RESUMO
Genetic alterations and deregulation of the miRNA biogenesis pathway components have been reported in human tumors. Tissue-specific deletion of the Dicer gene, which encodes an essential miRNA processing enzyme, promotes carcinogenesis in animal models. These features indicate that aberrant miRNA biogenesis components are directly associated with cancer. For the present study, we conducted quantitative RT-PCR of 14 genes that are related to the miRNA biogenesis pathway in 47 paired samples of primary hepatocellular carcinoma (HCC) and matched non-cancerous liver. Expression of seven genes (Dgcr8, p68, p72, Dicer, Ago3, Ago4 and Piwil4) was significantly decreased in primary HCC, especially in non-viral HCC subtypes, compared to the non-cancerous liver. Combinations of decreased expression of the miRNA biogenesis components in non-cancerous liver were related to cigarette smoking, alcohol intake and diabetes, which are known to be risk factors for HCC, and were also associated with the occurrence of multicentric tumors. Reduction of two of these genes (Dicer and p68) in HCC was associated with poor prognosis. Trimethylation of histone H3 lysine 27 in the promoters is implicated in the deregulation of these miRNA-biogenesis-related genes in non-HBV genome integrated HCC cell lines. In conclusion, deregulation of the miRNA biogenesis pathway components is frequently observed in non-viral-associated HCC and is linked to etiological risk factors and poor prognosis. Our study further showed that epigenetic regulation could be implicated in the deregulation of these genes during hepatocarcinogenesis.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Adulto , Idoso , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células Hep G2 , Histonas/genética , Humanos , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas , Fatores de RiscoRESUMO
Melanoma is the most deadly tumor of the skin, and systemic therapies for the advanced stage are still limited. Recent genetic analyses have revealed the molecular diversity of melanoma and potential therapeutic targets. By screening a cohort of 142 primary nonepithelial tumors, we discovered that about 10% of melanoma cases (4/39) harbored an IDH1 or IDH2 mutation. These mutations were found to coexist with BRAF or KIT mutation, and all IDH1 mutations were detected in metastatic lesions. BRAF-mutated melanoma cells, additionally expressing the cancer-related IDH1 mutant, acquired increased colony-forming and in vivo growth activities and showed enhanced activation of the MAPK and STAT3 pathways. Genome-wide gene expression profiling demonstrated that mutant IDH1 affected the expression of a set of genes. Especially, it caused the induction of growth-related transcriptional regulators (Jun, N-myc, Atf3) and the reduction of Rassf1 and two dehydrogenase genes (Dhrs1 and Adh5), which may be involved in the carcinogenesis of IDH1-mutated tumors. Our analyses demonstrate that IDH1 mutation works with other oncogenic mutations and could contribute to the metastasis in melanoma.
Assuntos
Isocitrato Desidrogenase/metabolismo , Melanoma/genética , Melanoma/patologia , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Análise Mutacional de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Melanoma/enzimologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.
Assuntos
Proteínas Culina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/metabolismo , Transporte Ativo do Núcleo Celular , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Modelos Moleculares , Mutação/genética , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Neoplasias/genética , Estresse Oxidativo , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Transcrição Gênica/genéticaRESUMO
The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome-wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine-treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine-treated recurrence cases, patients were categorized into "effective" and "non-effective" groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five "effective" cases and all four (100%) "non-effective" cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias do Sistema Biliar/tratamento farmacológico , Desoxicitidina/análogos & derivados , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias do Sistema Biliar/metabolismo , Desoxicitidina/uso terapêutico , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Proteínas Associadas aos Microtúbulos , Proteínas de Neoplasias , Organismos Livres de Patógenos Específicos , GencitabinaRESUMO
The tyrosine kinase (TK) family is an important regulator of signaling pathways that control a variety of physiological and pathological conditions, and a substantial proportion of TK genes are genetically altered in cancer. To clarify the somatic mutation profile of TK genes and discover potential targets for gastric cancer (GC) therapy, we undertook a systematic screening of mutations in the kinase domains of all human TK genes (636 exons of 90 genes) in 17 GC cell lines and 52 microdissected primary GCs with poorly differentiated histology. We identified 26 non-synonymous alterations (22 genes in total) that included 11 sequence alterations in cell lines and 15 somatic mutations in primary tumors. Recurrent mutations were found in four genes including a known oncogene (NTRK3), the Src kinase family (LTK and CSK) and a potential Wnt signal activator (ROR2). In addition, we analyzed copy number alterations of all the TK gene loci in the same cohort samples by array-based comparative genomic hybridization analysis and identified 24 high-level amplifications and two homozygous deletions. Both sequence alteration and frequent copy number aberration were detected in two TK genes (HCK and ERBB2), strongly suggesting that they encode potential oncogenes in GC. Our focused and integrated analyses of systemic resequencing and gene copy number have revealed the novel onco-kinome profile of GC and pave the way to a comprehensive understanding of the GC genome.
