Transcriptome Analysis Identifies Novel EZH2 Target Genes And Altered Signaling Pathways In Primary Hyperparathyroidism

Document Type

Conference Proceeding

Publication Date

9-27-2024

Publication Title

J Bone Miner Res

Abstract

BACKGROUND Primary hyperparathyroidism (PHPT) is characterized by hypercalcemia and elevated parathyroid hormone levels. Epigenetic modifications identified as a regulator in parathyroid tumorigenesis. EZH2, a critical epigenetic modifier catalyzes trimethylation of lysine (K) 27 (H3K27me3) thereby regulate gene expression but its role is obscured in PHPT. This study was designed to analyze the gene and protein expression of EZH2 in PHPT patients followed by in-vitro identification of novel EZH2 target genes and pathways for the advanced therapeutic strategies. METHODOLOGY- 40 PHPT (30 parathyroid tumors, 10 aggressive parathyroid tumors) and 10 normal parathyroid tissues were recruited. Aggressive parathyroid tumors include minimally invasive parathyroid carcinoma (06) and carcinoma (04) cases. mRNA and protein expression for EZH2 was performed by qRT-PCR and immunohistochemistry. GSK343 against EZH2 was used for target gene identification in parathyroid cell line (PTHC-1) by using high-throughput mRNA sequencing (Novaseq6000 platform). Then, bioinformatics viz; STRING-cytohubba modules, kyoto encyclopedia of genes and genomics (KEGG) and Reactome was applied to identify EZH2 novel target genes and pathways. RESULTS-We found that EZH2 mRNA expression was significantly high in parathyroid tumors and highest in aggressive parathyroid tumors compared to controls [mean±S.E; (5.7±2.7 and 12.2±2.6 vs 1.0 ±0.3, p=0.005)]. EZH2 mRNA expression is weakly correlated with serum calcium (r=0.24, p=0.0002) and moderately with tumor weight (r=0.61, p=0.005). Protein expression is also consistent with gene expression having 82% nuclear positivity in aggressive and 40% in parathyroid tumors compared to controls (negative staining). GSK343 treated cells showed inhibition of EZH2 mediated H3K27me3 with nanomolar potency. mRNA sequencing of GSK343 treated vs untreated cells identified ∼4000 differential expressed genes (DEGs) using R-package with fold change (≥±1.5, p<0.05). The top upregulated genes are VDR, PTEN, ENO1 with (>5 fold change) and downregulated CDC6 with (-4 fold change). Functional enrichment identifies these top genes in enriched protein-protein networks with high confidence score >0.7 (STRING 12.0 software). Further, cell cycle and MAPK pathway predicted as highly enriched pathways in parathyroid tumors. CONCLUSIONS- We confirms elevated levels of H3K27me3-mediated EZH2 expression in parathyroid tumors, with the highest in aggressive parathyroid tumors. mRNA sequencing following GSK343 treatment identified potential EZH2 target genes (VDR, CDC6, PTEN, ENO1) and pathways (cell cycle, MAPK) with highest fold enrichment those could act as novel therapeutic targets in treatment of parathyroid tumors.

Volume

39

First Page

129-130

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