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Sample records for p27kip1 reveals cancer

  1. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    PubMed

    Ibañez, Irene L; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L; Policastro, Lucía L; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27

  2. Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

    PubMed Central

    Ibañez, Irene L.; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L.; Policastro, Lucía L.; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1. PMID

  3. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    PubMed

    Ibañez, Irene L; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L; Policastro, Lucía L; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27

  4. Cdk inhibitor p27Kip1 and hormone dependence in breast cancer.

    PubMed

    Arteaga, Carlos L

    2004-01-01

    p27Kip1 is an important regulator of the G1 to S transition. While a potent inhibitor of cyclin-dependent-kinase (Cdk)2, p27 is also involved in assembly of cyclin D/Cdk4 complexes. Although rarely mutated, p27 is functionally downregulated in many human cancers by mechanisms involving enhanced degradation, cytoplasmic mislocalization, and/or sequestration by cyclin D/Cdk complexes in response to oncogenic signals. Therefore, low levels and/or cytoplasmic localized p27 have been associated with enhanced malignancy and poor patient prognosis in many neoplasias including breast cancer. Recent data discussed below suggest that a threshold of p27 is required for response to antiestrogens and, conversely, that low levels predict for antiestrogen resistance. These results imply that hormone receptor-positive tumors with low and/or cytosolic p27 respond poorly to antiestrogens and should be considered for alternative therapeutic strategies.

  5. Prognostic role of p27Kip1 and apoptosis in human breast cancer

    PubMed Central

    Wu, J; Shen, Z-Z; Lu, J-S; Jiang, M; Han, Q-X; Fontana, J A; Barsky, S H; Shao, Z-M

    1999-01-01

    Human breast carcinoma is biologically heterogeneous, and its clinical course may vary from an indolent slowly progressive one to a course associated with rapid progression and metastatic spread. It is important to establish prognostic factors which will define subgroups of patients with low vs high risk of recurrence so as to better define the need for additional therapy. Additional characterization of the molecular make-up of breast cancer phenotypes should provide important insights into the biology of breast cancer. In the present study, we investigated apoptosis, expression of p27Kip1 and p53 retrospectively in 181 human breast cancer specimens. In addition, their relevance to the biological behaviour of breast cancer was examined. Our studies found a significant association among high histological grade, high p53, low apoptosis and low p27. Our results also demonstrated that, in human breast cancer, low levels of p27 and apoptotic index (AI) strongly correlated with the presence of lymph node metastasis and decreased patient survival. In node-negative patients, however, p27 also had prognostic value for relapse-free and overall survival in multivariate analysis. Furthermore p27 and AI had predictive value for the benefits of chemotherapy. These latter observations should prompt prospective randomized studies designed to investigate the predictive role of p27 and AI in determining who should receive chemotherapy in node-negative patients. © 1999 Cancer Research Campaign PMID:10188908

  6. Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation.

    PubMed

    Khan, Naim A; Nishimura, Kazuhiro; Aires, Virginie; Yamashita, Tomoko; Oaxaca-Castillo, David; Kashiwagi, Keiko; Igarashi, Kazuei

    2006-10-01

    Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

  7. Rheb promotes cancer cell survival through p27Kip1-dependent activation of autophagy.

    PubMed

    Campos, Tania; Ziehe, Javiera; Palma, Mario; Escobar, David; Tapia, Julio C; Pincheira, Roxana; Castro, Ariel F

    2016-02-01

    We previously found that the small GTPase Rheb regulates the cell-cycle inhibitor p27KIP1 (p27) in colon cancer cells by a mTORC1-independent mechanism. However, the biological function of the Rheb/p27 axis in cancer cells remains unknown. Here, we show that siRNA-mediated depletion of Rheb decreases survival of human colon cancer cells under serum deprivation. As autophagy can support cell survival, we analyzed the effect of Rheb on this process by detecting the modification of the autophagy marker protein LC3 by western blot and imunofluorescence. We found that Rheb promotes autophagy in several human cancer cell lines under serum deprivation. Accordingly, blocking autophagy inhibited the pro-survival effect of Rheb in colon cancer cells. We then analyzed whether p27 was involved in the biological effect of Rheb. Depletion of p27 inhibited colon cancer cell survival, and Rheb induction of autophagy. These results suggest that p27 has an essential role in the effect of Rheb in response to serum deprivation. In addition, we demonstrated that the role of p27 in autophagy stands on the N-terminal portion of the protein, where the CDK-inhibitory domain is located. Our results indicate that a Rheb/p27 axis accounts for the activation of autophagy that supports cancer cell survival. Our work therefore highlights a biological function of Rheb and prompts the need for future studies to address whether the mTORC1-independent Rheb/p27 axis could contribute to tumorigenesis and/or resistance to mTOR inhibitors.

  8. Genetic mosaics reveal both cell-autonomous and cell-nonautonomous function of murine p27Kip1

    PubMed Central

    Chien, Wei-Ming; Rabin, Stuart; Macias, Everardo; Miliani de Marval, Paula L.; Garrison, Kendra; Orthel, Jason; Rodriguez-Puebla, Marcelo; Fero, Matthew L.

    2006-01-01

    Loss of the cyclin-dependent kinase inhibitor p27Kip1 leads to an overall increase in animal growth, pituitary tumors, and hyperplasia of hematopoietic organs, yet it is unknown whether all cells function autonomously in response to p27Kip1 activity or whether certain cells take cues from their neighbors. In addition, there is currently no genetic evidence that tumor suppression by p27Kip1 is cell-autonomous because biallelic gene inactivation is absent from tumors arising in p27Kip1 hemizygous mice. We have addressed these questions with tissue-specific targeted mouse mutants and radiation chimeras. Our results indicate that the suppression of pars intermedia pituitary tumors by p27Kip1 is cell-autonomous and does not contribute to overgrowth or infertility phenotypes. In contrast, suppression of spleen growth and hematopoietic progenitor expansion is a consequence of p27Kip1 function external to the hematopoietic compartment. Likewise, p27Kip1 suppresses thymocyte hyperplasia through a cell-nonautonomous mechanism. The interaction of p27Kip1 loss with epithelial cell-specific cyclin-dependent kinase 4 overexpression identifies the thymic epithelium as a relevant site of p27Kip1 activity for the regulation of thymus growth. PMID:16537495

  9. Regulation of the p27Kip1 tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation

    PubMed Central

    le Sage, Carlos; Nagel, Remco; Egan, David A; Schrier, Mariette; Mesman, Elly; Mangiola, Annunziato; Anile, Corrado; Maira, Giulio; Mercatelli, Neri; Ciafrè, Silvia Anna; Farace, Maria Giulia; Agami, Reuven

    2007-01-01

    MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR-221 and miR-222 (miR-221&222) as potent regulators of p27Kip1, a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-221&222 to maintain low p27Kip1 levels and continuous proliferation. Interestingly, high levels of miR-221&222 appear in glioblastomas and correlate with low levels of p27Kip1 protein. Thus, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27Kip1. PMID:17627278

  10. Upregulation of p27Kip1 by demethylation sensitizes cisplatin-resistant human ovarian cancer SKOV3 cells.

    PubMed

    Zhao, Yan; Li, Qiaoyan; Wu, Xiaoying; Chen, Puxiang

    2016-08-01

    Ovarian cancer has a poor prognosis due to its chemoresistance, and p27Kip1 (p27) has been implicated in tumor prognosis and drug-resistance. However, the regulatory mechanisms of p27 in drug‑resistance in ovarian cancer remain unknown. The current study successfully established chemoresistant cell lines using paclitaxel (TAX), cisplatin (DDP) and carboplatin (CBP) in SKOV3 ovarian cancer cells. The results indicated that the expression levels of p27 were dramatically downregulated in chemoresistant cells. However, 5-aza-2'-deoxycytidine (5-aza) treatment restored p27 expression in DDP-resistant cells, and increased their sensitivity to DDP. In addition, it was observed that the methylation of DDP‑resistant cells, which was downregulated by 5‑aza treatment, was significantly higher compared with SKOV3 cells. Additionally, the overexpression of p27 arrested the cell cycle in S phase and promoted an apoptotic response to DDP. In conclusion, p27 was involved in chemoresistance of SKOV3 cells. Upregulated p27 expression induced by demethylation may enhance sensitivity to DDP through the regulation of the cell cycle. PMID:27314502

  11. p27Kip1 in Stage III Colon Cancer: Implications for Outcome Following Adjuvant Chemotherapy in CALGB 89803

    PubMed Central

    Bertagnolli, Monica M.; Warren, Robert S.; Niedzwiecki, Donna; Mueller, Elke; Compton, Carolyn C.; Redston, Mark; Hall, Margaret; Hahn, Hejin P.; Jewell, Scott D.; Mayer, Robert J.; Goldberg, Richard M.; Saltz, Leonard B.; Loda, Massimo

    2010-01-01

    Background In retrospective studies, loss of p27Kip1 (p27), a cyclin dependent kinase inhibitor, has been associated with poor prognosis following colorectal cancer treatment. In a prospective study, we validated this relationship in patients enrolled on a trial of adjuvant chemotherapy for Stage III colon cancer. Methods Cancer and Leukemia Group B (CALGB) protocol 89803 randomized 1264 stage III colon cancer patients to receive weekly bolus fluorouracil/leucovorin (5FU/LV) or weekly bolus irinotecan, fluorouracil, and leucovorin (IFL). The primary endpoint was overall survival (OS); disease-free survival (DFS) was a secondary endpoint. Expression of p27 and DNA mismatch repair (MMR) proteins were determined by immunohistochemistry (IHC) in primary tumor and normal tissue from paraffin blocks. Data were analyzed using logrank test. Results Of 601 tumors analyzed, 207 (34.4%) demonstrated p27 loss, 377 (62.8%) retained p27, and 17 (2.8%) were indeterminate. Patients with p27 negative tumors showed reduced OS (5-year 66%; 95%CI 0.59-0.72 vs. 75%; 95%CI 0.70-0.79, logrank p=0.021). This relationship was not influenced by treatment arm. Combination of p27 status with MMR status, however, identified a small subset of patients that may benefit from IFL (n=36; 5-year DFS 81%; 95%CI 0.64-0.98 vs. 47%; 95%CI 0.21-0.72, logrank p=0.042; 5-year OS 81%; 95%CI 0.64-0.98 vs. 60%; 95%CI 0.35-0.85; logrank p=0.128). Conclusions Loss of p27 is associated with reduced survival in stage III colon cancer, but by itself does not indicate a significant difference in outcome between patients treated IFL or 5FU-LV. PMID:19276255

  12. CCN5/WISP-2 promotes growth arrest of triple-negative breast cancer cells through accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation.

    PubMed

    Haque, I; Banerjee, S; De, A; Maity, G; Sarkar, S; Majumdar, M; Jha, S S; McGragor, D; Banerjee, S K

    2015-06-11

    The matricellular protein CCN5/WISP-2 represents a promising target in triple-negative breast cancer (TNBC) because treatment or induced activation of CCN5 in TNBC cells promotes cell growth arrest at the G0/G1 phase, reduces cell proliferation and delays tumor growth in the xenograft model. Our studies found that the p27(Kip1) tumor suppressor protein is upregulated and relocalized to the nucleus from cytoplasm by CCN5 in these cells and that these two events (upregulation and relocalization of p27(Kip1)) are critical for CCN5-induced growth inhibition of TNBC cells. In the absence of CCN5, p27(Kip1) resides mostly in the cytoplasm, which is associated with the aggressive nature of cancer cells. Mechanistically, CCN5 inhibits Skp2 expression, which seems to stabilize the p27(Kip1) protein in these cells. On the other hand, CCN5 also recruits FOXO3a to mediate the transcriptional regulation of p27(Kip1). The recruitment of FOXO3a is achieved by the induction of its expression and activity through shifting from cytoplasm to the nucleus. Our data indicate that CCN5 blocks PI3K/AKT signaling to dephosphorylate at S318, S253 and Thr32 in FOXO3a for nuclear relocalization and activation of FOXO3a. Moreover, inhibition of α6β1 receptors diminishes CCN5 action on p27(Kip1) in TNBC cells. Collectively, these data suggest that CCN5 effectively inhibits TNBC growth through the accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation, and thus, activation of CCN5 may have the therapeutic potential to kill TNBC.

  13. KDM4B and KDM4A promote endometrial cancer progression by regulating androgen receptor, c-myc, and p27kip1

    PubMed Central

    Kwan, Suet-Ying; Chen, Limo; Chen, Jin-Hong; Ying, Zuo-Lin; Zhou, Ye; Gu, Wei; Wang, Li-Hua; Cheng, Wei-Wei; Zeng, Jianfang; Wan, Xiao-Ping; Mok, Samuel C.; Wong, Kwong-Kwok; Bao, Wei

    2015-01-01

    Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27kip1. Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity. PMID:26397136

  14. KDM4B and KDM4A promote endometrial cancer progression by regulating androgen receptor, c-myc, and p27kip1.

    PubMed

    Qiu, Mei-Ting; Fan, Qiong; Zhu, Zhu; Kwan, Suet-Ying; Chen, Limo; Chen, Jin-Hong; Ying, Zuo-Lin; Zhou, Ye; Gu, Wei; Wang, Li-Hua; Cheng, Wei-Wei; Zeng, Jianfang; Wan, Xiao-Ping; Mok, Samuel C; Wong, Kwong-Kwok; Bao, Wei

    2015-10-13

    Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27kip1. Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity. PMID:26397136

  15. Down-regulation of both p21/Cip1 and p27/Kip1 produces a more aggressive prostate cancer phenotype

    PubMed Central

    Roy, Srirupa; Singh, Rana P.; Agarwal, Chapla; Siriwardana, Sunitha; Sclafani, Robert; Agarwal, Rajesh

    2009-01-01

    Roles of cyclin dependent kinase inhibitors, p21/Cip1 (p21) and p27/Kip1 (p27) in prostate cancer (PCa) progression is still not clear. Lower p27 protein expression in PCa tissues is often associated with poor prognosis, but prognostic significance of p21 is still controversial. Herein, we investigated the role of these molecules in determining PCa growth characteristics. We generated human PCa DU145 cell variants with knocked down levels of p21 (DU-p21) or p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective shRNAs and compared their various characteristics with empty vector-transduced DU145 (DU-EV) cells in vitro as well as in vivo. Knocking down either p21 or p27 did not show any significant change in doubling time, clonogenicity and cell cycle progression in DU145 cells, but simultaneous knock-down of both p21 and p27 significantly enhanced these parameters. In athymic mice, DU-p21+p27 tumors showed higher growth rate than the comparable growth of DU-EV, DU-p21 and DU-p27 tumors. Concurrently, DU-p21+p27 tumors had significantly higher proliferation rate, showing 54% and 48% increase in proliferating cell nuclear antigen (PCNA) and Ki-67-positive cells, respectively, compared to DU-EV tumors. DU-p21+p27 tumors also showed higher microvessel density and increased expression of vascular endothelial growth factor (VEGF). Proliferation and angiogenic status of DU-p21 and DU-p27 tumors was comparable to DU-EV tumors. Both in vitro and in vivo results implicate that p21 and p27 have compensatory roles in advanced prostate cancer cells, and ablation or down-modulation of both these molecules essentially enhances the aggressive prostate carcinoma phenotype. PMID:18583941

  16. Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

    PubMed Central

    Su, Liang-Cheng; Jiang, Shih Sheng; Chan, Tzu-Min; Chang, Chung-Ho; Chen, Li-Tzong; Kung, Hsing-Jien; Wang, Horng-Dar; Chuu, Chih-Pin

    2015-01-01

    Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1–3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4–2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1. PMID:25788262

  17. gamma-Linolenic acid blocks cell cycle progression by regulating phosphorylation of p27kip1 and p57kip2 and their interactions with other cycle regulators in cancer cells.

    PubMed

    Jiang, W G; Bryce, R P; Horrobin, D F; Mansel, R E

    1998-09-01

    gamma-Linolenic acid (gamma-LA), a n-6 essential fatty acid, has been previously shown to affect cell cycle and growth of cancer cells. This study examined the effects of gamma-LA on the cell cycle and cycle regulators in human colon cancer HT115 and breast cancer MCF7 cells. Brief treatment of cancer cells (<2 h) with gamma-LA resulted in a decrease in the phosphorylation of both cell cycle inhibitors, p27kip1 and p57kip2 as shown by immunoprecipitation and Western blotting. Protein levels of both inhibitors were increased following a prolonged culture of cells with the fatty acid. A co-precipitation study showed that in cells treated with gamma-LA there was an increase in the binding of these inhibitors with CDK4, CDC2, and cyclin E. Flow cytometry study indicated an inhibition of cell cycle progression by gamma-LA (G0/G1 -45.4%, S - 34.6%, G2+M - 20.0% in control, and 70.5%, 21.0%, and 8.5%, respectively, in gamma-LA treated cells). It is concluded that gamma-linolenic acid inhibits cell cycle progression in the cancer cell lines investigated, via its regulation of the phosphorylation and subsequent degradation of p27kip1 and p57kip2 and their interactions with other cycle regulators. PMID:9683802

  18. MiR-320a inhibits gastric carcinoma by targeting activity in the FoxM1-P27KIP1 axis

    PubMed Central

    Pan, Jianyong; Geng, Xue; Li, Lupeng; Wu, Jing; Song, Ping; Wang, Ying; Liu, Jilan; Wang, Lixiang

    2016-01-01

    MicroRNAs (miRNAs) regulate tumorigenesis by inhibiting gene expression. In this study, we showed that miR-320a expression is decreased in human gastric cancer tissues and correlates inversely with expression of FoxM1, a key cell cycle regulator involved in gastric carcinoma. By contrast, the expression of P27KIP1, a downstream effector of FoxM1, correlates positively with miR-320a levels. Luciferase assays indicate that miR-320a suppresses FoxM1 expression, and in vitro recovery tests using FoxM1 siRNA indicate miR-320a inhibits gastric cancer cell proliferation by suppressing activity in the FoxM1-P27KIP1 axis. In vivo, nude mice injected with BGC-823 gastric cancer cells expressing a miR-320a inhibitor exhibit faster tumor growth than mice injected with control cells. Analysis of FoxM1 and P27KIP1 expression in tumor tissues indicates that miR-320a suppression increases the tumor growth by enhancing FoxM1-P27KIP1 signaling. These results thus reveal the crucial role played by miR-320a in limiting gastric carcinoma by directly targeting FoxM1- P27KIP1 axis. PMID:27086911

  19. Keratin-17 Promotes p27KIP1 Nuclear Export and Degradation and Offers Potential Prognostic Utility.

    PubMed

    Escobar-Hoyos, Luisa F; Shah, Ruchi; Roa-Peña, Lucia; Vanner, Elizabeth A; Najafian, Nilofar; Banach, Anna; Nielsen, Erik; Al-Khalil, Ramsey; Akalin, Ali; Talmage, David; Shroyer, Kenneth R

    2015-09-01

    Keratins that are overexpressed selectively in human carcinomas may offer diagnostic and prognostic utility. In this study, we show that high expression of keratin-17 (K17) predicts poor outcome in patients with cervical cancer, at early or late stages of disease, surpassing in accuracy either tumor staging or loss of p27(KIP1) as a negative prognostic marker in this setting. We investigated the mechanistic basis for the biologic impact of K17 through loss- and gain-of-function experiments in human cervix, breast, and pancreatic cancer cells. Specifically, we determined that K17 functions as an oncoprotein by regulating the subcellular localization and degradation of p27(KIP1). We found that K17 was released from intermediate filaments and translocated into the nucleus via a nuclear localization signal (NLS), specific among keratins, where it bound p27(KIP1) during G1 phase of the cell cycle. p27(KIP1) lacks a nuclear export signal (NES) and requires an adaptor for CRM1 binding for nuclear export. In K17, we defined and validated a leucine-rich NES that mediated CRM1 binding for export. Cervical cancer cells expressing K17 mutations in its NLS or NES signals exhibited an increase in levels of nuclear p27(KIP1), whereas cells expressing wild-type K17 exhibited a depletion in total endogenous p27(KIP1). In clinical specimens of cervical cancer, we confirmed that the expressions of K17 and p27(KIP1) were inversely correlated, both across tumors and within individual tumors. Overall, our findings establish that K17 functions specially among keratins as an oncoprotein by controlling the ability of p27(KIP1) to influence cervical cancer pathogenesis.

  20. Alternate cyclin D1 mRNA splicing modulates p27KIP1 binding and cell migration.

    PubMed

    Li, Zhiping; Wang, Chenguang; Jiao, Xuanmao; Katiyar, Sanjay; Casimiro, Mathew C; Prendergast, George C; Powell, Michael J; Pestell, Richard G

    2008-03-14

    Cyclin D1 is an important cell cycle regulator, but in cancer its overexpression also increases cellular migration mediated by p27 KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N terminus as the canonical cyclin D1a isoform but a distinct C terminus. In this study we show that these different isoforms have unique properties with regard to the cellular migration function of cyclin D1. Although they displayed little difference in transcriptional co-repression assays on idealized reporter genes, microarray cDNA expression analysis revealed differential regulation of genes, including those that influence cellular migration. Additionally, whereas cyclin D1a stabilized p27 KIP1 and inhibited RhoA-induced ROCK kinase activity, promoting cellular migration, cyclin D1b failed to stabilize p27 KIP1 or inhibit ROCK kinase activity and had no effect on migration. Our findings argue that alternate splicing is an important determinant of the function of cyclin D1 in cellular migration.

  1. CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis

    PubMed Central

    Mao, Weiqun; Ahmed, Ahmed A.; Yang, Hailing; Zhou, Jinhua; Jennings, Nicholas; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Miranda, Roberto; Qiao, Wei; Baladandayuthapani, Veera; Li, Zongfang; Sood, Anil K.; Liu, Jinsong; Le, Xiao-Feng; Bast, Robert C.

    2015-01-01

    Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27Kip1 as well as TP53-dependent transcriptional induction of p21Cip1 in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells. PMID:26146988

  2. CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

    PubMed

    Zhang, Shu; Lu, Zhen; Mao, Weiqun; Ahmed, Ahmed A; Yang, Hailing; Zhou, Jinhua; Jennings, Nicholas; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Miranda, Roberto; Qiao, Wei; Baladandayuthapani, Veera; Li, Zongfang; Sood, Anil K; Liu, Jinsong; Le, Xiao-Feng; Bast, Robert C

    2015-01-01

    Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells. PMID:26146988

  3. Thrombin Induces Tumor Cell Cycle Activation and Spontaneous Growth by Down-regulation of p27Kip1, in Association with the Up-regulation of Skp2 and MiR-222

    PubMed Central

    Hu, Liang; Ibrahim, Sherif; Liu, Cynthia; Skaar, Jeffrey; Pagano, Michele; Karpatkin, Simon

    2009-01-01

    The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G0 and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27Kip1 and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27Kip1 with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27Kip1-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27Kip1, was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27Kip1. PMID:19351827

  4. p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress

    PubMed Central

    Cassimere, Erica K.; Mauvais, Claire; Denicourt, Catherine

    2016-01-01

    The DNA damage response (DDR) is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140), which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A) significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks. PMID:27611996

  5. p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress.

    PubMed

    Cassimere, Erica K; Mauvais, Claire; Denicourt, Catherine

    2016-01-01

    The DNA damage response (DDR) is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140), which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A) significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks. PMID:27611996

  6. Associations and prognostic significance of p27Kip1, Jab1 and Skp2 in non-Hodgkin lymphoma

    PubMed Central

    Ma, Yan; Yan, Meijuan; Huang, Hua; Zhang, Li; Wang, Qian; Zhao, Yaqi; Zhao, Jianmei

    2016-01-01

    Non-Hodgkin lymphoma (NHL) is a primary tumor arising in lymph nodes and lymphoid tissue. The incidence of NHL is increasing at an annual rate of 3%. The human Jun activation domain-binding protein 1/COP9 signalosome subunit 5 (Jab1/CSN5) is a negative regulator of the cell cycle inhibitor p27Kip1 and abnormal expression of Jab1 is correlated with reduced p27 expression and associated with advanced tumor stage and poor prognosis in several human cancers. F-box protein S-phase kinase-interacting protein-2 (Skp2), the substrate recognition subunit of the Skp1-Cul1-F-box protein ubiquitin protein ligase complex, is required for the ubiquitination and consequent degradation of p27. The Skp2 protein is overexpressed in several human cancers and is associated with the degree of differentiation and the prognosis. The aim of the present study was to investigate the expression status of p27Kip1, Jab1 and Skp2 by immunohistochemistry, and assess their prognostic significance in patients with NHL. Immunohistochemical analysis revealed an inverse association between Jab1 and p27 in NHL tissue samples. Kaplan-Meier analysis demonstrated that Jab1 overexpression, Skp2 overexpression and low p27 expression were significantly associated with poor prognosis. Among clinicopathological parameters, overexpression of Jab1 was significantly associated with tumor size and International Prognostic Index (IPI), whereas Skp2 expression was significantly associated with metastasis and IPI. These findings suggest that the overexpression of Jab1 or Skp2 plays an important role in the pathogenesis of NHL. Thus, the expression of p27Kip1, Jab1 and Skp2 provided a clinical reference for the treatment of NHL.

  7. Regulating the stability and localization of CDK inhibitor p27(Kip1) via CSN6-COP1 axis.

    PubMed

    Choi, Hyun Ho; Guma, Sergei; Fang, Lekun; Phan, Liem; Ivan, Cristina; Baggerly, Keith; Sood, Anil; Lee, Mong-Hong

    2015-01-01

    The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27(Kip1) stability, and that COP1 is a negative regulator of p27(Kip1). Ectopic expression of CSN6 can decrease the expression of p27(Kip1), while CSN6 knockdown leads to p27(Kip1) stabilization. Mechanistic studies show that CSN6 interacts with p27(Kip1) and facilitates ubiquitin-mediated degradation of p27(Kip1). CSN6-mediated p27 degradation depends on the nuclear export of p27(Kip1), which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes. PMID:25945542

  8. Status of p53 and p27KIP1 in Iranian Patients With Oral Squamous Cell Carcinoma

    PubMed Central

    Etemad-Moghadam, Shahroo; Keyhani, Amanollah; Yazdani, Kamran; Alaeddini, Mojgan

    2015-01-01

    Background: Alterations in p53 and p27KIP1 have been documented as important events in the carcinogenesis of various cancers, but their prognostic role in oral squamous cell carcinoma (OSCC) remains controversial. Objectives: The present investigation aimed to evaluate the clinicopathologic and prognostic significance of p53 and p27KIP1 expression in a group of Iranian patients with OSCC. Patients and Methods: In this analytical cross-sectional study, medical records of patients with primary OSCC, diagnosed from 1994 to 2004 were reviewed and 28 subjects were selected based on the inclusion/exclusion criteria. Immunohistochemical staining using monoclonal antibodies against p53 and p27KIP1 was performed on representative archival paraffin blocks. Demographic data along with information on p53 and p27KIP1 expression, recurrence, and tumor grade was statistically analyzed using the Fischer exact test. Prognostic factors for overall survival were determined by Cox regression analysis (P < 0.05). Results: p53 and p27KIP1 expression were found in 28.57% (8 positive versus 20 negative) and 67.85% (19 positive versus 9 negative) of OSCC cases, respectively. There was no significant association between these two proteins (P = 0.371), and neither of them showed a significant relationship with the studied clinicopathologic variables (P > 0.05). In survival analysis, only histopathologic differentiation (17 low and moderate, 11 poor) demonstrated a significant correlation with overall survival (P = 0.048). Conclusions: Despite the fact that abnormalities in p53 and p27KIP1 may be involved in the development of OSCC, their clinical significance in the studied population seems limited. Further investigation on the combined p53/p27KIP1 expression may be helpful in predicting the biologic behavior of this tumor. PMID:26568852

  9. p27Kip1 expression as a prognostic marker for squamous cell carcinoma of the head and neck

    PubMed Central

    DE ALMEIDA, MIGUEL REIS; PÉREZ-SAYÁNS, MARIO; SUÁREZ-PEÑARANDA, JOSÉ MANUEL; SOMOZA-MARTÍN, JOSÉ MANUEL; GARCÍA-GARCÍA, ABEL

    2015-01-01

    Regulation of the cell cycle is essential for carcinogenesis. The cell cycle is controlled by cyclin-dependent kinases (CDKs), which are upregulated by cyclins and downregulated by CDK inhibitors (CDKIs). Decreased p27Kip1 expression has been associated with survival rate, tumor size, histological differentiation and the presence of lymph node metastasis in patients with various types of cancer. The aim of the current study is to provide a literature review on the association between p27Kip1 expression and the clinical and pathological aspects of head and neck squamous cell carcinoma (HNSCC), and the expression of other CDKIs of the Cip/Kip family and cyclins. Throughout the literature, different methodologies were used to determine the immunohistochemical expression of p27Kip1; thus, results concerning p27Kip1 expression in HNSCC vary widely. However, it has now been confirmed that p27Kip1 is underexpressed in SCC cells. p27 may be a promising marker for determining the prognosis of HNSCC, despite the marked variability of the results obtained. An association between p27 expression and survival rate, time to recurrence and tumor stage has been observed. Based on the information currently available, it is premature to recommend the analysis of p27Kip1 expression in guiding HNSCC treatment planning. However, although relatively unstudied, the correlation between p27Kip1 expression and other tumor suppressor genes may turn out to be important in determining the prognosis of HNSCC. Further prospective studies utilizing standardized laboratory methodologies and statistics that facilitate meta-analyses are required to confirm this proposal. PMID:26722226

  10. p27KIP1 is abnormally expressed in Diffuse Large B-Cell Lymphomas and is associated with an adverse clinical outcome

    PubMed Central

    Sáez, Al; Sánchez, E; Sánchez-Beato, M; Cruz, M A; Chacón, I; Muñoz, E; Camacho, F I; Martínez-Montero, J C; Mollejo, M; Garcia, J F; Piris, M A

    1999-01-01

    Cell cycle progression is regulated by the combined action of cyclins, cyclin-dependent kinases (CDKs), and CDK-inhibitors (CDKi), which are negative cell cycle regulators. p27KIP1 is a CDKi key in cell cycle regulation, whose degradation is required for G1/S transition. In spite of the absence of p27KIP1 expression in proliferating lymphocytes, some aggressive B-cell lymphomas have been reported to show an anomalous p27KIP1 staining. We analysed p27KIP1 expression in a series of Diffuse Large B-cell Lymphoma (DLBCL), correlating it with the proliferative index and clinical outcome, to characterize the implications of this anomalous staining in lymphomagenesis in greater depth. For the above mentioned purposes, an immunohistochemical technique in paraffin-embedded tissues was employed, using commercially available antibodies, in a series of 133 patients with known clinical outcomes. Statistical analysis was performed in order to ascertain which clinical and molecular variables may influence outcome, in terms of disease-free survival (DFS) and overall survival (OS). The relationships between p27KIP1 and MIB-1 (Ki-67) were also tested. An abnormally high expression of p27KIP1 was found in lymphomas of this type. The overall correlation between p27KIP1 and MIB-1 showed there to be no significant relationship between these two parameters, this differing from observations in reactive lymphoid and other tissues. Analysis of the clinical relevance of these findings showed that a high level of p27KIP1 expression in this type of tumour is an adverse prognostic marker, in both univariate and multivariate analysis. These results show that there is abnormal p27KIP1 expression in DLBCL, with adverse clinical significance, suggesting that this anomalous p27KIP1 protein may be rendered non-functional through interaction with other cell cycle regulator proteins. © 1999 Cancer Research Campaign PMID:10424746

  11. Ablation of galectin-3 induces p27KIP1-dependent premature senescence without oncogenic stress

    PubMed Central

    Kim, S-J; Lee, H-W; Gu Kang, H; La, S-H; Choi, Il Ju; Ro, J Y; Bresalier, R S; Song, J; Chun, K-H

    2014-01-01

    Premature senescence induced by oncogenic stimuli or tumor suppressor activation plays opposing roles in tumorigenesis. Here, we propose that galectin-3, a β-galactoside-binding lectin, regulates premature senescence without oncogenic stress. We detected premature senescence, decreased Skp2, and increased p27KIP1 expression in galectin-3 knockout MEFs and galectin-3-depleted gastric cancer cells. Interestingly, galectin-3 depletion did not affect other senescence inducers such as p14ARF, p16INK4A, and p21WAF1/CIP1, suggesting that galectin-3-regulated senescence is p27KIP1 dependent. We demonstrate that galectin-3 depletion decreases retinoblastoma protein (Rb) phosphorylation (Ser780, Ser807/811), cyclin D1 and CDK4 expression, and E2F1 transcriptional activation. Galectin-3 directly interacts with the cyclin D1/CDK4 complex and promotes hyperphosphorylation of Rb. It also blocks the inhibition of E2F1 transcription, thereby increasing the expression of Skp2 and reducing the stability of p27KIP1 to promote the proliferation of gastric cancer cells. Xenograft mice with galectin-3-depleted gastric cancer cells display tumor growth retardation that is reversed by Skp2 overexpression. Increased expression of galectin-3 is also associated with the advanced TNM (tumor, lymph node, metastasis) system, clinicopathological stage, and lymph node metastases. The probability of survival was significantly decreased in gastric cancer patients with galectin-3high p27KIP1-lowcells. Taken together, our results show that galectin-3 may accelerate gastric tumorigenesis by inhibiting premature senescence. PMID:24971481

  12. The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

    PubMed Central

    Lai, Tai-Yu; Yang, Yu-San; Hong, Wei-Fu; Chiang, Chi-Wu

    2016-01-01

    The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role. PMID:26684356

  13. Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation

    PubMed Central

    Zakaria, Siti Mariam; Frings, Oliver; Fahlén, Sara; Nilsson, Helén; Goodwin, Jacob; von der Lehr, Natalie; Su, Yingtao; Lüscher, Bernhard; Castell, Alina; Larsson, Lars-Gunnar

    2016-01-01

    The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27Kip1 (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157 - a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27KIP1 potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc. PMID:26701207

  14. Expression of p27Kip1 and E-cadherin in Head and Neck Squamous Cell Carcinoma of Indonesian Patients.

    PubMed

    E I, Auerkari; V, Joewono; D R, Handjari; A T, Sarwono; A W, Suhartono; K, Eto; M A, Ikeda

    2014-01-01

    Cancer cells exhibit characteristic damage of DNA and its expression. The expression of the tumor suppressors E-cadherin and p27(Kip1) has been tested on 57 head and neck squamous cell carcinomas (HNSCC) of Indonesian subjects. HNSCC tumor samples including both primary and (unrelated) nodal cases were obtained from the archives of Indonesian hospitals, in accordance with acknowledged ethical requirements. Only modest correlation was found between reduced expression of E-cadherin or p27(Kip1) with increased malignancy of primary and nodal growth. The observed strong correlation regardless of malignancy between the expressed levels of E-cadherin and p27(Kip1) suggests that also in combination these would not help to better predict the outcome of HNSCC.

  15. p27Kip1 modulates cell migration through the regulation of RhoA activation.

    PubMed

    Besson, Arnaud; Gurian-West, Mark; Schmidt, Anja; Hall, Alan; Roberts, James M

    2004-04-15

    The tumor suppressor p27(Kip1) is an inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes and plays a crucial role in cell cycle regulation. However, p27(Kip1) also has cell cycle-independent functions. Indeed, we find that p27(Kip1) regulates cell migration, as p27(Kip1)-null fibroblasts exhibit a dramatic decrease in motility compared with wild-type cells. The regulation of motility by p27(Kip1) is independent of its cell-cycle regulatory functions, as re-expression of both wild-type p27(Kip1) and a mutant p27(Kip1) (p27CK(-)) that cannot bind to cyclins and CDKs rescues migration of p27(-/-) cells. p27(-/-) cells have increased numbers of actin stress fibers and focal adhesions. This is reminiscent of cells in which the Rho pathway is activated. Indeed, active RhoA levels were increased in cells lacking p27(Kip1). Moreover, inhibition of ROCK, a downstream effector of Rho, was able to rescue the migration defect of p27(-/-) cells in response to growth factors. Finally, we found that p27(Kip1) binds to RhoA, thereby inhibiting RhoA activation by interfering with the interaction between RhoA and its activators, the guanine-nucleotide exchange factors (GEFs). Together, the data suggest a novel role for p27(Kip1) in regulating cell migration via modulation of the Rho pathway.

  16. Nanog induces suppression of senescence through downregulation of p27KIP1 expression

    PubMed Central

    Münst, Bernhard; Thier, Marc Christian; Winnemöller, Dirk; Helfen, Martina; Thummer, Rajkumar P.; Edenhofer, Frank

    2016-01-01

    ABSTRACT A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated β-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27KIP1 (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27KIP1 gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene. PMID:26795560

  17. Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

    PubMed

    Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

    2014-01-01

    The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression. PMID:25486569

  18. Oligodendrocyte Regeneration after Neonatal Hypoxia Requires FoxO1-Mediated p27Kip1 Expression

    PubMed Central

    Jablonska, Beata; Scafidi, Joseph; Aguirre, Adan; Vaccarino, Flora; Nguyen, Vien; Borok, Erzsebet; Horvath, Tamas L.; Rowitch, David H.; Gallo, Vittorio

    2012-01-01

    Diffuse white matter injury (DWMI) caused by hypoxia is associated with permanent neurodevelopmental disabilities in preterm infants. The cellular and molecular mechanisms producing DWMI are poorly defined. Using a mouse model of neonatal hypoxia, we demonstrate a biphasic effect on oligodendrocyte development, resulting in hypomyelination. Oligodendrocyte death and oligodendrocyte progenitor cell (OPC) proliferation during the week after hypoxia were followed by delayed oligodendrocyte differentiation and abnormal myelination, as demonstrated by electron microscopy. Cdk2 activation was essential for the regenerative OPC response after hypoxia and was accompanied by reduced FoxO1-dependent p27 Kip1 expression. p27 Kip1 was also reduced in OPCs in human infant white matter lesions after hypoxia. The negative effects of hypoxia on oligodendrogenesis and myelination were more pronounced in p27 Kip1-null mice; conversely, overexpression of FoxO1 or p27 Kip1 in OPCs after hypoxia promoted oligodendrogenesis. Our studies demonstrate for the first time that neonatal hypoxia affects the Foxo1/p27 Kip1 pathway during white matter development. We also show that molecular manipulation of this pathway enhances oligodendrocyte regeneration during a critical developmental time window after DWMI. Thus, FoxO1 and p27 Kip1 may serve as promising target molecules for promoting timely oligodendrogenesis in neonatal DWMI. PMID:23077062

  19. Skp2 Deficiency Inhibits Chemical Skin Tumorigenesis Independent of p27Kip1 Accumulation

    PubMed Central

    Sistrunk, Christopher; Kim, Sun Hye; Wang, Xian; Lee, Sung Hyun; Kim, Yongbaek; Macias, Everardo; Rodriguez-Puebla, Marcelo L.

    2014-01-01

    S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp–Cullin–F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21Cip1, p27Kip1, p57Kip2, and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27Kip1 levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27Kip1 with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2−/− mice. An analysis of mouse keratinocytes indicates that increased p27Kip1 levels in Skp2−/− epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2−/−/p27−/− compound mice. In contrast, we establish that a p27Kip1 deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2−/− mice. In addition, Skp2−/− epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27Kip1 accumulation is responsible for the hypoplasia observed in normal tissues of Skp2−/− mice but does not have a preponderant function in reducing skin tumorigenesis. PMID:23474082

  20. Zinc deficiency impairs the renewal of hippocampal neural stem cells in adult rats: involvement of FoxO3a activation and downstream p27(kip1) expression.

    PubMed

    Han, Jingling; Zhao, Jianya; Jiang, Junkang; Ma, Xia; Liu, Xinhang; Wang, Cheng; Jiang, Shengyang; Wan, Chunhua

    2015-09-01

    Zinc plays an important role in the development and maintenance of central neural system. Zinc deficiency has been known to alter normal brain function, whose molecular mechanism remains largely elusive. In the present study, we established a zinc deficiency-exposed rat model, and, using western blot and immunohistochemical analyses, found that the expression of FoxO3a and p27(kip1) was remarkably up-regulated in the rat brain hippocampus. Immunofluorescence assay showed that FOXO3a and p27(kip1) were significantly co-localized with nestin, the marker of neural stem cells (NSCs). Furthermore, we identified that the proportion of proliferating NSCs was markedly decreased in zinc-deficient rat hippocampaus. Using C17.2 neural stem cells, it was revealed that exposure to zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethy) ethylenediamine induced the expression of FoxO3a and p27(kip1) , which coincided with reduced NSC proliferation. Furthermore, depletion of FoxO3a inhibited p27(kip1) expression and restored the growth of NSCs. On the basis of these data, we concluded that FoxO3a/p27(kip1) signaling might play a significant role in zinc deficiency-induced growth impairment of NSCs and consequent neurological disorders. We describe here that zinc deficiency induces the proliferative impairment of hippocampal neural stem cells partially through the activation of FOXO3a-p27 axis in rats. Neural progenitor cells exhibited significantly up-regulated expression of FOXO3a and p27 after zinc deficiency in vivo and in vitro. Depletion of FOXO3a ameliorates zinc deficiency-induced expression of p27 and growth impairment of neural stem cells. We provide novel insight into the mechanisms underlying zinc deficiency-induced neurological deficits.

  1. Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway

    PubMed Central

    Rahmutulla, Bahityar; Matsushita, Kazuyuki; Satoh, Mamoru; Seimiya, Masanori; Tsuchida, Sachio; Kubo, Shuji; Shimada, Hideaki; Ohtsuka, Masayuki; Miyazaki, Masaru; Nomura, Fumio

    2014-01-01

    The far-upstream element-binding protein-interacting repressor (FIR) is a c-myc transcriptional suppressor. FIR is alternatively spliced to lack the transcriptional repression domain within exon 2 (FIRΔexon2) in colorectal cancers. FIR and FIRΔexon2 form homo- or heterodimers that complex with SAP155. SAP155, a subunit of the essential splicing factor 3b subcomplex in the spliceosome, is required for proper P27Kip1 pre-mRNA splicing, and P27Kip1 arrests cells at G1. In contrast, FIR was co-immunoprecipitated with Ku86 and DNA-PKcs. siRNA against Ku86/Ku70 decreased FIR and P27Kip1 expression, whereas siRNA against FIR decreased Ku86/XRCC5 and P27Kip1 expression. Thus the mechanical interaction of FIR/FIRΔexon2/SAP155 bridges c-myc and P27Kip1 expression, potentially integrates cell-cycle progression and c-myc transcription in cell. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. Because DNA breaks generate the recruitment of Ku86/Ku70 to bind to the broken DNA ends, the possible involvement of FIR and Ku86/Ku70 interaction in the BLM-induced DNA damage repair response was investigated in this study. First, BLM treatment reduced SAP155 expression and increased FIR and FIRΔexon2 mRNA expression as well as the ratio of FIRΔexon2:FIR in hepatoblastoma cells (HLE and HLF). Second, FIR or FIRΔexon2 adenovirus vectors (Ad-FIR or Ad-FIRΔexon2) increased Ku86/Ku70 and P27Kip1 expression in vitro. Third, BLM decreased P27Kip1 protein expression, whereas increased P27Kip1 and γH2AX expression with Ad-FIRΔexon2. Together, the interaction of FIR/SAP155 modulates FIR splicing and involves in cell-cycle control or cell fate via P27Kip1 and c-myc in BLM-induced DNA damage pathway. This novel function of FIR splicing will contribute to clinical studies of cancer management through elucidating the mechanical interaction of FIR/FIRΔexon2/SAP155 as a potential target for cancer treatment. PMID:24811221

  2. Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway.

    PubMed

    Kawada, M; Yamagoe, S; Murakami, Y; Suzuki, K; Mizuno, S; Uehara, Y

    1997-08-01

    While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal fibroblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and significantly shortened the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27Kip1. These results suggest that the loss of substrate attachment induces the cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2.

  3. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

    SciTech Connect

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum; Nam, Hyo Jung; Kang, Jin Ku; Park, Mi Jung; Lee, Ik Hwan; Seok, Heon; Lee, Dong Gun; Hwang, Jae Sam; Kim, Ho

    2013-07-19

    Highlights: •CopA3 peptide isolated from the Korean dung beetle has antimicrobial activity. •Our study reported that CopA3 has anticancer and immunosuppressive effects. •We here demonstrated that CopA3 has neurotropic and neuroprotective effects. •CopA3 degrades p27Kip1 protein and this mediates effects of CopA3 on neuronal cells. -- Abstract: We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

  4. Overexpression of TRIP6 promotes tumor proliferation and reverses cell adhesion-mediated drug resistance (CAM-DR) via regulating nuclear p27(Kip1) expression in non-Hodgkin's lymphoma.

    PubMed

    Miao, Xiaobing; Xu, Xiaohong; Wu, Yaxun; Zhu, Xinghua; Chen, Xudong; Li, Chunsun; Lu, Xiaoyun; Chen, Yali; Liu, Yushan; Huang, Jieyu; Wang, Yuchan; He, Song

    2016-01-01

    Recent studies have identified that thyroid hormone receptor-interacting protein 6 (TRIP6) is implicated in tumorigenesis. However, the functional role of TRIP6 in non-Hodgkin's lymphoma (NHL) has never been elucidated. In this study, we demonstrated that TRIP6 is reversely correlated with the clinical outcomes of NHL patients. Western blot and immunohistochemical analysis revealed that TRIP6 expression is lower in indolent lymphoma than in progressive lymphoma. Kaplan-Meier survival curves indicated that the upregulation of TRIP6 is significantly associated with poor overall survival. Moreover, patients with higher expression of TRIP6 are prone to shorter time to recurrence. Furthermore, we also found that TRIP6 can promote the proliferation of NHL cells via regulating cell cycle progression. In addition, adhesion of lymphoma cells to fibronectin (FN) decreased TRIP6 expression, which led to the upregulation of nuclear p27(Kip1) expression by decreasing phosphorylation of p27(Kip1) at T157. Importantly, overexpression of TRIP6 can reverse cell adhesion-mediated drug resistance (CAM-DR) phenotype in NHL. In summary, these results suggest that TRIP6 is a novel prognostic indicator for NHL patients and may shed new insights into the important role of TRIP6 in cancer development.

  5. Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2.

    PubMed

    Tapia, Julio C; Bolanos-Garcia, Victor M; Sayed, Muhammed; Allende, Catherine C; Allende, Jorge E

    2004-04-01

    The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity.

  6. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1.

    PubMed

    Luo, Jian; Chen, Yongjun; Li, Qiang; Wang, Bing; Zhou, Yanqiong; Lan, Hongzhen

    2016-08-01

    Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin‑dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III-IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments.

  7. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1

    PubMed Central

    Luo, Jian; Chen, Yongjun; Li, Qiang; Wang, Bing; Zhou, Yanqiong; Lan, Hongzhen

    2016-01-01

    Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin-dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III–IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments. PMID:27279267

  8. H2O2 scavenging inhibits G1/S transition by increasing nuclear levels of p27KIP1.

    PubMed

    Ibañez, Irene L; Policastro, Lucía L; Tropper, Ivanna; Bracalente, Candelaria; Palmieri, Mónica A; Rojas, Paola A; Molinari, Beatriz L; Durán, Hebe

    2011-06-01

    The aim of the present study was to evaluate cell cycle regulation by scavenging H(2)O(2) in tumor cells. A significant arrest in the G1 phase of the cell cycle was demonstrated in CH72-T4 carcinoma cells exposed to catalase, associated with a decrease in cyclin D1 and an increase in the CDK inhibitory protein p27(KIP1). Moreover, we found a differential intracellular distribution of p27(KIP1), which remained in the nucleus after catalase treatment. In vivo experiments showed an increase in nuclear levels of p27(KIP1) associated with the inhibition of tumor growth by H(2)O(2) scavenging, confirming in vitro results. To conclude, H(2)O(2) scavenging may induce cell cycle arrest through the modulation of cyclin D1 and p27(KIP1) levels and nuclear localization of p27(KIP1). To our knowledge, this is the first report that demonstrates that the modulation of ROS alters the intracellular localization of a key regulatory protein of G1/S transition.

  9. In vivo regulation of colonic cell proliferation, differentiation, apoptosis, and P27Kip1 by dietary fish oil and butyrate in rats.

    PubMed

    Hong, Mee Young; Turner, Nancy D; Murphy, Mary E; Carroll, Raymond J; Chapkin, Robert S; Lupton, Joanne R

    2015-11-01

    We have shown that dietary fish oil is protective against experimentally induced colon cancer, and the protective effect is enhanced by coadministration of pectin. However, the underlying mechanisms have not been fully elucidated. We hypothesized that fish oil with butyrate, a pectin fermentation product, protects against colon cancer initiation by decreasing cell proliferation and increasing differentiation and apoptosis through a p27(Kip1)-mediated mechanism. Rats were provided diets of corn or fish oil, with/without butyrate, and terminated 12, 24, or 48 hours after azoxymethane (AOM) injection. Proliferation (Ki-67), differentiation (Dolichos Biflorus Agglutinin), apoptosis (TUNEL), and p27(Kip1) (cell-cycle mediator) were measured in the same cell within crypts in order to examine the coordination of cell cycle as a function of diet. DNA damage (N(7)-methylguanine) was determined by quantitative IHC analysis. Dietary fish oil decreased DNA damage by 19% (P = 0.001) and proliferation by 50% (P = 0.003) and increased differentiation by 56% (P = 0.039) compared with corn oil. When combined with butyrate, fish oil enhanced apoptosis 24 hours after AOM injection compared with a corn oil/butyrate diet (P = 0.039). There was an inverse relationship between crypt height and apoptosis in the fish oil/butyrate group (r = -0.53, P = 0.040). The corn oil/butyrate group showed a positive correlation between p27(Kip1) expression and proliferation (r = 0.61, P = 0.035). These results indicate the in vivo effect of butyrate on apoptosis and proliferation is dependent on dietary lipid source. These results demonstrate the presence of an early coordinated colonocyte response by which fish oil and butyrate protects against colon tumorigenesis.

  10. The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms.

    PubMed

    Croft, Daniel R; Olson, Michael F

    2006-06-01

    The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.

  11. CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27Kip1 mRNA translation

    PubMed Central

    Galardi, Silvia; Petretich, Massimo; Pinna, Guillaume; D’Amico, Silvia; Loreni, Fabrizio; Michienzi, Alessandro; Groisman, Irina; Ciafrè, Silvia Anna

    2016-01-01

    The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3′ untranslated regions (3′UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27Kip1 by specifically targeting its 3′UTR, and competes with miR-221/222 binding at an overlapping site in the 3′UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27Kip1 3′UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27Kip1 mRNA. This leads to higher levels of p27Kip1 in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma. PMID:27142352

  12. Depletion of p18/LAMTOR1 promotes cell survival via activation of p27(kip1) -dependent autophagy under starvation.

    PubMed

    Zada, Sahib; Noh, Hae Sook; Baek, Seon Mi; Ha, Ji Hye; Hahm, Jong Ryeal; Kim, Deok Ryong

    2015-11-01

    The MAPK and mTOR signal pathways in endosomes or lysosomes play a crucial role in cell survival and death. They are also closely associated with autophagy, a catabolic process highly regulated under various cellular stress or nutrient deprivation. Recently we have isolated a protein, named p18/LAMTOR1, that specifically regulates the ERK or mTOR pathway in lysosomes. p18/LAMTOR1 also interacts with p27(kip1) . Here we examined how p18/LAMTOR1 plays a role in autophagy under nutrient deprivation. The p18(+/+) MEF cells were more susceptible to cell death under starvation or in the presence of AICAR in comparison with p18(-/-) MEF cells. Cleavage of caspase-3 was increased in p18(+/+) MEF cells under starvation, and phosphorylation at the threonine 198 of p27(kip1) was highly elevated in starved p18(-/-) MEF cells. Furthermore, LC3-II formation and other autophagy-associated proteins were largely increased in p18-deficient cells, and suppression of p27(kip1) expression in p18(-/-) MEF cells mitigated starvation-induced cell death. These data suggest that ablation of p18/LAMTOR1 suppresses starvation-induced cell death by stimulating autophagy through modulation of p27(kip1) activity.

  13. Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

    DOE PAGESBeta

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; Ramanathan, Arvind; Zhang, Weixing; Shelat, Anang A.; Zuo, Jian; Kriwacki, Richard W.

    2015-10-28

    In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups ofmore » small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less

  14. Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27(kip1) translocation and retinoblastoma protein phosphorylation by 1'-acetoxychavicol acetate.

    PubMed

    Unahara, Y; Kojima-Yuasa, A; Higashida, M; Kennedy, D O; Murakami, A; Ohigashi, H; Matsui-Yuasa, I

    2007-09-01

    1'-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27(kip1), which is an important regulator of the mammalian cell cycle, we estimated the amount of p27(kip1) levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27(kip1) levels, but caused a decrease in phosphorylated p27(kip1) and an increase in unphosphorylated p27(kip1) as well as an increase in the nuclear localization of p27(kip1). These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27(kip1) phosphorylation and an increase in nuclear localization of p27(kip1), and these events are dependent on the cellular thiol status.

  15. Protection of p27(Kip1) mRNA by quaking RNA binding proteins promotes oligodendrocyte differentiation.

    PubMed

    Larocque, Daniel; Galarneau, André; Liu, Hsueh-Ning; Scott, Michelle; Almazan, Guillermina; Richard, Stéphane

    2005-01-01

    The quaking (Qk) locus expresses a family of RNA binding proteins, and the expression of several alternatively spliced isoforms coincides with the development of oligodendrocytes and the onset of myelination. Quaking viable (Qk(v)) mice harboring an autosomal recessive mutation in this locus have uncompacted myelin in the central nervous system owing to the inability of oligodendrocytes to properly mature. Here we show that the expression of two QKI isoforms, absent from oligodendrocytes of Qk(v) mice, induces cell cycle arrest of primary rat oligodendrocyte progenitor cells and differentiation into oligodendrocytes. Injection of retroviruses expressing QKI into the telencephalon of mouse embryos induced differentiation and migration of multipotential neural progenitor cells into mature oligodendrocytes localized in the corpus callosum. The mRNA encoding the cyclin-dependent kinase (CDK)-inhibitor p27(Kip1) was bound and stabilized by QKI, leading to an increased accumulation of p27(Kip1) protein in oligodendrocytes. Our findings demonstrate that QKI is upstream of p27(Kip1) during oligodendrocyte differentiation.

