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• W3009261305 abstract "Article4 March 2020Open Access Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells Cristian Prieto-Garcia Cristian Prieto-Garcia Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Oliver Hartmann Oliver Hartmann Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Michaela Reissland Michaela Reissland Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Fabian Braun Fabian Braun Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Thomas Fischer Thomas Fischer Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Department for Radiotherapy, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Susanne Walz Susanne Walz Core Unit Bioinformatics, Comprehensive Cancer Centre Mainfranken, University of Würzburg, Würzburg, Germany Search for more papers by this author Christina Schülein-Völk Christina Schülein-Völk Core Unit High-Content Microscopy, Biocenter, University of Würzburg, Würzburg, Germany Search for more papers by this author Ursula Eilers Ursula Eilers Core Unit High-Content Microscopy, Biocenter, University of Würzburg, Würzburg, Germany Search for more papers by this author Carsten P Ade Carsten P Ade Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Department of Biochemistry and Molecular Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Marco A Calzado Marco A Calzado Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Córdoba, Spain Hospital Universitario Reina Sofía, Córdoba, Spain Search for more papers by this author Amir Orian Amir Orian Faculty of Medicine, TICC, Technion Haifa, Israel Search for more papers by this author Hans M Maric Hans M Maric Rudolf-Virchow-Center for Experimental Biomedicine, Würzburg, Germany Search for more papers by this author Christian Münch Christian Münch Institute of Biochemistry II, Goethe University, Frankfurt, Germany Search for more papers by this author Mathias Rosenfeldt Mathias Rosenfeldt Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Institute for Pathology, University of Würzburg, Würzburg, Germany Search for more papers by this author Martin Eilers Martin Eilers orcid.org/0000-0002-0376-6533 Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Department of Biochemistry and Molecular Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Markus E Diefenbacher Corresponding Author Markus E Diefenbacher [email protected] orcid.org/0000-0002-7402-7949 Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Cristian Prieto-Garcia Cristian Prieto-Garcia Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Oliver Hartmann Oliver Hartmann Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Michaela Reissland Michaela Reissland Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Fabian Braun Fabian Braun Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Thomas Fischer Thomas Fischer Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Department for Radiotherapy, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Susanne Walz Susanne Walz Core Unit Bioinformatics, Comprehensive Cancer Centre Mainfranken, University of Würzburg, Würzburg, Germany Search for more papers by this author Christina Schülein-Völk Christina Schülein-Völk Core Unit High-Content Microscopy, Biocenter, University of Würzburg, Würzburg, Germany Search for more papers by this author Ursula Eilers Ursula Eilers Core Unit High-Content Microscopy, Biocenter, University of Würzburg, Würzburg, Germany Search for more papers by this author Carsten P Ade Carsten P Ade Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Department of Biochemistry and Molecular Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Marco A Calzado Marco A Calzado Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Córdoba, Spain Hospital Universitario Reina Sofía, Córdoba, Spain Search for more papers by this author Amir Orian Amir Orian Faculty of Medicine, TICC, Technion Haifa, Israel Search for more papers by this author Hans M Maric Hans M Maric Rudolf-Virchow-Center for Experimental Biomedicine, Würzburg, Germany Search for more papers by this author Christian Münch Christian Münch Institute of Biochemistry II, Goethe University, Frankfurt, Germany Search for more papers by this author Mathias Rosenfeldt Mathias Rosenfeldt Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Institute