Abstract 1279: SUMOylation upregulates EZH2 expression by modifying transcriptional factor E2F1

Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which modifies histone H3 lysine 27 trimethylation (H3K27me3) at target promoters for gene silencing. High level of EZH2 expression is often observed in cancers, however the regulation of EZH2 expression is not well understood. We conducted genome wide RNA profiling in colorectal cancer cell line HCT116 with inducible knockdown of SUMO activating enzyme subunit 2 (SAE2). RNA-sequencing results showed EZH2 mRNA decreased and GSEA analysis indicated suppression of downstream genes of H3K27me3 upon SAE2 knockdown. Real-time PCR and western blot confirmed that knockdown of SAE2 decreased EZH2 mRNA and protein level as well as H3K27me3. The same effects were observed in breast cancer cell lines MB-MDA-231 and HCC1937. EZH2 promoter reporter assay demonstrated that SUMOylation enhanced EZH2 promoter transcriptional activity. SiRNA knockdown of SUMO E1 enzyme SAE2 or SUMO E2 enzyme UBC9 resulted in induction of CDH1, an target gene silenced by EZH2. EZH2 promoter luciferase reporter assay identified E2F transcription factor 1 (E2F1) as a major transcriptional activator of EZH2. The enhancement of EZH2 promoter transcriptional activity of E2F1 was induced by co-expression of SAE2 or SUMO1 but suppressed by expression of deSUMOylation enzyme SENP1. E2F1 SUMOylation site mutation (K266R) significantly decreased its transcriptional activation as measured by the EZH2 promoter reporter assay. SAE2 knockdown decreased the occupancy of E2F1 on EZH2 promoter as measured by Chromatin immunoprecipitation (ChIP) assay. The binding of E2F1 to EZH2 promoter region was significantly decreased when E2F1 SUMOyaltion site was mutated, which suggests that SUMOylation is required for E2F1 binding to EZH2 promoter. Western blot of tumor tissues from xenograft mouse model generated by colorectal cancer cell line HCT116 showed consistent results that knockdown of SAE2 decreased EZH2 mRNA and protein level in vivo. EZH2 protein level was also reduced in patient-derived xenograft (PDX) primary colorectal cancer cells when SAE2 was knockdown by siRNA transfection. Patient specimen data analysis showed SAE2 expression is associated with human colon and breast cancer patient survival and there is a strong positive correlation between EZH2 and SAE2 expression in tumor specimen from colon cancer and breast cancer patients. This data from patient samples supports our findings made in cellular and animal models. Of note, EZH2 and its partner SUZ12 are substrates of SUMOylation. However, their SUMOylation does not appear to impact the functions of PRC2. In conclusion, our results demonstrate that SUMOylation up-regulates EZH2 expression through regulating its gene expression via E2F19s transcriptional activity. Our findings reveal a novel mechanism of EZH2 regulation, indicating that SUMO E1 inhibitor could be synergistic with EZH2 inhibitor in colon and breast cancer therapy. Citation Format: Li Du, Yuan Chen, Steven Rosen. SUMOylation upregulates EZH2 expression by modifying transcriptional factor E2F1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1279.