RESUMO
To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Modelos Biológicos , Metástase Neoplásica/patologia , Metástase Neoplásica/fisiopatologia , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Tamanho Celular , Sobrevivência Celular , Simulação por Computador , HumanosRESUMO
PURPOSE: In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. EXPERIMENTAL DESIGN: One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001). RESULTS: In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.
Assuntos
Núcleo Celular/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/patologia , Mapeamento de Epitopos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Prognóstico , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/imunologia , Análise de SobrevidaRESUMO
We investigated the involvement of Rho GTPases in the secretory process of PC12 cells. Overexpression of wild-type RhoA, Rac1, or Cdc42 did affect exocytosis. In contrast, secretion elicited by depolarizing K(+) concentrations was enhanced by the dominant negative mutants RhoA(N19), Rac1(N17), and Cdc42(N17) and was diminished by the constitutively active mutants RhoA(V14), Rac1(V12), and Cdc42(V12). The inhibition observed in the presence of RhoA(V14) was likely a result of the activation of ROK(alpha), since the catalytic domain of this kinase was able to mimic both the reorganization of the actin cytoskeleton and the decrease in exocytosis induced by the RhoA mutant. Part of the effect of Rac1(V12) may be due to POR1 activation. Thus, overexpression of full-length POR1 diminished K(+)-stimulated exocytosis, and a point mutation in the effector domain of Rac1(V12) that prevents the interaction with POR1 abolished the inhibitory effect of the GTPase. We also searched for the Cdc42(V12) target but overexpression of the Cdc42 effector WASP did not mimic the inhibition of exocytosis observed in cells transfected with the activated GTPase. Our findings indicate that different signaling cascades resulting in the activation of RhoA, Rac1, or Cdc42 can modulate the exocytotic process of neuroendocrine cells.