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 counteracted 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 2018 American Association for Cancer Research. cycle 20 related 1, aka CDH1) and CDC20 play crucial roles in cell

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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 in ovarian oncogenesis (24). In colorectal sing myc-tagged FZR1 (wild-type and mutant) were provided by cancer, low-grade patients had higher AURKA expression than Sangon. Plasmids and siRNA were transfected into cells with high-grade patients (25). A retrospective study of metastatic Lipofectamine 3000. Lentiviral systems for overexpression and colorectal cancer revealed that high AURKA copy number pre- knockdown were provided by GenePharma and were infected dicted longer overall survival (26). Hence, precise roles of AURKA into cells following the provider's instructions. Interference sequences used were: control: 50-TTCTCCGAACGTGTCACGT- in tumorigenesis and mechanisms underlying its regulation 0 0 0 0 deserve further study. 3 ; PRL-3: 5 -GGTGGAGGTGAGCTACAAACA-3 ; AURKA-1: 5 - GGTCTTGTGTCCTTCAAATTC-30; AURKA-2: 50-GCTACCA- Here, by performing phenotypic and mechanistic investigations, 0 0 – GAGTCTACCTAATT-3 ; FZR1: 5 -GCAACGAUGUGUCUCC- we elucidated the essential role of PRL-3 regulated phenotypes in 0 controlling colorectal cancer progression through its interplay CUATT-3 . with AURKA and FZR1, and revealed PRL-3 as a phosphatase of Live-cell imaging FZR1 to promote AURKA ubiquitination and destabilization. After transfection with GFP-PRL-3 or GFP for 36 hours, cells were cultured in Microscope Slide Coverslip system (Nunk Lab- Materials and Methods Tek, 5 103 cells/chamber). Selected fields were continually Ethics statement scanned every 5 minutes for 24 hours with UltraVIEW VOX system Experiments using patient specimens (provided by Department (PerkinElmer) and analyzed with Volocity 6.1.1. For each group, of Pathology, Peking University Cancer Hospital, Beijing, China) 70 GFP-positive cells were selected for analysis. were approved by the Institutional Ethics Committee. Written Animal experiments informed consent was obtained from all patients. Animal exper- Cells were subcutaneously injected into the bilateral armpits of imentation were conducted with the approval of an Institutional 5-week-old female BALB/c nude mice (purchased from HFK Bio- Animal Care and Use Committee and followed internationally 6 Technology) as a 150 mL suspension (5 10 cells/mL). Changes recognized ARRIVE (Animal Research: Reporting of In Vivo of mice weight and xenograft volume were assessed every 3 days. Experiments) guidelines. After 21 days, mice were sacrificed and xenografts were stripped for analysis. Cell lines, antibodies, and reagents Colorectal cancer cell lines HCT116, HT29, and SW480 were Immunofluorescence staining obtained from ATCC. Human embryonic intestinal mucosa cell Cells were precultured on coverslips (5 105/mL) 24 hours line CCC-HIE-2 was obtained from National Infrastructure of Cell before assay, then fixed by 4% paraformaldehyde, permeabilized Line Resource (Beijing, China). RFP-H2B–labeled HeLa cells in 0.1% Triton X-100 for 5 minutes, and blocked by 5% goat

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Zhang et al.

