Christy R. Hagan

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 G1/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, HSD11β2, 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.

Molecular determinants of context-dependent progesterone receptor action in breast cancer

The ovarian steroid hormone, progesterone, and its nuclear receptor, the progesterone receptor, are implicated in the progression of breast cancer. Clinical trial data on the effects of hormone replacement therapy underscore the importance of understanding how progestins influence breast cancer growth. The progesterone receptor regulation of distinct target genes is mediated by complex interactions between the progesterone receptor and other regulatory factors that determine the context-dependent transcriptional action of the progesterone receptor. These interactions often lead to post-translational modifications to the progesterone receptor that can dramatically alter receptor function, both in the normal mammary gland and in breast cancer. This review highlights the molecular components that regulate progesterone receptor transcriptional action and describes how a better understanding of the complex interactions between the progesterone receptor and other regulatory factors may be critical to enhancing the clinical efficacy of anti-progestins for use in the treatment of breast cancer.

Progesterone receptor action: defining a role in breast cancer

The ovarian steroid hormones, estradiol and progesterone, and their nuclear receptors (estrogen receptor [ER] and progesterone receptor [PR]), are involved in breast cancer development. As ER-positive/PR-positive tumors progress, they are likely to become steroid hormone-resistant/independent, yet often retain expression of their steroid receptors. Notably, up to 40% of women with steroid receptor-positive tumors exhibit de novo resistance or eventually fail on estrogen- or ERα-blocking therapies (acquired resistance). Indeed, most of the research on this topic has centered on mechanisms of ER ‘escape’ from endocrine therapy and the design of better ER-blocking strategies; signaling pathways that mediate endocrine (i.e., anti-estrogen) resistance are also excellent therapeutic targets. However, serious consideration of PR isoforms as important drivers of early breast cancer progression and ER modulators is timely and significant. Indeed, progress has been hindered by ER-centric experimental approaches. This article will focus on defining a role for PR in breast cancer with hopes of providing a refreshing PR-focused perspective.

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.

Scaffolding actions of membrane-associated progesterone receptors

Progesterone is an ovarian steroid hormone that is essential for normal breast development. The actions of progesterone are largely mediated through binding to its cognate steroid hormone receptor, the progesterone receptor (PR). PR isoforms exist in the nucleus and transcriptionally activate genes necessary for proliferation and survival (classical role). Cytoplasmic or membrane-associated PR exists in the cytoplasm where it participates in protein complexes with signaling molecules and other steroid hormone receptors capable of rapid activation of cytoplasmic protein kinase cascades. This review details the extra nuclear scaffolding actions of PR with c-Src and MEK1, the upstream components of MAP kinase modules.

A Common Docking Domain in Progesterone Receptor-B links DUSP6 and CK2 signaling to proliferative transcriptional programs in breast cancer cells

Progesterone receptors (PR) are transcription factors relevant to breast cancer biology. Herein, we describe an N-terminal common docking (CD) domain in PR-B, a motif first described in mitogen-activated protein kinases. Binding studies revealed PR-B interacts with dual-specificity phosphatase 6 (DUSP6) via the CD domain. Mutation of the PR-B CD domain (mCD) attenuated cell cycle progression and expression of PR-B target genes (including STAT5A and Wnt1); mCD PR-B failed to undergo phosphorylation on Ser81, a ck2-dependent site required for expression of these genes. PR-B Ser81 phosphorylation was dependent on binding with DUSP6 and required for recruitment of a transcriptional complex consisting of PR-B, DUSP6 and ck2 to an enhancer region upstream of the Wnt1 promoter. STAT5 was present at this site in the absence or presence of progestin. Furthermore, phospho-Ser81 PR-B was recruited to the STAT5A gene upon progestin treatment, suggestive of a feed-forward mechanism. Inhibition of JAK/STAT-signaling blocked progestin-induced STAT5A and Wnt1 expression. Our studies show that DUSP6 serves as a scaffold for ck2-dependent PR-B Ser81 phosphorylation and subsequent PR-B-specific gene selection in coordination with STAT5. Coregulation of select target genes by PR-B and STAT5 is likely a global mechanism required for growth promoting programs relevant to mammary stem cell biology and 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.

