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Companion Diagnostics and Cancer Biomarkers

Circadian Clock Gene CRY2 Degradation IsInvolved in Chemoresistance of Colorectal CancerLekun Fang1,2, Zihuan Yang1, Junyi Zhou1, Jung-Yu Tung3, Chwan-Deng Hsiao3,Lei Wang1, Yanhong Deng4, Puning Wang1, Jianping Wang1, and Mong-Hong Lee2

Abstract

Biomarkers for predicting chemotherapy response are impor-tant to the treatment of colorectal cancer patients. Cryptochrome2 (CRY2) is a circadian clock protein involved in cell cycle, but thebiologic consequences of this activity in cancer are poorly under-stood.We set up biochemical and cell biology analyses to analyzeCRY2 expression and chemoresistance. Here, we report that CRY2is overexpressed in chemoresistant colorectal cancer samples, andCRY2 overexpression is correlated with poor patient survival.Knockdown of CRY2 increased colorectal cancer sensitivity tooxaliplatin in colorectal cancer cells. We also identify FBXW7 as anovel E3 ubiquitin ligase for targeting CRY2 through proteasomaldegradation. Mechanistic studies show that CRY2 is regulated by

FBXW7, in which FBXW7 binds directly to phosphorylatedThr300 of CRY2. Furthermore, FBXW7 expression leads to deg-radation of CRY2 through enhancing CRY2 ubiquitination andaccelerating the CRY2's turnover rate. High FBXW7 expressiondownregulates CRY2 and increases colorectal cancer cells' sensi-tivity to chemotherapy. Low FBXW7 expression is correlated withhigh CRY2 expression in colorectal cancer patient samples. Also,low FBXW7 expression is correlated with poor patient survival.Taken together, our findings indicate that the upregulation ofCRY2 caused by downregulation of FBXW7 may be a novelprognostic biomarker andmay represent a new therapeutic targetin colorectal cancer. Mol Cancer Ther; 14(6); 1476–87. �2015 AACR.

IntroductionColorectal cancer is one of the leading causes of cancer mor-

tality. 5-Fluorouracil (5-FU)–based chemotherapy is routinelyused to treat those patients who are at high risk of developingrecurrence or those with advanced or metastatic disease. As aconsequence of early diagnosis, prevention, and adjuvant che-motherapy, death rate of colorectal cancer has decreased by 40%in the past four decades (1). However, a significant proportion ofpatients receiving chemotherapy become chemoresistant (2).Therefore, understanding the mechanisms underlying chemore-sistance can help us to identify a subgroup of patients who maybenefit from chemotherapy and avoid overtreatment. Studieshave shown that multiple cellular processes, including DNArepair, cell apoptosis, and proliferation, may play important rolein chemoresistance (3–6). Several clinical studies have been

performed in an attempt to find biomarkers that predict thebenefit from chemotherapy. For example, BRAFV660Emutationsconfer poor prognosis, but limited data suggest lack of antit-umor activity from anti-EGFR monoclonal antibodies in thepresence of a BRAF mutation status (7–9). However, exceptKRAS/NRAS-activating mutations on exon2 and other RASmutations (10–12), only few of the studied markers have animpact on the clinical management of colorectal cancer so far.There is an urgent need for discovering better markers that canenhance the prognostic strength.

Circadian clock is an endogenous biochemical mechanismshared by most organisms, which can influence nearly all aspectsof physiology andbehavior.Deregulationof circadian rhythmhasbeen found to acceleratemalignant growth and increase the risk ofcertain kinds of cancer, such as breast cancer, prostate cancer, andcolorectal cancer, implying the involvement of the circadian clockin cancer development and tumor progression (13–19). Circadi-an genes are also related to the clinical outcome of cancer patients(14, 18). Several studies have shown that anticancer drugs weremore efficacious at a certain circadian time, indicating that chro-notherapy, a circadian-based chemotherapy, may be consideredwith patient treatment (20–23). It has been suggested that pre-vention of chemotherapy-induced circadian disruption mightreduce toxicity and improve efficacy in cancer patients (22).Cryptochrome 2 (CRY2), one of the identified circadian clockproteins, has been shown to be involved in DNA-damage check-point control and regulating important cell-cycle progressiongenes (24, 25). In addition, CRY mutation can increase the cellsensitivity to apoptosis induced by genotoxic agents and alsoprotect p53-mutantmice from the early onset of cancer (26). Also,breast cancer cells with reduced CRY2 have accumulated greatermutagen-induced DNA damage (15, 26, 27). These studies indi-cated that CRY2 might correlate with DNA damage and chemo-therapeutic outcome of patients. Understanding these molecularalterations can help improve clinical care.

