Progesterone receptors act as sensors for mitogenic protein kinases in breast cancer models.

Progesterone receptors (PR), members of the nuclear receptor superfamily, function as ligand-activated transcription factors and initiators of c-Src kinase and mitogen-activated protein kinase signaling. Bidirectional cross-talk between PR and mitogenic protein kinases results in changes in PR post-translational modification, leading to alterations in PR transcriptional activity and promoter selectivity. PR-induced rapid activation of cytoplasmic protein kinases insures precise regulatory input to downstream cellular processes that are dependent upon nuclear PR, such as cell-cycle progression, and pro-survival signaling. Here, we review interactions between PR and mitogenic protein kinases and discuss the consequences of specific post-translational modifications on PR action in breast cancer cell-line models.

Integrated actions of progesterone receptor and cell cycle machinery regulate breast cancer cell proliferation.

Multiple laboratories have investigated progesterone receptor (PR) involvement in breast cancer cell cycle progression. There is now a growing body of evidence demonstrating complex interactions between PR and cell cycle regulatory proteins. Here we review the current literature linking PR to cell cycle control and discuss gaps in the current knowledge. A more complete understanding of the relationships between PR and cell cycle regulatory molecules may reveal additional avenues for prevention and treatment of steroid receptor positive breast cancers.

Identification of membrane progestin receptors in human breast cancer cell lines and biopsies and their potential involvement in breast cancer.

Novel membrane progestin receptors (mPRs) coupled to G proteins recently identified in several species, including humans, are potential intermediaries in rapid, nongenomic progestin actions observed in a wide variety of tissues. Here we demonstrate mPR mRNA and protein expression and specific membrane-associated progestin binding in MCF-7 and SK-BR-3 human breast cancer cells. Interestingly, human mPRalpha mRNA expression was higher in breast tumor biopsies than in normal tissue from the same breast. Recent studies indicate intracellular signaling pathways initiated by the mPRs are broadly similar to those induced during breast cancer growth and development. Taken together these results suggest a potential involvement of mPRs during the development or progression of breast cancer.

Progesterone receptor-cyclin D1 complexes induce cell cycle-dependent transcriptional programs in breast cancer cells.

The progesterone receptor (PR) and its coactivators are direct targets of activated cyclin-dependent kinases (CDKs) in response to peptide growth factors, progesterone, and deregulation of cell cycle inhibitors. Herein, using the T47D breast cancer model, we probed mechanisms of cell cycle-dependent PR action. In the absence of exogenous progestin, the PR is specifically phosphorylated during the G2/M phase. Accordingly, numerous PR target genes are cell cycle regulated, including HSPB8, a heat-shock protein whose high expression is associated with tamoxifen resistance. Progestin-induced HSPB8 expression required cyclin D1 and was insensitive to antiestrogens but blocked by antiprogestins or inhibition of specificity factor 1 (SP1). HSPB8 expression increased with or without ligand when cells were G2/M synchronized or contained high levels of cyclin D1. Knockdown of PRs abrogated ligand-independent HSPB8 expression in synchronized cells. Notably, PRs and cyclin D1 copurified in whole-cell lysates of transiently transfected COS-1 cells and in PR-positive T47D breast cancer cells expressing endogenous cyclin D1. PRs, cyclin D1, and SP1 were recruited to the HSPB8 promoter in progestin-treated T47D breast cancer cells. Mutation of PR Ser345 to Ala (S345A) or inhibition of CDK2 activity using roscovitine disrupted PR/cyclin D1 interactions with DNA and blocked HSPB8 mRNA expression. Interaction of phosphorylated PRs with SP1 and cyclin D1 provides a mechanism for targeting transcriptionally active PRs to selected gene promoters relevant to breast cancer progression. Understanding the functional linkage between PRs and cell cycle regulatory proteins will provide keys to targeting novel PR/cyclin D1 cross talk in both hormone-responsive disease and HSPB8-high refractory disease with high HSPB8 expression.

Membrane progesterone receptors (mPRs) mediate progestin induced antimorbidity in breast cancer cells and are expressed in human breast tumors.

Membrane progesterone receptors (mPRs) have been detected in breast cancer cells and tissues, but their roles in cancer progression remain unclear. Here, we demonstrate the localization, signaling, and antiapoptotic actions of mPRs in two nuclear progesterone receptor (PR)-negative breast cancer cell lines, SKBR3 and MDA-MB-468 (MB468), and mPR expression in human breast tumor biopsies. mPRα, mPRß, and mPRγ subtypes were detected in both cell lines as well as in breast tumor tissues from 13 individuals irrespective of nuclear steroid receptor expression. Competitive receptor binding studies with a selective PR ligand, R5020, and an mPR agonist, Org OD 02-0 confirmed the presence of functional mPRs on both cancer cell lines. Progesterone treatment of either cell line caused rapid activation of an inhibitory G protein, as well as activation of p42/44 MAP kinase. Treatment with progesterone or Org OD 02-0 significantly decreased cell death and apoptosis in response to serum starvation, whereas testosterone, 17ß-estradiol, dexamethasone, and R5020 and RU486 were ineffective. Progesterone treatment of MB468 cells also increased mitochondrial membrane potential and Akt activity, but no decrease in caspase 3 activity was observed. Knockdown of mPRα expression in MB468 cells by siRNA transfection blocked the inhibitory effects of progesterone on cell death. The results indicate that progesterone can act through mPRs to inhibit apoptosis in breast cancer cells. The involvement of mPRs in the development or progression of breast tumor growth through inhibition of cell death is an intriguing possibility and requires further investigation.

