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• W4212816453 abstract "Article22 February 2022free access Source DataTransparent process USP33 deubiquitinates and stabilizes HIF-2alpha to promote hypoxia response in glioma stem cells Aili Zhang Aili Zhang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Data curation, Formal analysis, Validation, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Zhi Huang Zhi Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Validation, Methodology Search for more papers by this author Weiwei Tao Weiwei Tao Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Validation, Methodology Search for more papers by this author Kui Zhai Kui Zhai Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Methodology Search for more papers by this author Qiulian Wu Qiulian Wu Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Contribution: Methodology Search for more papers by this author Jeremy N Rich Jeremy N Rich Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Wenchao Zhou Corresponding Author Wenchao Zhou [email protected] orcid.org/0000-0002-6650-1831 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Shideng Bao Corresponding Author Shideng Bao [email protected] orcid.org/0000-0002-4236-2662 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Aili Zhang Aili Zhang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Data curation, Formal analysis, Validation, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Zhi Huang Zhi Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Validation, Methodology Search for more papers by this author Weiwei Tao Weiwei Tao Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Validation, Methodology Search for more papers by this author Kui Zhai Kui Zhai Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Methodology Search for more papers by this author Qiulian Wu Qiulian Wu Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Contribution: Methodology Search for more papers by this author Jeremy N Rich Jeremy N Rich Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Wenchao Zhou Corresponding Author Wenchao Zhou [email protected] orcid.org/0000-0002-6650-1831 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Shideng Bao Corresponding Author Shideng Bao [email protected] orcid.org/0000-0002-4236-2662 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Aili Zhang1, Zhi Huang1, Weiwei Tao1, Kui Zhai1, Qiulian Wu2, Jeremy N Rich2, Wenchao Zhou *,1 and Shideng Bao *,1,3,4 1Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 2Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA 3Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA 4Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA *Corresponding author. Tel: +1 2166360631; E-mail: [email protected] *Corresponding author (lead contact). Tel: +1 2166351009; E-mail: [email protected] The EMBO Journal (2022)41:e109187https://doi.org/10.15252/embj.2021109187 See also: A Pietras (April 2022) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Hypoxia regulates tumor angiogenesis, metabolism, and therapeutic response in malignant cancers including glioblastoma, the most lethal primary brain tumor. The regulation of HIF transcriptional factors by the ubiquitin–proteasome system is critical in the hypoxia response, but hypoxia-inducible deubiquitinases that counteract the ubiquitination remain poorly defined. While the activation of ERK1/2 also plays an important role in hypoxia response, the relationship between ERK1/2 activation and HIF regulation remains elusive. Here, we identified USP33 as essential deubiquitinase that stabilizes HIF-2alpha protein in an ERK1/2-dependent manner to promote hypoxia response in cancer cells. USP33 is preferentially induced in glioma stem cells by hypoxia and interacts with HIF-2alpha, leading to its stabilization through deubiquitination. The activation of ERK1/2 upon hypoxia promoted HIF-2alpha phosphorylation, enhancing its interaction with USP33. Silencing of USP33 disrupted glioma stem cells maintenance, reduced tumor vascularization, and inhibited glioblastoma growth. Our findings highlight USP33 as an essential regulator of hypoxia response in cancer stem cells, indicating a novel potential therapeutic target for brain tumor treatment. Synopsis While hypoxia induced by tumor growth is recognized as a key selective pressure in solid cancers, the molecular mechanisms underlying control of HIF signaling remain unclear. This work identifies deubiquitinating enzyme USP33 as regulator