Journal Article

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

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

2021

Overview

Background The 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. Methods Ovariectomized 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. Results Significant 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 . Conclusions PR 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Alternative RNA splicing / Alternative splicing / Alternative Splicing - drug effects / Analysis / Animals / Antagonists / Binding sites / Biomedical and Life Sciences / Biomedicine / Breast cancer / Cancer / Cancer Research / Cancer therapies / Cell Proliferation - drug effects / Cell surface / E-cadherin / Epithelial cells / Epithelial Cells - cytology / Epithelial Cells - drug effects / Epithelial Cells - metabolism / Epithelial-mesenchymal transition / Epithelial-Mesenchymal Transition - drug effects / Epithelial-Mesenchymal Transition - genetics / Estrogen / estrogen and progesterone / Estrogens / Estrogens - metabolism / Estrogens - pharmacology / Exon skipping / Female / Flow cytometry / Fluorides / Gene expression / Genes / Hormone Antagonists - pharmacology / Hormones / Intermediates / LRP2 protein / LRP5 protein / Luminal progenitor cells / Mammary gland / Mammary Glands, Animal - cytology / Mammary Glands, Animal - drug effects / Mammary Glands, Animal - metabolism / Mammary stem cells / Matrilysin / Mesenchyme / Mice / Mifepristone / Multivariate analysis / Oncology / Ovariectomy / Ovaries / Paracrine signalling / Population / Prevention / Progesterone / Progesterone - metabolism / Progesterone - pharmacology / Progesterone receptor modulators / Receptors, Progesterone - antagonists & inhibitors / Research Article / RNA / RNA Splicing Factors - genetics / RNA-Binding Proteins - genetics / Signal transduction / Signal Transduction - drug effects / Signal Transduction - genetics / Snail protein / Stem cells / Stem Cells - cytology / Stem Cells - drug effects / Stem Cells - metabolism / Steroids / Surface markers / Surgical Oncology / Thrombolytic drugs / TRANCE protein / Ulipristal acetate / Wnt protein