 13-cis RA resulted in the enhanced growth inhibition, differentiation, and migration of neuroblastoma cells. In summary, our data suggest that a combination of GSIs with 13-cis RA offers a therapeutic advantage over a single agent, indicating a potential novel therapy for neuroblastoma.

Tumor-infiltrating T lymphocytes improve clinical outcome of therapy-resistant neuroblastoma.

PubMed

Mina, Marco; Boldrini, Renata; Citti, Arianna; Romania, Paolo; D'Alicandro, Valerio; De Ioris, Maretta; Castellano, Aurora; Furlanello, Cesare; Locatelli, Franco; Fruci, Doriana

2015-09-01

Neuroblastoma grows within an intricate network of different cell types including epithelial, stromal and immune cells. The presence of tumor-infiltrating T cells is considered an important prognostic indicator in many cancers, but the role of these cells in neuroblastoma remains to be elucidated. Herein, we examined the relationship between the type, density and organization of infiltrating T cells and clinical outcome within a large collection of neuroblastoma samples by quantitative analysis of immunohistochemical staining. We found that infiltrating T cells have a prognostic value greater than, and independent of, the criteria currently used to stage neuroblastoma. A variable in situ structural organization and different concurrent infiltration of T-cell subsets were detected in tumors with various outcomes. Low-risk neuroblastomas were characterized by a higher number of proliferating T cells and a more structured T-cell organization, which was gradually lost in tumors with poor prognosis. We defined an immunoscore based on the presence of CD3 + , CD4 + and CD8 + infiltrating T cells that associates with favorable clinical outcome in MYCN-amplified tumors, improving patient survival when combined with the v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) status. These findings support the hypothesis that infiltrating T cells influence the behavior of neuroblastoma and might be of clinical importance for the treatment of patients.

Proliferate and survive: cell division cycle and apoptosis in human neuroblastoma.

PubMed

Borriello, Adriana; Roberto, Roberta; Della Ragione, Fulvio; Iolascon, Achille

2002-02-01

Neuroblastoma is one of the most frequent childhood cancers and a major cause of death from neoplasias of infancy. Although a wealth of studies on its molecular bases have been carried out, little conclusive information about its origin and evolution is available. Some intriguing findings have correlated neuroblastoma development with aberrations of two pivotal cellular processes generally altered in human cancers, namely cell division cycle and apoptosis. Indeed, it has been reported that neuroblastoma cell lines show accumulation of Id2 protein, a factor which is able to hamper the pRb protein antiproliferative activity. The increased Id2 is due to N-myc gene amplification and overexpression, a phenomenon frequently observed in neuroblastoma and an important independent negative marker. Moreover, neuroblastoma cells are frequently characterized by increased levels of survivin, an inhibitor of the apoptotic response, and by a deficiency of procaspase 8, a key intermediate of the programmed cell death cascade. These two events, probably, make neuroblastomas more resistant to programmed cell death. These recent findings might suggest that neuroblastoma cells have acquired the capability to proliferate easily and die difficultly. The mechanistic meaning of these data will be discussed in the present review. Moreover, we will suggest new therapeutic scenarios opened up by the described alterations of cell cycle and apoptosis engines.

SPARC Overexpression Inhibits Cell Proliferation in Neuroblastoma and Is Partly Mediated by Tumor Suppressor Protein PTEN and AKT

PubMed Central

Bhoopathi, Praveen; Gorantla, Bharathi; Sailaja, G. S.; Gondi, Christopher S.; Gujrati, Meena; Klopfenstein, Jeffrey D.; Rao, Jasti S.

2012-01-01

Secreted protein acidic and rich in cysteine (SPARC) is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell–cell and cell–matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo. PMID:22567126

Targeting gastrin-releasing peptide as a new approach to treat aggressive refractory neuroblastomas.

PubMed

Paul, Pritha; Gillory, Lauren A; Kang, JungHee; Qiao, Jingbo; Chung, Dai H

2011-03-01

The overall survival for neuroblastoma remains dismal, in part due to the emergence of resistance to chemotherapeutic drugs. We have demonstrated that gastrin-releasing peptide (GRP), a gut peptide secreted by neuroblastoma, acts as an autocrine growth factor. We hypothesized that knockdown of GRP will induce apoptosis in neuroblastoma cells and potentiate the cytotoxic effects of chemotherapeutic agents. The human neuroblastoma cell lines (JF, SK-N-SH) were transfected with small interfering (si) RNA targeted at GRP. Apoptosis was assessed by DNA fragmentation assay. Immunoblotting was used to confirm molecular markers of apoptosis, and flow cytometry was performed to determine cell cycle arrest after GRP knockdown. siGRP resulted in an increase in apoptosis in the absence of chemotherapeutic interventions. A combination of GRP silencing and chemotherapeutic drugs resulted in enhanced apoptosis when compared to either of the treatments alone. GRP silencing led to increased expression of proapoptotic proteins, p53 and p21. Silencing of GRP induces apoptosis in neuroblastoma cells; it acts synergistically with chemotherapeutic effects of etoposide and vincristine. GRP knockdown-mediated apoptosis appears to be associated with upregulation of p53 in neuroblastoma cells. Targeting GRP may be postulated as a potential novel agent for combinational treatment to treat aggressive neuroblastomas. Copyright © 2011 Mosby, Inc. All rights reserved.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

PubMed

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells

PubMed Central

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

2016-01-01

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible. PMID:26942051

Customized oligonucleotide microarray gene expression-based classification of neuroblastoma patients outperforms current clinical risk stratification.

PubMed

Oberthuer, André; Berthold, Frank; Warnat, Patrick; Hero, Barbara; Kahlert, Yvonne; Spitz, Rüdiger; Ernestus, Karen; König, Rainer; Haas, Stefan; Eils, Roland; Schwab, Manfred; Brors, Benedikt; Westermann, Frank; Fischer, Matthias

2006-11-01

To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease. Two hundred fifty-one neuroblastoma specimens were analyzed using a customized oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifier's predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States. The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival [EFS] 0.86 +/- 0.03 [favorable; n = 115] v 0.52 +/- 0.07 [unfavorable; n = 59] and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P < .0001) and separated risk groups of current neuroblastoma trials into subgroups with divergent outcome (NB2004: low-risk 3-year EFS 0.86 +/- 0.04 v 0.25 +/- 0.15, P < .0001; intermediate-risk 1.00 v 0.57 +/- 0.19, P = .018; high-risk 0.81 +/- 0.10 v 0.56 +/- 0.08, P = .06). In a multivariate Cox regression model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 [95% CI, 2.544 to 8.893]). Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.

Targeting of the MYCN Protein with Small Molecule c-MYC Inhibitors

PubMed Central

Müller, Inga; Larsson, Karin; Frenzel, Anna; Oliynyk, Ganna; Zirath, Hanna; Prochownik, Edward V.; Westwood, Nicholas J.; Henriksson, Marie Arsenian

2014-01-01

Members of the MYC family are the most frequently deregulated oncogenes in human cancer and are often correlated with aggressive disease and/or poorly differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a poor prognosis, targeting MYCN using small molecule inhibitors could represent a promising therapeutic approach. We have previously demonstrated that the small molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma tumor models with MYCN overexpression. Our previous work also revealed that MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of lipid droplets in neuroblastoma cells. To expand our understanding of how small molecules interfere with MYCN, we have now analyzed the direct binding of 10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon resonance. Using a proximity ligation assay, we found reduced interaction between MYCN and MAX after treatment with all molecules except for the 10058-F4 metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 10058-F4 were the most efficient in inducing neuronal differentiation and lipid accumulation in MYCN-amplified neuroblastoma cells. Together our data demonstrate MYCN-binding properties for a selection of small molecules, and provide functional information that could be of importance for future development of targeted therapies against MYCN-amplified neuroblastoma. PMID:24859015

Human Xenografts Are Not Rejected in a Naturally Occurring Immunodeficient Porcine Line: A Human Tumor Model in Pigs

PubMed Central

Basel, Matthew T.; Balivada, Sivasai; Beck, Amanda P.; Kerrigan, Maureen A.; Pyle, Marla M.; Dekkers, Jack C.M.; Wyatt, Carol R.; Rowland, Robert R.R.; Anderson, David E.; Bossmann, Stefan H.

2012-01-01

Abstract Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments; however, therapies tested in such models often fail to translate into clinical settings. Therefore, a better preclinical model for cancer treatment testing is needed. Here we demonstrate that an immunodeficient line of pigs can host and support the growth of xenografted human tumors and has the potential to be an effective animal model for cancer therapy. Wild-type and immunodeficient pigs were injected subcutaneously in the left ear with human melanoma cells (A375SM cells) and in the right ear with human pancreatic carcinoma cells (PANC-1). All immunodeficient pigs developed tumors that were verified by histology and immunohistochemistry. Nonaffected littermates did not develop tumors. Immunodeficient pigs, which do not reject xenografted human tumors, have the potential to become an extremely useful animal model for cancer therapy because of their similarity in size, anatomy, and physiology to humans. PMID:23514746

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

PubMed

Matossian, Margarite D; Burks, Hope E; Bowles, Annie C; Elliott, Steven; Hoang, Van T; Sabol, Rachel A; Pashos, Nicholas C; O'Donnell, Benjamen; Miller, Kristin S; Wahba, Bahia M; Bunnell, Bruce A; Moroz, Krzysztof; Zea, Arnold H; Jones, Steven D; Ochoa, Augusto C; Al-Khami, Amir A; Hossain, Fokhrul; Riker, Adam I; Rhodes, Lyndsay V; Martin, Elizabeth C; Miele, Lucio; Burow, Matthew E; Collins-Burow, Bridgette M

2018-06-01

Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Heterotypic mouse models of canine osteosarcoma recapitulate tumor heterogeneity and biological behavior

PubMed Central

Tomiyasu, Hirotaka; Garbe, John R.; Cornax, Ingrid; Amaya, Clarissa; O'Sullivan, M. Gerard; Subramanian, Subbaya

2016-01-01

ABSTRACT Osteosarcoma (OS) is a heterogeneous and rare disease with a disproportionate impact because it mainly affects children and adolescents. Lamentably, more than half of patients with OS succumb to metastatic disease. Clarification of the etiology of the disease, development of better strategies to manage progression, and methods to guide personalized treatments are among the unmet health needs for OS patients. Progress in managing the disease has been hindered by the extreme heterogeneity of OS; thus, better models that accurately recapitulate the natural heterogeneity of the disease are needed. For this study, we used cell lines derived from two spontaneous canine OS tumors with distinctly different biological behavior (OS-1 and OS-2) for heterotypic in vivo modeling that recapitulates the heterogeneous biology and behavior of this disease. Both cell lines demonstrated stability of the transcriptome when grown as orthotopic xenografts in athymic nude mice. Consistent with the behavior of the original tumors, OS-2 xenografts grew more rapidly at the primary site and had greater propensity to disseminate to lung and establish microscopic metastasis. Moreover, OS-2 promoted formation of a different tumor-associated stromal environment than OS-1 xenografts. OS-2-derived tumors comprised a larger percentage of the xenograft tumors than OS-1-derived tumors. In addition, a robust pro-inflammatory population dominated the stromal cell infiltrates in OS-2 xenografts, whereas a mesenchymal population with a gene signature reflecting myogenic signaling dominated those in the OS-1 xenografts. Our studies show that canine OS cell lines maintain intrinsic features of the tumors from which they were derived and recapitulate the heterogeneous biology and behavior of bone cancer in mouse models. This system provides a resource to understand essential interactions between tumor cells and the stromal environment that drive the progression and metastatic propensity of OS. PMID:27874835

In vivo bioluminescence imaging using orthotopic xenografts towards patient's derived-xenograft Medulloblastoma models.

PubMed

Asadzadeh, Fatemeh; Ferrucci, Veronica; DE Antonellis, Pasqualino; Zollo, Massimo

2017-03-01

Medulloblastoma is a cerebellar neoplasia of the central nervous system. Four molecular subgrups have been identified (MBWNT, MBSHH, MBgroup3 and MBgroup4) with distinct genetics and clinical outcome. Among these, MBgroup3-4 are highly metastatic with the worst prognosis. The current standard therapy includes surgery, radiation and chemotherapy. Thus, specific treatments adapted to cure those different molecular subgroups are needed. The use of orthotopic xenograft models, together with the non-invasive in vivo biolumiscence imaging (BLI) technology, is emerging during preclinical studies to test novel therapeutics for medulloblastoma treatment. Orthotopic MB xenografts were performed by injection of Daoy-luc cells, that had been previously infected with lentiviral particles to stably express luciferase gene, into the fourth right ventricle of the cerebellum of ten nude mice. For the implantation, specific stereotactic coordinates were used. Seven days after the implantation the mice were imaged by acquisitions of bioluminescence imaging (BLI) using IVIS 3D Illumina Imaging System (Xenogen). Tumor growth was evaluated by quantifying the bioluminescence signals using the integrated fluxes of photons within each area of interest using the Living Images Software Package 3.2 (Xenogen-Perkin Elmer). Finally, histological analysis using hematoxylin-eosin staining was performed to confirm the presence of tumorigenic cells into the cerebellum of the mice. We describe a method to use the in vivo bioluminescent imaging (BLI) showing the potential to be used to investigate the potential antitumorigenic effects of a drug for in vivo medulloblastoma treatment. We also discuss other studies in which this technology has been applied to obtain a more comprehensive knowledge of medulloblastoma using orthotopic xenograft mouse models. There is a need to develop patient's derived-xenograft (PDX) model systems to test novel drugs for medulloblastoma treatment within each molecular sub-groups with a higher predictive value. Here we show how this technology should be applied with hopes on generations of new treatments to be applied then in human.

Heterotypic mouse models of canine osteosarcoma recapitulate tumor heterogeneity and biological behavior.

PubMed

Scott, Milcah C; Tomiyasu, Hirotaka; Garbe, John R; Cornax, Ingrid; Amaya, Clarissa; O'Sullivan, M Gerard; Subramanian, Subbaya; Bryan, Brad A; Modiano, Jaime F

2016-12-01

Osteosarcoma (OS) is a heterogeneous and rare disease with a disproportionate impact because it mainly affects children and adolescents. Lamentably, more than half of patients with OS succumb to metastatic disease. Clarification of the etiology of the disease, development of better strategies to manage progression, and methods to guide personalized treatments are among the unmet health needs for OS patients. Progress in managing the disease has been hindered by the extreme heterogeneity of OS; thus, better models that accurately recapitulate the natural heterogeneity of the disease are needed. For this study, we used cell lines derived from two spontaneous canine OS tumors with distinctly different biological behavior (OS-1 and OS-2) for heterotypic in vivo modeling that recapitulates the heterogeneous biology and behavior of this disease. Both cell lines demonstrated stability of the transcriptome when grown as orthotopic xenografts in athymic nude mice. Consistent with the behavior of the original tumors, OS-2 xenografts grew more rapidly at the primary site and had greater propensity to disseminate to lung and establish microscopic metastasis. Moreover, OS-2 promoted formation of a different tumor-associated stromal environment than OS-1 xenografts. OS-2-derived tumors comprised a larger percentage of the xenograft tumors than OS-1-derived tumors. In addition, a robust pro-inflammatory population dominated the stromal cell infiltrates in OS-2 xenografts, whereas a mesenchymal population with a gene signature reflecting myogenic signaling dominated those in the OS-1 xenografts. Our studies show that canine OS cell lines maintain intrinsic features of the tumors from which they were derived and recapitulate the heterogeneous biology and behavior of bone cancer in mouse models. This system provides a resource to understand essential interactions between tumor cells and the stromal environment that drive the progression and metastatic propensity of OS. © 2016. Published by The Company of Biologists Ltd.

Chronic anti-inflammatory drug therapy inhibits gel-forming mucin production in a murine xenograft model of human pseudomyxoma peritonei.

PubMed

Choudry, Haroon Asif; Mavanur, Arun; O'Malley, Mark E; Zeh, Herbert J; Guo, Z Sheng; Bartlett, David L

2012-05-01

Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP. The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays. Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count. Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.

Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model: in situ detection of 2-HG and α-KG

PubMed Central

2013-01-01

Background Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level. Results LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis. Conclusions The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations. PMID:24252742

Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model: in situ detection of 2-HG and α-KG.

PubMed

Navis, Anna C; Niclou, Simone P; Fack, Fred; Stieber, Daniel; van Lith, Sanne; Verrijp, Kiek; Wright, Alan; Stauber, Jonathan; Tops, Bastiaan; Otte-Holler, Irene; Wevers, Ron A; van Rooij, Arno; Pusch, Stefan; von Deimling, Andreas; Tigchelaar, Wikky; van Noorden, Cornelis J F; Wesseling, Pieter; Leenders, William P J

2013-05-29

Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level. LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis. The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations.

[Inhibitory effect of Biejiajian pills on HepG2 cell xenograft growth and expression of β-catenin and Tbx3 in nude mice].

PubMed

Wen, Bin; Sun, Hai-Tao; He, Song-Qi; LA, Lei; An, Hai-Yan; Pang, Jie

2016-02-01

To explore the molecular mechanism by which Biejiajian pills inhibit hepatocellular carcinoma in a nude mouse model bearing HepG2 cell xenograft. The inhibitory effect of Biejiajian pills on the growth of HepG2 cell xenograft in nude mice was observed. Immunohistochemical method was used to examine proliferating cell nuclear antigen (PCNA) expression in HepG2 cell xenograft, and TUNEL method was employed to detect the cell apoptosis; the expression levels of β-catenin and Tbx3 were measured by Western blotting. Biejiajian pills significantly suppressed the growth of HepG2 cell xenograft in nude mice. The tumor-bearing mice treated with a high and a moderate dose of Biejiajian pills showed significantly increased apoptosis rate of the tumor cells [(22.9±1.220)% and (14.7±0.50)%, respectively] compared with the control group [(5.5±0.90)%, P<0.05]. Treatment with Biejiajian pills significantly decreased the expressions of PNCA, β-catenin, and Tbx3 in the cell xenograft (P<0.05). Biejiajian pills can inhibit the growth of HepG2 cell xenograft in nude mice and promote tumor cell apoptosis possibly by inhibiting PNCA expression and the Wnt/β-catenin signaling pathway.

Neuroblastoma Screening (PDQ®)—Patient Version

Cancer.gov

Neuroblastoma screening does reduce the chance of dying from the disease. Not all screening tests are helpful and most have risks. Learn more about neuroblastoma and the potential benefits and harms of screening in this expert-reviewed summary.

Neuroblastoma Screening (PDQ®)—Health Professional Version

Cancer.gov

Neuroblastoma screening, according to solid evidence, does not lead to decreased mortality and exposes infants to potential serious harms. Get detailed information about neuroblastoma and the potential benefits and harms of screening in this summary for clinicians.

Infection of neuroblastoma cells by rabies virus is modulated by the virus titer.

PubMed

Fuoco, Natalia Langenfeld; Dos Ramos Silva, Sandriana; Fernandes, Elaine Raniero; Luiz, Fernanda Guedes; Ribeiro, Orlando Garcia; Katz, Iana Suly Santos

2018-01-01

Rabies is a lethal viral infection that can affect almost all mammals, including humans. To better understand the replication of Rabies lyssavirus, we investigated if the viral load in brains naturally infected with rabies influences viral internalization and viral growth kinetics in neuroblastoma cells, and if the viral load affects mortality in mice after intradermal infection. We noted that high initial viral loads in brains (group II) were unfavourable for increasing viral titers during serial passages in neuroblastoma cells when compared to low initial viral loads in brains (group I). In addition, group I strains showed higher viral growth and enhanced internalization efficiency in neuroblastoma cells than group II strains. However, we observed that the dominant virus subpopulation in group II promoted efficient viral infection in the central nervous system in the new host, providing a selective advantage to the virus. Our data indicate that rabies infection in animal models depends on not only the virus strain but also the amount of virus. This study may serve as a basis for understanding the biologic proprieties of Rabies lyssavirus strains with respect to the effects on viral replication and the impact on pathogenesis, improving virus yields for use in vaccine development. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Offerdahl, Danielle K.

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranesmore » to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60–100 nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20–30 nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. - Highlights: •First electron tomography of Zika virus cytoarchitecture. •Comparison of Zika virus infection in human neuroblastoma and mosquito cells. •Ultrastructure of Zika virus infection in human neuroblastoma and mosquito cells.« less

Environment-Mediated Drug Resistance in Neuroblastoma

DTIC Science & Technology

2014-10-01

AD_________________ Award Number: W81XWH-12-1-0572 TITLE: Environment-Mediated Drug Resistance in Neuroblastoma PRINCIPAL INVESTIGATOR: Yu...Resistance in Neuroblastoma 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0572 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Yu, Hua E 5d. PROJECT...collaborative experiments have demonstrated that monocytes collaborate with MSC in inducing STAT3-dependent drug resistance in neuroblastoma (Task 1), that S1P

Lack of associations between AURKA gene polymorphisms and neuroblastoma susceptibility in Chinese children.

PubMed

Tang, Jue; Qian, Yuanmin; Zhu, Jinhong; Zhang, Jiao; Wang, Feng-Hua; Zeng, Jia-Hang; Liang, Jiang-Hua; Wang, Hui; Xia, Huimin; He, Jing; Liu, Wei

2018-05-29

Previous studies have demonstrated that polymorphisms in the AURKA gene are associated with various types of cancer. In neuroblastoma, AURKA protein product regulates N-myc protein levels and plays a critical role in tumorigenesis. To investigate the association between three AURKA polymorphisms (rs1047972 C>T, rs2273535 T>A, and rs8173 G>C) and neuroblastoma susceptibility in Chinese populations, we performed this two-center case-control study including 393 neuroblastoma cases and 812 controls. Two study populations were recruited from two different regions in China. No significant associations were identified amongst any of the three AURKA polymorphisms and the risk of neuroblastoma. Similar observations were found in the stratified analysis. In conclusion, our results indicate that none of the AURKA polymorphisms are associated with neuroblastoma susceptibility in two distinct Chinese populations. Further studies with larger sample sizes and different ethnicities are warranted to validate our results. © 2018 The Author(s).

CASC15-S is a tumor suppressor lncRNA at the 6p22 neuroblastoma susceptibility locus

PubMed Central

Russell, Mike R.; Penikis, Annalise; Oldridge, Derek A.; Alvarez-Dominguez, Juan R.; McDaniel, Lee; Diamond, Maura; Padovan, Olivia; Raman, Pichai; Li, Yimei; Wei, Jun S.; Zhang, Shile; Gnanchandran, Janahan; Seeger, Robert; Asgharzadeh, Shahab; Khan, Javed; Diskin, Sharon J.; Maris, John M.; Cole, Kristina A.

2015-01-01

Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single nucleotide polymorphism (SNP) associations reside within CASC15, a long non-coding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression. PMID:26100672

Preclinical and clinical aspects on the use of amifostine as chemoprotector in neuroblastoma patients.

PubMed

Fulda, S; Fichtner, I; Hero, B; Berthold, F

2001-01-01

In several studies in adults, amifostine (WR-2721) and its active metabolite WR-1065 have shown protection against myelo- and nephrotoxicity of chemotherapeutic agents without compromising cytotoxic efficacy to the tumor. In the present study, the effect of amifostine and WR-1065 on neuroblastoma tumor growth and its protective potential for hematotoxicity were investigated. Neither amifostine nor WR-1065 reduced the cytotoxic effect of six drugs commonly used for this tumor when tested on neuroblastoma cells in vitro. In mice carrying human xeno-transplanted neuroblastoma, tumor growth and antitumor activity of chemotherapy were unaffected by amifostine. In addition, hematotoxicity of alkylators was relieved in some cases. In patients with neuroblastoma, amifostine only slightly reduced bone marrow toxicity and was highly emetogenic. Therefore, amifostine warrants further investigation before its widespread clinical use in the treatment of children with neuroblastoma.

Identification of the anti-tumor activity and mechanisms of nuciferine through a network pharmacology approach

PubMed Central

Qi, Quan; Li, Rui; Li, Hui-ying; Cao, Yu-bing; Bai, Ming; Fan, Xiao-jing; Wang, Shu-yan; Zhang, Bo; Li, Shao

2016-01-01

Aim: Nuciferine is an aporphine alkaloid extracted from lotus leaves, which is a raw material in Chinese medicinal herb for weight loss. In this study we used a network pharmacology approach to identify the anti-tumor activity of nuciferine and the underlying mechanisms. Methods: The pharmacological activities and mechanisms of nuciferine were identified through target profile prediction, clustering analysis and functional enrichment analysis using our traditional Chinese medicine (TCM) network pharmacology platform. The anti-tumor activity of nuciferine was validated by in vitro and in vivo experiments. The anti-tumor mechanisms of nuciferine were predicted through network target analysis and verified by in vitro experiments. Results: The nuciferine target profile was enriched with signaling pathways and biological functions, including “regulation of lipase activity”, “response to nicotine” and “regulation of cell proliferation”. Target profile clustering results suggested that nuciferine to exert anti-tumor effect. In experimental validation, nuciferine (0.8 mg/mL) markedly inhibited the viability of human neuroblastoma SY5Y cells and mouse colorectal cancer CT26 cells in vitro, and nuciferine (0.05 mg/mL) significantly suppressed the invasion of 6 cancer cell lines in vitro. Intraperitoneal injection of nuciferine (9.5 mg/mL, ip, 3 times a week for 3 weeks) significantly decreased the weight of SY5Y and CT26 tumor xenografts in nude mice. Network target analysis and experimental validation in SY5Y and CT26 cells showed that the anti-tumor effect of nuciferine was mediated through inhibiting the PI3K-AKT signaling pathway and IL-1 levels in SY5Y and CT26 cells. Conclusion: By using a TCM network pharmacology method, nuciferine is identified as an anti-tumor agent against human neuroblastoma and mouse colorectal cancer in vitro and in vivo, through inhibiting the PI3K-AKT signaling pathways and IL-1 levels. PMID:27180984

Biological effects of induced MYCN hyper-expression in MYCN-amplified neuroblastomas.

PubMed

Torres, Jaime; Regan, Paul L; Edo, Robby; Leonhardt, Payton; Jeng, Eric I; Rappaport, Eric F; Ikegaki, Naohiko; Tang, Xao X

2010-10-01

Neuroblastoma is a childhood malignancy of the sympathetic nervous system. The tumor exhibits two different phenotypes: favorable and unfavorable. MYCN amplification is associated with rapid tumor progression and the worst neuroblastoma disease outcome. We have previously reported that inhibitors of histone deacetylase (HDAC) and proteasome enhance favorable neuroblastoma gene expression in neuroblastoma cell lines and inhibit growth of these cells. In this study, we investigated the effect of trichostatin A or TSA (an HDAC inhibitor), and epoxomycin (a proteasome inhibitor) on MYCN and p53 expression in MYCN-amplified neuroblastoma cells. It was found that TSA down-regulated MYCN expression, but Epoxomycin and the TSA/Epoxomycin combination led to MYCN hyper-expression in MYCN-amplified neuroblastoma cell lines. Despite their contrasting effects on MYCN expression, TSA and Epoxomycin caused growth suppression and cell death of the MYCN-amplified cell lines examined. Consistent with these data, forced hyper-expression of MYCN in MYCN-amplified IMR5 cells via transfection resulted in growth suppression and the increased expression of several genes known to suppress growth or induce cell death. Furthermore, Epoxomycin as a single agent and its combination with TSA enhance p53 expression in the MYCN-amplified neuroblastoma cell lines. Unexpectedly, co-transfection of TP53 and MYCN in IMR5 cells resulted in high p53 expression but a reduction of MYCN expression. Together our data suggest that either down regulation or hyper-expression of MYCN results in growth inhibition and/or apoptosis of MYCN-amplified neuroblastoma cells. In addition, elevated p53 expression has a suppressive effect on MYCN expression in these cells.

Gastrin-releasing peptide-induced down-regulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) in neuroblastomas.

PubMed

Qiao, Jingbo; Kang, Junghee; Cree, Jeremy; Evers, B Mark; Chung, Dai H

2005-05-01

To evaluate whether aggressive, undifferentiated neuroblastomas express tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) and to examine the effects of gastrin-releasing peptide (GRP) on PTEN gene and protein expression. We have previously shown that neuroblastomas secrete GRP, which binds to its cell surface receptor (GRP-R) to stimulate cell growth in an autocrine fashion. However, the effects of GRP on expression of the tumor suppressor gene PTEN have not been elucidated in neuroblastomas. Paraffin-embedded sections from human neuroblastomas were analyzed for PTEN and phospho-Akt protein expression by immunohistochemistry. Human neuroblastoma cell lines (SK-N-SH and SH-SY5Y) were stably transfected with the plasmid pEGFP-GRP-R to establish GRP-R overexpression cell lines, and the effects of GRP on PTEN gene and protein expression were determined. A decrease in the ratio of PTEN to phospho-Akt protein expression was identified in poorly differentiated neuroblastomas. An increase in GRP binding capacity was confirmed in GRP-R overexpressing cells, which demonstrated an accelerated constitutive cell growth rate. PTEN gene and protein expression was significantly decreased in GRP-R overexpressing cells when compared with controls. Our findings demonstrate decreased expression of the tumor suppressor protein PTEN in more aggressive undifferentiated neuroblastomas. An increase in GRP binding capacity, as a result of GRP-R overexpression, down-regulates PTEN expression. These findings suggest that an inhibition of the tumor suppressor gene PTEN may be an important regulatory mechanism involved in GRP-induced cell proliferation in neuroblastomas.

Principal component analysis for the comparison of metabolic profiles from human rectal cancer biopsies and colorectal xenografts using high-resolution magic angle spinning 1H magnetic resonance spectroscopy

PubMed Central

Seierstad, Therese; Røe, Kathrine; Sitter, Beathe; Halgunset, Jostein; Flatmark, Kjersti; Ree, Anne H; Olsen, Dag Rune; Gribbestad, Ingrid S; Bathen, Tone F

2008-01-01

Background This study was conducted in order to elucidate metabolic differences between human rectal cancer biopsies and colorectal HT29, HCT116 and SW620 xenografts by using high-resolution magnetic angle spinning (MAS) magnetic resonance spectroscopy (MRS) and for determination of the most appropriate human rectal xenograft model for preclinical MR spectroscopy studies. A further aim was to investigate metabolic changes following irradiation of HT29 xenografts. Methods HR MAS MRS of tissue samples from xenografts and rectal biopsies were obtained with a Bruker Avance DRX600 spectrometer and analyzed using principal component analysis (PCA) and partial least square (PLS) regression analysis. Results and conclusion HR MAS MRS enabled assignment of 27 metabolites. Score plots from PCA of spin-echo and single-pulse spectra revealed separate clusters of the different xenografts and rectal biopsies, reflecting underlying differences in metabolite composition. The loading profile indicated that clustering was mainly based on differences in relative amounts of lipids, lactate and choline-containing compounds, with HT29 exhibiting the metabolic profile most similar to human rectal cancers tissue. Due to high necrotic fractions in the HT29 xenografts, radiation-induced changes were not detected when comparing spectra from untreated and irradiated HT29 xenografts. However, PLS calibration relating spectral data to the necrotic fraction revealed a significant correlation, indicating that necrotic fraction can be assessed from the MR spectra. PMID:18439252

Patient-derived tumor xenografts of lung squamous cell carcinoma alter long non-coding RNA profile but not responsiveness to cisplatin.

PubMed

Lu, Dapeng; Luo, Peng; Zhang, Ju; Ye, Yuanyuan; Wang, Qi; Li, Ming; Zhou, Hangcheng; Xie, Mingran; Wang, Baolong

2018-06-01

Lung squamous cell carcinoma (LSCC), the second most common type of lung cancer, has received limited attention. Patient-derived tumor xenografts (PDTXs) are useful preclinical models to reproduce the diverse heterogeneity of cancer, but it is important to identify potential variations during their establishment. A total of 18 PDTXs were established from 37 the surgical specimens and 16 were serially passaged to third generation. Second- and third-generation xenografts had a faster growth rate in mice. The tumor implantation success rate was associated with poorer differentiation, larger tumor volume and higher expression of Ki-67. The xenografts largely retained histological and key immunophenotypic features (including p53, p63, cytokeratin5/6, and E-cadherin). However, increased Ki-67 expression was identified in partial xenografts. Long non-coding RNA (lncRNA) and mRNA expression in third-generation xenografts differed from that of matched primary tumors. Gene Ontology and pathway analysis showed that mRNAs involved in cell cycle, and metabolism regulation were generally upregulated in xenografts, while those associated with immune responses were typically downregulated. Furthermore, the responses of xenografts to cisplatin were consistent with clinical outcome. In the present study, PDTXs of SCC were successfully established, and closely resembled their original tumor regarding their immunophenotype and response to cisplatin. Overall, PDTXS of LSCC altered the lncRNA profile and increased the proliferative activity of cancer cells, whilst retaining responsiveness to cisplatin.

Transplantation of Tissue-Engineered Cartilage in an Animal Model (Xenograft and Autograft): Construct Validation.

PubMed

Nemoto, Hitoshi; Watson, Deborah; Masuda, Koichi

2015-01-01

Tissue engineering holds great promise for cartilage repair with minimal donor-site morbidity. The in vivo maturation of a tissue-engineered construct can be tested in the subcutaneous tissues of the same species for autografts or of immunocompromised animals for allografts or xenografts. This section describes detailed protocols for the surgical transplantation of a tissue-engineered construct into an animal model to assess construct validity.

Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model

NASA Astrophysics Data System (ADS)

Lwin, Thinzar M.; Miyake, Kentaro; Murakami, Takashi; DeLong, Jonathan C.; Yazaki, Paul J.; Shivley, John E.; Clary, Bryan; Hoffman, Robert M.; Bouvet, Michael

2018-03-01

Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.

Neuroblastoma Treatment (PDQ®)—Health Professional Version

Cancer.gov

Neuroblastoma treatment generally is based on whether the tumor is low, intermediate, or high risk. Treatment options include surgery, observation, radiation therapy, chemotherapy, stem cell rescue, and targeted therapy. Get detailed neuroblastoma treatment information in this summary for clinicians.

Human tumor xenografts in mouse as a model for evaluating therapeutic efficacy of monoclonal antibodies or antibody-drug conjugate targeting receptor tyrosine kinases.

PubMed

Feng, Liang; Wang, Wei; Yao, Hang-Ping; Zhou, Jianwei; Zhang, Ruiwen; Wang, Ming-Hai

2015-01-01

Targeting receptor tyrosine kinases by therapeutic monoclonal antibodies and antibody-drug conjugates has met with tremendous success in clinical oncology. Currently, numerous therapeutic monoclonal antibodies are under preclinical development. The potential for moving candidate antibodies into clinical trials relies heavily on therapeutic efficacy validated by human tumor xenografts in mice. Here we describe methods used to determine therapeutic efficacy of monoclonal antibodies or antibody-drug conjugates specific to human receptor tyrosine kinase using human tumor xenografts in mice as the model. The end point of the study is to determine whether treatment of tumor-bearing mice with a monoclonal antibody or antibody-drug conjugates results in significant delay of tumor growth.

Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen.

PubMed

Michaelis, Martin; Agha, Bishr; Rothweiler, Florian; Löschmann, Nadine; Voges, Yvonne; Mittelbronn, Michel; Starzetz, Tatjana; Harter, Patrick N; Abhari, Behnaz A; Fulda, Simone; Westermann, Frank; Riecken, Kristoffer; Spek, Silvia; Langer, Klaus; Wiese, Michael; Dirks, Wilhelm G; Zehner, Richard; Cinatl, Jaroslav; Wass, Mark N; Cinatl, Jindrich

2015-02-03

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.

Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen

PubMed Central

Michaelis, Martin; Agha, Bishr; Rothweiler, Florian; Löschmann, Nadine; Voges, Yvonne; Mittelbronn, Michel; Starzetz, Tatjana; Harter, Patrick N.; Abhari, Behnaz A.; Fulda, Simone; Westermann, Frank; Riecken, Kristoffer; Spek, Silvia; Langer, Klaus; Wiese, Michael; Dirks, Wilhelm G.; Zehner, Richard; Cinatl, Jaroslav; Wass, Mark N.; Cinatl, Jindrich

2015-01-01

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells. PMID:25644037

Stephen L. Gans overseas lecture. Mass screening for neuroblastoma in Japan: lessons learned and future directions.

PubMed

Suita, Sachiyo

2002-07-01

Since 1985, a nationwide mass screening program (MS) for neuroblastoma has been conducted for 6-month-old infants throughout Japan, resulting in the detection of more than 1,900 cases of neuroblastoma. The outcome of these patients has been excellent: more than 97% of them are alive. Yet, several reports suggest that the number of advanced-stage neuroblastoma patients over 1 year of age has not changed substantially. The current report focuses on the 15-year experience with MS of the Kyushu Pediatric Oncology Study Group. The clinical and biological features of neuroblastoms detected (n = 320) and not detected by MS (n = 245) were compared. Regional and national statistics for neuroblastoma before and after 1985 were analyzed using standard epidemiologic measures for the occurrence of disease. The majority of the MS-positive cases were biologically favorable and had an excellent outcome. In contrast, the majority of non-MS patients in whom neuroblastoma later developed had advanced-stage, unfavorable-prognosis tumors. The overall mortality rate of neuroblastoma in the Kyushu area was not improved by MS. The optimal time for screening is the point at which neuroblastomas regressing spontaneously can no longer be detected, but more aggressive disease can be found. A birth cohort study could determine the optimal timing for a second screening. Identification of other new prognostic factors may be required. Copyright 2002, Elsevier Science (USA). All rights reserved.

Survival of high-risk pediatric neuroblastoma patients in a developing country.

PubMed

Easton, Joseph C; Gomez, Sergio; Asdahl, Peter H; Conner, J Michael; Fynn, Alcira B; Ruiz, Claudia; Ojha, Rohit P

2016-09-01

Little information is available about survival of high-risk pediatric neuroblastoma patients in developing countries. We aimed to assess survival among high-risk pediatric neuroblastoma patients in La Plata, Argentina. Individuals eligible for our cohort were aged <20 yr when diagnosed with high-risk neuroblastoma and received cancer-directed therapy including stem cell transplantation at Hospital de Niños Sor Maria Ludovica between February 1999 and February 2015. We estimated overall survival probabilities using an extended Kaplan-Meier approach. Our study population comprised 39 high-risk neuroblastoma patients, of whom 39% were aged >4 yr at diagnosis, 54% were male, and 62% had adrenal neuroblastoma. We observed 18 deaths, and the median survival time of our study population was 1.7 yr. The five-yr overall survival probability was 24% (95% CL: 10%, 41%). In contrast, five-yr survival of high-risk neuroblastoma patients ranges between 23% and 76% in developed countries. Survival among high-risk neuroblastoma patients is generally poor regardless of geographic location, but our results illustrate dramatically worse survival for patients in a developing country. We speculate that the observed survival differences could be attenuated or eliminated with improvements in treatment and supportive care, but addressing these issues will require creative solutions because of resource limitations. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma

PubMed Central

Carpenter, EL; Haglund, EA; Mace, EM; Deng, D; Martinez, D; Wood, AC; Chow, AK; Weiser, DA; Belcastro, LT; Winter, C; Bresler, SC; Asgharzadeh, S; Seeger, RC; Zhao, H; Guo, R; Christensen, JG; Orange, JS; Pawel, BR; Lemmon, MA; Mossé, YP

2013-01-01

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies–as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK. PMID:22266870

Synthetic high-density lipoprotein nanoconjugate targets neuroblastoma stem cells, blocking migration and self-renewal.

PubMed

Subramanian, Chitra; White, Peter T; Kuai, Rui; Kalidindi, Avinaash; Castle, Valerie P; Moon, James J; Timmermann, Barbara N; Schwendeman, Anna; Cohen, Mark S

2018-05-09

Pathways critical for neuroblastoma cancer stem cell function are targeted by 4,19,27-triacetyl withalongolide A (WGA-TA). Because neuroblastoma cells and their cancer stem cells highly overexpress the scavenger receptor class B type 1 receptor that binds to synthetic high-density lipoprotein, we hypothesized that a novel mimetic synthetic high-density lipoprotein nanoparticle would be an ideal carrier for the delivery of 4,19,27-triacetyl withalongolide to neuroblastoma and neuroblastoma cancer stem cells. Expression of scavenger receptor class B type 1 in validated human neuroblastoma cells was evaluated by quantitative polymerase chain reaction (qPCR) and Western blot. In vitro cellular uptake of synthetic high-density lipoprotein nanoparticles was observed with a fluorescence microscope. In vivo biodistribution of synthetic high-density lipoprotein nanoparticles was investigated with IVIS imaging. Self-renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Viability was analyzed by CellTiter-Glo assay. Cancer stem cell markers were evaluated by flow cytometry. qPCR and Western blot analysis revealed a higher level of scavenger receptor class B type 1 expression and drug uptake in N-myc amplified neuroblastoma cells. In vitro uptake of synthetic high-density lipoprotein was almost completely blocked by excess synthetic high-density lipoprotein. The synthetic high-density lipoprotein nanoparticles mainly accumulated in the tumor and liver, but not in other organs. Synthetic HDL-4,19,27-triacetyl withalongolide showed a 1,000-fold higher potency than the carrier (synthetic high-density lipoprotein) alone (P < .01) to kill neuroblastoma cells. Additionally, a dose-dependent decrease in sphere formation, invasion, migration, and cancer stem cell markers was observed after treatment of neuroblastoma cells with synthetic high-density lipoprotein-4,19,27-triacetyl withalongolide A. Synthetic high-density lipoprotein is a promising platform to improve the delivery of anticancer drug 4,19,27-triacetyl withalongolide A to neuroblastomas and neuroblastoma cancer stem cells through SR-B1 targeting in vitro and in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

Neuroblastoma Treatment (PDQ®)—Health Professional Version

Cancer.gov

Neuroblastoma treatment depends on the assigned risk category (low, intermediate, high, stage 4S). Get detailed information about the genomic/biologic features, presentation, diagnosis/staging, risk groups, prognosis and treatment of newly diagnosed and recurrent neuroblastoma in this summary for clinicians.

Rapid in vivo testing of drug response in multiple myeloma made possible by xenograft to turkey embryos

PubMed Central

Farnoushi, Y; Cipok, M; Kay, S; Jan, H; Ohana, A; Naparstek, E; Goldstein, R S; Deutsch, V R

2011-01-01

Background: The best current xenograft model of multiple myeloma (MM) in immune-deficient non-obese diabetic/severe-combined immunodeficient mice is costly, animal maintenance is complex and several weeks are required to establish engraftment and study drug efficacy. More practical in vivo models may reduce time and drug development cost. We recently described a rapid low-cost xenograft model of human blood malignancies in pre-immune turkey. Here, we report application of this system for studying MM growth and the preclinical assessment of anticancer therapies. Methods: Cell lines and MM patient cells were injected intravenously into embryonic veins on embryonic day 11 (E11). Engraftment of human cells in haematopoietic organs was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry and circulating free light chain. Results: Engraftment was detected after 1 week in all embryos injected with cell lines and in 50% of those injected with patient cells. Injection of bortezomib or lenalinomide 48 h after cell injection at therapeutic levels that were not toxic to the bone marrow dramatically reduced MM engraftment. Conclusion: The turkey embryo provides a practical, xenograft system to study MM and demonstrates the utility of this model for rapid and affordable testing therapeutics in vivo. With further development, this model may enable rapid, inexpensive personalised drug screening. PMID:22045188

FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

PubMed Central

Pirou, Caroline; Montazer-Torbati, Fatemeh; Jah, Nadège; Delmas, Elisabeth; Lasbleiz, Christelle; Mignotte, Bernard; Renaud, Flore

2017-01-01

Neuroblastoma, a sympathetic nervous system tumor, accounts for 15% of cancer deaths in children. In contrast to most human tumors, p53 is rarely mutated in human primary neuroblastoma, suggesting impaired p53 activation in neuroblastoma. Various studies have shown correlations between fgf1 expression levels and both prognosis severity and tumor chemoresistance. As we previously showed that fibroblast growth factor 1 (FGF1) inhibited p53-dependent apoptosis in neuron-like PC12 cells, we initiated the study of the interaction between the FGF1 and p53 pathways in neuroblastoma. We focused on the activity of either extracellular FGF1 by adding recombinant rFGF1 in media, or of intracellular FGF1 by overexpression in human SH-SY5Y and mouse N2a neuroblastoma cell lines. In both cell lines, the genotoxic drug etoposide induced a classical mitochondrial p53-dependent apoptosis. FGF1 was able to inhibit p53-dependent apoptosis upstream of mitochondrial events in SH-SY5Y cells by both extracellular and intracellular pathways. Both rFGF1 addition and etoposide treatment increased fgf1 expression in SH-SY5Y cells. Conversely, rFGF1 or overexpressed FGF1 had no effect on p53-dependent apoptosis and fgf1 expression in neuroblastoma N2a cells. Using different FGF1 mutants (that is, FGF1K132E, FGF1S130A and FGF1S130D), we further showed that the C-terminal domain and phosphorylation of FGF1 regulate its intracrine anti-apoptotic activity in neuroblastoma SH-SY5Y cells. This study provides the first evidence for a role of an intracrine growth factor pathway on p53-dependent apoptosis in neuroblastoma, and could lead to the identification of key regulators involved in neuroblastoma tumor progression and chemoresistance. PMID:29048426

Iodine-131 metaiodobenzylguanidine therapy for neuroblastoma: reports so far and future perspective.

PubMed

Kayano, Daiki; Kinuya, Seigo

2015-01-01

Neuroblastoma, which derives from neural crest, is the most common extracranial solid cancer in childhood. The tumors express the norepinephrine (NE) transporters on their cell membrane and take in metaiodobenzylguanidine (MIBG) via a NE transporter. Since iodine-131 (I-131) MIBG therapy was firstly reported, many trails of MIBG therapy in patients with neuroblastoma were performed. Though monotherapy with a low dose of I-131 MIBG could achieve high-probability pain reduction, the objective response was poor. In contrast, more than 12 mCi/kg I-131 MIBG administrations with or without hematopoietic cell transplantation (HCT) obtain relatively good responses in patients with refractory or relapsed neuroblastoma. The combination therapy with I-131 MIBG and other modalities such as nonmyeloablative chemotherapy and myeloablative chemotherapy with HCT improved the therapeutic response in patients with refractory or relapsed neuroblastoma. In addition, I-131 MIBG therapy incorporated in the induction therapy was proved to be feasible in patients with newly diagnosed neuroblastoma. To expand more the use of MIBG therapy for neuroblastoma, further studies will be needed especially in the use at an earlier stage from diagnosis, in the use with other radionuclide formations of MIBG, and in combined use with other therapeutic agents.

Antitumor Effect of Burchellin Derivatives Against Neuroblastoma.

PubMed

Kurita, Masahiro; Takada, Tomomi; Wakabayashi, Noriko; Asami, Satoru; Ono, Shinichi; Uchiyama, Taketo; Suzuki, Takashi

2018-02-01

Neuroblastoma is one of the most commonly encountered malignant solid tumors in the pediatric age group. We examined the antitumor effects of five burchellin derivatives against human neuroblastoma cell lines. We evaluated cytotoxicity by the MTT assay for four human neuroblastoma and two normal cell lines. We also performed analysis of the apoptotic induction effect by flow cytometry, and examined the expression levels of apoptosis- and cell growth-related proteins by western blot analysis. We found that one of the burchellin derivatives (compound 4 ) exerted cytotoxicity against the neuroblastoma cell lines. Compound 4 induced caspase-dependent apoptosis via a mitochondrial pathway. The apoptosis mechanisms induced by compound 4 involved caspase-3, -7 and -9 activation and poly (ADP-ribose) polymerase cleavage. In addition, compound 4 induced cell death through inhibition of the cell growth pathway (via extracellular signal-regulated kinase 1 and 2, AKT8 virus oncogene cellular homolog, and signal transducer and activator of transcription 3). Compound 4 exerted cellular cytotoxicity against neuroblastoma cells via induction of caspase-dependent apoptosis, and may offer promise for further development as a useful drug for the treatment of advanced neuroblastoma. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Phosphoproteomics reveals ALK promote cell progress via RAS/ JNK pathway in neuroblastoma.

PubMed

Chen, Kai; Lv, Fan; Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

2016-11-15

Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500-and quantitatively analyzed approximately 10,000-phosphorylation sites from each cell line, ultimately detecting 450-790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma.

Inhibition of H3K9 Methyltransferase G9a Repressed Cell Proliferation and Induced Autophagy in Neuroblastoma Cells

PubMed Central

Ke, Xiao-Xue; Zhang, Dunke; Zhu, Shunqin; Xia, Qingyou; Xiang, Zhonghuai; Cui, Hongjuan

2014-01-01

Histone methylation plays an important role in gene transcription and chromatin organization and is linked to the silencing of a number of critical tumor suppressor genes in tumorigenesis. G9a is a histone methyltransferase (HMTase) for histone H3 lysine 9. In this study, we investigated the role of G9a in neuroblastoma tumor growth together with the G9a inhibitor BIX01294. The exposure of neuroblastoma cells to BIX01294 resulted in the inhibition of cell growth and proliferation, and BIX01294 treatment resulted in the inhibition of the tumorigenicity of neuroblastoma cells in NOD/SCID mice. Therefore, G9a may be a potential therapeutic target in neuroblastoma. Moreover, we found several specific characteristics of autophagy after BIX01294 treatment, including the appearance of membranous vacuoles and microtubule-associated protein light chain 3 (LC3B). Similar results were observed in G9a-knockdown cells. In conclusion, our results demonstrated that G9a is a prognostic marker in neuroblastoma, and revealed a potential role of G9a in regulating the autophagy signaling pathway in neuroblastoma. PMID:25198515

Evaluation of Cytarabine Against Ewing Sarcoma Xenografts by the Pediatric Preclinical Testing Program

PubMed Central

Houghton, Peter J.; Morton, Christopher L.; Kang, Min; Reynolds, C. Patrick; Billups, Catherine A.; Favours, Edward; Payne-Turner, Debbie; Tucker, Chandra; Smith, Malcolm A.

2015-01-01

Treatment with the nucleoside analog cytarabine has been shown to mimic changes in gene expression associated with down-regulation of the EWS-FLI1 oncogene in Ewing sarcoma cell lines, selectively inhibit their growth in vitro, and cause tumor regression in athymic nude mice. For this report cytarabine was studied in vitro against a panel of 23 pediatric cancer cell lines and in vivo against 6 Ewing sarcoma xenografts. Acute lymphoblastic leukemia cell lines were the most sensitive to cytarabine in vitro (median IC50 9 nM), while Ewing sarcoma cell lines showed intermediate sensitivity (median IC50 232 nM). Cytarabine at a dose of 150 mg/kg administered daily 5× failed to significantly inhibit growth of five xenograft models, but reduced growth rate of the A673 xenograft by 50%. Cytarabine shows no differential in vitro activity against Ewing sarcoma cell lines and is ineffective in vivo against Ewing sarcoma xenografts at the dose and schedule studied. PMID:20979180

Neuroblastoma and MYCN

PubMed Central

Huang, Miller; Weiss, William A.

2013-01-01

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to originate from undifferentiated neural crest cells. Amplification of the MYC family member, MYCN, is found in ∼25% of cases and correlates with high-risk disease and poor prognosis. Currently, amplification of MYCN remains the best-characterized genetic marker of risk in neuroblastoma. This article reviews roles for MYCN in neuroblastoma and highlights recent identification of other driver mutations. Strategies to target MYCN at the level of protein stability and transcription are also reviewed. PMID:24086065

The use of an investigational radiopharmaceutical in neuroblastoma: A nursing perspective

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelly, J.U.

Children with advanced-stage neuroblastoma usually have a poor prognosis. While conventional treatment with surgery, chemotherapy, and radiation may provide some palliation, long-term survival is rare. A number of investigational therapies are being performed nationwide in an attempt to improve the prognosis for children with neuroblastoma. One such treatment is the use of {sup 131}I-metaiodobenzylguanidine. This article will review the pathophysiology of neuroblastoma, give an overview of this investigational treatment, and discuss the nursing care associated with radioactive treatment.

A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E.; Pertsemlidis, Alexander; Du, Liqin

2014-01-01

Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy. PMID:24811707

Treatment and outcome of adult-onset neuroblastoma.

PubMed

Suzuki, Maya; Kushner, Brian H; Kramer, Kim; Basu, Ellen M; Roberts, Stephen S; Hammond, William J; LaQuaglia, Michael P; Wolden, Suzanne L; Cheung, Nai-Kong V; Modak, Shakeel

2018-03-25

Adult-onset neuroblastoma is rare and little is known about its biology and clinical course. There is no established therapy for adult-onset neuroblastoma. Anti-GD2 immunotherapy is now standard therapy in children with high-risk neuroblastoma; however, its use has not been reported in adults. Forty-four adults (18-71 years old) diagnosed with neuroblastoma between 1979 and 2015 were treated at Memorial Sloan Kettering Cancer Center. Five, 1, 5 and 33 patients had INSS stage 1, 2, 3 and 4 diseases, respectively. Genetic abnormalities included somatic ATRX (58%) and ALK mutations (42%) but not MYCN-amplification. In the 11 patients with locoregional disease, 10-year progression-free (PFS) and overall survival (OS) was 35.4 ± 16.1% and 61.4 ± 15.3%, respectively. Among 33 adults with stage 4 neuroblastoma, 7 (21%) achieved complete response (CR) after induction chemotherapy and/or surgery. Seven patients with primary refractory neuroblastoma (all with osteomedullary but no soft tissue disease) received anti-GD2 antibodies, mouse or humanized 3F8. Antibody-related adverse events were similar to those in children, response rate being 71.4%. In patients with stage 4 disease at diagnosis, 5-year PFS was 9.7± 5.3% and most patients who were alive with disease at 5 years died of neuroblastoma over the next 5 years, 10-year OS being only 19.0 ± 8.2%. Patients who achieved CR after induction had superior PFS and OS (p = 0.006, p = 0.031, respectively). Adult-onset neuroblastoma appeared to have different biology from pediatric or adolescent NB, and poorer outcome. Complete disease control appeared to improve long-term survival. Anti-GD2 immunotherapy was well tolerated and might be beneficial. © 2018 UICC.

Congenital Adrenal Neuroblastoma With and Without Cystic Change: Differentiating Features With an Emphasis on the of Value of Ultrasound.

PubMed

Hwang, Sook Min; Yoo, So-Young; Kim, Ji Hye; Jeon, Tae Yeon

2016-11-01

The purpose of this study was to compare the features of congenital adrenal neuroblastomas with and without cystic changes and to emphasize the value of ultrasound in the diagnostic evaluation of cystic congenital adrenal neuroblastoma. A total of 41 patients with surgically confirmed congenital adrenal neuroblastoma were enrolled. We divided the patients into two groups according to presence or absence of cystic change in the tumor, as determined from the initial ultrasound findings. Clinical and laboratory findings, disease stage, and patient outcome were investigated with a statistical comparison between the two groups. The imaging findings for cystic congenital adrenal neuroblastoma were reviewed to compare the additional diagnostic value of CT and MRI when paired with ultrasound. There were 22 patients (54%) in the group without cystic changes and 19 patients (46%) in the group with cystic changes. Prenatal detection and absence of metastasis were significantly more common in the cystic group than in the noncystic group (p < 0.05). Sensitivities of tumor marker levels were also significantly lower in the cystic group. Patient outcome was excellent, and there was no significant difference between the groups. With regard to imaging of cystic congenital adrenal neuroblastoma, in the 15 cases in which CT or MRI was paired with ultrasound, no additional diagnostic information was discerned with CT or MRI. Nearly one-half of congenital adrenal neuroblastomas are cystic, and these tumors have clinical and laboratory features that distinguish them from noncystic congenital adrenal neuroblastoma. Diagnostic tests, including CT, MRI, and assessment of tumor markers, have low diagnostic value in the evaluation of cystic congenital adrenal neuroblastoma.

Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akter, Jesmin; Takatori, Atsushi, E-mail: atakatori@chiba-cc.jp; Islam, Md. Sazzadul

2014-10-10

Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus duringmore » ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.« less

Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas

PubMed Central

Westermann, Frank; Muth, Daniel; Benner, Axel; Bauer, Tobias; Henrich, Kai-Oliver; Oberthuer, André; Brors, Benedikt; Beissbarth, Tim; Vandesompele, Jo; Pattyn, Filip; Hero, Barbara; König, Rainer; Fischer, Matthias; Schwab, Manfred

2008-01-01

Background Amplified MYCN oncogene resulting in deregulated MYCN transcriptional activity is observed in 20% of neuroblastomas and identifies a highly aggressive subtype. In MYCN single-copy neuroblastomas, elevated MYCN mRNA and protein levels are paradoxically associated with a more favorable clinical phenotype, including disseminated tumors that subsequently regress spontaneously (stage 4s-non-amplified). In this study, we asked whether distinct transcriptional MYCN or c-MYC activities are associated with specific neuroblastoma phenotypes. Results We defined a core set of direct MYCN/c-MYC target genes by applying gene expression profiling and chromatin immunoprecipitation (ChIP, ChIP-chip) in neuroblastoma cells that allow conditional regulation of MYCN and c-MYC. Their transcript levels were analyzed in 251 primary neuroblastomas. Compared to localized-non-amplified neuroblastomas, MYCN/c-MYC target gene expression gradually increases from stage 4s-non-amplified through stage 4-non-amplified to MYCN amplified tumors. This was associated with MYCN activation in stage 4s-non-amplified and predominantly c-MYC activation in stage 4-non-amplified tumors. A defined set of MYCN/c-MYC target genes was induced in stage 4-non-amplified but not in stage 4s-non-amplified neuroblastomas. In line with this, high expression of a subset of MYCN/c-MYC target genes identifies a patient subtype with poor overall survival independent of the established risk markers amplified MYCN, disease stage, and age at diagnosis. Conclusions High MYCN/c-MYC target gene expression is a hallmark of malignant neuroblastoma progression, which is predominantly driven by c-MYC in stage 4-non-amplified tumors. In contrast, moderate MYCN function gain in stage 4s-non-amplified tumors induces only a restricted set of target genes that is still compatible with spontaneous regression. PMID:18851746

Integrated imaging using MRI and 123I metaiodobenzylguanidine scintigraphy to improve sensitivity and specificity in the diagnosis of pediatric neuroblastoma.

PubMed

Pfluger, Thomas; Schmied, Christoph; Porn, Ute; Leinsinger, Gerda; Vollmar, Christian; Dresel, Stefan; Schmid, Irene; Hahn, Klaus

2003-10-01

The objectives of this study were to compare MRI and iodine-123 ((123)I) metaiodobenzylguanidine (MIBG) scintigraphy in the detection of neuroblastoma lesions in pediatric patients and to assess the additional value of combined imaging. Fifty MRI and 50 (123)I MIBG examinations (mean interval, 6.4 days) were analyzed retrospectively with regard to suspected or proven neuroblastoma lesions (n = 193) in 28 patients. MRI and MIBG scans were reviewed by two independent observers each. Separate and combined analyses of MRI and MIBG scintigraphy were compared with clinical and histologic findings. With regard to the diagnosis of neuroblastoma lesion, MIBG scintigraphy, MRI, and combined analysis showed a sensitivity of 69%, 86%, and 99% and a specificity of 85%, 77%, and 95%, respectively. On MRI, 15 false-positive findings were recorded: posttherapeutic reactive changes (n = 10), benign adrenal tumors (n = 3), and enlarged lymph nodes (n = 2). On MIBG scintigraphy, 10 false-positive findings occurred: ganglioneuromas (n = 2), benign liver tumors (n = 2), and physiologic uptake (n = 6). Thirteen neuroblastoma metastases and two residual masses under treatment with chemotherapy were judged to be false-negative findings on MRI. Two primary or residual neuroblastomas and one orbital metastasis were misinterpreted as Wilms' tumor, reactive changes after surgery, and rhabdomyosarcoma on MRI. Thirty-two bone metastases, six other neuroblastoma metastases, and one adrenal neuroblastoma showed no MIBG uptake. On combined imaging, one false-negative (bone metastasis) and three false-positive (two ganglioneuromas and one pheochromocytoma) findings remained. In the assessment of neuroblastoma lesions in pediatric patients, MRI showed a higher sensitivity and MIBG scintigraphy a higher specificity. However, integrated imaging showed an increase in both sensitivity and specificity.

Convection-Enhanced Delivery (CED) in an Animal Model of Malignant Peripheral Nerve Sheath Tumors and Plexiform Neurofibromas

DTIC Science & Technology

2013-02-01

successfully establish the xenograft within the sciatic nerve. Convection-Enhanced Delivery ( CED ), Malignant Peripheral Nerve Sheath ( MPNST ), Plexiform...intraneural PNs and MPNST via CED . Design: Orthotopic xenograft models of sciatic intraneural NF1 MPNST and PNs in scid mice as described by Perrin et...using convection-enhanced delivery ( CED ). Relative Growth of MPNST cells in vivo treated with rapamycin, imatinib or erlotinib: Elotinib

miR-138 overexpression is more powerful than hTERT knockdown to potentiate apigenin for apoptosis in neuroblastoma in vitro and in vivo

PubMed Central

Chakrabarti, Mrinmay; Banik, Naren L.; Ray, Swapan K.

2013-01-01

Decrease in expression of the tumor suppressor microRNA-138 (miR-138) correlates well with an increase in telomerase activity in many human cancers. The ability of almost all human cancer cells to grow indefinitely is dependent on presence of telomerase activity. The catalytic component of human telomerase reverse transcriptase (hTERT) regulates telomerase activity in most of the human cancers including malignant neuroblastoma. We observed an indirect increase in the expression of miR-138 after the transfection with hTERT short hairpin RNA (shRNA) plasmid in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines. Transfection with hTERT shRNA plasmid followed by treatment with the flavonoid apigenin (APG) further increased expression of miR-138. Direct transfection with miR-138 mimic was more powerful than transfection with hTERT shRNA plasmid in potentiating efficacy of APG for decreasing cell viability and colony formation capability of both cell lines. Upregulation of miR-138 was also more effective than down regulation of hTERT in enhancing efficacy of APG for induction of apoptosis in malignant neuroblastoma cells in vitro and in vivo. We delineated that apoptosis occurred with induction of molecular components of the extrinsic and intrinsic pathways in SK-N-DZ and SK-N-BE2 cells both in vitro and in vivo. In conclusion, these results demonstrate that direct miR-138 overexpression is more powerful than hTERT down regulation in enhancing pro-apoptotic effect of APG for controlling growth of human malignant neuroblastoma in cell culture and animal models. PMID:23562653

Optimization of liposomal topotecan for use in treating neuroblastoma.

PubMed

Chernov, Lina; Deyell, Rebecca J; Anantha, Malathi; Dos Santos, Nancy; Gilabert-Oriol, Roger; Bally, Marcel B

2017-06-01

The purpose of this work was to develop an optimized liposomal formulation of topotecan for use in the treatment of patients with neuroblastoma. Drug exposure time studies were used to determine that topotecan (Hycamtin) exhibited great cytotoxic activity against SK-N-SH, IMR-32 and LAN-1 neuroblastoma human cell lines. Sphingomyelin (SM)/cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes were prepared using extrusion methods and then loaded with topotecan by pH gradient and copper-drug complexation. In vitro studies showed that SM/Chol liposomes retained topotecan significantly better than DSPC/Chol liposomes. Decreasing the drug-to-lipid ratio engendered significant increases in drug retention. Dose-range finding studies on NRG mice indicated that an optimized SM/Chol liposomal formulation of topotecan prepared with a final drug-to-lipid ratio of 0.025 (mol: mol) was better tolerated than the previously described DSPC/Chol topotecan formulation. Pharmacokinetic studies showed that the optimized SM/Chol liposomal topotecan exhibited a 10-fold increase in plasma half-life and a 1000-fold increase in AUC 0-24 h when compared with Hycamtin administered at equivalent doses (5 mg/kg). In contrast to the great extension in exposure time, SM/Chol liposomal topotecan increased the life span of mice with established LAN-1 neuroblastoma tumors only modestly in a subcutaneous and systemic model. The extension in exposure time may still not be sufficient and the formulation may require further optimization. In the future, liposomal topotecan will be assessed in combination with high-dose radiotherapy such as 131 I-metaiodobenzylguanidine, and immunotherapy treatment modalities currently used in neuroblastoma therapy. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Human nasal polyp microenvironments maintained in a viable and functional state as xenografts in NOD-scid IL2rgamma(null) mice.

PubMed

Bernstein, Joel M; Brooks, Stephen P; Lehman, Heather K; Pope, Liza; Sands, Amy; Shultz, Leonard D; Bankert, Richard B

2009-12-01

The objective was to develop a model with which to study the cellular and molecular events associated with nasal polyp progression. To accomplish this, we undertook to develop a system in which nondisrupted human nasal polyp tissue could be successfully implanted into severely immunocompromised mice, in which the histopathology of the original nasal polyp tissue, including inflammatory lymphocytes, epithelial and goblet cell hyperplasia, and subepithelial fibrosis, could be preserved for prolonged periods. Small, non-disrupted pieces of human nasal polyp tissues were subcutaneously implanted into NOD-scid IL2rgamma(null) mice. Xenografts at 8 to 12 weeks after implantation were examined histologically and immunohistochemically to identify human inflammatory leukocytes and to determine whether the characteristic histopathologic characteristics of the nasal polyps were maintained for a prolonged period. The xenografts, spleen, lung, liver, and kidneys were examined histologically and immunohistochemically and were evaluated for changes in volume. The sera of these mice were assayed for human cytokines and immunoglobulin. Xenografts of human nasal polyp tissues were established after their subcutaneous implantation into NOD-scid IL2rgamma(null) mice. The xenografts were maintained in a viable and functional state for up to 3 months, and retained a histopathologic appearance similar to that of the original tissue, with a noticeable increase in goblet cell hyperplasia and marked mucus accumulation in the submucosal glands compared to the original nasal polyp tissue. Inflammatory lymphocytes present in the polyp microenvironment were predominantly human CD8+ T cells with an effector memory phenotype. Human CD4+ T cells, CD138+ plasma cells, and CD68+ macrophages were also observed in the xenografts. Human immunoglobulin and interferon-gamma were detected in the sera of xenograft-bearing mice. The polyp-associated lymphocytes proliferated and were found to migrate from the xenografts to the spleens of the recipient mice, resulting in a significant splenomegaly. A progressive increase in the volume of the xenografts was observed with little or no evidence of mouse cell infiltration into the human leukocyte antigen-positive human tissue. An average twofold increase in polyp volume was found at 3 months after engraftment. The use of innate and adaptive immunodeficient NOD-scid mice homozygous for targeted mutations in the interleukin-2 receptor gamma-chain locus NOD-scid IL2rgamma(null) for establishing xenografts of nondisrupted pieces of human nasal polyp tissues represents a significant improvement over the previously reported xenograft model that used partially immunoincompetent CB17-scid mice as tissue recipients. The absence of the interleukin-2 receptor gamma-chain results in complete elimination of natural killer cell development, as well as severe impairments in T and B cell development. These mice, lacking both innate and adaptive immune responses, significantly improve upon the long-term engraftment of human nasal polyp tissues and provide a model with which to study how nasal polyp-associated lymphocytes and their secreted biologically active products contribute to the histopathology and progression of this chronic inflammatory disease.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

PubMed

Marklein, Diana; Graab, Ulrike; Naumann, Ivonne; Yan, Tiandong; Ridzewski, Rosalie; Nitzki, Frauke; Rosenberger, Albert; Dittmann, Kai; Wienands, Jürgen; Wojnowski, Leszek; Fulda, Simone; Hahn, Heidi

2012-01-01

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

PI3K Inhibition Enhances Doxorubicin-Induced Apoptosis in Sarcoma Cells

PubMed Central

Marklein, Diana; Graab, Ulrike; Naumann, Ivonne; Yan, Tiandong; Ridzewski, Rosalie; Nitzki, Frauke; Rosenberger, Albert; Dittmann, Kai; Wienands, Jürgen; Wojnowski, Leszek; Fulda, Simone; Hahn, Heidi

2012-01-01

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines. PMID:23300809

Unusual Differentiation to Pheochromocytoma-Like Cells in an Adrenal Neuroblastoma After Chemotherapy: A Case Report and Literature Review.

