Deubiquitylating enzyme USP9x regulates radiosensitivity in glioblastoma cells by Mcl-1-dependent and -independent mechanisms

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.Along with surgery, radiotherapy, and chemotherapy are the main treatment options of tumors. While the former aims to remove the tumor bulk mass, the latter two intend to neutralize remaining tumor cells. Ionizing radiation (IR) exerts its cytotoxic effects by inducing cell death. One form of specific cell death induced by IR is intrinsic apoptosis, which is regulated by members of the B-cell leukemia (Bcl)-2 protein family.¹The 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 The activation of pro-apoptotic multidomain proteins Bax and Bak is essential to induce mitochondrial outer membrane permeabilization, resulting in the release of cytochrome C and other apoptotic factors into the cytosol where, in turn, caspases become activated. Antiapoptotic Bcl-2 family members prevent the activation of Bax and Bak either by direct interaction or indirectly by sequestering pro-apoptotic BH3-only proteins Bim and Bid that are required to activate Bax and Bak. Other BH3-only proteins are also able to bind to antiapoptotic proteins, thereby releasing Bax and Bak from their inhibitory complexes with antiapoptotic proteins. Changing the balance between anti- and pro-apoptotic Bcl-2 family members can shift the cells toward survival or apoptosis, depending on whether the protective or the detrimental proteins dominate.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. Blocking the antiapoptotic function of Bcl-2/Bcl-xL by inhibitors mimicking BH3-only proteins, such as ABT737 and ABT263, can induce apoptosis in cells with low Mcl-1 levels but has no effect on cells with high Mcl-1 levels.^{5, 6, 7} In contrast, specific inhibitors targeting Mcl-1 have been insufficiently described until now. However, Mcl-1 availability might be modulated by targeting pathways that regulate Mcl-1 stability.In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a relatively short-lived protein.^{8, 9} Usually, Mcl-1 is quickly ubiquitylated by specific ubiquitin ligases and targeted for proteasomal degradation. Phosphorylation of Mcl-1, for example by glycogen synthase kinase GSK-3β, can accelerate this degrading process,^{10, 11} whereas deubiquitinases counteract it by removing the polyubiquitin chain, thereby stabilizing the short-lived protein. The ubiquitin-specific protease 9x (USP9x) was recently identified as a Mcl-1 specific deubiquitinase.¹² However, the circumstances under which USP9x regulates Mcl-1 stability are not well understood. Schwickart et al.¹² showed that USP9x levels correlated with Mcl-1 levels, suggesting a constitutive regulation of Mcl-1 levels by the deubiquitinase. In contrast, 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.In the present study, we aimed to analyze the impact of USP9x on Mcl-1 and cell survival in glioblastoma cell lines. Glioblastoma is not only the most common but also a very aggressive form of brain tumor that are primarily removed by surgery as radically as possible and consecutively treated with radiochemotherapy, if the patient''s condition allows for adjuvant therapy.¹³ Despite the multimodal treatment, the median patient survival is below 1.5 years. Comparing human grade III astrocytoma with grade IV glioblastoma samples, we could show that Mcl-1 and USP9x are upregulated during tumor progression. Furthermore, we examined four established (A172, U373, Ln229, T98G) and two primary (LKI, WKI) glioblastoma cell lines that differ in their ability to downregulate Mcl-1 and induce apoptosis in response to IR. Analyzing A172 and U373 cells more closely, we detected an increased Mcl-1 ubiquitylation that correlated with a reduced Mcl-1 stability 48 h after irradiation in U373 cells, but not in A172 cells. Moreover, Mcl-1 knockdown sensitized A172, Ln229, and T98G cells to IR-induced apoptosis, suggesting that Mcl-1 is an important factor increasing glioblastoma cell survival after irradiation. In contrast, USP9x knockdown slightly increased apoptosis in IR-resistant A172 cells and significantly in Ln229 cells and reduced clonogenic survival after irradiation only on these two cell lines. Although USP9x knockdown reduced Mcl-1 levels and increased apoptosis in A172 cells, USP9x regulated radiosensitivity independently of Mcl-1 in Ln229 cells.Our results show a different requirement of USP9x in the control of glioblastoma cell survival and radiosensitivity.