The Mechanism Study Of Succinate On The Regulation Of YAP/TAZ And Hepatocellular Carcinoma Cells Growth

IntroductionMetabolism dysregulation is one of the main causes of hepatocellular carcinoma(HCC).Loss-of-function or mutations of enzymes in the tricarboxylic acid(TCA)cycle contributes to tumorigenesis of HCC.Succinate dehydrogenase(SDH),an important mitochondrial enzyme,catalyzes succinate oxidation in TCA cycle.Several lines of evidence revealed that SDH A and SDHB would be downregulated in HCC tumor samples.Nevertheless,how the dysregulation of SDHA and SDHB is involved in the HCC development remains largely unknown.YAP/TAZ(Yes-associated protein and transcriptional co-activator with PDZ-binding motif),two cofactors for transcription factors TEADs,are aberrantly activated in HCC.But whether and how this activation of YAP/TAZ pathway is elicited by metabolic deregulation in HCC need to be clarified.Therefore,the exploration of regulatory mechanism underlying the metabolic stress of succinate and YAP/TAZ signaling in HCC is essential to extend our knowledge about HCC.The current study establishes the negative correlation between SDH and YAP/TAZ by utilizing the HCC model induced by DEN(N-ethylnitrosamine)and CCl4(Carbon tetrachloride);clarifies the regulatory mechanism imposed on YAP/TAZ protein stability through the succinate-deprived neddylation on Cullin1,using the HCC cellular models and a variety of molecular biology methods.These results would provide novel directions and hints for the discovery of potential targets for anti-HCC drugs based on the regulation on SDH-YAP/TAZ axis.ObjectiveThe purpose of this study was to investigate the role and mechanism of SDH in regulating the growth of HCC by regulating YAP/TAZ.Using a variety of HCC cells and HCC patient samples to establish the relationship between SDH and YAP/TAZ;to clarify the effects of SDH on the function and protein expression of YAP/TAZ and the mechanism of the regulation of YAP/TAZ by SDH.MethodsMouse HCC model induced by DEN and CCI4 and Hepatocellular carcinoma cell lines were used in this study.(1)H&E staining was used to detect the tumor and normal tissue;(2)RNA-seq was used to detect the differentially regulated genes and pathways in mouse HCC model;(3)Western blot assay was used to detect the expression levels of proteins;(4)Immunohistochemistry staining was used to analyze SDHB and YAP/TAZ expression in mouse model and HCC patients;(5)Quantitative real-time PCR assay was used to analyze mRNA levels of genes in cell lines and liver tissues;(6)Immunoprecipitation was used to detect the protein-protein interaction between TAZ,ubiquitin or SCFβTrCP;(8)SRB(Sulforhodamine B)assay and colony formation assay was used to detect the proliferation of SDHB-overexpression/knockdown or succinate followed by YAP/TAZ knockdown on HCC cell lines.Results(1)Kaplan-Meier Plotter analysis showed that SDHB and SDHA high expression of liver cancer patients significantly prolonged survival compared with low expression patients;SDHA and SDHB expression were decreased and YAP/TAZ pathway was activation in DEN and CCl4-induced mouse HCC model;the RNA-seq analysis revealed significant changes in the Hippo pathway with significant up-regulation of downstream target genes;qRT-PCR results showed that YAP/TAZ target genes were significantly upregulated;immunohistochemistry results showed that there was a negative correlation between the expression of SDHB and YAP/TAZ protein;the above results indicated that SDHA and SDHB might be the negative regulatory protein of YAP/TAZ.(2)SDHA and SDHB negatively regulated YAP/TAZ protein expression and the mRNA level of YAP/TAZ target genes in HCC cells;dimethyl malonate(DMM)and succinate led to upregulation of YAP/TAZ protein and the regulation of succinate and SDHA/B on YAP/TAZ protein is independent of LATS1/2.(3)Succinate decreased Cullinl neddylation,thereby reducing YAP/TAZ ubiquitin proteasome degradation and stabilizing YAP/TAZ protein.(4)shYAP/TAZ reversed the proliferation effect of SDH and succinate on HCC cell line.(5)The expressions of SDHB and YAP in HCC samples were analyzed by immunohistochemical analysis,and the correlation coefficient between SDHB and YAP was calculated to be-0.310(p=0.002).ConclusionLoss of function of SDH during HCC development leads to succinate accumulation.The accumulation of succinate leads to the down-regulation of Cullin1 neddylation,which leads to the decrease of YAP/TAZ ubiquitination level and the accumulation of YAP/TAZ protein.Thereby,the accumulation of YAP/TAZ protein activates the downstream target genes to promote the growth of HCC cells.Our research revealed a novel pathway regulating YAP/TAZ independent of LATS 1/2 and provided new ideas and directions for the discovery of new targets for anti-HCC drugs based on YAP/TAZ.