PubMed

Tatekawa, Yukihiro

2015-01-01

The authors present a case of 3-year-old female with Stage 4 neuroblastoma originating from the left adrenal gland. Biopsy of the left adrenal tumor showed neuroblastoma. After three courses of chemotherapy, the left adrenal gland including the left adrenal tumor was surgically removed. Pathological findings of the resected tumor revealed that most of the neuroblastoma tissues changed to pheochromocytoma-like cells. The tumor cells were arranged in well-defined nests surrounded by a delicate fibrovascular stroma and had granular eosinophilic cytoplasm, and round to oval nuclei. Immunohistological analysis of the biopsy samples showed strongly positive Ganglioside GD2-staining cells, whereas almost all of the tumor cells in the resected specimen were Ganglioside GD2-negative; cells were very weakly stained. The authors suggest that a part of the neuroblastoma in the left adrenal gland exhibited unusual differentiation toward pheochromocytic lineage Ganglioside GD2-negative neuroblastoma in a patient who had been treated with intensive chemotherapy.

Growth, progression and chromosome instability of Neuroblastoma: a new scenario of tumorigenesis?

PubMed

Tonini, Gian Paolo

2017-01-05

Neuroblastoma is a pediatric cancer with a low survival rate of patients with metastatic stage 4 disease. Tumor aggressiveness and progression have been associated with structural copy number variations (CNVs) that are observed in malignant cells. In contrast, localized Neuroblastomas, which are associated with a low number of structural CNVs but frequent numerical CNVs, are less aggressive, and patients have good outcomes. Finally, whole-genome and whole-exome sequencing of Neuroblastoma tissues have shown few damaging mutations in these tumors. In the present report it is proposed that chromosome instability (CIN) plays a major role in Neuroblastoma tumorigenesis and that CIN is already present in the early phases of tumor development. High CIN can promote several types of chromosomal damage including chromothripsis, gene deletion, amplification and rearrangements, which deregulate gene expression. Indeed, gene rearrangements have been reported as a new scenario in the development of Neuroblastoma, which supports the hypothesis that CIN is an early step preliminary to the late catastrophic events leading to tumor development.

Therapeutic Innovations for Targeting Childhood Neuroblastoma: Implications of the Neurokinin-1 Receptor System.

PubMed

Berger, Michael; VON Schweinitz, Dietrich

2017-11-01

Neuroblastoma is the most common solid extracranial malignant tumor in children. Despite recent advances in the treatment of this heterogenous tumor with surgery and chemotherapy, the prognosis in advanced stages remains poor. Interestingly, neuroblastoma is one of the few solid tumors, to date, in which an effect for targeted immunotherapy has been proven in controlled clinical trials, giving hope for further advances in the treatment of this and other tumors by targeted therapy. A large array of novel therapeutic options for targeted therapy of neuroblastoma is on the horizon. To this repεrtoirε, the neurokinin-1 receptor (NK1R) system was recently added. The present article explores the most recent developments in targeting neuroblastoma cells via the NK1R and how this new knowledge could be helpful to create new anticancer therapies agains neuroblastoma and other cancers. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Predicting survival times for neuroblastoma patients using RNA-seq expression profiles.

PubMed

Grimes, Tyler; Walker, Alejandro R; Datta, Susmita; Datta, Somnath

2018-05-30

Neuroblastoma is the most common tumor of early childhood and is notorious for its high variability in clinical presentation. Accurate prognosis has remained a challenge for many patients. In this study, expression profiles from RNA-sequencing are used to predict survival times directly. Several models are investigated using various annotation levels of expression profiles (genes, transcripts, and introns), and an ensemble predictor is proposed as a heuristic for combining these different profiles. The use of RNA-seq data is shown to improve accuracy in comparison to using clinical data alone for predicting overall survival times. Furthermore, clinically high-risk patients can be subclassified based on their predicted overall survival times. In this effort, the best performing model was the elastic net using both transcripts and introns together. This model separated patients into two groups with 2-year overall survival rates of 0.40±0.11 (n=22) versus 0.80±0.05 (n=68). The ensemble approach gave similar results, with groups 0.42±0.10 (n=25) versus 0.82±0.05 (n=65). This suggests that the ensemble is able to effectively combine the individual RNA-seq datasets. Using predicted survival times based on RNA-seq data can provide improved prognosis by subclassifying clinically high-risk neuroblastoma patients. This article was reviewed by Subharup Guha and Isabel Nepomuceno.

Copper Induces Apoptosis of Neuroblastoma Cells Via Post-translational Regulation of the Expression of Bcl-2-family Proteins and the tx Mouse is a Better Model of Hepatic than Brain Cu Toxicity.

PubMed

Chan, Hsien W; Liu, Tianbing; Verdile, Giuseppe; Bishop, Glenda; Haasl, Ryan J; Smith, Mark A; Perry, George; Martins, Ralph N; Atwood, Craig S

2008-01-01

The basic mechanism(s) by which altered Cu homeostasis is toxic to hepatocytes and neurons, the two major cell types affected in copper storage diseases such as Wilson's disease (WD), remain unclear. Using human M17 neuroblastoma cells as a model to examine Cu toxicity, we found that there was a time- and concentration-dependent induction of neuronal death, such that at 24 h there was a approximately 50 % reduction in viability with 25 muM Cu-glycine(2). Cu-glycine(2) (25:50 muM) treatment for 24 h significantly altered the expression of 296 genes, including 8 genes involved with apoptosis (BCL2-associated athanogene 3, BCL2/adenovirus E1B 19kDa interacting protein caspase 5, regulator of Fas-induced apoptosis, V-jun sarcoma virus 17 oncogene homolog, claudin 5, prostaglandin E receptor 3 and protein tyrosine phosphatase, non-receptor type 6). Surprisingly, changes in the expression of more 'traditional' apoptotic genes (Bcl-2, Bax, Bak and Bad) did not vary more than 20 %. To test whether the induction of apoptosis in neuroblastoma cells was via post-translational mechanisms, we measured the protein expression of these apoptotic markers in M17 neuroblastoma cells treated with Cu-glycine(2) (0-100 muM) for 24-48 h. Compared with glycine treated cells, Cu-glycine(2) reduced Bcl-2 expression by 50 %, but increased Bax and Bak expression by 130% and 400 %, respectively. To assess whether Cu also induced apoptotic cell death in a mouse model of WD, we measured the expression of these apoptotic markers in the liver and brain of mice expressing an ATP7b gene mutation (tx(J) mice) at 10 months of age (near the end of their lives when overt liver pathology is displayed). Changes in the liver expression of these apoptotic markers in tx(J) mice compared to background mice mirrored those of Cu treated neuroblastoma cells. In contrast, few changes in apoptotic protein expression were detected in the brain between tx(J) and background mice, indicating the tx(J) mouse is a good model of hepatic, but not brain, Cu toxicity. Our results indicate that Cu-induction of neuronal apoptosis does not require de novo synthesis or degradation of apoptotic genes, and that Cu accumulation in the aged tx(J) mouse brain is insufficient to induce apoptosis.

Favorable prognostic role of tropomodulins in neuroblastoma.

PubMed

Bettinsoli, Paola; Ferrari-Toninelli, Giulia; Bonini, Sara Anna; Guarienti, Michela; Cangelosi, Davide; Varesio, Luigi; Memo, Maurizio

2018-06-05

Neuroblastoma is a pediatric tumor of the sympatoadrenal lineage of the neural crest characterized by high molecular and clinical heterogeneity, which are the main causes of the poor response to standard multimodal therapy. The identification of new and selective biomarkers is important to improve our knowledge on the mechanisms of neuroblastoma progression and to find the targets for innovative cancer therapies. This study identifies a positive correlation among tropomodulins (TMODs) proteins expression and neuroblastoma progression. TMODs bind the pointed end of actin filaments, regulate polymerization and depolymerization processes modifying actin cytoskeletal dynamic and influencing neuronal development processes. Expression levels of TMODs genes were analyzed in 17 datasets comprising different types of tumors, including neuroblastoma, and it was demonstrated that high levels of tropomodulin1 ( TMOD1 ) and tropomodulin 2 ( TMOD2 ) correlate positively with high survival probability and with favorable clinical and molecular characteristics. Functional studies on neuroblastoma cell lines, showed that TMOD1 knockin induced cell cycle arrest, cell proliferation arrest and a mature functional differentiation. TMOD1 overexpression was responsible for particular cell morphology and biochemical changes which directed cells towards a neuronal favorable differentiation profile. TMOD1 downregulation also induced cell proliferation arrest but caused the loss of mature cell differentiation and promoted the development of neuroendocrine cellular characteristics, delineating an aggressive and unfavorable tumor behavior. Overall, these data indicated that TMODs are favorable prognostic biomarkers in neuroblastoma and we believe that they could contribute to unravel a new pathophysiological mechanism of neuroblastoma resistance contributing to the design of personalized therapeutics opportunities.

Improved therapy for neuroblastoma using a combination approach: superior efficacy with vismodegib and topotecan

PubMed Central

Chaturvedi, Nagendra K.; McGuire, Timothy R.; Coulter, Don W.; Shukla, Ashima; McIntyre, Erin M.; Sharp, John Graham; Joshi, Shantaram S.

2016-01-01

Aberrant activation/expression of pathways/molecules including NF-kB, mTOR, hedgehog and polo-like-kinase-1 (PLK1) are correlated with poor-prognosis neuroblastoma. Therefore, to identify a most efficacious treatment for neuroblastoma, we investigated the efficacy of NF-kB/mTOR dual-inhibitor 13-197, hedgehog inhibitor vismodegib and PLK1 inhibitor BI2536 alone or combined with topotecan against high-risk neuroblastoma. The in vitro efficacy of the inhibitors alone or combined with topotecan on cell growth/apoptosis and molecular mechanism(s) were investigated. Results showed that as single agents 13-197, BI2536 and vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways/molecules. In combination with topotecan, 13-197 did not show significant additive/synergistic effects against neuroblastoma. However, BI2536 or vismodegib further significantly decreased neuroblastoma cell growth/survival. These results clearly showed that vismodegib combination with topotecan was synergistic and more efficacious compared with BI2536 in combination. Together, in vitro data demonstrated that vismodegib was most efficacious in potentiating topotecan-induced antineuroblastoma effects. Therefore, we tested the combined efficacy of vismodegib and topotecan against neuroblastoma in vivo using NSG mice. This resulted in significantly (p<0.001) reduced tumor growth and increased survival of mice. Together, the combination of vismodegib and topotecan showed a significant enhanced antineuroblastoma efficacy by targeting associated pathways/molecules which warrants further preclinical evaluation for translation to the clinic. PMID:26934655

Suppression of miR-19b enhanced the cytotoxic effects of mTOR inhibitors in human neuroblastoma cells.

PubMed

Chen, Yun; Tsai, Ya-Hui; Tseng, Bor-Jiun; Pan, Hsin-Yen; Tseng, Sheng-Hong

2016-11-01

Mammalian target of rapamycin (mTOR) inhibitors exert significant antitumor effects on several cancer cell types. In this study, we investigated the effects of mTOR inhibitors, in particular the regulation of the microRNA, in neuroblastoma cells. AZD8055 (a new mTOR inhibitor)- or rapamycin-induced cytotoxic effects on neuroblastoma cells were studied. Western blotting was used to investigate the expression of various proteins in the mTOR pathway. MicroRNA precursors and antagomirs were transfected into cells to manipulate the expression of target microRNA. AZD8055 exerted stronger cytotoxic effects than rapamycin in neuroblastoma cells (p<0.03). In addition, AZD8055 suppressed the mTOR pathway and increased the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the neuroblastoma cells. AZD8055 significantly decreased miR-19b expression (p<0.005); in contrast, rapamycin increased miR-19b expression (p<0.05). Transfection of miR-19b antagomir into the neuroblastoma cells mimicked the effects of AZD8055 treatment, whereas miR-19b overexpression reversed the effects of AZD8055. Combination of miR-19b knockdown and rapamycin treatment significantly improved the sensitivity of neuroblastoma cells to rapamycin (p<0.02). Suppression of miR-19b may enhance the cytotoxic effects of mTOR inhibitors in neuroblastoma cells. Copyright © 2016 Elsevier Inc. All rights reserved.

RAN/RANBP2 polymorphisms and neuroblastoma risk in Chinese children: a three-center case-control study.

PubMed

Wang, Juxiang; Zhuo, Zhenjian; Chen, Min; Zhu, Jinhong; Zhao, Jie; Zhang, Jiao; Chen, Shanshan; He, Jing; Zhou, Haixia

2018-04-28

The genetic etiology of sporadic neuroblastoma remains largely obscure. RAN and RANBP2 genes encode Ras-related nuclear protein and Ran-binding protein 2, respectively. These two proteins form Ran-RanBP2 complex that regulate various cellular activities including nuclear transport. Aberrant functions of the two proteins are implicated in carcinogenesis. Given the unknown role of RAN/RANBP2 single nucleotide polymorphisms (SNPs) in neuroblastoma risk, we performed a multi-center case-control study in Chinese children to assess the association of the RAN/RANBP2 SNPs with neuroblastoma risk. We analyzed three potentially functional SNPs in RAN gene (rs56109543 C>T, rs7132224 A>G, rs14035 C>T) and one in RANBP2 (rs2462788 C>T) in 429 cases and 884 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to access the association between these four polymorphisms and neuroblastoma risk. No single variant was found to statistically significantly associate with neuroblastoma risk. However, individuals with 3 protective genotypes were less likely to develop neuroblastoma, in comparison to non-carriers (adjusted OR=0.33; 95% CI=0.12-0.96; P =0.042), as well as those with 0-2 protective genotypes (adjusted OR=0.33; 95% CI=0.11-0.94; P =0.038). Stratified analysis revealed no significant association for any of the four polymorphisms. Further studies are warranted to validate the weak impact of RAN/RANBP2 SNPs on neuroblastoma risk.

MiR-181a/b induce the growth, invasion, and metastasis of neuroblastoma cells through targeting ABI1.

PubMed

Liu, Xiaodan; Peng, Hongxia; Liao, Wang; Luo, Ailing; Cai, Mansi; He, Jing; Zhang, Xiaohong; Luo, Ziyan; Jiang, Hua; Xu, Ling

2018-05-26

Neuroblastoma is a pediatric malignancy, and the clinical phenotypes range from localized tumors with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40%, despite intensive therapy. Emerging evidence suggests that aberrant miRNA regulation plays a role in neuroblastoma, but the miRNA functions and mechanisms remain unknown. miR-181 family members were detected in 32 neuroblastoma patients, and the effects of miR-181a/b on cell viability, invasion, and migration were evaluated in vitro and in vivo. A parallel global mRNA expression profile was obtained for neuroblastoma cells overexpressing miR-181a. The potential targets of miR-181a/b were validated. miR-181a/b expression levels were positively associated with MYCN amplification and neuroblastoma aggressiveness. Moreover, ectopic miR-181a/b expression significantly induced the growth and invasion of neuroblastoma cells in vitro and in vivo. Microarray analysis revealed that mRNAs were consistently downregulated after miR-181a overexpression, leading to cell migration. In addition, the expression of ABI1 was suppressed by miR-181a/b, and ABI1 was validated as a direct target of miR-181a/b. We concluded that miR-181a/b were significantly upregulated in aggressive neuroblastoma, which enhanced its tumorigenesis and progression by suppressing the expression of ABI1. © 2018 Wiley Periodicals, Inc.

Tumorigenic and Antiproliferative Properties of the TALE-Transcription Factors MEIS2D and MEIS2A in Neuroblastoma.

PubMed

Groß, Anja; Schulz, Catrine; Kolb, Jasmine; Koster, Jan; Wehner, Sibylle; Czaplinski, Sebastian; Khilan, Abdulghani; Rohrer, Hermann; Harter, Patrick N; Klingebiel, Thomas; Langer, Julian D; Geerts, Dirk; Schulte, Dorothea

2018-04-15

Neuroblastoma is one of only a few human cancers that can spontaneously regress even after extensive dissemination, a poorly understood phenomenon that occurs in as many as 10% of patients. In this study, we identify the TALE-homeodomain transcription factor MEIS2 as a key contributor to this phenomenon. We identified MEIS2 as a MYCN-independent factor in neuroblastoma and showed that in this setting the alternatively spliced isoforms MEIS2A and MEIS2D exert antagonistic functions. Specifically, expression of MEIS2A was low in aggressive stage 4 neuroblastoma but high in spontaneously regressing stage 4S neuroblastoma. Moderate elevation of MEIS2A expression reduced proliferation of MYCN -amplified human neuroblastoma cells, induced neuronal differentiation and impaired the ability of these cells to form tumors in mice. In contrast, MEIS2A silencing or MEIS2D upregulation enhanced the aggressiveness of the tumor phenotype. Mechanistically, MEIS2A uncoupled a negative feedback loop that restricts accumulation of cellular retinoic acid, an effective agent in neuroblastoma treatment. Overall, our results illuminate the basis for spontaneous regression in neuroblastoma and identify an MEIS2A-specific signaling network as a potential therapeutic target in this common pediatric malignancy. Significance: This study illuminates the basis for spontaneous regressions that can occur in a common pediatric tumor, with implications for the development of new treatment strategies. Cancer Res; 78(8); 1935-47. ©2018 AACR . ©2018 American Association for Cancer Research.

Both serum and tissue Galectin-1 levels are associated with adverse clinical features in neuroblastoma.

PubMed

Chen, Kai; Cai, Yuanxia; Zhang, Min; Wu, Zhixiang; Wu, Yeming

2018-05-24

Neuroblastoma is one of the most common pediatric solid tumors. Although the 5-year overall survival rate has increased over the past few decades, high-risk patients still have a poor prognosis due to a lack of biomonitoring therapy. This study was performed to investigate the role of Galectin-1 in neuroblastoma biomonitoring therapy. A tissue microarray containing 37 neuroblastoma tissue samples was used to evaluate the correlation between Galectin-1 expression and clinical features. Blood samples were examined to better understand whether serum Galectin-1 (sGalectin-1) could be used for biomonitoring therapy. Kaplan-Meier analysis and ROC analysis was conducted to distinguish the outcome associated with high or low expression of Galectin-1 in patients with neuroblastoma. Increased Galectin-1 expression was found in neuroblastoma and it was further demonstrated that elevated tissue Galectin-1 expression was related to INSS stage, histology, bone marrow metastasis, and poor survival. sGalectin-1 levels were higher in newly diagnosed patients with neuroblastoma than healthy subjects. Patients with elevated sGalectin-1 through treatment cycles correlated with the poor chemo-responses and tended to have worse outcomes, such as metastasis or stable tumor size, whereas gradually decreasing sGalectin-1 levels correlated with no observed progression in clinical symptoms. Tissue and serum Galectin-1 levels were associated with adverse clinical features in patients with neuroblastoma, and sGalectin-1 could be a potential biomarker for monitoring therapy. © 2018 Wiley Periodicals, Inc.

Neuroblastoma | Office of Cancer Genomics

Cancer.gov

The TARGET Neuroblastoma projects elucidate comprehensive molecular characterization to determine the genetic changes that drive the initiation and progression of high-risk or hard-to-treat childhood cancers. Neuroblastoma (NBL) is a cancer that arises in immature nerve cells of the sympathetic nervous system, primarily affecting infants and children.

Fred Hutchinson Cancer Research Center (FHCRC-1): Identification of drug targets for combination therapy with Retinoic Acid in Neuroblastoma | Office of Cancer Genomics

Cancer.gov

Retinoic Acid (RA) is employed in the clinic during the “consolidation” phase of treatment regimens for high-risk neuroblastoma.  While the addition of RA has greatly increased the survival of children with neuroblastoma, there is still a high frequency of relapse.  With the goal of identifying novel drug combinations that would enhance the effect of RA on neuroblastoma, an siRNA screen in the presence or absence of sub-lethal concentrations of RA was carried out.

Fred Hutchinson Cancer Research Center (FHCRC1): Identification of drug targets for combination therapy with Retinoic Acid in Neuroblastoma | Office of Cancer Genomics

Cancer.gov

Retinoic Acid (RA) is employed in the clinic during the “consolidation” phase of treatment regimens for high-risk neuroblastoma.  While the addition of RA has greatly increased the survival of children with neuroblastoma, there is still a high frequency of relapse.  With the goal of identifying novel drug combinations that would enhance the effect of RA on neuroblastoma, an siRNA screen in the presence or absence of sub-lethal concentrations of RA was carried out.

The role of genetic and epigenetic alterations in neuroblastoma disease pathogenesis

PubMed Central

Domingo-Fernandez, Raquel; Watters, Karen; Piskareva, Olga; Bray, Isabella

2013-01-01

Neuroblastoma is a highly heterogeneous tumor accounting for 15 % of all pediatric cancer deaths. Clinical behavior ranges from the spontaneous regression of localized, asymptomatic tumors, as well as metastasized tumors in infants, to rapid progression and resistance to therapy. Genomic amplification of the MYCN oncogene has been used to predict outcome in neuroblastoma for over 30 years, however, recent methodological advances including miR-NA and mRNA profiling, comparative genomic hybridization (array-CGH), and whole-genome sequencing have enabled the detailed analysis of the neuroblastoma genome, leading to the identification of new prognostic markers and better patient stratification. In this review, we will describe the main genetic factors responsible for these diverse clinical phenotypes in neuroblastoma, the chronology of their discovery, and the impact on patient prognosis. PMID:23274701

On the significance of germline cytogenetic rearrangements at MYCN locus in neuroblastoma

PubMed Central

2013-01-01

Background MYCN oncogene amplification is the most important prognostic factor in neuroblastoma. 25% neuroblastoma tumors have somatic amplifications at this locus but little is known about its constitutional aberrations and their potential role in carcinogenesis. Here, we have performed an array-CGH and qPCR characterization of two patients with constitutional partial 2p trisomy including MYCN genomic region. Results One of the patients had congenital neuroblastoma and showed presence of minute areas of gains and losses within the common fragile site FRA2C at 2p24 encompassing MYCN. The link between 2p24 germline rearrangements and neuroblastoma development was reassessed by reviewing similar cases in the literature. Conclusions It appears that constitutional rearrangements involving chromosome 2p24 may play role in NB development. PMID:24131700

Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse

PubMed Central

Corrias, Maria Valeria; Gambini, Claudio; Gregorio, Andrea; Croce, Michela; Barisione, Gaia; Cossu, Claudia; Rossello, Armando; Ferrini, Silvano; Fabbi, Marina

2010-01-01

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system. Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively. Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype. Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up. PMID:20208136

Iodine-131 Metaiodobenzylguanidine Therapy for Neuroblastoma: Reports So Far and Future Perspective

PubMed Central

Kayano, Daiki

2015-01-01

Neuroblastoma, which derives from neural crest, is the most common extracranial solid cancer in childhood. The tumors express the norepinephrine (NE) transporters on their cell membrane and take in metaiodobenzylguanidine (MIBG) via a NE transporter. Since iodine-131 (I-131) MIBG therapy was firstly reported, many trails of MIBG therapy in patients with neuroblastoma were performed. Though monotherapy with a low dose of I-131 MIBG could achieve high-probability pain reduction, the objective response was poor. In contrast, more than 12 mCi/kg I-131 MIBG administrations with or without hematopoietic cell transplantation (HCT) obtain relatively good responses in patients with refractory or relapsed neuroblastoma. The combination therapy with I-131 MIBG and other modalities such as nonmyeloablative chemotherapy and myeloablative chemotherapy with HCT improved the therapeutic response in patients with refractory or relapsed neuroblastoma. In addition, I-131 MIBG therapy incorporated in the induction therapy was proved to be feasible in patients with newly diagnosed neuroblastoma. To expand more the use of MIBG therapy for neuroblastoma, further studies will be needed especially in the use at an earlier stage from diagnosis, in the use with other radionuclide formations of MIBG, and in combined use with other therapeutic agents. PMID:25874239

Persistent positive metaiodobenzylguanidine scans after autologous peripheral blood stem cell transplantation may indicate maturation of stage 4 neuroblastoma.

PubMed

Okamoto, Yasuhiro; Kodama, Yuichi; Nishikawa, Takuro; Rindiarti, Almitra; Tanabe, Takayuki; Nakagawa, Shunsuke; Yoshioka, Takako; Takumi, Koji; Kaji, Tatsuru; Kawano, Yoshifumi

2017-04-01

Metaiodobenzylguanidine (MIBG) scans are sensitive testing tools for neuroblastoma. Persistent positive MIBG scans in patients with stage 3 neuroblastoma have previously been found to indicate maturation rather than regression. We assessed the significance of this finding in stage 4 neuroblastoma in the present study. Fifteen consecutive pediatric patients with stage 4 neuroblastoma treated between 2004 and 2014 at the Kagoshima University Hospital were retrospectively examined. Treatment involved a combination of multiagent chemotherapy, resection, autologous peripheral blood stem cell transplantation (PBSCT), radiotherapy, and maintenance therapy with retinoic acid. The MIBG uptake in each patient during treatment was assessed using a Curie score. The 5-year event-free and overall survival rates in 15 patients were 38.9% and 58.7%, respectively. Four patients with persistent positive MIBG scans who underwent autologous PBSCT but experienced decreased 123 I-MIBG uptake during the clinical course survived without progression, and their event-free survival (EFS) was significantly superior to that of patients who showed negative MIBG scans after PBSCT (5-year EFS rate: 18.2%, p = 0.0176). Therefore, persistent positive MIBG scans with gradually decreased uptake after PBSCT do not always indicate neuroblastoma progression, and may instead indicate tumor maturation in some selected cases, if not all cases, of stage 4 neuroblastoma.

Notch ligand Delta-like 1 as a novel molecular target in childhood neuroblastoma.

PubMed

Bettinsoli, P; Ferrari-Toninelli, G; Bonini, S A; Prandelli, C; Memo, M

2017-05-19

Neuroblastoma is the most common extracranial solid malignancy in childhood, responsible for 15% of all pediatric cancer deaths. It is an heterogeneous disease that does not always respond to classical therapy; so the identification of new and specific molecular targets to improve existing therapy is needed. We have previously demonstrated the involvement of the Notch pathway in the onset and progression of neuroblastoma. In this study we further investigated the role of Notch signaling and identified Delta-like 1 (DLL1) as a novel molecular target in neuroblastoma cells with a high degree of MYCN amplification, which is a major oncogenic driver in neuroblastoma. The possibility to act on DLL1 expression levels by using microRNAs (miRNAs) was assessed. DLL1 mRNA and protein expression levels were measured in three different neuroblastoma cell lines using quantitative real-time PCR and Western Blot analysis, respectively. Activation of the Notch pathway as a result of increased levels of DLL1 was analyzed by Immunofluorescence and Western Blot methods. In silico tools revealed the possibility to act on DLL1 expression levels with miRNAs, in particular with the miRNA-34 family. Neuroblastoma cells were transfected with miRNA-34 family members, and the effect of miRNAs transfection on DLL1 mRNA expression levels, on cell differentiation, proliferation and apoptosis was measured. In this study, the DLL1 ligand was identified as the Notch pathway component highly expressed in neuroblastoma cells with MYCN amplification. In silico analysis demonstrated that DLL1 is one of the targets of miRNA-34 family members that maps on chromosome regions that are frequently deregulated or deleted in neuroblastoma. We studied the possibility to use miRNAs to target DLL1. Among all miRNA-34 family members, miRNA-34b is able to significantly downregulate DLL1 mRNA expression levels, to arrest cell proliferation and to induce neuronal differentiation in malignant neuroblastoma cells. Targeted therapies have emerged as new strategies for cancer treatment. This study identified the Notch ligand DLL1 as a novel and attractive molecular target in childhood neuroblastoma and its results could help to devise a targeted therapy using miRNAs.

Reversal of multidrug resistance in xenograft nude-mice by magnetic Fe(3)O(4) nanoparticles combined with daunorubicin and 5-bromotetrandrine.

PubMed

Wu, Ya-Nan; Chen, Bao-An; Cheng, Jian; Gao, Feng; Xu, Wen-Lin; Ding, Jia-Hua; Gao, Chong; Sun, Xin-Chen; Li, Guo-Hong; Chen, Wen-Ji; Liu, Li-Jie; Li, Xiao-Mao; Wang, Xue-Mei

2009-02-01

This study was aimed to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe(3)O(4) (Fe(3)O(4)-MNPs) combined with DNR in vivo. The xenograft leukemia model with stable multiple drug resistance in nude mice was established. The two sub-clones of K562 and K562/A02 cells were respectively inoculated subcutaneously into back of athymic nude mice (1 x 10(7) cells/each) to establish the leukemia xenograft models. Drug resistant and the sensitive tumor-bearing nude mice were both assigned randomly into 5 groups: group A was treated with NS; group B was treated with DNR; group C was treated with nanoparticle of Fe(3)O(4) combined with DNR; group D was treated with 5-BrTet combined with DNR; group E was treated with 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR. The incidence of tumor formation, growth characteristics, weight and volume of tumor were observed. The histopathologic examination of tumors and organs were carried out. The protein levels of BCL-2, BAX, and Caspase-3 in resistant tumors were detected by Western blot. The results indicated that 5-BrTet and magnetic nanoparticle of Fe(3)O(4) combined with DNR significantly suppressed growth of K562/A02 cell xenograft tumor, histopathologic examination of tumors showed the tumors necrosis obviously. Application of 5-BrTet and magnetic nanoparticle of Fe(3)O(4) inhibited the expression of BCL-2 protein and up-regulated the expression of BAX, and Caspase-3 protein in K562/A02 cell xenograft tumor. It is concluded that 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR have significant tumor-suppressing effect on MDR leukemia cell xenograft model.

Comparison of DNA aneuploidy, chromosome 1 abnormalities, MYCN amplification and CD44 expression as prognostic factors in neuroblastoma.

PubMed

Christiansen, H; Sahin, K; Berthold, F; Hero, B; Terpe, H J; Lampert, F

1995-01-01

A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.

High Phosphohistone H3 Expression Correlates with Adverse Clinical, Biological, and Pathological Factors in Neuroblastomas

PubMed Central

Ramani, Pramila; Taylor, Scott; Miller, Elizabeth; Sowa-Avugrah, Emile; May, Margaret T.

2015-01-01

Phosphohistone H3 (pHH3), a biomarker of the late G2- and M-phase of the cell cycle, provides a powerful indication of the proliferative state of many cancers. We investigated the prognostic significance of pHH3 by immunostaining 80 neuroblastomas and counting the average number of strongly stained nuclei and mitotic figures. The median and 75th percentile pHH3 proliferation indices (PIs) were 0.54% and 1.06% (range, 0.01% to 2.23%) respectively. pHH3 expression was significantly higher in neuroblastomas from patients with adverse clinical characteristics, all unfavorable pathological factors including high mitosis karyorrhexis index (MKI), and adverse biological factors including MYCN oncogene amplification. High pHH3-PIs, at 1% threshold, were significantly associated with a shorter overall survival (OS) and event-free survival (EFS) in the univariable Cox regression analyses. In the multivariable models, high pHH3 counts were significantly associated with worse OS after adjustment for age but were not independent of either high MKI or MYCN amplification. In children less than 18 months of age, high MKIs and high PHH3-PIs were associated with significantly worse OS and EFS. In conclusion, high pHH3 expression correlates strongly with high MKI and MYCN amplification and indicates poor prognosis in neuroblastomas. PMID:25711230

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

PubMed

Blattmann, Claudia; Thiemann, Markus; Stenzinger, Albrecht; Roth, Eva K; Dittmar, Anne; Witt, Hendrik; Lehner, Burkhard; Renker, Eva; Jugold, Manfred; Eichwald, Viktoria; Weichert, Wilko; Huber, Peter E; Kulozik, Andreas E

2015-04-30

Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Neuronal differentiation and long-term culture of the human neuroblastoma line SH-SY5Y.

