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Ubiquitin-Specific Protease 4-Mediated Deubiquitination and Stabilization of PRL-3 Is Required for Potentiating Colorectal Oncogenesis

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Ubiquitin-Specific Protease 4-Mediated Deubiquitination and Stabilization of PRL-3 Is Required for Potentiating Colorectal Oncogenesis Published OnlineFirst December 15, 2015; DOI: 10.1158/0008-5472.CAN-14-3595 Cancer Molecular and Cellular Pathobiology Research Ubiquitin-Specific Protease 4-Mediated Deubiquitination and Stabilization of PRL-3 Is Required for Potentiating Colorectal Oncogenesis Cheng Xing1, Xing-Xing Lu2, Peng-Da Guo2, Tong Shen2, Shen Zhang2, Xiao-Shun He2, Wen-Juan Gan2, Xiu-Ming Li2, Jing-Ru Wang2, Yuan-Yuan Zhao2, Hua Wu2, and Jian-Ming Li1,2 Abstract Ubiquitin specific protease 4 (USP4) is a deubiquitinating Mechanistically, we observed that USP4 interacted with and enzyme with key roles in the regulation of p53 and TGFb stabilized PRL-3 via deubiquitination. This resulted in activa- signaling, suggesting its importance in tumorigenesis. Howev- tion of Akt and reduction of E-cadherin, critical regulators of er, the mechanisms and regulatory roles of USP4 in cancer, cancer cell growth and metastasis. Examination of clinical including colorectal cancer, remain largely elusive. Here, we samples confirmed that USP4 expression positively correlates present the first evidence that USP4 regulates the growth, with PRL-3 protein expression, but not mRNA transcript levels. invasion, and metastasis of colorectal cancer. USP4 expression Taken together, our results demonstrate that aberrant expres- was significantly elevated in colorectal cancer tissues and was sion of USP4 contributes to the development and progression significantly associated with tumor size, differentiation, distant of colorectal cancer and reveal a critical mechanism underlying metastasis, and poor survival. Knockdown of USP4 diminished USP4-mediated oncogenic activity. These observations suggest colorectal cancer cell growth, colony formation, migration, and that the potential of harnessing proteolytic degradation pro- invasion in vitro and metastasis in vivo. Importantly, we found cesses for therapeutic manipulationmayofferamuch-needed that phosphatase of regenerating liver-3 (PRL-3) is indispens- new approach for improving colorectal cancer treatment able for USP4-mediated oncogenic activity in colorectal cancer. strategies. Cancer Res; 76(1); 1–13. Ó2015 AACR. Introduction cell-cycle progression, transcriptional regulation, and signal trans- duction (3–5). However, deubiquitination, the reverse process Colorectal cancer is the third leading cause of death from cancer mediated by deubiquitinating enzymes (DUB), has gained worldwide (1). Although some advances have been made in the increasing attention as an important regulatory mechanism in treatment of colorectal cancer, including surgical techniques, che- controlling protein turnover (6, 7). Currently, approximately 100 motherapy, and radiation therapy, the overall survival rate of putative DUBs have been identified (8), but most have not been patients with colorectal cancer has not improved dramatically. This functionally characterized. The ubiquitin-specific proteases (USP) is due to recurrence and metastasis (2). Altered regulation of some represent the largest subclass of DUBs. It has more than 50 proteins and signaling pathways in the development of colorectal members (8). Like other USPs, USP4 also mediates the removal cancer has been described, but identification of novel regulatory and processing of ubiquitin. USP4 binds TRAF2, TRAF6, and mechanisms contributing to the development and progression of TAK1 for deubiquitination and negatively regulates TNFa- colorectal cancer remains a subject of significant interest. induced NF-kB activation (9, 10). USP4 binds directly to and Ubiquitination is an essential posttranslational modification. It stabilizes ARF-BP1 via deubiquitination, subsequently promoting plays critical roles in a diverse array of cellular processes, including ARF-BP1–dependent p53 ubiquitination and degradation (11). USP4 also directly interacts with and deubiquitinates TGFb type I receptor (TbRI), regulating TGFb signaling by controlling TbRI 1Department of Pathology, Nanfang Hospital, Southern Medical Uni- versity, Guangzhou, China. 