Targeting DNA Methylation Depletes Uterine Leiomyoma Stem Cell–enriched Population by Stimulating Their Differentiation

Abstract

Uterine leiomyoma is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires maintenance and proliferation of a stem cell population. Dysregulated DNA methylation has been reported in leiomyoma, but its role in leiomyoma stem cell regulation remains unclear. Here, we FACS sorted cells from human leiomyoma tissues into three populations: stem-cell like cells (LSC, 5%), intermediate cells (LIC, 7%), and differentiated cells (LDC, 88%) and analyzed the transcriptome and epigenetic landscape of leiomyoma cells at different differentiation stages. LSC harbored a unique methylome, with 8862 differentially methylated regions compared to LIC and 9444 compared to LDC, most of which were hypermethylated. Consistent with global hypermethylation, transcript levels of TET1 and TET3 methylcytosine dioxygenases were lower in LSC. Integrative analyses revealed an inverse relationship between methylation and gene expression changes during LSC differentiation. In LSC, hypermethylation suppressed genes important for myometrium- and leiomyoma-associated functions, including muscle contraction and hormone action, to maintain stemness. The hypomethylating drug, 5'-Aza stimulated LSC differentiation, depleting the stem cell population and inhibiting tumor initiation. Our data suggest that DNA methylation maintains the pool of LSC, which is critical for the regeneration of leiomyoma tumors.

Progesterone receptor-DNA methylation crosstalk regulates depletion of uterine leiomyoma stem cells: A potential therapeutic target

“…We performed PGR chromatin immunoprecipitation sequencing (ChIP-seq), searched for PGR-binding sites that were only present in all 5 LM tissues, and identified 6,893 regions. We then integrated the PGR cistrome with published methylome data and found that, among the three populations, LSCs had the highest overall DNA methylation level in regions at or adjacent to (<1 kb) the 6,893 PGR-binding sites ( Figure 2 A) ( Liu et al., 2020 ). K-means clustering pinpointed a cohort of PGR-binding regions (675 of 6,893, 9.8%) that were markedly hypermethylated in LSCs compared with LICs and LDCs ( Figures 2 B and 2C).…”

“…Next, we investigated whether DNA methylation influences PGR recruitment to its target gene loci to modulate gene transcription. As a proof-of-concept, we selected several candidate genes, including TIMP3, ROR2, GREB1, MYH11, and WNT5A, which showed the lowest gene expression by RNA-seq and harbor hypermethylated PGR-binding sites in LSCs ( Liu et al., 2020 ). These genes have established roles in hormone signaling, stem cell regulation, and carcinogenesis or LM pathogenesis ( Cheng et al., 2018 ; Goikuria et al., 2018 ; Jackson et al., 2015 ; Tarfiei et al., 2011 ; Zhou et al., 2017 ).…”

“…RNA-seq (on three samples with knockdown efficiency >70%) showed a significant transcriptome shift caused by PGR knockdown, identifying 2,832 genes regulated by both siPGR-1 and siPGR-2, with 1,601 genes downregulated and 1,231 genes upregulated ( Figures 3 B and 3C). Focusing on the genes previously identified as differentially expressed between LSCs and LICs/LDCs, principal-component analysis (PCA) indicated that, upon PGR knockdown, the transcriptome of total primary LM cells shifted toward that of LSCs ( Figure 3 D) ( Liu et al., 2020 ). Based on PCA and hierarchical clustering analyses, one outlier that had significantly higher PGR expression than the other two samples (~1.5- and ~3-fold higher before and after knockdown, respectively) was excluded from this integration assay ( Figures S3 A–S3C).…”

“…Focusing on this pathway, we discovered PGR binds to two key DNA demethylases, TET1 and TET2, and upregulates their expression ( Figures S3 F and 3 F). Transcript levels of TET methylcytosine dioxygenases were found to be lower in LSCs versus LICs and LDCs, which may account for the global hypermethylation identified in the stem cell population ( Liu et al., 2020 ). Given the important role of TETs in actively regulating DNA methylation, these findings prompted us to investigate whether PGR influences DNA methylation in LM cells.…”

“…Previous studies demonstrated dysregulated DNA methylation in LM versus normal myometrium (MM) ( Navarro et al., 2012 ) ( Maekawa et al., 2013 ). We recently demonstrated that LSCs harbor a unique DNA methylome, which suppresses key genes important for differentiation ( Liu et al., 2020 ). In this report, we examined how DNA methylation in LSCs affects PGR function and alters LSC differentiation and disease progression.…”