Abstract 4P
Background
Glioblastoma, lethal brain tumor arising from glial cells. Our lab, have identified the oncogenic role of FAT1 gene in glioblastoma, regulating inflammatory, hypoxic microenvironment and migratory/invasive properties of the tumor cells. The elimination of cancer cells may also be mediated by autophagic cell death. Here we have established the effect of FAT1 on cellular autophagy and cancer cell death.
Methods
Glioblastoma cells U87MG and LN229 were cultured under normoxia & hypoxia. FAT1 was knocked out by CRISPR Cas9. Cell death were analyzed by MTT assay. Expression of autophagy markers were analyzed by qPCR & Westernblot. Modulation of autophagy was done by EBSS and Bafilomycin. Subcellular expression of autophagy marker was analyzed using confocal microscopy. Expression correlation was done in Glioblastoma patient tumor samples (n=30) collected from Neurosurgery OT, AIIMS, by qPCR and IHC.
Results
FAT1 (∼500kDa protien) was knocked out successfully with ∼95% efficiency in U87MG and LN229 cells. We observed significant cell death with deprived serum condition in FAT1 knockout cells. Expression of autophagy markers (Beclin1, LC3b, ULK1, ATG13, ATG7 and ATG5) found significantly increased at mRNA and protein level in FAT1 knockout cells. On autophagy modulation we found further increased in expression of autophagy markers at mRNA and protein level in FAT1 knockout cells, as well as significantly increased subcellular expression of LC3b was observed. There was significant negative correlation between the expression of FAT1 and autophagy markers were observed in Glioblastoma patient tumor samples.
Conclusions
On FAT1 knockout, under tumor mimic conditions (serum-deprivation & hypoxia) glioblastoma cells were led to autophagic cell death. Significantly increased expression of autophagy markers on FAT1 knockout and its combination with autophagic modulation substantiates the autophagic cell death in glioblastoma cells. Increased subcellular localization of LC3 validates the accumulation of autophagic vacuoles on FAT1 knockout in glioblastoma cells. Conclusively, our glioblastoma tumor data and in-vitro findings strongly suggest the role of FAT1 in negative regulating the expression of autophagy markers, thereby protecting the tumor cells from autophagic cell death.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Governance by Kunzang Chosdol, AIIMS, New Delhi, ICMR, New Delhi.
Funding
AIIMS, New Delhi, ICMR, New Delhi.
Disclosure
All authors have declared no conflicts of interest.