MEN1/menin deficiency suppresses hepatocellular carcinogenesis via disrupting mitophagy-mediated mitochondrial homeostasis

Hepatocellular carcinoma (HCC) is a highly lethal liver Cancer with complex pathogenesis intertwined with metabolic and mitochondrial dysfunction. MEN1/menin is a protein with context-dependent functions in liver diseases. While MEN1 has been linked to HCC progression and mitochondrial homeostasis, its precise regulatory mechanism in these processes remains incompletely understood. Here, we report that MEN1 localizes to the outer mitochondrial membrane (OMM) in HCC cells, which is mediated by its N-terminal mitochondrial targeting sequence and the TOMM20 translocase complex. In a genetically engineered DEN- and CCl₄-induced HCC mouse model, hepatocyte-specific men1 deficiency significantly suppressed tumorigenesis, a phenotype associated with impaired mitochondrial homeostasis. Mechanistically, MEN1 deficiency disrupted mitochondrial function by manifesting as promoted mitochondrial fission, impaired Oxidative Phosphorylation, reduced ATP levels, and elevated Reactive Oxygen Species during energy stress. Critically, MEN1 loss inhibited Mitophagy via downregulating the PINK1-PRKN/Parkin pathway, which impaired clearance of dysfunctional mitochondria and promotes their cytotoxic accumulation. Moreover, MEN1 expression was upregulated in human HCC tissues, correlated with poor clinical outcomes and was positively associated with Autophagy signatures. Notably, pharmacological activation of Mitophagy reversed the tumor-suppressive effects of MEN1 deficiency in vitro and in vivo. These findings identified a noncanonical role of mitochondrial MEN1 in driving HCC progression via regulating Mitophagy homeostasis, and highlight the MEN1-mitophagy axis as a potential therapeutic target for HCC.Abbreviations: Alb-Cre: albumin promoter-driven recombinase Cre; Baf A1: bafilomycin A₁; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; CCl₄: carbon tetrachloride; Co-IP: co-immunoprecipitation; CQ: chloroquine; DEN: diethylnitrosamine; DNM1L: Dynamin 1 like; DQ-BSA: self-quenched BODIPY-conjugated bovine serum albumin; Gal: galactose; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvic transaminase; HCC: hepatocellular carcinoma; WT: wild type; HMKO: hepatocyte-specific men1 knockout; IF: immunofluorescence; IHC: immunohistochemistry; IMM: inner mitochondrial membrane; KEGG: Kyoto Encyclopedia of Genes and Genomes; MEFs: mouse embryonic fibroblasts; MEN1-FL: full-length MEN1; MFF: mitochondrial fission factor; MFN1: mitofusin 1; MTS: mitochondrial targeting sequence; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced kinase1; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKN: parkin RBR E3 ubiquitin protein ligase; qPCR: RNA extraction and quantitative polymerase chain reaction; RNA-seq: RNA-sequencing; ROS: reactive oxygen species; shMEN1: small hairpin RNA-mediated MEN1 knockdown; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20.