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1.
Cancer Sci ; 111(6): 2183-2195, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32237253

RESUMO

Molecular targeted therapies against EGFR and ALK have improved the quality of life of lung adenocarcinoma patients. However, targetable driver mutations are mainly found in thyroid transcription factor-1 (TTF-1)/NK2 homeobox 1 (NKX2-1)-positive terminal respiratory unit (TRU) types and rarely in non-TRU types. To elucidate the molecular characteristics of the major subtypes of non-TRU-type adenocarcinomas, we analyzed 19 lung adenocarcinoma cell lines (11 TRU types and 8 non-TRU types). A characteristic of non-TRU-type cell lines was the strong expression of TFF-1 (trefoil factor-1), a gastric mucosal protective factor. An immunohistochemical analysis of 238 primary lung adenocarcinomas resected at Jichi Medical University Hospital revealed that TFF-1 was positive in 31 cases (13%). Expression of TFF-1 was frequently detected in invasive mucinous (14/15, 93%), enteric (2/2, 100%), and colloid (1/1, 100%) adenocarcinomas, less frequent in acinar (5/24, 21%), papillary (7/120, 6%), and solid (2/43, 5%) adenocarcinomas, and negative in micropapillary (0/1, 0%), lepidic (0/23, 0%), and microinvasive adenocarcinomas or adenocarcinoma in situ (0/9, 0%). Expression of TFF-1 correlated with the expression of HNF4-α and MUC5AC (P < .0001, P < .0001, respectively) and inversely correlated with that of TTF-1/NKX2-1 (P < .0001). These results indicate that TFF-1 is characteristically expressed in non-TRU-type adenocarcinomas with gastrointestinal features. The TFF-1-positive cases harbored KRAS mutations at a high frequency, but no EGFR or ALK mutations. Expression of TFF-1 correlated with tumor spread through air spaces, and a poor prognosis in advanced stages. Moreover, the knockdown of TFF-1 inhibited cell proliferation and soft-agar colony formation and induced apoptosis in a TFF-1-high and KRAS-mutated lung adenocarcinoma cell line. These results indicate that TFF-1 is not only a biomarker, but also a potential molecular target for non-TRU-type lung adenocarcinomas.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Fator Trefoil-1/metabolismo , Adenocarcinoma de Pulmão/classificação , Adenocarcinoma de Pulmão/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade
2.
Cancer Sci ; 110(9): 2973-2981, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31293054

RESUMO

Every year, approximately 1.2 million cases of colorectal carcinoma (CRC) are newly diagnosed worldwide. Although metastases to distant organs are often fatal complications of CRC, little information is known as to how such metastatic lesions are formed. To reveal the genetic profiles for CRC metastasis, we conducted whole-exome RNA sequencing on CRC tumors with liver metastasis (LM) (group A, n = 12) and clinical stage-matched larger tumors without LM (group B, n = 16). While the somatic mutation profiles were similar among the primary tumors and LM lesions in group A and the tumors in group B, the A-to-C nucleotide change in the context of "AAG" was only enriched in the LM regions in group A, suggesting the presence of a DNA damage process specific to metastasis. Genes already known to be associated with CRC were mutated in all groups at a similar frequency, but we detected somatic nonsynonymous mutations in a total of 707 genes in the LM regions, but not in the tumors without LM. Signaling pathways linked to such "LM-associated" genes were overrepresented for extracellular matrix-receptor interaction or focal adhesion. Further, fusions of the ADAP1 (ArfGAP with dual PH domain 1) were newly identified in our cohort (3 out of 28 patients), which activated ARF6, an ADAP1-substrate. Infrequently, mutated genes may play an important role in metastasis formation of CRC. Additionally, recurrent ADAP1 fusion genes were unexpectedly discovered. As these fusions activate small GTPase, further experiments are warranted to examine their contribution to CRC carcinogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Fusão Gênica , Neoplasias Hepáticas/genética , Proteínas do Tecido Nervoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Sequenciamento do Exoma
3.
Cancer Sci ; 110(9): 3006-3011, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301084