of HIF-2alpha in glioma, shedding new light on the complexities of brain tumor growth. Hypoxia induces deubiquitinase USP33 expression in glioma stem cells (GSCs). USP33 deubiquitinates and stabilizes HIF-2alpha in GSCs in response to hypoxia. ERK1/2-mediated phosphorylation of S484 HIF-2alpha enhances binding to USP33. Depletion of USP33 impairs GSC maintenance and tumor sphere formation in vitro as well as glioblastoma growth in mice. Introduction Hypoxia promotes malignant progression of solid tumors by activating neovascularization, altering metabolism, enhancing therapeutic resistance, increasing pro-tumor inflammation, and supporting the maintenance of cancer stem cells (Li et al, 2009, 2014; Rankin & Giaccia, 2016; Semenza, 2016; Stegen et al, 2019). Cellular response to hypoxia is dominantly regulated by the hypoxia-inducible factors (HIFs; Palazon et al, 2012, 2017; Fan et al, 2014; Shukla et al, 2017). The ubiquitin–proteasome system plays a central role in hypoxia response and HIF protein stabilization (LaGory & Giaccia, 2016). Deubiquitinases (DUBs) are the main enzymes responsible for protein deubiquitination and stabilization, but the functional regulation of DUBs in response to hypoxia remains elusive. In the hypoxic niches in glioblastoma (GBM), glioma stem cells (GSCs) are enriched to promote therapeutic resistance and malignant progression (Li et al, 2009; Seidel et al, 2010; Man et al, 2018). Whereas HIF1α is universally induced in glioma cells under hypoxic conditions, HIF2α is preferentially elevated in GSCs (Li et al, 2009; Seidel et al, 2010; Man et al, 2018), suggesting that GSCs may have developed unique adaptive mechanisms including the induction of DUBs in response to hypoxia. There are around 100 DUBs in human cells, and the largest subgroup, the ubiquitin-specific protease (USP), consists of approximately 60 members (Clague et al, 2019; Mennerich et al, 2019). The relatively small number of DUBs counterbalance approximately 600 E3 ubiquitin ligases in regulating the ubiquitin–proteasome system, highlighting the importance of DUBs in both normal and cancer cells (Clague et al, 2019; Mennerich et al, 2019). Several studies have suggested that DUBs play crucial roles in the maintenance of normal and cancer stem cells. In neural progenitor cells (NPCs), USP7 stabilizes the repressor element 1 silencing transcription factor (REST) through deubiquitination and is required for maintaining neural progenitor cells (NPCs; Huang et al, 2011). Likewise, USP13 reverses the polyubiquitination of c-Myc in GSCs to promote GSC self-renewal and GBM growth (Fang et al, 2017). However, little is known about the DUBs involved in the hypoxia response in caner stem cells such as GSCs. In response to hypoxic stress, the HIF family of transcriptional factors predominantly direct cellular adaption to hypoxia. HIFs function as heterodimers containing an oxygen-sensitive α unit and a constitutively expressed β unit (Semenza, 2012). HIF1α is ubiquitously expressed in all extant metazoan species, whereas HIF2α expression is restricted to certain tissues and cell types (Tian et al, 1997; Li et al, 2009; Loenarz et al, 2011). Although HIF1α and HIF2α bind to similar hypoxia-responsive elements (HREs), they occupy distinct genomic sites and are not functionally interchangeable (Forsythe et al, 1996; Hu et al, 2003; Branco-Price et al, 2012). Normoxia promotes hydroxylation of the HIFα unit to facilitate the binding of the von Hippel-Lindau (VHL) E3 ubiquitin ligase, resulting in proteasomal degradation of HIFα proteins (Kaelin & Ratcliffe, 2008). Hypoxia abrogates the hydroxylation of HIFα proteins and the consequent VHL binding to increase the stability of HIFs (Kaelin & Ratcliffe, 2008). As the reverse process of ubiquitination, deubiquitination of HIFα has not been defined. A handful of DUBs, such as USP20, have been discovered to catalyze deubiquitination of HIF1α (Li et al, 2005; Troilo et al, 2014; Wu et al, 2016; Sun et al, 2018). However, most of these DUBs seem to promote deubiquitination of HIF1α in a hypoxia-independent manner (Altun et al, 2012; Flugel et al, 2012; Bremm et al, 2014; Troilo et al, 2014; Wu et al, 2016). Moreover, our previous study suggested that HIF1α is only stabilized under acute hypoxic conditions, but HIF2α is induced even under modest hypoxia (Li et al, 2009), indicating that HIF2α and HIF1α may be regulated through different mechanisms. Beside the upregulation of HIF proteins, the activation of ERK1/2 kinases