RESUMO
PURPOSE: We sought to define the prevalence and co-occurrence of actionable genomic alterations in patients with high-grade bladder cancer to serve as a platform for therapeutic drug discovery. PATIENTS AND METHODS: An integrative analysis of 97 high-grade bladder tumors was conducted to identify actionable drug targets, which are defined as genomic alterations that have been clinically validated in another cancer type (eg, BRAF mutation) or alterations for which a selective inhibitor of the target or pathway is under clinical investigation. DNA copy number alterations (CNAs) were defined by using array comparative genomic hybridization. Mutation profiling was performed by using both mass spectroscopy-based genotyping and Sanger sequencing. RESULTS: Sixty-one percent of tumors harbored potentially actionable genomic alterations. A core pathway analysis of the integrated data set revealed a nonoverlapping pattern of mutations in the RTK-RAS-RAF and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathways and regulators of G1-S cell cycle progression. Unsupervised clustering of CNAs defined two distinct classes of bladder tumors that differed in the degree of their CNA burden. Integration of mutation and copy number analyses revealed that mutations in TP53 and RB1 were significantly more common in tumors with a high CNA burden (P < .001 and P < .003, respectively). CONCLUSION: High-grade bladder cancer possesses substantial genomic heterogeneity. The majority of tumors harbor potentially tractable genomic alterations that may predict for response to target-selective agents. Given the genomic diversity of bladder cancers, optimal development of target-specific agents will require pretreatment genomic characterization.
Assuntos
Variações do Número de Cópias de DNA , Mutação , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Classe I de Fosfatidilinositol 3-Quinases , Fator de Transcrição E2F3/genética , Feminino , Amplificação de Genes , Genes erbB-2 , Genes p53 , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genéticaRESUMO
Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Sirolimo/análogos & derivados , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Ensaios Clínicos Fase II como Assunto , Códon sem Sentido , Intervalo Livre de Doença , Everolimo , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Terapia de Alvo Molecular , Complexos Multiproteicos , Metástase Neoplásica , Neurofibromina 2/genética , Proteínas/antagonistas & inibidores , Deleção de Sequência , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Alelos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Cetuximab , Neoplasias Colorretais/patologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Genes ras/genética , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Panitumumabe , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)RESUMO
PURPOSE: To compare the mutational and copy number profiles of primary and metastatic colorectal carcinomas (CRCs) using both unpaired and paired samples derived from primary and metastatic disease sites. PATIENTS AND METHODS: We performed a multiplatform genomic analysis of 736 fresh frozen CRC tumors from 613 patients. The cohort included 84 patients in whom tumor tissue from both primary and metastatic sites was available and 31 patients with pairs of metastases. Tumors were analyzed for mutations in the KRAS, NRAS, BRAF, PIK3CA, and TP53 genes, with discordant results between paired samples further investigated by analyzing formalin-fixed, paraffin-embedded tissue and/or by 454 sequencing. Copy number aberrations in primary tumors and matched metastases were analyzed by comparative genomic hybridization (CGH). RESULTS: TP53 mutations were more frequent in metastatic versus primary tumors (53.1% v 30.3%, respectively; P < .001), whereas BRAF mutations were significantly less frequent (1.9% v 7.7%, respectively; P = .01). The mutational status of the matched pairs was highly concordant (> 90% concordance for all five genes). Clonality analysis of array CGH data suggested that multiple CRC primary tumors or treatment-associated effects were likely etiologies for mutational and/or copy number profile differences between primary tumors and metastases. CONCLUSION: For determining RAS, BRAF, and PIK3CA mutational status, genotyping of the primary CRC is sufficient for most patients. Biopsy of a metastatic site should be considered in patients with a history of multiple primary carcinomas and in the case of TP53 for patients who have undergone interval treatment with radiation or cytotoxic chemotherapies.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/secundário , Variações do Número de Cópias de DNA , Mutação , Feminino , Perfilação da Expressão Gênica , Genes p53 , Humanos , Masculino , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
UNLABELLED: Effective oncoprotein-targeted therapies have not yet been developed for ovarian cancer. To explore the role of PI3 kinase/AKT signaling in this disease, we performed a genetic and functional analysis of ovarian cancer cell lines and tumors. PI3K pathway alterations were common in both, but the spectrum of mutational changes differed. Genetic activation of the pathway was necessary, but not sufficient, to confer sensitivity to selective inhibition of AKT and cells with RAS pathway alterations or RB1 loss were resistant to AKT inhibition, whether or not they had coexistent PI3K/AKT pathway activation. Inhibition of AKT1 caused growth arrest in a subset of ovarian cell lines, but not in those with AKT3 expression, which required pan-AKT inhibition. Thus, a subset of ovarian tumors are sensitive to AKT inhibition, but the genetic heterogeneity of the disease suggests that effective treatment with AKT pathway inhibitors will require a detailed molecular analysis of each patient's tumor. SIGNIFICANCE: A subset of ovarian cancers exhibits AKT pathway activation and is sensitive to selective AKT inhibition. Ovarian tumors exhibit significant genetic heterogeneity and thus an individualized approach based on real-time, detailed genomic and proteomic characterization of individual tumors will be required for the successful application of PI3K/AKT pathway inhibitors in this disease.
