cancer research - prl-3 promotes ubiquitination and ......here, we report that overexpressed prl-3...

Molecular Cell Biology

PRL-3 Promotes Ubiquitination and Degradationof AURKA and Colorectal Cancer Progression viaDephosphorylation of FZR1Cheng 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 inmetastatic colorectal cancer but not in nonmetastatic colorec-tal cancer or noncolorectal cancermetastatic cancers. Althoughthe proinvasive capacity of PRL-3 has been validated in mul-tiple types of cancer, its impact on colorectal cancer progres-sion and the underlying mechanisms remain poorly under-stood. Here, we report that overexpressed PRL-3 stimulatesG2–M arrest, chromosomal instability (CIN), self-renewal,and growth of colorectal cancer cells in xenograft models,while colorectal cancer cell proliferation is decreased. PRL-3–inducedG2–Marrest was associatedwith decreased expressionof Aurora kinase A (AURKA). PRL-3–promoted slow prolifer-ation, CIN, self-renewal, and growth in xenografts were coun-teracted by ectopic expression of AURKA. Conversely, knock-down of PRL-3 resulted in low proliferation, S-phase arrest,impaired self-renewal, increased apoptosis, and diminishedxenograft growth independently of AURKA. Analysis of colo-

rectal cancer specimens showed that expression of PRL-3 wasassociated with high status of CIN and poor prognosis, whichwere antagonized by expression of AURKA. PRL-3 enhancedAURKA ubiquitination and degradation in a phosphatase-dependent fashion. PRL-3 interacted with AURKA and FZR1,a regulatory component of the APC/CFZR1 complex. Destabi-lization of AURKAby PRL-3 required PRL-3-mediated dephos-phorylation of FZR1 and assembly of the APC/CFZR1 complex.Our study suggests that PRL-3–regulated colorectal cancerprogression is collectively determined by distinct malignantphenotypes and further reveals PRL-3 as an essential regulatorof APC/CFZR1 in controlling the stability of AURKA.

Significance: Dephosphorylation of FZR1 by PRL-3 facil-itates the activity of APC/CFZR1 by destabilizing AURKA, thusinfluencing aggressive characteristics and overall progressionof colorectal cancer.

IntroductionAlthough the incidence andmortality rates of colorectal cancer

have been declining for decades, it remains a major healthproblem (1). Approximately 50% of patients with colorectalcancer develop liver metastases during the course of diseaseprogression and 80% to 90% of metastatic lesions are not resect-able (2). Deeper understanding of the biological characteristics

and the identification of molecular determinants of metastaticcolorectal cancermay benefit the prevention and treatment of thisdisease. The dual-specificity phosphatase PRL-3 (phosphatase ofregenerating liver-3, PTP4A3) was originally found to be over-expressed in metastatic lesions of colorectal cancer in liver (3, 4),but not in nonmetastatic colorectal cancer or noncolorectalcancer metastatic cancers. To date, the proinvasive capacity ofPRL-3 has been well documented (4). Curiously, despite ofPRL-30s phosphatase activity, only limited proteins were identi-fied as its substrates, whereas phosphorylation of several criticalsignaling factors was found to be enhanced by PRL-3 (4–8). PRL-3could reprogram the secretome of colorectal cancer cells (5),inhibit apoptosis (6, 7), promote epithelial mesenchymal tran-sition (EMT; ref. 8), and modulate cell-cycle progression (7, 9).The oncogenic function of PRL-3 was further verified by mousemodels, in which colitis-associated colon tumorigenesis wasreduced by PRL-3 knockout (10, 11), or was exacerbated byPRL-3 transgene (12). Colon tumors from PRL-3 knockout micehad impaired clonogenicity (11), implying that PRL-3 plays anessential role inmaintaining self-renewal of tumor-initiating cells.These findings underline PRL-30s roles in controlling a widespectrum of biological events during tumorigenesis. However,the functional associations among these PRL-3–regulated phe-notypes and their contribution to colorectal cancer progressionremain unclear.

As the substrate recognition elements for the APC/C (anaphase-promoting complex/cyclosome), FZR1 (fizzy and cell divisioncycle 20 related 1, akaCDH1) andCDC20play crucial roles in cell

1Department of Biochemistry and Molecular Biology, Key Laboratory of Carci-nogenesis and Translational Research (Ministry of Education/Beijing), PekingUniversity Cancer Hospital & Institute, Beijing, China. 2Department of Gastro-intestinal Oncology, Key Laboratory of Carcinogenesis and TranslationalResearch (Ministry of Education/Beijing), Peking University Cancer Hospital &Institute, Beijing, China. 3Department of Pathology, Key Laboratory of Carci-nogenesis and Translational Research (Ministry of Education/Beijing), PekingUniversity Cancer Hospital & Institute, Beijing, China. 4Department of Gastro-enterology, Beijing Friendship Hospital, Capital Medical University, NationalClinical Research Center for Digestive Disease, Beijing Digestive Disease Center,Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing,China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Like Qu, Department of Biochemistry and MolecularBiology, Peking University Cancer Hospital & Institute, 52 Fucheng Road,Beijing 100142, China. Phone: 8610-8819-6769; Fax: 8610-8812-2437; E-mail:[email protected]; and Lin Shen, [email protected]

doi: 10.1158/0008-5472.CAN-18-0520

�2018 American Association for Cancer Research.

