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1.
Mutations in BRAF and KRAS converge on activation of the mitogen-activated protein kinase pathway in lung cancer mouse models 总被引:4,自引:0,他引:4
Ji H Wang Z Perera SA Li D Liang MC Zaghlul S McNamara K Chen L Albert M Sun Y Al-Hashem R Chirieac LR Padera R Bronson RT Thomas RK Garraway LA Jänne PA Johnson BE Chin L Wong KK 《Cancer research》2007,67(10):4933-4939
Mutations in the BRAF and KRAS genes occur in approximately 1% to 2% and 20% to 30% of non-small-cell lung cancer patients, respectively, suggesting that the mitogen-activated protein kinase (MAPK) pathway is preferentially activated in lung cancers. Here, we show that lung-specific expression of the BRAF V600E mutant induces the activation of extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway and the development of lung adenocarcinoma with bronchioloalveolar carcinoma features in vivo. Deinduction of transgene expression led to dramatic tumor regression, paralleled by dramatic dephosphorylation of ERK1/2, implying a dependency of BRAF-mutant lung tumors on the MAPK pathway. Accordingly, in vivo pharmacologic inhibition of MAPK/ERK kinase (MEK; MAPKK) using a specific MEK inhibitor, CI-1040, induced tumor regression associated with inhibition of cell proliferation and induction of apoptosis in these de novo lung tumors. CI-1040 treatment also led to dramatic tumor shrinkage in murine lung tumors driven by a mutant KRas allele. Thus, somatic mutations in different signaling intermediates of the same pathway induce exquisite dependency on a shared downstream effector. These results unveil a potential common vulnerability of BRAF and KRas mutant lung tumors that potentially affects rational deployment of MEK targeted therapies to non-small-cell lung cancer patients. 相似文献
2.
目的 在中国人群小细胞肺癌(SCLC)标本中检测BRAF/KRAS以及PIK3CA基因突变频率,分析这些基因突变的基因特征和临床特征。方法 2009-2014年共收集557例单纯SCLC患者组织样本。利用双脱氧测序法进行BRAF、KRAS、PIK3CA、NRAS、MEK1基因突变检测。χ2检验分析临床因素与基因突变的相关性,Kaplan-Meier法生存分析,Cox模型多因素预后分析。结果 在557例标本中检测到13例BRAF突变,突变类型包括V600E (n=5)、V600A (n=2)、V600M (n=1)、D594G (n=1)、G464E (n=1)、K601R (n=2)、S605N (n=1)。6例KRAS突变,突变类型包括G12C (n=3)、G12A (n=1)、G12D (n=1)、G13D (n=1)。4例PIK3CA突变,突变类型包括E545G (n=2)、H1047R (n=2)。另外1例NRAS突变(Q61R)和1例MEK1突变(D61Y)。这些突变基因与患者年龄、性别、吸烟状态、临床分期均无相关性。单因素生存分析结果显示基因突变组患者的生存时间比无此类突变者生存时间差,中位生存时间分别为(10.30±0.75)个月(95%CI为8.83~11.77个月)和(12.80±0.54)个月(95%CI为11.74~13.86)(P=0.011)。结论 在单纯SCLC中存在小比例的BRAF/KRAS、PIK3CA基因突变群体,这些基因突变与患者的临床特征无明显统计学相关性,但单因素生存分析显示与患者生存预后呈负相关。 相似文献
3.
Mutated BRAF is detected in approximately 45% of papillary thyroid carcinomas (PTC). To model PTC, we bred mice with adult-onset, thyrocyte-specific expression of BRAF(V600E). One month following BRAF(V600E) expression, mice displayed increased thyroid size, widespread alterations in thyroid architecture, and dramatic hypothyroidism. Over 1 year, without any deliberate manipulation of tumor suppressor genes, all mice developed PTC displaying nuclear atypia and marker expression characteristic of the human disease. Pharmacologic inhibition of MEK1/2 led to decreased thyroid size, restoration of thyroid form and function, and inhibition of tumorigenesis. Mice with BRAF(V600E)-induced PTC will provide an excellent system to study thyroid tumor initiation and progression and the evaluation of inhibitors of oncogenic BRAF signaling. 相似文献
4.
Collisson EA Trejo CL Silva JM Gu S Korkola JE Heiser LM Charles RP Rabinovich BA Hann B Dankort D Spellman PT Phillips WA Gray JW McMahon M 《Cancer discovery》2012,2(8):685-693
KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA. 相似文献
5.