PubMed

Constantinescu, R; Constantinescu, A T; Reichmann, H; Janetzky, B

2007-01-01

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in industrialized countries. Present cell culture models for PD rely on either primary cells or immortal cell lines, neither of which allow for long-term experiments on a constant population, a crucial requisite for a realistic model of slowly progressing neurodegenerative diseases. We differentiated SH-SY5Y human dopaminergic neuroblastoma cells to a neuronal-like state in a perfusion culture system using a combination of retinoic acid and mitotic inhibitors. The cells could be cultivated for two months without the need for passage. We show, by various means, that the differentiated cells exhibit, at the molecular level, many neuronal properties not characteristic to the starting line. This approach opens the possibility to develop chronic models, in which the effect of perturbations and putative counteracting strategies can be monitored over long periods of time in a quasi-stable cell population.

Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model.

PubMed

Koskimaki, Jacob E; Karagiannis, Emmanouil D; Tang, Benjamin C; Hammers, Hans; Watkins, D Neil; Pili, Roberto; Popel, Aleksander S

2010-02-01

Angiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays. One family of peptides with high activity is derived from the alpha-fibrils of type IV collagen. Based on the results from the in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the alpha5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line. Pentastatin-1 decreased the invasion of vessels into angioreactors in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model. The peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer.

Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis

PubMed Central

Guerquin, Marie-Justine; Matilionyte, Gabriele; Kilcoyne, Karen; N’Tumba-Byn, Thierry; Messiaen, Sébastien; Deceuninck, Yoann; Pozzi-Gaudin, Stéphanie; Benachi, Alexandra; Livera, Gabriel; Antignac, Jean-Philippe; Mitchell, Rod; Rouiller-Fabre, Virginie

2018-01-01

Background Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models. Methods Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks. Results With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice. Conclusions Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures. PMID:29385186

Monitoring the development of xenograft triple-negative breast cancer models using diffusion-weighted magnetic resonance imaging.

PubMed

Stephen, Renu M; Pagel, Mark D; Brown, Kathy; Baker, Amanda F; Meuillet, Emmanuelle J; Gillies, Robert J

2012-11-01

Evaluations of tumor growth rates and molecular biomarkers are traditionally used to assess new mouse models of human breast cancers. This study investigated the utility of diffusion weighted (DW)-magnetic resonance imaging (MRI) for evaluating cellular proliferation of new tumor models of triple-negative breast cancer, which may augment traditional analysis methods. Eleven human breast cancer cell lines were used to develop xenograft tumors in severe combined immunodeficient mice, with two of these cell lines exhibiting sufficient growth to be serially passaged. DW-MRI was performed to measure the distributions of the apparent diffusion coefficient (ADC) in these two tumor xenograft models, which showed a correlation with tumor growth rates and doubling times during each passage. The distributions of the ADC values were also correlated with expression of Ki67, a biomarker of cell proliferation, and hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor receptor-2 (VEGFR2), which are essential proteins involved in regulating aerobic glycolysis and angiogenesis that support tumor cell proliferation. Although phosphatase and tensin homolog (PTEN) levels were different between the two xenograft models, AKT levels did not differ nor did they correlate with tumor growth. This last result demonstrates the complexity of signaling protein pathways and the difficulty in interpreting the effects of protein expression on tumor cell proliferation. In contrast, DW-MRI may be a more direct assessment of tumor growth and cancer cell proliferation.

Exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancer progression

PubMed Central

Lasorsa, Vito Alessandro; Formicola, Daniela; Pignataro, Piero; Cimmino, Flora; Calabrese, Francesco Maria; Mora, Jaume; Esposito, Maria Rosaria; Pantile, Marcella; Zanon, Carlo; De Mariano, Marilena; Longo, Luca; Hogarty, Michael D.; de Torres, Carmen; Tonini, Gian Paolo; Iolascon, Achille; Capasso, Mario

2016-01-01

The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma. Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines. We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9, a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK. Other genes (PTK2, NAV3, NAV1, FZD1 and ATRX), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%. Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression. Notably BARD1, CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants. In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression. PMID:27009842

Exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancer progression.

PubMed

Lasorsa, Vito Alessandro; Formicola, Daniela; Pignataro, Piero; Cimmino, Flora; Calabrese, Francesco Maria; Mora, Jaume; Esposito, Maria Rosaria; Pantile, Marcella; Zanon, Carlo; De Mariano, Marilena; Longo, Luca; Hogarty, Michael D; de Torres, Carmen; Tonini, Gian Paolo; Iolascon, Achille; Capasso, Mario

2016-04-19

The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma.Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines.We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9, a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK.Other genes (PTK2, NAV3, NAV1, FZD1 and ATRX), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%.Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression.Notably BARD1, CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants.In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression.

Lung Metastases in Neuroblastoma at Initial Diagnosis: A Report from the International Neuroblastoma Risk Group (INRG) Project

PubMed Central

DuBois, Steven G.; London, Wendy B.; Zhang, Yang; Matthay, Katherine K.; Monclair, Tom; Ambros, Peter F.; Cohn, Susan L.; Pearson, Andrew; Diller, Lisa

2009-01-01

Background Neuroblastoma is the most common extracranial pediatric solid cancer. Lung metastasis is rarely detected in children with newly diagnosed neuroblastoma. We aimed to describe the incidence, clinical characteristics, and outcome of patients with lung metastasis at initial diagnosis using a large international database. Procedure The subset of patients from the International Neuroblastoma Risk Group database with INSS stage 4 neuroblastoma and known data regarding lung metastasis at diagnosis was selected for analysis. Clinical and biological characteristics were compared between patients with and without lung metastasis. Survival for patients with and without lung metastasis was estimated by Kaplan-Meier methods. Cox proportional hazards methods were used to determine the independent prognostic value of lung metastasis at diagnosis. Results Of the 2,808 patients with INSS stage 4 neuroblastoma diagnosed between 1990 and 2002, 100 patients (3.6%) were reported to have lung metastasis at diagnosis. Lung metastasis was more common among patients with MYCN amplified tumors, adrenal primary tumors, or elevated lactate dehydrogenase (LDH) levels (p < 0.02 in each case). Five-year overall survival ± standard error for patients with lung metastasis was 34.5% ± 6.8% compared to 44.7% ± 1.3% for patients without lung metastasis (p=0.0002). However, in multivariable analysis, the presence of lung metastasis was not independently predictive of outcome. Conclusions Lung metastasis at initial diagnosis of neuroblastoma is associated with MYCN amplification and elevated LDH levels. Although lung metastasis at diagnosis was not independently predictive of outcome in this analysis, it remains a useful prognostic marker of unfavorable outcome. PMID:18649370

LINC00673 rs11655237 C>T confers neuroblastoma susceptibility in Chinese population.

PubMed

Zhang, Zhuorong; Chang, Yitian; Jia, Wei; Zhang, Jiao; Zhang, Ruizhong; Zhu, Jinhong; Yang, Tianyou; Xia, Huimin; Zou, Yan; He, Jing

2018-02-28

Neuroblastoma, which accounts for approximately 10% of all pediatric cancer-related deaths, has become a therapeutic challenge and global burden attributed to poor outcomes and mortality rates of its high-risk form. Previous genome-wide association studies (GWASs) identified the LINC00673 rs11655237 C>T polymorphism to be associated with the susceptibility of several malignant tumors. However, the association between this polymorphism and neuroblastoma susceptibility is not clear. We genotyped LINC00673 rs11655237 C>T in 393 neuroblastoma patients in comparison with 812 age-, gender-, and ethnicity-matched healthy controls. We found a significant association between the LINC00673 rs11655237 C>T polymorphism and neuroblastoma risk (TT compared with CC: adjusted odds ratio (OR) =1.80, 95% confidence interval (CI) =1.06-3.06, P =0.029; TT/CT compared with CC: adjusted OR =1.31, 95% CI =1.02-1.67, P =0.033; and T compared with C: adjusted OR =1.29, 95% CI =1.06-1.58, P =0.013). Furthermore, stratified analysis indicated that the rs11655237 T allele carriers were associated with increased neuroblastoma risk for patients with tumor originating from the adrenal gland (adjusted OR =1.51, 95% CI =1.06-2.14, P =0.021) and International Neuroblastoma Staging System (INSS) stage IV disease (adjusted OR =1.60, 95% CI =1.12-2.30, P =0.011). In conclusion, we verified that the LINC00673 rs11655237 C>T polymorphism might be associated with neuroblastoma susceptibility. Prospective studies with a large sample size and different ethnicities are needed to validate our findings. © 2018 The Author(s).

[Establishment of an iRFP and luciferase dual-color fluorescence-traced hepatocellular carcinoma transplantation model in nude mice].

PubMed

Li, Hongjun; Yang, Tianhua; Huang, Yanping; Liu, Mingzhu; Qin, Zhongqiang; Chu, Fei; Li, Zhenghong; Li, Yonghai

2017-11-01

Objective To establish a hepatocellular carcinoma xenograft model in nude mice which could stably express gene and be monitored dynamically. Methods We first constructed the lentiviral particles containing luciferase (Luc) and near-infrared fluorescent protein (iRFP) and puromycin resistance gene, and then transduced them into the HepG2 hepatoma cells. The cell line stably expressing Luc and iRFP genes were screened and inoculated into nude mice to establish xenograft tumor model. Tumor growth was monitored using in vivo imaging system. HE staining and immunohistochemistry were used to evaluate the pathological features and tumorigenic ability. Results HepG2 cells stably expressing iRFP and Luc were obtained; with the engineered cell line, xenograft model was successfully established with the features of proper tumor developing time and high rate of tumor formation as well as typical pathological features as showed by HE staining and immunohistochemistry. Conclusion Hepatocellular carcinoma model in nude mice with the features of stable gene expression and dynamical monitoring has been established successfully with the HepG2-iRFP-Luc cell line.

Patient Derived Xenograft Models: An Emerging Platform for Translational Cancer Research

PubMed Central

Hidalgo, Manuel; Amant, Frederic; Biankin, Andrew V.; Budinská, Eva; Byrne, Annette T.; Caldas, Carlos; Clarke, Robert B.; de Jong, Steven; Jonkers, Jos; Mælandsmo, Gunhild Mari; Roman-Roman, Sergio; Seoane, Joan; Trusolino, Livio; Villanueva, Alberto

2014-01-01

Recently, there has been increasing interest in the development and characterization of patient derived tumor xenograft (PDX) models for cancer research. PDX models mostly retain the principal histological and genetic characteristics of their donor tumor and remain stable across passages. These models have been shown to be predictive of clinical outcomes and are being used for preclinical drug evaluation, biomarker identification, biological studies, and personalized medicine strategies. This paper summarizes the current state of the art in this field including methodological issues, available collections, practical applications, challenges and shortcoming, and future directions, and introduces a European consortium of PDX models. PMID:25185190

Differentiation of human SH-SY5Y neuroblastoma cells by all-trans retinoic acid activates the interleukin-18 system.

PubMed

Sallmon, Hannes; Hoene, Victoria; Weber, Sven C; Dame, Christof

2010-02-01

The clinical prognosis of children with high-stage neuroblastoma is still poor. Therapeutic approaches include surgery and cellular differentiation by retinoic acid, but also experimental interleukin-based immune modulation. However, the molecular mechanisms of all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells are incompletely understood. Herein, we examined the effect of ATRA on the activity of the interleukin-18 (IL-18) system in human SH-SY5Y neuroblastoma cells. It is shown that SH-SY5Y cells express IL-18 receptor (IL-18R) and the secreted antagonist IL-18-binding protein (IL-18BP), but no IL-18. SH-SY5Y cells are highly sensitive to ATRA treatment and react by cellular differentiation from a neuroblastic toward a more neuronal phenotype. This was associated with induction of IL-18 and reduction of IL-18BP expression, while IL-18R expression remained stable. Thereby, we identified the IL-18 system as a novel target of ATRA in neuroblastoma cells that might contribute to the therapeutic properties of retinoids in treatment of neuroblastoma.

Advances in Risk Classification and Treatment Strategies for Neuroblastoma

PubMed Central

Pinto, Navin R.; Applebaum, Mark A.; Volchenboum, Samuel L.; Matthay, Katherine K.; London, Wendy B.; Ambros, Peter F.; Nakagawara, Akira; Berthold, Frank; Schleiermacher, Gudrun; Park, Julie R.; Valteau-Couanet, Dominique; Pearson, Andrew D.J.

2015-01-01

Risk-based treatment approaches for neuroblastoma have been ongoing for decades. However, the criteria used to define risk in various institutional and cooperative groups were disparate, limiting the ability to compare clinical trial results. To mitigate this problem and enhance collaborative research, homogenous pretreatment patient cohorts have been defined by the International Neuroblastoma Risk Group classification system. During the past 30 years, increasingly intensive, multimodality approaches have been developed to treat patients who are classified as high risk, whereas patients with low- or intermediate-risk neuroblastoma have received reduced therapy. This treatment approach has resulted in improved outcome, although survival for high-risk patients remains poor, emphasizing the need for more effective treatments. Increased knowledge regarding the biology and genetic basis of neuroblastoma has led to the discovery of druggable targets and promising, new therapeutic approaches. Collaborative efforts of institutions and international cooperative groups have led to advances in our understanding of neuroblastoma biology, refinements in risk classification, and stratified treatment strategies, resulting in improved outcome. International collaboration will be even more critical when evaluating therapies designed to treat small cohorts of patients with rare actionable mutations. PMID:26304901

Transcription instability in high-risk neuroblastoma is associated with a global perturbation of chromatin domains.

PubMed

Zanon, Carlo; Tonini, Gian Paolo

2017-11-01

Chromosome instability has a pivotal role among the hallmarks of cancer, but its transcriptional counterpart is rarely considered a relevant factor in cell destabilization. To examine transcription instability (TIN), we first devised a metric we named TIN index and used it to evaluate TIN on a dataset containing more than 500 neuroblastoma samples. We found that metastatic tumors from high-risk (HR) patients are characterized by significantly different TIN index values compared to low/intermediate-risk patients. Our results indicate that the TIN index is a good predictor of neuroblastoma patient's outcome, and a related TIN index gene signature (TIN-signature) is also able to predict the neuroblastoma patient's outcome with high confidence. Interestingly, we find that TIN-signature genes have a strong positional association with superenhancers in neuroblastoma tumors. Finally, we show that TIN is linked to chromatin structural domains and interferes with their integrity in HR neuroblastoma patients. This novel approach to gene expression analysis broadens the perspective of genome instability investigations to include functional aspects. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Expression of Truncated Neurokinin-1 Receptor in Childhood Neuroblastoma is Independent of Tumor Biology and Stage.

PubMed

Pohl, Alexandra; Kappler, Roland; Mühling, Jakob; VON Schweinitz, Dietrich; Berger, Michael

2017-11-01

Neuroblastoma is an embryonal malignancy arising from the aberrant growth of neural crest progenitor cells of the sympathetic nervous system. The tachykinin receptor 1 (TACR1) - substance P complex is associated with tumoral angiogenesis and cell proliferation in a variety of cancer types. Inhibition of TACR1 was recently described to impede growth of NB cell lines. However, the relevance of TACR1 in clinical settings is unknown. We investigated gene expression levels of full-length and truncated TACR1 in 59 neuroblastomas and correlated these data with the patients' clinical parameters such as outcome, metastasis, International Neuroblastoma Staging System (INSS) status, MYCN proto-oncogene, bHLH transcription factor (MYCN) status, gender and age. Our results indicated that TACR1 is ubiquitously expressed in neuroblastoma but expression levels are independent of clinical parameters. Our data suggest that TACR1 might serve as a potent anticancer target in a large variety of patients with neuroblastoma, independent of tumor biology and clinical stage. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

TIAM1 variants improve clinical outcome in neuroblastoma.

PubMed

Sanmartín, Elena; Yáñez, Yania; Fornés-Ferrer, Victoria; Zugaza, José L; Cañete, Adela; Castel, Victoria; Font de Mora, Jaime

2017-07-11

Identification of tumor driver mutations is crucial for improving clinical outcome using a personalized approach to the treatment of cancer. Neuroblastoma is a tumor of the peripheral sympathetic nervous system for which only a few driver alterations have been described including MYCN amplification and ALK mutations. We assessed 106 primary neuroblastoma tumors by next generation sequencing using a customized amplicon-based gene panel. Our results reveal that genetic variants in TIAM1 gene associate with better clinical outcome, suggesting a role for these TIAM1 variants in preventing progression of this disease. The detected variants are located within the different domains of TIAM1 that signal to the upstream regulator RAS and downstream effector molecules MYC and RAC, which are all implicated in neuroblastoma etiology and progression. Clinical outcome was improved in tumors where a TIAM1 variant was present concomitantly with either ALK mutation or MYCN amplification. Given the function of these signaling molecules in cell survival, proliferation, differentiation and neurite outgrowth, our data suggest that the TIAM1-mediated network is essential to neuroblastoma and thus, inhibiting TIAM1 reflects a rational strategy for improving therapy efficacy in neuroblastoma.

Constitutional 11q14-q22 chromosome deletion syndrome in a child with neuroblastoma MYCN single copy.

PubMed

Passariello, Annalisa; De Brasi, Daniele; Defferrari, Raffaella; Genesio, Rita; Tufano, Maria; Mazzocco, Katia; Capasso, Maria; Migliorati, Roberta; Martinsson, Tommy; Siani, Paolo; Nitsch, Lucio; Tonini, Gian Paolo

2013-11-01

Constitutional 11q deletion is a chromosome imbalance possibly found in MCA/MR patients analyzed for chromosomal anomalies. Its role in determining the phenotype depends on extension and position of deleted region. Loss of heterozygosity of 11q (region 11q23) is also associated with neuroblastoma, the most frequent extra cranial cancer in children. It represents one of the most frequent cytogenetic abnormalities observed in the tumor of patients with high-risk disease even if germline deletion of 11q in neuroblastoma is rare. Hereby, we describe a 18 months old girl presenting with trigonocephaly and dysmorphic facial features, including hypotelorism, broad depressed nasal bridge, micrognathia, synophrys, epicanthal folds, and with a stage 4 neuroblastoma without MYCN amplification, carrying a germline 11q deletion (11q14.1-q22.3), outside from Jacobsen syndrome and from neuroblastoma 11q critical regions. The role of 11q deletion in determining the clinical phenotype and its association with neuroblastoma development in the patient are discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Prostate-targeted biodegradable nanoparticles loaded with androgen receptor silencing constructs eradicate xenograft tumors in mice

PubMed Central

Yang, Jun; Xie, Sheng-Xue; Huang, Yiling; Ling, Min; Liu, Jihong; Ran, Yali; Wang, Yanlin; Thrasher, J Brantley; Berkland, Cory; Li, Benyi

2012-01-01

Background Prostate cancer is the major cause of cancer death in men and the androgen receptor (AR) has been shown to play a critical role in the progression of the disease. Our previous reports showed that knocking down the expression of the AR gene using a siRNA-based approach in prostate cancer cells led to apoptotic cell death and xenograft tumor eradication. In this study, we utilized a biodegradable nanoparticle to deliver the therapeutic AR shRNA construct specifically to prostate cancer cells. Materials & methods The biodegradable nanoparticles were fabricated using a poly(dl-lactic-co-glycolic acid) polymer and the AR shRNA constructs were loaded inside the particles. The surface of the nanoparticles were then conjugated with prostate-specific membrane antigen aptamer A10 for prostate cancer cell-specific targeting. Results A10-conjugation largely enhanced cellular uptake of nanoparticles in both cell culture- and xenograft-based models. The efficacy of AR shRNA encapsulated in nanoparticles on AR gene silencing was confirmed in PC-3/AR-derived xenografts in nude mice. The therapeutic property of A10-conjugated AR shRNA-loaded nanoparticles was evaluated in xenograft models with different prostate cancer cell lines: 22RV1, LAPC-4 and LNCaP. Upon two injections of the AR shRNA-loaded nanoparticles, rapid tumor regression was observed over 2 weeks. Consistent with previous reports, A10 aptamer conjugation significantly enhanced xenograft tumor regression compared with nonconjugated nanoparticles. Discussion These data demonstrated that tissue-specific delivery of AR shRNA using a biodegradable nanoparticle approach represents a novel therapy for life-threatening prostate cancers. PMID:22583574

Inhibition of gamma-secretase activity impedes uterine serous carcinoma growth in a human xenograft model.

PubMed

Groeneweg, Jolijn W; Hall, Tracilyn R; Zhang, Ling; Kim, Minji; Byron, Virginia F; Tambouret, Rosemary; Sathayanrayanan, Sriram; Foster, Rosemary; Rueda, Bo R; Growdon, Whitfield B

2014-06-01

Uterine serous carcinoma (USC) represents an aggressive subtype of endometrial cancer. We sought to understand Notch pathway activity in USC and determine if pathway inhibition has anti-tumor activity. Patient USC tissue blocks were obtained and used to correlate clinical outcomes with Notch1 expression. Three established USC cell lines were treated with gamma-secretase inhibitor (GSI) in vitro. Mice harboring cell line derived or patient derived USC xenografts (PDXs) were treated with vehicle, GSI, paclitaxel and carboplatin (P/C), or combination GSI and P/C. Levels of cleaved Notch1 protein and Hes1 mRNA were determined in GSI treated samples. Statistical analysis was performed using the Wilcoxon rank sum and Kaplan-Meier methods. High nuclear Notch1 protein expression was observed in 58% of USC samples with no correlation with overall survival. GSI induced dose-dependent reductions in cell number and decreased levels of cleaved Notch1 protein and Hes1 mRNA in vitro. Treatment of mice with GSI led to decreased Hes1 mRNA expression in USC xenografts. In addition, GSI impeded tumor growth of cell line xenografts as well as UT1 USC PDXs. When GSI and P/C were combined, synergistic anti-tumor activity was observed in UT1 xenografts. Notch1 is expressed in a large subset of USC. GSI-mediated Notch pathway inhibition led to both reduced cell numbers in vitro and decreased tumor growth of USC some xenograft models. When combined with conventional chemotherapy, GSI augmented anti-tumor activity in one USC PDX line suggesting that targeting of the Notch signaling pathway is a potential therapeutic strategy for future investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts.

PubMed

Li, Li-Hua; Guo, Zi-Jian; Yan, Ling-Ling; Yang, Ji-Cheng; Xie, Yu-Feng; Sheng, Wei-Hua; Huang, Zhao-Hui; Wang, Xue-Hao

2007-12-21

To study the effectiveness and mechanisms of anti- human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis, oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7,721 and, subsequently, polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively, the growth of cells in nude mice and angiogenesis were observed. VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7,721 cells and xenografted tumor. Compared to the control group, the transfected cells grew slower in cell cultures and xenografts, and the xenograft formation was delayed as well. In addition, the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstrated by microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation, differentiation and tumori-genesis in hepatocarcinoma.

123I-Mibg scintigraphy and 18F-Fdg-Pet imaging for diagnosing neuroblastoma

PubMed Central

Bleeker, Gitta; Tytgat, Godelieve Am; Adam, Judit A; Caron, Huib N; Kremer, Leontien Cm; Hooft, Lotty; van Dalen, Elvira C

2015-01-01

Background Neuroblastoma is an embryonic tumour of childhood that originates in the neural crest. It is the second most common extracranial malignant solid tumour of childhood. Neuroblastoma cells have the unique capacity to accumulate Iodine-123-metaiodobenzylguanidine (123I-MIBG), which can be used for imaging the tumour. Moreover, 123I-MIBG scintigraphy is not only important for the diagnosis of neuroblastoma, but also for staging and localization of skeletal lesions. If these are present, MIBG follow-up scans are used to assess the patient's response to therapy. However, the sensitivity and specificity of 123I-MIBG scintigraphy to detect neuroblastoma varies according to the literature. Prognosis, treatment and response to therapy of patients with neuroblastoma are currently based on extension scoring of 123I-MIBG scans. Due to its clinical use and importance, it is necessary to determine the exact diagnostic accuracy of 123I-MIBG scintigraphy. In case the tumour is not MIBG avid, fluorine-18-fluorodeoxy-glucose (18F-FDG) positron emission tomography (PET) is often used and the diagnostic accuracy of this test should also be assessed. Objectives Primary objectives: 1.1 To determine the diagnostic accuracy of 123I-MIBG (single photon emission computed tomography (SPECT), with or without computed tomography (CT)) scintigraphy for detecting a neuroblastoma and its metastases at first diagnosis or at recurrence in children from 0 to 18 years old. 1.2 To determine the diagnostic accuracy of negative 123I-MIBG scintigraphy in combination with 18F-FDG-PET(-CT) imaging for detecting a neuroblastoma and its metastases at first diagnosis or at recurrence in children from 0 to 18 years old, i.e. an add-on test. Secondary objectives: 2.1 To determine the diagnostic accuracy of 18F-FDG-PET(-CT) imaging for detecting a neuroblastoma and its metastases at first diagnosis or at recurrence in children from 0 to 18 years old. 2.2 To compare the diagnostic accuracy of 123I-MIBG (SPECT-CT) and 18F-FDG-PET(-CT) imaging for detecting a neuroblastoma and its metastases at first diagnosis or at recurrence in children from 0 to 18 years old. This was performed within and between included studies. 123I-MIBG (SPECT-CT) scintigraphy was the comparator test in this case. Search methods We searched the databases of MEDLINE/PubMed (1945 to 11 September 2012) and EMBASE/Ovid (1980 to 11 September 2012) for potentially relevant articles. Also we checked the reference lists of relevant articles and review articles, scanned conference proceedings and searched for unpublished studies by contacting researchers involved in this area. Selection criteria We included studies of a cross-sectional design or cases series of proven neuroblastoma, either retrospective or prospective, if they compared the results of 123I-MIBG (SPECT-CT) scintigraphy or 18F-FDG-PET(-CT) imaging, or both, with the reference standards or with each other. Studies had to be primary diagnostic and report on children aged between 0 to 18 years old with a neuroblastoma of any stage at first diagnosis or at recurrence. Data collection and analysis One review author performed the initial screening of identified references. Two review authors independently performed the study selection, extracted data and assessed the methodological quality. We used data from two-by-two tables, describing at least the number of patients with a true positive test and the number of patients with a false negative test, to calculate the sensitivity, and if possible, the specificity for each included study. If possible, we generated forest plots showing estimates of sensitivity and specificity together with 95% confidence intervals. Main results Eleven studies met the inclusion criteria. Ten studies reported data on patient level: the scan was positive or negative. One study reported on all single lesions (lesion level). The sensitivity of 123I-MIBG (SPECT-CT) scintigraphy (objective 1.1), determined in 608 of 621 eligible patients included in the 11 studies, varied from 67% to 100%. One study, that reported on a lesion level, provided data to calculate the specificity: 68% in 115 lesions in 22 patients. The sensitivity of 123I-MIBG scintigraphy for detecting metastases separately from the primary tumour in patients with all neuroblastoma stages ranged from 79% to 100% in three studies and the specificity ranged from 33% to 89% for two of these studies. One study reported on the diagnostic accuracy of 18F-FDG-PET(-CT) imaging (add-on test) in patients with negative 123I-MIBG scintigraphy (objective 1.2). Two of the 24 eligible patients with proven neuroblastoma had a negative 123I-MIBG scan and a positive 18F-FDG-PET(-CT) scan. The sensitivity of 18F-FDG-PET(-CT) imaging as a single diagnostic test (objective 2.1) and compared to 123I-MIBG (SPECT-CT) (objective 2.2) was only reported in one study. The sensitivity of 18F-FDG-PET(-CT) imaging was 100% versus 92% of 123I-MIBG (SPECT-CT) scintigraphy. We could not calculate the specificity for both modalities. Authors' conclusions The reported sensitivities of 123-I MIBG scintigraphy for the detection of neuroblastoma and its metastases ranged from 67 to 100% in patients with histologically proven neuroblastoma. Only one study in this review reported on false positive findings. It is important to keep in mind that false positive findings can occur. For example, physiological uptake should be ruled out, by using SPECT-CT scans, although more research is needed before definitive conclusions can be made. As described both in the literature and in this review, in about 10% of the patients with histologically proven neuroblastoma the tumour does not accumulate 123I-MIBG (false negative results). For these patients, it is advisable to perform an additional test for staging and assess response to therapy. Additional tests might for example be 18F-FDG-PET(-CT), but to be certain of its clinical value, more evidence is needed. The diagnostic accuracy of 18F-FDG-PET(-CT) imaging in case of a negative 123I-MIBG scintigraphy could not be calculated, because only very limited data were available. Also the detection of the diagnostic accuracy of index test 18F-FDG-PET(-CT) imaging for detecting a neuroblastoma tumour and its metastases, and to compare this to comparator test 123I-MIBG (SPECT-CT) scintigraphy, could not be calculated because of the limited available data at time of this search. At the start of this project, we did not expect to find only very limited data on specificity. We now consider it would have been more appropriate to use the term "the sensitivity to assess the presence of neuroblastoma" instead of "diagnostic accuracy" for the objectives. PLAIN LANGUAGE SUMMARY 123I-MIBG- and 18F-FDG-PET-imaging, two nuclear imaging methods for diagnosing neuroblastoma tumours Background and rationale Neuroblastoma is a childhood tumour that can be visualized by a specific nuclear imaging compound, called metaiodobenzylguanidine (123I -MIBG). 123I-MIBG-imaging is not only important for the diagnosis of neuroblastoma, but also for localization of metastases (spread of the disease to other organs). Sometimes, the neuroblastoma does not take up 123I-MIBG and as a result the neuroblastoma is not visible on the scan. In that case, another type of nuclear imaging might be useful to visualize the neuroblastoma: fluoro-deoxy-glucose – positron emission tomography (18F-FDG-PET)-imaging. In the literature the ability to discriminate between neuroblastoma and non-neuroblastoma lesions for these two types of nuclear imaging methods vary. Prognosis, treatment and response to therapy of patients with neuroblastoma are currently based on scoring the amount of metastases per body segment visible on 123I-MIBG scans. Therefore, it is important to determine the exact ability to discriminate between neuroblastoma and non-neuroblastoma on 123I-MIBG-imaging and 18F-FDG-PET-imaging. We reviewed the evidence about the accuracy of 123I-MIBG-imaging and 18F-FDG-PET-imaging for the detection of a neuroblastoma in children suspected of this disease. Study characteristics We searched scientific databases for clinical studies comparing 123I-MIBG or 18F-FDG-PET imaging, or both, with microscopic examination of tissue suspected of neuroblastoma (histopathology). The evidence is current up to 11 September 2012. We identified 11 eligible studies including 621 children that fulfilled our inclusion criteria: children < 18 years old with a neuroblastoma and 123I-MIBG or 18F-FDG-PET imaging or both. All studies included proven neuroblastoma. Quality of the evidence All 11 included studies had methodological limitations. Only one included study provided data on specificity (the ability of a test to correctly classify an individual as 'disease-free') and therefore we could not perform all of the planned analyses. Key results When compared to histopathological results the sensitivity (the ability of a test to correctly classify an individual person as 'diseased') of 123I-MIBG imaging varied from 67% to 100% in patients with histologically proven neuroblastoma. This means that in 100 children with proven neuroblastoma 123I-MIBG imaging will correctly identify 67 to 100 of the neuroblastoma cases. Only one study, that reported on a lesion level, provided data to calculate the specificity (the ability of a test to correctly classify an individual as 'disease-free'): 68% in 115 lesions. This means that of 100 disease-free lesions in patients with proven neuroblastoma 123I-MIBG imaging will correctly identify 68 lesions. So, in about 10% of the cases the neuroblastoma is not visible on 123I-MIBG imaging (false negative results). For these cases, it is advisable to perform an additional test like 18F-FDG-PET imaging, but to be certain of its clinical value, more evidence is needed. Only one included study reported on false positive findings. This means that 123I-MIBG imaging and 18F-FDG-PET imaging incorrectly identified neuroblastoma lesions in patients which might result in wrongly classifying a patient with metastatic disease. It is important to keep in mind that false positive findings can occur, although more research is needed before definitive conclusions can be made. We could not determine the diagnostic accuracy of 18F-FDG-PET imaging, in case the neuroblastoma was incorrectly not identified with 123I-MIBG, due to limited data. Also, we could not calculate the diagnostic accuracy of 18F-FDG-PET imaging for detecting a neuroblastoma and compare this to 123I-MIBG imaging because of the limited available data. PMID:26417712

Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression

PubMed Central

Candolfi, Marianela; Curtin, James F.; Stephen Nichols, W.; Muhammad, AKM. G.; King, Gwendalyn D.; Elizabeth Pluhar, G.; McNiel, Elizabeth A.; Ohlfest, John R.; Freese, Andrew B.; Moore, Peter F.; Lerner, Jonathan; Lowenstein, Pedro R.