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fate decision and tumorigenesis (13, 14). APC/CCDC20 is activatedfrom prometaphase to telophase, while APC/CFZR1 is activatedfrom late anaphase to next G1 phase (15). APC/CFZR1 promotesmitotic exit by catalyzing ubiquitination-dependent proteolysisof cyclins, CDC20, PLK1, and Aurora kinases (13, 16). Mutation-al-loss or impaired expression of FZR1 was found in varioushuman tumors (17), while FZR1 homozygous deletion resultsin chromosomal instability (CIN) andmouse embryonic lethality(18). Both APC/CFZR1 and APC/CCDC20 are tightly controlled bycofactor binding and posttranslational modifications to ensurethe proper progression of cell cycle and to maintain the chromo-somal integrity (13–18). CDK/cyclin-mediated phosphorylationof FZR1 prevents its recruitment to the APC/C core complex andinactivates APC/CFZR1 from late G1 to mitotic exit (13, 16, 18).FZR1 is also phosphorylated by ERK, thereby stabilizing a subsetof oncogenic APC/CFZR1 substrates to support melanomagenesis(19). Conversely, dephosphorylation of FZR1 by PP2AB55 orCDC14 facilitates the assembly and subsequent activation ofAPC/CFZR1 (16, 17). Independently of the phosphatase activity,nuclear PTENpromotes the formation of APC/CFZR1 complex andenhances its tumor-suppressive function (20). Other phospha-tases targeting FZR1 are yet to be identified.

During anaphase, APC/CFZR1 promotes AURKA ubiquitinationand proteolysis, thereby controlling mitotic spindle reorganiza-tion and mitotic exit (13, 21). AURKA amplification and over-expression were implicated in mitotic disturbance, CIN induc-tion, and neoplastic progression (21–23). Although beingreported as oncogenic, controversial findings addressing AURKA'sprognostic indications existed. Activation and overexpression ofAURKA were preferentially detected in early-stage/low-grade aswell as noninvasive ovarian tumors, suggesting its alternationcould be an early event in ovarian oncogenesis (24). In colorectalcancer, low-grade patients had higher AURKA expression thanhigh-grade patients (25). A retrospective study of metastaticcolorectal cancer revealed that high AURKA copy number pre-dicted longer overall survival (26). Hence, precise roles of AURKAin tumorigenesis and mechanisms underlying its regulationdeserve further study.

Here, byperformingphenotypic andmechanistic investigations,we elucidated the essential role of PRL-3–regulated phenotypes incontrolling colorectal cancer progression through its interplaywith AURKA and FZR1, and revealed PRL-3 as a phosphatase ofFZR1 to promote AURKA ubiquitination and destabilization.

Materials and MethodsEthics statement

Experiments usingpatient specimens (providedbyDepartmentof Pathology, Peking University Cancer Hospital, Beijing, China)were approved by the Institutional Ethics Committee. Writteninformed consent was obtained from all patients. Animal exper-imentation were conducted with the approval of an InstitutionalAnimal Care and Use Committee and followed internationallyrecognized ARRIVE (Animal Research: Reporting of In VivoExperiments) guidelines.

Cell lines, antibodies, and reagentsColorectal cancer cell lines HCT116, HT29, and SW480 were

obtained from ATCC. Human embryonic intestinal mucosa celllineCCC-HIE-2was obtained fromNational Infrastructure of CellLine Resource (Beijing, China). RFP-H2B–labeled HeLa cells

were kindly given by Prof. Xuemin Zhang (National Center ofBiomedical Analysis, Beijing, China). mCherry-H2B–labeledHCT116 cells were kindly given by Prof. Qinghua Shi (Universityof Science & Technology of China, Beijing, China). Cell lines weremaintained in RPMI1640 or high glucose DMEM (Invitrogen)medium supplemented with 10% FCS (Invitrogen) and 0.1%gentamicin (Invitrogen). All cell lines were authenticated by shorttandem repeat profiling.Mycoplasma test was performed monthlyby qPCR amplification of M. hyorhinis p37 and Hochest33258staining.

Antibodies for pT288-AURKA (#3079), CDC2 (#28439), PLK1(#4513), pY15-CDC2 (#4539), phospho-tyrosine (#9411), pS10-H3 (#53348), and ubiquitin (#3936) were purchased from CellSignaling Technology. Antibodies for AURKA (ab52973), cyclinA2 (ab181591), cyclin B1 (ab32053), cyclinD1 (ab16663), cyclinE1 (ab33911), FZR1 (ab3242), and TOP2A (ab52934) werepurchased from Abcam. Antibodies for APC1 (BS1611, Bio-world), CDC27 (610455, BD), phospho-Serine/Threonine(612548, BD), myc-tag (AB103, TianGen), and GAPDH(60004, Proteintech) were also purchased. Monoclonal antibodyagainst PRL-3 was generated and characterized previously (12).

Nocodazole and MG132 were obtained from Selleck. Cyclo-heximide was from Cell Signaling Technology. ProTAME wasfrom R&D Systems. Recombinant human PRL-3 protein wasobtained fromOrigene. Lipofectamine 3000was from Invitrogen.FITC-phalloidin was from Sigma-Aldrich.

Plasmids and RNA interferencePlasmids for wild-type PRL-3, PRL-M (C104S, phosphatase

activity deficient), and PRL-D (DCAAX-motif deleted) were pre-viously constructed and verified (27). pcDNA3.1 plasmids expres-sing myc-tagged FZR1 (wild-type and mutant) were provided bySangon. Plasmids and siRNA were transfected into cells withLipofectamine 3000. Lentiviral systems for overexpression andknockdown were provided by GenePharma and were infectedinto cells following the provider's instructions. Interferencesequences used were: control: 50-TTCTCCGAACGTGTCACGT-30; PRL-3: 50-GGTGGAGGTGAGCTACAAACA-30; AURKA-1: 50-GGTCTTGTGTCCTTCAAATTC-30; AURKA-2: 50-GCTACCA-GAGTCTACCTAATT-30; FZR1: 50-GCAACGAUGUGUCUCC-CUATT-30.