RESUMO

Decreased cell adhesion has been reported as a significant negative prognostic factor of lung cancer. However, the molecular mechanisms responsible for the cell incohesiveness in lung cancer have not yet been elucidated in detail. We herein describe a rare histological variant of lung adenocarcinoma consisting almost entirely of individual cancer cells spreading in alveolar spaces in an incohesive pattern. A whole exome analysis of this case showed no genomic abnormalities in CDH1 or other genes encoding cell adhesion molecules. However, whole mRNA sequencing revealed that this case had an extremely high expression level of mucin 21 (MUC21), a mucin molecule that was previously shown to inhibit cell-cell and cell-matrix adhesion. The strong membranous expression of MUC21 was found on cancer cells using mAbs recognizing different O-glycosylated forms of MUC21. An immunohistochemical analysis of an unselected series of lung adenocarcinoma confirmed that the strong membranous expression of MUC21 correlated with incohesiveness. Thus, MUC21 could be a promising biomarker with potential diagnostic and therapeutic applications for lung adenocarcinoma showing cell incohesiveness.


Assuntos
Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Adenocarcinoma de Pulmão/diagnóstico por imagem , Adenocarcinoma de Pulmão/genética , Idoso , Antígenos CD/genética , Caderinas/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , Tomografia Computadorizada por Raios X , Sequenciamento do Exoma
4.
Clin Cancer Res ; 25(1): 378-389, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279230

RESUMO

PURPOSE: Colorectal cancers with microsatellite instability-high (MSI-H) status, due to mismatch repair deficiency, are associated with poor patient outcomes after relapse. We aimed to identify novel therapeutic targets for them. EXPERIMENTAL DESIGN: We performed MSI analyses of over 2,800 surgically resected colorectal tumors obtained from consecutive patients treated in Japan from 1998 through June 2016. Whole-exome sequencing, transcriptome sequencing, and methylation analyses were performed on 149 of 162 tumors showing MSI in BAT25 and BAT26 loci. We analyzed patient survival times using Bonferroni-adjusted log-rank tests. RESULTS: Sporadic MSI-H colorectal cancers with promoter methylation of MLH1 (called MM) had a clinicopathological profile that was distinct from that of colorectal cancers of patients with germline mutations (Lynch syndrome, LS-associated) or somatic, Lynch-like mutations in mismatch repair genes. MM tumors had more insertions and deletions and more recurrent mutations in BRAF and RNF43 than LS-associated or Lynch-like MSI-H tumors. Eleven fusion kinases were exclusively detected in MM MSI-H colorectal cancers lacking oncogenic KRAS/BRAF missense mutations and were associated with worse post-relapse prognosis. We developed a simple method to identify MM tumors and applied it to a validation cohort of 28 MSI-H colorectal cancers, identifying 16 MM tumors and 2 fusion kinases. CONCLUSIONS: We discovered that fusion kinases are frequently observed among sporadic MM MSI-H colorectal cancers. The new method to identify MM tumors enables us to straightforwardly group MSI-H patients into candidates of LS or fusion kinase carriers.


Assuntos
Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Instabilidade de Microssatélites , Recidiva Local de Neoplasia/genética , Idoso , Biomarcadores Tumorais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Metilação de DNA/genética , Intervalo Livre de Doença , Feminino , Marcadores Genéticos , Genômica , Mutação em Linhagem Germinativa/genética , Humanos , Japão/epidemiologia , Masculino , Repetições de Microssatélites , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Ubiquitina-Proteína Ligases/genética
5.
Oncogene ; 37(21): 2757-2772, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29511349