in response to hypoxia has been noticed for a while (Minet et al, 2000; Li et al, 2008; Lee et al, 2015). In fact, the concomitant elevation of HIF and ERK1/2 activities converge to promote malignant phenotypes in cancers (Franovic et al, 2009; Zhuo et al, 2019). The activation of ERK1/2 is closely associated with the upregulation of HIFs under hypoxic conditions. ERK1/2 activation seems to be an earlier event in hypoxia response and often a prerequisite for HIF1α accumulation (Li et al, 2008; Choi et al, 2010). In line with a possible direct effect of ERK1/2 on HIF proteins, HIF1α is phosphorylated under hypoxia in an ERK1/2-dependent manner (Minet et al, 2000). However, the potential role of ERK1/2 in regulating HIF2α in cancer stem cells in response to hypoxia remains elusive. Due to the critical impact of hypoxia on malignant tumors and the central role of the ubiquitination–deubiquitination system in regulating hypoxia response, the DUBs participating in hypoxia response in cancer stem cells could be promising druggable targets. In this study, we aim to identify the key hypoxia-inducible DUB for HIF2α stabilization in GSCs. We found that USP33 was markedly upregulated in GSCs in response to hypoxia, and validated the preferential expression of USP33 in GSCs in the hypoxic niches in GBM tumors. We further demonstrated that USP33 interacted with HIF2α to deubiquitinate and stabilize HIF2α upon hypoxia. Interestingly, the activation of ERK1/2 upon hypoxia executes phosphorylation of S484 on HIF2α to promote the interaction between USP33 and HIF2α, resulting in deubiquitination of HIF2α. Importantly, silencing USP33 in GSCs abolished hypoxia response, disrupted GSC maintenance, and inhibited GBM tumor growth, leading to a markedly increased survival of tumor-bearing animals. The discovery of USP33 as an upstream DUB directly regulating HIF2α in an ERK1/2-dependent manner not only offers new insights into the initiation of hypoxia response but may also have translational impact on cancer treatment. Results USP33 is a hypoxia-inducible deubiquitinase preferentially expressed in GSCs To identify potential hypoxia-inducible deubiquitinases in GSCs, we cultured GSCs under hypoxic and normoxic conditions and examined the change in protein levels of a cohort of deubiquitinases. Our previous studies showed that HIF2α is upregulated specifically in GSCs, whereas HIF1α is elevated in both GSCs and the matched non-stem tumor cells (NSTCs) in response to hypoxia, suggesting that the induction of HIF2α rather than HIF1α may reflect the unique hypoxic responses in GSCs (Li et al, 2009). We initially determined HIF2α protein levels in GSCs cultured at different oxygen concentrations and found that the maximal upregulation of HIF2α was achieved under 5% oxygen, whereas the HIF1α induction occurred under oxygen concentrations lower than 3% (Fig EV1A). Therefore, we examined the potential induction of deubiquitinases in GSCs at 5% oxygen. Immunoblot and qPCR analyses showed that only USP33 but no other tested deubiquitinases was induced by the hypoxic condition (5% oxygen) in GSCs (Figs 1A and EV1B, Appendix Fig S1A–D). Interestingly, USP33 protein levels achieved maximal upregulation within 12 h under 5% oxygen in most tested GSC lines, whereas HIF2α protein levels were slightly induced in 12 h but markedly upregulated in 24 h under the same hypoxic condition (Fig 1A), suggesting that hypoxia induction of USP33 was prior to that of HIF2α in GSCs. Similar to HIF2α, USP33 was induced by a wide range of hypoxic conditions (1–5% oxygen) (Figs 1A and B, and EV1A). To examine the hypoxia-induced upregulation of USP33 in GSCs in human GBMs, we performed immunofluorescent staining of USP33, the hypoxia marker CA9, and the GSC marker SOX2 on primary human GBM sections (Fig 1C). While only a fraction of CA9- or SOX2-positive cells showed USP33 staining, the majority of CA9/SOX2 double-positive cells had strong USP33 staining (Fig 1C and D), indicating that USP33 was upregulated in those GSCs under hypoxia in human GBMs. To further confirm the upregulation of USP33 in the hypoxic niches in GBM, we treated mice bearing intracranial GSC-derived GBM tumors with pimonidazole HCl to label the hypoxic cells and then examined USP33 protein levels. Two hours after tail vein injection of pimonidazole HCl, mice were sacrificed and GBM tumors were subjected to analysis. As expected, immunofluorescent staining showed that pimonidazole-positive areas were enriched with HIF1α and