serum. Antigens were stained with primary and FITC- or TRITC- decreased by PRL-3 overexpression or enhanced by PRL-3 conjugated secondary antibodies. Nuclei were stained with DAPI knockdown (Fig. 1F), supporting the antiapoptosis function (1 mg/mL). Alternatively, F-actin was stained with 1 mg/mL of observed in leukemia and breast cancer (6, 7). phalloidin-FITC. Images were acquired by the Zeiss LSM780 laser According to flow cytometry, G2–M-phase was elevated after confocal microscope (60 oil, NA 1.40 Plan-ApoChromat, PRL-3 overexpression or decreased after PRL-3 knockdown, while including two HyD detectors) at fixed exposure settings at room S-phase was increased by PRL-3 knockdown or decreased by PRL- temperature. 3 overexpression (Fig. 1G). These results were supported by analyzing the markers for G1/early-S-phase (cyclin E1), S/G2- In vitro phosphatase assay – phase (cyclin A2), and G2 M-phase (pY15-CDC2; Fig. 1A), indi- FZR1 protein was immunoprecipitated overnight at 4 C from cating the concomitant proliferation halt induced by PRL-3 over- m m HCT116 cell lysates (1,000 g) with 1 g anti-FZR1 antibody plus expression or knockdown were respectively associated with dis- protein G-Sepharose. After washing 4 times with lysis buffer [50 tinct cell-cycle changes. In addition, live-cell imaging showed that mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, mitotic length was significantly prolonged upon GFP-PRL-3 10% glycerol, 2 mmol/L dithiothreitol, 1 protease cocktail expression in HCT116 (Fig. 1H) and HeLa cells (Supplementary (Roche)] and twice with 1 dephosphorylation buffer Fig. S1), confirming PRL-3–induced G2–M-phase arrest. Above (50 mmol/L Tris-HCl pH 7.5, 0.1% NP-40, 150 mmol/L NaCl, results demonstrated that PRL-3 could regulate diverse malignant 2 mmol/L dithiothreitol, 1 mmol/L MgCl2, 0.1 mmol/L MnCl2), phenotypes of colorectal cancer cells. the precipitates (substrate) were resuspended in 20 mL1 dephos- m phorylation buffer. For AURKA dephosphorylation, 10 L PRL-3 is a negative regulator of AURKA m m HCT116 cell lysates (50 g) were mixed with 10 Lof2 After PRL-3 overexpression, cyclin B1/AURKA/pY15-CDC2 dis- dephosphorylation buffer and used as substrate. Recombinant played an evident trend of mitotic arrest after being released from PRL-3 (0.5 mg) was incubated with substrate at room temperature nocodazole-induced G2–M synchronization (Fig. 2A), and these for 30 minutes. For control, EDTA (10 mmol/L) was added to changes were opposite upon PRL-3 knockdown (Fig. 2B). Para- inhibit phosphatase activity. The reaction was terminated by doxically, canonical G –M-phase marker pS10-H3 was not upre- 2 boiling in 2 loading buffer and the phosphorylated protein gulated upon G –M arrest (Fig. 1A, 2A and B). We noted that fi fi 2 was detected by the pan-speci c (for FZR1) or site-speci c (for protein levels of mitotic kinase AURKA were negatively affected by AURKA) phospho-antibodies. PRL-3 (Fig. 1A). After being released from nocodazole-induced synchronization, the profiles of AURKA and pS10-H3 were similar Statistical analysis and formatting (Fig. 2A and B), which was consistent with the role of AURKA in Values represented mean SD of at least three independent phosphorylating Histone H3 (28). In addition, PRL-3 overexpres- experiments with duplicate or triplicate samples. Differences sion-induced changes of pS10-H3 and pY15-CDC2 were recapit- between scattered values in two groups were compared with ulated by AURKA knockdown (Fig. 2B). Hence, AURKA may play Student t test. Correlations between expression levels and a role in PRL-3–regulated cell-cycle progression. clinical variables were measured with c2 or Fisher exact test. PRL-3–AURKA cross-talk was then investigated. Serum starva- Prognostic significance was assessed with Kaplan–Meier sur- tion, nocodazole treatment, and subsequent release were per- vival analysis. All statistics in this study was performed by SPSS formed and levels of PRL-3/AURKA were assayed by immuno- 21.0, and formatted by Graphpad Prism 5 or Excel. , P < 0.05; fluorescence staining. In HCT116 (Fig. 2C), SW480 and HT29 , P < 0.01; , P < 0.001. ns, no significance. cells (Supplementary Fig. S2A and S2B), interphase cells had the Additional methods can be found in the Supplementary highest PRL-3 and lowest AURKA, whereas mitotic cells had the Material. lowest PRL-3 and highest AURKA. Both the protein and transcript levels of PRL-3 remained largely unchanged after siRNA-mediated Results AURKA interference in HCT116 and SW480 cells (Fig. 2D). Con- PRL-3 regulates distinct malignant phenotypes of colorectal versely, AURKA protein was dose-dependently decreased by PRL- cancer cells 3 overexpression (Fig. 2E) or enhanced by PRL-3 knockdown To evaluate PRL-30s role in colorectal cancer progression, we (Fig. 2F). Yet, transcript levels of AURKA were unaffected by PRL-3 performed PRL-3 stable overexpression and knockdown in (Fig. 2E and F), which was validated by comparing transcripts of colorectal cancer cell lines (Fig.1A).Consistentwiththeknown multiple cell-cycle regulators (including AURKA) in large-sample proinvasive capacity (4), cell migration and invasion were colorectal cancer datasets stratified with PRL-3 (Fig. 2G). PRL-30s increased by overexpressed PRL-3 or decreased by PRL-3 knock- negative effect on AURKA protein was also observed in primary down (Fig. 1B). In addition, PRL-3 increased plate colony intestinal mucosa cell line CCC-HIE-2 (Fig. 2H). These results formation (Fig. 1C). In soft-agar colony formation assay, underscored PRL-30s capacity to downregulate AURKA protein PRL-3 displayed a potential to enhance anchorage-independent expression. growth(Fig.1D),emphasizingits role in maintaining self- renewalofcolorectalcancercells.PRL-3–increased clonogeni- AURKA determines PRL-3–regulated phenotypes of colorectal city is in line with the study using colon tumors established cancer cells in vitro from PRL-3 knockout mice (11) and further verifies its onco- To verify the contribution of AURKA downregulation to PRL-3– genic property observed in different systems (4, 6, 10–12). regulated cell-cycle and other malignant phenotypes, we per- Interestingly, both PRL-3 overexpression and knockdown formed AURKA overexpression or knockdown in HCT116 cells resulted in decreased colorectal cancer cell proliferation with PRL-3 overexpression or knockdown (Fig. 3A). We found (Fig. 1E), which was consistent with our previous observation that PRL-3 overexpression-induced G2–M-phase accumulation (12). Annexin V/7-AAD staining showed that apoptotic rate was was reversed by ectopic AURKA (Fig. 3B). Correlated with this

