RESUMEN
Inactivating mutations in the PTEN tumor suppressor gene occur in approximately 30-50% of endometrial carcinomas. PTEN is a phosphatase that negatively regulates the phosphoinositide 3-kinase signaling pathway, including the downstream effector AKT. To evaluate the role of PTEN in endometrial growth regulation, we expressed wild-type or mutant PTEN in endometrial carcinoma cell lines. As expected, expression of exogenous PTEN decreased levels of activated AKT in all cell lines examined. However, PTEN induced a G(1) cell cycle arrest specifically in endometrial carcinoma cells that lack endogenous wild-type PTEN. Growth of cells containing wild-type PTEN was unaffected by exogenous PTEN expression. Growth arrest required a functional phosphatase domain but not the PDZ interaction motif of PTEN. Overall levels of CIP/KIP and INK4 family members, the known inhibitory regulators of the G(1) phase of the cell cycle, were unchanged. However, PTEN induced a specific reduction of cyclin D3 levels and an associated increase in the amount of the inhibitor p27(KIP1) complexed with CDK2. Enforced expression of cyclin D3 abrogated the PTEN-induced cell cycle arrest. Although PTEN signaling directly regulates p27(KIP1) levels in some settings, in endometrial carcinoma cells, PTEN expression indirectly regulated p27(KIP1) activity by modulating levels of cyclin D3. These data support multiple mechanisms of PTEN-induced cell cycle arrest.
Asunto(s)
Adenocarcinoma/patología , Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Ciclinas/metabolismo , Neoplasias Endometriales/patología , Fase G1/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Supresoras de Tumor , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Ciclina D3 , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Activación Enzimática , Femenino , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Despite recent studies, the molecular genetic events responsible for the development of uterine endometrioid carcinoma (UEC) remain incompletely characterized. Mutations in the beta-catenin (CTNNB1) gene have been recently reported in a small percentage of UECs and in the endometrioid variant of ovarian carcinoma suggesting that the Wnt signal transduction pathway is involved in the development of female genital tract tumors with endometrioid morphology. The Wnt pathway is a critical pathway in the development of colorectal cancer (CRC) with mutations occurring in the beta-catenin (CTNNB1) or adenomatous polyposis coli (APC) genes in 10 to 15% and 85% of cases, respectively. Because UEC and CRC share other molecular genetic alterations and histologic features and previous studies of UEC have not reported an analysis of the APC gene, we chose to further elucidate the role of the Wnt pathway in UEC. To this end, we analyzed 32 cases of UEC for mutations of the CTNNB1 and APC genes. Mutations of CTNNB1 were present in six of 32 (18%) cases: four grade 1 carcinomas, one grade 2, and one grade 3 carcinoma. Five missense mutations were identified, three involving Ser/Thr phosphorylation sites and two adjacent to a Ser phosphorylation site. One case contained a deletion encompassing codons 34 to 37, which includes a Ser phosphorylation site. No mutations resulting in truncation of the APC protein were found. Our results support a role for the Wnt signaling pathway via mutation of CTNNB1, but not APC, in the development of a subset of UECs.
Asunto(s)
Carcinoma Endometrioide/genética , Proteínas del Citoesqueleto/genética , Genes APC , Transactivadores , Neoplasias Uterinas/genética , Western Blotting , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patología , Proteínas del Citoesqueleto/metabolismo , Análisis Mutacional de ADN , Cartilla de ADN/química , ADN de Neoplasias/análisis , Femenino , Humanos , Mutación Puntual , Reacción en Cadena de la Polimerasa , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patología , beta CateninaRESUMEN
Mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene or the beta-catenin gene are present in most colon cancers and less frequently in other tumor types. In this study, we screened 24 human breast cancer cell lines and three immortalized human breast epithelial cell lines for alterations in beta- and gamma-catenin and APC by western blotting, protein truncation assay and DNA sequence analysis. In one cell line (DU 4475), an APC mutation was identified (E1577stop) that resulted in expression of truncated APC. This mutation was associated with elevated cytosolic beta-catenin levels, probably due to loss of APC function, as in colon cancers. No mutations were found in exon 3 of the beta- or gamma-catenin genes. We conclude that APC mutations and beta-catenin upregulation may occur with low frequency in human breast cancer cells.