Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

PubMed

Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

2015-12-15

Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

PubMed

Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P < 0.05). More colonies were grown 10 days after incubating pcDI-[12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma.

PubMed

Mainardi, Sara; Mijimolle, Nieves; Francoz, Sarah; Vicente-Dueñas, Carolina; Sánchez-García, Isidro; Barbacid, Mariano

2014-01-07

Ubiquitous expression of a resident K-Ras(G12V) oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-Ras(G12V) expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-Ras(G12V) expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras(G12V) oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC(+) alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras(G12V) expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10(+) Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC(+) ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-Ras(G12V)-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC(+) ATII lesions. Finally, activation of K-Ras(G12V) during embryonic development under the control of a Sca1 promoter yielded CC10(+), but not SPC(+), hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC(+) ATII cells were able to yield malignant adenocarcinomas.

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

PubMed Central

Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

2014-01-01

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1–3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner. PMID:24256730

K-ras mutations and HLA-DR expression in large bowel adenomas.

PubMed Central

Norheim Andersen, S.; Breivik, J.; Løvig, T.; Meling, G. I.; Gaudernack, G.; Clausen, O. P.; Schjölberg, A.; Fausa, O.; Langmark, F.; Lund, E.; Rognum, T. O.

1996-01-01

A total of 72 sporadic colorectal adenomas in 56 patients were studied for the presence of point mutations in codons 12 and 13 of the K-ras gene and for HLA-DR antigen expression related to clinicopathological variables. Forty K-ras mutations in 39 adenomas were found (54%): 31 (77%) in codon 12 and nine (23%) in codon 13. There was a strong relationship between the incidence of K-ras mutations and adenoma type, degree of dysplasia and sex. The highest frequency of K-ras mutations was seen in large adenomas of the villous type with high-grade dysplasia. Fourteen out of 15 adenomas obtained from 14 women above 65 years of age carried mutations. HLA-DR positivity was found in 38% of the adenomas, large tumours and those with high-grade dysplasia having the strongest staining. Coexpression of K-ras mutations and HLA-DR was found significantly more frequently in large and highly dysplastic adenomas, although two-way analysis of variance showing size and grade of dysplasia to be the most important variable. None of the adenomas with low-grade dysplasia showed both K-ras mutation and HLA-DR positivity (P = 0.004). K-ras mutation is recognised as an early event in colorectal carcinogenesis. The mutation might give rise to peptides that may be presented on the tumour cell surface by class II molecules, and thereby induce immune responses against neoplastic cells. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8679466

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

PubMed

Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko

2015-05-26

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

Retinoblastoma protein (pRB) was significantly phosphorylated through a Ras-to-MAPK pathway in mutant K-ras stably transfected human adrenocortical cells.

PubMed

Chen, Y-F; Chiu, H-H; Wu, C-H; Wang, J-Y; Chen, F-M; Tzou, W-H; Shin, S-J; Lin, S-R

2003-10-01

Our previous studies have shown that the cell proliferation rate, mRNA levels of p450scc, p450c17, and 3betaHSD, and secretion of cortisol were significantly increased in human adrenocortical cells stably transfected with mutated K-ras expression plasmid "pK568MRSV" after being inducted with IPTG. In addition, the increased level was a time-dependent manner. However, the levels of p450, p450scc, p450c17, 3betaHSD, cortisol, and cell proliferation rate were inhibited by a MEK phospholation inhibitor, PD098059. The above results prove that mutated K-ras oncogene is able to regulate tumorigenesis and steroidogenesis through a Ras-RAF-MEK-MAPK signal transduction pathway. The aim of this study was to investigate regulated factors in this pathway and also examine whether the other signal transduction pathways or other moles involved in tumorigenesis or steroidogenesis. In the first year, we analyzed gene profiles of mutant K-ras-transfected adrenocortical cells by DNA microarray to determine the gene expression related to cell cycle, signal transduction, apoptosis, tumorigenesis, steroidogenesis, and other expressed sequence tag. After being affected by the K-ras mutant, gene expression was significantly increased in some upregulated genes. Human zinc-finger protein 22 increased by 28.5 times, Osteopontin increased by 5.8 times, LIM domain Kinase 2 (LIMK2) increased by 3.3 times, Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated Kinase 2 (DYRK2) increased by 2.2 times, and human syntaxin 3 increased by two times. On the other hand, significant decreases in gene expression were also observed in some downregulated genes. Retinoblastoma binding protein 1 (RBBP1) decreased by four times, Homo sapiens craniofacial development protein 1 (CFDP1) decreased by 2.4 times, DAP Kinase-related apoptosis-inducing protein Kinase 1 (DRAK1) decreased by 2.3 times, SKI-interacting protein (SKIP) decreased by 2.2 times, and human poly(A)-Binding protein (PABP) decreased

Menin determines K-RAS proliferative outputs in endocrine cells

PubMed Central

Chamberlain, Chester E.; Scheel, David W.; McGlynn, Kathleen; Kim, Hail; Miyatsuka, Takeshi; Wang, Juehu; Nguyen, Vinh; Zhao, Shuhong; Mavropoulos, Anastasia; Abraham, Aswin G.; O’Neill, Eric; Ku, Gregory M.; Cobb, Melanie H.; Martin, Gail R.; German, Michael S.

2014-01-01

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors. PMID:25133424

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

PubMed

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P < 0.05). However, RafCAAX mutant can enhance pcDI-[12Asp]K-ras4B cell growth (P < 0.05). (1) [12Asp]K-ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

PubMed

Pamonsinlapatham, Perayot; Gril, Brunilde; Dufour, Sylvie; Hadj-Slimane, Réda; Gigoux, Véronique; Pethe, Stéphanie; L'hoste, Sébastien; Camonis, Jacques; Garbay, Christiane; Raynaud, Françoise; Vidal, Michel

2008-11-01

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Epigenetic therapy with inhibitors of histone methylation suppresses DNA damage signaling and increases glioma cell radiosensitivity.

PubMed

Gursoy-Yuzugullu, Ozge; Carman, Chelsea; Serafim, Rodolfo Bortolozo; Myronakis, Marios; Valente, Valeria; Price, Brendan D

2017-04-11

Radiation therapy is widely used to treat human malignancies, but many tumor types, including gliomas, exhibit significant radioresistance. Radiation therapy creates DNA double-strand breaks (DSBs), and DSB repair is linked to rapid changes in epigenetic modifications, including increased histone methylation. This increased histone methylation recruits DNA repair proteins which can then alter the local chromatin structure and promote repair. Consequently, combining inhibitors of specific histone methyltransferases with radiation therapy may increase tumor radiosensitivity, particularly in tumors with significant therapeutic resistance. Here, we demonstrate that inhibitors of the H4K20 methyltransferase SETD8 (UNC-0379) and the H3K9 methyltransferase G9a (BIX-01294) are effective radiosensitizers of human glioma cells. UNC-0379 blocked H4K20 methylation and reduced recruitment of the 53BP1 protein to DSBs, although this loss of 53BP1 caused only limited changes in radiosensitivity. In contrast, loss of H3K9 methylation through G9a inhibition with BIX-01294 increased radiosensitivity of a panel of glioma cells (SER2Gy range: 1.5 - 2.9). Further, loss of H3K9 methylation reduced DSB signaling dependent on H3K9, including reduced activation of the Tip60 acetyltransferase, loss of ATM signaling and reduced phosphorylation of the KAP-1 repressor. In addition, BIX-0194 inhibited DSB repair through both the homologous recombination and nonhomologous end-joining pathways. Inhibition of G9a and loss of H3K9 methylation is therefore an effective approach for increasing radiosensitivity of glioma cells. These results suggest that combining inhibitors of histone methyltransferases which are critical for DSB repair with radiation therapy may provide a new therapeutic route for sensitizing gliomas and other tumors to radiation therapy.

BMI-1 suppression increases the radiosensitivity of oesophageal carcinoma via the PI3K/Akt signaling pathway.

PubMed

Yang, Xing-Xiao; Ma, Ming; Sang, Mei-Xiang; Zhang, Xue-Yuan; Liu, Zhi-Kun; Song, Heng; Zhu, Shu-Chai

2018-02-01

B-cell‑specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose- and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/M phase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1 phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclin D2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of

Enhanced MET Translation and Signaling Sustains K-Ras-Driven Proliferation under Anchorage-Independent Growth Conditions.

PubMed

Fujita-Sato, Saori; Galeas, Jacqueline; Truitt, Morgan; Pitt, Cameron; Urisman, Anatoly; Bandyopadhyay, Sourav; Ruggero, Davide; McCormick, Frank

2015-07-15

Oncogenic K-Ras mutation occurs frequently in several types of cancers, including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage-independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage-independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage-independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage-independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global downregulation of mRNA translation during anchorage-independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras-mediated Met expression drives "K-Ras addiction" in anchorage-independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage-independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. ©2015 American Association for Cancer Research.

Enhanced MET translation and signaling sustains K-Ras driven proliferation under anchorage-independent growth conditions

PubMed Central

Fujita-Sato, Saori; Galeas, Jacqueline; Truitt, Morgan; Pitt, Cameron; Urisman, Anatoly; Bandyopadhyay, Sourav; Ruggero, Davide; McCormick, Frank

2015-01-01

Oncogenic K-Ras mutation occurs frequently in several types of cancers including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global down-regulation of mRNA translation during anchorage independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras mediated Met expression drives “K-Ras addiction” in anchorage independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. PMID:25977330

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras.

PubMed

Xu, Shenyuan; Long, Brian N; Boris, Gabriel H; Chen, Anqi; Ni, Shuisong; Kennedy, Michael A

2017-12-01

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras-GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

PubMed

Krygowska, Agata Adelajda; Castellano, Esther

2018-06-01

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Andrographolide Sensitizes Ras-Transformed Cells to Radiation in vitro and in vivo

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

Hung, Shih-Kai; Department of Radiation Oncology, Buddhist Dalin Tzu Chi General Hospital, Chiayi, Taiwan; Tzu Chi University School of Medicine, Hualian, Taiwan

2010-07-15

Purpose: Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization. Methods and Materials: An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-{kappa}B) and Akt inmore » andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting. Results: Concurrent andrographolide treatment (10 {mu}M, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 {+-} 2.5 days) compared with radiation alone (22 {+-} 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-{kappa}B activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-{kappa}B activity. Conclusion: Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-{kappa}B activity. These observations indicate that andrographolide

K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

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

Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer

2011-12-01

Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival ofmore » wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.« less

Mutation-Specific RAS Oncogenicity Explains N-RAS Codon 61 Selection in Melanoma

PubMed Central

Burd, Christin E.; Liu, Wenjin; Huynh, Minh V.; Waqas, Meriam A.; Gillahan, James E.; Clark, Kelly S.; Fu, Kailing; Martin, Brit L.; Jeck, William R.; Souroullas, George P.; Darr, David B.; Zedek, Daniel C.; Miley, Michael J.; Baguley, Bruce C.; Campbell, Sharon L.

2014-01-01

N-RAS mutation at codon 12, 13 or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an N-RasQ61R knock-in allele to similarly designed K-RasG12D and N-RasG12D alleles. With concomitant p16INK4a inactivation, K-RasG12D or N-RasQ61R expression efficiently promoted melanoma in vivo, whereas N-RasG12D did not. Additionally, N-RasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of N-RasQ61R and N-RasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, N-RasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity and increased stability when compared to N-RasG12D. This work identifies a faithful model of human N-RAS mutant melanoma, and suggests that the increased melanomagenecity of N-RasQ61R over N-RasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. PMID:25252692

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

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

Xu, Shenyuan; Long, Brian N.; Boris, Gabriel H.

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes amore » significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.« less

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

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

Kim, Han-Soo; Kim, Ju Won; Gang, Jingu

2006-09-15

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain themore » differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.« less

Cancer stem cell drugs target K-ras signaling in a stemness context

PubMed Central

Najumudeen, A K; Jaiswal, A; Lectez, B; Oetken-Lindholm, C; Guzmán, C; Siljamäki, E; Posada, I M D; Lacey, E; Aittokallio, T; Abankwa, D

2016-01-01

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC. PMID:26973241

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

PubMed Central

Pathan, Akbar Ali Khan; Panthi, Bhavana; Khan, Zahid; Koppula, Purushotham Reddy; Alanazi, Mohammed Saud; Sachchidanand; Parine, Narasimha Reddy; Chourasia, Mukesh

2016-01-01

Objective Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results Interestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. Conclusion Thus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein. PMID:27217775

Overexpression of K-p21Ras play a prominent role in lung cancer

NASA Astrophysics Data System (ADS)

Zhang, Peng-bo; Zhou, Xin-liang; Yang, Ju-lun

2018-06-01

The proto-oncogene ras product, p21Ras, has been found overexpression in many human tumors. However, the subtypes of overexpressed p21Ras still remain unclear. The purpose of this study was to investigate overexpressed isoforms of p21Ras and their roles in the progress of lung cancer. Method: The expression of total p21Ras in normal lung tissues and lung cancers was determined by immunohistochemically staining with monoclonal antibody (Mab) KGHR-1 which could recognize and broad spectrum reaction with the (K/H/N) ras protein. Then, the isoforms of p21Ras was examined by specific Mab for each p21Ras subtypes. Results: Low expression of total p21Ras was found in 26.67% (8/30) of normal lung tissues, and 81.31% (87/107) of adenocarcinoma harbored overexpressed total p21Ras. Besides, 70.00% (35/50) of squamous cell carcinoma were detected overexpressed total p21Ras. In addition, 122 lung cancer tissues from overexpression of total p21Ras protein were selected to detect the expression of each subtype. And all the 122 lung cancer tissues were K-p21Ras overexpression. Moreover, there was a statistical significance difference between the expression level of total p21Ras and differentiation, and the same results were observed between the expression level of total p21Ras and lymph node metastasis (P<0.05). However, there was no correlation between the expression level of total p21Ras and gender, age, tumor size (P>0.05). Conclusions: Overexpression of K-p21Ras plays a prominent role in the progress of lung cancer and it is suggested that the p21Ras could serve as a promising treatment target in lung cancer.

Impact of Nrf2 on tumour growth and drug sensitivity in oncogenic K-ras-transformed cells in vitro and in vivo.

PubMed

Shao, Jiajia; Glorieux, Christophe; Liao, Jianwei; Chen, Ping; Lu, Wenhua; Liang, Zhenhao; Wen, Shijun; Hu, Yumin; Huang, Peng

2018-06-01

K-ras is one of the most common oncogenes in human cancers, and its aberrant activation may lead to malignant transformation associated with oxidative stress and activation of the transcription factor Nrf2 that regulates multiple detoxification enzymes. The purpose of this research was to use gene editing technology to evaluate the role of Nrf2 in affecting tumour growth and drug sensitivity of K-ras G12V -transformed cells. We showed that induction of K-ras G12V caused a significant activation of Nrf2 associated with increased expression of its target genes NAD(P)H:quinone oxidoreductase 1 (NQO1) and haem oxygenase-1 (HO-1). Interestingly, knock-out of Nrf2 by CRISPR/Cas9 in K-ras G12V -expressing cells only impacted the expression of NQO1 but not HO-1. We also found that Nrf2 knock-out caused high reactive oxygen species (ROS) stress, suppression of cell proliferation, increased apoptosis in vitro, and a decrease of tumour growth in vivo. Furthermore, abrogation of Nrf2 significantly increased the sensitivity of K-ras G12V cells to multiple anticancer agents including phenethyl isothiocyanate (PEITC), doxorubicin, etoposide, and cisplatin. These results show that genetic abrogation of Nrf2 impairs the malignant phenotype of K-Ras G12V -transformed cells in vitro and in vivo, and demonstrates the critical role of Nrf2 in promoting cell survival and drug resistance in cells harbouring oncogenic K-ras. As such, inhibition of Nrf2 would be an attractive strategy to increase the therapeutic effect and overcome drug resistance in cancer with oncogenic K-ras activation.

Prx I Suppresses K-ras-Driven Lung Tumorigenesis by Opposing Redox-Sensitive ERK/Cyclin D1 Pathway

PubMed Central

Park, Young-Ho; Kim, Sun-Uk; Lee, Bo-Kyoung; Kim, Hyun-Sun; Song, In-Sung; Shin, Hye-Jun; Han, Ying-Hao; Chang, Kyu-Tae; Kim, Jin-Man; Lee, Dong-Seok; Kim, Yeul-Hong; Choi, Chang-Min; Kim, Bo-Yeon

2013-01-01

Abstract Aims: Coupled responses of mutated K-ras and oxidative stress are often an important etiological factor in non–small-cell lung cancer (NSCLC). However, relatively few studies have examined the control mechanism of oxidative stress in oncogenic K-ras-driven NSCLC progression. Here, we studied whether the redox signaling pathway governed by peroxiredoxin I (Prx I) is involved in K-rasG12D-mediated lung adenocarcinogenesis. Results: Using human-lung adenocarcinoma tissues and lung-specific K-rasG12D-transgenic mice, we found that Prx I was significantly up-regulated in the tumor regions via activation of nuclear erythroid 2-related factor 2 (Nrf2) transcription. Interestingly, the increased reactive oxygen species (ROS) by null mutation of Prx I greatly promoted K-rasG12D-driven lung tumorigenesis in number and size, which appeared to require the activation of the ROS-dependent extracellular signal-regulated kinase (ERK)/cyclin D1 pathway. Innovation: Taken together, these results suggest that Prx I functions as an Nrf2-dependently inducible tumor suppressant in K-ras-driven lung adenocarcinogenesis by opposing ROS/ERK/cyclin D1 pathway activation. Conclusion: These findings provide a better understanding of oxidative stress-mediated lung tumorigenesis. Antioxid. Redox Signal. 19, 482–496. PMID:23186333

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology

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

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya

Amino-acid mutations of Gly{sup 12} (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH{sub 2}) as a consensus sequence. KRpep-2 showedmore » more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K{sub D} and IC{sub 50} values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH{sub 2}) that inhibited enzyme activity of K-Ras(G12D) with IC{sub 50} = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. - Highlights: • The first K-Ras(G12D)-selective inhibitory peptides were generated. • These peptides showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D) in compared to wild type K-Ras. • The peptide KRpep-2d suppressed downstream signal of K-Ras(G12D) and cell proliferations of cancer cell line A427.« less

SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a

PubMed Central

Wisner, Stephanie A; Chen, Xiao; Spiegelman, Nicole A; Linder, Maurine E

2017-01-01

Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation. PMID:29239724

K-ras Mutations as the Earliest Driving Force in a Subset of Colorectal Carcinomas

PubMed Central

MARGETIS, NIKOLAOS; KOULOUKOUSSA, MYRSINI; PAVLOU, KYRIAKI; VRAKAS, SPYRIDON; MARIOLIS-SAPSAKOS, THEODOROS

2017-01-01

K-ras oncogene is a key factor in colorectal cancer. Based on published and our data we propose that K-ras could be the oncogene responsible for the inactivation of the tumor-suppressor gene APC, currently considered as the initial step in colorectal tumorigenesis. K-ras fulfills the criteria of the oncogene-induced DNA damage model, as it can provoke well- established causes for inactivating tumor-suppressors, i.e. DNA double-strand breaks (causing allele deletion) and ROS production (responsible for point mutation). The model we propose is a variation of the currently existing model and hypothesizes that, in a subgroup of colorectal carcinomas, K-ras mutation may precede APC inactivation, representing the earliest driving force and, probably, an early biomarker of colorectal carcinogenesis. This observation is clinically useful, since it may modify the preventive colorectal cancer strategy, restricting numerically patients undergoing colonoscopies to those bearing K-ras mutation in their colorectum, either in benign polyps or the normal accompanying mucosa. PMID:28652417

Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions

NASA Astrophysics Data System (ADS)

Vatansever, Sezen; Gümüş, Zeynep H.; Erman, Burak

2016-11-01

K-Ras is the most frequently mutated oncogene in human cancers, but there are still no drugs that directly target it in the clinic. Recent studies utilizing dynamics information show promising results for selectively targeting mutant K-Ras. However, despite extensive characterization, the mechanisms by which K-Ras residue fluctuations transfer allosteric regulatory information remain unknown. Understanding the direction of information flow can provide new mechanistic insights for K-Ras targeting. Here, we present a novel approach -conditional time-delayed correlations (CTC) - using the motions of all residue pairs of a protein to predict directionality in the allosteric regulation of the protein fluctuations. Analyzing nucleotide-dependent intrinsic K-Ras motions with the new approach yields predictions that agree with the literature, showing that GTP-binding stabilizes K-Ras motions and leads to residue correlations with relatively long characteristic decay times. Furthermore, our study is the first to identify driver-follower relationships in correlated motions of K-Ras residue pairs, revealing the direction of information flow during allosteric modulation of its nucleotide-dependent intrinsic activity: active K-Ras Switch-II region motions drive Switch-I region motions, while α-helix-3L7 motions control both. Our results provide novel insights for strategies that directly target mutant K-Ras.

Simultaneous Inhibition of EGFR and PI3K Enhances Radiosensitivity in Human Breast Cancer

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

Li Ping; Zhang Qing; Torossian, Artour

2012-07-01

Purpose: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells. Methods and Materials: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used tomore » investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect. Results: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF{sub SF2}) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF{sub SF2} at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts. Conclusions: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

PubMed

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

2017-03-11

Amino-acid mutations of Gly 12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH 2 ) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K D and IC 50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH 2 ) that inhibited enzyme activity of K-Ras(G12D) with IC 50  = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of K-ras gene mutations in feces by magnetic nanoprobe in patients with pancreatic cancer: A preliminary study.

PubMed

Wang, Xiaoguang; Wang, Jingshuai; Chen, Fei; Zhong, Zhengxiang; Qi, Lifeng

2018-01-01

The present study aimed to investigate the feasibility and effectiveness of detecting K-ras mutation by using magnetic nanoparticles in fecal samples of patients with pancreatic cancer at different stages. The novel methodology of K-ras mutation detection was compared to the existing methodology of cancer antigen (CA)19-9 examination. Patients with pancreatic cancer (n=88), pancreatic benign diseases who displayed chronic pancreatitis (n=35), pancreatic mucinous cyst neoplasms (n=10) and pancreatic serous cyst (n=9) admitted to the Department of Surgery, Jiaxing Second Hospital were enrolled in the present study. Fecal samples were collected from all patients, DNA was extracted and magnetic nanoprobe was then used to detect K-ras mutation. The results obtained using the novel magnetic nanoprobe detection technique showed a K-ras mutation rate of 81.8% (72/88) in the patients with pancreatic cancer and 18.5% (10/54) in patients with pancreatic benign diseases. In patients with pancreatic cancer, the K-ras mutation rate was comparable in stages I + IIA and IIB + III + IV (78.9 vs. 84.0%; P>0.05). The sensitivity and specificity of K-ras mutation for detection of pancreatic cancer was 81.8 and 81.5%, respectively. Sixty-eight pancreatic cancer patients had >37 U/ml CA99 with a sensitivity and specificity for pancreatic cancer detection of 77.3 and 77.8%, which was not significantly lower than detection by the fecal K-ras mutations (P>0.05). Combinational detection of fecal K-ras mutations and serum CA19-9 significantly increased the sensitivity regarding pancreatic cancer detection to 97.7% (P<0.05), while the specificity was not enhanced (80.9%; P>0.05) compared with fecal K-ras mutations or CA19-9 alone. The findings showed that the magnetic nanoprobe is able to detect fecal K-ras mutations in different stages of pancreatic cancer, with comparable sensitivity and specificity to CA19-9 examination for differentiating pancreatic cancer. Furthermore, combined detection

Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

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

Yu, S.-H.; Wang, T.-H.; Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 11227, Taiwan

2009-01-09

Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU {yields} GUU) at the GU dinucleotide while left the wild-type (WT)more » K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.« less

Ras inhibitors display an anti-metastatic effect by downregulation of lysyl oxidase through inhibition of the Ras-PI3K-Akt-HIF-1α pathway.

PubMed

Yoshikawa, Yoko; Takano, Osamu; Kato, Ichiro; Takahashi, Yoshihisa; Shima, Fumi; Kataoka, Tohru

2017-12-01

Metastasis stands as the major obstacle for the survival from cancers. Nonetheless most existing anti-cancer drugs inhibit only cell proliferation, and discovery of agents having both anti-proliferative and anti-metastatic properties would be more beneficial. We previously reported the discovery of small-molecule Ras inhibitors, represented by Kobe0065, that displayed anti-proliferative activity on xenografts of human colorectal cancer (CRC) cell line SW480 carrying the K-ras G12V gene. Here we show that treatment of cancer cells carrying the activated ras genes with Kobe0065 or a siRNA targeting Ras downregulates the expression of lysyl oxidase (LOX), which has been implicated in metastasis. LOX expression is enhanced by co-expression of Ras G12V through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and concomitant accumulation of hypoxia-inducible factor (HIF)-1α. Furthermore, Kobe0065 effectively inhibits not only migration and invasion of cancer cells carrying the activated ras genes but also lung metastasis of human CRC cell line SW620 carrying the K-ras G12V gene. Collectively, these results indicate that Kobe0065 prevents metastasis through inhibition of the Ras-PI3K-Akt-HIF-1α-LOX signaling and suggest that Ras inhibitors in general might exhibit both anti-proliferative and anti-metastatic properties toward cancer cells carrying the activated ras genes. Copyright © 2017 Elsevier B.V. All rights reserved.

The Significance of Ras Activity in Pancreatic Cancer Initiation.

PubMed

Logsdon, Craig D; Lu, Weiqin

2016-01-01

The genetic landscape of pancreatic cancer shows nearly ubiquitous mutations of K-RAS. However, oncogenic K-Ras(mt) alone is not sufficient to lead to pancreatic ductal adenocarcinoma (PDAC) in either human or in genetically modified adult mouse models. Many stimulants, such as high fat diet, CCK, LPS, PGE2 and others, have physiological effects at low concentrations that are mediated in part through modest increases in K-Ras activity. However, at high concentrations, they induce inflammation that, in the presence of oncogenic K-Ras expression, substantially accelerates PDAC formation. The mechanism involves increased activity of oncogenic K-Ras(mt). Unlike what has been proposed in the standard paradigm for the role of Ras in oncogenesis, oncogenic K-Ras(mt) is now known to not be constitutively active. Rather, it can be activated by standard mechanisms similar to wild-type K-Ras, but its activity is sustained for a prolonged period. Furthermore, if the level of K-Ras activity exceeds a threshold at which it begins to generate its own activators, then a feed-forward loop is formed between K-Ras activity and inflammation and pathological processes including oncogenesis are initiated. Oncogenic K-Ras(mt) activation, a key event in PDAC initiation and development, is subject to complex regulatory mechanisms. Reagents which inhibit inflammation, such as the Cox2 inhibitor celecoxib, block the feed-forward loop and prevent induction of PDAC in models with endogenous oncogenic K-Ras(mt). Increased understanding of the role of activating and inhibitory mechanisms on oncogenic K-Ras(mt) activity is of paramount importance for the development of preventive and therapeutic strategies to fight against this lethal disease.

K-ras mutation promotes ionizing radiation-induced invasion and migration of lung cancer in part via the Cathepsin L/CUX1 pathway.

PubMed

Wang, Long; Zhao, Yifan; Xiong, Yajie; Wang, Wenjuan; Fei, Yao; Tan, Caihong; Liang, Zhongqin

2018-01-15

K-ras mutation is involved in cancer progression including invasion and migration, but the underlying mechanism is not yet clear. Cathepsin L is a lysosomal cysteine protease and has recently been associated with invasion and migration in human cancers when it is overexpressed. Our recent studies have shown that ionizing radiation (IR) enhanced expression of cathepsin L and increased invasion and migration of tumor cells, but the molecular mechanism is still unclear. In the present study, the effects of K-ras mutation and IR induced invasion and migration of lung cancer as well as the underlying mechanisms were investigated both in vitro and in vivo. Firstly, the levels of cathepsin L and epithelial mesenchymal transition (EMT) marker proteins remarkably changed in A549 (K-ras mutant) after irradiation compared with H1299 (K-ras wild), thereby promoting invasion and migration. Additionally, cathepsin L and its downstream transcription factor CUX1/p110 were increased after irradiation in A549 transfected with CUX1/p200, and the proteolytic processing of CUX1 by cathepsin L was remarkably increased after co-transfection of CUX1/p200 and cathepsin L-lentivirus in H1299. In addition, delivery of a mutant K-ras (V12) into HEK 293 cells stimulated EMT after irradiation due to the accumulation of cathepsin L. Moreover, mutated K-ras was associated with IR-induced cathepsin L and EMT in BALB/c nude mice. Finally, the level of cathepsin L expression was higher in samples carrying a K-ras mutation than in wild-type K-ras samples and the mesenchymal markers were upregulated in the samples of mutant K-ras, whereas the epithelial marker E-cadherin was downregulated in non-small cell lung cancers tissues. In conclusion, the findings demonstrated that mutated K-ras promotes cathepsin L expression and plays a pivotal role in EMT of human lung cancer. The regulatory effect of IR-induced cathepsin L on lung cancer invasion and migration was partially attributed to the Cathepsin L

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2

PubMed Central

Cho, Kwang-jin; Casteel, Darren E.; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A.; Kim, Choel; Capon, Robert J.; Lacey, Ernest; Cunha, Shane R.; Gorfe, Alemayehu A.

2016-01-01

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function. PMID:27697864

K-Ras(V14I) -induced Noonan syndrome predisposes to tumour development in mice.

PubMed

Hernández-Porras, Isabel; Schuhmacher, Alberto J; Garcia-Medina, Raquel; Jiménez, Beatriz; Cañamero, Marta; de Martino, Alba; Guerra, Carmen

2016-06-01

The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock-in mouse model that carries one of the most frequent KRAS-NS-related mutations, the K-Ras(V14I) substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K-Ras(V14I) mutation is a mild activating K-Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K-Ras(G12V) oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K-Ras(V14I) mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K-Ras(V14I) activating protein is able to induce cancer, although at a much lower level than the classical K-Ras(G12V) oncogene, and that it can be significantly modulated by both genetic and non-genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

[Clinical relevance of the K-ras oncogene in colorectal cancer: experience in a Mexican population].

PubMed

Cabrera-Mendoza, F; Gainza-Lagunes, S; Castañeda-Andrade, I; Castro-Zárate, A

2014-01-01

Colorectal cancer is frequent in the developed countries, with a cancer-specific mortality rate of 33%. Different biomarkers are associated with overall survival and the prediction of monoclonal treatment effectiveness. The presence of mutations in the K-ras oncogene alters the response to target therapy with cetuximab and could be an independent prognostic factor. To analyze the difference in survival between patients with mutated K-ras and those with K-ras wild-type status. Thirty-one clinical records were retrospectively analyzed of patients presenting with colorectal cancer that underwent K-ras sequencing through real-time polymerase chain reaction within the time frame of 2009 to 2012 at the Hospital de Alta Especialidad de Veracruz of the Instituto para la Salud y Seguridad Social de los Trabajadores del Estado (HAEV-ISSSTE). Survival analysis for patients with and without K-ras mutation was performed using the Kaplan Meier method. Contrast of covariates was performed using logarithmic transformations. No statistically significant difference was found in relation to survival in the patients with mutated K-ras vs. those with K-ras wild-type (P=.416), nor were significant differences found when analyzing the covariants and survival in the patients with mutated K-ras: ECOG scale (P=.221); age (less than, equal to or greater than 65years, P=.441); clinical stage according to the AJCC (P=.057), and primary lesion site (P=.614). No relation was found between the K-ras oncogene mutation and reduced survival, in contrast to what has been established in the international medical literature. Further studies that include both a larger number of patients and those receiving monoclonal treatment, need to be conducted. There were only 5 patients in the present study that received cetuximab, resulting in a misleading analysis. Copyright © 2013 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

PubMed Central

Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

2013-01-01

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

K-ras mutations in benzotrichloride-induced lung tumors of A/J mice.

PubMed

You, M; Wang, Y; Nash, B; Stoner, G D

1993-06-01

Benzotrichloride (BTC) is used extensively as a chemical intermediate in the synthesis of benzoyl chloride and benzoyl peroxide. Epidemiological data suggest that BTC is a human lung carcinogen. BTC is also a carcinogen in the A/J mouse lung tumor bioassay. Activated K-ras protooncogenes were detected in BTC-induced lung tumors from A/J mice. The polymerase chain reaction was used to amplify specific DNA segments likely to contain activating mutations, and the amplified DNAs were sequenced to identify the mutation. The activating mutation present in the K-ras gene from all BTC-induced lung tumors (24/24) was a GC-->AT transition in codon 12. Thus, BTC may exert its carcinogenic action by activation of the K-ras protooncogene through a genotoxic mechanism.

p21ras independent down-regulation of ras-induced increases in natural antibody binding during tumor progression.