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Figure 1. PRL-3 regulates distinct malignant phenotypes of colorectal cancer cells. A, Protein levels of indicated cell-cycle regulators in HCT116 and SW480 cells stably expressing GFP-PRL-3 or knockdown of endogenous PRL-3 with lentiviral systems. B–G, Migration and invasion assays (B), plate colony formation assay (C), soft-agar colony formation assay (D), proliferation (E), Annexin V/7-AAD staining (F), and cell-cycle proﬁles (G) of indicated cells. H, Live-cell observation of HCT116-mCherry-H2B cells expressing GFP or GFP-PRL-3. Left, representative images. Red ﬂuorescence, chromatin. Right, average length of mitosis (sum of prophase, metaphase, anaphase, and telophase).

result, PRL-3-decreased cell proliferation was also negated indicating that PRL-3 could promote cytokinesis failure and (Fig. 3C). However, ectopic AURKA failed to counteract PRL-3 validating PRL-30s role in driving CIN (12). Consistent with knockdown-induced S-phase accumulation (Fig. 3B) or prolifer- AURKA's role in safeguarding the genomic integrity (21, 31, ation halt (Fig. 3C). Moreover, PRL-3 and AURKA exhibited 32), its knockdown increased CIN. Importantly, PRL-3 overex- similar trends in opposing apoptosis upon their overexpression pression-induced CIN was alleviated by ectopic AURKA or was or inducing apoptosis upon knockdown (Fig. 3D), confirming the aggravated by AURKA knockdown (Fig. 3F), highlighting an studies utilizing chemical inhibitor/RNA interference against PRL- essential role of AURKA downregulation in PRL-3–induced CIN. 3 (6, 7, 29) or AURKA (30, 31). Both PRL-3 and AURKA regulate cancer cells motility (4, 29, 31), In soft-agar colony formation assay, the basal and PRL-3– which was verified by migration and invasion assays (Supple- promoted self-renewal was prevented either by AURKA overex- mentary Fig. S3B). Unlike the inhibitory effects of AURKA on PRL- pression or by AURKA knockdown (Fig. 3E), implying that 3–induced G2–M arrest, proliferation halt, and CIN (Fig. 3B, C, AURKA is also a critical determinant of PRL-30s oncogenic func- and F), PRL-3-promoted invasiveness was increased by ectopic tion. Upon GFP-PRL-3 overexpression, live-cell observation AURKA or decreased by AURKA knockdown (Supplementary Fig. detected increased ratio of multi-nucleated cells (Supplementary S3B). Therefore, PRL-3 and AURKA might interdependently drive Fig. S3A), which was confirmed by F-actin/DAPI staining (Fig. 3F), colorectal cancer cell motility.

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AURKA determines PRL-3–regulated malignancy and assay (Fig. 3E). In dissected xenograft tumors, expression proﬁles prognosis of colorectal cancer of PRL-3 and AURKA (Supplementary Fig. S4) were similar to With the same cells used for in vitro assays (Fig. 3A), we carried those in cultured cells (Fig. 3A). Immunohistochemical evalua- out xenograft assay. Overexpression or knockdown of AURKA tion of these tumors showed that the labeling index of self- blocked PRL-3-promoted xenograft tumor growth, and reinforced renewal marker CD133 was increased by PRL-3 overexpression PRL-3 knockdown-induced xenograft growth inhibition (Fig. 4A or decreased by PRL-3 knockdown, while both basal and PRL-3- and B), which supported results of soft-agar colony formation promoted CD133 were reduced by overexpression or knockdown

Figure 2. PRL-3 is a negative regulator of AURKA. A and B, Levels of indicated proteins in HCT116 and SW480 cells overexpressing PRL-3 (A) or knockdown for PRL-3 or AURKA (B) after being released from nocodazole (NOC, 100 ng/mL, 12 h)-induced synchronization. C, AURKA and PRL-30s time-scaled distributions in HCT116 were assessed by synchronization and released after serum starvation (SS) for 24 hours or NOC treatment for 12 hours. Arrows, mitotic cells. D, Protein (top) and mRNA (bottom) levels of PRL-3 after transient knockdown (48 hours) of AURKA in colorectal cancer cells. E and F, Levels of AURKA protein (top) and mRNA (bottom) in PRL-3 overexpression (þ/þþ/þþþ ¼ 0.25/1/4 mg plasmid; E) or knockdown (F) colorectal cancer cells. G, Comparison of mRNA levels of indicated cell-cycle regulators in PRL-3–low and PRL-3–high cases of datasets GSE41258 (highest 150 vs. lowest 150) and GSE40967 (highest 200 vs. lowest 200). H, Levels of AURKA protein in CCC-HIE-2 cells with PRL-3 overexpression (2 mg plasmid) or knockdown. ns, nonsigniﬁcant.