2008-01-01

Although rodent glioblastoma (GBM) models have been used for over 30 years, the extent to which they recapitulate the characteristics encountered in human GBMs remains controversial. We studied the histopathological features of dog GBM and human xenograft GBM models in immune-deficient mice (U251 and U87 GBM in nude Balb/c), and syngeneic GBMs in immune-competent rodents (GL26 cells in C57BL/6 mice, CNS-1 cells in Lewis rats). All GBMs studied exhibited neovascularization, pleomorphism, vimentin immunoreactivity, and infiltration of T-cells and macrophages. All the tumors showed necrosis and hemorrhages, except the U87 human xenograft, in which the most salient feature was its profuse neovascularization. The tumors differed in the expression of astrocytic intermediate filaments: human and dog GBMs, as well as U251 xenografts expressed glial fibrillary acidic protein (GFAP) and vimentin, while the U87 xenograft and the syngeneic rodent GBMs were GFAP− and vimentin+. Also, only dog GBMs exhibited endothelial proliferation, a key feature that was absent in the murine models. In all spontaneous and implanted GBMs we found histopathological features compatible with tumor invasion into the non-neoplastic brain parenchyma. Our data indicate that murine models of GBM appear to recapitulate several of the human GBM histopathological features and, considering their reproducibility and availability, they constitute a valuable in vivo system for preclinical studies. Importantly, our results indicate that dog GBM emerges as an attractive animal model for testing novel therapies in a spontaneous tumor in the context of a larger brain. PMID:17874037

Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

PubMed

Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

2016-02-01

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

PubMed

Burchill, Susan A; Beiske, Klaus; Shimada, Hiroyuki; Ambros, Peter F; Seeger, Robert; Tytgat, Godelieve A M; Brock, Penelope R; Haber, Michelle; Park, Julie R; Berthold, Frank

2017-04-01

The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society. © 2016 American Cancer Society.

Localization of m-lodo(/sup 131/I)benzylguanidine in neuroblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hattner, R.S.; Huberty, J.P.; Engelstad, B.L.

1984-08-01

Patient survival and the therapeutic strategy for treatment of neuroblastoma are highly dependent on the stage of the tumor at presentation. For routine staging, the Children's Cancer Study group currently recommends a chest radiograph, abdominal CT scan, radionuclide bone scan, bone marrow biopsy, catecholamine metabolite estimations, and surgical determination of tumor extent. A noninvasive method for detectiton of neuroblastoma that avoids surgery and bone marrow biopsy would be a most welcome addition to the armamentarium of the pediatric oncologist. A case of neuroblastoma demonstrated with m-iodo(/sup 131/I)benzylguanidine (MIBG) scintigraphy is reported.

Effective Targeting of the P53/MDM2 Axis in Preclinical Models of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

PubMed Central

Richmond, Jennifer; Carol, Hernan; Evans, Kathryn; High, Laura; Mendomo, Agnes; Robbins, Alissa; Meyer, Claus; Venn, Nicola C.; Marschalek, Rolf; Henderson, Michelle; Sutton, Rosemary; Kurmasheva, Raushan T.; Kees, Ursula R.; Houghton, Peter J.; Smith, Malcolm A.; Lock, Richard B.

2015-01-01

Purpose While the overall cure rate for pediatric acute lymphoblastic leukemia (ALL) approaches 90%, infants with ALL harboring translocations in the mixed-lineage leukemia (MLL) oncogene (infant MLL-ALL) experience shorter remission duration and lower survival rates (∼50%). Mutations in the p53 tumor suppressor gene are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be beneficial. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenografts. Experimental Design Eight MLL-ALL patient-derived xenografts were established in immune-deficient mice, and their molecular features compared with B-lineage ALL and T-ALL xenografts. The sensitivity of MLL-ALL xenografts to RG7112 was assessed in vitro and in vivo, and the ability of RG7112 to induce p53, cell cycle arrest and apoptosis in vivo was evaluated. Results Gene expression analysis revealed that MLL-ALL, B-lineage ALL and T-ALL xenografts clustered according to subtype. Moreover, genes previously reported to be over-expressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA family, were significantly up-regulated in MLL-ALL xenografts, confirming their ability to recapitulate the clinical disease. Exposure of MLL-ALL xenografts to RG7112 in vivo caused p53 up-regulation, cell cycle arrest and apoptosis. RG7112 as a single agent induced significant regressions in infant MLL-ALL xenografts. Therapeutic enhancement was observed when RG7112 was assessed using combination treatment with an induction-type regimen (vincristine/dexamethasone/L-asparaginase) against an MLL-ALL xenograft. Conclusion The utility of targeting the p53-MDM2 axis in combination with established drugs for the management of infant MLL-ALL warrants further investigation. PMID:25573381

Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation.

PubMed

Akter, Jesmin; Takatori, Atsushi; Islam, Md Sazzadul; Nakazawa, Atsuko; Ozaki, Toshinori; Nagase, Hiroki; Nakagawara, Akira

2014-10-10

We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas. Copyright © 2014 Elsevier Inc. All rights reserved.

Breakpoint Features of Genomic Rearrangements in Neuroblastoma with Unbalanced Translocations and Chromothripsis

PubMed Central

Daveau, Romain; Combaret, Valérie; Pierre-Eugène, Cécile; Cazes, Alex; Louis-Brennetot, Caroline; Schleiermacher, Gudrun; Ferrand, Sandrine; Pierron, Gaëlle; Lermine, Alban; Frio, Thomas Rio; Raynal, Virginie; Vassal, Gilles; Barillot, Emmanuel; Delattre, Olivier; Janoueix-Lerosey, Isabelle

2013-01-01

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis. PMID:23991058

Upregulation of LYAR induces neuroblastoma cell proliferation and survival.

PubMed

Sun, Yuting; Atmadibrata, Bernard; Yu, Denise; Wong, Matthew; Liu, Bing; Ho, Nicholas; Ling, Dora; Tee, Andrew E; Wang, Jenny; Mungrue, Imran N; Liu, Pei Y; Liu, Tao

2017-09-01

The N-Myc oncoprotein induces neuroblastoma by regulating gene transcription and consequently causing cell proliferation. Paradoxically, N-Myc is well known to induce apoptosis by upregulating pro-apoptosis genes, and it is not clear how N-Myc overexpressing neuroblastoma cells escape N-Myc-mediated apoptosis. The nuclear zinc finger protein LYAR has recently been shown to modulate gene expression by forming a protein complex with the protein arginine methyltransferase PRMT5. Here we showed that N-Myc upregulated LYAR gene expression by binding to its gene promoter. Genome-wide differential gene expression studies revealed that knocking down LYAR considerably upregulated the expression of oxidative stress genes including CHAC1, which depletes intracellular glutathione and induces oxidative stress. Although knocking down LYAR expression with siRNAs induced oxidative stress, neuroblastoma cell growth inhibition and apoptosis, co-treatment with the glutathione supplement N-acetyl-l-cysteine or co-transfection with CHAC1 siRNAs blocked the effect of LYAR siRNAs. Importantly, high levels of LYAR gene expression in human neuroblastoma tissues predicted poor event-free and overall survival in neuroblastoma patients, independent of the best current markers for poor prognosis. Taken together, our data suggest that LYAR induces proliferation and promotes survival of neuroblastoma cells by repressing the expression of oxidative stress genes such as CHAC1 and suppressing oxidative stress, and identify LYAR as a novel co-factor in N-Myc oncogenesis.

Neuroblastoma.

PubMed

Nakagawara, Akira; Li, Yuanyuan; Izumi, Hideki; Muramori, Katsumi; Inada, Hiroko; Nishi, Masanori

2018-03-01

Neuroblastoma is one of the most common solid tumors in children and has a diverse clinical behavior that largely depends on the tumor biology. Neuroblastoma exhibits unique features, such as early age of onset, high frequency of metastatic disease at diagnosis in patients over 1 year of age and the tendency for spontaneous regression of tumors in infants. The high-risk tumors frequently have amplification of the MYCN oncogene as well as segmental chromosome alterations with poor survival. Recent advanced genomic sequencing technology has revealed that mutation of ALK, which is present in ~10% of primary tumors, often causes familial neuroblastoma with germline mutation. However, the frequency of gene mutations is relatively small and other aberrations, such as epigenetic abnormalities, have also been proposed. The risk-stratified therapy was introduced by the Japan Neuroblastoma Study Group (JNBSG), which is now moving to the Neuroblastoma Committee of Japan Children's Cancer Group (JCCG). Several clinical studies have facilitated the reduction of therapy for children with low-risk neuroblastoma disease and the significant improvement of cure rates for patients with intermediate-risk as well as high-risk disease. Therapy for patients with high-risk disease includes intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy. The JCCG aims for better cures and long-term quality of life for children with cancer by facilitating new approaches targeting novel driver proteins, genetic pathways and the tumor microenvironment.

Establishment and characterization of a human gastrointestinal stromal tumour (GIST) xenograft in athymic nude mice.

PubMed

Revheim, Mona-Elisabeth; Seierstad, Therese; Berner, Jeanne-Marie; Bruland, Oyvind Sverre; Røe, Kathrine; Ohnstad, Hege Oma; Bjerkehagen, Bodil; Bach-Gansmo, Tore

2009-11-01

The majority of gastrointestinal stromal tumours (GISTs) contain oncogenic KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) or platelet-derived growth factor-alpha (PDGFRA) receptor tyrosine kinase (TK) mutations and are initially, but only temporarily sensitive to TK inhibitors. The aim of this study was to establish and characterize a human GIST xenograft that could be used for evaluating various molecularly targeted therapies. GIST tissue from four patients was implanted under the skin of athymic nude mice. In one case a tumour line was established. The xenograft showed characteristic GIST morphology and exhibited the same mutation profile as that of the patient. A human GIST xenograft with mutation in KIT exons 11 and 17 has been established and maintained in nude mice for 3 years (13 passages). This model will enable further studies on mechanisms of resistance, combination therapies and allow testing of novel targeted therapies.

Clinically Relevant Cytotoxic Immune Cell Signatures and Clonal Expansion of T Cell Receptors in High-risk MYCN-not-amplified Human Neuroblastoma.

PubMed

Wei, Jun S; Kuznetsov, Igor B; Zhang, Shile; Song, Young K; Asgharzadeh, Shahab; Sindiri, Sivasish; Wen, Xinyu; Patidar, Rajesh; Nagaraj, Sushma; Walton, Ashley; Guidry Auvil, Jaime M; Gerhard, Daniela S; Yuksel, Aysen; Catchpoole, Daniel R; Hewitt, Stephen M; Sondel, Paul M; Seeger, Robert C; Maris, John M; Khan, Javed

2018-05-21

High-risk neuroblastoma is an aggressive disease. DNA sequencing studies have revealed a paucity of actionable genomic alterations and a low mutation burden, posing challenges to develop effective novel therapies. We used RNA sequencing (RNA-seq) to investigate the biology of this disease including a focus on tumor-infiltrating lymphocytes (TILs). We performed deep RNA-seq on pre-treatment diagnostic tumors from 129 high-risk and 21 low- or intermediate-risk patients with neuroblastomas. We used single-sample gene set enrichment analysis to detect gene expression signatures of TILs in tumors and examined their association with clinical and molecular parameters including patient outcome. The expression profiles of 190 additional pre-treatment diagnostic neuroblastomas, a neuroblastoma tissue microarray, and T-cell receptor (TCR) sequencing were used to validate our findings. We found that MYCN -not-amplified ( MYCN -NA) tumors had significant higher cytotoxic TIL signatures compared to MYCN -amplified ( MYCN -A) tumors. A reported MYCN-activation-signature was significantly associated with poor outcome for high-risk patients with MYCN -NA tumors; however, a subgroup of these patients who had elevated activated NK cells, CD8+ T-cells, and cytolytic signatures showed improved outcome and expansion of infiltrating T-cell receptor (TCR) clones. Furthermore, we observed up-regulation of immune exhaustion marker genes, indicating an immune suppressive microenvironment in these neuroblastomas. Conclusions: This study provides evidence that RNA signatures of cytotoxic TIL are associated with the presence of activated NK-/T-cells and improved outcomes in high-risk neuroblastoma patients harboring MYCN -NA tumors. Our findings suggest that these high-risk patients with MYCN -NA neuroblastoma may benefit from additional immunotherapies incorporated into the current therapeutic strategies. Copyright ©2018, American Association for Cancer Research.

Case control study of neuroblastoma in west-Germany after the Chernobyl accident.

PubMed

Michaelis, J; Haaf, H G; Zöllner, J; Kaatsch, P; Krummenauer, F; Berthold, F

1996-01-01

To explore possible causes of a 1988 incidence peak of infant neuroblastoma in west German regions which were contaminated with more than 6000 Bq/m2 Cs137 from the Chernobyl accident. The primary working hypothesis was that parents of the diseased children had been contaminated by an excessive intake of locally produced food, especially mushrooms or deer. Case control study with 1:2 (cases:controls) matching. Data were collected from the children's parents by questionnaires and telephone interviews. Nation-wide study (former FRG) based on the German Childhood Cancer Registry. Cases born in 1988 and reported with a neuroblastoma to the registry until March 1992. Population-based healthy controls, matched for age, sex and residence at time of diagnosis. The working hypothesis could not be confirmed by the study, because the parents of cases tended to eat less locally grown food than the parents of controls (RR = 0.63, 95% CI:0.20-1.97). Possible influence factors which previously have been described to be associated with neuroblastoma incidence could not be confirmed by the study. Parental exposure to herbicides and pesticides was associated with the occurrence of neuroblastoma (RR = 4.2, 95% CI:1.4-12.9). Neuroblastoma stage distribution in the contaminated regions was shifted towards lower stages as compared to the less contaminated regions and previous age cohorts. The study does not show additional evidence that the observed increase in neuroblastoma incidence might have been caused by exposure to fallout from the Chernobyl accident. The observed shift towards lower clinical stages may rather indicate increased diagnostic awareness. The association between neuroblastoma and parental exposure with herbicides and pesticides resulted from an extensive exploratory data analysis and needs to be confirmed in further studies.

Identification of ALK as the Major Familial Neuroblastoma Predisposition Gene

PubMed Central

Mossë, Yalë P; Laudenslager, Marci; Longo, Luca; Cole, Kristina A; Wood, Andrew; Attiyeh, Edward F; Laquaglia, Michael J; Sennett, Rachel; Lynch, Jill E; Perri, Patrizia; Laureys, Geneviève; Speleman, Frank; Hakonarson, Hakon; Torkamani, Ali; Schork, Nicholas J; Brodeur, Garrett M; Tonini, Gian Paolo; Rappaport, Eric; Devoto, Marcella; Maris, John M

2009-01-01

SUMMARY Survival rates for the childhood cancer neuroblastoma have not substantively improved despite dramatic escalation in chemotherapy intensity. Like most human cancers, this embryonal malignancy can be inherited, but the genetic etiology of familial and sporadically occurring neuroblastoma was largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase gene (ALK) explain the majority of hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at the short arm of chromosome 2 (maximum nonparametric LOD=4.23 at rs1344063) using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate missense mutations in the tyrosine kinase domain of ALK (G1128A, R1192P and R1275Q) that segregated with the disease in eight separate families. Examination of 491 sporadically occurring human neuroblastoma samples showed that the ALK locus was gained in 22.8%, and highly amplified in an additional 3.3%, and that these aberrations were highly associated with death from disease (P=0.0003). Resequencing of 194 high-risk neuroblastoma samples showed somatically acquired mutations within the tyrosine kinase domain in 12.4%. Nine of the ten mutations map to critical regions of the kinase domain and were predicted to be oncogenic drivers with high probability. Mutations resulted in constitutive phosphorylation consistent with activation, and targeted knockdown of ALK mRNA resulted in profound growth inhibition of 4 of 4 cell lines harboring mutant or amplified ALK, as well as 2 of 6 wild type for ALK. Our results demonstrate that heritable mutations of ALK are the major cause of familial neuroblastoma, and that germline or acquired activation of this cell surface kinase is a tractable therapeutic target for this lethal pediatric malignancy. PMID:18724359

Cross-Cohort Analysis Identifies a TEAD4-MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma.

PubMed

Rajbhandari, Presha; Lopez, Gonzalo; Capdevila, Claudia; Salvatori, Beatrice; Yu, Jiyang; Rodriguez-Barrueco, Ruth; Martinez, Daniel; Yarmarkovich, Mark; Weichert-Leahey, Nina; Abraham, Brian J; Alvarez, Mariano J; Iyer, Archana; Harenza, Jo Lynne; Oldridge, Derek; De Preter, Katleen; Koster, Jan; Asgharzadeh, Shahab; Seeger, Robert C; Wei, Jun S; Khan, Javed; Vandesompele, Jo; Mestdagh, Pieter; Versteeg, Rogier; Look, A Thomas; Young, Richard A; Iavarone, Antonio; Lasorella, Anna; Silva, Jose M; Maris, John M; Califano, Andrea

2018-05-01

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN -amplified ( MYCN Amp ) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCN Amp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582-99. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 517 . ©2018 American Association for Cancer Research.

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression

PubMed Central

Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M.; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

2017-01-01

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma. PMID:28696319

Telomerase activation by genomic rearrangements in high-risk neuroblastoma

PubMed Central

Peifer, Martin; Hertwig, Falk; Roels, Frederik; Dreidax, Daniel; Gartlgruber, Moritz; Menon, Roopika; Krämer, Andrea; Roncaioli, Justin L.; Sand, Frederik; Heuckmann, Johannes M.; Ikram, Fakhera; Schmidt, Rene; Ackermann, Sandra; Engesser, Anne; Kahlert, Yvonne; Vogel, Wenzel; Altmüller, Janine; Nürnberg, Peter; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Mariappan, Aruljothi; Heynck, Stefanie; Mariotti, Erika; Henrich, Kai-Oliver; Glöckner, Christian; Bosco, Graziella; Leuschner, Ivo; Schweiger, Michal R.; Savelyeva, Larissa; Watkins, Simon C.; Shao, Chunxuan; Bell, Emma; Höfer, Thomas; Achter, Viktor; Lang, Ulrich; Theissen, Jessica; Volland, Ruth; Saadati, Maral; Eggert, Angelika; de Wilde, Bram; Berthold, Frank; Peng, Zhiyu; Zhao, Chen; Shi, Leming; Ortmann, Monika; Büttner, Reinhard; Perner, Sven; Hero, Barbara; Schramm, Alexander; Schulte, Johannes H.; Herrmann, Carl; O’Sullivan, Roderick J.; Westermann, Frank; Thomas, Roman K.; Fischer, Matthias

2016-01-01

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system1. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2–4. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type1,2,5. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours. PMID:26466568

PHOX2B reliably distinguishes neuroblastoma among small round blue cell tumours.

PubMed

Hung, Yin P; Lee, John P; Bellizzi, Andrew M; Hornick, Jason L

2017-11-01

Neuroblastoma shows considerable histological overlap with other small round blue cell tumours. PHOX2B, a transcription factor that is essential for autonomic nervous system development, has been reported as an immunohistochemical marker for neuroblastoma. The aim of this study was to validate the specificity and diagnostic utility of PHOX2B for peripheral neuroblastic tumours. We evaluated 240 cases (133 in whole-tissue sections; 107 in tissue microarrays), including 76 peripheral neuroblastic tumours (median age 2 years; including four adults) and 164 other tumours: 44 Wilms tumours; 20 Ewing sarcomas; 10 each of CIC-rearranged round cell sarcomas, poorly differentiated synovial sarcomas, lymphoblastic lymphomas, alveolar rhabdomyosarcomas, embryonal rhabdomyosarcomas, mesenchymal chondrosarcomas, Merkel cell carcinomas, olfactory neuroblastomas, and melanomas; and five each of NUT midline carcinomas and desmoplastic small round cell tumours. Immunohistochemistry for PHOX2B was performed with a rabbit monoclonal antibody. PHOX2B positivity was defined as the presence of nuclear immunoreactivity in ≥5% of cells. PHOX2B was positive in 70 (92%) peripheral neuroblastic tumours, including 68 of 72 (94%) paediatric and two of four (50%) adult cases. Furthermore, PHOX2B was consistently negative in all non-peripheral neuroblastic tumours, with staining being absent in 160 cases and limited in four cases. PHOX2B is a highly sensitive and specific immunohistochemical marker for peripheral neuroblastic tumours, including neuroblastoma. PHOX2B reliably distinguishes neuroblastoma from histological mimics such as Wilms tumour, Ewing sarcoma, and CIC-rearranged round cell sarcoma. PHOX2B negativity in two of four adult neuroblastoma cases raises the possibility that some adult neuroblastomas are of a different lineage than paediatric cases. © 2017 John Wiley & Sons Ltd.

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression.

PubMed

Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

2017-07-25

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

Osteochondroma in long-term survivors of high-risk neuroblastoma.

PubMed

Kushner, Brian H; Roberts, Stephen S; Friedman, Danielle N; Kuk, Deborah; Ostrovnaya, Irina; Modak, Shakeel; Kramer, Kim; Basu, Ellen M; Cheung, Nai-Kong V

2015-06-15

Osteochondromas are benign bony protrusions that can be spontaneous or associated with radiotherapy (RT). Current treatment of high-risk neuroblastoma includes dose-intensive chemotherapy, local RT, an anti-GD2 monoclonal antibody (MoAb), and isotretinoin. Late effects are emerging. The authors examined osteochondromas in 362 patients who were aged <10 years when diagnosed with neuroblastoma, had received a MoAb plus isotretinoin since 2000, and had survived >24 months from the time of the first dose of the MoAb. The incidence rate of osteochondroma was determined using the competing risks approach, in which the primary event was osteochondroma calculated from the date of neuroblastoma diagnosis and the competing event was death without osteochondroma. A total of 21 osteochondroma cases were found among 14 patients who were aged 5.7 to 15.3 years (median, 10.4 years) and 3.1 to 11.2 years (median, 8.2 years) from the time of neuroblastoma diagnosis. The cumulative incidence rate was 0.6% at 5 years and 4.9% at 10 years from the neuroblastoma diagnosis. Nine osteochondromas were revealed incidentally during assessments of neuroblastoma disease status or bone age. Thirteen osteochondromas were detected outside RT portals and had characteristics of spontaneous forms. Complications were limited to pain necessitating surgical resection in 3 patients, but follow-up was short at 0.3 to 7.7 years (median, 3.5 years). Osteochondromas in long-term survivors of neuroblastoma should be expected because these benign growths can be related to RT and these patients undergo radiologic studies over years, are monitored for late toxicities through and beyond adolescence, and receive special attention (because of concerns about disease recurrence) if they develop a bony protuberance. A pathogenic role for chemotherapy, anti-GD2 MoAbs, or isotretinoin remains speculative. © 2015 American Cancer Society.

Suppression of RRM2 inhibits cell proliferation, causes cell cycle arrest and promotes the apoptosis of human neuroblastoma cells and in human neuroblastoma RRM2 is suppressed following chemotherapy.

PubMed

Li, Junfeng; Pang, Jinglin; Liu, Yongdong; Zhang, Jing; Zhang, Chuanguang; Shen, Gang; Song, Lili

2018-07-01

Ribonucleotide reductase regulatory subunit M2 (RRM2) is a rate‑limiting enzyme for DNA synthesis and repair. RRM2 has vital roles in controlling the progression of cancer. In the present study, we investigated the RRM2 level in neuroblastoma tissues, analyzed its relationship with clinicopathological characteristics of neuroblastoma patients, and explored the effect of RRM2 on the biological functions of neuroblastoma cells. RRM2 levels in 67 pairs of neuroblastoma and matched adjacent non‑cancerous tissues were detected by qRT‑PCR, and its association with patient clinicopathological features was assessed. Using RRM2 siRNA, the role of RRM2 in cell viability was detected by CCK‑8 assay, and the effects on cell cycle distribution and cell apoptosis were detected by flow cytometry. Hoechst 33342 staining was also performed. For RRM2 protein detection in cells and tissues, western blot analyses were employed. Our results revealed that RRM2 expression was significant higher in neuroblastoma tissues than that noted in adjacent non‑cancerous tissues at both the mRNA and protein levels. The increased RRM2 level was significantly associated with clinical stage. RRM2 levels were suppressed in stage III and IV tumors in the chemotherapy subgroup, compared with levels noted in tumors in the preoperative non‑chemotherapy subgroup. RRM2 siRNA significantly inhibited cell viability in the SH‑5Y5Y cells, induced cell arrest in the G0/G1 phase, and enhanced cell apoptosis. Taken together, overexpression of RRM2 is associated with the genesis and progression of neuroblastoma, and may be a potential chemotherapeutic target.

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma.

PubMed

Vieira, Gabriella Cunha; Chockalingam, S; Melegh, Zsombor; Greenhough, Alexander; Malik, Sally; Szemes, Marianna; Park, Ji Hyun; Kaidi, Abderrahmane; Zhou, Li; Catchpoole, Daniel; Morgan, Rhys; Bates, David O; Gabb, Peter David; Malik, Karim

2015-11-24

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma

PubMed Central

Melegh, Zsombor; Greenhough, Alexander; Malik, Sally; Szemes, Marianna; Park, Ji Hyun; Kaidi, Abderrahmane; Zhou, Li; Catchpoole, Daniel; Morgan, Rhys; Bates, David O.; Gabb, Peter J.; Malik, Karim

2015-01-01

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5. PMID:26517508

LINC00673 rs11655237 C>T confers neuroblastoma susceptibility in Chinese population

PubMed Central

Zhang, Zhuorong; Chang, Yitian; Jia, Wei; Zhang, Jiao; Zhang, Ruizhong; Zhu, Jinhong; Yang, Tianyou

2018-01-01

Neuroblastoma, which accounts for approximately 10% of all pediatric cancer-related deaths, has become a therapeutic challenge and global burden attributed to poor outcomes and mortality rates of its high-risk form. Previous genome-wide association studies (GWASs) identified the LINC00673 rs11655237 C>T polymorphism to be associated with the susceptibility of several malignant tumors. However, the association between this polymorphism and neuroblastoma susceptibility is not clear. We genotyped LINC00673 rs11655237 C>T in 393 neuroblastoma patients in comparison with 812 age-, gender-, and ethnicity-matched healthy controls. We found a significant association between the LINC00673 rs11655237 C>T polymorphism and neuroblastoma risk (TT compared with CC: adjusted odds ratio (OR) =1.80, 95% confidence interval (CI) =1.06–3.06, P=0.029; TT/CT compared with CC: adjusted OR =1.31, 95% CI =1.02–1.67, P=0.033; and T compared with C: adjusted OR =1.29, 95% CI =1.06–1.58, P=0.013). Furthermore, stratified analysis indicated that the rs11655237 T allele carriers were associated with increased neuroblastoma risk for patients with tumor originating from the adrenal gland (adjusted OR =1.51, 95% CI =1.06–2.14, P=0.021) and International Neuroblastoma Staging System (INSS) stage IV disease (adjusted OR =1.60, 95% CI =1.12–2.30, P=0.011). In conclusion, we verified that the LINC00673 rs11655237 C>T polymorphism might be associated with neuroblastoma susceptibility. Prospective studies with a large sample size and different ethnicities are needed to validate our findings. PMID:29339420

Ternary copper(II) complex: NCI60 screening, toxicity studies, and evaluation of efficacy in xenograft models of nasopharyngeal carcinoma.

PubMed

Ahmad, Munirah; Suhaimi, Shazlan-Noor; Chu, Tai-Lin; Abdul Aziz, Norazlin; Mohd Kornain, Noor-Kaslina; Samiulla, D S; Lo, Kwok-Wai; Ng, Chew-Hee; Khoo, Alan Soo-Beng

2018-01-01

Copper(II) ternary complex, [Cu(phen)(C-dmg)(H2O)]NO3 was evaluated against a panel of cell lines, tested for in vivo efficacy in nasopharyngeal carcinoma xenograft models as well as for toxicity in NOD scid gamma mice. The Cu(II) complex displayed broad spectrum cytotoxicity against multiple cancer types, including lung, colon, central nervous system, melanoma, ovarian, and prostate cancer cell lines in the NCI-60 panel. The Cu(II) complex did not cause significant induction of cytochrome P450 (CYP) 3A and 1A enzymes but moderately inhibited CYP isoforms 1A2, 2C9, 2C19, 2D6, 2B6, 2C8 and 3A4. The complex significantly inhibited tumor growth in nasopharyngeal carcinoma xenograft bearing mice models at doses which were well tolerated without causing significant or permanent toxic side effects. However, higher doses which resulted in better inhibition of tumor growth also resulted in toxicity.

THE HUMAN FETAL LUNG XENOGRAFT: VALIDATION AS MODEL OF MICROVASCULAR REMODELING IN THE POSTGLANDULAR LUNG

PubMed Central

De Paepe, Monique E.; Chu, Sharon; Hall, Susan; Heger, Nicholas; Thanos, Chris; Mao, Quanfu

2012-01-01

Background Coordinated remodeling of epithelium and vasculature is essential for normal postglandular lung development. The value of the human-to-rodent lung xenograft as model of fetal microvascular development remains poorly defined. Aim The aim of this study was to determine the fate of the endogenous (human-derived) microvasculature in fetal lung xenografts. Methods Lung tissues were obtained from spontaneous pregnancy losses (14–22 weeks’ gestation) and implanted in the renal subcapsular or dorsal subcutaneous space of SCID-beige mice (T, B and NK-cell-deficient) and/or nude rats (T-cell-deficient). Informed parental consent was obtained. Lung morphogenesis, microvascular angiogenesis and epithelial differentiation were assessed at two and four weeks post-transplantation by light microscopy, immunohistochemical and gene expression studies. Archival age-matched postmortem lungs served as control. Results The vascular morphology, density and proliferation of renal subcapsular grafts in SCID-beige mice were similar to age-matched control lungs, with preservation of the physiologic association between epithelium and vasculature. The microvasculature of subcutaneous grafts in SCID-beige mice was underdeveloped and dysmorphic, associated with significantly lower VEGF, endoglin, and angiopoietin-2 mRNA expression than renal grafts. Grafts at both sites displayed mild airspace dysplasia. Renal subcapsular grafts in nude rats showed frequent infiltration by host lymphocytes and obliterating bronchiolitis-like changes, associated with markedly decreased endogenous angiogenesis. Conclusion This study demonstrates the critical importance of host and site selection to ensure optimal xenograft development. When transplanted to severely immune suppressed, NK-cell-deficient hosts and engrafted in the renal subcapsular site, the human-to-rodent fetal lung xenograft provides a valid model of postglandular microvascular lung remodeling. PMID:22811288

Genomewide analysis of gene expression associated with Tcof1 in mouse neuroblastoma.

PubMed

Mogass, Michael; York, Timothy P; Li, Lin; Rujirabanjerd, Sinitdhorn; Shiang, Rita

2004-12-03

Mutations in the Treacher Collins syndrome gene, TCOF1, cause a disorder of craniofacial development. We manipulated the levels of Tcof1 and its protein treacle in a murine neuroblastoma cell line to identify downstream changes in gene expression using a microarray platform. We identified a set of genes that have similar expression with Tcof1 as well as a set of genes that are negatively correlated with Tcof1 expression. We also showed that the level of Tcof1 and treacle expression is downregulated during differentiation of neuroblastoma cells into neuronal cells. Inhibition of Tcof1 expression by siRNA induced morphological changes in neuroblastoma cells that mimic differentiation. Thus, expression of Tcof1 and treacle synthesis play an important role in the proliferation of neuroblastoma cells and we have identified genes that may be important in this pathway.

LMO1 Synergizes with MYCN to Promote Neuroblastoma Initiation and Metastasis.

PubMed

Zhu, Shizhen; Zhang, Xiaoling; Weichert-Leahey, Nina; Dong, Zhiwei; Zhang, Cheng; Lopez, Gonzalo; Tao, Ting; He, Shuning; Wood, Andrew C; Oldridge, Derek; Ung, Choong Yong; van Ree, Janine H; Khan, Amish; Salazar, Brittany M; Lummertz da Rocha, Edroaldo; Zimmerman, Mark W; Guo, Feng; Cao, Hong; Hou, Xiaonan; Weroha, S John; Perez-Atayde, Antonio R; Neuberg, Donna S; Meves, Alexander; McNiven, Mark A; van Deursen, Jan M; Li, Hu; Maris, John M; Look, A Thomas

2017-09-11

A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell Survival Signaling in Neuroblastoma

PubMed Central

Megison, Michael L.; Gillory, Lauren A.; Beierle, Elizabeth A.

2013-01-01

Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Neuroblastoma tumorigenesis and malignant transformation is driven by overexpression and dominance of cell survival pathways and a lack of normal cellular senescence or apoptosis. Therefore, manipulation of cell survival pathways may decrease the malignant potential of these tumors and provide avenues for the development of novel therapeutics. This review focuses on several facets of cell survival pathways including protein kinases (PI3K, AKT, ALK, and FAK), transcription factors (NF-κB, MYCN and p53), and growth factors (IGF, EGF, PDGF, and VEGF). Modulation of each of these factors decreases the growth or otherwise hinders the malignant potential of neuroblastoma, and many therapeutics targeting these pathways are already in the clinical trial phase of development. Continued research and discovery of effective modulators of these pathways will revolutionize the treatment of neuroblastoma. PMID:22934706

[The reaction of the neuroblastoma cells in the culture on the influence of tretionine and neurotoxine].

PubMed

Magakian, Iu A; Karalian, Z A; Karalova, E M; Abroian, L O; Akopian, L A; Avetisian, A C; Semerdzhian, Z B

2011-01-01

Effect of the tretionine (retinoid) and aluminum chloride (neurotoxin) on the growth and differentiation of neuroblastoma cells in culture after their introduction into the medium separately and in combination was studied. The introduction of these substances creates a new information field in the medium, which becomes apparent by the reactions of neuroblastoma found on the populational and cellular levels of its organization. The presence of tretionine stimulates proliferation and induces differentiation of the cells into astrocytes. Aluminum chloride inhibits cell proliferation and enhances the process of their destruction in the monolayer. The variety of the reactions of neuroblastoma cells to the presence of these substances in the medium indicates the existence and functioning of a mechanism that selects from the information introduced only the portion which may contribute to adaptation of neuroblastoma cells to the changed culture conditions.

