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Imbalance in ALR ubiquitination accelerates the progression of nonalcoholic steatohepatitis to hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer. Accumulating evidence indicates that non-alcoholic steatohepatitis (NASH) is a key predisposing factor for HCC occurrence. However, the precise mechanisms driving NASH transition to HCC remain largely obscure. Augmenter of liver regeneration (ALR) is a sulfhydryl oxidase and cytochrome c reductase that functions as an important regulator of mitochondrial dynamics. In this study, we focused on ALR ubiquitination-mediated degradation and its potential contribution to NASH-driven HCC progression at the mitochondrial level. Hepatic ALR expression in HCC patients was determined using immunohistochemical analysis. Mice with liver-specific deletion of ALR (ALRCKO) and ALRWT mice were fed a western diet (WD) and high-sugar solution for induction of NASH. HCC in animals was induced via peritoneal administration of CCl4. ALR expression was markedly decreased in liver tissues of patients with NASH and HCC compared with non-NASH and non-tumor tissues. Similarly, in ALRWT mice, the ALR level in tumor tissue was reduced relative to that in para-tumor tissue. In the ALRCKO group, mice fed WD plus CCl4 developed HCC starting at week 12 while ALRWT mice fed WD plus CCl4 developed HCC at week 24. Analysis of protein posttranslational modifications revealed ubiquitylation (Ub) and deubiquitination (DUb) of ALR by murine double minute 2 (MDM2) and ubiquitin-specific protease 36 (USP36), respectively. Imbalance between Ub and DUb of ALR resulted in profound ALR degradation, which appeared to be reversibly associated with Edmondson-Steiner tumor grade. Rescue of ALR levels via gene transfection abolished tumor malignant features to a certain extent in vitro. Notably, ALR deletion substantially enhanced mitochondrial fission by activating Drp1 phosphorylation at Ser616, thus disrupting the balance of mitochondrial dynamics between fission and fusion and severely impairing oxidative phosphorylation (OXPHOS) and ATP synthesis, instead enhancing anaerobic metabolism, which might be attributed to steatotic hepatocyte transition into the malignant HCC phenotype. Hepatic ALR depletion via dysregulation of ubiquitination is a critical aggravator of NASH-HCC progression and represents a promising therapeutic target for related liver diseases.

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Fig. 1: Hepatic ALR expression is decreased in NASH and HCC patients.
Fig. 2: NASH-HCC progression in mice treated with WD plus CCl4.
Fig. 3: Interactions between ALR and USP36/MDM2.
Fig. 4: Regulation of ALR by the E3 ubiquitin ligase MDM2 and deubiquitinating enzyme USP36.
Fig. 5: Expression of ALR, USP36 and MDM2 proteins in liver.
Fig. 6: ALR expression is correlated with MDM2 and USP36 in HCC.
Fig. 7: ALR deletion accelerates NASH development to HCC by exacerbating mitochondrial fission and inducing alterations in mitochondrial OXPHOS.

Data availability

The RNA-seq raw data of this study are available from the Sequence Read Archive repository BioProject (https://www.ncbi.nlm.nih.gov/bioproject/) with accession number PRJNA841174. Immunopurification and Mass spectrometry raw data have been deposited to the PRIDE database (https://www.iprox.cn/page/SSV024.html;url=1651495320815MEBa, the accession number is IPX0004307003. Please access the raw files with password “bzme”). Other materials, data, and any associated protocols that support the findings of this study are available from the corresponding authors on reasonable request.

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Funding

This work was supported by the National Natural Science Foundation of China (NSFC) (No. 32071128).

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Study concept and design: XP, AW; acquisition of patient specimens: ZMZ, GYY; acquisition of data: ZMZ, AZW, CYJ; analysis and interpretation of data: ZMZ, XP; drafting of the manuscript: ZMZ; study supervision and manuscript revision: AW; PI of project and lead contact: AW.

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Correspondence to Ping Xie or Wei An.

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Zheng, M., Ai, Z., Guo, Y. et al. Imbalance in ALR ubiquitination accelerates the progression of nonalcoholic steatohepatitis to hepatocellular carcinoma. Oncogene 42, 308–321 (2023). https://doi.org/10.1038/s41388-022-02549-7

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