HIF2α signals (Fig EV1C–F), demonstrating the correct labeling of hypoxic niches by pimonidazole in vivo. Consistently, USP33 staining was highly enriched in pimonidazole-positive areas (Fig 1E and F), indicating that USP33 upregulation mainly occurred in hypoxic niches in GBM tumors. Moreover, treatment with CoCl2 that mimics hypoxic conditions also upregulated USP33 protein levels in multiple GSC lines (Fig EV1G). These data indicate that hypoxia upregulates USP33 protein levels in GSCs in GBMs. Click here to expand this figure. Figure EV1. USP33 is a hypoxia-inducible deubiquitinase that is preferentially expressed in GSCs Immunoblot analysis of HIF2α and HIF1α protein levels in GSCs cultured under different concentrations of oxygen. GSCs were cultured under 20%, 5%, 3%, or 1% oxygen for 24 h. Full induction of HIF2α was observed under 5% oxygen, whereas the strongest induction of HIF1α was observed under 1% oxygen. Immunoblot analysis of the induction of a cohort of deubiquitylases in GSCs in response to low oxygen. GSCs were cultured under 20% or 5% oxygen for 24 h. No deubiquitylases other than USP33 was induced in GSCs in response to 5% oxygen. Immunofluorescent analyses of HIF1α (red) and the hypoxic cell population labeled by pimonidazole (green) in intracranial xenografts derived from T3359 GSCs. Mice bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of pimonidazole and were sacrificed 2 h post-injection. Hypoxic cells were labeled with the anti-pimonidazole, FITC-conjugated mouse monoclonal antibody. Scale bar, 80 µm. Statistical quantification of (C) showing the percentage of HIF1α+ cells in the pimonidazole+ hypoxic and the pimonidazole- normoxic cells. The majority of hypoxic cell population in GSC-derived xenografts were positively stained for HIF1α. (n = 5 tumors; ***P < 0.001; mean ± s.e.m.; two-tailed unpaired t-test). Immunofluorescent analyses of HIF2α (red) and the hypoxic cell population labeled by pimonidazole (green) in intracranial xenografts derived from T3359 GSCs. Mice bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of pimonidazole and were sacrificed 2 h post-injection. Hypoxic cells were labeled with the anti-pimonidazole, FITC-conjugated mouse monoclonal antibody. Scale bar, 80 µm. Statistical quantification of (E) showing the percentage of HIF2α+ cells in the pimonidazole+ hypoxic and the pimonidazole- normoxic cells. The majority of hypoxic cell population in GSC-derived xenografts were positively stained for HIF2α. (n = 5 tumors; ***P < 0.001; mean ± s.e.m.; two-tailed unpaired t-test). Immunoblot analysis of USP33 and HIF2α levels in GSCs in response to low oxygen or CoCl2-mimicking hypoxia. GSCs were cultured under 5% oxygen for 24 h or treated with CoCl2 (300 µM) for 12 h. Both 5% oxygen and CoCl2 treatment elevated USP33 and HIF2α levels in GSCs. Immunoblot analysis of USP33 and HIF2α levels in GSCs and matched NSTCs in normoxia. High levels of USP33 and HIF2α expression were observed in GSCs relative to NSTCs. Immunoblot analysis of USP33 and HIF2α levels in NSTCs in response to low oxygen or CoCl2-mimicking hypoxia. NSTCs were cultured under 5% oxygen for 24 h or treated with CoCl2 (300 µM) for 12 h. Neither 5% oxygen nor CoCl2 treatment elevated USP33 or HIF2α levels in NSTCs, although the induction of HIF1α was observed. Immunoblot analysis of USP33 and HIF2α levels in neural progenitor cells (NPCs) in response to low oxygen or CoCl2-mimicking hypoxia. NPCs were cultured under 5% oxygen for 24 h or treated with CoCl2 (300 µM) for 12 h. Neither 5% oxygen nor CoCl2 treatment elevated USP33 or HIF2α levels in NPCs, although the induction of HIF1α was observed. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. USP33 is a hypoxia-inducible deubiquitinase that is preferentially expressed in GSCs Immunoblot analysis of USP33 and HIF2α protein levels in GSCs cultured under 5% oxygen for different times. GSCs (T387, T3359, and T4121) were cultured under 5% oxygen for 0, 12, or 24 h before harvest. Dramatic induction of USP33 was observed at 12 h under 5% oxygen. HIF2α protein levels were slightly upregulated at 12 h and further elevated at 24 h under 5% oxygen. Immunoblot analysis of USP33 and HIF2α protein levels in GSCs cultured in different concentrations of oxygen. GSCs were cultured under 20%, 5%, 3%, or 1% oxygen for 24 h before harvest. Induction of USP33 was observed under 5%, 3%, and 1% oxygen. Immunofluorescent analyses of USP33 (red), the hypoxia marker CA9 (green), and the stem cell