Rationale: Protein palmitoylation is tightly related to tumorigenesis or tumor progression as many oncogenes or tumor suppressors are palmitoylated. AEG-1, an oncogene, is commonly elevated in a variety of human malignancies, including hepatocellular carcinoma (HCC). Although AEG-1 was suggested to be potentially modified by protein palmitoylation, the regulatory roles of AEG-1 palmitoylation in tumor progression of HCC has not been explored. Methods: Techniques as Acyl-RAC assay and point mutation were used to confirm that AEG-1 is indeed palmitoylated. Moreover, biochemical experiments and immunofluorescent microscopy were applied to examine the cellular functions of AEG-1 palmitoylation in several cell lines. Remarkably, genetically modified knock-in (AEG-1-C75A) and knockout (Zdhhc6-KO) mice were established and subjected to the treatment of DEN to induce the HCC mice model, through which the roles of AEG-1 palmitoylation in HCC is directly addressed. Last, HCQ, a chemical compound, was introduced to prove in principal that elevating the level of AEG-1 palmitoylation might benefit the treatment of HCC in xenograft mouse model. Results: We showed that AEG-1 undergoes palmitoylation on a conserved cysteine residue, Cys-75. Blocking AEG-1 palmitoylation exacerbates the progression of DEN-induced HCC in vivo. Moreover, it was demonstrated that AEG-1 palmitoylation is dynamically regulated by zDHHC6 and PPT1/2. Accordingly, suppressing the level of AEG-1 palmitoylation by the deletion of Zdhhc6 reproduces the enhanced tumor-progression phenotype in DEN-induced HCC mouse model. Mechanistically, we showed that AEG-1 palmitoylation adversely regulates its protein stability and weakens AEG-1 and staphylococcal nuclease and tudor domain containing 1 (SND1) interaction, which might contribute to the alterations of the RISC activity and the expression of tumor suppressors. For intervention, HCQ, an inhibitor of PPT1, was applied to augment the level of AEG-1 palmitoylation, which retards the tumor growth of HCC in xenograft model. Conclusion: Our study suggests an unknown mechanism that AEG-1 palmitoylation dynamically manipulates HCC progression and pinpoints that raising AEG-1 palmitoylation might confer beneficial effect on the treatment of HCC.

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