RESUMEN
The role of estrogen receptor-α (ER) in breast cancer development, and as a primary clinical marker for breast cancer prognosis, has been well documented. In this study, we identified the oncogenic protein, TWIST1 (Twist), which is overexpressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was performed using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high expressing cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly downregulate ER promoter activity in vitro. Functionally, Twist overexpression caused estrogen-independent proliferation of breast cells, and promoted hormone resistance to the selective estrogen receptor modulator tamoxifen and selective estrogen receptor down-regulator fulvestrant. Importantly, this effect was reversible on downregulating Twist. In addition, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area, as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter, leading to a significantly higher degree of ER promoter methylation compared with parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using the demethylating agent, 5-azacytidine, and the HDAC inhibitor, valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for the loss of ER activity observed in breast tumors, and may contribute to the generation of hormone-resistant, ER-negative breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/genética , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Acetilación/efectos de los fármacos , Animales , Azacitidina/farmacología , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Tumoral , Cromatina/efectos de los fármacos , Cromatina/genética , Cromatina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de los fármacos , Regulación hacia Abajo/genética , Elementos E-Box/efectos de los fármacos , Elementos E-Box/genética , Estrógenos/metabolismo , Femenino , Histona Desacetilasa 1/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Ratones , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , Tamoxifeno/farmacología , Proteína 1 Relacionada con Twist/deficiencia , Proteína 1 Relacionada con Twist/genética , Ácido Valproico/farmacología , ADN Metiltransferasa 3BRESUMEN
Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.
