RESUMO
In tumor cells, stepwise oncogenic deregulation of signaling cascades induces alterations of cellular morphology and promotes the acquisition of malignant traits. Here, we identified a set of 21 genes, including FGF9, as determinants of tumor cell morphology by an RNA interference phenotypic screen in SW480 colon cancer cells. Using a panel of small molecular inhibitors, we subsequently established phenotypic effects, downstream signaling cascades, and associated gene expression signatures of FGF receptor signals. We found that inhibition of FGF signals induces epithelial cell adhesion and loss of motility in colon cancer cells. These effects are mediated via the mitogen-activated protein kinase (MAPK) and Rho GTPase cascades. In agreement with these findings, inhibition of the MEK1/2 or JNK cascades, but not of the PI3K-AKT signaling axis also induced epithelial cell morphology. Finally, we found that expression of FGF9 was strong in a subset of advanced colon cancers, and overexpression negatively correlated with patients' survival. Our functional and expression analyses suggest that FGF receptor signals can contribute to colon cancer progression.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Progressão da Doença , Fatores de Crescimento de Fibroblastos/metabolismo , Testes Genéticos , Interferência de RNA , Transdução de Sinais , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias do Colo/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Fenótipo , Transdução de Sinais/genética , Análise de Sobrevida , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Functional analysis of multiple genes is key to understanding gene regulatory networks controlling embryonic development. We have developed an integrated vector system for inducible gene silencing by shRNAmir-mediated RNA interference in mouse embryos, as a fast method for dissecting mammalian gene function. For validation of the vector system, we generated mutant phenotypes for Brachyury, Foxa2 and Noto, transcription factors which play pivotal roles in embryonic development. Using a series of Brachyury shRNAmir vectors of various strengths we generated hypomorphic and loss of function phenotypes allowing the identification of Brachyury target genes involved in trunk development. We also demonstrate temporal control of gene silencing, thus bypassing early embryonic lethality. Importantly, off-target effects of shRNAmir expression were not detectable. Taken together, the system allows the dissection of gene function at unprecedented detail and speed, and provides tight control of the genetic background minimizing intrinsic variation.