PubMed

Tough, D F; Feng, X; Chow, D A

1995-01-01

Selective outgrowth of v-H-ras-infected 10T1/2 cells based on the cointroduction of a gene for resistance to geneticin (G418), yielded cells which exhibited an increased capacity to bind polyclonal serum natural antibody (NAb). This demonstrated an NAb-susceptible phase of tumor development which would be a basic requirement for NAb-mediated surveillance of tumors. The ras-oncogene dependence of the high-NAb-binding phenotype provided a model for assessing NAb resistance against ras transformants in vivo and for a comparative analysis of phenotypic and genetic alterations contributing to the progression of ras transformants. Variants were developed through in vitro and in vivo models of tumor progression. T24-H-ras and v-H-ras transformants were isolated in vitro through more rigorous growth conditions, focus formation in the presence of untransformed cells with no selecting drug. These clones expressed p21ras but exhibited little or no increase in NAb binding. Variants recovered following growth from intravenous or threshold subcutaneous (s.c.) inocula of high-NAb-binding ras transformants in syngeneic C3H/HeN mice exhibited decreases in NAb binding but no uniform change in p21ras. Concurring inverse correlations between NAb binding and s.c. tumorigenicity were exhibited by the T24-H-ras transformant clones, the ras transformants grown in vivo, and the v-H-ras-transformed clones isolated in the presence versus the absence of untransformed cells. This consistent inverse correlation, together with the reduced NAb binding of the ras transformants grown in vivo, provides strong evidence that NAb participates in the defense against ras-transformed cells in vivo. The lack of any direct correlation between p21ras expression and the reduction in NAb binding or the increase in tumorigenicity of cells generated through progression in vivo suggested the regulatory action of additional genes. Hybridization studies between high- and low-NAb-binding clones implicated the

K-RAS GTPase- and B-RAF kinase-mediated T-cell tolerance defects in rheumatoid arthritis.

PubMed

Singh, Karnail; Deshpande, Pratima; Li, Guangjin; Yu, Mingcan; Pryshchep, Sergey; Cavanagh, Mary; Weyand, Cornelia M; Goronzy, Jörg J

2012-06-19

Autoantibodies to common autoantigens and neoantigens, such as IgG Fc and citrullinated peptides, are immunological hallmarks of rheumatoid arthritis (RA). We examined whether a failure in maintaining tolerance is mediated by defects in T-cell receptor activation threshold settings. RA T cells responded to stimulation with significantly higher ERK phosphorylation (P < 0.001). Gene expression arrays of ERK pathway members suggested a higher expression of KRAS and BRAF, which was confirmed by quantitative PCR (P = 0.003), Western blot, and flow cytometry (P < 0.01). Partial silencing of KRAS and BRAF lowered activation-induced phosphorylated ERK levels (P < 0.01). In individual cells, levels of these signaling molecules correlated with ERK phosphorylation, attesting that their concentrations are functionally important. In confocal studies, B-RAF/K-RAS clustering was increased in RA T cells 2 min after T-cell receptor stimulation (P < 0.001). Overexpression of B-RAF and K-RAS in normal CD4 T cells amplified polyclonal T-cell proliferation and facilitated responses to citrullinated peptides. We propose that increased expression of B-RAF and K-RAS lowers T-cell activation thresholds in RA T cells, enabling responses to autoantigens.

Long-term follow-up of chronic pancreatitis patients with K-ras mutation in the pancreatic juice.

PubMed

Kamisawa, Terumi; Takuma, Kensuke; Tabata, Taku; Egawa, Naoto; Yamaguchi, Toshikazu

2011-01-01

Pancreatic cancer is known to occur during the course of chronic pancreatitis in some patients. This study aimed to identify a high risk group for developing pancreatic cancer associated with chronic pancreatitis, particularly the presence of K-ras mutations in the pancreatic juice. K-ras mutation was analyzed by enriched polymerase chain reaction-enzyme linked mini-sequence assay in endoscopically-collected pancreatic juice of 21 patients with chronic pancreatitis between 1995 and 2000. All of them were followed-up for 6.0 +/- 3.8 (mean +/- SD) years (range, 2.1-14.2 years). K-ras point mutation was observed in the pancreatic juice of 11 patients with chronic pancreatitis (2+, n=2; 1+, n=6; +/-, n=3). Of these, 2 chronic pancreatitis patients with 2+K-ras point mutation developed pancreatic cancer 4.5 and 10.8 years, respectively, after the examination. Two chronic pancreatitis patients with K-ras mutation developed pancreatic cancer 4.5 and 10.8 years later. Semiquantitative analysis of K-ras mutation in endoscopically-collected pancreatic juice appears to be a useful tool for identifying chronic pancreatitis patients at high risk for developing pancreatic cancer.

The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting.

PubMed

Jonckheere, Nicolas; Vasseur, Romain; Van Seuningen, Isabelle

2017-03-01

RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives. Copyright © 2017 Elsevier B.V. All rights reserved.

A Raf-competitive K-Ras binder can fail to functionally antagonize signaling.

PubMed

Kauke, Monique J; Tisdale, Alison W; Kelly, Ryan L; Braun, Christian J; Hemann, Michael T; Wittrup, K Dane

2018-05-02

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically-encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacological efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Copyright ©2018, American Association for Cancer Research.

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

PubMed

Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to

Loss of Cbl-PI3K interaction enhances osteoclast survival due to p21-Ras mediated PI3K activation independent of Cbl-b.

PubMed

Adapala, Naga Suresh; Barbe, Mary F; Tsygankov, Alexander Y; Lorenzo, Joseph A; Sanjay, Archana

2014-07-01

Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases and adaptor proteins, which play important roles in bone-resorbing osteoclasts. Loss of Cbl in mice decreases osteoclast migration, resulting in delayed bone development where as absence of Cbl-b decreases bone volume due to hyper-resorptive osteoclasts. A major structural difference between Cbl and Cbl-b is tyrosine 737 (in YEAM motif) only on Cbl, which upon phosphorylation interacts with the p85 subunit of phosphatidylinositol-3 Kinase (PI3K). In contrast to Cbl(-/-) and Cbl-b(-/-) , mice lacking Cbl-PI3K interaction due to a Y737F (tyrosine to phenylalanine, YF) mutation showed enhanced osteoclast survival, but defective bone resorption. To investigate whether Cbl-PI3K interaction contributes to distinct roles of Cbl and Cbl-b in osteoclasts, mice bearing CblY737F mutation in the Cbl-b(-/-) background (YF/YF;Cbl-b(-/-) ) were generated. The differentiation and survival were augmented similarly in YF/YF and YF/YF;Cbl-b(-/-) osteoclasts, associated with enhanced PI3K signaling suggesting an exclusive role of Cbl-PI3K interaction, independent of Cbl-b. In addition to PI3K, the small GTPase Ras also regulates osteoclast survival. In the absence of Cbl-PI3K interaction, increased Ras GTPase activity and Ras-PI3K binding were observed and inhibition of Ras activation attenuated PI3K mediated osteoclast survival. In contrast to differentiation and survival, increased osteoclast activity observed in Cbl-b(-/-) mice persisted even after introduction of the resorption-defective YF mutation in YF/YF;Cbl-b(-/-) mice. Hence, Cbl and Cbl-b play mutually exclusive roles in osteoclasts. Whereas Cbl-PI3K interaction regulates differentiation and survival, bone resorption is predominantly regulated by Cbl-b in osteoclasts. © 2014 Wiley Periodicals, Inc.

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

NASA Astrophysics Data System (ADS)

Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

2016-02-01

Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP-GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors.

PubMed

Loboda, Andrey; Nebozhyn, Michael; Klinghoffer, Rich; Frazier, Jason; Chastain, Michael; Arthur, William; Roberts, Brian; Zhang, Theresa; Chenard, Melissa; Haines, Brian; Andersen, Jannik; Nagashima, Kumiko; Paweletz, Cloud; Lynch, Bethany; Feldman, Igor; Dai, Hongyue; Huang, Pearl; Watters, James

2010-06-30

Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

PubMed Central

2010-01-01

Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast

Differential Expression of IL-17, 22 and 23 in the Progression of Colorectal Cancer in Patients with K-ras Mutation: Ras Signal Inhibition and Crosstalk with GM-CSF and IFN-γ

PubMed Central

Petanidis, Savvas; Anestakis, Doxakis; Argyraki, Maria; Hadzopoulou-Cladaras, Margarita; Salifoglou, Athanasios

2013-01-01

Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future. PMID:24040001

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.

PubMed

Chen, Yen-Ling; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei

2009-06-26

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

PubMed Central

Sasaki, Atsuo T.; Chun, Cheryl; Takeda, Kosuke; Firtel, Richard A.

2004-01-01

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal. PMID:15534002

[miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

PubMed

Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

2016-12-23

Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P <0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P <0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P <0.05) but higher than the miR-143 mimic group significantly ( P <0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control

Computational studies of Ras and PI3K

NASA Technical Reports Server (NTRS)

Ren, Lei; Cucinotta, Francis A.

2004-01-01

Until recently, experimental techniques in molecular cell biology have been the primary means to investigate biological risk upon space radiation. However, computational modeling provides an alternative theoretical approach, which utilizes various computational tools to simulate proteins, nucleotides, and their interactions. In this study, we are focused on using molecular mechanics (MM) and molecular dynamics (MD) to study the mechanism of protein-protein binding and to estimate the binding free energy between proteins. Ras is a key element in a variety of cell processes, and its activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. Different computational approaches for this particular study are presented to calculate the solvation energies and binding free energies of H-Ras and PI3K. The goal of this study is to establish computational methods to investigate the roles of different proteins played in the cellular responses to space radiation, including modification of protein function through gene mutation, and to support the studies in molecular cell biology and theoretical kinetics models for our risk assessment project.

Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study.

PubMed

Xiong, Jinjun; He, Mai; Jackson, Cynthia; Ou, Joyce J; Sung, C James; Breese, Virgina; Steinhoff, Margaret M; Quddus, M Ruhul; Tejada-Berges, Trevor; Lawrence, W Dwayne

2013-09-01

K-ras gene product in the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway is critical in the development of certain types of malignancies. K-ras mutation-associated pancreatic and ovarian carcinomas often display mucinous differentiation. Previous studies have shown that k-ras mutation is found in 10% to 30% of endometrial carcinomas. We investigated k-ras mutations in several morphologic subtypes of endometrial carcinomas with particular emphasis on various degrees of mucinous differentiation. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections. Polymerase chain reaction amplification for k-ras codons 12 and 13 were performed, followed by sequencing using capillary electrophoresis. The Fisher exact test is used to compare the prevalent difference of k-ras mutation among the groups. P < 0.05 was considered significant. K-ras mutations were detected in 8 (80%) of 10 mucinous carcinomas, 12 (67%) of 18 endometrioid carcinomas (ECs) with significant mucinous differentiation (ECMD), 4 (25%) of 16 ECs, and 1 (9%) of 11 serous carcinomas. The differences were statistically significant between mucinous carcinomas versus EC (P < 0.01) and ECMD versus EC (P < 0.05). The findings suggest that mucinous carcinoma and endometrioid carcinoma with significant mucinous component are more likely to be associated with k-ras mutation. Potential clinical implications of k-ras mutation lies in the management of recurrent or higher-stage endometrial mucinous tumors, which would not be responsive to treatment protocols containing epidermal growth factor receptor inhibitors.

K-Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

EPA Science Inventory

K-Ras mutant fraction (MF) was measured to examine the default assumption of low dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of ten male A/J mice (7-9 weeks-old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P, and were sacrifi...

Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy.

PubMed

Wang, Xiaoying; Shu, Guofang; Gao, Chanchan; Yang, Yu; Xu, Qian; Tang, Meng

2014-12-01

An electrochemical biosensor based on functional composite nanofibers for hybridization detection of specific K-ras gene that is highly associated with colorectal cancer via multiple signal amplification strategy has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped nylon 6 (PA6) composite nanofibers (MWCNTs-PA6) was prepared using electrospinning, which served as the nanosized backbone for thionine (TH) electropolymerization. The functional composite nanofibers [MWCNTs-PA6-PTH, where PTH is poly(thionine)] used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. Through the hybridization reaction, a sandwich format of ssDNA1/K-ras gene/gold nanoparticle-labeled ssDNA2 (AuNPs-ssDNA2) was fabricated, and the AuNPs offered excellent electrochemical signal transduction. The signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles (TA/AuNPs). A significant sensitivity enhancement was obtained; the detection limit was down to 30fM, and the discriminations were up to 54.3 and 51.9% between the K-ras gene and the one-base mismatched sequences including G/C and A/T mismatched bases, respectively. The amenability of this method to the analyses of K-ras gene from the SW480 colorectal cancer cell lysates was demonstrated. The results are basically consistent with those of the K-ras Kit (HRM: high-resolution melt). The method holds promise for the diagnosis and management of cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering

PubMed Central

Blaževitš, Olga; Mideksa, Yonatan G.; Šolman, Maja; Ligabue, Alessio; Ariotti, Nicholas; Nakhaeizadeh, Hossein; Fansa, Eyad K.; Papageorgiou, Anastassios C.; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Abankwa, Daniel

2016-01-01

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling. PMID:27087647

The Significance of Ras Activity in Pancreatic Cancer Initiation

PubMed Central

Logsdon, Craig D.; Lu, Weiqin

2016-01-01

The genetic landscape of pancreatic cancer shows nearly ubiquitous mutations of K-RAS. However, oncogenic K-Rasmt alone is not sufficient to lead to pancreatic ductal adenocarcinoma (PDAC) in either human or in genetically modified adult mouse models. Many stimulants, such as high fat diet, CCK, LPS, PGE2 and others, have physiological effects at low concentrations that are mediated in part through modest increases in K-Ras activity. However, at high concentrations, they induce inflammation that, in the presence of oncogenic K-Ras expression, substantially accelerates PDAC formation. The mechanism involves increased activity of oncogenic K-Rasmt. Unlike what has been proposed in the standard paradigm for the role of Ras in oncogenesis, oncogenic K-Rasmt is now known to not be constitutively active. Rather, it can be activated by standard mechanisms similar to wild-type K-Ras, but its activity is sustained for a prolonged period. Furthermore, if the level of K-Ras activity exceeds a threshold at which it begins to generate its own activators, then a feed-forward loop is formed between K-Ras activity and inflammation and pathological processes including oncogenesis are initiated. Oncogenic K-Rasmt activation, a key event in PDAC initiation and development, is subject to complex regulatory mechanisms. Reagents which inhibit inflammation, such as the Cox2 inhibitor celecoxib, block the feed-forward loop and prevent induction of PDAC in models with endogenous oncogenic K-Rasmt. Increased understanding of the role of activating and inhibitory mechanisms on oncogenic K-Rasmt activity is of paramount importance for the development of preventive and therapeutic strategies to fight against this lethal disease. PMID:26929740

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

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

Naumov, Inna; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; Kazanov, Dina

2012-01-15

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1more » and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.« less

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

NASA Astrophysics Data System (ADS)

Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

2017-01-01

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

K-RasV14I recapitulates Noonan syndrome in mice

PubMed Central

Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

2014-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

PubMed

Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2018-05-01

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

The Frequency and Type of K-RAS Mutations in Mexican Patients With Colorectal Cancer: A National Study.

PubMed

Cárdenas-Ramos, Susana G; Alcázar-González, Gregorio; Reyes-Cortés, Luisa M; Torres-Grimaldo, Abdiel A; Calderón-Garcidueñas, Ana L; Morales-Casas, José; Flores-Sánchez, Patricia; De León-Escobedo, Raúl; Gómez-Díaz, Antonio; Moreno-Bringas, Carmen; Sánchez-Guillén, Jorge; Ramos-Salazar, Pedro; González-de León, César; Barrera-Saldaña, Hugo A

2017-06-01

Current metastatic colorectal cancer (mCRC) therapy uses monoclonal antibodies against the epidermal growth factor receptor. This treatment is only useful in the absence of K-RAS gene mutations; therefore the study of such mutations is part of a personalized treatment. The aim of this work is to determine the frequency and type of the most common K-RAS mutations in Mexican patients with metastatic disease by nucleotide sequencing. We studied 888 patients with mCRC from different regions of Mexico. The presence of mutations in exon 2, codons 12 and 13, of the K-RAS gene was determined by nucleotide sequencing. Patients exhibited K-RAS gene mutations in 35% (310/888) of cases. Mutation frequency of codons 12 and 13 was 71% (221/310) and 29% (89/310), respectively. The most common mutation (45.7%) in codon 12 was c.35G>A (p.G12D), whereas the one in codon 13 was c.38G>A (p.G13D) (78.7%). Given the frequency of K-RAS mutations in Mexicans, making a genetic study before deciding to treat mCRC patients with monoclonal antibodies is indispensable.

Activation of the N-Ras-PI3K-Akt-mTOR Pathway by Hepatitis C Virus: Control of Cell Survival and Viral Replication

PubMed Central

Mannová, Petra; Beretta, Laura

2005-01-01

The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV. PMID:15994768

Loss of Cbl–PI3K Interaction Enhances Osteoclast Survival due to p21-Ras Mediated PI3K Activation Independent of Cbl-b

PubMed Central

Adapala, Naga Suresh; Barbe, Mary F.; Tsygankov, Alexander Y.; Lorenzo, Joseph A.; Sanjay, Archana

2015-01-01

Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases and adaptor proteins, which play important roles in bone-resorbing osteoclasts. Loss of Cbl in mice decreases osteoclast migration, resulting in delayed bone development where as absence of Cbl-b decreases bone volume due to hyper-resorptive osteoclasts. A major structural difference between Cbl and Cbl-b is tyrosine 737 (in YEAM motif) only on Cbl, which upon phosphorylation interacts with the p85 subunit of phosphatidylinositol-3 Kinase (PI3K). In contrast to Cbl−/− and Cbl-b−/−, mice lacking Cbl–PI3K interaction due to a Y737F (tyrosine to phenylalanine, YF) mutation showed enhanced osteoclast survival, but defective bone resorption. To investigate whether Cbl–PI3K interaction contributes to distinct roles of Cbl and Cbl-b in osteoclasts, mice bearing CblY737F mutation in the Cbl-b−/− background (YF/YF;Cbl-b−/−) were generated. The differentiation and survival were augmented similarly in YF/YF and YF/YF;Cbl-b−/− osteoclasts, associated with enhanced PI3K signaling suggesting an exclusive role of Cbl–PI3K interaction, independent of Cbl-b. In addition to PI3K, the small GTPase Ras also regulates osteoclast survival. In the absence of Cbl–PI3K interaction, increased Ras GTPase activity and Ras–PI3K binding were observed and inhibition of Ras activation attenuated PI3K mediated osteoclast survival. In contrast to differentiation and survival, increased osteoclast activity observed in Cbl-b−/− mice persisted even after introduction of the resorption-defective YF mutation in YF/YF;Cbl-b−/− mice. Hence, Cbl and Cbl-b play mutually exclusive roles in osteoclasts. Whereas Cbl–PI3K interaction regulates differentiation and survival, bone resorption is predominantly regulated by Cbl-b in osteoclasts. PMID:24470255

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

PubMed

Fiordalisi, James J; Holly, Stephen P; Johnson, Ronald L; Parise, Leslie V; Cox, Adrienne D

2002-03-29

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first

The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

PubMed

Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

2016-02-01

K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. © 2016 Federation of European Biochemical Societies.

Activation of Bmp2-Smad1 Signal and Its Regulation by Coordinated Alteration of H3K27 Trimethylation in Ras-Induced Senescence

PubMed Central

Kaneda, Atsushi; Fujita, Takanori; Anai, Motonobu; Yamamoto, Shogo; Nagae, Genta; Morikawa, Masato; Tsuji, Shingo; Oshima, Masanobu; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence. PMID:22072987

K-ras mutation in colorectal cancer: relations to patient age, sex and tumour location.

PubMed Central

Breivik, J.; Meling, G. I.; Spurkland, A.; Rognum, T. O.; Gaudernack, G.

1994-01-01

DNA from 251 primary tumours obtained from 123 male and 125 female Norwegian patients with colorectal carcinoma was analysed for the presence of K-ras point mutations at codons 12 and 13. Mutations were found in 99 (39%) of the samples. The frequency of K-ras mutations was significantly related to age and sex of the patients, and to the location of the tumours (overall: P = 0.008). K-ras mutations were much less frequent in colonic tumours from male than female patients at younger ages (< 40 years, odds ratio < 0.014). The low frequency might indicate that a different, ras-independent, pathway to neoplasia is dominating in the colon of younger males. In contrast, older men had more mutations than older women (e.g. 90 years, odds ratio = 5.8). An inverse but less pronounced relationship was seen for rectal tumours. The type of mutation was found to be associated to sex of patient and location of tumour. G-->C transversions accounted for 35% of the mutations in rectal tumours from females, in contrast to only 2.5% in the rest of the material (P = 0.0005). This may indicate that there are specific carcinogens acting in this location. PMID:8297737

A New Strategy to Control and Eradicate "Undruggable" Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology.

PubMed

Van Sciver, Robert E; Lee, Michael P; Lee, Caroline Dasom; Lafever, Alex C; Svyatova, Elizaveta; Kanda, Kevin; Colliver, Amber L; Siewertsz van Reesema, Lauren L; Tang-Tan, Angela M; Zheleva, Vasilena; Bwayi, Monicah N; Bian, Minglei; Schmidt, Rebecca L; Matrisian, Lynn M; Petersen, Gloria M; Tang, Amy H

2018-05-14

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely "undruggable". Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

PubMed

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Detection of K-ras gene mutation by liquid biopsy in patients with pancreatic cancer.

PubMed

Kinugasa, Hideaki; Nouso, Kazuhiro; Miyahara, Koji; Morimoto, Yuki; Dohi, Chihiro; Tsutsumi, Koichiro; Kato, Hironari; Matsubara, Takehiro; Okada, Hiroyuki; Yamamoto, Kazuhide

2015-07-01

Cell-free circulating tumor DNA (ctDNA) in serum has been considered to be a useful candidate for noninvasive cancer diagnosis. The current study was designed to estimate the clinical usefulness of genetic analysis for ctDNA by digital polymerase chain reaction in patients with pancreatic cancer. The authors compared K-ras mutations detected in endoscopic ultrasound-guided fine-needle aspiration biopsy tissue DNA and in ctDNA from 75 patients with pancreatic cancer. K-ras mutations in the serum of 66 independent, consecutive patients with pancreatic cancer were also analyzed and the authors compared the results with survival rates. The frequencies of the mutations in tissue samples at G12V, G12D, and G12R in codon 12 were 28 of 75 samples (37.3%), 22 of 75 samples (29.3%), and 6 of 75 samples (8.0%), respectively. Conversely, the rates of the mutations in ctDNA were 26 of 75 samples (34.6%), 29 of 75 samples (38.6%), and 4 of 75 samples (5.3%), respectively. Overall, the K-ras mutation rates in tissue and ctDNA were 74.7% and 62.6%, respectively, and the concordance rate between them was 58 of 75 samples (77.3%). Survival did not appear to differ by the presence of K-ras mutations in tissue DNA, but the survival of patients with K-ras mutations in ctDNA was significantly shorter than that of patients without mutations in both a development set (P = .006) and an independent validation set (P = .002). The difference was especially evident in cases with a G12V mutation. Analysis of ctDNA is a new useful procedure for detecting mutations in patients with pancreatic cancer. This noninvasive method may have great potential as a new strategy for the diagnosis of pancreatic cancer as well as for predicting survival. © 2015 American Cancer Society.

K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors

PubMed Central

Cornejo, Melanie G.; Scholl, Claudia; Liu, Jianing; Leeman, Dena S.; Haydu, J. Erika; Fröhling, Stefan; Lee, Benjamin H.; Gilliland, D. Gary

2008-01-01

To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways. PMID:18663146

A New Strategy to Control and Eradicate “Undruggable” Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology

PubMed Central

Lee, Michael P.; Lee, Caroline Dasom; Lafever, Alex C.; Svyatova, Elizaveta; Kanda, Kevin; Collier, Amber L.; Siewertsz van Reesema, Lauren L.; Tang-Tan, Angela M.; Zheleva, Vasilena; Bwayi, Monicah N.; Bian, Minglei; Schmidt, Rebecca L.; Petersen, Gloria M.

2018-01-01

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely “undruggable”. Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future. PMID:29757973

Epidermal growth factor receptor and K-Ras in non-small cell lung cancer-molecular pathways involved and targeted therapies

PubMed Central

de Mello, Ramon Andrade; Marques, Dânia Sofia; Medeiros, Rui; Araújo, António MF

2011-01-01

Lung cancer is currently the leading cause of cancer death in Western nations. Non-small cell lung cancer (NSCLC) represents 80% of all lung cancers, and adenocarcinoma is the predominant histological type. Despite the intensive research carried out on this field and therapeutic advances, the overall prognosis of these patients remains unsatisfactory, with a 5-year overall survival rate of less than 15%. Nowadays, pharmacogenetics and pharmacogenomics represent the key to successful treatment. Recent studies suggest the existence of two distinct molecular pathways in the carcinogenesis of lung adenocarcinoma: one associated with smoking and activation of the K-Ras oncogene and the other not associated with smoking and activation of the epidermal growth factor receptor (EGFR). The K-ras mutation is mainly responsible for primary resistance to new molecules which inhibit tyrosine kinase EGFR (erlotinib and gefitinib) and most of the EGFR mutations are responsible for increased tumor sensitivity to these drugs. This article aims to conduct a systematic review of the literature regarding the molecular pathways involving the EGFR, K-Ras and EGFR targeted therapies in NSCLC tumor behavior. PMID:22087435

Specific Activation of K-RasG12D Allele in the Bladder Urothelium Results in Lung Alveolar and Vascular Defects

PubMed Central

Kanasaki, Megumi; Vong, Sylvia; Rovira, Carlota; Kalluri, Raghu

2014-01-01

K-ras is essential for embryogenesis and its mutations are involved in human developmental syndromes and cancer. To determine the consequences of K-ras activation in urothelium, we used uroplakin-II (UPK II) promoter driven Cre recombinase mice and generated mice with mutated KrasG12D allele in the urothelium (UPK II-Cre;LSL-K-rasG12D). The UPK II-Cre;LSL-K-rasG12D mice died neonatally due to lung morphogenesis defects consisting of simplification with enlargement of terminal air spaces and dysmorphic pulmonary vasculature. A significant alteration in epithelial and vascular basement membranes, together with fragmentation of laminin, points to extracellular matrix degradation as the causative mechanism of alveolar and vascular defects. Our data also suggest that altered protease activity in amniotic fluid might be associated with matrix defects in lung of UPK II-Cre;LSL-K-rasG12. These defects resemble those observed in early stage human neonatal bronchopulmonary dysplasia (BPD), although the relevance of this new mouse model for BPD study needs further investigation. PMID:24760005

Targeting the RAS oncogene

PubMed Central

Takashima, Asami

2013-01-01

Introduction The Ras proteins (K-Ras, N-Ras, H-Ras) are GTPases that function as molecular switches for a variety of critical cellular activities and their function is tightly and temporally regulated in normal cells. Oncogenic mutations in the RAS genes, which create constitutively-active Ras proteins, can result in uncontrolled proliferation or survival in tumor cells. Areas covered The paper discusses three therapeutic approaches targeting the Ras pathway in cancer: 1) Ras itself, 2) Ras downstream pathways, and 3) synthetic lethality. The most adopted approach is targeting Ras downstream signaling, and specifically the PI3K-AKT-mTOR and Raf-MEK pathways, as they are frequently major oncogenic drivers in cancers with high Ras signaling. Although direct targeting of Ras has not been successful clinically, newer approaches being investigated in preclinical studies, such as RNA interference-based and synthetic lethal approaches, promise great potential for clinical application. Expert opinion The challenges of current and emerging therapeutics include the lack of “tumor specificity” and their limitation to those cancers which are “dependent” upon aberrant Ras signaling for survival. While the newer approaches have the potential to overcome these limitations, they also highlight the importance of robust preclinical studies and bidirectional translational research for successful clinical development of Ras-related targeted therapies. PMID:23360111

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

PubMed

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd

Sphingosine-1-Phosphate Receptor Subtype 3: A Novel Therapeutic Target of K-Ras Mutant Driven Non-Small Cell Lung Carcinoma

DTIC Science & Technology

2015-10-01

Breeding of LSL K-RasG12D transgenic mice (Projective: months 5-8; Actual: 100% completion) 2b. Nasal instillation of adenoviral particles carrying...1a. Regulatory review and approval of animal protocol (Projective: Months 1-2; Actual: 100% completion). 1b. Mice acquisition and breeding of...S1P3-/-:LSL-K-RasG12D and S1P3+/+:LSL-K-RasG12D bi- transgenic mice (Projective: Months 3-4; Actual: 100% completion). 1c. Nasal instillation of

Assessment of K-Ras mutant frequency and micronucleus incidence in the mouse duodenum following 90-days of exposure to Cr(VI) in drinking water.

PubMed

O'Brien, Travis J; Ding, Hao; Suh, Mina; Thompson, Chad M; Parsons, Barbara L; Harris, Mark A; Winkelman, William A; Wolf, Jeffrey C; Hixon, J Gregory; Schwartz, Arnold M; Myers, Meagan B; Haws, Laurie C; Proctor, Deborah M

2013-06-14

Chronic exposure to high concentrations of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) in drinking water induces duodenal tumors in mice, but the mode of action (MOA) for these tumors has been a subject of scientific debate. To evaluate the tumor-site-specific genotoxicity and cytotoxicity of SDD in the mouse small intestine, tissue pathology and cytogenetic damage were evaluated in duodenal crypt and villus enterocytes from B6C3F1 mice exposed to 0.3-520mg/L SDD in drinking water for 7 and 90 days. Allele-competitive blocker PCR (ACB-PCR) was used to investigate the induction of a sensitive, tumor-relevant mutation, specifically in vivo K-Ras codon 12 GAT mutation, in scraped duodenal epithelium following 90 days of drinking water exposure. Cytotoxicity was evident in the villus as disruption of cellular arrangement, desquamation, nuclear atypia and blunting. Following 90 days of treatment, aberrant nuclei, occurring primarily at villi tips, were significantly increased at ≥60mg/L SDD. However, in the crypt compartment, there were no dose-related effects on mitotic and apoptotic indices or the formation of aberrant nuclei indicating that Cr(VI)-induced cytotoxicity was limited to the villi. Cr(VI) caused a dose-dependent proliferative response in the duodenal crypt as evidenced by an increase in crypt area and increased number of crypt enterocytes. Spontaneous K-Ras codon 12 GAT mutations in untreated mice were higher than expected, in the range of 10(-2) to 10(-3); however no treatment-related trend in the K-Ras codon 12 GAT mutation was observed. The high spontaneous background K-Ras mutant frequency and Cr(VI) dose-related increases in crypt enterocyte proliferation, without dose-related increase in K-Ras mutant frequency, micronuclei formation, or change in mitotic or apoptotic indices, are consistent with a lack of genotoxicity in the crypt compartment, and a MOA involving accumulation of mutations late in carcinogenesis as a

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

PubMed

Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

PubMed Central

Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

2014-01-01

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

Mutations of the EGFR, K-ras, EML4-ALK, and BRAF genes in resected pathological stage I lung adenocarcinoma.

PubMed

Ohba, Taro; Toyokawa, Gouji; Osoegawa, Atsushi; Hirai, Fumihiko; Yamaguchi, Masafumi; Taguchi, Ken-Ichi; Seto, Takashi; Takenoyama, Mitsuhiro; Ichinose, Yukito; Sugio, Kenji

2016-09-01

The EGFR, K-ras, EML4-ALK, and BRAF genes are oncogenic drivers of lung adenocarcinoma. We conducted this study to analyze the mutations of these genes in stage I adenocarcinoma. The subjects of this retrospective study were 256 patients with resected stage I lung adenocarcinoma. We analyzed mutations of the EGFR, K-ras, and BRAF genes, and the EML4-ALK fusion gene. We also assessed disease-free survival (DFS) to evaluate the prognostic value and overall survival (OS) to evaluate the predictive value of treatment after recurrence. Mutations of the EGFR, K-ras, EML4-ALK, and BRAF genes were detected in 120 (46.8 %), 14 (5.5 %), 6 (2.3 %), and 2 (0.8 %) of the 256 tumors. Two tumors had double mutations (0.8 %). The incidence of EGFR mutations was significantly higher in women than in men. The EML4-ALK fusion gene was detected only in younger patients. The DFS and OS of the K-ras mutant group were significantly worse than those of the EGFR mutant group, the EML4-ALK fusion gene group, and the wild-type group. Six of the seven patients with the EML4-ALK fusion gene are still alive without recurrent disease. In patients with stage I adenocarcinoma, mutation of the K-ras gene was a poor prognostic factor for recurrence. The presence of a mutation of the EGFR or EML4-ALK gene was not a prognostic factor.

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

PubMed Central

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Kirkwood, John; Avogadri-Connors, Francesca; Cutler Jr, Richard E.; Lalani, Alshad S.; Dent, Paul

2018-01-01

ABSTRACT The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins. PMID:29219657

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib.

PubMed

Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2018-02-01

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation.

PubMed

Ni, Duan; Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-10-26

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras-NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras.

The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

PubMed Central

Kötting, Carsten; Kallenbach, Angela; Suveyzdis, Yan; Wittinghofer, Alfred; Gerwert, Klaus

2008-01-01

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved “arginine finger” of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras·RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling “off” to the signaling “on” state with a rate of 3 s−1. The next step is the movement of the “arginine finger” into the active site of Ras with a rate of k2 = 0.8 s−1. Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the “off” state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 = 0.1 s−1, the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the “arginine finger” movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate. PMID:18434546

Geranylgeranyl Diphosphate Synthase Modulates Fetal Lung Branching Morphogenesis Possibly through Controlling K-Ras Prenylation.