Assuntos
Mutação , Proteínas Tirosina Quinases/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Éxons , Dosagem de Genes , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Receptores Proteína Tirosina Quinases/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptor trkC/genética , Análise de Sequência , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Quinases da Família src/genéticaRESUMO
BACKGROUND & AIMS: Biliary tract cancer (BTC) is a highly malignant tumor, and identification of effective therapeutic targets to improve prognosis is urgently required. Oncogenic activation of survival genes is important for cancer cells to overcome oxidative stresses induced by their microenvironments that include chronic inflammation or exposure to anticancer drugs. We attempted to examine whether deregulation of Nrf2, a master transcriptional factor of various cytoprotective genes against oxidative stress, plays a role in the carcinogenesis of BTC. METHODS: We screened genetic alteration of Keap1, a negative regulator of Nrf2, in BTC including tumors originated from gallbladder and extra- and intrahepatic bile ducts. Functional analysis of cancer-related mutant Keap1 in Nrf2 repression and the association between Nrf2 activation and resistance to 5-fluorouracil (5-FU) were investigated. RESULTS: Recurrent (in 1/11 cell lines and 6/53 primary tumors) Keap1 gene alterations were observed in BTC and were especially frequent (4/13, 30.7%) in gallbladder cancer (GBC). These alterations led to a considerable loss of Nrf2 repression activity, caused constitutive activation of Nrf2, and promoted cell proliferation. Down-regulation of Nrf2 activity by either Keap1 complementation or Nrf2 short interference RNA increased sensitivity to 5-FU in Keap1-altered BTC cells. CONCLUSIONS: Keap1 mutation occurs frequently in GBC. Aberrant Nrf2 activation provoked by Keap1 alteration is one of the molecular mechanisms for chemotherapeutic resistance in GBC and will be a novel therapeutic target as an enhancer of sensitivity to 5-FU-based regimens.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias do Sistema Biliar/tratamento farmacológico , Neoplasias do Sistema Biliar/genética , Neoplasias do Sistema Biliar/metabolismo , Linhagem Celular Tumoral , DNA Complementar , Regulação para Baixo/fisiologia , Neoplasias da Vesícula Biliar/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Mutação , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND & AIMS: The biological aggressiveness of hepatocellular carcinoma (HCC) and the lack of optimal therapeutic strategies have rendered the disease a major challenge. Highly heterogeneous genetic alteration profiles of HCC have made it difficult to identify effective tailor-made molecular therapeutic targets. Therefore, classification of HCC into genetically homogeneous subclasses would be of great worth to develop novel therapeutic strategies. METHODS: We clarified genome-scale chromosomal copy number alteration profiles and mutational statuses of p53 and beta-catenin in 87 HCC tumors. We investigated the possibility that HCC might be classifiable into a number of homogeneous subclasses based solely on their genetic alteration profiles. We also explored putative molecular therapeutic targets specific for each HCC subgroup. RESULTS: Unsupervised hierarchical cluster analysis based on chromosomal alteration profiles suggested that HCCs with heterogeneous genetic backgrounds are divisible into homogeneous subclasses that are highly associated with a range of clinicopathologic features of the tumors and moreover with clinical outcomes of the patients (P < .05). These genetically homogeneous subclasses could be characterized distinctively by pathognomonic chromosomal amplifications (eg, c-Myc-induced HCC, 6p/1q-amplified HCC, and 17q-amplified HCC). An in vitro experiment raised a possibility that Rapamycin would significantly inhibit the proliferative activities of HCCs with 17q amplification. CONCLUSIONS: HCC is composed of several genetically homogeneous subclasses, each of which harbors characteristic genetic alterations that can be putative tailor-made molecular therapeutic targets for HCCs with specific genetic backgrounds. Our results offer an opportunity for developing novel individualized therapeutic modalities for distinctive genome types of HCC.