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Figure 3. AURKA determines PRL-3–regulated phenotypes of colorectal cancer cells in vitro. A, Veriﬁcation of PRL-3 and AURKA overexpression or knockdown in HCT116 cells. B–F, Cell-cycle proﬁles (B), proliferation indexes (C), apoptotic rates (D), soft-agar colony formation (50-cell counts; E), and ratio of multinucleated cells (red arrowheads, 500-cell counts; F) of indicated groups.

of AURKA (Fig. 4C), which were also consistent with results of further emphasizing the role of PRL-3–AURKA interplay in soft-agar colony formation assay (Fig. 3E). In agreement with the affecting colorectal cancer progression. in vitro apoptosis assay (Fig. 3D), similar anti-apoptosis functions of PRL-3 and AURKA were confirmed by examining the labeling PRL-3-promoted AURKA degradation is dependent on index of apoptotic marker cleaved-caspase3 (Fig. 4D). phosphatase activity and ubiquitin–proteasome system We further analyzed PRL-3 and AURKA expression in 267 PRL-3 possesses dual-specificity phosphatase activity and is consecutive colorectal cancer tissue sections (Fig. 4E). Due to the also subjected to prenylation on its carboxyl-terminal CAAX motif low positive rate of PRL-3 (18%), AURKA positivity in PRL-3 to facilitate membrane targeting (4). By using catalytic inactive negative and positive groups were 63% vs 58%, respectively (PRL-M/PM) and CAAX motif-deleted (PRL-D/PD) mutants, we (Fig. 4F). When only considering PRL-3 positive group, positive found that PRL-M-induced AURKA inhibition was weaker than rates of AURKA significantly dropped from 62% to 45% as PRL-3 that of wild-type PRL-3, however PRL-D elicited comparable expression levels elevated from medium (þþ) to high (þþþ) changes as wild-type PRL-3 did (Fig. 5A), suggesting that phos- (Fig. 4F), suggesting an inverse correlation. 249 cases paired with phatase activity of PRL-3, instead of its modification by prenyla- clinical variables were used for further analysis. Indicators of tion, was required for inhibiting AURKA. Moreover, PRL-M's chromosomal mis-segregation and CIN, including anaphase capability in inducing G2–M arrest, proliferation halt, plate col- bridge, unaligned chromosome, and multipolar mitosis, were ony formation, and soft-agar colony formation was significantly more frequently detected in PRL-3 positive tissues and signifi- abolished (Supplementary Fig. S5A–S5D), supporting the func- cantly decreased by positive expression of AURKA (Fig. 4G), tional requirement of PRL-30s phosphatase activity in tumorigenic which confirmed the role of AURKA downregulation in PRL-3– clustering. Furthermore, treatment with proteasome inhibitor induced CIN. PRL-3/AURKA's correlations with clinical indica- MG132 counteracted PRL-3-induced AURKA downregulation tions in this cohort were calculated (Supplementary Table (Fig. 5B) and stabilized AURKA to similar levels in control and S1-S3). Despite of insignificant log-rank P values, PRL-3 dis- PRL-3 knockdown cells (Fig. 5C). In cycloheximide-treated cells, played the potential as an adverse prognostic factor, while half-life of AURKA was markedly decreased by PRL-3 overexpres- AURKA was related with a favorable prognosis (Fig. 4H). As sion or prolonged by PRL-3 knockdown (Fig. 5D). Consistently, shown by stratification, PRL-3-negative-AURKA-positive group AURKA ubiquitination was enhanced by PRL-3 overexpression or correlated with the most favorable prognosis and the best reduced by PRL-3 knockdown (Fig. 5E). Importantly, wild-type clinical indications, but PRL-3-positive-AURKA-negative group PRL-3 displayed stronger activity in promoting AURKA ubiquiti- correlated with the worst (Fig. 4H; Supplementary Table S3), nation than PRL-M did (Fig. 5E). These results suggested that

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Figure 4. AURKA determines PRL-3–regulated malignancy and prognosis of colorectal cancer. A, Changes of mice body weight (top) and xenograft tumor volumes weights (bottom). B, Macroscopic recording of xenograft tumors and comparison of tumor weights. C and D, IHC staining of CD133 (C) and cleaved caspase-3 (D) of indicated xenograft tumor sections. Labeling index (%) of positive cells of each section is shown. E, Representative IHC staining of AURKA/PRL-3 in two pairs of consecutive colorectal cancer tissue samples. F, Summary of IHC staining of AURKA/PRL-3 in 267 pairs of consecutive colorectal cancer tissue sections. N/P, negative/positive. G, Left, representative normal and aberrant mitosis in colorectal cancer sections stained with anti–PRL-3. Right, ratio of aberrant mitosis in colorectal cancer samples stratiﬁed with PRL-3/AURKA staining. H, Effects of PRL-3 and AURKA expression on prognosis of 249 patients with colorectal cancer.

