Organelle contact reorganization drives calcium-dependent autophagy under proteostatic stress.

Disruption of proteostasis is a defining feature of cancer and other chronic diseases. The AAA+ ATPase VCP/p97 (valosin containing protein) is a key regulator of proteostasis by disassembling ubiquitinated substrates for degradation. VCP overexpression supports cancer cell survival and correlates with poor prognosis, promoting the development of VCP inhibitors as anti-cancer agents. However, the molecular basis for cancer-selective vulnerability of VCP inhibition remains unclear. Here, we demonstrate that allosteric VCP inhibition triggers cell-type specific macroautophagy/autophagy through dynamic reorganization of organelle contact sites. In human umbilical vein endothelial cells (HUVECs), VCP inhibition induces adaptive autophagy through coordinated reorganization of plasma membrane (PM)-ER-mitochondria contacts. Controlled opening of the mitochondrial permeability transition pore (mPTP) releases calcium into the cytosol, activating AMP-activated protein kinase (AMPK) and TFEB pathways, collectively enhancing autophagic flux and sustaining endothelial survival. Critically, calcium-activated kinase inhibitor or calcium chelators blocked VCP inhibitor-induced autophagy in HUVECs, confirming calcium signaling as the central mediator of adaptive autophagy. In contrast, HCT116 colon cancer cells fail to maintain calcium homeostasis under VCP inhibition, leading to mitochondrial calcium overload, defective autophagy, and cell death. Together, our findings identify organelle contact reorganization and calcium homeostasis as key determinants of cell fate under conditions of proteotoxic stress, revealing how VCP inhibition selectively suppresses tumor progression while preserving vascular integrity that could enhance drug delivery and reduce tumor hypoxia.Abbreviations: ATF4: activating transcription factor 4; ATG3: autophagy related 3; ATG7: autophagy related 7; ATP: adenosine triphosphate; CAMKK2: calcium/calmodulin dependent protein kinase 2; CETSA: cellular thermal shift assay; CQ: chloroquine; CTS: cryptotanshinone; DDIT3/CHOP: DNA damage inducible transcript 3; DQ-BSA: dye quenched-bovine serum albumin; EIF2A: eukaryotic initiation factor 2A; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated protein degradation; HCT116: human colon carcinoma cell line; HSPA5: heat shock protein family A (Hsp70) member 5; HUVECs: human umbilical vein endothelial cells; ITPR1/IP₃R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; MT-ND1: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; MTOR: mechanistic target of rapamycin kinase; ORAI1: ORAI calcium release-activated calcium modulator 1; PLA: proximity ligation assay; PM: plasma membrane; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit alpha; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STIM1: stromal interaction molecule 1; TFEB: transcription factor EB; U87MG: human glioblastoma astrocytoma cell line; FAF2/UBXD8: Fas associated factor family member 2; UPR: unfolded protein response; VCP/p97: valosin containing protein; VDAC1: voltage dependent anion channel 1.