Assuntos
Carcinoma Hepatocelular/classificação , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/classificação , Neoplasias Hepáticas/genética , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Caspase 3/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Impressões Digitais de DNA , Feminino , Fatores de Transcrição Forkhead , Dosagem de Genes , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Família Multigênica/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , beta Catenina/genéticaRESUMO
BACKGROUND: High-resolution computed tomography (HRCT) of lung adenocarcinoma at early stage shows pure ground-glass opacity (GGO) and most cases of pure GGO remain stable during follow-up. There is no consensus on the strategy for follow-up. Identification of the molecular mechanisms that are associated with the natural history of lung adenocarcinoma should provide useful information. METHODS: Twenty-three lung adenocarcinomas that were followed-up for more than 6 months pre-operatively by HRCT were included in this study. Patterns of radiological changes during the follow-up period were classified into three groups; type 1, pure GGO without consolidation; type 2, appearance or increase in consolidation within pure GGO; type 3, consolidation without pure GGO. Mutational analysis of the epidermal growth factor receptor (EGFR) and K-ras genes and immunohistochemical staining of p53 protein were performed. RESULTS: EGFR mutations were found in 17 cases (74%), and there was no K-ras mutation. Positive staining of p53 was found in 8 cases (35%). As for radiological findings during the follow-up period, the frequencies of EGFR mutations and positive p53 staining were 67 and 0% in type 1 (n = 9), 89 and 44% in type 2 (n = 9) and 60 and 80% in type 3 (n = 5). CONCLUSIONS: EGFR mutations were frequently found in lung adenocarcinoma with GGO on HRCT in this study. Inactivation of p53 may be associated with the appearance of central consolidation within pure GGO on HRCT which reflects invasive features and may be useful as a molecular marker during the follow-up of pure GGO.
Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Receptores ErbB/genética , Neoplasias Pulmonares/patologia , Mutação , Tomografia Computadorizada Espiral , Proteína Supressora de Tumor p53/metabolismo , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/genética , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Seguimentos , Inativação Gênica , Genes ras , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estudos RetrospectivosRESUMO
The array-based comparative genomic hybridization using microarrayed bacterial artificial chromosome clones allows high-resolution analysis of genome-wide copy number changes in tumors. To analyze the genetic alterations of primary lung adenocarcinoma in a high-throughput way, we used laser-capture microdissection of cancer cells and array comparative genomic hybridization focusing on 800 chromosomal loci containing cancer-related genes. We identified a large number of chromosomal numerical alterations, including frequent amplifications on 7p12, 11q13, 12q14-15, and 17q21, and two homozygous deletions on 9p21 and one on 8p23. Unsupervised hierarchical clustering analysis of multiple alterations revealed three subgroups of lung adenocarcinoma that were characterized by the accumulation of distinct genetic alterations and associated with smoking history and gender. The mutation status of the epidermal growth factor receptor (EGFR) gene was significantly associated with specific genetic alterations and supervised clustering analysis based on EGFR gene mutations elucidated a subgroup including all EGFR gene mutated tumors, which showed significantly shorter disease-free survival. Our results suggest that there exist multiple molecular carcinogenesis pathways in lung adenocarcinoma that may associate with smoking habits and gender, and that genetic cancer profiling will reveal previously uncharacterized genetic heterogeneity of cancer and be beneficial in estimating patient prognosis and discovering novel cancer-related genes including therapeutic targets.