NEMO, a transcriptional target of estrogen and progesterone, is linked to tumor suppressor PML in breast cancer

The beneficial versus detrimental roles of estrogen plus progesterone (E+P) in breast cancer remains controversial. Here we report a beneficial mechanism of E+P treatment in breast cancer cells driven by transcriptional upregulation of the NFκB modulator NEMO, which in turn promotes expression of the tumor suppressor protein promyelocytic leukemia (PML). E+P treatment of patient-derived epithelial cells derived from ductal carcinoma in situ (DCIS) increased secretion of the proinflammatory cytokine IL6. Mechanistic investigations indicated that IL6 upregulation occurred as a result of transcriptional upregulation of NEMO, the gene that harbored estrogen receptor (ER) binding sites within its promoter. Accordingly, E+P treatment of breast cancer cells increased ER binding to the NEMO promoter, thereby increasing NEMO expression, NFκB activation, and IL6 secretion. In two mouse xenograft models of DCIS, we found that RNAi-mediated silencing of NEMO increased tumor invasion and progression. This seemingly paradoxical result was linked to NEMO-mediated regulation of NFκB and IL6 secretion, increased phosphorylation of STAT3 on Ser727, and increased expression of PML, a STAT3 transcriptional target. In identifying NEMO as a pivotal transcriptional target of E+P signaling in breast cancer cells, our work offers a mechanistic explanation for the paradoxical antitumorigenic roles of E+P in breast cancer by showing how it upregulates the tumor suppressor protein PML. Cancer Res; 77(14); 3802-13. ©2017 AACR.

Meeting review: Extra-nuclear steroid receptors-Integration with multiple signaling pathways

The FASEB Summer Research Meeting entitled “Extra-Nuclear Steroid Receptors: Integration with Multiple Signaling Pathways,” was held from July 27 through August 1, 2008 in Carefree, Arizona. The meeting served as an excellent opportunity for scientists interested in both nuclear and extra-nuclear steroid signaling to exchange ideas in a relatively small, informal setting. The meeting began with a short talk by Richard Pietras (University of California at Los Angeles) on the history of extra-nuclear, or nongenomic, steroid signaling. His presentation covered work from the 1970s up to the present, and served as a wonderful introduction to the field. After Dr. Pietras, Margaret Shupnik (University of Virginia) presented the keynote lecture on the integration of membrane and nuclear receptor functionality. The talk focused on the role of membrane-initiated kinase cascades in regulating nuclear steroid receptor signaling, and introduced many important concepts that were addressed throughout the meeting.

Abstract 1868A: Progesterone receptor promotes inflammatory gene programs in breast cancer

Mounting clinical data implicates progesterone and its receptor (progesterone receptor; PR) in breast cancer progression. Upon diagnosis, nearly 70% of breast cancers express PR and the estrogen receptor (ER). In contrast, only 7-10% of normal luminal epithelial cells express ER and PR. Mitogenic protein kinases (MAPK, ck2) known to modify and activate PR have been shown to be dysregulated in breast cancer. This creates a context wherein PR is inappropriately activated by aberrant kinases in the absence of hormones. We hypothesize that ck2-dependent phosphorylation of PR Serine 81 (Ser81) “redirects” the modified receptor to a distinct subset of target genes, including STAT5A, a gene required for mammary gland development and key to mediating inflammatory/interferon responses. Microarray data sets were analyzed using Gene Set Enrichment Analysis (GSEA) from progestin (R5020)-treated T47D breast cancer cells stably expressing either wt or Ser81 phospho-mutant PR (S79/81A PR). GSEA revealed that genes from multiple JAK/STAT, inflammatory and interferon-regulated gene sets were significantly enriched in our T47D-wt PR dataset. Enrichment of these gene sets was lost in cells lacking PR Ser81 phosphorylation (S79/81A PR), suggesting that Ser81 phosphorylation is necessary for their PR-dependent regulation. Leading Edge (LE) analysis revealed a significant enrichment in inflammatory/interferon-regulated pathways, again dependent upon PR Ser81 phosphorylation. Regulation of individual genes identified as being highly associated with the overlapping gene sets (LE gene list) was impaired in cells expressing S79/81A PR relative to cells expressing wt PR. Of note, many of these genes are transcriptional targets of interferon signaling pathways and key mediators of inflammation (i.e. IFITs, STATs, CXCL10, CXCL2, OAS1/2). Moreover, CEAS (cis-regulatory element annotation system) analysis of a publically available PR ChIP-chip data set (obtained from T47D breast cancer cells treated with progesterone) revealed significant enrichment of STAT5 binding motifs (GAS sites) within PR binding sites. Finally, using ChIP analysis we identified coordinate PR and STAT5 binding at key PR Ser81-regulated target genes; recruitment failed to occur in PR S79/81A-expressing cells. Cumulatively, these data support a model of coordinate regulation between PR and STAT5 on a select subset of Ser81 phosphorylation-dependent PR target genes. Many any of these genes are involved in cellular immune/inflammatory responses. We predict that direct interaction between STAT5 and PR at enhancer regions drives inflammatory gene programs in breast cancer. Understanding cross-talk between PR and inflammation is critical to developing better treatments, and could expose a novel mechanism through which PR drives early events in breast cancer development and progression that may be exploited using novel PR-based endocrine therapies. Citation Format: Christy R. Hagan, Todd P. Knutson, Carol A. Lange. Progesterone receptor promotes inflammatory gene programs in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1868A. doi:10.1158/1538-7445.AM2015-1868A