  16. Differential expression and role of p21cip/waf1 and p27kip1 in TNF-α-induced inhibition of proliferation in human glioma cells

    PubMed Central

    Kumar, Pabbisetty Sudheer; Shiras, Anjali; Das, Gowry; Jagtap, Jayashree C; Prasad, Vandna; Shastry, Padma

    2007-01-01

    Background The role of TNF-α in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-α, others provide evidence that endogenous TNF-α promotes growth and development of tumors. Understanding the mechanism(s) of TNF-α mediated growth arrest will be important in unraveling the contribution of tissue associated macrophages in tumor resistance. The aim of this study was to investigate the role of Cyclin Dependent Kinase Inhibitors (CDKI) – p21cip/waf1 and p27kip1 in TNF-α mediated responses in context with p53 and activation of NF-κB and Akt pathways. The study was done with human glioma cell lines -LN-18 and LN-229 cells, using monolayer cultures and Multicellular Spheroids (MCS) as in vitro models. Results TNF-α induced inhibition of proliferation and enhanced the expression of p21cip/waf1 and p27kip1 in LN-18 cells. p21 was induced on exposure to TNF-α, localized exclusively in the nucleus and functioned as an inhibitor of cell cycle but not as an antiapoptotic protein. In contrast, p27 was constitutively expressed, localized predominantly in the cytoplasm and was not involved in arrest of proliferation. Our data using IκBα mutant LN-18 cells and PI3K/Akt inhibitor-LY294002 revealed that the expression of p21 is regulated by NF-κB. Loss of IκBα function in LN-229 cells (p53 positive) did not influence TNF-α induced accumulation of pp53 (Ser-20 p53) suggesting that p53 was not down stream of NF-κB. Spheroidogenesis enhanced p27 expression and p21 induced by TNF-α was significantly increased in the MCS compared to monolayers. Conclusion This study demarcates the functional roles for CDKIs-p21cip/waf1 and p27kip1 during TNF-α stimulated responses in LN-18 glioma cells. Our findings provide evidence that TNF-α-induced p21 might be regulated by NF-κB or p53 independently. p21 functions as an inhibitor of cell proliferation and does not have a direct role in rendering the cells resistant

  17. Notch Signaling Activates Stem Cell Properties of Müller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27Kip1

    PubMed Central

    Parameswaran, Sowmya; Mathews, Saumi; Xia, Xiaohuan; Zheng, Li; Neville, Andrew J.; Ahmad, Iqbal

    2016-01-01

    Müller glia (MG), the sole glial cells generated by retinal progenitors, have emerged as a viable cellular target for therapeutic regeneration in degenerative blinding diseases, as they possess dormant stem cell properties. However, the mammalian MG does not display the neurogenic potential of their lower vertebrate counterparts, precluding their practical clinical use. The answer to this barrier may be found in two interlinked processes underlying the neurogenic potential, i.e., the activation of the dormant stem cell properties of MG and their differentiation along the neuronal lineage. Here, we have focused on the former and examined Notch signaling-mediated activation of MG. We demonstrate that one of the targets of Notch signaling is the cyclin-dependent kinase inhibitor (CKI), p27Kip1, which is highly expressed in quiescent MG. Notch signaling facilitates the activation of MG by inhibiting p27Kip1 expression. This is likely achieved through the Notch- p27Kip1 and Notch-Skp2-p27Kip1 axes, the former inhibiting the expression of p27Kip1 transcripts and the latter levels of p27Kip1 proteins by Skp2-mediated proteasomal degradation. Thus, Notch signaling may facilitate re-entry of MG into the cell cycle by inhibiting p27Kip1 expression both transcriptionally and post-translationally. PMID:27011052

  18. Notch Signaling Activates Stem Cell Properties of Müller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27Kip1.

    PubMed

    Del Debbio, Carolina Beltrame; Mir, Qulsum; Parameswaran, Sowmya; Mathews, Saumi; Xia, Xiaohuan; Zheng, Li; Neville, Andrew J; Ahmad, Iqbal

    2016-01-01

    Müller glia (MG), the sole glial cells generated by retinal progenitors, have emerged as a viable cellular target for therapeutic regeneration in degenerative blinding diseases, as they possess dormant stem cell properties. However, the mammalian MG does not display the neurogenic potential of their lower vertebrate counterparts, precluding their practical clinical use. The answer to this barrier may be found in two interlinked processes underlying the neurogenic potential, i.e., the activation of the dormant stem cell properties of MG and their differentiation along the neuronal lineage. Here, we have focused on the former and examined Notch signaling-mediated activation of MG. We demonstrate that one of the targets of Notch signaling is the cyclin-dependent kinase inhibitor (CKI), p27Kip1, which is highly expressed in quiescent MG. Notch signaling facilitates the activation of MG by inhibiting p27Kip1 expression. This is likely achieved through the Notch- p27Kip1 and Notch-Skp2-p27Kip1 axes, the former inhibiting the expression of p27Kip1 transcripts and the latter levels of p27Kip1 proteins by Skp2-mediated proteasomal degradation. Thus, Notch signaling may facilitate re-entry of MG into the cell cycle by inhibiting p27Kip1 expression both transcriptionally and post-translationally. PMID:27011052

  19. Rac1-dependent transcriptional up-regulation of p27Kip1 by homophilic cell-cell contact in vascular endothelial cells.

    PubMed

    Hirano, Mayumi; Kanaide, Hideo; Hirano, Katsuya

    2007-10-01

    The mechanism for the transcriptional up-regulation of p27Kip1 due to the formation of the cell-cell contact was investigated in vascular endothelial cells. The induction of the cell-cell contact by adding an extra number of endothelial cells activated Rac1, up-regulated p27Kip1 mRNA and protein, and also facilitated the cell cycle arrest. Transduction of the Rac1 inhibitor protein using the cell-penetrating peptide or treatment with a Rac1 inhibitor NSC23766 inhibited the p27Kip1 up-regulation and delayed the cell cycle arrest. Rac1 was therefore suggested to mediate the contact-induced transcriptional up-regulation of p27Kip1. The role of Rac1 in the regulation of the p27Kip1 promoter activity was next examined with a luciferase reporter assay. The promoter activity was increased by inducing the cell-cell contact, which was significantly inhibited by the Rac1 inhibitory protein and NSC23766. The evaluation of various truncated promoter regions determined region -620 to -573 nucleotides from the initiation codon to be responsible for the contact-induced, Rac1-dependent activation of the p27Kip1 promoter. The present study thus demonstrated for the first time that the activation of Rac1 due to the cell-cell contact plays a critical role in the transcriptional up-regulation of p27Kip1 in vascular endothelial cells.

  20. The cyclin-dependent kinase inhibitor p27 kip1 regulates radial stem cell quiescence and neurogenesis in the adult hippocampus.

    PubMed

    Andreu, Zoraida; Khan, Muhammad Amir; González-Gómez, Pilar; Negueruela, Santiago; Hortigüela, Rafael; San Emeterio, Juana; Ferrón, Sacri R; Martínez, Gloria; Vidal, Anxo; Fariñas, Isabel; Lie, Dieter Chichung; Mira, Helena

    2015-01-01

    Members of the cyclin-dependent kinase (CDK)-inhibitory protein (CIP)/kinase-inhibitory protein (KIP) family of cyclin-dependent kinase inhibitors regulate proliferation and cell cycle exit of mammalian cells. In the adult brain, the CIP/KIP protein p27(kip1) has been related to the regulation of intermediate progenitor cells located in neurogenic niches. Here, we uncover a novel function of p27(kip1) in the adult hippocampus as a dual regulator of stem cell quiescence and of cell-cycle exit of immature neurons. In vivo, p27(kip1) is detected in radial stem cells expressing SOX2 and in newborn neurons of the dentate gyrus. In vitro, the Cdkn1b gene encoding p27(kip1) is transcriptionally upregulated by quiescence signals such as BMP4. The nuclear accumulation of p27(kip1) protein in adult hippocampal stem cells encompasses the BMP4-induced quiescent state and its overexpression is able to block proliferation. p27(kip1) is also expressed in immature neurons upon differentiation of adult hippocampal stem cell cultures. Loss of p27(kip1) leads to an increase in proliferation and neurogenesis in the adult dentate gyrus, which results from both a decrease in the percentage of radial stem cells that are quiescent and a delay in cell cycle exit of immature neurons. Analysis of animals carrying a disruption in the cyclin-CDK interaction domain of p27(kip1) indicates that the CDK inhibitory function of the protein is necessary to control the activity of radial stem cells. Thus, we report that p27(kip1) acts as a central player of the molecular program that keeps adult hippocampal stem cells out of the cell cycle.

  1. Rat Protein Tyrosine Phosphatase η Suppresses the Neoplastic Phenotype of Retrovirally Transformed Thyroid Cells through the Stabilization of p27Kip1

    PubMed Central

    Trapasso, Francesco; Iuliano, Rodolfo; Boccia, Angelo; Stella, Antonella; Visconti, Roberta; Bruni, Paola; Baldassarre, Gustavo; Santoro, Massimo; Viglietto, Giuseppe; Fusco, Alfredo

    2000-01-01

    The r-PTPη gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPη (the human homolog of r-PTPη) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPη gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPη caused G1 growth arrest and increased the cyclin-dependent kinase inhibitor p27Kip1 protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPη tumor suppressor activity is mediated by p27Kip1 protein stabilization, because suppression of p27Kip1 protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPη. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27Kip1 protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPη regulated p27Kip1 stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation. PMID:11094075

  2. CKS1B, overexpressed in aggressive disease, regulates multiple myeloma growth and survival through SKP2- and p27Kip1-dependent and -independent mechanisms.

    PubMed

    Zhan, Fenghuang; Colla, Simona; Wu, Xiaosong; Chen, Bangzheng; Stewart, James P; Kuehl, W Michael; Barlogie, Bart; Shaughnessy, John D

    2007-06-01

    Overexpression of CKS1B, a gene mapping within a minimally amplified region between 153 to 154 Mb of chromosome 1q21, is linked to a poor prognosis in multiple myeloma (MM). CKS1B binds to and activates cyclin-dependent kinases and also interacts with SKP2 to promote the ubiquitination and proteasomal degradation of p27(Kip1). Overexpression of CKS1B or SKP2 contributes to increased p27(Kip1) turnover, cell proliferation, and a poor prognosis in many tumor types. Using 4 MM cell lines harboring MAF-, FGFR3/MMSET-, or CCND1-activating translocations, we show that lentiviral delivery of shRNA directed against CKS1B resulted in ablation of CKS1B mRNA and protein with concomitant stabilization of p27(Kip1), cell cycle arrest, and apoptosis. Although shRNA-mediated knockdown of SKP2 and forced expression of a nondegradable form of p27(Kip1) (p27(T187A)) led to cell cycle arrest, apoptosis was modest. Of importance, while knockdown of SKP2 or overexpression of p27(T187A) induced cell cycle arrest in KMS28PE, an MM cell line with biallelic deletion of CDKN1B/p27(Kip1), CKS1B ablation induced strong apoptosis. These data suggest that CKS1B influences myeloma cell growth and survival through SKP2- and p27(Kip1)-dependent and -independent mechanisms and that therapeutic strategies aimed at abolishing CKS1B function may hold promise for the treatment of high-risk disease for which effective therapies are currently lacking.

  3. Induction of anergy in Th1 cells associated with increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1.

    PubMed

    Jackson, S K; DeLoose, A; Gilbert, K M

    2001-01-15

    Th1 cells exposed to Ag and the G(1) blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21(Cip1) and p27(Kip1). Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21(Cip1) and p27(Kip1). However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21(Cip1) and p27(Kip1) when examined 24 h after Ag stimulation. The increase in p21(Cip1) observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27(Kip1) observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21(Cip1) and perhaps the downstream failure to maintain low levels of p27(Kip1). Increased levels of both p21(Cip1) and p27(Kip1) at the end of G(1) could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.

  4. Cooperative role between p21cip1/waf1 and p27kip1 in premature senescence in glandular proliferative lesions in mice.

    PubMed

    García-Fernández, R A; García-Palencia, P; Suarez, C; Sánchez, M A; Gil-Gómez, G; Sánchez, B; Rollán, E; Martín-Caballero, J; Flores, J M

    2014-03-01

    Cellular senescence has been considered a novel target for cancer therapy. It has also been pointed out that p21(cip1/waf1) and p27(kip1) cyclin-dependent kinase inhibitors (CKIs) play a role in cellular senescence in some tumor types. Therefore, in order to address the possibility of a cooperative role between p21 and p27 proteins in senescence in vivo we analyzed cellular senescence in spontaneous glandular proliferative lesions (adrenal, thyroid and pituitary glands) in a double-KO mice model, using γH2AX, p53, p16, PTEN and Ki67 as senescence markers. The results obtained showed that p21p27 double-null mice had the lowest number of γH2AX positive cells in glandular hyperplasias and benign tumors. Also, in this group, Ki67 proliferation index correlated with a lower immunohistochemical expression of γH2AX and p53. The expression of p16 and PTEN do not seem to cause synergism of senescence in the benign lesions analyzed in p21p27 double-KO mice. These observations suggest an intrinsic cooperation between p21 and p27 CKIs in the activation of stress-induced cellular senescence and tumor progression in vivo, which would be a physiological mechanism to prevent tumor cell proliferation.

  5. HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling.

    PubMed

    Hu, Jiao-Jiao; Song, Wei; Zhang, Shao-Dan; Shen, Xiao-Hui; Qiu, Xue-Mei; Wu, Hua-Zhang; Gong, Pi-Hai; Lu, Sen; Zhao, Zhu-Jiang; He, Ming-Liang; Fan, Hong

    2016-01-01

    It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC. PMID:27009634

  6. HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling

    PubMed Central

    Hu, Jiao-Jiao; Song, Wei; Zhang, Shao-Dan; Shen, Xiao-Hui; Qiu, Xue-Mei; Wu, Hua-Zhang; Gong, Pi-Hai; Lu, Sen; Zhao, Zhu-Jiang; He, Ming-Liang; Fan, Hong

    2016-01-01

    It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC. PMID:27009634

  7. Knockdown of AMPKα2 Promotes Pulmonary Arterial Smooth Muscle Cells Proliferation via mTOR/Skp2/p27Kip1 Signaling Pathway

    PubMed Central

    Ke, Rui; Liu, Lu; Zhu, Yanting; Li, Shaojun; Xie, Xinming; Li, Fangwei; Song, Yang; Yang, Lan; Gao, Li; Li, Manxiang

    2016-01-01

    It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27Kip1. Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27Kip1 reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27Kip1 downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27Kip1 axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension. PMID:27258250

  8. p27(Kip1) directly represses Sox2 during embryonic stem cell differentiation.

    PubMed

    Li, Han; Collado, Manuel; Villasante, Aranzazu; Matheu, Ander; Lynch, Cian J; Cañamero, Marta; Rizzoti, Karine; Carneiro, Carmen; Martínez, Gloria; Vidal, Anxo; Lovell-Badge, Robin; Serrano, Manuel

    2012-12-01

    The mechanisms responsible for the transcriptional silencing of pluripotency genes in differentiated cells are poorly understood. We have observed that cells lacking the tumor suppressor p27 can be reprogrammed into induced pluripotent stem cells (iPSCs) in the absence of ectopic Sox2. Interestingly, cells and tissues from p27 null mice, including brain, lung, and retina, present an elevated basal expression of Sox2, suggesting that p27 contributes to the repression of Sox2. Furthermore, p27 null iPSCs fail to fully repress Sox2 upon differentiation. Mechanistically, we have found that upon differentiation p27 associates to the SRR2 enhancer of the Sox2 gene together with a p130-E2F4-SIN3A repressive complex. Finally, Sox2 haploinsufficiency genetically rescues some of the phenotypes characteristic of p27 null mice, including gigantism, pituitary hyperplasia, pituitary tumors, and retinal defects. Collectively, these results demonstrate an unprecedented connection between p27 and Sox2 relevant for reprogramming and cancer and for understanding human pathologies associated with p27 germline mutations. PMID:23217425

  9. p27Kip1 Directly Represses Sox2 during Embryonic Stem Cell Differentiation

    PubMed Central

    Li, Han; Collado, Manuel; Villasante, Aranzazu; Matheu, Ander; Lynch, Cian J.; Cañamero, Marta; Rizzoti, Karine; Carneiro, Carmen; Martínez, Gloria; Vidal, Anxo; Lovell-Badge, Robin; Serrano, Manuel

    2012-01-01

    Summary The mechanisms responsible for the transcriptional silencing of pluripotency genes in differentiated cells are poorly understood. We have observed that cells lacking the tumor suppressor p27 can be reprogrammed into induced pluripotent stem cells (iPSCs) in the absence of ectopic Sox2. Interestingly, cells and tissues from p27 null mice, including brain, lung, and retina, present an elevated basal expression of Sox2, suggesting that p27 contributes to the repression of Sox2. Furthermore, p27 null iPSCs fail to fully repress Sox2 upon differentiation. Mechanistically, we have found that upon differentiation p27 associates to the SRR2 enhancer of the Sox2 gene together with a p130-E2F4-SIN3A repressive complex. Finally, Sox2 haploinsufficiency genetically rescues some of the phenotypes characteristic of p27 null mice, including gigantism, pituitary hyperplasia, pituitary tumors, and retinal defects. Collectively, these results demonstrate an unprecedented connection between p27 and Sox2 relevant for reprogramming and cancer and for understanding human pathologies associated with p27 germline mutations. PMID:23217425

  10. NSun2 delays replicative senescence by repressing p27 (KIP1) translation and elevating CDK1 translation

    PubMed Central

    Xing, Junyue; Liu, Zhenyun; Jiang, Bin; Dou, Yali; Gorospe, Myriam; Wang, Wengong

    2015-01-01

    A rise in the levels of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 is important for the growth arrest of senescent cells, but the mechanisms responsible for this increase are poorly understood. Here, we show that the tRNA methyltransferase NSun2 represses the expression of p27 in replicative senescence. NSun2 methylated the 5′-untranslated region (UTR) of p27 mRNA at cytosine C64 in vitro and in cells, thereby repressing the translation of p27. During replicative senescence, increased p27 protein levels were accompanied by decreased NSun2 protein levels. Knockdown of NSun2 in human diploid fibroblasts (HDFs) elevated p27 levels and reduced the expression of CDK1 (encoded by CDK1 mRNA, a previously reported target of NSun2), which in turn further repressed cell proliferation and accelerated replicative senescence, while overexpression of NSun2 exerted the opposite effect. Ectopic overexpression of the p27 5′UTR fragment rescued the effect of NSun2 overexpression in lowering p27, increasing CDK1, promoting cell proliferation, and delaying replicative senescence. Our findings indicate that NSun2-mediated mRNA methylation regulates p27 and CDK1 levels during replicative senescence. PMID:26687548

  11. Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

    PubMed

    Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

    2004-06-18

    Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

  12. TGF-β activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth.

    PubMed

    Pavlides, Savvas C; Lecanda, Jon; Daubriac, Julien; Pandya, Unnati M; Gama, Patricia; Blank, Stephanie; Mittal, Khushbakhat; Shukla, Pratibha; Gold, Leslie I

    2016-01-01

    We previously reported that aberrant TGF-β/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-β signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-β increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-β-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-β signaling.

  13. Forkhead Box O1 is present in quiescent pituitary cells during development and is increased in the absence of p27 Kip1.

    PubMed

    Majumdar, Sreeparna; Farris, Corrie L; Kabat, Brock E; Jung, Deborah O; Ellsworth, Buffy S

    2012-01-01

    Congenital pituitary hormone deficiencies have been reported in approximately one in 4,000 live births, however studies reporting mutations in some widely studied transcription factors account for only a fraction of congenital hormone deficiencies in humans. Anterior pituitary hormones are required for development and function of several glands including gonads, adrenals, and thyroid. In order to identify additional factors that contribute to human congenital hormone deficiencies, we are investigating the forkhead transcription factor, FOXO1, which has been implicated in development of several organs including ovary, testis, and brain. We find that FOXO1 is present in the nuclei of non-dividing pituitary cells during embryonic development, consistent with a role in limiting proliferation and/or promoting differentiation. FOXO1 is present in a subset of differentiated cells at e18.5 and in adult with highest level of expression in somatotrope cells. We detected FOXO1 in p27(Kip1)-positive cells at e14.5. In the absence of p27(Kip1) the number of pituitary cells containing FOXO1 is significantly increased at e14.5 suggesting that a feedback loop regulates the interplay between FOXO1 and p27(Kip1). PMID:23251696

  14. p27KIP1 loss promotes proliferation and phagocytosis but prevents epithelial–mesenchymal transition in RPE cells after photoreceptor damage

    PubMed Central

    ul Quraish, Reeshan; Sudou, Norihiro; Nomura-Komoike, Kaori; Sato, Fumi

    2016-01-01

    Purpose p27KIP1 (p27), originally identified as a cell cycle inhibitor, is now known to have multifaceted roles beyond cell cycle regulation. p27 is required for the normal histogenesis of the RPE, but the role of p27 in the mature RPE remains elusive. To define the role of p27 in the maintenance and function of the RPE, we investigated the effects of p27 deletion on the responses of the RPE after photoreceptor damage. Methods Photoreceptor damage was induced in wild-type (WT) and p27 knockout (KO) mice with N-methyl-N-nitrosourea (MNU) treatment. Damage-induced responses of the RPE were investigated with bromodeoxyuridine (BrdU) incorporation assays, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays at different stages after MNU treatment. Subcellular localization of p27 in the WT RPE was also analyzed in vivo and in vitro. Results MNU treatment induced photoreceptor-specific degeneration in the WT and KO retinas. BrdU incorporation assays revealed virtually no proliferation of RPE cells in the WT retinas while, in the KO retinas, approximately 16% of the RPE cells incorporated BrdU at day 2 after MNU treatment. The RPE in the KO retinas developed aberrant protrusions into the outer nuclear layer in response to photoreceptor damage and engulfed outer segment debris, as well as TUNEL-positive photoreceptor cells. Increased phosphorylation of myosin light chains and their association with rhodopsin-positive phagosomes were observed in the mutant RPE, suggesting possible deregulation of cytoskeletal dynamics. In addition, WT RPE cells exhibited evidence of the epithelial–mesenchymal transition (EMT), including morphological changes, induction of α-smooth muscle actin expression, and attenuated expression of tight junction protein ZO-1 while these changes were absent in the KO retinas. In the normal WT retinas, p27 was localized to the nuclei of RPE cells while nuclear and cytoplasmic p27 was detected in RPE cells

  15. Tuberous sclerosis complex suppression in cerebellar development and medulloblastoma: separate regulation of mTOR activity and p27Kip1 localization

    PubMed Central

    Bhatia, Bobby; Northcott, Paul A.; Hambardzumyan, Dolores; Govindarajan, Baskaran; Brat, Daniel J.; Arbiser, Jack L.; Holland, Eric C.; Taylor, Michael D.; Kenney, Anna Marie

    2009-01-01

    During development, proliferation of cerebellar granule neuron precursors (CGNPs), candidate cells-of-origin for the pediatric brain tumor medulloblastoma, requires signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF), whose pathways are also implicated in medulloblastoma. One of the consequences of IGF signaling is inactivation of the mTOR-suppressing Tuberous Sclerosis Complex (TSC), comprised of TSC1 and TSC2, leading to increased mRNA translation. We show that mice in which TSC function is impaired display increased mTOR pathway activation, enhanced CGNP proliferation, GSK-3α/β inactivation, and cytoplasmic localization of the cyclin-dependent kinase (cdk) inhibitor p27Kip1, which has been proposed to cause its inactivation or gain of oncogenic functions. We observed the same characteristics in wild-type primary cultures of CGNPs in which TSC1 and/or TSC2 were knocked down, and in mouse medulloblastomas induced by ectopic Shh pathway activation. Moreover, Shh-induced mouse medulloblastomas manifested Akt-mediated TSC2 inactivation, and the mutant TSC2 allele synergized with aberrant Shh signaling to increase medulloblastoma incidence in mice. Driving exogenous TSC2 expression in Shh-induced medulloblastoma cells corrected p27Kip1 localization and reduced proliferation. GSK-3α/β inactivation in the tumors in vivo and in primary CGNP cultures was mTOR-dependent, whereas p27Kip1 cytoplasmic localization was regulated upstream of mTOR, by TSC2. These results indicate that a balance between Shh mitogenic signaling and TSC function regulating new protein synthesis and cdk inhibition is essential for normal development and prevention of tumor formation or expansion. PMID:19738049

  16. HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1.

    PubMed

    Yao, Zhi Qiang; Eisen-Vandervelde, Audrey; Ray, Suma; Hahn, Young S

    2003-09-15

    Hepatitis C virus (HCV) is efficient in the establishment of persistent infection. We have previously shown that HCV core protein inhibits T cell proliferation through its interaction with the complement receptor, gC1qR. Here we show that HCV core-induced inhibition of T cell proliferation involves a G(0)/G(1) cell cycle arrest, which is reversible upon addition of anti-gC1qR antibody. Correspondingly, the expression of cyclin-dependent kinases (Cdk) 2/4 and cyclin E/D, as well as subsequent phosphorylation of retinoblastoma (pRb), is reduced in core-treated T cells in response to mitogenic stimulation. Remarkably, degradation of p27(Kip1), a negative regulator of both Cdk4/cyclin D and Cdk2/cyclin E complexes, is significantly diminished in T cells treated with HCV core upon mitogenic stimulation. These data indicate that the stability of p27(Kip1) by HCV core is associated with blocking activated T cells for the G(1) to S phase transition and inhibiting T cell proliferation.

  17. TM4SF5 accelerates G1/S phase progression via cytosolic p27Kip1 expression and RhoA activity.

    PubMed

    Kim, Hyeonjung; Kang, Minkyung; Lee, Sin-Ae; Kwak, Tae Kyoung; Jung, Oisun; Lee, Hyo Jeong; Kim, Sung-Hoon; Lee, Jung Weon

    2010-08-01

    Transmembrane 4 L six family member 5 (TM4SF5) causes epithelial-mesenchymal transition (EMT) for aberrant cell proliferation. However, the effects of TM4SF5 expression on cell cycle are unknown so far. In this study, using hepatocytes that either ectopically or endogenously express TM4SF5 and human hepatocarcinoma tissues, the role of TM4SF5 in G1/S phase progression was examined. We found that TM4SF5 expression accelerated G1/S phase progression with facilitated cyclin D1 and E expression and Rb phosphorylation. Furthermore, TM4SF5 enhanced trafficking of CDK4 and cyclin D1 into the nucleus and induced complex formation between them. However, TM4SF5-facilitated G1/S phase progression was blocked by silencing of p27Kip1 using siRNA or by infection of active RhoA. Pharmacological inhibition of ROCK accelerated the G1/S phase progression of control TM4SF5-unexpressing cells. Altogether, these observations suggest that TM4SF5 accelerates G1/S phase progression with facilitated CDK4/cyclin D1 entry into the nucleus, which might be supported by TM4SF5-mediated actin reorganization through cytosolic p27Kip1 expression and Rho GTPase activity.

  18. Loss of heterozygosity in the chromosomal region 12p12-13 is very common in childhood acute lymphoblastic leukemia and permits the precise localization of a tumor-suppressor gene distinct from p27KIP1.

    PubMed

    Cavé, H; Gérard, B; Martin, E; Guidal, C; Devaux, I; Weissenbach, J; Elion, J; Vilmer, E; Grandchamp, B

    1995-11-15

    Abnormalities of the short arm of chromosome 12 are relatively common in hematologic malignancies and deletions of the region. 12p12-13 are found in approximately 5% of the patients with acute lymphoblastic leukemia (ALL). As a potent inhibitor of cyclin-dependent kinases, p27KIP1 prevents the progression of the cell cycle and the gene encoding p27KIP1 represents a potential tumor-suppressor gene. Its recent assignment to the chromosomal region (12p12.3) prompted us to study the p27KIP1 gene in a series of 61 children with ALL. Microsatellite polymorphic markers flanking the p27KIP1 gene were analyzed to detect losses of heterozygosity (LOH). Eleven patients displayed LOH for at least one of the markers. The deleted are encompassed the p27KIP1 gene locus in 10 cases, but inactivation of the remaining allele by deletion, translocation, or mutation was never observed. In addition, in 1 patient, the p27KIP1 gene was situated outside of the region of LOH. Thus, p27KIP1 does not seem to be the target gene of 12p12-13 alterations. However, this study indicates that 12p12-13 alterations at the molecular level, which are present in about 27% of the children with B-lineage ALL, are much more common than had previously been reported by usual chromosome analysis. Moreover, LOH mapping allowed us to better define the location of a putative tumor-suppressor gene implicated in these malignancies and should therefore help in identifying this gene.

  19. The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

    PubMed Central

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G1 phase. Sustained overexpression of DYRK1A induced G0 cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome. PMID:24806449

  20. The Down syndrome-related protein kinase DYRK1A phosphorylates p27(Kip1) and Cyclin D1 and induces cell cycle exit and neuronal differentiation.

    PubMed

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27(Kip1) on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27(Kip1) Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.

  1. The Down syndrome-related protein kinase DYRK1A phosphorylates p27(Kip1) and Cyclin D1 and induces cell cycle exit and neuronal differentiation.

    PubMed

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27(Kip1) on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27(Kip1) Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome. PMID:24806449

  2. Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    PubMed Central

    Wang, Hui-Chen; Hsu, Sung-Po; Lee, Wen-Sen

    2015-01-01

    Previously, we demonstrated that progesterone (P4) at physiologic levels (5-500 nM) inhibited proliferation in cultured rat aortic smooth muscle cells (RASMCs) through a P4 receptor (PR)-dependent pathway. We also showed that P4-induced cell cycle arrest in RASMCs occurs when the cyclin-CDK2 system is inhibited just as p21cip1 and p27kip1 protein levels are augmented. In the present study, we further investigated the molecular mechanism underlying P4-induced up-regulations of p21cip1 and p27kip1 in RASMCs. We used pharmacological inhibitors as well as dominant negative constructs and conducted Western blot analyses to delineate the signaling pathway involved. Our data suggest that P4 up-regulated the expression of p21cip1 and p27kip1 in RASMCs through increasing the level of p53 protein mediated by activating the cSrc/Kras/Raf-1/AKT/ERK/p38/IκBα/NFκB pathway. The findings of the present study highlight the molecular mechanism underlying P4-induced up-regulations in p21cip1 and p27kip1 in RASMCs. PMID:25932965

  3. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

    PubMed

    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation.

  4. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

    PubMed

    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. PMID:25498792

  5. Cyclin D1, p16(INK) (4A) and p27(Kip1) in pancreatic adenocarcinoma: assessing prognostic implications through quantitative image analysis.

    PubMed

    Georgiadou, Despoina; Sergentanis, Theodoros N; Sakellariou, Stratigoula; Filippakis, George M; Zagouri, Flora; Vlachodimitropoulos, Dimitris; Psaltopoulou, Theodora; Lazaris, Andreas C; Patsouris, Efstratios; Zografos, George C

    2014-12-01

    The prognostic significance of cyclin D1, p16(INK) (4A) and p27(Kip1) expression has been documented in several human malignancies; however, their prognostic potential in pancreatic adenocarcinoma is still unclear. This study aimed to assess the correlation of the aforementioned molecules with clinicopathological parameters and prognosis. Sixty patients with pancreatic ductal adenocarcinoma underwent surgical resection at a single institution; immunohistochemical staining of the studied markers was quantified by Ιmage analysis system. Cyclin D1 overexpression was positively associated with grade, neural infiltration and vascular invasion, whereas p27 positively correlated with age. Higher cyclin D1 expression indicated poorer survival (adjusted HR = 9.75, 95%CI: 1.48-64.31, p = 0.018, increment: one unit in H-score), whereas a marginal trend toward an association between p16 positivity and improved survival was observed (adjusted HR = 0.58, 95%CI: 0.32-1.05, p = 0.072 regarding positive vs negative cases). No significant association with overall survival was noted regarding p27. In conclusion, cyclin D1 overexpression and possibly p16 loss of expression in pancreatic adenocarcinoma seem to be adverse prognostic factors, whereas p27 expression did not seem to possess such prognostic properties. Further validation of the present findings in studies encompassing larger samples seems to be needed.

  6. FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21(Waf1) and p27(Kip1).

    PubMed

    Dombrowski, Christian; Helledie, Torben; Ling, Ling; Grünert, Martin; Canning, Claire A; Jones, C Michael; Hui, James H; Nurcombe, Victor; van Wijnen, Andre J; Cool, Simon M

    2013-12-01

    Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential.

  7. p27 kip1 haplo-insufficiency improves cardiac function in early-stages of myocardial infarction by protecting myocardium and increasing angiogenesis by promoting IKK activation.

    PubMed

    Zhou, Ningtian; Fu, Yuxuan; Wang, Yunle; Chen, Pengsheng; Meng, Haoyu; Guo, Shouyu; Zhang, Min; Yang, Zhijian; Ge, Yingbin

    2014-08-07

    p27(kip1) (p27) is widely known as a potent cell cycle inhibitor in several organs, especially in the heart. However, its role has not been fully defined during the early phase of myocardial infarction (MI). In this study, we investigated the relationships between p27, vascular endothelial growth factor/hepatocyte growth factor (VEGF/HGF) and NF-κB in post-MI cardiac function repair both in vivo and in the hypoxia/ischemia-induced rat myocardiocyte model. In vivo, haplo-insufficiency of p27 improved cardiac function, diminished the infarct zone, protected myocardiocytes and increased angiogenesis by enhancing the production of VEGF/HGF. In vitro, the presence of conditioned medium from hypoxia/ischemia-induced p27 knockdown myocardiocytes reduced the injury caused by hypoxia/ischemia in myocardiocytes, and this effect was reversed by VEGF/HGF neutralizing antibodies, consistent with the cardioprotection being due to VEGF/HGF secretion. We also observed that p27 bound to IKK and that p27 haplo-insufficiency promoted IKK/p65 activation both in vivo and in vitro, thereby inducing the NF-κB downstream regulator, VEGF/HGF. Furthermore, IKKi and IKK inhibitor negated the effect of VEGF/HGF. Therefore, we conclude that p27 haplo-insufficiency protects against heart injury by VEGF/HGF mediated cardioprotection and increased angiogenesis through promoting IKK activation.

  8. Cdc6 protein activates p27KIP1-bound Cdk2 protein only after the bound p27 protein undergoes C-terminal phosphorylation.

    PubMed

    Uranbileg, Baasanjav; Yamamoto, Hanako; Park, Jung-ha; Mohanty, Atish R; Arakawa-Takeuchi, Shiho; Jinno, Shigeki; Okayama, Hiroto

    2012-02-24

    In mammalian cells Cdk2 activity during the G(1)-S transition is mainly controlled by p27(KIP1). Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21(CIP1)-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.

  9. Expression of p27Kip1, a cell cycle repressor protein, is inversely associated with potential carcinogenic risk in the genetic rodent models of obesity and long-lived Ames dwarf mice

    PubMed Central

    Eto, Isao

    2014-01-01

    Introduction The association of genetic rodent models of obesity and cancer still remains a controversial issue. Although this controversy has largely been resolved in recent years for homozygous leptin receptor-deficient obese Zucker rats and homozygous long-lived Ames dwarf mice, it is still unresolved for homozygous leptin-deficient obese ob/ob mice. Objective The objective of the present study described below was to investigate whether the expression of the cell cycle repressor protein p27(Kip1) is (a) down-regulated in the tumor-free homozygous leptin receptor-deficient obese Zucker rats as well as tumor-free homozygous leptin-deficient obese ob/ob mice and (b) up-regulated in the tumor-free homozygous long-lived Ames dwarf mice. Methods To achieve this objective, we first performed western immunoblot analysis of the hepatic expression of p27. We then performed western immunoblot analysis and proteomic analysis of the hepatic expression of the proteins involved in the upstream molecular signaling pathways for the expression of p27. Lastly, we analyzed the serum levels of glucose, insulin, and branched-chain amino acids, all of which have been shown to regulate, causally and inversely, the expression of p27. Results/Conclusions The results indicated that the hepatic expression of p27 was down-regulated in the homozygous leptin receptor-deficient obese Zucker rats and up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the hepatic expression of p27 was down-regulated in the homozygous leptin-deficient obese ob/ob mice. This last observation was not completely consistent with all of the results of the published studies where homozygous leptin-deficient obese ob/ob mice were used. PMID:23357529

  10. Long-term exposure to superantigen induces p27Kip1 and Bcl-2 expression in effector memory CD4+ T cells.

    PubMed

    Koyanagi, Madoka; Fukada, Kenji; Uchiyama, Takehiko; Yagi, Junji; Arimura, Yutaka

    2007-08-01

    The long-term exposure of mice to superantigen SEA using a mini-osmotic pump (SEA pump) induced a long-lasting expansion of Vbeta3+ CD4+ T cells with T helper (Th) 2 cell-type properties. Removal of the SEA pump 10 days after pump implantation did not significantly alter the level of Vbeta3+ CD4+ T cell expansion/maintenance. Furthermore, CFSE-labeled CD4+ T cells failed to divide when transferred to post-implantation day 15 mice. Thus, CD4+ T cells appeared to survive for at least 30 days in the absence of a sufficient amount of antigen to trigger cell division. STAT6 deficient mice, in which Th2 cell development is largely impaired, also exhibited a protracted cell expansion, similar to that observed in normal mice, suggesting that the Th2 cell property is dispensable for the maintenance of Vbeta3+ CD4+ T cell expansion. The expanded CD4+ T cells on post-implantation day 26 were arrested in the G0/G1 phase of the cell cycle and showed a lower level of cell division upon restimulation. The Cdk inhibitor p27(Kip1) was highly expressed, and Cdk2 was downregulated. Moreover, the CD4+ T cells were resistant to in vitro apoptosis induction in parallel with their level of Bcl-2 expression. Collectively, the Vbeta3+ CD4+ T cells appeared to develop into long-lived memory T cells with cell cycle arrest upon long-term exposure to SEA.

  11. Successive phosphorylation of p27(KIP1) protein at serine-10 and C terminus crucially controls its potency to inactivate Cdk2.

    PubMed

    Mohanty, Atish R; Kan, Qiuming; Srivastava, Saumya; Uranbileg, Baasanjav; Arakawa-Takeuchi, Shiho; Fujita, Naoya; Okayama, Hiroto

    2012-06-22

    During the G(1)-S transition, the activity of Cdk2 is regulated by its association with p27(KIP1), which in rodent fibroblasts undergoes phosphorylation mainly at serine 10, threonine 187, and C-terminal threonine 197 by KIS, Cdk2, and Pim or ROCK, respectively. Recently Cdc6 the AAA+ ATPase, identified initially to assemble pre-replicative complexes on origins of replication and later to activate p21(CIP1)-inactivated Cdk2, was found also to activate p27-bound Cdk2 but only after the bound p27 is C-terminally phosphorylated. On the other hand, the biological significance of the serine 10 phosphorylation remains elusive aside from its involvement in the stability of p27 itself. We report here that serine 10 phosphorylation is required for efficient C-terminal phosphorylation of its own by PIM and ROCK kinases and critically controls the potency of p27 as a Cdk2 inhibitor. In vitro, PIM1 and active ROCK1 efficiently phosphorylated free as well as Cdk2-bound p27 but only when the p27 was phosphorylated at Ser-10 in advance. Consistently, a Ser-10 nonphosphorylatable mutant p27 protein was not phosphorylated at the C terminus in vivo. Furthermore, when double-phosphorylated, free p27 was no longer a potent inhibitor of Cdk2, and Cdk2-bound p27 could be removed by Cdc6 to reactivate the Cdk2. Thus, phosphorylation at these two sites crucially controls the potency of this CDK inhibitor in two distinct modes.

  12. Molecular cloning and functional characterization of a cell-permeable superoxide dismutase targeted to lung adenocarcinoma cells. Inhibition cell proliferation through the Akt/p27kip1 pathway.

    PubMed

    Lu, Min; Gong, Xingguo; Lu, Yuwen; Guo, Jianjun; Wang, Chenhui; Pan, Yuanjiang

    2006-05-12

    In clinical oncology, many trials with superoxide dismutase (SOD) have failed to demonstrate antitumor ability and in many cases even caused deleterious effects because of low tumor-targeting ability. In the current research, the Nostoc commune Fe-SOD coding sequence was amplified from genomic DNA. In addition, the single chain variable fragment (ScFv) was constructed from the cDNA of an LC-1 hybridoma cell line secreting anti-lung adenocarcinoma monoclonal antibody. After modification, the SOD and ScFv were fused and co-expressed, and the resulting fusion protein produced SOD and LC-1 antibody activity. Tracing SOD-ScFv by fluorescein isothiocyanate and superoxide anions (O2*-) in SPC-A-1 cells showed that the fusion protein could recognize and enter SPC-A-1 cells to eliminate O2*-. The lower oxidative stress resulting from the decrease in cellular O2*- delayed the cell cycle at G1 and significantly slowed SPC-A-1 cell growth in association with the dephosphorylation of the serine-threonine protein kinase Akt and expression of p27kip1. The tumor-targeting fusion protein resulting from this research overcomes two disadvantages of SODs previously used in the clinical setting, the inability to target tumor cells or permeate the cell membrane. These findings lay the groundwork for development of an efficient antitumor drug targeted by the ScFv. PMID:16551617

  13. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery.

    PubMed Central

    Chen, D; Krasinski, K; Sylvester, A; Chen, J; Nisen, P D; Andrés, V

    1997-01-01

    Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty. PMID:9153274

  14. TReP-132 Controls Cell Proliferation by Regulating the Expression of the Cyclin-Dependent Kinase Inhibitors p21WAF1/Cip1 and p27Kip1

    PubMed Central

    Gizard, Florence; Robillard, Romain; Barbier, Olivier; Quatannens, Brigitte; Faucompré, Anne; Révillion, Françoise; Peyrat, Jean-Philippe; Staels, Bart; Hum, Dean W.

    2005-01-01

    The transcriptional regulating protein of 132 kDa (TReP-132) has been identified in steroidogenic tissues, where it acts as a coactivator of steroidogenic factor 1 (SF-1). We show here that TReP-132 plays a role in the control of cell proliferation. In human HeLa cells, TReP-132 knockdown by using small interfering RNA resulted in increased G1→S cell cycle progression. The growth-inhibitory effects of TReP-132 was further shown to be mediated by induction of G1 cyclin-dependent kinase inhibitors p21WAF1 (p21) and p27KIP1 (p27) expression levels. As a consequence, G1 cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G1 phase. The stimulatory effect of TReP-132 on p21 and p27 gene transcription involved interaction of TReP-132 with the transcription factor Sp1 at proximal Sp1-binding sites in their promoters. Moreover, in different breast tumor cell lines, endogenous TReP-132 expression was positively related with a lower proliferation rate. In addition, TReP-132 knockdown resulted in enhanced cell proliferation and lowered p21 and p27 mRNA levels in the steroid-responsive and nonresponsive T-47D and MDA-MB-231 cell lines, respectively. Finally, a statistic profiling of human breast tumor samples highlighted that expression of TReP-132 is correlated with p21 and p27 levels and is associated with lower tumor incidence and aggressiveness. Together, these results identify TReP-132 as a basal cell cycle regulatory protein acting, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters. PMID:15899840

  15. Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1.

    PubMed

    Alevizopoulos, K; Sanchez, B; Amati, B

    2000-04-13

    Ectopic expression of the CDK inhibitors (CKIs) p16INK4a and p27Kip1 in Rat1 fibroblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations affect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the EIA mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context. PMID:10803468

  16. Up-regulation of p27Kip1, p21WAF1/Cip1 and p16Ink4a is associated with, but not sufficient for, induction of squamous differentiation.

    PubMed

    Harvat, B L; Wang, A; Seth, P; Jetten, A M

    1998-05-01

    Irreversible growth arrest is an early and integral part of squamous cell differentiation in normal human epidermal keratinocytes (NHEKs) and is assumed to be linked to the control of expression of differentiation-specific genes. In this study, we examine the link between the molecular events associated with growth arrest and the expression of differentiation genes. NHEKs that have been induced to undergo growth arrest and differentiation by suspension culture contain populations in both G1 and G2/M of the cell cycle. The irreversible growth arrest state in NHEKs is characterized by an accumulation of the hypophosphorylated forms of Rb and p130, with subsequent down-regulation of levels of Rb, up-regulation of p130 and associated down-regulation of E2F-regulated genes such as cyclin A. These events correlate with an inhibition of G1 cdk activity, mediated in part by an increase in the cdk inhibitors p21(WAF1/Cip1), p27(Kip1) and p16(Ink4a). Flow cytometric and immunoblot analysis demonstrated that the timing of the up-regulation of p27, p16 and p130 corresponds closely with the induction of the squamous-specific genes cornifin alpha (SPRR-1) and transglutaminase type I, suggesting a close link between control of growth arrest and differentiation. However, growth arrest induced by over-expression of p27, p21 or p16 by recombinant adenovirus is not sufficient to induce expression of the differentiation genes, or to invoke the pattern of cell cycle regulatory protein expression characteristic of the differentiation-specific irreversible growth arrest. We conclude that growth arrest mediated by activation of the Rb pathway is not sufficient to trigger terminal squamous differentiation and additional signals which can be generated during suspension culture are required to promote the complete differentiation program. PMID:9547295

  17. The expression of the ubiquitin ligase subunit Cks1 in human breast cancer

    PubMed Central

    Slotky, Merav; Shapira, Ma'anit; Ben-Izhak, Ofer; Linn, Shai; Futerman, Boris; Tsalic, Medy; Hershko, Dan D

    2005-01-01

    Introduction Loss of the cell-cycle inhibitory protein p27Kip1 is associated with a poor prognosis in breast cancer. The decrease in the levels of this protein is the result of increased proteasome-dependent degradation, mediated and rate-limited by its specific ubiquitin ligase subunits S-phase kinase protein 2 (Skp2) and cyclin-dependent kinase subunit 1 (Cks1). Skp2 was recently found to be overexpressed in breast cancers, but the role of Cks1 in these cancers is unknown. The present study was undertaken to examine the role of Cks1 expression in breast cancer and its relation to p27Kip1 and Skp2 expression and to tumor aggressiveness. Methods The expressions of Cks1, Skp2, and p27Kip1 were examined immunohistochemically on formalin-fixed, paraffin-wax-embedded tissue sections from 50 patients with breast cancer and by immunoblot analysis on breast cancer cell lines. The relation between Cks1 levels and patients' clinical and histological parameters were examined by Cox regression and the Kaplan–Meier method. Results The expression of Cks1 was strongly associated with Skp2 expression (r = 0.477; P = 0.001) and inversely with p27Kip1 (r = -0.726; P < 0.0001). Overexpression of Cks1 was associated with loss of tumor differentiation, young age, lack of expression of estrogen receptors and of progesterone receptors, and decreased disease-free (P = 0.0007) and overall (P = 0.041) survival. In addition, Cks1 and Skp2 expression were increased by estradiol in estrogen-dependent cell lines but were down-regulated by tamoxifen. Conclusion These results suggest that Cks1 is involved in p27Kip1 down-regulation and may have an important role in the development of aggressive tumor behavior in breast cancer. PMID:16168119

  18. 6-Gingerol Inhibits Growth of Colon Cancer Cell LoVo via Induction of G2/M Arrest.

    PubMed

    Lin, Ching-Bin; Lin, Chun-Che; Tsay, Gregory J

    2012-01-01

    6-Gingerol, a natural component of ginger, has been widely reported to possess antiinflammatory and antitumorigenic activities. Despite its potential efficacy against cancer, the anti-tumor mechanisms of 6-gingerol are complicated and remain sketchy. In the present study, we aimed to investigate the anti-tumor effects of 6-gingerol on colon cancer cells. Our results revealed that 6-gingerol treatment significantly reduced the cell viability of human colon cancer cell, LoVo, in a dose-dependent manner. Further flow cytometric analysis showed that 6-gingerol induced significant G2/M phase arrest and had slight influence on sub-G1 phase in LoVo cells. Therefore, levels of cyclins, cyclin-dependent kinases (CDKs), and their regulatory proteins involved in S-G2/M transition were investigated. Our findings revealed that levels of cyclin A, cyclin B1, and CDK1 were diminished; in contrast, levels of the negative cell cycle regulators p27(Kip1) and p21(Cip1) were increased in response to 6-gingerol treatment. In addition, 6-gingerol treatment elevated intracellular reactive oxygen species (ROS) and phosphorylation level of p53. These findings indicate that exposure of 6-gingerol may induce intracellular ROS and upregulate p53, p27(Kip1), and p21(Cip1) levels leading to consequent decrease of CDK1, cyclin A, and cyclin B1 as result of cell cycle arrest in LoVo cells. It would be suggested that 6-gingerol should be beneficial to treatment of colon cancer.

  19. Sodium butyrate regulates androgen receptor expression and cell cycle arrest in human prostate cancer cells.

    PubMed

    Kim, Jeonga; Park, Hyeyoung; Im, Ji Young; Choi, Wahn Soo; Kim, Hyung Sik

    2007-01-01

    Histone deacetylase (HDAC) inhibitors have been shown to modify the expression of a variety of genes related to cell cycle regulation and apoptosis in several cancer cells. However, the precise mode of action of HDAC inhibitors in prostate cancer cells is not completely understood. This study examined whether an HDAC inhibitor affects cell death in human prostate cancer cells through the epigenetic regulation of androgen receptor (AR) expression. The molecular mechanism of the HDAC inhibitor, sodium butyrate, on the epigenetic alterations of cell cycle regulators was evaluated in androgen-dependent human prostate cancer LNCaP cells. The expression levels of acetylated histone H3 and H4 increased significantly after 48 h treatment with sodium butyrate. Sodium butyrate induced the expression of AR after 48 h treatment. In addition, immunofluorescence assay revealed the nuclear localization of the AR after sodium butyrate treatment. Sodium butyrate also significantly decreased the expression of the cell cycle regulatory proteins (cyclin D1/cyclin dependent kinase (CDK)4, CDK6, and cyclin E/CDK2) in the LNCaP cells after 48 h treatment. Furthermore, p21Waf1/Cip1 and p27Kip1 were upregulated as a result of the sodium butyrate treatment. These results suggest that sodium butyrate effectively inhibited cell proliferation and induced apoptosis of human prostate cancer cells by altering the expression of cell cycle regulators and AR. This study indicated that sodium butyrate may be a potential agent in prostate cancer treatment.

  20. Targeting prostate cancer based on signal transduction and cell cycle pathways

    PubMed Central

    Lee, John T.; Lehmann, Brian D.; Terrian, David M.; Chappell, William H.; Stivala, Franca; Libra, Massimo; Martelli, Alberto M.; Steelman, Linda S.

    2008-01-01

    Prostate cancer remains a leading cause of death in men despite increased capacity to diagnose at earlier stages. After prostate cancer has become hormone independent, which often occurs after hormonal ablation therapies, it is difficult to effectively treat. Prostate cancer may arise from mutations and dysregulation of various genes involved in regulation signal transduction (e.g., PTEN, Akt, etc.,) and the cell cycle (e.g., p53, p21Cip1, p27Kip1, Rb, etc.,). This review focuses on the aberrant interactions of signal transduction and cell cycle genes products and how they can contribute to prostate cancer and alter therapeutic effectiveness. PMID:18594202

  1. From microRNA functions to microRNA therapeutics: novel targets and novel drugs in breast cancer research and treatment (Review).

    PubMed

    Piva, Roberta; Spandidos, Demetrios A; Gambari, Roberto

    2013-10-01

    MicroRNAs (miRNAs or miRs) are a family of small non‑coding RNAs that regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences. miRNAs play a crucial role in cancer. In the case of breast tumors, several studies have demonstrated a correlation between: i) the expression profile of oncogenic miRNAs (oncomiRs) and tumor suppressor miRNAs; and ii) the tumorigenic potential of triple-negative [estrogen receptor (ER), progesterone receptor (PR) and Her2/neu] primary breast cancers. Among the miRNAs involved in breast cancer, miR-221 plays a crucial role for the following reasons: i) miR-221 is significantly overexpressed in triple-negative primary breast cancer; ii) the oncosuppressor p27Kip1, a validated miR-221 target is downregulated in aggressive cancer cell lines; and iii) the upregulation of a key transcription factor, Slug, appears to be crucial, since it binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. A Slug/miR-221 network has been suggested, linking miR-221 activity with the downregulation of a Slug repressor, leading to Slug/miR-221 upregulation and p27Kip1 downregulation. Interference with this process can be achieved using antisense miRNA (antagomiR) molecules targeting miR-221, inducing the downregulation of Slug and the upregulation of p27Kip1.

  2. Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression

    PubMed Central

    Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

    2016-01-01

    Background/Aims Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. Methods The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. Results The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27kip-1 increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Conclusions Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27kip-1. PMID:26470770

  3. HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells.

    PubMed

    Mardente, Stefania; Mari, Emanuela; Consorti, Fabrizio; Di Gioia, Cira; Negri, Rodolfo; Etna, Marilena; Zicari, Alessandra; Antonaci, Alfredo

    2012-12-01

    Experimental and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to cellular transformation. Inflammatory infiltrates in papillary thyroid cancer include lymphocytes, macrophages and cytokines. High-mobility group box 1 protein (HMGB1) is a late inflammatory cytokine that signals danger to the immune system through the receptor for advanced glycation end-products (RAGE) and Toll-like receptor. The activation of the above receptors results in the secretion of growth, chemotactic and angiogenic factors that contribute to chronic inflammation. In this study, we suggest that apart from the activation of signal transduction pathways by the activation of RAGE, the indirect inhibition of cell cycle regulators [such as phosphatase and tensin homolog (PTEN)] may also cause an increase in cell growth and motility. MicroRNAs (miRNAs) have increasingly been implicated in regulating the malignant progression of cancer. MiR-221 and miR-222 have been found to be deregulated in human papillary thyroid carcinomas. They are involved in cell proliferation through the inhibition of the cell cycle regulator, p27kip1, in human papillary carcinomas. In this study, we show that HMGB1 increases the expression of miR-221 and miR-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line (BC PAP). The overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 is associated with an increase in malignancy scores, namely cell growth and motility.

  4. Effects of an Indolocarbazole-Derived CDK4 Inhibitor on Breast Cancer Cells

    PubMed Central

    Sun, Yuan; Li, Ying-xia; Wu, Hai-jun; Wu, Si-hung; Wang, Y. Alan; Luo, Dian-zhong; Liao, D. Joshua

    2011-01-01

    Introduction: Cyclin D1 (D1) binds to cyclin-dependent kinases (CDK) 4 or 6 to form a holoenzyme that phosphorylates the Rb protein to promote cell cycle progression from G1 to S phase. Therefore, targeting CDK4/6 may be a good strategy for chemotherapy of cancer. We performed a proof-of-principle study to determine the effect of Naphtho [2, 1-α] pyrrolo [3, 4-c] carbazole-5, 7 (6H, 12H)-dione (NPCD), a novel CDK4 inhibitor, on breast cancer cell lines. Methods: NPCD was synthesized and purified to over 99% purity verified by HPLC. MCF7, MB231, MCF15, T47D and GI101Ap human breast cancer cells were analyzed for the efficacy of NPCD with MTT and clonogenic assays, with FACS and staining for ethidium bromide and acridine orange for cell death and cell cycle profile. Western blot, reverse transcription and PCR were used for studies of gene expression, and co-immunoprecipitation for protein-complex formation. Results: MTT assay showed that NPCD caused growth arrest and apoptosis of MCF7, MDA-MB231, T47D, MCF15 and GI101Ap cells with an IC50 ranging between 3 to 8 µM given as a single dose. The growth arrest persisted for many days after cessation of the treatment, as shown in a clonogenic assay. NPCD could induce or reduce the D1 and CDK4 protein levels, depending on the cell line, but this effect was not correlated with its efficacy. Phosphorylation of D1 at Thr286 was decreased but it unexpectedly did not correlate with the change in D1 level in the cell lines studied. Phosphorylation of the Rb protein was decreased as expected whereas the p27kip1 protein level was decreased unexpectedly. Protein levels of p21cip1, CDK2 and cyclin E were also decreased in some, but not all, of the cell lines, whereas the mRNA levels of D1, CDK4, cyclin E, CDK2, p27kip1 and p21cip1 were increased in different cell lines. Conclusions: NPCD can cause long-lasting growth arrest and cell death of breast cancer cell lines at an IC50 of 3-8 µM. Decreased phosphorylation of Rb by D1-CDK4

  5. Prevention of carcinogenesis and inhibition of breast cancer tumor burden by dietary stearate

    PubMed Central

    Li, Chuanyu; Zhao, Xiangmin; Toline, Eric C.; Siegal, Gene P.; Evans, Lynda M.; Ibrahim-Hashim, Arig; Desmond, Renee A.; Hardy, Robert W.

