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. 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. 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. 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.

中文翻译：

---

孕酮受体拮抗剂逆转小鼠乳腺干细胞扩增和孕酮作用的旁分泌效应物

卵巢激素雌激素和孕激素 (EP) 与乳腺癌的病因有关。孕酮暴露的一个具体后果是乳腺干细胞 (MSC) 和管腔祖细胞 (LP) 区室的扩张。我们假设在小鼠模型中施用孕酮受体 (PR) 拮抗剂可以消除这种效应及其分子促进剂。切除卵巢的 FVB 小鼠随机接受 14 天的治疗：假手术、EP、EP + 特拉司酮 (EP + TPA)、EP + 米非司酮 (EP + MFP)。然后处死小鼠，收获乳腺，并使用细胞表面标记通过流式细胞术分离乳腺上皮细胞谱系。对来自每个谱系的 RNA 进行测序，并使用 DESeq 分析差异基因表达。进行定量 PCR 以确认在 RNA seq 中发现的候选基因。使用 Tukey 事后分析进行方差分析以比较相对表达。使用 rMAT 多变量分析工具检查替代剪接事件。与对照相比，EP 处理后观察到 MSC 和管腔成熟 (LM) 细胞分数显着增加（分别为 p < 0.01 和 p < 0.05），而 LP 分数显着减少（p < 0.05）。这些激素诱导的作用在暴露于 TPA 和 MFP 后被逆转（两者 p < 0.01）。RNA 测序数据的 Gene Ontology 分析显示 EP 诱导的几种途径富集，对 MSC 中 Wnt 信号传导的影响最大，被 PR 抑制剂显着抑制。在 LP 细胞中，Wnt4 和 Rank1 以及 Wnt 途径中间体 Lrp2 和 Axin2（通过 qRTPCR 确认）的显着诱导被 TPA 和 MFP 逆转（p < 0.0001）。这些途径的下游信号中间体（Lrp5、Mmp7）显示出类似的效果。上皮间质转化标志物（Cdh1、Cdh3）的表达和 EMT 调节因子（Zeb1、Zeb2、Gli3、Snai1 和 Ptch2）的诱导对孕酮有显着反应。EP 处理与大规模选择性剪接事件相关，丰富了与 Srsf、Esrp 和 Rbfox 家族相关的基序。在 Cdh1、Enah 和 Brd4 中观察到外显子跳跃。PR 抑制逆转了乳腺中已知的致瘤途径，并抑制了孕酮对 RNA 剪接事件的先前未知的影响。总的来说，我们的结果加强了重新考虑 PR 抑制剂预防乳腺癌的理由。上皮间质转化标志物（Cdh1、Cdh3）的表达和 EMT 调节因子（Zeb1、Zeb2、Gli3、Snai1 和 Ptch2）的诱导对孕酮有显着反应。EP 处理与大规模选择性剪接事件相关，丰富了与 Srsf、Esrp 和 Rbfox 家族相关的基序。在 Cdh1、Enah 和 Brd4 中观察到外显子跳跃。PR 抑制逆转了乳腺中已知的致瘤途径，并抑制了孕酮对 RNA 剪接事件的先前未知的影响。总的来说，我们的结果加强了重新考虑 PR 抑制剂预防乳腺癌的理由。上皮间质转化标志物（Cdh1、Cdh3）的表达和 EMT 调节因子（Zeb1、Zeb2、Gli3、Snai1 和 Ptch2）的诱导对孕酮有显着反应。EP 处理与大规模选择性剪接事件相关，丰富了与 Srsf、Esrp 和 Rbfox 家族相关的基序。在 Cdh1、Enah 和 Brd4 中观察到外显子跳跃。PR 抑制逆转了乳腺中已知的致瘤途径，并抑制了孕酮对 RNA 剪接事件的先前未知的影响。总的来说，我们的结果加强了重新考虑 PR 抑制剂预防乳腺癌的理由。EP 处理与大规模选择性剪接事件相关，丰富了与 Srsf、Esrp 和 Rbfox 家族相关的基序。在 Cdh1、Enah 和 Brd4 中观察到外显子跳跃。PR 抑制逆转了乳腺中已知的致瘤途径，并抑制了孕酮对 RNA 剪接事件的先前未知的影响。总的来说，我们的结果加强了重新考虑 PR 抑制剂预防乳腺癌的理由。EP 处理与大规模选择性剪接事件相关，丰富了与 Srsf、Esrp 和 Rbfox 家族相关的基序。在 Cdh1、Enah 和 Brd4 中观察到外显子跳跃。PR 抑制逆转了乳腺中已知的致瘤途径，并抑制了孕酮对 RNA 剪接事件的先前未知的影响。总的来说，我们的结果加强了重新考虑 PR 抑制剂预防乳腺癌的理由。