NEDD4 degrades TUSC2 to promote glioblastoma progression
Tadas K Rimkus1, Austin B Arrigo1, Dongqin Zhu1
1Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, 27175, USA.
Abstract:
Whether tumor suppressor candidate 2 (TUSC2) plays an important role in glioblastoma (GBM) progression is largely unknown. Whether TUSC2 undergoes polyubiquitination is unknown. Herein, we report that TUSC2 protein expression is reduced/lost in GBM compared to normal brain due to protein destabilization; TUSC2 mRNA is equally expressed in both tissues. NEDD4 E3 ubiquitin ligase polyubiquitinates TUSC2 at residue K71, and the TUSC2-K71R mutant is resistant to NEDD4-mediated proteasomal degradation. Analysis of GBM specimens showed NEDD4 protein is highly expressed in GBM and the level is inversely correlated with TUSC2 protein levels. Furthermore, TUSC2 restoration induces apoptosis and inhibits patient-derived glioma stem cells (PD-GSCs) in vitro and in vivo. Conversely, TUSC2-knockout promotes PD-GSCs in vitro and in vivo. RNA-Seq analysis and subsequent validations showed GBM cells with TUSC2-knockout expressed increased Bcl-xL and were more resistant to apoptosis induced by a Bcl-xL-specific BH3 mimetic. A TUSC2-knockout gene signature created from the RNA-seq data predicts poor patient survival. Together, these findings establish that NEDD4-mediated polyubiquitination is a novel mechanism for TUSC2 degradation in GBM and that TUSC2 loss promotes GBM progression in part through Bcl-xL upregulation.
Insights
Tumor suppressor candidate 2 (TUSC2) protein is degraded in glioblastoma (GBM) by NEDD4-mediated polyubiquitination. Loss of TUSC2 promotes GBM progression, partly by upregulating Bcl-xL, predicting poor patient survival.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- The role of tumor suppressor candidate 2 (TUSC2) in glioblastoma (GBM) progression is unclear.
- The mechanism of TUSC2 regulation, including polyubiquitination, in GBM is unknown.
Purpose of the Study:
- To investigate the role of TUSC2 in GBM.
- To determine if TUSC2 undergoes polyubiquitination and identify the responsible E3 ligase.
- To elucidate the functional consequences of TUSC2 loss in GBM.
Main Methods:
- Western blotting to assess TUSC2 and NEDD4 protein levels in GBM and normal brain tissues.
- Site-directed mutagenesis to identify the polyubiquitination site on TUSC2.
- In vitro and in vivo experiments using patient-derived glioma stem cells (PD-GSCs) with TUSC2 restoration or knockout.
- RNA-Seq analysis to identify downstream targets of TUSC2.
- Correlation analysis of NEDD4 and TUSC2 protein levels in GBM specimens.
Main Results:
- TUSC2 protein is reduced in GBM due to destabilization, while mRNA levels are similar to normal brain.
- NEDD4 E3 ubiquitin ligase polyubiquitinates TUSC2 at K71, leading to proteasomal degradation.
- High NEDD4 expression in GBM inversely correlates with TUSC2 protein levels.
- TUSC2 restoration inhibits PD-GSCs and induces apoptosis; TUSC2 knockout promotes PD-GSCs.
- TUSC2 loss upregulates Bcl-xL, conferring resistance to apoptosis and predicting poor patient survival.
Conclusions:
- NEDD4-mediated polyubiquitination is a novel mechanism for TUSC2 degradation in GBM.
- TUSC2 functions as a tumor suppressor in GBM, inhibiting glioma stem cell growth and promoting apoptosis.
- TUSC2 loss contributes to GBM progression via Bcl-xL upregulation, serving as a potential therapeutic target.