Mutations in the ERK pathway occur in approximately one-third of all human cancers and most often involve production of mutant RAS or BRAF. Several studies, including our own, have shown that mutations in the BRAF and RAS genes are generally mutually exclusive. This study was performed to determine the relative oncogenic potential of the BRAF and RAS oncogenes. BRAF(V600E)-, H-RAS(G12V)-, and N-RAS(Q61R)-transfected mouse embryonic fibroblasts (MEFs) that lack p53 (p53(-/-)) or contain mutations at codon 172 (p53(R172H) and p53(R172P)) were able to induce morphologically transformed foci in p53(-/-) and p53(R172H) MEFs but not in p53(R172P) MEFs. Interestingly, BRAF(V600E) was less potent than mutant H-RAS(G12V) or N-RAS(Q61R) was in cooperating with mutant p53 as the numbers and sizes of foci induced by BRAF(V600E) were significantly lower and smaller. In vitro growth characteristics and anchorage-independent growth of transfected MEFs corroborated the transformed phenotype, and in vivo tumorigenesis confirmed the results. These results indicate that mutant BRAF(V600E) is weakly oncogenic compared with mutant RAS and that they both cooperate with p53(-/-) and p53(R172H) but not with p53(R172P) in oncogenic transformation. 相似文献
6.
Swati Bisht Firoz Ahmad Satyakam Sawaimoon Simi Bhatia Bibhu Ranjan Das 《Medical oncology (Northwood, London, England)》2014,31(9):1-13
Molecular evaluation of KRAS, BRAF, and PIK3CA mutation has become an important part in colorectal carcinoma evaluation, and their alterations may determine the therapeutic response to anti-EGFR therapy. The current study demonstrates the evaluation of KRAS, BRAF, and PIK3CA mutation using direct sequencing in 204 samples. The frequency of KRAS, BRAF, and PIK3CA mutations was 23.5, 9.8, and 5.9 %, respectively. Five different substitution mutations at KRAS codon 12 (G12S, G12D, G12A, G12V, and G12C) and one substitution type at codon 13 (G13D) were observed. KRAS mutations were significantly higher in patients who were >50 years, and were associated with moderate/poorly differentiated tumors and adenocarcinomas. All mutations in BRAF gene were of V600E type, which were frequent in patients who were ≤50 years. Unlike KRAS mutations, BRAF mutations were more frequent in well-differentiated tumors and right-sided tumors. PIK3CA–E545K was the most recurrent mutation while other mutations detected were T544I, Q546R, H1047R, G1049S, and D1056N. No significant association of PIK3CA mutation with age, tumor differentiation, location, and other parameters was noted. No concomitant mutation of KRAS and BRAF mutations was observed, while, interestingly, five cases showed concurrent mutation of KRAS and PIK3CA mutations. In conclusion, to our knowledge, this is the first study to evaluate the PIK3CA mutation in Indian CRC patients. The frequency of KRAS, BRAF, and PIK3CA was similar to worldwide reports. Furthermore, identification of molecular markers has unique strengths, and can provide insights into the pathogenic process and help optimize personalized prevention and therapy. 相似文献
7.
AZD6244 (ARRY 142886) is a potent and selective mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor currently in early clinical trials. We examined the activity of AZD6244 in a panel of non-small cell lung cancer and a panel of cell lines representing many cancer types using in vitro growth assays. AZD6244 induced G(0)-G(1) cell cycle arrest in sensitive cell lines that primarily included cells containing the BRAF V600E mutation. In these cells, G(0)-G(1) arrest is accompanied by the up-regulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1) and down-regulation of cyclin D1. In the majority of cell lines tested, including those with K-ras or non-V600E BRAF mutations, AZD6244 induced the accumulation of phospho-MEK, an effect not observed in the most sensitive BRAF V600E-containing cells. Accumulation of phospho-MEK in non-V600E-containing cell lines is due to abrogation of negative feedback pathways. BRAF V600E disrupts negative feedback signaling, which results in enhanced baseline phospho-MEK expression. Exogenous expression of BRAF V600E disrupts feedback inhibition but does not sensitize cells to AZD6244. Specific suppression of endogenous BRAF V600E does not confer resistance to AZD6244 but enhances sensitivity to AZD6244. Thus, our findings show that BRAF V600E marks cells with an in vitro requirement for MAPK signaling to support proliferation. These cells are exquisitely sensitive to AZD6244 (IC(50), <100 nmol/L), have high baseline levels of phospho-MEK, and lack feedback inhibition between ERK and Raf. These data suggest an approach to identifying cells that may be sensitive to AZD6244 and other MEK inhibitors. 相似文献
8.