    2011-01-01

    Previous studies have shown that stearate (C18:0), a dietary long-chain saturated fatty acid, inhibits breast cancer cell neoplastic progression; however, little is known about the mechanism modulating these processes. We demonstrate that stearate, at physiological concentrations, inhibits cell cycle progression in human breast cancer cells at both the G1 and G2 phases. Stearate also increases cell cycle inhibitor p21CIP1/WAF1 and p27KIP1 levels and concomitantly decreases cyclin-dependent kinase 2 (Cdk2) phosphorylation. Our data also show that stearate induces Ras– guanosine triphosphate formation and causes increased phosphorylation of extracellular signal-regulated kinase (pERK). The MEK1 inhibitor, PD98059, reversed stearate-induced p21CIP1/WAF1 upregulation, but only partially restored stearate-induced dephosphorylation of Cdk2. The Ras/mitogen-activated protein kinase/ERK pathway has been linked to cell cycle regulation but generally in a positive way. Interestingly, we found that stearate inhibits both Rho activation and expression in vitro. In addition, constitutively active RhoC reversed stearate-induced upregulation of p27KIP1, providing further evidence of Rho involvement. To test the effect of stearate in vivo, we used the N-Nitroso-N-methylurea rat breast cancer carcinogen model. We found that dietary stearate reduces the incidence of carcinogen-induced mammary cancer and reduces tumor burden. Importantly, mammary tumor cells from rats on a stearate diet had reduced expression of RhoA and B as well as total Rho compared with a low-fat diet. Overall, these data indicate that stearate inhibits breast cancer cell proliferation by inhibiting key check points in the cell cycle as well as Rho expression in vitro and in vivo and inhibits tumor burden and carcinogen-induced mammary cancer in vivo. PMID:21586513

  6. Male breast cancer is not congruent with the female disease.

    PubMed

    Fentiman, Ian S

    2016-05-01

    It has become customary to extrapolate from the results of treatment trials for female breastcancer and apply them to males with the disease. In the absence of results from national and international randomised trials for male breast cancer (MBC) this appears superficially to be an appropriate response. Closer examination of available data reveals that aspects of the aetiology and treatment of MBC do not fit the simplistic model that men usually have endocrine sensitive tumours which behave like those in postmenopausal women. Most females and males with breast cancer have none of the recognised risk factors, indicating the gaps in our knowledge of the epidemiology of this disease. Several studies have compared epidemiological risk factors for MBC and female breast cancer (FBC) but many have been blighted by small numbers. In comparison with FBC there is a larger proportion of BRCA2 tumours, (occurring in 10% of MBC), and underrepresentation of BRCA1 tumours (found in only 1%), suggesting significant differences in the genetic aetiology of MBC and FBC. Genome-wide association studies in FBC reported single nucleotide polymorphisms (SNPs) in 12 novel independent loci were consistently associated with disease but for MBC 2 SNPs had a significantly increased risk. Molecular profiles of matched cancers in males and females showed a gender-associated modulation of major processes including energy metabolism, regulation of translation, matrix remodelling and immune recruitment. Immunohistochemistry for kinase inhibitor proteins (KIPs) p27Kip1 and p21Waf1 indicate a significant difference in the immunostaining of tumours from male patients compared with females. MBC is almost always estrogen receptor positive (ER+ve) and so systemic treatment is usually endocrine. With evidence in FBC that aromatase inhibitors are more effective than tamoxifen in the postmenopausal it was seemingly logical that the same would be true for MBC. Results however suggest less efficacy with AIs

  7. RhoA regulates G1-S progression of gastric cancer cells by modulation of multiple INK4 family tumor suppressors.

    PubMed

    Zhang, Siyuan; Tang, Qiulin; Xu, Feng; Xue, Yan; Zhen, Zipeng; Deng, Yu; Liu, Ming; Chen, Ji; Liu, Surui; Qiu, Meng; Liao, Zhengyin; Li, Zhiping; Luo, Deyun; Shi, Fang; Zheng, Yi; Bi, Feng

    2009-04-01

    RhoA, a member of the Rho GTPase family, has been extensively studied in the regulation of cytoskeletal dynamics, gene transcription, cell cycle progression, and cell transformation. Overexpression of RhoA is found in many malignancies and elevated RhoA activity is associated with proliferation phenotypes of cancer cells. We reported previously that RhoA was hyperactivated in gastric cancer tissues and suppression of RhoA activity could partially reverse the proliferation phenotype of gastric cancer cells, but the underlying mechanism has yet to be elucidated. It has been reported that RhoA activation is crucial for the cell cycle G(1)-S procession through the regulation of Cip/Kip family tumor suppressors in benign cell lines. In this study, we found that selective suppression of RhoA or its effectors mammalian Diaphanous 1 and Rho kinase (ROCK) by small interfering RNA and a pharmacologic inhibitor effectively inhibited proliferation and cell cycle G(1)-S transition in gastric cancer lines. Down-regulation of RhoA-mammalian Diaphanous 1 pathway, but not RhoA-ROCK pathway, caused an increase in the expression of p21(Waf1/Cip1) and p27(Kip1), which are coupled with reduced expression and activity of CDK2 and a cytoplasmic mislocalization of p27(Kip1). Suppression of RhoA-ROCK pathway, on the other hand, resulted in an accumulation of p15(INK4b), p16(INK4a), p18(INK4c), and p19(INK4d), leading to reduced expression and activities of CDK4 and CDK6. Thus, RhoA may use two distinct effector pathways in regulating the G(1)-S progression of gastric cancer cells.

  8. Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1.

    PubMed

    Jia, Wei; He, Ming-Xiao; McLeod, Ian X; Guo, Jian; Ji, Dong; He, You-Wen

    2015-01-01

    The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8(+) T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.

  9. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation.

    PubMed

    Kato, J Y; Matsuoka, M; Polyak, K; Massagué, J; Sherr, C J

    1994-11-01

    Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme. PMID:7954814

  10. CRM1/Ran-Mediated Nuclear Export of p27Kip1 Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

    PubMed Central

    Connor, Michael K.; Kotchetkov, Rouslan; Cariou, Sandrine; Resch, Ansgar; Lupetti, Rafaella; Beniston, Richard G.; Melchior, Frauke; Hengst, Ludger; Slingerland, Joyce M.

    2003-01-01

    We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase. PMID:12529437

  11. CRM1/Ran-mediated nuclear export of p27(Kip1) involves a nuclear export signal and links p27 export and proteolysis.

    PubMed

    Connor, Michael K; Kotchetkov, Rouslan; Cariou, Sandrine; Resch, Ansgar; Lupetti, Rafaella; Beniston, Richard G; Melchior, Frauke; Hengst, Ludger; Slingerland, Joyce M

    2003-01-01

    We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase.

  12. Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

    PubMed Central

    2011-01-01

    Background We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways. Results Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2. Conclusion From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation. PMID:21999842

  13. Ligands for peroxisome proliferator-activated receptor gamma inhibit growth of pancreatic cancers both in vitro and in vivo.

    PubMed

    Itami, A; Watanabe, G; Shimada, Y; Hashimoto, Y; Kawamura, J; Kato, M; Hosotani, R; Imamura, M

    2001-11-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed largely in adipose tissues and plays an important role in adipocyte differentiation. Several studies have recently shown that ligands of PPARgamma could lead to growth inhibition in some malignancies. In our study, we focused on pancreatic cancers, because the prognosis of advanced pancreatic cancer has not significantly improved due to its resistance to various chemotherapeutic regimens, so that a novel strategy should be required. We show here that PPARgamma is expressed in 5 pancreatic cancer cell lines detected in both mRNA and protein level as well as in human primary and metastatic pancreatic carcinomas examined by immunohistochemical studies. A specific ligand of PPARgamma, troglitazone, led to G1 accumulation with the increase in p27(Kip1), but not p21(Waf1/Cip1) and inhibited cellular proliferation in a pancreatic cancer cell line, Panc-1. The overexpression of PPARgamma in a pancreatic cancer cell line, KMP-3, caused lipid accumulation, which suggested cell growth in some cancers might be inhibited, at least in part, through terminal differentiation in the adipogenic lineage. In addition, implanted Panc-1 tumors in nude mice showed significant inhibition of tumor growth, when treated with pioglitazone, another specific ligand of PPARgamma. Our results suggest that ligands of PPARgamma may be a novel therapeutic agent for the treatment of pancreatic carcinomas.

  14. Fangchinoline induces G1 arrest in breast cancer cells through cell-cycle regulation.

    PubMed

    Xing, Zhibo; Zhang, Youxue; Zhang, Xianyu; Yang, Yanmei; Ma, Yuyan; Pang, Da

    2013-12-01

    Fangchinoline, an alkaloid derived from the dry roots of Stephaniae tetrandrine S. Moore (Menispermaceae), has been shown to possess cytotoxic, anti-inflammatory, and antioxidant properties. In this study, we used Fangchinoline to inhibit breast cancer cell proliferation and to investigate its underlying molecular mechanisms. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were both used in this study. We found that Fangchinoline significantly decreased cell proliferation in a dose-dependent manner and induced G1-phase arrest in both cell lines. In addition, upon analysis of expression of cell cycle-related proteins, we found that Fangchinoline reduced expression of cyclin D1, cyclin D3, and cyclin E, and increased expression of the cyclin-dependent kinase (CDK) inhibitors, p21/WAF1, and p27/KIP1. Moreover, Fangchinoline also inhibited the kinase activities of CDK2, CDK4, and CDK6. These results suggest that Fangchinoline can inhibit human breast cancer cell proliferation and thus may have potential applications in cancer therapy. PMID:23401195

  15. Casticin induces ovarian cancer cell apoptosis by repressing FoxM1 through the activation of FOXO3a

    PubMed Central

    JIANG, LING; CAO, XIAO-CHENG; CAO, JIAN-GUO; LIU, FEI; QUAN, MEI-FANG; SHENG, XI-FENG; REN, KAI-QUN

    2013-01-01

    Casticin, a polymethoxyflavone, is reported to have anticancer activities. The aim of the present study was to examine the molecular mechanisms by which casticin induces apoptosis in ovarian cancer cells. The human ovarian cancer cell lines SKOV3 and A2780 were cultured in vitro. Various molecular techniques, including histone/DNA enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and gene transfection, were used to assess the expression of FOXO3a and forkhead box protein M1 (FoxM1) in casticin-treated ovarian cancer cell lines. Casticin-induced apoptotic cell death was accompanied by the activation of transcription factor FOXO3a, with a concomitant decrease in the expression levels of FoxM1 and its downstream target factors, namely survivin and polo-like kinase 1 (PLK1), and an increase in p27KIP1. A small inhibitory RNA (siRNA) knockout of FoxM1 potentiated casticin-induced apoptosis in ovarian cancer cells. Silencing FOXO3a expression using siRNA increased FoxM1 expression levels and clearly attenuated the induction of apoptosis by casticin treatment. These results show that casticin-induced apoptosis in ovarian cancer may be caused by the activation of FOXO3a, leading to FoxM1 inhibition. PMID:23761826

  16. Direct current electrical fields induce apoptosis in oral mucosa cancer cells by NADPH oxidase-derived reactive oxygen species.

    PubMed

    Wartenberg, Maria; Wirtz, Nina; Grob, Alexander; Niedermeier, Wilhelm; Hescheler, Jürgen; Peters, Saskia C; Sauer, Heinrich

    2008-01-01

    The presence of more than one dental alloy in the oral cavity often causes pathological galvanic currents and voltage resulting in superficial erosions of the oral mucosa and eventually in the emergence of oral cancer. In the present study the mechanisms of apoptosis of oral mucosa cancer cells in response to electromagnetic fields was investigated. Direct current (DC) electrical fields with field strengths between 2 and 16 V/m, applied for 24 h to UM-SCC-14-C oral mucosa cancer cells, dose-dependently resulted in decreased cell proliferation as evaluated by Ki-67 immunohistochemistry and upregulation of the cyclin-dependent kinase (CDK) inhibitors p21(cip1/waf1) and p27(kip1), which are associated with cell cycle arrest. Electrical field treatment (4 V/m, 24 h) increased apoptosis as evaluated by immunohistochemical analysis of cleaved caspase-3 and poly-(ADP-ribose)-polymerase-1 (PARP-1). Furthermore, robust reactive oxygen species (ROS) generation, increased expression of NADPH oxidase subunits as well as Hsp70 was observed. Electrical field treatment (4 V/m, 24 h) resulted in increased expression of Cu/Zn superoxide dismutase and decreased intracellular concentration of reduced glutathione (GSH), whereas the expression of catalase remained unchanged. Pre-treatment with the free radical scavenger N-acetyl cysteine (NAC) and the superoxide dismutase mimetic EUK-8 abolished caspase-3 and PARP-1 induction, suggesting that apoptosis in oral mucosa cancer cells is initated by ROS generation in response to DC electrical field treatment.

  17. Androgen suppresses the proliferation of androgen receptor-positive castration-resistant prostate cancer cells via inhibition of Cdk2, CyclinA, and Skp2.

    PubMed

    Kokontis, John M; Lin, Hui-Ping; Jiang, Shih Sheng; Lin, Ching-Yu; Fukuchi, Junichi; Hiipakka, Richard A; Chung, Chi-Jung; Chan, Tzu-Min; Liao, Shutsung; Chang, Chung-Ho; Chuu, Chih-Pin

    2014-01-01

    The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27(Kip1); and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3AR cells partially blocked accumulation of p27(Kip1) and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.

  18. Induction of cell cycle arrest in prostate cancer cells by the dietary compound isoliquiritigenin.

    PubMed

    Lee, Yeo Myeong; Lim, Do Young; Choi, Hyun Ju; Jung, Jae In; Chung, Won-Yoon; Park, Jung Han Yoon

    2009-02-01

    Isoliquiritigenin (ISL), a flavonoid chalcone that is present in licorice, shallot, and bean sprouts, is known to have antitumorigenic activities. The present study examined whether ISL alters prostate cancer cell cycle progression. DU145 human and MatLyLu (MLL) rat prostate cancer cells were cultured with various concentrations of ISL. In both DU145 and MLL cells treated with ISL, the percentage of cells in the G1 phase increased, and the incorporation of [(3)H]thymidine decreased. ISL decreased the protein levels of cyclin D1, cyclin E, and cyclin-dependent kinase (CDK) 4, whereas cyclin A and CDK2 expressions were unaltered in cells treated with ISL. The expression of the CDK inhibitor p27(KIP1) was increased in cells treated with 20 micromol/L ISL. In addition, treatment of cells with 20 micromol/L ISL for 24 hours led to G2/M cell cycle arrest. Cell division control (CDC) 2 protein levels remained unchanged. The protein levels of phospho-CDC2 (Tyr15) and cyclin B1 were increased, and the CDC25C level was decreased by ISL dose-dependently. We demonstrate that ISL promotes cell cycle arrest in DU145 and MLL cells, thereby providing insights into the mechanisms underlying its antitumorigenic activities.

  19. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity.

    PubMed

    Tolba, Mai F; Abdel-Rahman, Sherif Z

    2015-01-01

    Although colorectal cancer (CRC) treatment with 5-fluorouracil (5-FU) is the first line of therapy for this debilitating disease, treatment effectiveness is often hampered by the development of drug resistance and toxicity at high doses. ER-β can play an important role in CRC development and possibly in its response to therapy. Pterostilbene (PT) possesses antioxidant and anticancer effects that are mediated by ER-β. In the current study, we test the hypothesis that PT sensitizes colon cancer cells to 5-FU and we examine the underlying mechanism(s) by which PT exerts its cytotoxic effects in CRC cells. Our data indicate that PT exhibited a more potent cytotoxic effect in Caco-2 compared to HCT-116 cells. PT/5-FU co-treatment was more effective in Caco-2 cells. Our data indicate that ER-β is expressed at higher levels in Caco-2 cells and its levels are further boosted with PT treatment. PT significantly suppressed Akt and ERK phosphorylations, and enhanced FOXO-1 and p27(kip1) levels in Caco-2 cells. PT also induced a significant increase in Caco-2 cells at pre-G phase coupled with increased Bax/Bcl-2 ratio and PARP cleavage. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing ER-β protein. PMID:26472352

  20. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity.

    PubMed

    Tolba, Mai F; Abdel-Rahman, Sherif Z

    2015-01-01

    Although colorectal cancer (CRC) treatment with 5-fluorouracil (5-FU) is the first line of therapy for this debilitating disease, treatment effectiveness is often hampered by the development of drug resistance and toxicity at high doses. ER-β can play an important role in CRC development and possibly in its response to therapy. Pterostilbene (PT) possesses antioxidant and anticancer effects that are mediated by ER-β. In the current study, we test the hypothesis that PT sensitizes colon cancer cells to 5-FU and we examine the underlying mechanism(s) by which PT exerts its cytotoxic effects in CRC cells. Our data indicate that PT exhibited a more potent cytotoxic effect in Caco-2 compared to HCT-116 cells. PT/5-FU co-treatment was more effective in Caco-2 cells. Our data indicate that ER-β is expressed at higher levels in Caco-2 cells and its levels are further boosted with PT treatment. PT significantly suppressed Akt and ERK phosphorylations, and enhanced FOXO-1 and p27(kip1) levels in Caco-2 cells. PT also induced a significant increase in Caco-2 cells at pre-G phase coupled with increased Bax/Bcl-2 ratio and PARP cleavage. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing ER-β protein.

  1. Induction of cell cycle arrest in prostate cancer cells by the dietary compound isoliquiritigenin.

    PubMed

    Lee, Yeo Myeong; Lim, Do Young; Choi, Hyun Ju; Jung, Jae In; Chung, Won-Yoon; Park, Jung Han Yoon

    2009-02-01

    Isoliquiritigenin (ISL), a flavonoid chalcone that is present in licorice, shallot, and bean sprouts, is known to have antitumorigenic activities. The present study examined whether ISL alters prostate cancer cell cycle progression. DU145 human and MatLyLu (MLL) rat prostate cancer cells were cultured with various concentrations of ISL. In both DU145 and MLL cells treated with ISL, the percentage of cells in the G1 phase increased, and the incorporation of [(3)H]thymidine decreased. ISL decreased the protein levels of cyclin D1, cyclin E, and cyclin-dependent kinase (CDK) 4, whereas cyclin A and CDK2 expressions were unaltered in cells treated with ISL. The expression of the CDK inhibitor p27(KIP1) was increased in cells treated with 20 micromol/L ISL. In addition, treatment of cells with 20 micromol/L ISL for 24 hours led to G2/M cell cycle arrest. Cell division control (CDC) 2 protein levels remained unchanged. The protein levels of phospho-CDC2 (Tyr15) and cyclin B1 were increased, and the CDC25C level was decreased by ISL dose-dependently. We demonstrate that ISL promotes cell cycle arrest in DU145 and MLL cells, thereby providing insights into the mechanisms underlying its antitumorigenic activities. PMID:19298190

  2. The enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, is regulated by miR-101 in renal cancer cells

    SciTech Connect

    Sakurai, Toshihiko; Bilim, Vladimir N.; Ugolkov, Andrey V.; Yuuki, Kaori; Tsukigi, Masaaki; Motoyama, Teiichi; Tomita, Yoshihiko

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer EZH2 is overexpressed in the nuclei of renal cancer cells. Black-Right-Pointing-Pointer Nuclear EZH2 is associated with advanced stage and worse survival of RCC patients. Black-Right-Pointing-Pointer EZH2 is negatively regulated by miR-101 in renal cancer cells. Black-Right-Pointing-Pointer Depletion of EZH2 leads to re-expression of p27Kip1. Black-Right-Pointing-Pointer Reintroduction of miR-101 results in suppression of cell proliferation. -- Abstract: We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.

  3. Lycopene and Beta-Carotene Induce Growth Inhibition and Proapoptotic Effects on ACTH-Secreting Pituitary Adenoma Cells

    PubMed Central

    Leite de Oliveira, Felipe; Soares, Nathália; de Mattos, Rômulo Medina; Hecht, Fábio; Dezonne, Rômulo Sperduto; Vairo, Leandro; Goldenberg, Regina Coeli dos Santos; Gomes, Flávia Carvalho Alcântara; de Carvalho, Denise Pires; Gadelha, Mônica R.; Nasciutti, Luiz Eurico; Miranda-Alves, Leandro

    2013-01-01

    Pituitary adenomas comprise approximately 10–15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27kip1 in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27kip1 in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27kip1; and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing’s disease. PMID:23667519

  4. CSN6 deregulation impairs genome integrity in a COP1-dependent pathway

    PubMed Central

    Choi, Hyun Ho; Su, Chun-Hui; Fang, Lekun; Zhang, Jin; Yeung, Sai-Ching J.; Lee, Mong-Hong

    2015-01-01

    Understanding genome integrity and DNA damage response are critical to cancer treatment. In this study, we identify CSN6's biological function in regulating genome integrity. Constitutive photomorphogenic 1 (COP1), an E3 ubiquitin ligase regulated by CSN6, is downregulated by DNA damage, but the biological consequences of this phenomenon are poorly understood. p27Kip1 is a critical CDK inhibitor involved in cell cycle regulation, but its response to DNA damage remains unclear. Here, we report that p27Kip1 levels are elevated after DNA damage, with concurrent reduction of COP1 levels. Mechanistic studies showed that during DNA damage response COP1's function as an E3 ligase of p27 is compromised, thereby reducing the ubiquitin-mediated degradation of p27Kip1. Also, COP1 overexpression leads to downregulation of p27Kip1, thereby promoting the expression of mitotic kinase Aurora A. Overexpression of Aurora A correlates with poor survival. These findings provide new insight into CSN6-COP1-p27Kip1-Aurora A axis in DNA damage repair and tumorigenesis. PMID:25957415

  5. Nuclear export of proteins and drug resistance in cancer.

    PubMed

    Turner, Joel G; Dawson, Jana; Sullivan, Daniel M

    2012-04-15

    The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, p21(CIP), p27(KIP1), N-WASP/FAK, estradiol receptor and Tob, drug targets topoisomerase I and IIα and BCR-ABL, and the molecular chaperone protein Hsp90. Here, we review in detail the current processes and known structures involved in the export of a protein through the nuclear pore complex. We also discuss the export receptor molecule CRM1 and its binding to the leucine-rich nuclear export signal of the cargo protein and the formation of a nuclear export trimer with RanGTP. The therapeutic potential of various CRM1 inhibitors will be addressed, including leptomycin B, ratjadone, KOS-2464, and specific small molecule inhibitors of CRM1, N-azolylacrylate analogs, FOXO export inhibitors, valtrate, acetoxychavicol acetate, CBS9106, and SINE inhibitors. We will also discuss examples of how drug resistance may be reversed by targeting the exported proteins topoisomerase IIα, BCR-ABL, and galectin-3. As effective and less toxic CRM1 export inhibitors become available, they may be used as both single agents and in combination with current chemotherapeutic drugs. We believe that the future development of low-toxicity, small-molecule CRM1 inhibitors may provide a new approach to treating cancer.

  6. Identification and functional analysis of 9p24 amplified genes in human breast cancer

    PubMed Central

    Wu, J; Liu, S; Liu, G; Dombkowski, A; Abrams, J; Martin-Trevino, R; Wicha, MS; Ethier, SP; Yang, Z-Q

    2013-01-01

    Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2 Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16INK4a, p21Waf1/Cip1 and p27Kip1. Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis. PMID:21666724

  7. The anticancer effects of hispolon on lung cancer cells.

    PubMed

    Wu, Qiuge; Kang, Yan; Zhang, Hui; Wang, Hongmin; Liu, Yuanhua; Wang, Jing

    2014-10-24

    Hispolon is isolated from Phellinus igniarius and exhibits antitumor activity. Here, we explored the effects of hispolon on the lung cancer A549 and H661 cells. Cells were incubated with various concentrations of hispolon (0, 5, 10, 20, 40, 80 or 160μM) for 12, 24, 48 or 72h. Cell viability was examined by MTT assay. Cell cycle and apoptosis assay were assessed by flow cytometry. Hispolon decreased cell viability in a dose- and time-dependent manner. The cell cycle distribution showed that hispolon enhanced the accumulations of the cells in G0/G1 phase. Mechanically, hispolon decreased the expression of G1-S transition-related proteins: Cyclin D1, cyclin E, CDK2, CDK4 and CDK6, but increased the expression of CDK inhibitor p21(CIP1) and p27(KIP1). Moreover, hispolon induced cell apoptosis through activation of the mitochondrial pathway, evidenced by the loss of mitochondrial membrane potential, the release of cytochrome c into cytosol, and the cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) in hispolon-treated cells. Additionally, hispolon enhanced the expression of p53, specific silencing of which almost completely reversed hispolon-mediated antitumor activity. Moreover, hispolon treatment was more effective on H661 cells than on A549 cells in inhibiting cell viability and inducing cell apoptosis. Our results indicate that hispolon inhibits the cell viability, induces G0/G1 cell cycle arrest and apoptosis in lung cancer cells and p53 plays a critical role in hispolon-mediated antitumor activity. PMID:25268766

  8. Src family kinase inhibitor PP2 efficiently inhibits cervical cancer cell proliferation through down-regulating phospho-Src-Y416 and phospho-EGFR-Y1173.

    PubMed

    Kong, Lu; Deng, Zhihong; Shen, Haiying; Zhang, Yuxiang

    2011-02-01

    Tyrosine (Y) kinases inhibitors have been approved for targeted treatment of cancer. However, their clinical use is limited to some cancers and the mechanism of their action remains unclear. Previous study has indicated that PP2, a selective inhibitor of the Src family of non-receptor tyrosine kinases (nRTK), efficiently repressed cervical cancer growth in vitro and in vivo. In this regard, our aims are to explore the mechanism of PP2 on cervical cancer cell growth inhibition by investigating the suppressive divergence among PP1, PP2, and a negative control compound PP3. MTT results showed that three compounds had different inhibitory effects on proliferation of two cervical cancer cells, HeLa and SiHa, and PP2 was most efficient in a time- and dose-dependent manner. Moreover, we found 10 μM PP2 down-regulated pSrc-Y416 (P < 0.05), pEGFR-Y845 (P < 0.05), and -Y1173 (P < 0.05) expression levels, while 10 μM PP1 down-regulated pSrc-Y416 (P < 0.05) and pEGFR-Y845 (P < 0.05), but not pEGFR-Y1173; 10 μM PP3 down-regulated only pEGFR-Y1173 (P < 0.05). PP2 could modulate cell cycle arrest by up-regulating p21(Cip1) and p27(Kip1) in both HeLa and SiHa cells and down-regulating expression of cyclin A, and cyclin dependent kinase-2, -4 (Cdk-2, -4) in HeLa and of cyclin B and Cdk-2 in SiHa. Our results indicate that Src pathway and EGFR pathway play different roles in the proliferation of cervical cancer cells and PP2 efficiently reduces cervical cancer cell proliferation by reduction of both Src and EGFR activity.

  9. Augmentation of Sodium Butyrate-induced Apoptosis by Phosphatidylinositol 3-kinase Inhibition in the Human Cervical Cancer Cell-line

    PubMed Central

    Park, Jung Kyu; Cho, Chi Heum; Ramachandran, Sabarish; Shin, So Jin; Kwon, Sang Hoon; Kwon, Sun Young

    2006-01-01

    Purpose Sodium butyrate (NaBT) is principally a histone deacetylase (HDAC) inhibitor, and it has the potential to arrest HPV-positive carcinoma cells at the G1 to S phase transition of the cell cycle. The aim of study was to determine whether phosphatidylinositol 3-kinase (PI3K) inhibition can enhance the inhibitory effect of NaBT on a human cervical cancer cell line (HeLa). Materials and Methods Cervical cancer cells (HeLa) were treated with NaBT alone or in combination with the PI3K inhibitors wortmannin or LY294002. Cell viability analysis and FACS analysis were carried out. The expressions of the cell cycle related proteins were evaluated by Western-blot analysis. Results Inhibition of PI3K enhanced NaBT-mediated apoptosis and this decreased the HeLa cell viability. Either wortmannin or LY294002, combined with NaBT, enhanced the activation of caspase 3 and caspase 9, and this enhanced the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Cervical cancer cells were arrested in the subG1 and G2/M phase, as was detected by FACS analysis. NaBT treatment in combination with PI3K inhibitors showed the increased expression of the CDK inhibitors p21Cip1/Waf1 and p27Kip1, in a p53 dependent manner, and also the increased dephosphorylation of Rb whereas there was a reduction in the expression levels of cyclin A, cyclin D1 and cyclin B1. Conclusion The results demonstrate that inhibition of PI3K enhances NaBT-mediated cervical cancer cell apoptosis through the activation of the caspase pathway. Moreover, these findings will support future investigation using the PI3K inhibitors in combination with adjuvant treatment for treating carcinoma of the cervix. PMID:19771269

  10. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

    PubMed

    Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

    2015-12-01

    Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

  11. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

    PubMed Central

    Parker, Andrew; Cross, Sally H.; Jackson, Ian J.; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R.; Brown, Steve D. M.

    2015-01-01

    ABSTRACT Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. PMID:26542706

  12. GNA13 as a prognostic factor and mediator of gastric cancer progression

    PubMed Central

    Chen, Jie-Wei; Weng, Hui-Wen; Zheng, Zou-San; Chen, Cui; Xie, Dan; Ye, Sheng

    2016-01-01

    Guanine nucleotide binding protein (G protein), alpha 13 (GNA13) has been implicated as an oncogenic protein in several human cancers. In this study, GNA13 was characterized for its role in gastric cancer (GC) progression and underlying molecular mechanisms. The expression dynamics of GNA13 were examined by immunohistochemistry (IHC) in two independent cohorts of GC samples. A series of in-vivo and in-vitro assays was performed to elucidate the function of GNA13 in GC and its underlying mechanisms. In both two cohorts of GC samples, we observed that GNA13 was markedly overexpressed in GC tissues and associated closely with aggressive magnitude of GC progression and poor patients' survival. Further study showed that upregulation of GNA13 expression increased the proliferation and tumorigenicity of GC cells in vitro and in vivo, by promoting cell growth rate, colony formation, and tumor formation in nude mice. By contrast, knockdown of GNA13 effectively suppressed the proliferation and tumorigenicity of GC cells in vitro and in vivo. Our results also demonstrated that the molecular mechanisms of the effect of GNA13 in GC included promotion of G1/S cell cycle transition through upregulation of c-Myc, activation of AKT and ERK activity, suppression of FOXO1 activity, upregulation of cyclin-dependent kinase (CDK) regulator cyclin D1 and downregulation of CDK inhibitor p21Cip1 and p27Kip1. Our present study illustrated that GNA13 has an important role in promoting proliferation and tumorigenicity of GC, and may represent a novel prognostic biomarker and therapeutic target for this disease. PMID:26735177

  13. Apoptosis induced by 7-difluoromethoxyl-5,4'-di-n-octyl genistein via the inactivation of FoxM1 in ovarian cancer cells.

    PubMed

    Ning, Yingxia; Li, Qingxiu; Xiang, Honglin; Liu, Fei; Cao, Jianguo

    2012-06-01

    Genistein, 5,7,4'-trihydroxylisoflavone, a major component of soybean products, has been reported to possess anticancer activities. We examined the antitumor effects of 7-difluoromethoxyl-5,4'-di-n-octylgenistein (DFOG), a novel synthetic genistein derivative, on human ovarian cancer cells as well as the molecular mechanism. The growth-inhibitory effects of genistein and DFOG were determined using MTT assay and clonogenic assay in CoC1 and SKOV3 human ovarian cancer cells. Apoptotic activities of DFOG were observed using histone/DNA ELISA assay and flow cytometry with propidium iodide (PI) staining. Multiple molecular techniques, such as RT-PCR, western blot analysis, siRNA and cDNA transfection were used to explore the molecular mechanism. We demonstrated that nine of the genistein derivatives had a more effective antitumor activity than genistein. Among the afore-mentioned derivatives, DFOG presented with the strongest activity against CoC1 and SKOV3 cells in vitro. DFOG and genistein inhibited the growth of CoC1 and SKOV3 cells, accompanied by cell cycle arrest in the G2/M phase. DFOG caused apoptotic cell death with concomitant attenuation of Forkhead box protein M1 (FoxM1) and its downstream genes, such as survivin, cdc25B, cyclin B, and increased p27KIP1. Downregulation of FoxM1 by siRNA followed by DFOG treatment resulted in enhanced cell growth inhibition and induction of apoptosis. Upregulation of FoxM1 by cDNA transfection attenuated DFOG-induced cell growth inhibition and apoptotic cell death. Our results show that the molecular role of FoxM1 in mediating the biological effects of DFOG and genistein in human ovarian cancer cells suggests that FoxM1 could be a novel target for the treatment of human ovarian cancer.

  14. Somatostatin, somatostatin analogs and somatostatin receptor dynamics in the biology of cancer progression.

    PubMed

    Ruscica, M; Arvigo, M; Steffani, L; Ferone, D; Magni, P

    2013-05-01

    The pharmacological effects (i.e., inhibition of endocrine secretion and cell proliferation) mediated by the hormone somatostatin (SRIF) are derived from its universal high-affinity binding to five different G proteincoupled receptors (GPCRs), named sst1-5. However, SRIF has a half-life of less than 3 min, whereas the available mono- and bi-specific SRIF preferential analogs show prolonged half-life and increased potency. These compounds may control tumor development, cell proliferation and metastatization by direct actions, including cell division arrest in G0/G1 phase (i.e., induction of cyclin-dependent kinase inhibitor p27(kip1) or p21(Cip1)), induction of apoptosis (i.e., induction of p53 and Bax) and suppression of cell invasion. Along with these direct actions on the biology of cancer progression, in vivo SRIF analogs may also regulate tumor growth through indirect actions, by suppressing the secretion of growth-promoting hormones and growth factors and angiogenesis. Interestingly, when ssts are co-expressed, they may interact forming homo- or heterodimers, also with other GPCRs such as type 2 dopamine receptor and the μ-opioid receptor 1, altering their original pharmacological and functional properties. Dimers can be not only constitutive, but perhaps also ligandpromoted: hence, compounds with high affinity for different ssts isoforms may be used to achieve effects elicited by specific dimers. Future developments in the knowledge of ssts dynamics upon SRIF and SRIF analogs binding in neoplastic tissues may allow the full elucidation of the pathophysiological role of this system and the exploitation of the therapeutic potential of its modulation.

  15. Piperine, an alkaloid from black pepper, inhibits growth of human colon cancer cells via G1 arrest and apoptosis triggered by endoplasmic reticulum stress.

    PubMed

    Yaffe, Paul B; Power Coombs, Melanie R; Doucette, Carolyn D; Walsh, Mark; Hoskin, David W

    2015-10-01

    Piperine, a piperidine alkaloid present in black pepper, inhibits the growth of cancer cells, although the mechanism of action is not well understood. In this study, we show that piperine (75-150 µM) inhibited the growth of several colon cancer cell lines but had little effect on the growth of normal fibroblasts and epithelial cells. Piperine inhibited HT-29 colon carcinoma cell proliferation by causing G1 phase cell cycle arrest that was associated with decreased expression of cyclins D1 and D3 and their activating partner cyclin-dependent kinases 4 and 6, as well as reduced phosphorylation of the retinoblastoma protein and up-regulation of p21/WAF1 and p27/KIP1 expression. In addition, piperine caused hydroxyl radical production and apoptosis that was partially dependent on the production of reactive oxygen species. Piperine-treated HT-29 cells showed loss of mitochondrial membrane integrity and cleavage of poly (ADP-ribose) polymerase-1, as well as caspase activation and reduced apoptosis in the presence of the pan-caspase inhibitor zVAD-FMK. Increased expression of the endoplasmic reticulum stress-associated proteins inositol-requiring 1α protein, C/EBP homologous protein, and binding immunoglobulin protein, and activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, as well as decreased phosphorylation of Akt and reduced survivin expression were also observed in piperine-treated HT-29 cells. Furthermore, piperine inhibited colony formation by HT-29 cells, as well as the growth of HT-29 spheroids. Cell cycle arrest and endoplasmic reticulum stress-associated apoptosis following piperine treatment of HT-29 cells provides the first evidence that piperine may be useful in the treatment of colon cancer.

  16. The Topoisomerase 1 Inhibitor Austrobailignan-1 Isolated from Koelreuteria henryi Induces a G2/M-Phase Arrest and Cell Death Independently of p53 in Non-Small Cell Lung Cancer Cells

    PubMed Central

    Yang, Tsung-Ying; Li, Ya-Ling; Wen, Chi-Luan; Hsu, Shih-Lan; Chen, Tzu-Hsiu

    2015-01-01

    Koelreuteria henryi Dummer, an endemic plant of Taiwan, has been used as a folk medicine for the treatment of hepatitis, enteritis, cough, pharyngitis, allergy, hypertension, hyperlipidemia, and cancer. Austrobailignan-1, a natural lignan derivative isolated from Koelreuteria henryi Dummer, has anti-oxidative and anti-cancer properties. However, the effects of austrobailignan-1 on human cancer cells have not been studied yet. Here, we showed that austrobailignan-1 inhibited cell growth of human non-small cell lung cancer A549 and H1299 cell lines in both dose- and time-dependent manners, the IC50 value (48 h) of austrobailignan-1 were 41 and 22 nM, respectively. Data from flow cytometric analysis indicated that treatment with austrobailignan-1 for 24 h retarded the cell cycle at the G2/M phase. The molecular event of austrobailignan-1-mediated G2/M phase arrest was associated with the increase of p21Waf1/Cip1 and p27Kip1 expression, and decrease of Cdc25C expression. Moreover, treatment with 100 nM austrobailignan-1 for 48 h resulted in a pronounced release of cytochrome c followed by the activation of caspase-2, -3, and -9, and consequently induced apoptosis. These events were accompanied by the increase of PUMA and Bax, and the decrease of Mcl-1 and Bcl-2. Furthermore, our study also showed that austrobailignan-1 was a topoisomerase 1 inhibitor, as evidenced by a relaxation assay and induction of a DNA damage response signaling pathway, including ATM, and Chk1, Chk2, γH2AX phosphorylated activation. Overall, our results suggest that austrobailignan-1 is a novel DNA damaging agent and displays a topoisomerase I inhibitory activity, causes DNA strand breaks, and consequently induces DNA damage response signaling for cell cycle G2/M arrest and apoptosis in a p53 independent manner. PMID:26147394

  17. The Topoisomerase 1 Inhibitor Austrobailignan-1 Isolated from Koelreuteria henryi Induces a G2/M-Phase Arrest and Cell Death Independently of p53 in Non-Small Cell Lung Cancer Cells.

    PubMed

    Wu, Chun-Chi; Huang, Keh-Feng; Yang, Tsung-Ying; Li, Ya-Ling; Wen, Chi-Luan; Hsu, Shih-Lan; Chen, Tzu-Hsiu

    2015-01-01

    Koelreuteria henryi Dummer, an endemic plant of Taiwan, has been used as a folk medicine for the treatment of hepatitis, enteritis, cough, pharyngitis, allergy, hypertension, hyperlipidemia, and cancer. Austrobailignan-1, a natural lignan derivative isolated from Koelreuteria henryi Dummer, has anti-oxidative and anti-cancer properties. However, the effects of austrobailignan-1 on human cancer cells have not been studied yet. Here, we showed that austrobailignan-1 inhibited cell growth of human non-small cell lung cancer A549 and H1299 cell lines in both dose- and time-dependent manners, the IC50 value (48 h) of austrobailignan-1 were 41 and 22 nM, respectively. Data from flow cytometric analysis indicated that treatment with austrobailignan-1 for 24 h retarded the cell cycle at the G2/M phase. The molecular event of austrobailignan-1-mediated G2/M phase arrest was associated with the increase of p21Waf1/Cip1 and p27Kip1 expression, and decrease of Cdc25C expression. Moreover, treatment with 100 nM austrobailignan-1 for 48 h resulted in a pronounced release of cytochrome c followed by the activation of caspase-2, -3, and -9, and consequently induced apoptosis. These events were accompanied by the increase of PUMA and Bax, and the decrease of Mcl-1 and Bcl-2. Furthermore, our study also showed that austrobailignan-1 was a topoisomerase 1 inhibitor, as evidenced by a relaxation assay and induction of a DNA damage response signaling pathway, including ATM, and Chk1, Chk2, γH2AX phosphorylated activation. Overall, our results suggest that austrobailignan-1 is a novel DNA damaging agent and displays a topoisomerase I inhibitory activity, causes DNA strand breaks, and consequently induces DNA damage response signaling for cell cycle G2/M arrest and apoptosis in a p53 independent manner. PMID:26147394

  18. Vitamin E δ-tocotrienol prolongs survival in the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) transgenic mouse model of pancreatic cancer.

    PubMed

    Husain, Kazim; Centeno, Barbara A; Chen, Dung-Tsa; Hingorani, Sunil R; Sebti, Said M; Malafa, Mokenge P

    2013-10-01

    Previous work has shown that vitamin E δ-tocotrienol (VEDT) prolongs survival and delays progression of pancreatic cancer in the LSL-Kras(G12D)(/+);Pdx-1-Cre mouse model of pancreatic cancer. However, the effect of VEDT alone or in combination with gemcitabine in the more aggressive LSL-Kras(G12D)(/+);LSL-Trp53(R172H)(/+);Pdx-1-Cre (KPC) mouse model is unknown. Here, we studied the effects of VEDT and the combination of VEDT and gemcitabine in the KPC mice. KPC mice were randomized into four groups: (i) vehicle [olive oil, 1.0 mL/kg per os twice a day and PBS 1.0 mL/kg intrapertoneally (i.p.) twice a week], (ii) gemcitabine (100 mg/kg i.p. twice a week), (iii) VEDT (200 mg/kg per os twice a day), and (iv) gemcitabine + VEDT. Mice received treatment until they displayed symptoms of impending death from pancreatic cancer, at which point animals were euthanized. At 16 weeks, survival was 10% in the vehicle group, 30% in the gemcitabine group, 70% in the VEDT group (P < 0.01), and 90% in the VEDT combined with gemcitabine group (P < 0.05). VEDT alone and combined with gemcitabine resulted in reversal of epithelial-to-mesenchymal transition in tumors. Biomarkers of apoptosis (plasma CK18), PARP1 cleavage, and Bax expression were more greatly induced in tumors subjected to combined treatment versus individual treatment. Combined treatment induced cell-cycle inhibitors (p27(Kip1) and p21(Cip1)) and inhibited VEGF, vascularity (CD31), and oncogenic signaling (pAKT, pMEK, and pERK) greater than individual drugs. No significant differences in body weight gain between drug treatment and control mice were observed. These results strongly support further investigation of VEDT alone and in combination with gemcitabine for pancreatic cancer prevention and treatment.

  19. Chloroquine potentiates the anti-cancer effect of 5-fluorouracil on colon cancer cells

    PubMed Central

    2010-01-01

    Background Chloroquine (CQ), the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth and/or to induce cell death in various types of cancer. 5-Fluorouracil (5-FU) is the chemotherapeutic agent of first choice in colorectal cancer, but in most cases, resistance to 5-FU develops through various mechanisms. Here, we focused on the combination of CQ as a mechanism to potentiate the inhibitory effect of 5-FU on human colon cancer cells. Methods HT-29 cells were treated with CQ and/or 5-FU, and their proliferative ability, apoptosis and autophagy induction effects, and the affection of the cell cycle were evaluated. The proliferative ability of HT-29 was analyzed by the MTS assay. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. The cell cycle was evaluated by flow-cytometry after staining of cells with PI. Autophagy was quantified by flow-cytometry and Western blot analysis. Finally, to evaluate the fate of the cells treated with CQ and/or 5-FU, the colony formation assay was performed. Results 5-FU inhibited the proliferative activity of HT-29 cells, which was mostly dependent on the arrest of the cells to the G0/G1-phase but also partially on apoptosis induction, and the effect was potentiated by CQ pre-treatment. The potentiation of the inhibitory effect of 5-FU by CQ was dependent on the increase of p21Cip1 and p27Kip1 and the decrease of CDK2. Since CQ is reported to inhibit autophagy, the catabolic process necessary for cell survival under conditions of cell starvation or stress, which is induced by cancer cells as a protective mechanism against chemotherapeutic agents, we also analyzed the induction of autophagy in HT-29. HT-29 induced autophagy in response to 5-FU, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer

  20. Galbanic acid decreases androgen receptor abundance and signaling and induces G1 arrest in prostate cancer cells

    PubMed Central

    Zhang, Yong; Kim, Kwan-Hyun; Zhang, Wei; Guo, Yinglu; Kim, Sung-Hoon; Lü, Junxuan

    2011-01-01

    Androgen receptor (AR) signaling is crucial for the genesis and progression of prostate cancer (PCa). We compared the growth responses of AR(+) LNCaP and LNCaP C4-2 vs. AR(−) DU145 and PC-3 PCa cell lines to galbanic acid (GBA) isolated from the resin of medicinal herb Ferula assafoetida and assessed their connection to AR signaling and cell cycle regulatory pathways. Our results showed that GBA preferentially suppressed AR(+) PCa cell growth than AR(−) PCa cells. GBA induced a caspase-mediated apoptosis that was attenuated by a general caspase inhibitor. Subapoptotic GBA down-regulated AR protein in LNCaP cells primarily through promoting its proteasomal degradation, and inhibited AR-dependent transcription without affecting AR nuclear translocation. Whereas docking simulations predicted binding of GBA to the AR ligand binding domain with similarities and differences with the AR antagonist drug bicalutamide, LNCaP cell culture assays did not detect agonist activity of GBA. GBA and bicalutamide exerted greater than additive inhibitory effect on cell growth when used together. Subapoptotic GBA induced G1 arrest associated with an inhibition of cyclin/CDK4/6 pathway, especially cyclin D1 without the causal involvement of CDK inhibitory proteins P21Cip1 and P27Kip1. In summary, the novelty of GBA as an anti-AR compound resides in the distinction between GBA and bicalutamide with respect to AR protein turnover and a lack of agonist effect. Our observations of anti-AR and cell cycle arrest actions plus the anti-angiogenesis effect reported elsewhere suggest GBA as a multi-targeting drug candidate for the prevention and therapy of PCa. PMID:21328348

  1. Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

    PubMed Central

    Bian, Benjamin; Mongrain, Sébastien; Cagnol, Sébastien; Langlois, Marie‐Josée; Boulanger, Jim; Bernatchez, Gérald; Carrier, Julie C.; Boudreau, François

    2015-01-01

    Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non‐permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27Kip1 were observed in cathepsin B‐deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27Kip1 within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy. © 2015 The Authors. Molecular Carcinogenesis, published by Wiley Periodicals, Inc. PMID:25808857

  2. Phosphoproteome Integration Reveals Patient-Specific Networks in Prostate Cancer.

    PubMed

    Drake, Justin M; Paull, Evan O; Graham, Nicholas A; Lee, John K; Smith, Bryan A; Titz, Bjoern; Stoyanova, Tanya; Faltermeier, Claire M; Uzunangelov, Vladislav; Carlin, Daniel E; Fleming, Daniel Teo; Wong, Christopher K; Newton, Yulia; Sudha, Sud; Vashisht, Ajay A; Huang, Jiaoti; Wohlschlegel, James A; Graeber, Thomas G; Witte, Owen N; Stuart, Joshua M

    2016-08-11

    We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients. PMID:27499020

  3. A shoulder-hand syndrome revealing a lung cancer.

    PubMed

    Akasbi, N; Elidrissi, M; Tahiri, L; Shimi, M; Kadi, N; Elouazzani, F Z; Elmrini, A; Harzy, T

    2013-02-01

    Shoulder-hand syndrome is a reflex sympathetic dystrophy which is usually associated with minor trauma, fracture or surgical procedures on bones, or follows peripheral nerve injury. In the present report, we describe a patient who developed sympathetic dystrophy which revealed a lung cancer. Reflex sympathetic dystrophy, therefore, should be considered an occasional manifestation of a paraneoplastic syndrome warranting a thorough search for underlying malignancy.

  4. Sparse expression bases in cancer reveal tumor drivers.

    PubMed

    Logsdon, Benjamin A; Gentles, Andrew J; Miller, Chris P; Blau, C Anthony; Becker, Pamela S; Lee, Su-In

    2015-02-18

    We define a new category of candidate tumor drivers in cancer genome evolution: 'selected expression regulators' (SERs)-genes driving dysregulated transcriptional programs in cancer evolution. The SERs are identified from genome-wide tumor expression data with a novel method, namely SPARROW ( SPAR: se selected exp R: essi O: n regulators identified W: ith penalized regression). SPARROW uncovers a previously unknown connection between cancer expression variation and driver events, by using a novel sparse regression technique. Our results indicate that SPARROW is a powerful complementary approach to identify candidate genes containing driver events that are hard to detect from sequence data, due to a large number of passenger mutations and lack of comprehensive sequence information from a sufficiently large number of samples. SERs identified by SPARROW reveal known driver mutations in multiple human cancers, along with known cancer-associated processes and survival-associated genes, better than popular methods for inferring gene expression networks. We demonstrate that when applied to acute myeloid leukemia expression data, SPARROW identifies an apoptotic biomarker (PYCARD) for an investigational drug obatoclax. The PYCARD and obatoclax association is validated in 30 AML patient samples.

  5. Sparse expression bases in cancer reveal tumor drivers

    PubMed Central

    Logsdon, Benjamin A.; Gentles, Andrew J.; Miller, Chris P.; Blau, C. Anthony; Becker, Pamela S.; Lee, Su-In

    2015-01-01

    We define a new category of candidate tumor drivers in cancer genome evolution: ‘selected expression regulators’ (SERs)—genes driving dysregulated transcriptional programs in cancer evolution. The SERs are identified from genome-wide tumor expression data with a novel method, namely SPARROW (SPARse selected expRessiOn regulators identified With penalized regression). SPARROW uncovers a previously unknown connection between cancer expression variation and driver events, by using a novel sparse regression technique. Our results indicate that SPARROW is a powerful complementary approach to identify candidate genes containing driver events that are hard to detect from sequence data, due to a large number of passenger mutations and lack of comprehensive sequence information from a sufficiently large number of samples. SERs identified by SPARROW reveal known driver mutations in multiple human cancers, along with known cancer-associated processes and survival-associated genes, better than popular methods for inferring gene expression networks. We demonstrate that when applied to acute myeloid leukemia expression data, SPARROW identifies an apoptotic biomarker (PYCARD) for an investigational drug obatoclax. The PYCARD and obatoclax association is validated in 30 AML patient samples. PMID:25583238

  6. Most small bowel cancers are revealed by a complication

    PubMed Central

    Negoi, Ionut; Paun, Sorin; Hostiuc, Sorin; Stoica, Bodgan; Tanase, Ioan; Negoi, Ruxandra Irina; Beuran, Mircea

    2015-01-01

    ABSTRACT Objective To characterize the pattern of primary small bowel cancers in a tertiary East-European hospital. Methods A retrospective study of patients with small bowel cancers admitted to a tertiary emergency center, over the past 15 years. Results There were 57 patients with small bowel cancer, representing 0.039% of admissions and 0.059% of laparotomies. There were 37 (64.9%) men, mean age of 58 years; and 72 years for females. Out of 57 patients, 48 (84.2%) were admitted due to an emergency situation: obstruction in 21 (38.9%), perforation in 17 (31.5%), upper gastrointestinal bleeding in 8 (14.8%), and lower gastrointestinal bleeding in 2 (3.7%). There were 10 (17.5%) duodenal tumors, 21 (36.8%) jejunal tumors and 26 (45.6%) ileal tumors. The most frequent neoplasms were gastrointestinal stromal tumor in 24 patients (42.1%), adenocarcinoma in 19 (33.3%), lymphoma in 8 (14%), and carcinoids in 2 (3.5%). The prevalence of duodenal adenocarcinoma was 14.55 times greater than that of the small bowel, and the prevalence of duodenal stromal tumors was 1.818 time greater than that of the small bowel. Obstruction was the complication in adenocarcinoma in 57.9% of cases, and perforation was the major local complication (47.8%) in stromal tumors. Conclusion Primary small bowel cancers are usually diagnosed at advanced stages, and revealed by a local complication of the tumor. Their surgical management in emergency setting is associated to significant morbidity and mortality rates. PMID:26676271

  7. miR-3646 promotes cell proliferation, migration, and invasion via regulating G2/M transition in human breast cancer cells

    PubMed Central

    Tao, Shuang; Liu, Yao-Bang; Zhou, Zhi-Wei; Lian, Bin; Li, Hong; Li, Jin-Ping; Zhou, Shu-Feng

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that are often located in genomic breakpoint regions and play a critical role in regulating a variety of the cellular processes in human cancer. miR-3646 has been reported to take part in tumorigenic progression in breast and bladder cancer, but its potential functions and exact mechanistic roles in breast cancer are still unclear. The objective of this study was to investigate the role of miR-3646 in breast cancer growth and metastasis using both bioinformatic and experimental approaches. Before starting the bench work, we conducted a bioinformatic study to predict the target genes regulated by miR-3646 using a panel of different algorithms. The results showed that miR-3646 might regulate a large number of genes that are related to cell growth, proliferation, metabolis, transport, and apoptosis and some were cancer-related genes. We found that the expression level of miR-3646 was significantly upregulated in breast cancer cells and tissues compared with normal breast cells and no tumor tissues. Subsequently, the MTT and colony formation assay results showed that up-regulation of miR-3646 promoted the cell viability and proliferation. Our results also showed that down-regulation of miR-3646 arrested the cells in G2/M phase in MCF7 and MDA-MB-231 cells which was accompanied by the down-regulation of CDK1/CDC2 and cyclin B1 and upregulation of p21Waf1/Cip1, p27 Kip1, and p53, suggesting that down-regulation of miR-3646 induces G2/M arrest through activation of the p53/p21/CDC2/cyclin B1 pathway. In addition, overexpression of miR-3646 promoted migration and invasion of MCF7 and MDA-MB-231 cells. Taken together, miR-3646 is a potential oncogene in breast cancer and it may represent a new niomarker in the diagnosis and prediction of prognosis and therapeutic response. PMID:27186291

  8. Visual gene-network analysis reveals the cancer gene co-expression in human endometrial cancer

    PubMed Central

    2014-01-01

    Background Endometrial cancers (ECs) are the most common form of gynecologic malignancy. Recent studies have reported that ECs reveal distinct markers for molecular pathogenesis, which in turn is linked to the various histological types of ECs. To understand further the molecular events contributing to ECs and endometrial tumorigenesis in general, a more precise identification of cancer-associated molecules and signaling networks would be useful for the detection and monitoring of malignancy, improving clinical cancer therapy, and personalization of treatments. Results ECs-specific gene co-expression networks were constructed by differential expression analysis and weighted gene co-expression network analysis (WGCNA). Important pathways and putative cancer hub genes contribution to tumorigenesis of ECs were identified. An elastic-net regularized classification model was built using the cancer hub gene signatures to predict the phenotypic characteristics of ECs. The 19 cancer hub gene signatures had high predictive power to distinguish among three key principal features of ECs: grade, type, and stage. Intriguingly, these hub gene networks seem to contribute to ECs progression and malignancy via cell-cycle regulation, antigen processing and the citric acid (TCA) cycle. Conclusions The results of this study provide a powerful biomarker discovery platform to better understand the progression of ECs and to uncover potential therapeutic targets in the treatment of ECs. This information might lead to improved monitoring of ECs and resulting improvement of treatment of ECs, the 4th most common of cancer in women. PMID:24758163

  9. Comparative genomic analysis reveals bilateral breast cancers are genetically independent.

    PubMed

    Song, Fangfang; Li, Xiangchun; Song, Fengju; Zhao, Yanrui; Li, Haixin; Zheng, Hong; Gao, Zhibo; Wang, Jun; Zhang, Wei; Chen, Kexin

    2015-10-13

    Bilateral breast cancer (BBC) poses a major challenge for oncologists because of the cryptic relationship between the two lesions. The purpose of this study was to determine the origin of the contralateral breast cancer (either dependent or independent of the index tumor). Here, we used ultra-deep whole-exome sequencing and array comparative genomic hybridization (aCGH) to study four paired samples of BBCs with different tumor subtypes and time intervals between the developments of each tumor. We used two paired primary breast tumors and corresponding metastatic liver lesions as the control. We tested the origin independent nature of BBC in three ways: mutational concordance, mutational signature clustering, and clonality analysis using copy number profiles. We found that the paired BBC samples had near-zero concordant mutation rates, which were much lower than those of the paired primary/metastasis samples. The results of a mutational signature analysis also suggested that BBCs are independent of one another. A clonality analysis using aCGH data further revealed that paired BBC samples was clonally independent, in contrast to clonal related origin found for paired primary/metastasis samples. Our preliminary findings show that BBCs in Han Chinese women are origin independent and thus should be treated separately. PMID:26378809

  10. Tumor and host factors that may limit efficacy of chemotherapy in non-small cell and small cell lung cancer.

    PubMed

    Stewart, David J

    2010-09-01

    and expression of GST-pi, RB or p27kip1 may possibly confer resistance. While limited clinical data suggest that p53 mutations are associated with resistance to platinum-based therapies in NSCLC, data on p53 IHC positivity are equivocal. To date, resistance-modulating strategies have generally not proven clinically useful in lung cancer, although small randomized trials suggest a modest benefit of verapamil and related agents in NSCLC.

  11. TUMOR AND HOST FACTORS THAT MAY LIMIT EFFICACY OF CHEMOTHERAPY IN NON-SMALL CELL AND SMALL CELL LUNG CANCER

    PubMed Central

    Stewart, David J.

    2010-01-01

    expression of GST-pi, RB or p27kip1 may possibly confer resistance. While limited clinical data suggest that p53 mutations are associated with resistance to platinum-based therapies in NSCLC, data on p53 IHC positivity are equivocal. To date, resistance-modulating strategies have generally not proven clinically useful in lung cancer, although small randomized trials suggest a modest benefit of verapamil and related agents in NSCLC. PMID:20047843

  12. Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells

    PubMed Central

    Wang, Feng; Li, Hai; Yan, Xiao-Gang; Zhou, Zhi-Wei; Yi, Zhi-Gang; He, Zhi-Xu; Pan, Shu-Ting; Yang, Yin-Xue; Wang, Zuo-Zheng; Zhang, Xueji; Yang, Tianxing; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Pancreatic cancer is the most aggressive cancer worldwide with poor response to current therapeutics. Alisertib (ALS), a potent and selective Aurora kinase A inhibitor, exhibits potent anticancer effects in preclinical and clinical studies; however, the effect and underlying mechanism of ALS in the pancreatic cancer treatment remain elusive. This study aimed to examine the effects of ALS on cell growth, autophagy, and epithelial-to-mesenchymal transition (EMT) and to delineate the possible molecular mechanisms in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that ALS exerted potent cell growth inhibitory, pro-autophagic, and EMT-suppressing effects in PANC-1 and BxPC-3 cells. ALS remarkably arrested PANC-1 and BxPC-3 cells in G2/M phase via regulating the expression of cyclin-dependent kinases 1 and 2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. ALS concentration-dependently induced autophagy in PANC-1 and BxPC-3 cells, which may be attributed to the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinases 1 and 2 (Erk1/2) but activation of 5′-AMP-dependent kinase signaling pathways. ALS significantly inhibited EMT in PANC-1 and BxPC-3 cells with an increase in the expression of E-cadherin and a decrease in N-cadherin. In addition, ALS suppressed the expression of sirtuin 1 (Sirt1) and pre-B cell colony-enhancing factor/visfatin in both cell lines with a rise in the level of acetylated p53. These findings show that ALS induces cell cycle arrest and promotes autophagic cell death but inhibits EMT in pancreatic cancer cells with the involvement of PI3K/Akt/mTOR, p38 MAPK, Erk1/2, and Sirt1-mediated signaling pathways. Taken together, ALS may represent a promising anticancer drug for pancreatic cancer treatment. More studies are warranted to investigate other molecular targets and

  13. Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells.