Initiation and Characterization of Small Cell Lung Cancer Patient-Derived Xenografts from Ultrasound-Guided Transbronchial Needle Aspirates

PubMed Central

Anderson, Wade C.; Boyd, Michael B.; Aguilar, Jorge; Pickell, Brett; Laysang, Amy; Pysz, Marybeth A.; Bheddah, Sheila; Ramoth, Johanna; Slingerland, Brian C.; Dylla, Scott J.; Rubio, Edmundo R.

2015-01-01

Small cell lung cancer (SCLC) is a devastating disease with limited treatment options. Due to its early metastatic nature and rapid growth, surgical resection is rare. Standard of care treatment regimens remain largely unchanged since the 1980’s, and five-year survival lingers near 5%. Patient-derived xenograft (PDX) models have been established for other tumor types, amplifying material for research and serving as models for preclinical experimentation; however, limited availability of primary tissue has curtailed development of these models for SCLC. The objective of this study was to establish PDX models from commonly collected fine needle aspirate biopsies of primary SCLC tumors, and to assess their utility as research models of primary SCLC tumors. These transbronchial needle aspirates efficiently engrafted as xenografts, and tumor histomorphology was similar to primary tumors. Resulting tumors were further characterized by H&E and immunohistochemistry, cryopreserved, and used to propagate tumor-bearing mice for the evaluation of standard of care chemotherapy regimens, to assess their utility as models for tumors in SCLC patients. When treated with Cisplatin and Etoposide, tumor-bearing mice responded similarly to patients from whom the tumors originated. Here, we demonstrate that PDX tumor models can be efficiently established from primary SCLC transbronchial needle aspirates, even after overnight shipping, and that resulting xenograft tumors are similar to matched primary tumors in cancer patients by both histology and chemo-sensitivity. This method enables physicians at non-research institutions to collaboratively contribute to the rapid establishment of extensive PDX collections of SCLC, enabling experimentation with clinically relevant tissues and development of improved therapies for SCLC patients. PMID:25955027

Radiotherapy and chemotherapy change vessel tree geometry and metastatic spread in a small cell lung cancer xenograft mouse tumor model

PubMed Central

Bethge, Anja; Schumacher, Udo

2017-01-01

Background Tumor vasculature is critical for tumor growth, formation of distant metastases and efficiency of radio- and chemotherapy treatments. However, how the vasculature itself is affected during cancer treatment regarding to the metastatic behavior has not been thoroughly investigated. Therefore, the aim of this study was to analyze the influence of hypofractionated radiotherapy and cisplatin chemotherapy on vessel tree geometry and metastasis formation in a small cell lung cancer xenograft mouse tumor model to investigate the spread of malignant cells during different treatments modalities. Methods The biological data gained during these experiments were fed into our previously developed computer model “Cancer and Treatment Simulation Tool” (CaTSiT) to model the growth of the primary tumor, its metastatic deposit and also the influence on different therapies. Furthermore, we performed quantitative histology analyses to verify our predictions in xenograft mouse tumor model. Results According to the computer simulation the number of cells engrafting must vary considerably to explain the different weights of the primary tumor at the end of the experiment. Once a primary tumor is established, the fractal dimension of its vasculature correlates with the tumor size. Furthermore, the fractal dimension of the tumor vasculature changes during treatment, indicating that the therapy affects the blood vessels’ geometry. We corroborated these findings with a quantitative histological analysis showing that the blood vessel density is depleted during radiotherapy and cisplatin chemotherapy. The CaTSiT computer model reveals that chemotherapy influences the tumor’s therapeutic susceptibility and its metastatic spreading behavior. Conclusion Using a system biological approach in combination with xenograft models and computer simulations revealed that the usage of chemotherapy and radiation therapy determines the spreading behavior by changing the blood vessel geometry of the primary tumor. PMID:29107953

Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model.

PubMed

Drabsch, Yvette; He, Shuning; Zhang, Long; Snaar-Jagalska, B Ewa; ten Dijke, Peter

2013-11-07

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

Intracellular Doppler Signatures of Platinum Sensitivity Captured by Biodynamic Profiling in Ovarian Xenografts

NASA Astrophysics Data System (ADS)

Merrill, Daniel; An, Ran; Sun, Hao; Yakubov, Bakhtiyor; Matei, Daniela; Turek, John; Nolte, David

2016-01-01

Three-dimensional (3D) tissue cultures are replacing conventional two-dimensional (2D) cultures for applications in cancer drug development. However, direct comparisons of in vitro 3D models relative to in vivo models derived from the same cell lines have not been reported because of the lack of sensitive optical probes that can extract high-content information from deep inside living tissue. Here we report the use of biodynamic imaging (BDI) to measure response to platinum in 3D living tissue. BDI combines low-coherence digital holography with intracellular Doppler spectroscopy to study tumor drug response. Human ovarian cancer cell lines were grown either in vitro as 3D multicellular monoculture spheroids or as xenografts in nude mice. Fragments of xenografts grown in vivo in nude mice from a platinum-sensitive human ovarian cell line showed rapid and dramatic signatures of induced cell death when exposed to platinum ex vivo, while the corresponding 3D multicellular spheroids grown in vitro showed negligible response. The differences in drug response between in vivo and in vitro growth have important implications for predicting chemotherapeutic response using tumor biopsies from patients or patient-derived xenografts.

Development of resting membrane potentials in differentiating murine neuroblastoma cells (N1E-115) evaluated by flow cytometry.

PubMed

Kisaalita, W S; Bowen, J M

1997-09-01

With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential (V(m)) and forward angle light scatter (FALS) profiles of a differentiating/differentiated murine neuroblastoma cell line (N1E-115). Electrophysiological differentiation was characterized by V(m) establishment. The (V(m))-time profile was found to be seed cell concentration-dependent for cell densities of less than 2 × 10(4) cells/cm(2). At higher initial cell densities, under differentiating culture conditions, V(m) development commenced on day 2 and reached a steady-state on day 12. The relative distribution of differentiated cells between low and high FALS has been proposed as a potential culture electrophysiological differentiation state index. These experiments offer a general methodology to characterize cultured excitable cells of nervous system origin, with respect to electrophysiological differentiation. This information is valuable in studies employing neuroblastoma cells as in vitro screening models for safety/hazard evaluation and/or risk assessment of therapeutical and industrial chemicals under development.

Ethical Issues for Control-Arm Patients After Revelation of Benefits of Experimental Therapy: A Framework Modeled in Neuroblastoma

PubMed Central

Unguru, Yoram; Joffe, Steven; Fernandez, Conrad V.; Yu, Alice L.

2013-01-01

In 2009, the Children's Oncology Group (COG) phase III randomized controlled trial, ANBL0032, found that adding immunotherapy (Ch14.18) to standard therapy significantly improved outcomes in patients with high-risk neuroblastoma when administered within 110 days after autologous stem-cell transplantation (SCT). After careful deliberation and consultation, the COG Neuroblastoma Committee decided to offer Ch14.18 to prior trial participants who had been randomly assigned to the control arm (no immunotherapy), regardless of the time that had elapsed since SCT. This decision occurred in the context of a limited supply of Ch14.18 and no data regarding its role when administered beyond 110 days. In this article, we analyze the numerous ethical challenges highlighted by the ANBL0032 trial, including the limits of researchers' reciprocity-based obligations to study participants, post-trial access to beneficial therapies, and the balance between scientific knowledge and parental hope. These deliberations may be useful to other researchers when considering their ethical obligations to control-arm participants in the wake of a positive randomized trial. PMID:23295797

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia.

PubMed

Maugham, Michelle L; Thomas, Patrick B; Crisp, Gabrielle J; Philp, Lisa K; Shah, Esha T; Herington, Adrian C; Chen, Chen; Gregory, Laura S; Nelson, Colleen C; Seim, Inge; Jeffery, Penny L; Chopin, Lisa K

2017-03-28

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. Here, the long-term effects of a 23% Western high-fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1 -/- ) suitable for engraftment with human-derived tissue xenografts, and the effect of diet-induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1 -/- and NOD/SCID HFD-fed mice demonstrated diet-induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1 -/- mice developed significantly higher fasting insulin levels (2.16 ± 1.01 ng/ml, P = 0.01) and increased insulin resistance (6.70 ± 1.68 HOMA-IR, P = 0.01) compared to low-fat chow-fed mice (0.71 ± 0.12 ng/ml and 2.91 ± 0.42 HOMA-IR). This was not observed in the NOD/SCID strain. Hepatic steatosis was more extensive in Rag1 -/- HFD-fed mice compared to NOD/SCID mice. Intramyocellular lipid storage was increased in Rag1 -/- HFD-fed mice, but not in NOD/SCID mice. In Rag1 -/- HFD-fed mice, LNCaP xenograft tumours grew more rapidly compared to low-fat chow-fed mice. This is the first characterisation of the metabolic effects of long-term Western HFD in two mouse strains suitable for xenograft studies. We conclude that Rag1 -/- mice are an appropriate and novel xenograft model for studying the relationship between cancer and hyperinsulinaemia.

Intratumoral delivery of docetaxel enhances antitumor activity of Ad-p53 in murine head and neck cancer xenograft model.

PubMed

Yoo, George H; Subramanian, Geetha; Ezzat, Waleed H; Tulunay, Ozlem E; Tran, Vivian R; Lonardo, Fulvio; Ensley, John F; Kim, Harold; Won, Joshua; Stevens, Timothy; Zumstein, Louis A; Lin, Ho-Sheng

2010-01-01

The aim of this study is to determine the ability of intratumorally delivered docetaxel to enhance the antitumor activity of adenovirus-mediated delivery of p53 (Ad-p53) in murine head and neck cancer xenograft model. A xenograft head and neck squamous cell carcinoma mouse model was used. Mice were randomized into 4 groups of 6 mice receiving 6 weeks of biweekly intratumoral injection of (a) diluent, (b) Ad-p53 (1 x 10(10) viral particles per injection), (c) docetaxel (1 mg/kg per injection), and (d) combination of Ad-p53 (1 x 10(10) viral particles per injection) and docetaxel (1 mg/kg per injection). Tumor size, weight, toxicity, and overall and disease-free survival rates were determined. Intratumoral treatments with either docetaxel alone or Ad-p53 alone resulted in statistically significant antitumor activity and improved survival compared with control group. Furthermore, combined delivery of Ad-p53 and docetaxel resulted in a statistically significant reduction in tumor weight when compared to treatment with either Ad-p53 or docetaxel alone. Intratumoral delivery of docetaxel enhanced the antitumor effect of Ad-p53 in murine head and neck cancer xenograft model. The result of this preclinical in vivo study is promising and supports further clinical testing to evaluate efficacy of combined intratumoral docetaxel and Ad-p53 in treatment of head and neck cancer. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Orthotopic glioblastoma stem-like cell xenograft model in mice to evaluate intra-arterial delivery of bevacizumab: from bedside to bench.

PubMed

Burkhardt, Jan-Karl; Hofstetter, Christoph P; Santillan, Alejandro; Shin, Benjamin J; Foley, Conor P; Ballon, Douglas J; Pierre Gobin, Y; Boockvar, John A

2012-11-01

Bevacizumab (BV), a humanized monocolonal antibody directed against vascular endothelial growth factor (VEGF), is a standard intravenous (IV) treatment for recurrent glioblastoma multiforme (GBM), that has been introduced recently as an intra-arterial (IA) treatment modality in humans. Since preclinical models have not been reported, we sought to develop a tumor stem cell (TSC) xenograft model to investigate IA BV delivery in vivo. Firefly luciferase transduced patient TSC were injected into the cortex of 35 nude mice. Tumor growth was monitored weekly using bioluminescence imaging. Mice were treated with either intraperitoneal (IP) or IA BV, with or without blood-brain barrier disruption (BBBD), or with IP saline injection (controls). Tumor tissue was analyzed using immunohistochemistry and western blot techniques. Tumor formation occurred in 31 of 35 (89%) mice with a significant signal increase over time (p=0.018). Post mortem histology revealed an infiltrative growth of TSC xenografts in a similar pattern compared to the primary human GBM. Tumor tissue analyzed at 24 hours after treatment revealed that IA BV treatment with BBBD led to a significantly higher intratumoral BV concentration compared to IA BV alone, IP BV or controls (p<0.05). Thus, we have developed a TSC-based xenograft mouse model that allows us to study IA chemotherapy. However, further studies are needed to analyze the treatment effects after IA BV to assess tumor progression and overall animal survival. Copyright © 2012 Elsevier Ltd. All rights reserved.

Targeting Hypoxia-Inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment.

PubMed

Nigim, Fares; Cavanaugh, Jill; Patel, Anoop P; Curry, William T; Esaki, Shin-ichi; Kasper, Ekkehard M; Chi, Andrew S; Louis, David N; Martuza, Robert L; Rabkin, Samuel D; Wakimoto, Hiroaki

2015-07-01

Tissue hypoxia and necrosis represent pathophysiologic and histologic hallmarks of glioblastoma (GBM). Although hypoxia inducible factor 1α (HIF-1α) plays crucial roles in the malignant phenotypes of GBM, developing HIF-1α-targeted agents has been hampered by the lack of a suitable preclinical model that recapitulates the complex biology of clinical GBM. We present a new GBM model, MGG123, which was established from a recurrent human GBM. Orthotopic xenografting of stem-like MGG123 cells reproducibly generated lethal tumors that were characterized by foci of palisading necrosis, hypervascularity, and robust stem cell marker expression. Perinecrotic neoplastic cells distinctively express HIF-1α and are proliferative in both xenografts and the patient tissue. The xenografts contain scattered hypoxic foci that were consistently greater than 50 μm distant from blood vessels, indicating intratumoral heterogeneity of oxygenation. Hypoxia enhanced HIF-1α expression in cultured MGG123 cells, which was abrogated by the HIF-1α inhibitors digoxin or ouabain. In vivo, treatment of orthotopic MGG123 xenografts with digoxin decreased HIF-1α expression, vascular endothelial growth factor mRNA levels, and CD34-positive vasculature within the tumors, and extended survival of mice bearing the aggressive MGG123 GBM. This preclinical tumor model faithfully recapitulates the GBM-relevant hypoxic microenvironment and stemness and is a suitable platform for studying disease biology and developing hypoxia-targeted agents.

Incidental neuroblastoma with bilateral retinoblastoma: what are the chances?

PubMed

Roelofs, Kelsey; Shaikh, Furqan; Astle, William; Gallie, Brenda L; Soliman, Sameh E

2018-06-01

A child with bilateral familial retinoblastoma underwent staging MRI brain and orbit which identified subtle leptomeningeal enhancement, thus prompting an MRI whole body, which revealed a retroperitoneal mass, confirmed on laparoscopic biopsy to be neuroblastoma. This is the first reported case of these two rare embryonal non-central nervous system tumors occurring concurrently. The cause of this concurrence is unknown despite their pathogenic similarities with a chance of 4 cases per 10 billion children aged 1-4 years. Incidental neuroblastomas in infants can regress spontaneously but this child underwent systemic chemotherapy for his retinoblastoma that may have caused regression of the neuroblastoma.

11q deletion in neuroblastoma: a review of biological and clinical implications.

PubMed

Mlakar, Vid; Jurkovic Mlakar, Simona; Lopez, Gonzalo; Maris, John M; Ansari, Marc; Gumy-Pause, Fabienne

2017-06-29

Deletion of the long arm of chromosome 11 (11q deletion) is one of the most frequent events that occur during the development of aggressive neuroblastoma. Clinically, 11q deletion is associated with higher disease stage and decreased survival probability. During the last 25 years, extensive efforts have been invested to identify the precise frequency of 11q aberrations in neuroblastoma, the recurrently involved genes, and to understand the molecular mechanisms of 11q deletion, but definitive answers are still unclear. In this review, it is our intent to compile and review the evidence acquired to date on 11q deletion in neuroblastoma.

Advances in Risk Classification and Treatment Strategies for Neuroblastoma.

PubMed

Pinto, Navin R; Applebaum, Mark A; Volchenboum, Samuel L; Matthay, Katherine K; London, Wendy B; Ambros, Peter F; Nakagawara, Akira; Berthold, Frank; Schleiermacher, Gudrun; Park, Julie R; Valteau-Couanet, Dominique; Pearson, Andrew D J; Cohn, Susan L

2015-09-20

Risk-based treatment approaches for neuroblastoma have been ongoing for decades. However, the criteria used to define risk in various institutional and cooperative groups were disparate, limiting the ability to compare clinical trial results. To mitigate this problem and enhance collaborative research, homogenous pretreatment patient cohorts have been defined by the International Neuroblastoma Risk Group classification system. During the past 30 years, increasingly intensive, multimodality approaches have been developed to treat patients who are classified as high risk, whereas patients with low- or intermediate-risk neuroblastoma have received reduced therapy. This treatment approach has resulted in improved outcome, although survival for high-risk patients remains poor, emphasizing the need for more effective treatments. Increased knowledge regarding the biology and genetic basis of neuroblastoma has led to the discovery of druggable targets and promising, new therapeutic approaches. Collaborative efforts of institutions and international cooperative groups have led to advances in our understanding of neuroblastoma biology, refinements in risk classification, and stratified treatment strategies, resulting in improved outcome. International collaboration will be even more critical when evaluating therapies designed to treat small cohorts of patients with rare actionable mutations. © 2015 by American Society of Clinical Oncology.

A comprehensive characterization of rare mitochondrial DNA variants in neuroblastoma.

PubMed

Calabrese, Francesco Maria; Clima, Rosanna; Pignataro, Piero; Lasorsa, Vito Alessandro; Hogarty, Michael D; Castellano, Aurora; Conte, Massimo; Tonini, Gian Paolo; Iolascon, Achille; Gasparre, Giuseppe; Capasso, Mario

2016-08-02

Neuroblastoma, a tumor of the developing sympathetic nervous system, is a common childhood neoplasm that is often lethal. Mitochondrial DNA (mtDNA) mutations have been found in most tumors including neuroblastoma. We extracted mtDNA data from a cohort of neuroblastoma samples that had undergone Whole Exome Sequencing (WES) and also used snap-frozen samples in which mtDNA was entirely sequenced by Sanger technology. We next undertook the challenge of determining those mutations that are relevant to, or arisen during tumor development. The bioinformatics pipeline used to extract mitochondrial variants from matched tumor/blood samples was enriched by a set of filters inclusive of heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. Our in silico multistep workflow applied both on WES and Sanger-sequenced neuroblastoma samples, allowed us to identify a limited burden of somatic and germline mitochondrial mutations with a potential pathogenic impact. The few singleton germline and somatic mitochondrial mutations emerged, according to our in silico analysis, do not appear to impact on the development of neuroblastoma. Our findings are consistent with the hypothesis that most mitochondrial somatic mutations can be considered as 'passengers' and consequently have no discernible effect in this type of cancer.

L-DOPA therapy interferes with urine catecholamine analysis in children with suspected neuroblastoma: a case series.

PubMed

Kelly, Alison U; Srivastava, Rajeev; Dow, Ellie; Davidson, D Fraser

2017-09-01

Neuroblastoma is the most common solid extracranial malignancy diagnosed in childhood. Clinical presentation is variable, and metastatic disease is common at diagnosis. Analyses of urinary catecholamines and their metabolites are commonly requested as a first-line investigation when clinical suspicion exists. Levodopa (L-Dopa) therapy is utilized as a treatment for a number of disorders in childhood, including Dopa-responsive dystonia. Neuroblastoma may mimic some of the clinical features of this disorder. L-Dopa can interfere with analysis of urinary catecholamines and their metabolites and complicate the interpretation of results. We present the cases of three children who were prescribed L-dopa at the time of analysis of urinary catecholamines and metabolites as a screen for neuroblastoma, but who did not have the disease. Comparison of their results with those from cases with true neuroblastoma reveal that it is impossible to reliably distinguish true neuroblastoma from L-Dopa therapy using these tests. We recommend that patients should be off L-dopa therapy, if possible when these tests are performed. These cases illustrate the importance of providing clinical details and drug history to the laboratory in order to avoid diagnostic confusion.

Emerging importance of ALK in neuroblastoma

PubMed Central

Azarova, Anna M.; Gautam, Gargi; George, Rani E.

2011-01-01

Since the original descriptions of gain-of function mutations in anaplastic lymphoma kinase (ALK), interest in the role of this receptor tyrosine kinase in neuroblastoma development and as a potential therapeutic target has escalated. As a group, the activating point mutations in full-length ALK, found in approximately 8% of all neuroblastoma tumors, are distributed evenly across different clinical stages. However, the most frequent somatic mutation, F1174L, is associated with amplification of the MYCN oncogene. This combination of features appears to confer a worse prognosis than MYCN amplification alone, suggesting a cooperative effect on neuroblastoma formation by these two proteins. Indeed, F1174L has shown more potent transforming activity in vivo than the second most common activating mutation, R1275Q, and is responsible for innate and acquired resistance to crizotinib, a clinically relevant ALK inhibitor that will soon be commercially available. These advances cast ALK as a bona fide oncoprotein in neuroblastoma and emphasize the need to understand ALK-mediated signaling in this tumor. This review addresses many of the current issues surrounding the role of ALK in normal development and neuroblastoma pathogenesis, and discusses the prospects for clinically effective targeted treatments based on ALK inhibition. PMID:21945349

Genetically modified "obligate" anaerobic Salmonella typhimurium as a therapeutic strategy for neuroblastoma.

PubMed

Guo, Zhu-Ling; Yu, Bin; Ning, Bo-Tao; Chan, Shing; Lin, Qiu-Bin; Li, James Chun-Bong; Huang, Jian-Dong; Chan, Godfrey Chi-Fung

2015-08-19

Neuroblastoma currently has poor prognosis, therefore we proposed a new strategy by targeting neuroblastoma with genetically engineered anaerobic Salmonella (Sal-YB1). Nude and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) orthotopic mouse models were used, and Sal-YB1 was administered via tail vein. The therapeutic effectiveness, bio-safety, and mechanisms were studied. No mice died of therapy-related complications. Tumor size reduction was 70 and 30% in nude and NOD-SCID mice, respectively. No Salmonella was detected in the urine; 75% mice had positive stool culture if diaminopimelic acid was added, but all turned negative subsequently. Tumor tissues had more Sal-YB1 infiltration, necrosis, and shrinkage in Sal-YB1-treated mice. Significantly higher expression of TLR4, TNF-stimulated gene 6 protein (TSG6), and cleaved caspase 1, 3, 8, and 9 was found in the tumor masses of the Sal-YB1-treated group with a decrease of interleukin 1 receptor-associated kinase (IRAK) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα). There was a high release of TNFα both in human macrophages and mouse tumor tissues with Sal-YB1 treatment. The antitumor effect of the supernatant derived from macrophages treated with Sal-YB1 could be reversed with TNFα and pan-caspase inhibitors. This new approach in targeting neuroblastoma by bio-engineered Salmonella with the assistance of macrophages indirectly may have a clinical therapeutic impact in the future.

Identification of potential serum markers for nasopharyngeal carcinoma from a xenografted mouse model using Cy-dye labeling combined with three-dimensional fractionation.

PubMed

Wu, Chih-Ching; Peng, Pei-Hua; Chang, Ya-Ting; Huang, Yu-Shan; Chang, Kai-Ping; Hao, Sheng-Po; Tsang, Ngan-Ming; Yeh, Chau-Ting; Chang, Yu-Sun; Yu, Jau-Song

2008-09-01

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for improving NPC diagnosis, we established a proteomic platform for detecting aberrant serum proteins in nude mice bearing NPC xenografts. We first removed the three most abundant proteins from serum samples of tumor-bearing and control mice, and then labeled the samples with different fluorescent cyanine (Cy) dyes. The labeled serum proteins were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Differentially expressed proteins were identified by in-gel tryptic digestion and MALDI-TOF MS. We identified peroxiredoxin 2 (Prx-II) and carbonic anhydrase 2 (CA-II) as being elevated in the xenograft mouse model compared to controls. Western blot analysis confirmed up-regulation of Prx-II and CA-II in plasma from five NPC patients, and ELISA showed that plasma Prx-II levels were significantly higher in NPC patients (n = 84) versus healthy controls (n = 90) (3.03 +/- 4.47 versus 1.90 +/- 2.74 microg/mL, p = 0.047). In conclusion, Cy dye labeling combined with three-dimensional fractionation is a feasible strategy for identifying differentially expressed serum proteins in an NPC xenograft model, and Prx-II may represent a potential NPC biomarker.

[Establishment of a human bladder cancer cell line stably co-expressing hSPRY2 and luciferase genes and its subcutaneous tumor xenograft model in nude mice].

PubMed

Yin, Xiaotao; Li, Fanglong; Jin, Yipeng; Yin, Zhaoyang; Qi, Siyong; Wu, Shuai; Wang, Zicheng; Wang, Lin; Yu, Jiyun; Gao, Jiangping

2017-03-01

Objective To establish a human bladder cancer cell line stably co-expressing human sprouty2 (hSPRY2) and luciferase (Luc) genes simultaneously, and develop its subcutaneous tumor xenograft model in nude mice. Methods The hSPRY2 and Luc gene segments were amplified by PCR, and were cloned into lentiviral vector pCDH and pLVX respectively to produce corresponding lentivirus particles. The J82 human bladder cancer cells were infected with these two kinds of lentivirus particles, and then further screened by puromycin and G418. The expressions of hSPRY2 and Luc genes were detected by bioluminescence, immunofluorescence and Western blot analysis. The screened J82-hSPRY2/Luc cells were injected subcutaneously into BALB/c nude mice, and the growth of tumor was monitored dynamically using in vivo fluorescence imaging system. Results J82-hSPRY2/Luc cell line stably expressing hSPRY2 and Luc genes was established successfully. Bioluminescence, immunofluorescence and Western blot analysis validated the expressions of hSPRY2 and Luc genes. The in vivo fluorescence imaging system showed obvious fluorescence in subcutaneous tumor xenograft in nude mice. Conclusion The J82-hSPRY2/Luc bladder cancer cell line and its subcutaneous tumor xenograft model in nude mice have been established successfully.

Establishment and characterization of intraperitoneal xenograft models by co-injection of human tumor cells and extracellular matrix gel

PubMed Central

YAO, YUQIN; ZHOU, YONGJUN; SU, XIAOLAN; DAI, LEI; YU, LIN; DENG, HONGXIN; GOU, LANTU; YANG, JINLIANG

2015-01-01

Establishing a feasible intraperitoneal (i.p.) xenograft model in nude mice is a good strategy to evaluate the antitumor effect of drugs in vivo. However, the manipulation of human cancer cells in establishing a stable peritoneal carcinomatosis model in nude mice is problematic. In the present study, the ovarian and colorectal peritoneal tumor models were successfully established in nude mice by co-injection of human tumor cells and extracellular matrix gel. In ovarian tumor models, the mean number tumor nodes was significantly higher in the experimental group (intraperitoneal tumor cell co-injection with ECM gel) compared with the PBS control group on the 30th day (21.0±3.0 vs. 3.6±2.5; P<0.05). The same results were observed in the colorectal peritoneal tumor models on the 28th day. The colorectal peritoneal tumor model was further used to evaluate the chemotherapy effect of irinotecan (CPT-11). The mean weight of peritoneal tumor nodes in CPT-11 treatment group was significantly less than that of the control group (0.81±0.16 vs. 2.18±0.21 g; P<0.05). The results confirmed the value of these i.p. xenograft models in nude mice as efficient and feasible tools for preclinical evaluation. PMID:26788149

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

PubMed

Wang, Haiyan; Cai, Shanbao; Bailey, Barbara J; Reza Saadatzadeh, M; Ding, Jixin; Tonsing-Carter, Eva; Georgiadis, Taxiarchis M; Zachary Gunter, T; Long, Eric C; Minto, Robert E; Gordon, Kevin R; Sen, Stephanie E; Cai, Wenjing; Eitel, Jacob A; Waning, David L; Bringman, Lauren R; Wells, Clark D; Murray, Mary E; Sarkaria, Jann N; Gelbert, Lawrence M; Jones, David R; Cohen-Gadol, Aaron A; Mayo, Lindsey D; Shannon, Harlan E; Pollok, Karen E

2017-02-01

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

ODC1 is a critical determinant of MYCN oncogenesis and a therapeutic target in neuroblastoma

PubMed Central

Hogarty, Michael D.; Norris, Murray D.; Davis, Kim; Liu, Xueyuan; Evageliou, Nicholas F.; Hayes, Candace S.; Pawel, Bruce; Guo, Rong; Zhao, Huaqing; Sekyere, Eric; Keating, Joanna; Thomas, Wayne; Cheng, Ngan Ching; Murray, Jayne; Smith, Janice; Sutton, Rosemary; Venn, Nicola; London, Wendy B.; Buxton, Allan; Gilmour, Susan K.; Marshall, Glenn M; Haber, Michelle

2008-01-01

Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have co-ordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower risk tumors, suggesting this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor α-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically-engineered mice (TH-MYCN GEM) extended tumor latency and survival in homozygous mice, and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments anti-tumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and demonstrates initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors. PMID:19047152

ODC1 is a critical determinant of MYCN oncogenesis and a therapeutic target in neuroblastoma.

PubMed

Hogarty, Michael D; Norris, Murray D; Davis, Kimberly; Liu, Xueyuan; Evageliou, Nicholas F; Hayes, Candace S; Pawel, Bruce; Guo, Rong; Zhao, Huaqing; Sekyere, Eric; Keating, Joanna; Thomas, Wayne; Cheng, Ngan Ching; Murray, Jayne; Smith, Janice; Sutton, Rosemary; Venn, Nicola; London, Wendy B; Buxton, Allen; Gilmour, Susan K; Marshall, Glenn M; Haber, Michelle

2008-12-01

Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor alpha-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors.

International neuroblastoma staging system stage 1 neuroblastoma: a prospective study and literature review.

PubMed

Kushner, B H; Cheung, N K; LaQuaglia, M P; Ambros, P F; Ambros, I M; Bonilla, M A; Ladanyi, M; Gerald, W L

1996-07-01

To gain insight into the management of non-metastatic neuroblastoma by examining clinical and biologic features of International Neuroblastoma Staging System (INSS) stage 1 tumors. Patients were staged by both the INSS and the Evans staging system and were evaluated for biologic prognostic factors. Patients with INSS stage 1 received no cytotoxic therapy. The literature was reviewed for clinical and biologic data about INSS stage 1. We evaluated 10 consecutive patients (median age, 17.5 months) with INSS stage 1; all remain disease-free (median follow-up duration, > 5 years). Tumors were in the abdomen (n = 6), chest (n = 3), or pelvis (n = 1). Neuroblastoma involved margins of resection in six tumors. Poor-prognostic biologic findings included tumor-cell diploidy (n = 2) and unfavorable Shimada histopathology (n = 2). Two patients were to receive chemotherapy for, respectively, a tumor deemed unresectable and a tumor classified as Evans stage III; second opinions resulted in surgical management alone in each case. Published reports confirm that some INSS stage 1 patients (1) are at risk for overtreatment, and (2) have poor-prognostic biologic findings yet do well. Surgery alone suffices for INSS stage 1 neuroblastoma, even if biologic prognostic factors are unfavorable, microscopic disease remains after surgery, and tumor size is suggestive of "advanced-stage" status in other staging systems. Attempts to resect regionally confined neuroblastomas should take precedence over immediate use of cytotoxic therapy; otherwise, some patients may receive chemotherapy or radiotherapy unnecessarily.

Multidisciplinary management of cervical neuroblastoma in infants.

PubMed

Csanády, Miklós; Vass, Gábor; Bartyik, Katalin; Majoros, Valéria; Rovó, László

2014-12-01

Neuroblastoma is the most common malignancy in infancy, it is a histologically and genetically heterogeneous tumor, the therapy and outcome of which is influenced by age, histological variant and genetic background as well. We present two consecutive infant patients with neuroblastoma of the neck discussing the etiology, the diagnosis and the surgical and oncological treatment of the tumor, which was observed in a relatively rare manifestation in the head-neck region. Our first patient (age: 5.5 months) was MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived) negative, INSS (International Neuroblastoma Staging System) Stage 3 and INRGSS (International Neuroblastoma Risk Group Staging System) Stage 3 because of the contralateral lymph node involvement while the complete gross resection of the primary tumor mass was feasible. The patient is tumor free after three years of follow-up. Our second patient (age: 5 months) was MYCN negative, INSS Stage 2 and INRGSS Stage 1, as both the primary tumor and the ipsilateral lymph nodes were totally removed via a modified radical neck dissection. The patient is tumor free after three years of follow-up. For MYCN negative patients, especially in early age, the prognosis of neuroblastoma is good, surgical resection and chemotherapy together is an adequate treatment protocol (as in our two patients). While MYCN-amplified patients require a combined and aggressive treatment with surgery, chemotherapy, radiotherapy, and immunotherapy to be able to obtain a favorable survival rate according to the literature. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Silencing of Y-box binding protein-1 by RNA interference inhibits proliferation, invasion, and metastasis, and enhances sensitivity to cisplatin through NF-κB signaling pathway in human neuroblastoma SH-SY5Y cells.

