USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

Abstract

Background Glioblastomas (GBMs) are grade IV central nervous system tumors characterized by a poor prognosis and a short median overall survival. Effective induction of GBM cell death is difficult because the GBM cell population is genetically unstable, resistant to chemotherapy and highly angiogenic. In recent studies, ubiquitin-specific protease 7 (USP7) is shown to scavenge ubiquitin from oncogenic protein substrates, so effective inhibition of USP7 may be a potential key treatment for GBM. Methods Immunohistochemistry and western blotting were used to detect the expression of USP7 in GBM tissues. In vitro apoptosis assay of USP7 inhibition was performed by western blotting, immunofluorescence, and flow cytometry. Anti-apoptotic substrates of USP7 were defined by Co-IP and TMT proteomics. Western blotting and IP were used to verify the relationship between USP7 and its substrate. In an in vivo experiment using an intracranial xenograft model in nude mice was constructed to assess the therapeutic effect of target USP7. Results Immunohistochemistry and western blotting confirmed that USP7 was significantly upregulated in glioblastoma samples. In in vitro experiments, inhibition of USP7 in GBM induced significant apoptosis. Co-IP and TMT proteomics identified a key anti-apoptotic substrate of USP7, ADP-ribosylation factor 4 (ARF4). Western blotting and IP confirmed that USP7 interacted directly with ARF4 and catalyzed the removal of the K48-linked polyubiquitinated chain that binded to ARF4. In addition, in vivo experiments revealed that USP7 inhibition significantly suppressed tumor growth and promoted the expression of apoptotic genes. Conclusions Targeted inhibition of USP7 enhances the ubiquitination of ARF4 and ultimately mediates the apoptosis of GBM cells. In a clinical sense, P5091 as a novel specific inhibitor of USP7 may be an effective approach for the treatment of GBM.

Ubiquitin-Specific Peptidase 7: A Novel Deubiquitinase That Regulates Protein Homeostasis and Cancers

“…Schauer et al reported the first irreversible USP7 inhibitor with high efficacy (sub-nanomolar potency) and high selectivity (112). Moreover, P5091 has been shown to have efficacy and very low toxicity in tumor treatment in vivo (113)(114)(115). In sum, all of the issues are not insurmountable for the development of more ideal inhibitors.…”

Highlights in USP7 inhibitors for cancer treatment

“…It was reported to overcome bortezomib resistance, to trigger synergistic anti-MM activity in combination with Lenalidomide, Suberoylanilide Hydroxamic Acid or Dexamethasone and to synergize with DNA damaging drugs such as etoposide and doxorubicin for killing MM cells in vitro ( Chauhan et al, 2012 ). P5091 has also been used in other xenograft models including colorectal cancer ( An et al, 2017 ), glioblastoma ( Pan et al, 2021 ) and esophageal squamous cell carcinoma ( Hu et al, 2019 ), among others.…”

Recent insights into USP7: Construct, pathophysiology, and inhibitors

“…In addition, ADP-ribosylation factor 4 (ARF4) is a small guanine nucleotide-binding protein, which belongs to Ras superfamily of small G proteins. Previous studies have demonstrated that ARF4 is an anti-apoptotic protein, which acts as a BAX inhibitor, and its function is indispensable in glioblastoma tumorgenesis [52,115] . Interestingly, USP7 has been shown to be significantly upregulated in glioblastoma patient samples.…”

“…Structure and function of USP7 polyubiquitinated chain from ARF4, and promotes malignant progression of glioblastoma. In addition, in vivo experiments revealed that treatment of USP7 specific inhibitor, P5091, can significantly suppress the growth of tumor models through ARF4 degradation [52] .…”