PO-125 FAT1 on salvador-warts-hippo (SWH) pathway in human glioblastoma

Abstract

IntroductionGlioblastoma(GBM) is an aggressive brain tumour arising from glial cells. Our lab has identified the oncogenic role of FAT1 gene in GBM, regulating inflammatory and hypoxic microenvironment of the tumour as well as migratory/invasive properties of the tumour cells. In Drosophila, fat, the ortholog of FAT1, is known to regulate the Salvador-Warts-Hippo (SWH) pathway, but its role in human is not clear. Here, we have analysed the effect of FAT1 on SWH pathway in glioma.Material and methodsGlioma cell lines (U87MG, U373, A172, GOS3 and SW1088) were transfected with FAT1 specific siRNA/control siRNA and analysed the expression of SWH pathway molecules by qPCR/Western blot. Protein-protein interactions were analysed by Co-immunoprecipitation (Co-IP) after overexpression of YAP1 (wild-type and mutated) and TEAD1 with and without FAT1 knockdown. Sub-cellular localization of proteins was analysed by Confocal microscopy.Results and discussionsThe mRNA expression of FAT1 and SWH pathway molecules (MST1, LATS1, LATS2, YAP1 and TEAD1) was highest in U87MG cells followed by A172, U373MG and GOS3. After FAT1 knockdown, the mRNA expression of MST1 and BIRC2 were significantly decreased with no change in the levels of LATS1, LATS2, YAP1, TEAD1 and BIRC5. At protein level, increased YAP1 and phospho-YAP1 was observed after FAT1 knockdown with increased total as well as phospho-YAP1 in the cytoplasmic extract as compared to the nuclear extract. There was significant reduction in the interaction between YAP1 and TEAD1 in siFAT1 treated cells as compared to siControl treated cells.ConclusionKnockdown of FAT1 (i) increases the YAP1 protein level, could be by increasing the protein stability as no change was observed at the mRNA level (ii) it relieves the inhibitory effect on YAP1 phosphorylation, thereby increasing the phospho-YAP1 level (iii) it affects the sub-cellular localization of YAP1 by retaining YAP1 in the cytosol and thereby (iv) decrease in the YAP1:TEAD1 interaction with decreased expression of their target gene Birc2.This finding of the effect of FAT1 on YAP1 in GBM is novel with features pointing towards the oncogenic role of FAT1 by regulating YAP1 sub-cellular localization and co-transcriptional activity independent of SWH pathway.