PubMed

Wang, Hong; Sun, Ruowen; Chi, Zuofei; Li, Shuang; Hao, Liangchun

2017-09-01

Y-box binding protein-1 (YB-1), a member of Y-box protein family binding DNA and RNA, has been proposed as a novel marker in multiple malignant tumors and found to be associated with tumor malignancy. Neuroblastoma is an embryonal tumor arising from neuroblast cells of the autonomic nervous system, which is the most common cancer diagnosed in infants. It has been reported that YB-1 is highly expressing in various human tumors including nasopharynx, thyroid, lung, breast, colon, ovary, and prostate cancers. This study aimed to investigate the functional role of YB-1 in neuroblastoma by silencing YB-1 using RNA interference (shRNA) in neuroblastoma SH-SY5Y cells. We found that silencing of YB-1 decreased the proliferation, migration, and invasion of SH-SY5Y cells. At molecular level, inhibition of YB-1 decreased the expression level of PCNA as well as MMP-2 in neuroblastoma SH-SY5Y cells. Also, we discovered that YB-1 silencing sensitized SH-SY5Y cells to cisplatin and promoted the apoptosis induced by cisplatin due to down-regulation of multidrug resistance (MDR) 1 protein via NF-κB signaling pathway. Therefore, we consider that targeting YB-1 is promising for neuroblastoma treatment and for overcoming its cisplatin resistance in the development of new neuroblastoma therapeutic strategies.

Vitrification and xenografting of human ovarian tissue.

PubMed

Amorim, Christiani Andrade; Dolmans, Marie-Madeleine; David, Anu; Jaeger, Jonathan; Vanacker, Julie; Camboni, Alessandra; Donnez, Jacques; Van Langendonckt, Anne

2012-11-01

To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. Pilot study. Gynecology research unit in a university hospital. Ovarian biopsies were obtained from seven women aged 30-41 years. Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)-warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

[Inhibitory effect of VEGF antisense phosphorothioate oligodeoxynucleotides on the growth of human salivary adenoid cystic carcinoma xenografts in nude mice].

PubMed

Li, Xiao-guang; Wang, Xu-xia; Li, Teng-yu; Wang, Yan-xiu; Gao, Jing; Ni, Chun-xiao

2012-12-01

To investigate the inhibitory effect of VEGF antisense phosphorothioate oligodeoxynucleoiides on the growth of human salivary adenoid cystic carcinoma (SACC) xenografts in nude mice. The VEGF-ASODN was synthesised artificially. After the model of human SACC xenografts in nude mice was established, they were random1y divided into three groups: antisense group, scrambled group and normal saline group. A control group without cancer was also established. Antisense(66 μg), scrambled sequence(66 μg) and normal saline(once every 3 days and 7 times in all) were injected in three experimental groups, respectively. Two days after therapy, the mice were sacrificed. Serums were used for detection of VEGF protein. All tumors were measured and weighted. The quantity of VEGF mRNA and protein and PLI, MVD was detected by hybridization in situ and immunohistochemistry. SPSS13.0 software package was used for statistical analysis. The VEGF-ASODN could suppress the expression of VEGF in human SACC xenografts in nude mice and reduce VEGF protein in serum of nude mice significantly. It cou1d also reduce the volume and weight of xenografts and could reduce the expression of VEGF mRNA and its protein, PCNA and CD34. By inhibiting the expression of VEGF, VEGF-ASODN can inhabit proliferation of human SACC xenografts in nude mice.

Long-term psychological and educational outcomes for survivors of neuroblastoma: A report from the Childhood Cancer Survivor Study.

PubMed

Zheng, Daniel J; Krull, Kevin R; Chen, Yan; Diller, Lisa; Yasui, Yutaka; Leisenring, Wendy; Brouwers, Pim; Howell, Rebecca; Lai, Jin-Shei; Balsamo, Lyn; Oeffinger, Kevin C; Robison, Leslie L; Armstrong, Gregory T; Kadan-Lottick, Nina S

2018-06-11

Neuroblastoma survivors may be at elevated risk for psychological impairments because of their young age at diagnosis and neurotoxic treatment, but this is not well described. A total of 859 ≥5-year survivors of neuroblastoma younger than 18 years (diagnosed in 1970-1999), who had a median age at diagnosis of 0.8 years (range: 0.0-7.3 years) and a median follow-up of 13.3 years (range: 8.0-17.9 years), were compared with 872 siblings of childhood cancer survivors who were younger than 18 years with the parent-reported Behavior Problem Index (BPI) for psychological functioning. Age- and sex-adjusted multivariate log-binomial models were used to identify factors associated with impairment in BPI domains (scores worse than the sibling 10th percentile). The impact of psychological impairment on educational outcomes was examined among survivors. Compared with siblings, neuroblastoma survivors had an increased prevalence of impairment in the domains of anxiety/depression (19% vs 14%; P = .003), headstrong behavior (19% vs 13%; P < .001), attention deficits (21% vs 13%; P < .001), peer conflict/social withdrawal (26% vs 17%; P < .001), and antisocial behavior (16% vs 12%; P = .01). Common treatment exposures (vincristine, cisplatin, and retinoic acid) were not associated with impairment. Having 2 or more chronic health conditions predicted impairment in 4 domains (P < .001). Specifically, pulmonary disease predicted impairment in all 5 domains (P ≤ .004). Endocrine disease (P ≤ .004) and peripheral neuropathy (P ≤ .02) each predicted impairment in 3 domains. Psychological impairment was associated with special education service usage and educational attainment less than college. Neuroblastoma survivors are at elevated risk for psychological impairment, which is associated with special education service usage and lower adult educational attainment. Those with chronic health conditions may represent a high-risk group for targeted screening and intervention. Cancer 2018. © 2018 American Cancer Society. © 2018 American Cancer Society.

Delayed xenograft rejection.

PubMed

Hancock, W W

1997-01-01

The triumph of genetic engineering in overcoming hyperacute rejection (HAR) of a discordant organ xenograft is clear, but the promise of clinical application of xenotransplantation remains unfulfilled as further immunologic barriers are defined that lead to rejection of a vascularized xenograft within days of transplantation. This report describes the features of this second set of immunologic responses, collectively termed delayed xenograft rejection (DXR). DXR is a syndrome seen in xenograft recipients in which HAR has been avoided or suppressed by antibody depletion or blockade of complement activation. DXR may result, at least in part, from the persisting activation of those pathways first encountered during the HAR phase. Serial studies over several days after transplant show that, histologically, xenografts undergoing DXR demonstrate varying combinations of (1) progressive infiltration by activated macrophages and natural killer (NK) cells, (2) platelet aggregation and fibrin deposition throughout the microvasculature, and (3) endothelial activation. In various experimental models, DXR is T cell-independent and can occur in the absence of demonstrable xenoreactive antibodies. Hence DXR is probably best regarded as arising from the activation of innate host defense mechanisms coupled with failure of normal regulatory mechanisms due to manifold molecular incompatibilities. Although DXR-like features can be seen in concordant models, T cell involvement in the latter is probably requisite. Similarly, in a much muted form, aspects of a DXR-like process may contribute to numerous inflammatory processes, including allograft rejection. The importance of DXR in xenotransplantation is that its development appears resistant to all but the most dense and toxic forms of immunosuppression, which prolong xenograft survival at the expense of inducing host leukopenia, thrombocytopenia, and coagulopathies. It is likely that until the basis of DXR is more clearly understood there can be no further significant progress toward clinical xenotransplantation. However, as the mechanisms responsible for DXR are dissected and understood, still further genetic engineering of donor pigs, involving the introduction of additional or multiple genes to regulate macrophage and NK cell responses, local coagulation, and endothelial cell activation, may once again prove to be an attractive, practical, powerful therapeutic option.

Cellular High-Energy Cavitation Trauma - Description of a Novel In Vitro Trauma Model in Three Different Cell Types.

PubMed

Cao, Yuli; Risling, Mårten; Malm, Elisabeth; Sondén, Anders; Bolling, Magnus Frödin; Sköld, Mattias K

2016-01-01

The mechanisms involved in traumatic brain injury have yet to be fully characterized. One mechanism that, especially in high-energy trauma, could be of importance is cavitation. Cavitation can be described as a process of vaporization, bubble generation, and bubble implosion as a result of a decrease and subsequent increase in pressure. Cavitation as an injury mechanism is difficult to visualize and model due to its short duration and limited spatial distribution. One strategy to analyze the cellular response of cavitation is to employ suitable in vitro models. The flyer-plate model is an in vitro high-energy trauma model that includes cavitation as a trauma mechanism. A copper fragment is accelerated by means of a laser, hits the bottom of a cell culture well causing cavitation, and shock waves inside the well and cell medium. We have found the flyer-plate model to be efficient, reproducible, and easy to control. In this study, we have used the model to analyze the cellular response to microcavitation in SH-SY5Y neuroblastoma, Caco-2, and C6 glioma cell lines. Mitotic activity in neuroblastoma and glioma was investigated with BrdU staining, and cell numbers were calculated using automated time-lapse imaging. We found variations between cell types and between different zones surrounding the lesion with these methods. It was also shown that the injured cell cultures released S-100B in a dose-dependent manner. Using gene expression microarray, a number of gene families of potential interest were found to be strongly, but differently regulated in neuroblastoma and glioma at 24 h post trauma. The data from the gene expression arrays may be used to identify new candidates for biomarkers in cavitation trauma. We conclude that our model is useful for studies of trauma in vitro and that it could be applied in future treatment studies.

The delivery of poly(lactic acid)-poly(ethylene glycol) nanoparticles loaded with non-toxic drug to overcome drug resistance for the treatment of neuroblastoma

NASA Astrophysics Data System (ADS)

Dhulekar, Jhilmil

Neuroblastoma is a rare cancer of the sympathetic nervous system. A neuroblastoma tumor develops in the nerve tissue and is diagnosed in infants and children. Approximately 10.2 per million children under the age of 15 are affected in the United States and is slightly more common in boys. Neuroblastoma constitutes 6% of all childhood cancers and has a long-term survival rate of only 15%. There are approximately 700 new cases of neuroblastoma each year in the United States. With such a low rate of survival, the development of more effective treatment methods is necessary. A number of therapies are available for the treatment of these tumors; however, clinicians and their patients face the challenges of systemic side effects and drug resistance of the tumor cells. The application of nanoparticles has the potential to provide a safer and more effective method of delivery drugs to tumors. The advantage of using nanoparticles for drug delivery is the ability to specifically or passively target tumors while reducing the harmful side effects of chemotherapeutics. Drug delivery via nanoparticles can also allow for lower dosage requirements with controlled release of the drugs, which can further reduce systemic toxicity. The aim of this research was to develop a polymeric nanoparticle drug delivery system for the treatment of high-risk neuroblastoma. Nanoparticles composed of a poly(lactic acid)-poly(ethylene glycol) block copolymer were formulated to deliver a non-toxic drug in combination with Temozolomide, a commonly used chemotherapeutic drug for the treatment of neuroblastoma. The non-toxic drug acts as an inhibitor to the DNA-repair protein present in neuroblastoma cells that is responsible for inducing drug resistance in the cells, which would potentially allow for enhanced temozolomide activity. A variety of studies were completed to prove the nanoparticles' low toxicity, loading abilities, and uptake into cells. Additionally, studies were performed to determine the individual effect on cell toxicity of each drug and in combination. Finally, nanoparticles were loaded with the non-toxic drug and delivered with free temozolomide to determine the overall efficacy of the drugs in reducing neuroblastoma cell viability.

Zebrafish xenograft models of cancer and metastasis for drug discovery.

PubMed

Brown, Hannah K; Schiavone, Kristina; Tazzyman, Simon; Heymann, Dominique; Chico, Timothy Ja

2017-04-01

Patients with metastatic cancer suffer the highest rate of cancer-related death, but existing animal models of metastasis have disadvantages that limit our ability to understand this process. The zebrafish is increasingly used for cancer modelling, particularly xenografting of human cancer cell lines, and drug discovery, and may provide novel scientific and therapeutic insights. However, this model system remains underexploited. Areas covered: The authors discuss the advantages and disadvantages of the zebrafish xenograft model for the study of cancer, metastasis and drug discovery. They summarise previous work investigating the metastatic cascade, such as tumour-induced angiogenesis, intravasation, extravasation, dissemination and homing, invasion at secondary sites, assessing metastatic potential and evaluation of cancer stem cells in zebrafish. Expert opinion: The practical advantages of zebrafish for basic biological study and drug discovery are indisputable. However, their ability to sufficiently reproduce and predict the behaviour of human cancer and metastasis remains unproven. For this to be resolved, novel mechanisms must to be discovered in zebrafish that are subsequently validated in humans, and for therapeutic interventions that modulate cancer favourably in zebrafish to successfully translate to human clinical studies. In the meantime, more work is required to establish the most informative methods in zebrafish.

Solidago Virgaurea for Prostate Cancer Therapy

DTIC Science & Technology

2010-04-01

CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON Kounosuke Watabe Ph.D. a. REPORT Unclassified b...We have purified 48KD protein as described above and examined the efficacy of this protein on tumorigenesis using a xenograft ...clinically more relevant than xenograft model, and most importantly, we will examine the preventive effect of this protein. For this purpose, we need to

A Study of Pemetrexed in Children With Recurrent Cancer

ClinicalTrials.gov

2011-02-23

Osteosarcoma; Medulloblastoma; Sarcoma, Ewing's; Neuroblastoma (Measurable Disease); Neuroblastoma (Metaiodobenzylguanidine; Positive Evaluable); Rhabdomyosarcoma; Ependymoma; Non-brainstem High-grade Glioma

Intrathecal transplantation of neuroblastoma cells decreases heat hyperalgesia and cold allodynia in a rat model of neuropathic pain.

PubMed

De la Calle, J L; Mena, M A; González-Escalada, J R; Paíno, C L

2002-11-30

Intrathecal grafting of cells as biological pumps to deliver monoamines, endorphins, and/or trophic factors, has been shown to be effective in treating chronic pain both in experimental animals and in clinical trials. We have tested whether intrathecal implantation of neuroblastoma cells reduces heat hyperalgesia and cold allodynia in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Behavioral tests and cerebrospinal fluid (CSF) collection were performed before CCI, 1 week later (after which, vehicle or NB69 cells were intrathecally injected) and at 4, 7, and 14 days post-injection. Both CSF sampling and injection of the cells were performed by direct lumbar puncture. Intrathecal grafting of 4 x 10(6) NB69 neuroblastoma cells reduced to basal levels the nociceptive response to heat in nerve-injured hindpaws, while the response of control limbs remained unchanged. Similarly, the allodynic response to cold elicited by acetone evaporation decreased in the animals implanted with NB69 cells. An increase in the concentrations of dopamine and serotonin metabolites of around 150% was observed in the CSF of animals that received grafts of NB69 cells. These data suggest that the monoamines released by NB69 cells in the intrathecal space produce analgesia to neuropathic pain in rats. Copyright 2002 Elsevier Science Inc.

Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research.

PubMed

Cheung, Yuen-Ting; Lau, Way Kwok-Wai; Yu, Man-Shan; Lai, Cora Sau-Wan; Yeung, Sze-Chun; So, Kwok-Fai; Chang, Raymond Chuen-Chung

2009-01-01

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.

Molecular Pathways: Disrupting polyamine homeostasis as a therapeutic strategy for neuroblastoma

PubMed Central

Evageliou, Nicholas F.; Hogarty, Michael D.

2009-01-01

MYC genes are deregulated in a plurality of human cancers. Through direct and indirect mechanisms the MYC network regulates the expression of >15% of the human genome, including both protein-coding and non-coding RNAs. This complexity has complicated efforts to define the principal pathways mediating MYC’s oncogenic activity. MYC plays a central role providing for the bioenergetic and biomass needs of proliferating cells, and polyamines are essential cell constituents supporting many of these functions. The rate-limiting enzyme in polyamine biosynthesis, ODC, is a bona fide MYC target, as are other regulatory enzymes in this pathway. A wealth of data link enhanced polyamine biosynthesis to cancer progression, and polyamine-depletion may limit malignant transformation of pre-neoplastic lesions. Studies using transgenic cancer models also supports that the effect of MYC on tumor initiation and progression can be attenuated through repression of polyamine production. High-risk neuroblastomas (an often lethal embryonal tumor in which MYC activation is paramount) deregulate numerous polyamine enzymes to promote expansion of intracellular polyamine pools. Selective inhibition of key enzymes in this pathway, e.g., using DFMO and/or SAM486, reduces tumorigenesis and synergizes with chemotherapy to regress tumors in pre-clinical models. Here we review the potential clinical application of these and additional polyamine-depletion agents to neuroblastoma and other advanced cancers in which MYC is operative. PMID:19789308

Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility.

PubMed

Knudsen, Erik S; Balaji, Uthra; Mannakee, Brian; Vail, Paris; Eslinger, Cody; Moxom, Christopher; Mansour, John; Witkiewicz, Agnieszka K

2018-03-01

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities. 27 primary PDAC tumours were employed for genetic analysis and development of tumour models. Tumour tissue was used for derivation of xenografts and cell lines. Exome sequencing was performed on the originating tumour and developed models. RNA sequencing, histological and functional analyses were employed to determine the relationship of the patient-derived models to clinical presentation of PDAC. The cohort employed captured the genetic diversity of PDAC. From most cases, both cell lines and xenograft models were developed. Exome sequencing confirmed preservation of the primary tumour mutations in developed cell lines, which remained stable with extended passaging. The level of genetic conservation in the cell lines was comparable to that observed with patient-derived xenograft (PDX) models. Unlike historically established PDAC cancer cell lines, patient-derived models recapitulated the histological architecture of the primary tumour and exhibited metastatic spread similar to that observed clinically. Detailed genetic analyses of tumours and derived models revealed features of ex vivo evolution and the clonal architecture of PDAC. Functional analysis was used to elucidate therapeutic vulnerabilities of relevance to treatment of PDAC. These data illustrate that with the appropriate methods it is possible to develop cell lines that maintain genetic features of PDAC. Such models serve as important substrates for analysing the significance of genetic variants and create a unique biorepository of annotated cell lines and xenografts that were established simultaneously from same primary tumour. These models can be used to infer genetic and empirically determined therapeutic sensitivities that would be germane to the patient. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K., E-mail: swapan.ray@uscmed.sc.edu

Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly inmore » SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-{kappa}B), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network formation ability of cells was significantly inhibited by survivin silencing and completely by combination of survivin silencing and EGCG treatment. Collectively, survivin silencing potentiated anti-cancer effects of EGCG in human malignant neuroblastoma cells having survivin overexpression. -- Highlights: Black-Right-Pointing-Pointer Survivin shRNA + EGCG controlled growth of human malignant neuroblastoma cells. Black-Right-Pointing-Pointer Survivin knockdown induced neuronal differentiation in neuroblastoma cells. Black-Right-Pointing-Pointer Survivin shRNA + EGCG induced morphological and biochemical features of apoptosis. Black-Right-Pointing-Pointer Combination therapy inhibited invasion, proliferation, and angiogenesis as well. Black-Right-Pointing-Pointer So, combination therapy showed multiple anti-cancer mechanisms in neuroblastoma.« less

Conservative treatment of massive hemothorax in a girl with neuroblastoma.

PubMed

Shiokawa, Naohiro; Okamoto, Yasuhiro; Kodama, Yuichi; Nishikawa, Takuro; Tanabe, Takayuki; Mukai, Motoi; Yoshioka, Takako; Kawano, Yoshifumi

2016-10-01

We report the case of a 1-year-old girl with stage 4 neuroblastoma who developed massive hemothorax due to tumor invasion before treatment. She presented with tachypnea, worsening anemia, and oxygen desaturation. Hemothorax was diagnosed based on chest radiography, ultrasonography, and diagnostic thoracic puncture results. High neuron-specific enolase, vanillylmandelic acid, and homovanillic acid as well as computed tomography strongly supported a diagnosis of neuroblastoma. Chemotherapy along with intermittent puncture drainage, oxygen, and blood transfusion reduced the accumulated blood, and hemothorax disappeared within 1 week. Thus, it is possible to avoid invasive treatment for massive hemothorax by initiating chemotherapy for chemosensitive solid tumors, including neuroblastoma. © 2016 Japan Pediatric Society.

Flavonoids from Potentilla parvifolia Fisch. and Their Neuroprotective Effects in Human Neuroblastoma SH-SY5Y Cells in vitro.

PubMed

Yuan, Zhenzhen; Luan, Guangxiang; Wang, Zhenhua; Hao, Xueyan; Li, Ji; Suo, Yourui; Li, Gang; Wang, Honglun

2017-06-01

Potentilla parvifolia Fisch. (Rosaceae) is a traditional medicinal plant in P. R. China. In this study, seven flavonoids, ayanin (1), tricin (2), quercetin (3), tiliroside (4), miquelianin (5), isoquercitrin (6), and astragalin (7), were separated and purified from ethyl acetate extractive fractions from ethanol extracts of P. parvifolia using a combination of sevaral chromatographic methods. The human neuroblastoma SH-SY5Y cells were differentiated with all trans-retinoic acid and treated with okadaic acid to induce tau protein phosphorylation and synaptic atrophy, which could establish an Alzheimer's disease cell model. The neuroprotective effects of these flavonoids in cellular were evaluated in vitro by this cell model. Results from the Western blot and morphology analysis suggested that compounds 3 and 4 had the better neuroprotective effects. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

[Effect of bufalin on proliferation and apoptosis through ERK/RSK2 pathway in human esophageal carcinoma cell line xenografts in nude mice].

PubMed

Yue, M; Liu, X J; Ding, Y; Wang, X L; Yang, H C; Liu, Y P

2016-05-23

To investigate the effect of bufalin on proliferation and apoptosis through ERK/RSK2 pathway in esophageal squamous cell carcinoma xenografts in nude mice. The subcutaneous xenograft model of esophageal cancer ECA109 cells in nude mice was established. The mice were divided into the model group, low-dose bufalin group, medium-dose bufalin group, high-dose bufalin group, PD98059 group and combination group to evaluate the effect of bufalin on the xenografts. The morphology of xenografts was observed by microscopy. The cell apoptosis index of xenografts was detected by TUNEL assay. The expression of ERK and RSK2 mRNA of human ECA109 cell transplantation tumor in nude mice was examined by real-time quantitative PCR. The protein levels of ERK, p-ERK, RSK2, p-RSK2, GSK3β, p-GSK3β, Bad and p-Bad in the xenografts were examined by Western blot and Immunohistochemistry. The tumor size of nude mice in the model group, low-dose bufalin group (BL), medium -dose bufalin group (BM), high-dose bufalin group (BH), PD98059 group and combined therapy group (BP) was (1.758±0.181) cm(3,) (1.680±0.150) cm(3,) (1.285±0.134) cm(3,) (0.873±0.095) cm(3,) (0.815±0.108) cm(3) and (0.530±0.104) cm(3,) respectively. Histological examination showed that the xenografts of each group had varying degrees of necrosis, and the most extensive necrosis was observed in the BP group. The TUNEL assay showed that the cell apoptosis index of xenografts in the model, BL, BM, BH, PD98059 and BP groups was (6.0±0.6)%, (11.0±0.7)%, (19.1±0.9)%, (25.1±1.4)%, (20.0±1.2)% and (17.1±0.7)%, respectively, which is highest in the BH group. The real-time quantitative PCR results showed that the ΔCT values of ERK mRNA in the model, BL, BM, BH, PD98059 and BP groups were 0.270±0.084, 0.293±0.081, 0.596±0.224, 0.857±0.183, 0.868±0.187 and 1.313±0.282, respectively. The ΔCT values of RSK2 mRNA in the model, BL, BM, BH, PD98059 and BP groups were 0.340±0.062, 0.337±0.071, 0.642±0.226, 0.915±0.170, 0.923±0.176 and 1.413±0.269, respectively. The relative expression of ERK and RSK2 mRNA was gradually decreased. Western blot and immunohistochemistry results showed that the protein levels of ERK, RSK2 and Bad in each group were not significantly different (P>0.05). The protein levels of p-ERK in the model, BL, BM, BH, PD98059 and BP groups were 0.721±0.094, 0.695±0.095, 0.555±0.080, 0.388±0.052, 0.341±0.060, 0.235± 0.056, respectively. The median immunoreactivity scores of p-ERK in each group were 8, 8, 6, 4, 5 and 3. The protein levels of p-RSK2 in the model, BL, BM, BH, PD98059 and BP groups were 0.613±0.085, 0.612±0.084, 0.427±0.089, 0.305±0.056, 0.258±0.051, 0.158±0.058, respectively. The median immunoreactivity scores of p-RSK in each group were 8, 8, 5, 3, 3 and 1. The protein level of GSK3β in the model, BL, BM, BH, PD98059 and BP groups were increased gradually, while the protein level of p-GSK3β and p-Bad were decreased gradually. Bufalin exerts significant inhibitory effect on the esophageal squamous cell carcinoma xenogragts in nude mice. Bufalin may suppress the growth of xenogragts in nude mice by down-regulating the level of ERK and RSK2 phosphorylation, inhibit the proliferation of xenogragts via inactivating GSK3β and promote apoptosis through down-regulation of p-Bad.

Role of genomic architecture in the expression dynamics of long noncoding RNAs during differentiation of human neuroblastoma cells.

PubMed

Batagov, Arsen O; Yarmishyn, Aliaksandr A; Jenjaroenpun, Piroon; Tan, Jovina Z; Nishida, Yuichiro; Kurochkin, Igor V

2013-10-16

Mammalian genomes are extensively transcribed producing thousands of long non-protein-coding RNAs (lncRNAs). The biological significance and function of the vast majority of lncRNAs remain unclear. Recent studies have implicated several lncRNAs as playing important roles in embryonic development and cancer progression. LncRNAs are characterized with different genomic architectures in relationship with their associated protein-coding genes. Our study aimed at bridging lncRNA architecture with dynamical patterns of their expression using differentiating human neuroblastoma cells model. LncRNA expression was studied in a 120-hours timecourse of differentiation of human neuroblastoma SH-SY5Y cells into neurons upon treatment with retinoic acid (RA), the compound used for the treatment of neuroblastoma. A custom microarray chip was utilized to interrogate expression levels of 9,267 lncRNAs in the course of differentiation. We categorized lncRNAs into 19 architecture classes according to their position relatively to protein-coding genes. For each architecture class, dynamics of expression of lncRNAs was studied in association with their protein-coding partners. It allowed us to demonstrate positive correlation of lncRNAs with their associated protein-coding genes at bidirectional promoters and for sense-antisense transcript pairs. In contrast, lncRNAs located in the introns and downstream of the protein-coding genes were characterized with negative correlation modes. We further classified the lncRNAs by the temporal patterns of their expression dynamics. We found that intronic and bidirectional promoter architectures are associated with rapid RA-dependent induction or repression of the corresponding lncRNAs, followed by their constant expression. At the same time, lncRNAs expressed downstream of protein-coding genes are characterized by rapid induction, followed by transcriptional repression. Quantitative RT-PCR analysis confirmed the discovered functional modes for several selected lncRNAs associated with proteins involved in cancer and embryonic development. This is the first report detailing dynamical changes of multiple lncRNAs during RA-induced neuroblastoma differentiation. Integration of genomic and transcriptomic levels of information allowed us to demonstrate specific behavior of lncRNAs organized in different genomic architectures. This study also provides a list of lncRNAs with possible roles in neuroblastoma.

N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification

PubMed Central

Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K.

2013-01-01

Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification. PMID:23941992

Molecular mechanisms and therapeutic targets in neuroblastoma.

PubMed

Johnsen, John Inge; Dyberg, Cecilia; Fransson, Susanne; Wickström, Malin

2018-05-01

Neuroblastoma is the most common extracranical tumor of childhood and the most deadly tumor of infancy. It is characterized by early age onset and high frequencies of metastatic disease but also the capacity to spontaneously regress. Despite intensive therapy, the survival for patients with high-risk neuroblastoma and those with recurrent or relapsed disease is low. Hence, there is an urgent need to develop new therapies for these patient groups. The molecular pathogenesis based on high-throughput omics technologies of neuroblastoma is beginning to be resolved which have given the opportunity to develop personalized therapies for high-risk patients. Here we discuss the potential of developing targeted therapies against aberrantly expressed molecules detected in sub-populations of neuroblastoma patients and how these selected targets can be drugged in order to overcome treatment resistance, improve survival and quality of life for these patients and also the possibilities to transfer preclinical research into clinical testing. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evaluation of a functional epigenetic approach to identify promoter region methylation in phaeochromocytoma and neuroblastoma

PubMed Central

Margetts, Caroline D E; Morris, Mark; Astuti, Dewi; Gentle, Dean C; Cascon, Alberto; McRonald, Fiona E; Catchpoole, Daniel; Robledo, Mercedes; Neumann, Hartmut P H; Latif, Farida; Maher, Eamonn R

2008-01-01

The molecular genetics of inherited phaeochromocytoma have received considerable attention, but the somatic genetic and epigenetic events that characterise tumourigenesis in sporadic phaeochromocytomas are less well defined. Previously, we found considerable overlap between patterns of promoter region tumour suppressor gene (TSG) hypermethylation in two neural crest tumours, neuroblastoma and phaeochromocytoma. In order to identify candidate biomarkers and epigenetically inactivated TSGs in phaeochromocytoma and neuroblastoma, we characterised changes in gene expression in three neuroblastoma cell lines after treatment with the demethylating agent 5-azacytidine. Promoter region methylation status was then determined for 28 genes that demonstrated increased expression after demethylation. Three genes HSP47, homeobox A9 (HOXA9) and opioid binding protein (OPCML) were methylated in >10% of phaeochromocytomas (52, 17 and 12% respectively). Two of the genes, epithelial membrane protein 3 (EMP3) and HSP47, demonstrated significantly more frequent methylation in neuroblastoma than phaeochromocytoma. These findings extend epigenotype of phaeochromocytoma and identify candidate genes implicated in sporadic phaeochromocytoma tumourigenesis. PMID:18499731

N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification.

PubMed

Hossain, Md Motarab; Banik, Naren L; Ray, Swapan K

2013-10-15

Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification. © 2013 Elsevier B.V. All rights reserved.

A Comparison of Raman Spectral Features of Frozen and Deparaffinized Tissues in Neuroblastoma and Ganglioneuroma

NASA Astrophysics Data System (ADS)

Devpura, Suneetha; Thakur, Jagdish S.; Poulik, Janet M.; Rabah, Raja; Naik, Vaman M.; Naik, Ratna

2012-02-01

We have investigated the cellular regions in neuroblastoma and ganglioneuroma using Raman spectroscopy and compared their spectral characteristics with those of normal adrenal gland. Thin sections from both frozen and deparaffinized tissues, obtained from the same tissue specimen, were studied in conjunction with the pathological examination of the tissues. We found a significant difference in the spectral features of frozen sections of normal adrenal gland, neuroblastoma, and ganglioneuroma when compared to deparaffinized tissues. The quantitative analysis of the Raman data using chemometric methods of principal component analysis and discriminant function analysis obtained from the frozen tissues show a sensitivity and specificity of 100% each. The biochemical identification based on the spectral differences shows that the normal adrenal gland tissues have higher levels of carotenoids, lipids, and cholesterol compared to the neuroblastoma and ganglioneuroma frozen tissues. However, deparaffinized tissues show complete removal of these biochemicals in adrenal tissues. This study demonstrates that Raman spectroscopy combined with chemometric methods can successfully distinguish neuroblastoma and ganglioneuroma at cellular level.

Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol A treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells

PubMed Central

Ma, Hongda; Yao, Yao; Wang, Changli; Zhang, Liyu; Cheng, Long; Wang, Yiren; Wang, Tao; Liang, Erguang; Jia, Hui; Ye, Qinong; Hou, Mingxiao; Feng, Fan

2016-01-01

Many kinds of endocrine-disrupting chemicals (EDCs), for example, the environmental estrogens bisphenol A and nonylphenol, may regulate the activity of estrogen receptor α (ERα) and therefore induce potential disruption of normal endocrine function. However, the involvement of EDCs in human cancers, especially in endocrine-related cancer neuroblastoma regulation, is not very clear. In this work, results showed that upon bisphenol A or nonylphenol treatment, the transcription factor activity of ERα was significantly increased in neuroblastoma cell line SH-SY5Y. Bisphenol A and nonylphenol could enhance ERα activity via recruiting it to the target gene promoter. Furthermore, treatment of bisphenol A and nonylphenol enhanced the in vitro proliferation, invasion, and migration ability of neuroblastoma cells. By investigating the role of EDC-induced ERα upregulation, our data extend the understanding of the function of EDCs and further suggest that ERα might be a potential therapeutic target in human neuroblastoma treatment. PMID:27366082

The genetic landscape of high-risk neuroblastoma | Office of Cancer Genomics

Cancer.gov

Abstract: Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative.

The “Neuro” of Neuroblastoma: Neuroblastoma as a Neurodevelopmental Disorder

PubMed Central

Ratner, Nancy; Brodeur, Garrett M.; Dale, Russell C.; Schor, Nina F.

