Mechanisms of RANKL Gene Overexpression in Uterine Leiomyoma

Uterine leiomyoma (LM) is the most common benign tumor in women. In contrast to its severity, there are few medical treatments available for LM. Understanding how LM develops is essential for identifying new non-surgical treatments. Previously, we demonstrated that the receptor activator of nuclear factor kappa-B ligand (RANKL), a progesterone receptor (PR) target gene, was significantly upregulated in LM tissue compared to adjacent normal myometrial (MM) tissue and played a tumor-promoting role by inducing proliferation and inhibiting apoptosis in LM cells. But the regulation of RANKL in LM remains unclear. Here, we aimed to investigate the mechanisms by which RANKL is regulated in LM versus MM tissue. Using LM and MM patient samples, we found that RANKL gene expression was more robustly induced by R5020 (P4 agonist) treatment in cultured LM tissue explants than in MM explants. PR expression was similar between the two tissues, however, PR binding to the RANKL proximal promoter and distal enhancer regions was markedly higher in LM vs. MM tissues (P<0.01). Regression analysis indicated that recruitment of PR to the enhancer region was significant and positively correlated with RANKL mRNA levels. Using MethylCap-Seq, we demonstrated that the RANKL enhancer region was hypermethylated in MM tissue (P<0.05). Treatment with DNA methylation inhibitor, 5’-aza reduced the DNA methylation level of the enhancer region and stimulated RANKL expression in both MM and LM explants, but the change in MM was much more dramatic (n=5). The effect of DNA methylation on the transcriptional regulatory function of the RANKL enhancer region was further confirmed by luciferase reporter assay, which demonstrated that the enhancer region showed significant transcription-enhancing activity in LM and MM cells (n=3) and in vitro methylation of this region blocked its regulatory function (n=3). In addition, ChIP assay revealed that active histone modification marks, H3K27Ac and H3K4me3, were more enriched in the RANKL gene enhancer and promoter regions in LM tissues (n=5). Given the prevalence and importance of MED12 mutation in LM pathogenesis, we also examined whether MED12 mutation affected RANKL gene expression. We found that RANKL mRNA levels are upregulated in LM tissue that carries the MED12 G44D mutation compared to WT as well as G44R and G44V MED12 mutation subtypes. Interestingly, MED12 mutation changed the binding activity of endogenous MED12 to RANKL promoter and enhancer regions. Particularly, G44D MED12 mutants exhibited significantly higher binding activity than G44R mutants whereas WT MED12 (n=3) did not. In summary, our findings suggest that epigenetic regulation and MED12 mutation together constitute a complicated regulatory network to affect P4/PR-mediated RANKL gene transcriptional activity. Our studies represent a key step towards the better understanding of mechanisms underlying the pathogenesis of specific LM subtypes.