The similarity of results from the colony forming and MTT assays suggested that the effects of FOXA1 in mediating cell proliferation of EC cells were mediated through AR. AR is not required for FOXA1 enhanced migration and invasion of EC cells Our immunohistochemistry results revealed that pa tients with myometrial invasion displayed higher FOXA1 http://www.selleckchem.com/products/Vandetanib.html expression. With this observation in mind, we hypothe sized that functional expression of FOXA1 might induce tumor metastasis in EC. To explore the role of FOXA1 in the regulation of metastatic function and to determine whether AR is involved in FOXA1 mediated regulation of metastatic function, we examined the migration and invasion ability of MFE 296 shFOXA1 and AN3CA exFOXA1 cells after exAR or siAR cotransfection using transwell migration and invasion assays.

MFE 296 shFOXA1 cells displayed a decreased rate of migration compared to MFE 296 NC cells. However, cotransfection of MFE 296 shFOXA1 cells with exAR did not rescue the migration to the levels observed in MFE 296 NC or untransfected cells. Furthermore, AN3CA exFOXA1 cells exhibited a high migration rate as com pared with AN3CA NC cells, but cotransfection with siAR did not significantly attenuate the migration rate. Consistent with these findings, the invasion rate was significantly reduced in MFE 296 shFOXA1 cells, but the reduction was not reversed upon transfection with exAR. Likewise, the invasion rate was en hanced in AN3CA exFOXA1 cells, but this enhance ment was not attenuated upon transfection with siAR.

These results demonstrated a functional role for FOXA1 in mediating migration and invasion in EC cells and suggested a mechanism by which AR might not con tribute to FOXA1 mediated metastasis of EC. Oncogenic role of FOXA1 in a tumor xenograft model Tumors generated by subcutaneous implantation of MFE 296 cells were used to evaluate the effect of FOXA1 on proliferation in a mouse tumor xenograft model. We measured tumor volumes in xenografted mice over a 6 week period following injection of untransfected MFE 296, stably transfected with shFOXA1 or NC. These measurements indicated that tumors in the MFE 296 shFOXA1 group grew significantly slower than those in the MFE 296 NC group and the MFE 296 group. Six weeks after injection, tumors were removed from the mice. The final mean weight and volume of tumors in the MFE 296 shFOXA1 group were significantly lower than those in the MFE 296 NC group.

Tumor tissues were then embedded in paraffin, stained with hematoxylin and eosin, and immunohisto chemically twice stained with antibodies against FOXA1, AR, Notch1, Hes1, Ki67, or PCNA. Lower FOXA1 expres sion in the MFE 296 shFOXA1 group also led to re duced staining for AR, indicating that FOXA1 also affected AR expression in vivo, in accordance with the results in vitro.