    PubMed

    Wang, Feng; Li, Hai; Yan, Xiao-Gang; Zhou, Zhi-Wei; Yi, Zhi-Gang; He, Zhi-Xu; Pan, Shu-Ting; Yang, Yin-Xue; Wang, Zuo-Zheng; Zhang, Xueji; Yang, Tianxing; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Pancreatic cancer is the most aggressive cancer worldwide with poor response to current therapeutics. Alisertib (ALS), a potent and selective Aurora kinase A inhibitor, exhibits potent anticancer effects in preclinical and clinical studies; however, the effect and underlying mechanism of ALS in the pancreatic cancer treatment remain elusive. This study aimed to examine the effects of ALS on cell growth, autophagy, and epithelial-to-mesenchymal transition (EMT) and to delineate the possible molecular mechanisms in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that ALS exerted potent cell growth inhibitory, pro-autophagic, and EMT-suppressing effects in PANC-1 and BxPC-3 cells. ALS remarkably arrested PANC-1 and BxPC-3 cells in G2/M phase via regulating the expression of cyclin-dependent kinases 1 and 2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. ALS concentration-dependently induced autophagy in PANC-1 and BxPC-3 cells, which may be attributed to the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinases 1 and 2 (Erk1/2) but activation of 5'-AMP-dependent kinase signaling pathways. ALS significantly inhibited EMT in PANC-1 and BxPC-3 cells with an increase in the expression of E-cadherin and a decrease in N-cadherin. In addition, ALS suppressed the expression of sirtuin 1 (Sirt1) and pre-B cell colony-enhancing factor/visfatin in both cell lines with a rise in the level of acetylated p53. These findings show that ALS induces cell cycle arrest and promotes autophagic cell death but inhibits EMT in pancreatic cancer cells with the involvement of PI3K/Akt/mTOR, p38 MAPK, Erk1/2, and Sirt1-mediated signaling pathways. Taken together, ALS may represent a promising anticancer drug for pancreatic cancer treatment. More studies are warranted to investigate other molecular targets and

  14. Analysis of cancer genomes reveals basic features of human aging and its role in cancer development

    PubMed Central

    Podolskiy, Dmitriy I.; Lobanov, Alexei V.; Kryukov, Gregory V.; Gladyshev, Vadim N.

    2016-01-01

    Somatic mutations have long been implicated in aging and disease, but their impact on fitness and function is difficult to assess. Here by analysing human cancer genomes we identify mutational patterns associated with aging. Our analyses suggest that age-associated mutation load and burden double approximately every 8 years, similar to the all-cause mortality doubling time. This analysis further reveals variance in the rate of aging among different human tissues, for example, slightly accelerated aging of the reproductive system. Age-adjusted mutation load and burden correlate with the corresponding cancer incidence and precede it on average by 15 years, pointing to pre-clinical cancer development times. Behaviour of mutation load also exhibits gender differences and late-life reversals, explaining some gender-specific and late-life patterns in cancer incidence rates. Overall, this study characterizes some features of human aging and offers a mechanism for age being a risk factor for the onset of cancer. PMID:27515585

  15. Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

    PubMed Central

    Lin, I-Hsuan; Chen, Dow-Tien; Chang, Yi-Feng; Lee, Yu-Ling; Su, Chia-Hsin; Cheng, Ching; Tsai, Yi-Chien; Ng, Swee-Chuan; Chen, Hsiao-Tan; Lee, Mei-Chen; Chen, Hong-Wei; Suen, Shih-Hui; Chen, Yu-Cheng; Liu, Tze-Tze; Chang, Chuan-Hsiung; Hsu, Ming-Ta

    2015-01-01

    Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation. PMID:25706888

  16. Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells

    PubMed Central

    Wang, Feng; Wang, Qi; Zhou, Zhi-Wei; Yu, Song-Ning; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Yin-Xue; Yang, Tianxing; Sun, Tao; Li, Min; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of

  17. Subclonal diversification of primary breast cancer revealed by multiregion sequencing

    PubMed Central

    Yates, Lucy R; Gerstung, Moritz; Knappskog, Stian; Desmedt, Christine; Gundem, Gunes; Loo, Peter Van; Aas, Turid; Alexandrov, Ludmil B; Larsimont, Denis; Davies, Helen; Li, Yilong; Ju, Young Seok; Ramakrishna, Manasa; Haugland, Hans Kristian; Lilleng, Peer Kaare; Nik-Zainal, Serena; McLaren, Stuart; Butler, Adam; Martin, Sancha; Glodzik, Dominic; Menzies, Andrew; Raine, Keiran; Hinton, Jonathan; Jones, David; Mudie, Laura J; Jiang, Bing; Vincent, Delphine; Greene-Colozzi, April; Adnet, Pierre-Yves; Fatima, Aquila; Maetens, Marion; Ignatiadis, Michail; Stratton, Michael R; Sotiriou, Christos; Richardson, Andrea L; Lønning, Per Eystein; Wedge, David C; Campbell, Peter J

    2015-01-01

    Sequencing cancer genomes may enable tailoring of therapeutics to the underlying biological abnormalities driving a particular patient’s tumor. However, sequencing-based strategies rely heavily on representative sampling of tumors. To understand the subclonal structure of primary breast cancer, we applied whole genome and targeted sequencing to multiple samples from each of 50 patients’ tumors (total 303). The extent of subclonal diversification varied among cases and followed spatial patterns. No strict temporal order was evident, with point mutations and rearrangements affecting the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumors and late in others. In 13/50 cancers, potentially targetable mutations were subclonal. Landmarks of disease progression, such as resisting chemotherapy and acquiring invasive or metastatic potential, arose within detectable subclones of antecedent lesions. These findings highlight the importance of including analyses of subclonal structure and tumor evolution in clinical trials of primary breast cancer. PMID:26099045

  18. The investigational Aurora kinase A inhibitor alisertib (MLN8237) induces cell cycle G2/M arrest, apoptosis, and autophagy via p38 MAPK and Akt/mTOR signaling pathways in human breast cancer cells

    PubMed Central

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Chen, Xiao-Wu; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Alisertib (ALS) is an investigational potent Aurora A kinase inhibitor currently undergoing clinical trials for the treatment of hematological and non-hematological malignancies. However, its antitumor activity has not been tested in human breast cancer. This study aimed to investigate the effect of ALS on the growth, apoptosis, and autophagy, and the underlying mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. In the current study, we identified that ALS had potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects in MCF7 and MDA-MB-231 cells. ALS arrested the cells in G2/M phase in MCF7 and MDA-MB-231 cells which was accompanied by the downregulation of cyclin-dependent kinase (CDK)1/cell division cycle (CDC) 2, CDK2, and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53, suggesting that ALS induces G2/M arrest through modulation of p53/p21/CDC2/cyclin B1 pathways. ALS induced mitochondria-mediated apoptosis in MCF7 and MDA-MB-231 cells; ALS significantly decreased the expression of B-cell lymphoma 2 (Bcl-2), but increased the expression of B-cell lymphoma 2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and increased the expression of cleaved caspases 3 and 9. ALS significantly increased the expression level of membrane-bound microtubule-associated protein 1 light chain 3 (LC3)-II and beclin 1 and induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways in MCF7 and MDA-MB-231 cells as indicated by their altered phosphorylation, contributing to the pro-autophagic activities of ALS. Furthermore, treatment with wortmannin markedly downregulated ALS-induced p38 MAPK activation and LC3 conversion. In addition, knockdown of the p38 MAPK gene by ribonucleic acid interference upregulated Akt activation and resulted in LC3-II accumulation. These findings indicate that ALS promotes cellular

  19. An integrative and comparative study of pan-cancer transcriptomes reveals distinct cancer common and specific signatures

    PubMed Central

    Cao, Zhen; Zhang, Shihua

    2016-01-01

    To investigate the commonalities and specificities across tumor lineages, we perform a systematic pan-cancer transcriptomic study across 6744 specimens. We find six pan-cancer subnetwork signatures which relate to cell cycle, immune response, Sp1 regulation, collagen, muscle system and angiogenesis. Moreover, four pan-cancer subnetwork signatures demonstrate strong prognostic potential. We also characterize 16 cancer type-specific subnetwork signatures which show diverse implications to somatic mutations, somatic copy number aberrations, DNA methylation alterations and clinical outcomes. Furthermore, some of them are strongly correlated with histological or molecular subtypes, indicating their implications with tumor heterogeneity. In summary, we systematically explore the pan-cancer common and cancer type-specific gene subnetwork signatures across multiple cancers, and reveal distinct commonalities and specificities among cancers at transcriptomic level. PMID:27633916

  20. An integrative and comparative study of pan-cancer transcriptomes reveals distinct cancer common and specific signatures.

    PubMed

    Cao, Zhen; Zhang, Shihua

    2016-01-01

    To investigate the commonalities and specificities across tumor lineages, we perform a systematic pan-cancer transcriptomic study across 6744 specimens. We find six pan-cancer subnetwork signatures which relate to cell cycle, immune response, Sp1 regulation, collagen, muscle system and angiogenesis. Moreover, four pan-cancer subnetwork signatures demonstrate strong prognostic potential. We also characterize 16 cancer type-specific subnetwork signatures which show diverse implications to somatic mutations, somatic copy number aberrations, DNA methylation alterations and clinical outcomes. Furthermore, some of them are strongly correlated with histological or molecular subtypes, indicating their implications with tumor heterogeneity. In summary, we systematically explore the pan-cancer common and cancer type-specific gene subnetwork signatures across multiple cancers, and reveal distinct commonalities and specificities among cancers at transcriptomic level. PMID:27633916

  1. TCGA Bladder Cancer Study Reveals Potential Drug Targets - TCGA

    Cancer.gov

    Investigators with the TCGA Research Network have identified new potential therapeutic targets for a major form of bladder cancer, including important genes and pathways that are disrupted in the disease.

  2. TCGA bladder cancer study reveals potential drug targets

    Cancer.gov

    Investigators with TCGA have identified new potential therapeutic targets for a major form of bladder cancer, including important genes and pathways that are disrupted in the disease. They also discovered that, at the molecular level, some subtypes of bla

  3. Epstein-Barr virus latent membrane protein 2A enhances MYC-driven cell cycle progression in a mouse model of B lymphoma

    PubMed Central

    Fish, Kamonwan; Chen, Jia

    2014-01-01

    Elevated expression of MYC is a shared property of many human cancers. Epstein-Barr virus (EBV) has been associated with lymphoid malignancies, yet collaborative roles between MYC and EBV in lymphomagenesis are unclear. EBV latent membrane protein 2A (LMP2A) functions as a B-cell receptor (BCR) mimic known to provide survival signals to infected B cells. Co-expression of human MYC and LMP2A in mice (LMP2A/λ-MYC) accelerates B lymphoma onset compared with mice expressing human MYC alone (λ-MYC mice). Here we show a novel role of LMP2A in potentiating MYC to promote G1-S transition and hyperproliferation by downregulating cyclin-dependent kinase inhibitor p27kip1 in a proteasome-dependent manner. Expressing a gain-of-function S10A mutant of p27kip1 has minor effect on tumor latency. However, pretumor B cells from λ-MYC mice expressing homozygous S10A mutant show a significant decrease in the percentage of S-phase cells. Interestingly, LMP2A is able to counteract the antiproliferative effect of the S10A mutant to promote S-phase entry. Finally, we show that LMP2A expression correlates with higher levels of MYC expression and suppression of p27kip1 before lymphoma onset. Our study demonstrates a novel function of EBV LMP2A in maximizing MYC expression, resulting in hyperproliferation and cellular transformation into cancer cells in vivo. PMID:24174629

  4. Revealing the Complexity of Breast Cancer by Next Generation Sequencing

    PubMed Central

    Verigos, John; Magklara, Angeliki

    2015-01-01

    Over the last few years the increasing usage of “-omic” platforms, supported by next-generation sequencing, in the analysis of breast cancer samples has tremendously advanced our understanding of the disease. New driver and passenger mutations, rare chromosomal rearrangements and other genomic aberrations identified by whole genome and exome sequencing are providing missing pieces of the genomic architecture of breast cancer. High resolution maps of breast cancer methylomes and sequencing of the miRNA microworld are beginning to paint the epigenomic landscape of the disease. Transcriptomic profiling is giving us a glimpse into the gene regulatory networks that govern the fate of the breast cancer cell. At the same time, integrative analysis of sequencing data confirms an extensive intertumor and intratumor heterogeneity and plasticity in breast cancer arguing for a new approach to the problem. In this review, we report on the latest findings on the molecular characterization of breast cancer using NGS technologies, and we discuss their potential implications for the improvement of existing therapies. PMID:26561834

  5. Genomic Interaction Profiles in Breast Cancer Reveal Altered Chromatin Architecture

    PubMed Central

    Zeitz, Michael J.; Ay, Ferhat; Heidmann, Julia D.; Lerner, Paula L.

    2013-01-01

    Gene transcription can be regulated by remote enhancer regions through chromosome looping either in cis or in trans. Cancer cells are characterized by wholesale changes in long-range gene interactions, but the role that these long-range interactions play in cancer progression and metastasis is not well understood. In this study, we used IGFBP3, a gene involved in breast cancer pathogenesis, as bait in a 4C-seq experiment comparing normal breast cells (HMEC) with two breast cancer cell lines (MCF7, an ER positive cell line, and MDA-MB-231, a triple negative cell line). The IGFBP3 long-range interaction profile was substantially altered in breast cancer. Many interactions seen in normal breast cells are lost and novel interactions appear in cancer lines. We found that in HMEC, the breast carcinoma amplified sequence gene family (BCAS) 1–4 were among the top 10 most significantly enriched regions of interaction with IGFBP3. 3D-FISH analysis indicated that the translocation-prone BCAS genes, which are located on chromosomes 1, 17, and 20, are in close physical proximity with IGFBP3 and each other in normal breast cells. We also found that epidermal growth factor receptor (EGFR), a gene implicated in tumorigenesis, interacts significantly with IGFBP3 and that this interaction may play a role in their regulation. Breakpoint analysis suggests that when an IGFBP3 interacting region undergoes a translocation an additional interaction detectable by 4C is gained. Overall, our data from multiple lines of evidence suggest an important role for long-range chromosomal interactions in the pathogenesis of cancer. PMID:24019942

  6. Subclonal diversification of primary breast cancer revealed by multiregion sequencing

    SciTech Connect

    Yates, Lucy R.; Gerstung, Moritz; Knappskog, Stian; Desmedt, Christine; Gundem, Gunes; Van Loo, Peter; Aas, Turid; Alexandrov, Ludmil B.; Larsimont, Denis; Davies, Helen; Li, Yilong; Ju, Young Seok; Ramakrishna, Manasa; Haugland, Hans Kristian; Lilleng, Peer Kaare; Nik-Zainal, Serena; McLaren, Stuart; Butler, Adam; Martin, Sancha; Glodzik, Dominic; Menzies, Andrew; Raine, Keiran; Hinton, Jonathan; Jones, David; Mudie, Laura J.; Jiang, Bing; Vincent, Delphine; Greene-Colozzi, April; Adnet, Pierre -Yves; Fatima, Aquila; Maetens, Marion; Ignatiadis, Michail; Stratton, Michael R.; Sotiriou, Christos; Richardson, Andrea L.; Lønning, Per Eystein; Wedge, David C.; Campbell, Peter J.

    2015-06-22

    Sequencing cancer genomes may enable tailoring of therapeutics to the underlying biological abnormalities driving a particular patient's tumor. However, sequencing-based strategies rely heavily on representative sampling of tumors. To understand the subclonal structure of primary breast cancer, we applied whole-genome and targeted sequencing to multiple samples from each of 50 patients' tumors (303 samples in total). The extent of subclonal diversification varied among cases and followed spatial patterns. No strict temporal order was evident, with point mutations and rearrangements affecting the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumors and late in others. In 13 out of 50 cancers, potentially targetable mutations were subclonal. Landmarks of disease progression, such as resistance to chemotherapy and the acquisition of invasive or metastatic potential, arose within detectable subclones of antecedent lesions. These findings highlight the importance of including analyses of subclonal structure and tumor evolution in clinical trials of primary breast cancer.

  7. Subclonal diversification of primary breast cancer revealed by multiregion sequencing

    DOE PAGESBeta

    Yates, Lucy R.; Gerstung, Moritz; Knappskog, Stian; Desmedt, Christine; Gundem, Gunes; Van Loo, Peter; Aas, Turid; Alexandrov, Ludmil B.; Larsimont, Denis; Davies, Helen; et al

    2015-06-22

    Sequencing cancer genomes may enable tailoring of therapeutics to the underlying biological abnormalities driving a particular patient's tumor. However, sequencing-based strategies rely heavily on representative sampling of tumors. To understand the subclonal structure of primary breast cancer, we applied whole-genome and targeted sequencing to multiple samples from each of 50 patients' tumors (303 samples in total). The extent of subclonal diversification varied among cases and followed spatial patterns. No strict temporal order was evident, with point mutations and rearrangements affecting the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumors and latemore » in others. In 13 out of 50 cancers, potentially targetable mutations were subclonal. Landmarks of disease progression, such as resistance to chemotherapy and the acquisition of invasive or metastatic potential, arose within detectable subclones of antecedent lesions. These findings highlight the importance of including analyses of subclonal structure and tumor evolution in clinical trials of primary breast cancer.« less

  8. Subclonal diversification of primary breast cancer revealed by multiregion sequencing.

    PubMed

    Yates, Lucy R; Gerstung, Moritz; Knappskog, Stian; Desmedt, Christine; Gundem, Gunes; Van Loo, Peter; Aas, Turid; Alexandrov, Ludmil B; Larsimont, Denis; Davies, Helen; Li, Yilong; Ju, Young Seok; Ramakrishna, Manasa; Haugland, Hans Kristian; Lilleng, Peer Kaare; Nik-Zainal, Serena; McLaren, Stuart; Butler, Adam; Martin, Sancha; Glodzik, Dominic; Menzies, Andrew; Raine, Keiran; Hinton, Jonathan; Jones, David; Mudie, Laura J; Jiang, Bing; Vincent, Delphine; Greene-Colozzi, April; Adnet, Pierre-Yves; Fatima, Aquila; Maetens, Marion; Ignatiadis, Michail; Stratton, Michael R; Sotiriou, Christos; Richardson, Andrea L; Lønning, Per Eystein; Wedge, David C; Campbell, Peter J

    2015-07-01

    The sequencing of cancer genomes may enable tailoring of therapeutics to the underlying biological abnormalities driving a particular patient's tumor. However, sequencing-based strategies rely heavily on representative sampling of tumors. To understand the subclonal structure of primary breast cancer, we applied whole-genome and targeted sequencing to multiple samples from each of 50 patients' tumors (303 samples in total). The extent of subclonal diversification varied among cases and followed spatial patterns. No strict temporal order was evident, with point mutations and rearrangements affecting the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumors and late in others. In 13 out of 50 cancers, potentially targetable mutations were subclonal. Landmarks of disease progression, such as resistance to chemotherapy and the acquisition of invasive or metastatic potential, arose within detectable subclones of antecedent lesions. These findings highlight the importance of including analyses of subclonal structure and tumor evolution in clinical trials of primary breast cancer.

  9. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells.

    PubMed

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5' AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression of

  10. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells

    PubMed Central

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5′ AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression

  11. Metformin Induced AMPK Activation, G0/G1 Phase Cell Cycle Arrest and the Inhibition of Growth of Esophageal Squamous Cell Carcinomas In Vitro and In Vivo.

    PubMed

    Cai, Xianbin; Hu, Xi; Tan, Xiaojun; Cheng, Weijie; Wang, Qinjia; Chen, Xiaofeng; Guan, Yinghong; Chen, Chong; Jing, Xubin

    2015-01-01

    Esophageal squamous cell carcinomas (ESCC) have become a severe threat to health and the current treatments for ESCC are frequently not effective. Recent epidemiological studies suggest that the anti-hyperglycemic agent metformin may reduce the risk of developing cancer, including ESCC, among diabetic patients. However, the antitumor effects of metformin on ESCC and the mechanisms underlying its cell cycle regulation remain elusive. The findings reported herein show that the anti-proliferative action of metformin on ESCC cell lines is partially mediated by AMPK. Moreover, we observed that metformin induced G0/G1 phase arrest accompanied by the up-regulation of p21CIP1 and p27KIP1. In vivo experiments further showed that metformin inhibited tumor growth in a ESCC xenograft model. Most importantly, the up-regulation of AMPK, p53, p21CIP1, p27KIP1 and the down-regulation of cyclinD1 are involved in the anti-tumor action of metformin in vivo. In conclusion, metformin inhibits the growth of ESCC cells both in cell cultures and in an animal model. AMPK, p53, p21CIP1, p27KIP1 and cyclinD1 are involved in the inhibition of tumor growth that is induced by metformin and cell cycle arrest in ESCC. These findings indicate that metformin has the potential for use in the treatment of ESCC.

  12. PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

    PubMed Central

    Li, Da-Ming; Sun, Hong

    1998-01-01

    PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27KIP1 and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27KIP1. PMID:9860981

  13. TReP-132 Is a Novel Progesterone Receptor Coactivator Required for the Inhibition of Breast Cancer Cell Growth and Enhancement of Differentiation by Progesterone

    PubMed Central

    Gizard, Florence; Robillard, Romain; Gross, Barbara; Barbier, Olivier; Révillion, Françoise; Peyrat, Jean-Philippe; Torpier, Gérard; Hum, Dean W.; Staels, Bart

    2006-01-01

    The sex steroid progesterone is essential for the proliferation and differentiation of the mammary gland epithelium during pregnancy. In relation to this, in vitro studies using breast carcinoma T47D cells have demonstrated a biphasic progesterone response, consisting of an initial proliferative burst followed by a sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the progesterone effects on mammary cell growth and differentiation remain to be determined. Recently, it has been demonstrated that the transcriptional regulating protein of 132 kDa (TReP-132), initially identified as a regulator of steroidogenesis, is also a cell growth suppressor. Similar to progesterone-bound PR, TReP-132 acts by inducing the gene expression of the G1 cyclin-dependent kinase inhibitors p21WAF1/Cip1 (p21) and p27Kip1 (p27). The putative interaction between TReP-132 and progesterone pathways in mammary cells was therefore analyzed in the present study. Our results show that TReP-132 interacts in vitro and in T47D cells with progesterone-activated PR. TReP-132 synergizes with progesterone-bound PR to trans activate the p21 and p27 gene promoters at proximal Sp1-binding sites. Moreover, TReP-132 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. As a consequence, TReP-132 knockdown also resulted in the loss of the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. Furthermore, the knockdown of TReP-132 expression also prevented the induction of both early and terminal markers of breast cell differentiation which had been previously identified as progesterone target genes. As well, the progesterone-induced accumulation of lipid vacuoles was inhibited in the TReP-132-depleted cells. Finally, TReP-132 gene expression levels increased following progesterone treatment, indicating the

  14. Identification of a GαGβγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition.

    PubMed

    Ouelaa-Benslama, Radia; De Wever, Olivier; Hendrix, An; Sabbah, Michèle; Lambein, Kathleen; Land, David; Prévost, Grégoire; Bracke, Marc; Hung, Mien-Chie; Larsen, Annette K; Emami, Shahin; Gespach, Christian

    2012-07-01

    The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype associated with chemotherapy resistance in epithelial cancers. However, the mechanisms underlying the EMT and its associated signaling dysfunctions are still poorly understood. In two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation, we investigated the status of several signaling elements downstream of G-protein receptors (GPR) and their functional roles in the invasive growth potential. We report that the E-cadherin repressors Slug, Zeb1/2 and Twist are overexpressed in these EMT cells characterized by a triple negative phenotype (loss of estrogen ERα and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the alternative GPR30 estrogen receptor and induction of the invasive growth in collagen type I gels. Ectopic Snail expression suppressed WISP-2 transcripts and down-regulated WISP-2 gene promoter expression in transfected cells. Accordingly, WISP-2 transcripts and Wisp-2 protein were depleted in these two convergent models of BC cell EMT. The EMT caused dominance of several proinvasive pathways downstream of GPR, including GαGβγ subunits, PKCα, AKT and c-Jun induction, constitutive activation of the actin-remodeling GTPase Rac1, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27kip1/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting GαGβγ subunits (BIM-46187, gallein), PKCα (Gö6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin) alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signaling independence to inhibitors of HER family tyrosine kinases and the mitogen- and stress-activated protein kinases. Our study suggests that the signaling protagonists GαGβγ, PKCα and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the

  15. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin.

    PubMed

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Hwang, Jae Sam; Seok, Heon; Choi, Hyemin; Lee, Dong Gun; Kim, Jae Il; Kim, Ho

    2014-06-01

    We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH2-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson's disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27(Kip1) protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27(Kip1) significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27(Kip1) degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin. PMID:24796676

  16. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

    PubMed

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J; Adams, David J; Leung, Hing Y

    2016-07-19

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition. PMID:27357679

  17. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

    PubMed

    Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Gingras, Marie-Claude; Muthuswamy, Lakshmi B; Johns, Amber L; Miller, David K; Wilson, Peter J; Patch, Ann-Marie; Wu, Jianmin; Chang, David K; Cowley, Mark J; Gardiner, Brooke B; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J; Gill, Anthony J; Pinho, Andreia V; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R Scott; Humphris, Jeremy L; Kaplan, Warren; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chou, Angela; Chin, Venessa T; Chantrill, Lorraine A; Mawson, Amanda; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Daly, Roger J; Merrett, Neil D; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M; Fisher, William E; Brunicardi, F Charles; Hodges, Sally E; Reid, Jeffrey G; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R; Dinh, Huyen; Buhay, Christian J; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E; Yung, Christina K; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A; Petersen, Gloria M; Gallinger, Steven; Hruban, Ralph H; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Schulick, Richard D; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A; Mann, Karen M; Jenkins, Nancy A; Perez-Mancera, Pedro A; Adams, David J; Largaespada, David A; Wessels, Lodewyk F A; Rust, Alistair G; Stein, Lincoln D; Tuveson, David A; Copeland, Neal G; Musgrove, Elizabeth A; Scarpa, Aldo; Eshleman, James R; Hudson, Thomas J; Sutherland, Robert L; Wheeler, David A; Pearson, John V; McPherson, John D; Gibbs, Richard A; Grimmond, Sean M

    2012-11-15

    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

  18. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer

    PubMed Central

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G.; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J.; Adams, David J.; Leung, Hing Y.

    2016-01-01

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition. PMID:27357679

  19. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

    PubMed

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J; Adams, David J; Leung, Hing Y

    2016-07-19

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition.

  20. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes

    PubMed Central

    Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Gingras, Marie-Claude; Muthuswamy, Lakshmi B.; Johns, Amber L.; Miller, David K.; Wilson, Peter J.; Patch, Ann-Marie; Wu, Jianmin; Chang, David K.; Cowley, Mark J.; Gardiner, Brooke B.; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J.; Gill, Anthony J.; Pinho, Andreia V.; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J. Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R. Scott; Humphris, Jeremy L.; Kaplan, Warren; Jones, Marc D.; Colvin, Emily K.; Nagrial, Adnan M.; Humphrey, Emily S.; Chou, Angela; Chin, Venessa T.; Chantrill, Lorraine A.; Mawson, Amanda; Samra, Jaswinder S.; Kench, James G.; Lovell, Jessica A.; Daly, Roger J.; Merrett, Neil D.; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q.; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M.; Fisher, William E.; Brunicardi, F. Charles; Hodges, Sally E.; Reid, Jeffrey G.; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R.; Dinh, Huyen; Buhay, Christian J.; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E.; Yung, Christina K.; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A.; Petersen, Gloria M.; Gallinger, Steven; Hruban, Ralph H.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Schulick, Richard D.; Wolfgang, Christopher L.; Morgan, Richard A.; Lawlor, Rita T.; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A.; Mann, Karen M.; Jenkins, Nancy A.; Perez-Mancera, Pedro A.; Adams, David J.; Largaespada, David A.; Wessels, Lodewyk F. A.; Rust, Alistair G.; Stein, Lincoln D.; Tuveson, David A.; Copeland, Neal G.; Musgrove, Elizabeth A.; Scarpa, Aldo; Eshleman, James R.; Hudson, Thomas J.; Sutherland, Robert L.; Wheeler, David A.; Pearson, John V.; McPherson, John D.; Gibbs, Richard A.; Grimmond, Sean M.

    2012-01-01

    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis. PMID:23103869

  1. Intravital imaging reveals new ancillary mechanisms co-opted by cancer cells to drive tumor progression

    PubMed Central

    Lucas, Morghan C.; Timpson, Paul

    2016-01-01

    Intravital imaging is providing new insights into the dynamics of tumor progression in native tissues and has started to reveal the layers of complexity found in cancer. Recent advances in intravital imaging have allowed us to look deeper into cancer behavior and to dissect the interactions between tumor cells and the ancillary host niche that promote cancer development. In this review, we provide an insight into the latest advances in cancer biology achieved by intravital imaging, focusing on recently discovered mechanisms by which tumor cells manipulate normal tissue to facilitate disease progression. PMID:27239290

  2. Quantitative Proteomic Profiling of Prostate Cancer Reveals a Role for miR-128 in Prostate Cancer*

    PubMed Central

    Khan, Amjad P.; Poisson, Laila M.; Bhat, Vadiraja B.; Fermin, Damian; Zhao, Rong; Kalyana-Sundaram, Shanker; Michailidis, George; Nesvizhskii, Alexey I.; Omenn, Gilbert S.; Chinnaiyan, Arul M.; Sreekumar, Arun

    2010-01-01

    Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of biomarkers of cancer invasion and disease aggressiveness. Although alterations in gene expression have been extensively quantified during neoplastic progression, complementary analyses of proteomic changes have been limited. Here we interrogate the proteomic alterations in a cohort of 15 prostate-derived tissues that included five each from adjacent benign prostate, clinically localized prostate cancer, and metastatic disease from distant sites. The experimental strategy couples isobaric tags for relative and absolute quantitation with multidimensional liquid phase peptide fractionation followed by tandem mass spectrometry. Over 1000 proteins were quantified across the specimens and delineated into clinically localized and metastatic prostate cancer-specific signatures. Included in these class-specific profiles were both proteins that were known to be dysregulated during prostate cancer progression and new ones defined by this study. Enrichment analysis of the prostate cancer-specific proteomic signature, to gain insight into the functional consequences of these alterations, revealed involvement of miR-128-a/b regulation during prostate cancer progression. This finding was validated using real time PCR analysis for microRNA transcript levels in an independent set of 15 clinical specimens. miR-128 levels were elevated in benign prostate epithelial cell lines compared with invasive prostate cancer cells. Knockdown of miR-128 induced invasion in benign prostate epithelial cells, whereas its overexpression attenuated invasion in prostate cancer cells. Taken together, our profiles of the proteomic alterations of prostate cancer progression revealed miR-128 as a potentially important negative regulator of prostate cancer cell invasion. PMID:19955085

  3. A Conserved Gammaherpesvirus Cyclin Specifically Bypasses Host p18INK4c To Promote Reactivation from Latency

    PubMed Central

    Williams, Lisa M.; Niemeyer, Brian F.; Franklin, David S.; Clambey, Eric T.

    2015-01-01

    ABSTRACT Gammaherpesviruses (GHVs) carry homologs of cellular genes, including those encoding a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitors in vitro; however, the in vivo significance of this is unclear. Here, we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors p27Kip1 and p18INK4c, two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, it was dispensable for reactivation in mice lacking p27Kip1 and p18INK4c. Further analysis revealed that genetic ablation of only p18INK4c alleviated the requirement for the viral cyclin for reactivation from latency. p18INK4c regulated reactivation in a dose-dependent manner so that the viral cyclin was dispensable in p18INK4c heterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18INK4c function in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data show that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin-dependent kinase inhibitor p18INK4c, revealing an unanticipated specificity between a GHV cyclin and a single cyclin-dependent kinase inhibitor. IMPORTANCE The gammaherpesviruses (GHVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many GHVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the GHVs resist this inhibition. We used a mouse GHV that either contained or lacked a viral cyclin to test whether the viral cyclin lost importance when these inhibitory

  4. Potential microRNA-mediated oncogenic intercellular communication revealed by pan-cancer analysis

    NASA Astrophysics Data System (ADS)

    Li, Yue; Zhang, Zhaolei

    2014-11-01

    Carcinogenesis consists of oncogenesis and metastasis, and intriguingly microRNAs (miRNAs) are involved in both processes. Although aberrant miRNA activities are prevalent in diverse tumor types, the exact mechanisms for how they regulate cancerous processes are not always clear. To this end, we performed a large-scale pan-cancer analysis via a novel probabilistic approach to infer recurrent miRNA-target interactions implicated in 12 cancer types using data from The Cancer Genome Atlas. We discovered ~20,000 recurrent miRNA regulations, which are enriched for cancer-related miRNAs/genes. Notably, miRNA 200 family (miR-200/141/429) is among the most prominent miRNA regulators, which is known to be involved in metastasis. Importantly, the recurrent miRNA regulatory network is not only enriched for cancer pathways but also for extracellular matrix (ECM) organization and ECM-receptor interactions. The results suggest an intriguing cancer mechanism involving miRNA-mediated cell-to-cell communication, which possibly involves delivery of tumorigenic miRNA messengers to adjacent cells via exosomes. Finally, survival analysis revealed 414 recurrent-prognostic associations, where both gene and miRNA involved in each interaction conferred significant prognostic power in one or more cancer types. Together, our comprehensive pan-cancer analysis provided not only biological insights into metastasis but also brought to bear the clinical relevance of the proposed recurrent miRNA-gene associations.

  5. An international survey of physician attitudes and practice in regard to revealing the diagnosis of cancer.

    PubMed

    Holland, J C; Geary, N; Marchini, A; Tross, S

    1987-01-01

    In 1984, questionnaires were sent to members of the International Psycho-Oncology Society concerning the practice in their country with regard to revealing the diagnosis of cancer to patients, their opinion about the effect of their policy, and their impression of local trends and attitudes toward cancer. Data from 90 respondents from 20 countries revealed that use of the word "cancer" was often avoided in discussions with the patient. Words commonly substituted for cancer were those that implied a "swelling" (e.g., tumor, growth, lump), and "inflammation," or a pathophysiologic change (blood disease, precancerous, unclean tissue). Oncologists estimated that a low percentage (less than 40%) of their colleagues revealed the word cancer in Africa, France, Hungary, Italy, Japan, Panama, Portugal, and Spain. Oncologists from Austria, Denmark Finland, The Netherlands, New Zealand, Norway, Sweden, and Switzerland, estimated the percentage to be high (greater than 80%). However, in all countries, the majority of physicians tell the family the diagnosis. The majority (90%) reported a trend toward increased telling of the diagnosis, due to greater patient information and expectations, and increased physician openess in using the word cancer. Most (68%) felt that the overall effect of revealing the diagnosis was positive. While emotional distress was transiently greater when patients were told, there were positive effects concerning coping, compliance, tolerance of treatment, planning for future, communication with physicians and others, and improved prognosis. The transient negative effects were depression, anxiety, and anger. It is important to recognize that efforts to find the "correct" position about revealing or concealing cancer diagnosis must recognize that the language between doctor and patient is constrained by cultural norms. Communication is likely to be far less dependent upon the specific words used then upon the meaning that is conveyed by the doctor. PMID

  6. Trespassing cancer cells: ‘fingerprinting’ invasive protrusions reveals metastatic culprits

    PubMed Central

    Klemke, Richard L.

    2012-01-01

    Metastatic cancer cells produce invasive membrane protrusions called invadopodia and pseudopodia, which play a central role in driving cancer cell dissemination in the body. Malignant cells use these structures to attach to and degrade extracellular matrix proteins, generate force for cell locomotion, and to penetrate the vasculature. Recent work using unique subcellular fractionation methodologies combined with spatial genomic, proteomic, and phosphoproteomic profiling has provided insight into the invadopodiome and pseudopodiome signaling networks that control the protrusion of invasive membranes. Here I highlight how these powerful spatial “omics” approaches reveal important signatures of metastatic cancer cells and possible new therapeutic targets aimed at treating metastatic disease. PMID:22980730

  7. A Balanced Tissue Composition Reveals New Metabolic and Gene Expression Markers in Prostate Cancer.

    PubMed

    Tessem, May-Britt; Bertilsson, Helena; Angelsen, Anders; Bathen, Tone F; Drabløs, Finn; Rye, Morten Beck

    2016-01-01

    Molecular analysis of patient tissue samples is essential to characterize the in vivo variability in human cancers which are not accessible in cell-lines or animal models. This applies particularly to studies of tumor metabolism. The challenge is, however, the complex mixture of various tissue types within each sample, such as benign epithelium, stroma and cancer tissue, which can introduce systematic biases when cancers are compared to normal samples. In this study we apply a simple strategy to remove such biases using sample selections where the average content of stroma tissue is balanced between the sample groups. The strategy is applied to a prostate cancer patient cohort where data from MR spectroscopy and gene expression have been collected from and integrated on the exact same tissue samples. We reveal in vivo changes in cancer-relevant metabolic pathways which are otherwise hidden in the data due to tissue confounding. In particular, lowered levels of putrescine are connected to increased expression of SRM, reduced levels of citrate are attributed to upregulation of genes promoting fatty acid synthesis, and increased succinate levels coincide with reduced expression of SUCLA2 and SDHD. In addition, the strategy also highlights important metabolic differences between the stroma, epithelium and prostate cancer. These results show that important in vivo metabolic features of cancer can be revealed from patient data only if the heterogeneous tissue composition is properly accounted for in the analysis. PMID:27100877

  8. A Balanced Tissue Composition Reveals New Metabolic and Gene Expression Markers in Prostate Cancer

    PubMed Central

    Tessem, May-Britt; Bertilsson, Helena; Angelsen, Anders; Bathen, Tone F.; Drabløs, Finn; Rye, Morten Beck

    2016-01-01

    Molecular analysis of patient tissue samples is essential to characterize the in vivo variability in human cancers which are not accessible in cell-lines or animal models. This applies particularly to studies of tumor metabolism. The challenge is, however, the complex mixture of various tissue types within each sample, such as benign epithelium, stroma and cancer tissue, which can introduce systematic biases when cancers are compared to normal samples. In this study we apply a simple strategy to remove such biases using sample selections where the average content of stroma tissue is balanced between the sample groups. The strategy is applied to a prostate cancer patient cohort where data from MR spectroscopy and gene expression have been collected from and integrated on the exact same tissue samples. We reveal in vivo changes in cancer-relevant metabolic pathways which are otherwise hidden in the data due to tissue confounding. In particular, lowered levels of putrescine are connected to increased expression of SRM, reduced levels of citrate are attributed to upregulation of genes promoting fatty acid synthesis, and increased succinate levels coincide with reduced expression of SUCLA2 and SDHD. In addition, the strategy also highlights important metabolic differences between the stroma, epithelium and prostate cancer. These results show that important in vivo metabolic features of cancer can be revealed from patient data only if the heterogeneous tissue composition is properly accounted for in the analysis. PMID:27100877

  9. Genome-wide transcript profiling reveals novel breast cancer-associated intronic sense RNAs.

    PubMed

    Kim, Sang Woo; Fishilevich, Elane; Arango-Argoty, Gustavo; Lin, Yuefeng; Liu, Guodong; Li, Zhihua; Monaghan, A Paula; Nichols, Mark; John, Bino

    2015-01-01

    Non-coding RNAs (ncRNAs) play major roles in development and cancer progression. To identify novel ncRNAs that may identify key pathways in breast cancer development, we performed high-throughput transcript profiling of tumor and normal matched-pair tissue samples. Initial transcriptome profiling using high-density genome-wide tiling arrays revealed changes in over 200 novel candidate genomic regions that map to intronic regions. Sixteen genomic loci were identified that map to the long introns of five key protein-coding genes, CRIM1, EPAS1, ZEB2, RBMS1, and RFX2. Consistent with the known role of the tumor suppressor ZEB2 in the cancer-associated epithelial to mesenchymal transition (EMT), in situ hybridization reveals that the intronic regions deriving from ZEB2 as well as those from RFX2 and EPAS1 are down-regulated in cells of epithelial morphology, suggesting that these regions may be important for maintaining normal epithelial cell morphology. Paired-end deep sequencing analysis reveals a large number of distinct genomic clusters with no coding potential within the introns of these genes. These novel transcripts are only transcribed from the coding strand. A comprehensive search for breast cancer associated genes reveals enrichment for transcribed intronic regions from these loci, pointing to an underappreciated role of introns or mechanisms relating to their biology in EMT and breast cancer. PMID:25798919

  10. Metabolomic profiling reveals potential markers and bioprocesses altered in bladder cancer progression.

    PubMed

    Putluri, Nagireddy; Shojaie, Ali; Vasu, Vihas T; Vareed, Shaiju K; Nalluri, Srilatha; Putluri, Vasanta; Thangjam, Gagan Singh; Panzitt, Katrin; Tallman, Christopher T; Butler, Charles; Sana, Theodore R; Fischer, Steven M; Sica, Gabriel; Brat, Daniel J; Shi, Huidong; Palapattu, Ganesh S; Lotan, Yair; Weizer, Alon Z; Terris, Martha K; Shariat, Shahrokh F; Michailidis, George; Sreekumar, Arun

    2011-12-15

    Although alterations in xenobiotic metabolism are considered causal in the development of bladder cancer, the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in bladder cancer. This metabolic signature distinguished both normal and benign bladder from bladder cancer. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing bladder cancer from controls and also nonmuscle from muscle-invasive bladder cancer. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in bladder cancer. In particular, we validated tumor-associated hypermethylation in the cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) promoters of bladder cancer tissues by bisulfite sequence analysis and methylation-specific PCR and also by in vitro treatment of T-24 bladder cancer cell line with the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of bladder cancer specimens compared with matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of bladder cancer, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression. PMID:21990318

  11. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    PubMed

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-01

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy.

  12. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    PubMed

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-01

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy. PMID:25556991

  13. Exome Analysis Reveals Differentially Mutated Gene Signatures of Stage, Grade and Subtype in Breast Cancers

    PubMed Central

    Li, You; Wang, Xiaosheng; Vural, Suleyman; Mishra, Nitish K.; Cowan, Kenneth H.; Guda, Chittibabu

    2015-01-01

    Breast cancers exhibit highly heterogeneous molecular profiles. Although gene expression profiles have been used to predict the risks and prognostic outcomes of breast cancers, the high variability of gene expression limits its clinical application. In contrast, genetic mutation profiles would be more advantageous than gene expression profiles because genetic mutations can be stably detected and the mutational heterogeneity widely exists in breast cancer genomes. We analyzed 98 breast cancer whole exome samples that were sorted into three subtypes, two grades and two stages. The sum deleterious effect of all mutations in each gene was scored to identify differentially mutated genes (DMGs) for this case-control study. DMGs were corroborated using extensive published knowledge. Functional consequences of deleterious SNVs on protein structure and function were also investigated. Genes such as ERBB2, ESP8, PPP2R4, KIAA0922, SP4, CENPJ, PRCP and SELP that have been experimentally or clinically verified to be tightly associated with breast cancer prognosis are among the DMGs identified in this study. We also identified some genes such as ARL6IP5, RAET1E, and ANO7 that could be crucial for breast cancer development and prognosis. Further, SNVs such as rs1058808, rs2480452, rs61751507, rs79167802, rs11540666, and rs2229437 that potentially influence protein functions are observed at significantly different frequencies in different comparison groups. Protein structure modeling revealed that many non-synonymous SNVs have a deleterious effect on protein stability, structure and function. Mutational profiling at gene- and SNV-level revealed differential patterns within each breast cancer comparison group, and the gene signatures correlate with expected prognostic characteristics of breast cancer classes. Some of the genes and SNVs identified in this study show high promise and are worthy of further investigation by experimental studies. PMID:25803781

  14. Base changes in tumour DNA have the power to reveal the causes and evolution of cancer

    DOE PAGESBeta

    Hollstein, M.; Alexandrov, L. B.; Wild, C. P.; Ardin, M.; Zavadil, J.

    2016-06-06

    Next-generation sequencing (NGS) technology has demonstrated that the cancer genomes are peppered with mutations. Although most somatic tumour mutations are unlikely to have any role in the cancer process per se, the spectra of DNA sequence changes in tumour mutation catalogues have the potential to identify the mutagens, and to reveal the mutagenic processes responsible for human cancer. Very recently, a novel approach for data mining of the vast compilations of tumour NGS data succeeded in separating and precisely defining at least 30 distinct patterns of sequence change hidden in mutation databases. At least half of these mutational signatures canmore » be readily assigned to known human carcinogenic exposures or endogenous mechanisms of mutagenesis. A quantum leap in our knowledge of mutagenesis in human cancers has resulted, stimulating a flurry of research activity. We trace here the major findings leading first to the hypothesis that carcinogenic insults leave characteristic imprints on the DNA sequence of tumours, and culminating in empirical evidence from NGS data that well-defined carcinogen mutational signatures are indeed present in tumour genomic DNA from a variety of cancer types. The notion that tumour DNAs can divulge environmental sources of mutation is now a well-accepted fact. This approach to cancer aetiology has also incriminated various endogenous, enzyme-driven processes that increase the somatic mutation load in sporadic cancers. The tasks now confronting the field of molecular epidemiology are to assign mutagenic processes to orphan and newly discovered tumour mutation patterns, and to determine whether avoidable cancer risk factors influence signatures produced by endogenous enzymatic mechanisms. As a result, innovative research with experimental models and exploitation of the geographical heterogeneity in cancer incidence can address these challenges.« less

  15. Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer

    PubMed Central

    Liu, Jinfeng; McCleland, Mark; Stawiski, Eric W.; Gnad, Florian; Mayba, Oleg; Haverty, Peter M.; Durinck, Steffen; Chen, Ying-Jiun; Klijn, Christiaan; Jhunjhunwala, Suchit; Lawrence, Michael; Liu, Hanbin; Wan, Yinan; Chopra, Vivek; Yaylaoglu, Murat B.; Yuan, Wenlin; Ha, Connie; Gilbert, Houston N.; Reeder, Jens; Pau, Gregoire; Stinson, Jeremy; Stern, Howard M.; Manning, Gerard; Wu, Thomas D.; Neve, Richard M.; de Sauvage, Frederic J.; Modrusan, Zora; Seshagiri, Somasekar; Firestein, Ron; Zhang, Zemin

    2014-01-01

    Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK. PMID:24807215

  16. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    NASA Astrophysics Data System (ADS)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  17. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    PubMed Central

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-01-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy. PMID:26893143

  18. Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer*

    PubMed Central

    Chen, Chien-Lun; Chung, Ting; Wu, Chih-Ching; Ng, Kwai-Fong; Yu, Jau-Song; Tsai, Cheng-Han; Chang, Yu-Sun; Liang, Ying; Tsui, Ke-Hung; Chen, Yi-Ting

    2015-01-01

    More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins—SLC3A2, STMN1, and TAGLN2—in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant

  19. Structure–function insights reveal the human ribosome as a cancer target for antibiotics

    PubMed Central

    Myasnikov, Alexander G.; Kundhavai Natchiar, S.; Nebout, Marielle; Hazemann, Isabelle; Imbert, Véronique; Khatter, Heena; Peyron, Jean-François; Klaholz, Bruno P.

    2016-01-01

    Many antibiotics in clinical use target the bacterial ribosome by interfering with the protein synthesis machinery. However, targeting the human ribosome in the case of protein synthesis deregulations such as in highly proliferating cancer cells has not been investigated at the molecular level up to now. Here we report the structure of the human 80S ribosome with a eukaryote-specific antibiotic and show its anti-proliferative effect on several cancer cell lines. The structure provides insights into the detailed interactions in a ligand-binding pocket of the human ribosome that are required for structure-assisted drug design. Furthermore, anti-proliferative dose response in leukaemic cells and interference with synthesis of c-myc and mcl-1 short-lived protein markers reveals specificity of a series of eukaryote-specific antibiotics towards cytosolic rather than mitochondrial ribosomes, uncovering the human ribosome as a promising cancer target. PMID:27665925

  20. Identification of lncRNA-associated competing triplets reveals global patterns and prognostic markers for cancer

    PubMed Central

    Wang, Peng; Ning, Shangwei; Zhang, Yunpeng; Li, Ronghong; Ye, Jingrun; Zhao, Zuxianglan; Zhi, Hui; Wang, Tingting; Guo, Zheng; Li, Xia

    2015-01-01

    Recent studies have suggested that long non-coding RNAs (lncRNAs) can interact with microRNAs (miRNAs) and indirectly regulate miRNA targets though competing interactions. However, the molecular mechanisms underlying these interactions are still largely unknown. In this study, these lncRNA–miRNA–gene interactions were defined as lncRNA-associated competing triplets (LncACTs), and an integrated pipeline was developed to identify lncACTs that are active in cancer. Competing lncRNAs had sponge features distinct from non-competing lncRNAs. In the lncACT cross-talk network, disease-associated lncRNAs, miRNAs and coding-genes showed specific topological patterns indicative of their competence and control of communication within the network. The construction of global competing activity profiles revealed that lncACTs had high activity specific to cancers. Analyses of clustered lncACTs revealed that they were enriched in various cancer-related biological processes. Based on the global cross-talk network and cluster analyses, nine cancer-specific sub-networks were constructed. H19- and BRCA1/2-associated lncACTs were able to discriminate between two groups of patients with different clinical outcomes. Disease-associated lncACTs also showed variable competing patterns across normal and cancer patient samples. In summary, this study uncovered and systematically characterized global properties of human lncACTs that may have prognostic value for predicting clinical outcome in cancer patients. PMID:25800746

  1. Splicing mutation analysis reveals previously unrecognized pathways in lymph node-invasive breast cancer

    PubMed Central

    Dorman, Stephanie N.; Viner, Coby; Rogan, Peter K.

    2014-01-01

    Somatic mutations reported in large-scale breast cancer (BC) sequencing studies primarily consist of protein coding mutations. mRNA splicing mutation analyses have been limited in scope, despite their prevalence in Mendelian genetic disorders. We predicted splicing mutations in 442 BC tumour and matched normal exomes from The Cancer Genome Atlas Consortium (TCGA). These splicing defects were validated by abnormal expression changes in these tumours. Of the 5,206 putative mutations identified, exon skipping, leaky or cryptic splicing was confirmed for 988 variants. Pathway enrichment analysis of the mutated genes revealed mutations in 9 NCAM1-related pathways, which were significantly increased in samples with evidence of lymph node metastasis, but not in lymph node-negative tumours. We suggest that comprehensive reporting of DNA sequencing data should include non-trivial splicing analyses to avoid missing clinically-significant deleterious splicing mutations, which may reveal novel mutated pathways present in genetic disorders. PMID:25394353

  2. Revealing Glycoproteins in the Secretome of MCF-7 Human Breast Cancer Cells

    PubMed Central

    Tan, Aik-Aun; Phang, Wai-Mei; Gopinath, Subash C. B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2015-01-01

    Breast cancer is one of the major issues in the field of oncology, reported with a higher prevalence rate in women worldwide. In attempt to reveal the potential biomarkers for breast cancer, the findings of differentially glycosylated haptoglobin and osteonectin in previous study have drawn our attention towards glycoproteins of secretome from the MCF-7 cancer cell line. In the present study, further analyses were performed on the medium of MCF-7 cells by subjecting it to two-dimensional analyses followed by image analysis in contrast to the medium of human mammary epithelial cells (HMEpC) as a negative control. Carboxypeptidase A4 (CPA4), alpha-1-antitrypsin (AAT), haptoglobin (HP), and HSC70 were detected in the medium of MCF-7, while only CPA4 and osteonectin (ON) were detected in HMEpC medium. In addition, CPA4 was detected as upregulated in the MCF-7 medium. Further analysis by lectin showed that CPA4, AAT, HP, and HSC70 were secreted as N-glycan in the medium of MCF-7, with HP also showing differentially N-glycosylated isoforms. For the HMEpC, only CPA4 was detected as N-glycan. No O-glycan was detected in the medium of HMEpC but MCF-7 expressed O-glycosylated CPA4 and HSC70. All these revealed that glycoproteins could be used as glycan-based biomarkers for the prognosis of breast cancer. PMID:26167486

  3. Solutions to Peto's paradox revealed by mathematical modelling and cross-species cancer gene analysis

    PubMed Central

    Caulin, Aleah F.; Graham, Trevor A.; Wang, Li-San; Maley, Carlo C.

    2015-01-01

    Whales have 1000-fold more cells than humans and mice have 1000-fold fewer; however, cancer risk across species does not increase with the number of somatic cells and the lifespan of the organism. This observation is known as Peto's paradox. How much would evolution have to change the parameters of somatic evolution in order to equalize the cancer risk between species that differ by orders of magnitude in size? Analysis of previously published models of colorectal cancer suggests that a two- to three-fold decrease in the mutation rate or stem cell division rate is enough to reduce a whale's cancer risk to that of a human. Similarly, the addition of one to two required tumour-suppressor gene mutations would also be sufficient. We surveyed mammalian genomes and did not find a positive correlation of tumour-suppressor genes with increasing body mass and longevity. However, we found evidence of the amplification of TP53 in elephants, MAL in horses and FBXO31 in microbats, which might explain Peto's paradox in those species. Exploring parameters that evolution may have fine-tuned in large, long-lived organisms will help guide future experiments to reveal the underlying biology responsible for Peto's paradox and guide cancer prevention in humans. PMID:26056366

  4. Solutions to Peto's paradox revealed by mathematical modelling and cross-species cancer gene analysis.

    PubMed

    Caulin, Aleah F; Graham, Trevor A; Wang, Li-San; Maley, Carlo C

    2015-07-19

    Whales have 1000-fold more cells than humans and mice have 1000-fold fewer; however, cancer risk across species does not increase with the number of somatic cells and the lifespan of the organism. This observation is known as Peto's paradox. How much would evolution have to change the parameters of somatic evolution in order to equalize the cancer risk between species that differ by orders of magnitude in size? Analysis of previously published models of colorectal cancer suggests that a two- to three-fold decrease in the mutation rate or stem cell division rate is enough to reduce a whale's cancer risk to that of a human. Similarly, the addition of one to two required tumour-suppressor gene mutations would also be sufficient. We surveyed mammalian genomes and did not find a positive correlation of tumour-suppressor genes with increasing body mass and longevity. However, we found evidence of the amplification of TP53 in elephants, MAL in horses and FBXO31 in microbats, which might explain Peto's paradox in those species. Exploring parameters that evolution may have fine-tuned in large, long-lived organisms will help guide future experiments to reveal the underlying biology responsible for Peto's paradox and guide cancer prevention in humans.

  5. Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value.

    PubMed

    Stirzaker, Clare; Zotenko, Elena; Song, Jenny Z; Qu, Wenjia; Nair, Shalima S; Locke, Warwick J; Stone, Andrew; Armstong, Nicola J; Robinson, Mark D; Dobrovic, Alexander; Avery-Kiejda, Kelly A; Peters, Kate M; French, Juliet D; Stein, Sandra; Korbie, Darren J; Trau, Matt; Forbes, John F; Scott, Rodney J; Brown, Melissa A; Francis, Glenn D; Clark, Susan J

    2015-02-02

    Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.

  6. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin.

