ANO6 Scaffolds ER Mitochondria Contacts to Govern Spatial Lipid Compartmentalization and Ferroptosis Evasion in Chemoresistant Breast Cancer

Abstract

Background: Metabolic glycerophospholipid remodeling promotes tumor persistence under neoadjuvant chemotherapy in breast cancer; however, how resistant cells evade lipotoxicity from accumulating peroxidizable lipids presents an intriguing metabolic conundrum. The underlying organelle-level spatial mechanisms remain poorly defined. Methods: Integrated multi-omic profiling of paired pre- and post-NAC clinical specimens was performed. Engineered ANO6 models (wild-type, knockout, and conformational mutants) were characterized using subcellular lipidomics of purified mitochondria-associated ER membranes (MAMs), electron microscopy, in situ proximity ligation assays, and preclinical xenograft models. Results: Chemoresistant lesions exhibited marked ANO6 upregulation and polyunsaturated phospholipid (PUFA-PL) accumulation without enhanced apoptosis. Beyond its canonical role as a plasma membrane scramblase, ANO6 operates as an organelle contact scaffold at MAMs, leveraging its conformational activation to coordinate inter-organellar tethering. ANO6 directly recruits vimentin (VIM) and VAPB to assemble a VIM-ANO6-VAPB-PTPIP51 tethering platform, structurally bridging intermediate filaments to organelle contact sites. This complex tightens ER-mitochondria coupling, establishing a specialized structural platform that routes and sequesters peroxidizable lipids into protective cytoplasmic lipid droplets, thereby shielding functional membrane bilayers from ferroptotic peroxidation. Functional dissection revealed that the active scaffolding conformation (I612A) sustains MAMs integrity and lipid packaging, whereas scaffold deficiency (D703R) compromises contact architecture. Pharmacologically perturbing this platform with niclosamide destabilized lipid sequestration, selectively resensitizing chemoresistant cells to synthetic lethal ferroptosis in vitro and in vivo. Conclusions: ANO6-mediated contact scaffolding governs spatial lipid compartmentalization, establishing the ANO6-MAMs structural axis as a potential target to exploit lipid metabolic vulnerabilities in refractory breast cancer.