1DepartmentofSurgery,GuangdongKeyLaboratoryofColorectal andPelvic Floor Disease, The Sixth Affiliated Hospital of Sun Yat-SenUniversity, Guangzhou, China. 2Department of Molecular and CellularOncology,The University of Texas MDAnderson Cancer Center, Hous-ton, Texas. 3Institute of Molecular Biology, Academia Sinica, Taipei,Taiwan. 4Department of Oncology, Guangdong GastroenterologyInstitute, The Sixth Affiliated Hospital of Sun Yat-Sen University,Guangzhou, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

L. Fang and Z. Yang contributed equally to this article.

Corresponding Authors: Jianping Wang, Department of Surgery, GuangdongGastroenterology Institute, The Sixth Affiliated Hospital of Sun Yat-sen Univer-sity, Guangzhou 510655, China, E-mail: [email protected]; and Mong-HongLee, Department of Molecular and Cellular Oncology, Unit 79, The University ofTexas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030;Phone: 713-794-1323; Fax: 713-792-6059; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0030

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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on May 14, 2020. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 8, 2015; DOI: 10.1158/1535-7163.MCT-15-0030

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FBXW7 (F-box and WD repeat domain-containing 7 or FBW7)is a component of conserved SCF (complex of SKP1, CUL1, andF-box protein)-type ubiquitin ligase (28, 29). SCFFBXW7 is knownto degrade several proto-oncogenes that function in cellulargrowth and division pathways, including cyclin E (30), AuroraB (31), Aurora A (32), MYC (33, 34), JUN (35, 36), and Notch(37). Loss-of-function of tumor-suppressor FBXW7 increasedprosurvival protein MCL1 levels and conferred resistance toTaxol-induced cell death, which indicated that FBXW7 is alsoinvolved in drug resistance (38). Interestingly, the ubiquitin-mediated protein degradation pathway plays an essential role inmaintaining normal circadian clock function (39, 40). It is notclear whether FBXW7 and CRY2 are involved in chemoresistance.

In this study, we found that CRY2 was overexpressed in che-moresistant colorectal cancer patient samples. We have shownthat FBXW7 negatively regulated CRY2 via the ubiquitinationpathway and sensitized colorectal cancer cells to chemotherapy.Low FBWX7 expression was correlated with CRY2 overexpressionin colorectal cancer patient samples. Our studies provide impor-tant insight into the signal deregulation of the FBXW7–CRY2axis in the chemosensitivity of colon cancer and help elucidateCRY2's role as a therapeutic intervention target in colorectalcancer treatment.

Materials and MethodsHuman tissue

First, 16 patients with stage III primary rectal cancer whoconsecutively underwent neoadjuvant chemotherapy (fluoroura-cil–leucovorin–oxaliplatin) were randomly selected from the Bio-bank of the Department of Colorectal Surgery, the Sixth AffiliatedHospital of Sun Yat-Sen University (Guangzhou, China). Tumorspecimens were obtained by colonoscopy prior to neoadjuvanttherapy. The effect of chemotherapy on the tumor was assessedas the three-dimensional volume reduction rate or tumor re-sponse rate. The tumor response was evaluated by the ResponseEvaluation Criteria in Solid Tumors (RECIST), which is definedas the following: complete response (CR; disappearance of thedisease), partial response (PR; reduction of�30%), stable disease(SD; reduction <30% or enlargement �20%), or progressivedisease (PD; enlargement �20%). Of these, 8 patients weredefined as CR/PR and others were defined as SD/PD. Paraffin-embedded samples of primary colorectal adenocarcinomas wereincluded from 289 patients from the First Affiliated Hospitalof Sun Yat-Sen University. Our study protocol was approved bythe ethics committee. Overall survival was the endpoint of thisstudy. Survival time was calculated from the date of surgery tothe date of death or the last follow-up time. Written informedconsent from all patients regarding tissue sampling was obtained.

