H3K4me3 mediates uterine leiomyoma pathogenesis via neuronal processes, synapsis components, proliferation, and Wnt/ß-catenin and TGF-ß pathways.

BACKGROUND: Uterine leiomyomas (UL) are the most common benign tumor in women of reproductive age. Their pathology remains unclear, which hampers the development of safe and effective treatments. Raising evidence suggests epigenetics as a main mechanism involved in tumor development. Histone modification is a key component in the epigenetic regulation of gene expression. Specifically, the histone mark H3K4me3, which promotes gene expression, is altered in many tumors. In this study, we aimed to identify if the histone modification H3K4me3 regulates the expression of genes involved in uterine leiomyoma pathogenesis. METHODS: Prospective study integrating RNA-seq (n = 48) and H3K4me3 CHIP-seq (n = 19) data of uterine leiomyomas versus their adjacent myometrium. Differentially expressed genes (FDR < 0.01, log2FC > 1 or < - 1) were selected following DESeq2, edgeR, and limma analysis. Their differential methylation and functional enrichment (FDR < 0.05) were respectively analyzed with limma and ShinyGO. RESULTS: CHIP-seq data showed a global suppression of H3K4me3 in uterine leiomyomas versus their adjacent myometrial tissue (p-value< 2.2e-16). Integrating CHIP-seq and RNA-seq data highlighted that transcription of 696/922 uterine leiomyoma-related differentially expressed genes (DEG) (FDR < 0.01, log2FC > 1 or < - 1) was epigenetically mediated by H3K4me3. Further, 50 genes were differentially trimethylated (FDR < 0.05), including 33 hypertrimethylated/upregulated, and 17 hypotrimethylated/downregulated genes. Functional enrichment analysis of the latter showed dysregulation of neuron-related processes and synapsis-related cellular components in uterine leiomyomas, and a literature review study of these DEG found additional implications with tumorigenesis (i.e. aberrant proliferation, invasion, and dysregulation of Wnt/ß-catenin, and TGF-ß pathways). Finally, SATB2, DCX, SHOX2, ST8SIA2, CAPN6, and NPTX2 proto-oncogenes were identified among the hypertrimethylated/upregulated DEG, while KRT19, ABCA8, and HOXB4 tumor suppressor genes were identified among hypotrimethylated/downregulated DEG. CONCLUSIONS: H3K4me3 instabilities alter the expression of oncogenes and tumor suppressor genes, inducing aberrant proliferation, and dysregulated Wnt/ß-catenin, and TGF-ß pathways, that ultimately promote uterine leiomyoma progression. The reversal of these histone modifications may be a promising new therapeutic alternative for uterine leiomyoma patients.

Deciphering the Role of Histone Modifications in Uterine Leiomyoma: Acetylation of H3K27 Regulates the Expression of Genes Involved in Proliferation, Cell Signaling, Cell Transport, Angiogenesis and Extracellular Matrix Formation.

Uterine leiomyoma (UL) is a benign tumor arising from myometrium (MM) with a high prevalence and unclear pathology. Histone modifications are altered in tumors, particularly via histone acetylation which is correlated with gene activation. To identify if the acetylation of H3K27 is involved in UL pathogenesis and if its reversion may be a therapeutic option, we performed a prospective study integrating RNA-seq (n = 48) and CHIP-seq for H3K27ac (n = 19) in UL vs MM tissue, together with qRT-PCR of SAHA-treated UL cells (n = 10). CHIP-seq showed lower levels of H3K27ac in UL versus MM (p-value < 2.2 × 10−16). From 922 DEGs found in UL vs. MM (FDR < 0.01), 482 presented H3K27ac. A differential acetylation (FDR < 0.05) was discovered in 82 of these genes (29 hyperacetylated/upregulated, 53 hypoacetylated/downregulated). Hyperacetylation/upregulation of oncogenes (NDP,HOXA13,COL24A1,IGFL3) and hypoacetylation/downregulation of tumor suppressor genes (CD40,GIMAP8,IL15,GPX3,DPT) altered the immune system, the metabolism, TGFß3 and the Wnt/ß-catenin pathway. Functional enrichment analysis revealed deregulation of proliferation, cell signaling, transport, angiogenesis and extracellular matrix. Inhibition of histone deacetylases by SAHA increased expression of hypoacetylated/downregulated genes in UL cells (p < 0.05). Conclusively, H3K27ac regulates genes involved in UL onset and maintenance. Histone deacetylation reversion upregulates the expression of tumor suppressor genes in UL cells, suggesting targeting histone modifications as a therapeutic approach for UL.

