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.

Progesterone and Breast Cancer: an NCI Workshop Report

“…PR-A is a potent driver of stem cell expansion in breast cancer models relative to PR-B, while of the two isoforms, is a stronger driver of proliferation in soft agar (6). These recently published data demonstrated that phosphorylation of PR Ser294 is required for breast cancer stem cell outgrowth as measured in tumorsphere assays and using a variety of breast cancer stem cell markers including CD44+/CD24-and ALDH (6).…”

“…PR-A is a potent driver of stem cell expansion in breast cancer models relative to PR-B, while of the two isoforms, is a stronger driver of proliferation in soft agar (6). These recently published data demonstrated that phosphorylation of PR Ser294 is required for breast cancer stem cell outgrowth as measured in tumorsphere assays and using a variety of breast cancer stem cell markers including CD44+/CD24-and ALDH (6). PR Ser294 phosphorylation is elevated when the same cells are subjected to 3D relative to 2D culture, as measured using specific antibodies and flow cytometry of permeabilized cells to detect minority PR+ subpopulations (AR Dwyer, TH Truong, and CA Lange, unpublished results).…”

“…PR Ser294 phosphorylation is mediated by either P4 or growth factor (i.e., EGF) induced activation of MAPKs. Exogenously added P4 is not required for tumorsphere (mammosphere) formation (i.e., number) but contributes to increased tumorsphere size in defined media (6). While the combination of E2 + P4 inhibited cell growth in soft agar, these hormones (alone or in combination) have no effect on secondary tumorsphere formation or the expression of stem cell markers (i.e., the ALDH+ and CD44+/CD24-low cell populations).…”

“…It is not known if both PR isoforms A and B are equally expressed in basal-like cancer stem cells in human breast cancer specimens. However, PR-B is a more potent proliferative isoform (in the absence of PR-A), while PR-A (in the absence of PR-B) is a more potent driver of stem cell-like phenotypes (6). While both PR isoforms can drive the formation of secondary mammospheres (also called tumorspheres) that are CD44+/ CD24-low and ALDH+ in vitro, PR-A+ breast cancer cell lines grown as spheres appear more basal-like (i.e., are more CD49f+), while PR-B+ breast cancer cell lines form fewer spheres that arẽ 30% larger and more luminal-like (CD49f-low) (6).…”

“…However, PR-B is a more potent proliferative isoform (in the absence of PR-A), while PR-A (in the absence of PR-B) is a more potent driver of stem cell-like phenotypes (6). While both PR isoforms can drive the formation of secondary mammospheres (also called tumorspheres) that are CD44+/ CD24-low and ALDH+ in vitro, PR-A+ breast cancer cell lines grown as spheres appear more basal-like (i.e., are more CD49f+), while PR-B+ breast cancer cell lines form fewer spheres that arẽ 30% larger and more luminal-like (CD49f-low) (6). Evidence suggests that the ratio of PR-A:PR-B is disrupted in early carcinogenesis; atypical ductal hyperplasia, DCIS, and invasive disease all have higher PR-A expression relative to PR-B (7)(8)(9).…”