Nathan J. Charles

Membrane progesterone receptor expression in mammalian tissues: A review of regulation and physiological implications

The recent discovery of a novel, membrane localized progestin receptor (mPR) unrelated to the classical progesterone receptor (PR) in fishes and its subsequent identification in mammals suggests a potential mediator of non-traditional progestin actions, particularly in tissues where PR is absent. While early studies on mPR focused on final oocyte maturation in fishes, more current studies have examined mPRs in multiple mammalian systems in both reproductive and non-reproductive tissues as well as in diseased tissues. Here we review the current data on mPR in mammalian systems including male and female reproductive tracts, liver, neuroendocrine tissues, the immune system and breast and ovarian cancer. We also provide new data demonstrating mPR expression in the RAW 264.7 immune cell line and bone marrow-derived macrophages as well as mPR expression and downstream gene regulation in ovarian cancer cells.

Progesterone receptors induce FOXO1-dependent senescence in ovarian cancer cells.

Loss of nuclear progesterone receptors (PR) and low circulating progesterone levels are associated with increased ovarian cancer (OC) risk. However, PR are abundantly expressed in a significant percentage of serous and endometrioid ovarian tumors; patients with PR+ tumors typically experience longer progression-free survival relative to those with PR-null tumors. The molecular mechanisms of these protective effects are poorly understood. To study PR action in OC in the absence of added estrogen (i.e., needed to induce robust PR expression), we created ES-2 OC cells stably expressing vector control or GFP-tagged PR-B (GFP-PR). Progestin (R5020) stimulation of ES-2 cells stably expressing GFP-PR induced cellular senescence characterized by altered cellular morphology, prolonged survival, senescence-associated β-galactosidase activity, G1 cell cycle arrest and upregulation of the cell cycle inhibitor, p21, as well as the Forkhead-box transcription factor, FOXO1; these results repeated in unmodified ER+/PR+ PEO4 OC cells. PR-B and FOXO1 were detected within the same PRE-containing regions of the p21 upstream promoter. Knockdown of p21 resulted in molecular compensation via FOXO1-dependent upregulation of numerous FOXO1 target genes (p15, p16, p27) and an increased rate of senescence. Inhibition of FOXO1 (with AS1842856) or stable FOXO1 knockdown inhibited progestin-induced p21 expression and blocked progestin-induced senescence. Overall, these findings support a role for PR as a tumor suppressor in OC cells, which exhibits inhibitory effects by inducing FOXO1-dependent cellular senescence. Clinical “priming” of the PR-FOXO1-p21 signaling pathway using PR agonists may provide a useful strategy to induce irreversible cell cycle arrest and thereby sensitize OC cells to existing chemotherapies as part of combination “two-step” therapies.

Expression of membrane progesterone receptors (mPR/PAQR) in ovarian cancer cells: implications for progesterone-induced signaling events.

The high mortality rates associated with ovarian cancer are largely due to a lack of highly effective treatment options for advanced stage disease; a time when initial diagnosis most commonly occurs. Recent evidence suggests that the steroid hormone, progesterone, may possess anti-tumorigenic properties. With the discovery of a new class of membrane-bound progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor (PAQR) gene family in the ovary, there are undefined mechanisms by which progesterone may inhibit tumor progression. Therefore, our goal was to define potential mPR-dependent signaling mechanisms operative in ovarian cancer cells. We detected abundant mPRα (PAQR7), mPRβ (PAQR8), and mPRγ (PAQR5), but not classical nuclear PR (A or B isoforms) mRNA expression and mPRα protein expression in a panel of commonly used ovarian cancer cell lines. In contrast to mPR action in breast cancer cells, progesterone alone failed to induce changes in cyclic adenosine monophosphate (cAMP) levels in ovarian cancer cells. However, progesterone enhanced cAMP production by β1,2-adrenergic receptors and increased isoproterenol-induced transcription from a cAMP response element (CRE)-driven reporter gene. Independently of β-adrenergic signaling, we additionally observed activation of both JNK1/2 and p38 MAPK in response to progesterone alone. This finding was supported by the results of a screen for potential mPR gene targets. Progesterone induced a significant increase in transcription of the pro-apoptotic marker BAX, whose activity and expression has been linked to JNK1/2 and p38 signaling. Inhibitors of JNK, but not p38, blocked progesterone-induced BAX expression. Taken together, these observations implicate at least two distinct signaling pathways that may be utilized by mPRs in ovarian cancer cells that exhibit regulatory genomic changes. These studies on mPR signaling in ovarian cancer lay the foundation for future work aimed at understanding how progesterone exerts its anti-tumorigenic effects in the ovary and suggest that pharmacologic activation of mPRs, abundantly expressed in ovarian cancers, may provide a new treatment option for patients with advanced stage disease.

