USP7 Inhibition Induces Apoptosis in Glioblastoma by Stabilizing ARF4

Abstract

Background: Glioblastomas (GBMs) are grade IV central nervous system tumors characterized by a poor prognosis and a median overall survival of 15 months. The glioblastoma cell population is more genetically unstable, resistant to chemotherapy, more angiogenic, and more malignant. Because ubiquitin-specific proteases (USPs) remove ubiquitin from oncogenic protein substrates, effective inhibition of ubiquitin-specific protease 7 (USP7), which is highly expressed in GBMs, is a potentially critical therapeutic approach. Methods: Immunohistochemistry and Western blotting were used to detect high expression of USP7 in GBM. in vitro studies were performed by Western blotting, immunofluorescence, and flow cytometry to detect apoptosis following inhibition of USP7. Anti-apoptotic substrates of USP7 were defined by Co-IP and TMT proteomics. Nude mouse intracranial xenograft models were constructed to verify whether inhibition of USP7 inhibited the proliferation rate of tumours.Results: USP7 is significantly upregulated in glioblastoma samples. Interfering with USP7 in GBMs induced significant apoptosis, which also occurred after treatment with P5091, a novel small molecule inhibitor of USP7. Mechanistically, apoptosis of GBMs after interference with USP7 function is achieved by stabilizing a key anti-apoptotic protein, ADP-ribosylation factor 4 (ARF4).Furthermore, USP7 interacts directly with ARF4 and catalyzes the removal of the K48-linked polyubiquitinated chain that binds to ARF4, thereby stabilizing the protein. In in vivo experiments, P5091 significantly inhibited tumor growth and promoted the expression of apoptotic genes. Conclusion: Targeted inhibition of USP7 enhances the ubiquitination of ARF4 and ultimately mediates the apoptosis of GBM cells. In a clinical sense, P5091 as a specific inhibitor of USP7 may be an effective approach for the treatment of GBM.