Assuntos
Adenocarcinoma/classificação , Adenocarcinoma/genética , Receptores ErbB/genética , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/genética , Hibridização de Ácido Nucleico , Adulto , Idoso , Cromossomos Artificiais Bacterianos , Cromossomos Humanos/genética , DNA de Neoplasias/genética , Feminino , Amplificação de Genes , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes erbB-1/fisiologia , Humanos , Hibridização in Situ Fluorescente , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Mutação/genéticaRESUMO
A hypothesis of multistep carcinogenesis of lung adenocarcinoma from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma through bronchioloalveolar carcinoma (BAC) has been proposed. However, the genetic alterations that play a role during these processes are not yet clear. Recently, somatic mutations of the epidermal growth factor receptor (EGFR) gene were found in lung adenocarcinoma. We examined the status of EGFR mutations in AAH and BAC to elucidate the role they play during multistage of lung adenocarcinoma. We found somatic EGFR mutations in 3% (1/35) of AAH, 10.8% (4/37) of BAC and 41.9% (13/31) of invasive adenocarcinoma. Sixteen of 18 EGFR mutations were found in exon 19 and two were in exon 21. Among the 16 EGFR mutations in exon 19, 13 were deletions of 15bp and one was an insertion/duplication of 18bp. Mutations of the K-ras gene were detected in 26.7% (8/30) of AAH, 16.7% (5/30) of BAC and 10% (3/30) of invasive adenocarcinoma. None of the tumors with EGFR mutations had K-ras mutation simultaneously. Patients who had invasive adenocarcinoma with EGFR mutations were younger than those without mutations (60.6 versus 67.4 years, p=0.03). These results suggest that tumors with EGFR mutations may progress more rapidly and develop into invasive cancer faster than those without mutations. Alternatively it is also possible that some invasive adenocarcinomas with EGFR mutations may not follow the AAH-adenocarcinoma sequence. We analyzed 24 patients with multiple lung lesions and 13 patients had at least one lesion that had either an EGFR or K-ras mutation. In all cases each lesion had a different mutation status. This finding suggests that the genetic alterations responsible for the development of lung adenocarcinoma occur randomly even under exposure to the same carcinogen.
Assuntos
Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma/genética , Transformação Celular Neoplásica/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/fisiopatologia , Adenocarcinoma Bronquioloalveolar/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinógenos , Análise Mutacional de DNA , Feminino , Humanos , Hiperplasia/genética , Hiperplasia/fisiopatologia , Pulmão/patologia , Neoplasias Pulmonares/fisiopatologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Esophageal squamous cancer (ESC) is one of the most aggressive tumors of the gastrointestinal tract. A combination of chemotherapy and radiation therapy (CRT) has improved the clinical outcome, but the molecular background determining the effectiveness of therapy remains unknown. NRF2 is a master transcriptional regulator of stress adaptation, and gain of-function mutation of NRF2 in cancer confers resistance to stressors including anticancer therapy. Direct resequencing analysis revealed that Nrf2 gain-of-function mutation occurred recurrently (18/82, 22%) in advanced ESC tumors and ESC cell lines (3/10). The presence of Nrf2 mutation was associated with tumor recurrence and poor prognosis. Short hairpin RNA-mediated down-regulation of NRF2 in ESC cells that harbor only mutated Nrf2 allele revealed that themutant NRF2 conferred increased cell proliferation, attachment-independent survival, and resistance to 5-fluorouracil and γ-irradiation. Based on the Nrf2 mutation status, gene expression signatures associated with NRF2 mutation were extracted from ESC cell lines, and their potential utility for monitoring and prognosis was examined in a cohort of 33 pre-CRT cases of ESC. The molecular signatures of NRF2 mutation were significantly predictive and prognostic for CRT response. In conclusion, recurrent NRF2 mutation confers malignant potential and resistance to therapy in advanced ESC, resulting in a poorer outcome. Molecular signatures of NRF2 mutation can be applied as predictive markers of response to CRT, and efficient inhibition of aberrant NRF2 activation could be a promising approach in combination with CRT.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Fator 2 Relacionado a NF-E2/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sequência de Bases , Biomarcadores Tumorais , Carcinoma de Células Escamosas/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Quimiorradioterapia , Regulação para Baixo , Neoplasias Esofágicas/patologia , Feminino , Fluoruracila/farmacologia , Raios gama , Humanos , Masculino , Mutação , Recidiva Local de Neoplasia , Prognóstico , Interferência de RNA , RNA Interferente Pequeno , Tolerância a Radiação/genética , Análise de Sequência de DNA , Resultado do TratamentoRESUMO
In multicellular organisms, adaptive responses to oxidative stress are regulated by NF-E2-related factor 2 (NRF2), a master transcription factor of antioxidant genes and phase II detoxifying enzymes. Aberrant activation of NRF2 by either loss-of-function mutations in the Keap1 gene or gain-of-function mutations in the Nrf2 gene occurs in a wide range of human cancers, but details of the biological consequences of NRF2 activation in the cancer cells remain unclear. Here, we report that mutant NRF2 induces epithelial cell proliferation, anchorage-independent growth, and tumorigenicity and metastasis in vivo. Genome-wide gene expression profiling revealed that mutant NRF2 affects diverse molecular pathways including the mammalian target of rapamycin (mTOR) pathway. Mutant NRF2 upregulates RagD, a small G-protein activator of the mTOR pathway, which was also overexpressed in primary lung cancer. Consistently, Nrf2-mutated lung cancer cells were sensitive to mTOR pathway inhibitors (rapamycin and NVP-BEZ235) in both in vitro and an in vivo xenograft model. The gene expression signature associated with mutant NRF2 was a marker of poor prognosis in patients with carcinoma of the head and neck region and lung. These results show that oncogenic Nrf2 mutation induces dependence on the mTOR pathway during carcinogenesis. Our findings offer a rationale to target NRF2 as an anticancer strategy, and they suggest NRF2 activation as a novel biomarker for personalized molecular therapies or prognostic assessment.