Live-cell imagingAfter transfection with GFP-PRL-3 or GFP for 36 hours, cells

were cultured in Microscope Slide Coverslip system (Nunk Lab-Tek, 5 � 103 cells/chamber). Selected fields were continuallyscanned every 5minutes for 24hourswithUltraVIEWVOXsystem(PerkinElmer) and analyzed with Volocity 6.1.1. For each group,70 GFP-positive cells were selected for analysis.

Animal experimentsCells were subcutaneously injected into the bilateral armpits of

5-week-old female BALB/c nude mice (purchased from HFK Bio-Technology) as a 150 mL suspension (5� 106 cells/mL). Changesof mice weight and xenograft volume were assessed every 3 days.After 21 days, mice were sacrificed and xenografts were strippedfor analysis.

Immunofluorescence stainingCells were precultured on coverslips (5 � 105/mL) 24 hours

before assay, then fixed by 4% paraformaldehyde, permeabilizedin 0.1% Triton X-100 for 5 minutes, and blocked by 5% goat

PRL-3 as an APC/CFZR1 Activator

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serum. Antigens were stained with primary and FITC- or TRITC-conjugated secondary antibodies. Nuclei were stained with DAPI(1 mg/mL). Alternatively, F-actin was stained with 1 mg/mL ofphalloidin-FITC. Images were acquired by the Zeiss LSM780 laserconfocal microscope (60 � oil, NA 1.40 Plan-ApoChromat,including two HyD detectors) at fixed exposure settings at roomtemperature.

In vitro phosphatase assayFZR1 protein was immunoprecipitated overnight at 4�C from

HCT116 cell lysates (1,000 mg) with 1 mg anti-FZR1 antibody plusprotein G-Sepharose. After washing 4 times with lysis buffer [50mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1% Triton X-100,10% glycerol, 2 mmol/L dithiothreitol, 1� protease cocktail(Roche)] and twice with 1� dephosphorylation buffer(50 mmol/L Tris-HCl pH 7.5, 0.1% NP-40, 150 mmol/L NaCl,2 mmol/L dithiothreitol, 1 mmol/L MgCl2, 0.1 mmol/L MnCl2),the precipitates (substrate)were resuspended in 20mL 1�dephos-phorylation buffer. For AURKA dephosphorylation, 10 mLHCT116 cell lysates (50 mg) were mixed with 10 mL of 2�dephosphorylation buffer and used as substrate. RecombinantPRL-3 (0.5 mg) was incubated with substrate at room temperaturefor 30 minutes. For control, EDTA (10 mmol/L) was added toinhibit phosphatase activity. The reaction was terminated byboiling in 2� loading buffer and the phosphorylated proteinwas detected by the pan-specific (for FZR1) or site-specific (forAURKA) phospho-antibodies.

Statistical analysis and formattingValues represented mean � SD of at least three independent

experiments with duplicate or triplicate samples. Differencesbetween scattered values in two groups were compared withStudent t test. Correlations between expression levels andclinical variables were measured with c2 or Fisher exact test.Prognostic significance was assessed with Kaplan–Meier sur-vival analysis. All statistics in this study was performed by SPSS21.0, and formatted by Graphpad Prism 5 or Excel. �, P < 0.05;��, P < 0.01; ��, P < 0.001. ns, no significance.

Additional methods can be found in the SupplementaryMaterial.

ResultsPRL-3 regulates distinct malignant phenotypes of colorectalcancer cells

To evaluate PRL-30s role in colorectal cancer progression, weperformed PRL-3 stable overexpression and knockdown incolorectal cancer cell lines (Fig. 1A). Consistent with the knownproinvasive capacity (4), cell migration and invasion wereincreased by overexpressed PRL-3 or decreased by PRL-3 knock-down (Fig. 1B). In addition, PRL-3 increased plate colonyformation (Fig. 1C). In soft-agar colony formation assay,PRL-3 displayed a potential to enhance anchorage-independentgrowth (Fig. 1D), emphasizing its role in maintaining self-renewal of colorectal cancer cells. PRL-3–increased clonogeni-city is in line with the study using colon tumors establishedfrom PRL-3 knockout mice (11) and further verifies its onco-genic property observed in different systems (4, 6, 10–12).Interestingly, both PRL-3 overexpression and knockdownresulted in decreased colorectal cancer cell proliferation(Fig. 1E), which was consistent with our previous observation(12). Annexin V/7-AAD staining showed that apoptotic rate was

decreased by PRL-3 overexpression or enhanced by PRL-3knockdown (Fig. 1F), supporting the antiapoptosis functionobserved in leukemia and breast cancer (6, 7).

According to flow cytometry, G2–M-phase was elevated afterPRL-3 overexpression or decreased after PRL-3 knockdown, whileS-phase was increased by PRL-3 knockdown or decreased by PRL-3 overexpression (Fig. 1G). These results were supported byanalyzing the markers for G1/early-S-phase (cyclin E1), S/G2-phase (cyclin A2), and G2–M-phase (pY15-CDC2; Fig. 1A), indi-cating the concomitant proliferation halt induced by PRL-3 over-expression or knockdown were respectively associated with dis-tinct cell-cycle changes. In addition, live-cell imaging showed thatmitotic length was significantly prolonged upon GFP-PRL-3expression in HCT116 (Fig. 1H) and HeLa cells (SupplementaryFig. S1), confirming PRL-3–induced G2–M-phase arrest. Aboveresults demonstrated that PRL-3 could regulate diverse malignantphenotypes of colorectal cancer cells.