TMA construction and immunohistochemistryTissue microarrays were constructed; in brief, paraffin-embed-

ded tissue blocks were overlaid with corresponding slides ofhematoxylin and eosin–stained tissue sections for tissue micro-array sampling. Duplicate 0.6-mm-diameter cylinders werepunched from representative tumor areas of an individual donortissue block and reembedded into a recipient paraffin block atpredefined positions using a tissue-arraying instrument (BeecherInstruments). Immunohistochemical (IHC) staining was per-formed on 5-mm tissue sections. The sections were placed onpoly-lysine–coated slides, deparaffinized in xylene, and then

rehydrated using graded ethanol. The slides were then placedin 3% hydrogen peroxide to quench for endogenous peroxidaseand then processed for antigen retrieval by microwave heatingfor 10minutes in 10mmol/L citrate buffer (pH 6.0). The primaryantibody against CRY2 (1:1,000; Abcam) or FBXW7 (1:1,000;Invitrogen) was diluted in phosphate-buffered saline (PBS) con-taining 1% bovine serum albumin and incubated at 4�C over-night. The next day, immunostaining was performed using theInvitrogen SuperPicture 3rd Gen IHC Detection Kit, whichresulted in a brown precipitate at the antigen site. Then, the slideswere counterstained with hematoxylin (Zymed Laboratories),mounted in nonaqueous mounting medium, and coverslipped.The original IHC slides were scanned with the Advanced CCDImaging Spectrometer that captured digital images of the immu-nostained slides. The ACIS calculates a score for regions selectedby the pathologist. The receiver operating characteristic (ROC)curve was used to define the cutoff point.

Data miningThe CRY2 gene mRNA expressions in colon tissue were obtain-

ed by the Gene Expression Omnibus. Cohort: GSE-14333 con-sisting of 223 colon cancer patients.

DNA constructs and reagentsPCR-amplified human CRY2 was cloned into PCMV5 or

PDEST15. CRY2 T300A mutant was made by the Herculase IIFusion Enzyme Kit (Agilent Technologies). siRNA oligos weredesigned and manufactured by Ruibo, targeting CRY2, sense:50-GAACGAAUGAAGCAGAUUU. Flag-FBXW7 has been previ-ously described (31). Cycloheximide and MG132 were obtainedfrom Sigma. Ni-NTA Agarose was obtained from Invitrogen(#R901-15). Antibodies used were: Flag (M2 monoclonal anti-body; Sigma; F3165), CRY2 (Abcam), and myc (Santa CruzBiotechnology; sc-40).

Cell culture and transfectionThe colon cancer cell lines DLD-1 and SW480 were purchased

from the ATCC (authenticated by short-tandem repeat analysis).Cells were maintained in DMEM/F12 (Gibco), supplementedwith 10% (v/v) fetal calf serum (FCS). HCT116 FBXW7þ/þ andHCT116 FBXW7�/� (a kind gift from Dr. Bert Vogelstein, TheJohns Hopkins Medical Institutions, Baltimore, MD; authenti-cated with Western blot analysis routinely) were cultured inMcCoy's 5A media (Hyclone) supplemented with 10% (v/v)FCS. All cells were incubated in a humidified atmosphere of 5%CO2 at 37�C. Plasmids and CRY2 siRNA were transfected usingLipofectamine 2000 (Invitrogen) according to the manufac-turer's instructions. Cells were harvested 48 hours after trans-fection to assay for transfection efficiency by qPCR.

RNA isolation and quantitationRNA samples were isolated from harvested cells using TRizol

reagent (Invitrogen) according to themanufacturer's instructions.A total of 1 mg of RNA from each sample was reversed transcrib-ed using oligo-dT primers and the ReverAid First Strand cDNASynthesis Kit (Fermentas, EU). Quantitative real-time PCR wasperformed using the Thunderbird SYBR qPCR Mix (TOYOBO)in the Applied Biosystems 7500 Real-Time PCR system (ABI).The primers used for CRY2 amplification were: forward: GTCC-TGCAGTGCTTTCTTCC; reverse: CCACACAGGAAGGGACAGAT.mRNA quantity was normalized using GAPDH content, and

FBXW7 and CRY2 in Chemoresistance of Colorectal Cancer

www.aacrjournals.org Mol Cancer Ther; 14(6) June 2015 1477




fold change of expression was calculated according to the DDCT

method.