PRL-3 suppresses AURKA by enhancing ubiquitin/proteasome (Fig. 2G), protein levels of several APC/CFZR1 substrates (13), system-mediated proteolysis, and PRL-30s phosphatase activity including AURKA, cyclin A2, cyclin B1, cyclin D1, and PLK1, were was required for this regulation. all decreased by PRL-3 overexpression or increased by PRL-3 In addition, phosphorylation of T288-AURKA, which is the knockdown (Fig. 1A). In contrast, levels of TOP2A, another auto-phosphorylation site critical for the kinase activity (21), was APC/CFZR1 substrate (33), remained stable (Fig. 1A), suggesting also negatively affected by PRL-3 and the trends of its alteration that PRL-3 may speciﬁcally exert inhibition on a subset of cell- were similar to those of total AURKA (Fig. 1A). To exclude the cycle regulators. Besides PRL-3-AURKA colocalization (Fig. 2C; inﬂuence of AURKA's stability, an in vitro phosphatase assay was Supplementary Fig. S2A and S2B), PRL-3-FZR1 and AURKA-FZR1 performed and no obvious effect of PRL-3 on pT288-AURKA colocalizations were observed in interphase and mitotic colorec- levels was detected (Fig. 5F); therefore, PRL-3-induced AURKA tal cancer cells (Fig. 6A). Coimmunoprecipitation assay further inhibition was unrelated to AURKA's kinase activity. revealed the existence of AURKA–FZR1–PRL-3 complex (Fig. 6B). Importantly, FZR1 interference or APC/C inhibitor proTAME PRL-3 promotes AURKA proteolysis by dephosphorylating diminished PRL-3 overexpression-induced AURKA destabiliza- FZR1 tion (Fig. 6C–D), while PRL-3–induced AURKA ubiquitination Although transcript levels of several cell-cycle-related factors was overridden by FZR1 interference (Fig. 6E). Therefore, APC/ were mostly unaffected by PRL-3 in colorectal cancer datasets CFZR1 mediates PRL-3–induced AURKA degradation.

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PRL-3 as an APC/CFZR1 Activator

Figure 5. PRL-3–promoted AURKA degradation is dependent on phosphatase activity and ubiquitin–proteasome system. A, Impact of GFP-tagged wild-type PRL-3, PRL-M (PM), and PRL-D (PD) on AURKA protein levels. B and C, Impact of PRL-3 overexpression (B) or knockdown (C) on AURKA expression after MG132 treatment (10 mmol/L, 4 hours). D, The degradation rates of indicated proteins in PRL-3 overexpression or knockdown cells after cycloheximide (CHX; 50 mg/mL) administration. E, Effects of PRL-3 overexpression or knockdown on AURKA ubiquitination. Cells were pretreated with MG132 (10 mmol/L, 4 hours) and lysates (1,000 mg) were immunoprecipitated with antibody against AURKA. F, In vitro phosphatase assay of recombinant PRL-30s effect on pT288-AURKA in HCT116 lysates. R-PRL-3, recombinant PRL-3.