RESUMO

Tumor microenvironment plays a pivotal role in cancer progression; however, little is known regarding how differences in the microenvironment affect characteristics of cancer cells. Here, we investigated the effects of tumor microenvironment on cancer cells by using mouse tumor models. After three cycles of inoculation and extraction of human pancreatic cancer cells, including SUIT-2 and Panc-1 cells, from tumors, distinct cancer cell lines were established: 3P cells from the pancreas obtained using the orthotopic tumor model and 3sc cells from subcutaneous tissue obtained using the subcutaneous tumor model. On re-inoculation of these cells, the 3sc cells and, more prominently, the 3P cells, exhibited higher tumorigenic activity than the parental cells. The 3P cells specifically exhibited low E-cadherin expression and high invasiveness, suggesting that they were endowed with the highest malignant characteristics. RNA-sequence analysis demonstrated that distinct signaling pathways were activated in each cell line and that the 3P cells acquired a cancer stem cell-like phenotype. Among cancer stem cell-related genes, those specifically expressed in the 3P cells, including NES, may be potential new targets for cancer therapy. The mechanisms underlying the development of highly malignant cancer cell lines were investigated. Individual cell clones within the parental cells varied in tumor-forming ability, indicating the presence of cellular heterogeneity. Moreover, the tumor-forming ability and the gene expression profile of each cell clone were altered after serial orthotopic inoculations. The present study thus suggests that both selection and education processes by tumor microenvironment are involved in the development of highly malignant cancer cells.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Análise de Sequência de RNA , Transdução de Sinais
6.
Int J Cancer ; 142(8): 1627-1639, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29193056

RESUMO

Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA-seq data from TGS-01 human glioma-initiating cells, and identified a novel fusion gene (HMGA2-EGFR), encoding a protein comprising the N-terminal region of the high-mobility group AT-hook protein 2 (HMGA2) fused to the C-terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor-forming capacity in cell culture and in vivo. Mechanistically, HMGA2-EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in-frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2-EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS-01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in-frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma.


Assuntos
Receptores ErbB/genética , Glioblastoma/genética , Proteína HMGA2/genética , Proteínas de Fusão Oncogênica/genética , Idoso , Animais , Linhagem Celular , Linhagem Celular Tumoral , Éxons/genética , Feminino , Amplificação de Genes/genética , Deleção de Genes , Glioma/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação/genética , Transdução de Sinais/genética
7.
Cancer Sci ; 108(9): 1888-1896, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28677170

RESUMO

The major driver mutations of lung cancer, EGFR mutations and EML4-ALK fusion, are mainly detected in terminal respiratory unit (TRU)-type lung adenocarcinomas, which typically show lepidic and/or papillary patterns, but are rarely associated with a solid or invasive mucinous morphology. In order to elucidate the key genetic events in non-TRU-type lung cancer, we carried out whole-exome sequencing on 43 non-TRU-type lung adenocarcinomas based on morphology (17 acinar, nine solid, and two enteric adenocarcinomas, and 15 adenocarcinomas with a mucinous morphology). Our analysis identified mutations in TP53 (16/43, 37.2%), KRAS (13/43, 30.2%), and NKX2-1/TTF-1 (7/43; 16.3%) as the top three significantly mutated genes, while the EGFR mutation was rare (1/43, 2.3%) in this cohort. Eight NKX2-1/TTF-1 mutations (five frameshift, two nonsense, and one missense) were identified, with one case harboring two distinct NKX2-1/TTF-1 mutations (one missense and one frameshift). Functional assays with the NK2 homeobox 1 (NKX2-1)/thyroid transcription factor 1 (TTF-1) mutants revealed that none of them retain the activity as a transcriptional factor. Histologically, invasive mucinous adenocarcinomas accounted for most of the NKX2-1/TTF-1 mutations (five cases), as well as one enteric and one acinar adenocarcinoma. Immunohistochemistry showed that the cohort was largely divided into TTF-1-postive/hepatocyte nuclear factor 4-α (HNF4-α)-negative and TTF-1-negative/HNF4-α-positive groups. NKX2-1/TTF-1 mutations were exclusively found in the latter, in which the gastrointestinal markers, mucin 5AC and cytokeratin 20, were frequently expressed. Bisulfite sequencing revealed that the NKX2-1/TTF-1 gene body was highly methylated in NKX2-1/TTF-1-negative cases, including those without the NKX2-1/TTF-1 mutations. The genetic or epigenetic inactivation of NKX2-1/TTF-1 may play an essential role in the development and aberrant differentiation of non-TRU-type lung adenocarcinomas.