Assuntos
Cistadenocarcinoma Seroso/genética , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/enzimologia , Cistadenocarcinoma Seroso/patologia , Feminino , Genômica , Humanos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transplante HeterólogoRESUMO
Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.
Assuntos
Processamento Alternativo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Éxons/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/química , Sulfonamidas/farmacologia , VemurafenibRESUMO
FGFR3 mutations are common in low-grade urothelial carcinoma and represent a potential therapeutic target in this disease. Their incidence and functional role in high-grade urothelial carcinoma (HGUC), which displays an increased propensity for recurrence and muscularis propria invasion, is less well defined. We developed a mass spectrometry-based genotyping assay to define the incidence of FGFR3 mutations in a large clinically annotated set of urothelial carcinomas. FGFR3 mutations were found in 17% of HGUC versus 84% of low-grade lesions. Retrospective pathological review of the class of FGFR3 mutant HGUC revealed unique histological features, characterized by a bulky, exophytic component with branching papillary architecture as well as irregular nuclei with a koilocytoid appearance. The predictive value of this histological appearance was confirmed using a prospective set of 49 additional HGUCs. Prospective histological review was able to correctly predict for the presence of an FGFR3 mutation in 13/24 HGUC specimens that exhibited the distinct morphology (54%). All 25 specimens lacking the defined histological features were FGFR3 wild-type for a negative predictive value of 100%. Macrodissection of individual tumours confirmed the presence of the FGFR3 mutant allele in non-invasive and invasive, low and high-grade regions of individual tumours and in the lymph node metastases of patients whose tumours possessed the characteristic morphological signature, suggesting that FGFR3 mutations are not restricted to the more clinically indolent regions of HGUCs. These data suggest that histological screening of HGUCs followed by confirmatory genotyping can be used to enrich for the population of HGUCs most likely to harbour activating mutations in the FGFR-3 receptor tyrosine kinase. Histological review could thus aid in the development of targeted inhibitors of FGFR-3 by facilitating the identification of the subset of patients most likely to harbour activating mutations in the FGFR3 gene.
Assuntos
Mutação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Prognóstico , Estudos Retrospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise de Sobrevida , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Éxons , Genes ras , Mutação , Adenocarcinoma/enzimologia , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Hibridização Genômica Comparativa , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genótipo , Humanos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/biossíntese , Proteínas ras/genéticaRESUMO
Mutation of the gene PARK2, which encodes an E3 ubiquitin ligase, is the most common cause of early-onset Parkinson's disease. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2-q27 in cancer. Here we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes at the same residues, as the germline mutations causing familial Parkinson's disease. Cancer-specific mutations abrogate the growth-suppressive effects of the PARK2 protein. PARK2 mutations in cancer decrease PARK2's E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2-q27. Thus, PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells.
Assuntos
Neoplasias do Colo/genética , Glioblastoma/genética , Mutação , Ubiquitina-Proteína Ligases/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Dosagem de Genes , Variação Genética , Genótipo , Glioblastoma/patologia , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Doença de Parkinson/genética , Estrutura Terciária de Proteína , Transplante Heterólogo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1_G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET-positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors.
Assuntos
Carcinoma Papilar, Variante Folicular/genética , Radioisótopos do Iodo/uso terapêutico , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Carcinoma Papilar, Variante Folicular/diagnóstico por imagem , Carcinoma Papilar, Variante Folicular/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Progressão da Doença , Perfilação da Expressão Gênica , Genótipo , Humanos , Mutação/fisiologia , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/genética , Cintilografia , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Neoplasias da Glândula Tireoide/patologia , Falha de TratamentoRESUMO
Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Neoplasias Pulmonares/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/genéticaRESUMO
Survival signaling by RAF occurs through largely unknown mechanisms. Here we provide evidence for the first time that RAF controls cell survival by maintaining permissive levels of mitochondrial reactive oxygen species (ROS) and Ca(2+). Interleukin-3 (IL-3) withdrawal from 32D cells resulted in ROS production, which was suppressed by activated C-RAF. Oncogenic C-RAF decreased the percentage of apoptotic cells following treatment with staurosporine or the oxidative stress-inducing agent tert-butyl hydroperoxide. However, it was also the case that in parental 32D cells growing in the presence of IL-3, inhibition of RAF signaling resulted in elevated mitochondrial ROS and Ca(2+) levels. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca(2+), which was absent from cells expressing transforming C-RAF. Prevention of mitochondrial Ca(2+) overload after IL-3 deprivation increased cell viability. MEK was essential for the mitochondrial effects of RAF. In summary, our data show that survival control by C-RAF involves controlling ROS production, which otherwise perturbs mitochondrial Ca(2+) homeostasis.