Temporal regulation of phosphorylation/dephosphorylation is FZR1 determines PRL-3–regulated malignancy and prognosis essential for timely assembly and activation of APC/CFZR1 (13– of colorectal cancer 18). FZR10s association with APC/C core subunit CDC27 (APC3) Phenotypically, after expression of ectopic FZR1 (Fig. 6I), PRL-3 was enhanced by PRL-3 (Fig. 6F), suggesting that PRL-3 promotes overexpression-induced G2–M-phase arrest was only rescued by assembly of APC/CFZR1 complex. Although PRL-3 did not affect FZR1-D (Fig. 7A). Although PRL-3 knockdown-increased S-phase FZR10s protein levels (Fig. 1A, 6C and D) or stability (Fig. 5D), failed to be abrogated by AURKA overexpression or ablation FZR10s phospho-serine/threonine and -tyrosine levels were (Fig. 3B), it was reversed by both wild-type and mutant FZR1 reduced by PRL-3 overexpression or increased by PRL-3 knock- (Fig. 7A), suggesting the involvement of APC/CFZR1 substrates down, however PRL-M exerted no obvious effects (Fig. 6G). More- other than AURKA in PRL-3-regulated S-phase. Consistent with over, phospho-levels of immunoprecipitated FZR1 were evidently FZR10s tumor-suppressive role (13, 18), FZR1 ablation exacer- suppressed by coincubation with recombinant PRL-3 protein bated PRL-3–promoted soft-agar colony formation (Fig. 7B). in vitro (Fig. 6H). Thus, PRL-3 is a phosphatase targeting FZR1. Conversely, PRL-3–promoted self-renewal was significantly The six phospho-serine/threonine sites (S32/S36/S40/T121/ blocked by ectopic FZR1, but this activity was lost in the case of S151/S163) closely related with FZR10s co-factor function (13, FZR1-A (Fig. 7C). 14, 19) were mutated to construct myc-tagged FZR1-A and FZR1- By analyzing large-sample datasets (GSE40967/GSE41258) D, which respectively mimicked the dephosphorylated/activated based on the mRNA levels of PRL-3/FZR1/AURKA, we found and phosphorylated/inactivated FZR1. In control cells, FZR1-A that AURKA- or FZR1-high groups were correlated with favor- elicited the strongest AURKA destabilization and ubiquitination, able prognosis and better clinical indications (i.e., T/N/M/ while FZR1-D elicited the weakest (Fig. 6I). AURKA destablization stage), while PRL-3 displayed an adverse prognostic trend and and ubiquitination in PRL-3 overexpression cells were alleviated poor indications (Supplementary Fig. S6A; Supplementary by FZR1-D, meanwhile FZR1-A displayed highest inhibition on Table S4-S9). Next, we performed stratification analysis. For AURKA in PRL-3 knockdown cells (Fig. 6I and J). Changes of other PRL-3/AURKA stratification, PRL-3-low-AURKA-high group two APC/CFZR1 substrates, cyclin B1 and cyclin D1, were similar to displayed better prognosis and clinical indications than PRL- those of AURKA in all these settings (Fig. 6I). These results 3-high-AURKA-low group, while double-low/double-high identified PRL-3 as a phosphatase of FZR1 capable of facilitating groups were in-between (Fig. 7D; Supplementary Tables S10 the E3 ligase activity of APC/CFZR1 in ubiquitination and desta- and S11), confirming the results of immunohistochemical bilization of AURKA (Fig. 6K). analysis of colorectal cancer samples (Fig. 4H; Supplementary

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Zhang et al.

Figure 6. PRL-3 promotes AURKA proteolysis by dephosphorylating FZR1. A, Coimmunoﬂuorescent staining of PRL-3, FZR1, and AURKA in HCT116 and SW480 cells. Arrowheads, mitotic cells. B, Endogenous interactions among PRL-3/AURKA/FZR1 in HCT116 and SW480 cells. Cell lysates (500 mg) were immunoprecipitated with antibody to AURKA or FZR1. Input, 50 mg of lysates. C, Impact of FZR1 knockdown on PRL-3–induced AURKA destabilization. D, Effect of proTAME (12 mmol/L, 12 h) on PRL-3–induced AURKA destabilization. E, Impact of FZR1 knockdown on PRL-3–induced AURKA ubiquitination in HCT116, as performed in Fig. 5E. F, Effect of PRL-3 overexpression on FZR1-CDC27 interaction in HCT116. G, Levels of phosphorylated FZR1 upon PRL-3/PRL-M overexpression or PRL-3 knockdown. H, In vitro dephosphorylation of immunoprecipitated FZR1 by recombinant PRL-3. I, Effects of FZR1 (wt), FZR1-A, and FZR1-D on PRL-3–induced changes of indicated cell-cycle markers. J, Effects of FZR1/FZR1-A/FZR1-D on PRL-3–induced AURKA ubiquitination in HCT116 cells. K, Model of PRL-3–promoted AURKA destabilization through FZR1 dephosphorylation and APC/CFZR1complex formation.

Table S3). Similarly, for PRL-3/FZR1 stratification, PRL-3-low- Discussion FZR1-high group displayed better prognosis and clinical indi- Tumorigenesis is a dynamic and multistep process determined cations than PRL-3-high-FZR1-low group (Fig. 7D; Supplemen- by many factors. Acquisition of an optimal level of CIN may tary Table S12 and S13). For AURKA/FZR1 stratification, confer survival advantage for cancer cells, while drastic CIN would double-high group had better prognosis and clinical indica- be deleterious (34, 35). As a master regulator of mitosis, AURKA tions, while those of double-low group were inferior (Fig. 7D; precisely coordinates mitotic chromosomal events and spindle Supplementary Table S14 and S15). For PRL-3/FZR1/AURKA- formation to ensure accurate cell division (21). Both AURKA triple stratifications, PRL-3-low-AURKA-high-FZR1-high pre- ablation and chemical inhibition could generate CIN, validating dicted the best prognosis, but PRL-3-high-AURKA-low-FZR1- the pivotal role of AURKA in maintaining genomic integrity (21, low group had the worst (Supplementary Fig. S6B). Important- 31, 32). Consistent with this concept, tumor-suppressive role of ly, the prognostic diversities between PRL-3-low-AURKA-high þ AURKA has been demonstrated in AURKA / mice and Drosoph- and PRL-3-high-AURKA-low groups were larger in FZR1-high ila expressing loss-of-function mutant AURKA (32, 36). Previous- groups than in FZR1-low groups, as marked by higher HR ly, we found that PRL-3 could dissociate shelterin components values (Fig. 7E), emphasizing that FZR1 was required to RAP1 and TRF2 from telomeric DNA, thereby eliciting telomere strengthen the PRL-3–AURKA cross-talk and the subsequent dysfunction and CIN (12). Here, we uncovered PRL-3-promoted oncogenic impacts on colorectal cancer.

