SMAD4/DPC4 acts as a transcriptional corepressor for estrogen receptor alpha and mediates crosstalk between TGF-beta and estrogen signaling

Estrogen is one of the most important physiological regulators in the reproductive system, in bone metabolism, and in the maintenance of the cardiovascular and central nervous systems. Estrogen also has a mitogenic effect in breast and endometrial tissues and is associated pathologically with an increased risk for tumors developed from these organs. Consequently, the estrogen receptors (ERs) have been found to play an essential role in the initiation and development of most breast and endometrial cancers. Transforming growth factor (TGF)-betas, on the other hand, are plausible tumor suppressors in breast cancer. Downstream signaling of TGF-betas involves a family of effector molecules known as Smads. Smad4 is the common partner Smad that form complexes with the receptor-phosphorylated Smads and activate target gene transcription in the nucleus. This study investigated the mechanism by which TGF-beta inhibits estrogen signaling through Smad4-mediated crosstalk with ERalpha. ERalpha, the nuclear transcription factor for estrogen signaling, was found to associate with Smad4 in yeast and mammalian cells. Deletional analysis revealed that the interaction involves the N-terminal activation function 1 (AF1) domain of ERalpha and the MH1 domain and linker region of Smad4. In vitro binding and reporter assays demonstrated that transient expression of Smad4 interacts with ERalpha and suppresses the 17beta-estradiol (E2)-induced ERE activity. Further, in the presence of antiestrogens Smad4 and ERalpha can form a complex together with the estrogen responsive element located at the 5'-flanking region from -295 to -54 of cathepsin D (CATD), an important marker gene for breast cancer. Smad3, which was identified as a transcriptional coactivator for androgen receptor (AR) and ERalpha, can comodulate the ERalpha transactivity together with Smad4 and confer an inhibitory TGF-beta signal to the estrogen-mediated transcription. However, Smad4 was found to be essential for the suppressive function of Smad3/4 and TGF-beta in the estrogen target gene expression. This dissertation also reports a novel mechanism for the disruption of TGF-beta inhibition in the breast cancer cells. We found that the overexpression of oncoprotein Ski in the breast cancer cells can prevent the nuclear translocation of Smad4 in the presence of TGF-beta. The ectopic Ski enhances the estrogen activity on ERalpha-mediated gene transcription. The last part of this dissertation discusses the potential role of Smad4 in the other signaling pathways of estrogen and implicates a connection between the role as a tumor suppressor and the corepressor for ERalpha.