Savvas C. Pavlides

Inhibitors of SCF-Skp2/Cks1 E3 Ligase Block Estrogen-Induced Growth Stimulation and Degradation of Nuclear p27kip1: Therapeutic Potential for Endometrial Cancer

In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3μM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%-62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

Estrogen and progesterone regulate p27kip1 levels via the ubiquitin-proteasome system: pathogenic and therapeutic implications for endometrial cancer.

The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA.

Exogenous calreticulin improves diabetic wound healing.

A serious consequence of diabetes mellitus is impaired wound healing, which largely resists treatment. We previously reported that topical application of calreticulin (CRT), an endoplasmic reticulum chaperone protein, markedly enhanced the rate and quality of wound healing in an experimental porcine model of cutaneous repair. Consistent with these in vivo effects, in vitro CRT induced the migration and proliferation of normal human cells critical to the wound healing process. These functions are particularly deficient in poor healing diabetic wounds. Using a genetically engineered diabetic mouse (db/db) in a full‐thickness excisional wound healing model, we now show that topical application of CRT induces a statistically significant decrease in the time to complete wound closure compared with untreated wounds by 5.6 days (17.6 vs. 23.2). Quantitative analysis of the wounds shows that CRT increases the rate of reepithelialization at days 7 and 10 and increases the amount of granulation tissue at day 7 persisting to day 14. Furthermore, CRT treatment induces the regrowth of pigmented hair follicles observed on day 28. In vitro, fibroblasts isolated from diabetic compared with wild‐type mouse skin and human fibroblasts cultured under hyperglycemic compared with normal glucose conditions proliferate and strongly migrate in response to CRT compared with untreated controls. The in vitro effects of CRT on these functions are consistent with CRTs potent effects on wound healing in the diabetic mouse. These studies implicate CRT as a potential powerful topical therapeutic agent for the treatment of diabetic and other chronic wounds.

Hormonal and Growth Regulation of Epithelial and Stromal Cells From the Normal and Malignant Endometrium by Pigment Epithelium-Derived Factor

&NA; We discovered that pigment epithelium‐derived factor (PEDF)‐null mice have endometrial hyperplasia, the precursor to human type I endometrial cancer (ECA), which is etiologically linked to unopposed estrogen (E2), suggesting that this potent antiangiogenic factor might contribute to dysregulated growth and the development of type I ECA. Treatment of both ECA cell lines and primary ECA cells with recombinant PEDF dose dependently decreased cellular proliferation via an autocrine mechanism by blocking cells in G1 and G2 phases of the cell cycle. Consistent with the known opposing effects of E2 and progesterone (Pg) on endometrial proliferation, Pg increases PEDF protein synthesis and release, whereas E2 has the converse effect. Using PEDF luciferase promoter constructs containing two Pg and one E2 response elements, E2 reduced and Pg increased promoter activity due to distal response elements. Furthermore, E2 decreases and Pg increases PEDF secretion into conditioned media (CM) by both normal endometrial stromal fibroblasts (ESFs) and cancer‐associated fibroblasts (CAFs), but only CM from ESFs mediated growth‐inhibitory activity of primary endometrial epithelial cells (EECs). In addition, in cocultures with primary EECs, Pg‐induced growth inhibition is mediated by ESFs, but not CAFs. This is consistent with reduced levels of Pg receptors on CAFs surrounding human malignant glands in vivo. Taken together, the data suggest that PEDF is a hormone‐regulated negative autocrine mediator of endometrial proliferation, and that paracrine growth inhibition by soluble factors, possibly PEDF, released by ESFs in response to Pg, but not CAFs, exemplifies a tumor microenvironment that contributes to the pathogenesis of ECA.