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PRL-3 as an APC/CFZR1 Activator

Figure 7. FZR1 determines PRL-3–regulated malignancy and prognosis of colorectal cancer. A, Effects of FZR1/FZR1-A/FZR1-D on PRL-3–regulated HCT116 cell-cycle profiles. B, Effects of FZR1 knockdown on PRL-3–promoted soft-agar colony formation. C, Effects of FZR1/FZR1-A/FZR1-D on PRL-3–promoted HCT116 soft-agar colony formation. D, Effects of double stratification on prognosis in two colorectal cancer GEO datasets (GSE41258 and GSE40967). P, PRL-3; A, AURKA; F, FZR1. þ, high expression; , low expression. E, Effects of FZR1 transcript levels on PRL-3/AURKA–stratified prognosis. F, Summary of PRL-30s role in colorectal cancer progression through its effects on different phenotypes.

AURKA destabilization, which may represent a novel mechanism However, PRL-3 knockdown-induced S-phase arrest was unaf- of PRL-3-induced CIN. Considering that telomere dysfunction- fected by AURKA expression or knockdown, but was abrogated by induced fusion-breakage-bridge cycles require intimate coordi- ectopic FZR1. PRL-3 could dephosphorylate FZR1 and enhance nation with spindle machinery to generate numerical/structural APC/CFZR1 assembly, implying that PRL-3 may contribute to APC/ abnormalities of chromosomes (37), and that mitotic disturbance CFZR1 activation, as functioned by other two known phospha- may jeopardize telomere homeostasis (38, 39), it is necessary to tases, i.e. PP2AB55 and CDC14 (16, 17). FZR1 ablation also evaluate the functional linkage between telomere dysfunction induced S-phase accumulation (17, 18); thus, PRL-3 knockdown and AURKA destabilization in PRL-3-induced CIN. Notably, could partially phenocopy FZR1 inactivation in S-phase control. despite PRL-3-induced CIN was neutralized by ectopic AURKA Contrary to loss of FZR1-induced CIN (18), PRL-3 knockdown or phenocopied by AURKA knockdown, apoptosis was only did not provoke CIN (12), as proven by the present study, so PRL- induced by AURKA knockdown, but not by PRL-3 overexpression. 3-dependent APC/CFZR1 activation might be restricted to limited Thus, PRL-3 has a capability to resist loss of AURKA-induced circumstances. It is possible that PRL-3 differentially ﬁne-tunes apoptosis, thereby maintaining high level of CIN. the activity of APC/CFZR1 to regulate the stabilities of mitotic Several factors, for example, PI3K-AKT, p53-p21, FOXO3a, proteins and DNA replication proteins, which requires further were implicated in PRL-3-regulated cell-cycle (9), while the pre- exploration. cise mechanism has been a mystery. Moreover, PRL-30s impact on It is seemingly counterintuitive that PRL-3 overexpression cell-cycle progression seemed to be cell type-dependent (9). inhibited colorectal cancer cell proliferation. Uncontrolled cell Herein we found that PRL-3-promoted G2–M-phase accumula- proliferation is one of the hallmarks of cancer and has been tion in colorectal cancer cells was overturned by ectopic AURKA. extensively studied as a surrogate to evaluate the phenotypes of

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Zhang et al.