for Pathology, University of Würzburg, Würzburg, Germany Search for more papers by this author Martin Eilers Martin Eilers orcid.org/0000-0002-0376-6533 Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Department of Biochemistry and Molecular Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Markus E Diefenbacher Corresponding Author Markus E Diefenbacher [email protected] orcid.org/0000-0002-7402-7949 Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany Comprehensive Cancer Centre Mainfranken, Würzburg, Germany Search for more papers by this author Author Information Cristian Prieto-Garcia1,2, Oliver Hartmann1,2, Michaela Reissland1,2, Fabian Braun1,2, Thomas Fischer1,3, Susanne Walz4, Christina Schülein-Völk5, Ursula Eilers5, Carsten P Ade2,6, Marco A Calzado7,8,9, Amir Orian10, Hans M Maric11, Christian Münch12, Mathias Rosenfeldt2,13, Martin Eilers2,6 and Markus E Diefenbacher *,1,2 1Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany 2Comprehensive Cancer Centre Mainfranken, Würzburg, Germany 3Department for Radiotherapy, University Hospital Würzburg, Würzburg, Germany 4Core Unit Bioinformatics, Comprehensive Cancer Centre Mainfranken, University of Würzburg, Würzburg, Germany 5Core Unit High-Content Microscopy, Biocenter, University of Würzburg, Würzburg, Germany 6Department of Biochemistry and Molecular Biology, University of Würzburg, Würzburg, Germany 7Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain 8Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Córdoba, Spain 9Hospital Universitario Reina Sofía, Córdoba, Spain 10Faculty of Medicine, TICC, Technion Haifa, Israel 11Rudolf-Virchow-Center for Experimental Biomedicine, Würzburg, Germany 12Institute of Biochemistry II, Goethe University, Frankfurt, Germany 13Institute for Pathology, University of Würzburg, Würzburg, Germany *Corresponding author. Tel: +49 0931 31 88167; Fax: +49 0931 31 84113; E-mail: [email protected] EMBO Mol Med (2020)12:e11101https://doi.org/10.15252/emmm.201911101 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 The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours. Synopsis The study reveals that squamous tumours are dependent on the expression of the deubiquitylase USP28. Inhibition of USP28 destabilises ΔNp63 protein abundance and enables therapeutic targeting of squamous tumours of various origins, such as head and neck, lung, cervix and pancreas. USP28 protein was upregulated in squamous tumours. USP28 modulated the expression of essential squamous genes by regulating ΔNp63 protein abundance. Pharmacologic inhibition of USP28 activity was well tolerated in vivo and negatively affected squamous tumour growth. The paper explained Problem Squamous cell carcinomas (SCCs) are among the genetically most complex and heterogeneous entities. While driver mutations can vary widely, all SCC have in common their intricate dependency on ∆Np63 expression. Previous work has unequivocally demonstrated that ∆Np63 is a master transcription factor that establishes SCC cell identity. In several SCC tumour models, it was demonstrated that tumours are addicted to ∆Np63 expression. Therefore, targeting ∆Np63, either directly or by altering its protein abundance, appears to be a promising strategy to tackle SCC tumours. Results In our study, we report that the deubiquitylase USP28 directly interacts and stabilizes ∆Np63 in SCC. Depletion of USP28 in human tumour cell lines affected proliferation and epithelial cell identity of SCC cells. This effect is mediated directly by destabilization of ∆Np63 protein upon loss of USP28. We document the dependence of SCC on USP28 using both cancer cell lines and in vivo murine lung tumour models. We determined that both proteins directly interact and that the enzymatic activity of USP28 is required to deubiquitylate, and stabilize, ∆Np63. In vivo, we could demonstrate that in a mouse model of lung SCC, loss of Usp28 during tumour initiation abolished SCC formation entirely. Similar effects could be demonstrated in an orthotopic lung SCC transplant model, where knock-down or inhibition of Usp28 was sufficient to hinder tumour growth. Finally, using pharmacologic inhibitors of USP28, we were able to specifically target SCC tumours by destabilizing ∆Np63. Impact Our work thus identifies for the first time a deubiquitylase, USP28, regulating ∆Np63 protein abundance. USP28 is druggable, and its modulation, in vitro and in vivo, negatively affected SCC in a ∆Np63-dependent manner; hence, USP28 can function as a druggable surrogate target for ∆Np63 in SCC. Our findings, by using currently available USP28 inhibitors, serve as a proof of concept that targeting SCC by targeting the DUB USP28 is a promising selective therapeutic strategy. If proved safe and efficient in humans, USP28 inhibitors could expand the current limited available portfolio of applicable SCC therapeutic agents. Introduction Each year, around 2 million patients are diagnosed and approximately 1.76 million succumb to lung cancer, making this tumour entity the leading cause of cancer-related death for men and women alike (Bray et al, 2018). According to the current WHO classification, lung cancer is classified into two major subtypes depending on marker expression: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), causing around 85 or 15% of disease incidences, respectively (Inamura, 2017). NSCLC can be further subdivided, according to marker expression and prevalent mutational aberrations, into adenocarcinomas (ADC) and squamous carcinomas (SCCs). Comprehensive analyses of the mutational landscape show that lung SCC is one of the genetically most complex tumours (Cancer Genome Atlas Research, 2012). As a consequence, little is known about therapeutic targetable vulnerabilities of this disease. A key regulatory protein in SCC is the p53-related transcription factor ∆Np63, encoded by the TP63 gene (Su et al, 2013). ∆Np63 is highly expressed in lung SCC as well as in SCCs of the skin, head and neck, and oesophagus, in part due to gene amplification (Hibi et al, 2000, Tonon et al, 2005; Cancer Genome Atlas Research N, 2012). The TP63 locus encodes multiple mRNAs that give rise to functionally distinct proteins. Notably, transcription from two different promoters produces N-terminal variants either containing or lacking the transactivation domain: TAp63 or ΔNp63 (Deyoung & Ellisen, 2007). The major p63 isoform expressed in squamous epithelium and SCC is ΔNp63α (Rocco et al, 2006; Koster et al, 2007), which is a master transcription factor that establishes epithelial cell identity, including cytokeratin 5/6 and 14 (Rocco et al, 2006; Deyoung & Ellisen, 2007; Su et al, 2013; Hamdan & Johnsen, 2018; Somerville et al, 2018). In addition, ∆Np63 binds to and thereby inactivates TP53 at promoters of pro-apoptotic genes, suppressing their expression (Westfall et al, 2003; Craig et al, 2010). ∆Np63 is essential for the survival of skin and pancreatic SCC cells, since established murine skin SCCs are exquisitely dependent on ∆Np63; acute deletion of TP63 in advanced, invasive SCC induced rapid and dramatic apoptosis and tumour regression (Rocco et al, 2006; Galli et al, 2010; Ramsey et al, 2013; Su et al, 2013; Somerville et al, 2018). Collectively, these findings raise the possibility that ∆Np63 is a therapeutic target in SCC tumours. ∆Np63 is an unstable protein that is continuously turned over by the proteasome upon ubiquitination by E3 ligases, such as the FBXW7 ubiquitin ligase (Galli et al, 2010). FBXW7 is frequently mutated or deleted in SCC tumours (cervix 13.15%, HNSC 7.55%, lung 6.4% and oesophagus 7.29%; cBioPortal, Galli et al, 2010; Ruiz et al, 2019). Intriguingly, it has been shown previously that the degradation of many targets of FBXW7 is counteracted by the deubiquitylase (DUB) USP28 (Popov et al, 2007b). This is in part due to the fact that USP28 exploits binding to FBXW7 to interact with its substrates (Schulein-Volk et al, 2014). However, USP28 can also recognize the phosphodegron that is required for the binding of FBXW7 to its substrates in an FBXW7-independent manner (Diefenbacher et al, 2015). Loss of USP28 counteracts the loss of Fbxw7 in a murine colon tumour model (Diefenbacher et al, 2015; Cremona et al, 2016), and acute deletion of USP28 in established tumours increases survival in the APCmin∆/+ colorectal tumour model (Diefenbacher et al, 2014), while not affecting tissue homeostasis in non-transformed cells (Schulein-Volk et al, 2014). Together, these data argue that targeting USP28 may destabilize ∆Np63 and suggest that this strategy may have therapeutic efficacy in SCC. Results USP28 is highly abundant in