Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
1.
Int J Clin Oncol ; 28(5): 654-663, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36856908

RESUMO

BACKGROUND: Oncogenic mutations in BRAF genes are found in approximately 5-10% of colorectal cancers. The majority of BRAF mutations are located within exons 11-15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation. METHODS: HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis. RESULTS: The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance. CONCLUSIONS: We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.


Assuntos
Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas c-akt , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Células HEK293 , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Mutação , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
2.
Br J Cancer ; 117(10): 1450-1458, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-28972961

RESUMO

BACKGROUND: Patients with BRAFV600E-mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAFV600E (BRAFnon-V600E mutations) contribute to anti-EGFR antibody resistance. METHODS: This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. RESULTS: In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAFV600E (6.0%), and 7 patients with BRAFnon-V600E mutations (4.7%), respectively. The response rates in RAS, BRAFV600E, and BRAFnon-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAFnon-V600E mutations was 2.4 months, similar to that in RAS or BRAFV600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). CONCLUSIONS: Although BRAFnon-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAFnon-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adulto , Idoso , Anticorpos Monoclonais/uso terapêutico , Cetuximab/uso terapêutico , Estudos de Coortes , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Intervalo Livre de Doença , Receptores ErbB/antagonistas & inibidores , Feminino , Genômica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Panitumumabe
3.
Cancer Sci ; 104(7): 896-903, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23578175

RESUMO

Rearrangements of the proto-oncogene RET are newly identified potential driver mutations in lung adenocarcinoma (LAD). However, the absence of cell lines harboring RET fusion genes has hampered the investigation of the biological relevance of RET and the development of RET-targeted therapy. Thus, we aimed to identify a RET fusion positive LAD cell line. Eleven LAD cell lines were screened for RET fusion transcripts by reverse transcription-polymerase chain reaction. The biological relevance of the CCDC6-RET gene products was assessed by cell growth, survival and phosphorylation of ERK1/2 and AKT with or without the suppression of RET expression using RNA interference. The efficacy of RET inhibitors was evaluated in vitro using a culture system and in an in vivo xenograft model. Expression of the CCDC6-RET fusion gene in LC-2/ad cells was demonstrated by the mRNA and protein levels, and the genomic break-point was confirmed by genomic DNA sequencing. Mutations in KRAS and EGFR were not observed in the LC-2/ad cells. CCDC6-RET was constitutively active, and the introduction of a siRNA targeting the RET 3' region decreased cell proliferation by downregulating RET and ERK1/2 phosphorylation. Moreover, treatment with RET-inhibitors, including vandetanib, reduced cell viability, which was accompanied by the downregulation of the AKT and ERK1/2 signaling pathways. Vandetanib exhibited anti-tumor effects in the xenograft model. Endogenously expressing CCDC6-RET contributed to cell growth. The inhibition of kinase activity could be an effective treatment strategy for LAD. LC-2/ad is a useful model for developing fusion RET-targeted therapy.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Proteínas do Citoesqueleto/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Mutação , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Quinazolinas/farmacologia , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
4.
Biomed Rep ; 11(4): 171-180, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31565223

RESUMO

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR: 4.30, 95% CI: 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR: 0.23, 95% CI: 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR: 4.43, 95% CI: 1.87-10.49; and P=0.0003, OR: 5.51, 95% CI: 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of <3 years allows the extraction of genomic DNA suitable for NGS.

5.
Genome Biol ; 16: 66, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25887790

RESUMO

BACKGROUND: To understand the heterogeneous behaviors of individual cancer cells, it is essential to investigate gene expression levels as well as their divergence between different individual cells. Recent advances in next-generation sequencing-related technologies have enabled us to conduct a single-cell RNA-Seq analysis of a series of lung adenocarcinoma cell lines. RESULTS: We analyze a total of 336 single-cell RNA-Seq libraries from seven cell lines. The results are highly robust regarding both average expression levels and the relative gene expression differences between individual cells. Gene expression diversity is characteristic depending on genes and pathways. Analyses of individual cells treated with the multi-tyrosine kinase inhibitor vandetanib reveal that, while the ribosomal genes and many other so-called house-keeping genes reduce their relative expression diversity during the drug treatment, the genes that are directly targeted by vandetanib, the EGFR and RET genes, remain constant. Rigid transcriptional control of these genes may not allow plastic changes of their expression with the drug treatment or during the cellular acquisition of drug resistance. Additionally, we find that the gene expression patterns of cancer-related genes are sometimes more diverse than expected based on the founder cells. Furthermore, we find that this diversity is occasionally latent in a normal state and initially becomes apparent after the drug treatment. CONCLUSIONS: Characteristic patterns in gene expression divergence, which would not be revealed by transcriptome analysis of bulk cells, may also play important roles when cells acquire drug resistance, perhaps by providing a cellular reservoir for gene expression programs.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Genes Essenciais , Humanos , Neoplasias Pulmonares/metabolismo , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Quinazolinas/farmacologia , RNA/genética , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma
6.
PLoS One ; 8(9): e73484, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069199

RESUMO

We analyzed whole-exome sequencing data from 97 Japanese lung adenocarcinoma patients and identified several putative cancer-related genes and pathways. Particularly, we observed that cancer-related mutation patterns were significantly different between different ethnic groups. As previously reported, mutations in the EGFR gene were characteristic to Japanese, while those in the KRAS gene were more frequent in Caucasians. Furthermore, during the course of this analysis, we found that cancer-specific somatic mutations can be detected without sequencing normal tissue counterparts. 64% of the germline variants could be excluded using a total of 217 external Japanese exome datasets. We also show that a similar approach may be used for other three ethnic groups, although the discriminative power depends on the ethnic group. We demonstrate that the ATM gene and the PAPPA2 gene could be identified as cancer prognosis related genes. By bypassing the sequencing of normal tissue counterparts, this approach provides a useful means of not only reducing the time and cost of sequencing but also analyzing archive samples, for which normal tissue counterparts are not available.


Assuntos
Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Adenocarcinoma de Pulmão , Povo Asiático/genética , Feminino , Humanos , Masculino , Mutação/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA