Abstract Our previous studies show that the cyclin-dependent kinase inhibitor (CKI), p27kip1 (p27), is absent from the glandular epithelia in estrogen-induced endometriod type I endometrial adenocarcinoma (ECA). The absence of p27 is due to its ubiquitylation and high rate of proteasomal degradation, which we propose is a major mechanism involved in the pathogenesis of ECA. These results suggest that maintaining nuclear p27 levels might have therapeutic application for this disease. To this point, TGF-β signaling through Smads (direct downstream transcription factors of TGF-β receptor signaling) dose and time-dependently increase nuclear p27 in synchronized HEC-1A cells (ECA cell line). By knocking down p27 (p27-siRNA), we further showed that p27 is the sole mediator of the growth inhibitory action of TGF-β by blocking Cdk2 activity in HEC-1A cells. The mechanism by which TGF-β increases nuclear p27 is by preventing its ubiquitin-mediated degradation through the downregulation of Skp2 and Cks1 proteins [but not mRNA], the rate limiting components of the SCF complex that target p27 for degradation. This decrease in nuclear Cks1 was blocked by the TGF-β Receptor I kinase inhibitor, SD208 and the proteasome inhibitor, lactacystin, suggesting that TGF-β regulation of Cks1 protein is by proteasomal degradation. The goal of the present study was to determine how TGF-β decreases Skp2 and Cks1 to preserve the level of nuclear p27. We now show in HEC-1A and ECC-1 cell lines that TGF-β time and dose-dependently increases nuclear Cdh1, an E3 ligase that ubiquitylates Cks1 and Skp2, when bound to anaphase promoting complex (APC), in early G1 phase of the cell cycle. TGF-β induction of Cdh1 was blocked by SD208 thereby causing an increase in nuclear Cks1. Furthermore, TGF-β-mediated growth inhibition of these cell lines is abrogated by knocking down Cdh1 (Cdh1 si RNA) with a concomitant decrease in nuclear p27 and an increase in nuclear Cks1. These studies show that TGF-β signaling increases Cdh1 [bound to APC] causing a decrease in Cks1 and Skp2 to permit nuclear accumulation of p27 for growth arrest in G1. Therefore, for the first time TGF-β is shown to regulate growth by solely affecting the levels of cell cycle proteins through manipulation of the ubiquitin-proteasome pathway in a chain of events that ultimately preserves and accumulates nuclear p27. As p27 appears to be a critical target for endometrial hyperplasia and carcinoma, these data implicate the preservation of nuclear p27 by proteasome inhibitors or specific inhibitors of Cks1 and Skp2 [or inducers of Cdh1] with therapeutic potential for ECA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1089.