human squamous tumours and correlates with poor prognosis To investigate the mutational as well as the expression status of USP28 in lung cancer, we analysed publicly available datasets of human tumours (Figs 1A and B, and EV1A, B and D). USP28 is rarely lost or mutated, but frequently transcriptionally upregulated in human SCC compared to healthy lung tissue or ADC (adenocarcinoma) patient samples (Figs 1A and B, and EV1A and B). Similarly, the expression of TP63 was significantly upregulated in SCC samples compared to non-transformed tissue or to ADC samples (Figs 1A and EV1A and B). Figure 1. USP28 is highly abundant in human squamous tumours and correlates with poor prognosis A. Expression of USP28 (left) and TP63 (right) in human lung squamous cell carcinomas (SCC, n = 498), adenocarcinomas (ADC, n = 513) and normal non-transformed tissue (normal SCC = 338, normal ADC = 348). Xena UCSC software. In box plots, the centre line reflects the median, the cross represents the mean, and the upper and lower box limits indicate the first and third quartiles. Whiskers extend 1.5× the IQR, and outliers are marked as dots. B. Correlation of mRNA expression of USP28 and TP63 in lung SCC (left, n = 498), ADC (right, n = 513) and normal non-transformed tissue (normal SCC = 338, normal ADC = 348). R: Spearman's correlation coefficient; m = Slope. Xena UCSC software. C. IHC analysis of USP28 and ∆Np63 protein abundance in lung cancer and non-transformed human samples (n = 300). The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists. In box plots, the centre line reflects the median, the cross represents the mean, and the upper and lower box limits indicate the first and third quartiles. Whiskers extend 1.5× the IQR, and outliers are marked as dots. P-values were calculated using two-tailed t-test. D. Kaplan–Meier estimator of NSCLC patients stratified by USP28 (left, n = 1,145) and TP63 (right, n = 1,926) expression. P-values were calculated using log-rank test. HR: hazard ratio. KmPlot software. E. Kaplan–Meier estimator of lung SCC patients stratified by USP28 expression (n = 271). The P-value was calculated using a log-rank test. HR: hazard ratio. KmPlot software. Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001. See also Fig EV1 and Appendix Table S3 (exact P-values and statistical test used). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. USP28 and ΔNp63 mRNA and protein expression in public datasets, TMA and patient material A. Analysis of occurring genetic alterations in USP28 and TP63 in lung cancer (CBioPortal). B. USP28 and TP63 gene expression heatmap in ADC (n = 364) and SCC (n = 527) lung cancer samples (Xena UCSC software). C. Representative IHC grading scores of endogenous USP28 and ΔNp63 in lung tissue samples (left panel, low magnification, scale bar 200 μm; right panel high magnification, scale bar 50 μm). D. Genetic alterations of USP28 in human lung SCC. Each column represents a tumour sample (n = 179 LSCC). Disease-free survival of USP28 mutant lung SCC patients. Data from TCGA were analysed using cBioPortal software. Download figure Download PowerPoint Next, we determined the abundance of USP28 protein via immunohistochemistry (IHC) on tissue microarrays and tumour sections of a total of 300 human lung tumour samples. Relative to non-transformed tissue, all samples from different human lung tumour subtypes expressed elevated levels of USP28, with SCC presenting the highest levels (Figs 1C and EV1C), confirming the USP28 mRNA expression data (Fig 1A and B). TP63 protein abundance was evaluated within the same cohort and, like USP28, exhibited the highest protein abundance in SCC tumours compared to ADC and SCLC samples and normal tissue (Figs 1C and EV1C). To evaluate the relevance of both proteins for tumour development, we used publicly available datasets to correlate mRNA expression data with patient survival. Patients with an increased expression of either ∆Np63 or USP28 showed a significantly shortened overall survival (Fig 1D). Importantly, this correlation was not a secondary consequence of a generally shorter survival of SCC patients, since USP28 expression correlated with worse prognosis even when only SCC patients were analysed (Fig 1E). Finally, we noted that 3% of lung SCC patients display mutations in USP28 or a deletion of