Cell proliferation assayCell proliferation was determined using the MTT assay as

previously described (41). Briefly, 5 � 103 cells per well wereplated into 96-well plates, incubated at 37�C, 5% CO2 overnight.After transfection, cells were treated with different concentrationsof fluorouracil or oxaliplatin (Sigma) for 72 hours. Cells wereincubated in medium containing 0.1 mg/mL MTT (Sigma) for anadditional 4 hours. The cells were then lysed in 150-mL DMSO(Sigma). Cell viability was determined by measuring the absor-bance at 550 nm using a 680 BioRad Microplate AbsorbanceReader (Bio-Rad).

Annexin V/PI stainingThe cell was seeded on 96-well plates at a density of 105cells per

well and cultured overnight. Cells were transfected with 100nmol/L negative control or CRY2 siRNAs. After 48 hours oftreatment with oxaliplatin, cells were collected and washed twicewith PBS by spinning at 1,000 rpm for 10 minutes. Cell pelletswere resuspended in a FITC-labeled Annexin V and propidiumiodide (PI) staining solution (BD Bioscience) and incubated for15minutes at room temperature. The sampleswere then analyzedon a FACSCalibur (FACSCanton II; BD).

TUNEL assayApoptosis was determined by the terminal deoxynucleotidyl

transferase–mediated dUTP nick end labeling (TUNEL) methodusing the TMR Red kit (Roche) according to the manufacturer'sprotocol. Briefly, cells were rinsed with PBS, fixed in 4% parafor-maldehyde, and permeabilized with 0.1% Triton X-100. Cellswere then incubated in TUNEL reaction mixture at 37�C for60 minutes, protected from light. After three rinses with PBS, thesamples were analyzed under a fluorescence microscope (LeicaDMI4000B) using an excitation wavelength of 540 nm andemission wavelength of 580 nm. The number of cells was deter-mined by DAPI staining. At least 200 cells were counted for eachexperiment.

Immunoprecipitation and immunoblottingTotal cell lysates were processed as described previously

(42–44). Cell lysates for Western blot analysis or immunopre-cipitationwere collected from tissue culture dishes after two rinseswith cold PBS. Cells were centrifuged at low speed for 10minutesand supernatants were discarded. Pellets were then lysed with200-mL 1� lysis buffer [0.5 L batch:7.5-g 1mol/L Tris (Fisher), 15-mL 5 mol/L NaCl (Fisher), 0.5-mL NP-40 (USB Corp.), 0.5-mLTriton X-100 (Sigma), and 1-mL 0.5 mol/L EDTA (Fisher)] for 20minutes at 4�C. Lysis buffer also contained a cocktail of protease/phosphatase inhibitors: 5mmol/L NaV, 1mmol/L NaF, 1 mmol/Ldithiothreitol (DTT), 0.1 mg/mL Pepstatin A, 1 mmol/L phenyl-methylsulfonylfluoride (PMSF), and 1,000� Complete CocktailProtease Inhibitor (Roche). Lysates were immunoblotted withindicated antibodies. For immunoprecipitations, cell lysates wereprepared and standardized as before, and 1-mg protein wasimmunoprecipitated with appropriately diluted antibody in lysisbuffer overnight. Antibody was pulled down with 50 mL of eitherProtein A or G beads (Santa Cruz Biotechnology) for 1 hour.Beads were centrifuged at low speed for 10 minutes, and super-natants were discarded. Dried beads were mixed with 2� loading

dye and boiled for 5 minutes. Lysate samples were loaded ontogels, and SDS-PAGE was performed as before.

In vitro binding assayFlag-FBXW7 andmyc-CRY2 were prepared by in vitro transcrip-

tion and translation using the TNT coupled system (Promega).TNT products were mixed and immunoprecipitated followed byimmunoblotting as described in the legend to Figure 3.