cancer cells and predict the course of cancer progression. For Recently, CIN was also demonstrated to drive metastasis (48). instance, the positive correlation between proliferation and However, without further evidence, it is still premature to con- aggressiveness has been found in breast cancer (40) and lym- clude that low proliferation, increased self-renewal ability and phoma (41). But independent studies found that colorectal motility might be the further effects, that is, tertiary effects, cancer seems to be an exception, because both the metastatic following PRL-3–promoted CIN. colorectal cancer in liver and the primary colorectal cancer with The opposing functions of AURKA and PRL-3 were further the ability to metastasis had reduced proliferation index (42, supported by prognosis analysis of colorectal cancer patient 43). Several mechanisms were proposed to explain the acqui- specimens and GEO datasets, as low-PRL-3 and high-AURKA sition of low cellular proliferation during the course of colo- predicted better survival. At present, the prognostic value of FZR1 rectal cancer progression from primary lesion to metastatic foci in cancer was poorly reported. We revealed that PRL-3-AURKA- (42). These mechanisms include: (i) CIN, as CIN generates a determined colorectal cancer prognosis was affected by the pres- proliferative disadvantage in both yeast and mammalian cells ence of FZR1, thus confirming an essential role of FZR1 in (44); (ii) stemness, because cancer stem cells are always slow- mediating PRL-3-AURKA cross-talk. These findings indicated that cycling (45, 46); (iii) EMT, as cancer cells in the process of EMT better understandings of the PRL-3–FZR1–AURKA interplay are are less proliferative (46). It is of note that PRL-3 was initially demanded to expand current cognition of colorectal cancer devel- found to be highly expressed in metastatic colorectal cancer, opment and progression. but not in nonmetastatic primary colorectal cancer lesions or In summary, we found a critical role of PRL-3 in downregulat- noncolorectal cancer metastatic lesions (3, 4). The uncoupling ing AURKA, through which to influence diverse malignant phe- of proliferation and motility/colony formation/anchorage- notypes of colorectal cancer cells as well as the prognosis of independent growth/xenograft growth upon PRL-3 overexpres- patients with colorectal cancer. We further identified PRL-3 as a sion in colorectal cancer cells, as shown in this study, plus its phosphatase of FZR1 capable of facilitating the activity of APC/ ability to promote CIN (ref. 12 and this study), stemness (11), CFZR1 in destabilizing AURKA. These results expand current and EMT (8), suggest that PRL-3 could be responsible for the knowledge regarding the contributions of PRL-3, AURKA, and low proliferation during colorectal cancer progression. This FZR1 to colorectal cancer progression. particular phenotype of colorectal cancer raised a serious issue that targeting fast-cycling cells may not be the reasonable Disclosure of Potential Conflicts of Interest approach for the treatment of colorectal cancer at advanced No potential conflicts of interest were disclosed. stage, while this would be overcame by strategies aiming at PRL-3 or PRL-3–FZR1–AURKA pathway. On the basis of the results of the current study, PRL-3– Authors' Contributions regulated colorectal cancer progression would be determined Conception and design: C. Zhang, L. Qu, L. Shen, C. Shou by six aspects of mechanisms: (i) CIN; (ii) cell-cycle; (iii) Development of methodology: C. Zhang, L. Qu, S. Lian proliferation; (iv) stemness; (v) cell death; and (vi) motility. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Zhang, S. Lian, L. Meng, L. Min, J. Liu, Q. Song Overexpressed PRL-3 promoted CIN, self-renewal, and motil- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, – ity; meanwhile, it inhibited proliferation, induced G2 Marrest, computational analysis): C. Zhang, L. Qu, L. Min, C. Shou and decreased apoptosis. The slow proliferation was eventually Writing, review, and/or revision of the manuscript: C. Zhang, L. Qu, L. Min, surpassed by enhanced CIN, self-renewal ability, motility, and L. Shen, C. Shou low apoptosis, resulting in colorectal cancer progression. Administrative, technical, or material support (i.e., reporting or organizing Among these phenotypes, effects of PRL-3 on CIN, G –Marrest, data, constructing databases): C. Zhang, L. Meng, J. Liu, L. Shen 2 Study supervision: C. Zhang, L. Qu, L. Shen, C. Shou and proliferation were counteracted by ectopic AURKA, or mimicked by AURKA knockdown, which underscored the Acknowledgments functional significance of PRL-3–induced AURKA destabiliza- We deeply appreciated Drs. Xuemin Zhang (National Center of Biomedical tion. In this sense, these three phenotypes may represent the Analysis, China) and Qinghua Shi (University of Science & Technology of secondary effects following AURKA destabilization (Fig. 7F). China) for sharing critical reagents, Drs. Bin Dong, Caiyun Liu, Chuanke Zhao, Conversely, in the setting of PRL-3 deficiency, decreased pro- and Jing Gao (Peking University Cancer Hospital & Institute) for generous help liferation, S-phase arrest, impaired self-renewal, increased apo- in experimental suggestions and IHC staining and evaluation. This work was ptosis and diminished motility would collectively restrict colo- supported by the National Basic Research Program of China (no. 2015CB553906 to C. Shou), the National Natural Science Foundation of China rectal cancer progression (Fig. 7F). Despite of PRL-3 ablation- (no. 81230046 to C. Shou; no. 81672732 to L. Qu), and China Postdoctoral enhanced AURKA expression, concomitant AURKA knockdown Science Foundation (2018M631281 to C. Zhang). failed to reverse these phenotypic changes, thus the effects of PRL-3 loss on colorectal cancer progression might be largely The costs of publication of this article were defrayed in part by the AURKA independent. payment of page charges. This article must therefore be hereby marked advertisement Furthermore, the interplays among individual mechanisms in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. could not be excluded. As shown by previous studies, CIN could impair cell proliferation (44); meanwhile, CIN conferred growth Received March 5, 2018; revised August 8, 2018; accepted November 21, advantage of stem cells through enhanced genetic diversity (47). 2018; published first November 29, 2018.

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940 Cancer Res; 79(5) March 1, 2019 Cancer Research

PRL-3 Promotes Ubiquitination and Degradation of AURKA and Colorectal Cancer Progression via Dephosphorylation of FZR1

Cheng Zhang, Like Qu, Shenyi Lian, et al.

Cancer Res 2019;79:928-940. Published OnlineFirst November 29, 2018.

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