Theranostics 2019; 9(9):2424-2438. doi:10.7150/thno.30941

Research Paper

EBV(LMP1)-induced metabolic reprogramming inhibits necroptosis through the hypermethylation of the RIP3 promoter

Feng Shi1,2,3, Min Zhou1,2,3, Li Shang4, Qianqian Du1,2,3, Yueshuo Li1,2,3, Longlong Xie1,2,3, Xiaolan Liu1,2,3, Min Tang1,2,3, Xiangjian Luo1,2,3, Jia Fan5, Jian Zhou5, Qiang Gao5, ShuangJian Qiu5, Weizhong Wu5, Xin Zhang6, Ann M. Bode7, Ya Cao1,2,3,8,9✉

1. Key Laboratory of Carcinogenesis and Invasion, Chinese Ministry of Education, Xiangya Hospital, Central South University, Changsha 410078, China
2. Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha 410078, China
3. Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha 410078, China
4. Department of Pathology, Xiangya Hospital, Central South University, Changsha 410078, China
5. Key Laboratory for Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Zhongshan Hospital, Shanghai Medical School, Fudan University, Shanghai 200000, China
6. Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha 410078, China
7. The Hormel Institute, University of Minnesota, Austin, MN 55912, USA
8. Research Center for Technologies of Nucleic Acid-Based Diagnostics and Therapeutics Hunan Province, Changsha 410078, China
9. National Joint Engineering Research Center for Genetic Diagnostics of Infectious Diseases and Cancer, Changsha 410078, China

Abstract

EBV infection is a recognized epigenetic driver of carcinogenesis. We previously showed that EBV could protect cancer cells from TNF-induced necroptosis. This study aims to explore the epigenetic mechanisms allowing cancer cells with EBV infection to escape from RIP3-dependent necroptosis.

Methods: Data from the TCGA database were used to evaluate the prognostic value of RIP3 promoter methylation and its expression. Western blotting, real-time PCR, and immunochemistry were conducted to investigate the relationship between LMP1 and RIP3 in cell lines and NPC tissues. BSP, MSP and hMeDIP assays were used to examine the methylation level. Induction of necroptosis was detected by cell viability assay, p-MLKL, and Sytox Green staining.

Results: RIP3 promoter hypermethylation is an independent prognostic factor of poorer disease-free and overall survival in HNSCC patients, respectively. RIP3 is down-regulated in NPC (a subtype of HNSCC). EBV(LMP1) suppresses RIP3 expression by hypermethylation of the RIP3 promoter. RIP3 protein expression was inversely correlated with LMP1 expression in NPC tissues. Restoring RIP3 expression in EBV(LMP1)-positive cells inhibits xenograft tumor growth. The accumulation of fumarate and reduction of α-KG in EBV(LMP1)-positive cells led to RIP3 silencing due to the inactivation of TETs. Decreased FH activity caused fumarate accumulation, which might be associated with its acetylation. Incubating cells with fumarate protected NPC cells from TNF-induced necroptosis.

Conclusion: These results demonstrate a pathway by which EBV(LMP1)-associated metabolite changes inhibited necroptosis signaling by DNA methylation, and shed light on the mechanism underlying EBV-related carcinogenesis, which may provide new options for cancer diagnosis and therapy.

Keywords: Epstein-Barr virus, Nasopharyngeal carcinoma, Necroptosis, Receptor-interacting protein 3, Fumarate.

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How to cite this article:
Shi F, Zhou M, Shang L, Du Q, Li Y, Xie L, Liu X, Tang M, Luo X, Fan J, Zhou J, Gao Q, Qiu S, Wu W, Zhang X, Bode AM, Cao Y. EBV(LMP1)-induced metabolic reprogramming inhibits necroptosis through the hypermethylation of the RIP3 promoter. Theranostics 2019; 9(9):2424-2438. doi:10.7150/thno.30941. Available from http://www.thno.org/v09p2424.htm