Schönleben F Qiu W Bruckman KC Ciau NT Li X Lauerman MH Frucht H Chabot JA Allendorf JD Remotti HE Su GH 《Cancer letters》2007,249(2):242-248
The Raf/MEK/ERK (MAPK) signal transduction is an important mediator of a number of cellular fates including growth, proliferation, and survival. The BRAF gene is activated by oncogenic RAS, leading to cooperative effects in cells responding to growth factor signals. Our study was performed to elucidate a possible role of BRAF in the development of IPMN (Intraductal Papillary Mucinous Neoplasm) and IPMC (Intraductal Papillary Mucinous Carcinoma) of the pancreas. Mutations of BRAF and KRAS were evaluated in 36 IPMN/IPMC samples and two mucinous cystadenomas by direct genomic sequencing. Exons 1 for KRAS, and 5, 11, and 15 for BRAF were examined. Totally we identified 17 (47%) KRAS mutations in exon 1, codon 12 and one missense mutation (2.7%) within exon 15 of BRAF. The mutations appear to be somatic since the same alterations were not detected in the corresponding normal tissues. Our data provide evidence that oncogenic properties of BRAF contribute to the tumorigenesis of IPMN/IPMC, but at a lower frequency than KRAS. 相似文献
9.
Smalley KS Xiao M Villanueva J Nguyen TK Flaherty KT Letrero R Van Belle P Elder DE Wang Y Nathanson KL Herlyn M 《Oncogene》2009,28(1):85-94
Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity. 相似文献
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Cho NY Choi M Kim BH Cho YM Moon KC Kang GH 《International journal of cancer. Journal international du cancer》2006,119(8):1858-1862
Constitutive activation of the kinase cascade involving RAS, RAF, MEK and ERK is common to human cancers, and mutations of KRAS and BRAF are mutually exclusive and serve as alternatives to activate the RAS/RAF/ERK signaling pathway. RAS mutations are known to occur in prostate adenocarcinomas, but little is known about BRAF mutations in these tumors. In the present study, BRAF and KRAS mutations were characterized in 206 prostate adenocarcinomas by enhanced PCR-RFLP and direct sequencing. The identified KRAS and BRAF mutations were then analyzed with respect to preoperative serum PSA levels, Gleason scores and tumor stages. Mutations in codon 600 of BRAF were identified in 21 (10.2%) of 206 prostate adenocarcinomas. KRAS mutations in codons 12 or 13 were found in 15 (7.3%) of 206 prostate adenocarcinomas. However, no tumor specimen contained both BRAF and KRAS mutations. Prostate adenocarcinomas with a BRAF mutation tended to show higher preoperative serum PSA levels, Gleason scores and tumor stages than prostate adenocarcinomas with a KRAS mutation. The results obtained show that BRAF mutations are as uncommon as KRAS mutations in prostate adenocarcinoma. Although BRAF and KRAS are members of the same RAS/ERK signaling pathway, prostate adenocarcinomas with a BRAF mutation showed clinicopathologic features that differed from those of prostate adenocarcinoma with a KRAS mutation. 相似文献
13.
Eisenhardt AE Olbrich H Röring M Janzarik W Anh TN Cin H Remke M Witt H Korshunov A Pfister SM Omran H Brummer T 《International journal of cancer. Journal international du cancer》2011,129(9):2297-2303
Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gain-of-function mutations. To investigate the mutation spectrum within the proto-oncogene encoding the Ser/Thr-kinase B-Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well-known BRAF(V600E) mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAF(V600E) mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3-bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras-dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B-Raf(insT) ) and another B-Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B-Raf(V600E) . Notably, replacement of threonines by valine residues had similar effects on B-Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B-Raf(insT) and B-Raf(V600E) , but not B-Raf(wt) , provoke drastic morphological alterations in human astrocytes. 相似文献
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Su F Bradley WD Wang Q Yang H Xu L Higgins B Kolinsky K Packman K Kim MJ Trunzer K Lee RJ Schostack K Carter J Albert T Germer S Rosinski J Martin M Simcox ME Lestini B Heimbrook D Bollag G 《Cancer research》2012,72(4):969-978
A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance. 相似文献
16.
