Phosphorylated progesterone receptor isoforms mediate opposing stem cell and proliferative breast cancer cell fates

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⁺/CD24², and CD49f⁺/CD24² 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 PRA–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.