Assuntos
Adenocarcinoma/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Metilação de DNA , Análise Mutacional de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Humanos , Neoplasias Pulmonares/patologia , Mutação , Fator Nuclear 1 de Tireoide
8.
PLoS Genet ; 13(6): e1006853, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28636652

RESUMO

Triple-negative breast cancer (TNBC) cells do not express estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2. Currently, apart from poly ADP-ribose polymerase inhibitors, there are few effective therapeutic options for this type of cancer. Here, we present comprehensive characterization of the genetic alterations in TNBC performed by high coverage whole genome sequencing together with transcriptome and whole exome sequencing. Silencing of the BRCA1 gene impaired the homologous recombination pathway in a subset of TNBCs, which exhibited similar phenotypes to tumors with BRCA1 mutations; they harbored many structural variations (SVs) with relative enrichment for tandem duplication. Clonal analysis suggested that TP53 mutations and methylation of CpG dinucleotides in the BRCA1 promoter were early events of carcinogenesis. SVs were associated with driver oncogenic events such as amplification of MYC, NOTCH2, or NOTCH3 and affected tumor suppressor genes including RB1, PTEN, and KMT2C. Furthermore, we identified putative TGFA enhancer regions. Recurrent SVs that affected the TGFA enhancer region led to enhanced expression of the TGFA oncogene that encodes one of the high affinity ligands for epidermal growth factor receptor. We also identified a variety of oncogenes that could transform 3T3 mouse fibroblasts, suggesting that individual TNBC tumors may undergo a unique driver event that can be targetable. Thus, we revealed several features of TNBC with clinically important implications.


Assuntos
Proteína BRCA1/genética , Transcriptoma/genética , Neoplasias de Mama Triplo Negativas/genética , Proteína Supressora de Tumor p53/genética , Células 3T3 , Animais , Metilação de DNA/genética , Exoma/genética , Feminino , Amplificação de Genes , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Recombinação Homóloga/genética , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Neoplasias de Mama Triplo Negativas/patologia
9.
Oncotarget ; 8(25): 41474-41486, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28467813

RESUMO

BACKGROUND: A germline deletion in the BIM (BCL2L11) gene has been shown to impair the apoptotic response to tyrosine kinase inhibitors (TKIs) in vitro but its association with poor outcomes in TKI-treated non-small cell lung cancer (NSCLC) patients remains unclear. We conducted a systematic review and meta-analysis on both aggregate and individual patient data to address this issue. RESULTS: In an aggregate data meta-analysis (n = 1429), the BIM deletion was associated with inferior PFS (HR = 1.51, 95%CI = 1.06-2.13, P = 0.02). Using individual patient data (n = 1200), we found a significant interaction between the deletion and ethnicity. Amongst non-Koreans, the deletion was an independent predictor of shorter PFS (Chinese: HR = 1.607, 95%CI = 1.251-2.065, P = 0.0002; Japanese: HR = 2.636, 95%CI = 1.603-4.335, P = 0.0001), and OS (HR = 1.457, 95% CI = 1.063-1.997, P = 0.019). In Kaplan-Meier analyses, the BIM deletion was associated with shorter survival in non-Koreans (PFS: 8.0 months v 11.1 months, P < 0.0005; OS: 25.7 v 30.0 months, P = 0.042). In Koreans, the BIM deletion was not predictive of PFS or OS. MATERIALS AND METHODS: 10 published and 3 unpublished studies that reported survival outcomes in NSCLC patients stratified according to BIM deletion were identified from PubMed and Embase. Summary risk estimates were calculated from aggregate patient data using a random-effects model. For individual patient data, Kaplan-Meier analyses were supported by multivariate Cox regression to estimate hazard ratios (HRs) for PFS and OS. CONCLUSIONS: In selected populations, the BIM deletion is a significant predictor of shorter PFS and OS on EGFR-TKIs. Further studies to determine its effect on response to other BIM-dependent therapeutic agents are needed, so that alternative treatment strategies may be devised.