Assuntos
Sinalização do Cálcio/fisiologia , Mitocôndrias/metabolismo , Proteínas Oncogênicas v-raf/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Homeostase , Humanos , Interleucina-3/farmacologia , Camundongos , Microscopia Confocal , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Proteínas Oncogênicas v-raf/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Estaurosporina/farmacologia , Superóxido Dismutase/genética , Superóxido Dismutase/fisiologia , terc-Butil Hidroperóxido/farmacologiaRESUMO
Mutations leading to activation of the RAF-mitogen-activated protein kinase/extracellular signal-regulated (ERK) kinase (MEK)-ERK pathway are key events in the pathogenesis of human malignancies. In a screen of 82 acute myeloid leukemia (AML) samples, 45 (55%) showed activated ERK and thus were further analyzed for mutations in B-RAF and C-RAF. Two C-RAF germ-line mutations, S427G and I448V, were identified in patients with therapy-related AML in the absence of alterations in RAS and FLT3. Both exchanges were located within the kinase domain of C-RAF. In vitro and in vivo kinase assays revealed significantly increased activity for (S427G)C-RAF but not for (I448V)C-RAF. The involvement of the S427G C-RAF mutation in constitutive activation of ERK was further confirmed through demonstration of activating phosphorylations on C-RAF, MEK, and ERK in neoplastic cells, but not in nonneoplastic cells. Transformation and survival assays showed oncogenic and antiapoptotic properties for both mutations. Screening healthy individuals revealed a <1/400 frequency of these mutations and, in the case of I448V, inheritance was observed over three generations with another mutation carrier suffering from cancer. Taken together, these data are the first to relate C-RAF mutations to human malignancies. As both mutations are of germ-line origin, they might constitute a novel tumor-predisposing factor.
Assuntos
Transformação Celular Neoplásica/genética , Mutação em Linhagem Germinativa , Leucemia Mieloide/genética , Segunda Neoplasia Primária/genética , Proteínas Proto-Oncogênicas c-raf/genética , Doença Aguda , Adulto , Idoso , Sequência de Aminoácidos , Animais , Apoptose/genética , Sequência de Bases , Células COS , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Leucêmica da Expressão Gênica/genética , Genes ras , Células HL-60 , Humanos , Leucemia Mieloide/enzimologia , Leucemia Mieloide/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Segunda Neoplasia Primária/enzimologia , Segunda Neoplasia Primária/patologia , Linhagem , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Alinhamento de Sequência , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Cancer results from genetic alterations in intracellular signaling pathways, which normally orchestrate the execution of developmental programs and the organismic response to extrinsic factors. Mutations in upstream activators and components of the cytoplasmic (Ras-Raf MEK-ERK) cascade frequently occur in tumors. In vitro and in vivo studies have shown that isolated activation of this pathway is both, necessary and sufficient for transformation. During the last years two new groups of related kinases have joined the ranks of mitogen-activated protein kinases, stress-activated protein kinases/Jun N-terminal kinases and p38. Their activation not only occurs during cellular responses to unphysiological stimuli but also downstream of cytokine and pathogen receptors and has been observed in tumors. In this article we will review the role of stress kinases in cancer, and discuss the mechanisms through which they regulate the transformation process.
Assuntos
Proteínas de Transporte/fisiologia , Transformação Celular Neoplásica , Proteínas de Choque Térmico/fisiologia , Neoplasias/enzimologia , Transdução de Sinais/fisiologia , MutaçãoRESUMO
Cell survival is maintained by growth factors and critically depends on sufficient energy supply. New evidence suggests that a rise in cellular energy production is not merely a homeostatic response to increased demand but subject to regulation by extrinsic factors. The mechanisms operating in this control are largely enigmatic. Work on transformed cells identified direct targeting of glycolytic enzymes by signaling proteins as one possibility. But mitochondrial oxidative phosphorylation and biogenesis may also be subject to regulation by growth and survival factors. Both, positive and negative regulators of cell survival impinge on the processes of cellular energy production to regulate growth and survival versus death.