Abstract 2603: Investigating TUSC2 for its tumor suppressive functions in glioblastoma and its role in gliomagenesis

Abstract

Abstract The role of Tumor Suppressor Candidate 2 (TUSC2) in glioblastoma (GBM) is still poorly understood. Our recent study (Cancer Letters, 2022) reported, for the first time, that TUSC2 functions as a novel tumor suppressor for GBM. In this report, we found TUSC2 protein expression, not mRNA expression, to be significantly decreased in GBM as compared to normal brain. TUSC2 protein is destabilized in GBM due to polyubiquitination by E3 ligase, neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4), and degradation by the proteasome. We further identified that NEDD4 targets TUSC2 on lysine residue K71. We reported that TUSC2 overexpression reduces colony formation, neurosphere formation and promotes apoptosis in vitro, and suppresses tumor growth in vivo. Conversely, GBM cells with CRISPR-mediated knockout (KO) of TUSC2 were highly aggressive in vitro and in vivo, demonstrating the role of TUSC2 as a novel tumor suppressor for GBM. We further performed RNA-seq analysis using our stable Control gRNA and TUSC2-KO GBM cell lines and discovered over 1,200 significantly differentially expressed genes. Since TUSC2 is not a transcription factor, we hypothesized that TUSC2 alters the GBM transcriptome through protein-protein interaction with transcriptional regulators. In the current study, we tested this hypothesis by performing immunoprecipitation-mass spectrometry analysis to identify TUSC2-interacting proteins in both normal human astrocytes and GBM cell lines. Results showed 95 and 88 TUSC2-interacing proteins in astrocytes and GBM cell lines respectively. 27 proteins were found to interact with TUSC2 in both astrocytes and GBM. Ongoing efforts are focused to validate the interaction of TUSC2 with transcriptional regulators identified within the immunoprecipitation-mass spectrometry analysis. Since the expression of TUSC2 is lost in GBM, we hypothesized that TUSC2 loss is an important event during gliomagenesis. To test this hypothesis, we generated a novel conditional transgenic mouse model to investigate the role of TUSC2 loss in gliomagenesis. GFAP-Cre C57/BL6 mice were crossed with TUSC2flx/flx mice, mice containing loxp-flanked TUSC2, to specifically delete TUSC2 from mouse astrocytes. The recombination would introduce firefly luciferase to allow for bioluminescence imaging to detect TUSC2 knockout. Ongoing efforts are to monitor TUSC2flx/flx/GFAP-Cre (TUSC2-KO) mice for glioma development using IVIS imaging and MRI. Taken together, our study is the first step in elucidating the functions of a novel GBM tumor suppressor TUSC2 and investigating the role that TUSC2 plays in gliomagenesis. Citation Format: Austin Arrigo, Mariana Najjar, Angelina Regua, Hui-Wen Lo. Investigating TUSC2 for its tumor suppressive functions in glioblastoma and its role in gliomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2603.