Mitofusin 2 orchestrates mitochondrial metabolic homeostasis and suppresses cutaneous squamous cell carcinoma by stabilizing ER-mitochondria contacts.

This study aimed to elucidate the biological role of Mitofusin 2 (Mfn2) in cutaneous squamous cell carcinoma (cSCC) and evaluate how its regulation of mitochondria-associated membranes (MAMs) may suppress tumor progression by reshaping mitochondrial metabolic homeostasis and apoptosis. We compared Mfn2 expression between normal keratinocytes (HaCaT) and cSCC cells (A431), establishing a stable Mfn2-overexpressing (Mfn2-OE) A431 model. Structural impacts on ER-mitochondrial contacts and MAMs were evaluated using immunofluorescence, STED super-resolution microscopy, TEM, and subcellular fractionation (VDAC1/IP3R/Grp75). Functional assays assessed ER-mitochondrial calcium transport (probes), lipid peroxidation/ROS (C11-BODIPY/DCFH-DA), mitochondrial membrane potential, and ATP levels (JC-1/ATP assays). Mitochondria-dependent apoptosis (Bcl-2/p53/Cytochrome c/Caspase-3) was analyzed via flow cytometry, TUNEL, and Western blot. Phenotypes were assessed using CCK-8, EdU, colony formation, and Transwell assays. In vivo xenografts in nude mice were monitored, with H&E/IHC analysis. Proteomics and untargeted metabolomics (LC-MS) were performed (n = 6/group). Mfn2 protein levels were lower in A431 cSCC cells than in HaCaT keratinocytes. Mfn2-OE enhanced ER-mitochondrial contacts, increased MAMs, and stabilized the VDAC1-Grp75-IP3R complex. This enhanced calcium transport, reduced ROS/lipid peroxidation, stabilized MMP, and decreased ATP. Mfn2-OE inhibited Bcl-2, promoted p53 mitochondrial translocation and Cytochrome c release, activating Caspase cascades. Both in vitro and in vivo, Mfn2-OE suppressed proliferation, invasion, and tumor growth. Proteomics/metabolomics revealed metabolic reprogramming involving energy metabolism, lipid metabolism, and redox balance. Mfn2 may act as a tumor-suppressive regulator in the A431 cSCC model by stabilizing MAMs, remodeling mitochondrial metabolic homeostasis, and activating mitochondria-dependent apoptosis.