CM Ardito BM Grüner KK Takeuchi C Lubeseder-Martellato N Teichmann PK Mazur KE Delgiorno ES Carpenter CJ Halbrook JC Hall D Pal T Briel A Herner M Trajkovic-Arsic B Sipos GY Liou P Storz NR Murray DW Threadgill M Sibilia MK Washington CL Wilson RM Schmid EW Raines HC Crawford JT Siveke 《Cancer cell》2012,22(3):304-317
Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction. Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in?vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation. 相似文献
17.
Yang H Higgins B Kolinsky K Packman K Bradley WD Lee RJ Schostack K Simcox ME Kopetz S Heimbrook D Lestini B Bollag G Su F 《Cancer research》2012,72(3):779-789
The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation. 相似文献
18.
Dominic Leiser Michaela Medová Kei Mikami Lluís Nisa Deborah Stroka Andree Blaukat Friedhelm Bladt Daniel M. Aebersold Yitzhak Zimmer 《Molecular oncology》2015,9(7):1434-1446
The MET receptor tyrosine kinase is often deregulated in human cancers and several MET inhibitors are evaluated in clinical trials. Similarly to EGFR, MET signals through the RAS‐RAF‐ERK/MAPK pathway which plays key roles in cell proliferation and survival. Mutations of genes encoding for RAS proteins, particularly in KRAS, are commonly found in various tumors and are associated with constitutive activation of the MAPK pathway. It was shown for EGFR, that KRAS mutations render upstream EGFR inhibition ineffective in EGFR‐positive colorectal cancers. Currently, there are no clinical studies evaluating MET inhibition impairment due to RAS mutations. To test the impact of RAS mutations on MET targeting, we generated tumor cells responsive to the MET inhibitor EMD1214063 that express KRAS G12V, G12D, G13D and HRAS G12V variants. We demonstrate that these MAPK‐activating RAS mutations differentially interfere with MET‐mediated biological effects of MET inhibition. We report increased residual ERK1/2 phosphorylation indicating that the downstream pathway remains active in presence of MET inhibition. Consequently, RAS variants counteracted MET inhibition‐induced morphological changes as well as anti‐proliferative and anchorage‐independent growth effects. The effect of RAS mutants was reversed when MET inhibition was combined with MEK inhibitors AZD6244 and UO126. In an in vivo mouse xenograft model, MET‐driven tumors harboring mutated RAS displayed resistance to MET inhibition. Taken together, our results demonstrate for the first time in details the role of KRAS and HRAS mutations in resistance to MET inhibition and suggest targeting both MET and MEK as an effective strategy when both oncogenic drivers are expressed. 相似文献
19.
Naghibalhossaini F Hosseini HM Mokarram P Zamani M 《Pathology oncology research : POR》2011,17(4):819-825
Gene silencing due to DNA hypermethylation is a major mechanism for loss of tumor suppressor genes function in colorectal cancer. Activating V600E mutation in BRAF gene has been linked with widespread methylation of CpG islands in sporadic colorectal cancers. The aim of the present study was to evaluate the methylation status of three cancer-related genes, APC2, p14ARF, and ECAD in colorectal carcinogenesis and their association with the mutational status of BRAF and KRAS among Iranian colorectal cancer patients. DNA from 110 unselected series of sporadic colorectal cancer patients was examined for BRAF V600E mutation by PCR-RFLP. Promoter methylation of genes in tumors was determined by methylation specific PCR. The frequency of APC2, E-CAD, and p14 methylation was 92.6%, 40.4% and 16.7%, respectively. But, no V600E mutation was identified in the BRAF gene in any sample. No association was found in cases showing epigenetic APC, ECAD, and p14 abnormality with the clinicopathological parameters under study. The association between KRAS mutations and the so called methylator phenotype was previously reported. Therefore, we also analyzed the association between the hot spot KRAS gene mutations in codons of 12 and 13 with genes' promoter hypermethylation in a subset of this group of patients. Out of 86 tumors, KRAS was mutated in 24 (28%) of tumors, the majority occurring in codon 12. KRAS mutations were not associated with genes' methylation in this tumor series. These findings suggest a distinct molecular pathway for methylation of APC2, p14, and ECAD genes from those previously described for colorectal cancers with BRAF or KRAS mutations. 相似文献
20.
Rachel N. Grisham MD Gopa Iyer MD Karuna Garg MD Deborah DeLair MD David M. Hyman MD Qin Zhou MA Alexia Iasonos PhD Michael F. Berger PhD Fanny Dao BA David R. Spriggs MD Douglas A. Levine MD Carol Aghajanian MD David B. Solit MD 《Cancer》2013,119(3):548-554