Abstract 4050: Progesterone receptor signaling induces cellular senescence in ovarian cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite major advancements in surgical techniques and chemotherapeutics, over 90% of women with advanced ovarian cancer die with recurrent disease, due to the emergence of chemo-resistant tumors (Bukowski et al 2007). In recent years, the progesterone receptor (PR) has become an attractive target in ovarian cancer. PR is typically expressed in normal ovarian surface epithelial (OSE) cells, but the detection of PR mRNA and protein decreases during the transformation to malignancy. However, up to 35% of ovarian tumors express abundant PR. Several independent studies have indicated that the expression of PR in ovarian tumors is associated with longer progression-free survival in ovarian cancer patients (Hempling et al 1998, Munstedt et al 2000, Sinn et al 2011). The detailed molecular mechanisms of PR expression in OSE cells and its anti-tumorigenic effects remain poorly understood. To study the suppressive role of PR in ovarian cancer in the absence of added estrogen (i.e. needed to stimulate PR expression), we created ES-2 ovarian cancer cells stably expressing vector control or GFP-tagged PR-B. Unmodified ER+/PR+ PEO4 ovarian cancer cells were included to validate our findings. Progestin stimulation (R5020; 10 nM) of ES-2 cells stably expressing GFP-PR inhibited the formation of large colonies in soft-agar assays, but yielded a significant increase in the number of viable, very small colonies relative to vehicle-treated and PR-null cohorts. Continuous treatment with R5020 induced cellular senescence characterized by altered cellular morphology, senescence-associated β-galactosidase activity, irreversible G1 cell-cycle arrest, and upregulation of the cell-cycle inhibitor, p21, as well as the Forkhead-box transcription factor, FOXO1. Notably, both PR-B and FOXO1 were detected within the same PRE-containing regions of the p21 upstream promoter. Stable knock-down using lentiviral shRNAs targeting FOXO1 inhibited progestin-induced p21 expression and blocked the development of senescence, suggesting that progestin-induced cellular senescence in PR+ ovarian cancer cells is mediated by FOXO1-dependent p21 expression. Overall, these findings support the concept of PR as a tumor suppressor in ovarian cancer cells that exhibits its inhibitory effects by inducing cellular senescence. Clinical targeting of the PR-FOXO1-p21 signaling pathway may provide a useful strategy to induce irreversible cell cycle arrest and sensitize ovarian cancer cells to existing chemotherapies as part of combination therapy. (This work was supported by grants from the Minnesota Ovarian Cancer Alliance (MOCA), the Cancer Biology Training Grant (NIH T32 CA009138), and the University of Minnesota Clinical and Translational Science Institute (CTSI) F&T Pilot Grant.) Citation Format: Caroline H. Diep, Nathan J. Charles, C. Blake Gilks, Steve E. Kalloger, Peter A. Argenta, Carol A. Lange. Progesterone receptor signaling induces cellular senescence in ovarian cancer cells. [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 4050. doi:10.1158/1538-7445.AM2013-4050