RESUMEN
Genetic changes in colon cancer are known to parallel the tissue abnormalities associated with the disease, namely adenoma and adenocarcinoma. The role of microRNA dysregulation in dysplastic progression, however, is not well understood. Here, we show that miR-182 and miR-503 undergo sequential up-regulation and drive the progression of colon adenoma to adenocarcinoma by cooperatively down-regulating the tumour suppressor FBXW7. We identified that increased expression of miR-182 is a feature of adenomas. A subsequent increase in miR-503 expression works cooperatively with miR-182 to induce transformation of an adenoma to adenocarcinoma. We show that introducing miR-503 into AAC1 cells, which are derived from a benign adenoma, confers tumourigenic potential. We also demonstrated that blocking both miR-182 and miR-503 in HCT116 colon cancer cells resulted in increased FBXW7 expression and significantly reduced tumour size in xenograft models. We confirmed relevance of these results in patients by examining the expression levels of miR-182 and miR-503 in over 200 colon cancer patients with 12 year survival outcome data. Decreased patient survival was correlated with elevated expression of both miRNAs, suggesting that elevated levels of both miR-182 and miR-503 define a novel prognostic biomarker for colon cancer patients. In conclusion, we show that a sequential expression of miR-182 and miR-503 in benign adenoma cooperatively regulates the tumour suppressor FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma and miR-182 and miR-503 may prove to be novel therapeutic targets. Array data are available at: http://www.oncomir.umn.edu/
Asunto(s)
Adenocarcinoma/genética , Adenoma/genética , Proteínas de Ciclo Celular/biosíntesis , Transformación Celular Neoplásica/genética , Neoplasias del Colon/genética , Proteínas F-Box/biosíntesis , MicroARNs/genética , Ubiquitina-Proteína Ligasas/biosíntesis , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenoma/mortalidad , Adenoma/patología , Animales , Proteínas de Ciclo Celular/genética , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Estimación de Kaplan-Meier , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Modelos de Riesgos Proporcionales , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The CD4 gene is regulated in a stage-specific manner during T cell development, being repressed in CD4(-)CD8(-) double-negative (DN) and CD8 cells, but expressed in CD4(+)CD8(+) double-positive (DP) and CD4 cells. Furthermore, the expression/repression pattern is reversible in developing (DN and DP) thymocytes, but irreversible in mature (CD4 and CD8) T cells. Here, we explored the molecular mechanisms underlying this complex mode of regulation by examining the nucleoprotein structure of the CD4 locus throughout T cell development and in DN cells lacking the CD4 silencer. In DN cells, the CD4 enhancer is preloaded with multiple transcription activators, but p300 recruitment is impaired by the silencer that is associated with the repressor Runx1. DP cells achieve high-level CD4 expression via a combination of CD4 derepression and true activation, but Runx1 remains bound to the silencer that retains an open chromatin configuration. In CD4 cells, Runx1 dissociates from the silencer that has become less accessible, and CD4 transcription appears to be achieved via a mechanism distinct from that operating in DP cells. In CD8 cells, the CD4 promoter becomes incorporated into heterochromatin-like structure. Our data shed light on the molecular basis of CD4 regulation and provide a conceptual framework for understanding how the same regulatory elements can mediate both reversible and irreversible CD4 regulation.
Asunto(s)
Antígenos CD4/genética , Regulación de la Expresión Génica , Nucleoproteínas/química , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Histonas/metabolismo , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Regiones Promotoras Genéticas/genética , Unión Proteica , Elementos Silenciadores Transcripcionales/genética , Factores de Transcripción/metabolismoRESUMEN
We have developed a general strategy for creating littermates bearing either a tissue-specific point mutation or deletion in any target gene, and used the method to dissect the roles of Brg, the ATPase subunit of the chromatin-remodeling Brg-associated factor (BAF) complex, in early thymocyte development. We found that a point mutation that inactivates the Brg ATPase recapitulates multiple defects previously described for Brg deletion (Chi, T.H., M. Wan, P.P. Lee, K. Akashi, D. Metzger, P. Chambon, C.B. Wilson, and G.R. Crabtree. 2003. Immunity. 19:169-182). However, the point mutant helps reveal unexpected roles of Brg in CD25 repression and CD4 activation. Surprisingly, CD4 activation occurs independently of the Brg ATPase and is perhaps mediated by physical interactions between Brg and the CD4 locus. Our study thus suggests that the BAF complex harbors novel activities that can be necessary and even sufficient for stimulating transcription from an endogenous chromatin template in the absence of Brg-dependent remodeling of that template. We conclude that conditional point mutants, rarely used in mammalian genetics, can help uncover important gene functions undetectable or overlooked in deletion mutants.