AR is required for FOXA1 enhanced Notch pathway activation of EC cells Pathway analysis in liver cancer shows that FOXA1 AR dual target genes are most involved in the cellular growth proliferation pathway. compound libraries Notch pathway acti vation appears to affect proliferation in many cancers. In EC, the Notch pathway has also been shown to be in volved in cell proliferation. Thus, we considered that the interaction between FOXA1 and AR might be related with the Notch pathway. We used western blot analysis to assess the levels of Notch1 and the Notch pathway target protein, Hes1, in MFE 296 shFOXA1 and AN3CA exFOXA1 cells after exAR or siAR cotrans fection, respectively. Cotransfection with exAR rescued the decreased expression of Notch1 and Hes1 caused by FOXA1 downregulation in MFE 296 shFOXA1 cells.

Furthermore, cotransfection with siAR at tenuated the increased expression of Notch1 and Hes1 caused by upregulation of FOXA1 in AN3CA exFOXA1 cells. These results suggested that the effects of FOXA1 on Notch pathway activation were mediated by AR. In order to determine whether AR was required for FOXA1 enhanced Notch pathway activation, we over expressed AR expression in AN3CA cells, which has low level of AR. We assessed the levels of Notch1 and Hes1 in untransfected AN3CA cells as well as AN3CA cells transfected with NC, exAR, or exAR together with siFOXA1. AN3CA exAR cells exhibited a substantial in crease in AR expression as compared to AN3CA NC cells, accompanied by over expression of Notch1 and Hes1. Furthermore, cotransfection with siFOXA1 did not rescue the activation of Notch1 and Hes1 caused by AR upregulation in AN3CA exAR cells.

These results suggested a mechanism, where AR might be a necessary medium in FOXA1 enhanced Notch pathway activation in AN3CA cells. FOXA1 promotes proliferation of human EC cells To examine the role of FOXA1 in EC cell proliferation, we assessed the effect of FOXA1 in colony forming and MTT assays. In the colony forming assay, MFE 296 cell transfected with shFOXA1 showed significantly decreased colony forming ability when compared with MFE 296 cells transfected with NC. Moreover, upre gualtion of FOXA1 in AN3CA cells showed increased colony forming ability compared with NC cells. In the MTT assay, downregulation of FOXA1 in MFE 296 cells resulted in poor cell viability, and upregu lation of FOXA1 in AN3CA cells caused increased cell viability.

These data indicated that FOXA1 promoted cell proliferation. AR is required for FOXA1 enhanced proliferation of EC cells To directly address whether the effects of FOXA1 in promoting EC cell proliferation can be attributed to its activation of AR, a rescue experiment in MFE 296 cells was performed. In the colony forming assay, ABT888 cotransfec tion with exAR rescued the decreased rate of cell growth caused by FOXA1 downregulation in shFOXA1 cells.