RESUMO
SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis.
Assuntos
Neoplasias do Colo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Adesão Celular/genética , Linhagem Celular Tumoral , Polaridade Celular/genética , Proliferação de Células/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células Epiteliais , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Fenótipo , Proteína Proto-Oncogênica c-ets-1/genética , Transdução de Sinais , Transfecção , Regulação para Cima , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
SPROUTY2 (SPRY2) is an intracellular regulator of receptor tyrosine kinase signaling involved in cell growth, differentiation and tumorigenesis. Here, we show that SPRY2 is a target gene of the Wnt/ß-catenin pathway that is abnormally activated in more than 90% of colon carcinomas. In human colon cancer cells, SPRY2 expression is induced by ß-catenin in co-operation with the transcription factor FOXO3a instead of lymphoid enhancer factor/T-cell factor proteins. We found binding of ß-catenin to the SPRY2 promoter at FOXO3a response elements. In vivo, cells marked by nuclear ß-catenin and FOXO3a express SPRY2 in proliferative epithelial tissues, such as intestinal mucosa and epidermis. Consistently, inducible ß-catenin deletion in mice reduced Spry2 expression in the small intestine. Moreover, SPRY2 protein expression correlated with nuclear ß-catenin and FOXO3a colocalization in human colon carcinomas. Importantly, the amount of SPRY2 protein correlated with shorter overall survival of colon cancer patients. Our data reveal SPRY2 as a novel Wnt/ß-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer.
Assuntos
Neoplasias do Colo/genética , Fatores de Transcrição Forkhead/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , beta Catenina/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Animais , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Imunofluorescência , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Microscopia Confocal , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/metabolismoRESUMO
SPROUTY-2 (SPRY2) regulates receptor tyrosine kinase signalling and therefore cell growth and differentiation. In this study, we show that SPRY2 expression in colon cancer cells is inhibited by the active vitamin D metabolite 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) through E-cadherin-dependent and -independent mechanisms. In turn, SPRY2 represses both basal and 1,25(OH)(2)D(3)-induced E-cadherin expression. In line with this, SPRY2 induces ZEB1 RNA and protein, but not that of other epithelial-to-mesenchymal transition inducers that repress the CDH1/E-cadherin promoter. Consistently, SPRY2 and E-cadherin protein levels inversely correlate in colon cancer cell lines and xenografted tumours. Moreover, SPRY2 knockdown by small hairpin RNA increases CDH1/E-cadherin expression and, reciprocally, CDH1/E-cadherin knockdown increases that of SPRY2. In colon cancer patients, SPRY2 is upregulated in undifferentiated high-grade tumours and at the invasive front of low-grade carcinomas. Quantification of protein expression in 34 tumours confirmed an inverse correlation between SPRY2 and E-cadherin. Our data demonstrate a tumourigenic action of SPRY2 that is based on the repression of E-cadherin, probably by the induction of ZEB1, and a reciprocal regulation of SPRY2 and E-cadherin that dictates cell phenotype. We propose SPRY2 as a candidate novel marker for high-grade tumours and a target of therapeutic intervention in colon cancer.