PubMed

Jia, Wen-Jun; Jiang, Shan; Tang, Qiao-Li; Shen, Di; Xue, Bin; Ning, Wen; Li, Chao-Jun

2016-06-01

G proteins play essential roles in regulating fetal lung development, and any defects in their expression or function (eg, activation or posttranslational modification) can lead to lung developmental malformation. Geranylgeranyl diphosphate synthase (GGPPS) can modulate protein prenylation that is required for protein membrane-anchoring and activation. Here, we report that GGPPS regulates fetal lung branching morphogenesis possibly through controlling K-Ras prenylation during fetal lung development. GGPPS was continuously expressed in lung epithelium throughout whole fetal lung development. Specific deletion of geranylgeranyl diphosphate synthase 1 (Ggps1) in lung epithelium during fetal lung development resulted in neonatal respiratory distress syndrome-like disease. The knockout mice died at postnatal day 1 of respiratory failure, and the lungs showed compensatory pneumonectasis, pulmonary atelectasis, and hyaline membranes. Subsequently, we proved that lung malformations in Ggps1-deficient mice resulted from the failure of fetal lung branching morphogenesis. Further investigation revealed Ggps1 deletion blocked K-Ras geranylgeranylation and extracellular signal-related kinase 1 or 2/mitogen-activated protein kinase signaling, which in turn disturbed fibroblast growth factor 10 regulation on fetal lung branching morphogenesis. Collectively, our data suggest that GGPPS is essential for maintaining fetal lung branching morphogenesis, which is possibly through regulating K-Ras prenylation. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The Function of Embryonic Stem Cell-expressed RAS (E-RAS), a Unique RAS Family Member, Correlates with Its Additional Motifs and Its Structural Properties.

PubMed

Nakhaei-Rad, Saeideh; Nakhaeizadeh, Hossein; Kordes, Claus; Cirstea, Ion C; Schmick, Malte; Dvorsky, Radovan; Bastiaens, Philippe I H; Häussinger, Dieter; Ahmadian, Mohammad Reza

2015-06-19

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

K-Ras mutation detection in liquid biopsy and tumor tissue as prognostic biomarker in patients with pancreatic cancer: a systematic review with meta-analysis.

PubMed

Li, Tao; Zheng, Yuanting; Sun, Hong; Zhuang, Rongyuan; Liu, Jing; Liu, Tianshu; Cai, Weimin

2016-07-01

K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95 % confidence interval (CI) 1.36-1.90; p < 0.01] in K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95 % CI 1.20-1.57; p < 0.01) and 3.16 (95 % CI 2.1-4.71; p < 0.01), respectively. In addition, the HR was higher when K-Ras mutations were detected in fresh frozen samples (HR = 2.01, 95 % CI 1.28-3.16, p = 0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR = 1.29, 95 % CI 1.12-1.49, p < 0.01). Though K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples.

¹H, ¹³C and ¹⁵N resonance assignment for the human K-Ras at physiological pH.

PubMed

Vo, Uybach; Embrey, Kevin J; Breeze, Alexander L; Golovanov, Alexander P

2013-10-01

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras

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

Shin, Ki-Hyuk, E-mail: kshin@dentistry.ucla.edu; Dental Research Institute, University of California, Los Angeles, CA 90095; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095

2011-01-28

Research highlights: {yields} MicroRNA-181a (miR-181a) was frequently downregulated in oral squamous cell carcinoma (OSCC). {yields} Overexpression of miR-181a suppressed OSCC growth. {yields} K-ras is a novel target of miR-181a. {yields} Decreased miR-181a expression is attributed to its lower promoter activity in OSCC. -- Abstract: MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biologicalmore » role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3'-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.« less

Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation

PubMed Central

Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-01-01

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras–NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras. PMID:29072601

Activation of RAS family genes in urothelial carcinoma.

PubMed

Boulalas, I; Zaravinos, A; Karyotis, I; Delakas, D; Spandidos, D A

2009-05-01

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras

PubMed Central

Acin, Sergio; Li, Zhongyou; Mejia, Olga; Roop, Dennis R; El-Naggar, Adel K; Caulin, Carlos

2015-01-01

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53. PMID:21952947

NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

PubMed

Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

2018-05-02

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

Realgar bioleaching solution suppress ras excessive activation by increasing ROS in Caenorhabditis elegans.

PubMed

Zhi, De Juan; Feng, Na; Liu, Dong Ling; Hou, Rong Li; Wang, Mei Zu; Ding, Xiao Xia; Li, Hong Yu

2014-03-01

Although realgar bioleaching solution (RBS) has been proved to be a potential candidate for cancer therapy, the mechanisms of RBS anticancer are still far from being completely understood. Dosed with RBS in C. elegans, the multivulva phenotype resulting from oncogenic ras gain-of-function was inhibited in a dose dependent manner. It could be abrogated by concurrent treatment C. elegans with RBS and the radical scavenger DMSO. However, RBS could not induce DAF-16 nuclear translocation in TJ356 or the increase of HSP 16.2 expression in CL2070, which both could be aroused visible GFP fluorescent variation to represent for oxidative stress generation. Treatment C. elegans with superoxide anion generator paraquat, similar results were also obtained. Our results indicated that RBS suppress excessive activated ras by increasing reactive oxygen species (ROS) in C. elegans. Secondly, ROS induced by RBS significantly accumulated on a higher level in C. elegans with a mutational ras than that with wild ras, thus leading to oxidative stress on ras gain-of-function background rather than on normal ras context. Our results firstly demonstrated that using C. elegans as a model organism for evaluating prooxidant drug candidates for cancer therapy.

Influence of K-ras status and anti-tumour treatments on complications due to colorectal self-expandable metallic stents: a retrospective multicentre study.

PubMed

Fuccio, Lorenzo; Correale, Loredana; Arezzo, Alberto; Repici, Alessandro; Manes, Gianpiero; Trovato, Cristina; Mangiavillano, Benedetto; Manno, Mauro; Cortelezzi, Claudio Camillo; Dinelli, Marco; Cennamo, Vincenzo; de Bellis, Mario

2014-06-01

This study aimed to explore the relationship between K-ras status, anti-tumour treatments, and the complications of colorectal self-expandable metallic stenting in colorectal cancer. This is a retrospective, multicentre study of 91 patients with obstructive advanced colorectal cancer palliated with enteral stents between 2007 and 2011. K-ras wild-type tumours were diagnosed in 44 patients (48.4%); 82 (90.1%) received chemotherapy and 45 (49.4%) had additional biological therapy (34 bevacizumab, 11 cetuximab). Twenty-one (23.1%) experienced stent-related complications: 11 (52.4%) occurred in the K-ras mutant group (P=0.9). K-ras wild-type patients were not less likely to develop adverse events than K-ras mutant patients (OR, 0.99; 95% CI: 0.4-2.7). Overall mean time to complication was 167.6 days (range 4-720 days), with no difference between the two groups (141 vs. 197 days; P=0.5). Chemotherapy did not influence the risk of complications (OR, 0.56; 95% CI: 0.14-2.9), and there was no evidence that patients treated with chemotherapy and cetuximab were more likely to experience stent-related complications than patients treated with chemotherapy alone, or untreated (OR, 1.2; 95% CI: 0.2-5.9). Although perforation rates were higher with bevacizumab-based treatment (11.8% vs. 7%), this result was not statistically significant (P=0.69). K-ras mutation status, chemotherapy, and biological treatments should not influence colorectal stent-related complication rates. Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Caveolae regulate the nanoscale organization of the plasma membrane to remotely control Ras signaling

PubMed Central

Ariotti, Nicholas; Fernández-Rojo, Manuel A.; Zhou, Yong; Hill, Michelle M.; Rodkey, Travis L.; Inder, Kerry L.; Tanner, Lukas B.; Wenk, Markus R.

2014-01-01

The molecular mechanisms whereby caveolae exert control over cellular signaling have to date remained elusive. We have therefore explored the role caveolae play in modulating Ras signaling. Lipidomic and gene array analyses revealed that caveolin-1 (CAV1) deficiency results in altered cellular lipid composition, and plasma membrane (PM) phosphatidylserine distribution. These changes correlated with increased K-Ras expression and extensive isoform-specific perturbation of Ras spatial organization: in CAV1-deficient cells K-RasG12V nanoclustering and MAPK activation were enhanced, whereas GTP-dependent lateral segregation of H-Ras was abolished resulting in compromised signal output from H-RasG12V nanoclusters. These changes in Ras nanoclustering were phenocopied by the down-regulation of Cavin1, another crucial caveolar structural component, and by acute loss of caveolae in response to increased osmotic pressure. Thus, we postulate that caveolae remotely regulate Ras nanoclustering and signal transduction by controlling PM organization. Similarly, caveolae transduce mechanical stress into PM lipid alterations that, in turn, modulate Ras PM organization. PMID:24567358

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

PubMed Central

Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-01-01

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

Downregulation of Ras C-terminal processing by JNK inhibition

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

Mouri, Wataru; Department of Neurosurgery, Yamagata University School of Medicine, Yamagata 990-9585; Biology Division, National Cancer Center Research Institute, Tokyo 104-0045

2008-06-27

After translation, Ras proteins undergo a series of modifications at their C-termini. This post-translational C-terminal processing is essential for Ras to become functional, but it remains unknown whether and how Ras C-terminal processing is regulated. Here we show that the C-terminal processing and subsequent plasma membrane localization of H-Ras as well as the activation of the downstream signaling pathways by H-Ras are prevented by JNK inhibition. Conversely, JNK activation by ultraviolet irradiation resulted in promotion of C-terminal processing of H-Ras. Furthermore, increased cell density promoted C-terminal processing of H-Ras most likely through an autocrine/paracrine mechanism, which was also blocked undermore » JNK-inhibited condition. Ras C-terminal processing was sensitive to JNK inhibition in the case of H- and N-Ras but not K-Ras, and in a variety of cell types. Thus, our results suggest for the first time that Ras C-terminal processing is a regulated mechanism in which JNK is involved.« less

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

PubMed

Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

2000-05-01

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

TRIM67 Protein Negatively Regulates Ras Activity through Degradation of 80K-H and Induces Neuritogenesis*

PubMed Central

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-01-01

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis. PMID:22337885

TRIM67 protein negatively regulates Ras activity through degradation of 80K-H and induces neuritogenesis.

PubMed

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-04-06

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways.

PubMed

Vasseur, Romain; Skrypek, Nicolas; Duchêne, Belinda; Renaud, Florence; Martínez-Maqueda, Daniel; Vincent, Audrey; Porchet, Nicole; Van Seuningen, Isabelle; Jonckheere, Nicolas

2015-12-01

The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene.

PubMed

Goodrow, T; Reynolds, S; Maronpot, R; Anderson, M

1990-08-01

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a

[Enhanced growth inhibition by combined two pathway inhibitors on K-ras mutated non-small cell lung cancer cells].

PubMed

Yang, Zhenli; Li, Zhanwen; Feng, Hailiang; Bian, Xiaocui; Liu, Yanyan; Liu, Yuqin

2014-09-01

To evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms. A549 cells were treated with different concentrations of two inhibitors. Growth inhibition was determined by MTT assay. According to the results of MTT test, the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941,0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244+0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244+5.0 µmol/L GDC-0941). The cell cycle and apoptosis were analyzed by flow cytometry. The expression of proteins related to apoptosis was tested with Western blot. Both GDC-0941 and AZD6244 inhibited the cell proliferation. The combination group II led to a stronger growth inhibition. The combination group I showed an antagonistic effect and combination group II showed an additive or synergistic effect. Compared with the control group, the combination group I led to reduced apoptotic rate [(20.70 ± 0.99)% vs. (18.65 ± 0.92 )%, P > 0.05]; Combination group II exhibited enhanced apoptotic rate [(37.85 ± 3.18)% vs. (52.27 ± 4.36)%, P < 0.01]. In addition, in the combination group II, more A549 cells were arrested in G0/G1 phase and decreased S phase (P < 0.01), due to the reduced expressions of CyclinD1 and Cyclin B1, the increased cleaved PARP and the diminished ratio of Bcl-2/Bax. For single K-ras mutated NSCLC cell line A549 cells, combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses. Double pathways targeted therapy may be beneficial for these patients.

Nicotine does not enhance tumorigenesis in mutant K-ras-driven mouse models of lung cancer.

PubMed

Maier, Colleen R; Hollander, M Christine; Hobbs, Evthokia A; Dogan, Irem; Linnoila, R Ilona; Dennis, Phillip A

2011-11-01

Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiologic levels of nicotine on lung tumor formation, tumor growth, or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Tumors that develop in this model have mutations in K-ras, which is commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size and did not affect overall survival. Likewise, in a syngeneic model using lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable with those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiologic data showing that NRT does not enhance lung cancer risk in former smokers.

Association of folate intake, dietary habits, smoking and COX-2 promotor -765G>C polymorphism with K-ras mutation in patients with colorectal cancer.

PubMed

Kamal, Manal M; Youssef, Omar Z; Lotfy, Ahmed N; Elsaed, Eman T; Fawzy, May M T

2012-09-01

Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G>C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, -765G>C. The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) -765G>C polymorphism were determined by PCR-RFLP. The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96±51.3 ng/ml) than the negative group (216.6±166.4 ng/ml), (P<0.01). Higher folate levels were found in males than females (median=173 ng/ml and 85 ng/ml; respectively, P=0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P=0.0018) followed by gender (P=0.036) contributed significantly in the discrimination between patients prone to develop K-ras

Dual PI3K/mTOR inhibitors, GSK2126458 and PKI-587, suppress tumor progression and increase radiosensitivity in nasopharyngeal carcinoma.

PubMed

Liu, Tongxin; Sun, Quanquan; Li, Qi; Yang, Hua; Zhang, Yuqin; Wang, Rong; Lin, Xiaoshan; Xiao, Dong; Yuan, Yawei; Chen, Longhua; Wang, Wei

2015-02-01

Although combined chemoradiotherapy has provided considerable improvements for nasopharyngeal carcinoma (NPC), recurrence and metastasis are still frequent. The PI3K/Akt/mTOR pathway plays a critical role in tumor formation and tumor cell survival after radiation-induced DNA damage. In the present study, we evaluated whether inhibition of PI3K/mTOR by two novel dual inhibitors, GSK2126458 and PKI-587, could suppress tumor progression and sensitize NPC cells to radiation. Four NPC cell lines (CNE-1, CNE-2, 5-8F, and 6-10B) were used to analyze the effects of GSK216458 and PKI-587 on cell proliferation, migration, invasion, clonogenic survival, amount of residual γ-H2AX foci, cell cycle, and apoptosis after radiation. A 5-8F xenograft model was used to evaluate the in vivo effects of the two compounds in combination with ionizing radiation (IR). Both GSK216458 and PKI-587 effectively inhibited cell proliferation and motility in NPC cells and suppressed phosphorylation of Akt, mTOR, S6, and 4EBP1 proteins in a concentration- and time-dependent manner. Moreover, both compounds sensitized NPC cells to IR by increasing DNA damage, enhancing G2-M cell-cycle delay, and inducing apoptosis. In vivo, the combination of IR with GSK2126458 or PKI-587 significantly inhibited tumor growth. Antitumor effect was correlated with induction of apoptosis and suppression of the phosphorylation of mTOR, Akt, and 4EBP1. These new findings suggest the usefulness of PI3K/mTOR dual inhibition for antitumor and radiosensitizing. The combination of IR with a dual PI3K/mTOR inhibitor, GSK2126458 or PKI-587, might be a promising therapeutic strategy for NPC. ©2014 American Association for Cancer Research.

In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.

PubMed

Qian, Jiaying; Niu, Jiangong; Li, Ming; Chiao, Paul J; Tsao, Ming-Sound

2005-06-15

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

Cell-specific radiosensitization by gold nanoparticles at megavoltage radiation energies.

PubMed

Jain, Suneil; Coulter, Jonathan A; Hounsell, Alan R; Butterworth, Karl T; McMahon, Stephen J; Hyland, Wendy B; Muir, Mark F; Dickson, Glenn R; Prise, Kevin M; Currell, Fred J; O'Sullivan, Joe M; Hirst, David G

2011-02-01

Gold nanoparticles (GNPs) have been shown to cause sensitization with kilovoltage (kV) radiation. Differences in the absorption coefficient between gold and soft tissue, as a function of photon energy, predict that maximum enhancement should occur in the kilovoltage (kV) range, with almost no enhancement at megavoltage (MV) energies. Recent studies have shown that GNPs are not biologically inert, causing oxidative stress and even cell death, suggesting a possible biological mechanism for sensitization. The purpose of this study was to assess GNP radiosensitization at clinically relevant MV X-ray energies. Cellular uptake, intracellular localization, and cytotoxicity of GNPs were assessed in normal L132, prostate cancer DU145, and breast cancer MDA-MB-231 cells. Radiosensitization was measured by clonogenic survival at kV and MV photon energies and MV electron energies. Intracellular DNA double-strand break (DSB) induction and DNA repair were determined and GNP chemosensitization was assessed using the radiomimetic agent bleomycin. GNP uptake occurred in all cell lines and was greatest in MDA-MB-231 cells with nanoparticles accumulating in cytoplasmic lysosomes. In MDA-MB-231 cells, radiation sensitizer enhancement ratios (SERs) of 1.41, 1.29, and 1.16 were achieved using 160 kVp, 6 MV, and 15 MV X-ray energies, respectively. No significant effect was observed in L132 or DU145 cells at kV or MV energies (SER 0.97-1.08). GNP exposure did not increase radiation-induced DSB formation or inhibit DNA repair; however, GNP chemosensitization was observed in MDA-MB-231 cells treated with bleomycin (SER 1.38). We have demonstrated radiosensitization in MDA-MB-231 cells at MV X-ray energies. The sensitization was cell-specific with comparable effects at kV and MV energies, no increase in DSB formation, and GNP chemopotentiation with bleomycin, suggesting a possible biological mechanism of radiosensitization. Copyright © 2011 Elsevier Inc. All rights reserved.

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

PubMed Central

Hocker, Harrison J.; Cho, Kwang-Jin; Chen, Chung-Ying K.; Rambahal, Nandini; Sagineedu, Sreenivasa Rao; Shaari, Khozirah; Stanslas, Johnson; Hancock, John F.; Gorfe, Alemayehu A.

2013-01-01

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)—a bicyclic diterpenoid lactone isolated from Andrographis paniculata—and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP–GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP–GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras. PMID:23737504

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

PubMed

Sarkar, F H; Valdivieso, M; Borders, J; Yao, K L; Raval, M M; Madan, S K; Sreepathi, P; Shimoyama, R; Steiger, Z; Visscher, D W

1995-12-01

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional

Association of p21ras with phosphatidylinositol 3-kinase.

PubMed Central

Sjölander, A; Yamamoto, K; Huber, B E; Lapetina, E G

1991-01-01

In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21ras proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (PtdIns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity of this association with p21ras was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off. These data indicate that PtdIns 3-kinase activity is an important step in the cascade of reactions in p21ras signal transduction, suggesting that the alterations of the cytoskeleton and growth in ras-transformed cells could be mediated by PtdIns 3-kinase activity. Images PMID:1716764

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras.

PubMed

Akutagawa, J; Huang, T Q; Epstein, I; Chang, T; Quirindongo-Crespo, M; Cottonham, C L; Dail, M; Slusher, B S; Friedman, L S; Sampath, D; Braun, B S

2016-06-01

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation because of NRAS, KRAS, PTPN11, CBL and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3'-OH kinase (PI3K)/Akt cascades. Mx1-Cre, Kras(D12) and Mx1-Cre, Nf1(flox/)(-) mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, Kras(D12) mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients.

FcγR-induced production of superoxide and inflammatory cytokines is differentially regulated by SHIP through its influence on PI3K and/or Ras/Erk pathways

PubMed Central

Ganesan, Latha P.; Joshi, Trupti; Fang, Huiqing; Kutala, Vijay Kumar; Roda, Julie; Trotta, Rossana; Lehman, Amy; Kuppusamy, Periannan; Byrd, John C.; Carson, William E.; Caligiuri, Michael A.; Tridandapani, Susheela

2006-01-01

Phagocytosis of IgG-coated particles via FcγR is accompanied by the generation of superoxide and inflammatory cytokines, which can cause collateral tissue damage in the absence of regulation. Molecular mechanisms regulating these phagocytosis-associated events are not known. SHIP is an inositol phosphatase that downregulates PI3K-mediated activation events. Here, we have examined the role of SHIP in FcγR-induced production of superoxide and inflammatory cytokines. We report that primary SHIP-deficient bone marrow macrophages produce elevated levels of superoxide upon FcγR clustering. Analysis of the molecular mechanism revealed that SHIP regulates upstream Rac-GTP binding, an obligatory event for superoxide production. Likewise, SHIP-deficient macrophages displayed enhanced IL-1β and IL-6 production in response to FcγR clustering. Interestingly, whereas IL-6 production required activation of both PI3K and Ras/Erk pathways, IL-1β production was dependent only on Ras/Erk activation, suggesting that SHIP may also regulate the Ras/Erk pathway in macrophages. Consistently, SHIP-deficient macrophages displayed enhanced activation of Erk upon FcγR clustering. Inhibition of Ras/Erk or PI3K suppressed the enhanced production of IL-6 in SHIP-deficient macrophages. In contrast, inhibition of Ras/Erk, but not PI3K, suppressed IL-1β production in these cells. Together, these data demonstrate that SHIP regulates phagocytosis-associated events through the inhibition of PI3K and Ras/Erk pathways. PMID:16543474

Rapid Development of Salivary Gland Carcinomas upon Conditional Expression of K-ras Driven by the Cytokeratin 5 Promoter

PubMed Central

Raimondi, Ana R.; Vitale-Cross, Lynn; Amornphimoltham, Panomwat; Gutkind, J. Silvio; Molinolo, Alfredo

2006-01-01

We have used a recently described model in which a ras oncogene is expressed in cytokeratin 5 (K5)-expressing cells on doxycycline administration to explore the effects of this oncogene in salivary glands of adult mice. Inducible expression of a mutated K-ras gene under the control of the K5 promoter led to the development of hyperplastic and dysplastic epithelial lesions and carcinomas, with an incidence of 100% and a minimum latency of a week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the cytokeratin 5-positive basal cell compartment. Myoepithelial cells participated in the hyperplasias but not in carcinomas, because the tumors are negative for smooth muscle actin. Carcinomas did not accumulate immunoreactive p53 but are positive for p63, as assayed by immunohistochemistry using an antibody against the N terminus of ΔN p63, a splice variant of p63 that can inhibit p53 transcriptional activity. In this study, we provide evidence that the ras oncogene, targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis. PMID:16651631

An innovative modeling approach using Qual2K and HEC-RAS integration to assess the impact of tidal effect on River Water quality simulation.

PubMed

Fan, Chihhao; Ko, Chun-Han; Wang, Wei-Shen

2009-04-01

Water quality modeling has been shown to be a useful tool in strategic water quality management. The present study combines the Qual2K model with the HEC-RAS model to assess the water quality of a tidal river in northern Taiwan. The contaminant loadings of biochemical oxygen demand (BOD), ammonia nitrogen (NH(3)-N), total phosphorus (TP), and sediment oxygen demand (SOD) are utilized in the Qual2K simulation. The HEC-RAS model is used to: (i) estimate the hydraulic constants for atmospheric re-aeration constant calculation; and (ii) calculate the water level profile variation to account for concentration changes as a result of tidal effect. The results show that HEC-RAS-assisted Qual2K simulations taking tidal effect into consideration produce water quality indices that, in general, agree with the monitoring data of the river. Comparisons of simulations with different combinations of contaminant loadings demonstrate that BOD is the most import contaminant. Streeter-Phelps simulation (in combination with HEC-RAS) is also performed for comparison, and the results show excellent agreement with the observed data. This paper is the first report of the innovative use of a combination of the HEC-RAS model and the Qual2K model (or Streeter-Phelps equation) to simulate water quality in a tidal river. The combination is shown to provide an alternative for water quality simulation of a tidal river when available dynamic-monitoring data are insufficient to assess the tidal effect of the river.

Computational Equilibrium Thermodynamic and Kinetic Analysis of K-Ras Dimerization through an Effector Binding Surface Suggests Limited Functional Role.

PubMed

Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Hancock, John F; Gorfe, Alemayehu A

2016-08-25

Dimer formation is believed to have a substantial impact on regulating K-Ras function. However, the evidence for dimerization and the molecular details of the process are scant. In this study, we characterize a K-Ras pseudo-C2-symmetric dimerization interface involving the effector interacting β2-strand. We used structure matching and all-atom molecular dynamics (MD) simulations to predict, refine, and investigate the stability of this interface. Our MD simulation suggested that the β2-dimer is potentially stable and remains relatively close to its initial conformation due to the presence of a number of hydrogen bonds, ionic salt bridges, and other favorable interactions. We carried out potential of mean force calculations to determine the relative binding strength of the interface. The results of these calculations indicated that the β2 dimerization interface provides a weak binding free energy in solution and a dissociation constant that is close to 1 mM. Analyses of Brownian dynamics simulations suggested an association rate kon ≈ 10(5)-10(6) M(-1) s(-1). Combining these observations with available literature data, we propose that formation of auto-inhibited β2 K-Ras dimers is possible but its fraction in cells is likely very small under normal physiologic conditions.

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

PubMed

Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

Ras, an Actor on Many Stages

PubMed Central

Arozarena, Imanol; Calvo, Fernando; Crespo, Piero

2011-01-01

Among the wealth of information that we have gathered about Ras in the past decade, the introduction of the concept of space in the field has constituted a major revolution that has enabled many pieces of the Ras puzzle to fall into place. In the early days, it was believed that Ras functioned exclusively at the plasma membrane. Today, we know that within the plasma membrane, the 3 Ras isoforms—H-Ras, K-Ras, and N-Ras—occupy different microdomains and that these isoforms are also present and active in endomembranes. We have also discovered that Ras proteins are not statically associated with these localizations; instead, they traffic dynamically between compartments. And we have learned that at these localizations, Ras is under site-specific regulatory mechanisms, distinctively engaging effector pathways and switching on diverse genetic programs to generate different biological responses. All of these processes are possible in great part due to the posttranslational modifications whereby Ras proteins bind to membranes and to regulatory events such as phosphorylation and ubiquitination that Ras is subject to. As such, space and these control mechanisms act in conjunction to endow Ras signals with an enormous signal variability that makes possible its multiple biological roles. These data have established the concept that the Ras signal, instead of being one single, homogeneous entity, results from the integration of multiple, site-specified subsignals, and Ras has become a paradigm of how space can differentially shape signaling. PMID:21779492

Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation

PubMed Central

Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Jaiswal, Alok; Zhou, Yong; Liang, Hong; Lectez, Benoit; Kopra, Kari; Guzmán, Camilo; Härmä, Harri; Hancock, John F; Aittokallio, Tero; Abankwa, Daniel

2015-01-01

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer. DOI: http://dx.doi.org/10.7554/eLife.08905.001 PMID:26274561

RAP/RAS workshop.

DOT National Transportation Integrated Search

2013-01-01

: RAP & RAS increases mix stiffness : : Most WMA additives decrease stiffness : : Tear-Off shingles are stiffer than Man-waste shingles : : Using multiple recycled bins improves consistency : : Finer RAS material improves consiste...

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras

PubMed Central

Akutagawa, Jon; Huang, Tannie Q.; Epstein, Inbal; Chang, Tiffany; Quirindongo-Crespo, Maricel; Cottonham, Charisa L.; Dail, Monique; Slusher, Barbara S.; Friedman, Lori S.; Sampath, Deepak; Braun, Benjamin S.

2016-01-01

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation due to NRAS, KRAS, PTPN11, CBL, and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3´-OH kinase (PI3K)/Akt cascades. Mx1-Cre, KrasD12 and Mx1-Cre, Nf1flox/− mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, KrasD12 mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia, and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients. PMID:26965285

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

PubMed

Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

2017-01-01

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

PubMed Central

Yuen, Hiu-Fung; Chan, Ka-Kui; Grills, Claire; Murray, James T.; Platt-Higgins, Angela; Eldin, Osama Sharaf; O’Byrne, Ken; Janne, Pasi; Fennell, Dean A.; Johnston, Patrick G.; Rudland, Philip S.; El-Tanani, Mohamed

2011-01-01

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown compared to normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK and PI3K/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry (PI and Annexin V staining) and MTT assay in cancer cells grown under different conditions after knockdown of Ran.. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. KRas mutated, c-Met amplified and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of KRas or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. PMID:22090358

RAS mutations affect pattern of metastatic spread and increase propensity for brain metastasis in colorectal cancer.

PubMed

Yaeger, Rona; Cowell, Elizabeth; Chou, Joanne F; Gewirtz, Alexandra N; Borsu, Laetitia; Vakiani, Efsevia; Solit, David B; Rosen, Neal; Capanu, Marinela; Ladanyi, Marc; Kemeny, Nancy

2015-04-15

RAS and PIK3CA mutations in metastatic colorectal cancer (mCRC) have been associated with worse survival. We sought to evaluate the impact of RAS and PIK3CA mutations on cumulative incidence of metastasis to potentially curable sites of liver and lung and other sites such as bone and brain. We performed a computerized search of the electronic medical record of our institution for mCRC cases genotyped for RAS or PIK3CA mutations from 2008 to 2012. Cases were reviewed for patient characteristics, survival, and site-specific metastasis. Among the 918 patients identified, 477 cases were RAS wild type, and 441 cases had a RAS mutation (394 at KRAS exon 2, 29 at KRAS exon 3 or 4, and 18 in NRAS). RAS mutation was significantly associated with shorter median overall survival (OS) and on multivariate analysis independently predicted worse OS (HR, 1.6; P < .01). RAS mutant mCRC exhibited a significantly higher cumulative incidence of lung, bone, and brain metastasis and on multivariate analysis was an independent predictor of involvement of these sites (HR, 1.5, 1.6, and 3.7, respectively). PIK3CA mutations occurred in 10% of the 786 cases genotyped, did not predict for worse survival, and did not exhibit a site-specific pattern of metastatic spread. The metastatic potential of CRC varies with the presence of RAS mutation. RAS mutation is associated with worse OS and increased incidence of lung, bone, and brain metastasis. An understanding of this site-specific pattern of spread may help to inform physicians' assessment of symptoms in patients with mCRC. © 2014 American Cancer Society.

Cognitive Dysfunctions in Intellectual Disabilities: The Contributions of the Ras-MAPK and PI3K-AKT-mTOR Pathways.

PubMed

Borrie, Sarah C; Brems, Hilde; Legius, Eric; Bagni, Claudia

2017-08-31

The Ras-MAPK and PI3K-AKT-mTOR signaling cascades were originally identified as cancer regulatory pathways but have now been demonstrated to be critical for synaptic plasticity and behavior. Neurodevelopmental disorders arising from mutations in these pathways exhibit related neurological phenotypes, including cognitive dysfunction, autism, and intellectual disability. The downstream targets of these pathways include regulation of transcription and protein synthesis. Other disorders that affect protein translation include fragile X syndrome (an important cause of syndromal autism), and other translational regulators are now also linked to autism. Here, we review how mechanisms of synaptic plasticity have been revealed by studies of mouse models for Ras-MAPK, PI3K-AKT-mTOR, and translation regulatory pathway disorders. We discuss the face validity of these mouse models and review current progress in clinical trials directed at ameliorating cognitive and behavioral symptoms.

Ras mutations are rare in solitary cold and toxic thyroid nodules.

PubMed

Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

High frequency of TP53 but not K-ras gene mutations in Bolivian patients with gallbladder cancer.

PubMed

Asai, Takao; Loza, Ernesto; Roig, Guido Villa-Gomez; Ajioka, Yoichi; Tsuchiya, Yasuo; Yamamoto, Masaharu; Nakamura, Kazutoshi

2014-01-01

Although genetic characteristics are considered to be a factor influencing the geographic variation in the prevalence of gallbladder cancer (GBC), they have not been well studied in Bolivia, which has a high prevalence rate of GBC. The purpose of this study was to examine the frequency of TP53 and K-ras mutations in Bolivian patients with GBC and to compare them with our previous data obtained in other high-GBC-prevalence countries, namely Japan, Chile, and Hungary. DNA was extracted from cancer sites in paraffin-embedded tissue from 36 patients using a microdissection technique. TP53 mutations at exons 5 to 8 and K-ras mutations at codons 12, 13 and 61 were examined using direct sequencing techniques. The data obtained were compared with those in the other high-GBC-prevalence countries. Of the 36 patients, 18 (50.0%) had a TP53 mutation (one mutation in each of 17 patients and three mutations in one patient), and only one (2.8%) had a K-ras mutation. Of the 20 TP53 mutations, 12 were of the transition type (60.0%). This rate was significantly lower than that in Chile (12/12, P<0.05). In addition, three mutations were of the CpG transition type (15.0%), which is a feature of endogenous mutation. All three were found in the hot spot region of the TP53 gene. In contrast, G:C to T:A transversion was found in Bolivia, suggesting the presence of exogenous carcinogens. Our findings suggest that the development of GBC in Bolivia is associated with both exogenous carcinogens and endogenous mechanisms. The identification of an environmental risk factor for GBC is needed to confirm these findings.

