Interaction between DNA methylation and progesterone action during uterine leiomyoma stem cell development

Liu, Shimeng

doi:https://doi.org/10.21985/n2-h1qr-2e18

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Interaction between DNA methylation and progesterone action during uterine leiomyoma stem cell development

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Uterine leiomyoma (LM), the most common tumor of women, causes severe morbidity. LM cells can be separated into three molecularly and functionally distinct cell populations based on the expression pattern of CD34 and CD49b: stem (LSC, CD34+/CD49b+), intermediate (LIC, CD34+/CD49b-), and differentiated cells (LDC, CD34-/CD49b-). Progesterone via progesterone receptor (PR/PGR) expressed in differentiated LM cells stimulate paracrine signaling that targets PR-deficient stem cells to induce their proliferation, leading to tumor growth. Antiprogestins shrink LM but tumors regrow after treatment cessation possibly due to persisting stem cells. Thus, the molecular mechanism underlying LSC regulation is critical for understanding the LM tumorigenesis process and identifying potential novel therapeutic avenue for LM patients. In this study, we analyzed the transcriptome landscape of LM cells at the three differentiation stages. The Receptor Activator of NF-κB Ligand (RANKL) was identified as one of the most significant differentially expressed genes among the three LM populations. As a novel progesterone/PR-responsive gene, we discovered that RANKL plays an important role in mediating the progesterone signals to the stem cell population via paracrine action through its receptor RANK. Upon progesterone stimulation, RANKL specifically upregulates LM stem cell proliferation through activation of Cyclin D1. Within the LM and normal myometrium (MM) system, RANKL expression was governed by the crosstalk among steroid hormone, genetic, and epigenetic signals. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent MM tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited the 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, we discovered that PR physically interacts with MED12 protein, which is mutated in 70% 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. These 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. Due to the importance of the crosstalk between DNA methylation and progesterone signaling on the RANKL gene regulation, we further investigated this interaction on a genome-wide scale by profiling DNA methylome in each LM population and progesterone receptor cistrome in LM tissues. Via vigorous bioinformatic analysis of PR ChIP-seq and DNA methylation data, we found that PR target genes were highly involved in the differentiation process and PR-deficient LM stem cells harbored a unique DNA methylation landscape with hypermethylation at the PGR gene locus and its genome-wide target regions, thereby suppressing the stem cell population’s direct response to progesterone and maintaining its stemness. The DNA methylation inhibitor 5'-Aza upregulated the expression of PR, stimulated PR binding to its target genes, significantly depleted the LM stem cell population via accelerating its differentiation, and extensively reduced the tumor-initiating capacity of LM primary cells. Furthermore, PGR upregulated the expression of TET methylcytosine dioxygenases, suggesting a potential feedback loop between DNA methylation and hormone signaling to facilitate stem cell differentiation. Targeting stem cells using 5'-Aza also sensitized LM tissue to antiprogestins, which may lead to tumor eradication. This study provides new mechanistic insights into the stem cell differentiation process of hormone-sensitive tumors and may serve as an efficient method to identify potential treatment targeting the stem cell population.

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