RESUMO
We have identified a new variant of human Stat5a, found at higher ratios to full-length Stat5a in invasive ductal carcinoma versus contiguous normal tissue. The variant, missing exon 5, inhibits p21 and Bax production and increases cell number. After prolactin stimulation, only full-length Stat5a interacts with the vitamin D and retinoid X receptors, whereas only Δ5 Stat5a interacts with activating protein 1-2 and specificity protein 1. Prolactin also oppositely regulates interaction of the two Stat5a forms with ß-catenin. We propose that a change in splicing leading to upregulation of this new isoform is a pathogenic aspect of invasive ductal carcinoma.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Western Blotting , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Humanos , Dados de Sequência Molecular , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Based on their content of prolactin receptors, osteosarcoma cells were predicted to be responsive to prolactin (PRL), but whether PRL would be beneficial or contribute to pathogenesis was unclear. 1,25(OH)(2) vitamin D(3) [1alpha,25(OH)(2)D(3)] has antiproliferative effects on osteosarcoma cells, and a complex interregulatory situation exists between PRL and 1alpha,25(OH)(2)D(3). Using osteosarcoma cells, Western blot, real time RT-PCR, and promoter-luciferase assays, we have examined the interaction between PRL and 1alpha,25(OH)(2)D(3) and demonstrated that physiological concentrations of PRL block increased osteocalcin and vitamin D receptor (VDR) expression in response to 1alpha,25(OH)(2)D(3.) This blockade was shown to be the result of lack of nuclear accumulation of the VDR in response to 1alpha,25(OH)(2)D(3). Although inhibition of proteasomic degradation with MG132 had no effect on the VDR itself in a 30-min time frame, it relieved the blockade by PRL. Analysis of ubiquitinated proteins brought down by immunoprecipitation with anti-VDR showed PRL regulation of a 250-kDa protein-VDR complex. P250 was identified as the breast cancer tumor suppressor gene product, BRCA1, by Western blot of the VDR immunoprecipitate and confirmed by immunoprecipitation with anti-BRCA1 and blotting for the VDR in the absence and presence of PRL. Knockdown of BRCA1 inhibited nuclear translocation of the VDR and the ability of 1alpha,25(OH)(2)D(3) to induce the VDR. This, to our knowledge, is the first demonstration of a role for BRCA1 in nuclear accumulation of a steroid hormone and the first demonstration that PRL has the potential to affect the cell cycle through effects on BRCA1.