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

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

Bigdeli, Bahareh, E-mail: bhr.bigdeli@ut.ac.ir

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay.more » Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel

Breast Tumors with Elevated Expression of 1q Candidate Genes Confer Poor Clinical Outcome and Sensitivity to Ras/PI3K Inhibition

PubMed Central

Viveka Thangaraj, Soundara; Periasamy, Jayaprakash; Bhaskar Rao, Divya; Barnabas, Georgina D.; Raghavan, Swetha; Ganesan, Kumaresan

2013-01-01

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA–drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors. PMID:24147022

Boldine Improves Kidney Damage in the Goldblatt 2K1C Model Avoiding the Increase in TGF-β.

PubMed

Gómez, Gonzalo I; Velarde, Victoria

2018-06-25

Boldine, a major aporphine alkaloid found in the Chilean boldo tree, is a potent antioxidant. Oxidative stress plays a detrimental role in the pathogenesis of kidney damage in renovascular hypertension (RVH). The activation of the renin-angiotensin system (RAS) is crucial to the development and progression of hypertensive renal damage and TGF-β is closely associated with the activation of RAS. In the present study, we assessed the effect of boldine on the progression of kidney disease using the 2K1C hypertension model and identifying mediators in the RAS, such as TGF-β, that could be modulated by this alkaloid. Toward this hypothesis, rats ( n = 5/group) were treated with boldine (50 mg/kg/day, gavage) for six weeks after 2K1C surgery (pressure ≥ 180 mmHg). Kidney function was evaluated by measuring of proteinuria/creatininuria ratio (U prot/U Crea), oxidative stress (OS) by measuring thiobarbituric acid reactive substances (TBARS). The evolution of systolic blood pressure (SBP) was followed weekly. Alpha-smooth muscle actin (α-SMA) and Col III were used as markers of kidney damage; ED-1 and osteopontin (OPN) were used as markers of inflammation. We also explored the effect in RAS mediators, such as ACE-1 and TGF-β. Boldine treatment reduced the UProt/UCrea ratio, plasma TBARS, and slightly reduced SBP in 2K1C hypertensive rats, producing no effect in control animals. In 2K1C rats treated with boldine the levels of α-SMA, Col III, ED-1, and OPN were lower when compared to 2K1C rats. Boldine prevented the increase in ACE-1 and TGF-β in 2K1C rats, suggesting that boldine reduces kidney damage. These results suggest that boldine could potentially be used as a nutraceutic.

RAS Initiative - Community Outreach

Cancer.gov

Through community and technical collaborations, workshops and symposia, and the distribution of reference reagents, the RAS Initiative seeks to increase the sharing of knowledge and resources essential to defeating cancers caused by mutant RAS genes.

MEK1/2 inhibition enhances the radiosensitivity of cancer cells by downregulating survival and growth signals mediated by EGFR ligands

PubMed Central

CHUNG, EUN JOO; URICK, MARY ELLEN; KURSHAN, NAAMIT; SHIELD, WILLIAM; ASANO, HIROAKI; SMITH, PAUL D.; SCROGGINS, BRADLEY S.; BURKEEN, JEFFREY; CITRIN, DEBORAH E.

2013-01-01

The inhibition of the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway through the suppression of mutated Ras or MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) has been shown to sensitize tumor cells to ionizing radiation (IR). The molecular mechanisms of this sensitization however, are not yet fully understood. In this study, we investigated the role of transforming growth factor-α (TGF-α) in the radiosensitizing effects of selumetinib, a selective inhibitor of MEK1/2. The expression of epidermal growth factor receptor (EGFR) ligands was assessed by ELISA in both Ras wild-type and Ras mutant cells that were exposed to radiation with or without selumetinib. The effects of selumetinib on the TGF-α/EGFR signaling cascade in response to radiation were examined by western blot analysis, clonogenic assay and by determing the yield of mitotic catastrophe. The treatment of cells with selumetinib reduced the basal and IR-induced secretion of TGF-α in both Ras wild-type and Ras mutant cell lines in vitro and in vivo. The reduction of TGF-α secretion was accompanied with a reduction in phosphorylated tumor necrosis factor-α converting enzyme (TACE) in the cells treated with selumetinib with or without IR. The treatment of cells with selumetinib with or without IR inhibited the phosphorylation of EGFR and check-point kinase 2 (Chk2), and reduced the expression of survivin. Supplementation with exogenous TGF-α partially rescued the selumetinib-treated cells from IR-induced cell death, restored EGFR and Chk2 phosphorylation and increased survivin expression. These data suggest that the inhibition of MEK1/2 with selumetinib may provide a mechanism to sensitize tumor cells to IR in a fashion that prevents the activation of the TGF-α autocrine loop following IR. PMID:23588995

RAS diseases in children

PubMed Central

Niemeyer, Charlotte M.

2014-01-01

RAS genes encode a family of 21 kDa proteins that are an essential hub for a number of survival, proliferation, differentiation and senescence pathways. Signaling of the RAS-GTPases through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade is essential in development. A group of genetic syndromes, named “RASopathies”, had been identified which are caused by heterozygosity for germline mutations in genes that encode protein components of the RAS/MAPK pathway. Several of these clinically overlapping disorders, including Noonan syndrome, Noonan-like CBL syndrome, Costello syndrome, cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type I, and Legius syndrome, predispose to cancer and abnormal myelopoiesis in infancy. This review focuses on juvenile myelomonocytic leukemia (JMML), a malignancy of early childhood characterized by initiating germline and/or somatic mutations in five genes of the RAS/MAPK pathway: PTPN11, CBL, NF-1, KRAS and NRAS. Natural courses of these five subtypes differ, although hematopoietic stem cell transplantation remains the only curative therapy option for most children with JMML. With whole-exome sequencing studies revealing few secondary lesions it will be crucial to better understand the RAS/MAPK signaling network with its crosstalks and feed-back loops to carefully design early clinical trials with novel pharmacological agents in this still puzzling leukemia. PMID:25420281

Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

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

Tsubaki, Masanobu; Satou, Takao; Itoh, Tatsuki

Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion,more » and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma. -- Highlights: ► We investigated whether YM529/ONO-5920 inhibited tumor metastasis in osteosarcoma. ► YM529/ONO-5920 inhibited metastasis, cell migration, invasion, and adhesion. ► YM529/ONO-5920 suppressed Ras signalings. ► YM529

Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

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

Buhrman, Greg; O; #8242

2012-09-17

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond themore » active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.« less

Comparison of microwave and magnetic nanoparticle hyperthermia radiosensitization in murine breast tumors

NASA Astrophysics Data System (ADS)

Giustini, Andrew J.; Petryk, Alicia A.; Hoopes, Paul J.

2011-03-01

Hyperthermia has been shown to be an effective radiosensitizer. Its utility as a clinical modality has been limited by a minimally selective tumor sensitivity and the inability to be delivered in a tumor-specific manner. Recent in vivo studies (rodent and human) have shown that cancer cell-specific cytotoxicity can be effectively and safely delivered via iron oxide magnetic nanoparticles (mNP) and an appropriately matched noninvasive alternating magnetic field (AMF). To explore the tumor radiosensitization potential of mNP hyperthermia we used a syngeneic mouse breast cancer model, dextran-coated 110 nm hydrodynamic diameter mNP and a 169 kHz / 450 Oe (35.8 kA/m) AMF. Intradermally implanted (flank) tumors (150 +/- 40 mm3) were treated by injection of 0.04 ml mNP (7.5 mg Fe) / cm3 into the tumor and an AMF (35.8 kA/m and 169 kHz) exposure necessary to achieve a CEM (cumulative equivalent minute) thermal dose of 60 (CEM 60). Tumors were treated with mNP hyperthermia (CEM 60), radiation alone (15 Gy, single dose) and in combination. Compared to the radiation and heat alone treatments, the combined treatment resulted in a greater than two-fold increase in tumor regrowth tripling time (tumor treatment efficacy). None of the treatments resulted in significant normal tissue toxicity or morbidity. Studies were also conducted to compare the radiosensitization effect of mNP hyperthermia with that of microwave-induced hyperthermia. The effects of incubation of nanoparticles within tumors (to allow nanoparticles to be endocytosed) before application of AMF and radiation were determined. This preliminary information suggests cancer cell specific hyperthermia (i.e. antibody-directed or anatomically-directed mNP) is capable of providing significantly greater radiosensitization / therapeutic ratio enhancement than other forms of hyperthermia delivery.

Ras and relatives--job sharing and networking keep an old family together.

PubMed

Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

2002-10-01

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line, LM217.

PubMed

Murata, Yasuhiko; Hashimoto, Takuma; Urushihara, Yusuke; Shiga, Soichiro; Takeda, Kazuya; Jingu, Keiichi; Hosoi, Yoshio

2018-01-22

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines. LM217 and HepG2 cells were used to examine the effects of hypoxia and nutrient starvation on cellular radiosensitivity, mTORC1 pathway including AMPK, ATM, and HIF-1α, which are known as regulators of mTORC1 activity, and glycogen storage, which is induced by HIF-1 and HIF-2 under hypoxia and promotes cell survival. Under hypoxia and nutrient starvation, AMPK activity and ATM expression were increased in LM217 cells and decreased in HepG2 cells compared with AMPK activity under nutrient starvation alone or ATM expression under hypoxia alone. Under hypoxia and nutrient starvation, radiosensitivity was decreased in LM217 cells and increased in HepG2 cells compared with radiosensitivity under hypoxia alone. Under hypoxia and nutrient starvation, knockdown of AMPK decreased ATM activity and increased radiation sensitivity in LM217 cells. In both cell lines, mTORC1 activity was decreased under hypoxia and nutrient starvation. Under hypoxia alone, knockdown of mTOR slightly increased ATM

Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK

PubMed Central

Wang, Qin; Downey, Gregory P.; McCulloch, Christopher A.

2011-01-01

In connective tissue cells, IL-1-induced ERK activation leading to matrix metalloproteinase (MMP)-3 expression is dependent on cooperative interactions between focal adhesions and the endoplasmic reticulum (ER). As Ras can be activated on the ER, we investigated the role of Ras in IL-1 signaling and focal adhesion formation. We found that constitutively active H-Ras, K-Ras or N-Ras enhanced focal adhesion maturation and β1-integrin activation. IL-1 promoted the accumulation of Ras isoforms in ER and focal adhesion fractions, as shown in cells cotransfected with GFP-tagged Ras isoforms and YFP-ER protein and by analysis of subcellular fractions enriched for ER or focal adhesion proteins. Dominant-negative H-Ras or K-Ras reduced accumulation of H-Ras and K-Ras in focal adhesions induced by IL-1 and also blocked ERK activation and focal adhesion maturation. Ras-GRF was enriched constitutively in focal adhesion fractions and was required for Ras recruitment to focal adhesions. We conclude that Ras activation and IL-1 signaling are interactive processes that regulate the maturation of focal adhesions, which, in turn, is required for ERK activation.—Wang, Q., Downey, G. P., McCulloch, C. A. Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK. PMID:21719512

Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

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

Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Instituto 'Reina Sofia' de Investigacion Nefrologica, Universidad de Salamanca, 37007 Salamanca

2006-07-01

Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms andmore » from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.« less

Absence of ras-gene hot-spot mutations in canine fibrosarcomas and melanomas.

PubMed

Murua Escobar, Hugo; Günther, Kathrin; Richter, Andreas; Soller, Jan T; Winkler, Susanne; Nolte, Ingo; Bullerdiek, Jörn

2004-01-01

Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.

Inhibitors of Ras-SOS Interactions.

PubMed

Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

2016-04-19

Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous detection of 19 K-ras mutations by free-solution conjugate electrophoresis of ligase detection reaction products on glass microchips

PubMed Central

Albrecht, Jennifer Coyne; Kotani, Akira; Lin, Jennifer S.; Soper, Steven A.; Barron, Annelise E.

2015-01-01

We demonstrate here the power and flexibility of free-solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild-type DNA. Here, four large drag-tags are used to achieve free-solution electrophoretic separation of 19 LDR products ranging in size from 42–66 nt that correspond to mutations in the K-ras oncogene. LDR-FSCE enabled electrophoretic resolution of these 19 LDR-FSCE products by CE in 13.5 minutes (E = 310 V/cm) and by microchip electrophoresis in 140 seconds (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free-solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR-FSCE products were separated in < 70 seconds with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K-ras mutations on integrated “sample-in/answer-out” devices with amplification, LDR, and detection all on one platform. PMID:23192597

Ras-Related Small GTPases RalA and RalB Regulate Cellular Survival After Ionizing Radiation

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

Kidd, Ambrose R.; Snider, Jared L.; Martin, Timothy D.

2010-09-01

Purpose: Oncogenic activation of Ras renders cancer cells resistant to ionizing radiation (IR), but the mechanisms have not been fully characterized. The Ras-like small GTPases RalA and RalB are downstream effectors of Ras function and are critical for both tumor growth and survival. The Ral effector RalBP1/RLIP76 mediates survival of mice after whole-body irradiation, but the role of the Ral GTPases themselves in response to IR is unknown. We have investigated the role of RalA and RalB in cellular responses to IR. Methods and Materials: RalA, RalB, and their major effectors RalBP1 and Sec5 were knocked down by stable expressionmore » of short hairpin RNAs in the K-Ras-dependent pancreatic cancer-derived cell line MIA PaCa-2. Radiation responses were measured by standard clonogenic survival assays for reproductive survival, {gamma}H2AX expression for double-strand DNA breaks (DSBs), and poly(ADP-ribose)polymerase (PARP) cleavage for apoptosis. Results: Knockdown of K-Ras, RalA, or RalB reduced colony-forming ability post-IR, and knockdown of either Ral isoform decreased the rate of DSB repair post-IR. However, knockdown of RalB, but not RalA, increased cell death. Surprisingly, neither RalBP1 nor Sec5 suppression affected colony formation post-IR. Conclusions: Both RalA and RalB contribute to K-Ras-dependent IR resistance of MIA PaCa-2 cells. Sensitization due to suppressed Ral expression is likely due in part to decreased efficiency of DNA repair (RalA and RalB) and increased susceptibility to apoptosis (RalB). Ral-mediated radioresistance does not depend on either the RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.« less

DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, is a putative tumor suppressor.

PubMed

Kanno, Emiri; Kawasaki, Osamu; Takahashi, Kazuya; Takano, Kazunori; Endo, Takeshi

2018-01-01

Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf-MEK-ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Ras regulation of DNA-methylation and cancer

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

Patra, Samir Kumar

2008-04-01

Genome wide hypomethylation and regional hypermethylation of cancer cells and tissues remain a paradox, though it has received a convincing confirmation that epigenetic switching systems, including DNA-methylation represent a fundamental regulatory mechanism that has an impact on genome maintenance and gene transcription. Methylated cytosine residues of vertebrate DNA are transmitted by clonal inheritance through the strong preference of DNA methyltransferase, DNMT1, for hemimethylated-DNA. Maintenance of methylation patterns is necessary for normal development of mice, and aberrant methylation patterns are associated with many human tumours. DNMT1 interacts with many proteins during cell cycle progression, including PCNA, p53, EZH2 and HP1. Rasmore » family of GTPases promotes cell proliferation by its oncogenic nature, which transmits signals by multiple pathways in both lipid raft dependent and independent fashion. DNA-methylation-mediated repression of DNA-repair protein O6-methylguanine DNA methyltransferase (MGMT) gene and increased rate of K-Ras mutation at codon for amino acids 12 and 13 have been correlated with a secondary role for Ras-effector homologues (RASSFs) in tumourigenesis. Lines of evidence suggest that DNA-methylation associated repression of tumour suppressors and apoptotic genes and ceaseless proliferation of tumour cells are regulated in part by Ras-signaling. Control of Ras GTPase signaling might reduce the aberrant methylation and accordingly may reduce the risk of cancer development.« less

The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF*

PubMed Central

Baljuls, Angela; Beck, Matthias; Oenel, Ayla; Robubi, Armin; Kroschewski, Ruth; Hekman, Mirko; Rudel, Thomas; Rapp, Ulf R.

2012-01-01

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity. PMID:22605333

Differences in K-ras and mitochondrial DNA mutations and microsatellite instability between colorectal cancers of Vietnamese and Japanese patients.

PubMed

Miwata, Tomohiro; Hiyama, Toru; Quach, Duc Trong; Le, Huy Minh; Hua, Ha Ngoc Thi; Oka, Shiro; Tanaka, Shinji; Arihiro, Koji; Chayama, Kazuaki

2014-11-30

The incidence of early-onset (under 50 years of age) colorectal cancer (CRC) in the Vietnamese has been reported to be quite higher than that in the Japanese. To clarify the differences in genetic alterations between Vietnamese and Japanese CRCs, we investigated mutations in K-ras and mitochondrial DNA (mtDNA) and high-frequency microsatellite instability (MSI-H) in the CRCs of Vietnamese and Japanese patients. We enrolled 60 Vietnamese and 233 Japanese patients with invasive CRCs. DNA was extracted from formalin-fixed, paraffin-embedded tissue sections. K-ras mutations were examined with PCR-single-strand conformation polymorphism analysis. mtDNA mutations and MSI-H were examined with microsatellite analysis using D310 and BAT-26, respectively. K-ras mutations were examined in 60 Vietnamese and 45 Japanese CRCs. The frequency of the mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (8 of 24 [33%] vs 5 of 45 [11%], p =0.048). MSI-H was examined in 60 Vietnamese and 130 Japanese CRCs. The frequency of MSI-H in the Vietnamese CRCs was also significantly higher than that in the Japanese CRCs (6 of 27 [22%] vs 10 of 130 [8%], p =0.030). mtDNA mutations were examined in 60 Vietnamese and 138 Japanese CRCs. The frequency of mtDNA mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (19 of 44 [43%] vs 11 of 133 [9%], p <0.001). There were no significant differences in clinicopathologic characteristics, such as age, sex, tumour location, and depth, in terms of tumours with/without each genetic alteration in the CRCs of the Vietnamese and Japanese patients. These results indicate that the developmental pathways of CRCs in the Vietnamese may differ from those of CRCs in the Japanese.

Cisplatin radiosensitizes radioresistant human mesenchymal stem cells.

PubMed

Rühle, Alexander; Perez, Ramon Lopez; Glowa, Christin; Weber, Klaus-Josef; Ho, Anthony D; Debus, Jürgen; Saffrich, Rainer; Huber, Peter E; Nicolay, Nils H

2017-10-20

Cisplatin-based chemo-radiotherapy is widely used to treat cancers with often severe therapy-associated late toxicities. While mesenchymal stem cells (MSCs) were shown to aid regeneration of cisplatin- or radiation-induced tissue lesions, the effect of the combined treatment on the stem cells remains unknown. Here we demonstrate that cisplatin treatment radiosensitized human bone marrow-derived MSCs in a dose-dependent manner and increased levels of radiation-induced apoptosis. However, the defining stem cell properties of MSCs remained largely intact after cisplatin-based chemo-radiation, and stem cell motility, adhesion, surface marker expression and the characteristic differentiation potential were not significantly influenced. The increased cisplatin-mediated radiosensitivity was associated with a cell cycle shift of MSCs towards the radiosensitive G2/M phase and increased residual DNA double-strand breaks. These data demonstrate for the first time a dose-dependent radiosensitization effect of MSCs by cisplatin. Clinically, the observed increase in radiation sensitivity and subsequent loss of regenerative MSCs may contribute to the often severe late toxicities observed after cisplatin-based chemo-radiotherapy in cancer patients.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats.

PubMed

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-05-10

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats

PubMed Central

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-01-01

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function. PMID:28489060

Radiosensitization of Cancer Cells by Hydroxychalcones

PubMed Central

Pruitt, Rory; Sasi, Nidhish; Freeman, Michael L.; Sekhar, Konjeti R.

2010-01-01

Radiation sensitization is significantly increased by proteotoxic stress, such as a heat shock. We undertook an investigation, seeking to identify natural products that induced proteotoxic stress and then determined if a compound exhibited radiosensitizing properties. The hydroxychalcones, 2′,5′-dihydroxychalcone (D-601) and 2,2′-dihydroxychalcone (D-501), were found to activate heat shock factor 1 (Hsf1) and exhibited radiation sensitization properties in colon and pancreatic cancer cells. The radiosensitization ability of D-601 was blocked by pretreatment with α-napthoflavone (ANF), a specific inhibitor of cytochrome P450 1A2 (CYP1A2), suggesting that the metabolite of D-601 is essential for radiosensitization. The study demonstrated the ability of hydroxychalcones to radiosensitize cancer cells and provides new leads for developing novel radiation sensitizers. PMID:20826087

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation

PubMed Central

Lim, Chinten James; Spiegelman, George B.; Weeks, Gerald

2001-01-01

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC– cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC– cells stimulated by 2′-deoxy-cAMP, but is produced in response to GTPγS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC– cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC– cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC– cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC– cells, suggesting that AleA may activate RasC. PMID:11500376

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

PubMed

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Ras Signaling Regulates Stem Cells and Amelogenesis in the Mouse Incisor.

PubMed

Zheng, X; Goodwin, A F; Tian, H; Jheon, A H; Klein, O D

2017-11-01

The role of Ras signaling during tooth development is poorly understood. Ras proteins-which are activated by many upstream pathways, including receptor tyrosine kinase cascades-signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor-which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells-provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.

Ras history

PubMed Central

2010-01-01

Although the roots of Ras sprouted from the rich history of retrovirus research, it was the discovery of mutationally activated RAS genes in human cancer in 1982 that stimulated an intensive research effort to understand Ras protein structure, biochemistry and biology. While the ultimate goal has been developing anti-Ras drugs for cancer treatment, discoveries from Ras have laid the foundation for three broad areas of science. First, they focused studies on the origins of cancer to the molecular level, with the subsequent discovery of genes mutated in cancer that now number in the thousands. Second, elucidation of the biochemical mechanisms by which Ras facilitates signal transduction established many of our fundamental concepts of how a normal cell orchestrates responses to extracellular cues. Third, Ras proteins are also founding members of a large superfamily of small GTPases that regulate all key cellular processes and established the versatile role of small GTP-binding proteins in biology. We highlight some of the key findings of the last 28 years. PMID:21686117

Zidovudine, abacavir and lamivudine increase the radiosensitivity of human esophageal squamous cancer cell lines.

PubMed

Chen, Xuan; Wang, Cong; Guan, Shanghui; Liu, Yuan; Han, Lihui; Cheng, Yufeng

2016-07-01

Telomerase is a type of reverse transcriptase that is overexpressed in almost all human tumor cells, but not in normal tissues, which provides an opportunity for radiosensitization targeting telomerase. Zidovudine, abacavir and lamivudine are reverse transcriptase inhibitors that have been applied in clinical practice for several years. We sought to explore the radiosensitization effect of these three drugs on human esophageal cancer cell lines. Eca109 and Eca9706 cells were treated with zidovudine, abacavir and lamivudine for 48 h before irradiation was administered. Samples were collected 1 h after irradiation. Clonal efficiency assay was used to evaluate the effect of the combination of these drugs with radiation doses of 2, 4, 6 and 8 Gy. DNA damage was measured by comet assay. Telomerase activity (TA) and relative telomere length (TL) were detected and evaluated by real-time PCR. Apoptosis rates were assessed by flow cytometric analysis. The results showed that all the drugs tested sensitized the esophageal squamous cell carcinoma (ESCC) cell lines to radiation through an increase in radiation-induced DNA damage and cell apoptosis, deregulation of TA and decreasing the shortened TL caused by radiation. Each of the drugs investigated (zidovudine, abacavir and lamivudine) could be used for sensitizing human esophageal cancer cell lines to radiation. Consequently, the present study supports the potential of these three drugs as therapeutic agents for the radiosensitization of esophageal squamous cell cancer.

Radiosensitization of cancer cells by hydroxychalcones.

PubMed

Pruitt, Rory; Sasi, Nidhish; Freeman, Michael L; Sekhar, Konjeti R

2010-10-15

Radiation sensitization is significantly increased by proteotoxic stress, such as a heat shock. We undertook an investigation, seeking to identify natural products that induced proteotoxic stress and then determined if a compound exhibited radiosensitizing properties. The hydroxychalcones, 2',5'-dihydroxychalcone (D-601) and 2,2'-dihydroxychalcone (D-501), were found to activate heat shock factor 1 (Hsf1) and exhibited radiation sensitization properties in colon and pancreatic cancer cells. The radiosensitization ability of D-601 was blocked by pretreatment with α-napthoflavone (ANF), a specific inhibitor of cytochrome P450 1A2 (CYP1A2), suggesting that the metabolite of D-601 is essential for radiosensitization. The study demonstrated the ability of hydroxychalcones to radiosensitize cancer cells and provides new leads for developing novel radiation sensitizers. Copyright © 2010 Elsevier Ltd. All rights reserved.

Silencing the Girdin gene enhances radio-sensitivity of hepatocellular carcinoma via suppression of glycolytic metabolism.

PubMed

Yu, Li; Sun, Yifan; Li, Jingjing; Wang, Yan; Zhu, Yuxing; Shi, Yong; Fan, Xiaojun; Zhou, Jianda; Bao, Ying; Xiao, Jie; Cao, Ke; Cao, Peiguo

2017-08-15

Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

PubMed Central

Cheong, Jit Kong; Zhang, Fuquan; Chua, Pei Jou; Bay, Boon Huat; Thorburn, Andrew; Virshup, David M.

2015-01-01

Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS–induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS–driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS–driven cancers. PMID:25798617

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.

PubMed

Tikoo, A; Cutler, H; Lo, S H; Chen, L B; Maruta, H

1999-01-01

For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK

Targeting Phosphatidylinositol 4-Kinase IIIα for Radiosensitization: A Potential Model of Drug Repositioning Using an Anti-Hepatitis C Viral Agent

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

Kwon, Jeanny; Kim, Dan Hyo; Park, Ji Min

Purpose: To investigate which isotype of phosphatidylinositol 4-kinase (PI4K) may affect radiosensitivity and examine whether anti–hepatitis C viral (HCV) agents, some of which have been shown to inhibit PI4K IIIα activity, could be repositioned as a radiosensitizer in human cancer cells. Methods and Materials: U251, BT474, and HepG2 cell lines and normal human astrocyte were used. Ribonucleic acid interference, clonogenic assays, Western blotting, immunofluorescence, annexin V assay, lysotracker staining, and β-galactosidase assay were performed. Results: Of the 4 PI4K isotypes, specific inhibition of IIIα increased radiosensitivity. For pharmacologic inhibition of PI4K IIIα, we screened 9 anti-HCV agents by half-maximal inhibitorymore » concentration assay. Simeprevir was selected, and its inhibition of PI4K IIIα activity was confirmed. Combination of simeprevir treatment and radiation significantly attenuated expression of phospho-phospho-PKC and phospho-Akt and increased radiation-induced cell death in tested cell lines. Pretreatment with simeprevir prolonged γH2AX foci formation and down-regulation of phospho-DNA-PKcs, indicating impairment of nonhomologous end-joining repair. Cells pretreated with simeprevir exhibited mixed modes of cell death, including apoptosis and autophagy. Conclusion: These data demonstrate that targeting PI4K IIIα using an anti-HCV agent is a viable approach to enhance the therapeutic efficacy of radiation therapy in various human cancers, such as glioma, breast, and hepatocellular carcinoma.« less

Comparative Roles of Overexpressed and Mutated H- and K-ras in Mammary Carcinogenesis.

DTIC Science & Technology

1996-08-01

transgene of these tumors. 14. SUBJECT TERMS 15. NUMBER OF PAGES Breast Cancer , mammary carcinogenesis, oncogenes, ras genes, 44 replication defective...27 Appendix 5 29 Appendix 6 31 Appendix 7 33 Appendix 8 35 Appendix 9 37 Appendix 10 39 Introduction Breast cancer development involves multiple poorly...understood steps (25). Currently, several genes that may participate in breast cancer development are under investigation. The ras family of genes

Ras Dimer Formation as a New Signaling Mechanism and Potential Cancer Therapeutic Target

PubMed Central

Chen, Mo; Peters, Alec; Huang, Tao; Nan, Xiaolin

2016-01-01

The K-, N-, and HRas small GTPases are key regulators of cell physiology and are frequently mutated in human cancers. Despite intensive research, previous efforts to target hyperactive Ras based on known mechanisms of Ras signaling have been met with little success. Several studies have provided compelling evidence for the existence and biological relevance of Ras dimers, establishing a new mechanism for regulating Ras activity in cells additionally to GTP-loading and membrane localization. Existing data also start to reveal how Ras proteins dimerize on the membrane. We propose a dimer model to describe Ras-mediated effector activation, which contrasts existing models of Ras signaling as a monomer or as a 5-8 membered multimer. We also discuss potential implications of this model in both basic and translational Ras biology. PMID:26423697

Targeting Bcl-2 stability to sensitize cells harboring oncogenic ras.

PubMed

Peng, Bo; Ganapathy, Suthakar; Shen, Ling; Huang, Junchi; Yi, Bo; Zhou, Xiaodong; Dai, Wei; Chen, Changyan

2015-09-08

The pro-survival factor Bcl-2 and its family members are critical determinants of the threshold of the susceptibility of cells to apoptosis. Studies are shown that cells harboring an oncogenic ras were extremely sensitive to the inhibition of protein kinase C (PKC) and Bcl-2 could antagonize this apoptotic process. However, it remains unrevealed how Bcl-2 is being regulated in this apoptotic process. In this study, we investigate the role of Bcl-2 stability in sensitizing the cells harboring oncogenic K-ras to apoptosis triggered by PKC inhibitor GO6976. We demonstrated that Bcl-2 in Swiss3T3 cells ectopically expressing or murine lung cancer LKR cells harboring K-ras rapidly underwent ubiquitin-dependent proteasome pathway after the treatment of GO6976, accompanied with induction of apoptosis. In this process, Bcl-2 formed the complex with Keap-1 and Cul3. The mutation of serine-17 and deletion of BH-2 or 4 was required for Bcl-2 ubiquitination and degradation, which elevate the signal threshold for the induction of apoptosis in the cells following PKC inhibition. Thus, Bcl-2 appears an attractive target for the induction of apoptosis by PKC inhibition in cancer cells expressing oncogenic K-ras.

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells

Ras signaling in aging and metabolic regulation.

PubMed

Slack, Cathy

2017-12-07

Aberrant signal transduction downstream of the Ras GTPase has a well-established role in tumorigenesis. Mutations that result in hyperactivation of Ras are responsible for a third of all human cancers. Hence, small molecule inhibitors of the Ras signal transduction cascade have been under intense focus as potential cancer treatments. In both invertebrate and mammalian models, emerging evidence has also implicated components of the Ras signaling pathway in aging and metabolic regulation. Here, I review the current evidence for Ras signaling in these newly discovered roles highlighting the interactions between the Ras pathway and other longevity assurance mechanisms. Defining the role of Ras signaling in maintaining age-related health may have important implications for the development of interventions that could not only increase lifespan but also delay the onset and/or progression of age-related functional decline.

TAN-1813, a novel Ras-farnesyltransferase inhibitor produced by Phoma sp. taxonomy, fermentation, isolation and biological activities in vitro and in vivo.

PubMed

Ishii, T; Hayashi, K; Hida, T; Yamamoto, Y; Nozaki, Y

2000-08-01

A novel Ras-farnesyltransferase inhibitor designated TAN-1813 was isolated from the culture broth of a fungus strain, FL-41510, isolated as a plant endophyte. The producer was taxonomically characterized as Phoma sp. FL-41510. TAN-1813 inhibited rat brain farnesyltransferase and geranylgeranyltransferase I activity with IC50 values of 23 microg/ml and 47/microg/ml, respectively. TAN-1813 showed mixed-type inhibition with respect to farnesylpyrophosphate and noncompetitive inhibition with respect to a K-Ras C-terminal peptide. It also inhibited the in situ farnesylation of cellular Ras proteins in a K-ras transformant (NIH3T3/K-ras) of mouse embryonic fibroblast cell line NIH3T3. TAN- 1813 inhibited the proliferation of various human cancer cells, some of which harbor activated ras alleles, with IC50 values of 15 approximately 110 ng/ml as well as that of NIH3T3 and NIH3T3/K-ras cells with IC50S of 540 and 310 ng/ml, respectively. Flow cytometric analysis indicated that TAN-1813 arrests NIH3T3/K-ras cells at both G1 and G2/M phases of the cell cycle. In addition, TAN-1813 was found to induce morphological reversion of NIH3T3/K-ras cells from the transformed phenotype. Antitumor activity of TAN-1813 against human fibrosarcoma HT-1080 and NIH3T3/K-ras tumors in nude mice was also verified.

Interaction between Wnt/β-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway.

PubMed

Jeong, Woo-Jeong; Ro, Eun Ji; Choi, Kang-Yell

2018-01-01

Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli ( APC ) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

Study Illuminates K-Ras4B Activation, Which May Help Predict Drug Resistance | Poster

Cancer.gov

Until recently, researchers studying RAS, a family of proteins involved in transmitting signals within cells, believed that the exchange of guanosine 5’-diphosphate (GDP) by guanosine triphosphate (GTP) was sufficient to activate the protein. Once activated, RAS can cause unintended and overactive signaling in cells, which can lead to cell division and, ultimately, cancer.

Association Between Increased Vascular Density and Loss of Protective RAS in Early-stage NPDR

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan; Raghunandan, Sneha; Vyas, Ruchi J.; Vu, Amanda C.; Bryant, Douglas; Yaqian, Duan; Knecht, Brenda E.; Grant, Maria B.; Chalam, K. V.; Parsons-Wingerter, Patricia

2016-01-01

Our hypothesis predicts that retinal blood vessels increase in density during early-stage progression to moderate nonproliferative diabetic retinopathy (NPDR). The renin-angiotensin system (RAS) is implicated in the pathogenesis of DR and in the function of circulating angiogenic cells (CACs), a critical bone marrow-derived population that is instrumental in vascular repair.

CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

PubMed Central

Zheng, Ze-Yi; Cheng, Chiang-Min; Fu, Xin-Rong; Chen, Liuh-Yow; Xu, Lizhong; Terrillon, Sonia; Wong, Stephen T.; Bar-Sagi, Dafna; Songyang, Zhou; Chang, Eric C.

2011-01-01

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling. PMID:22231449

The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.

PubMed

Akino, Kimishige; Toyota, Minoru; Suzuki, Hiromu; Mita, Hiroaki; Sasaki, Yasushi; Ohe-Toyota, Mutsumi; Issa, Jean-Pierre J; Hinoda, Yuji; Imai, Kohzoh; Tokino, Takashi

2005-07-01

Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells. The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing. Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas. RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.

Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

PubMed Central

2014-01-01

Background Ras is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression. Results Here, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrix-degrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable. Conclusion MSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals. PMID:24383791

The RAS Initiative

Cancer.gov

NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

PubMed Central

Iwig, Jeffrey S; Vercoulen, Yvonne; Das, Rahul; Barros, Tiago; Limnander, Andre; Che, Yan; Pelton, Jeffrey G; Wemmer, David E; Roose, Jeroen P; Kuriyan, John

2013-01-01

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI: http://dx.doi.org/10.7554/eLife.00813.001 PMID:23908768

[Analysis of prevalence of point mutations in codon 12 of oncogene K-ras from non-cancerous samples of cervical cytology positive for type 16 or 18 PVH].

PubMed

Golijow, C D; Mourón, S A; Gómez, M A; Dulout, F N

1999-12-01

Ninety-one non cancerous samples from genital specimens positives for VPH 16 or 18 and 27 non-infected samples as controls were studied. Mutations at codon 12 in K-ras gene was analyzed using enriched alelic PCR technique. Among the samples studied 17.58% showed mutations in this codon. Significant differences were observed between the control group (negative DNA-HPV) and positives DNA-HPV samples (p < 0.01). No differences were found between both viral types in relation to the mutation frequency. The presence of mutations in the K-ras gene in non cancerous cytological samples point out new questions about the role of mutations in proto-oncogenes and the development of cervical cancer.

Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions.

PubMed

Bache, Matthias; Zschornak, Martin P; Passin, Sarina; Kessler, Jacqueline; Wichmann, Henri; Kappler, Matthias; Paschke, Reinhard; Kaluđerović, Goran N; Kommera, Harish; Taubert, Helge; Vordermark, Dirk

2011-09-09

Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied. In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses. Under normoxic conditions, a half maximal inhibitory concentration (IC50) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions. Our results suggest that BA is capable of

Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery?

PubMed Central

Cox, Adrienne D.; Der, Channing J.; Philips, Mark R.

2015-01-01

RAS proteins require membrane association for their biological activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTIs) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anti-cancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated post-translational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. PMID:25878363

Comparison of the Effects of RAS vs. Kain-Fritsch Convective Schemes on Katrina Forecasts with GEOS-5

NASA Technical Reports Server (NTRS)

Miller, Timothy L.; Cohen, Charles; Paxton, Jessica; Robertson, F. R. (Pete)

2009-01-01

Global forecasts were made with the 0.25-degree latitude version of GEOS-5, with the RAS scheme and with the Kain-Fritsch scheme. Examination was made of the Katrina (2005) hurricane simulation. Replacement of the RAS convective scheme with the K-F scheme results in a much more vigorous Katrina, closer to reality. Still, the result is not as vigorous as reality. In terms of wind maximum, the gap was closed by 50%. The result seems to be due to the RAS scheme drying out the boundary layer, thus hampering the grid-scale secondary circulation and attending cyclone development. The RAS case never developed a full warm core, whereas the K-F case did. Not shown here: The K-F scheme also resulted in a more vigorous storm than when GEOS-5 is run with no convective parameterization. Also not shown: An experiment in which the RAS firing level was moved up by 3 model levels resulted in a stronger, warm-core storm, though not as strong as the K-F case. Effects on storm track were noticed, but not studied.

Enhanced G2 chromatid radiosensitivity, an early stage in the neoplastic transformation of human epidermal keratinocytes in culture

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

Gantt, R.; Sanford, K.K.; Parshad, R.

1987-03-01

A deficiency in DNA repair, manifest as enhanced chromatid radiosensitivity during the G2 phase of the cell cycle, together with a proliferative stimulus such as that provided by active oncogenes may be necessary and sufficient for the malignant neoplastic transformation of human keratinocytes in culture. Normal epidermal keratinocytes established as continuous cell lines by transfection with pSV3-neo or infection with adeno 12-SV40 hybrid virus developed enhanced G2 chromatid radiosensitivity after 18 passages in culture. In contrast to cells from primary or secondary culture, these cells could be transformed to malignant neoplastic cells by infection with Kirsten murine sarcoma virus containingmore » the Ki-ras oncogene or in one line by the chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine; both of these agents produced a marked proliferative response. Cytological heterogeneity and karyotypic instability characterized the cells during their progression to neoplasia. These results are interpreted in terms of a mechanism for neoplastic transformation.« less

Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner*

PubMed Central

Luhtala, Natalie; Aslanian, Aaron; Yates, John R.; Hunter, Tony

2017-01-01

Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states. PMID:27909058

MO-FG-BRA-02: Modulation of Clinical Orthovoltage X-Ray Spectrum Further Enhances Radiosensitization of Cancer Cells Targeted with Gold Nanoparticles

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

Wolfe, T; Reynoso, F; Cho, J

2015-06-15

Purpose: To assess the potential to amplify radiosensitization of cancer cells targeted with gold nanoparticles by augmenting selective spectral components of X-ray beam. Methods: Human prostate cancer cells were treated for 24h with gold nanorods conjugated to goserelin acetate or pegylated, systematically washed and irradiated with 250 kVp X-rays (25mA, 0.25mm Cu- filter, 8x8cm{sup 2} field size, 50cm SSD) with or without an additional 0.25 mm Erbium (Er) filter. As demonstrated in a companion Monte Carlo study, Er-filter acted as an external target to feed Erbium K-shell X-ray fluorescence photons (∼50 keV) into the 250 kVp beam. After irradiation, wemore » performed measurements of clonogenic viability with doses between 0 -6Gy, irreparable DNA damage assay to measure double-strand breaks via γH2AX-foci staining, and production of stable reactive oxygen species (ROS). Results: The clonogenic assay for the group treated with conjugated nanoparticles showed radiosensitization enhancement factor (REF), calculated at the 10% survival fraction aisle, of (1.62±0.07) vs. (1.23±0.04) with/without the Er-filter in the 250 kVp beam, respectively. The group treated with pegylated nanoparticles, albeit retained in modest amounts within the cells, also showed statistically significant REF (1.13±0.09) when the Erbium filter was added to the beam. No significant radiosensitization was observed for other groups. Measurements of ROS levels showed increments of (1.9±0.2) vs. (1.4±0.1) for combined treatment with targeted nanoparticles and Er-filtered beam. γH2AX-foci showed 50% increase for the same treatment combination, confirming the enhanced radiosensitization in a consistent fashion. Conclusion: Our study demonstrates the feasibility of enhancing radiosensitization of cancer cells by combining actively targeted gold nanoparticles and modulating the X-ray spectrum in the desired energy range. The established technique will not only help develop strategies to

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

PubMed Central

2012-01-01

Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents. PMID:22917272

Daily rhythms of radiosensitivity of animals and several determining causes

NASA Technical Reports Server (NTRS)

Druzhinin, Y. P.; Malyutina, T. S.; Seraya, V. M.; Rodina, G. P.; Vatsek, A.; Rakova, A.

1974-01-01

Daily rhythms of radiosensitivity in rats and mice were determined by survival rates after acute total radiation at the same dosage at different times of the day. Radiosensitivity differed in animals of different species and varieties. Inbred mice exhibited one or two increases in radiosensitivity during the dark, active period of the day. These effects were attributed to periodic changes in the state of stem hematopoietic cells.

Ras oncogenes in oral cancer: the past 20 years.

PubMed

Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan; Tsuchida, Nobuo

2012-05-01

Oral squamous cell carcinoma (OSCC) of head and neck is associated with high morbidity and mortality in both Western and Asian countries. Several risk factors for the development of oral cancer are very well established, including tobacco chewing, betel quid, smoking, alcohol drinking and human papilloma virus (HPV) infection. Apart from these risk factors, many genetic factors such as oncogenes, tumor suppressor genes and regulatory genes are identified to involve in oral carcinogenesis with these risk factors dependent and independent manner. Ras is one of the most frequently genetically deregulated oncogene in oral cancer. In this review, we analyze the past 22years of literature on genetic alterations such as mutations and amplifications of the isoforms of the ras oncogene in oral cancer. Further, we addressed the isoform-specific role of the ras in oral carcinogenesis. We also discussed how targeting the Akt and MEK, downstream effectors of the PI3K/Akt and MAPK pathways, respectively, would probably pave the possible molecular therapeutic target for the ras driven tumorigenesis in oral cancer. Analysis of these ras isoforms may critically enlighten specific role of a particular ras isoform in oral carcinogenesis, enhance prognosis and pave the way for isoform-specific molecular targeted therapy in OSCC. Copyright © 2011 Elsevier Ltd. All rights reserved.

TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

PubMed

Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

2013-11-01

The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John

Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3.

PubMed

Czikora, Agnes; Kedei, Noemi; Kalish, Heather; Blumberg, Peter M

2017-12-01

RasGRP comprises a family of guanine nucleotide exchange factors, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP1 possesses REM (Ras exchange), GEF (catalytic), EF-hand, C1, SuPT (suppressor of PT), and PT (plasma membrane-targeting) domains, among which the C1 domain drives membrane localization in response to diacylglycerol or phorbol ester and the PT domain recognizes phosphoinositides. The homologous family member RasGRP3 shows less plasma membrane localization. The objective of this study was to explore the role of the different domains of RasGRP3 in membrane translocation in response to phorbol esters. The full-length RasGRP3 shows limited translocation to the plasma membrane in response to PMA, even when the basic hydrophobic cluster in the PT domain, reported to be critical for RasGRP1 translocation to endogenous activators, is mutated to resemble that of RasGRP1. Moreover, exchange of the C-termini (SuPT-PT domain) of the two proteins had little effect on their plasma membrane translocation. On the other hand, while the C1 domain of RasGRP3 alone showed partial plasma membrane translocation, truncated RasGRP3 constructs, which contain the PT domain and are missing the REM, showed stronger translocation, indicating that the REM of RasGRP3 was a suppressor of its membrane interaction. The REM of RasGRP1 failed to show comparable suppression of RasGRP3 translocation. The marked differences between RasGRP3 and RasGRP1 in membrane interaction necessarily will contribute to their different behavior in cells and are relevant to the design of selective ligands as potential therapeutic agents. Published by Elsevier B.V.

Ras mutation cooperates with β-catenin activation to drive bladder tumourigenesis.

PubMed

Ahmad, I; Patel, R; Liu, Y; Singh, L B; Taketo, M M; Wu, X-R; Leung, H Y; Sansom, O J

2011-03-03

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras(Q61L) or K-Ras(G12D)) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.

ACB-PCR MEASUREMENT OF K-RAS CODON 12 MUTATION IN A/J MOUSE LUNG EXPOSED TO BENZO[A]PYRENE: A DOSE-RESPONSE ASSESSMENT

EPA Science Inventory

Benzo[a]pyrene (B[a]P) is a known human carcinogen and environmental contaminant. The direct measurement of K-Ras mutant fraction (MF) was developed as a metric with which to examine the default assumption of low dose linearity in the mutational response to B...

DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

EPA Science Inventory

Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
<...

Anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize.

PubMed

Adams, Stephen R; Yang, Howard C; Savariar, Elamprakash N; Aguilera, Joe; Crisp, Jessica L; Jones, Karra A; Whitney, Michael A; Lippman, Scott M; Cohen, Ezra E W; Tsien, Roger Y; Advani, Sunil J

2016-10-04

Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery.

Anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize

PubMed Central

Adams, Stephen R.; Yang, Howard C.; Savariar, Elamprakash N.; Aguilera, Joe; Crisp, Jessica L.; Jones, Karra A.; Whitney, Michael A.; Lippman, Scott M.; Cohen, Ezra E. W.; Tsien, Roger Y.; Advani, Sunil J.

2016-01-01

Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery. PMID:27698471

Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling

PubMed Central

Dhawan, Neil S.; scopton, Alex P.; Dar, Arvin C.

2016-01-01

Deregulation of the Ras–mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies1. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes2. Kinase suppressor of Ras (KSR) is a MAPK scaffold3–5 that is subject to allosteric regulation through dimerization with RAF6,7. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras–MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers. PMID:27556948

RAS Insight

Cancer.gov

David Heimbrook, now CEO of the Frederick National Laboratory for Cancer Research, played a major role in a large pharma as it tried to develop an anti-RAS drug. Lessons from that failure inform the RAS Initiative today.

Radiosensitivity in plants

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

Nauman, A F

1979-01-01

The report presents a compilation of available data on the sensitivity of plants to ionizing radiation, and provides basic information on methods of determining such sensitivities, or of estimating radiosensitivities by calcuation of the nuclear factors upon which they depend. The scope of the data presented here is necessarily limited to the most generally useful radiobiological end points and to the most commonly-used types of radiation. Many of the factors which influence radiosensitivity, particularly nuclear factors, will be discussed. Emphasis will be upon whole-plant studies done at Brookhaven National Laboratory by A.H. Sparrow and his associates, since these studies aremore » the source of most of the available radiosensitivity data and of all the sensitivity predictions listed here. Data presented here include summaries of experimentally-determined radiosensitivities at various end points for both herbaceous and woody higher plants, and for a few species of ferns and lower plants. The algae and fungi have not been considered here due to space limitations.« less

Change in radiosensitivity of rats during hypokinetic stress

NASA Technical Reports Server (NTRS)

Chernov, I. P.

1980-01-01

The laws governing stress modification of radiation sickness in relation to hypokinetic stress were investigated. It was found that gamma irradiation (800 rad) of rats on the third day of exposure to hypokinesia increased the radiosensitivity of the animals which was determined by the survival rate and the dynamics of body weight and the weight of some internal organs. The same radiation dose was given on the 20th day of hypokinesia and on the third day of recovery from the 20 day hypokinesia decreased the radiosensitivity of rats. It is concluded that the variations in the radiosensitivity observed may be due to a stress effect of hypokinesia.

Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

PubMed Central

Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

2013-01-01

The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

Effects of low-level chronic irradiation on radiosensitivity of mammals: modeling and experimental studies

NASA Astrophysics Data System (ADS)

Smirnova, O. A.; Yonezawa, M.

Effects of low dose rate chronic irradiation on radiosensitivity of mammals mice are studied by experimental and modeling methods Own and reference experiments show that priming chronic low-level short-term and long-term exposures to radiation induce respectively elevated radiosensitivity and lowered radiosensitivity radioresistance in mice The manifestation of these radiosensitization and radioprotection effects are respectively increased and decreased mortality of preirradiated specimens after challenge acute irradiation in comparison with those for previously unexposed ones Taking into account that the reason of the animal death in the experiments was the hematopoietic syndrome the biophysical models of the critical body system hematopoiesis are used to simulate the dynamics of the major hematopoietic lines in mice exposed to challenge acute irradiation following the chronic one Juxtaposition of the modeling results obtained and the relevant experimental data shows that the radiosensitization effect of chronic low-level short-term less than 1 month preirradiation on mice is due to increased radiosensitivity of lymphopoietic granulocytopoietic and erythropoietic systems accompanied by increased or close to the normal level radiosensitivity of thrombocytopoietic system which are induced by the above-indicated exposure In turn the radioprotection effect of chronic low-level long-term more than 1 month preirradiation on mice is caused by decreased radiosensitivity radioresistance of the granulocytopoietic system which

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

PubMed

Fang, Bingliang

2016-01-01

Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

Studying the Immunomodulatory Effects of Small Molecule Ras-Inhibitors in Animal Models of Rheumatoid Arthritis

DTIC Science & Technology

2015-10-01

Models of Rheumatoid Arthritis PRINCIPAL INVESTIGATOR: Yoel Kloog RECIPIENT: Tel Aviv University TEL AVIV 69978 Israel REPORT DATE: October...TITLE AND SUBTITLE Studying the Immunomodulatory Effects of Small Molecule Ras- Inhibitors in Animal Models of Rheumatoid Arthritis 5a. CONTRACT NUMBER... Rheumatoid Arthritis (RA) display augmented activation of the Ras/Raf/MEK/ERK1/2 signaling pathway, and accordingly overexpression of active K-RAS in

RasGRP3 regulates the migration of glioma cells via interaction with Arp3

PubMed Central

Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

2015-01-01

Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

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

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

DOE PAGES

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.; ...

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling.

PubMed

Erickson, Keesha E; Rukhlenko, Oleksii S; Posner, Richard G; Hlavacek, William S; Kholodenko, Boris N

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

PubMed Central

Shantz, Lisa M

2004-01-01

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation. PMID:14519103

In TCR-Stimulated T-cells, N-ras Regulates Specific Genes and Signal Transduction Pathways

PubMed Central

Lynch, Stephen J.; Zavadil, Jiri; Pellicer, Angel

2013-01-01

It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. PMID:23755101

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance

PubMed Central

McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Franklin, Richard A.; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.

2012-01-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance. PMID:23085539

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance.

PubMed

McCubrey, James A; Steelman, Linda S; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M

2012-10-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance.

Radiosensitivity study and radiation effects on morphology characterization of grey oyster mushroom Pleurotus sajor-caju

NASA Astrophysics Data System (ADS)

Rashid, Rosnani Abdul; Daud, Fauzi; Senafi, Sahidan; Awang, Mat Rasol; Mohamad, Azhar; Mutaat, Hassan Hamdani; Maskom, Mohd Meswan

2014-09-01

Radiosensitive dosage and morphology characterization of irradiated grey oyster mushroom Pleurotus sajor-caju by gamma rays was investigated due to effects of irradiation. In order to establish the effect, mycelium of P. sajor-caju was irradiated by gamma rays at dose 0.1 to 8.0 kGy with dose rate 0.227 Gy sec-1. The irradiation of mycelia was carried out at the radiation facility in Malaysian Nuclear Agency. The radiosensitivity study was performed by evaluating the percentage of survival irradiated mycelia. The lethal dose of the mycelium P. sajor-caju was determined at 4.0 kGy and LD50 to be equal at 2.2 kGy. The radiation effects on morphology were evaluated based on growth rate of irradiated mycelia, mycelia types, colonization period on substrate, morphology of fruit bodies and yields. The results shown growth rate of irradiated mycelium was slightly lower than the control and decreased as the dose increased. Irradiation was found can induced the primordia formation on PDA and the BE of irradiated seed is higher than to control. The irradiation is proven to be useful for generating new varieties of mushroom with commercial value to the industry.

RAS - Target Identification - Informatics

Cancer.gov

The RAS Informatics lab group develops tools to track and analyze “big data” from the RAS Initiative, as well as analyzes data from external projects. By integrating internal and external data, this group helps improve understanding of RAS-driven cancers.

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

PubMed

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-05-22

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration*

PubMed Central

Haeussler, Dagmar J.; Pimentel, David R.; Hou, Xiuyun; Burgoyne, Joseph R.; Cohen, Richard A.; Bachschmid, Markus M.

2013-01-01

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling. PMID:23548900

RAS Initiative - Events

Cancer.gov

The NCI RAS Initiative has organized multiple events with outside experts to discuss how the latest scientific and technological breakthroughs can be applied to discover vulnerabilities in RAS-driven cancers.

Deconstructing Ras Signaling in the Thymus

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Pinski, John M.; Alexander, Clayton P.; Merrill, Robert K.; Li, Wenmei; Love, Paul E.

2012-01-01

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance. PMID:22586275

Bacterial radiosensitization by using radiation processing in combination with essential oil: Mechanism of action

NASA Astrophysics Data System (ADS)

Lacroix, Monique; Caillet, Stéphane; Shareck, Francois

2009-07-01

Spice extracts under the form of essential oils were tested for their efficiency to increase the relative radiosensitivity of Listeria monocytogenes and Escherichia coli O157H7 in culture media. The two pathogens were treated by gamma-irradiation alone or in combination with oregano essential oil to evaluate their mechanism of action. The membrane murein composition, and the intracellular and extracellular concentration of ATP was determined. The bacterial strains were treated with two irradiation doses: 1.2 kGy to induce cell damage and 3.5 kGy to cause cell death for L. monocytogenes. A dose of 0.4 kGy to induce cell damages, 1.1 kGy to obtain viable but nonculturable (VBNC) state and 1.3 kGy to obtain a lethal dose was also applied on E. coli O157H7. Oregano essential oil was used at 0.020% and 0.025% (w/v), which is the minimum inhibitory concentration (MIC) for L. monocytogenes. For E. coli O157H7, a concentration of 0.006% and 0.025% (w/v) which is the minimum inhibitory concentration was applied. The use of essential oils in combination with irradiation has permitted an increase of the bacterial radiosensitization by more than 3.1 times. All treatments had also a significant effect ( p⩽0.05) on the murein composition, although some muropeptides did not seem to be affected by the treatment. Each treatment influenced differently the relative percentage and number of muropeptides. There was a significant ( p⩽0.05) correlation between the reduction of intracellular ATP and increase in extracellular ATP following treatment of the cells with oregano oil. The reduction of intracellular ATP was even more important when essential oil was combined with irradiation, but irradiation of L. monocytogenes alone induced a significant decrease ( p⩽0.05) of the internal ATP without affecting the external ATP.

Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa–induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways

PubMed Central

Hou, Yuanyuan; Nie, Yan; Cheng, Binfeng; Tao, Jin; Ma, Xiaoyao; Jiang, Min; Gao, Jie; Bai, Gang

2016-01-01

Gram-negative pathogen–induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa–induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively. PMID:27175332

Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling | Office of Cancer Genomics

Cancer.gov

In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras.

Mutations that abolish the ability of Ha-Ras to associate with Raf-1.

PubMed

Shirouzu, M; Koide, H; Fujita-Yoshigaki, J; Oshio, H; Toyama, Y; Yamasaki, K; Fuhrman, S A; Villafranca, E; Kaziro, Y; Yokoyama, S

1994-08-01

Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

Cancer.gov

The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

Synthesis and radiosensitization properties of hydrogen peroxide and sodium hyaluronate complex

NASA Astrophysics Data System (ADS)

Rosli, Nur Ratasha Alia Md.; Mohamed, Faizal; Heng, Cheong Kai; Rahman, Irman Abdul; Ahmad, Ainee Fatimah; Mohamad, Hur Munawar Kabir

2014-09-01

Cancer cells which are large in size are resistant towards radiation therapy due to the presence of large amount of anti-oxidative enzymes and hypoxic cancer cells. Thus radiosensitizer agents have been developed to enhance the therapeutic effect of radiotherapy by increasing the sensitivity of these cancer cells towards radiation. This study is conducted to investigate the radiosensitization properties of radiosensitizer complex containing hydrogen peroxide and sodium hyaluronate. Combination with sodium hyaluronate may decrease reactivity of hydrogen peroxide but maintain the oxygen concentration needed for radiosensitizing effect. HepG2 cancer cells are cultured as the mean of test subject. Cancer cell samples which are targeted and not targeted with these radiosensitizers are irradiated with 2Gy single fractionated dose. Results obtained shows that the cancer cells which are not targeted with radiosensitizers has a cell viability of 98.80±0.37% after a time interval of 48 hours and has even repopulated over 100% after a 72 hour time interval. This shows that the cancer cells are resistant towards radiation. However, when the cancer cells are targeted with radiosensitizers prior to irradiation, there is a reduction of cell viability by 25.50±10.81% and 10.30±5.10% at time intervals of 48 and 72 hours respectively. This indicates that through the use of these radiosensitizers, cancer cells are more sensitive towards radiation.

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes.

PubMed

Mendoza, Pilar; Martínez-Martín, Nuria; Bovolenta, Elena R; Reyes-Garau, Diana; Hernansanz-Agustín, Pablo; Delgado, Pilar; Diaz-Muñoz, Manuel D; Oeste, Clara L; Fernández-Pisonero, Isabel; Castellano, Ester; Martínez-Ruiz, Antonio; Alonso-Lopez, Diego; Santos, Eugenio; Bustelo, Xosé R; Kurosaki, Tomohiro; Alarcón, Balbino

2018-05-29

Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell-intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Differentiated State of Initiating Tumor Cells Is Key to Distinctive Immune Responses Seen in H-RasG12V-Induced Squamous Tumors.

PubMed

Podolsky, Michael A; Bailey, Jacob T; Gunderson, Andrew J; Oakes, Carrie J; Breech, Kyle; Glick, Adam B

2017-03-01

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-Ras G12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1 -/- background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1 +/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1 +/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198-210. ©2017 AACR . ©2017 American Association for Cancer Research.

Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

PubMed Central

Benanti, Jennifer A.; Galloway, Denise A.

2004-01-01

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated β-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS. PMID:15024073

Dragging ras back in the ring.

PubMed

Stephen, Andrew G; Esposito, Dominic; Bagni, Rachel K; McCormick, Frank

2014-03-17

Ras proteins play a major role in human cancers but have not yielded to therapeutic attack. Ras-driven cancers are among the most difficult to treat and often excluded from therapies. The Ras proteins have been termed "undruggable," based on failures from an era in which understanding of signaling transduction, feedback loops, redundancy, tumor heterogeneity, and Ras' oncogenic role was poor. Structures of Ras oncoproteins bound to their effectors or regulators are unsolved, and it is unknown precisely how Ras proteins activate their downstream targets. These knowledge gaps have impaired development of therapeutic strategies. A better understanding of Ras biology and biochemistry, coupled with new ways of targeting undruggable proteins, is likely to lead to new ways of defeating Ras-driven cancers. Copyright © 2014 Elsevier Inc. All rights reserved.

RAS - Screens & Assays

Cancer.gov

A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

Synthesis and radiosensitization properties of hydrogen peroxide and sodium hyaluronate complex

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

Rosli, Nur Ratasha Alia Md.; Mohamed, Faizal; Heng, Cheong Kai

2014-09-03

Cancer cells which are large in size are resistant towards radiation therapy due to the presence of large amount of anti-oxidative enzymes and hypoxic cancer cells. Thus radiosensitizer agents have been developed to enhance the therapeutic effect of radiotherapy by increasing the sensitivity of these cancer cells towards radiation. This study is conducted to investigate the radiosensitization properties of radiosensitizer complex containing hydrogen peroxide and sodium hyaluronate. Combination with sodium hyaluronate may decrease reactivity of hydrogen peroxide but maintain the oxygen concentration needed for radiosensitizing effect. HepG2 cancer cells are cultured as the mean of test subject. Cancer cell samplesmore » which are targeted and not targeted with these radiosensitizers are irradiated with 2Gy single fractionated dose. Results obtained shows that the cancer cells which are not targeted with radiosensitizers has a cell viability of 98.80±0.37% after a time interval of 48 hours and has even repopulated over 100% after a 72 hour time interval. This shows that the cancer cells are resistant towards radiation. However, when the cancer cells are targeted with radiosensitizers prior to irradiation, there is a reduction of cell viability by 25.50±10.81% and 10.30±5.10% at time intervals of 48 and 72 hours respectively. This indicates that through the use of these radiosensitizers, cancer cells are more sensitive towards radiation.« less

MEK-1 Activates C-Raf Through a Ras-Independent Mechanism

PubMed Central

Leicht, Deborah T.; Balan, Vitaly; Zhu, Jun; Kaplun, Alexander; Bronisz, Agnieszka; Rana, Ajay; Tzivion, Guri

2013-01-01

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the downregulation of RKIP and MST2. PMID:23360980

Radiosensitivity study and radiation effects on morphology characterization of grey oyster mushroom Pleurotus sajor-caju

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

Rashid, Rosnani Abdul; Awang, Mat Rasol; Mohamad, Azhar

2014-09-03

Radiosensitive dosage and morphology characterization of irradiated grey oyster mushroom Pleurotus sajor-caju by gamma rays was investigated due to effects of irradiation. In order to establish the effect, mycelium of P. sajor-caju was irradiated by gamma rays at dose 0.1 to 8.0 kGy with dose rate 0.227 Gy sec{sup −1}. The irradiation of mycelia was carried out at the radiation facility in Malaysian Nuclear Agency. The radiosensitivity study was performed by evaluating the percentage of survival irradiated mycelia. The lethal dose of the mycelium P. sajor-caju was determined at 4.0 kGy and LD{sub 50} to be equal at 2.2 kGy.more » The radiation effects on morphology were evaluated based on growth rate of irradiated mycelia, mycelia types, colonization period on substrate, morphology of fruit bodies and yields. The results shown growth rate of irradiated mycelium was slightly lower than the control and decreased as the dose increased. Irradiation was found can induced the primordia formation on PDA and the BE of irradiated seed is higher than to control. The irradiation is proven to be useful for generating new varieties of mushroom with commercial value to the industry.« less

TU-F-CAMPUS-T-03: Enhancing the Tumor Specific Radiosensitization Using Molecular Targeted Gold Nanorods

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

Diagaradjane, P; Deorukhkar, A; Sankaranarayanapillai, M

2015-06-15

Purpose: Gold nanoparticle (GNP) mediated radiosensitization has gained significant attention in recent years. However, the widely used passive targeting strategy requires high concentration of GNPs to induce the desired therapeutic effect, thus dampening the enthusiasm for clinical translation. The purpose of this study is to utilize a molecular targeting strategy to minimize the concentration of GNPs injected while simultaneously enhancing the tumor specific radiosensitization for an improved therapeutic outcome. Methods: Cetuximab (antibody specific to the epidermal growth factor receptor that is over-expressed in tumors) conjugated gold nanorods (cGNRs) was used for the tumor targeting. The binding affinity, internalization, and inmore » vitro radiosensitization were evaluated using dark field microscopy, transmission electron microscopy, and clonogenic cell survival assay, respectively. In vivo biodistribution in tumor (HCT116-colorectal cancer cells) bearing mice were quantified using inductively coupled plasma mass spectrometry. In vivo radiosensitization potential was tested using 250-kVp x-rays and clinically relevant 6-MV radiation beams. Results: cGNRs displayed excellent cell-surface binding and internalization (∼31,000 vs 12,000/cell) when compared to unconjugated GNRs (pGNRs). In vitro, the dose enhancement factor at 10% survival (DEF10) was estimated as 1.06 and 1.17, respectively for both 250-kVp and 6-MV beams. In vivo biodistribution analysis revealed enhanced uptake of cGNRs in tumor (1.3 µg/g of tumor tissue), which is ∼1000-fold less than the reported values using passive targeting strategy. Nonetheless, significant radiosensitization was observed in vivo with cGNRs when compared to pGNRs, when irradiated with 250-kVp (tumor volume doubling time 35 days vs 25 days; p=0.002) and 6 MV (17 days vs 13 days; p=0.0052) beams. Conclusion: The enhanced radiosensitization effect observed with very low intratumoral concentrations of gold and

Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

NASA Technical Reports Server (NTRS)

Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

2000-01-01

The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

PubMed

Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S; Janssen, Axel B; White, Katharine A; Amini, Mojtaba; Barber, Diane L; Kuriyan, John; Roose, Jeroen P

2017-09-27

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles.

PubMed

Chu, Sheng-Hua; Karri, Surya; Ma, Yan-Bin; Feng, Dong-Fu; Li, Zhi-Qiang

2013-07-01

Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs. In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model. Nano-HAPs enhanced each cell line's radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation. These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe.

[Changes in cellular radiosensitivity after low dose irradiation].

PubMed

Pelevina, I I; Aleshchenko, A V; Antoshchina, M M; Kudriashova, O V; Riabchenko, N I; Akleev, A V

2012-01-01

When the adaptive response (AR) was studied on human blood lymphocytes, a new dependence was discovered. This dependence defines the direction of the radiosensitivity change after a low dose of irradiation. Using micronucleus (MN) test with cytochalasin B the dependence between the cell reaction after low level irradiation and radiosensititvity (the effect after irradiation at the dose of 1 Gy) was observed. The negative correlation between the frequency of AR manifestation, sensibilization, intermediate links and radiosensitivity was discovered. This regularity is observed in the population of Moscow, Obninsk, Chelyabinsk region (irradiated and control) inhabitants, Chernobyl accident liquidators, Moscow children, in individuals with Hodgkin's lymphoma before and during treatment. The negative correlation is also noted by AR determination with two irradiation schemes: in one or two different cell cycle phases (G1-G1 or G1-G2). Similar links are observed using the chromosome methaphase analysis (the frequency of cells with chromosome aberrations). So, the results of the experiments conducted allow us to suppose that the connection between the cell radiosensitivity and a different type of reaction after low dose irradiation--from AR to the increase in radiosensitivity (sensibilization) is a general regularity. AR is induced by low level irradiation and high cell radiosensitivity, while sensibilization is induced by low radiosensitivity. Since AR and sensibilization can be induced not only by irradiation, but many different chemicals and physical agents, the described correlation can be observed in the case of different exposures. Cellular AR and sensibilization are integral indexes depending on many genetic and epigenetic factors, as well as on the initiation of a large number of events. However, the discovered mechanisms of interrelations are still difficult to explain.