PRL-3 is a negative regulator of AURKAAfter PRL-3 overexpression, cyclin B1/AURKA/pY15-CDC2 dis-

played an evident trend of mitotic arrest after being released fromnocodazole-induced G2–M synchronization (Fig. 2A), and thesechanges were opposite upon PRL-3 knockdown (Fig. 2B). Para-doxically, canonical G2–M-phase marker pS10-H3 was not upre-gulated upon G2–M arrest (Fig. 1A, 2A and B). We noted thatprotein levels ofmitotic kinaseAURKAwere negatively affected byPRL-3 (Fig. 1A). After being released from nocodazole-inducedsynchronization, theprofiles of AURKAandpS10-H3were similar(Fig. 2A and B), which was consistent with the role of AURKA inphosphorylating HistoneH3 (28). In addition, PRL-3 overexpres-sion-induced changes of pS10-H3 and pY15-CDC2 were recapit-ulated by AURKA knockdown (Fig. 2B). Hence, AURKAmay playa role in PRL-3–regulated cell-cycle progression.

PRL-3–AURKA cross-talk was then investigated. Serum starva-tion, nocodazole treatment, and subsequent release were per-formed and levels of PRL-3/AURKA were assayed by immuno-fluorescence staining. In HCT116 (Fig. 2C), SW480 and HT29cells (Supplementary Fig. S2A and S2B), interphase cells had thehighest PRL-3 and lowest AURKA, whereas mitotic cells had thelowest PRL-3 and highest AURKA. Both the protein and transcriptlevels of PRL-3 remained largely unchanged after siRNA-mediatedAURKA interference in HCT116 and SW480 cells (Fig. 2D). Con-versely, AURKA protein was dose-dependently decreased by PRL-3 overexpression (Fig. 2E) or enhanced by PRL-3 knockdown(Fig. 2F). Yet, transcript levels of AURKAwere unaffected by PRL-3(Fig. 2E and F), which was validated by comparing transcripts ofmultiple cell-cycle regulators (including AURKA) in large-samplecolorectal cancer datasets stratified with PRL-3 (Fig. 2G). PRL-30snegative effect on AURKA protein was also observed in primaryintestinal mucosa cell line CCC-HIE-2 (Fig. 2H). These resultsunderscored PRL-30s capacity to downregulate AURKA proteinexpression.

AURKA determines PRL-3–regulated phenotypes of colorectalcancer cells in vitro

To verify the contribution of AURKAdownregulation to PRL-3–regulated cell-cycle and other malignant phenotypes, we per-formed AURKA overexpression or knockdown in HCT116 cellswith PRL-3 overexpression or knockdown (Fig. 3A). We foundthat PRL-3 overexpression-induced G2–M-phase accumulationwas reversed by ectopic AURKA (Fig. 3B). Correlated with this

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result, PRL-3-decreased cell proliferation was also negated(Fig. 3C). However, ectopic AURKA failed to counteract PRL-3knockdown-induced S-phase accumulation (Fig. 3B) or prolifer-ation halt (Fig. 3C). Moreover, PRL-3 and AURKA exhibitedsimilar trends in opposing apoptosis upon their overexpressionor inducing apoptosis upon knockdown (Fig. 3D), confirming thestudies utilizing chemical inhibitor/RNA interference against PRL-3 (6, 7, 29) or AURKA (30, 31).

In soft-agar colony formation assay, the basal and PRL-3–promoted self-renewal was prevented either by AURKA overex-pression or by AURKA knockdown (Fig. 3E), implying thatAURKA is also a critical determinant of PRL-30s oncogenic func-tion. Upon GFP-PRL-3 overexpression, live-cell observationdetected increased ratio of multi-nucleated cells (SupplementaryFig. S3A),whichwas confirmedby F-actin/DAPI staining (Fig. 3F),

indicating that PRL-3 could promote cytokinesis failure andvalidating PRL-30s role in driving CIN (12). Consistent withAURKA's role in safeguarding the genomic integrity (21, 31,32), its knockdown increased CIN. Importantly, PRL-3 overex-pression-induced CIN was alleviated by ectopic AURKA or wasaggravated by AURKA knockdown (Fig. 3F), highlighting anessential role of AURKA downregulation in PRL-3–induced CIN.Both PRL-3 and AURKA regulate cancer cells motility (4, 29, 31),which was verified by migration and invasion assays (Supple-mentary Fig. S3B). Unlike the inhibitory effects of AURKAonPRL-3–induced G2–M arrest, proliferation halt, and CIN (Fig. 3B, C,and F), PRL-3-promoted invasiveness was increased by ectopicAURKA or decreased by AURKA knockdown (Supplementary Fig.S3B). Therefore, PRL-3 and AURKAmight interdependently drivecolorectal cancer cell motility.

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 stablyexpressing 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 ofHCT116-mCherry-H2B cells expressing GFP or GFP-PRL-3. Left, representative images. Red fluorescence, chromatin. Right, average length of mitosis (sum ofprophase, metaphase, anaphase, and telophase).