Assuntos
Proteína 11 Semelhante a Bcl-2/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Intervalo Livre de Doença , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Mutação , Polimorfismo Genético , Resultado do Tratamento
10.
Cancer Res ; 76(7): 1814-24, 2016 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-26921333

RESUMO

ELF4 (also known as MEF) is a member of the ETS family of transcription factors. An oncogenic role for ELF4 has been demonstrated in hematopoietic malignancies, but its function in epithelial tumors remains unclear. Here, we show that ELF4 can function as a tumor suppressor and is somatically inactivated in a wide range of human tumors. We identified a missense mutation affecting the transactivation potential of ELF4 in oral squamous cell carcinoma cells. Restoration of the transactivation activity through introduction of wild-type ELF4 significantly inhibited cell proliferation in vitro and tumor xenograft growth. Furthermore, we found that ELF1 and ELF2, closely related transcription factors to ELF4, also exerted antiproliferative effects in multiple cancer cell lines. Mutations in ELF1 and ELF2, as in ELF4, were widespread across human cancers, but were almost all mutually exclusive. Moreover, chromatin immunoprecipitation coupled with high-throughput sequencing revealed ELF4-binding sites in genomic regions adjacent to genes related to cell-cycle regulation and apoptosis. Finally, we provide mechanistic evidence that the antiproliferative effects of ELF4 were mediated through the induction of HRK, an activator of apoptosis, and DLX3, an inhibitor of cell growth. Collectively, our findings reveal a novel subtype of human cancer characterized by inactivating mutations in the ELF subfamily of proteins, and warrant further investigation of the specific settings where ELF restoration may be therapeutically beneficial. Cancer Res; 76(7); 1814-24. ©2016 AACR.


Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias/genética , Fatores de Transcrição/genética , Humanos , Mutação
11.
Acta Neuropathol ; 131(6): 865-75, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26757737

RESUMO

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to the central nervous system (CNS), and majority of PCNSL is pathologically classified as diffuse large B-cell lymphoma (DLBCL). We have now performed whole-exome sequencing for 41 tumor tissues of DLBCL-type PCNSL and paired normal specimens and also RNA-sequencing for 30 tumors, revealing a very high frequency of nonsynonymous somatic mutations in PIM1 (100 %), BTG2 (92.7 %), and MYD88 (85.4 %). Many genes in the NF-κB pathway are concurrently mutated within the same tumors. Further, focal deletion or somatic mutations in the HLA genes are associated with poor prognosis. Copy number amplification and overexpression of genes at chromosome 7q35 were both found to predict short progression-free survival as well. Oncogenic mutations in GRB2 were also detected, the effects of which in cultured cells were attenuated by inhibitors of the downstream kinases MAP2K1 and MAP2K2. Individuals with tumors positive for MYD88 mutations also harbored the same mutations at a low frequency in peripheral blood mononuclear cells, suggesting that MYD88 mutation-positive precancerous cells originate outside of the CNS and develop into lymphoma after additional genetic hits that confer adaptation to the CNS environment.


Assuntos
Neoplasias do Sistema Nervoso Central/genética , Regulação Neoplásica da Expressão Gênica/genética , Linfoma Difuso de Grandes Células B/genética , Mutação/genética , Intervalo Livre de Doença , Genômica/métodos , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma Difuso de Grandes Células B/patologia , NF-kappa B/genética , Sistema Nervoso/patologia
13.
Cancer Sci ; 106(12): 1687-92, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26432419