    PubMed

    Hoadley, Katherine A; Yau, Christina; Wolf, Denise M; Cherniack, Andrew D; Tamborero, David; Ng, Sam; Leiserson, Max D M; Niu, Beifang; McLellan, Michael D; Uzunangelov, Vladislav; Zhang, Jiashan; Kandoth, Cyriac; Akbani, Rehan; Shen, Hui; Omberg, Larsson; Chu, Andy; Margolin, Adam A; Van't Veer, Laura J; Lopez-Bigas, Nuria; Laird, Peter W; Raphael, Benjamin J; Ding, Li; Robertson, A Gordon; Byers, Lauren A; Mills, Gordon B; Weinstein, John N; Van Waes, Carter; Chen, Zhong; Collisson, Eric A; Benz, Christopher C; Perou, Charles M; Stuart, Joshua M

    2014-08-14

    Recent genomic analyses of pathologically defined tumor types identify "within-a-tissue" disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head and neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multiplatform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All data sets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies.

  7. Solutions to Peto's paradox revealed by mathematical modelling and cross-species cancer gene analysis.

    PubMed

    Caulin, Aleah F; Graham, Trevor A; Wang, Li-San; Maley, Carlo C

    2015-07-19

    Whales have 1000-fold more cells than humans and mice have 1000-fold fewer; however, cancer risk across species does not increase with the number of somatic cells and the lifespan of the organism. This observation is known as Peto's paradox. How much would evolution have to change the parameters of somatic evolution in order to equalize the cancer risk between species that differ by orders of magnitude in size? Analysis of previously published models of colorectal cancer suggests that a two- to three-fold decrease in the mutation rate or stem cell division rate is enough to reduce a whale's cancer risk to that of a human. Similarly, the addition of one to two required tumour-suppressor gene mutations would also be sufficient. We surveyed mammalian genomes and did not find a positive correlation of tumour-suppressor genes with increasing body mass and longevity. However, we found evidence of the amplification of TP53 in elephants, MAL in horses and FBXO31 in microbats, which might explain Peto's paradox in those species. Exploring parameters that evolution may have fine-tuned in large, long-lived organisms will help guide future experiments to reveal the underlying biology responsible for Peto's paradox and guide cancer prevention in humans. PMID:26056366

  8. Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

    PubMed Central

    2014-01-01

    Background KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. Methods We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. Results KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. Conclusions Our findings indicate that KIAA1199 may play an important role in breast

  9. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells.

    PubMed

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  10. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    PubMed Central

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  11. Unmasking risk loci: DNA methylation illuminates the biology of cancer predisposition: analyzing DNA methylation of transcriptional enhancers reveals missed regulatory links between cancer risk loci and genes.

    PubMed

    Aran, Dvir; Hellman, Asaf

    2014-02-01

    Paradoxically, DNA sequence polymorphisms in cancer risk loci rarely correlate with the expression of cancer genes. Therefore, the molecular mechanism underlying an individual's susceptibility to cancer has remained largely unknown. However, recent evaluations of the correlations between DNA methylation and gene expression levels across healthy and cancerous genomes have revealed enrichment of disease-related DNA methylation variations within disease-associated risk loci. Moreover, it appears that transcriptional enhancers embedded in cancer risk loci often contain DNA methylation sites that closely define the expression of prominent cancer genes, despite the lack of significant correlations between gene expression levels and the surrounding disease-associated polymorphic sequences. We suggest that DNA methylation variations may obscure the effect of co-residing risk sequence alleles. Analysis of enhancer methylation data may help to reveal the regulatory circuits underlying predisposition to cancers and other common diseases.

  12. Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells

    NASA Astrophysics Data System (ADS)

    Dvir, Liron; Nissim, Ronen; Alvarez-Elizondo, Martha B.; Weihs, Daphne

    2015-04-01

    Metastasis formation is a major cause of mortality in cancer patients and includes tumor cell relocation to distant organs. A metastatic cell invades through other cells and extracellular matrix by biochemical attachment and mechanical force application. Force is used to move on or through a 2- or 3-dimensional (3D) environment, respectively, or to penetrate a 2D substrate. We have previously shown that even when a gel substrate is impenetrable, metastatic breast cancer cells can still indent it by applying force. Cells typically apply force through the acto-myosin network, which is mechanically connected to the nucleus. We develop a 3D image-analysis to reveal relative locations of the cell elements, and show that as cells apply force to the gel, a coordinated process occurs that involves cytoskeletal remodeling and repositioning of the nucleus. Our approach shows that the actin and microtubules reorganize in the cell, bringing the actin to the leading edge of the cell. In parallel, the nucleus is transported behind the actin, likely by the cytoskeleton, into the indentation dimple formed in the gel. The nucleus volume below the gel surface correlates with indentation depth, when metastatic breast cancer cells indent gels deeply. However, the nucleus always remains above the gel in benign cells, even when small indentations are observed. Determining mechanical processes during metastatic cell invasion can reveal how cells disseminate in the body and can uncover targets for diagnosis and treatment.

  13. Network modelling reveals the mechanism underlying colitis-associated colon cancer and identifies novel combinatorial anti-cancer targets.

    PubMed

    Lu, Junyan; Zeng, Hanlin; Liang, Zhongjie; Chen, Limin; Zhang, Liyi; Zhang, Hao; Liu, Hong; Jiang, Hualiang; Shen, Bairong; Huang, Ming; Geng, Meiyu; Spiegel, Sarah; Luo, Cheng

    2015-10-08

    The connection between inflammation and tumourigenesis has been well established. However, the detailed molecular mechanism underlying inflammation-associated tumourigenesis remains unknown because this process involves a complex interplay between immune microenvironments and epithelial cells. To obtain a more systematic understanding of inflammation-associated tumourigenesis as well as to identify novel therapeutic approaches, we constructed a knowledge-based network describing the development of colitis-associated colon cancer (CAC) by integrating the extracellular microenvironment and intracellular signalling pathways. Dynamic simulations of the CAC network revealed a core network module, including P53, MDM2, and AKT, that may govern the malignant transformation of colon epithelial cells in a pro-tumor inflammatory microenvironment. Furthermore, in silico mutation studies and experimental validations led to a novel finding that concurrently targeting ceramide and PI3K/AKT pathway by chemical probes or marketed drugs achieves synergistic anti-cancer effects. Overall, our network model can guide further mechanistic studies on CAC and provide new insights into the design of combinatorial cancer therapies in a rational manner.

  14. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces

    PubMed Central

    Engin, H. Billur; Kreisberg, Jason F.; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10−4) and oncogenes (Odds Ratio 1.17, P-value < 10−3). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10−8). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer

  15. Bioinformatic and metabolomic analysis reveals miR-155 regulates thiamine level in breast cancer.

    PubMed

    Kim, Sinae; Rhee, Je-keun; Yoo, Hyun Ju; Lee, Hee Jin; Lee, Eun Ji; Lee, Jong Won; Yu, Jong Han; Son, Byung Ho; Gong, Gyungyup; Kim, Sung Bae; Singh, Shree Ram; Ahn, Sei Hyun; Chang, Suhwan

    2015-02-28

    microRNA-155 (miR-155) is one of the well-known oncogenic miRNA implicated in various types of tumors. Thiamine, commonly known as vitamin B1, is one of critical cofactors for energy metabolic enzymes including pyruvate dehydrogenase, alpha ketoglutarate dehydrogenase, and transketolase. Here we report a novel role of miR-155 in cancer metabolism through the up-regulation of thiamine in breast cancer cells. A bioinformatic analysis of miRNA array and metabolite-profiling data from NCI-60 cancer cell panel revealed thiamine as a metabolite positively correlated with the miR-155 expression level. We confirmed it in MCF7, MDA-MB-436 and two human primary breast cancer cells by showing reduced thiamine levels upon a knock-down of miR-155. To understand how the miR-155 controls thiamine level, a set of key molecules for thiamine homeostasis were further analyzed after the knockdown of miR-155. The results showed the expression of two thiamine transporter genes (SLC19A2, SLC25A19) as well as thiamine pyrophosphokinase-1 (TPK1) were decreased in both RNA and protein level in miR-155 dependent manner. Finally, we confirm the finding by showing a positive correlation between miR-155 and thiamine level in 71 triple negative breast tumors. Taken altogether, our study demonstrates a role of miR-155 in thiamine homeostasis and suggests a function of this oncogenic miRNA on breast cancer metabolism.

  16. Quantitative Proteomics Reveals That Enzymes of the Ketogenic Pathway Are Associated with Prostate Cancer Progression*

    PubMed Central

    Saraon, Punit; Cretu, Daniela; Musrap, Natasha; Karagiannis, George S.; Batruch, Ihor; Drabovich, Andrei P.; van der Kwast, Theodorus; Mizokami, Atsushi; Morrissey, Colm; Jarvi, Keith; Diamandis, Eleftherios P.

    2013-01-01

    Prostate cancer is the most common malignancy and the second leading cause of cancer-related deaths in men. One common treatment is androgen-deprivation therapy, which reduces symptoms in most patients. However, over time, patients develop tumors that are androgen-independent and ultimately fatal. The mechanisms that cause this transition remain largely unknown, and as a result, there are no effective treatments against androgen-independent prostate cancer. As a model platform, we used the LNCaP cell line and its androgen-independent derivative, LNCaP-SF. Utilizing stable isotope labeling with amino acids in cell culture coupled to mass spectrometry, we assessed the differential global protein expression of the two cell lines. Our proteomic analysis resulted in the quantification of 3355 proteins. Bioinformatic prioritization resulted in 42 up-regulated and 46 down-regulated proteins in LNCaP-SF cells relative to LNCaP cells. Our top candidate, HMGCS2, an enzyme involved in ketogenesis, was found to be 9-fold elevated in LNCaP-SF cells, based on peptide ratios. After analyzing the remaining enzymes of this pathway (ACAT1, BDH1, HMGCL, and OXCT1), we observed increased expression of these proteins in the LNCaP-SF cells, which was further verified using Western blotting. To determine whether these enzymes were up-regulated in clinical samples, we performed quantitative PCR and immunohistochemistry on human prostate cancer tissues, from which we observed significantly increased transcript and protein levels in high-grade cancer (Gleason grade ≥ 8). In addition, we observed significant elevation of these enzymes in the LuCaP 96AI castration-resistant xenograft. Further assessment of ACAT1 on human castration-resistant metastatic prostate cancer tissues revealed substantially elevated expression of ACAT1 in these specimens. Taken together, our results indicate that enzymes of the ketogenic pathway are up-regulated in high-grade prostate cancer and could serve as

  17. Quantitative proteomics reveals that enzymes of the ketogenic pathway are associated with prostate cancer progression.

    PubMed

    Saraon, Punit; Cretu, Daniela; Musrap, Natasha; Karagiannis, George S; Batruch, Ihor; Drabovich, Andrei P; van der Kwast, Theodorus; Mizokami, Atsushi; Morrissey, Colm; Jarvi, Keith; Diamandis, Eleftherios P

    2013-06-01

    Prostate cancer is the most common malignancy and the second leading cause of cancer-related deaths in men. One common treatment is androgen-deprivation therapy, which reduces symptoms in most patients. However, over time, patients develop tumors that are androgen-independent and ultimately fatal. The mechanisms that cause this transition remain largely unknown, and as a result, there are no effective treatments against androgen-independent prostate cancer. As a model platform, we used the LNCaP cell line and its androgen-independent derivative, LNCaP-SF. Utilizing stable isotope labeling with amino acids in cell culture coupled to mass spectrometry, we assessed the differential global protein expression of the two cell lines. Our proteomic analysis resulted in the quantification of 3355 proteins. Bioinformatic prioritization resulted in 42 up-regulated and 46 down-regulated proteins in LNCaP-SF cells relative to LNCaP cells. Our top candidate, HMGCS2, an enzyme involved in ketogenesis, was found to be 9-fold elevated in LNCaP-SF cells, based on peptide ratios. After analyzing the remaining enzymes of this pathway (ACAT1, BDH1, HMGCL, and OXCT1), we observed increased expression of these proteins in the LNCaP-SF cells, which was further verified using Western blotting. To determine whether these enzymes were up-regulated in clinical samples, we performed quantitative PCR and immunohistochemistry on human prostate cancer tissues, from which we observed significantly increased transcript and protein levels in high-grade cancer (Gleason grade ≥ 8). In addition, we observed significant elevation of these enzymes in the LuCaP 96AI castration-resistant xenograft. Further assessment of ACAT1 on human castration-resistant metastatic prostate cancer tissues revealed substantially elevated expression of ACAT1 in these specimens. Taken together, our results indicate that enzymes of the ketogenic pathway are up-regulated in high-grade prostate cancer and could serve as

  18. Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients

    PubMed Central

    Wang, Chih-Yang; Lai, Ming-Derg; Phan, Nam Nhut; Sun, Zhengda; Lin, Yen-Chang

    2015-01-01

    Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment. PMID:26147197

  19. Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients.

    PubMed

    Wang, Chih-Yang; Lai, Ming-Derg; Phan, Nam Nhut; Sun, Zhengda; Lin, Yen-Chang

    2015-01-01

    Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment.

  20. Endogenous molecular network reveals two mechanisms of heterogeneity within gastric cancer

    PubMed Central

    Li, Site; Zhu, Xiaomei; Liu, Bingya; Wang, Gaowei; Ao, Ping

    2015-01-01

    Intratumor heterogeneity is a common phenomenon and impedes cancer therapy and research. Gastric cancer (GC) cells have generally been classified into two heterogeneous cellular phenotypes, the gastric and intestinal types, yet the mechanisms of maintaining two phenotypes and controlling phenotypic transition are largely unknown. A qualitative systematic framework, the endogenous molecular network hypothesis, has recently been proposed to understand cancer genesis and progression. Here, a minimal network corresponding to such framework was found for GC and was quantified via a stochastic nonlinear dynamical system. We then further extended the framework to address the important question of intratumor heterogeneity quantitatively. The working network characterized main known features of normal gastric epithelial and GC cell phenotypes. Our results demonstrated that four positive feedback loops in the network are critical for GC cell phenotypes. Moreover, two mechanisms that contribute to GC cell heterogeneity were identified: particular positive feedback loops are responsible for the maintenance of intestinal and gastric phenotypes; GC cell progression routes that were revealed by the dynamical behaviors of individual key components are heterogeneous. In this work, we constructed an endogenous molecular network of GC that can be expanded in the future and would broaden the known mechanisms of intratumor heterogeneity. PMID:25962957

  1. Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer

    PubMed Central

    Arthur, Janelle C.; Gharaibeh, Raad Z.; Mühlbauer, Marcus; Perez-Chanona, Ernesto; Uronis, Joshua M.; McCafferty, Jonathan; Fodor, Anthony A.; Jobin, Christian

    2014-01-01

    Enterobacteria, especially Escherichia coli, are abundant in patients with inflammatory bowel disease or colorectal cancer (CRC). However, it is unclear whether cancer is promoted by inflammation-induced expansion of E. coli and/or changes in expression of specific microbial genes. Here we use longitudinal (2, 12 and 20 weeks) 16S rRNA sequencing of luminal microbiota from ex-germ free mice to show that inflamed Il10−/− mice maintain a higher abundance of Enterobacteriaceae than healthy wild-type mice. Experiments with mono-colonized Il10−/− mice reveal that host inflammation is necessary for E. coli cancer-promoting activity. RNA-sequence analysis indicates significant changes in E. coli gene catalogue in Il10−/− mice, with changes mostly driven by adaptation to the intestinal environment. Expression of specific genes present in the tumor-promoting E. coli pks island are modulated by inflammation/CRC development. Thus, progression of inflammation in Il10−/− mice supports Enterobacteriaceae and alters a small subset of microbial genes important for tumor development. PMID:25182170

  2. Integrative Molecular Profiling Reveals Asparagine Synthetase Is a Target in Castration-Resistant Prostate Cancer

    PubMed Central

    Sircar, Kanishka; Huang, Heng; Hu, Limei; Cogdell, David; Dhillon, Jasreman; Tzelepi, Vassiliki; Efstathiou, Eleni; Koumakpayi, Ismaël H.; Saad, Fred; Luo, Dijun; Bismar, Tarek A.; Aparicio, Ana; Troncoso, Patricia; Navone, Nora; Zhang, Wei

    2013-01-01

    The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells. PMID:22245216

  3. Singularity analysis of the AKT signaling pathway reveals connections between cancer and metabolic diseases.

    PubMed

    Wang, Guanyu

    2010-01-01

    Connections between cancer and metabolic diseases may consist in the complex network of interactions among a common set of biomolecules. By applying singularity and bifurcation analysis, the phenotypes constrained by the AKT signaling pathway are identified and mapped onto the parameter space, which include cancer and certain metabolic diseases. By considering physiologic properties (sensitivity, robustness and adaptivity) the AKT pathway must possess in order to efficiently sense growth factors and nutrients, the region of normal responses is located. To optimize these properties, the intracellular concentration of the AKT protein must be sufficiently high to saturate its enzymes; the strength of the positive feedback must be stronger than that of the negative feedback. The analysis illuminates the parameter space and reveals system-level mechanisms in regulating biological functions (cell growth, survival, proliferation and metabolism) and how their deregulation may lead to the development of diseases. The analytical expressions summarize the synergistic interactions among many molecules, which provides valuable insights into therapeutic interventions. In particular, a strategy for overcoming the limitations of mTOR inhibition is proposed for cancer therapy.

  4. Endogenous Voltage Potentials and the Microenvironment: Bioelectric Signals that Reveal, Induce and Normalize Cancer

    PubMed Central

    Chernet, Brook; Levin, Michael

    2014-01-01

    Cancer may be a disease of geometry: a misregulation of the field of information that orchestrates individual cells’ activities towards normal anatomy. Recent work identified molecular mechanisms underlying a novel system of developmental control: bioelectric gradients. Endogenous spatio-temporal differences in resting potential of non-neural cells provide instructive cues for cell regulation and complex patterning during embryogenesis and regeneration. It is now appreciated that these cues are an important layer of the dysregulation of cell: cell interactions that leads to cancer. Abnormal depolarization of resting potential (Vmem) is a convenient marker for neoplasia and activates a metastatic phenotype in genetically-normal cells in vivo. Moreover, oncogene expression depolarizes cells that form tumor-like structures, but is unable to form tumors if this depolarization is artificially prevented by misexpression of hyperpolarizing ion channels. Vmem triggers metastatic behaviors at considerable distance, mediated by transcriptional and epigenetic effects of electrically-modulated flows of serotonin and butyrate. While in vivo data on voltages in carcinogenesis comes mainly from the amphibian model, unbiased genetic screens and network profiling in rodents and human tissues reveal several ion channel proteins as bona fide oncogene and promising targets for cancer drug development. However, we propose that a focus on specific channel genes is just the tip of the iceberg. Bioelectric state is determined by post-translational gating of ion channels, not only from genetically-specified complements of ion translocators. A better model is a statistical dynamics view of spatial Vmem gradients. Cancer may not originate at the single cell level, since gap junctional coupling results in multi-cellular physiological networks with multiple stable attractors in bioelectrical state space. New medical applications await a detailed understanding of the mechanisms by which organ

  5. HPV-18 transformed cells fail to arrest in G1 in response to quercetin treatment.

    PubMed

    Beniston, R G; Campo, M S

    2005-05-01

    Previous work with primary human keratinocytes demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 with concomitant elevation of the cyclin-dependent kinase inhibitor (cdki) p27Kip1. Expression of the human papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins, under transcriptional control of a heterologous promoter, in transformed keratinocytes failed to abrogate this arrest [Beniston, R., Campo, M.S., 2003. Quercetin elevates p27Kip1 and arrests both primary and HPV-16 E6/E7 transformed human keratinocytes in G1. Oncogene 22, 5504-5514]. Given the link between papillomavirus infection, bracken fern in the diet and cancer of the oesophagus in humans, we wished to investigate further whether cells transformed by the whole genome of HPV-16 or HPV-18, with E6 and E7 under the transcriptional control of their respective homologous promoters, would be similarly arrested in G1 by quercetin. In agreement with earlier work, quercetin arrested HPV-16 transformed cells in G1 with an increase in the cyclin-dependent kinase inhibitor p27Kip1. However, HPV-18 transformed cells did not arrest after quercetin treatment. The failure of HPV-18 transformed cells to arrest in G1 was linked to the up-regulation of the HPV-18 long control region (LCR) by quercetin, maintaining high expression of the viral transforming proteins. Transcriptional up-regulation of the HPV-18 LCR was mediated by a "quercetin responsive element" homologous to the one identified previously in the bovine papillomavirus type 4 (BPV-4) LCR.

  6. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells.

    PubMed

    Liu, Xiang; Du, Liying; Feng, Renqing

    2013-07-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells. Here, we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Western blot analysis demonstrated the down-regulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2. Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3β). Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKT pathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression. The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity, whereas the p27 Kip1 expression was increased. In addition, knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2, AKT, and GSK3β. After c-Src depletion by siRNAs, we observed significant down-regulation of cyclin D1 and cyclin E, and up-regulation of p27 Kip1. These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  7. Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process

    PubMed Central

    Chandran, Uma R; Ma, Changqing; Dhir, Rajiv; Bisceglia, Michelle; Lyons-Weiler, Maureen; Liang, Wenjing; Michalopoulos, George; Becich, Michael; Monzon, Federico A

    2007-01-01

    Background Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets. Methods Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors. Results The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1). Conclusion We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer. PMID:17430594

  8. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer.

    PubMed

    Zauri, Melania; Berridge, Georgina; Thézénas, Marie-Laëtitia; Pugh, Kathryn M; Goldin, Robert; Kessler, Benedikt M; Kriaucionis, Skirmantas

    2015-08-01

    Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues.

  9. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer.

    PubMed

    Zauri, Melania; Berridge, Georgina; Thézénas, Marie-Laëtitia; Pugh, Kathryn M; Goldin, Robert; Kessler, Benedikt M; Kriaucionis, Skirmantas

    2015-08-01

    Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues. PMID:26200337

  10. Transcriptomic profiling of urine extracellular vesicles reveals alterations of CDH3 in prostate cancer

    PubMed Central

    Sanchez-Mosquera, Pilar; Ugalde-Olano, Aitziber; González, Esperanza; Cortazar, Ana R.; Palomo, Laura; Fernández-Ruiz, Sonia; Lacasa-Viscasillas, Isabel; Berdasco, Maria; Sutherland, James D.; Barrio, Rosa; Zabala-Letona, Amaia; Martín-Martín, Natalia; Arruabarrena-Aristorena, Amaia; Valcarcel-Jimenez, Lorea; Caro-Maldonado, Alfredo; Gonzalez-Tampan, Jorge; Cachi-Fuentes, Guido; Esteller, Manel; Aransay, Ana M.; Unda, Miguel

    2016-01-01

    Extracellular vesicles (EV) are emerging structures with promising properties for intercellular communication. In addition, the characterization of EV in biofluids is an attractive source of non-invasive diagnostic, prognostic and predictive biomarkers. Here we show that urinary EV (uEV) from prostate cancer (PCa) patients exhibit genuine and differential physical and biological properties compared to benign prostate hyperplasia (BPH). Importantly, transcriptomics characterization of uEVs led us to define the decreased abundance of Cadherin 3, type 1 (CDH3) transcript in uEV from PCa patients. Tissue and cell line analysis strongly suggested that the status of CDH3 in uEVs is a distal reflection of changes in the expression of this cadherin in the prostate tumor. CDH3 was negatively regulated at the genomic, transcriptional, and epigenetic level in PCa. Our results reveal that uEVs could represent a non-invasive tool to inform about the molecular alterations in PCa. PMID:26771841

  11. An acute adrenal insufficiency revealing pituitary metastases of lung cancer in an elderly patient

    PubMed Central

    Marmouch, Hela; Arfa, Sondes; Mohamed, Saoussen Cheikh; Slim, Tensim; Khochtali, Ines

    2016-01-01

    Metastases of solid tumors to the pituitary gland are often asymptomatic or appereas as with diabetes insipid us. Pituitary metastases more commonly affect the posterior lobe and the infundibulum than the anterior lobe. The presentation with an acute adrenal insufficiency is a rare event. A 69-year-old men presented with vomiting, low blood pressure and hypoglycemia. Hormonal exploration confirmed a hypopituitarism. Appropriate therapy was initiated urgently. The hypothalamic-pituitary MRI showed a pituitary hypertrophy, a nodular thickening of the pituitary stalk. The chest X Rays revealed pulmonary opacity. Computed tomography scan of the chest showed a multiples tumors with mediastinal lymphadenopathy. Bronchoscopy and biopsy demonstrated a pulmonary adenocarcinoma. Hence we concluded to a lung cancer with multiple pituitary and adrenal gland metastases. This case emphasizes the need for an etiological investigation of acute adrenal insufficiency after treatment of acute phase. PMID:27200139

  12. T-Cell-Specific Deletion of Map3k1 Reveals the Critical Role for Mekk1 and Jnks in Cdkn1b-Dependent Proliferative Expansion

    PubMed Central

    Suddason, Tesha; Anwar, Saba; Charlaftis, Nikolaos; Gallagher, Ewen

    2016-01-01

    Summary MAPK signaling is important for T lymphocyte development, homeostasis, and effector responses. To better understand the role of Mekk1 (encoded by Map3k1) in T cells, we conditionally deleted Map3k1 in LckCre/+Map3k1f/f mice, and these display larger iNKT cell populations within the liver, spleen, and bone marrow. Mekk1 signaling controls splenic and liver iNKT cell expansion in response to glycolipid antigen. LckCre/+Map3k1f/f mice have enhanced liver damage in response to glycolipid antigen. Mekk1 regulates Jnk activation in iNKT cells and binds and transfers Lys63-linked poly-ubiquitin onto Carma1. Map3k1 is critical for the regulation of p27Kip1 (encoded by Cdkn1b). PMID:26774476

  13. An Atlas of the Human Kinome Reveals the Mutational Landscape Underlying Dysregulated Phosphorylation Cascades in Cancer.

    PubMed

    Olow, Aleksandra; Chen, Zhongzhong; Niedner, R Hannes; Wolf, Denise M; Yau, Christina; Pankov, Aleksandr; Lee, Evelyn Pei Rong; Brown-Swigart, Lamorna; van 't Veer, Laura J; Coppé, Jean-Philippe

    2016-04-01

    Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular subnetworks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks that drive tumorigenesis. Cancer Res; 76(7); 1733-45. ©2016 AACR. PMID:26921330

  14. Sustained conditional knockdown reveals intracellular bone sialoprotein as essential for breast cancer skeletal metastasis.

    PubMed

    Kovacheva, Marineta; Zepp, Michael; Berger, Stefan M; Berger, Martin R

    2014-07-30

    Increased bone sialoprotein (BSP) serum levels are related to breast cancer skeletal metastasis, but their relevance is unknown. We elucidated novel intracellular BSP functions by a conditional knockdown of BSP. Conditional MDA-MB-231 subclones were equipped with a novel gene expression cassette containing a tet-reg-ulated miRNA providing knockdown of BSP production. These clones were used to assess the effect of BSP on morphology, proliferation, migration, colony formation and gene expression in vitro, and on soft tissue and osteolytic le-sions in a xenograft model by three imaging methods. BSP knockdown caused significant anti-proliferative, anti-migratory and anti-clonogenic effects in vitro (p<0.001). In vivo, significant de-creases of soft tissue and osteolytic lesions (p<0.03) were recorded after 3 weeks of miRNA treatment, leading to complete remission within 6 weeks. Microarray data revealed that 0.3% of genes were modulated in response to BSP knockdown. Upregulated genes included the endoplasmic reticulum stress genes ATF3 and DDIT3, the tumor suppressor gene EGR1, ID2 (related to breast epithelial differentiation), c-FOS and SERPINB2, whereas the metastasis associated genes CD44 and IL11 were downregulated. Also, activation of apoptotic pathways was demonstrated. These results implicate that intracellular BSP is essential for breast cancer skeletal metastasis and a target for treating these lesions.

  15. Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells.

    PubMed

    Oikawa, Tsunekazu; Wauthier, Eliane; Dinh, Timothy A; Selitsky, Sara R; Reyna-Neyra, Andrea; Carpino, Guido; Levine, Ronald; Cardinale, Vincenzo; Klimstra, David; Gaudio, Eugenio; Alvaro, Domenico; Carrasco, Nancy; Sethupathy, Praveen; Reid, Lola M

    2015-01-01

    The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells--newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies.

  16. Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells.

    PubMed

    Oikawa, Tsunekazu; Wauthier, Eliane; Dinh, Timothy A; Selitsky, Sara R; Reyna-Neyra, Andrea; Carpino, Guido; Levine, Ronald; Cardinale, Vincenzo; Klimstra, David; Gaudio, Eugenio; Alvaro, Domenico; Carrasco, Nancy; Sethupathy, Praveen; Reid, Lola M

    2015-01-01

    The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells--newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies. PMID:26437858

  17. TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer.

    PubMed

    Nyquist, Michael D; Li, Yingming; Hwang, Tae Hyun; Manlove, Luke S; Vessella, Robert L; Silverstein, Kevin A T; Voytas, Daniel F; Dehm, Scott M

    2013-10-22

    Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC.

  18. Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells

    PubMed Central

    Oikawa, Tsunekazu; Wauthier, Eliane; Dinh, Timothy A.; Selitsky, Sara R.; Reyna-Neyra, Andrea; Carpino, Guido; Levine, Ronald; Cardinale, Vincenzo; Klimstra, David; Gaudio, Eugenio; Alvaro, Domenico; Carrasco, Nancy; Sethupathy, Praveen; Reid, Lola M.

    2015-01-01

    The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells—newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies. PMID:26437858

  19. Breast cancer proteomics reveals correlation between estrogen receptor status and differential phosphorylation of PGRMC1

    PubMed Central

    Neubauer, Hans; Clare, Susan E; Wozny, Wojciech; Schwall, Gerhard P; Poznanović, Slobodan; Stegmann, Werner; Vogel, Ulrich; Sotlar, Karl; Wallwiener, Diethelm; Kurek, Raffael; Fehm, Tanja; Cahill, Michael A

    2008-01-01

    Introduction Breast tumors lacking the estrogen receptor-α (ER-α) have increased incidence of resistance to therapy and poorer clinical prognosis. Methods Whole tissue sections from 16 cryopreserved breast cancer tumors that were either positive or negative for the ER (eight ER positive and eight ER negative) were differentially analyzed by multiplex imaging of two-dimensional PAGE gels using 54 cm isoelectric focusing. Differentially detected spots of Progesterone Receptor Membrane Component 1 (PGRMC1) were shown to differ in phosphorylation status by differential two dimensional polyacrylamide gel electrophoresis of phosphatase-treated tumor proteins. Site directed mutagenesis was used to create putative phosphorylation site point mutants in PGRMC1. Stable transfectants of these mutants in MCF7 cells were assayed for their survival after oxidative stress, and for AKT kinase phosphorylation. Immune fluorescence using anti-PGRMC1 monoclonal antibody 5G7 was performed on breast cancer tissue microarrays. Results Proteins significantly differentially abundant between estrogen receptor negative and estrogen receptor positive tumors at the 0.1% level were consistent with published profiles, suggesting an altered keratin pool, and increased inflammation and wound responses in estrogen receptor negative tumors. Two of three spots of PGRMC1 were more abundant in estrogen receptor negative tumors. Phosphatase treatment of breast tumor proteins indicated that the PGRMC1 isoforms differed in their phosphorylation status. Simultaneous mutation of PGRMC1 serine-56 and serine-181 fully abrogated the sensitivity of stably transfected MCF7 breast cancer cells to peroxide-induced cell death. Immune fluorescence revealed that PGRMC1 was primarily expressed in ER-negative basal epithelial cells of mammary ductules. Even in advanced tumors, high levels of ER or PGRMC1 were almost mutually exclusive in individual cells. In five out of five examined ductal in situ breast cancers of

  20. Catabolism of exogenous lactate reveals it as a legitimate metabolic substrate in breast cancer.

    PubMed

    Kennedy, Kelly M; Scarbrough, Peter M; Ribeiro, Anthony; Richardson, Rachel; Yuan, Hong; Sonveaux, Pierre; Landon, Chelsea D; Chi, Jen-Tsan; Pizzo, Salvatore; Schroeder, Thies; Dewhirst, Mark W

    2013-01-01

    Lactate accumulation in tumors has been associated with metastases and poor overall survival in cancer patients. Lactate promotes angiogenesis and metastasis, providing rationale for understanding how it is processed by cells. The concentration of lactate in tumors is a balance between the amount produced, amount carried away by vasculature and if/how it is catabolized by aerobic tumor or stromal cells. We examined lactate metabolism in human normal and breast tumor cell lines and rat breast cancer: 1. at relevant concentrations, 2. under aerobic vs. hypoxic conditions, 3. under conditions of normo vs. hypoglucosis. We also compared the avidity of tumors for lactate vs. glucose and identified key lactate catabolites to reveal how breast cancer cells process it. Lactate was non-toxic at clinically relevant concentrations. It was taken up and catabolized to alanine and glutamate by all cell lines. Kinetic uptake rates of lactate in vivo surpassed that of glucose in R3230Ac mammary carcinomas. The uptake appeared specific to aerobic tumor regions, consistent with the proposed "metabolic symbiont" model; here lactate produced by hypoxic cells is used by aerobic cells. We investigated whether treatment with alpha-cyano-4-hydroxycinnamate (CHC), a MCT1 inhibitor, would kill cells in the presence of high lactate. Both 0.1 mM and 5 mM CHC prevented lactate uptake in R3230Ac cells at lactate concentrations at ≤ 20 mM but not at 40 mM. 0.1 mM CHC was well-tolerated by R3230Ac and MCF7 cells, but 5 mM CHC killed both cell lines ± lactate, indicating off-target effects. This study showed that breast cancer cells tolerate and use lactate at clinically relevant concentrations in vitro (± glucose) and in vivo. We provided additional support for the metabolic symbiont model and discovered that breast cells prevailingly take up and catabolize lactate, providing rationale for future studies on manipulation of lactate catabolism pathways for therapy.

  1. Exome sequencing of a colorectal cancer family reveals shared mutation pattern and predisposition circuitry along tumor pathways

    PubMed Central

    Suleiman, Suleiman H.; Koko, Mahmoud E.; Nasir, Wafaa H.; Elfateh, Ommnyiah; Elgizouli, Ubai K.; Abdallah, Mohammed O. E.; Alfarouk, Khalid O.; Hussain, Ayman; Faisal, Shima; Ibrahim, Fathelrahamn M. A.; Romano, Maurizio; Sultan, Ali; Banks, Lawrence; Newport, Melanie; Baralle, Francesco; Elhassan, Ahmed M.; Mohamed, Hiba S.; Ibrahim, Muntaser E.

    2015-01-01

    The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed hMSH2 loss of function SNV consistent with Lynch syndrome. Sets of genes harboring insertions–deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions–deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values (p-value 5e-6). NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins. PMID:26442106

  2. FT-IR Microspectrometry Reveals the Variation of Membrane Polarizability due to Epigenomic Effect on Epithelial Ovarian Cancer

    PubMed Central

    Hsu, Morris M. H.; Huang, Pei-Yu; Lee, Yao-Chang; Fang, Yuang-Chuen; Chan, Michael W. Y.; Lee, Cheng-I

    2014-01-01

    Ovarian cancer, as well as other cancers, is primarily caused by methylation at cytosines in CpG islands, but the current marker for ovarian cancer is low in sensitivity and failed in early-stage detection. Fourier transform infrared (FT-IR) spectroscopy is powerful in analysis of functional groups within molecules, and infrared microscopy illustrates the location of specific groups within single cells. In this study, we applied HPLC and FT-IR microspectrometry to study normal epithelial ovarian cell line immortalized ovarian surface epithelium (IOSE), two epithelial ovarian cell lines (A2780 and CP70) with distinct properties, and the effect of a cancer drug 5-aza-2'-deoxycytidine (5-aza) without labeling. Our results reveal that inhibition of methylation on cytosine with 5-aza initiates the protein expression. Furthermore, paraffin-adsorption kinetic study allows us to distinguish hypermethylated and hypomethyated cells, and this assay can be a potential diagnosis method for cancer screening. PMID:25299694

  3. FT-IR microspectrometry reveals the variation of membrane polarizability due to epigenomic effect on epithelial ovarian cancer.

    PubMed

    Hsu, Morris M H; Huang, Pei-Yu; Lee, Yao-Chang; Fang, Yuang-Chuen; Chan, Michael W Y; Lee, Cheng-I

    2014-10-08

    Ovarian cancer, as well as other cancers, is primarily caused by methylation at cytosines in CpG islands, but the current marker for ovarian cancer is low in sensitivity and failed in early-stage detection. Fourier transform infrared (FT-IR) spectroscopy is powerful in analysis of functional groups within molecules, and infrared microscopy illustrates the location of specific groups within single cells. In this study, we applied HPLC and FT-IR microspectrometry to study normal epithelial ovarian cell line immortalized ovarian surface epithelium (IOSE), two epithelial ovarian cell lines (A2780 and CP70) with distinct properties, and the effect of a cancer drug 5-aza-2'-deoxycytidine (5-aza) without labeling. Our results reveal that inhibition of methylation on cytosine with 5-aza initiates the protein expression. Furthermore, paraffin-adsorption kinetic study allows us to distinguish hypermethylated and hypomethyated cells, and this assay can be a potential diagnosis method for cancer screening.

  4. Metabolomic Profiling Reveals Potential Markers and Bioprocesses Altered in Bladder Cancer Progression

    PubMed Central

    Putluri, Nagireddy; Shojaie, Ali; Vasu, Vihas T; Vareed, Shaiju K.; Nalluri, Srilatha; Putluri, Vasanta; Thangjam, Gagan Singh; Panzitt, Katrin; Tallman, Christopher T.; Butler, Charles; Sana, Theodore R.; Fischer, Steven M.; Sica, Gabriel; Brat, Daniel J.; Shi, Huidong; Palapattu, Ganesh S; Lotan, Yair; Weizer, Alon Z.; Terris, Martha K.; Shariat, Shahrokh F.; Michailidis, George; Sreekumar, Arun

    2011-01-01

    While alterations in xenobiotic metabolism are considered causal in the development of bladder cancer (BCa), the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in BCa. This metabolic signature distinguished both normal and benign bladder from BCa. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing BCa from controls, and also non-muscle from muscle-invasive BCa. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in BCa. In particular, we validated tumor-associated hypermethylation in the CYP1A1 and CYP1B1 promoters of BCa tissues by bisulfite sequence analysis and methylation-specific PCR, and also by in vitro treatment of T-24 BCa cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Further, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of BCa specimens compared to matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of BCa, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression. PMID:21990318

  5. Power-Law Modeling of Cancer Cell Fates Driven by Signaling Data to Reveal Drug Effects

    PubMed Central

    Zhang, Fan; Wu, Min; Kwoh, Chee Keong; Zheng, Jie

    2016-01-01

    Extracellular signals are captured and transmitted by signaling proteins inside a cell. An important type of cellular responses to the signals is the cell fate decision, e.g., apoptosis. However, the underlying mechanisms of cell fate regulation are still unclear, thus comprehensive and detailed kinetic models are not yet available. Alternatively, data-driven models are promising to bridge signaling data with the phenotypic measurements of cell fates. The traditional linear model for data-driven modeling of signaling pathways has its limitations because it assumes that the a cell fate is proportional to the activities of signaling proteins, which is unlikely in the complex biological systems. Therefore, we propose a power-law model to relate the activities of all the measured signaling proteins to the probabilities of cell fates. In our experiments, we compared our nonlinear power-law model with the linear model on three cancer datasets with phosphoproteomics and cell fate measurements, which demonstrated that the nonlinear model has superior performance on cell fates prediction. By in silico simulation of virtual protein knock-down, the proposed model is able to reveal drug effects which can complement traditional approaches such as binding affinity analysis. Moreover, our model is able to capture cell line specific information to distinguish one cell line from another in cell fate prediction. Our results show that the power-law data-driven model is able to perform better in cell fate prediction and provide more insights into the signaling pathways for cancer cell fates than the linear model. PMID:27764199

  6. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

    PubMed Central

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-01-01

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

  7. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold.

    PubMed

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-06-12

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.

  8. Zebrafish reporter lines reveal in vivo signaling pathway activities involved in pancreatic cancer.

    PubMed

    Schiavone, Marco; Rampazzo, Elena; Casari, Alessandro; Battilana, Giusy; Persano, Luca; Moro, Enrico; Liu, Shu; Leach, Steve D; Tiso, Natascia; Argenton, Francesco

    2014-07-01

    Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRAS(G12D) during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRAS(G12D) expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRAS(G12D) line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-KRAS(G12D) showed normal exocrine pancreas development until 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degrees of acinar lesions, characterized by an increase in mesenchymal cells and mixed acinar/ductal features, followed by progressive bowel and liver infiltrations and, finally, highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of KRAS-positive cells and progressive activation of TGFβ and Notch pathways. Increase in TGFβ, following KRAS(G12D) activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma, indicating a tissue-specific regulation of cell signaling pathways. Moreover, our results show that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool for

  9. Cross-Cancer Genome-Wide Analysis of Lung, Ovary, Breast, Prostate, and Colorectal Cancer Reveals Novel Pleiotropic Associations.

    PubMed

    Fehringer, Gordon; Kraft, Peter; Pharoah, Paul D; Eeles, Rosalind A; Chatterjee, Nilanjan; Schumacher, Fredrick R; Schildkraut, Joellen M; Lindström, Sara; Brennan, Paul; Bickeböller, Heike; Houlston, Richard S; Landi, Maria Teresa; Caporaso, Neil; Risch, Angela; Amin Al Olama, Ali; Berndt, Sonja I; Giovannucci, Edward L; Grönberg, Henrik; Kote-Jarai, Zsofia; Ma, Jing; Muir, Kenneth; Stampfer, Meir J; Stevens, Victoria L; Wiklund, Fredrik; Willett, Walter C; Goode, Ellen L; Permuth, Jennifer B; Risch, Harvey A; Reid, Brett M; Bezieau, Stephane; Brenner, Hermann; Chan, Andrew T; Chang-Claude, Jenny; Hudson, Thomas J; Kocarnik, Jonathan K; Newcomb, Polly A; Schoen, Robert E; Slattery, Martha L; White, Emily; Adank, Muriel A; Ahsan, Habibul; Aittomäki, Kristiina; Baglietto, Laura; Blomquist, Carl; Canzian, Federico; Czene, Kamila; Dos-Santos-Silva, Isabel; Eliassen, A Heather; Figueroa, Jonine D; Flesch-Janys, Dieter; Fletcher, Olivia; Garcia-Closas, Montserrat; Gaudet, Mia M; Johnson, Nichola; Hall, Per; Hazra, Aditi; Hein, Rebecca; Hofman, Albert; Hopper, John L; Irwanto, Astrid; Johansson, Mattias; Kaaks, Rudolf; Kibriya, Muhammad G; Lichtner, Peter; Liu, Jianjun; Lund, Eiliv; Makalic, Enes; Meindl, Alfons; Müller-Myhsok, Bertram; Muranen, Taru A; Nevanlinna, Heli; Peeters, Petra H; Peto, Julian; Prentice, Ross L; Rahman, Nazneen; Sanchez, Maria Jose; Schmidt, Daniel F; Schmutzler, Rita K; Southey, Melissa C; Tamimi, Rulla; Travis, Ruth C; Turnbull, Clare; Uitterlinden, Andre G; Wang, Zhaoming; Whittemore, Alice S; Yang, Xiaohong R; Zheng, Wei; Buchanan, Daniel D; Casey, Graham; Conti, David V; Edlund, Christopher K; Gallinger, Steven; Haile, Robert W; Jenkins, Mark; Le Marchand, Loïc; Li, Li; Lindor, Noralene M; Schmit, Stephanie L; Thibodeau, Stephen N; Woods, Michael O; Rafnar, Thorunn; Gudmundsson, Julius; Stacey, Simon N; Stefansson, Kari; Sulem, Patrick; Chen, Y Ann; Tyrer, Jonathan P; Christiani, David C; Wei, Yongyue; Shen, Hongbing; Hu, Zhibin; Shu, Xiao-Ou; Shiraishi, Kouya; Takahashi, Atsushi; Bossé, Yohan; Obeidat, Ma'en; Nickle, David; Timens, Wim; Freedman, Matthew L; Li, Qiyuan; Seminara, Daniela; Chanock, Stephen J; Gong, Jian; Peters, Ulrike; Gruber, Stephen B; Amos, Christopher I; Sellers, Thomas A; Easton, Douglas F; Hunter, David J; Haiman, Christopher A; Henderson, Brian E; Hung, Rayjean J

    2016-09-01

    Identifying genetic variants with pleiotropic associations can uncover common pathways influencing multiple cancers. We took a two-stage approach to conduct genome-wide association studies for lung, ovary, breast, prostate, and colorectal cancer from the GAME-ON/GECCO Network (61,851 cases, 61,820 controls) to identify pleiotropic loci. Findings were replicated in independent association studies (55,789 cases, 330,490 controls). We identified a novel pleiotropic association at 1q22 involving breast and lung squamous cell carcinoma, with eQTL analysis showing an association with ADAM15/THBS3 gene expression in lung. We also identified a known breast cancer locus CASP8/ALS2CR12 associated with prostate cancer, a known cancer locus at CDKN2B-AS1 with different variants associated with lung adenocarcinoma and prostate cancer, and confirmed the associations of a breast BRCA2 locus with lung and serous ovarian cancer. This is the largest study to date examining pleiotropy across multiple cancer-associated loci, identifying common mechanisms of cancer development and progression. Cancer Res; 76(17); 5103-14. ©2016 AACR.

  10. Cross-Cancer Genome-Wide Analysis of Lung, Ovary, Breast, Prostate, and Colorectal Cancer Reveals Novel Pleiotropic Associations.

    PubMed

    Fehringer, Gordon; Kraft, Peter; Pharoah, Paul D; Eeles, Rosalind A; Chatterjee, Nilanjan; Schumacher, Fredrick R; Schildkraut, Joellen M; Lindström, Sara; Brennan, Paul; Bickeböller, Heike; Houlston, Richard S; Landi, Maria Teresa; Caporaso, Neil; Risch, Angela; Amin Al Olama, Ali; Berndt, Sonja I; Giovannucci, Edward L; Grönberg, Henrik; Kote-Jarai, Zsofia; Ma, Jing; Muir, Kenneth; Stampfer, Meir J; Stevens, Victoria L; Wiklund, Fredrik; Willett, Walter C; Goode, Ellen L; Permuth, Jennifer B; Risch, Harvey A; Reid, Brett M; Bezieau, Stephane; Brenner, Hermann; Chan, Andrew T; Chang-Claude, Jenny; Hudson, Thomas J; Kocarnik, Jonathan K; Newcomb, Polly A; Schoen, Robert E; Slattery, Martha L; White, Emily; Adank, Muriel A; Ahsan, Habibul; Aittomäki, Kristiina; Baglietto, Laura; Blomquist, Carl; Canzian, Federico; Czene, Kamila; Dos-Santos-Silva, Isabel; Eliassen, A Heather; Figueroa, Jonine D; Flesch-Janys, Dieter; Fletcher, Olivia; Garcia-Closas, Montserrat; Gaudet, Mia M; Johnson, Nichola; Hall, Per; Hazra, Aditi; Hein, Rebecca; Hofman, Albert; Hopper, John L; Irwanto, Astrid; Johansson, Mattias; Kaaks, Rudolf; Kibriya, Muhammad G; Lichtner, Peter; Liu, Jianjun; Lund, Eiliv; Makalic, Enes; Meindl, Alfons; Müller-Myhsok, Bertram; Muranen, Taru A; Nevanlinna, Heli; Peeters, Petra H; Peto, Julian; Prentice, Ross L; Rahman, Nazneen; Sanchez, Maria Jose; Schmidt, Daniel F; Schmutzler, Rita K; Southey, Melissa C; Tamimi, Rulla; Travis, Ruth C; Turnbull, Clare; Uitterlinden, Andre G; Wang, Zhaoming; Whittemore, Alice S; Yang, Xiaohong R; Zheng, Wei; Buchanan, Daniel D; Casey, Graham; Conti, David V; Edlund, Christopher K; Gallinger, Steven; Haile, Robert W; Jenkins, Mark; Le Marchand, Loïc; Li, Li; Lindor, Noralene M; Schmit, Stephanie L; Thibodeau, Stephen N; Woods, Michael O; Rafnar, Thorunn; Gudmundsson, Julius; Stacey, Simon N; Stefansson, Kari; Sulem, Patrick; Chen, Y Ann; Tyrer, Jonathan P; Christiani, David C; Wei, Yongyue; Shen, Hongbing; Hu, Zhibin; Shu, Xiao-Ou; Shiraishi, Kouya; Takahashi, Atsushi; Bossé, Yohan; Obeidat, Ma'en; Nickle, David; Timens, Wim; Freedman, Matthew L; Li, Qiyuan; Seminara, Daniela; Chanock, Stephen J; Gong, Jian; Peters, Ulrike; Gruber, Stephen B; Amos, Christopher I; Sellers, Thomas A; Easton, Douglas F; Hunter, David J; Haiman, Christopher A; Henderson, Brian E; Hung, Rayjean J

    2016-09-01

    Identifying genetic variants with pleiotropic associations can uncover common pathways influencing multiple cancers. We took a two-stage approach to conduct genome-wide association studies for lung, ovary, breast, prostate, and colorectal cancer from the GAME-ON/GECCO Network (61,851 cases, 61,820 controls) to identify pleiotropic loci. Findings were replicated in independent association studies (55,789 cases, 330,490 controls). We identified a novel pleiotropic association at 1q22 involving breast and lung squamous cell carcinoma, with eQTL analysis showing an association with ADAM15/THBS3 gene expression in lung. We also identified a known breast cancer locus CASP8/ALS2CR12 associated with prostate cancer, a known cancer locus at CDKN2B-AS1 with different variants associated with lung adenocarcinoma and prostate cancer, and confirmed the associations of a breast BRCA2 locus with lung and serous ovarian cancer. This is the largest study to date examining pleiotropy across multiple cancer-associated loci, identifying common mechanisms of cancer development and progression. Cancer Res; 76(17); 5103-14. ©2016 AACR. PMID:27197191

  11. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer

    PubMed Central

    Zauri, Melania; Berridge, Georgina; Thézénas, Marie-Laëtitia; Pugh, Kathryn M.; Goldin, Robert; Kessler, Benedikt M.; Kriaucionis, Skirmantas

    2015-01-01

    Summary Cells require nucleotides to support DNA replication and to repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Since epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands1-3, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2′deoxycytidine (5mdC) has been investigated before4-6, currently it remains unknown how cells deal with the recently identified oxidised forms of 5mdC – 5-hydroxymethyl-2′deoxycytidine (5hmdC), 5-formy-2′deoxycytidine (5fdC) and 5-carboxyl-2′deoxycytidine (5cadC)7-10. Here we demonstrate that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Interestingly, by screening cancer cell lines for growth defects following exposure to 5hmdC, we unexpectedly identify a subset of cell lines where 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches we discover that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidised epigenetic bases, and suggest a therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment

  12. A common promoter hypomethylation signature in invasive breast, liver and prostate cancer cell lines reveals novel targets involved in cancer invasiveness

    PubMed Central

    Yi, Cao; Li, Chen Chen; Yu, Patricia; Arakelian, Ani; Tanvir, Imrana; Khan, Haseeb Ahmed; Rabbani, Shafaat

    2015-01-01

    Cancer invasion and metastasis is the most morbid aspect of cancer and is governed by different cellular mechanisms than those driving the deregulated growth of tumors. We addressed here the question of whether a common DNA methylation signature of invasion exists in cancer cells from different origins that differentiates invasive from non-invasive cells. We identified a common DNA methylation signature consisting of hyper- and hypomethylation and determined the overlap of differences in DNA methylation with differences in mRNA expression using expression array analyses. A pathway analysis reveals that the hypomethylation signature includes some of the major pathways that were previously implicated in cancer migration and invasion such as TGF beta and ERBB2 triggered pathways. The relevance of these hypomethylation events in human tumors was validated by identification of the signature in several publicly available databases of human tumor transcriptomes. We shortlisted novel invasion promoting candidates and tested the role of four genes in cellular invasiveness from the list C11orf68, G0S2, SHISA2 and TMEM156 in invasiveness using siRNA depletion. Importantly these genes are upregulated in human cancer specimens as determined by immunostaining of human normal and cancer breast, liver and prostate tissue arrays. Since these genes are activated in cancer they constitute a group of targets for specific pharmacological inhibitors of cancer invasiveness. SUMMARY Our study provides evidence that common DNA hypomethylation signature exists between cancer cells derived from different tissues, pointing to a common mechanism of cancer invasiveness in cancer cells from different origins that could serve as drug targets. PMID:26427334

  13. Differential secretome analysis reveals CST6 as a suppressor of breast cancer bone metastasis

    PubMed Central

    Jin, Lei; Zhang, Yan; Li, Hui; Yao, Ling; Fu, Da; Yao, Xuebiao; Xu, Lisa X; Hu, Xiaofang; Hu, Guohong

    2012-01-01

    Bone metastasis is a frequent complication of breast cancer and a common cause of morbidity and mortality from the disease. During metastasis secreted proteins play crucial roles in the interactions between cancer cells and host stroma. To characterize the secreted proteins that are associated with breast cancer bone metastasis, we preformed a label-free proteomic analysis to compare the secretomes of four MDA-MB-231 (MDA231) derivative cell lines with varied capacities of bone metastasis. A total of 128 proteins were found to be consistently up-/down-regulated in the conditioned medium of bone-tropic cancer cells. The enriched molecular functions of the altered proteins included receptor binding and peptidase inhibition. Through additional transcriptomic analyses of breast cancer cells, we selected cystatin E/M (CST6), a cysteine protease inhibitor down-regulated in bone-metastatic cells, for further functional studies. Our results showed that CST6 suppressed the proliferation, colony formation, migration and invasion of breast cancer cells. The suppressive function against cancer cell motility was carried out by cancer cell-derived soluble CST6. More importantly, ectopic expression of CST6 in cancer cells rescued mice from overt osteolytic metastasis and deaths in the animal study, while CST6 knockdown markedly enhanced cancer cell bone metastasis and shortened animal survival. Overall, our study provided a systemic secretome analysis of breast cancer bone tropism and established secreted CST6 as a bona fide suppressor of breast cancer osteolytic metastasis. PMID:22688893

  14. In Search of a Cure for Proteostasis-Addicted Cancer: A AAA Target Revealed.

    PubMed

    Xia, Di; Ye, Yihong

    2015-11-01

    Tumorigenesis is often associated with an unbalanced protein homeostasis (proteostasis) network, which sensitizes cancer cells to drugs targeting protein quality control (PQC) regulators. In this issue of Cancer Cell, Anderson and colleagues investigated the anti-cancer activity of a new class of inhibitor against a multi-functional ATPase essential for proteostasis maintenance. PMID:26555170

  15. In Search of a Cure for Proteostasis-Addicted Cancer: A AAA Target Revealed.

    PubMed

    Xia, Di; Ye, Yihong

    2015-11-01

    Tumorigenesis is often associated with an unbalanced protein homeostasis (proteostasis) network, which sensitizes cancer cells to drugs targeting protein quality control (PQC) regulators. In this issue of Cancer Cell, Anderson and colleagues investigated the anti-cancer activity of a new class of inhibitor against a multi-functional ATPase essential for proteostasis maintenance.

