Mechanism Study For Interaction Of Smad4 With ERα Inhibiting Breast Tumorigenesis

Estrogens regulate differentiation and maintenance of reproductive, skeletal and other tissues by activating their estrogen receptors (ER). Estrogens also act as mitogens to promote cell prolifERαtion in both normal breast tissue and breast carcinomas. In contrast, TGF-P is an inhibitor of cell cycle progression in epithelial cells and antagonizes the effects of ERα mitogenic effect. We have identified that Smad4, a common TGF-P signal transducer, functions as an ERα transcriptional corepressor. Here we examined the function of Smad4 intERαction with ERα in breast epithelial cells.In order to fulfill our goal, we chose 4 common breast cancer cell lines (MCF-7, T47D, MDA-MB-231 and MDA-MB-468) to compare their profile for further study. We have genERαted two recombinant retroviruses that mediate expression of △μ.3 Flag-Smad4 and green florescence protein (GFP) and got 8 kinds of stable cell lines (MCF-7-GFP and MCF-7-Smad4; T47D-GFP and T47D-Smad4; MDA-MB-231-GFP and MDA-MB-231-Smad4; MDA-MB-468-GFP and MDA-MB-468-Smad4). Also we have genERαted another two recombinant retroviruses that mediate expression of pMSCV-ERα and GFP that were introduced into MDA-MB-231 -ERα or MDA-MB-231-GFP). Hoechst 33342 staining and Annexin V stainingof Flow Cytometry were conducted for apoptosis positive cells. A nude mouse is used as a model to evaluate function of Smad4 inhibits tumorigenesis. Immunofluorescence staining was conducted for Smad4 and ERa colocalization. Western Blot Analysis and inmmunochemistry stain was used to determine apoptosis markers in all cell lines and xenograft from tumor.In this study we found that MCF-7, T47D and MDA-MB-231 have Smad4 protein expression, MCF-7 and T47D have ERa protein expression. MDA-MB-468 has no Smad4 and ERa protein expression.The 4 cell lines were infected Smad4 by retrovirus showed that Smad4 only induced apoptosis in ERa positive cells and not in ERa negative cells, which suggested a potential function of the interaction between Smad4 and ERa.From these results we chose MCF-7 and MDA-MB-231 to do further study. MCF-7 ERa positive and MDA-MB-231 ERa negative cells were infected with a retrovirus encoding Smad4. Hoechst 33342 staining and Flow Cytometry analysis showed that Smad4 induced significant apoptotic cells in MCF-7, not in MDA-MB-231 cells, within 48h of infection. Furthermore, stable Smad4 expression cell line induces proapoptosis marker protein expression including BIM and Bax and Cytochrome-c release in MCF-7, again not in MDA-MB-231 ERa negative cells. Also stable Smad4 expression cell line decreases anti-apoptosis marker protein expression includingBcl-XL and Bcl-2 in MCF-7, again not in MDA-MB-231 ERa negative cells. To confirm that ERa is required, we expressed ERa siRNA in MCF-7 cells, which significantly reduced Smad4-induced Bax expression, cytochrome-c release and apoptosis. For the same purpose, ERa was stably introduced in MDA-MB-231 cells with retrovirus, and Smad4 was able to induce apoptosis in MDA-MB-231 cells with acquired ERa expression. These results indicate that ERa is required for Smad4-induced cell apoptosis.On the other hand, we examined ERa interaction with Smad4 may facilitate Smad4 into nuclear by Immunofluorescence staining. We found that ERa and exogenous Smad4 are enriched in nuclei in MCF-7 cells. ERa showed good co-localization with Smad4, suggesting that ERa interacted with Smad4 may facilitate complex to reside in the nuclear and induce the function of apoptosis. For ERa negative cell, overexpression of Smad4 was predominately localized on cytoplasm.We then examined whether Smad4 also induces apoptosis in ERa positive cells in nude mice. MCF-7 ERa positive and MDA-MB-231 ERa negative cells were stably transfected with Smad4 or GFP, and were inoculated in nude mice. The tumor volumes were measured every two days. The tumor sizes of MCF-7 breast cancer cells stably transfected with Smad4 are only one tenth of those expressing GFP, and Tunnel assays showed apoptosis positive cells were significantly induced in Smad4 tumor xenograft compared with its GFP control.Whereas in MDA-MB-231, rumor sizes in Smad4 transfected cells are not significantly different from GFP transfected cells.These tumor xenografts were further analyzed with Western blot and immunihistochemical stain for apoptotic biomarkers, which showed that MCF-7 with Smad4 expression induced pro-apoptosis protein expression including BIM and Bax, but induction of these pro-apoptotic markers was not observed in nude mice bearing MDA-MB-231 tumor xenografts. These results indicate that presence of ERa is required in Smad4 inducing apoptosis in breast cancer cells. Western blotting and immunohistochemical staining showed that Smad4 promotes induction of BIM and Bax expression and release of cytochrome c only in MCF-7 ERa positive tumor xenografts. In the same staining, we observe that overexpression of Smad4 predominantly was localized in nuclear in MCF-7 cells whereas Smad4 predominantly was localized in cytoplasm in MDA-MB-231 cell.These results is consistent with our in vitro data which further evidence that ERa interaction with Smad4 may facilitate Smad4 into nuclear and induce apoptosis.In conclusion, (1) This result first elucidated the function of Smad4 as ERa corepressor is able to induce cell apoptosis in ERa positive breast cancer cells; (2) these results clearly indicate that Smad4-induced breast cancer cell apoptosis is ERa-required. These may provide one explanation for Smad4 as tumor suppressor is the celltype specify; (3) Smad4 as ERoc corepressor is ligand independent effect to induce apoptosis in vitro; (4) Nuclear localization of Smad4 is associated with ERa, which suggests that ERa interacted with Smad4 may facilitate complex to reside in the nuclear and trigger apoptosis; Immunohistchemistry staining in xenograft have the same results; (5) Also Smad4 overexpression induced tumor growth inhibition by inducing apoptosis only in ERa-positive MCF-7 cells in nude mice with estrogen pellet implantation. These suggest that Smad4 as ERa corepressor in ERa positive breast cancer cell may be regulated by estrogen in vivo.Our data is consistent with clinic data that Estrogen receptor (ER)-positive breast cancers generally have a better prognosis and are often responsive to anti-estrogen therapy; ER-a negative breast cancers are more aggressive and unresponsive to anti-estrogens. Therefore, our findings may help to understand the mechanism of SERMs and provide a potential therapeutical target to suppress growth by inducing cell apoptosis of ERa positive tumor cells.