Molecular docking of CRY2–FBXW7 complex structureThe information-driven docking program HADDOCK (version

2.0; ref. 45) was used to generate the Cry2–FBXW7 complexmodel. The starting structures for docking were an X-ray crystalstructure of FBXW7 (PDBID: 2ovp; ref. 30) and the homologymodel of Cry2. The Cry2 model was constructed using the crystalstructure of Cryptochrome-2 from Mus musculus (PDBID: 4i6g)as the template model, and the modeled residues were fromresidues 22 to 511. On the basis of the Skp1–FBXW7–CyclinE peptide complex structure (PDB: 2ovp), specific degron motif(TPXXS) binding to narrow face of the WD40 domain ofFBXW7 was used to generate this complex model. The WD40domain of FBXW7 structure contained sets that included the activeresidues W425, T439, S462, T463, R465, R479, Y519, Y545, andS585, and the neighbors of these active residues were selected aspassive residues. The Cry2 model contained sets that included theactive residues from R275 to S304, and the neighbors of theseactive residueswere selected as passive residues. The active residues

Table 1. Correlation between expression of CRY2 and clinicopathologic featuresin 289 case of colorectal cancer

CRY2All cases Low expression Overexpression P

Gender 0.813a

Male 147 (50.9) 85 (51.5) 62 (50.0)Female 142 (49.1) 80 (48.5) 62 (50.0)

Ageb 0.073a

<59 130 (45.0) 82 (49.7) 48 (38.7)�59 159 (55.0) 83 (50.3) 76 (61.3)

Histologic grade 0.233a

G1 20 (6.9) 12 (7.3) 8 (6.5)G2 230 (79.6) 126 (76.4) 104 (83.9)G3 39 (13.5) 27 (16.4) 12 (9.7)

pT status 0.111a

T1 7 (2.4) 7 (4.2) 0 (0.0)T2 39 (13.5) 24 (14.5) 15 (12.1)T3 237 (82.0) 131 (79.4) 106 (85.5)T4 6 (2.1) 3 (1.8) 3 (2.4)

pN status 0.226a

N0 173 (59.9) 104 (63.0) 69 (55.6)N1 116 (40.1) 61 (37.0) 55 (44.4)

pM status 0.246a

M0 259 (89.6) 151 (91.5) 108 (87.1)M1 30 (10.4) 14 (8.5) 16 (12.9)

Clinical stage 0.430a

I 30 (10.4) 20 (12.1) 10 (8.1)II 124 (42.9) 73 (44.2) 51 (41.1)III 105 (36.3) 58 (35.2) 47 (37.9)IV 30 (10.4) 14 (8.5) 16 (12.9)

Chemotherapy 0.221a

No 211 (73.0) 136 (75.6) 75 (68.8)Yes 78 (27.0) 44 (24.4) 34 (31.2)

aP values were calculated in SPSS16.0 using a chi-square test. P values <0.05were considered to indicate statistical significance.bMedian age.

Fang et al.

Mol Cancer Ther; 14(6) June 2015 Molecular Cancer Therapeutics1478




were chosen on the basis of the Skp1–FBXW7–Cyclin E peptidecomplex structure (PDB: 2ovp). During the rigid body energyminimization, 10,000 structures were calculated, and the 162 bestsolutions based on the intermolecular energy were used for thesemiflexible, simulated annealing. The best 162 docked modelswere clustered a cutoff of 3.5Å with a minimum of four structuresin each cluster, which yielded seven clusters. In terms of HAD-DOCK score and the basis of the FBXW7–CycE peptides complexbinding motif, the best structure was selected as a final model ofCry2–FBXW7 complex structure.

Turnover assayThe cells were transfected with indicated plasmids and incu-

bated in the 37�C with 5% (v/v) CO2 for 24 hours. Turnover ratewas analyzed as previously described (46–48). Then, cyclohexi-mide was added into the media at 60 mg/mL of final concentra-tion. The cells were harvested at the indicated time points aftercycloheximide treatment. The protein levels were analyzed byimmunoblotting.