AURKA determines PRL-3–regulated malignancy andprognosis of colorectal cancer

With the same cells used for in vitro assays (Fig. 3A), we carriedout xenograft assay. Overexpression or knockdown of AURKAblocked PRL-3-promoted xenograft tumor growth, and reinforcedPRL-3 knockdown-induced xenograft growth inhibition (Fig. 4Aand B), which supported results of soft-agar colony formation

assay (Fig. 3E). In dissected xenograft tumors, expression profilesof PRL-3 and AURKA (Supplementary Fig. S4) were similar tothose in cultured cells (Fig. 3A). Immunohistochemical evalua-tion of these tumors showed that the labeling index of self-renewal marker CD133 was increased by PRL-3 overexpressionor decreased by PRL-3 knockdown, while both basal and PRL-3-promoted CD133were 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 orAURKA (B) after being released from nocodazole (NOC, 100 ng/mL, 12 h)-induced synchronization. C,AURKA and PRL-30s time-scaled distributions in HCT116were assessed by synchronization and released after serum starvation (SS) for 24 hours or NOC treatment for 12 hours. Arrows, mitotic cells. D, Protein (top) andmRNA (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/þþþ¼ 0.25/1/4 mg plasmid; E) or knockdown (F) colorectal cancer cells. G, Comparison of mRNA levels of indicatedcell-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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of AURKA (Fig. 4C), which were also consistent with results ofsoft-agar colony formation assay (Fig. 3E). In agreement with thein vitro apoptosis assay (Fig. 3D), similar anti-apoptosis functionsof PRL-3 and AURKA were confirmed by examining the labelingindex of apoptotic marker cleaved-caspase3 (Fig. 4D).

We further analyzed PRL-3 and AURKA expression in 267consecutive colorectal cancer tissue sections (Fig. 4E). Due to thelow positive rate of PRL-3 (18%), AURKA positivity in PRL-3negative and positive groups were 63% vs 58%, respectively(Fig. 4F). When only considering PRL-3 positive group, positiverates of AURKA significantly dropped from 62% to 45% as PRL-3expression levels elevated from medium (þþ) to high (þþþ)(Fig. 4F), suggesting an inverse correlation. 249 cases paired withclinical variables were used for further analysis. Indicators ofchromosomal mis-segregation and CIN, including anaphasebridge, unaligned chromosome, and multipolar mitosis, weremore frequently detected in PRL-3 positive tissues and signifi-cantly decreased by positive expression of AURKA (Fig. 4G),which confirmed the role of AURKA downregulation in PRL-3–induced CIN. PRL-3/AURKA's correlations with clinical indica-tions in this cohort were calculated (Supplementary TableS1-S3). Despite of insignificant log-rank P values, PRL-3 dis-played the potential as an adverse prognostic factor, whileAURKA was related with a favorable prognosis (Fig. 4H). Asshown by stratification, PRL-3-negative-AURKA-positive groupcorrelated with the most favorable prognosis and the bestclinical indications, but PRL-3-positive-AURKA-negative groupcorrelated with the worst (Fig. 4H; Supplementary Table S3),

further emphasizing the role of PRL-3–AURKA interplay inaffecting colorectal cancer progression.

PRL-3-promoted AURKA degradation is dependent onphosphatase activity and ubiquitin–proteasome system

PRL-3 possesses dual-specificity phosphatase activity and isalso subjected to prenylation on its carboxyl-terminal CAAXmotifto facilitate membrane targeting (4). By using catalytic inactive(PRL-M/PM) and CAAX motif-deleted (PRL-D/PD) mutants, wefound that PRL-M-induced AURKA inhibition was weaker thanthat of wild-type PRL-3, however PRL-D elicited comparablechanges as wild-type PRL-3 did (Fig. 5A), suggesting that phos-phatase activity of PRL-3, instead of its modification by prenyla-tion, was required for inhibiting AURKA. Moreover, PRL-M'scapability in inducing G2–M arrest, proliferation halt, plate col-ony formation, and soft-agar colony formation was significantlyabolished (Supplementary Fig. S5A–S5D), supporting the func-tional requirement of PRL-30s phosphatase activity in tumorigenicclustering. Furthermore, treatment with proteasome inhibitorMG132 counteracted PRL-3-induced AURKA downregulation(Fig. 5B) and stabilized AURKA to similar levels in control andPRL-3 knockdown cells (Fig. 5C). In cycloheximide-treated cells,half-life of AURKA was markedly decreased by PRL-3 overexpres-sion or prolonged by PRL-3 knockdown (Fig. 5D). Consistently,AURKA ubiquitination was enhanced by PRL-3 overexpression orreduced by PRL-3 knockdown (Fig. 5E). Importantly, wild-typePRL-3 displayed stronger activity in promoting AURKA ubiquiti-nation than PRL-M did (Fig. 5E). These results suggested that

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 HCT116cells. 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.






PRL-3 suppresses AURKA by enhancing ubiquitin/proteasomesystem-mediated proteolysis, and PRL-30s phosphatase activitywas required for this regulation.

In addition, phosphorylation of T288-AURKA, which is theauto-phosphorylation site critical for the kinase activity (21), wasalso negatively affected by PRL-3 and the trends of its alterationwere similar to those of total AURKA (Fig. 1A). To exclude theinfluence of AURKA's stability, an in vitro phosphatase assay wasperformed and no obvious effect of PRL-3 on pT288-AURKAlevels was detected (Fig. 5F); therefore, PRL-3-induced AURKAinhibition was unrelated to AURKA's kinase activity.

