Abstract
Glioblastoma is a very aggressive form of brain tumor with limited therapeutic options. Usually, glioblastoma is treated with ionizing radiation (IR) and chemotherapy after surgical removal. However, radiotherapy is frequently unsuccessful, among others owing to resistance mechanisms the tumor cells have developed. Antiapoptotic B-cell leukemia (Bcl)-2 family members can contribute to radioresistance by interfering with apoptosis induction in response to IR. Bcl-2 and the closely related Bcl-xL and Mcl-1 are often overexpressed in glioblastoma cells. In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a short-lived protein whose stability is closely regulated by ubiquitylation-dependent proteasomal degradation. Although ubiquitin ligases facilitate degradation, the deubiquitylating enzyme ubiquitin-specific protease 9x (USP9x) interferes with degradation by removing polyubiquitin chains from Mcl-1, thereby stabilizing this protein. Thus, an inability to downregulate Mcl-1 by enhanced USP9x activity might contribute to radioresistance. Here we analyzed the impact of USP9x on Mcl-1 levels and radiosensitivity in glioblastoma cells. Correlating Mcl-1 and USP9x expressions were significantly higher in human glioblastoma than in astrocytoma. Downregulation of Mcl-1 correlated with apoptosis induction in established glioblastoma cell lines. Although Mcl-1 knockdown by siRNA increased apoptosis induction after irradiation in all glioblastoma cell lines, USP9x knockdown significantly improved radiation-induced apoptosis in one of four cell lines and slightly increased apoptosis in another cell line. In the latter two cell lines, USP9x knockdown also increased radiation-induced clonogenic death. The massive downregulation of Mcl-1 and apoptosis induction in A172 cells transfected with USP9x siRNA shows that the deubiquitinase regulates cell survival by regulating Mcl-1 levels. In contrast, USP9x regulated radiosensitivity in Ln229 cells without affecting Mcl-1 levels. We conclude that USP9x can control survival and radiosensitivity in glioblastoma cells by Mcl-1-dependent and Mcl-1-independent mechanisms.
Highlights
The B-cell leukemia (Bcl)-2 protein family consists of protective antiapoptotic and pro-apoptotic members, which keep each other in check by antagonizing each other’s function.[2]
In the first set of experiments, we examined the expression of myeloid cell lymphoma-1 (Mcl-1) and ubiquitin-specific protease 9x (USP9x) in astrocytoma (WHO grade III) and glioblastoma (WHO grade IV) (Figure 1)
Immunohistochemical analysis shows that number of Mcl-1- and USP9x-positive cells and staining intensity were
Summary
The Bcl-2 protein family consists of protective antiapoptotic and pro-apoptotic members, which keep each other in check by antagonizing each other’s function.[2]. Bcl-2 itself, Bcl-xL, and myeloid cell lymphoma-1 (Mcl-1) belong to the antiapoptotic proteins of the Bcl-2 family. They are often overexpressed in tumor cells and are associated with increased resistance to apoptosis induction in response to radio- and chemotherapy.[3,4] As more than one of the protective proteins can be upregulated in tumors, the neutralization of all antiapoptotic proteins is needed to successfully induce apoptosis. Our recent results showed no effect of USP9x on Mcl-1 levels in healthy Jurkat cells, but an accelerated IR-induced Mcl-1 degradation was detected when USP9x was knocked down.[9] This indicates that the association of USP9x with Mcl-1 is regulated by a yet unknown mechanism in response to irradiation
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