Atypical cadherin FAT1 promotes tumorigenesis by suppressing autophagic cell death in glioblastoma under hypoxia or nutrient stress.

Background: Autophagy, a conserved intracellular degradation process, plays dual roles in cancer, promoting survival under stress or mediating cell death through deregulated autophagy. Atypical cadherin FAT1 functions as an oncogene or tumor suppressor in a context-dependent manner. Our previous work identifies the oncogenic role of FAT1 in glioblastoma. Deregulated autophagy has been documented in glioma. Here, we investigated the role of FAT1 in regulating autophagy and its implications for glioblastoma growth and progression.

Methods: CRISPR-Cas9 mediated FAT1 knockout was generated in glioblastoma (U87MG and LN229) and other cancers such as hepatocellular carcinoma (HepG2 and HUH7) and pancreatic adenocarcinoma (MIAPaca-2 and Panc-1) cells. The cell viability and growth under hypoxia ± serum deprivation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and Annexin V-FITC assays. Autophagy markers were assessed by quantitative polymerase chain reaction (qPCR), Western blot, immunocytochemistry (ICC), and immunohistochemistry (IHC). Autophagosomes were visualized by transmission electron microscopy (TEM), and puncta formation was analyzed by transfecting the cells with pEGFP-LC3. Autophagy flux was evaluated by analyzing p62/SQSTM1 levels, and the GFP/RFP ratio using pMRX-IP-GFP-LC3-RFP-LC3ΔG. In vivo, FAT1-knockout U87MG xenografts in nude mice were analyzed for tumor growth and autophagy marker expression. Surgically resected glioblastoma tumors from our hospital and The Cancer Genome Atlas (TCGA) dataset were analyzed for autophagy marker expression and patient survival correlations.

Results: FAT1-knockout glioblastoma (U87MG and LN229) cells demonstrated reduced survival and colony numbers under normoxia and hypoxia with serum deprivation, facilitated by autophagy-dependent cell death. These cells exhibited upregulated autophagy markers, increased LC3 puncta, autophagosomes, and autophagy flux. FAT1-knockout glioblastoma cells showed decreased total and phospho-mTOR levels. FAT1-knockout xenografts showed reduced tumor progression with increased LC3II, Beclin1, and autophagosomes. Human glioblastoma tumors and TCGA glioblastoma data revealed an inverse expression correlation of FAT1 with LC3B/Beclin1, tumors with high-FAT1/low-LC3B expression were associated with poor patient survival. FAT1 also regulated autophagy in hepatocellular and pancreatic cancers.

Conclusion: Our findings indicate that FAT1 mediates pro-tumorigenic function by suppressing autophagic cell death in glioblastoma and other cancers. FAT1 may serve as a potential therapeutic adjuvant along with standard therapeutic regimens for treating cancers with high FAT1 expression having an oncogenic role.