Assuntos
Perfilação da Expressão Gênica/métodos , Mutação , Fator 2 Relacionado a NF-E2/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Carga Tumoral/genéticaRESUMO
Lung cancer is one of the most prevalent cancers worldwide. This study focused on small cell lung cancer (SCLC), which has a poor clinical prognosis, and attempted to elucidate potential therapeutic molecular targets. A target-specific mutational search revealed mutation of the PIK3CA gene in three of 13 SCLC cell lines and two of 15 primary SCLCs. By introducing these mutant PIK3CA cDNAs, we established artificial "PIK3CA-addicted" cells and found that Tricribine, a small-molecule inhibitor of AKT signaling that is located downstream from PIK3CA, significantly inhibited the growth and colony formation activity of these cells. Using cancer cell lines, we further showed that PIK3CA-mutated SCLC cells are more sensitive to Tricribine than PIK3CA wild-type cells. Additionally, we found that a cisplatin-resistant subclone of PIK3CA-mutant SCLC cells was equally sensitive to Tricribine. This study for the first time uncovered PIK3CA alterations in SCLC, and our findings suggest that anti-AKT molecular therapy could be effective for a subgroup of SCLC, which shows activation of specific genes, such as PIK3CA mutation, and that genetic stratification of SCLC according to the activation status of individual therapeutic target pathways could be clinically beneficial, especially for chemotherapy-resistant/relapsing tumors.
Assuntos
Antineoplásicos/farmacologia , Clorpropamida/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Fosfatidilinositol 3-Quinases/genética , Carcinoma de Pequenas Células do Pulmão/genética , Sequência de Bases , Linhagem Celular Tumoral , Clorpropamida/farmacologia , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológicoRESUMO
OBJECTIVES: Pancreatic cancer is one of the most intractable of cancers. However, the comprehensive view of somatic mutations in this tumor is far from clear. The tyrosine kinase (TK) gene family, which encodes important regulators of various signal transduction pathways, is one of the most frequently altered gene families in human cancer. METHODS: To clarify the somatic mutation profile of TKs in pancreatic cancer, we performed a systematic screening of mutations in the kinase domains of all human TK genes (636 exons of 90 genes in total) in 11 pancreatic cancer cell lines and 29 microdissected primary tumors. RESULTS: We identified 15 nonsynonymous alterations that included 9 DNA alterations in cell lines and 6 somatic mutations in primary tumors. In particular, we identified the previously reported pathogenic mutation of NTRK3 in a KRAS/BRAF wild-type tumor and 2 somatic mutations in the Src family of kinases (YES1 and LYN) that would be expected to cause structural changes. CONCLUSIONS: Our genome-wide resequencing approach revealed novel oncogenic pathways in pancreatic cancers.
