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The Deubiquitinating Enzyme USP17 Is Highly Expressed in Tumor Biopsies, Is Cell Cycle Regulated, and Is Required For

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The Deubiquitinating Enzyme USP17 Is Highly Expressed in Tumor Biopsies, Is Cell Cycle Regulated, and Is Required For Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-4152 Tumor and Stem Cell Biology Cancer Research The Deubiquitinating Enzyme USP17 Is Highly Expressed in Tumor Biopsies, Is Cell Cycle Regulated, and Is Required for G1-S Progression Cheryl McFarlane1, Alyson A. Kelvin1, Michelle de la Vega1, Ureshnie Govender1, Christopher J. Scott2, James F. Burrows1,2, and James A. Johnston1 Abstract Ubiquitination is a reversible posttranslational modification that is essential for cell cycle control, and it is becoming increasingly clear that the removal of ubiquitin from proteins by deubiquitinating enzymes (DUB) is equally important. In this study, we have identified high levels of the DUB USP17 in several tumor-derived cell lines and primary lung, colon, esophagus, and cervix tumor biopsies. We also report that USP17 is tightly regulated during the cell cycle in all the cells examined, being abundantly evident in G1 and absent in S phase. Moreover, regulated USP17 expression was necessary for cell cycle progression because its depletion signifi- cantly impaired G1-S transition and blocked cell proliferation. Previously, we have shown that USP17 regulates the intracellular translocation and activation of the GTPase Ras by controlling Ras-converting enzyme 1 (RCE1) activation. RCE1 also regulates the processing of other proteins with a CAAX motif, including Rho family GTPases. We now show that USP17 depletion blocks Ras and RhoA localization and activation. More- over, our results confirm that USP17-depleted cells have constitutively elevated levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, known downstream targets of Ras and RhoA signaling. These observa- tions clearly show that USP17 is tightly regulated during cell division and that its expression is necessary to coordinate cell cycle progression, and thus, it may be considered a promising novel cancer therapeutic target. Cancer Res; 70(8); 3329–39. ©2010 AACR. Introduction tant role in the cell cycle as they help maintain the dynamic balance between ubiquitination and deubiquitination (3, 4). Ubiquitination is an important mechanism for regulating Recently, several DUBs have been identified that regulate cell cycle progression. Numerous components of the cell cycle the cell cycle and control mitotic and spindle assembly machinery, including various cyclins and cyclin-dependent ki- checkpoints. For instance, anaphase initiation is facilitated nase inhibitors (CDKI), are modified by ubiquitination (1). by USP44, which regulates mitosis by deubiquitinating Ubiquitination is relatively well characterized and is me- cdc20 (5). In addition, mitotic entry is facilitated by the gene diated by either the E3 ligases SKP1-CUL-F-box complex responsible for cylindromatosis (CYLD), which modulates or the anaphase-promoting complex/cyclosome at distinct polo-like kinase 1 (Plk1) activity (6). Moreover, chromosomal stages of the cell cycle (2). Similar to other covalent modifica- alignment and segregation during mitosis is also regulated by tions, such as phosphorylation or methylation, ubiquitination is the deubiquitination of survivin by USP9 (7). a reversible posttranslational modification. Deubiquitinating DNA damage checkpoints are regulated by USP1 and USP7. enzymes (DUBs) catalyze the removal of ubiquitin by hydrolyz- USP1 catalyzes the deubiquitination of two important compo- ing the isopeptide bond linking ubiquitin to its substrate. nents of DNA damage repair pathways: FANCD2 and prolifer- Accumulating evidence suggests that DUBs also play an impor- ating cell nuclear antigen (PCNA; refs. 8, 9). USP7 (HAUSP) deubiquitinates Mdm2, and Mdmx, which alters the stability Authors' Affiliations: 1Centre for Infection and Immunity, School of 2 and activity of p53 (10). DUBs can also modulate chromatin Medicine, Dentistry and Biomedical Sciences; Molecular Therapeutics, – School of Pharmacy, Faculty of Medicine, Health and Life Sciences, structure, thus altering gene transcription in a cell cycle Queen's University Belfast, Belfast, United Kingdom specific manner. Histone H2A is deubiquitinated by USP16 Note: Supplementary data for this article are available at Cancer during mitosis and enables histone phosphorylation and chro- Research Online (http://cancerres.aacrjournals.org/). mosomal segregation (11). Additionally, USP3 catalyzes the re- Corresponding Author: James A. Johnston, Centre for Infection and moval of ubiquitin from histones in a cell cycle–dependent Immunity, School of Medicine, Dentistry and Biomedical Sciences, Whitla Medical