Feng Shi 1 , 2 , 3 , Min Zhou 1 , 2 , 3 , Li Shang 4 , Qianqian Du 1 , 2 , 3 , Yueshuo Li 1 , 2 , 3 , Longlong Xie 1 , 2 , 3 , Xiaolan Liu 1 , 2 , 3 , Min Tang 1 , 2 , 3 , Xiangjian Luo 1 , 2 , 3 , Jia Fan 5 , Jian Zhou 5 , Qiang Gao 5 , ShuangJian Qiu 5 , Weizhong Wu 5 , Xin Zhang 6 , Ann M. Bode 7 , Ya Cao 1 , 2 , 3 , 8 , 9 , ✉
13 April 2019
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.