Assuntos
Mutação , Neoplasias Pancreáticas/genética , Proteínas Tirosina Quinases/genética , Análise de Sequência de DNA/métodos , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Predisposição Genética para Doença/genética , Testes Genéticos , Genoma Humano/genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Família Multigênica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas Proto-Oncogênicas p21(ras) , Receptor trkC/genética , Proteínas ras/genética , Quinases da Família src/genéticaRESUMO
We examined the genome-wide expression profiles of 86 primary lung adenocarcinomas and compared them with the mutation status of the four key molecules (EGFR, ERBB2, KRAS and BRAF) in the EGFR/KRAS/BRAF pathway. Unsupervised classification revealed two subtypes (the bronchial type and the alveolar type) of lung adenocarcinoma. Mutually exclusive somatic mutations of the epidermal growth factor receptor (EGFR) gene (36/86, 41.8%), K-ras gene (11/86, 12.8%) and BRAF gene (1/86, 1.1%) were detected. KRAS mutations were observed significantly frequently in bronchial-type tumors, whereas the frequencies of EGFR mutations were similar in both the alveolar and bronchial types. Twenty-seven genes showed increased expression in EGFR-mutated tumors and these included molecules that function in the EGFR/KRAS/BRAF pathway (EGFR, AKT1 and BCR). In particular, expression of BCR, which is required for EGFR protein degradation, was induced by EGF stimulation, suggesting a negative feedback loop in lung cancer. A subgroup of the alveolar type tumors showed significantly better prognosis than other tumors. Integrated analysis of genetic and gene expression profiling aimed to delineate inherent oncogenic pathways in cancer will be valuable not only for the understanding of molecular pathogenesis, but also for discovering novel biomarkers and predicting clinical outcome.
Assuntos
Adenocarcinoma/genética , Receptores ErbB/genética , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Linhagem Celular Tumoral , Feminino , Genes ras , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Mutação , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We analyzed the subchromosomal numerical aberrations of 44 surgically resected pancreatic adenocarcinomas by array-based comparative genomic hybridization. The aberration profile ranged widely between cases, suggesting the presence of multiple or complementary mechanisms of evolution in pancreatic cancer, and was associated with lymph node metastasis and venous or serosal invasion. A large number of small loci, previously uncharacterized in pancreatic cancer, showed non-random loss or gain. Frequent losses at 1p36, 4p16, 7q36, 9q34, 11p15, 11q13, 14q32-33, 16p13, 17p11-13, 17q11-25, 18q21-tel, 19p13, 21q22 and 22q11-12, and gains at 1q25, 2p16, 2q21-37, 3q25, 5p14, 5q11-13, 7q21, 7p22, 8p22, 8q21-23, 10q21, 12p13, 13q22, 15q13-22 and 18q11 were identified. Sixteen loci were amplified recurrently. We identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Gain of LUNX, HCK, E2F1 and DNMT3b at 20q11, loss of p73 at 1p36 and gain of PPM1D at 17q23 independently predicted patient outcome. Expression profiling of amplified genes identified Smurf1 and TRRAP at 7q22.1, BCAS1 at 20q13.2-3, and VCL at 10q22.1 as potential novel oncogenes. Our results contribute to a complete description of genomic structural aberrations and the identification of potential therapeutic targets and genetic indicators that predict patient outcome in pancreatic adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Aberrações Cromossômicas , Genoma Humano , Neoplasias Pancreáticas/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Cromossomos Humanos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização Genética , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
The aim of this study was to clarify the association between the epigenetic instability phenotype and the chromosomal instability phenotype in primary hepatocellular carcinoma (HCC). Sixty primary HCC tumors were examined. Methylation status for nine CpG islands (the p16, COX2, GSTP1, RASSF1A, E-cadherin, and APC gene promoters, and the MINT 1, 25, and 31 clones) was evaluated by methylation-specific polymerase chain reaction. Chromosomal structural alterations of these 60 HCC tumors were characterized in our previous study by using whole genomic array-based comparative genomic hybridization. We found that the epigenetic instability phenotype and the chromosomal instability phenotype are not mutually exclusive in hepatocarcinogenesis and that they do not show a simple cause-and-effect relationship. Hepatitis virus infection in the background liver was significantly associated with these instability phenotypes. Furthermore, we identified an epigenetic instability-dependent HCC that shows frequent epigenetic aberrations without chromosomal instability. It was noteworthy that epigenetic instability-positive and -negative HCCs displayed distinctive combinations of chromosomal structural alterations. In summary, by combined analyses of genetic and epigenetic aberration profiles in HCC, we obtained a comprehensive view of genomic alterations in hepatocarcinogenesis. Our results have clinical relevance because epigenetic instability-dependent HCCs may respond well to methylation inhibitory therapies.