Building, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom. manner and is essential to promote S-phase entry (12). Phone: 44-289-097-2260; Fax: 44-289-032-5839; E-mail: jim.johnston@ The importance of DUBs for cell cycle control has been qub.ac.uk. underscored by the strong association with tumorigenesis. doi: 10.1158/0008-5472.CAN-09-4152 Mutations in CYLD have been associated with cylindromato- ©2010 American Association for Cancer Research. sis, Brooke-Spiegler syndrome, and familial trichoepithelioma, www.aacrjournals.org 3329 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-4152 McFarlane et al. all of which are predisposed to multiple skin tumors of the (10 μg/mL) with RNase A (250 μg/mL), incubated at 37°C for head and neck (13). Additionally, downregulation of USP7 30 min, and analyzed by FACSCanto II (BD Biosciences) and has been linked with non–small cell lung carcinoma, and in FlowJo. conjunction with p53 status, USP7 was shown to be a prog- Immunoblotting and immunoprecipitation. Cells were nostic marker for adenocarcinoma patients (14). lysed in radioimmunoprecipitation assay buffer, and whole- The DUB/USP17 family of cytokine-inducible DUBs was cell lysates or immunoprecipitates were prepared as described initially identified in murine hematopoietic cells. There are previously (21). Antibodies were as follows: anti-USP17 (Fusion several murine family members (DUB-1, DUB-1A, DUB-2, Antibodies); anti–pan-Ras, anti–extracellular signal-regulated and DUB-2A) and a human family member (DUB-3/USP17, kinase (ERK) 1/2, anti–phospho-ERK1/2, anti–mitogen- subsequently referred to as USP17). USP17 family members activated protein kinase (MAPK)/ERK kinase (MEK) 1/2, and are immediate-early genes, which are rapidly induced follow- anti–phospho-MEK1/2 (Cell Signaling); anti-RhoA, anti–cyclin ing cytokine stimulation (15–18). Several lines of evidence D1, cyclin A, and cyclin E (Calbiochem); anti–cyclin B1 (Santa have implicated these enzymes in the regulation of cell Cruz Biotechnology); anti-p21cip1 (Upstate Antibodies); and growth and survival. Constitutive expression of murine anti-p27kip1 (Calbiochem). Densitometry was carried out us- DUB-1 has been shown to induce G1 cell cycle arrest, whereas ing ImageJ software (NIH). DUB-2 expression can markedly inhibit apoptosis following Confocal microscopy. Cells were fixed with 10% trichlor- cytokine withdrawal (15, 17, 19, 20). In addition, we have con- oacetic acid (TCA) as described previously (23). Ras and firmed that constitutive overexpression of human USP17 RhoA were stained according to antibody manufacturer's in- blocks growth factor–dependent cell proliferation (20). More structions and detected with tetramethylrhodamine isothio- recently, we have also shown that USP17 regulates the activity cyanate (TRITC)–conjugated donkey anti-mouse (Jackson of the GTPase CAAX-box processing enzyme Ras-converting ImmunoResearch). Nuclei were counterstained with 300 enzyme 1 (RCE1) to alter Ras processing and membrane local- nmol/L 4′,6-diamidino-2-phenylindole (DAPI; Molecular ization, and confirmed that the ability of USP17 to block cell Probes). Slides were mounted using Vectashield (Vector Lab- proliferation was RCE1 dependent (21). oratories) and then viewed with LSM 510 META NLO confo- In this study, we report high levels of USP17 expression in cal microscope (Zeiss). many primary tumor biopsies and tumor-derived cell lines. GTPase activation assays. Ras and RhoA activation was In addition, we find that USP17 expression is tightly regulat- determined by pull-downs using glutathione S-transferase ed during the cell cycle and plays a fundamental role in me- (GST)–Raf–RBD and GST-Rhotekin, respectively, as de- diating cell cycle progression. USP17 depletion markedly scribed previously (21, 24). inhibits cell proliferation and prevents the passage of cells RNA extraction and reverse transcription-PCR. RNA was from G1 into S phase. We report that USP17 depletion blocks extracted using STAT-60 according to the manufacturer's in- the translocation and proper activation of Ras and RhoA in structions (Biogenesis). Reverse transcription-PCR (RT-PCR) cip1 G1, resulting in the accumulation of the CDKIs p21 and was performed on 1 μgoftotalRNAusingOneStepRT- kip1 p27 , which we suggest is the underlying cause of the G1 PCR kit (Qiagen) as described previously (25). The following block. Our findings indicate that tight regulation of USP17 primers were used: USP17, 5′-CAGTGAATTCGTGGGAATG- expression is essential for G1-S transition and that targeting GAGGACGACTCACTCTAC-3′ (forward) and 5′-AGTCATC- USP17 could be a novel anticancer approach. GATCTGGCACACAAGCATAGCCCTC-3′ (reverse); β-actin, 5′-GGACTTCGAGCAAGAGATGG-3′ (forward) and Materials and Methods 5′-AGCACTGTGTTGGCGTACAG-3′ (reverse). Immunohistochemistry. Immunohistochemical staining Results of
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