Ubiquitination assayUbiquitination was processed as previously described

(34, 43). DLD-1 cells were cotransfected with indicated plas-mids. At 24 hours after transfection, cells were treated with50 mg/mL of MG132 for 6 hours. The ubiquitinated proteinswere pulled down by the Ni-NTA. The protein complexes werethen resolved by SDS-PAGE and probed with indicated anti-body to observe the ubiquitinated level.

Statistical analysisStatistical analyses were performed using SPSS (standard

version 16.0; SPSS). The correlation between CRY2 expressionand clinicopathologic features of colorectal cancer patients wasanalyzed by the x2 test or the Fisher exact test. For univariatesurvival analysis, survival curves were obtained by the Kaplan–Meier method, and differences between survival curves wereassessed with the log-rank test. The Cox proportional hazardsregression model was used to identify the independent prog-nostic factors. All in vitro experiments were performed intriplicate. The results are described as mean � SD. Statisticalanalysis was performed by the one-way analysis of variance(ANOVA), and comparisons among groups were performed bythe independent-sample t test. Results were considered signif-icant at a P value less than 0.05.

ResultsCRY2 expression is correlated with chemoresistance and pooroutcome in colorectal cancer patients

To investigate whether CRY2 expression relates to chemosen-sitivity in colorectal cancer patients, we first compared the expres-sion of CRY2 levels in colorectal cancer colonoscopy samplesfrom 16 rectal cancer patients who subsequently underwentneoadjuvant chemotherapy. IHC revealed that the level of CRY2expression was higher in cancer tissue samples from stable/pro-gressive patients than in samples from complete/partial responsepatients (Fig. 1A). Statistical analysis revealed that IHC scorebetween two groupswas significantly different (P¼ 0.001; Fig. 1B).

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P = 0.015

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Figure 1.Chemoresistant colorectal cancersamples express high level of CRY2.A, representative IHC staining forCRY2 in chemosensitive andchemoresistant colorectal cancersamples from colonoscopy. Inchemoresistant patient sections,strong CRY2 signals were detected. Inchemosensitive patient sections,weak staining was detected. Top,�100; bottom, �400. Scale bars,100 mm (top), 50 mm (bottom). B,statistical analysis revealed that IHCscore between two groups wassignificantly different (P ¼ 0.001).C, the Kaplan–Meier survival curvesof overall survival duration based onCRY2 expression in the colorectalcancer tissues. The ROC curve wasused to define the cutoff, andlog-rank analysis was used to testfor significance.






DLD-1 SW480

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Fang et al.





To determine the clinical relevance, we subjected a tissuemicroarray containing 289 human colorectal cancer specimensto IHC staining for CRY2. The clinicopathologic characteristicsof the colorectal cancer patients are summarized in Table 1.Among the colorectal cancer patients, 9 of 30 metastatic colo-rectal cancer patients (30%) received the oxaliplatin-basedchemotherapy, and 69 of 259 patients (26.7%) without metas-tasis received the chemotherapy. To assess statistical signifi-cance, the ROC curve was plotted to determine cutoff scoresfor CRY2 expression. We divided the colorectal cancer patientsinto high and low CRY2 expression groups according tocutoff scores. In the cohort, high expression of CRY2 was foundin 124 of 289 (42.9%) of colorectal cancer patients. There wasno significant association between CRY2 expression and clin-icopathologic features, such as patient gender, age, T classifi-cation, N classification, distant metastasis, and clinical stage(Table 1). The expressions of CRY2 as well as other clinico-pathologic factors were further examined by the multivariateCox regression analysis. Results indicated that histologicgrade, N stage, M stage, and CRY2 overexpression were inde-pendent prognostic factors for colorectal cancer patients (Sup-plementary Table S1). Importantly, the Kaplan–Meier analysisshowed that high expression of CRY2 was correlated with poorsurvival, especially in colorectal cancer patients who receivedadjuvant chemotherapy (Fig. 1C). Therefore, CRY2 can beidentified as a biomarker that can predict outcomes of colo-rectal cancer patients.