Differentiation and radiosensitivity of hemopoietic stem cells of mice during hypokinesia

NASA Technical Reports Server (NTRS)

Shvets, V. N.

1980-01-01

The potential for differentiation and radiosensitivity of the stem hemopoietic cells (KOE) under conditions of initial and later hypokinesia is examined. It is established that in the initial period of hypokinesia (3 days) when a stress reaction prevails, changes occur in the erythroid differentiation and radiosensitivity of KOE. This effect is associated with redistribution of T-lymphocytes that increase in number in the bone marrow of mice during hypokinesia. At later periods of hypokinesia (30 days) when changes in the organism are related to hypokinesia proper, differentiation and radiosensitivity of KOE were normalized.

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice

PubMed Central

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-01-01

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR–Ras–Raf–MEK–ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [3H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras–MAPK activity could be important in its anticancer activity. PMID:24853419

Repurposing cephalosporin antibiotics as pro-senescent radiosensitizers.

PubMed

Labay, Edwardine; Mauceri, Helena J; Efimova, Elena V; Flor, Amy C; Sutton, Harold G; Kron, Stephen J; Weichselbaum, Ralph R

2016-06-07

Radiation therapy remains a significant therapeutic modality in the treatment of cancer. An attractive strategy would be to enhance the benefits of ionizing radiation (IR)with radiosensitizers. A high-content drug repurposing screen of approved and investigational agents, natural products and other small molecules has identified multiple candidates that blocked repair of IR damage in vitro. Here, we validated a subset of these hits in vitro and then examined effects on tumor growth after IR in a murine tumor model. Based on robust radiosensitization in vivo and other favorable properties of cephalexin, we conducted additional studies with other beta-lactam antibiotics. When combined with IR, each cephalosporin tested increased DNA damage and slowed tumor growth without affecting normal tissue toxicity. Our data implicate reactive oxygen species in the mechanism by which cephalosporins augment the effects of IR. This work provides a rationale for using commonly prescribed beta-lactam antibiotics as non-toxic radiosensitizers to enhance the therapeutic ratio of radiotherapy.

Repurposing cephalosporin antibiotics as pro-senescent radiosensitizers

PubMed Central

Flor, Amy C.; Sutton, Harold G.; Kron, Stephen J.; Weichselbaum, Ralph R.

2016-01-01

Radiation therapy remains a significant therapeutic modality in the treatment of cancer. An attractive strategy would be to enhance the benefits of ionizing radiation (IR)with radiosensitizers. A high-content drug repurposing screen of approved and investigational agents, natural products and other small molecules has identified multiple candidates that blocked repair of IR damage in vitro. Here, we validated a subset of these hits in vitro and then examined effects on tumor growth after IR in a murine tumor model. Based on robust radiosensitization in vivo and other favorable properties of cephalexin, we conducted additional studies with other beta-lactam antibiotics. When combined with IR, each cephalosporin tested increased DNA damage and slowed tumor growth without affecting normal tissue toxicity. Our data implicate reactive oxygen species in the mechanism by which cephalosporins augment the effects of IR. This work provides a rationale for using commonly prescribed beta-lactam antibiotics as non-toxic radiosensitizers to enhance the therapeutic ratio of radiotherapy. PMID:27129153

Mutations and Deregulation of Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascades Which Alter Therapy Response

PubMed Central

McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Montalto, Giuseppe; Cervello, Melchiorre; Nicoletti, Ferdinando; Fagone, Paolo; Malaponte, Grazia; Mazzarino, Maria C.; Candido, Saverio; Libra, Massimo; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Cocco, Lucio; Evangelisti, Camilla; Chiarini, Francesca; Martelli, Alberto M.

2012-01-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Certain components of these pathways, RAS, NF1, BRAF, MEK1, DUSP5, PP2A, PIK3CA, PIK3R1, PIK3R4, PIK3R5, IRS4, AKT, NFKB1, MTOR, PTEN, TSC1, and TSC2 may also be activated/inactivated by mutations or epigenetic silencing. Upstream mutations in one signaling pathway or even in downstream components of the same pathway can alter the sensitivity of the cells to certain small molecule inhibitors. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of components of these cascades can contribute to: resistance to other pathway inhibitors, chemotherapeutic drug resistance, premature aging as well as other diseases. This review will first describe these pathways and discuss how genetic mutations and epigenetic alterations can result in resistance to various inhibitors. PMID:23006971

RasGRP1 opposes proliferative EGFR–SOS1–Ras signals and restricts intestinal epithelial cell growth

PubMed Central

Depeille, Philippe; Henricks, Linda M.; van de Ven, Robert A. H.; Lemmens, Ed; Wang, Chih-Yang; Matli, Mary; Werb, Zena; Haigis, Kevin M.; Donner, David; Warren, Robert; Roose, Jeroen P.

2015-01-01

The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR–SOS1–Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras–ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR–RasGRP1 and EGFR–SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma. PMID:26005835

Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

PubMed Central

Bunda, Severa; Burrell, Kelly; Heir, Pardeep; Zeng, Lifan; Alamsahebpour, Amir; Kano, Yoshihito; Raught, Brian; Zhang, Zhong-Yin; Zadeh, Gelareh; Ohh, Michael

2015-01-01

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously ‘undruggable' oncogenic or hyperactive Ras. PMID:26617336

Pleiotrophin mediates hematopoietic regeneration via activation of RAS

PubMed Central

Himburg, Heather A.; Yan, Xiao; Doan, Phuong L.; Quarmyne, Mamle; Micewicz, Eva; McBride, William; Chao, Nelson J.; Slamon, Dennis J.; Chute, John P.

2014-01-01

Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation–mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation–induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner. PMID:25250571

Pleiotrophin mediates hematopoietic regeneration via activation of RAS.

PubMed

Himburg, Heather A; Yan, Xiao; Doan, Phuong L; Quarmyne, Mamle; Micewicz, Eva; McBride, William; Chao, Nelson J; Slamon, Dennis J; Chute, John P

2014-11-01

Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation-mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation-induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner.

In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles

PubMed Central

Chu, Sheng-Hua; Karri, Surya; Ma, Yan-Bin; Feng, Dong-Fu; Li, Zhi-Qiang

2013-01-01

Background Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs. Methods In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model. Results Nano-HAPs enhanced each cell line's radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation. Conclusions These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe. PMID:23519742

Suppression of autophagy augments the radiosensitizing effects of STAT3 inhibition on human glioma cells

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

Yuan, Xiaopeng; Du, Jie; Hua, Song

Radiotherapy is an essential component of the standard therapy for newly diagnosed glioblastoma. To increase the radiosensitivity of glioma cells is a feasible solution to improve the therapeutic effects. It has been suggested that inhibition of signal transducer and activator of transcription 3 (STAT3) can radiosensitize glioma cells, probably via the activation of mitochondrial apoptotic pathway. In this study, human malignant glioma cells, U251 and A172, were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The radiosensitizing effects of STAT3 inhibition were confirmed in glioma cells. Intriguingly,more » combination of ionizing radiation exposure and STAT3 inhibition triggered a pronounced increase of autophagy flux. To explore the role of autophagy, glioma cells were treated with 3-methyladenine or siRNA for autophagy-related gene 5, and it was demonstrated that inhibition of autophagy further strengthened the radiosensitizing effects of STAT3 inhibition. Accordingly, more apoptotic cells were induced by the dual inhibition of autophagy and STAT3 signaling. In conclusion, our data revealed a protective role of autophagy in the radiosensitizing effects of STAT3 inhibition, and inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells. - Highlights: • Inactivation of STAT3 signaling radiosensitizes malignant glioma cells. • STAT3 inhibition triggers a significant increase of autophagy flux induced by ionizing radiation in glioma cells. • Suppression of autophagy further strengthens the radiosensitizing effects of STAT3 inhibition in glioma cells. • Dual inhibition of autophagy and STAT3 induce massive apoptotic cells upon exposure to ionizing radiation.« less

Ras-related tumorigenesis is suppressed by BNIP3-mediated autophagy through inhibition of cell proliferation.

PubMed

Wu, Shan-Ying; Lan, Sheng-Hui; Cheng, Da-En; Chen, Wei-Kai; Shen, Cheng-Huang; Lee, Ying-Ray; Zuchini, Roberto; Liu, Hsiao-Sheng

2011-12-01

Autophagy plays diverse roles in Ras-related tumorigenesis. H-ras(val12) induces autophagy through multiple signaling pathways including Raf-1/ERK pathway, and various ERK downstream molecules of autophagy have been reported. In this study, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) is identified as a downstream transducer of the Ras/Raf/ERK signaling pathway to induce autophagy. BNIP3 was upregulated by H-ras(val12) at the transcriptional level to compete with Beclin 1 for binding with Bcl-2. H-ras(val12)-induced autophagy suppresses cell proliferation demonstrated both in vitro and in vivo by expression of ectopic BNIP3, Atg5, or interference RNA of BNIP3 (siBNIP3) and Atg5 (shAtg5) using mouse NIH3T3 and embryo fibroblast cells. H-ras(val12) induces different autophagic responses depending on the duration of Ras overexpression. After a short time (48 hours) of Ras overexpression, autophagy inhibits cell proliferation. In contrast, a longer time (2 weeks) of Ras overexpression, cell proliferation was enhanced by autophagy. Furthermore, overexpression of mutant Ras, BNIP3, and LC3-II was detected in bladder cancer T24 cells and the tumor parts of 75% of bladder cancer specimens indicating a positive correlation between autophagy and tumorigenesis. Taken together, our mouse model demonstrates a balance between BNIP3-mediated autophagy and H-ras(val12)-induced tumor formation and reveals that H-ras(val12) induces autophagy in a BNIP3-dependent manner, and the threshold of autophagy plays a decisive role in H-ras(val12)-induced tumorigenesis. Our findings combined with others' reports suggest a new therapeutic strategy against Ras-related tumorigenesis by negative or positive regulation of autophagic activity, which is determined by the level of autophagy and tumor progression stages.

Reduced signaling of PI3K-Akt and RAS-MAPK pathways are the key targets for weight loss-induced cancer prevention by dietary calorie restriction and/or physical activity

PubMed Central

Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

2014-01-01

Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics, and lipidomics were employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into 4 groups for 10 weeks: ad lib-fed sedentary control, ad lib-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE), and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics, and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67, and 110 genes were significantly changed in DCR, PE, and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including upregulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears the reduction of both Ras-MAPK and PI3K-Akt signaling pathways are cancer preventive targets that have been consistently demonstrated by three bioinformatics approaches. PMID:25283328

About the RAS Initiative

Cancer.gov

The RAS Initiative, a "hub and spoke" model, connects researchers to better understand and target the more than 30% of cancers driven by mutations in RAS genes. Includes oversight and contact information.

Inhibiting DNA-PK{sub CS} radiosensitizes human osteosarcoma cells

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

Mamo, Tewodros; Mladek, Ann C.; Shogren, Kris L.

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PK{sub CS}), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PK{sub CS} in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PK{sub CS} inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PK{submore » CS} was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PK{sub CS} inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. - Highlights: • DNA-PKcs is consistently expressed in human osteosarcoma tissue and cell lines. • The DNA-PKcs inhibitor, KU60648, effectively radiosensitizes osteosarcoma cells. • Combining KU60648 with radiation increases G2/M accumulation and DNA damage.« less

Drugging the undruggable Ras: mission possible?

PubMed Central

Cox, Adrienne D.; Fesik, Stephen W.; Kimmelman, Alec C.; Luo, Ji; Der, Channing J.

2015-01-01

Despite more than three decades of intensive effort, no effective pharmacologic inhibitors of the Ras oncoproteins have reached the clinic, prompting the widely held perception that Ras proteins are “undruggable”. However, there is renewed hope that this is not the case. In this review, we summarize the progress and promise of five key directions. First, we focus on the prospects of direct inhibitors of Ras. Second, we revisit the issue of whether blocking Ras membrane association is a viable approach. Third, we assess the status of targeting Ras downstream effector signalling, arguably the most favourable current direction. Fourth, we address whether the search for synthetic lethal interactors of mutant RAS still holds promise. Finally, Ras-mediated changes in cell metabolism have recently been described. Can these changes be exploited for new therapeutic directions? We conclude with perspectives on how additional complexities, not yet fully understood, may impact each of these approaches. PMID:25323927

RAS one-equation turbulence model with non-singular eddy-viscosity coefficient

NASA Astrophysics Data System (ADS)

Rahman, M. M.; Agarwal, R. K.; Siikonen, T.

2016-02-01

A simplified consistency formulation for Pk/ε (production to dissipation ratio) is devised to obtain a non-singular Cμ (coefficient of eddy-viscosity) in the explicit algebraic Reynolds stress model of Gatski and Speziale. The coefficient Cμ depends non-linearly on both rotational/irrotational strains and is used in the framework of an improved RAS (Rahman-Agarwal-Siikonen) one-equation turbulence model to calculate a few well-documented turbulent flows, yielding predictions in good agreement with the direct numerical simulation and experimental data. The strain-dependent Cμ assists the RAS model in constructing the coefficients and functions such as to benefit complex flows with non-equilibrium turbulence. Comparisons with the Spalart-Allmaras one-equation model and the shear stress transport k-ω model demonstrate that Cμ improves the response of RAS model to non-equilibrium effects.

WE-G-BRE-08: Radiosensitization by Olaparib Eluting Nanospheres

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

Tangutoori, S; Kumar, R; Sridhar, S

2014-06-15

Purpose: Permanent prostate brachytherapy often uses inert bio-absorbable spacers to achieve the desired geometric distribution of sources within the prostate. Transforming these spacers into implantable nanoplatforms for chemo-radiation therapy (INCeRT) provides a means of providing sustained in-situ release of radiosensitizers in the prostate to enhance the therapeutic ratio of the procedure. Olaparib, a PARP inhibitor, suppresses DNA repair processes present during low dose rate continuous irradiation. This work investigates the radiosensitizing/DNA damage repair inhibition by NanoOlaparib eluting nanospheres. Methods: Human cell line PC3 (from ATCC), was maintained in F12-k medium supplemented with fetal bovine serum. Clonogenic assay kit (from Fischermore » Scientific) was used to fix and stain the cells to determine the long term effects of irradiation. Nanoparticle size and zeta potential of nanospheres were determined using a Zeta particle size analyzer. The incorporation of Olaparib in nanospheres was evaluated by HPLC. Irradiation was performed in a small animal irradiator operating at 220 KeV.The long term effects of radio-sensitization with olaparib and nanoolaparib was determined using the clonogenic assay at 2 Gy and 4 Gy doses. The cells were allowed to grow for around 10 doubling cycles, The colonies were fixed and stained using clonogenic assay kit. The excess stain was washed off using DI water and the images were taken using a digital camera. Results: Radiosensitization studies were carried out in prostate cancer cell line, PC3 radiation at 0, 2 and 4Gy doses. Strongest dose response was observed with nanoolaparib treated cells compared to untreated cells. Conclusion: A two stage drug release of drug eluting nanospheres from a biodegradable spacer has been suggested for sustained in-situ release of Olaparib to suppress DNA repair processes during prostate brachytherapy. The Olaparib eluting nanospheres had the same in-vitro radiosensitizing

Ras plasma membrane signalling platforms

PubMed Central

2005-01-01

The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction. PMID:15954863

Glutamate increases pancreatic cancer cell invasion and migration via AMPA receptor activation and Kras-MAPK signaling.

PubMed

Herner, Alexander; Sauliunaite, Danguole; Michalski, Christoph W; Erkan, Mert; De Oliveira, Tiago; Abiatari, Ivane; Kong, Bo; Esposito, Irene; Friess, Helmut; Kleeff, Jörg

2011-11-15

Glutamate has been implicated in tumorigenesis through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR). However, the function of a glutamate-to-AMPAR signal in pancreatic ductal adenocarcinoma (PDAC) has remained elusive. We now show that glutamate-mediated AMPA receptor activation increases invasion and migration of pancreatic cancer cells via activation of the classical MAPK pathway. Glutamate levels were increased in pancreatic cancer accompanied by downregulation of GluR subunits 1, 2, and 4. In pancreatic cancer precursor lesions, pancreatic intraepithelial neoplasia (PanIN), GluR1 subunit levels were strikingly and step-wise increased but its expression was rare in PDAC. Pharmacological inhibition or RNAi-mediated suppression of GluR1 or GluR2 did not affect cancer cell growth but significantly decreased invasion. In a K-ras wildtype cell line, AMPA receptor activation enhanced K-ras activity and--further downstream--phosphorylation of p38 and of p44/42. Preemptive blockade of AMPA receptors in a mouse model of pancreatic cancer inhibited tumor cell settling. AMPA receptor activation thus not only activates MAPK signalling but also directly increases activity of K-ras. Glutamate might serve as a molecular switch that decreases the threshold of K-ras-induced oncogenic signalling and increases the chance of malignant transformation of pancreatic cancer precursor lesions. Copyright © 2011 UICC.

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery.

PubMed

Buckel, Lisa; Savariar, Elamprakash N; Crisp, Jessica L; Jones, Karra A; Hicks, Angel M; Scanderbeg, Daniel J; Nguyen, Quyen T; Sicklick, Jason K; Lowy, Andrew M; Tsien, Roger Y; Advani, Sunil J

2015-04-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. ©2015 American Association for Cancer Research.

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery

PubMed Central

Crisp, Jessica L.; Jones, Karra A.; Hicks, Angel M.; Scanderbeg, Daniel J.; Nguyen, Quyen T.; Sicklick, Jason K.; Lowy, Andrew M.; Tsien, Roger Y.; Advani, Sunil J.

2015-01-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell penetrating peptide targeting matrix metalloproteinases and RGD binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule and dose dependent manner correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with non-targeted free MMAE or tumor targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell penetrating peptides. PMID:25681274

Ras proteins have multiple functions in vegetative cells of Dictyostelium.

PubMed

Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

2010-11-01

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

PubMed Central

Kriegs, Malte; Kasten-Pisula, Ulla; Riepen, Britta; Hoffer, Konstantin; Struve, Nina; Myllynen, Laura; Braig, Friederike; Binder, Mascha; Rieckmann, Thorsten; Grénman, Reidar; Petersen, Cordula; Dikomey, Ekkehard; Rothkamm, Kai

2016-01-01

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells. PMID:27281611

Reduced signaling of PI3K-Akt and RAS-MAPK pathways is the key target for weight-loss-induced cancer prevention by dietary calorie restriction and/or physical activity.

PubMed

Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

2014-12-01

Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches. Copyright © 2014 Elsevier Inc. All rights reserved.

Epac activation sensitizes rat sensory neurons through activation of Ras.

PubMed

Shariati, Behzad; Thompson, Eric L; Nicol, Grant D; Vasko, Michael R

2016-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. Copyright © 2015 Elsevier Inc. All rights reserved.

Epac activation sensitizes rat sensory neurons via activation of Ras

PubMed Central

Shariati, Behzad; Thompson, Eric L.; Nicol, Grant D.; Vasko, Michael R.

2015-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2′-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. PMID:26596174

Fibroblast growth factor 2 restrains Ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence.

PubMed

Costa, Erico T; Forti, Fábio L; Matos, Tatiana G F; Dermargos, Alexandre; Nakano, Fábio; Salotti, Jacqueline; Rocha, Kátia M; Asprino, Paula F; Yoshihara, Celina K; Koga, Marianna M; Armelin, Hugo A

2008-08-01

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

Superiority of Low Energy 160 KV X-Rays Compared to High Energy 6 MV X-Rays in Heavy Element Radiosensitization for Cancer Treatment

NASA Astrophysics Data System (ADS)

Lim, Sara N.; Pradhan, Anil K.; Nahar, Sultana N.; Barth, Rolf F.; Yang, Weilian; Nakkula, Robin J.; Palmer, Alycia; Turro, Claudia

2013-06-01

High energy X-rays in the MeV range are generally employed in conventional radiation therapy from linear accelerators (LINAC) to ensure sufficient penetration depths. However, lower energy X-rays in the keV range may be more effective when coupled with heavy element (high-Z or HZ) radiosensitizers. Numerical simulations of X-ray energy deposition for tumor phantoms sensitized with HZ radiosensitizers were performed using the Monte Carlo code Geant4. The results showed enhancement in energy deposition to radiosensitized phantoms relative to unsensitized phantoms for low energy X-rays in the keV range. In contrast, minimal enhancement was seen using high energy X-rays in the MeV range. Dose enhancement factors (DEFs) were computed and showed radiosensitization only in the low energy range < 200 keV, far lower than the energy of the majority of photons in the LINAC energy range. In vitro studies were carried to demonstrate the tumoricidal effects of HZ sensitized F98 rat glioma cells following irradiation with both low energy 160 kV and high energy 6 MV X-ray sources. The platinum compound, pyridine terpyridine Pt(II) nitrate, was initially used because it was 7x less toxic that an equivalent amount of carboplatin in vitro studies. This would allow us to separate the radiotoxic and the chemotoxic effects of HZ sensitizers. Results from this study showed a 10-fold dose dependent reduction in surviving fractions (SF) of radiosensitized cells treated with low energy 160 kV X-rays compared to those treated with 6 MV X-rays. This is in agreement with our simulations that show an increase in dose deposition in radiosensitized tumors for low energy X-rays. Due to unforeen in vivo toxicity, however, another in vitro study was performed using the commonly used, Pt-based chemotherapeutic drug carboplatin which confirmed earlier results. This lays the ground work for a planned in vivo study using F98 glioma bearing rats. This study demonstrates that while high energy X-rays are

Roadmap to clinical use of gold nanoparticles for radiosensitization

PubMed Central

Schuemann, J.; Berbeco, R.; Chithrani, B. D.; Cho, S.; Kumar, R.; McMahon, S.; Sridhar, S.; Krishnan, S.

2015-01-01

The past decade has seen a dramatic increase in interest in the use of Gold Nanoparticles (GNPs) as radiation sensitizers for radiotherapy. This interest was initially driven by their strong absorption of ionizing radiation and the resulting ability to increase dose deposited within target volumes even at relatively low concentrations. These early observations are supported by extensive experimental validation, showing GNPs’ efficacy at sensitizing tumors in both in vitro and in vivo systems to a range of types of ionizing radiation, including kilovoltage and megavoltage X-rays as well as charged particles. Despite this experimental validation, there has been limited translation of GNP-mediated radiosensitization to a clinical setting. One of the key challenges in this area is the wide range of experimental systems that have been investigated, spanning a range of particle sizes, shapes and preparations. As a result, mechanisms of uptake and radiosensitization have remained difficult to clearly identify. This has proven a significant impediment to the identification of optimal GNP formulations which strike a balance among their radiosensitizing properties, their specificity to the tumors, their biocompatibility, and their imageability in vivo. This white paper reviews the current state of knowledge in each of the areas concerning the use of GNPs as radiosensitizers, and outlines the steps which will be required to advance GNP-enhanced radiation therapy from their current pre-clinical setting to clinical trials and eventual routine usage. PMID:26700713

Trabecular meshwork ECM remodeling in glaucoma: could RAS be a target?

PubMed

Agarwal, Puneet; Agarwal, Renu

2018-06-14

Disturbances of extracellular matrix (ECM) homeostasis in trabecular meshwork (TM) cause increased aqueous outflow resistance leading to elevated intraocular pressure (IOP) in glaucomatous eyes. Therefore, restoration of ECM homeostasis is a rational approach to prevent disease progression. Since renin-angiotensin system (RAS) inhibition positively alters ECM homeostasis in cardiovascular pathologies involving pressure and volume overload, it is likely that RAS inhibitors reduce IOP primarily by restoring ECM homeostasis. Areas covered: Current evidence showing the presence of RAS components in ocular tissue and its role in regulating aqueous humor dynamics is briefly summarized. The role of RAS in ECM remodeling is discussed both in terms of its effects on ECM synthesis and its breakdown. The mechanisms of ECM remodeling involving interactions of RAS with transforming growth factor-β, Wnt/β-catenin signaling, bone morphogenic proteins, connective tissue growth factor, and matrix metalloproteinases in ocular tissue are discussed. Expert opinion: Current literature strongly indicates a significant role of RAS in ECM remodeling in TM of hypertensive eyes. Hence, IOP-lowering effect of RAS inhibitors may primarily be attributed to restoration of ECM homeostasis in aqueous outflow pathways rather than its vascular effects. However, the mechanistic targets for RAS inhibitors have much wider distribution and consequences, which remain relatively unexplored in TM.

Mechanism of radiosensitization by porphyrins.

PubMed

Luksiene, Zivile; Labeikyte, Danute; Juodka, Benediktas; Moan, Johan

2006-01-01

According to our previous data, hematoporphyrin dimethyl ether (HPde) at concentrations useful for photodynamic therapy can radiosensitize aggressive Ehrlich ascite carcinoma (EAT) to 2Gy irradiation inducing total tumour growth inhibition. The aim of this study was to further investigate the possible mechanism of radiosensitization of EAT by dicarboxylic porphyrin-HPde. Our results reveal that HPde is inducing several rearrangements in the EAT cells: 1.2 x 10-6 M of the photosensitizer diminishes the number of cells in mitosis by a factor of 3, increases the number of cells in the S phase of the cell cycle, modifies the activities of antioxidant enzymes glutation S-transferase (GST) and DT-diaphorase (DTD), and eventually induces slight apoptosis. Moreover, it was shown that HPde is a ligand of peripheral benzodiazepine receptor (PBR). Named "house keeper," PBR is usually responsible for all these perturbations, which, in our case, act in concert with the following ionizing radiation, producing the interaction of two antiproliferative/destructive factors.

Ovarian expression of cellular Ki-ras p21 varies with physiological status.

PubMed Central

Palejwala, S; Goldsmith, L T

1992-01-01

To elucidate the potential role of the ras protooncogene proteins in a specific tissue, the present study determined the levels of individual c-ras-encoded p21 proteins in the rat ovary during various stages of physiological function. p21 protein was extracted from ovaries taken from immature normal female rats, mature nonpregnant animals in the metestrus stage of the estrus cycle, rats at various stages of pregnancy, and actively lactating animals. Levels of individual p21s were evaluated by immunoblot analysis with specific antibodies to the p21 proteins encoded by the Kirsten, Harvey, and neuroblastoma c-ras protooncogenes, c-Ki-ras, c-Ha-ras, and N-ras. Results showed that c-Ki-ras p21 is at its lowest level in the immature ovary and increases with development of the corpora lutea to its highest levels at day 16 of pregnancy, after which levels decline and then rise again during lactation. This pattern, which mimics that of circulating progesterone levels, suggests that ovarian c-Ki-ras p21 levels are regulated and that c-Ki-ras p21 plays a role in the differentiated function of the rat ovary, likely the luteal compartment. In contrast, levels of c-N-ras p21 did not appear to vary with changes in the physiological function of the ovary but appeared to be constitutive. A preferential role for the c-Ki-ras p21 may be due to the documented unique differences in the structure of the carboxyl terminus of this particular c-ras p21. Images PMID:1570348

Radiosensitization: enhancing the radiation inactivation of foodborne bacteria

NASA Astrophysics Data System (ADS)

Borsa, J.; Lacroix, M.; Ouattara, B.; Chiasson, F.

2004-09-01

Irradiation of meat products to kill pathogens can be limited by radiation-induced detriment of sensory quality. Since such detriment is directly related to dose, one approach to reduce it is by devising means to lower the dose of radiation required for processing. Increasing the radiation sensitivity of the target microorganisms would lower the dose required for a given level of microbial kill. In this work, the radiation sensitivities of inoculated Escherichia coli and Salmonella typhi in ground beef were examined under a variety of conditions. Results showed that specific manipulations of treatment conditions significantly increased the radiation sensitivity of the test organisms, ranging from a few percent to several-fold reduction in D10. In particular, radiation sensitization could be effected by certain additives, including carvacrol, thymol and trans-cinnamaldehyde, and also by certain compositions of modified atmosphere in the package headspace. A combination of additives and modified atmosphere effected a greater radiosensitization effect than could be achieved by either factor applied alone. Radiosensitization could be demonstrated with irradiation of either fresh or frozen ground meat. The radiosensitization phenomenon may be of practical utility in enhancing the technical effectiveness and feasibility of irradiation of a variety of meat and other food products.

5-(Halomethyl)uridine derivatives as potential antitumor radiosensitizers: A DFT study

NASA Astrophysics Data System (ADS)

Wang, Shoushan; Zhang, Min; Liu, Peng; Xie, Shilei; Cheng, Faliang; Wang, Lishi

2018-01-01

Considering the fact that the efficiency of the uridine-5-methyl radical in producing cytotoxic DNA intrastrand cross-link lesions is greatly higher than that of the uridine-5-yl radical, the radiosensitizing action of 5-(halomethyl)uridines (5-XCH2U, X = F, Cl, or Br) is studied in the present work. It is found that 5-XCH2U has sufficient electron affinity to capture a pre-hydrated or a hydrated electron, and electron attachment leads to significantly facile X- elimination forming the uridine-5-methyl radical. All these three halogenated uridine derivatives are shown to be potential radiosensitizers, with their radiosensitizing abilities increased in an order 5-FCH2U < 5-ClCH2U ≈ 5-BrCH2U.

Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

PubMed

DeSmet, Marsha L; Fleet, James C

2017-10-01

High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of N-acetylglucosaminyltransferase V enhances the cetuximab-induced radiosensitivity of nasopharyngeal carcinoma cells likely through EGFR N-glycan alterations.

PubMed

Huang, Xiaomin; Liu, Ting; Wang, Qiongyao; Zhu, Weiliang; Meng, Hui; Guo, Linlang; Wei, Ting; Zhang, Jian

2017-05-23

N-acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyses the formation of the N-linked β-1-6 branching of oligosaccharides, is related to the radiosensitivity of nasopharyngeal carcinoma (NPC). Cetuximab (C225) is an epidermal growth factor receptor (EGFR) inhibitor used as a radiosensitizer in the treatment of NPC. In this study, we used GnT-V as a molecular target to further sensitize cetuximab-treated NPC cells to radiation. The results from two NPC cell lines (CNE1 and CNE2) revealed that the silencing of GnT-V enhanced cetuximab-induced radiosensitivity by decreasing the β-1-6 branching of oligosaccharides on the EGFR. GnT-V down-regulation combined with cetuximab decreased the survival fraction, healing rate and cell viability and increased the apoptosis rate. Concomitantly, the combination of cetuximab and irradiation did not change the EGFR mRNA and protein levels and decreased the β-1-6 branching on the EGFR. Subsequently, we further explored the signalling downstream of EGF, particularly the PI3K/Akt signalling pathway, and discovered that treatment consisting of GnT-V down-regulation, irradiation and cetuximab was negatively correlated with phospho-Akt and phspho-PI3K. Finally, an in vivo experiment with radiotherapy revealed that the combination of GnT-V down-regulation and cetuximab decelerated tumour growth. In summary, our study demonstrated that the combination of decreased GnT-V activity and cetuximab enhanced NPC radiosensitivity, and the possible mechanism underlying this effect might involve the N-linked β1-6 branching of the EGFR. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of low-level chronic irradiation on the radiosensitivity of mammals: Modeling studies

NASA Astrophysics Data System (ADS)

Smirnova, O. A.

Mathematical models of the major hematopoietic lines are used to study the modifying effects of low-level chronic preirradiation on radiosensitivity of mammals which resulted in their reduced radiosensitivity (acquired radioresistance) and elevated radiosensitivity (hypersensitivity) to the subsequent radiation exposure. These effects of preirradiation manifest themselves, respectively, in decreased and increased mortality of preirradiated experimental animals (mice) after challenge acute exposure in comparison with that for previously nonirradiated ones. Analysis of the modeling results reveals the biological mechanisms of these radioprotection and radiosensitization effects, and enables one to estimate the ranges of dose rate and duration of chronic preirradiation where these effects are realized. Juxtapositions of the modeling predictions with the relevant experimental data show their qualitative agreement. All this testifies to the importance of accounting the nonlinear effect of low-level chronic irradiation on radiosensitivity of the hematopoiesis system and organism as a whole, when the radiation risk for astronauts on long-term space missions is estimated. The developed models of hematopoiesis can be used, after appropriate identification, as a component of the mathematical tools for radiation risk assessment.

w09, a novel autophagy enhancer, induces autophagy-dependent cell apoptosis via activation of the EGFR-mediated RAS-RAF1-MAP2K-MAPK1/3 pathway.