RESUMO

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts downstream of the phosphatidylinositol 3-kinase signaling pathway and regulates a wide range of cellular functions including transcription, translation, proliferation, apoptosis, and autophagy. Whereas genetic alterations that result in mTOR activation are frequently present in human cancers, whether the mTOR gene itself becomes an oncogene through somatic mutation has remained unclear. We have now identified a somatic non-synonymous mutation of mTOR that results in a leucine-to-valine substitution at amino acid position 2209 in a specimen of large cell neuroendocrine carcinoma. The mTOR(L2209V) mutant manifested marked transforming potential in a focus formation assay with mouse 3T3 fibroblasts, and it induced the phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eukaryotic translation initiation factor 4E-binding protein 1 in these cells. Examination of additional tumor specimens as well as public and in-house databases of cancer genome mutations identified another 28 independent non-synonymous mutations of mTOR in various cancer types, with 12 of these mutations also showing transforming ability. Most of these oncogenic mutations cluster at the interface between the kinase domain and the FAT (FRAP, ATM, TRRAP) domain in the 3-D structure of mTOR. Transforming mTOR mutants were also found to promote 3T3 cell survival, and their oncogenic activity was sensitive to rapamycin. Our data thus show that mTOR acquires transforming activity through genetic changes in cancer, and they suggest that such tumors may be candidates for molecularly targeted therapy with mTOR inhibitors.


Assuntos
Transformação Celular Neoplásica/genética , Mutação , Neoplasias/genética , Oncogenes/genética , Serina-Treonina Quinases TOR/genética , Células 3T3 , Animais , Humanos , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Cancer Sci ; 106(9): 1137-42, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26094954

RESUMO

BIRC2 and BIRC3 are closely related members of the inhibitor of apoptosis (IAP) family of proteins and play pivotal roles in regulation of nuclear factor-κB (NF-κB) signaling and apoptosis. Copy number loss for and somatic mutation of BIRC2 and BIRC3 have been frequently detected in lymphoid malignancies, with such genetic alterations being thought to contribute to carcinogenesis through activation of the noncanonical NF-κB signaling pathway. Here we show that BIRC2 and BIRC3 mutations are also present in a wide range of epithelial tumors and that most such nonsense or frameshift mutations confer direct transforming potential. This oncogenic function of BIRC2/3 mutants is largely independent of their ability to activate NF-κB signaling. Rather, all of the transforming mutants lack an intact RING finger domain, with loss of ubiquitin ligase activity being essential for transformation irrespective of NF-κB regulation. The serine-threonine kinase NIK was found to be an important, but not exclusive, mediator of BIRC2/3-driven carcinogenesis, although this function was independent of NF-κB activation. Our data thus suggest that, in addition to the BIRC2/3-NIK-NF-κB signaling pathway, BIRC2/3-NIK signaling targets effectors other than NF-κB and thereby contributes directly to carcinogenesis. Identification of these effectors may provide a basis for the development of targeted agents for the treatment of lymphoid malignancies and other cancers with BIRC2/3 alterations.


Assuntos
Carcinogênese/genética , Mutação da Fase de Leitura/genética , Proteínas Inibidoras de Apoptose/genética , NF-kappa B/genética , Ubiquitina-Proteína Ligases/genética , Células 3T3 , Animais , Apoptose/genética , Linhagem Celular , Células HEK293 , Humanos , Linfócitos/patologia , Camundongos , Proteínas Serina-Treonina Quinases/genética , Domínios RING Finger/genética , Transdução de Sinais/genética
15.
Sci Rep ; 5: 10434, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-26000717

RESUMO

Age-associated accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for the age-associated mitochondrial respiration defects found in elderly human subjects. We carried out reprogramming of human fibroblast lines derived from elderly subjects by generating their induced pluripotent stem cells (iPSCs), and examined another possibility, namely that these aging phenotypes are controlled not by mutations but by epigenetic regulation. Here, we show that reprogramming of elderly fibroblasts restores age-associated mitochondrial respiration defects, indicating that these aging phenotypes are reversible and are similar to differentiation phenotypes in that both are controlled by epigenetic regulation, not by mutations in either the nuclear or the mitochondrial genome. Microarray screening revealed that epigenetic downregulation of the nuclear-coded GCAT gene, which is involved in glycine production in mitochondria, is partly responsible for these aging phenotypes. Treatment of elderly fibroblasts with glycine effectively prevented the expression of these aging phenotypes.