2Pathology Center and Department of levels at the plasma membrane (12). USP4 is significantly over- Pathology, Soochow University, Suzhou, China. expressed in many human cancers, such as urinary, prostate, Note: Supplementary data for this article are available at Cancer Research thyroid, and liver cancer (11, 13). Unfortunately, other studies Online (http://cancerres.aacrjournals.org/). show conflicting observations. USP4 protein expression is down- Corresponding Authors: Jian-Ming Li, Department of Pathology, Nanfang regulated in lung cancer cell lines (14). USP4, which is located on Hospital, Southern Medical University, Guangzhou 510515, China. Phone: 86- chromosome 3 (3p21.3), is frequently deleted in human epithe- 512-65882673; Fax: 86-512-65882673; E-mail: lixinyue@fimmu.com; and Hua lial cancers, including those of the breast, kidney, and lung Wu, Pathology Center and Department of Pathology, Soochow University, (15–17). However, USP4's mechanism of regulation in cancer, Suzhou 215123, China. Phone: 86-512-65882673; Fax: 86-512-65882673; E-mail: including colorectal cancer, has not been well established. [email protected] Phosphatase of regenerating liver-3 (PRL-3, also called doi: 10.1158/0008-5472.CAN-14-3595 PTP4A3), a member of the protein tyrosine phosphatase family Ó2015 American Association for Cancer Research. (PTPs), plays an important role in cancer progression, including www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst December 15, 2015; DOI: 10.1158/0008-5472.CAN-14-3595 Xing et al. cell proliferation, migration, invasion, metastasis, and angiogen- tissues were purchased from Shanghai Outdo Biotech Co., Ltd., esis (18–20). The expression of PRL-3 is frequently upregulated in collected between 2006 and 2007. A total of 167 patients were human tumors, such as breast cancer (21), liver cancer (22), involved in the study. The clinical characteristics of all patients are ovarian cancer (23), gastric cancer (24), and colorectal cancer listed in Supplementary Table S1. The study was approved by (25). This expression is associated with cancer progression and Soochow University for Biomedical Research Ethics Committee, poor survival. PRL-3 modulates multiple signaling pathways, and all of the patients provided informed consent. Tissues were including PI3K/AKT, SRC, Rho GTPases, MAPK/ERK, and EGFR probed using the indicated antibodies. in various cancer cells (19, 26, 27). Previous findings, including The staining score was evaluated using two blinded individuals. ours, clearly indicate that PRL-3 plays a critical role in the epi- The staining scoring criteria were as follows: 0, 1%–4% of the cells thelial–mesenchymal transition (EMT; refs. 28, 29). Recently, a stained positive; 1, 5%–25% of the cells stained positive; 2, 26%– new functional role of PRL-3 in epigenetic regulation was dis- 50% of the cells stained positive; 3, 51%–75% of the cells stained covered. PRL-3 probably affects the activity of JMJD1B and positive; and 4, 75% of the cells stained positive. The staining JMJD2B, two histone demethylases, thus participating in the intensity of cancer cells was scored as 0 (no staining), 1 (weak regulation of histone methylation (30). Despite PRL-3's impor- staining), 2 (intermediate staining), and 3 (strong staining). An tance as a regulatory of tumorigenesis, the mechanism leading to intensity score of 2 with at least 50% of USP4-positive cells was the aberrant upregulation of PRL-3 in tumors remains unclear. regarded as high expression, and <50% of USP4-positive cells or In this study, results showed USP4 expression to be elevated in <2 in intensity score was considered low expression. The multiple tumor tissues sampled from colorectal cancer patients. A series of of the intensity and extent score was used as the final score (0–12) experiments in vitro and in vivo revealed that stable knockdown of for USP4. USP4 expression in colorectal cancer cells significantly impaired the capacity for growth, migration, invasion, and metastasis. In Generation of stable cell lines addition, the mechanism by which USP4 promotes development HCT116 and SW480 cell lines stably expressing USP4-specific and progression of colorectal cancer was investigated. USP4 shRNA (USP4/shRNA) or scrambled shRNA control (USP4/Ctr) activates PI3K/AKT signaling and downregulates E-cadherin were constructed using a lentiviral shRNA technique. Oligonu- expression by binding to and deubiquitinating PRL-3, thereby cleotides were constructed in GV248 lentiviral RNAi vector preventing degradation of PRL-3. Collectively, these results sug- (GeneChem). SW480 and HCT116 cells were transduced with gest that USP4 is a bona fide DUB of PRL-3 and plays a pivotal role serial dilutions of lentiviral supernatant and selected for using in colorectal cancer. 5 mg/mL puromycin for 3 to 4 weeks. The human USP4 shRNA target sequences are listed in Supplementary Table S2. Materials and Methods siRNA Antibody and reagents Human USP4 and PRL-3 siRNA and siRNA nonspecific control Antibodies against USP4 (#2651), p-Akt (#9271), and Akt were synthesized by RiboBio. Colorectal cancer cells were tran- (#9272) were purchased from Cell Signaling Technology. siently transfected with these siRNAs using Lipofectamine 2000. E-cadherin (ab53033) and PRL-3 (ab50276) were purchased The human USP4 and PRL-3 siRNA target sequences are listed in from Abcam. HA (F-7) and C-myc (9E10) were purchased from Supplementary Table S3. Santa
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