PRL-3 promotes AURKA proteolysis by dephosphorylatingFZR1

Although transcript levels of several cell-cycle-related factorswere mostly unaffected by PRL-3 in colorectal cancer datasets

(Fig. 2G), protein levels of several APC/CFZR1 substrates (13),including AURKA, cyclin A2, cyclin B1, cyclin D1, and PLK1, wereall decreased by PRL-3 overexpression or increased by PRL-3knockdown (Fig. 1A). In contrast, levels of TOP2A, anotherAPC/CFZR1 substrate (33), remained stable (Fig. 1A), suggestingthat PRL-3 may specifically exert inhibition on a subset of cell-cycle regulators. Besides PRL-3-AURKA colocalization (Fig. 2C;Supplementary Fig. S2A and S2B), PRL-3-FZR1 and AURKA-FZR1colocalizations were observed in interphase and mitotic colorec-tal cancer cells (Fig. 6A). Coimmunoprecipitation assay furtherrevealed the existence of AURKA–FZR1–PRL-3 complex (Fig. 6B).Importantly, FZR1 interference or APC/C inhibitor proTAMEdiminished PRL-3 overexpression-induced AURKA destabiliza-tion (Fig. 6C–D), while PRL-3–induced AURKA ubiquitinationwas overridden by FZR1 interference (Fig. 6E). Therefore, APC/CFZR1 mediates PRL-3–induced AURKA degradation.

Figure 4.

AURKA determines PRL-3–regulated malignancy and prognosis of colorectal cancer. A, Changes of mice body weight (top) and xenograft tumor volumesweights (bottom). B, Macroscopic recording of xenograft tumors and comparison of tumor weights. C and D, IHC staining of CD133 (C) and cleavedcaspase-3 (D) of indicated xenograft tumor sections. Labeling index (%) of positive cells of each section is shown. E, Representative IHC staining ofAURKA/PRL-3 in two pairs of consecutive colorectal cancer tissue samples. F, Summary of IHC staining of AURKA/PRL-3 in 267 pairs of consecutivecolorectal cancer tissue sections. N/P, negative/positive. G, Left, representative normal and aberrant mitosis in colorectal cancer sections stained withanti–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 onprognosis of 249 patients with colorectal cancer.

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Temporal regulation of phosphorylation/dephosphorylation isessential for timely assembly and activation of APC/CFZR1 (13–18). FZR10s association with APC/C core subunit CDC27 (APC3)was enhanced by PRL-3 (Fig. 6F), suggesting that PRL-3 promotesassembly of APC/CFZR1 complex. Although PRL-3 did not affectFZR10s protein levels (Fig. 1A, 6C and D) or stability (Fig. 5D),FZR10s phospho-serine/threonine and -tyrosine levels werereduced by PRL-3 overexpression or increased by PRL-3 knock-down, however PRL-M exerted no obvious effects (Fig. 6G).More-over, phospho-levels of immunoprecipitated FZR1were evidentlysuppressed by coincubation with recombinant PRL-3 proteinin vitro (Fig. 6H). Thus, PRL-3 is a phosphatase targeting FZR1.

The six phospho-serine/threonine sites (S32/S36/S40/T121/S151/S163) closely related with FZR10s co-factor function (13,14, 19) were mutated to construct myc-tagged FZR1-A and FZR1-D, which respectively mimicked the dephosphorylated/activatedand phosphorylated/inactivated FZR1. In control cells, FZR1-Aelicited the strongest AURKA destabilization and ubiquitination,while FZR1-D elicited theweakest (Fig. 6I). AURKAdestablizationand ubiquitination in PRL-3 overexpression cells were alleviatedby FZR1-D, meanwhile FZR1-A displayed highest inhibition onAURKA inPRL-3 knockdown cells (Fig. 6I and J). Changes of othertwoAPC/CFZR1 substrates, cyclin B1 and cyclinD1,were similar tothose of AURKA in all these settings (Fig. 6I). These resultsidentified PRL-3 as a phosphatase of FZR1 capable of facilitatingthe E3 ligase activity of APC/CFZR1 in ubiquitination and desta-bilization of AURKA (Fig. 6K).

FZR1 determines PRL-3–regulated malignancy and prognosisof colorectal cancer

Phenotypically, after expressionof ectopic FZR1 (Fig. 6I), PRL-3overexpression-induced G2–M-phase arrest was only rescued byFZR1-D (Fig. 7A). Although PRL-3 knockdown-increased S-phasefailed to be abrogated by AURKA overexpression or ablation(Fig. 3B), it was reversed by both wild-type and mutant FZR1(Fig. 7A), suggesting the involvement of APC/CFZR1 substratesother than AURKA in PRL-3-regulated S-phase. Consistent withFZR10s tumor-suppressive role (13, 18), FZR1 ablation exacer-bated PRL-3–promoted soft-agar colony formation (Fig. 7B).Conversely, PRL-3–promoted self-renewal was significantlyblocked by ectopic FZR1, but this activity was lost in the case ofFZR1-A (Fig. 7C).

By analyzing large-sample datasets (GSE40967/GSE41258)based on the mRNA levels of PRL-3/FZR1/AURKA, we foundthat AURKA- or FZR1-high groups were correlated with favor-able prognosis and better clinical indications (i.e., T/N/M/stage), while PRL-3 displayed an adverse prognostic trend andpoor indications (Supplementary Fig. S6A; SupplementaryTable S4-S9). Next, we performed stratification analysis. ForPRL-3/AURKA stratification, PRL-3-low-AURKA-high groupdisplayed better prognosis and clinical indications than PRL-3-high-AURKA-low group, while double-low/double-highgroups were in-between (Fig. 7D; Supplementary Tables S10and S11), confirming the results of immunohistochemicalanalysis of colorectal cancer samples (Fig. 4H; Supplementary

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 MG132treatment (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 HCT116lysates. R-PRL-3, recombinant PRL-3.