Assuntos
Carcinoma Hepatocelular/genética , Instabilidade Cromossômica/genética , Neoplasias Hepáticas/genética , Fenótipo , Adulto , Idoso , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/virologia , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Feminino , Hepatite A/complicações , Hepatite A/virologia , Hepatite B/complicações , Hepatite B/virologia , Humanos , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Regiões Promotoras GenéticasRESUMO
Although activated Wnt/beta-catenin pathway is considered to be one of the main driving forces of hepatocarcinogenesis, no somatic mutations of the adenomatous polyposis coli (APC) gene have been found in sporadic hepatocellular carcinoma (HCC) to date. Here we present a case of a sporadic nodule-in-nodule-type HCC that provides the first evidence that biallelic genetic inactivation of the APC gene contributed to the development of the tumor. High-density array-based comparative genomic hybridization (aCGH) was performed to clarify genome-wide chromosomal structural alteration profiles of both early and advanced components of this nodule-in-nodule HCC. aCGH analysis revealed a chromosomal loss of the APC gene locus only in the inner advanced component of this tumor. Direct sequencing of the remaining allele revealed a nonsense mutation at codon 682 in the Armadillo repeats, resulting in a truncated protein that lacked all of the beta-catenin-binding motifs. Nonsense mutations at this location are rare among other types of cancer. In conclusion, combined with an immunohistochemical analysis of the beta-catenin protein, this case provides the first evidence that genetic inactivation of the APC gene can play a significant role in the progression of sporadic HCC, probably by activating the Wnt/beta-catenin pathway.
Assuntos
Carcinoma Hepatocelular/genética , Inativação Gênica , Genes APC , Neoplasias Hepáticas/genética , Alelos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 17/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , beta Catenina/metabolismoRESUMO
BACKGROUND/AIMS: We conducted an analysis of chromosomal numerical aberrations and their clinical significance in hepatocellular carcinoma. METHODS: We analyzed 87 hepatocellular carcinomas by array-based comparative genomic hybridization with an array containing 800 bacterial artificial chromosome clones. RESULTS: Frequent (>30%) chromosomal losses on 1p36.1, 4q21-25, 4q34-35.1, 8p23.3b-11.1, 13q14.1-14.3, 16p13.3, 16q22.1-24.3b, 17p13.3-13.1 and 17p13.3-11, and gains on 1q21-44f, 2q21.2, 2q34, 3q11.2, 5p14.2, 5q13.2-14, 7p22, 7p14.2, 7q21.1, 7q22.3, 7q34, 8q12-24.3 and 17q23, were observed. Recurrent (>5%) amplifications were detected on 1q25, 8q11 and 11q11, and we discovered a novel homozygous deletion at 14q32.11. The extent of chromosomal aberrations correlated significantly with various clinicopathological characteristics of the tumors, and increased in a stepwise manner with the progression of hepatocellular carcinoma. We also identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Multivariate analysis revealed that both chromosomal loss on 17p13.3 and gain on 8q11 are independent prognostic indicators. CONCLUSIONS: Our results contribute to a complete description of genomic structural aberrations in relation to hepatocarcinogenesis and provide a valuable basis from which we can begin to understand the characteristics of tumors, predict patient outcomes and discover novel therapeutic targets for hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/genética , Aberrações Cromossômicas , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Adulto , Idoso , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Cromossomos Humanos , Progressão da Doença , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Prognóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
We examined a large number of primary high-grade neuroendocrine tumors of the lung (10 small cell lung carcinomas and 31 large cell neuroendocrine carcinomas) by using array-based comparative genomic hybridization using microarrays spotted with 800 bacterial artificial chromosome clones containing tumor-related genes from throughout the human genome. We identified the genome-wide copy number alteration profiles of these tumors, including recurrent amplifications located at 2q21.2, 3q21-27, 3q26, 3q27-29, 5p14.2, 5p13, 7q21.1, 8q21, and 8q24 and homozygous deletions at 1p36, 4p16, 4p16.3, 9p21.3, 9p21, 19p13.3, and 20q13. Our results revealed that small cell lung carcinomas and large cell neuroendocrine carcinomas have multiple characteristic chromosomal alterations in common, but that distinctive alterations also exist between the two subtypes. Moreover, we found that the two subtypes undergo different processes of accumulating these genetic alterations during tumor development. By comparing the genetic profiles with the clinicopathological features, we discovered many chromosomal loci whose alterations were significantly associated with clinical stage and patient prognosis. These results will be valuable for evaluating clinical status, including patient prognosis, and for identifying novel molecular targets for effective therapies.