Assuntos
Aciltransferases/genética , Envelhecimento , Epigênese Genética , Glicina Hidroximetiltransferase/genética , Lipase/genética , Mitocôndrias/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Idoso de 80 Anos ou mais , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Criança , DNA Mitocondrial/análise , Fibroblastos/citologia , Fibroblastos/metabolismo , Dosagem de Genes , Glicina/biossíntese , Glicina Hidroximetiltransferase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Lipase/antagonistas & inibidores , Lipase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Consumo de Oxigênio , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
16.
Jpn J Clin Oncol ; 44(6): 593-6, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24688086

RESUMO

It is widely recognized that the risk of secondary neoplasms increases as childhood cancer survivors progress through adulthood. These are mainly hematological malignancies, and recurrent chromosome translocations are commonly detected in such cases. On the other hand, while secondary epithelial malignancies have sometimes been reported, chromosome translocations in these epithelial malignancies have not. A 33-year-old man who had been diagnosed with acute lymphoblastic leukemia and treated with chemotherapy almost 20 years earlier was diagnosed with lung adenocarcinoma. After chromosomal rearrangement of echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene was detected in this adenocarcinoma, he responded to treatment with crizotinib. It was therefore concluded that this echinoderm microtubule-associated protein-like 4 gene-anaplastic lymphoma kinase gene-positive lung adenocarcinoma was a secondary epithelial malignancy.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Terapia de Alvo Molecular , Segunda Neoplasia Primária/tratamento farmacológico , Proteínas de Fusão Oncogênica/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Adenocarcinoma/química , Adenocarcinoma de Pulmão , Adulto , Crizotinibe , Humanos , Neoplasias Pulmonares/química , Masculino , Sobreviventes , Resultado do Tratamento
17.
BMC Cancer ; 13: 262, 2013 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-23714228

RESUMO

BACKGROUND: The EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion oncogene represents a novel molecular target in a small subset of non-small-cell lung cancers (NSCLCs). The EML4-ALK fusion gene occurs generally in NSCLC without mutations in epidermal growth factor receptor (EGFR) and KRAS. CASE PRESENTATION: We report that a case of EML4-ALK-positive NSCLC with EGFR mutation had a response of stable disease to both an EGFR tyrosine kinase inhibitor (EGFR-TKI) and ALK inhibitor. CONCLUSIONS: We described the first clinical report of a patient with EML4-ALK-positive NSCLC with EGFR mutation that had a response of stable disease to both single-agent EGFR-TKI and ALK inhibitor. EML4-ALK translocation may be associated with resistance to EGFR-TKI, and EGFR signaling may contribute to resistance to ALK inhibitor in EML4-ALK-positive NSCLC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Quinase do Linfoma Anaplásico , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/antagonistas & inibidores , Feminino , Genes erbB-1 , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores
18.
Cancer Sci ; 104(8): 1002-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23659359

RESUMO

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a 5-year survival rate of ~50%. With the use of a custom cDNA-capture system coupled with massively parallel sequencing, we have now investigated transforming mechanisms for this malignancy. The cDNAs of cancer-related genes (n = 906) were purified from a human HNSCC cell line (T3M-1 Cl-10) and subjected to high-throughput resequencing, and the clinical relevance of non-synonymous mutations thus identified was evaluated with luciferase-based reporter assays. A CASP8 (procaspase-8) cDNA with a novel G-to-C point mutation that results in the substitution of alanine for glycine at codon 325 was identified, and the mutant protein, CASP8 (G325A), was found to activate nuclear factor-κB (NF-κB) signaling to an extent far greater than that achieved with the wild-type protein. Moreover, forced expression of wild-type CASP8 suppressed the growth of T3M-1 Cl-10 cells without notable effects on apoptosis. We further found that most CASP8 mutations previously detected in various epithelial tumors also increase the ability of the protein to activate NF-κB signaling. Such NF-κB activation was shown to be mediated through the COOH-terminal region of the second death effector domain of CASP8. Although CASP8 mutations associated with cancer have been thought to promote tumorigenesis as a result of attenuation of the proapoptotic function of the protein, our results now show that most such mutations, including the novel G325A identified here, separately confer a gain of function with regard to activation of NF-κB signaling, indicating another role of CASP8 in the transformation of human malignancies including HNSCC.