Abstract 3410: Inhibitors of Skp2 E3ligase stabilize nuclear p27kip1 for regain of growth regulation in cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In many human cancers, the tumor suppressor, p27kip1 (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization; lack of nuclear p27 causes aberrant cell cycle progression and cytoplasmic p27 can mediate cell migration/metastasis. We previously showed that estrogen (E2) induces Skp2-dependent degradation of p27 and cell proliferation in primary endometrial epithelial cells (EECs) and endometrial carcinoma (ECA) cell lines (e.g., ECC-1) suggesting a pathogenic mechanism for type I endometrial carcinoma (ECA), an estrogen (E2)-linked cancer. The current studies show that treatment of ECC-1 cells with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) increase protein levels of p27, increase p27 half-life by 6 hours and inhibit cell proliferation (IC50 10μM). Two of five SKP2E3LIs, designated as C2 and C20 specifically increase p27 in the nucleus while decreasing p27 in the cytoplasm in both ECC-1 cells and primary ECA cells. In addition, C2 and C20 prevent both E2-induced proliferation and degradation of nuclear p27. These data suggest that Skp2E3LIs can function in the nucleus to prevent E2-induced degradation of p27 in primary ECA cells to regain growth control by directly blocking p27 ubiquitylation and subsequent degradation. Skp2E3LIs enable a chemical biology approach to understanding the functional significance of the ubiquitin pathway in p27-mediated cell cycle regulation and importantly, are the first specific proteasome inhibitors that pharmacologically target the binding interaction between the E3ligase, SCF-Skp2/Cks1 and p27 to prevent its ubiquitylation and subsequent degradation. These targeted inhibitors represent a major therapeutic advancement over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of p27. Citation Format: Savvas C. Pavlides, Kuang-Tzu Huang, Lily Wu, Bo R. Rueda, Stephanie V. Blank, Khushbakhat R. Mittal, Timothy Cardozo, Leslie I. Gold. Inhibitors of Skp2 E3ligase stabilize nuclear p27kip1 for regain of growth regulation in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2013-3410

Abstract 545: Autocrine growth regulation by PEDF is mediated by ovarian steroid hormones: Implications in the pathogenesis of endometrial carcinoma

The angiogenic inhibitor and neurotrophic factor, pigment epithelium-derived factor (PEDF) was first shown in the eye and is now shown to be expressed in a number of tissues and to function in apoptosis and growth inhibition. The importance of PEDF in growth regulation of the endometrium is reflected in PEDF null mice, which develop endometrial hyperplasia. Therefore, we hypothesized that PEDF is decreased in human endometrial carcinoma with loss of growth inhibition. We show by immunostaining (n=44) that PEDF expression is decreased in both stromal and epithelial cells in atypical endometrial hyperplasia (AEH), the precursor to type I endometrial carcinoma (ECA; 80% of all ECAs) and ECA. Whereas PEDF is highly expressed by primary endometrial epithelial cells (EECs) and stromal cells, ECA cells synthesize less protein and stromal cells isolated from malignant endometrium (cancer associated fibroblasts; CAFs) synthesize 80% less PEDF mRNA (n=35) by quantitative real time PCR and less PEDF protein. In addition, whereas exogenous recombinant PEDF inhibits proliferation of ECA cell lines (e.g. HEC-1B by 40%; peak response at 4nM), PEDF antibodies and PEDF siRNA stimulate proliferation suggesting PEDF inhibits cell proliferation in an autocrine manner in the endometrium. Estrogen (E2) stimulates and progesterone (Pg) inhibits proliferation of the endometrium in a cyclical manner. Due to these hormonal growth regulatory effects, E2 induces AEH and Type I ECA and Pg is a successful therapeutic agent for these diseases. Therefore, we further hypothesized that E2 decreases PEDF causing dysreglated growth and conversely, that Pg might inhibit growth by increasing PEDF. Indeed, we show that treatment of the ECA cell lines, HEC-1A, HEC-1B, KLE and primary EECs with E2 decreased PEDF mRNA and protein expression, which was blocked by the estrogen receptor (ER) antagonist, ICI 182,780. In direct contrast, Pg increased PEDF mRNA and protein, which was blocked by the antagonist, RU486. It is notable that the PEDF promoter contains two putative PREs and one ERE thereby justifying these responses. Taken together, our data implicate PEDF as an autocrine hormone-regulated inhibitor of endometrial growth and suggest that the E2-driven decrease in PEDF leads to dysregulated endometrial growth as an important molecular target in the pathogenesis of type I ECA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 545. doi:1538-7445.AM2012-545

Abstract 3885: Mechanisms that mediate the increase in p27kip1 by progestins: Therapeutic implications for endometrial carcinoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.

Abstract 1089: TGF-β increases Cdh1 to prevent proteasomal degradation of the cyclin-dependent kinase inhibitor, p27kip1, a significant molecular target for growth arrest in the endometrium

Our previous studies show that the cyclin-dependent kinase inhibitor (CKI), p27 kip1 (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.