Table S3). Similarly, for PRL-3/FZR1 stratification, PRL-3-low-FZR1-high group displayed better prognosis and clinical indi-cations than PRL-3-high-FZR1-low group (Fig. 7D; Supplemen-tary Table S12 and S13). For AURKA/FZR1 stratification,double-high group had better prognosis and clinical indica-tions, while those of double-low group were inferior (Fig. 7D;Supplementary Table S14 and S15). For PRL-3/FZR1/AURKA-triple stratifications, PRL-3-low-AURKA-high-FZR1-high pre-dicted the best prognosis, but PRL-3-high-AURKA-low-FZR1-low group had the worst (Supplementary Fig. S6B). Important-ly, the prognostic diversities between PRL-3-low-AURKA-highand PRL-3-high-AURKA-low groups were larger in FZR1-highgroups than in FZR1-low groups, as marked by higher HRvalues (Fig. 7E), emphasizing that FZR1 was required tostrengthen the PRL-3–AURKA cross-talk and the subsequentoncogenic impacts on colorectal cancer.

DiscussionTumorigenesis is a dynamic and multistep process determined

by many factors. Acquisition of an optimal level of CIN mayconfer survival advantage for cancer cells, while drastic CINwouldbe deleterious (34, 35). As a master regulator of mitosis, AURKAprecisely coordinates mitotic chromosomal events and spindleformation to ensure accurate cell division (21). Both AURKAablation and chemical inhibition could generate CIN, validatingthe pivotal role of AURKA in maintaining genomic integrity (21,31, 32). Consistent with this concept, tumor-suppressive role ofAURKA has been demonstrated in AURKAþ/�mice andDrosoph-ila expressing loss-of-functionmutant AURKA (32, 36). Previous-ly, we found that PRL-3 could dissociate shelterin componentsRAP1 and TRF2 from telomeric DNA, thereby eliciting telomeredysfunction and CIN (12). Here, we uncovered PRL-3-promoted

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 immunoprecipitatedwith 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 performedin 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-3knockdown. H, In vitro dephosphorylation of immunoprecipitated FZR1 by recombinant PRL-3. I, Effects of FZR1 (wt), FZR1-A, and FZR1-D on PRL-3–inducedchanges 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–promotedAURKA destabilization through FZR1 dephosphorylation and APC/CFZR1complex formation.

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AURKA destabilization, which may represent a novel mechanismof PRL-3-induced CIN. Considering that telomere dysfunction-induced fusion-breakage-bridge cycles require intimate coordi-nation with spindle machinery to generate numerical/structuralabnormalities of chromosomes (37), and thatmitotic disturbancemay jeopardize telomere homeostasis (38, 39), it is necessary toevaluate the functional linkage between telomere dysfunctionand AURKA destabilization in PRL-3-induced CIN. Notably,despite PRL-3-induced CIN was neutralized by ectopic AURKAor phenocopied by AURKA knockdown, apoptosis was onlyinduced byAURKAknockdown, but not by PRL-3 overexpression.Thus, PRL-3 has a capability to resist loss of AURKA-inducedapoptosis, thereby maintaining high level of CIN.

Several factors, for example, PI3K-AKT, p53-p21, FOXO3a,were implicated in PRL-3-regulated cell-cycle (9), while the pre-cise mechanism has been amystery. Moreover, PRL-30s impact oncell-cycle progression seemed to be cell type-dependent (9).Herein we found that PRL-3-promoted G2–M-phase accumula-tion in colorectal cancer cells was overturned by ectopic AURKA.

However, PRL-3 knockdown-induced S-phase arrest was unaf-fected by AURKA expression or knockdown, but was abrogated byectopic FZR1. PRL-3 could dephosphorylate FZR1 and enhanceAPC/CFZR1 assembly, implying that PRL-3may contribute toAPC/CFZR1 activation, as functioned by other two known phospha-tases, i.e. PP2AB55 and CDC14 (16, 17). FZR1 ablation alsoinduced S-phase accumulation (17, 18); thus, PRL-3 knockdowncould partially phenocopy FZR1 inactivation in S-phase control.Contrary to loss of FZR1-induced CIN (18), PRL-3 knockdowndid not provoke CIN (12), as proven by the present study, so PRL-3-dependent APC/CFZR1 activation might be restricted to limitedcircumstances. It is possible that PRL-3 differentially fine-tunesthe activity of APC/CFZR1 to regulate the stabilities of mitoticproteins and DNA replication proteins, which requires furtherexploration.

It is seemingly counterintuitive that PRL-3 overexpressioninhibited colorectal cancer cell proliferation. Uncontrolled cellproliferation is one of the hallmarks of cancer and has beenextensively studied as a surrogate to evaluate the phenotypes of

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-cycleprofiles. 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-agarcolony 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 cancerprogression through its effects on different phenotypes.