PubMed

Zhang, Pinghu; Zheng, Zuguo; Ling, Li; Yang, Xiaohui; Zhang, Ni; Wang, Xue; Hu, Maozhi; Xia, Yu; Ma, Yiwen; Yang, Haoran; Wang, Yunyi; Liu, Hongqi

2017-07-03

The EGFR (epidermal growth factor receptor) signaling pathway is frequently deregulated in many malignancies. Therefore, targeting the EGFR pathway is regarded as a promising strategy for anticancer drug discovery. Herein, we identified a 2-amino-nicotinonitrile compound (w09) as a novel autophagy enhancer, which potently induced macroautophagy/autophagy and consequent apoptosis in gastric cancer cells. Mechanistic studies revealed that EGFR-mediated activation of the RAS-RAF1-MAP2K-MAPK1/3 signaling pathway played a critical role in w09-induced autophagy and apoptosis of gastric cancer cells. Inhibition of the MAPK1/3 pathway with U0126 or blockade of autophagy by specific chemical inhibitors markedly attenuated the effect of w09-mediated growth inhibition and caspase-dependent apoptosis. Furthermore, these conclusions were supported by knockdown of ATG5 or knockout of ATG5 and/or ATG7. Notably, w09 increased the expression of SQSTM1 by transcription, and knockout of SQSTM1 or deleting the LC3-interaction region domain of SQSTM1, significantly inhibited w09-induced PARP1 cleavage, suggesting the central role played by SQSTM1 in w09-induced apoptosis. In addition, in vivo administration of w09 effectively inhibited tumor growth of SGC-7901 xenografts. Hence, our findings not only suggested that activation of the EGFR-RAS-RAF1-MAP2K-MAPK1/3 signaling pathway may play a critical role in w09-induced autophagy and apoptosis, but also imply that induction of autophagic cancer cell death through activation of the EGFR pathway may be a potential therapeutic strategy for EGFR-disregulated gastric tumors.

Ras trafficking, localization and compartmentalized signalling

PubMed Central

Prior, Ian A.; Hancock, John F.

2012-01-01

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes. PMID:21924373

Cytogenetic, ras, and p53: studies in cases of canine neoplasms (hemangiopericytoma, mastocytoma, histiocytoma, chloroma).

PubMed

Mayr, B; Reifinger, M; Brem, G; Feil, C; Schleger, W

1999-01-01

Four case reports of mesenchymal neoplasms showing chromosomal abnormalities are presented. In a case of hemangiopericytoma trisomy 2 and centric fusion 19;21 were present. In a mastocytoma a deleted chromosome 35 was seen. A homogeneously staining region (HSR) on chromosome 1 was detected in a histiocytoma. Trisomy 5 and monosomy 31 were observed in a case of granulocytic sarcoma (chloroma). The lack of mutations in exons 1 and 2 of oncogenes N-ras, K-ras, and H-ras and exons 5, 6, 7, and 8 of tumor suppressor gene p53 in these four patients and in a larger series of investigated dogs (25 hemangiopericytomas, 12 mastocytomas, and 8 histiocytomas) is highlighted.

Exploring the interactions of the RAS family in the human protein network and their potential implications in RAS-directed therapies

PubMed Central

Bueno, Anibal; Morilla, Ian; Diez, Diego; Moya-Garcia, Aurelio A.; Lozano, José; Ranea, Juan A.G.

2016-01-01

RAS proteins are the founding members of the RAS superfamily of GTPases. They are involved in key signaling pathways regulating essential cellular functions such as cell growth and differentiation. As a result, their deregulation by inactivating mutations often results in aberrant cell proliferation and cancer. With the exception of the relatively well-known KRAS, HRAS and NRAS proteins, little is known about how the interactions of the other RAS human paralogs affect cancer evolution and response to treatment. In this study we performed a comprehensive analysis of the relationship between the phylogeny of RAS proteins and their location in the protein interaction network. This analysis was integrated with the structural analysis of conserved positions in available 3D structures of RAS complexes. Our results show that many RAS proteins with divergent sequences are found close together in the human interactome. We found specific conserved amino acid positions in this group that map to the binding sites of RAS with many of their signaling effectors, suggesting that these pairs could share interacting partners. These results underscore the potential relevance of cross-talking in the RAS signaling network, which should be taken into account when considering the inhibitory activity of drugs targeting specific RAS oncoproteins. This study broadens our understanding of the human RAS signaling network and stresses the importance of considering its potential cross-talk in future therapies. PMID:27713118

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3*

PubMed Central

Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

2015-01-01

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. PMID:26109071

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

PubMed

Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

2015-08-07

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

An orthosteric inhibitor of the RAS-SOS interaction.

PubMed

Nickerson, Seth; Joy, Stephen T; Arora, Paramjit S; Bar-Sagi, Dafna

2013-01-01

Rat sarcoma (RAS) proteins are signaling nodes that transduce extracellular cues into precise alterations in cellular physiology by engaging effector pathways. RAS signaling thus regulates diverse cell processes including proliferation, migration, differentiation, and survival. Owing to this central role in governing mitogenic signals, RAS pathway components are often dysregulated in human diseases. Targeted therapy of RAS pathways has generally not been successful, largely because of the robust biochemistry of the targets and their multifaceted network of molecular regulators. The rate-limiting step of RAS activation is Son of Sevenless (SOS)-mediated nucleotide exchange involving a single evolutionarily conserved catalytic helix from SOS. Structure function data of this mechanism provided a strong platform to design an SOS-derived, helically constrained peptide mimic as an inhibitor of the RAS-SOS interaction. In this chapter, we review RAS-SOS signaling dynamics and present evidence supporting the novel paradigm of inhibiting their interaction as a therapeutic strategy. We then describe a method of generating helically constrained peptide mimics of protein surfaces, which we have employed to inhibit the RAS-SOS active site interaction. The biochemical and functional properties of this SOS mimic support the premise that inhibition of RAS-nucleotide exchange can effectively block RAS activation and downstream signaling. © 2013 Elsevier Inc. All rights reserved.

RAS - Screens & Assays - Drug Discovery

Cancer.gov

The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

Multivalent small molecule pan-RAS inhibitors

PubMed Central

Welsch, Matthew E.; Kaplan, Anna; Chambers, Jennifer M.; Stokes, Michael E.; Bos, Pieter H.; Zask, Arie; Zhang, Yan; Sanchez-Martin, Marta; Badgley, Michael A.; Huang, Christine S.; Tran, Timothy H.; Akkiraju, Hemanth; Brown, Lewis M.; Nandakumar, Renu; Cremers, Serge; Yang, Wan S.; Tong, Liang; Olive, Kenneth P.; Ferrando, Adolfo; Stockwell, Brent R.

2017-01-01

SUMMARY Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, have potential use as chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers, and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins. PMID:28235199

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.

PubMed

Philips, M R; Pillinger, M H; Staud, R; Volker, C; Rosenfeld, M G; Weissmann, G; Stock, J B

1993-02-12

In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of β-catenin signaling.

PubMed

Shin, Wookjin; Lee, Sang-Kyu; Hwang, Jeong-Ha; Park, Jong-Chan; Cho, Yong-Hee; Ro, Eun Ji; Song, Yeonhwa; Seo, Haeng Ran; Choi, Kang-Yell

2018-06-06

Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/β-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both β-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegradable mutant form of β-catenin and the need to determine an alternative mechanism of Ras degradation, we designed a cell-based system to screen compounds that degrade Ras independent of the Wnt/β-catenin signaling pathway. A cell-based high-content screening (HCS) system that monitors the levels of EGFP-K-Ras G12V was established using HCT-116 cells harboring a nondegradable mutant CTNNB1 (ΔS45). Through HCS of a chemical library composed of 10,000 compounds and subsequent characterization of hits, we identified several compounds that degrade Ras without affecting the β-catenin levels. KY7749, one of the most effective compounds, inhibited the proliferation and transformation of CRC cells, especially KRAS-mutant cells that are resistant to the EGFR monoclonal antibody cetuximab. Small molecules that degrade Ras independent of β-catenin may able to be used in treatments for cancers caused by aberrant EGFR and Ras.

H-Ras Exerts Opposing Effects on Type I Interferon Responses Depending on Its Activation Status.

PubMed

Chen, Guann-An; Lin, Yun-Ru; Chung, Hai-Ting; Hwang, Lih-Hwa

2017-01-01

Using shRNA high-throughput screening, we identified H-Ras as a regulator of antiviral activity, whose depletion could enhance Sindbis virus replication. Further analyses indicated that depletion of H-Ras results in a robust increase in vesicular stomatitis virus infection and a decrease in Sendai virus (SeV)-induced retinoic acid-inducible gene-I-like receptor (RLR) signaling. Interestingly, however, ectopic expression of wild-type H-Ras results in a biphasic mode of RLR signaling regulation: while low-level expression of H-Ras enhances SeV-induced RLR signaling, high-level expression of H-Ras significantly inhibits this signaling. The inhibitory effects correlate with the activation status of H-Ras. As a result, oncogenic H-Ras, H-RasV12, strongly inhibits SeV-induced IFN-β promoter activity and type I interferon signaling. Conversely, the positive effects exerted by H-Ras on RLR signaling are independent of its signaling activity, as a constitutively inactive form of H-Ras, H-RasN17, also positively regulates RLR signaling. Mechanistically, we demonstrate that depletion of H-Ras reduces the formation of MAVS-TNF receptor-associated factor 3 signaling complexes. These results reveal that the H-Ras protein plays a role in promoting MAVS signalosome assembly in the mitochondria, whereas oncogenic H-Ras exerts a negative effect on type I IFN responses.

R-Ras contributes to LTP and contextual discrimination.

PubMed

Darcy, M J; Jin, S-X; Feig, L A

2014-09-26

The ability to discriminate between closely related contexts is a specific form of hippocampal-dependent learning that may be impaired in certain neurodegenerative disorders such as Alzheimer's and Down Syndrome. However, signaling pathways regulating this form of learning are poorly understood. Previous studies have shown that the calcium-dependent exchange factor Ras-GRF1, an activator of Rac, Ras and R-Ras GTPases, is important for this form of learning and memory. Moreover, the ability to discriminate contexts was linked to the ability of Ras-GRF1 to promote high-frequency stimulation long-term potentiation (HFS-LTP) via the activation of p38 Map kinase. Here, we show that R-Ras is involved in this form of learning by using virally-delivered miRNAs targeting R-Ras into the CA1 region of the dorsal hippocampus and observing impaired contextual discrimination. Like the loss of GRF1, knockdown of R-Ras in the CA1 also impairs the induction of HFS-LTP and p38 Map kinase. Nevertheless, experiments indicate that this involvement of R-Ras in HFS-LTP that is required for contextual discrimination is independent of Ras-GRF1. Thus, R-Ras is a novel regulator of a form of hippocampal-dependent LTP as well as learning and memory that is affected in certain forms of neurodegenerative diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

R-Ras Contributes to LTP and Contextual Discrimination

PubMed Central

Darcy, Michael J.; Jin, Shan-Xue; Feig, Larry A.

2014-01-01

The ability to discriminate between closely related contexts is a specific form of hippocampal-dependent learning that may be impaired in certain neurodegenerative disorders such as Alzheimer's and Down Syndrome. However, signaling pathways regulating this form of learning are poorly understood. Previous studies have shown that the calcium-dependent exchange factor Ras-GRF1, an activator of Rac, Ras and R-Ras GTPases, is important for this form of learning and memory. Moreover, the ability to discriminate contexts was linked to the ability of Ras-GRF1 to promote high-frequency stimulation (HFS)-LTP via the activation of p38 Map kinase. Here, we show that R-Ras is involved in this form of learning by using virally-delivered miRNAs targeting R-Ras into the CA1 region of dorsal hippocampus and observing impaired contextual discrimination. Like the loss of GRF1, knockdown of R-Ras in the CA1 also impairs the induction of HFS-LTP and p38 Map kinase. Nevertheless, experiments indicate that this involvement of R-Ras in HFS-LTP that is required for contextual discrimination is independent of Ras-GRF1. Thus, R-Ras is a novel regulator of a form of hippocampal-dependent LTP as well as learning and memory that is affected in certain forms of neurodegenerative diseases. PMID:25043327

RAS and sex differences in diabetic nephropathy.

PubMed

Clotet, Sergi; Riera, Marta; Pascual, Julio; Soler, Maria José

2016-03-09

The incidence and progression of kidney diseases are influenced by sex. The renin-angiotensin system (RAS) is an important regulator of cardiovascular and renal function. Sex differences in the renal response to RAS blockade have been demonstrated. Circulating and renal RAS has been shown to be altered in type 1 and type 2 diabetes; this enzymatic cascade plays a critical role in the development of diabetic nephropathy (DN). Angiotensin converting enzyme (ACE) and ACE2 are differentially regulated depending on its localization within the diabetic kidney. Furthermore, clinical and experimental studies have shown that circulating levels of sex hormones are clearly modulated in the context of diabetes, suggesting that sex-dependent RAS regulation may be also be affected in these individuals. The effect of sex hormones on circulating and renal RAS may be involved in the sex differences observed in DN progression. In this paper we will review the influence of sex hormones on RAS expression and its relation to diabetic kidney disease. A better understanding of the sex dimorphism on RAS might provide a new approach for diabetic kidney disease treatment. Copyright © 2015, American Journal of Physiology - Renal Physiology.

C/EBPbeta represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19(Arf).

PubMed

Ewing, S J; Zhu, S; Zhu, F; House, J S; Smart, R C

2008-11-01

CCAAT/enhancer-binding protein-beta (C/EBPbeta) is a mediator of cell survival and tumorigenesis. When C/EBPbeta(-/-) mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19(Arf) and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19(Arf) is dramatically elevated in C/EBPbeta(-/-) epidermis and that C/EBPbeta represses a p19(Arf) promoter reporter. To determine whether p19(Arf) is responsible for the proapoptotic phenotype in C/EBPbeta(-/-) mice, C/EBPbeta(-/-);p19(Arf-/-) mice were generated. C/EBPbeta(-/-);p19(Arf-/-) mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19(Arf) is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPbeta(-/-) epidermis, we generated K14-ER:Ras;C/EBPbeta(-/-) mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPbeta(-/-) mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPbeta represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPbeta may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.

Vascular endothelial growth factor-C enhances radiosensitivity of lymphatic endothelial cells

PubMed Central

Kesler, Cristina T.; Kuo, Angera; Wong, Hon-Kit; Masuck, David J.; Shah, Jennifer L.; Kozak, Kevin; Held, Kathryn D.; Padera, Timothy P.

2013-01-01

Radiation therapy after lymph node dissection increases the risk of developing painful and incurable lymphedema in breast cancer patients. Lymphedema occurs when lymphatic vessels become unable to maintain proper fluid balance. The sensitivity of lymphatic endothelial cells (LECs) to ionizing radiation has not been reported to date. Here, the radiosensitivity of LECs in vitro has been determined using clonogenic survival assays. The ability of various growth factors to alter LEC radiosensitivity was also examined. Vascular endothelial growth factor (VEGF)-C enhanced radiosensitivity when LECs were treated prior to radiation. VEGF-C-treated LECs exhibited higher levels of entry into the cell cycle at the time of radiation, with a greater number of cells in the S and G2/M phases. These LECs showed higher levels of H2A.X—an indicator of DNA damage—after radiation. VEGF-C did not increase cell death as a result of radiation. Instead, it increased the relative number of quiescent LECs. These data suggest that abundant VEGF-C or lymphangiogenesis may predispose patients to radiation-induced lymphedema by impairing lymphatic vessel repair through induction of LEC quiescence. PMID:24201897

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

PubMed

Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

2007-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

Corynebacterium parvum-induced radiosensitivity and cycling changes of hematopoietic spleen colony-forming units

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

Maruyama, Y.; Magura, C.; Feola, J.

1977-07-01

Ten days after total-body irradiation with 550 rads of /sup 60/Co, spleen colonies were observed in adult C57BL mice. A change in radiosensitivity induced by Corynebacterium parvum, as measured by increased numbers of colony-forming units that survived the 550 rads, began shortly after C. parvum stimulation and extended for at least 7 days before irradiation. C. parvum given 4-24 hours before, followed by high specific activity (/sup 3/H)thymidine (HSATT) 1 hour before total-body irradiation greatly reduced survival of the stem cells that formed spleen colonies (CFU/sub s/) and CFU/sub s/ radiosensitivity to control levels. The HSATT sensitivity by ''suicide'' assaymore » in vivo and the time-response change in radiosensitivity corresponded with the decrease in radiosensitivity, which showed that CFU/sub s/ were stimulated by C. parvum administration and entered the S-phase shortly after stimulation. The data indicated a resting population close to the S-phase. After stimulation, this population entered S-phase. Syngeneic mouse lymphoma cells injected iv 24 hours earlier did not elicit any effect as a stimulus to CFU/sub s/ radiosensitivity change.« less

Characterization of c-Ki-ras and N-ras oncogenes in aflatoxin B sub 1 -induced rat liver tumors

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

McMahon, G.; Davis, E.F.; Huber, L.J.

c-Ki-ras and N-ras oncogenes have been characterized in aflatoxin B{sub 1}-induced hepatocellular carcinomas. Detection of different protooncogene and oncogene sequences and estimation of their frequency distribution were accomplished by polymerase chain reaction, cloning, and plaque screening methods. Two c-Ki-ras oncogene sequences were identified in DNA from liver tumors that contained nucleotide changes absent in DNA from livers of untreated control rats. Sequence changes involving G{center dot}C to T{center dot}A or G{center dot}C to A{center dot}T nucleotide substitutions in codon 12 were scored in three of eight tumor-bearing animals. Distributions of c-Ki-ras sequences in tumors and normal liver DNA indicated thatmore » the observed nucleotide changes were consistent with those expected to result from direct mutagenesis of the germ-line protooncogene by aflatoxin B{sub 1}. N-ras oncogene sequences were identified in DNA from two of eight tumors. Three N-ras gene regions were identified, one of which was shown to be associated with an oncogene containing a putative activating amino acid residing at codon 13. All three N-ras sequences, including the region detected in N-ras oncogenes, were present at similar frequencies in DNA samples from control livers as well as liver tumors. The presence of a potential germ-line oncogene may be related to the sensitivity of the Fischer rat strain to liver carcinogenesis by aflatoxin B{sub 1} and other chemical carcinogens.« less

Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells

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

Hirai, Takahisa; Division of Chemotherapy and Clinical Cancer Research, National Cancer Center Research Institute, Chuo-ku, Tokyo; Saito, Soichiro

The poly(ADP-ribose) polymerase (PARP)-1 regulates DNA damage responses and promotes base excision repair. PARP inhibitors have been shown to enhance the cytotoxicity of ionizing radiation in various cancer cells and animal models. We have demonstrated that the PARP inhibitor (PARPi) AZD2281 is also an effective radiosensitizer for carbon-ion radiation; thus, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. Institutes for biological experiments using proton beam are limited worldwide. This study was performed as a cooperative research at heavy ion medical accelerator in Chiba (HIMAC) in Nationalmore » Institute of Radiological Sciences. HIMAC can generate various ion beams; this enabled us to compare the radiosensitization effect of the PARPi on cells subjected to proton and carbon-ion beams from the same beam line. After physical optimization of proton beam irradiation, the radiosensitization effect of the PARPi was assessed in the human lung cancer cell line, A549, and the pancreatic cancer cell line, MIA PaCa-2. The effect of the PARPi, AZD2281, on radiosensitization to Bragg peak was more significant than that to entrance region. The PARPi increased the number of phosphorylated H2AX (γ-H2AX) foci and enhanced G2/M arrest after proton beam irradiation. This result supports our hypothesis that a PARPi could be applied to a wide therapeutic range of LET radiation by blocking the DNA repair response. - Highlights: • Effective radiosensitizers for particle radiation therapy have not been reported. • PARP inhibitor treatment radiosensitized after proton beam irradiation. • The sensitization at Bragg peak was greater than that at entrance region. • DSB induction and G2/M arrest is involved in the sensitization mechanism.« less

Resistance of R-Ras knockout mice to skin tumour induction

PubMed Central

May, Ulrike; Prince, Stuart; Vähätupa, Maria; Laitinen, Anni M.; Nieminen, Katriina; Uusitalo-Järvinen, Hannele; Järvinen, Tero A. H.

2015-01-01

The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment. PMID:26133397

Genomic structure of two ras family genes in the slime mold Physarum polycephalum.

PubMed

Trzcińska-Danielewicz, Joanna; Kozlowski, Piotr; Gierdal, Katarzyna; Wiejak, Jolanta; Jagielski, Adam; Toczko, Kazimierz; Fronk, Jan

2002-08-01

Genomic structure of two Physarum polycephalum ras family genes, Ppras2 and Pprap1, has been determined, including the upstream region of the latter. The genes are interrupted by three and four introns, respectively. The first intron of Ppras2 has the same location within the coding sequence as the first intron in another ras homolog from this organism, Ppras1 [Trzcińska-Danielewicz, J., Kozlowski, P., and Toczko, K. (1996). "Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene", Gene 169, pp. 143-144]. All introns, ranging from 53 to ca. 460 base pairs, have the canonical 5' and 3' ends, are greatly enriched in pyrimidines in the coding strand and have frequent pyrimidines-only tracts. These latter features seem to be responsible for the difficulties in cloning and sequencing of parts of these genes. Short sequences shared with P. polycephalum transposon-like repeats are common in the introns, indicating a possible role of transposition in intron evolution. In all three ras family genes phase zero introns are located mostly between sequences coding for regular protein secondary structure elements.

Transposon mutagenesis identifies chromatin modifiers cooperating with Ras in thyroid tumorigenesis and detects ATXN7 as a cancer gene.

PubMed

Montero-Conde, Cristina; Leandro-Garcia, Luis J; Chen, Xu; Oler, Gisele; Ruiz-Llorente, Sergio; Ryder, Mabel; Landa, Iñigo; Sanchez-Vega, Francisco; La, Konnor; Ghossein, Ronald A; Bajorin, Dean F; Knauf, Jeffrey A; Riordan, Jesse D; Dupuy, Adam J; Fagin, James A

2017-06-20

Oncogenic RAS mutations are present in 15-30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with Hras G12V in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in Tpo-Cre/homozygous FR-Hras G12V mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway. ATXN7 , a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with RAS or NF1 mutations. Expression of ATXN7 mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to ATXN7 as a previously unrecognized cancer gene.

Transposon mutagenesis identifies chromatin modifiers cooperating with Ras in thyroid tumorigenesis and detects ATXN7 as a cancer gene

PubMed Central

Montero-Conde, Cristina; Leandro-Garcia, Luis J.; Chen, Xu; Oler, Gisele; Ruiz-Llorente, Sergio; Ryder, Mabel; Landa, Iñigo; Sanchez-Vega, Francisco; La, Konnor; Ghossein, Ronald A.; Bajorin, Dean F.; Knauf, Jeffrey A.; Riordan, Jesse D.; Dupuy, Adam J.; Fagin, James A.

2017-01-01

Oncogenic RAS mutations are present in 15–30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with HrasG12V in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in Tpo-Cre/homozygous FR-HrasG12V mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway. ATXN7, a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with RAS or NF1 mutations. Expression of ATXN7 mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to ATXN7 as a previously unrecognized cancer gene. PMID:28584132

Enhancement of radiosensitivity of melanoma cells by pegylated gold nanoparticles under irradiation of megavoltage electrons.

PubMed

Mousavi, Mehdi; Nedaei, Hassan Ali; Khoei, Samideh; Eynali, Samira; Khoshgard, Karim; Robatjazi, Mostafa; Iraji Rad, Rasoul

2017-02-01

Gold nanoparticles (GNP) have significant potential as radiosensitizer agents due to their distinctive properties. Several studies have shown that the surface modification of nanoparticles with methyl polyethylene glycol (mPEG) can increase their biocompatibility. However, the present study investigated the radiosensitization effects of mPEG-coated GNP (mPEG-GNP) in B16F10 murine melanoma cells under irradiation of 6 MeV Electron beam. The synthesized GNP were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy, and zeta potential. Enhancement of radiosensitization was evaluated by the clonogenic assay at different radiation doses of megavoltage electron beams. It was observed that mPEG-GNP with a hydrodynamic size of approximately 50 nm are almost spherical and cellular uptake occurred at all concentrations. Both proliferation efficiency and survival fraction decreased with increasing mPEG-GNP concentration. Furthermore, significant GNP sensitization occurred with a maximum dose enhancement factor of 1.22 at a concentration of 30 μM. Pegylated-GNP are taken up by B16F10 cancer cells and cause radiosensitization in the presence of 6 MeV electrons. The radiosensitization effects of GNP may probably be due to biological processes. Therefore, the underlying biological mechanisms beyond the physical dose enhancement need to be further clarified.

Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

PubMed Central

Drosten, Matthias; Sum, Eleanor Y. M.; Lechuga, Carmen G.; Simón-Carrasco, Lucía; Jacob, Harrys K. C.; García-Medina, Raquel; Huang, Sidong; Beijersbergen, Roderick L.; Bernards, Rene; Barbacid, Mariano

2014-01-01

The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N- and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism. PMID:25288756

Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage.

PubMed

Bayart, Emilie; Pouzoulet, Frédéric; Calmels, Lucie; Dadoun, Jonathan; Allot, Fabien; Plagnard, Johann; Ravanat, Jean-Luc; Bridier, André; Denozière, Marc; Bourhis, Jean; Deutsch, Eric

2017-01-01

Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2'-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 μM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy.

Inhibition of Ras for cancer treatment: the search continues

PubMed Central

Baines, Antonio T.; Xu, Dapeng; Der, Channing J.

2012-01-01

Background The RAS oncogenes (HRAS, NRAS and KRAS) comprise the most frequently mutated class of oncogenes in human cancers (33%), stimulating intensive effort in developing anti-Ras inhibitors for cancer treatment. Discussion Despite intensive effort, to date no effective anti-Ras strategies have successfully made it to the clinic. We present an overview of past and ongoing strategies to inhibit oncogenic Ras in cancer. Conclusions Since approaches to directly target mutant Ras have not been successful, most efforts have focused on indirect approaches to block Ras membrane association or downstream effector signaling. While inhibitors of effector signaling are currently under clinical evaluation, genome-wide unbiased genetic screens have identified novel directions for future anti-Ras drug discovery. PMID:22004085

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

PubMed

Ge, Y; Li, Z H; Marshall, M S; Broxmeyer, H E; Lu, L

1998-06-01

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

Differential requirement of RasGRP1 for γδ T cell development and activation

PubMed Central

Chen, Yong; Ci, Xinxin; Gorentla, Balachandra; Sullivan, Sarah A.; Stone, James C.; Zhang, Weiguo; Pereira, Pablo; Lu, Jianxin; Zhong, Xiao-Ping

2012-01-01

γδ T cells (γδT) belong to a distinct T cell lineage that performs immune functions different from αβ T cells (αβT). Previous studies have established that Erk1/2 MAPKs are critical for positive selection of αβT cells. Additional evidence also suggests that elevated Erk1/2 activity promotes γδT cell generation. RasGRP1, a guanine nucleotide releasing factor for Ras, plays an important role in positive selection of αβT cells by activating the Ras-Erk1/2 pathway. In this report, we demonstrate that RasGRP1 is critical for TCR-induced Erk1/2 activation in γδT cells but exerts different roles for γδT cell generation and activation. Deficiency of RasGRP1 does not obviously affect γδT cell numbers in the thymus but leads to increased γδT cells, particularly CD4−CD8+ γδT cells, in the peripheral lymphoid organs. The virtually unhindered γδT cell development in the RasGRP1−/− thymus proved to be cell intrinsic, while the increase in CD8+ γδT cells is caused by non-cell-intrinsic mechanisms. Our data provides genetic evidence that decreased Erk1/2 activation in the absence of RasGRP1 is compatible for γδT cell generation. Although RasGRP1 is dispensable for γδT cell generation, RasGRP1-deficient γδT cells are defective in proliferation following TCR stimulation. Additionally, RasGRP1-deficient γδT cells are impaired to produce IL-17 but not IFNγ. Together, these observations have revealed that RasGRP1 plays differential roles for γδ and αβ T cell development but is critical for γδT cell proliferation and production of IL-17. PMID:22623331

EphA2 Drives the Segregation of Ras-Transformed Epithelial Cells from Normal Neighbors.

PubMed

Porazinski, Sean; de Navascués, Joaquín; Yako, Yuta; Hill, William; Jones, Matthew Robert; Maddison, Robert; Fujita, Yasuyuki; Hogan, Catherine

2016-12-05

In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells) are detected by normal neighbors and as a result are often extruded from the tissue [1-6]. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton [2, 6]. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. Here we identify differential EphA2 signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. We also show that Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells in vivo, suggesting that our in vitro findings are conserved in evolution. We propose that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium. Copyright © 2016 Elsevier Ltd. All rights reserved.

Does Harvey-Ras gene expression lead to oral squamous cell carcinoma? A clinicopathological aspect

PubMed Central

Krishna, Akhilesh; Singh, Shraddha; Singh, Vineeta; Kumar, Vijay; Singh, Uma Shankar; Sankhwar, Satya Narayan

2018-01-01

Background: Harvey-Ras (H-Ras) is an important guanosine triphosphatase protein for the regulation of cellular growth and survival. Altered Ras signaling has been observed in different types of cancer either by gene amplification and/or mutation. The H-Ras oncogene mutations are well reported, but expression of the H-Ras gene is still unknown. Objective: This study aimed to examine both protein and messenger-RNA (mRNA) expressions of H-Ras in oral squamous cell carcinoma (OSCC) and analyzed the association with risk habits and the clinicopathological profile of cases. Methodology: A total of 65 tissue specimens of OSCC (case group) and equal number of normal tissues (control group) were included in this study. H-Ras protein and mRNA expressions were analyzed using immunohistochemical and quantitative real time-polymerase chain reaction techniques, respectively. Results: The H-Ras protein was significantly overexpressed in the oral carcinoma group compared to the normal group (P = 0.03). Most of the OSCC cases showed positive staining with moderate expression, while negative and moderate staining was high in the control group. The majority of H-Ras positive cases were found in individuals with multiple risk habits including tobacco chewing. The risk of H-Ras positivity was 1.46 times higher in smokers than non-smokers. H-Ras positivity increased in cases affected with buccal mucosa site and higher grade of carcinoma. Relative mRNA level of H-Ras was significantly elevated in oral carcinoma as compared with the control group (P ≤ 0.001). Protein and mRNA levels of H-Ras in case group was poorly correlated. Conclusion: H-Ras oncogene expression was markedly higher in oral carcinoma, and it can be a prognostic marker and target for an effective molecular therapy. PMID:29731559

Effect of blood estrogen and progesterone on severity of minor RAS

NASA Astrophysics Data System (ADS)

Utami, S.; Rahardjo, T. W. B.; Baziad, A.; Alwadris, T. T.; Auerkari, E. I.

2018-05-01

Recurrent Aphthous Stomatitis (RAS) is a chronic inflammatory disease that attacks oral mucosa. Estrogen stimulates proliferation and buccal mucosa cornification and generally acts as an immunoinhibitor that can increase antibody synthesis through humoral response, while progesterone has immunosuppressive potential. This study aimed to examine the levels of blood estrogen and progesterone in females with Minor RAS of varying severity. Subjects were 42 women, 17–40 years old, with Minor RAS but without hormonal therapy or hysterectomy. They were investigated to see whether the severity of pain was related to condition of onset, recurrence and site or amount of lesions. Blood was taken on the 21st – 22nd day of the menstrual cycle to test both hormone levels, using Microparticle Enzyme Immunoassay for the estrogen level, and Competitive Radioimmunoassay for the progesterone level. There was no significant relation between estrogen (p=0.530) or progesterone (p=0.717) level and the severity of Minor RAS. There is a tendency of normal estrogen level on both mild (62.5%) and severe (37.5%) Minor RAS. However, the progesterone level tends to be low both in mild (61.3%) and severe (38.7%) Minor RAS. It is assumed that the decrease of progesterone level also decreases the anti-inflammation function.

Ras-Association Domain of Sorting Nexin 27 Is Critical for Regulating Expression of GIRK Potassium Channels

PubMed Central

Bodhinathan, Karthik; Taura, Jaume J.; Taylor, Natalie M.; Nettleton, Margaret Y.; Ciruela, Francisco; Slesinger, Paul A.

2013-01-01

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability. PMID:23536889

BMP suppresses PTEN expression via RAS/ERK signaling.

PubMed

Beck, Stayce E; Carethers, John M

2007-08-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

Association Between Increased Vascular Density and Loss of Protective RAS in Early-Stage NPDR

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan; Raghunandan, Sneha; Vyas, Ruchi J.; Vu, Amanda C.; Bryant, Douglas; Yaqian, Duan; Knecht, Brenda E.; Grant, Maria B.; Chalam, K . V.; Parsons-Wingerter, Patricia

2016-01-01

Our hypothesis predicts that retinal blood vessels increase in density during early-stage progression to moderate nonproliferative diabetic retinopathy (NPDR). The prevailing paradigm of NPDR progression is that vessels drop out prior to abnormal, vision-impairing regrowth at late-stage proliferative diabetic retinopathy (DR). However, surprising results for our previous preliminary study 1 with NASA's VESsel GENeration Analysis (VESGEN) software showed that vessels proliferated considerably during moderate NPDR compared to drop out at both mild and severe NPDR. Validation of our hypothesis will support development of successful early-stage regenerative therapies such as vascular repair by circulating angiogenic cells (CACs). The renin-angiotensin system (RAS)is implicated in the pathogenesis of DR and in the function of CACs, a critical bone marrow-derived population that is instrumental in vascular repair.

The RAS mutation status predicts survival in patients undergoing hepatic resection for colorectal liver metastases: The results from a genetic analysis of all-RAS.