Role of phosphorylation in progesterone receptor signaling and specificity.

Progesterone receptors (PR), in concert with peptide growth factor-initiated signaling pathways, initiate massive expansion of the epithelial cell compartment associated with the process of alveologenesis in the developing mammary gland. PR-dependent signaling events also contribute to inappropriate proliferation observed in breast cancer. Notably, PR-B isoform-specific cross talk with growth factor-driven pathways is required for the proliferative actions of progesterone. Indeed, PRs act as heavily phosphorylated transcription factor "sensors" for mitogenic protein kinases that are often elevated and/or constitutively activated in invasive breast cancers. In addition, phospho-PR-target genes frequently include the components of mitogenic signaling pathways, revealing a mechanism for feed-forward signaling that confers increased responsiveness of, PR +mammary epithelial cells to these same mitogenic stimuli. Understanding the mechanisms and isoform selectivity of PR/kinase interactions may yield further insight into targeting altered signaling networks in breast and other hormonally responsive cancers (i.e. lung, uterine and ovarian) in the clinic. This review focuses on PR phosphorylation by mitogenic protein kinases and mechanisms of PR-target gene selection that lead to increased cell proliferation.

ck2-dependent phosphorylation of progesterone receptors (PR) on Ser81 regulates PR-B isoform-specific target gene expression in breast cancer cells.

Progesterone receptors (PR) are critical mediators of mammary gland development and contribute to breast cancer progression. Progestin-induced rapid activation of cytoplasmic protein kinases leads to selective regulation of growth-promoting genes by phospho-PR species. Herein, we show that phosphorylation of PR Ser81 is ck2 dependent and progestin regulated in intact cells but also occurs in the absence of PR ligands when cells enter the G(1)/S phase of the cell cycle. T47D breast cancer cells stably expressing a PR-B mutant receptor that cannot be phosphorylated at Ser79/81 (S79/81A) formed fewer soft agar colonies. Regulation of selected genes by PR-B, but not PR-A, also required Ser79/81 phosphorylation for basal and/or progestin-regulated (BIRC3, HSD11ß2, and HbEGF) expression. Additionally, wild-type (wt) PR-B, but not S79/81A mutant PR, was robustly recruited to a progesterone response element (PRE)-containing transcriptional enhancer region of BIRC3; abundant ck2 also associated with this region in cells expressing wt but not S79/81A PR. We conclude that phospho-Ser81 PR provides a platform for ck2 recruitment and regulation of selected PR-B target genes. Understanding how ligand-independent PRs function in the context of high levels of kinase activities characteristic of breast cancer is critical to understanding the basis of tumor-specific changes in gene expression and will speed the development of highly selective treatments.

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.

Progestin signaling through mPRα in Atlantic croaker granulosa/theca cell cocultures and its involvement in progestin inhibition of apoptosis.

Although there is substantial evidence that membrane progestin receptors (mPRs) perform a critical physiological role in meiotic maturation of fish oocytes, it is unknown whether they are also intermediaries in progestin signaling in the surrounding follicular cells. Here, we show that mPRα protein is located on the plasma membranes of both granulosa and theca cells (G/T cells) isolated from Atlantic croaker ovaries and is associated with the presence of a single high affinity, limited capacity, pertussis toxin-sensitive, specific progestin [17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S)] membrane binding site with the characteristics of mPRα. Treatment of G/T cells with 20ß-S caused rapid G protein activation and a transient, pertussis toxin-sensitive, decrease in cAMP levels, whereas the selective nuclear progesterone receptor agonist, R5020, did not cause G protein activation, consistent with previous reports on mPRα signaling. 20ß-S treatment decreased serum starvation-induced cell death in both G/T cells and in seatrout mPRα-transfected MDA-MB-231 cells, whereas R5020 was ineffective. Moreover, a selective mPRα agonist, 10-ethenyl-19-norprogesterone, mimicked the protective action of 20ß-S against cell death, which was lost upon knockdown of mPRα protein but not after progesterone receptor knockdown, further demonstrating an involvement of mPRα. Signaling molecules involved in inhibition of apoptosis, Erk and serine-threonine kinase, were activated in G/T cells by 20ß-S, which suggests a potential mechanism for mPRα inhibition of apoptosis. This is the first study to demonstrate endogenous mPR signaling in the ovarian follicle and to suggest a novel physiological role for mPRα in mediating the antiapoptotic actions of progestins in ovarian follicle cells.