marker SOX2 (gray) in human primary GBMs. Arrows indicate the USP33+/CA9+/SOX2+ cells. Scale bar, 80 μm. Statistical quantification of (C) showing the ratio of USP33+ cells in SOX2+, CA9+, or SOX2+/CA9+ cells in human primary GBMs. USP33 expression was detected in a fraction of GSCs (SOX2+) or CA9+ hypoxic cells, but the majority of the hypoxic GSCs (SOX2+/CA9+ cells) were USP33+, indicating that USP33 was induced in GSCs in response to hypoxia. (n = 5 sections; ***P < 0.001; mean ± s.e.m.; two-tailed unpaired t-test). Immunofluorescent analyses of USP33 (red) and the hypoxic cell population labeled by pimonidazole (green) in intracranial xenografts derived from T3359 GSCs. Mice bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of pimonidazole and were sacrificed 2 h post-injection. Hypoxic cells were labeled with the FITC-conjugated mouse monoclonal anti-pimonidazole antibody. Scale bar, 80 μm. Statistical quantification of (E) showing the percentage of USP33+ cells in the pimonidazole+ hypoxic and the pimonidazole- normoxic cells. The majority of hypoxic cell population in GSC-derived xenografts were positively stained for USP33. (n = 5 tumors; ***P < 0.001; mean ± s.e.m.; two tailed unpaired t-test). Immunoblot analysis of USP33, HIF2α, and HIF1α protein levels in GSCs, NSTCs, and NPCs in normoxic and hypoxic conditions. Hypoxia (5% O2) relative to normoxia (20% O2) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs. The mild hypoxia of 5% O2 showed negligible induction of HIF1α in all cells. Source data are available online for this figure. Source Data for Figure 1 [embj2021109187-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Previous reports showed that GSCs had higher HIF2α expression than matched NSTCs even under regular culture conditions and that HIF2α was required for the GSC maintenance (Li et al, 2009). Consistently, immunoblot analyses of paired GSCs and NSTCs cultured under normoxic conditions demonstrated relatively higher protein levels of both USP33 and HIF2α in GSCs (Fig EV1H), suggesting that higher expression of this hypoxia-inducible DUB may also be required for GSC maintenance. As HIF2α is preferentially induced in GSCs but not in NSTCs, hypoxia-responsive signaling may be cell type specific (Li et al, 2009). Similarly, hypoxia-induced upregulation of USP33 was clearly detected in GSCs but not in NSTCs or NPCs (Figs 1G and EV1G,I,J). Taken together, USP33 is preferentially expressed by GSCs under normoxic condition and further upregulated under hypoxia in GSCs in vitro and in vivo. USP33 stabilizes HIF2α proteins in GSCs in response to hypoxia Because USP33 is induced under hypoxia preferentially in GSCs, we speculated that USP33 functioning as a deubiquitinase may stabilize its downstream targets specifically in GSCs. USP33 shares an approximately 59% identity with the HIF1α deubiquitinase USP20, indicating the possible regulation of HIFs by USP33 (Li et al, 2005). Our previous studies showed that HIF1α was induced in both GSCs and NSTCs, but HIF2α expression was elevated preferentially in GSCs in response to hypoxia (Li et al, 2009). The preferential induction of USP33 in GSCs rather than NSTCs or NPCs is consistent with the HIF2α induction pattern in response to hypoxia (Figs 1G and EV1G,I and J). In addition, 5% oxygen mildly upregulated HIF2α mRNA levels but dramatically increased HIF2α protein levels (Fig 1A, Appendix Fig S2A), suggesting a potential post-translational stabilization of HIF2α in GSCs in response to hypoxia at 5% oxygen. Moreover, the USP33 protein levels increased ahead of HIF2α induction in response to hypoxia at 5% oxygen (Fig 1A) but declined behind HIF2α degradation when returning to normoxia (Appendix Fig S2B), suggesting a longer turnover of USP33 relative to HIF2α proteins during the hypoxia response. Thus, we sought to explore the possible association between USP33 and HIF2α. We initially determined the correlation between HIF2α and USP33 expression in human primary GBMs. Immunofluorescent analyses showed that the majority of HIF2α-positive cells had USP33 staining (Fig 2A and B), suggesting a positive correlation between HIF2α and USP33. We then disrupted the endogenous USP33 in GSCs and examined the induction of HIF2α under hypoxic conditions. Immunoblot analyses showed that disruption of USP33 almost abolished HIF2α induction in response to either 5% oxygen or CoCl2 treatment (Fig 2C and D, Appendix Fig S2C–F), indicating that USP33 is required for HIF2α induction under hypoxic