  16. Aberrant gene expression in mucosa adjacent to tumor reveals a molecular crosstalk in colon cancer

    PubMed Central

    2014-01-01

    Background A colorectal tumor is not an isolated entity growing in a restricted location of the body. The patient’s gut environment constitutes the framework where the tumor evolves and this relationship promotes and includes a complex and tight correlation of the tumor with inflammation, blood vessels formation, nutrition, and gut microbiome composition. The tumor influence in the environment could both promote an anti-tumor or a pro-tumor response. Methods A set of 98 paired adjacent mucosa and tumor tissues from colorectal cancer (CRC) patients and 50 colon mucosa from healthy donors (246 samples in total) were included in this work. RNA extracted from each sample was hybridized in Affymetrix chips Human Genome U219. Functional relationships between genes were inferred by means of systems biology using both transcriptional regulation networks (ARACNe algorithm) and protein-protein interaction networks (BIANA software). Results Here we report a transcriptomic analysis revealing a number of genes activated in adjacent mucosa from CRC patients, not activated in mucosa from healthy donors. A functional analysis of these genes suggested that this active reaction of the adjacent mucosa was related to the presence of the tumor. Transcriptional and protein-interaction networks were used to further elucidate this response of normal gut in front of the tumor, revealing a crosstalk between proteins secreted by the tumor and receptors activated in the adjacent colon tissue; and vice versa. Remarkably, Slit family of proteins activated ROBO receptors in tumor whereas tumor-secreted proteins transduced a cellular signal finally activating AP-1 in adjacent tissue. Conclusions The systems-level approach provides new insights into the micro-ecology of colorectal tumorogenesis. Disrupting this intricate molecular network of cell-cell communication and pro-inflammatory microenvironment could be a therapeutic target in CRC patients. PMID:24597571

  17. Exome sequencing reveals AMER1 as a frequently mutated gene in colorectal cancer

    PubMed Central

    Sanz-Pamplona, Rebeca; Lopez-Doriga, Adriana; Paré-Brunet, Laia; Lázaro, Kira; Bellido, Fernando; Alonso, M. Henar; Aussó, Susanna; Guinó, Elisabet; Beltrán, Sergi; Castro-Giner, Francesc; Gut, Marta; Sanjuan, Xavier; Closa, Adria; Cordero, David; Morón-Duran, Francisco D.; Soriano, Antonio; Salazar, Ramón; Valle, Laura; Moreno, Victor

    2015-01-01

    PURPOSE Somatic mutations occur at early stages of adenoma and accumulate throughout colorectal cancer (CRC) progression. The aim of this study was to characterize the mutational landscape of stage II tumors and to search for novel recurrent mutations likely implicated in CRC tumorigenesis. DESIGN The exomic DNA of 42 stage II, microsatellite stable, colon tumors and their paired mucosae were sequenced. Other molecular data available in the discovery dataset (gene expression, methylation, and CNV) was used to further characterize these tumors. Additional datasets comprising 553 CRC samples were used to validate the discovered mutations. RESULTS As a result, 4,886 somatic single nucleotide variants (SNVs) were found. Almost all SNVs were private changes, with few mutations shared by more than one tumor, thus revealing tumor-specific mutational landscapes. Nevertheless, these diverse mutations converged into common cellular pathways such as cell cycle or apoptosis. Among this mutational heterogeneity, variants resulting in early stop-codons in the AMER1 (also known as FAM123B or WTX) gene emerged as recurrent mutations in CRC. Loses of AMER1 by other mechanisms apart from mutations such as methylation and copy number aberrations were also found. Tumors lacking this tumor suppressor gene exhibited a mesenchymal phenotype characterized by inhibition of the canonical Wnt pathway. CONCLUSION In silico and experimental validation in independent datasets confirmed the existence of functional mutations in AMER1 in approximately 10% of analyzed CRC tumors. Moreover, these tumors exhibited a characteristic phenotype. PMID:26071483

  18. Photoactivation of Mutant Isocitrate Dehydrogenase 2 Reveals Rapid Cancer-Associated Metabolic and Epigenetic Changes

    PubMed Central

    2016-01-01

    Isocitrate dehydrogenase is mutated at a key active site arginine residue (Arg172 in IDH2) in many cancers, leading to the synthesis of the oncometabolite (R)-2-hydroxyglutarate (2HG). To investigate the early events following acquisition of this mutation in mammalian cells we created a photoactivatable version of IDH2(R172K), in which K172 is replaced with a photocaged lysine (PCK), via genetic code expansion. Illumination of cells expressing this mutant protein led to a rapid increase in the levels of 2HG, with 2HG levels reaching those measured in patient tumor samples, within 8 h. 2HG accumulation is closely followed by a global decrease in 5-hydroxymethylcytosine (5-hmC) in DNA, demonstrating that perturbations in epigenetic DNA base modifications are an early consequence of mutant IDH2 in cells. Our results provide a paradigm for rapidly and synchronously uncloaking diverse oncogenic mutations in live cells to reveal the sequence of events through which they may ultimately cause transformation. PMID:26761588

  19. Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

    PubMed

    Ohmura, Mitsuyo; Hishiki, Takako; Yamamoto, Takehiro; Nakanishi, Tsuyoshi; Kubo, Akiko; Tsuchihashi, Kenji; Tamada, Mayumi; Toue, Sakino; Kabe, Yasuaki; Saya, Hideyuki; Suematsu, Makoto

    2015-04-30

    CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue. PMID:25461272

  20. Profiling of Discrete Gynecological Cancers Reveals Novel Transcriptional Modules and Common Features Shared by Other Cancer Types and Embryonic Stem Cells

    PubMed Central

    Jacob-Hirsch, Jasmine; Amariglio, Ninette; Vlachos, George D.; Loutradis, Dimitrios; Anagnou, Nicholas P.

    2015-01-01

    Studies on individual types of gynecological cancers (GCs), utilizing novel expression technologies, have revealed specific pathogenetic patterns and gene markers for cervical (CC), endometrial (EC) and vulvar cancer (VC). Although the clinical phenotypes of the three types of gynecological cancers are discrete, the fact they originate from a common embryological origin, has led to the hypothesis that they might share common features reflecting regression to early embryogenesis. To address this question, we performed a comprehensive comparative analysis of their profiles. Our data identified both common features (pathways and networks) and novel distinct modules controlling the same deregulated biological processes in all three types. Specifically, four novel transcriptional modules were discovered regulating cell cycle and apoptosis. Integration and comparison of our data with other databases, led to the identification of common features among cancer types, embryonic stem (ES) cells and the newly discovered cell population of squamocolumnar (SC) junction of the cervix, considered to host the early cancer events. Conclusively, these data lead us to propose the presence of common features among gynecological cancers, other types of cancers, ES cells and the pre-malignant SC junction cells, where the novel E2F/NFY and MAX/CEBP modules play an important role for the pathogenesis of gynecological carcinomas. PMID:26559525

  1. Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells

    PubMed Central

    Wu, Xing-Zhong; Zhang, Li; Shi, Bi-Zhi; Hu, Ping

    2005-01-01

    AIM: To investigate the effect of N-(4-hydrophenyl) retinamide (4-HPR), the derivative of retinoic acid, on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatocellular carcinoma cells (HCCs) and malignant melanoma cells. METHODS: 4-HPR was chemically synthesized. Cellular migration and invasion were assayed by Borden chamber experiment. Cell growth was assayed by MTT chromometry. Apoptosis effect was measured using Hoechst 32258 staining and flow cytometry. Gene transfection was performed with lipofectamine. RESULTS: We observed that the migration of HCC and melanoma cells was significantly suppressed by 4-HPR and the migration cells were reduced to 585.03 (control 20127.2, P < 0.05, n = 4) in SMMC 7721-k3 HCC, and to 25425.04 (control 30230.1, P < 0.05, n = 4) in melanoma cells after 6-h incubation with 4-HPR. The invasion through reconstituted basement membrane was also significantly reduced by 4-HPR treatment to 11.23.3 in SMMC 7721-k3 HCC (control 2713.1), and to 24.33.2 in melanoma cells (control 67.510.1, P < 0.05, n = 3). Cell growth, especially in melanoma cells, was also significantly inhibited. Furthermore, 3 mmol/L of 4-HPR induced apoptosis in B16 melanoma cells (37.110.94%) more significantly than all-trans retinoic acid (P < 0.05), but it failed to induce apoptosis in SMMC 7721-k3 HCC. The mechanism for 4-HPR-induced apoptosis was not clear, but we observed that 4-HPR could regulate p27kip1, and overexpression of cerebroside sulfotransferase (CST) diminished the apoptosis induced by 4-HPR in melanoma cells. CONCLUSION: 4-HPR is a potent inhibitor of HCC migration and inducer of melanoma cell apoptosis. CST and p27kip1expression might be associated with 4-HPR-induced apoptosis. PMID:16270382

  2. The lncRNA landscape of breast cancer reveals a role for DSCAM-AS1 in breast cancer progression

    PubMed Central

    Niknafs, Yashar S.; Han, Sumin; Ma, Teng; Speers, Corey; Zhang, Chao; Wilder-Romans, Kari; Iyer, Matthew K.; Pitchiaya, Sethuramasundaram; Malik, Rohit; Hosono, Yasuyuki; Prensner, John R.; Poliakov, Anton; Singhal, Udit; Xiao, Lanbo; Kregel, Steven; Siebenaler, Ronald F.; Zhao, Shuang G.; Uhl, Michael; Gawronski, Alexander; Hayes, Daniel F.; Pierce, Lori J.; Cao, Xuhong; Collins, Colin; Backofen, Rolf; Sahinalp, Cenk S.; Rae, James M.; Chinnaiyan, Arul M.; Feng, Felix Y.

    2016-01-01

    Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise <1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1. We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer. PMID:27666543

  3. In-Depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer

    PubMed Central

    Lee, Deanna; Das Gupta, Jaydip; Gaughan, Christina; Steffen, Imke; Tang, Ning; Luk, Ka-Cheung; Qiu, Xiaoxing; Urisman, Anatoly; Fischer, Nicole; Molinaro, Ross; Broz, Miranda; Schochetman, Gerald; Klein, Eric A.; Ganem, Don; DeRisi, Joseph L.; Simmons, Graham; Hackett, John; Silverman, Robert H.; Chiu, Charles Y.

    2012-01-01

    XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection. PMID:23028701

  4. In-depth investigation of archival and prospectively collected samples reveals no evidence for XMRV infection in prostate cancer.

    PubMed

    Lee, Deanna; Das Gupta, Jaydip; Gaughan, Christina; Steffen, Imke; Tang, Ning; Luk, Ka-Cheung; Qiu, Xiaoxing; Urisman, Anatoly; Fischer, Nicole; Molinaro, Ross; Broz, Miranda; Schochetman, Gerald; Klein, Eric A; Ganem, Don; Derisi, Joseph L; Simmons, Graham; Hackett, John; Silverman, Robert H; Chiu, Charles Y

    2012-01-01

    XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.

  5. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  6. Integrated Analysis of Whole Genome and Transcriptome Sequencing Reveals Diverse Transcriptomic Aberrations Driven by Somatic Genomic Changes in Liver Cancers

    PubMed Central

    Shiraishi, Yuichi; Fujimoto, Akihiro; Furuta, Mayuko; Tanaka, Hiroko; Chiba, Ken-ichi; Boroevich, Keith A.; Abe, Tetsuo; Kawakami, Yoshiiku; Ueno, Masaki; Gotoh, Kunihito; Ariizumi, Shun-ichi; Shibuya, Tetsuo; Nakano, Kaoru; Sasaki, Aya; Maejima, Kazuhiro; Kitada, Rina; Hayami, Shinya; Shigekawa, Yoshinobu; Marubashi, Shigeru; Yamada, Terumasa; Kubo, Michiaki; Ishikawa, Osamu; Aikata, Hiroshi; Arihiro, Koji; Ohdan, Hideki; Yamamoto, Masakazu; Yamaue, Hiroki; Chayama, Kazuaki; Tsunoda, Tatsuhiko; Miyano, Satoru; Nakagawa, Hidewaki

    2014-01-01

    Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV)-related hepatocellular carcinomas (HCCs) and their matched controls. Comparison of whole genome sequence (WGS) and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3), and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome. PMID:25526364

  7. Kinome RNAi Screens Reveal Synergistic Targeting of MTOR and FGFR1 Pathways for Treatment of Lung Cancer and HNSCC.

    PubMed

    Singleton, Katherine R; Hinz, Trista K; Kleczko, Emily K; Marek, Lindsay A; Kwak, Jeff; Harp, Taylor; Kim, Jihye; Tan, Aik Choon; Heasley, Lynn E

    2015-10-15

    The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non-small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. PMID:26359452

  8. The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region.

    PubMed

    Jeon, Woo-Kwang; Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Young K; Lim, Seunghwan; Kim, Jae-Hong; Letterio, John J; Liu, Fang; Kim, Seong-Jin; Kim, Byung-Chul

    2015-12-01

    Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-β1 (TGF-β1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-β1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-β1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-β1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-β1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth. PMID:26497676

  9. Urinary proteome alterations in HER2 enriched breast cancer revealed by multipronged quantitative proteomics.

    PubMed

    Gajbhiye, Akshada; Dabhi, Raju; Taunk, Khushman; Vannuruswamy, Garikapati; RoyChoudhury, Sourav; Adhav, Ragini; Seal, Shubhendu; Mane, Anupama; Bayatigeri, Santhakumari; Santra, Manas K; Chaudhury, Koel; Rapole, Srikanth

    2016-09-01

    Globally, breast cancer is the second most common cancer among women. Although biomarker discoveries through various proteomic approaches of tissue and serum samples have been studied in breast cancer, urinary proteome alterations in breast cancer are least studied. Urine being a noninvasive biofluid and a significant source of proteins, it has the potential in early diagnosis of breast cancer. This study used complementary quantitative gel-based and gel-free proteomic approaches to find a panel of urinary protein markers that could discriminate HER2 enriched (HE) subtype breast cancer from the healthy controls. A total of 183 differentially expressed proteins were identified using three complementary approaches, namely 2D-DIGE, iTRAQ, and sequential window acquisition of all theoretical mass spectra. The differentially expressed proteins were subjected to various bioinformatics analyses for deciphering the biological context of these proteins using protein analysis through evolutionary relationships, database for annotation, visualization and integrated discovery, and STRING. Multivariate statistical analysis was undertaken to identify the set of most significant proteins, which could discriminate HE breast cancer from healthy controls. Immunoblotting and MRM-based validation in a separate cohort testified a panel of 21 proteins such as zinc-alpha2-glycoprotein, A2GL, retinol-binding protein 4, annexin A1, SAP3, SRC8, gelsolin, kininogen 1, CO9, clusterin, ceruloplasmin, and α1-antitrypsin could be a panel of candidate markers that could discriminate HE breast cancer from healthy controls. PMID:27324523

  10. Characterization of single disseminated prostate cancer cells reveals tumor cell heterogeneity and identifies dormancy associated pathways.

    PubMed

    Chéry, Lisly; Lam, Hung-Ming; Coleman, Ilsa; Lakely, Bryce; Coleman, Roger; Larson, Sandy; Aguirre-Ghiso, Julio A; Xia, Jing; Gulati, Roman; Nelson, Peter S; Montgomery, Bruce; Lange, Paul; Snyder, Linda A; Vessella, Robert L; Morrissey, Colm

    2014-10-30

    Cancer dormancy refers to the prolonged clinical disease-free time between removal of the primary tumor and recurrence, which is common in prostate cancer (PCa), breast cancer, esophageal cancer, and other cancers. PCa disseminated tumor cells (DTC) are detected in both patients with no evidence of disease (NED) and advanced disease (ADV). However, the molecular and cellular nature of DTC is unknown. We performed a first-in-field study of single DTC transcriptomic analyses in cancer patients to identify a molecular signature associated with cancer dormancy. We profiled eighty-five individual EpCAM⁺/CD45⁻ cells from the bone marrow of PCa patients with NED or ADV. We analyzed 44 DTC with high prostate-epithelial signatures, and eliminated 41 cells with high erythroid signatures and low prostate epithelial signatures. DTC were clustered into 3 groups: NED, ADV_1, and ADV_2, in which the ADV_1 group presented a distinct gene expression pattern associated with the p38 stress activated kinase pathway. Additionally, DTC from the NED group were enriched for a tumor dormancy signature associated with head and neck squamous carcinoma and breast cancer. This study provides the first clinical evidence of the p38 pathway as a potential biomarker for early recurrence and an attractive target for therapeutic intervention.

  11. Cytogenomic profiling of breast cancer brain metastases reveals potential for repurposing targeted therapeutics.

    PubMed

    Bollig-Fischer, Aliccia; Michelhaugh, Sharon K; Wijesinghe, Priyanga; Dyson, Greg; Kruger, Adele; Palanisamy, Nallasivam; Choi, Lydia; Alosh, Baraa; Ali-Fehmi, Rouba; Mittal, Sandeep

    2015-06-10

    Breast cancer brain metastases remain a significant clinical problem. Chemotherapy is ineffective and a lack of treatment options result in poor patient outcomes. Targeted therapeutics have proven to be highly effective in primary breast cancer, but lack of molecular genomic characterization of metastatic brain tumors is hindering the development of new treatment regimens. Here we contribute to fill this void by reporting on gene copy number variation (CNV) in 10 breast cancer metastatic brain tumors, assayed by array comparative genomic hybridization (aCGH). Results were compared to a list of cancer genes verified by others to influence cancer. Cancer gene aberrations were identified in all specimens and pathway-level analysis was applied to aggregate data, which identified stem cell pluripotency pathway enrichment and highlighted recurring, significant amplification of SOX2, PIK3CA, NTRK1, GNAS, CTNNB1, and FGFR1. For a subset of the metastatic brain tumor samples (n = 4) we compared patient-matched primary breast cancer specimens. The results of our CGH analysis and validation by alternative methods indicate that oncogenic signals driving growth of metastatic tumors exist in the original cancer. This report contributes support for more rapid development of new treatments of metastatic brain tumors, the use of genomic-based diagnostic tools and repurposed drug treatments.

  12. Regulators Associated with Clinical Outcomes Revealed by DNA Methylation Data in Breast Cancer

    PubMed Central

    Ung, Matthew H.; Varn, Frederick S.; Lou, Shaoke; Cheng, Chao

    2015-01-01

    The regulatory architecture of breast cancer is extraordinarily complex and gene misregulation can occur at many levels, with transcriptional malfunction being a major cause. This dysfunctional process typically involves additional regulatory modulators including DNA methylation. Thus, the interplay between transcription factor (TF) binding and DNA methylation are two components of a cancer regulatory interactome presumed to display correlated signals. As proof of concept, we performed a systematic motif-based in silico analysis to infer all potential TFs that are involved in breast cancer prognosis through an association with DNA methylation changes. Using breast cancer DNA methylation and clinical data derived from The Cancer Genome Atlas (TCGA), we carried out a systematic inference of TFs whose misregulation underlie different clinical subtypes of breast cancer. Our analysis identified TFs known to be associated with clinical outcomes of p53 and ER (estrogen receptor) subtypes of breast cancer, while also predicting new TFs that may also be involved. Furthermore, our results suggest that misregulation in breast cancer can be caused by the binding of alternative factors to the binding sites of TFs whose activity has been ablated. Overall, this study provides a comprehensive analysis that links DNA methylation to TF binding to patient prognosis. PMID:25996148

  13. Exome sequencing reveals frequent deleterious germline variants in cancer susceptibility genes in women with invasive breast cancer undergoing neoadjuvant chemotherapy.

    PubMed

    Ellingson, Marissa S; Hart, Steven N; Kalari, Krishna R; Suman, Vera; Schahl, Kimberly A; Dockter, Travis J; Felten, Sara J; Sinnwell, Jason P; Thompson, Kevin J; Tang, Xiaojia; Vedell, Peter T; Barman, Poulami; Sicotte, Hugues; Eckel-Passow, Jeanette E; Northfelt, Donald W; Gray, Richard J; McLaughlin, Sarah A; Moreno-Aspitia, Alvaro; Ingle, James N; Moyer, Ann M; Visscher, Daniel W; Jones, Katie; Conners, Amy; McDonough, Michelle; Wieben, Eric D; Wang, Liewei; Weinshilboum, Richard; Boughey, Judy C; Goetz, Matthew P

    2015-09-01

    When sequencing blood and tumor samples to identify targetable somatic variants for cancer therapy, clinically relevant germline variants may be uncovered. We evaluated the prevalence of deleterious germline variants in cancer susceptibility genes in women with breast cancer referred for neoadjuvant chemotherapy and returned clinically actionable results to patients. Exome sequencing was performed on blood samples from women with invasive breast cancer referred for neoadjuvant chemotherapy. Germline variants within 142 hereditary cancer susceptibility genes were filtered and reviewed for pathogenicity. Return of results was offered to patients with deleterious variants in actionable genes if they were not aware of their result through clinical testing. 124 patients were enrolled (median age 51) with the following subtypes: triple negative (n = 43, 34.7%), HER2+ (n = 37, 29.8%), luminal B (n = 31, 25%), and luminal A (n = 13, 10.5%). Twenty-eight deleterious variants were identified in 26/124 (21.0%) patients in the following genes: ATM (n = 3), BLM (n = 1), BRCA1 (n = 4), BRCA2 (n = 8), CHEK2 (n = 2), FANCA (n = 1), FANCI (n = 1), FANCL (n = 1), FANCM (n = 1), FH (n = 1), MLH3 (n = 1), MUTYH (n = 2), PALB2 (n = 1), and WRN (n = 1). 121/124 (97.6%) patients consented to return of research results. Thirteen (10.5%) had actionable variants, including four that were returned to patients and led to changes in medical management. Deleterious variants in cancer susceptibility genes are highly prevalent in patients with invasive breast cancer referred for neoadjuvant chemotherapy undergoing exome sequencing. Detection of these variants impacts medical management. PMID:26296701

  14. Generation of 2,000 breast cancer metabolic landscapes reveals a poor prognosis group with active serotonin production.

    PubMed

    Leoncikas, Vytautas; Wu, Huihai; Ward, Lara T; Kierzek, Andrzej M; Plant, Nick J

    2016-01-01

    A major roadblock in the effective treatment of cancers is their heterogeneity, whereby multiple molecular landscapes are classified as a single disease. To explore the contribution of cellular metabolism to cancer heterogeneity, we analyse the Metabric dataset, a landmark genomic and transcriptomic study of 2,000 individual breast tumours, in the context of the human genome-scale metabolic network. We create personalized metabolic landscapes for each tumour by exploring sets of active reactions that satisfy constraints derived from human biochemistry and maximize congruency with the Metabric transcriptome data. Classification of the personalized landscapes derived from 997 tumour samples within the Metabric discovery dataset reveals a novel poor prognosis cluster, reproducible in the 995-sample validation dataset. We experimentally follow mechanistic hypotheses resulting from the computational study and establish that active serotonin production is a major metabolic feature of the poor prognosis group. These data support the reconsideration of concomitant serotonin-specific uptake inhibitors treatment during breast cancer chemotherapy. PMID:26813959

  15. Generation of 2,000 breast cancer metabolic landscapes reveals a poor prognosis group with active serotonin production

    PubMed Central

    Leoncikas, Vytautas; Wu, Huihai; Ward, Lara T.; Kierzek, Andrzej M.; Plant, Nick J.

    2016-01-01

    A major roadblock in the effective treatment of cancers is their heterogeneity, whereby multiple molecular landscapes are classified as a single disease. To explore the contribution of cellular metabolism to cancer heterogeneity, we analyse the Metabric dataset, a landmark genomic and transcriptomic study of 2,000 individual breast tumours, in the context of the human genome-scale metabolic network. We create personalized metabolic landscapes for each tumour by exploring sets of active reactions that satisfy constraints derived from human biochemistry and maximize congruency with the Metabric transcriptome data. Classification of the personalized landscapes derived from 997 tumour samples within the Metabric discovery dataset reveals a novel poor prognosis cluster, reproducible in the 995-sample validation dataset. We experimentally follow mechanistic hypotheses resulting from the computational study and establish that active serotonin production is a major metabolic feature of the poor prognosis group. These data support the reconsideration of concomitant serotonin-specific uptake inhibitors treatment during breast cancer chemotherapy. PMID:26813959

  16. A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer.

    PubMed

    O'Donnell, Kathryn A; Keng, Vincent W; York, Brian; Reineke, Erin L; Seo, Daekwan; Fan, Danhua; Silverstein, Kevin A T; Schrum, Christina T; Xie, Wei Rose; Mularoni, Loris; Wheelan, Sarah J; Torbenson, Michael S; O'Malley, Bert W; Largaespada, David A; Boeke, Jef D

    2012-05-22

    The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy. PMID:22556267

  17. Computational imaging reveals mitochondrial morphology as a biomarker of cancer phenotype and drug response

    NASA Astrophysics Data System (ADS)

    Giedt, Randy J.; Fumene Feruglio, Paolo; Pathania, Divya; Yang, Katherine S.; Kilcoyne, Aoife; Vinegoni, Claudio; Mitchison, Timothy J.; Weissleder, Ralph

    2016-09-01

    Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations’ morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response.

  18. Computational imaging reveals mitochondrial morphology as a biomarker of cancer phenotype and drug response.

    PubMed

    Giedt, Randy J; Fumene Feruglio, Paolo; Pathania, Divya; Yang, Katherine S; Kilcoyne, Aoife; Vinegoni, Claudio; Mitchison, Timothy J; Weissleder, Ralph

    2016-01-01

    Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations' morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response. PMID:27609668

  19. Global transcription network incorporating distal regulator binding reveals selective cooperation of cancer drivers and risk genes

    PubMed Central

    Kim, Kwoneel; Yang, Woojin; Lee, Kang Seon; Bang, Hyoeun; Jang, Kiwon; Kim, Sang Cheol; Yang, Jin Ok; Park, Seongjin; Park, Kiejung; Choi, Jung Kyoon

    2015-01-01

    Global network modeling of distal regulatory interactions is essential in understanding the overall architecture of gene expression programs. Here, we developed a Bayesian probabilistic model and computational method for global causal network construction with breast cancer as a model. Whereas physical regulator binding was well supported by gene expression causality in general, distal elements in intragenic regions or loci distant from the target gene exhibited particularly strong functional effects. Modeling the action of long-range enhancers was critical in recovering true biological interactions with increased coverage and specificity overall and unraveling regulatory complexity underlying tumor subclasses and drug responses in particular. Transcriptional cancer drivers and risk genes were discovered based on the network analysis of somatic and genetic cancer-related DNA variants. Notably, we observed that the risk genes were functionally downstream of the cancer drivers and were selectively susceptible to network perturbation by tumorigenic changes in their upstream drivers. Furthermore, cancer risk alleles tended to increase the susceptibility of the transcription of their associated genes. These findings suggest that transcriptional cancer drivers selectively induce a combinatorial misregulation of downstream risk genes, and that genetic risk factors, mostly residing in distal regulatory regions, increase transcriptional susceptibility to upstream cancer-driving somatic changes. PMID:26001967

  20. Computational imaging reveals mitochondrial morphology as a biomarker of cancer phenotype and drug response

    PubMed Central

    Giedt, Randy J.; Fumene Feruglio, Paolo; Pathania, Divya; Yang, Katherine S.; Kilcoyne, Aoife; Vinegoni, Claudio; Mitchison, Timothy J.; Weissleder, Ralph

    2016-01-01

    Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations’ morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response. PMID:27609668

  1. Gene expression data reveal common pathways that characterize the unifocal nature of ovarian cancer

    PubMed Central

    MARCHION, Douglas C.; XIONG, Yin; CHON, Hye Sook; SAWAH, Entidhar AL; ZGHEIB, Nadim BOU; RAMIREZ, Ingrid J.; ABBASI, Forough; STICKLES, Xiaomang B.; JUDSON, Patricia L.; HAKAM, Ardeshir; GONZALEZ-BOSQUET, Jesus; WENHAM, Robert M.; APTE, Sachin M.; BERGLUND, Anders E.; LANCASTER, Johnathan M.

    2013-01-01

    Objectives To evaluate the biologic validity of ovarian cancer (OVCA) screening and early detection efforts and to characterize signaling pathways associated with human cancer metastasis and patient survival. Study Design Using genome-wide expression profiling and DNA sequencing, we compared pelvic and matched extra-pelvic implants from 30 patients with advanced-stage OVCA for expression of molecular signaling pathways and p53 gene mutations. Differentially expressed pathways were further evaluated in a series of primary or early-stage versus metastatic or recurrent cancer samples from 389 ovarian, prostate, and oral cancer patients. Metastasis pathways were also evaluated for associations with survival in nine independent clinico-genomic datasets from 1,691 ovarian, breast, colon, brain, and lung cancer and leukemia patients. The inhibitory effects of one pathway (TGF-WNT) on in-vitro OVCA cell migration were studied. Results Pelvic and extra-pelvic OVCA implants demonstrated similar patterns of signaling pathway expression and identical p53 mutations. However, we identified 3 molecular pathways/cellular processes that were differentially expressed between pelvic and extra-pelvic OVCA samples and between primary/early-stage and metastatic/advanced or recurrent ovarian, oral, and prostate cancers. Furthermore, their expression was associated with overall survival from ovarian cancer (P=0.006), colon cancer (1 pathway at P=0.005), and leukemia (P=0.05). Artesunate-induced TGF-WNT pathway inhibition impaired OVCA cell migration. Conclusions Advanced-stage OVCA has a unifocal origin in the pelvis, supporting validity of early detection/screening efforts. Molecular pathways associated with extra-pelvic OVCA spread are also associated with metastasis from other human cancers and with overall patient survival. Such pathways represent appealing therapeutic targets for patients with metastatic disease. PMID:23933223

  2. Co-evolution of cancer microenvironment reveals distinctive patterns of gastric cancer invasion: laboratory evidence and clinical significance

    PubMed Central

    2010-01-01

    Background Cancer invasion results from constant interactions between cancer cells and their microenvironment. Major components of the cancer microenvironment are stromal cells, infiltrating inflammatory cells, collagens, matrix metalloproteinases (MMP) and newly formed blood vessels. This study was to determine the roles of MMP-9, MMP-2, type IV collagen, infiltrating macrophages and tumor microvessels in gastric cancer (GC) invasion and their clinico-pathological significance. Methods Paraffin-embedded tissue sections from 37 GC patients were studied by Streptavidin-Peroxidase (SP) immunohistochemical technique to determine the levels of MMP-2, MMP-9, type IV collagen, macrophages infiltration and microvessel density (MVD). Different invasion patterns were delineated and their correlation with major clinico-pathological information was explored. Results MMP2 expression was higher in malignant gland compared to normal gland, especially nearby the basement membrane (BM). High densities of macrophages at the interface of cancer nests and stroma were found where BM integrity was destroyed. MMP2 expression was significantly increased in cases with recurrence and distant metastasis (P = 0.047 and 0.048, respectively). Infiltrating macrophages were correlated with serosa invasion (P = 0.011) and TNM stage (P = 0.001). MVD was higher in type IV collagen negative group compared to type IV collagen positive group (P = 0.026). MVD was related to infiltrating macrophages density (P = 0.040). Patients with negative MMP9 expression had better overall survival (OS) compared to those with positive MMP9 expression (Median OS 44.0 vs 13.5 mo, P = 0.036). Median OS was significantly longer in type IV collagen positive group than negative group (Median OS 25.5 vs 10.0 mo, P = 0.044). The cumulative OS rate was higher in low macrophages density group than in high macrophages density group (median OS 40.5 vs 13.0 mo, P = 0.056). Median OS was significantly longer in low MVD group than

  3. Genomic and Proteomic Profiles Reveal the Association of Gelsolin to TP53 Status and Bladder Cancer Progression

    PubMed Central

    Sanchez-Carbayo, Marta; Socci, Nicholas D.; Richstone, Lee; Corton, Marta; Behrendt, Nille; Wulkfuhle, Julia; Bochner, Bernard; Petricoin, Emmanuel; Cordon-Cardo, Carlos

    2007-01-01

    Bladder cancer transformation and immortalization require the inactivation of key regulatory genes, including TP53. Genotyping of a large cohort of bladder cancer patients (n = 256) using the TP53 GeneChip showed mutations in 103 cases (40.2%), the majority of them mapping to the DNA-binding core domain. TP53 mutation status was significantly associated with tumor stage (P = 0.0001) and overall survival for patients with advanced disease (P = 0.01). Transcript profiling using oligonucleotide arrays was performed on a subset of these cases (n = 46). Supervised analyses identified genes differentially expressed between invasive bladder tumors with wild-type (n = 24) and mutated TP53 (n = 22). Pathway analyses of top-ranked genes supported the central role of TP53 in the functional network of such gene patterns. A proteomic strategy using reverse phase arrays with protein extracts of bladder cancer cell lines validated the association of identified differentially expressed genes, such as gelsolin, to TP53 status. Immunohistochemistry on tissue microarrays (n = 294) revealed that gelsolin was associated with tumor stage and overall survival, correlating positively with TP53 status in a subset of these patients. This study further reveals that TP53 mutations are frequent events in bladder cancer progression and identified gelsolin related to TP53 status, tumor staging, and clinical outcome by independent high-throughput strategies. PMID:17982131

  4. Genome-wide analysis in human colorectal cancer cells reveals ischemia-mediated expression of motility genes via DNA hypomethylation.

    PubMed

    Skowronski, Karolina; Skowronki, Karolina; Andrews, Joseph; Rodenhiser, David I; Coomber, Brenda L

    2014-01-01

    DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumours, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes.

  5. Clinical significance of CDC25A and CDC25B expression in squamous cell carcinomas of the oesophagus

    PubMed Central

    Nishioka, K; Doki, Y; Shiozaki, H; Yamamoto, H; Tamura, S; Yasuda, T; Fujiwara, Y; Yano, M; Miyata, H; Kishi, K; Nakagawa, H; Shamma, A; Monden, M

    2001-01-01

    CDC25A, CDC25B and CDC25C belong to a family of protein phosphatases which activate the cyclin-dependent kinase at different points of the cell cycle. According to accumulating evidence, CDC25A and CDC25B seem to possess oncogenic properties. We have analysed these expressions by immunohistochemistry, western blot and RT-PCR in a series of 100 patients with squamous cell carcinoma of the oesophagus. When compared with non-cancerous cells, CDC25A and CDC25B were strongly expressed in the cytoplasm of cancer cells, with positive (+) classification in 46% (46 cases) and 48% (48 cases), respectively. There was no significant correlation between CDC25A and CDC25B expression, nor was there any association with the expression of other cell cycle-regulating molecules, including cyclin D1, Rb, p16INK4, p27KIP1 and PCNA (proliferating cell nuclear antigen). CDC25A (+), as well as CDC25B (+), was more frequently found in patients with deeper tumour invasion and lymph node metastasis, while tumour size was correlated only with CDC25A expression. Postoperative survival was significantly poorer for CDC25A (+) patients than CDC25A (–) patients, but was not affected by the CDC25B status. Nuclear localization of CDC25A was observed in 51 cases (51%), regardless of its cytoplasmic expression, and was not associated with clinico-pathological factors or prognosis. Multivariate analysis revealed only the CDC25A status to be an independent significant prognostic factor among these biological and clinico-pathological factors. CDC25A but not CDC25B may be a new prognostic factor for squamous cell carcinoma of the oesophagus. Thus, regulation of the G1 checkpoint in the cell cycle may be important in oesophageal carcinogenesis, which may also involve many other oncogenes. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11487274

  6. Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

    PubMed

    Sacco, Francesca; Silvestri, Alessandra; Posca, Daniela; Pirrò, Stefano; Gherardini, Pier Federico; Castagnoli, Luisa; Mann, Matthias; Cesareni, Gianni

    2016-03-23

    Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin. PMID:27135362

  7. Multi-platform analysis of 12 cancer types reveals molecular classification within and across tissues-of-origin

    PubMed Central

    Hoadley, Katherine A.; Yau, Christina; Wolf, Denise M.; Cherniack, Andrew D.; Tamborero, David; Ng, Sam; Leiserson, Max D.M.; Niu, Beifang; McLellan, Michael D.; Uzunangelov, Vladislav; Zhang, Jiashan; Kandoth, Cyriac; Akbani, Rehan; Shen, Hui; Omberg, Larsson; Chu, Andy; Margolin, Adam A.; van’t Veer, Laura J.; Lopez-Bigas, Nuria; Laird, Peter W.; Raphael, Benjamin J.; Ding, Li; Robertson, A. Gordon; Byers, Lauren A.; Mills, Gordon B.; Weinstein, John N.; Van Waes, Carter; Chen, Zhong; Collisson, Eric A.

    2014-01-01

    Recent genomic analyses of pathologically-defined tumor types identify “within-a-tissue” disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head & neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multi-platform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All datasets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies. PMID:25109877

  8. Integrated analysis of breast cancer cell lines reveals unique signaling pathways

    SciTech Connect

    Heiser, Laura M.; Wang, Nicholas J.; Talcott, Carolyn L.; Laderoute, Keith R.; Knapp, Merrill; Guan, Yinghui; Hu, Zhi; Ziyad, Safiyyah; Weber, Barbara L.; Laquerre, Sylvie; Jackson, Jeffrey R.; Wooster, Richard F.; Kuo, Wen-Lin; Gray, Joe W.; Spellman, Paul T.

    2009-03-31

    Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EGFR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EGFR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EGFR-MEK signaling. This model was comprised of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype specific subnetworks, including one that suggested PAK1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that PAK1 overexpressing cell lines would have increased sensitivity to MEK inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three MEK inhibitors. We found that PAK1 over-expressing luminal breast cancer cell lines are significantly more sensitive to MEK inhibition as compared to those that express PAK1 at low levels. This indicates that PAK1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to MEK inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.

  9. Multiparametric profiling of non–small-cell lung cancers reveals distinct immunophenotypes

    PubMed Central

    Lizotte, Patrick H.; Ivanova, Elena V.; Awad, Mark M.; Jones, Robert E.; Keogh, Lauren; Liu, Hongye; Dries, Ruben; Herter-Sprie, Grit S.; Santos, Abigail; Feeney, Nora B.; Paweletz, Cloud P.; Kulkarni, Meghana M.; Bass, Adam J.; Rustgi, Anil K.; Yuan, Guo-Cheng; Kufe, Donald W.; Jänne, Pasi A.; Hammerman, Peter S.; Sholl, Lynette M.; Hodi, F. Stephen; Richards, William G.; Bueno, Raphael; English, Jessie M.; Bittinger, Mark A.

    2016-01-01

    BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non–small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling identified an immunologically “hot” cluster with abundant CD8+ T cells expressing high levels of PD-1 and TIM-3 and an immunologically “cold” cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation.

  10. Integrated Classification of Prostate Cancer Reveals a Novel Luminal Subtype with Poor Outcome.

    PubMed

    You, Sungyong; Knudsen, Beatrice S; Erho, Nicholas; Alshalalfa, Mohammed; Takhar, Mandeep; Al-Deen Ashab, Hussam; Davicioni, Elai; Karnes, R Jeffrey; Klein, Eric A; Den, Robert B; Ross, Ashley E; Schaeffer, Edward M; Garraway, Isla P; Kim, Jayoung; Freeman, Michael R

    2016-09-01

    Prostate cancer is a biologically heterogeneous disease with variable molecular alterations underlying cancer initiation and progression. Despite recent advances in understanding prostate cancer heterogeneity, better methods for classification of prostate cancer are still needed to improve prognostic accuracy and therapeutic outcomes. In this study, we computationally assembled a large virtual cohort (n = 1,321) of human prostate cancer transcriptome profiles from 38 distinct cohorts and, using pathway activation signatures of known relevance to prostate cancer, developed a novel classification system consisting of three distinct subtypes (named PCS1-3). We validated this subtyping scheme in 10 independent patient cohorts and 19 laboratory models of prostate cancer, including cell lines and genetically engineered mouse models. Analysis of subtype-specific gene expression patterns in independent datasets derived from luminal and basal cell models provides evidence that PCS1 and PCS2 tumors reflect luminal subtypes, while PCS3 represents a basal subtype. We show that PCS1 tumors progress more rapidly to metastatic disease in comparison with PCS2 or PCS3, including PSC1 tumors of low Gleason grade. To apply this finding clinically, we developed a 37-gene panel that accurately assigns individual tumors to one of the three PCS subtypes. This panel was also applied to circulating tumor cells (CTC) and provided evidence that PCS1 CTCs may reflect enzalutamide resistance. In summary, PCS subtyping may improve accuracy in predicting the likelihood of clinical progression and permit treatment stratification at early and late disease stages. Cancer Res; 76(17); 4948-58. ©2016 AACR.

  11. Multiparametric profiling of non–small-cell lung cancers reveals distinct immunophenotypes

    PubMed Central

    Lizotte, Patrick H.; Ivanova, Elena V.; Awad, Mark M.; Jones, Robert E.; Keogh, Lauren; Liu, Hongye; Dries, Ruben; Herter-Sprie, Grit S.; Santos, Abigail; Feeney, Nora B.; Paweletz, Cloud P.; Kulkarni, Meghana M.; Bass, Adam J.; Rustgi, Anil K.; Yuan, Guo-Cheng; Kufe, Donald W.; Jänne, Pasi A.; Hammerman, Peter S.; Sholl, Lynette M.; Hodi, F. Stephen; Richards, William G.; Bueno, Raphael; English, Jessie M.; Bittinger, Mark A.

    2016-01-01

    BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non–small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling identified an immunologically “hot” cluster with abundant CD8+ T cells expressing high levels of PD-1 and TIM-3 and an immunologically “cold” cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation. PMID:27699239

  12. Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability

    PubMed Central

    Markkanen, Enni; Fischer, Roman; Ledentcova, Marina; Kessler, Benedikt M.; Dianov, Grigory L.

    2015-01-01

    Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell ‘hallmarks’. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells. PMID:25800737

  13. Mammary Stem Cell Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation

    PubMed Central

    Zhang, Zheng; Christin, John R.; Wang, Chunhui; Ge, Kai; Oktay, Maja H.; Guo, Wenjun

    2016-01-01

    SUMMARY Cancer genomics have provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic GEMMs (genetically engineered mouse models). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study established a robust in vivo platform for functional cancer genomics and discovered functional breast cancer mutations. PMID:27653681

  14. Mammary-Stem-Cell-Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation.

    PubMed

    Zhang, Zheng; Christin, John R; Wang, Chunhui; Ge, Kai; Oktay, Maja H; Guo, Wenjun

    2016-09-20

    Cancer genomics has provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic genetically engineered mouse models (GEMMs). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study has established a robust in vivo platform for functional cancer genomics and has discovered functional breast cancer mutations. PMID:27653681

  15. Integrative analysis of mutational and transcriptional profiles reveals driver mutations of metastatic breast cancers.

    PubMed

    Lee, Ji-Hyun; Zhao, Xing-Ming; Yoon, Ina; Lee, Jin Young; Kwon, Nam Hoon; Wang, Yin-Ying; Lee, Kyung-Min; Lee, Min-Joo; Kim, Jisun; Moon, Hyeong-Gon; In, Yongho; Hao, Jin-Kao; Park, Kyung-Mii; Noh, Dong-Young; Han, Wonshik; Kim, Sunghoon

    2016-01-01

    Despite the explosion in the numbers of cancer genomic studies, metastasis is still the major cause of cancer mortality. In breast cancer, approximately one-fifth of metastatic patients survive 5 years. Therefore, detecting the patients at a high risk of developing distant metastasis at first diagnosis is critical for effective treatment strategy. We hereby present a novel systems biology approach to identify driver mutations escalating the risk of metastasis based on both exome and RNA sequencing of our collected 78 normal-paired breast cancers. Unlike driver mutations occurring commonly in cancers as reported in the literature, the mutations detected here are relatively rare mutations occurring in less than half metastatic samples. By supposing that the driver mutations should affect the metastasis gene signatures, we develop a novel computational pipeline to identify the driver mutations that affect transcription factors regulating metastasis gene signatures. We identify driver mutations in ADPGK, NUP93, PCGF6, PKP2 and SLC22A5, which are verified to enhance cancer cell migration and prompt metastasis with in vitro experiments. The discovered somatic mutations may be helpful for identifying patients who are likely to develop distant metastasis. PMID:27625789

  16. Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival

    PubMed Central

    Kawamura, Tatsuro; Kawatani, Makoto; Muroi, Makoto; Kondoh, Yasumitsu; Futamura, Yushi; Aono, Harumi; Tanaka, Miho; Honda, Kaori; Osada, Hiroyuki

    2016-01-01

    Since recent publications suggested that the survival of cancer cells depends on MTH1 to avoid incorporation of oxidized nucleotides into the cellular DNA, MTH1 has attracted attention as a potential cancer therapeutic target. In this study, we identified new purine-based MTH1 inhibitors by chemical array screening. However, although the MTH1 inhibitors identified in this study targeted cellular MTH1, they exhibited only weak cytotoxicity against cancer cells compared to recently reported first-in-class inhibitors. We performed proteomic profiling to investigate the modes of action by which chemically distinct MTH1 inhibitors induce cancer cell death, and found mechanistic differences among the first-in-class MTH1 inhibitors. In particular, we identified tubulin as the primary target of TH287 and TH588 responsible for the antitumor effects despite the nanomolar MTH1-inhibitory activity in vitro. Furthermore, overexpression of MTH1 did not rescue cells from MTH1 inhibitor–induced cell death, and siRNA-mediated knockdown of MTH1 did not suppress cancer cell growth. Taken together, we conclude that the cytotoxicity of MTH1 inhibitors is attributable to off-target effects and that MTH1 is not essential for cancer cell survival. PMID:27210421

  17. Direct analysis reveals an absence of gamma-carboxyglutamic acid in cancer procoagulant from human tissues.

    PubMed

    Kaplinska, Katarzyna; Mielicki, Wojciech P

    2009-07-01

    Additional carboxylation of glutamic acid by vitamin K-dependent gamma-carboxylase is a common posttranslational modification of many proteins, including some of blood clotting factors. Vitamin K-antagonists, such as warfarin, are often included in the therapy of malignant disease, decreasing the blood coagulation potential. Cancer procoagulant, a direct blood coagulation factor X activator from malignant tissue, is considered as a vitamin K-dependent protein, so it could serve as one of possible targets for the therapy with warfarin. However, there is still no experimental data demonstrating directly the presence of gamma-carboxyglutamic acid (Gla) in a cancer procoagulant molecule. The presence of Gla in cancer procoagulant isolated from human amnion-chorion membranes and from human malignant melanoma WM 115 cell line was analyzed directly, using specific anti-Gla monoclonal antibodies. There was no detectable amount of Gla in cancer procoagulant isolated from fetal or malignant tissue. Cancer procoagulant from human tissues does not contain Gla-rich domain. The finding indicates that cancer procoagulant is rather a poor target for warfarin therapy of malignant disease.

  18. Integration of Metabolomics and Transcriptomics Reveals Major Metabolic Pathways and Potential Biomarker Involved in Prostate Cancer.

    PubMed

    Ren, Shancheng; Shao, Yaping; Zhao, Xinjie; Hong, Christopher S; Wang, Fubo; Lu, Xin; Li, Jia; Ye, Guozhu; Yan, Min; Zhuang, Zhengping; Xu, Chuanliang; Xu, Guowang; Sun, Yinghao

    2016-01-01

    Prostate cancer is a highly prevalent tumor affecting millions of men worldwide, but poor understanding of its pathogenesis has limited effective clinical management of patients. In addition to transcriptional profiling or transcriptomics, metabolomics is being increasingly utilized to discover key molecular changes underlying tumorigenesis. In this study, we integrated transcriptomics and metabolomics to analyze 25 paired human prostate cancer tissues and adjacent noncancerous tissues, followed by further validation of our findings in an additional cohort of 51 prostate cancer patients and 16 benign prostatic hyperplasia patients. We found several altered pathways aberrantly expressed at both metabolic and transcriptional levels, including cysteine and methionine metabolism, nicotinamide adenine dinucleotide metabolism, and hexosamine biosynthesis. Additionally, the metabolite sphingosine demonstrated high specificity and sensitivity for distinguishing prostate cancer from benign prostatic hyperplasia, particularly for patients with low prostate specific antigen level (0-10 ng/ml). We also found impaired sphingosine-1-phosphate receptor 2 signaling, downstream of sphingosine, representing a loss of tumor suppressor gene and a potential key oncogenic pathway for therapeutic targeting. By integrating metabolomics and transcriptomics, we have provided both a broad picture of the molecular perturbations underlying prostate cancer and a preliminary study of a novel metabolic signature, which may help to discriminate prostate cancer from normal tissue and benign prostatic hyperplasia.

  19. Integrative analysis of mutational and transcriptional profiles reveals driver mutations of metastatic breast cancers

    PubMed Central

    Lee, Ji-Hyun; Zhao, Xing-Ming; Yoon, Ina; Lee, Jin Young; Kwon, Nam Hoon; Wang, Yin-Ying; Lee, Kyung-Min; Lee, Min-Joo; Kim, Jisun; Moon, Hyeong-Gon; In, Yongho; Hao, Jin-Kao; Park, Kyung-Mii; Noh, Dong-Young; Han, Wonshik; Kim, Sunghoon

    2016-01-01

    Despite the explosion in the numbers of cancer genomic studies, metastasis is still the major cause of cancer mortality. In breast cancer, approximately one-fifth of metastatic patients survive 5 years. Therefore, detecting the patients at a high risk of developing distant metastasis at first diagnosis is critical for effective treatment strategy. We hereby present a novel systems biology approach to identify driver mutations escalating the risk of metastasis based on both exome and RNA sequencing of our collected 78 normal-paired breast cancers. Unlike driver mutations occurring commonly in cancers as reported in the literature, the mutations detected here are relatively rare mutations occurring in less than half metastatic samples. By supposing that the driver mutations should affect the metastasis gene signatures, we develop a novel computational pipeline to identify the driver mutations that affect transcription factors regulating metastasis gene signatures. We identify driver mutations in ADPGK, NUP93, PCGF6, PKP2 and SLC22A5, which are verified to enhance cancer cell migration and prompt metastasis with in vitro experiments. The discovered somatic mutations may be helpful for identifying patients who are likely to develop distant metastasis.

  20. Pan-cancer transcriptome analysis reveals long noncoding RNAs with conserved function

    PubMed Central

    Cabanski, Christopher R; White, Nicole M; Dang, Ha X; Silva-Fisher, Jessica M; Rauck, Corinne E; Cicka, Danielle; Maher, Christopher A

    2015-01-01

    A growing number of gene-centric studies have highlighted the emerging significance of lncRNAs in cancer. However, these studies primarily focus on a single cancer type. Therefore, we conducted a pan-cancer analysis of lncRNAs comparing tumor and matched normal expression levels using RNA-Seq data from ∼ 3,000 patients in 8 solid tumor types. While the majority of differentially expressed lncRNAs display tissue-specific expression we discovered 229 lncRNAs with outlier or differential expression across multiple cancers, which we refer to as 'onco-lncRNAs'. Due to their consistent altered expression, we hypothesize that these onco-lncRNAs may have conserved oncogenic and tumor suppressive functions across cancers. To address this, we associated the onco-lncRNAs in biological processes based on their co-expressed protein coding genes. To validate our predictions, we experimentally confirmed cell growth dependence of 2 novel oncogenic lncRNAs, onco-lncRNA-3 and onco-lncRNA-12, and a previously identified lncRNA CCAT1. Overall, we discovered lncRNAs that may have broad oncogenic and tumor suppressor roles that could significantly advance our understanding of cancer lncRNA biology. PMID:25864709

  1. Integrative analysis of mutational and transcriptional profiles reveals driver mutations of metastatic breast cancers

    PubMed Central

    Lee, Ji-Hyun; Zhao, Xing-Ming; Yoon, Ina; Lee, Jin Young; Kwon, Nam Hoon; Wang, Yin-Ying; Lee, Kyung-Min; Lee, Min-Joo; Kim, Jisun; Moon, Hyeong-Gon; In, Yongho; Hao, Jin-Kao; Park, Kyung-Mii; Noh, Dong-Young; Han, Wonshik; Kim, Sunghoon

    2016-01-01

    Despite the explosion in the numbers of cancer genomic studies, metastasis is still the major cause of cancer mortality. In breast cancer, approximately one-fifth of metastatic patients survive 5 years. Therefore, detecting the patients at a high risk of developing distant metastasis at first diagnosis is critical for effective treatment strategy. We hereby present a novel systems biology approach to identify driver mutations escalating the risk of metastasis based on both exome and RNA sequencing of our collected 78 normal-paired breast cancers. Unlike driver mutations occurring commonly in cancers as reported in the literature, the mutations detected here are relatively rare mutations occurring in less than half metastatic samples. By supposing that the driver mutations should affect the metastasis gene signatures, we develop a novel computational pipeline to identify the driver mutations that affect transcription factors regulating metastasis gene signatures. We identify driver mutations in ADPGK, NUP93, PCGF6, PKP2 and SLC22A5, which are verified to enhance cancer cell migration and prompt metastasis with in vitro experiments. The discovered somatic mutations may be helpful for identifying patients who are likely to develop distant metastasis. PMID:27625789

  2. A Systematic Analysis Reveals Heterogeneous Changes in the Endocytic Activities of Cancer Cells

    PubMed Central

    Elkin, Sarah R.; Bendris, Nawal; Reis, Carlos R.; Zhou, Yunyun; Xie, Yang; Huffman, Kenneth E.; Minna, John D.; Schmid, Sandra L.

    2016-01-01

    Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed versus normal counterparts. However, when we screened 29 independently isolated non–small cell lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. Interestingly, the two clusters also differed significantly in clathrin-independent internalization and surface expression of CD44 and CD59. Taken together, our results suggest that endocytotic alterations in cancer cells that affect cell surface expression of critical molecules have a significant influence on cancer-relevant phenotypes, with potential implications for interventions to control cancer by modulating endocytic dynamics. PMID:26359453

  3. Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation.

    PubMed

    Vosper, Jonathan; Masuccio, Alessia; Kullmann, Michael; Ploner, Christian; Geley, Stephan; Hengst, Ludger

    2015-02-20

    Statins, such as lovastatin, can induce a cell cycle arrest in the G1 phase. This robust antiproliferative activity remains intact in many cancer cells that are deficient in cell cycle checkpoints and leads to an increased expression of CDK inhibitor proteins p27Kip1 and p21Cip1. The molecular details of this statin-induced growth arrest remains unclear. Here we present evidence that lovastatin can induce the degradation of Skp2, a subunit of the SCFSkp2 ubiquitin ligase that targets p27Kip1 and p21Cip1 for proteasomal destruction. The statin-induced degradation of Skp2 is cell cycle phase independent and does not require its well characterised degradation pathway mediated by APC/CCdh1- or Skp2 autoubiquitination. An N-terminal domain preceding the F-box of Skp2 is both necessary and sufficient for its statin mediated degradation. The degradation of Skp2 results from statin induced depletion of geranylgeranyl isoprenoid intermediates of cholesterol biosynthesis. Inhibition of geranylgeranyl-transferase-I also promotes APC/CCdh1- independent degradation of Skp2, indicating that de-modification of a geranylgeranylated protein triggers this novel pathway of Skp2 degradation.

  4. MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing

    PubMed Central

    Zhang, Yuanwei; Xu, Bo; Zhou, Jun; Fan, Song; Hao, Zongyao; Shi, Haoqiang; Zhang, Xiansheng; Kong, Rui; Xu, Lingfan; Gao, Jingjing; Zou, Duohong; Liang, Chaozhao

    2015-01-01

    Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights

  5. Co-modulation analysis of gene regulation in breast cancer reveals complex interplay between ESR1 and ERBB2 genes

    PubMed Central

    2015-01-01

    Background Gene regulation is dynamic across cellular conditions and disease subtypes. From the aspect of regulation under modulation, regulation strength between a pair of genes can be modulated by (dependent on) expression abundance of another gene (modulator gene). Previous studies have demonstrated the involvement of genes modulated by single modulator genes in cancers, including breast cancer. However, analysis of multi-modulator co-modulation that can further delineate the landscape of complex gene regulation is, to our knowledge, unexplored previously. In the present study we aim to explore the joint effects of multiple modulator genes in modulating global gene regulation and dissect the biological functions in breast cancer. Results To carry out the analysis, we proposed the Covariability-based Multiple Regression (CoMRe) method. The method is mainly built on a multiple regression model that takes expression levels of multiple modulators as inputs and regulation strength between genes as output. Pairs of genes were divided into groups based on their co-modulation patterns. Analyzing gene expression profiles from 286 breast cancer patients, CoMRe investigated ten candidate modulator genes that interacted and jointly determined global gene regulation. Among the candidate modulators, ESR1, ERBB2, and ADAM12 were found modulating the most numbers of gene pairs. The largest group of gene pairs was composed of ones that were modulated by merely ESR1. Functional annotation revealed that the group was significantly related to tumorigenesis and estrogen signaling in breast cancer. ESR1−ERBB2 co-modulation was the largest group modulated by more than one modulators. Similarly, the group was functionally associated with hormone stimulus, suggesting that functions of the two modulators are performed, at least partially, through modulation. The findings were validated in majorities of patients (> 99%) of two independent breast cancer datasets. Conclusions We have

  6. Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer

    PubMed Central

    Ulz, Peter; Belic, Jelena; Graf, Ricarda; Auer, Martina; Lafer, Ingrid; Fischereder, Katja; Webersinke, Gerald; Pummer, Karl; Augustin, Herbert; Pichler, Martin; Hoefler, Gerald; Bauernhofer, Thomas; Geigl, Jochen B.; Heitzer, Ellen; Speicher, Michael R.

    2016-01-01

    Genomic alterations in metastatic prostate cancer remain incompletely characterized. Here we analyse 493 prostate cancer cases from the TCGA database and perform whole-genome plasma sequencing on 95 plasma samples derived from 43 patients with metastatic prostate cancer. From these samples, we identify established driver aberrations in a cancer-related gene in nearly all cases (97.7%), including driver gene fusions (TMPRSS2:ERG), driver focal deletions (PTEN, RYBP and SHQ1) and driver amplifications (AR and MYC). In serial plasma analyses, we observe changes in focal amplifications in 40% of cases. The mean time interval between new amplifications was 26.4 weeks (range: 5–52 weeks), suggesting that they represent rapid adaptations to selection pressure. An increase in neuron-specific enolase is accompanied by clonal pattern changes in the tumour genome, most consistent with subclonal diversification of the tumour. Our findings suggest a high plasticity of prostate cancer genomes with newly occurring focal amplifications as a driving force in progression. PMID:27328849

  7. Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells.

