ck2-Dependent Phosphorylation of Progesterone Receptors (PR) on Ser81 Regulates PR-B Isoform-Specific Target Gene Expression in Breast Cancer Cells

Abstract

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 progestinregulated (BIRC3, HSD11␤2, 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.The ovarian steroid hormone progesterone acts by binding to and activating progesterone receptor (PR) A, B, and C isoforms expressed in target tissues. In the normal breast, PR-A and PR-B are typically expressed in a minority population (7 to 10%) of luminal epithelial cells. PR-B is required for mammary gland development during puberty and pregnancy and acts by contributing to lobulo-alveolar proliferation and ductal side branching (8, 46). Studies from PR-knockout mice show that these mice have significant defects in mammary gland morphology (primarily PR-B dependent) and reproductive abnormalities (primarily PR-A driven) (46, 54). Additionally, the presence of PR was shown to be required for the formation of mammary tumors in a carcinogen-induced mouse model of breast cancer (47). Finally, recent clinical data have shown that women taking hormone replacement therapy (HRT) whose regimens included both estrogen and a progestin, but not estrogen alone, experienced increased breast tumor numbers and sizes (1,5,12). Interestingly, the effect of combined HRT on breast cancer risk was reversible (5, 13), suggestive of epigenetic events.In the absence of progesterone, PR molecules rapidly shuttle between the cytoplasm and the nucleus; cytoplasmic PRs contain membrane-associated species capable of direct binding and signaling to mitogenic protein kinases (c-Src, MAPK, PI3K) (3,7,25,50). Following ligand binding, PRs dissociate from heat shock protein-containing chaperone complexes, undergo dimerization, and are largely retained in the nucleus. Nuclear receptors activate transcription of ...

Recent advances in structure of progestins and their binding to progesterone receptors

“…The stimulation of this enzyme and other kinases, could enhance the phosphorylation of PR and its co-activators in the presence of these antagonists (11)(12)(13). In fact, progestins bind to the PR, in cells' cytoplasm or membrane, thus inducing a cell signaling cascades that promotes phosphorylation of different proteins that could be involved in transcription 7,10,12 .…”

FOXA1 Promotes Tumor Cell Proliferation through AR Involving the Notch Pathway in Endometrial Cancer

“…CK2 is a ubiquitously expressed kinase often up-regulated in many different types of cancer, including breast [55][56][57]. We and others have shown that CK2 phosphorylates PR on Ser81, a site that is basally phosphorylated; however, Ser81 phosphorylation levels increase markedly in response to ligand (or when cells enter S phase in the absence of ligand) [16,58]. PR phosphorylation at Ser81 is associated with a specific gene expression profile, which is correlated with pathways altered in breast cancer, including genes implicated in mammary stem cell maintenance and renewal [8,16].…”

“…Target gene selectivity is achieved not only through differential recruitment of PR [8,16], but also through associated transcriptional co-activators and repressors that are critical to PR function [9,10,46]. For example, pioneer factors are specialized subsets of transcription factors that open defined regions of chromatin, making it accessible for other transcription factors, like SRs (reviewed in [47,48]).…”

“…PRs are highly post-translationally modified, primarily through N-terminal phosphorylation (select phosphorylation sites most relevant to breast cancer biology are highlighted in Figure 1), acetylation, SUMOylation, and ubiquitination [9,[11][12][13][14][15][16][17]. These receptor modifications dramatically alter PR function, receptor localization and turnover, and promoter selectivity.…”

“…These receptor modifications dramatically alter PR function, receptor localization and turnover, and promoter selectivity. The PR can be phosphorylated basally in the absence of the hormonal ligand, but is potently modified after ligand treatment, in response to local growth factors or in a cell cycle-dependent manner [12,13,[15][16][17] (G. Dressing and C. Lange, unpublished data). Mitogenic protein kinases -such as CDK2, CK2, and MAPK -have been shown to phosphorylate PR and subsequently modify PR action.…”