conditions in GSCs. Consistently, ectopic overexpression of USP33 in GSCs elevated endogenous HIF2α protein levels under normoxic conditions (Fig 2E), suggesting that USP33 is able to upregulate HIF2α levels in GSCs. We further explored the USP33-mediated stabilization of HIF2α proteins by ectopic expression of USP33 and HIF2α under the control of CMV promoter in 293T cells, which excluded the involvement of transcriptional regulation. Immunoblot analyses showed that USP33 expression increased HIF2α protein levels in a dose-dependent manner (Fig 2F), indicating that USP33 upregulates HIF2α at the protein level but not at the mRNA level. To further clarify whether USP33 affects HIF2α protein synthesis or degradation, 293T cells expressing ectopic HIF2α with or without USP33 were treated with the translation inhibitor cycloheximide. Immunoblot analyses demonstrated that overexpression of USP33 delayed the decline in HIF2α protein levels, while HIF2α protein levels declined rapidly after cycloheximide treatment in the control cells without USP33 expression (Appendix Fig S2G), suggesting that USP33 controls HIF2α protein stability. We next determined whether the deubiquitinase activity of USP33 is required for HIF2α stabilization. Whereas ectopic expression of the wild-type USP33 upregulated the protein levels of HIF2α, ectopic expression of the catalytically dead USP33 (C194S/H673Q) mutant (Berthouze et al, 2009; Niu et al, 2020) largely lost the capacity to elevate HIF2α levels (Fig 2G), indicating that the USP33 deubiquitinase activity is essential for the USP33-mediated stabilization of HIF2α protein in GSCs. Of note, ectopic overexpression of USP33 in NSTCs upregulated the levels of HIF2α protein to a much less extent than that in GSCs (Fig 2E, Appendix Fig S2H), indicating that USP33 may have a stronger potency to stabilize HIF2α proteins in GSCs. Finally, treatment with the proteasome inhibitor MG132 markedly restored the HIF2α protein levels in GSCs transduced with shUSP33 shRNAs under hypoxia (Appendix FigS2I and J). Taken together, our data demonstrate that USP33 stabilizes HIF2α protein in GSCs in response to hypoxia. Figure 2. USP33 stabilizes HIF2α protein in GSCs A, B. Representative images (A) and statistical quantification (B) of immunofluorescent analyses of USP33 (red) and HIF2α (green) in human primary GBMs. Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α, and counterstained with DAPI to show nuclei (blue). The percentage of USP33+ cells in the HIF2α+ population was quantified. The majority of HIF2α+ cells were positively stained for USP33 in human GBMs. Scale bar, 40 μm. (n = 5 sections; mean ± s.e.m). C. Immunoblot analysis of HIF2α induction in T4121 GSCs expressing shUSP33 or shNT in response to low oxygen. Twenty-four hours post-lentiviral transduction of shUSP33 or shNT, GSCs were incubated under 20% or 5% oxygen for 24 h and then harvested for the immunoblot analysis. Disruption of USP33 abrogated HIF2α induction in GSCs in response to 5% oxygen. D. Immunoblot analysis of HIF2α induction in T4121 GSCs expressing shNT or shUSP33 in response to CoCl2-mimicking hypoxia. Twenty-four hours post-lentiviral transduction of shUSP33 or shNT, GSCs were treated with CoCl2 (300 μM) for 12 h and then harvested for the immunoblot analysis. Disruption of USP33 abrogated CoCl2-induced HIF2α expression in GSCs. E. Immunoblot analysis of HIF2α protein levels in GSCs expressing ectopic Flag-tagged USP33 in normoxia. Cells were transduced with lentiviruses carrying Flag-tagged USP33 or the control vector, followed by two rounds of puromycin selection. Ectopic expression of USP33 elevated HIF2α protein levels in GSCs cultured under 20% oxygen. F. Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33. 0.5 μg of HA-tagged HIF2α plasmids together with the increased amounts of GFP-tagged USP33 plasmids (0, 0.1, 0.2, or 0.3 μg) were introduced into 293T cells. Forty-eight hours post-transfection, levels of ectopic HIF2α-HA and GFP-USP33 were analyzed by immunoblot analysis. Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33. G. Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild-type (WT) USP33 or the catalytically dead (CD) USP33. HA-tagged HI" @default.
• W4212816453 created "2022-02-24" @default.
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• W4212816453 title "USP33 deubiquitinates and stabilizes HIF‐2alpha to promote hypoxia response in glioma stem cells" @default.
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