USP28, and those showed a much better disease-free survival compared to USP28 wild-type patients (Fig EV1D). These data indicate that USP28 is upregulated in NSCLC, and high expression of USP28 negatively correlates with overall patient survival in SCC tumours. Additionally, we were able to detect a strong correlation between USP28 and ∆Np63 abundance in lung SCC, indicating a potential crosstalk between both proteins. ∆Np63 stability is regulated by USP28 via its catalytic activity To test whether USP28 controls ∆Np63 protein abundance, we initially expressed HA-tagged USP28 and FLAG-tagged ∆Np63 in HEK293 cells by transient transfection. Immunofluorescence staining using antibodies against USP28 and ∆Np63 revealed that both proteins localize to the nucleus of transfected cells (Appendix Fig S1A). Co-immunoprecipitation experiments showed that ∆Np63 binds to USP28 and vice versa, indicating an interaction of both molecules in cells (Fig 2A). Upon co-expression of His-tagged ubiquitin, ∆Np63 was ubiquitylated, as demonstrated by pulldown of His-tagged ubiquitin followed by immunoblot using a ∆Np63-specific antibody, and co-expression of USP28 resulted in the deubiquitylation of ∆Np63 (Fig 2B). To test the chain specificity of substrate deubiquitylation by USP28 on ∆Np63, we ectopically co-expressed a His-tagged ubiquitin that carries a single lysine residue either at position K48 or K63. Upon His-ubiquitin pulldown, K48- as well as K63-linked poly-ubiquitin chains could be detected on ∆Np63, as previously reported (Galli et al, 2010; Peschiaroli et al, 2010; Fig 2C). Upon overexpression of USP28, only K48-linked ubiquitin chains were removed from ∆Np63, whereas K63-linked chains were resistant to USP28 (Fig 2C). To test whether USP28 catalytic activity is required for deubiquitination of ∆Np63, we used a catalytic inactive mutant of USP28, USP28C171A (Fig 2D–F; Popov et al, 2007b; Diefenbacher et al, 2014, 2015; Schulein-Volk et al, 2014). Immunoprecipitation of transfected cells using an ∆Np63-specific antibody revealed that USP28C171A was able to bind to ∆Np63 (Fig 2D). While overexpression of the wild-type form of USP28 deubiquitylated ∆Np63 (Fig 2E), USP28C171A failed to do so, demonstrating that the catalytically active cysteine of USP28 is required for deubiquitylation of ∆Np63 (Fig 2E). Figure 2. ∆Np63 stability is regulated by USP28 via its catalytic activity A. Co-immunoprecipitation of exogenous HA-USP28 and FLAG-ΔNp63 in HEK293 cells. Either HA-USP28 or FLAG-ΔNp63 were precipitated and blotted against FLAG-ΔNp63 or HA-USP28. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control). B. Ni-NTA His-ubiquitin pulldown in control-transfected or HA-USP28-overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization. C. Ni-NTA His-ubiquitin pulldown K48 or K63 in control and HA-USP28-overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization. D. Co-immunoprecipitation of exogenous FLAG-USP28 C171A and FLAG-ΔNp63 in HEK293 cells. ΔNp63 was precipitated and blotted against FLAG-USP28 or ΔNp63. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control). E. Ni-NTA His-ubiquitin pulldown in control-, FLAG-USP28- or FLAG-USP28 C171A-transfected HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization. F. CHX chase assay (100 μg/ml) of control-, FLAG-USP28- or FLAG-USP28 C171A-transfected HEK293 cells for indicated time points. Representative immunoblot analysis of FLAG (USP28) and ∆Np63 as well as quantification of relative protein abundance (ACTIN as loading control). G. Immunoblot of USP28 and ∆Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR-619 for 24 h. Relative protein abundance was calculated ACTIN as loading control. Data information: Western blots shown are representative of three independent experiments (n = 3). All quantitative graphs are represented as mean ± SD of three experiments (n = 3). P-values were calculated using two-tailed t-test statistical analysis; *P < 0.05; **P < 0.01; see also Appendix Fig S1 and Appendix Table S3 (exact P-values and statistical test used). Download figure Download PowerPoint K48-linked ubiquitin chains target proteins to the proteasome for degradation (Grice & Nathan, 2016). Since USP28 is able to counteract K48-linked ubiquitylation of ∆Np63, we investigated the ability of USP28 to modulate ∆Np63 protein turnover. To do so, we co-expressed ∆Np63 with either wild-type USP28 or USP28C171A in HEK293 cells. Twenty-four hours post-transfection, cells were treated with 100 μg/ml cycloheximide (CHX) to block protein synthesis. Co-expression of wild-type USP28, but not of the catalytically inactive mutant, strongly stabilized ∆Np63 protein (Fig 2F). As ∆Np63 protein stability was enhanced by USP28, but not via the catalytic inactive C171A mutant, we tested whether a pharmacologic inhibitor of DUBs, PR-619, would also affect overall protein abundance of ∆Np63. Therefore, we expressed ∆Np63 in HEK293 cells and, 24 h post-transfection, treated cells with either DMSO or increasing amounts of PR-619 for additional 24 h (Fig 2G). While ∆Np63 was not degraded in control cells treated with DMSO, cells exposed to PR-619 showed a shortened half-life of 8 h for ∆Np63 protein (PR-619 IC50 of < 5 μM, Fig 2G). In control-treated cells, the protein abundance of USP28 was not affected; however, upon addition of the pan-DUB inhibitor PR-619, USP28 protein was reduced in a dose-dependent fashion (Fig 2G). This is in line with previous observations that the enzymatic activity of DUBs is required to enhance their own stability (de Bie & Ciechanover, 2011; Wang et al, 2017). Collectively, these data demonstrate that USP28 can interact with and stabilize the ∆Np63 protein by removing K48-linked ubiquitin chains and that the catalytic domain of USP28 is required for this activity. USP28 stabilizes ∆Np63 independently of FBXW7 Previous reports highlighted the regulation of ∆Np63 protein stability by the E3 ligase FBXW7 (Galli et al, 2010), which is commonly mutated or lost in human SCC of various origins (Appendix Fig S2A and B). To identify via which protein domain ∆Np63 interacts with USP28, we performed peptide spot interaction studies (Appendix Fig S1B, Materials and Methods) and were able to identify, apart from several lysine-containing domains, the Fbxw7 phosphodegron as a putative interaction site for USP28 (Appendix Fig S1B). To investigate whether USP28 interacts with ∆Np63 in a FBXW7-dependent fashion and whether the phosphodegron motive is required to facilitate the interaction, we made use of a ∆Np63 point mutant, ∆Np63S383A, which is not phosphorylated by GSK3β and abolishes binding to FBXW7 (Galli et al, 2010). Ectopic expression of USP28 and ∆Np63S383A in HEK293 cells showed that USP28 was able to increase ∆Np63S383A abundance (Appendix Fig S1C). Furthermore, by co-immunoprecipitation experiments with exogenous USP28 and ∆Np63S383A in HEK293 cells, we were able to detect that USP28 binds to ∆Np63S383A (Appendix Fig S1D), This interaction resulted in a decreased ubiquitylation of ∆Np63S383A (Appendix Fig S1E). Furthermore, overexpression of USP28 was able to increase protein half-life (Appendix Fig S1F) and treatment of cells with PR-619 affected ∆Np63S383A protein stability, albeit to a somewhat lesser extent compared to wild-type ∆Np63 (Appendix Fig S1G). To determine whether endogenous USP28 regulates the abundance and stability of ∆Np63, we used a human SCC cell line (A-431). These cells are homozygous for the S462Y mutation in FBXW7, which is thought to inactivate substrate recognition (Appendix Fig S2C and D; Yeh et al, 2016). FBXW7, USP28 and ∆Np63 were readily detectable in the nucleus of these cells by immunofluorescence (Appendix Fig S2C and E). Furthermore, immunoprecipitation of endogenous USP28 co-immunoprecipitated endogenous ∆Np63, and vice versa (Fig 3A). In contrast, antibodies against USP25, a ubiquitin-specific protease that is structurally very similar to USP28 (Appendix Fig S2F; Gersch et al, 2019; Sauer et al, 2019), did not co-immunoprecipitate ∆Np63 although USP25 is readily detectable in A-431 cells. Correspondingly, antibodies against ∆Np63 did not co-immunoprecipitate endogenous" @default.
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• W3009261305 title "Maintaining protein stability of ∆Np63 via <scp>USP</scp> 28 is required by squamous cancer cells" @default.
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• W3009261305 doi "https://doi.org/10.15252/emmm.201911101" @default.
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• W3009261305 hasPublicationYear "2020" @default.
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