Abstract 3885: Mechanisms that mediate the increase in p27kip1 by progestins: Therapeutic implications for endometrial carcinoma
Authors: Pavlides Savvas C. , Lecanda Jon , Blank Stephanie V. , Mittal Khushbakhat R. , Gold Leslie I. 
Published: February 2011

Abstract
The steroid hormones, estrogen (E2) and progestins (Pg) regulate growth of the endometrium. E2 stimulates and Pg inhibits endometrial epithelial cell (EEC) proliferation. The importance of these hormones in controlling EEC growth is underscored by E2-induction of type I endometrial cancer (ECA; 85% of all ECAs), and by the therapeutic effect of Pg on causing regression of both endometrial hyperplasia and ECA. The cyclin-dependent kinase inhibitor, p27kip1 (p27) arrests cells in G1 phase of the cell cycle by blocking Cdk2 activity. We previously showed that nuclear p27 is lost in the glands of ECA tissue due to a high rate of p27 degradation via the ubiquitin-proteasome pathway. In vitro, using primary EECs derived from hysterectomies, we also showed that E2 induces proteasomal degradation of p27. Therefore, together with the lack of nuclear p27 in endometrial hyperplasia tissue, p27 degradation appears to be an early critical molecular event involved in the pathogenesis of E2-induced ECA. In contrast and importantly, we show in vitro that 100 nM Pg markedly increases p27 in primary normal EECs and ECA cells. Moreover, the PR antagonist, RU486, completely blocks the increase in nuclear p27 and sequesters p27 in the cytoplasm in EECs suggesting that Pg might act by enabling nuclear-cytoplasmic shuttling, which is abrogated by RU486. In addition, we propose that the therapeutic effect of Pg on endometrial hyperplasia and ECA is through Pg-induction and nuclear localization of p27. Indeed we show that patients responding to Pg [Megace®] therapy with histological regression of disease demonstrate a concomitant increase in nuclear p27. To understand the mechanisms involved in upregulation of p27 by Pg, we investigated mRNA and protein regulation of p27 by Pg in EECs. By real-time PCR, Pg does not increase mRNA transcription of p27 (time points=0.5 to 12 hr). However, Pg induced Cdh1 protein, the E3 ligase for Cks1 and thus, decreased Cks1, a rate limiting co-factor that mediates proteasomal degradation of p27, thereby simultaneously increasing nuclear p27. These effects were reversed by RU486 and the decrease in Cks1 was reversed by the proteasome inhibitor, lactacystin. Thus, we show that through the PR, Pg increases p27 by preventing its degradation by Cks1 through Pg-induced upregulation of Cdh1. Finally, whereas Pg induced growth inhibition of EECs by 25%, the addition of normal stromal cells (NSFs) in co-culture further mediated growth inhibition of EECs by 75% over untreated controls. In contrast, co-cultures of stromal cells derived from ECA tissue (CAFs) did not have this effect. Similarly, conditioned media from CAFs, but not NSFs stimulated EEC growth with a concomitant decrease in p27 and increase in Cks1. These studies reveal mechanisms of how Pg acts as a therapeutic agent to control endometrial growth through increased p27 nuclear localization involving participation by normal adjacent stroma.
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 3885.