2017-01-01

Neuroblastoma is a childhood cancer derived from cells of neural crest origin. The hallmarks of its enigmatic character include its propensity for spontaneous regression under some circumstances and its association with paraneoplastic opsoclonus, myoclonus, and ataxia. The neurodevelopmental underpinnings of its origins may provide important clues for development of novel therapeutic and preventive agents for this frequently fatal malignancy and for the associated paraneoplastic syndromes. PMID:27043043

A comprehensive characterization of rare mitochondrial DNA variants in neuroblastoma

PubMed Central

Pignataro, Piero; Lasorsa, Vito Alessandro; Hogarty, Michael D.; Castellano, Aurora; Conte, Massimo; Tonini, Gian Paolo; Iolascon, Achille; Gasparre, Giuseppe; Capasso, Mario

2016-01-01

Background Neuroblastoma, a tumor of the developing sympathetic nervous system, is a common childhood neoplasm that is often lethal. Mitochondrial DNA (mtDNA) mutations have been found in most tumors including neuroblastoma. We extracted mtDNA data from a cohort of neuroblastoma samples that had undergone Whole Exome Sequencing (WES) and also used snap-frozen samples in which mtDNA was entirely sequenced by Sanger technology. We next undertook the challenge of determining those mutations that are relevant to, or arisen during tumor development. The bioinformatics pipeline used to extract mitochondrial variants from matched tumor/blood samples was enriched by a set of filters inclusive of heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. Results Our in silico multistep workflow applied both on WES and Sanger-sequenced neuroblastoma samples, allowed us to identify a limited burden of somatic and germline mitochondrial mutations with a potential pathogenic impact. Conclusions The few singleton germline and somatic mitochondrial mutations emerged, according to our in silico analysis, do not appear to impact on the development of neuroblastoma. Our findings are consistent with the hypothesis that most mitochondrial somatic mutations can be considered as ‘passengers’ and consequently have no discernible effect in this type of cancer. PMID:27351283

Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin-related protein 14-mediated autophagy.

PubMed

Zhen, Zijun; Yang, Kaibin; Ye, Litong; You, Zhiyao; Chen, Rirong; Liu, Ying; He, Youjian

2017-07-01

Paclitaxel is not as effective for neuroblastoma as most of the front-line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel-associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy-associated proteins were assessed by western blot. Autophagy was induced and the autophagy-associated proteins LC3-I, LC3-II, Beclin 1, and thioredoxin-related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1-mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel-induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel-induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

A Promyelocytic Leukemia Protein-Thrombospondin-2 Axis and the Risk of Relapse in Neuroblastoma.

PubMed

Dvorkina, Maria; Nieddu, Valentina; Chakelam, Shalini; Pezzolo, Annalisa; Cantilena, Sandra; Leite, Ana Paula; Chayka, Olesya; Regad, Tarik; Pistorio, Angela; Sementa, Angela Rita; Virasami, Alex; Barton, Jack; Montano, Ximena; Lechertier, Tanguy; Brindle, Nicola; Morgenstern, Daniel; Lebras, Morgane; Burns, Alan J; Saunders, Nigel J; Hodivala-Dilke, Kairbaan; Bagella, Luigi; De The, Hugues; Anderson, John; Sebire, Neil; Pistoia, Vito; Sala, Arturo; Salomoni, Paolo

2016-07-01

Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system with a complex biology, prone to metastasize and relapse. High-risk, metastatic cases are explained in part by amplification or mutation of oncogenes, such as MYCN and ALK, and loss of tumor suppressor genes in chromosome band 1p. However, it is fundamental to identify other pathways responsible for the large portion of neuroblastomas with no obvious molecular alterations. Neuroblastoma cell lines were used for the assessment of tumor growth in vivo and in vitro Protein expression in tissues and cells was assessed using immunofluorescence and IHC. The association of promyelocytic leukemia (PML) expression with neuroblastoma outcome and relapse was calculated using log-rank and Mann-Whitney tests, respectively. Gene expression was assessed using chip microarrays. PML is detected in the developing and adult sympathetic nervous system, whereas it is not expressed or is low in metastatic neuroblastoma tumors. Reduced PML expression in patients with low-risk cancers, that is, localized and negative for the MYCN proto-oncogene, is strongly associated with tumor recurrence. PML-I, but not PML-IV, isoform suppresses angiogenesis via upregulation of thrombospondin-2 (TSP2), a key inhibitor of angiogenesis. Finally, PML-I and TSP2 expression inversely correlates with tumor angiogenesis and recurrence in localized neuroblastomas. Our work reveals a novel PML-I-TSP2 axis for the regulation of angiogenesis and cancer relapse, which could be used to identify patients with low-risk, localized tumors that might benefit from chemotherapy. Clin Cancer Res; 22(13); 3398-409. ©2016 AACR. ©2016 American Association for Cancer Research.

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiao, Lan; Department of Pharmaceutical Sciences, Jilin University, Changchun 130021; Paul, Pritha

2013-05-31

Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated thatmore » GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.« less

TAZ promotes epithelial to mesenchymal transition via the upregulation of connective tissue growth factor expression in neuroblastoma cells.

PubMed

Wang, Qiang; Xu, Zhilin; An, Qun; Jiang, Dapeng; Wang, Long; Liang, Bingxue; Li, Zhaozhu

2015-02-01

Neuroblastoma (NB) is a neuroendocrine cancer that occurs most commonly in infants and young children. The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP). The effect of TAZ on the metastatic progression of neuroblastoma and the underlying mechanisms involved remain elusive. In the current study, it was determined by western blot analysis that the migratory and invasive properties of SK‑N‑BE(2) human neuroblastoma cells are associated with high expression levels of TAZ. Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay. In contrast, overexpression of TAZ in SK‑N‑SH human neuroblastoma cells was shown by Transwell migration and invasion assays, and western blot analysis, to result in epithelial‑mesenchymal transition (EMT) and increased invasiveness. Mechanistically, the overexpression of TAZ was demonstrated to upregulate the expression levels of connective tissue growth factor (CTGF), by western blot analysis and chromatin immunoprecipitation assay, while the knockdown of TAZ downregulated it. Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway. In conclusion, the present study is, to the best of our knowledge, the first to demonstrate that the overexpression of TAZ induces EMT, increasing the invasive abilities of neuroblastoma cells. This suggests that TAZ may serve as a potential target in the development of novel therapies for the treatment of neuroblastoma.

Pediatric Sarcomas Are Targetable by MR-Guided High Intensity Focused Ultrasound (MR-HIFU): Anatomical Distribution and Radiological Characteristics.

PubMed

Shim, Jenny; Staruch, Robert M; Koral, Korgun; Xie, Xian-Jin; Chopra, Rajiv; Laetsch, Theodore W

2016-10-01

Despite intensive therapy, children with metastatic and recurrent sarcoma or neuroblastoma have a poor prognosis. Magnetic resonance guided high intensity focused ultrasound (MR-HIFU) is a noninvasive technique allowing the delivery of targeted ultrasound energy under MR imaging guidance. MR-HIFU may be used to ablate tumors without ionizing radiation or target chemotherapy using hyperthermia. Here, we evaluated the anatomic locations of tumors to assess the technical feasibility of MR-HIFU therapy for children with solid tumors. Patients with sarcoma or neuroblastoma with available cross-sectional imaging were studied. Tumors were classified based on the location and surrounding structures within the ultrasound beam path as (i) not targetable, (ii) completely or partially targetable with the currently available MR-HIFU system, and (iii) potentially targetable if a respiratory motion compensation technique was used. Of the 121 patients with sarcoma and 61 patients with neuroblastoma, 64% and 25% of primary tumors were targetable at diagnosis, respectively. Less than 20% of metastases at diagnosis or relapse were targetable for both sarcoma and neuroblastoma. Most targetable lesions were located in extremities or in the pelvis. Respiratory motion compensation may increase the percentage of targetable tumors by 4% for sarcomas and 10% for neuroblastoma. Many pediatric sarcomas are localized at diagnosis and are targetable by current MR-HIFU technology. Some children with neuroblastoma have bony tumors targetable by MR-HIFU at relapse, but few newly diagnosed children with neuroblastoma have tumors amenable to MR-HIFU therapy. Clinical trials of MR-HIFU should focus on patients with anatomically targetable tumors. © 2016 Wiley Periodicals, Inc.

Antagonism of cytotoxic chemotherapy in neuroblastoma cell lines by 13-cis-retinoic acid is mediated by the anti-apoptotic Bcl-2 family proteins

PubMed Central

Hadjidaniel, Michael Daniel; Reynolds, Charles Patrick

2010-01-01

13-cis-retinoic acid (13-cis-RA), is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. We investigated the effect of combining 13-cis-RA with cytotoxic agents employed in neuroblastoma therapy using a panel of 6 neuroblastoma cell lines. The effect of 13-cis-RA on the mitochondrial apoptotic pathway, was studied by flow cytometry, cytotoxicity by DIMSCAN, and protein expression by immuoblotting. Pre-treatment and direct combination of 13-cis-RA with etoposide, topotecan, cisplatin, melphalan, or doxorubicin markedly antagonized the cytotoxicity of those agents in 4 out of 6 tested neuroblastoma cell lines, increasing fractional cell survival by 1 to 3 logs. The inhibitory concentration of drugs (IC99) increased from clinically achievable levels to non-achievable levels: > 5-fold (cisplatin) to > 7-fold (etoposide). In SMS-KNCR neuroblastoma cells, 13-cis-RA upregulated expression of Bcl-2 and Bcl-xL RNA and protein, and this was associated with protection from etoposide-mediated apoptosis at the mitochondrial level. A small molecule inhibitor of the Bcl-2 family of proteins (ABT-737) restored mitochondrial membrane potential loss and apoptosis in response to cytotoxic agents in 13-cis-RA treated cells. Prior selection for resistance to RA did not diminish the response to cytotoxic treatment. Thus, combining 13-cis-RA with cytotoxic chemotherapy significantly reduced the cytotoxiciity for neuroblastoma in vitro, mediated at least in part via the anti-apoptotic Bcl-2 family of proteins. PMID:21159604

Radiolabeling and initial biological evaluation of [18F]KBM-1 for imaging RAR-α receptors in neuroblastoma.

PubMed

Solingapuram Sai, Kiran Kumar; Das, Bhaskar C; Sattiraju, Anirudh; Almaguel, Frankis G; Craft, Suzanne; Mintz, Akiva

2017-03-15

Retinoic acid receptor alpha (RAR-α) plays a significant role in a number of diseases, including neuroblastoma. Children diagnosed with high-risk neuroblastoma are treated13-cis-retinoic acid, which reduces risk of cancer recurrence. Neuroblastoma cell death is mediated via RAR-α, and expression of RAR-α is upregulated after treatment. A molecular imaging probe that binds RAR-α will help clinicians to diagnose and stratify risk for patients with neuroblastoma, who could benefit from retinoid-based therapy. In this study, we report the radiolabeling, and initial in vivo evaluation of [ 18 F]KBM-1, a novel RAR-α agonist. The radiochemical synthesis of [ 18 F]KBM-1 was carried out through KHF 2 assisted substitution of [ 18 F] - from aryl-substituted pinacolatoesters-based retinoid precursor. In vitro cell uptake assay in human neuroblastoma cell line showed that the uptake of [ 18 F]KBM-1 was significantly inhibited by all three blocking agents (KBM-1, ATRA, BD4) at all the selected incubation times. Standard biodistribution in mice bearing neuroblastoma tumors demonstrated increased tumor uptake from 5min to 60min post radiotracer injection and the uptake ratios for target to non-target (tumor: muscle) increased 2.2-fold to 3.7-fold from 30min to 60min post injection. Tumor uptake in subset of 30min blocking group was 1.7-fold lower than unblocked. These results demonstrate the potential utility of [ 18 F]KBM-1 as a RAR-α imaging agent. Copyright © 2017 Elsevier Ltd. All rights reserved.

Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

PubMed

Florea, Ana-Maria; Varghese, Elizabeth; McCallum, Jennifer E; Mahgoub, Safa; Helmy, Irfan; Varghese, Sharon; Gopinath, Neha; Sass, Steffen; Theis, Fabian J; Reifenberger, Guido; Büsselberg, Dietrich

2017-04-04

Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1-10 μM) or TOPO (0.1 nM-1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.

RFC-1 80G>A polymorphism in case-mother/control-mother dyads is associated with risk of nephroblastoma and neuroblastoma.

PubMed

Montalvão-de-Azevedo, Rafaela; Vasconcelos, Gisele M; Vargas, Fernando R; Thuler, Luiz Claudio; Pombo-de-Oliveira, Maria S; de Camargo, Beatriz

2015-02-01

Embryonic tumors are associated with an interruption during normal organ development; they may be related to disturbances in the folate pathway involved in DNA synthesis, methylation, and repair. Prenatal supplementation with folic acid is associated with a decreased risk of neuroblastoma, brain tumors, retinoblastoma, and nephroblastoma. The aim of this study was to investigate the association between MTHFR rs1801133 (C677T) and RFC-1 rs1051266 (G80A) genotypes with the risk of developing nephroblastoma and neuroblastoma. Case-mother/control-mother dyad study. Samples from Brazilian children with nephroblastoma (n=80), neuroblastoma (n=66), healthy controls (n=453), and their mothers (case n=93; control n=75) were analyzed. Genomic DNA was isolated from peripheral blood cells and/or buccal cells and genotyped to identify MTHFR C677T and RFC-1 G80A polymorphisms. Differences in genotype distribution between patients and controls were tested by multiple logistic regression analysis. Risk for nephroblastoma and neuroblastoma was two- to fourfold increased among children with RFC-1 polymorphisms. An increased four- to eightfold risk for neuroblastoma and nephroblastoma was seen when the child and maternal genotypes were combined. Our results suggest that mother and child RFC-1 G80A genotypes play a role on the risk of neuroblastoma and nephroblastoma since this polymorphism may impair the intracellular levels of folate, through carrying fewer folate molecules to the cell interior, and thus, the intracellular concentration is not enough to maintain regular DNA synthesis and methylation pathways.

The role of bone marrow evaluation in the staging of patients with otherwise localized, low-risk neuroblastoma.

PubMed

Russell, Heidi V; Golding, Laurie A; Suell, Mary Nell; Nuchtern, Jed G; Strother, Douglas R

2005-12-01

Bone marrow aspirations and biopsies are standard staging procedures for neuroblastoma because the tumor frequently metastasizes to the bone marrow. The presence of bone marrow metastases indicates stage 4 or 4S neuroblastoma by International Neuroblastoma Staging System (INSS) criteria; these stages are also associated with other metastatic sites of disease. We questioned whether bone marrow studies changed the staging or treatment of children with localized, completely resected tumors if there was no other evidence of metastatic spread. If stage of disease rarely changed with bone marrow results, it might be possible to avoid this procedure in a subset of patients with neuroblastoma. The staging studies of patients with INSS stage 1 (n = 29), 4 (n = 60), and 4S (n = 13) neuroblastoma from two institutions were reviewed. There were no patients upstaged from stage 1 to 4 or 4S by bone marrow metastases alone. Fifty-nine of 60 stage 4 patients had other sites of metastases on imaging studies, the remaining patient had an unresectable primary tumor and marrow disease. All subjects with stage 4S disease had liver metastases. Bone marrow studies did not contribute data that changed the stage of patients who had surgically resectable tumors and no evidence of metastatic spread on imaging studies. When present, metastatic spread to the marrow was associated with advanced local tumors or other sites of metastatic disease. Given the relatively small size of our study population, further studies are warranted that investigate the utility of bone marrow studies for patients who otherwise have INSS stage 1 neuroblastoma. 2005 Wiley-Liss, Inc.

Kinin and Purine Signaling Contributes to Neuroblastoma Metastasis.

PubMed

Ulrich, Henning; Ratajczak, Mariusz Z; Schneider, Gabriela; Adinolfi, Elena; Orioli, Elisa; Ferrazoli, Enéas G; Glaser, Talita; Corrêa-Velloso, Juliana; Martins, Poliana C M; Coutinho, Fernanda; Santos, Ana P J; Pillat, Micheli M; Sack, Ulrich; Lameu, Claudiana

2018-01-01

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca 2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.

Downregulation of N‑Myc inhibits neuroblastoma cell growth via the Wnt/β‑catenin signaling pathway.

PubMed

Wang, Yingge; Gao, Shan; Wang, Weiguang; Xia, Yuting; Liang, Jingyan

2018-05-03

Neuroblastoma, one of the most common types of cancer in childhood, is commonly treated with surgery, radiation and chemotherapy. However, prognosis and survival remain poor for children with high‑risk neuroblastoma. Therefore, the identification of novel, effective therapeutic targets is necessary. N‑Myc, a proto‑oncogene protein encoded by the v‑myc avial myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) gene, is associated with tumorigenesis. In the present study, the effect of N‑Myc silencing on MYCN‑amplified CHP134 and BE‑2C neuroblastoma cells was evaluated, and the underlying molecular mechanism was investigated. N‑Myc was successfully knocked down using an N‑Myc‑specific small interfering RNA, the efficacy of interference efficiency confirmed by reverse transcription‑quantitative polymerase chain reaction and western blotting. Cell viability was evaluated by MTT assay and apoptosis was measured by ELISA assay. The results indicated that MYCN silencing significantly decreased cell viability and promoted apoptosis. Subsequently, the expression levels of key Wnt/β‑catenin signaling pathway proteins were detected by western blotting, and MYCN silencing was demonstrated to inhibit Wnt/β‑catenin signaling, decreasing the expression ofanti‑apoptosis proteins and increasing the expression of pro‑apoptosis protein. This suggested that N‑Myc regulated survival and growth of CHP134 and BE‑2C neuroblastoma cells, potentially through Wnt/β‑catenin signaling. Furthermore, associated proteins, N‑Myc and STAT interactor and dickkopf Wnt signaling pathway inhibitor 1, were demonstrated to be involved in this regulation. Therefore, N‑Myc and its downstream targets may provide novel therapeutic targets for the treatment of neuroblastoma.

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

PubMed Central

Suemizu, Hiroshi; Kawai, Kenji; Higuchi, Yuichiro; Hashimoto, Haruo; Ogura, Tomoyuki; Itoh, Toshio; Sasaki, Erika; Nakamura, Masato

2013-01-01

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging. PMID:24358218

MIBG scintiscan

MedlinePlus

Adrenal medullary imaging; Meta-iodobenzylguanidine scintiscan; Pheochromocytoma - MIBG; Neuroblastoma - MIBG; Carcinoid MIBG ... answer. It is also used to help diagnose neuroblastoma and can be used for carcinoid tumors.

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D.; Sung, Derek C.; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D.; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-01-01

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation. PMID:27764804

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells.

PubMed

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D; Sung, Derek C; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-11-29

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3'UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

Natural killer T cell facilitated engraftment of rat skin but not islet xenografts in mice.

PubMed

Gordon, Ethel J; Kelkar, Vinaya

2009-01-01

We have studied cellular components required for xenograft survival mediated by anti-CD154 monoclonal antibody (mAb) and a transfusion of donor spleen cells and found that the elimination of CD4(+) but not CD8(+) cells significantly improves graft survival. A contribution of other cellular components, such as natural killer (NK) cells and natural killer T (NKT) cells, for costimulation blockade-induced xenograft survival has not been clearly defined. We therefore tested the hypothesis that NK or NKT cells would promote rat islet and skin xenograft acceptance in mice. Lewis rat islets or skin was transplanted into wild type B6 mice or into B6 mice that were Jalpha18(null), CD1(null), or beta2 microglobulin (beta2M)(null) NK 1.1 depleted, or perforin(null). Graft recipients were pretreated with an infusion of donor derived spleen cells and a brief course of anti-CD154 mAb treatments. Additional groups received mAb or cells only. We first observed that the depletion of NK1.1 cells does not significantly interfere with graft survival in C57BL/6 (B6) mice. We used NKT cell deficient B6 mice to test the hypothesis that NKT cells are involved in islet and skin xenograft survival in our model. These mice bear a null mutation in the gene for the Jalpha18 component of the T-cell receptor. The component is uniquely associated with NKT cells. We found no difference in islet xenograft survival between Jalpha18(null) and wild type B6 mice. In contrast, median skin graft survival appeared shorter in Jalpha18(null) recipients. These data imply a role for Jalpha18(+) NKT cells in skin xenograft survival in treated mice. In order to confirm this inference, we tested skin xenograft survival in B6 CD1(null) mice because NKT cells are CD1 restricted. Results of these trials demonstrate that the absence of CD1(+) cells adversely affects rat skin graft survival. An additional assay in beta2M(null) mice demonstrated a requirement for major histocompatibility complex (MHC) class I expression in the graft host, and we demonstrate that CD1 is the requisite MHC component. We further demonstrated that, unlike reports for allograft survival, skin xenograft survival does not require perforin-secreting NK cells. We conclude that MHC class I(+) CD1(+) Jalpha18(+) NKT cells promote the survival of rat skin but not rat islet xenografts. These studies implicate different mechanisms for inducing and maintaining islet vs. skin xenograft survival in mice treated with donor antigen and anti-CD154 mAb, and further indicate a role for NKT cells but not NK cells in skin xenograft survival.

Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.

PubMed

Verginelli, Fabio; Perconti, Silvia; Vespa, Simone; Schiavi, Francesca; Prasad, Sampath Chandra; Lanuti, Paola; Cama, Alessandro; Tramontana, Lorenzo; Esposito, Diana Liberata; Guarnieri, Simone; Sheu, Artenca; Pantalone, Mattia Russel; Florio, Rosalba; Morgano, Annalisa; Rossi, Cosmo; Bologna, Giuseppina; Marchisio, Marco; D'Argenio, Andrea; Taschin, Elisa; Visone, Rosa; Opocher, Giuseppe; Veronese, Angelo; Paties, Carlo T; Rajasekhar, Vinagolu K; Söderberg-Nauclér, Cecilia; Sanna, Mario; Lotti, Lavinia Vittoria; Mariani-Costantini, Renato

2018-05-01

Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.

Heterogeneous Binding and Central Nervous System Distribution of the Multitargeted Kinase Inhibitor Ponatinib Restrict Orthotopic Efficacy in a Patient-Derived Xenograft Model of Glioblastoma.

PubMed

Laramy, Janice K; Kim, Minjee; Gupta, Shiv K; Parrish, Karen E; Zhang, Shuangling; Bakken, Katrina K; Carlson, Brett L; Mladek, Ann C; Ma, Daniel J; Sarkaria, Jann N; Elmquist, William F

2017-11-01

This study investigated how differences in drug distribution and free fraction at different tumor and tissue sites influence the efficacy of the multikinase inhibitor ponatinib in a patient-derived xenograft model of glioblastoma (GBM). Efficacy studies in GBM6 flank (heterotopic) and intracranial (orthotopic) models showed that ponatinib is effective in the flank but not in the intracranial model, despite a relatively high brain-to-plasma ratio. In vitro binding studies indicated that flank tumor had a higher free (unbound) drug fraction than normal brain. The total and free drug concentrations, along with the tissue-to-plasma ratio (Kp) and its unbound derivative (Kp,uu), were consistently higher in the flank tumor than the normal brain at 1 and 6 hours after a single dose in GBM6 flank xenografts. In the orthotopic xenografts, the intracranial tumor core displayed higher Kp and Kp,uu values compared with the brain-around-tumor (BAT). The free fractions and the total drug concentrations, hence free drug concentrations, were consistently higher in the core than in the BAT at 1 and 6 hours postdose. The delivery disadvantages in the brain and BAT were further evidenced by the low total drug concentrations in these areas that did not consistently exceed the in vitro cytotoxic concentration (IC 50 ). Taken together, the regional differences in free drug exposure across the intracranial tumor may be responsible for compromising efficacy of ponatinib in orthotopic GBM6. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

The growth-inhibitory and apoptosis-inducing effect of deferoxamine combined with arsenic trioxide on HL-60 xenografts in nude mice.

PubMed

Yu, Runhong; Wang, Dao; Ren, Xiuhua; Zeng, Li; Liu, Yufeng

2014-09-01

The aim of this study is to investigate the growth-inhibitory and apoptosis-inducing effect of deferoxamine (DFO) combined with arsenic trioxide (ATO) on the human HL-60 xenografts in nude mice and its mechanism. The highly tumorigenic leukemia cell line HL-60 cells were inoculated subcutaneously into nude mice to establish a human leukemia xenograft model. The HL-60 xenograft nude mice models were randomly divided into four groups: control (Normal saline, NS), 50mg/kg DFO, 3mg/kg ATO, the combined treatment (50mg/kg DFO+1.5mg/kg ATO) once HL-60 cells were inoculated. Tumor sizes, growth curves, inhibitory rates, cell apoptosis, and the expression of apoptosis related markers were measured to evaluate the tumor growth. Xenografted tumors were observed in all nude mice since the 5th day of inoculation. The inhibitory rates of tumor weight were 2.67%, 10.69%, and 25.57% in DFO, ATO and combination therapy groups, respectively. The combination of DFO with ATO induces significantly more tumor cell apoptosis than either agent alone (p<0.05). The expression of NF-κBp65 and survivin proteins decreased significantly while the expression of Caspase-3 and Bax increased in the combination therapy group (p<0.05). Double immunofluorescence for Caspase-3 and NFκBp65 demonstrated an inverse relationship between Caspase-3-positive areas and NFκBp65-positive areas, as well as the co-localization of Bax and survivin in xenografted tumor cells. Combination of DFO and ATO has synergistic effects on tumor growth inhibition and apoptosis-inducing in vivo with no significant side effects. The DFO and ATO can up-regulate the expression of Caspase-3 and Bax, and down-regulate the expression of NF-κBp65 and survivin, especially for their combination. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sequential Exposure of Bortezomib and Vorinostat is Synergistic in Multiple Myeloma Cells

PubMed Central

Nanavati, Charvi; Mager, Donald E.

2018-01-01

Purpose To examine the combination of bortezomib and vorinostat in multiple myeloma cells (U266) and xenografts, and to assess the nature of their potential interactions with semi-mechanistic pharmacodynamic models and biomarkers. Methods U266 proliferation was examined for a range of bortezomib and vorinostat exposure times and concentrations (alone and in combination). A non-competitive interaction model was used with interaction parameters that reflect the nature of drug interactions after simultaneous and sequential exposures. p21 and cleaved PARP were measured using immunoblotting to assess critical biomarker dynamics. For xenografts, data were extracted from literature and modeled with a PK/PD model with an interaction parameter. Results Estimated model parameters for simultaneous in vitro and xenograft treatments suggested additive drug effects. The sequence of bortezomib preincubation for 24 hours, followed by vorinostat for 24 hours, resulted in an estimated interaction term significantly less than 1, suggesting synergistic effects. p21 and cleaved PARP were also up-regulated the most in this sequence. Conclusions Semi-mechanistic pharmacodynamic modeling suggests synergistic pharmacodynamic interactions for the sequential administration of bortezomib followed by vorinostat. Increased p21 and cleaved PARP expression can potentially explain mechanisms of their enhanced effects, which require further PK/PD systems analysis to suggest an optimal dosing regimen. PMID:28101809

Melatonin exerts anti-oral cancer effect via suppressing LSD1 in patient-derived tumor xenograft models

PubMed Central

Yang, Cheng-Yu; Lin, Chih-Kung; Tsao, Chang-Huei; Hsieh, Cheng-Chih; Lin, Gu-Jiun; Ma, Kuo-Hsing; Shieh, Yi-Shing; Sytwu, Huey-Kang; Chen, Yuan-Wu

2017-01-01

Aberrant activation of histone lysine-specific demethylase (LSD1) increases tumorigenicity; hence, LSD1 is considered a therapeutic target for various human cancers. Although melatonin, an endogenously produced molecule, may defend against various cancers, the precise mechanism involved in its anti-oral cancer effect remains unclear. Patient-derived tumor xenograft (PDTX) models are preclinical models that can more accurately reflect human tumor biology compared with cell line xenograft models. Here, we evaluated the anticancer activity of melatonin by using LSD1-overexpressing oral cancer PDTX models. By assessing oral squamous cell carcinoma (OSCC) tissue arrays through immunohistochemistry, we examined whether aberrant LSD1 overexpression in OSCC is associated with poor prognosis. We also evaluated the action mechanism of melatonin against OSCC with lymphatic metastases by using the PDTX models. Our results indicated that melatonin, at pharmacological concentrations, significantly suppresses cell proliferation in a dose- and time-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of LSD1 in oral cancer PDTXs and oral cancer cell lines. In conclusion, we determined that the beneficial effects of melatonin in reducing oral cancer cell proliferation are associated with reduced LSD1 expression in vivo and in vitro. PMID:28422711

Exosome-Mediated Transfer of microRNAs Within the Tumor Microenvironment and Neuroblastoma Resistance to Chemotherapy

PubMed Central

Challagundla, Kishore B.; Wise, Petra M.; Neviani, Paolo; Chava, Haritha; Murtadha, Mariam; Xu, Tong; Kennedy, Rebekah; Ivan, Cristina; Zhang, Xinna; Vannini, Ivan; Fanini, Francesca; Amadori, Dino; Calin, George A.; Hadjidaniel, Michael; Shimada, Hiroyuki; Jong, Ambrose; Seeger, Robert C.; Asgharzadeh, Shahab; Goldkorn, Amir

2015-01-01

Background: How exosomic microRNAs (miRNAs) contribute to the development of drug resistance in the context of the tumor microenvironment has not been previously described in neuroblastoma (NBL). Methods: Coculture experiments were performed to assess exosomic transfer of miR-21 from NBL cells to human monocytes and miR-155 from human monocytes to NBL cells. Luciferase reporter assays were performed to assess miR-155 targeting of TERF1 in NBL cells. Tumor growth was measured in NBL xenografts treated with Cisplatin and peritumoral exosomic miR-155 (n = 6 mice per group) CD163, miR-155, and TERF1 levels were assessed in 20 NBL primary tissues by Human Exon Arrays and quantitative real-time polymerase chain reaction. Student’s t test was used to evaluate the differences between treatment groups. All statistical tests were two-sided. Results: miR-21 mean fold change (f.c.) was 12.08±0.30 (P < .001) in human monocytes treated with NBL derived exosomes for 48 hours, and miR-155 mean f.c. was 4.51±0.25 (P < .001) in NBL cells cocultured with human monocytes for 48 hours. TERF1 mean luciferase activity in miR-155 transfected NBL cells normalized to scrambled was 0.36 ± 0.05 (P <.001). Mean tumor volumes in Dotap-miR-155 compared with Dotap-scrambled were 322.80±120mm3 and 76.00±39.3mm3, P = .002 at day 24, respectively. Patients with high CD163 infiltrating NBLs had statistically significantly higher intratumoral levels of miR-155 (P = .04) and lower levels of TERF1 mRNA (P = .02). Conclusions: These data indicate a unique role of exosomic miR-21 and miR-155 in the cross-talk between NBL cells and human monocytes in the resistance to chemotherapy, through a novel exosomic miR-21/TLR8-NF-кB/exosomic miR-155/TERF1 signaling pathway. PMID:25972604

Exosome-mediated transfer of microRNAs within the tumor microenvironment and neuroblastoma resistance to chemotherapy.

PubMed

Challagundla, Kishore B; Wise, Petra M; Neviani, Paolo; Chava, Haritha; Murtadha, Mariam; Xu, Tong; Kennedy, Rebekah; Ivan, Cristina; Zhang, Xinna; Vannini, Ivan; Fanini, Francesca; Amadori, Dino; Calin, George A; Hadjidaniel, Michael; Shimada, Hiroyuki; Jong, Ambrose; Seeger, Robert C; Asgharzadeh, Shahab; Goldkorn, Amir; Fabbri, Muller

2015-07-01

How exosomic microRNAs (miRNAs) contribute to the development of drug resistance in the context of the tumor microenvironment has not been previously described in neuroblastoma (NBL). Coculture experiments were performed to assess exosomic transfer of miR-21 from NBL cells to human monocytes and miR-155 from human monocytes to NBL cells. Luciferase reporter assays were performed to assess miR-155 targeting of TERF1 in NBL cells. Tumor growth was measured in NBL xenografts treated with Cisplatin and peritumoral exosomic miR-155 (n = 6 mice per group) CD163, miR-155, and TERF1 levels were assessed in 20 NBL primary tissues by Human Exon Arrays and quantitative real-time polymerase chain reaction. Student's t test was used to evaluate the differences between treatment groups. All statistical tests were two-sided. miR-21 mean fold change (f.c.) was 12.08±0.30 (P < .001) in human monocytes treated with NBL derived exosomes for 48 hours, and miR-155 mean f.c. was 4.51±0.25 (P < .001) in NBL cells cocultured with human monocytes for 48 hours. TERF1 mean luciferase activity in miR-155 transfected NBL cells normalized to scrambled was 0.36 ± 0.05 (P <.001). Mean tumor volumes in Dotap-miR-155 compared with Dotap-scrambled were 322.80±120mm(3) and 76.00±39.3mm(3), P = .002 at day 24, respectively. Patients with high CD163 infiltrating NBLs had statistically significantly higher intratumoral levels of miR-155 (P = .04) and lower levels of TERF1 mRNA (P = .02). These data indicate a unique role of exosomic miR-21 and miR-155 in the cross-talk between NBL cells and human monocytes in the resistance to chemotherapy, through a novel exosomic miR-21/TLR8-NF-кB/exosomic miR-155/TERF1 signaling pathway. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Hyperpolarized 13C MR Markers of Renal Tumor Aggressiveness

DTIC Science & Technology

2015-12-01

production in two human glioblastoma xenograft models where the blood–brain barrier (BBB) was disrupted relative to normal brain, suggesting that HP...rodent mammary adenocarcinoma and murine lymphoma xenografts ) has shown ample conversion to leucine.98 In this preclinical study, SNR and contrast were...4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner, Oncogene 33 (2013) 4433–4441. Real