RESUMO
We developed inducible and constitutive expression systems of Ha-RasV12 in HEK 293 cells to examine early oncogenic RasV12 signaling. Inducible expression of oncogenic Ras-triggered growth arrest, early senescence, and later apoptosis. Gene expression profile analysis revealed early Ras proliferation and cell cycle genes like c-fos, cyclin E, cdk2, cell-cell contact, and signaling like integrin a6, MEK5, and free radical signaling genes, like proline oxidase. Therefore, Ras-mediated signaling is a fine regulated process both positively and negatively influencing cell cycle, senescence, and apoptosis pathways. Novel early RAS-target genes could be potentially exploited in cancer diagnostics and therapeutics.
Assuntos
Apoptose/genética , Transformação Celular Neoplásica/genética , Senescência Celular/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes ras , Proteínas ras/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ativação Enzimática , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Tempo , Transfecção , Proteínas ras/metabolismoRESUMO
In colorectal cancer, BRAF and KRAS oncogenes are mutated in about 15% and 35% respectively at approximately the same stage of the adenoma-carcinoma sequence. Since these two mutations rarely coexist, further analysis to dissect their function of transformation in colon cancer is required. Caco-2 human colon adenocarcinoma cells were stably transfected with BRAF(V600E) (Caco-BR cells) or KRAS(G12V) (Caco-K cells) oncogenes. BRAF(V600E) is more efficient in transforming Caco-2 cells and altering their morphology. The dominant nature of BRAF(V600E) is evident by its ability to render Caco-2 cells tumorigenic in vivo all be it through selective extracellular signal-related kinase (ERK) 2 phosphorylation and high levels of cyclin D1. As a consequence, the cell cycle distribution of parental cells is altered and microsatellite instability is introduced. Attenuated ERK activation observed correlated with KSR downregulation by BRAF(V600E) without further implications to signaling. Highly activated ERK in case of KRAS(G12V) (Caco-K cells) leads to mild transformation causing Caco-K cells to express premature senescence-related markers and acquire growth factor-dependent viability. Interestingly, BRAF(WT)gets equally activated by upstream KRAS mutations present in colon adenocarcinoma cells such as DLD-1 and SW620. Taken together, these results suggest that the two oncogenes have different transforming capability in colon cancer, although they both use the mitogen-activated protein (MAP) kinase pathway to carry out their effect. In general, BRAF(V600E) presents greater potential in mediating tumorigenic effect as compared to KRAS(G12V) both in vivo and in vitro. These findings may have implications in personalised diagnosis and targeted therapeutics.
Assuntos
Transformação Celular Neoplásica/genética , Senescência Celular/genética , Neoplasias do Colo/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Biomarcadores Tumorais/análise , Western Blotting , Células CACO-2 , Citometria de Fluxo , Imunofluorescência , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Instabilidade de Microssatélites , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Sporadic colorectal cancer is a major cause of death worldwide. Development takes place in a sequential manner from benign adenomas leading to carcinomas. In 90% of tumours bearing a Ras mutation it is Ki-Ras that is mutated. We have developed a model cell system to study oncogenic Ras mutations in colorectal cancer cell lines. In this analysis two Caco-2 derived cell lines expressing Ha-RasV12 (Caco-H) and Ki-RasV12 (Caco-K), respectively, have been used in large-scale microarray profiling against a Caco-2 control. This was carried out using an Illumina microarray containing 24,000 genes. Genes have been identified as differentially expressed in each isoform as well as commonly regulated. In addition the Caco-H cell line has a strong epithelial-mesenchymal phenotype that is reflected in many of its differentially expressed genes. These include the known EMT markers Vimentin, E-cadherin and Slug. Other genes of interest include several members of the Claudin family, Forkhead transcription factors and GATA-factors. The Caco-K cell line shows strong downregulation of the Dickkopf transcriptional repressor implicating it in WNT signalling. Pathway and functional analysis has also been carried out for the differentially expressed genes for both cell lines using Ingenuity software. This genome wide microarray analysis has provided a molecular signature for EMT in a Caco-H colon cancer cell line. It has also revealed a number of key genes for Caco-K expression and identified novel markers for Ras expression that have been verified by PCR analysis.
Assuntos
Neoplasias do Colo/patologia , Epitélio/patologia , Mesoderma/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células CACO-2 , Neoplasias do Colo/genética , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
Sporadic colon cancer is a major cause of death throughout the world. Multistage development of the disease has been associated with remarkable genetic events, mainly at the level of oncogenes and oncosuppressor genes, most notably APC (adenomatous polyposis coli), ras and p53. Despite all of these efforts, the development of a sensitive and convenient diagnostic system for detecting colorectal cancers at the early stage is still in progress. In recent years, cDNA and oligonucleotide microarray technologies have made the analysis of gene expression profiles of colorectal tumours at the genomic level possible and have identified signatures of gene expression associated with pre-cancerous phenotypes, cancers of the early stage and/or metastatic cancer. The contribution of this powerful technology in identification of novel important genes for prognosis, diagnosis and therapy of sporadic colorectal will be discussed.