Progesterone receptor antagonists reverse stem cell expansion and the paracrine effectors of progesterone action in the mouse mammary gland

Manish Ranjan,Susan E Clare,Seema A Khan,Elnaz Mirzaei Mehrabad,Robert Chatterton,Benjamin T Spike,Shivangi Yadav,J Julie Kim,Zexian Zeng,Oukseub Lee,Gannon Cottone

doi:10.1186/s13058-021-01455-2

Manish Ranjan, Susan E Clare + Show 9 more

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https://doi.org/10.1186/s13058-021-01455-2

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Abstract

BackgroundThe ovarian hormones estrogen and progesterone (EP) are implicated in breast cancer causation. A specific consequence of progesterone exposure is the expansion of the mammary stem cell (MSC) and luminal progenitor (LP) compartments. We hypothesized that this effect, and its molecular facilitators, could be abrogated by progesterone receptor (PR) antagonists administered in a mouse model.MethodsOvariectomized FVB mice were randomized to 14 days of treatment: sham, EP, EP + telapristone (EP + TPA), EP + mifepristone (EP + MFP). Mice were then sacrificed, mammary glands harvested, and mammary epithelial cell lineages separated by flow cytometry using cell surface markers. RNA from each lineage was sequenced and differential gene expression was analyzed using DESeq. Quantitative PCR was performed to confirm the candidate genes discovered in RNA seq. ANOVA with Tukey post hoc analysis was performed to compare relative expression. Alternative splicing events were examined using the rMATs multivariate analysis tool.ResultsSignificant increases in the MSC and luminal mature (LM) cell fractions were observed following EP treatment compared to control (p < 0.01 and p < 0.05, respectively), whereas the LP fraction was significantly reduced (p < 0.05). These hormone-induced effects were reversed upon exposure to TPA and MFP (p < 0.01 for both). Gene Ontology analysis of RNA-sequencing data showed EP-induced enrichment of several pathways, with the largest effect on Wnt signaling in MSC, significantly repressed by PR inhibitors. In LP cells, significant induction of Wnt4 and Rankl, and Wnt pathway intermediates Lrp2 and Axin2 (confirmed by qRTPCR) were reversed by TPA and MFP (p < 0.0001). Downstream signaling intermediates of these pathways (Lrp5, Mmp7) showed similar effects. Expression of markers of epithelial-mesenchymal transition (Cdh1, Cdh3) and the induction of EMT regulators (Zeb1, Zeb2, Gli3, Snai1, and Ptch2) were significantly responsive to progesterone. EP treatment was associated with large-scale alternative splicing events, with an enrichment of motifs associated with Srsf, Esrp, and Rbfox families. Exon skipping was observed in Cdh1, Enah, and Brd4.ConclusionsPR inhibition reverses known tumorigenic pathways in the mammary gland and suppresses a previously unknown effect of progesterone on RNA splicing events. In total, our results strengthen the case for reconsideration of PR inhibitors for breast cancer prevention.

Highlights

The ovarian hormones estrogen and progesterone (EP) are implicated in breast cancer causation
mammary stem cell (MSC), located in a specialized niche in the basal epithelium of the mouse mammary gland [4], are capable of giving rise to basal and luminal cells [5,6,7]. This selfrenewing cell population increases several fold in the presence of steroid hormones despite a lack of estrogen receptor (ER) or progesterone receptor (PR) expression. This expansion has been shown to result from the paracrine effects of receptor activator of nuclear factor kappa-B ligand (Rankl) and Wnt4 produced in the luminal compartment [3, 8], which is comprised of luminal mature (LM) and luminal progenitor (LP) cells
MSC percent was significantly greater in the EP group compared to sham (p < 0.05), and this effect was reversed upon exposure to PR inhibitors telapristone acetate (TPA) and MFP (p < 0.01) (Fig. 1b)

Summary

Introduction

The ovarian hormones estrogen and progesterone (EP) are implicated in breast cancer causation. The role of the ovarian steroid hormone progesterone in the development of breast cancer has been increasingly recognized, supported by both murine models and human data [1] One dimension of this cancer-promoting effect relates to the finding that progesterone, in the presence of estrogen, generates a significant increase in the number of murine mammary stem cells (MSC) and influences the intrinsic ability of these cells to regenerate the mammary gland [2, 3]. MSC, located in a specialized niche in the basal epithelium of the mouse mammary gland [4], are capable of giving rise to basal and luminal cells [5,6,7] This selfrenewing cell population increases several fold in the presence of steroid hormones despite a lack of estrogen receptor (ER) or progesterone receptor (PR) expression. In a subsequent study using a different cell sorting strategy, these investigators demonstrated that exposure to EP in addition to increasing the number of MSC, increases the number of ER/PR negative luminal progenitor cells [8]

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