Abstract A021: Lysine methylation in EHMT1/GLP acts as a molecular switch to reprogram transcription networks to drive prostate cancer progression

• Published in: Cancer research (Chicago, Ill.) Ill.), 2022-12, Vol.82 (23_Supplement_2), p.A021-A021
• Main Authors: Besschetnova, Anna, Han, Wanting, Liu, Mingyu, Gao, Yanfei, Li, Muqing, Wang, Zifeng, Labaf, Maryam, Patalano, Susan, Venkataramani, Kavita, Muriph, Rachel, Macoska, Jill, Siegfried-Harris, Kellee, Evans, Jason, Balk, Steven, Gao, Shuai, Han, Dong, Cai, Changmeng
• Format: Article
• Language: English
• ISSN: 1538-7445; 1538-7445
• DOI: 10.1158/1538-7445.CancEpi22-A021

Background: Prostate Cancer (PCa) progresses to a metastatic form of cancer called Castration-Resistant Prostate Cancer (CRPC) after treatment with androgen deprivation therapies (ADTs). Epigenetic reprogramming through altered expression and activity of histone modifier proteins is one major mechanism of tumor resistance. In particular, histone lysine methyltransferases (KMTs) and demethylases (KDMs) are important epigenetic targets in CRPC. In this study, we have focused on studying the function and regulation of Euchromatic Histone Methyltransferase 1 protein (EHMT1/GLP), which is a well-known H3K9 methyltransferase and functions in repressing gene transcription. EHMT family genes are altered in ~2-3% of primary PCa and ~10% of CRPC (primarily gene amplification) and their expression levels are significantly increased in CRPC, suggesting these proteins may have oncogenic activities in driving CRPC progression. Methods: We conducted an integrated analysis of ChIP-seq and RNA-seq analysis in LNCaP PCa cells to characterize the transcription program of EHMT1. We then performed proteomics analyses in VCaP and LNCaP PCa cells to identify post-translational modifications of EHMT1 and functionally validated the findings by generating loss-of-function mutations. We performed a histone demethylase assay to discover if EHMT1 is a substrate of LSD1 (a member of the KDM family). Moreover, we also assessed the effects of EHMT1 silencing or inhibition (in multiple PCa cell lines) on cell proliferation and metastasis in vitro using cell cycle analysis and transwell migration assay, and tumor growth and metastasis in vivo using mouse subcutaneous injection and zebrafish embryo injection approaches. Results: Our data showed that EHMT1 can transcriptionally activate multiple oncogenic pathways, including E2F and MYC signaling. Proteomic analyses revealed that EHMT1 is methylated in PCa cell lines, with dual-lysine methylation occurring at the K450/K451 sites. The histone demethylase assay showed that methylated K450, but not K451 is a potential substrate of LSD1. By generating the K450R, K451R, and K450/451R mutants, we showed that the K450/451R mutant, but not any single lysine mutants, can greatly expand EHMT1 chromatin binding independent of its H3K9 methyltransferase activity. This mutant also significantly induced EHMT1 oncogenic activity by activating E2F and MYC pathways. Moreover, EHMT1 silencing, or inhibition can significantly suppress tumor growth and metastas