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Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem Cell and Proliferative Breast Cancer Cell Fates.

ABSTRACT: Progesterone receptors (PRs) are key modifiers of estrogen receptor (ER) target genes and drivers of luminal breast cancer progression. Total PR expression, rather than isoform-specific PR expression, is measured in breast tumors as an indicator of functional ER. We identified phenotypic differences between PR-A and PR-B in luminal breast cancer models with a focus on tumorsphere biology. Our findings indicated that PR-A is a dominant driver of cancer stem cell (CSC) expansion in T47D models, and PR-B is a potent driver of anchorage-independent proliferation. PR-A+ tumorspheres were enriched for aldehyde dehydrogenase (ALDH) activity, CD44+/CD24-, and CD49f+/CD24- cell populations relative to PR-B+ tumorspheres. Progestin promoted heightened expression of known CSC-associated target genes in PR-A+ but not PR-B+ cells cultured as tumorspheres. We report robust phosphorylation of PR-A relative to PR-B Ser294 and found that this residue is required for PR-A-induced expression of CSC-associated genes and CSC behavior. Cells expressing PR-A S294A exhibited impaired CSC phenotypes but heightened anchorage-independent cell proliferation. The PR target gene and coactivator, FOXO1, promoted PR phosphorylation and tumorsphere formation. The FOXO1 inhibitor (AS1842856) alone or combined with onapristone (PR antagonist), blunted phosphorylated PR, and tumorsphere formation in PR-A+ and PR-B+ T47D, MCF7, and BT474 models. Our data revealed unique isoform-specific functions of phosphorylated PRs as modulators of distinct and opposing pathways relevant to mechanisms of late recurrence. A clear understanding of PR isoforms, phosphorylation events, and the role of cofactors could lead to novel biomarkers of advanced tumor behavior and reveal new approaches to pharmacologically target CSCs in luminal breast cancer.

SUBMITTER: Truong TH

PROVIDER: S-EPMC6349004 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Project description:IntroductionProgesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression.MethodsGlobal gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature.Results'SUMO-sensitive' PR target genes primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (that is, MSX2, RGS2, MAP1A, and PDK4) with the steroid receptor coactivator, CREB-(cAMP-response element-binding protein)-binding protein (CBP), and mixed lineage leukemia 2 (MLL2), a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at 'closed' enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR target-genes and inhibited proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells.ConclusionsWe conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (that is, derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.

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Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem Cell and Proliferative Breast Cancer Cell Fates.

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