    PubMed

    Singh, Jagdeep K; Simões, Bruno M; Howell, Sacha J; Farnie, Gillian; Clarke, Robert B

    2013-01-01

    Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are purported to be responsible for tumor initiation, maintenance, metastases, and disease recurrence. Interleukin-8 (IL-8) is upregulated in breast cancer compared with normal breast tissue and is associated with poor prognosis. IL-8 is reported to promote breast cancer progression by increasing cell invasion, angiogenesis, and metastases and is upregulated in HER2-positive cancers. Recently, we and others have established that IL-8 via its cognate receptors, CXCR1 and CXCR2, is also involved in regulating breast CSC activity. Our work demonstrates that in metastatic breast CSCs, CXCR1/2 signals via transactivation of HER2. Given the importance of HER2 in breast cancer and in regulating CSC activity, a pathway driving the activation of these receptors would have important biological and clinical consequences, especially in tumors that express high levels of IL-8 and other CXCR1/2-activating ligands. Here, we review the IL-8 signaling pathway and the role of HER2 in maintaining an IL-8 inflammatory loop and discuss the potential of combining CXCR1/2 inhibitors with other treatments such as HER2-targeted therapy as a novel approach to eliminate CSCs and improve patient survival.

  8. Activity-Based Protein Profiling of Oncogene-Driven Changes in Metabolism Reveals Broad Dysregulation of PAFAH1B2 and 1B3 in Cancer

    PubMed Central

    Kohnz, Rebecca A.; Mulvihill, Melinda M.; Chang, Jae Won; Hsu, Ku-Lung; Sorrentino, Antonio; Cravatt, Benjamin F.; Bandyopadhyay, Sourav; Goga, Andrei; Nomura, Daniel K.

    2015-01-01

    Targeting dysregulated metabolic pathways is a promising therapeutic strategy for eradicating cancer. Understanding how frequently altered oncogenes regulate metabolic enzyme targets would be useful in identifying both broad-spectrum and targeted metabolic therapies for cancer. Here, we used activity-based protein profiling to identify serine hydrolase activities that were consistently upregulated by various human oncogenes. Through this profiling effort, we found oncogenic regulatory mechanisms for several cancer-relevant serine hydrolases and discovered that platelet activating factor acetylhydrolase 1B2 and 1B3 (PAFAH1B2 and PAFAH1B3) activities were consistently upregulated by several oncogenes, alongside previously discovered cancer-relevant hydrolases fatty acid synthase and monoacylglycerol lipase. While we previously showed that PAFAH1B2 and 1B3 were important in breast cancer our most recent profiling studies have revealed that these enzymes may be dysregulated broadly across many types of cancers. Here, we find that pharmacological blockade of both enzymes impairs cancer pathogenicity across multiple different types of cancer cells, including breast, ovarian, melanoma, and prostate cancer. We also show that pharmacological blockade of PAFAH1B2 and 1B3 cause unique changes in lipid metabolism, including heightened levels of tumor-suppressing lipids. Our results reveal oncogenic regulatory mechanisms of several cancer-relevant serine hydrolases using activity-based protein profiling and we show that PAFAH1B2 and 1B3 are important in maintaining cancer pathogenicity across a wide spectrum of cancer types. PMID:25945974

  9. Whole blood mRNA in prostate cancer reveals a four-gene androgen regulated panel.

    PubMed

    Thomas, Benjamin C; Kay, Jonathan D; Menon, Suraj; Vowler, Sarah L; Dawson, Sarah N; Bucklow, Laura J; Luxton, Hayley J; Johnston, Thomas; Massie, Charlie E; Pugh, Michelle; Warren, Anne Y; Barker, Peter; Burling, Keith; Lynch, Andy G; George, Anne; Burge, Johanna; Corcoran, Marie; Stearn, Sara; Lamb, Alastair D; Sharma, Naomi L; Shaw, Greg L; Neal, David E; Whitaker, Hayley C

    2016-10-01

    Due to increased sensitivity, the expression of circulating nucleotides is rapidly gaining popularity in cancer diagnosis. Whole blood mRNA has been used in studies on a number of cancers, most notably two separate studies that used whole blood mRNA to define non-overlapping signatures of prostate cancer that has become castration independent. Prostate cancer is known to rely on androgens for initial growth, and there is increasing evidence on the importance of the androgen axis in advanced disease. Using whole blood mRNA samples from patients with prostate cancer, we have identified the four-gene panel of FAM129A, MME, KRT7 and SOD2 in circulating mRNA that are differentially expressed in a discovery cohort of metastatic samples. Validation of these genes at the mRNA and protein level was undertaken in additional cohorts defined by risk of relapse following surgery and hormone status. All the four genes were downregulated at the mRNA level in the circulation and in primary tissue, but this was not always reflected in tissue protein expression. MME demonstrated significant differences in the hormone cohorts, whereas FAM129A is downregulated at the mRNA level but is raised at the protein level in tumours. Using published ChIP-seq data, we have demonstrated that this may be due to AR binding at the FAM129A and MME loci in multiple cell lines. These data suggest that whole blood mRNA of androgen-regulated genes has the potential to be used for diagnosis and monitoring of prostate cancer. PMID:27578825

  10. Transcriptome Sequencing of Tumor Subpopulations Reveals a Spectrum of Therapeutic Options for Squamous Cell Lung Cancer

    PubMed Central

    Barrett, Christian L.; Schwab, Richard B.; Jung, HyunChul; Crain, Brian; Goff, Daniel J.; Jamieson, Catriona H. M.; Thistlethwaite, Patricia A.; Harismendy, Olivier; Carson, Dennis A.; Frazer, Kelly A.

    2013-01-01

    Background The only therapeutic options that exist for squamous cell lung carcinoma (SCC) are standard radiation and cytotoxic chemotherapy. Cancer stem cells (CSCs) are hypothesized to account for therapeutic resistance, suggesting that CSCs must be specifically targeted. Here, we analyze the transcriptome of CSC and non-CSC subpopulations by RNA-seq to identify new potential therapeutic strategies for SCC. Methods We sorted a SCC into CD133− and CD133+ subpopulations and then examined both by copy number analysis (CNA) and whole genome and transcriptome sequencing. We analyzed The Cancer Genome Atlas (TCGA) transcriptome data of 221 SCCs to determine the generality of our observations. Results Both subpopulations highly expressed numerous mRNA isoforms whose protein products are active drug targets for other cancers; 31 (25%) correspond to 18 genes under active investigation as mAb targets and an additional 4 (3%) are of therapeutic interest. Moreover, we found evidence that both subpopulations were proliferatively driven by very high levels of c-Myc and the TRAIL long isoform (TRAILL) and that normal apoptotic responses to high expression of these genes was prevented through high levels of Mcl-1L and Bcl-xL and c-FlipL—isoforms for which drugs are now in clinical development. SCC RNA-seq data (n = 221) from TCGA supported our findings. Our analysis is inconsistent with the CSC concept that most cells in a cancer have lost their proliferative potential. Furthermore, our study suggests how to target both the CSC and non-CSC subpopulations with one treatment strategy. Conclusions Our study is relevant to SCC in particular for it presents numerous potential options to standard therapy that target the entire tumor. In so doing, it demonstrates how transcriptome sequencing provides insights into the molecular underpinnings of cancer propagating cells that, importantly, can be leveraged to identify new potential therapeutic options for cancers beyond what is

  11. Multi-study Integration of Brain Cancer Transcriptomes Reveals Organ-Level Molecular Signatures

    PubMed Central

    Sung, Jaeyun; Kim, Pan-Jun; Ma, Shuyi; Funk, Cory C.; Magis, Andrew T.; Wang, Yuliang; Hood, Leroy; Geman, Donald; Price, Nathan D.

    2013-01-01

    We utilized abundant transcriptomic data for the primary classes of brain cancers to study the feasibility of separating all of these diseases simultaneously based on molecular data alone. These signatures were based on a new method reported herein – Identification of Structured Signatures and Classifiers (ISSAC) – that resulted in a brain cancer marker panel of 44 unique genes. Many of these genes have established relevance to the brain cancers examined herein, with others having known roles in cancer biology. Analyses on large-scale data from multiple sources must deal with significant challenges associated with heterogeneity between different published studies, for it was observed that the variation among individual studies often had a larger effect on the transcriptome than did phenotype differences, as is typical. For this reason, we restricted ourselves to studying only cases where we had at least two independent studies performed for each phenotype, and also reprocessed all the raw data from the studies using a unified pre-processing pipeline. We found that learning signatures across multiple datasets greatly enhanced reproducibility and accuracy in predictive performance on truly independent validation sets, even when keeping the size of the training set the same. This was most likely due to the meta-signature encompassing more of the heterogeneity across different sources and conditions, while amplifying signal from the repeated global characteristics of the phenotype. When molecular signatures of brain cancers were constructed from all currently available microarray data, 90% phenotype prediction accuracy, or the accuracy of identifying a particular brain cancer from the background of all phenotypes, was found. Looking forward, we discuss our approach in the context of the eventual development of organ-specific molecular signatures from peripheral fluids such as the blood. PMID:23935471

  12. Kinome RNAi Screens Reveal Synergistic Targeting of MTOR and FGFR1 Pathways for Treatment of Lung Cancer and HNSCC

    PubMed Central

    Singleton, Katherine R.; Hinz, Trista K.; Kleczko, Emily K.; Marek, Lindsay A.; Kwak, Jeff; Harp, Taylor; Kim, Jihye; Tan, Aik Choon; Heasley, Lynn E.

    2015-01-01

    The fibroblast growth factor receptor FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with single TKI represents a logical step towards personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multi-kinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between Mammalian Target of Rapamycin (MTOR) and ponatinib in non-small cell lung carcinoma cells. Additionally, treatment with MTOR-targeting shRNAs and pharmacological inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Further, they suggest clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. PMID:26359452

  13. KIPase activity is a novel caspase-like activity associated with cell proliferation.

    PubMed

    Medina-Palazon, Cahora; Bernard, Emmanuelle; Frost, Victoria; Morley, Simon; Sinclair, Alison J

    2004-07-01

    A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27(KIP1), in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100-200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1-10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypothesis whereby KIPase is a currently unidentified caspase-like enzyme which regulates the abundance of p27(KIP1) in a proliferation-dependent manner.

  14. Transforming growth factor-β1 induces cell cycle arrest by activating atypical cyclin-dependent kinase 5 through up-regulation of Smad3-dependent p35 expression in human MCF10A mammary epithelial cells.

    PubMed

    Park, Seong Ji; Yang, Sun Woo; Kim, Byung-Chul

    2016-04-01

    Cyclin-dependent kinases (Cdks) play important roles in control of cell division. Cdk5 is an atypical member of Cdk family with non-cyclin-like regulatory subunit, p35, but its role in cell cycle progression is still unclear. In the present study, we investigated the role of Cdk5/p35 on transforming growth factor-β1 (TGF-β1)-induced cell cycle arrest. In human MCF10A mammary epithelial cells, TGF-β1 induced cell cycle arrest at G1 phase and increased p27KIP1 expression. Interestingly, pretreatment with roscovitine, an inhibitor of Cdk5, or transfection with small interfering (si) RNAs specific to Cdk5 and p35 significantly attenuated the TGF-β1-induced p27KIP1 expression and cell cycle arrest. TGF-β1 increased Cdk5 activity via up-regulation of p35 gene at transcriptional level, and these effects were abolished by transfection with Smad3 siRNA or infection of adenovirus carrying Smad3 mutant at the C-tail (3SA). Chromatin immunoprecipitation assay further revealed that wild type Smad3, but not mutant Smad3 (3SA), binds to the region of the p35 promoter region (-1000--755) in a TGF-β1-dependent manner. These results for the first time demonstrate a role of Cdk5/p35 in the regulation of cell cycle progression modulated by TGF-β1. PMID:26966064

  15. Transforming growth factor-β1 induces cell cycle arrest by activating atypical cyclin-dependent kinase 5 through up-regulation of Smad3-dependent p35 expression in human MCF10A mammary epithelial cells.

    PubMed

    Park, Seong Ji; Yang, Sun Woo; Kim, Byung-Chul

    2016-04-01

    Cyclin-dependent kinases (Cdks) play important roles in control of cell division. Cdk5 is an atypical member of Cdk family with non-cyclin-like regulatory subunit, p35, but its role in cell cycle progression is still unclear. In the present study, we investigated the role of Cdk5/p35 on transforming growth factor-β1 (TGF-β1)-induced cell cycle arrest. In human MCF10A mammary epithelial cells, TGF-β1 induced cell cycle arrest at G1 phase and increased p27KIP1 expression. Interestingly, pretreatment with roscovitine, an inhibitor of Cdk5, or transfection with small interfering (si) RNAs specific to Cdk5 and p35 significantly attenuated the TGF-β1-induced p27KIP1 expression and cell cycle arrest. TGF-β1 increased Cdk5 activity via up-regulation of p35 gene at transcriptional level, and these effects were abolished by transfection with Smad3 siRNA or infection of adenovirus carrying Smad3 mutant at the C-tail (3SA). Chromatin immunoprecipitation assay further revealed that wild type Smad3, but not mutant Smad3 (3SA), binds to the region of the p35 promoter region (-1000--755) in a TGF-β1-dependent manner. These results for the first time demonstrate a role of Cdk5/p35 in the regulation of cell cycle progression modulated by TGF-β1.

  16. Microfluidic Platform for Studying Chemotaxis of Adhesive Cells Revealed a Gradient-Dependent Migration and Acceleration of Cancer Stem Cells.

    PubMed

    Zou, Heng; Yue, Wanqing; Yu, Wai-Kin; Liu, Dandan; Fong, Chi-Chun; Zhao, Jianlong; Yang, Mengsu

    2015-07-21

    Recent studies reveal that solid tumors consist of heterogeneous cells with distinct phenotypes and functions. However, it is unclear how different subtypes of cancer cells migrate under chemotaxis. Here, we developed a microfluidic device capable of generating multiple stable gradients, culturing cells on-chip, and monitoring single cell migratory behavior. The microfluidic platform was used to study gradient-induced chemotaxis of lung cancer stem cell (LCSC) and differentiated LCSC (dLCSC) in real time. Our results showed the dynamic and differential response of both LCSC and dLCSC to chemotaxis, which was regulated by the β-catenin dependent Wnt signaling pathway. The microfluidic analysis showed that LCSC and dLCSC from the same origin behaved differently in the same external stimuli, suggesting the importance of cancer cell heterogeneity. We also observed for the first time the acceleration of both LCSC and dLCSC during chemotaxis caused by increasing local concentration in different gradients, which could only be realized through the microfluidic approach. The capability to analyze single cell chemotaxis under spatially controlled conditions provides a novel analytical platform for the study of cellular microenvironments and cancer cell metastasis.

  17. Multifaceted enrichment analysis of RNA–RNA crosstalk reveals cooperating micro-societies in human colorectal cancer

    PubMed Central

    Mazza, Tommaso; Mazzoccoli, Gianluigi; Fusilli, Caterina; Capocefalo, Daniele; Panza, Anna; Biagini, Tommaso; Castellana, Stefano; Gentile, Annamaria; De Cata, Angelo; Palumbo, Orazio; Stallone, Raffaella; Rubino, Rosa; Carella, Massimo; Piepoli, Ada

    2016-01-01

    Alterations in the balance of mRNA and microRNA (miRNA) expression profiles contribute to the onset and development of colorectal cancer. The regulatory functions of individual miRNA-gene pairs are widely acknowledged, but group effects are largely unexplored. We performed an integrative analysis of mRNA–miRNA and miRNA–miRNA interactions using high-throughput mRNA and miRNA expression profiles obtained from matched specimens of human colorectal cancer tissue and adjacent non-tumorous mucosa. This investigation resulted in a hypernetwork-based model, whose functional backbone was fulfilled by tight micro-societies of miRNAs. These proved to modulate several genes that are known to control a set of significantly enriched cancer-enhancer and cancer-protection biological processes, and that an array of upstream regulatory analyses demonstrated to be dependent on miR-145, a cell cycle and MAPK signaling cascade master regulator. In conclusion, we reveal miRNA-gene clusters and gene families with close functional relationships and highlight the role of miR-145 as potent upstream regulator of a complex RNA–RNA crosstalk, which mechanistically modulates several signaling pathways and regulatory circuits that when deranged are relevant to the changes occurring in colorectal carcinogenesis. PMID:27067546

  18. Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression.

    PubMed

    Stewart, Jonathan; James, Jacqueline; McCluggage, Glenn W; McQuaid, Stephen; Arthur, Kenneth; Boyle, David; Mullan, Paul; McArt, Darragh; Yan, Benedict; Irwin, Gareth; Harkin, D Paul; Zhengdeng, Lei; Ong, Chee-Wee; Yu, Jia; Virshup, David M; Salto-Tellez, Manuel

    2015-03-01

    The oncogenic role of WNT is well characterized. Wntless (WLS) (also known as GPR177, or Evi), a key modulator of WNT protein secretion, was recently found to be highly overexpressed in malignant astrocytomas. We hypothesized that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signaling such as ovarian, gastric, and breast cancers. Immunohistochemical analysis using a TMA platform revealed WLS overexpression in a subset of ovarian, gastric, and breast tumors; this overexpression was associated with poorer clinical outcomes in gastric cancer (P=0.025). In addition, a strong correlation was observed between WLS expression and human epidermal growth factor receptor 2 (HER2) overexpression. Indeed, 100% of HER2-positive intestinal gastric carcinomas, 100% of HER2-positive serous ovarian carcinomas, and 64% of HER2-positive breast carcinomas coexpressed WLS protein. Although HER2 protein expression or gene amplification is an established predictive biomarker for trastuzumab response in breast and gastric cancers, a significant proportion of HER2-positive tumors display resistance to trastuzumab, which may be in part explainable by a possible mechanistic link between WLS and HER2.

  19. Integrative analyses reveal a long noncoding RNA-mediated sponge regulatory network in prostate cancer

    PubMed Central

    Du, Zhou; Sun, Tong; Hacisuleyman, Ezgi; Fei, Teng; Wang, Xiaodong; Brown, Myles; Rinn, John L.; Lee, Mary Gwo-Shu; Chen, Yiwen; Kantoff, Philip W.; Liu, X. Shirley

    2016-01-01

    Mounting evidence suggests that long noncoding RNAs (lncRNAs) can function as microRNA sponges and compete for microRNA binding to protein-coding transcripts. However, the prevalence, functional significance and targets of lncRNA-mediated sponge regulation of cancer are mostly unknown. Here we identify a lncRNA-mediated sponge regulatory network that affects the expression of many protein-coding prostate cancer driver genes, by integrating analysis of sequence features and gene expression profiles of both lncRNAs and protein-coding genes in tumours. We confirm the tumour-suppressive function of two lncRNAs (TUG1 and CTB-89H12.4) and their regulation of PTEN expression in prostate cancer. Surprisingly, one of the two lncRNAs, TUG1, was previously known for its function in polycomb repressive complex 2 (PRC2)-mediated transcriptional regulation, suggesting its sub-cellular localization-dependent function. Our findings not only suggest an important role of lncRNA-mediated sponge regulation in cancer, but also underscore the critical influence of cytoplasmic localization on the efficacy of a sponge lncRNA. PMID:26975529

  20. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells.

    PubMed

    Lawson, Devon A; Bhakta, Nirav R; Kessenbrock, Kai; Prummel, Karin D; Yu, Ying; Takai, Ken; Zhou, Alicia; Eyob, Henok; Balakrishnan, Sanjeev; Wang, Chih-Yang; Yaswen, Paul; Goga, Andrei; Werb, Zena

    2015-10-01

    Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are initiated

  1. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells

    PubMed Central

    Lawson, Devon A.; Bhakta, Nirav R.; Kessenbrock, Kai; Prummel, Karin D.; Yu, Ying; Takai, Ken; Zhou, Alicia; Eyob, Henok; Balakrishnan, Sanjeev; Wang, Chih-Yang; Yaswen, Paul; Goga, Andrei; Werb, Zena

    2015-01-01

    Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality1. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours2–5. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown2. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are

  2. Systematic drug perturbations on cancer cells reveal diverse exit paths from proliferative state.

    PubMed

    Zhou, Joseph X; Isik, Zerrin; Xiao, Caide; Rubin, Irit; Kauffman, Stuart A; Schroeder, Michael; Huang, Sui

    2016-02-16

    During a cell state transition, cells travel along trajectories in a gene expression state space. This dynamical systems framework complements the traditional concept of molecular pathways that drive cell phenotype switching. To expose the structure that hinders cancer cells from exiting robust proliferative state, we assessed the perturbation capacity of a drug library and identified 16 non-cytotoxic compounds that stimulate MCF7 breast cancer cells to exit from proliferative state to differentiated state. The transcriptome trajectories triggered by these drugs diverged, then converged. Chemical structures and drug targets of these compounds overlapped minimally. However, a network analysis of targeted pathways identified a core signaling pathway--indicating common stress-response and down-regulation of STAT1 before differentiation. This multi-trajectory analysis explores the cells' state transition with a multitude of perturbations in combination with traditional pathway analysis, leading to an encompassing picture of the dynamics of a therapeutically desired cell-state switching.

  3. Systematic drug perturbations on cancer cells reveal diverse exit paths from proliferative state

    PubMed Central

    Xiao, Caide; Rubin, Irit; Kauffman, Stuart A.; Schroeder, Michael; Huang, Sui

    2016-01-01

    During a cell state transition, cells travel along trajectories in a gene expression state space. This dynamical systems framework complements the traditional concept of molecular pathways that drive cell phenotype switching. To expose the structure that hinders cancer cells from exiting robust proliferative state, we assessed the perturbation capacity of a drug library and identified 16 non-cytotoxic compounds that stimulate MCF7 breast cancer cells to exit from proliferative state to differentiated state. The transcriptome trajectories triggered by these drugs diverged, then converged. Chemical structures and drug targets of these compounds overlapped minimally. However, a network analysis of targeted pathways identified a core signaling pathway - indicating common stress-response and down-regulation of STAT1 before differentiation. This multi-trajectory analysis explores the cells' state transition with a multitude of perturbations in combination with traditional pathway analysis, leading to an encompassing picture of the dynamics of a therapeutically desired cell-state switching. PMID:26871731

  4. A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC

    PubMed Central

    Schell, Michael J.; Yang, Mingli; Teer, Jamie K.; Lo, Fang Yin; Madan, Anup; Coppola, Domenico; Monteiro, Alvaro N. A.; Nebozhyn, Michael V.; Yue, Binglin; Loboda, Andrey; Bien-Willner, Gabriel A.; Greenawalt, Danielle M.; Yeatman, Timothy J.

    2016-01-01

    Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC. PMID:27302369

  5. Systematic Analysis Reveals that Cancer Mutations Converge on Deregulated Metabolism of Arachidonate and Xenobiotics.

    PubMed

    Gatto, Francesco; Schulze, Almut; Nielsen, Jens

    2016-07-19

    Mutations are the basis of the clonal evolution of most cancers. Nevertheless, a systematic analysis of whether mutations are selected in cancer because they lead to the deregulation of specific biological processes independent of the type of cancer is still lacking. In this study, we correlated the genome and transcriptome of 1,082 tumors. We found that nine commonly mutated genes correlated with substantial changes in gene expression, which primarily converged on metabolism. Further network analyses circumscribed the convergence to a network of reactions, termed AraX, that involves the glutathione- and oxygen-mediated metabolism of arachidonic acid and xenobiotics. In an independent cohort of 4,462 samples, all nine mutated genes were consistently correlated with the deregulation of AraX. Among all of the metabolic pathways, AraX deregulation represented the strongest predictor of patient survival. These findings suggest that oncogenic mutations drive a selection process that converges on the deregulation of the AraX network. PMID:27396332

  6. Detection of gene communities in multi-networks reveals cancer drivers

    NASA Astrophysics Data System (ADS)

    Cantini, Laura; Medico, Enzo; Fortunato, Santo; Caselle, Michele

    2015-12-01

    We propose a new multi-network-based strategy to integrate different layers of genomic information and use them in a coordinate way to identify driving cancer genes. The multi-networks that we consider combine transcription factor co-targeting, microRNA co-targeting, protein-protein interaction and gene co-expression networks. The rationale behind this choice is that gene co-expression and protein-protein interactions require a tight coregulation of the partners and that such a fine tuned regulation can be obtained only combining both the transcriptional and post-transcriptional layers of regulation. To extract the relevant biological information from the multi-network we studied its partition into communities. To this end we applied a consensus clustering algorithm based on state of art community detection methods. Even if our procedure is valid in principle for any pathology in this work we concentrate on gastric, lung, pancreas and colorectal cancer and identified from the enrichment analysis of the multi-network communities a set of candidate driver cancer genes. Some of them were already known oncogenes while a few are new. The combination of the different layers of information allowed us to extract from the multi-network indications on the regulatory pattern and functional role of both the already known and the new candidate driver genes.

  7. Differential network analysis reveals the genome-wide landscape of estrogen receptor modulation in hormonal cancers

    PubMed Central

    Hsiao, Tzu-Hung; Chiu, Yu-Chiao; Hsu, Pei-Yin; Lu, Tzu-Pin; Lai, Liang-Chuan; Tsai, Mong-Hsun; Huang, Tim H.-M.; Chuang, Eric Y.; Chen, Yidong

    2016-01-01

    Several mutual information (MI)-based algorithms have been developed to identify dynamic gene-gene and function-function interactions governed by key modulators (genes, proteins, etc.). Due to intensive computation, however, these methods rely heavily on prior knowledge and are limited in genome-wide analysis. We present the modulated gene/gene set interaction (MAGIC) analysis to systematically identify genome-wide modulation of interaction networks. Based on a novel statistical test employing conjugate Fisher transformations of correlation coefficients, MAGIC features fast computation and adaption to variations of clinical cohorts. In simulated datasets MAGIC achieved greatly improved computation efficiency and overall superior performance than the MI-based method. We applied MAGIC to construct the estrogen receptor (ER) modulated gene and gene set (representing biological function) interaction networks in breast cancer. Several novel interaction hubs and functional interactions were discovered. ER+ dependent interaction between TGFβ and NFκB was further shown to be associated with patient survival. The findings were verified in independent datasets. Using MAGIC, we also assessed the essential roles of ER modulation in another hormonal cancer, ovarian cancer. Overall, MAGIC is a systematic framework for comprehensively identifying and constructing the modulated interaction networks in a whole-genome landscape. MATLAB implementation of MAGIC is available for academic uses at https://github.com/chiuyc/MAGIC. PMID:26972162

  8. A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis

    PubMed Central

    Wagenblast, Elvin; Soto, Mar; Gutiérrez-Ángel, Sara; Hartl, Christina A.; Gable, Annika L.; Maceli, Ashley R.; Erard, Nicolas; Williams, Alissa M.; Kim, Sun Y.; Dickopf, Steffen; Harrell, J. Chuck; Smith, Andrew D.; Perou, Charles M.; Wilkinson, John E.; Hannon, Gregory J.; Knott, Simon R. V.

    2015-01-01

    Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites1. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions2–5. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extra-vascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer. PMID:25855289

  9. Detection of gene communities in multi-networks reveals cancer drivers

    PubMed Central

    Cantini, Laura; Medico, Enzo; Fortunato, Santo; Caselle, Michele

    2015-01-01

    We propose a new multi-network-based strategy to integrate different layers of genomic information and use them in a coordinate way to identify driving cancer genes. The multi-networks that we consider combine transcription factor co-targeting, microRNA co-targeting, protein-protein interaction and gene co-expression networks. The rationale behind this choice is that gene co-expression and protein-protein interactions require a tight coregulation of the partners and that such a fine tuned regulation can be obtained only combining both the transcriptional and post-transcriptional layers of regulation. To extract the relevant biological information from the multi-network we studied its partition into communities. To this end we applied a consensus clustering algorithm based on state of art community detection methods. Even if our procedure is valid in principle for any pathology in this work we concentrate on gastric, lung, pancreas and colorectal cancer and identified from the enrichment analysis of the multi-network communities a set of candidate driver cancer genes. Some of them were already known oncogenes while a few are new. The combination of the different layers of information allowed us to extract from the multi-network indications on the regulatory pattern and functional role of both the already known and the new candidate driver genes. PMID:26639632

  10. Detection of gene communities in multi-networks reveals cancer drivers.

    PubMed

    Cantini, Laura; Medico, Enzo; Fortunato, Santo; Caselle, Michele

    2015-12-07

    We propose a new multi-network-based strategy to integrate different layers of genomic information and use them in a coordinate way to identify driving cancer genes. The multi-networks that we consider combine transcription factor co-targeting, microRNA co-targeting, protein-protein interaction and gene co-expression networks. The rationale behind this choice is that gene co-expression and protein-protein interactions require a tight coregulation of the partners and that such a fine tuned regulation can be obtained only combining both the transcriptional and post-transcriptional layers of regulation. To extract the relevant biological information from the multi-network we studied its partition into communities. To this end we applied a consensus clustering algorithm based on state of art community detection methods. Even if our procedure is valid in principle for any pathology in this work we concentrate on gastric, lung, pancreas and colorectal cancer and identified from the enrichment analysis of the multi-network communities a set of candidate driver cancer genes. Some of them were already known oncogenes while a few are new. The combination of the different layers of information allowed us to extract from the multi-network indications on the regulatory pattern and functional role of both the already known and the new candidate driver genes.

  11. Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel.

    PubMed

    Kharaziha, Pedram; Chioureas, Dimitris; Rutishauser, Dorothea; Baltatzis, George; Lennartsson, Lena; Fonseca, Pedro; Azimi, Alireza; Hultenby, Kjell; Zubarev, Roman; Ullén, Anders; Yachnin, Jeffrey; Nilsson, Sten; Panaretakis, Theocharis

    2015-08-28

    Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy.

  12. Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel

    PubMed Central

    Kharaziha, Pedram; Chioureas, Dimitris; Rutishauser, Dorothea; Baltatzis, George; Lennartsson, Lena; Fonseca, Pedro; Azimi, Alireza; Hultenby, Kjell; Zubarev, Roman; Ullén, Anders; Yachnin, Jeffrey; Nilsson, Sten; Panaretakis, Theocharis

    2015-01-01

    Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy. PMID:25844599

  13. A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells.

    PubMed

    Koseki, Jun; Matsui, Hidetoshi; Konno, Masamitsu; Nishida, Naohiro; Kawamoto, Koichi; Kano, Yoshihiro; Mori, Masaki; Doki, Yuichiro; Ishii, Hideshi

    2016-02-11

    Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

  14. A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells

    NASA Astrophysics Data System (ADS)

    Koseki, Jun; Matsui, Hidetoshi; Konno, Masamitsu; Nishida, Naohiro; Kawamoto, Koichi; Kano, Yoshihiro; Mori, Masaki; Doki, Yuichiro; Ishii, Hideshi

    2016-02-01

    Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

  15. Iroquois homeobox transcription factor (Irx5) promotes G1/S-phase transition in vascular smooth muscle cells by CDK2-dependent activation.

    PubMed

    Liu, Dong; Pattabiraman, Vaishnavi; Bacanamwo, Methode; Anderson, Leonard M

    2016-08-01

    The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3-14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2'-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27(kip1)), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis. PMID:27170637

  16. Resveratrol exerts growth inhibitory effects on human SZ95 sebocytes through the inactivation of the PI3-K/Akt pathway.

    PubMed

    Kim, So Young; Hyun, Moo Yeol; Go, Kyung Chan; Zouboulis, Christos C; Kim, Beom Joon

    2015-04-01

    Resveratrol is known to be a naturally produced polyphenol that is able to reduce cell proliferation in several types of cancer cells and adipocytes. However, the antiproliferative effects of resveratrol on the growth of human sebocytes are not yet clear. In the present study, we investigated possible cellular pathways associated with these growth inhibitory effects on human SZ95 sebocytes. Our results revealed that resveratrol inhibited the proliferation of sebocytes, and that this resulted in disruption of the cell cycle. The inactivation of extracellular signal-regulated protein kinase (ERK), Akt and peroxisome proliferator-activated receptor (PPAR)-γ was also shown to be involved in the inhibition of sebocyte growth by resveratrol. To examine the antiproliferative effects of resveratrol, we determined the levels of cell cycle control proteins. Resveratrol inhibited cyclin D1 synthesis, whereas it stimulated p21WAF1/CIP1 (p21) and p27KIP1 (p27) synthesis. In addition, we demonstrated that the resveratrol-mediated cell cycle arrest resulted in an increase in the proportion of cells in the sub-G0/G1 phase. Moreover, we found that the growth inhibitory effects of resveratrol were enhanced by treatment with LY294002 [a phosphatidylinositol 3-kinase (PI3-K) inhibitor] more so than by treatment with PD98059 (a MEK inhibitor), which indicates that resveratrol exerts its inhibitory effects on sebocyte proliferation through the inhibition of Akt. Linoleic acid (LA) is a well-established lipid inducer in sebocytes and is known to stimulate sebocyte differentiation through the upregulation of PPAR-γ. In this study, resveratrol was found to decrease the lipid content and PPAR-γ expression during LA-stimulated lipogenesis. Our results indicate that resveratrol plays a critical role in the inhibition of sebocyte growth through the inactivation of the Akt pathway. The present data suggest that resveratrol may be used as a therapeutic agent for the treatment of acne vulgaris.

  17. Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells

    PubMed Central

    Phang, Chung-Weng; Karsani, Saiful Anuar; Sethi, Gautam; Abd Malek, Sri Nurestri

    2016-01-01

    Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb. PMID:26859847

  18. Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells.

    PubMed

    Phang, Chung-Weng; Karsani, Saiful Anuar; Sethi, Gautam; Abd Malek, Sri Nurestri

    2016-01-01

    Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb. PMID:26859847

  19. Epitaxially Grown Collagen Fibrils Reveal Diversity in Contact Guidance Behavior among Cancer Cells

    PubMed Central

    2015-01-01

    Invasion of cancer cells into the surrounding tissue is an important step during cancer progression and is driven by cell migration. Cell migration can be random, but often it is directed by various cues such as aligned fibers composed of extracellular matrix (ECM), a process called contact guidance. During contact guidance, aligned fibers bias migration along the long axis of the fibers. These aligned fibers of ECM are commonly composed of type I collagen, an abundant structural protein around tumors. In this paper, we epitaxially grew several different patterns of organized type I collagen on mica and compared the morphology and contact guidance behavior of two invasive breast cancer cell lines (MDA-MB-231 and MTLn3 cells). Others have shown that these cells randomly migrate in qualitatively different ways. MDA-MB-231 cells exert large traction forces, tightly adhere to the ECM, and migrate with spindle-shaped morphology and thus adopt a mesenchymal mode of migration. MTLn3 cells exert small traction forces, loosely adhere to the ECM, and migrate with a more rounded morphology and thus adopt an amoeboid mode of migration. As the degree of alignment of type I collagen fibrils increases, cells become more elongated and engage in more directed contact guidance. MDA-MB-231 cells perceive the directional signal of highly aligned type I collagen fibrils with high fidelity, elongating to large extents and migrating directionally. Interestingly, behavior in MTLn3 cells differs. While highly aligned type I collagen fibril patterns facilitate spreading and random migration of MTLn3 cells, they do not support elongation or directed migration. Thus, different contact guidance cues bias cell migration differently and the fidelity of contact guidance is cell type dependent, suggesting that ECM alignment is a permissive cue for contact guidance, but requires a cell to have certain properties to interpret that cue. PMID:25531276

  20. Early development of cutaneous cancer revealed by intravital nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Lin, Wei-Chou; Chen, Yang-Fang; Chen, Shean-Jen; Lin, Sung-Jan; Dong, Chen-Yuan

    2010-09-01

    We performed intravital multiphoton microscopy to image and analyze normal and carcinogen treated skin tissues of nude mice in vivo. Using intravital images and the quantitative pixel to pixel ratiometric processing of multiphoton autofluorescence to second harmonic generation index (MAFSI), we can visualize the interaction between epithelial cells and extracellular matrix. We found that as the imaging depth increases, MAFSI has different distribution in normal and treated cutaneous specimens. Since the treated skin eventually became squamous cell carcinoma, our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy.

  1. High-Throughput Sequencing of miRNAs Reveals a Tissue Signature in Gastric Cancer and Suggests Novel Potential Biomarkers.

    PubMed

    Darnet, Sylvain; Moreira, Fabiano C; Hamoy, Igor G; Burbano, Rommel; Khayat, André; Cruz, Aline; Magalhães, Leandro; Silva, Artur; Santos, Sidney; Demachki, Samia; Assumpção, Monica; Assumpção, Paulo; Ribeiro-Dos-Santos, Ândrea

    2015-01-01

    Gastric cancer has a high incidence and mortality rate worldwide; however, the use of biomarkers for its clinical diagnosis remains limited. The microRNAs (miRNAs) are biomarkers with the potential to identify the risk and prognosis as well as therapeutic targets. We performed the ultradeep miRnomes sequencing of gastric adenocarcinoma and gastric antrum without tumor samples. We observed that a small set of those samples were responsible for approximately 80% of the total miRNAs expression, which might represent a miRNA tissue signature. Additionally, we identified seven miRNAs exhibiting significant differences, and, of these, hsa-miR-135b and hsa-miR-29c were able to discriminate antrum without tumor from gastric cancer regardless of the histological type. These findings were validated by quantitative real-time polymerase chain reaction. The results revealed that hsa-miR-135b and hsa-miR-29c are potential gastric adenocarcinoma occurrence biomarkers with the ability to identify individuals at a higher risk of developing this cancer, and could even be used as therapeutic targets to allow individualized clinical management. PMID:26157332

  2. Interstitial flows promote an amoeboid over mesenchymal motility of breast cancer cells revealed by a three dimensional microfluidic model

    PubMed Central

    Huang, Yu Ling; Tung, Chih-kuan; Zheng, Anqi; Kim, Beum Jun; Wu, Mingming

    2015-01-01

    Malignant tumors are often associated with an elevated fluid pressure due to the abnormal growth of vascular vessels, and thus an increased interstitial flow out of the tumor. Recent in vitro work revealed that interstitial flows critically regulated tumor cell migration within a three dimensional biomatrix, and breast cancer cell migration behavior depended sensitively on the cell seeding density, chemokine availability and flow rates. In this paper, we focus on roles of interstitial flows in modulating heterogeneity of cancer cell motility phenotype within a three dimensional biomatrix. Using a newly developed microfluidic model, we show that breast cancer cells (MDA-MB-231) embedded in a 3D type I collagen matrix exhibit both an amoeboid and a mesenchymal motility, and interstitial flows promote the cell population towards the amoeboid motility phenotype. Furthermore, the addition of exogenous adhesion molecules (fibronectin) within the extracellular matrix (type I collagen) partially rescues the mesenchymal phenotype in the presence of the flow. Quantitative analysis of cell tracks and cell shape shows distinct differential migration characteristics of amoeboid and mesenchymal cells. Notably, the fastest moving cells belong to the subpopulation of amoeboid cells. Together, these findings highlight the important roles of biophysical forces in modulating tumor cell migration heterogeneity and plasticity, as well as the suitability of microfluidic models in interrogating tumor cell dynamics at single-cell and subpopulation level. PMID:26235230

  3. High-Throughput Sequencing of miRNAs Reveals a Tissue Signature in Gastric Cancer and Suggests Novel Potential Biomarkers

    PubMed Central

    Darnet, Sylvain; Moreira, Fabiano C; Hamoy, Igor G; Burbano, Rommel; Khayat, André; Cruz, Aline; Magalhães, Leandro; Silva, Artur; Santos, Sidney; Demachki, Samia; Assumpção, Monica; Assumpção, Paulo; Ribeiro-dos-Santos, Ândrea

    2015-01-01

    Gastric cancer has a high incidence and mortality rate worldwide; however, the use of biomarkers for its clinical diagnosis remains limited. The microRNAs (miRNAs) are biomarkers with the potential to identify the risk and prognosis as well as therapeutic targets. We performed the ultradeep miRnomes sequencing of gastric adenocarcinoma and gastric antrum without tumor samples. We observed that a small set of those samples were responsible for approximately 80% of the total miRNAs expression, which might represent a miRNA tissue signature. Additionally, we identified seven miRNAs exhibiting significant differences, and, of these, hsa-miR-135b and hsa-miR-29c were able to discriminate antrum without tumor from gastric cancer regardless of the histological type. These findings were validated by quantitative real-time polymerase chain reaction. The results revealed that hsa-miR-135b and hsa-miR-29c are potential gastric adenocarcinoma occurrence biomarkers with the ability to identify individuals at a higher risk of developing this cancer, and could even be used as therapeutic targets to allow individualized clinical management. PMID:26157332

  4. Lymph node revealing solution: a new method for detection of minute axillary lymph nodes in breast cancer specimens.

    PubMed

    Koren, R; Kyzer, S; Paz, A; Veltman, V; Klein, B; Gal, R

    1997-11-01

    The staging and prognosis of patients with breast cancer is related to the presence or absence of axillary lymph node involvement. However, in some cases no lymph nodes or too small a number of lymph nodes are revealed by the traditional method of palpating and sectioning the axillary fat. In the present study we demonstrate the usefulness of the lymph node revealing solution (LNRS) in breast cancer. Specimens from 13 patients, in whom <10 lymph nodes were identified in the axilla by the traditional method, were included in the study. After excising the lymph nodes by the traditional method, axillary tissue was immersed in LNRS for 6-12 hours. Additional lymph nodes, which stood out as white chalky nodules, were excised and processed as usual. The LNRS increased the mean number of nodes per case from 6.0+/-2.5 found by the traditional method to 12.54+/-4.61 nodes per case (p < 0.01). The size of the nodes identified by the LNRS was significantly smaller (p < 0.01) than those detected by the traditional method. The LNRS changed the lymph node stage of the disease in four of the studied cases (30%). LNRS seems to be the technique of choice for detection of axillary lymph nodes in cases where the number of detected lymph nodes by the traditional method is too small for accurate staging. PMID:9351579

  5. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer.

    PubMed

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga; Savova, Virginia; Krzystanek, Marcin; Li, Lewyn; Marusyk, Andriy; Tabassum, Doris P; Zak, Alexander; Flacker, Mary Jo; Li, Mei; Lin, Jessica J; Sukumar, Saraswati; Suzuki, Hiromu; Long, Henry; Szallasi, Zoltan; Gimelbrant, Alexander; Maruyama, Reo; Polyak, Kornelia

    2015-06-16

    Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells. PMID:26051943

  6. Whole Transcriptome Sequencing Reveals Extensive Unspliced mRNA in Metastatic Castration-Resistant Prostate Cancer

    PubMed Central

    Sowalsky, Adam G.; Xia, Zheng; Wang, Liguo; Zhao, Hao; Chen, Shaoyong; Bubley, Glenn J.; Balk, Steven P.; Li, Wei

    2014-01-01

    Men with metastatic prostate cancer (PCa) who are treated with androgen deprivation therapies (ADT) usually relapse within 2–3 years with disease that is termed castration-resistant prostate cancer (CRPC). To identify the mechanism that drives these advanced tumors, paired-end RNA-sequencing (RNA-seq) was performed on a panel of CRPC bone marrow biopsy specimens. From this genome-wide approach, mutations were found in a series of genes with PCa relevance including: AR, NCOR1, KDM3A, KDM4A, CHD1, SETD5, SETD7, INPP4B, RASGRP3, RASA1, TP53BP1 and CDH1, and a novel SND1:BRAF gene fusion. Amongst the most highly-expressed transcripts were ten non-coding RNAs (ncRNAs), including MALAT1 and PABPC1, which are involved in RNA processing. Notably, a high percentage of sequence reads mapped to introns, which were determined to be the result of incomplete splicing at canonical splice junctions. Using quantitative PCR (qPCR) a series of genes (AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19) were confirmed to have a greater proportion of unspliced RNA in CRPC specimens than in normal prostate epithelium, untreated primary PCa, and cultured PCa cells. This inefficient coupling of transcription and mRNA splicing suggests an overall increase in transcription or defect in splicing. PMID:25189356

  7. Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

    PubMed Central

    Le, Anne; Stine, Zachary E.; Nguyen, Christopher; Afzal, Junaid; Sun, Peng; Hamaker, Max; Siegel, Nicholas M.; Gouw, Arvin M.; Kang, Byung-hak; Yu, Shu-Han; Cochran, Rory L.; Sailor, Kurt A.; Song, Hongjun; Dang, Chi V.

    2014-01-01

    Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity. PMID:25114222

  8. Cancer cell(s) cycle sequencing reveals universal mechanisms of apoptosis.

    PubMed

    Marretta, R M Ardito; Ales, F

    2010-12-01

    In this paper, cell cycle in higher eukaryotes and their molecular networks signals both in G1/S and G2/M transitions are replicated in silico. Biochemical kinetics, converted into a set of differential equations, and system control theory are employed to design multi-nested digital layers to simulate protein-to-protein activation and inhibition for cell cycle dynamics in the presence of damaged genomes. Sequencing and controlling the digital process of four micro-scale species networks (p53/Mdm2/DNA damage, p21mRNA/cyclin-CDK complex, CDK/CDC25/weel/SKP2/APC/CKI and apoptosis target genes system) not only allows the comprehension of the mechanisms of these molecule interactions but paves the way for unraveling the participants and their by-products, until now quite unclear, which have the task of carrying out (or not) cell death. Whatever the running simulations (e.g., different species signals, mutant cells and different DNA damage levels), the results of the proposed cell digital multi-layers give reason to believe in the existence of a universal apoptotic mechanism. As a consequence, we identified and selected cell check points, sizers, timers and specific target genes dynamic both for influencing mitotic process and avoiding cancer proliferation as much as for leading the cancer cell(s) to collapse into a steady stable apoptosis phase. PMID:21141676

  9. Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis

    PubMed Central

    Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter; Bianchi, Cristina; Johnstone, Megan E.; Donohoe, Dallas R.; Trott, Josephine F.; Aboud, Omran Abu; Stirdivant, Steven; Neri, Bruce; Wolfert, Robert; Stewart, Benjamin; Perego, Roberto; Hsieh, James J.; Weiss, Robert H.

    2015-01-01

    Kidney cancer (or renal cell carcinoma [RCC]) is known as “the internist’s tumor” because it has protean systemic manifestations suggesting it utilizes complex, non-physiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the VHL tumor suppressor, in characterizing higher grade tumors we found that the Warburg effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, while the β-oxidation pathway is inhibited leading to increased fatty acyl-carnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared to other metabolic pathways. Together, our results offer a rationale to evaluate novel anti-metabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC. PMID:25952651

  10. Conserved mechanisms across development and tumorigenesis revealed by a mouse development perspective of human cancers

    PubMed Central

    Kho, Alvin T.; Zhao, Qing; Cai, Zhaohui; Butte, Atul J.; Kim, John Y.H.; Pomeroy, Scott L.; Rowitch, David H.; Kohane, Isaac S.

    2004-01-01

    Identification of common mechanisms underlying organ development and primary tumor formation should yield new insights into tumor biology and facilitate the generation of relevant cancer models. We have developed a novel method to project the gene expression profiles of medulloblastomas (MBs)—human cerebellar tumors—onto a mouse cerebellar development sequence: postnatal days 1-60 (P1-P60). Genomically, human medulloblastomas were closest to mouse P1-P10 cerebella, and normal human cerebella were closest to mouse P30-P60 cerebella. Furthermore, metastatic MBs were highly associated with mouse P5 cerebella, suggesting that a clinically distinct subset of tumors is identifiable by molecular similarity to a precise developmental stage. Genewise, down- and up-regulated MB genes segregate to late and early stages of development, respectively. Comparable results for human lung cancer vis-a-vis the developing mouse lung suggest the generalizability of this multiscalar developmental perspective on tumor biology. Our findings indicate both a recapitulation of tissue-specific developmental programs in diverse solid tumors and the utility of tumor characterization on the developmental time axis for identifying novel aspects of clinical and biological behavior. PMID:15075291

  11. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  12. 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

  13. 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. PMID:27009842

  14. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling

    PubMed Central

    Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-01

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca2+). In the present study, we established conditions that allow the in vivo detection of Ca2+ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca2+ concentrations and, consequently, an increase in cell death in a p53-dependent pathway. PMID:25544762

  15. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling.

    PubMed

    Giorgi, Carlotta; Bonora, Massimo; Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-30

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca²⁺). In the present study, we established conditions that allow the in vivo detection of Ca²⁺ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca²⁺ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.

  16. mRNA Profiling Reveals Determinants of Trastuzumab Efficiency in HER2-Positive Breast Cancer

    PubMed Central

    von der Heyde, Silvia; Wagner, Steve; Czerny, Alexander; Nietert, Manuel; Ludewig, Fabian; Salinas-Riester, Gabriela; Arlt, Dorit; Beißbarth, Tim

    2015-01-01

    Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs) affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we called and analyzed

  17. mRNA profiling reveals determinants of trastuzumab efficiency in HER2-positive breast cancer.

    PubMed

    von der Heyde, Silvia; Wagner, Steve; Czerny, Alexander; Nietert, Manuel; Ludewig, Fabian; Salinas-Riester, Gabriela; Arlt, Dorit; Beißbarth, Tim

    2015-01-01

    Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs) affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we called and analyzed

  18. Actinomycin D binding mode reveals the basis for its potent HIV-1 and cancer activity

    NASA Astrophysics Data System (ADS)

    Paramanathan, Thayaparan; Vladescu, Ioana D.; McCauley, Micah J.; Rouzina, Ioulia; Williams, Mark C.

    2011-03-01

    Actinomycin D (ActD) is one of the most studied antibiotics, which has been used as an anti-cancer agent and also shown to inhibit HIV reverse transcription. Initial studies with ActD established that it intercalates double stranded DNA (dsDNA). However, recent studies have shown that ActD binds with even higher affinity to single stranded DNA (ssDNA). In our studies we use optical tweezers to stretch and hold single dsDNA molecule at constant force in the presence of varying ActD concentrations until the binding reaches equilibrium. The change in dsDNA length upon ActD binding measured as a function of time yields the rate of binding in addition to the equilibrium lengthening of DNA. The results suggest extremely slow kinetics, on the order of several minutes and 0.52 +/- 0.06 μ M binding affinity. Holding DNA at constant force while stretching and relaxing suggests that ActD binds to two single strands that are close to each other rather than to pure dsDNA or ssDNA. This suggests that biological activity of ActD that contributes towards the inhibition of cellular replication is due to its ability to bind at DNA bubbles during RNA transcription, thereby stalling the transcription process.

  19. Lipidomic Profiling of Adipose Tissue Reveals an Inflammatory Signature in Cancer-Related and Primary Lymphedema

    PubMed Central

    Sedger, Lisa M.; Tull, Dedreia L.; McConville, Malcolm J.; De Souza, David P.; Rupasinghe, Thusitha W. T.; Williams, Spencer J.; Dayalan, Saravanan; Lanzer, Daniel; Mackie, Helen; Lam, Thomas C.; Boyages, John

    2016-01-01

    Cancer-related and primary lymphedema (LE) are associated with the production of adipose tissue (AT). Nothing is known, however, about the lipid-based molecules that comprise LE AT. We therefore analyzed lipid molecules in lipoaspirates and serum obtained from LE patients, and compared them to lipoaspirates from cosmetic surgery patients and healthy control cohort serum. LE patient serum analysis demonstrated that triglycerides, HDL- and LDL-cholesterol and lipid transport molecules remained within the normal range, with no alterations in individual fatty acids. The lipidomic analysis also identified 275 lipid-based molecules, including triacylglycerides, diacylglycerides, fatty acids and phospholipids in AT oil and fat. Although the majority of lipid molecules were present in a similar abundance in LE and non-LE samples, there were several small changes: increased C20:5-containing triacylglycerides, reduced C10:0 caprinic and C24:1 nervonic acids. LE AT oil also contained a signature of increased cyclopropane-type fatty acids and inflammatory mediators arachidonic acid and ceramides. Interestingly C20:5 and C22:6 omega-3-type lipids are increased in LE AT, correlating with LE years. Hence, LE AT has a normal lipid profile containing a signature of inflammation and omega-3-lipids. It remains unclear, however, whether these differences reflect a small-scale global metabolic disturbance or effects within localised inflammatory foci. PMID:27182733

  20. Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers.

    PubMed

    McCreery, Melissa Q; Halliwill, Kyle D; Chin, Douglas; Delrosario, Reyno; Hirst, Gillian; Vuong, Peter; Jen, Kuang-Yu; Hewinson, James; Adams, David J; Balmain, Allan

    2015-12-01

    Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies. PMID:26523969

  1. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients.

    PubMed

    Chabon, Jacob J; Simmons, Andrew D; Lovejoy, Alexander F; Esfahani, Mohammad S; Newman, Aaron M; Haringsma, Henry J; Kurtz, David M; Stehr, Henning; Scherer, Florian; Karlovich, Chris A; Harding, Thomas C; Durkin, Kathleen A; Otterson, Gregory A; Purcell, W Thomas; Camidge, D Ross; Goldman, Jonathan W; Sequist, Lecia V; Piotrowska, Zofia; Wakelee, Heather A; Neal, Joel W; Alizadeh, Ash A; Diehn, Maximilian

    2016-01-01

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment. PMID:27283993

  2. Characterization of Breast Cancer Preclinical Models Reveals a Specific Pattern of Macrophage Polarization.

    PubMed

    Vallerand, David; Massonnet, Gérald; Kébir, Fatima; Gentien, David; Maciorowski, Zofia; De la Grange, Pierre; Sigal-Zafrani, Brigitte; Richardson, Marion; Humbert, Sandrine; Thuleau, Aurélie; Assayag, Franck; de Plater, Ludmilla; Nicolas, André; Scholl, Suzy; Marangoni, Elisabetta; Weigand, Stefan; Roman-Roman, Sergio; Savina, Ariel; Decaudin, Didier

    2016-01-01

    Drug discovery efforts have focused on the tumor microenvironment in recent years. However, few studies have characterized the stroma component in patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMs). In this study, we characterized the stroma in various models of breast cancer tumors in mice. We performed transcriptomic and flow cytometry analyses on murine populations for a series of 25 PDXs and the two most commonly used GEMs (MMTV-PyMT and MMTV-erBb2). We sorted macrophages from five models. We then profiled gene expression in these cells, which were also subjected to flow cytometry for phenotypic characterization. Hematopoietic cell composition, mostly macrophages and granulocytes, differed between tumors. Macrophages had a specific polarization phenotype related to their M1/M2 classification and associated with the expression of genes involved in the recruitment, invasion and metastasis processes. The heterogeneity of the stroma component of the models studied suggests that tumor cells modify their microenvironment to satisfy their needs. Our observations suggest that such models are of relevance for preclinical studies. PMID:27388901

  3. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients

    PubMed Central

    Chabon, Jacob J.; Simmons, Andrew D.; Lovejoy, Alexander F.; Esfahani, Mohammad S.; Newman, Aaron M.; Haringsma, Henry J.; Kurtz, David M.; Stehr, Henning; Scherer, Florian; Karlovich, Chris A.; Harding, Thomas C.; Durkin, Kathleen A.; Otterson, Gregory A.; Purcell, W. Thomas; Camidge, D. Ross; Goldman, Jonathan W.; Sequist, Lecia V.; Piotrowska, Zofia; Wakelee, Heather A.; Neal, Joel W.; Alizadeh, Ash A.; Diehn, Maximilian

    2016-01-01

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment. PMID:27283993

  4. Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA

    PubMed Central

    Togneri, Fiona S; Ward, Douglas G; Foster, Joseph M; Devall, Adam J; Wojtowicz, Paula; Alyas, Sofia; Vasques, Fabiana Ramos; Oumie, Assa; James, Nicholas D; Cheng, K K; Zeegers, Maurice P; Deshmukh, Nayneeta; O'Sullivan, Brendan; Taniere, Philippe; Spink, Karen G; McMullan, Dominic J; Griffiths, Mike; Bryan, Richard T

    2016-01-01

    Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions. PMID:26757983

  5. Characterization of Breast Cancer Preclinical Models Reveals a Specific Pattern of Macrophage Polarization

    PubMed Central

    Vallerand, David; Massonnet, Gérald; Kébir, Fatima; Gentien, David; Maciorowski, Zofia; De la Grange, Pierre; Sigal-Zafrani, Brigitte; Richardson, Marion; Humbert, Sandrine; Thuleau, Aurélie; Assayag, Franck; de Plater, Ludmilla; Nicolas, André; Scholl, Suzy; Marangoni, Elisabetta; Weigand, Stefan; Roman-Roman, Sergio; Savina, Ariel; Decaudin, Didier

    2016-01-01

    Drug discovery efforts have focused on the tumor microenvironment in recent years. However, few studies have characterized the stroma component in patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMs). In this study, we characterized the stroma in various models of breast cancer tumors in mice. We performed transcriptomic and flow cytometry analyses on murine populations for a series of 25 PDXs and the two most commonly used GEMs (MMTV-PyMT and MMTV-erBb2). We sorted macrophages from five models. We then profiled gene expression in these cells, which were also subjected to flow cytometry for phenotypic characterization. Hematopoietic cell composition, mostly macrophages and granulocytes, differed between tumors. Macrophages had a specific polarization phenotype related to their M1/M2 classification and associated with the expression of genes involved in the recruitment, invasion and metastasis processes. The heterogeneity of the stroma component of the models studied suggests that tumor cells modify their microenvironment to satisfy their needs. Our observations suggest that such models are of relevance for preclinical studies. PMID:27388901

  6. Analysis of laser-microdissected prostate cancer tissues reveals potential tumor markers.

    PubMed

    Shaikhibrahim, Zaki; Lindstrot, Andreas; Buettner, Reinhard; Wernert, Nicolas

    2011-10-01

    Prostate cancer (PCA) is a clinically heterogeneous and often multifocal disease with a clinical outcome difficult to predict. A deeper knowledge of the molecular basis of the disease may lead to a better prediction of prognosis. Therefore, in this study we investigated the molecular basis of PCA by identifying potential tumor markers in laser-microdisected PCA tissues. Among a group of PCA patients, quantitative RT-PCR analysis was performed to compare the expression of 70 genes. These genes were selected from the results of two microarrays which investigated the gene expression profile differences between moderately or poorly differentiated prostate carcinoma glands and the corresponding normal glands. Among the genes examined, CDKN2A, GATA3, CREBBP, ITGA2, NBL1 and TGM4 were down-regulated in the prostate carcinoma glands compared to the corresponding normal glands, whereas TFF3, TMPRSS2 and ERG were up-regulated. Our findings indicate that these genes may play roles as tumor suppressor genes or oncogenes in PCA, and may serve as potential tumor markers and novel therapeutic targets.

