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PRL-3 Promotes Ubiquitination and Degradation of AURKA And Published OnlineFirst November 29, 2018; DOI: 10.1158/0008-5472.CAN-18-0520 Cancer Molecular Cell Biology Research PRL-3 Promotes Ubiquitination and Degradation of AURKA and Colorectal Cancer Progression via Dephosphorylation of FZR1 Cheng Zhang1,2, Like Qu1, Shenyi Lian1,3, Lin Meng1, Li Min1,4, Jiafei Liu1, Qian Song1, Lin Shen2, and Chengchao Shou1 Abstract The oncogenic phosphatase PRL-3 is highly expressed in rectal cancer specimens showed that expression of PRL-3 was metastatic colorectal cancer but not in nonmetastatic colorec- associated with high status of CIN and poor prognosis, which tal cancer or noncolorectal cancer metastatic cancers. Although were antagonized by expression of AURKA. PRL-3 enhanced the proinvasive capacity of PRL-3 has been validated in mul- AURKA ubiquitination and degradation in a phosphatase- tiple types of cancer, its impact on colorectal cancer progres- dependent fashion. PRL-3 interacted with AURKA and FZR1, sion and the underlying mechanisms remain poorly under- a regulatory component of the APC/CFZR1 complex. Destabi- stood. Here, we report that overexpressed PRL-3 stimulates lization of AURKA by PRL-3 required PRL-3-mediated dephos- FZR1 G2–M arrest, chromosomal instability (CIN), self-renewal, phorylation of FZR1 and assembly of the APC/C complex. and growth of colorectal cancer cells in xenograft models, Our study suggests that PRL-3–regulated colorectal cancer while colorectal cancer cell proliferation is decreased. PRL-3– progression is collectively determined by distinct malignant induced G2–M arrest was associated with decreased expression phenotypes and further reveals PRL-3 as an essential regulator of Aurora kinase A (AURKA). PRL-3–promoted slow prolifer- of APC/CFZR1 in controlling the stability of AURKA. ation, CIN, self-renewal, and growth in xenografts were coun- teracted by ectopic expression of AURKA. Conversely, knock- Significance: Dephosphorylation of FZR1 by PRL-3 facil- down of PRL-3 resulted in low proliferation, S-phase arrest, itates the activity of APC/CFZR1 by destabilizing AURKA, thus impaired self-renewal, increased apoptosis, and diminished influencing aggressive characteristics and overall progression xenograft growth independently of AURKA. Analysis of colo- of colorectal cancer. Introduction and the identification of molecular determinants of metastatic colorectal cancer may benefit the prevention and treatment of this Although the incidence and mortality rates of colorectal cancer disease. The dual-specificity phosphatase PRL-3 (phosphatase of have been declining for decades, it remains a major health regenerating liver-3, PTP4A3) was originally found to be over- problem (1). Approximately 50% of patients with colorectal expressed in metastatic lesions of colorectal cancer in liver (3, 4), cancer develop liver metastases during the course of disease but not in nonmetastatic colorectal cancer or noncolorectal progression and 80% to 90% of metastatic lesions are not resect- cancer metastatic cancers. To date, the proinvasive capacity of able (2). Deeper understanding of the biological characteristics PRL-3 has been well documented (4). Curiously, despite of PRL-30s phosphatase activity, only limited proteins were identi- fi 1Department of Biochemistry and Molecular Biology, Key Laboratory of Carci- ed as its substrates, whereas phosphorylation of several critical nogenesis and Translational Research (Ministry of Education/Beijing), Peking signaling factors was found to be enhanced by PRL-3 (4–8). PRL-3 University Cancer Hospital & Institute, Beijing, China. 2Department of Gastro- could reprogram the secretome of colorectal cancer cells (5), intestinal Oncology, Key Laboratory of Carcinogenesis and Translational inhibit apoptosis (6, 7), promote epithelial mesenchymal tran- Research (Ministry of Education/Beijing), Peking University Cancer Hospital & sition (EMT; ref. 8), and modulate cell-cycle progression (7, 9). Institute, Beijing, China. 3Department of Pathology, Key Laboratory of Carci- The oncogenic function of PRL-3 was further verified by mouse nogenesis and Translational Research (Ministry of Education/Beijing), Peking University Cancer Hospital & Institute, Beijing, China. 