OR26-1 Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem-Like and Proliferative Breast Cancer Cell Fates

Progesterone receptors (PRs) are key modifiers of ER target gene selection and drivers of luminal breast cancer progression. In addition to Her2 and estrogen receptor (ER), total PRs rather than isoform-specific PR expression levels are measured in breast tumors as indicators of functional ER. Herein, we identified phenotypic differences between PR-A and PR-B in luminal breast cancer cell models with focus on tumorsphere biology. Our findings indicate that PR-A is a dominant driver of cancer stem and stem-like cell (CSC) expansion in T47D breast cancer models, whereas PR-B is a potent driver of anchorage-independent proliferation. PR-A+ tumorspheres were enriched for ALDH activity, CD44+/CD24-, and CD49f+/CD24- (i.e. basal-like) cell populations relative to PR-B+ tumorspheres. Progestin promoted heightened expression of known CSC-associated target genes (e.g. FOXO1, p21, KLF4, WNT4, PTGES, NOTCH2) in PR-A+ but not PR-B+ cells cultured in non-adherent tumorsphere (3D) conditions. In contrast to previous reports, we report robust phosphorylation of PR-A relative to PR-B Ser294 (i.e. Ser130 in PR-A) and show that this residue is required for PR-A-induced expression of CSC-associated genes and CSC behavior in vitro. Chromatin immunoprecipitation assays demonstrated that PR-A phosphorylation was required for receptor recruitment to PRE-containing promoter regions of CSC-associated genes (WNT4, KLF4, NOTCH2). Remarkably, cells expressing S294A-PR-A exhibited severely impaired CSC phenotypes but heightened anchorage-independent cell proliferation. The PR target gene and co-activator, FOXO1, promoted PR Ser294 phosphorylation and tumorsphere formation. The FOXO1 inhibitor (AS1842856) alone or in combination with onapristone (PR antagonist) blunted phospho-PR (Ser294) and tumorsphere formation in PR-A+ and PR-B+ T47D cells and unmodified ER+/PR+ MCF-7 and BT474 breast cancer models. Our data reveal unique isoform-specific functions of phospho-PRs as modulators of distinct and opposing pathways (i.e. CSC expansion versus proliferation) in luminal breast cancer models. A clear understanding of PR isoform-specific actions, phosphorylation events, and the role of co-factors such as FOXO1 may lead to novel biomarkers of advanced tumor behavior and reveal new approaches to pharmacologically target CSCs in luminal breast cancers.