P18.19.A STRESS ADAPTIVE ROLE OF THE ATYPICAL CADHERIN FAT1 TO SUPPORT TUMOR GROWTH IN GLIOBLASTOMA BY INHIBITING AUTOPHAGIC CELL DEATH

Abstract BACKGROUND Autophagy is a conserved degradation process that plays dual roles in cancer, either supporting survival under stress or inducing cell death when dysregulated. FAT1, an atypical cadherin, acts as a tumor suppressor or oncogene in a context-dependent manner. Our previous work identified FAT1 as oncogenic in glioblastoma (GBM). Given the role of dysregulated autophagy in GBM, this study explores how FAT1 modulates autophagy under stressful conditions (severe hypoxia and low serum), and studies the mechanism by which FAT1 regulates autophagy and glioma progression. MATERIAL AND METHODS Surgically resected GBMs (n=35), collected after taking institutional ethical clearance and informed written consent, and TCGA GBM datasets were analysed for the expression of autophagy markers and their correlation with patient survival. FAT1 knockout was generated in U87MG, LN229 cell lines using the CRISPR-Cas9 system for in-vitro study. Cell viability and growth were evaluated using MTT, colony formation, and apoptotic assays in FAT1 knockout cells maintained under hypoxia with or without serum deprivation. Autophagy-related markers were assessed by qPCR, Western blotting, immunocytochemistry (ICC), and immunohistochemistry (IHC). p62/SQSTM1 levels, pEGFP-LC3-RFP-LC3ΔG clearance, and autophagosome formation via transmission electron microscopy were used to determine the autophagic flux. For in-vivo study, FAT1-knockout U87MG xenografts in nude mice were analysed for tumor growth and the expression of autophagy markers and autophagosome formation. RESULTS GBM tissues and the TCGA GBM dataset revealed an inverse expression correlation of FAT1 with autophagy markers LC3B and Beclin1. High FAT1 expression and low LC3B expression are associated with poor patient survival. In in-vitro study, FAT1-knockout GBM cells (U87MG/LN229) exhibited significantly reduced cell survival and colony formation under normoxia/hypoxia with serum deprivation, which was driven by autophagy-dependent cell death. These cells showed upregulation of autophagy markers, increased LC3 puncta, enhanced autophagosome formation, and elevated autophagy flux. There was decreased total and phospho-mTOR levels in FAT1 knockout cells. In in-vivo, FAT1-knockout xenografts in nude mice exhibited slower tumor growth and lower tumor volume. FAT1-knockout xenografts exhibited increased LC3-II and Beclin1 levels and increased autophagosome formation. These findings indicate the role of FAT1 in regulating autophagy in a stressful tumor microenvironment and promoting tumor progression in GBM. CONCLUSION Our findings suggest that FAT1 promotes tumorigenesis by inhibiting autophagic cell death in glioblastoma under stressful microenvironments and promotes tumor cell survival and growth. FAT1 may serve as a potential prognostic/therapeutic marker in cancers with elevated FAT1 expression.