Assuntos
Carcinoma de Células Escamosas/genética , Caspase 8/genética , Neoplasias de Cabeça e Pescoço/genética , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Apoptose/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço
19.
Proc Natl Acad Sci U S A ; 110(8): 3029-34, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23382236

RESUMO

Members of the RAS superfamily of small guanosine triphosphatases (GTPases) transition between GDP-bound, inactive and GTP-bound, active states and thereby function as binary switches in the regulation of various cellular activities. Whereas HRAS, NRAS, and KRAS frequently acquire transforming missense mutations in human cancer, little is known of the oncogenic roles of other small GTPases, including Ras-related C3 botulinum toxin substrate (RAC) proteins. We show that the human sarcoma cell line HT1080 harbors both NRAS(Q61K) and RAC1(N92I) mutant proteins. Whereas both of these mutants were able to transform fibroblasts, knockdown experiments indicated that RAC1(N92I) may be the essential growth driver for this cell line. Screening for RAC1, RAC2, or RAC3 mutations in cell lines and public databases identified several missense mutations for RAC1 and RAC2, with some of the mutant proteins, including RAC1(P29S), RAC1(C157Y), RAC2(P29L), and RAC2(P29Q), being found to be activated and transforming. P29S, N92I, and C157Y mutants of RAC1 were shown to exist preferentially in the GTP-bound state as a result of a rapid transition from the GDP-bound state, rather than as a result of a reduced intrinsic GTPase activity. Activating mutations of RAC GTPases were thus found in a wide variety of human cancers at a low frequency; however, given their marked transforming ability, the mutant proteins are potential targets for the development of new therapeutic agents.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Mutação , Neoplasias/enzimologia , Proteínas rac de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/genética , Humanos , Modelos Moleculares , Proteínas rac de Ligação ao GTP/genética
20.
Clin Cancer Res ; 18(20): 5682-9, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22908099

RESUMO

PURPOSE: EML4-ALK is a lung cancer oncogene, and ALK inhibitors show marked therapeutic efficacy for tumors harboring this fusion gene. It remains unsettled, however, how the fusion gene should be detected in specimens other than formalin-fixed, paraffin-embedded tissue. We here tested whether reverse transcription PCR (RT-PCR)-based detection of EML4-ALK is a sensitive and reliable approach. EXPERIMENTAL DESIGN: We developed a multiplex RT-PCR system to capture ALK fusion transcripts and applied this technique to our prospective, nationwide cohort of non-small cell lung cancer (NSCLC) in Japan. RESULTS: During February to December 2009, we collected 916 specimens from 853 patients, quality filtering of which yielded 808 specimens of primary NSCLC from 754 individuals. Screening for EML4-ALK and KIF5B-ALK with our RT-PCR system identified EML4-ALK transcripts in 36 samples (4.46%) from 32 individuals (4.24%). The RT-PCR products were detected in specimens including bronchial washing fluid (n = 11), tumor biopsy (n = 8), resected tumor (n = 7), pleural effusion (n = 5), sputum (n = 4), and metastatic lymph node (n = 1). The results of RT-PCR were concordant with those of sensitive immunohistochemistry with ALK antibodies. CONCLUSIONS: Multiplex RT-PCR was confirmed to be a reliable technique for detection of ALK fusion transcripts. We propose that diagnostic tools for EML4-ALK should be selected in a manner dependent on the available specimen types. FISH and sensitive immunohistochemistry should be applied to formalin-fixed, paraffin-embedded tissue, but multiplex RT-PCR is appropriate for other specimen types.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas de Fusão Oncogênica/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Japão , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Estudos Prospectivos
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