Steroid hormones play a fundamental role in the development, treatment, and progression of breast cancer, often in a paradoxical manner. For example, breast cancer risk has been linked to exposure to hormones (i.e., number of estrous cycles) as well as loss of hormone exposure (i.e., menopausal status) (1). Early hormone treatments for breast cancer involved estrogen, progestin, and androgen analogs. Over time, however, targeting of estrogen activity with selective estrogen receptor (ER) modulators, degraders, or aromatase inhibitors evolved as the standard of care for ER-positive breast tumors. Targeting the progesterone receptor (PR) may also be efficacious and there is currently renewed interest in exploring PR therapeutics. One impediment to such an approach has been the divergent roles of PR in breast cancer.
The PR field has increasingly noted a dichotomy in progestin action that is summarized as “context dependent” (2). One confounding factor has been the use of a variety of ligands empirically categorized as “agonists” (i.e., progesterone, medroxyprogesterone acetate) or “antagonists” (i.e., onapristone, mifepristone), based mainly on effects on uterine physiology. Each of these ligands alters PR activity differentially, through changes in PR conformation, DNA binding, and posttranslational modifications (2). Clinically, agonists and antagonists have shown efficacy in a subset of patients [reviewed in Carroll et al. (3)]. Mechanistically, PR agonists, including the natural hormone progesterone, modify ER function, in part by promoting ER/PR interaction and redirecting ER to less mitogenic target genes (4, 5). Interestingly, a selective PR antagonist, CDB4124, was more potent than agonists at blocking estrogen-driven growth of breast cancer xenografts when given in combination with tamoxifen (2). Thus, selective PR antagonists may share similar ER-blocking functions as agonists, but may have additional benefits through other mechanisms.
One important aspect of PR biology that emerged in the last decade is its ability to expand breast cancer stem cells (CSCs). Such cells have increased tumor-initiating and tumorsphere-forming potential, drug insensitivity, and relative quiescence. Breast cancer cells that are CD44+/CD24−/low, ALDH1+, or CK5+ all correspond with increased breast CSC activity, and progestins increase each of these populations in ER+/PR+ breast cancer cell lines, whereas antiprogestins, where tested, block these actions [reviewed in Finlay-Schultz and Sartorius (6)]. To date, these studies have largely underappreciated the considerable differences between the actions of the two naturally occurring PR isoforms, PR-A and PR-B.
In their article, Truong et al. (7) have started to tease apart the seemingly divergent roles the two PR isoforms play in supporting CSC properties. The authors elegantly establish that PR is necessary for tumorsphere formation in ER+/PR+ breast cancer cells. They go on to show that cells that express PR-A only form more primary or secondary tumorspheres compared with cells expressing only PR-B. Notably, this occurred in the absence or presence of exogenously added PR and/or ER agonists. On the contrary, PR-B–expressing cells formed larger tumorspheres than their PR-A–expressing counterparts. These data suggest that PR-A supports increased stemness whereas PR-B supports increased proliferation. PR-B–driven proliferation is backed by a previous study that showed PR-B, but not PR-A, regulates progestin-induced reentry into the cell cycle after antiestrogen-induced cell cycle arrest (8).
A possible explanation for the divergent roles of PR isoforms may be their transcriptional control over overlapping but distinct gene sets, which is, in part, directed by posttranslational modifications. Indeed, Truong et al. (7) demonstrate that PR-A is preferentially recruited to promoters of CSC-associated genes such as Wnt4 and Klf4 and induces their expression relative to PR-B. Interestingly, PR-A phosphorylation at the activating Ser294 residue occurs more rapidly and is maintained longer than Ser294 PR-B phosphorylation. Again, the mere presence of PR-A was sufficient to increase tumorsphere number compared with PR-null cells. However, cells with phospho-Ser294–deficient PR-A become more like cells that express only PR-B: They have reduced recruitment to CSC-associated gene promoters, decreased tumorsphere formation, and increased anchorage-independent proliferation. These data indicate that this phosphorylation event is critical in differentiating the roles of PR-A and PR-B, particularly in regard to CSCs.
To better define how PR-A and PR-B might be regulating transcription of differential target genes, Truong et al. (7) investigated the role of FOXO1, a known transcriptional target and coactivator of PR. Interestingly, FOXO1 overexpression was sufficient to increase secondary tumorsphere formation in PR-A and PR-B cells, and this increase was reversed upon FOXO1 knock-down or by addition of a small molecule inhibitor of FOXO1. Inhibition of FOXO1 was also associated with reduced phosphorylation of both PR isoforms, proposed by the authors as due to disruption of the feed-forward signaling loop between FOXO1 and PRs that typically promotes a robust, sustained signal. The FOXO1 inhibitor also blunted tumorsphere formation in unmodified breast cancer cell lines expressing both endogenous PR isoforms, and was additive with the PR antagonist onapristone at blocking this effect (7). Taken together, these results provide evidence that FOXO1 cooperates with both PR isoforms, though more potently with PR-A, to sustain CSC properties through maintenance of PR phosphorylation and activation.
This work by Truong et al. (7) clearly demonstrates PR isoforms differentially regulate breast CSC properties. Clinically, tumors are only assessed for expression of total PR, not activated (phospho-Ser294) or isoform-specific expression. PR expression is measured in patient samples for the express purpose of determining ER functionality and response to endocrine therapy. These studies by Truong et al. (7) suggest that deeper assessment of PR isoform ratios or activation via phospho-Ser294 staining may be able to delineate patients who would benefit from therapeutic modulation of PR. Ligand choice is critical and may, under the right context, both attenuate ER function and inhibit expansion of therapy resistant CSCs, slowing tumor growth and preventing breast cancer recurrence.
Acknowledgments
Financial Support: This work was supported by National Institutes of Health Grants F31 CA210519 (to L.M.F.) and R01 CA140985 (to C.A.S.) and by Breast Cancer Research Foundation Grant 16-072 (to C.A.S.).
Disclosure Summary: The authors have nothing to disclose.
Glossary
Abbreviations:
- CSC
cancer stem cell
- ER
estrogen receptor
- PR
progesterone receptor
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