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Abstract
Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.
Highlights
Uterine leiomyomas (LM, a.k.a., fibroids) represent the most common gynecological tumors in women
We recently reported that RANKL is preferentially expressed and upregulated by R5020 in LIC [11], we went on investigating whether the same mechanism (DNA methylation difference in the differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) of RANKL gene) was involved
We demonstrated that RANKL expression is regulated by converging signals from steroid hormone (P4/ PR), genetic factors (MED12 mutation), and epigenetic modifications (DNA methylation and histone modification) and contributes to LM stem cell (LSC) proliferation and tumor formation
Summary
Uterine leiomyomas (LM, a.k.a., fibroids) represent the most common gynecological tumors in women. C ChIP-qPCR showing PR enrichment at the RANKL distal PR-binding site (PRBS, −87,184/−87,024 bp, Chr13: 43,061,107– 43,061,267) (means ± SEM, n = 10, ***P < 0.001, paired two-way ANOVA). E ChIP-qPCR showing PR enrichment at the RANKL proximal promoter (−1256/−1118 bp, Chr13: 43,147,035–43,147,173) (mean ± SEM, n = 10, **P < 0.01, paired two-way ANOVA). Right panel: 3C-qPCR (dot plot) showing RANKL promoter and distal enhancer interactions in MM and LM primary cells (means ± SEM, n = 3, *P < 0.05, **P < 0.01, paired two-way ANOVA). We used RANKL as a representative P4/PR target gene to study the genetic and epigenetic mechanisms underlying dysregulated RANKL expression in LM, which will shed mechanistic light on our understanding of RANKL gene regulation and PR action in breast cancer and other steroid-responsive tumors [5]
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