  7. Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers

    PubMed Central

    McCreery, Melissa Q; Halliwill, Kyle D; Chin, Douglas; Delrosario, Reyno; Hirst, Gillian; Vuong, Peter; Jen, Kuang-Yu; Hewinson, James; Adams, David J; Balmain, Allan

    2016-01-01

    Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies. PMID:26523969

  8. Integrated Analysis Reveals together miR-182, miR-200c and miR-221 Can Help in the Diagnosis of Prostate Cancer

    PubMed Central

    Qin, Xia; Chen, Panyu; Zou, Yi ming; Hu, Yanling

    2015-01-01

    Research has shown that microRNAs are promising biomarkers that can be used to promote a more accurate diagnosis of cancer. In this study, we developed an integrated multi-step selection process to analyze available high-throughput datasets to obtain information on microRNAs as cancer biomarkers. Applying this approach to the microRNA expression profiles of prostate cancer and the datasets in The Cancer Genome Atlas Data Portal, we identified miRNA-182, miRNA-200c and miRNA-221 as possible biomarkers for prostate cancer. The associations between the expressions of these three microRNAs with clinical parameters as well as their diagnostic capability were studied. Several online databases were used to predict the target genes of these three microRNAs, and the results were confirmed by significant statistical correlations. Comparing with the other 18 types of cancers listed in The Cancer Genome Atlas Data Portal, we found that the combination of both miRNA-182 and miRNA-200c being up-regulated and miRNA-221 being down-regulated only happens in prostate cancer. This provides a unique biological characteristic for prostate cancer that can potentially be used for diagnosis based on tissue testing. In addition, our study also revealed that these three microRNAs are associated with the pathological status of prostate cancer. PMID:26484677

  9. SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells

    PubMed Central

    2015-01-01

    Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca2+ sequestration, which confirmed the alteration of the ER pathway due to drug

  10. Boolean ErbB network reconstructions and perturbation simulations reveal individual drug response in different breast cancer cell lines

    PubMed Central

    2014-01-01

    Background Despite promising progress in targeted breast cancer therapy, drug resistance remains challenging. The monoclonal antibody drugs trastuzumab and pertuzumab as well as the small molecule inhibitor erlotinib were designed to prevent ErbB-2 and ErbB-1 receptor induced deregulated protein signalling, contributing to tumour progression. The oncogenic potential of ErbB receptors unfolds in case of overexpression or mutations. Dimerisation with other receptors allows to bypass pathway blockades. Our intention is to reconstruct the ErbB network to reveal resistance mechanisms. We used longitudinal proteomic data of ErbB receptors and downstream targets in the ErbB-2 amplified breast cancer cell lines BT474, SKBR3 and HCC1954 treated with erlotinib, trastuzumab or pertuzumab, alone or combined, up to 60 minutes and 30 hours, respectively. In a Boolean modelling approach, signalling networks were reconstructed based on these data in a cell line and time course specific manner, including prior literature knowledge. Finally, we simulated network response to inhibitor combinations to detect signalling nodes reflecting growth inhibition. Results The networks pointed to cell line specific activation patterns of the MAPK and PI3K pathway. In BT474, the PI3K signal route was favoured, while in SKBR3, novel edges highlighted MAPK signalling. In HCC1954, the inferred edges stimulated both pathways. For example, we uncovered feedback loops amplifying PI3K signalling, in line with the known trastuzumab resistance of this cell line. In the perturbation simulations on the short-term networks, we analysed ERK1/2, AKT and p70S6K. The results indicated a pathway specific drug response, driven by the type of growth factor stimulus. HCC1954 revealed an edgetic type of PIK3CA-mutation, contributing to trastuzumab inefficacy. Drug impact on the AKT and ERK1/2 signalling axes is mirrored by effects on RB and RPS6, relating to phenotypic events like cell growth or proliferation

  11. Systems Perturbation Analysis of a Large-Scale Signal Transduction Model Reveals Potentially Influential Candidates for Cancer Therapeutics

    PubMed Central

    Puniya, Bhanwar Lal; Allen, Laura; Hochfelder, Colleen; Majumder, Mahbubul; Helikar, Tomáš

    2016-01-01

    Dysregulation in signal transduction pathways can lead to a variety of complex disorders, including cancer. Computational approaches such as network analysis are important tools to understand system dynamics as well as to identify critical components that could be further explored as therapeutic targets. Here, we performed perturbation analysis of a large-scale signal transduction model in extracellular environments that stimulate cell death, growth, motility, and quiescence. Each of the model’s components was perturbed under both loss-of-function and gain-of-function mutations. Using 1,300 simulations under both types of perturbations across various extracellular conditions, we identified the most and least influential components based on the magnitude of their influence on the rest of the system. Based on the premise that the most influential components might serve as better drug targets, we characterized them for biological functions, housekeeping genes, essential genes, and druggable proteins. The most influential components under all environmental conditions were enriched with several biological processes. The inositol pathway was found as most influential under inactivating perturbations, whereas the kinase and small lung cancer pathways were identified as the most influential under activating perturbations. The most influential components were enriched with essential genes and druggable proteins. Moreover, known cancer drug targets were also classified in influential components based on the affected components in the network. Additionally, the systemic perturbation analysis of the model revealed a network motif of most influential components which affect each other. Furthermore, our analysis predicted novel combinations of cancer drug targets with various effects on other most influential components. We found that the combinatorial perturbation consisting of PI3K inactivation and overactivation of IP3R1 can lead to increased activity levels of apoptosis

  12. Systems Perturbation Analysis of a Large-Scale Signal Transduction Model Reveals Potentially Influential Candidates for Cancer Therapeutics.

    PubMed

    Puniya, Bhanwar Lal; Allen, Laura; Hochfelder, Colleen; Majumder, Mahbubul; Helikar, Tomáš

    2016-01-01

    Dysregulation in signal transduction pathways can lead to a variety of complex disorders, including cancer. Computational approaches such as network analysis are important tools to understand system dynamics as well as to identify critical components that could be further explored as therapeutic targets. Here, we performed perturbation analysis of a large-scale signal transduction model in extracellular environments that stimulate cell death, growth, motility, and quiescence. Each of the model's components was perturbed under both loss-of-function and gain-of-function mutations. Using 1,300 simulations under both types of perturbations across various extracellular conditions, we identified the most and least influential components based on the magnitude of their influence on the rest of the system. Based on the premise that the most influential components might serve as better drug targets, we characterized them for biological functions, housekeeping genes, essential genes, and druggable proteins. The most influential components under all environmental conditions were enriched with several biological processes. The inositol pathway was found as most influential under inactivating perturbations, whereas the kinase and small lung cancer pathways were identified as the most influential under activating perturbations. The most influential components were enriched with essential genes and druggable proteins. Moreover, known cancer drug targets were also classified in influential components based on the affected components in the network. Additionally, the systemic perturbation analysis of the model revealed a network motif of most influential components which affect each other. Furthermore, our analysis predicted novel combinations of cancer drug targets with various effects on other most influential components. We found that the combinatorial perturbation consisting of PI3K inactivation and overactivation of IP3R1 can lead to increased activity levels of apoptosis

  13. Genetic alterations and their clinical implications in gastric cancer peritoneal carcinomatosis revealed by whole-exome sequencing of malignant ascites

    PubMed Central

    Kim, Jeong-Hwan; Kwon, Woo Sun; Lee, Won Seok; Kim, Jeong Min; Park, Jun Yong; Kim, Hyo Song; Park, Kyu Hyun; Kim, Tae Soo; Park, Jong-Lyul; Chung, Hyun Cheol; Rha, Sun Young; Kim, Seon-Young

    2016-01-01

    Peritoneal carcinomatosis accompanied by malignant ascites is a major cause of death of advanced gastric cancer (GC). To comprehensively characterize the underlying genomic events involved in GC peritoneal carcinomatosis, we analyzed whole-exome sequences of normal gastric tissues, primary tumors, and malignant ascites from eight GC patients. We identified a unique mutational signature biased toward C-to-A substitutions in malignant ascites. In contrast, the patients who received treatment of adjuvant chemotherapy showed a high rate of C-to-T substitutions along with hypermutation in malignant ascites. Comparative analysis revealed several candidate mutations for GC peritoneal carcinomatosis: recurrent mutations in COL4A6, INTS2, and PTPN13; mutations in druggable genes including TEP1, PRKCD, BRAF, ERBB4, PIK3CA, HDAC9, FYN, FASN, BIRC2, FLT3, ROCK1, CD22, and PIK3C2B; and mutations in metastasis-associated genes including TNFSF12, L1CAM, DIAPH3, ROCK1, TGFBR1, MYO9B, NR4A1, and RHOA. Notably, gene ontology analysis revealed the significant enrichment of mutations in the Rho-ROCK signaling pathway-associated biological processes in malignant ascites. At least four of the eight patients acquired somatic mutations in the Rho-ROCK pathway components, suggesting the possible relevance of this pathway to GC peritoneal carcinomatosis. These results provide a genome-wide molecular understanding of GC peritoneal carcinomatosis and its clinical implications, thereby facilitating the development of effective therapeutics. PMID:26811494

  14. Genetic alterations and their clinical implications in gastric cancer peritoneal carcinomatosis revealed by whole-exome sequencing of malignant ascites.

    PubMed

    Lim, Byungho; Kim, Chan; Kim, Jeong-Hwan; Kwon, Woo Sun; Lee, Won Seok; Kim, Jeong Min; Park, Jun Yong; Kim, Hyo Song; Park, Kyu Hyun; Kim, Tae Soo; Park, Jong-Lyul; Chung, Hyun Cheol; Rha, Sun Young; Kim, Seon-Young

    2016-02-16

    Peritoneal carcinomatosis accompanied by malignant ascites is a major cause of death of advanced gastric cancer (GC). To comprehensively characterize the underlying genomic events involved in GC peritoneal carcinomatosis, we analyzed whole-exome sequences of normal gastric tissues, primary tumors, and malignant ascites from eight GC patients. We identified a unique mutational signature biased toward C-to-A substitutions in malignant ascites. In contrast, the patients who received treatment of adjuvant chemotherapy showed a high rate of C-to-T substitutions along with hypermutation in malignant ascites. Comparative analysis revealed several candidate mutations for GC peritoneal carcinomatosis: recurrent mutations in COL4A6, INTS2, and PTPN13; mutations in druggable genes including TEP1, PRKCD, BRAF, ERBB4, PIK3CA, HDAC9, FYN, FASN, BIRC2, FLT3, ROCK1, CD22, and PIK3C2B; and mutations in metastasis-associated genes including TNFSF12, L1CAM, DIAPH3, ROCK1, TGFBR1, MYO9B, NR4A1, and RHOA. Notably, gene ontology analysis revealed the significant enrichment of mutations in the Rho-ROCK signaling pathway-associated biological processes in malignant ascites. At least four of the eight patients acquired somatic mutations in the Rho-ROCK pathway components, suggesting the possible relevance of this pathway to GC peritoneal carcinomatosis. These results provide a genome-wide molecular understanding of GC peritoneal carcinomatosis and its clinical implications, thereby facilitating the development of effective therapeutics. PMID:26811494

  15. Metabolomics Reveals Metabolic Targets and Biphasic Responses in Breast Cancer Cells Treated by Curcumin Alone and in Association with Docetaxel

    PubMed Central

    2013-01-01

    Background Curcumin (CUR) has deserved extensive research due to its anti-inflammatory properties, of interest in human diseases including cancer. However, pleiotropic even paradoxical responses of tumor cells have been reported, and the mechanisms of action of CUR remain uncompletely elucidated. Methodology/Principal Findings 1H-NMR spectroscopy-based metabolomics was applied to get novel insight into responses of MCF7 and MDA-MB-231 breast cancer cells to CUR alone, and MCF7 cells to CUR in cotreatment with docetaxel (DTX). In both cell types, a major target of CUR was glutathione metabolism. Total glutathione (GSx) increased at low dose CUR (≤ 10 mg.l−1–28 µM-) (up to +121% in MCF7 cells, P<0.01, and +138% in MDA-MB-231 cells, P<0.01), but decreased at high dose (≥ 25 mg.l−1 −70 µM-) (−49%, in MCF7 cells, P<0.02, and −56% in MDA-MB-231 cells, P<0.025). At high dose, in both cell types, GSx-related metabolites decreased, including homocystein, creatine and taurine (−60 to −80%, all, P<0.05). Together with glutathione-S-transferase actvity, data established that GSx biosynthesis was upregulated at low dose, and GSx consumption activated at high dose. Another major target, in both cell types, was lipid metabolism involving, at high doses, accumulation of polyunsaturated and total free fatty acids (between ×4.5 and ×11, P<0.025), and decrease of glycerophospho-ethanolamine and -choline (about −60%, P<0.025). Multivariate statistical analyses showed a metabolic transition, even a biphasic behavior of some metabolites including GSx, between low and high doses. In addition, CUR at 10 mg.l−1 in cotreatment with DTX induced modifications in glutathione metabolism, lipid metabolism, and glucose utilization. Some of these changes were biphasic depending on the duration of exposure to CUR. Conclusions/Significance Metabolomics reveals major metabolic targets of CUR in breast cancer cells, and biphasic responses that challenge the widely accepted

  16. Comparative analysis of somatic copy-number alterations across different human cancer types reveals two distinct classes of breakpoint hotspots

    PubMed Central

    Li, Yudong; Zhang, Li; Ball, Robyn L.; Liang, Xinle; Li, Jianrong; Lin, Zhenguo; Liang, Han

    2012-01-01

    Somatic copy-number alterations (SCNAs) play a crucial role in the development of human cancer. However, it is not well understood what evolutionary mechanisms contribute to the global patterns of SCNAs in cancer genomes. Taking advantage of data recently available through The Cancer Genome Atlas, we performed a systematic analysis on genome-wide SCNA breakpoint data for eight cancer types. First, we observed a high degree of overall similarity among the SCNA breakpoint landscapes of different cancer types. Then, we compiled 19 genomic features and evaluated their effects on the observed SCNA patterns. We found that evolutionary indel and substitution rates between species (i.e. humans and chimpanzees) consistently show the strongest correlations with breakpoint frequency among all the surveyed features; whereas the effects of some features are quite cancer-type dependent. Focusing on SCNA breakpoint hotspots, we found that cancer-type-specific breakpoint hotspots and common hotspots show distinct patterns. Cancer-type-specific hotspots are enriched with known cancer genes but are poorly predicted from genomic features; whereas common hotspots show the opposite patterns. This contrast suggests that explaining high-frequency SCNAs in cancer may require different evolutionary models: positive selection driven by cancer genes, and non-adaptive evolution related to an intrinsically unstable genomic context. Our results not only present a systematic view of the effects of genetic factors on genome-wide SCNA patterns, but also provide deep insights into the evolutionary process of SCNAs in cancer. PMID:22899649

  17. Pan-Cancer Analyses Reveal Long Intergenic Non-Coding RNAs Relevant to Tumor Diagnosis, Subtyping and Prognosis.

    PubMed

    Ching, Travers; Peplowska, Karolina; Huang, Sijia; Zhu, Xun; Shen, Yi; Molnar, Janos; Yu, Herbert; Tiirikainen, Maarit; Fogelgren, Ben; Fan, Rong; Garmire, Lana X

    2016-05-01

    Long intergenic noncoding RNAs (lincRNAs) are a relatively new class of non-coding RNAs that have the potential as cancer biomarkers. To seek a panel of lincRNAs as pan-cancer biomarkers, we have analyzed transcriptomes from over 3300 cancer samples with clinical information. Compared to mRNA, lincRNAs exhibit significantly higher tissue specificities that are then diminished in cancer tissues. Moreover, lincRNA clustering results accurately classify tumor subtypes. Using RNA-Seq data from thousands of paired tumor and adjacent normal samples in The Cancer Genome Atlas (TCGA), we identify six lincRNAs as potential pan-cancer diagnostic biomarkers (PCAN-1 to PCAN-6). These lincRNAs are robustly validated using cancer samples from four independent RNA-Seq data sets, and are verified by qPCR in both primary breast cancers and MCF-7 cell line. Interestingly, the expression levels of these six lincRNAs are also associated with prognosis in various cancers. We further experimentally explored the growth and migration dependence of breast and colon cancer cell lines on two of the identified lncRNAs. In summary, our study highlights the emerging role of lincRNAs as potentially powerful and biologically functional pan-cancer biomarkers and represents a significant leap forward in understanding the biological and clinical functions of lincRNAs in cancers.

  18. Global detection of molecular changes reveals concurrent alteration of several biological pathways in nonsmall cell lung cancer cells

    PubMed Central

    Ju, Z.; Kapoor, M.; Newton, K; Cheon, K.; Ramaswamy, A.; Lotan, R.; Strong, L. C.; Koo, J. S.

    2006-01-01

    To identify the molecular changes that occur in non-small cell lung carcinoma (NSCLC), we compared the gene expression profile of the NCI-H292 (H292) NSCLC cell line with that of normal human tracheobronchial epithelial (NHTBE) cells. The NHTBE cells were grown in a three-dimensional organotypic culture system that permits maintenance of the normal pseudostratified mucociliary phenotype characteristic of bronchial epithelium in vivo. Microarray analysis using the Affymetrix oligonucleotide chip U95Av2 revealed that 1,683 genes showed a > 1.5-fold change in expression in the H292 cell line relative to the NHTBE cells. Specifically, 418 genes were downregulated and 1,265 were upregulated in the H292 cells. The expression data for selected genes were validated in several different NSCLC cell lines using quantitative real-time PCR and Western analysis. Further analysis of the differentially expressed genes indicated that WNT responses, apoptosis, cell cycle regulation and cell proliferation were significantly altered in the H292 cells. Functional analysis using fluorescence-activated cell sorting confirmed concurrent changes in the activity of these pathways in the H292 line. These findings show that (1) NSCLC cells display deregulation of the WNT, apoptosis, proliferation and cell cycle pathways, as has been found in many other types of cancer cells, and (2) that organotypically cultured NHTBE cells can be used as a reference to identify genes and pathways that are differentially expressed in tumor cells derived from bronchogenic epithelium. PMID:16049682

  19. Genomic and epigenomic analysis of high-risk prostate cancer reveals changes in hydroxymethylation and TET1

    PubMed Central

    Spans, Lien; Van den Broeck, Thomas; Smeets, Elien; Prekovic, Stefan; Thienpont, Bernard; Lambrechts, Diether; Karnes, R. Jeffrey; Erho, Nicholas; Alshalalfa, Mohammed; Davicioni, Elai; Helsen, Christine; Gevaert, Thomas; Tosco, Lorenzo; Haustermans, Karin; Lerut, Evelyne; Joniau, Steven; Claessens, Frank

    2016-01-01

    The clinical heterogeneity of prostate cancer (PCa) makes it difficult to identify those patients that could benefit from more aggressive treatments. As a contribution to a better understanding of the genomic changes in the primary tumor that are associated with the development of high-risk disease, we performed exome sequencing and copy number determination of a clinically homogeneous cohort of 47 high-risk PCas. We confirmed recurrent mutations in SPOP, PTEN and TP53 among the 850 point mutations we detected. In seven cases, we discovered genomic aberrations in the TET1 (Ten-Eleven Translocation 1) gene which encodes a DNA hydroxymethylase than can modify methylated cytosines in genomic DNA and thus is linked with gene expression changes. TET1 protein levels were reduced in tumor versus non-tumor prostate tissue in 39 of 40 cases. The clinical relevance of changes in TET1 levels was demonstrated in an independent PCa cohort, in which low TET1 mRNA levels were significantly associated with worse metastases-free survival. We also demonstrate a strong reduction in hydroxymethylated DNA in tumor tissue in 27 of 41 cases. Furthermore, we report the first exploratory (h)MeDIP-Seq analyses of eight high-risk PCa samples. This reveals a large heterogeneity in hydroxymethylation changes in tumor versus non-tumor genomes which can be linked with cell polarity. PMID:27014907

  20. Integrative Omics Analysis Reveals Post-Transcriptionally Enhanced Protective Host Response in Colorectal Cancers with Microsatellite Instability

    PubMed Central

    2015-01-01

    Microsatellite instability (MSI) is a frequent and clinically relevant molecular phenotype in colorectal cancer. MSI cancers have favorable survival compared with microsatellite stable cancers (MSS), possibly due to the pronounced tumor-infiltrating lymphocytes observed in MSI cancers. Consistent with the strong immune response that MSI cancers trigger in the host, previous transcriptome expression studies have identified mRNA signatures characteristic of immune response in MSI cancers. However, proteomics features of MSI cancers and the extent to which the mRNA signatures are reflected at the protein level remain largely unknown. Here, we performed a comprehensive comparison of global proteomics profiles between MSI and MSS colorectal cancers in The Cancer Genome Atlas (TCGA) cohort. We found that protein signatures of MSI are also associated with increased immunogenicity. To reliably quantify post-transcription regulation in MSI cancers, we developed a resampling-based regression method by integrative modeling of transcriptomics and proteomics data sets. Compared with the popular simple method, which detects post-transcriptional regulation by either identifying genes differentially expressed at the mRNA level but not at the protein level or vice versa, our method provided a quantitative, more sensitive, and accurate way to identify genes subject to differential post-transcriptional regulation. With this method, we demonstrated that post-transcriptional regulation, coordinating protein expression with key players, initiates de novo and enhances protective host response in MSI cancers. PMID:26680540

  1. Global Oct4 target gene analysis reveals novel downstream PTEN and TNC genes required for drug-resistance and metastasis in lung cancer.

    PubMed

    Tang, Yen-An; Chen, Chi-Hsin; Sun, H Sunny; Cheng, Chun-Pei; Tseng, Vincent S; Hsu, Han-Shui; Su, Wu-Chou; Lai, Wu-Wei; Wang, Yi-Ching

    2015-02-18

    Overexpression of Oct4, a stemness gene encoding a transcription factor, has been reported in several cancers. However, the mechanism by which Oct4 directs transcriptional program that leads to somatic cancer progression remains unclear. In this study, we provide mechanistic insight into Oct4-driven transcriptional network promoting drug-resistance and metastasis in lung cancer cell, animal and clinical studies. Through an integrative approach combining our Oct4 chromatin-immunoprecipitation sequencing and ENCODE datasets, we identified the genome-wide binding regions of Oct4 in lung cancer at promoter and enhancer of numerous genes involved in critical pathways which promote tumorigenesis. Notably, PTEN and TNC were previously undefined targets of Oct4. In addition, novel Oct4-binding motifs were found to overlap with DNA elements for Sp1 transcription factor. We provided evidence that Oct4 suppressed PTEN in an Sp1-dependent manner by recruitment of HDAC1/2, leading to activation of AKT signaling and drug-resistance. In contrast, Oct4 transactivated TNC independent of Sp1 and resulted in cancer metastasis. Clinically, lung cancer patients with Oct4 high, PTEN low and TNC high expression profile significantly correlated with poor disease-free survival. Our study reveals a critical Oct4-driven transcriptional program that promotes lung cancer progression, illustrating the therapeutic potential of targeting Oc4 transcriptionally regulated genes.

  2. Revealing a cancer diagnosis to patients: attitudes of patients, families, friends, nurses, and physicians in Lebanon—results of a cross-sectional study

    PubMed Central

    Farhat, F.; Othman, A.; el Baba, G.; Kattan, J.

    2015-01-01

    Background Disclosure of a cancer diagnosis to patients is a major problem for physicians in Lebanon. Our survey aimed to identify the attitudes of patients, families and friends, nurses, and physicians regarding disclosure of a cancer diagnosis. Methods Study participants included 343 physicians, nurses, cancer patients, families, and friends from clinics in two major hospitals in Lebanon. All completed a 29-item questionnaire that assessed, by demographic group, the information provided about cancer, opinions about the disclosure of the diagnosis to cancer patients, perceived consequences to patients, and the roles of family, friends, and religion. Results Overall, 7.8% of the patients were convinced that cancer is incurable. Nearly 82% preferred to be informed about their diagnosis. Similarly, 83% of physicians were in favour of disclosing a cancer diagnosis to their patients. However, only 14% of the physicians said that they revealed the truth to the patients themselves, with only 9% doing so immediately after confirmation of the diagnosis. Disclosure of a cancer diagnosis was preferred before the start of the treatment by 59% of the patients and immediately after confirmation of the diagnosis by 72% of the physicians. Overall, 86% of physicians, 51% of nurses, and 69% of patients and their families believed that religion helped with the acceptance of a cancer diagnosis. A role for family in accepting the diagnosis was reported by 74% of the patients, 56% of the nurses, and 88% of the physicians. All participants considered that fear was the most difficult feeling (63%) experienced by cancer patients, followed by pain (29%), pity (8%), and death (1%), with no statistically significant difference between the answers given by the participant groups. Conclusions The social background in Lebanese society is the main obstacle to revealing the truth to cancer patients. Lebanese patients seem to prefer direct communication of the truth, but families take the opposite

  3. Metabolomic profiling reveals a role for androgen in activating amino acid metabolism and methylation in prostate cancer cells.

    PubMed

    Putluri, Nagireddy; Shojaie, Ali; Vasu, Vihas T; Nalluri, Srilatha; Vareed, Shaiju K; Putluri, Vasanta; Vivekanandan-Giri, Anuradha; Byun, Jeman; Pennathur, Subramaniam; Sana, Theodore R; Fischer, Steven M; Palapattu, Ganesh S; Creighton, Chad J; Michailidis, George; Sreekumar, Arun

    2011-01-01

    Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer. PMID:21789170

  4. Metabolomic Profiling Reveals a Role for Androgen in Activating Amino Acid Metabolism and Methylation in Prostate Cancer Cells

    PubMed Central

    Putluri, Nagireddy; Shojaie, Ali; Vasu, Vihas T.; Nalluri, Srilatha; Vareed, Shaiju K.; Putluri, Vasanta; Vivekanandan-Giri, Anuradha; Byun, Jeman; Pennathur, Subramaniam; Sana, Theodore R.; Fischer, Steven M.; Palapattu, Ganesh S.; Creighton, Chad J.; Michailidis, George; Sreekumar, Arun

    2011-01-01

    Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer. PMID:21789170

  5. Monoclonal Antibodies Specific for STAT3β Reveal Its Contribution to Constitutive STAT3 Phosphorylation in Breast Cancer

    PubMed Central

    Bharadwaj, Uddalak; Kasembeli, Moses M.; Eckols, T. Kris; Kolosov, Mikhail; Lang, Paul; Christensen, Kurt; Edwards, Dean P.; Tweardy, David J.

    2014-01-01

    Since its discovery in mice and humans 19 years ago, the contribution of alternatively spliced Stat3, Stat3β, to the overall functions of Stat3 has been controversial. Tyrosine-phosphorylated (p) Stat3β homodimers are more stable, bind DNA more avidly, are less susceptible to dephosphorylation, and exhibit distinct intracellular dynamics, most notably markedly prolonged nuclear retention, compared to pStat3α homodimers. Overexpression of one or the other isoform in cell lines demonstrated that Stat3β acted as a dominant-negative of Stat3α in transformation assays; however, studies with mouse strains deficient in one or the other isoform indicated distinct contributions of Stat3 isoforms to inflammation. Current immunological reagents cannot differentiate Stat3β proteins derived from alternative splicing vs. proteolytic cleavage of Stat3α. We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to Stat3β (CT7) and do not cross-react with Stat3α. Immunoblotting studies revealed that levels of Stat3β protein, but not Stat3α, in breast cancer cell lines positively correlated with overall pStat3 levels, suggesting that Stat3β may contribute to constitutive Stat3 activation in this tumor system. The ability to unambiguously discriminate splice alternative Stat3β from proteolytic Stat3β and Stat3α will provide new insights into the contribution of Stat3β vs. Stat3α to oncogenesis, as well as other biological and pathological processes. PMID:25268166

  6. Cellular Metabolism and Dose Reveal Carnitine-Dependent and -Independent Mechanisms of Butyrate Oxidation in Colorectal Cancer Cells.

    PubMed

    Han, Anna; Bennett, Natalie; MacDonald, Amber; Johnstone, Megan; Whelan, Jay; Donohoe, Dallas R

    2016-08-01

    Dietary fiber has been suggested to suppress colorectal cancer development, although the mechanisms contributing to this beneficial effect remain elusive. Butyrate, a fermentation product of fiber, has been shown to have anti-proliferative and pro-apoptotic effects on colorectal cancer cells. The metabolic fate of butyrate in the cell is important in determining whether, it acts as an HDAC inhibitor or is consumed as a short-chain fatty acid. Non-cancerous colonocytes utilize butyrate as the primary energy source whereas cancerous colonocytes increase glucose utilization through the Warburg effect. In this study, we show that butyrate oxidation is decreased in cancerous colonocytes compared to non-cancerous colonocytes. We demonstrate that colorectal cancer cells utilize both a carnitine-dependent and carnitine-independent mechanism that contributes to butyrate oxidation. The carnitine-dependent mechanism is contingent on butyrate concentration. Knockdown of CPT1A in colorectal cancer cells abolishes butyrate oxidation. In terms of selectivity, the carnitine-dependent mechanism only regulated butyrate oxidation, as acetate and propionate oxidation were carnitine-independent. Carnitine decreased the action of butyrate as an HDAC inhibitor and suppressed induction of H3 acetylation by butyrate in colorectal cancer cells. Thus, diminished oxidation of butyrate is associated with decreased HDAC inhibition and histone acetylation. In relation to the mechanism, we find that dichloroacetate, which decreases phosphorylation of pyruvate dehydrogenase, increased butyrate oxidation and that this effect was carnitine-dependent. In conclusion, these data suggest that colorectal cancer cells decrease butyrate oxidation through inhibition of pyruvate dehydrogenase, which is carnitine-dependent, and provide insight into why butyrate shows selective effects toward colorectal cancer cells. J. Cell. Physiol. 231: 1804-1813, 2016. © 2015 Wiley Periodicals, Inc.

  7. Role of the p53 family in stabilizing the genome and preventing polyploidization.

    PubMed

    Talos, Flaminia; Moll, Ute M

    2010-01-01

    Cellular defects resulting in chromosomal instability and aneuploidy are the most common features of human cancers. As a major tumor suppressor and intrinsic part of several cellular checkpoints, p53 contributes to maintenance of the stability of the genetic material, both in quality (ensures faithful replication) and quantity (preservation of diploidy). Although the exact trigger of p53 in case of numerical chromosomal aberrations is unknown, the absence of p53 allows polyploid cells to proliferate and generate unstable aneuploid progeny. A more recent addition to the p53 family, p73, emerged as an important contributor to genomic integrity when p53 is inactivated. p73 loss in p53-null background leads to a rapid increase in polyploidy and aneuploidy, markedly exceeding that caused by p53 loss alone. Constitutive deregulation of Cyclin-Cdk and p27/Kip1 activities and excess failure of the G2/M DNA damage checkpoint are important deficiencies associated with p73 loss.

  8. A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair

    PubMed Central

    Shahar, Or David; Kalousi, Alkmini; Eini, Lital; Fisher, Benoit; Weiss, Amelie; Darr, Jonatan; Mazina, Olga; Bramson, Shay; Kupiec, Martin; Eden, Amir; Meshorer, Eran; Mazin, Alexander V.; Brino, Laurent; Goldberg, Michal; Soutoglou, Evi

    2014-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy. PMID:24682826

  9. A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair.

    PubMed

    Shahar, Or David; Kalousi, Alkmini; Eini, Lital; Fisher, Benoit; Weiss, Amelie; Darr, Jonatan; Mazina, Olga; Bramson, Shay; Kupiec, Martin; Eden, Amir; Meshorer, Eran; Mazin, Alexander V; Brino, Laurent; Goldberg, Michal; Soutoglou, Evi

    2014-05-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy.

  10. Quantitative Proteomic Analysis Reveals That Anti-Cancer Effects of Selenium-Binding Protein 1 In Vivo Are Associated with Metabolic Pathways

    PubMed Central

    Ying, Qi; Ansong, Emmanuel; Diamond, Alan M.; Lu, Zhaoxin; Yang, Wancai; Bie, Xiaomei

    2015-01-01

    Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways. PMID:25974208

  11. Quantitative proteomic analysis reveals that anti-cancer effects of selenium-binding protein 1 in vivo are associated with metabolic pathways.

    PubMed

    Ying, Qi; Ansong, Emmanuel; Diamond, Alan M; Lu, Zhaoxin; Yang, Wancai; Bie, Xiaomei

    2015-01-01

    Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.

  12. Measurement of PIP3 Levels Reveals an Unexpected Role for p110β in Early Adaptive Responses to p110α-Specific Inhibitors in Luminal Breast Cancer

    PubMed Central

    Costa, Carlotta; Ebi, Hiromichi; Martini, Miriam; Beausoleil, Sean A.; Faber, Anthony C.; Jakubik, Charles T.; Huang, Alan; Wang, Youzhen; Nishtala, Madhuri; Hall, Ben; Rikova, Klarisa; Zhao, Jean; Hirsch, Emilio; Benes, Cyril H.

    2016-01-01

    SUMMARY BYL719, which selectively inhibits the alpha isoform of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110α), is currently in clinical trials for the treatment of solid tumors, especially luminal breast cancers with PIK3CA mutations and/or HER2 amplification. This study reveals that, even among these sensitive cancers, the initial efficacy of p110α inhibition is mitigated by rapid re-accumulation of the PI3K product PIP3 produced by the p110β isoform. Importantly, the reactivation of PI3K mediated by p110β does not invariably restore AKT phosphorylation, demonstrating the limitations of using phospho-AKT as a surrogate to measure PI3K activation. Consistently, we show that the addition of the p110β inhibitor to BYL719 prevents the PIP3 rebound and induces greater antitumor efficacy in HER2-amplified and PIK3CA mutant cancers. PMID:25544637

  13. Integrative analysis reveals clinical phenotypes and oncogenic potentials of long non-coding RNAs across 15 cancer types

    PubMed Central

    Piccolo, Stephen R.; Zhang, Xiao-Qin; Li, Jun-Hao; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

    2016-01-01

    Long non-coding RNAs (lncRNAs) have been shown to contribute to tumorigenesis. However, surprisingly little is known about the comprehensive clinical and genomic characterization of lncRNAs across human cancer. In this study, we conducted comprehensive analyses for the expression profile, clinical outcomes, somatic copy number alterations (SCNAs) profile of lncRNAs in ~7000 clinical samples from 15 different cancer types. We identified significantly differentially expressed lncRNAs between tumor and normal tissues from each cancer. Notably, we characterized 47 lncRNAs which were extensively dysregulated in at least 10 cancer types, suggesting a conserved function in cancer development. We also analyzed the associations between lncRNA expressions and patient survival, and identified sets of lncRNAs that possessed significant prognostic values in specific cancer types. Our combined analysis of SCNA data and expression data uncovered 116 dysregulated lncRNAs are strikingly genomic altered across 15 cancer types, indicating their oncogenic potentials. Our study may lay the groundwork for future functional studies of lncRNAs and help facilitate the discovery of novel clinical biomarkers. PMID:27147563

  14. Integrative analysis of lung development-cancer expression associations reveals the roles of signatures with inverse expression patterns.

    PubMed

    Zhang, Chunlong; Li, Chunquan; Xu, Yanjun; Feng, Li; Shang, Desi; Yang, Xinmiao; Han, Junwei; Sun, Zeguo; Li, Yixue; Li, Xia

    2015-05-01

    Recent studies have focused on exploring the associations between organ development and malignant tumors; however, the clinical relevance of the development signatures was inadequately addressed in lung cancer. In this study, we explored the associations between lung development and lung cancer progression by analyzing a total of two development and seven cancer datasets. We identified representative expression patterns (continuously up- and down-regulated) from development and cancer profiles, and inverse pattern associations were observed at both the gene and functional levels. Furthermore, we dissected the biological processes dominating the associations, and found that proliferation and immunity were respectively involved in the two inverse development-cancer expression patterns. Through sub-pathway analysis of the signatures with inverse expression patterns, we finally identified a 13-gene risk signature from the cell cycle sub-pathway, and evaluated its predictive performance for lung cancer patient clinical outcome using independent cohorts. Our findings indicated that the integrative analysis of development and cancer expression patterns provided a framework for identifying effective molecular signatures for clinical utility.

  15. Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

    PubMed

    Krimmel, Jeffrey D; Schmitt, Michael W; Harrell, Maria I; Agnew, Kathy J; Kennedy, Scott R; Emond, Mary J; Loeb, Lawrence A; Swisher, Elizabeth M; Risques, Rosa Ana

    2016-05-24

    Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue. PMID:27152024

  16. The landscape of chromosomal aberrations in breast cancer mouse models reveals driver-specific routes to tumorigenesis

    PubMed Central

    Ben-David, Uri; Ha, Gavin; Khadka, Prasidda; Jin, Xin; Wong, Bang; Franke, Lude; Golub, Todd R.

    2016-01-01

    Aneuploidy and copy-number alterations (CNAs) are a hallmark of human cancer. Although genetically engineered mouse models (GEMMs) are commonly used to model human cancer, their chromosomal landscapes remain underexplored. Here we use gene expression profiles to infer CNAs in 3,108 samples from 45 mouse models, providing the first comprehensive catalogue of chromosomal aberrations in cancer GEMMs. Mining this resource, we find that most chromosomal aberrations accumulate late during breast tumorigenesis, and observe marked differences in CNA prevalence between mouse mammary tumours initiated with distinct drivers. Some aberrations are recurrent and unique to specific GEMMs, suggesting distinct driver-dependent routes to tumorigenesis. Synteny-based comparison of mouse and human tumours narrows critical regions in CNAs, thereby identifying candidate driver genes. We experimentally validate that loss of Stratifin (SFN) promotes HER2-induced tumorigenesis in human cells. These results demonstrate the power of GEMM CNA analysis to inform the pathogenesis of human cancer. PMID:27374210

  17. A new role of the Rac-GAP β2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression

    PubMed Central

    Casado-Medrano, Victoria; Barrio-Real, Laura; García-Rostán, Ginesa; Baumann, Matti; Rocks, Oliver; Caloca, María J.

    2016-01-01

    β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression. PMID:27058424

  18. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk.

    PubMed

    Leitner-Dagan, Yael; Sevilya, Ziv; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S; Schechtman, Edna; Freedman, Laurence; Rennert, Gad; Livneh, Zvi; Paz-Elizur, Tamar

    2014-12-01

    DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/μg protein (95% CI 15.3-16.3), significantly higher than in control subjects-15.1 (14.6-15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1-2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6-4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT.

  19. Atomic Force Microscopy Reveals a Role for Endothelial Cell ICAM-1 Expression in Bladder Cancer Cell Adherence

    PubMed Central

    Laurent, Valérie M.; Duperray, Alain; Sundar Rajan, Vinoth; Verdier, Claude

    2014-01-01

    Cancer metastasis is a complex process involving cell-cell interactions mediated by cell adhesive molecules. In this study we determine the adhesion strength between an endothelial cell monolayer and tumor cells of different metastatic potentials using Atomic Force Microscopy. We show that the rupture forces of receptor-ligand bonds increase with retraction speed and range between 20 and 70 pN. It is shown that the most invasive cell lines (T24, J82) form the strongest bonds with endothelial cells. Using ICAM-1 coated substrates and a monoclonal antibody specific for ICAM-1, we demonstrate that ICAM-1 serves as a key receptor on endothelial cells and that its interactions with ligands expressed by tumor cells are correlated with the rupture forces obtained with the most invasive cancer cells (T24, J82). For the less invasive cancer cells (RT112), endothelial ICAM-1 does not seem to play any role in the adhesion process. Moreover, a detailed analysis of the distribution of rupture forces suggests that ICAM-1 interacts preferentially with one ligand on T24 cancer cells and with two ligands on J82 cancer cells. Possible counter receptors for these interactions are CD43 and MUC1, two known ligands for ICAM-1 which are expressed by these cancer cells. PMID:24857933

  20. An integrated approach to reveal miRNAs' impacts on the functional consequence of copy number alterations in cancer.

    PubMed

    Li, Kening; Liu, Yongjing; Zhou, Yuanshuai; Zhang, Rui; Zhao, Ning; Yan, Zichuang; Zhang, Qiang; Zhang, Shujuan; Qiu, Fujun; Xu, Yan

    2015-01-01

    Copy number alteration (CNA) is known to induce gene expression changes mainly through dosage effect, and therefore affect the initiation and progression of tumor. However, tumor samples exhibit heterogeneity in gene dosage sensitivity due to the complicated mechanisms of transcriptional regulation. Currently, no high-throughput method has been available for identifying the regulatory factors affecting the functional consequences of CNA, and determining their effects on cancer. In view of the important regulatory role of miRNA, we investigated the influence of miRNAs on the dosage sensitivities of genes within the CNA regions. By integrating copy number, mRNA expression, miRNA expression profiles of three kinds of cancer, we observed a tendency for high dosage-sensitivity genes to be more targeted by miRNAs in cancer, and identified the miRNAs regulating the dosage sensitivity of amplified/deleted target genes. The results show that miRNAs can modulate oncogenic biological functions by regulating the genes within the CNA regions, and thus play a role as a trigger or balancer in cancer, affecting cancer processes, even survival. This work provided a framework for analyzing the regulation of dosage effect, which will shed a light on understanding the oncogenic and tumor suppressive mechanisms of CNA. Besides, new cancer-related miRNAs were identified. PMID:26099552

  1. An integrated approach to reveal miRNAs’ impacts on the functional consequence of copy number alterations in cancer

    PubMed Central

    Li, Kening; Liu, Yongjing; Zhou, Yuanshuai; Zhang, Rui; Zhao, Ning; Yan, Zichuang; Zhang, Qiang; Zhang, Shujuan; Qiu, Fujun; Xu, Yan

    2015-01-01

    Copy number alteration (CNA) is known to induce gene expression changes mainly through dosage effect, and therefore affect the initiation and progression of tumor. However, tumor samples exhibit heterogeneity in gene dosage sensitivity due to the complicated mechanisms of transcriptional regulation. Currently, no high-throughput method has been available for identifying the regulatory factors affecting the functional consequences of CNA, and determining their effects on cancer. In view of the important regulatory role of miRNA, we investigated the influence of miRNAs on the dosage sensitivities of genes within the CNA regions. By integrating copy number, mRNA expression, miRNA expression profiles of three kinds of cancer, we observed a tendency for high dosage-sensitivity genes to be more targeted by miRNAs in cancer, and identified the miRNAs regulating the dosage sensitivity of amplified/deleted target genes. The results show that miRNAs can modulate oncogenic biological functions by regulating the genes within the CNA regions, and thus play a role as a trigger or balancer in cancer, affecting cancer processes, even survival. This work provided a framework for analyzing the regulation of dosage effect, which will shed a light on understanding the oncogenic and tumor suppressive mechanisms of CNA. Besides, new cancer-related miRNAs were identified. PMID:26099552

  2. Raman Spectroscopic Analysis Reveals Abnormal Fatty Acid Composition in Tumor Micro- and Macroenvironments in Human Breast and Rat Mammary Cancer

    PubMed Central

    You, Sixian; Tu, Haohua; Zhao, Youbo; Liu, Yuan; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

    2016-01-01

    Fatty acids play essential roles in the growth and metastasis of cancer cells. To facilitate their avid growth and proliferation, cancer cells not only alter the fatty acid synthesis and metabolism intracellularly and extracellularly, but also in the macroenvironment via direct or indirect pathways. We report here, using Raman micro-spectroscopy, that an increase in the production of polyunsaturated fatty acids (PUFAs) was identified in both cancerous and normal appearing breast tissue obtained from breast cancer patients and tumor-bearing rats. By minimizing confounding effects from mixed chemicals and optimizing the signal-to-noise ratio of Raman spectra, we observed a large-scale transition from monounsaturated fatty acids to PUFAs in the tumor while only a small subset of fatty acids transitioned to PUFAs in the tumor micro- and macroenvironment. These data have important implications for further clarifying the macroenvironmental effect of cancer progression and provide new potential approaches for characterizing the tumor micro- and macroenvironment of breast cancer in both pre-clinical animal studies and clinical applications. PMID:27596041

  3. Co-expression networks revealed potential core lncRNAs in the triple-negative breast cancer.

    PubMed

    Yang, Fan; Liu, Ye-Huan; Dong, Si-Yang; Yao, Zhi-Han; Lv, Lin; Ma, Rui-Min; Dai, Xuan-Xuan; Wang, Jiao; Zhang, Xiao-Hua; Wang, Ou-Chen

    2016-10-15

    Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with unfavorable outcome. It is urgent to explore novel biomarkers and potential therapeutic targets in this malignancy. Increasing knowledge of long noncoding RNAs (lncRNAs) significantly deepens our understanding of cancer biology. Here, we sequenced eight paired TNBC tumor tissues and non-cancerous tissues, and validated significantly differentially expressed lncRNAs. Gene ontology (GO) and pathway analysis were used to investigate the function of differentially expressed mRNAs. Further, potential core lncRNAs in TNBC were identified by co-expression networks. Kaplan-Meier analysis also indicated that breast cancer patients with lower expression level of rhabdomyosarcoma 2 associated transcript (RMST), one of the potential core lncRNAs, had worse overall survival. To the best of our knowledge, it was the first report that RMST was involved in breast cancer. Our research provided a rich resource to the research community for further investigating lncRNAs functions and identifying lncRNAs with diagnostic and therapeutic potentials in TNBC.

  4. Raman Spectroscopic Analysis Reveals Abnormal Fatty Acid Composition in Tumor Micro- and Macroenvironments in Human Breast and Rat Mammary Cancer.

    PubMed

    You, Sixian; Tu, Haohua; Zhao, Youbo; Liu, Yuan; Chaney, Eric J; Marjanovic, Marina; Boppart, Stephen A

    2016-01-01

    Fatty acids play essential roles in the growth and metastasis of cancer cells. To facilitate their avid growth and proliferation, cancer cells not only alter the fatty acid synthesis and metabolism intracellularly and extracellularly, but also in the macroenvironment via direct or indirect pathways. We report here, using Raman micro-spectroscopy, that an increase in the production of polyunsaturated fatty acids (PUFAs) was identified in both cancerous and normal appearing breast tissue obtained from breast cancer patients and tumor-bearing rats. By minimizing confounding effects from mixed chemicals and optimizing the signal-to-noise ratio of Raman spectra, we observed a large-scale transition from monounsaturated fatty acids to PUFAs in the tumor while only a small subset of fatty acids transitioned to PUFAs in the tumor micro- and macroenvironment. These data have important implications for further clarifying the macroenvironmental effect of cancer progression and provide new potential approaches for characterizing the tumor micro- and macroenvironment of breast cancer in both pre-clinical animal studies and clinical applications. PMID:27596041

  5. HR-MAS MRS of the pancreas reveals reduced lipid and elevated lactate and taurine associated with early pancreatic cancer.

    PubMed

    Wang, Alan S; Lodi, Alessia; Rivera, Lee B; Izquierdo-Garcia, Jose L; Firpo, Matthew A; Mulvihill, Sean J; Tempero, Margaret A; Bergers, Gabriele; Ronen, Sabrina M

    2014-11-01

    The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five-year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS-detectable metabolic changes provide useful biomarkers for tumor detection and response-monitoring in other cancers. The goal of this study was to identify MRS-detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used (1) H high-resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide-induced inflammation, in pancreata with pre-cancerous metaplasia (4 week old p48-Cre;LSL-Kras(G12D) mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48-Cre LSL-Kras(G12D) mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre-cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p < 0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer-associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and

  6. Transcriptomics profiling study of breast cancer from Kingdom of Saudi Arabia revealed altered expression of Adiponectin and Fatty Acid Binding Protein4: Is lipid metabolism associated with breast cancer?

    PubMed Central

    2015-01-01

    Background Breast cancer incidence rates are increasing at an alarming rate among Saudi Arabian females. Most molecular genetic discoveries on breast cancer and other cancers have arisen from studies examining European and American patients. However, possibility of specific changes in molecular signature among cancer patients of diverse ethnic groups remains largely unexplored. We performed transcriptomic profiling of surgically-resected breast tumors from 45 patients based in the Western region of Saudi Arabia using Affymetrix Gene 1.0 ST chip. Pathway and biological function-based clustering was apparent across the tissue samples. Results Pathway analysis revealed canonical pathways that had not been previously implicated in breast cancer. Biological network analysis of differentially regulated genes revealed that Fatty acid binding protein 4, adipocyte (FABP4), adiponectin (ADIPOQ), and retinol binding protein 4 (RBP4) were most down regulated genes, sharing strong connection with the other molecules of lipid metabolism pathway. The marked biological difference in the signatures uncovered between the USA and Saudi samples underpins the importance of this study. Connectivity Map identified compounds that could reverse an observed gene expression signature Conclusions This study describes, to our knowledge, the first genome-wide profiling of breast cancer from Saudi ethnic females. We demonstrate the involvement of the lipid metabolism pathway in the pathogenesis of breast cancer from this region. This finding also highlights the need for strategies to curb the increasing rates of incidence of this disease by educating the public about life-style risk factors such as unhealthy diet and obesity. PMID:25923423

  7. Interleukin 6 -174G>C polymorphism and cancer risk: meta-analysis reveals a site dependent differential influence in Ancestral North Indians.

    PubMed

    Joshi, Narendra; Kannan, Sadhana; Kotian, Nirupama; Bhat, Shreyas; Kale, Mithila; Hake, Sujata

    2014-08-01

    In our earlier studies, single nucleotide polymorphisms (SNPs) associated with anti-inflammatory cytokines were found to influence risk for breast cancer in western Indian women. Analysis of Interleukin 6 (IL-6) -174G>C polymorphism in this cohort (patients = 182; controls = 236) suggested a protective role for IL-6 -174C allele associated with the lower expression of the cytokine (OR = 0.54; 95% CI 0.32-0.89, dominant model). Together these observations suggested that in comparison to Caucasians, inflammation associated-cytokine gene polymorphisms may have higher influence on risk for cancer in this population. To examine this possibility we analyzed data assessing influence of Interleukin 6 (IL-6) -174G>C polymorphism on risk for various cancers. Overall, there was a marginally higher risk for rare allele homozygotes compared to wild type homozygotes (OR = 1.07; 95% CI 1.00-1.15). Increased risks for genitourinary cancers and for skin cancer were also indicated. The ethnicity based analysis indicated a protective effect of the minor allele in Ancestral North Indians (OR = 0.73; 95% CI 0.55-0.97). Site by ethnicity analysis once again revealed a significant protection against breast cancer (OR = 0.51; 95% CI = 0.37-0.70; dominant model) but an opposite influence on the risk of genitourinary malignancies (OR = 2.51; 95% CI 1.59-3.96; recessive model) in this population alone. The observations imply that contribution of IL-6 to inflammation or effector immunity may depend on the site of malignancy. Assessment of available data in relation to prognosis in breast cancer patients also revealed trends that are compatible with the observations of the meta-analysis. Thus, IL-6 -174G>C polymorphism clearly represents a potential modulator of risk for malignant disorders with ethnicity and site dependent trends. The results also support the possibility of higher influence of inflammation related cytokine gene polymorphisms on the risk for cancers in Ancestral North Indians.

  8. A Multiplexed Cytokeratin Analysis Using Targeted Mass Spectrometry Reveals Specific Profiles in Cancer-Related Pleural Effusions.

    PubMed

    Domanski, Dominik; Perzanowska, Anna; Kistowski, Michal; Wojtas, Grzegorz; Michalak, Agata; Krasowski, Grzegorz; Dadlez, Michal

    2016-07-01

    Pleural effusion (PE), excess fluid in the pleural space, is often observed in lung cancer patients and also forms due to many benign ailments. Classifying it quickly is critical, but this remains an analytical challenge often lengthening the diagnosis process or exposing patients to unnecessary risky invasive procedures. We tested the analysis of PE using a multiplexed cytokeratin (CK) panel with targeted mass spectrometry-based quantitation for its rapid classification. CK markers are often assessed in pathological examinations for cancer diagnosis and guiding treatment course. We developed methods to simultaneously quantify 33 CKs in PE using peptide standards for increased analytical specificity and a simple CK enrichment method to detect their low amounts. Analyzing 121 PEs associated with a variety of lung cancers and noncancerous causes, we show that abundance levels of 10 CKs can be related to PE etiology. CK-6, CK-7, CK-8, CK-18, and CK-19 were found at significantly higher levels in cancer-related PEs. Additionally, elevated levels of vimentin and actin differentiated PEs associated with bacterial infections. A classifier algorithm effectively grouped PEs into cancer-related or benign PEs with 81% sensitivity and 79% specificity. A set of undiagnosed PEs showed that our method has potential to shorten PE diagnosis time. For the first time, we show that a cancer-relevant panel of simple-epithelial CK markers currently used in clinical assessment can also be quantitated in PEs. Additionally, while requiring less invasive sampling, our methodology demonstrated a significant ability to identify cancer-related PEs in clinical samples and thus could improve patient care in the future. PMID:27435923

  9. Genomic and protein expression analysis reveals Flap endonuclease 1 (FEN1) as a key biomarker in breast and ovarian cancer

    PubMed Central

    Abdel-Fatah, Tarek MA; Russell, Roslin; Albarakati, Nada; Maloney, David J; Dorjsuren, Dorjbal; Rueda, Oscar M; Moseley, Paul; Mohan, Vivek; Sun, Hongmao; Abbotts, Rachel; Mukherjee, Abhik; Agarwal, Devika; Illuzzi, Jennifer L.; Jadhav, Ajit; Simeonov, Anton; Ball, Graham; Chan, Stephen; Caldas, Carlos; Ellis, Ian O; Wilson, David M; Madhusudan, Srinivasan

    2015-01-01

    FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p=4.89 × 10−57), high mitotic index (p=5.25 × 10−28), pleomorphism (p=6.31 × 10−19), ER negative (p=9.02 × 10−35), PR negative (p=9.24 × 10−24), triple negative phenotype (p=6.67 × 10−21), PAM50.Her2 (p=5.19 × 10−13), PAM50.Basal (p=2.7 × 10−41), PAM50.LumB (p=1.56 × 10−26), integrative molecular cluster 1 (intClust.1) (p=7.47 × 10−12), intClust.5 (p=4.05 × 10−12) and intClust. 10 (p=7.59 × 10−38) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p= 4.4 × 10−16) and multivariate analysis (p= 9.19 × 10−7). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps<0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps<0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps<0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor sur