PubMed

Amikura, Katsumi; Akagi, Kiwamu; Ogura, Toshiro; Takahashi, Amane; Sakamoto, Hirohiko

2018-03-01

We investigated the impact of mutations in KRAS exons 3-4 and NRAS exons 2-3 in addition to KRAS exon 2, so-called all-RAS mutations, in patients with colorectal liver metastasis (CLM) undergoing hepatic resection. We analyzed 421 samples from CLM patients for their all-RAS mutation status to compare the overall survival rate (OS), recurrence-free survival rate (RFS), and the pattern of recurrence between the patients with and without RAS mutations. RAS mutations were detected in 191 (43.8%). Thirty-two rare mutations (12.2%) were detected in 262 patients with KRAS exon 2 wild-type. After excluding 79 patients who received anti-EGFR antibody therapy, 168 were classified as all-RAS wild-type, and 174 as RAS mutant-type. A multivariate analysis of factors associated with OS and RFS identified the RAS status as an independent factor (OS; hazard ratio [HR] = 1.672, P = 0.0031, RFS; HR = 1.703, P = 0.0024). Recurrence with lung metastasis was observed significantly more frequent in patients with RAS mutations than in patients with RAS wild-type (P = 0.0005). Approximately half of CLM patients may have a RAS mutation. CLM patients with RAS mutations had a significantly worse survival rate in comparison to patients with RAS wild-type, regardless of the administration of anti-EGFR antibody therapy. © 2017 Wiley Periodicals, Inc.

C/EBPβ represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19Arf

PubMed Central

Ewing, SJ; Zhu, S; Zhu, F; House, JS; Smart, RC

2013-01-01

CCAAT/enhancer-binding protein-β (C/EBPβ) is a mediator of cell survival and tumorigenesis. When C/EBPβ−/− mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19Arf and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19Arf is dramatically elevated in C/EBPβ−/− epidermis and that C/EBPβ represses a p19Arf promoter reporter. To determine whether p19Arf is responsible for the proapoptotic phenotype in C/EBPβ−/− mice, C/EBPβ−/−;p19Arf−/− mice were generated. C/EBPβ−/−;p19Arf−/− mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19Arf is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPβ−/− epidermis, we generated K14-ER:Ras; C/EBPβ−/− mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPβ−/− mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPβ represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPβ may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents. PMID:18636078

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

PubMed

Matheny, Sharon A; Chen, Chiyuan; Kortum, Robert L; Razidlo, Gina L; Lewis, Robert E; White, Michael A

2004-01-15

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

Mechanism of Ras Activation by TGFBeta

DTIC Science & Technology

2002-07-01

32P-labeled oligonucleotides to each reaction. The reactions were incubated at room temperature for 20 min. For supershift assays, 1 p\\ of antibodies ...formation of this TGFß3-inducible complex. In addition, as shown in Fig. 4A, left side, addition of either a pan-Fos or pan-Jun antibody completely blocked...addition to c-Jun 30770 A Ras/MAPK/Smads and TGFß1 Production RasNI7E3 -RasN17 +RiisN17 Antibodies TGFß I " - Jun Fits Ig(J

Exploiting the bad eating habits of Ras-driven cancers.

PubMed

White, Eileen

2013-10-01

Oncogenic Ras promotes glucose fermentation and glutamine use to supply central carbon metabolism, but how and why have only emerged recently. Ras-mediated metabolic reprogramming generates building blocks for growth and promotes antioxidant defense. To fuel metabolic pathways, Ras scavenges extracellular proteins and lipids. To bolster metabolism and mitigate stress, Ras activates cellular self-cannibalization and recycling of proteins and organelles by autophagy. Targeting these distinct features of Ras-driven cancers provides novel approaches to cancer therapy.

RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK-positive lung cancer.

PubMed

Hrustanovic, Gorjan; Olivas, Victor; Pazarentzos, Evangelos; Tulpule, Asmin; Asthana, Saurabh; Blakely, Collin M; Okimoto, Ross A; Lin, Luping; Neel, Dana S; Sabnis, Amit; Flanagan, Jennifer; Chan, Elton; Varella-Garcia, Marileila; Aisner, Dara L; Vaishnavi, Aria; Ou, Sai-Hong I; Collisson, Eric A; Ichihara, Eiki; Mack, Philip C; Lovly, Christine M; Karachaliou, Niki; Rosell, Rafael; Riess, Jonathan W; Doebele, Robert C; Bivona, Trever G

2015-09-01

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS-mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRAS(WT)) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK-positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

PubMed

Severyn, Bryan; Nguyen, Thi; Altman, Michael D; Li, Lixia; Nagashima, Kumiko; Naumov, George N; Sathyanarayanan, Sriram; Cook, Erica; Morris, Erick; Ferrer, Marc; Arthur, Bill; Benita, Yair; Watters, Jim; Loboda, Andrey; Hermes, Jeff; Gilliland, D Gary; Cleary, Michelle A; Carroll, Pamela M; Strack, Peter; Tudor, Matt; Andersen, Jannik N

2016-10-01

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway. © 2016 Society for Laboratory Automation and Screening.

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

PubMed Central

Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

2012-01-01

Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching.

PubMed

Harrison, Rane A; Lu, Jia; Carrasco, Martin; Hunter, John; Manandhar, Anuj; Gondi, Sudershan; Westover, Kenneth D; Engen, John R

2016-11-20

Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogen-deuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

PubMed

Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

2016-01-01

To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. © 2014 International Society for Diseases of the Esophagus.

Optimizing depuration of salmon in RAS

USDA-ARS?s Scientific Manuscript database

Fish cultured within water recirculating aquaculture systems (RAS) can acquire "earthy" or "musty" off-flavors due to bioaccumulation of the compounds geosmin and 2-methylisoborneol (MIB), respectively, which are produced by certain bacterial species present in RAS biosolids and microbial biofilms. ...

Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS.

PubMed

Yao, Zhan; Yaeger, Rona; Rodrik-Outmezguine, Vanessa S; Tao, Anthony; Torres, Neilawattie M; Chang, Matthew T; Drosten, Matthias; Zhao, Huiyong; Cecchi, Fabiola; Hembrough, Todd; Michels, Judith; Baumert, Hervé; Miles, Linde; Campbell, Naomi M; de Stanchina, Elisa; Solit, David B; Barbacid, Mariano; Taylor, Barry S; Rosen, Neal

2017-08-10

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

Radiosensitizing effects of neem (Azadirachta indica) oil.

PubMed

Kumar, Ashok; Rao, A R; Kimura, H

2002-02-01

Radiosensitization by neem oil was studied using Balbc/3T3 cells and SCID cells. Neem oil enhanced the radiosensitivity of the cells when applied both during and after x-irradiation under aerobic conditions. Neem oil completely inhibited the repair of sublethal damage and potentially lethal damage repair in Balbc/3T3 cells. The cytofluorimeter data show that neem oil treatment before and after x-irradiation reduced the G(2) + M phase, thus inhibiting the expression of the radiation induced arrest of cells in the G(2) phase of the cell cycle. However, SCIK cells (derived from the SCID mouse), deficient in DSB repair, treated with neem oil did not show any enhancement in the radiosensitivity. There was no effect of neem oil on SLD repair or its inhibition in SCIK cells. These results suggest that neem oil enhanced the radiosensitivity of cells by interacting with residual damage after x-irradiation, thereby converting the sublethal damage or potentially lethal damage into lethal damage, inhibiting the double-strand break repair or reducing the G(2) phase of the cell cycle. Copyright 2002 John Wiley & Sons, Ltd.

Short hairpin RNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.

PubMed

Flanagan, Sheryl A; Cooper, Kristin S; Mannava, Sudha; Nikiforov, Mikhail A; Shewach, Donna S

2012-12-01

To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. shRNA suppression of TS was compared with 5-fluoro-2'-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. TS shRNA produced profound (≥ 90%) and prolonged (≥ 8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, sh

Investigation of gold nanoparticle radiosensitization mechanisms using a free radical scavenger and protons of different energies.

PubMed

Jeynes, J C G; Merchant, M J; Spindler, A; Wera, A-C; Kirkby, K J

2014-11-07

Gold nanoparticles (GNPs) have been shown to sensitize cancer cells to x-ray radiation, particularly at kV energies where photoelectric interactions dominate and the high atomic number of gold makes a large difference to x-ray absorption. Protons have a high cross-section for gold at a large range of relevant clinical energies, and so potentially could be used with GNPs for increased therapeutic effect.Here, we investigate the contribution of secondary electron emission to cancer cell radiosensitization and investigate how this parameter is affected by proton energy and a free radical scavenger. We simulate the emission from a realistic cell phantom containing GNPs after traversal by protons and x-rays with different energies. We find that with a range of proton energies (1-250 MeV) there is a small increase in secondaries compared to a much larger increase with x-rays. Secondary electrons are known to produce toxic free radicals. Using a cancer cell line in vitro we find that a free radical scavenger has no protective effect on cells containing GNPs irradiated with 3 MeV protons, while it does protect against cells irradiated with x-rays. We conclude that GNP generated free radicals are a major cause of radiosensitization and that there is likely to be much less dose enhancement effect with clinical proton beams compared to x-rays.

Human Mut T Homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic RAS-induced ROS.

PubMed

Rai, Priyamvada

2012-01-01

Oncogenic RAS-induced reactive oxygen species (ROS) trigger barriers to cell transformation and cancer progression through tumor-suppressive responses such as cellular senescence or cell death. We have recently shown that oncogenic RAS-induced DNA damage and attendant premature senescence can be prevented by overexpressing human MutT Homolog 1 (MTH1), the major mammalian detoxifier of the oxidized DNA precursor, 8-oxo-dGTP. Paradoxically, RAS-induced ROS are also able to participate in tumor progression via transformative processes such as mitogenic signaling, the epithelial-mesenchymal transition (EMT), anoikis inhibition, and PI3K/Akt-mediated survival signaling. Here we provide a preliminary insight into the influence of MTH1 levels on the EMT phenotype and Akt activation in RAS-transformed HMLE breast epithelial cells. Within this context, we will discuss the implications of MTH1 upregulation in oncogenic RAS-sustaining cells as a beneficial adaptive change that inhibits ROS-mediated cell senescence and participates in the maintenance of ROS-associated tumor-promoting mechanisms. Accordingly, targeting MTH1 in RAS-transformed tumor cells will not only induce proliferative defects but also potentially enhance therapeutic cytotoxicity by shifting cellular response away from pro-survival mechanisms.

miR-11 regulates pupal size of Drosophila melanogaster via directly targeting Ras85D.

PubMed

Li, Yao; Li, Shengjie; Jin, Ping; Chen, Liming; Ma, Fei

2017-01-01

MicroRNAs play diverse roles in various physiological processes during Drosophila development. In the present study, we reported that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with the following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3'-untranslated region in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, our current work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis. Copyright © 2017 the American Physiological Society.

Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential

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

Dungey, Fiona A.; Loeser, Dana A.; Chalmers, Anthony J.

2008-11-15

Purpose: Current treatments for glioblastoma multiforme are inadequate and limited by the radiation sensitivity of normal brain. Because glioblastoma multiforme are rapidly proliferating tumors within nondividing normal tissue, the therapeutic ratio might be enhanced by combining radiotherapy with a replication-specific radiosensitizer. KU-0059436 (AZD2281) is a potent and nontoxic inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) undergoing a Phase II clinical trial as a single agent. Methods and Materials: Based on previous observations that the radiosensitizing effects of PARP inhibition are more pronounced in dividing cells, we investigated the mechanisms underlying radiosensitization of human glioma cells by KU-0059436, evaluating the replication dependence ofmore » this effect and its therapeutic potential. Results: KU-0059436 increased the radiosensitivity of four human glioma cell lines (T98G, U373-MG, UVW, and U87-MG). Radiosensitization was enhanced in populations synchronized in S phase and abrogated by concomitant exposure to aphidicolin. Sensitization was further enhanced when the inhibitor was combined with a fractionated radiation schedule. KU-0059436 delayed repair of radiation-induced DNA breaks and was associated with a replication-dependent increase in {gamma}H2AX and Rad51 foci. Conclusion: The results of our study have shown that KU-0059436 increases radiosensitivity in a replication-dependent manner that is enhanced by fractionation. A mechanism is proposed whereby PARP inhibition increases the incidence of collapsed replication forks after ionizing radiation, generating persistent DNA double-strand breaks. These observations indicate that KU-0059436 is likely to enhance the therapeutic ratio achieved by radiotherapy in the treatment of glioblastoma multiforme. A Phase I clinical trial is in development.« less

Iron modulates cell survival in a Ras- and MAPK-dependent manner in ovarian cells

PubMed Central

Bauckman, K A; Haller, E; Flores, I; Nanjundan, M

2013-01-01

Ovarian cancer is a leading cause of cancer death in women in the United States. While the majority of ovarian cancers are serous, some rarer subtypes (i.e. clear cell) are often associated with endometriosis, a benign gynecological disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian cancers and induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-independent, and Ras-independent manner. Knockdown of autophagy mediators in HEY ovarian cancer cells reversed FAC-induced LC3-II levels, but there was little effect on reversing the cell death response. Intriguingly, transmission electron microscopy of FAC-treated T80 cells demonstrated abundant lysosomes (confirmed using Lysotracker) rich in iron particles, which occurred in a Ras-independent manner. Although the mitogen-activated protein kinase (MAPK) inhibitor, U0126, reversed FAC-induced LC3-II/autophagic punctae and lysosomes in a Ras-independent manner, it was remarkable that U0126 reversed cell death in malignant ovarian cells associated with Ras mutations. Moreover, FAC increased heme oxygenase-1 expression in H-Ras-overexpressing T80 cells, which was associated with increased cell death when overexpressed in T80 cells. Disruption of intracellular iron levels, via chelation of intracellular iron (deferoxamine), was also detrimental to malignant ovarian cell survival; thus

Deconstruction of the Ras switching cycle through saturation mutagenesis

PubMed Central

Bandaru, Pradeep; Shah, Neel H; Bhattacharyya, Moitrayee; Barton, John P; Kondo, Yasushi; Cofsky, Joshua C; Gee, Christine L; Chakraborty, Arup K; Kortemme, Tanja; Ranganathan, Rama; Kuriyan, John

2017-01-01

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins. DOI: http://dx.doi.org/10.7554/eLife.27810.001 PMID:28686159

Targeting Phosphatidylinositide3-Kinase/Akt pathway by BKM120 for radiosensitization in hepatocellular carcinoma

PubMed Central

Liu, Wei-Lin; Gao, Ming; Tzen, Kai-Yuan; Tsai, Chiao-Ling; Hsu, Feng-Ming; Cheng, Ann-Lii; Cheng, Jason Chia-Hsien

2014-01-01

Tumor control of hepatocellular carcinoma by radiotherapy remains unsatisfactory. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in inhibiting cancer cell death. Elevated PI3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to determine whether the inhibition of PI3K/Akt activity by a PI3K inhibitor, BKM120, contributes to the increased sensitivity of liver cancer cells to irradiation. The hepatocellular carcinoma cell lines (Huh7 and BNL) were used to evaluate the in vitro synergism between BKM120 and irradiation. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy to assess the in vivo response. BKM120 increased cell killing by radiation, increased the expression of apoptotic markers, and suppressed the repair of radiation-induced DNA double-strand breaks. BKM120 pretreatment inhibited radiation-induced Akt phosphorylation and enhanced the tumor-suppressive effect and radiation-induced tumor cell apoptosis in ectopic xenografts. Inhibition of mTOR phosphorylation by rapamycin enhanced the radiosensitivity of BKM120-treated hepatocellular carcinoma cells. The synergism between BKM120 and irradiation likely inhibits the activation of Akt by radiation, leading to increased cell apoptosis and suppression of DNA-double-strand breaks repair in hepatocellular carcinoma cells. These data suggest that the BKM120/radiation combination may be a strategy worthy of clinical trials. PMID:25004403

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy

PubMed Central

Ramos-Kuri, Manuel; Rapti, Kleopatra; Mehel, Hind; Zhang, Shihong; Dhandapany, Perundurai S.; Liang, Lifan; García-Carrancá, Alejandro; Bobe, Regis; Fischmeister, Rodolphe; Adnot, Serge; Lebeche, Djamel; Hajjar, Roger J.; Lipskaia, Larissa; Chemaly, Elie R.

2015-01-01

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy. PMID:26260012

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

PubMed Central

Gawecka, Joanna E.; Griffiths, Genevieve S.; Ek-Rylander, Barbro; Ramos, Joe W.; Matter, Michelle L.

2010-01-01

Background Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins. Methods and Findings We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly. Conclusions These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration. PMID:20585650

Prostate-Specific Natural Health Products (Dietary Supplements) Radiosensitize Normal Prostate Cells

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

Hasan, Yasmin; Schoenherr, Diane; Martinez, Alvaro A.

Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines. Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity wasmore » assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing gammaH2AX. Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2mug/mL], Trinovin [10mug/mL], and Prostate Rx [50 mug/mL]). However, both Trinovin (10mug/mL) and Prostate Rx (6mug/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair. Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells.« less

The nitric oxide donor JS-K sensitizes U87 glioma cells to repetitive irradiation.

PubMed

Heckler, Max; Osterberg, Nadja; Guenzle, Jessica; Thiede-Stan, Nina Kristin; Reichardt, Wilfried; Weidensteiner, Claudia; Saavedra, Joseph E; Weyerbrock, Astrid

2017-06-01

As a potent radiosensitizer nitric oxide (NO) may be a putative adjuvant in the treatment of malignant gliomas which are known for their radio- and chemoresistance. The NO donor prodrug JS-K (O2-(2.4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1,2-diolate) allows cell-type specific intracellular NO release via enzymatic activation by glutathione-S-transferases overexpressed in glioblastoma multiforme. The cytotoxic and radiosensitizing efficacy of JS-K was assessed in U87 glioma cells in vitro focusing on cell proliferation, induction of DNA damage, and cell death. In vivo efficacy of JS-K and repetitive irradiation were investigated in an orthotopic U87 xenograft model in mice. For the first time, we could show that JS-K acts as a potent cytotoxic and radiosensitizing agent in U87 cells in vitro. This dose- and time-dependent effect is due to an enhanced induction of DNA double-strand breaks leading to mitotic catastrophe as the dominant form of cell death. However, this potent cytotoxic and radiosensitizing effect could not be confirmed in an intracranial U87 xenograft model, possibly due to insufficient delivery into the brain. Although NO donor treatment was well tolerated, neither a retardation of tumor growth nor an extended survival could be observed after JS-K and/or radiotherapy.

Efficacy of radiosensitizing doped titania nanoparticles under hypoxia and preparation of an embolic microparticle

PubMed Central

Morrison, Rachel A; Rybak-Smith, Malgorzata J; Thompson, James M; Thiebaut, Bénédicte; Hill, Mark A; Townley, Helen E

2017-01-01

The aim of this study was to develop a manufacturing protocol for large-scale production of doped titania radiosensitizing nanoparticles (NPs) to establish their activity under hypoxia and to produce a multimodal radiosensitizing embolic particle for cancer treatment. We have previously shown that radiosensitizing NPs can be synthesized from titania doped with rare earth elements, especially gadolinium. To translate this technology to the clinic, a crucial step is to find a suitable, scalable, high-throughput method. Herein, we have described the use of flame spray pyrolysis (FSP) to generate NPs from titanium and gadolinium precursors to produce titania NPs doped with 5 at% gadolinium. The NPs were fully characterized, and their capacity to act as radiosensitizers was confirmed by clonogenic assays. The integrity of the NPs in vitro was also ascertained due to the potentially adverse effects of free gadolinium in the body. The activity of the NPs was then studied under hypoxia since this is often a barrier to effective radiotherapy. In vitro radiosensitization experiments were performed with both the hypoxia mimetics deferoxamine and cobalt chloride and also under true hypoxia (oxygen concentration of 0.2%). It was shown that the radiosensitizing NPs were able to cause a significant increase in cell death even after irradiation under hypoxic conditions such as those found in tumors. Subsequently, the synthesized NPs were used to modify polystyrene embolization microparticles. The NPs were sintered to the surface of the microparticles by heating at 230°C for 15 minutes. This resulted in a good coverage of the surface and to generate embolization particles that were shown to be radiosensitizing. Such multimodal particles could therefore result in occlusion of the tumor blood vessels in conjunction with localized reactive oxygen species generation, even under hypoxic conditions such as those found in the center of tumors. PMID:28572729

Efficacy of radiosensitizing doped titania nanoparticles under hypoxia and preparation of an embolic microparticle.

PubMed

Morrison, Rachel A; Rybak-Smith, Malgorzata J; Thompson, James M; Thiebaut, Bénédicte; Hill, Mark A; Townley, Helen E

2017-01-01

The aim of this study was to develop a manufacturing protocol for large-scale production of doped titania radiosensitizing nanoparticles (NPs) to establish their activity under hypoxia and to produce a multimodal radiosensitizing embolic particle for cancer treatment. We have previously shown that radiosensitizing NPs can be synthesized from titania doped with rare earth elements, especially gadolinium. To translate this technology to the clinic, a crucial step is to find a suitable, scalable, high-throughput method. Herein, we have described the use of flame spray pyrolysis (FSP) to generate NPs from titanium and gadolinium precursors to produce titania NPs doped with 5 at% gadolinium. The NPs were fully characterized, and their capacity to act as radiosensitizers was confirmed by clonogenic assays. The integrity of the NPs in vitro was also ascertained due to the potentially adverse effects of free gadolinium in the body. The activity of the NPs was then studied under hypoxia since this is often a barrier to effective radiotherapy. In vitro radiosensitization experiments were performed with both the hypoxia mimetics deferoxamine and cobalt chloride and also under true hypoxia (oxygen concentration of 0.2%). It was shown that the radiosensitizing NPs were able to cause a significant increase in cell death even after irradiation under hypoxic conditions such as those found in tumors. Subsequently, the synthesized NPs were used to modify polystyrene embolization microparticles. The NPs were sintered to the surface of the microparticles by heating at 230°C for 15 minutes. This resulted in a good coverage of the surface and to generate embolization particles that were shown to be radiosensitizing. Such multimodal particles could therefore result in occlusion of the tumor blood vessels in conjunction with localized reactive oxygen species generation, even under hypoxic conditions such as those found in the center of tumors.

Effect of hydrocortisone on radiosensitivity of hemopoietic stem cells. [. gamma. rays; mice; bone marrow; spleen

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

Shvets, V.N.

Studies were made of the direction of differentiation and radiosensitivity of CFU (colony-forming units) of bone marrow and spleen for 1 month after single injection of 5 mg hydrocortisone (HC) per mouse. It was found that there was a sharp change in direction of differentiation of CFU from different sources. Bone marrow CFU enhanced erythropoiesis and CFU of the spleen enhanced myelopoiesis, which is not inherent in the same CFU of normal mice. Determination of radiosensitivity of CFU from different sources according to the spleen colony test failed to demonstrate any differences in value of D/sub 0/ and extrapolation number,more » whereas substantial changes in radiosensitivity were demonstrated in the bone marrow colony test. Radiosensitivity of marrow CFU diminished while that of the spleen increased, as compared to the control. It is assumed that these phenomena are due to redistribution of T lymphocytes in response to HC.« less

Effect of anemia on tumor radiosensitivity under normo and hyperbaric conditions

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

Rojas, A.; Stewart, F.A.; Smith, K.A.

1987-11-01

The effect of chronic anemia on tumor radiosensitivity in a murine tumor has been investigated. Anemia was induced by bilateral kidney irradiation given several months before tumor implantation. Anemic, anemic transfused, and normal non-anemic age-matched tumor bearing animals were irradiated with X rays (2 F/24 hr) either in air, air plus misonidazole, or under hyperbaric oxygen. The most resistant response was that of tumors grown in normal mice treated in air. Anemia produced an increase in radiosensitivity which was further enhanced by red blood cell replacement. The most sensitive overall response was seen in the anemic-transfused group treated with HBO.

Imaging of Ras/Raf activity induced by low energy laser irradiation in living cell using FRET

NASA Astrophysics Data System (ADS)

Wang, Fang; Chen, Tong-Sheng; Xing, Da

2005-01-01

Ras/Raf signaling pathway is an important signaling pathway that governs cell proliferation, differential and apoptosis. Low-energy laser irradiation (LELI) was found to modulate various processes. Generally, cell proliferation is induced by low doses LELI and apoptosis is induced by high doses LELI. Mechanism of biological effect of LELI has not been clear. Recently, activation of MEK (mitogen-activated protein kinase) and ERK (extracellular-signal-regulated kinase), which are downstream protein kinases of Ras/Raf, are observed during LELI-induced cell proliferation by immunoprecipitation and western blot analysis. RaichuRas reporter consisting of fusions of H-ras, the Ras-binding domain of Raf (RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). Therefore, intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) was transfected with the plasmid (pRaichuRas) and then treated with LELI at dose of 60J/cm2. Effect of LELI on Ras/Raf in physiological condition of living cells was observed by fluorescence resonance energy transfer (FRET) technique during lung adenocarcinoma cell apoptosis induced by high dose (60J/cm2) LELI. Experimental results showed that after high dose LELI treatment, the binding of Ras and Raf decreases obviously, Ras/Raf signaling pathway deregulates and cell apoptosis occurs.

Inhibiting CD146 by its Monoclonal Antibody AA98 Improves Radiosensitivity of Cervical Cancer Cells.

PubMed

Cheng, Huawen

2016-09-20

BACKGROUND Cervical cancer is one of the major causes of cancer death of females worldwide. Radiotherapy is considered effective for cervical cancer treatment, but the low radiosensitivity found in some cases severely affects therapeutic outcomes. This study aimed to reveal the role of CD146, an important adhesion molecule facilitating tumor angiogenesis, in regulating radiosensitivity of cervical cancer cells. MATERIAL AND METHODS CD146 protein expression was compared in normal cells, cervical cancer cells with lower radiosensitivity, and cervical cancer cells with higher sensitivity from cervical squamous cell carcinoma patients. Anti-CD146 monoclonal antibody AA98 was used to inhibit CD146 in human cervical cancer SiHa cells with relatively low radiosensitivity, and then the cell survival and apoptosis changes after radiation were detected by colony formation assay and flow cytometry. RESULTS CD146 protein was significantly up-regulated in cervical cancer cells (P<0.001), especially in cancer cells with lower radiosensitivity. The SiHa cells treated with AA98 showed more obvious inhibition in cell survival (P<0.05) and promotion in cell apoptosis (P<0.01) after radiation, compared to the untreated cells. More dramatic changes in apoptotic factors Caspase 3 and Bcl-XL were also detected in AA98-treated cells. CONCLUSIONS These results indicate that inhibiting CD146 improves the effect of radiation in suppressing SiHa cells. This study shows the potential of CD146 as a target for increasing radiosensitivity of cervical cancer cells, which might allow improvement in treatment outcome in cervical cancer. Further studies are necessary for understanding the detailed mechanism of CD146 in regulating radiosensitivity.

Exploring environmental causes of altered ras effects: fragmentation plus integration?

PubMed

Porta, Miquel; Ayude, Daniel; Alguacil, Juan; Jariod, Manuel

2003-02-01

Mutations in ras genes are the most common abnormality of oncogenes in human cancer and a major example of activation by point mutation. Experimental and epidemiological studies support the notion that Ki-ras activation and expression may be chemically related. We discuss the potential role of several environmental compounds in the induction or promotion of ras mutations in humans, with a focus on exocrine pancreatic cancer, the human tumor with the highest prevalence at diagnosis of Ki-ras mutations. Organochlorine compounds, organic solvents, and coffee compounds may play an indirect role in causing Ki-ras mutations, rather than as direct inducers of the mutations. Although for some organochlorine compounds the induction of point mutations in ras oncogenes cannot be excluded, it seems more likely that the effects of these compounds are mediated through nongenomic or indirectly genotoxic mechanisms of action. Organic solvents also may act via enzymatic induction of ras mutagens or by providing a proliferation advantage to ras-mutated cell clones. In exocrine pancreatic cancer, caffeine, other coffee compounds, or other factors with which coffee drinking is associated could modulate Ki-ras activation by interfering with DNA repair, cell-cycle checkpoints, and apoptosis. Asbestos, cigarette smoking, and some dietary factors also may be involved in the initiation or the promotion of Ki-ras mutations in lung and colon cancers. Further development of the mechanistic scenarios proposed here could contribute to a meaningful integration of biological, clinical, and environmental knowledge on the causes of altered ras effects. Copyright 2003 Wiley-Liss, Inc.

The Bcr Kinase Downregulates Ras Signaling by Phosphorylating AF-6 and Binding to Its PDZ Domain

PubMed Central

Radziwill, G.; Erdmann, R. A.; Margelisch, U.; Moelling, K.

2003-01-01

The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state. PMID:12808105

siRNA blocking the RAS signalling pathway and inhibits the growth of oesophageal squamous cell carcinoma in nude mice.

PubMed

Wang, Xinjie; Zheng, Yuling; Fan, Qingxia; Zhang, Xudong; Shi, Yonggang

2014-12-01

The aim of this study was to study RAS-siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS-siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS-siRNA group, paclitaxel group and RAS-siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group (p < 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS-siRNA group, and the number of apoptosis cells in the paclitaxel and RAS-siRNA group is significantly most than the paclitaxel group and RAS-siRNA group (p < 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS-siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group (p < 0.05). We therefore conclude that RAS-siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs. Copyright © 2014 John Wiley & Sons, Ltd.

Genetic analysis of Ras genes in epidermal development and tumorigenesis

PubMed Central

Drosten, Matthias; Lechuga, Carmen G; Barbacid, Mariano

2013-01-01

Proliferation and differentiation of epidermal keratinocytes are tightly controlled to ensure proper development and homeostasis of the epidermis. The Ras family of small GTPases has emerged as a central node in the coordination of cell proliferation in the epidermis. Recent genetic evidence from mouse models has revealed that the intensity of Ras signaling modulates the proliferative capacity of epidermal keratinocytes. Interfering with Ras signaling either by combined elimination of the 3 Ras genes from the basal layer of the epidermis or by overexpression of dominant-negative Ras isoforms caused epidermal thinning due to hypoproliferation of keratinocytes. In contrast, overexpression of oncogenic Ras mutants in different epidermal cell layers led to hyperproliferative phenotypes including the development of papillomas and squamous cell carcinomas. Here, we discuss the value of loss- and gain-of-function studies in mouse models to assess the role of Ras signaling in the control of epidermal proliferation. PMID:24150175

Celecoxib enhances radiosensitivity of hypoxic glioblastoma cells through endoplasmic reticulum stress

PubMed Central

Suzuki, Kenshi; Gerelchuluun, Ariungerel; Hong, Zhengshan; Sun, Lue; Zenkoh, Junko; Moritake, Takashi; Tsuboi, Koji

2013-01-01

Background Refractoriness of glioblastoma multiforme (GBM) largely depends on its radioresistance. We investigated the radiosensitizing effects of celecoxib on GBM cell lines under both normoxic and hypoxic conditions. Methods Two human GBM cell lines, U87MG and U251MG, and a mouse GBM cell line, GL261, were treated with celecoxib or γ-irradiation either alone or in combination under normoxic and hypoxic conditions. Radiosensitizing effects were analyzed by clonogenic survival assays and cell growth assays and by assessing apoptosis and autophagy. Expression of apoptosis-, autophagy-, and endoplasmic reticulum (ER) stress–related genes was analyzed by immunoblotting. Results Celecoxib significantly enhanced the radiosensitivity of GBM cells under both normoxic and hypoxic conditions. In addition, combined treatment with celecoxib and γ-irradiation induced marked autophagy, particularly in hypoxic cells. The mechanism underlying the radiosensitizing effect of celecoxib was determined to be ER stress loading on GBM cells. Conclusion Celecoxib enhances the radiosensitivity of GBM cells by a mechanism that is different from cyclooxygenase-2 inhibition. Our results indicate that celecoxib may be a promising radiosensitizing drug for clinical use in patients with GBM. PMID:23658321

Non-covalent interactions of the carcinogen (+)-anti-BPDE with exon 1 of the human K-ras proto-oncogene

NASA Astrophysics Data System (ADS)

Rodriguez, Jorge H.; Deligkaris, Christos

2013-03-01

Investigating the complementary, but different, effects of physical (non-covalent) and chemical (covalent) mutagen-DNA and carcinogen-DNA interactions is important for understanding possible mechanisms of development and prevention of mutagenesis and carcinogenesis. A highly mutagenic and carcinogenic metabolite of the polycyclic aromatic hydrocarbon benzo[ α]pyrene, namely (+)-anti-BPDE, is known to undergo both physical and chemical complexation with DNA. The major covalent adduct, a promutagenic, is known to be an external (+)-trans-anti-BPDE-N2-dGuanosine configuration whose origins are not fully understood. Thus, it is desirable to study the mechanisms of external non-covalent BPDE-DNA binding and their possible relationships to external covalent trans adduct formation. We present a detailed codon-by-codon computational study of the non-covalent interactions of (+)-anti-BPDE with DNA which explains and correctly predicts preferential (+)-anti-BPDE binding at minor groove guanosines. Due to its relevance to carcinogenesis, the interaction of (+)-anti-BPDE with exon 1 of the human K-ras gene has been studied in detail. Present address: Department of Physics, Drury University

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

PubMed Central

Jeong, Ji Hun; Park, Soon Ho; Park, Mi Jung; Kim, Moon Jin; Kim, Kyung Hee; Park, Pil Whan; Seo, Yiel Hea; Lee, Jae Hoon; Park, Jinny; Hong, Junshik

2013-01-01

Background N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. Methods We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. Results The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. Conclusions There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses. PMID:23667841