Integrative analysis of the DNA methylome and transcriptome in uterine leiomyoma shows altered regulation of genes involved in metabolism, proliferation, extracellular matrix, and vesicles.

Uterine leiomyomas (ULs) are the most common benign tumors in women of reproductive age. Despite the high prevalence, tumor pathology remains unclear, which hampers the development of safe and effective treatments. Epigenetic mechanisms appear to be involved in UL development, particularly via DNA methylation that regulates gene expression. We aimed to determine the relationship between DNA methylation and gene expression in UL compared with adjacent myometrium (MM) to identify molecular mechanisms involved in UL formation that are under epigenetic control. Our results showed a different DNA methylation profile between UL and MM, leading to hypermethylation of UL, and a different global transcriptome profile. Integration of DNA methylation and whole-transcriptome RNA-sequencing data identified 93 genes regulated by methylation, with 22 hypomethylated/upregulated and 71 hypermethylated/downregulated. Functional enrichment analysis showed dysregulated biological processes and molecular functions involved in metabolism and cell physiology, response to extracellular signals, invasion, and proliferation, as well as pathways related to uterine biology and cancer. Cellular components such as cell membranes, vesicles, extracellular matrix, and cell junctions were dysregulated in UL. In addition, we found hypomethylation/upregulation of oncogenes (PRL, ATP8B4, CEMIP, ZPMS2-AS1, RIMS2, TFAP2C) and hypermethylation/downregulation of tumor suppressor genes (EFEMP1, FBLN2, ARHGAP10, HTATIP2), which are related to proliferation, invasion, altered metabolism, deposition of extracellular matrix, and Wnt/ß-catenin pathway dysregulation. This confirms that key processes of UL development are under DNA methylation control. Finally, inhibition of DNA methyltransferases by 5-aza-2'-deoxycitidine increased the expression of hypermethylated/downregulated genes in UL cells in vitro. In conclusion, gene regulation by DNA methylation is implicated in UL pathogenesis, and reversion of this methylation could offer a therapeutic option for UL. © 2022 The Pathological Society of Great Britain and Ireland.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid: a therapeutic approach to treat human uterine leiomyoma.

OBJECTIVE: To evaluate the effect of inhibition of histone deacetylases (HDACs) by suberoylanilide hydroxamic acid (SAHA) treatment of human uterine leiomyoma primary (HULP) cells in vitro on cell proliferation, cell cycle, extracellular matrix (ECM) formation, and transforming growth factor ß3 (TGF-ß3) signaling. DESIGN: Prospective study comparing uterine leiomyoma (UL) vs. adjacent myometrium (MM) tissue and cells with or without SAHA treatment. SETTING: Hospital and university laboratories. PATIENT(S): Women with UL without any hormone treatment. INTERVENTION(S): Myomectomy or hysterectomy surgery in women for leiomyoma disease. MAIN OUTCOME MEASURE(S): HDAC activity was assessed by enzyme-linked immunosorbent assay, and gene expression was assessed by quantitative real-time polymerase chain reaction. Effects of SAHA on HULP cells were analyzed by CellTiter (Promega, Madison, Wisconsin), Western blot, and quantitative real-time polymerase chain reaction. RESULT(S): The expression of HDAC genes (HDAC1, fold change [FC] = 1.65; HDAC3, FC = 2.08; HDAC6, FC = 2.42) and activity (0.56 vs. 0.10 optical density [OD]/h/mg) was significantly increased in UL vs. MM tissue. SAHA decreased HDAC activity in HULP cells but not in MM cells. Cell viability significantly decreased in HULP cells (81.68% at 5 µM SAHA, 73.46% at 10 µM SAHA), but not in MM cells. Proliferating cell nuclear antigen expression was significantly inhibited in SAHA-treated HULP cells (5 µM SAHA, FC = 0.556; 10 µM SAHA, FC = 0.622). Cell cycle markers, including C-MYC (5 µM SAHA, FC = 0.828) and CCND1 (5 µM SAHA, FC = 0.583; 10 µM SAHA, FC = 0.482), were significantly down-regulated after SAHA treatment. SAHA significantly inhibited ECM protein expression, including FIBRONECTIN (5 µM SAHA, FC = 0.815; 10 µM SAHA, FC = 0.673) and COLLAGEN I (5 µM SAHA, FC = 0.599; 10 µM SAHA, FC = 0.635), in HULP cells. TGFß3 and MMP9 gene expression was also significantly down-regulated by 10 µM SAHA (TGFß3, FC = 0.596; MMP9, FC = 0.677). CONCLUSION(S): SAHA treatment inhibits cell proliferation, cell cycle, ECM formation, and TGF-ß3 signaling in HULP cells, suggesting that histone deacetylation may be useful for treatment of UL.