cancer cells and predict the course of cancer progression. Forinstance, the positive correlation between proliferation andaggressiveness has been found in breast cancer (40) and lym-phoma (41). But independent studies found that colorectalcancer seems to be an exception, because both the metastaticcolorectal cancer in liver and the primary colorectal cancer withthe ability to metastasis had reduced proliferation index (42,43). Several mechanisms were proposed to explain the acqui-sition of low cellular proliferation during the course of colo-rectal cancer progression from primary lesion to metastatic foci(42). These mechanisms include: (i) CIN, as CIN generates aproliferative disadvantage in both yeast and mammalian cells(44); (ii) stemness, because cancer stem cells are always slow-cycling (45, 46); (iii) EMT, as cancer cells in the process of EMTare less proliferative (46). It is of note that PRL-3 was initiallyfound to be highly expressed in metastatic colorectal cancer,but not in nonmetastatic primary colorectal cancer lesions ornoncolorectal cancer metastatic lesions (3, 4). The uncouplingof proliferation and motility/colony formation/anchorage-independent growth/xenograft growth upon PRL-3 overexpres-sion in colorectal cancer cells, as shown in this study, plus itsability to promote CIN (ref. 12 and this study), stemness (11),and EMT (8), suggest that PRL-3 could be responsible for thelow proliferation during colorectal cancer progression. Thisparticular phenotype of colorectal cancer raised a serious issuethat targeting fast-cycling cells may not be the reasonableapproach for the treatment of colorectal cancer at advancedstage, while this would be overcame by strategies aiming atPRL-3 or PRL-3–FZR1–AURKA pathway.

On the basis of the results of the current study, PRL-3–regulated colorectal cancer progression would be determinedby six aspects of mechanisms: (i) CIN; (ii) cell-cycle; (iii)proliferation; (iv) stemness; (v) cell death; and (vi) motility.Overexpressed PRL-3 promoted CIN, self-renewal, and motil-ity; meanwhile, it inhibited proliferation, induced G2–M arrest,and decreased apoptosis. The slow proliferation was eventuallysurpassed by enhanced CIN, self-renewal ability, motility, andlow apoptosis, resulting in colorectal cancer progression.Among these phenotypes, effects of PRL-3 on CIN, G2–M arrest,and proliferation were counteracted by ectopic AURKA, ormimicked by AURKA knockdown, which underscored thefunctional significance of PRL-3–induced AURKA destabiliza-tion. In this sense, these three phenotypes may represent thesecondary effects following AURKA destabilization (Fig. 7F).Conversely, in the setting of PRL-3 deficiency, decreased pro-liferation, S-phase arrest, impaired self-renewal, increased apo-ptosis and diminished motility would collectively restrict colo-rectal cancer progression (Fig. 7F). Despite of PRL-3 ablation-enhanced AURKA expression, concomitant AURKA knockdownfailed to reverse these phenotypic changes, thus the effects ofPRL-3 loss on colorectal cancer progression might be largelyAURKA independent.

Furthermore, the interplays among individual mechanismscould not be excluded. As shown by previous studies, CIN couldimpair cell proliferation (44); meanwhile, CIN conferred growthadvantage of stem cells through enhanced genetic diversity (47).

Recently, CIN was also demonstrated to drive metastasis (48).However, without further evidence, it is still premature to con-clude that low proliferation, increased self-renewal ability andmotility might be the further effects, that is, tertiary effects,following PRL-3–promoted CIN.

The opposing functions of AURKA and PRL-3 were furthersupported by prognosis analysis of colorectal cancer patientspecimens and GEO datasets, as low-PRL-3 and high-AURKApredicted better survival. At present, the prognostic value of FZR1in cancer was poorly reported. We revealed that PRL-3-AURKA-determined colorectal cancer prognosis was affected by the pres-ence of FZR1, thus confirming an essential role of FZR1 inmediating PRL-3-AURKA cross-talk. These findings indicated thatbetter understandings of the PRL-3–FZR1–AURKA interplay aredemanded to expand current cognition of colorectal cancer devel-opment and progression.

In summary, we found a critical role of PRL-3 in downregulat-ing AURKA, through which to influence diverse malignant phe-notypes of colorectal cancer cells as well as the prognosis ofpatients with colorectal cancer. We further identified PRL-3 as aphosphatase of FZR1 capable of facilitating the activity of APC/CFZR1 in destabilizing AURKA. These results expand currentknowledge regarding the contributions of PRL-3, AURKA, andFZR1 to colorectal cancer progression.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Zhang, L. Qu, L. Shen, C. ShouDevelopment of methodology: C. Zhang, L. Qu, S. LianAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Zhang, S. Lian, L. Meng, L. Min, J. Liu, Q. SongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Zhang, L. Qu, L. Min, C. ShouWriting, review, and/or revision of the manuscript: C. Zhang, L. Qu, L. Min,L. Shen, C. ShouAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Zhang, L. Meng, J. Liu, L. ShenStudy supervision: C. Zhang, L. Qu, L. Shen, C. Shou

AcknowledgmentsWe deeply appreciated Drs. Xuemin Zhang (National Center of Biomedical

Analysis, China) and Qinghua Shi (University of Science & Technology ofChina) for sharing critical reagents, Drs. Bin Dong, Caiyun Liu, Chuanke Zhao,and Jing Gao (Peking University Cancer Hospital & Institute) for generous helpin experimental suggestions and IHC staining and evaluation. This work wassupported by the National Basic Research Program of China (no.2015CB553906 to C. Shou), the National Natural Science Foundation of China(no. 81230046 to C. Shou; no. 81672732 to L. Qu), and China PostdoctoralScience Foundation (2018M631281 to C. Zhang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 5, 2018; revised August 8, 2018; accepted November 21,2018; published first November 29, 2018.

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2019;79:928-940. Published OnlineFirst November 29, 2018.Cancer Res Cheng Zhang, Like Qu, Shenyi Lian, et al. Colorectal Cancer Progression via Dephosphorylation of FZR1PRL-3 Promotes Ubiquitination and Degradation of AURKA and

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