4Department of Gastro- models, in which colitis-associated colon tumorigenesis was enterology, Beijing Friendship Hospital, Capital Medical University, National reduced by PRL-3 knockout (10, 11), or was exacerbated by Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, PRL-3 transgene (12). Colon tumors from PRL-3 knockout mice Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, had impaired clonogenicity (11), implying that PRL-3 plays an China. essential role in maintaining self-renewal of tumor-initiating cells. Note: Supplementary data for this article are available at Cancer Research These findings underline PRL-30s roles in controlling a wide Online (http://cancerres.aacrjournals.org/). spectrum of biological events during tumorigenesis. However, Corresponding Authors: Like Qu, Department of Biochemistry and Molecular the functional associations among these PRL-3–regulated phe- Biology, Peking University Cancer Hospital & Institute, 52 Fucheng Road, notypes and their contribution to colorectal cancer progression Beijing 100142, China. Phone: 8610-8819-6769; Fax: 8610-8812-2437; E-mail: remain unclear. [email protected]; and Lin Shen, [email protected] As the substrate recognition elements for the APC/C (anaphase- doi: 10.1158/0008-5472.CAN-18-0520 promoting complex/cyclosome), FZR1 (fizzy and cell division Ó2018 American Association for Cancer Research. cycle 20 related 1, aka CDH1) and CDC20 play crucial roles in cell 928 Cancer Res; 79(5) March 1, 2019 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 29, 2018; DOI: 10.1158/0008-5472.CAN-18-0520 PRL-3 as an APC/CFZR1 Activator fate decision and tumorigenesis (13, 14). APC/CCDC20 is activated were kindly given by Prof. Xuemin Zhang (National Center of from prometaphase to telophase, while APC/CFZR1 is activated Biomedical Analysis, Beijing, China). mCherry-H2B–labeled FZR1 from late anaphase to next G1 phase (15). APC/C promotes HCT116 cells were kindly given by Prof. Qinghua Shi (University mitotic exit by catalyzing ubiquitination-dependent proteolysis of Science & Technology of China, Beijing, China). Cell lines were of cyclins, CDC20, PLK1, and Aurora kinases (13, 16). Mutation- maintained in RPMI1640 or high glucose DMEM (Invitrogen) al-loss or impaired expression of FZR1 was found in various medium supplemented with 10% FCS (Invitrogen) and 0.1% human tumors (17), while FZR1 homozygous deletion results gentamicin (Invitrogen). All cell lines were authenticated by short in chromosomal instability (CIN) and mouse embryonic lethality tandem repeat profiling. Mycoplasma test was performed monthly (18). Both APC/CFZR1 and APC/CCDC20 are tightly controlled by by qPCR amplification of M. hyorhinis p37 and Hochest33258 cofactor binding and posttranslational modifications to ensure staining. the proper progression of cell cycle and to maintain the chromo- Antibodies for pT288-AURKA (#3079), CDC2 (#28439), PLK1 somal integrity (13–18). CDK/cyclin-mediated phosphorylation (#4513), pY15-CDC2 (#4539), phospho-tyrosine (#9411), pS10- of FZR1 prevents its recruitment to the APC/C core complex and H3 (#53348), and ubiquitin (#3936) were purchased from Cell FZR1 inactivates APC/C from late G1 to mitotic exit (13, 16, 18). Signaling Technology. Antibodies for AURKA (ab52973), cyclin FZR1 is also phosphorylated by ERK, thereby stabilizing a subset A2 (ab181591), cyclin B1 (ab32053), cyclin D1 (ab16663), cyclin of oncogenic APC/CFZR1 substrates to support melanomagenesis E1 (ab33911), FZR1 (ab3242), and TOP2A (ab52934) were (19). Conversely, dephosphorylation of FZR1 by PP2AB55 or purchased from Abcam. Antibodies for APC1 (BS1611, Bio- CDC14 facilitates the assembly and subsequent activation of world), CDC27 (610455, BD), phospho-Serine/Threonine APC/CFZR1 (16, 17). Independently of the phosphatase activity, (612548, BD), myc-tag (AB103, TianGen), and GAPDH nuclear PTEN promotes the formation of APC/CFZR1 complex and (60004, Proteintech) were also purchased. Monoclonal antibody enhances its tumor-suppressive function (20). Other phospha- against PRL-3 was generated and characterized previously (12). tases targeting FZR1 are yet to be identified. Nocodazole and MG132 were obtained from Selleck. Cyclo- During anaphase, APC/CFZR1 promotes AURKA ubiquitination heximide was from Cell Signaling Technology. ProTAME was and proteolysis, thereby controlling mitotic spindle reorganiza- from R&D Systems. Recombinant human PRL-3 protein was tion and mitotic exit (13, 21). AURKA amplification and over- obtained from Origene. Lipofectamine 3000 was from Invitrogen. expression were implicated in mitotic disturbance, CIN induc- FITC-phalloidin was from Sigma-Aldrich. tion, and neoplastic progression (21–23). Although being reported as oncogenic, controversial findings addressing AURKA's Plasmids and RNA interference prognostic indications existed. Activation and overexpression of Plasmids for wild-type PRL-3, PRL-M (C104S, phosphatase fi D AURKA were preferentially detected in early-stage/low-grade as activity de cient), and PRL-D ( CAAX-motif deleted) were pre- fi well as noninvasive ovarian tumors, suggesting its alternation viously constructed and veri ed (27). pcDNA3.1 plasmids expres- could be an early event
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