CRY2 knockdown leads to chemosensitivity of colorectalcancer cell lines

To examine further whether CRY2 expression has an impact onchemoresistance of colon cancer cells in vitro, CRY2 was down-regulatedby specific siRNA in two colorectal cancer cell linesDLD-1 and SW480. We found that DLD-1 and SW480 cells withreduced CRY2 were more sensitive to oxaliplatin compared withcontrol cells (Fig. 2A). Because CRY2 was involved in DNAdamage–specific control of cell death, we then examinedthe contribution of CRY2 to oxaliplatin-induced apoptosis inDLD-1 and SW480 cells. In the presence of 4-mmol/L oxaliplatin,CRY2 depletion significantly increased number of apoptotic cells(P < 0.001; Fig. 2B), as evident by Annexin V staining. Cells werealso analyzed for the presence of the cleaved form of poly-(ADP-ribose) polymerase cleavage (PARP), an apoptosis marker. CRY2knockdown cells showedmore PARP cleavage compared with thecontrol cells (Fig. 2C). Conversely, overexpression of CRY2 pre-vents oxaliplatin-induced PARP cleavage (Fig. 2C). In addition,CRY2 depletion by siRNA resulted in six times more apoptoticcells than the cells transfected with scramble siRNA, as evident by

TUNEL staining (Fig. 2D). Taken together, these results indicatethat CRY2 expression leads to chemoresistance.

FBXW7 negatively regulates CRY2Because FBXW7 can regulate the chemosensitivity of cancer

cells (38), we explored whether the FBXW7 is involved inCRY2-mediated chemosensitivity. CRY2 levels were decreasedwhen cells were transfected with FBXW7 in a dose-dependentmanner (Fig. 3A). Given that FBXW7 is involved in the desta-bilization of CRY2, it is possible that there is an interactionbetween FBXW7 and CRY2. To investigate this potential inter-action, we analyzed cell lysates cotransfected with FBXW7and CRY2. Indeed, exogenous expressed FBXW7 was able toassociate with endogenous CRY2 as assayed by Co-IP (Fig. 3B).In vitro binding assay confirmed the interaction betweenFBXW7 and CRY2 (Fig. 3B). FBXW7 preferentially binds totarget proteins with the degron TPXXS motifs (36). We foundthat CRY2 contains a consensus sequence (298NSTPPLSL305)that is highly similar to the FBXW7-binding sites in c-Myc,cyclin E, Notch1, and c-Jun (Fig. 3C). FBXW7-binding site onCRY2 is conserved in different species (Fig. 3C). To determinewhether the consensus sequence is critical in mediating thebinding between FBXW7 and CRY2, we constructed T300Asite mutant of CRY2 and assessed the binding by immunopre-cipatiation and immunoblotting. The result showed that CRY2T300A mutant reduced its binding to FBXW7 (Fig. 3C), sug-gesting that T300 of CRY2 is important site for mediatingFBXW7 binding.

To gain insight into the structural basis of CRY2 recognitionby FBXW7, we generated a model of the CRY2–FBXW7 complexusing HADDOCK docking protocol (45). Briefly, the crystalstructures of WD40 domain of FBXW7 and CRY2 were usedfor modeling the protein–protein complex. We defined struc-ture-based degron motif (f-X-f-f-f-pT/S-P-P-X-pS/T, with frepresenting a hydrophobic residue and X any amino acid) asactive bases in CRY2. Residues 298–305 (comprising degronmotif) are acid positions shared by degron motif in cyclin E,Notch-1, c-Myc, and c-Jun (Fig. 3C). Structures of the FBXW7–CycE peptide complexes show that the CycE peptide bindsto the narrow face of the WD40 domain of FBXW7 extendingfrom blades six and seven at one side of the surface acrossthe central channel. After the HADDOCK docking protocolsimilar to FBXW7–CycE peptide complexes, we have shownthe best structure interactions between CRY2 and WD40domain of FBXW7 (Fig. 3D). The degron motif of CRY2residues 298–305 is docked to the narrow face of WD40domain of FBXW7 (Fig. 3D). We also plotted the intermoleculeH-bonds and hydrophobic interactions observed in the

Figure 2.CRY2 regulates colon cancer chemoresistance. A, knockdown of CRY2 accelerates apoptosis induced by oxaliplatin. DLD-1 and SW480 cells were treatedwith increasing concentrations of oxaliplatin from 0.25 to 80 mmol/L after transfection with siCRY2 or control. The proportion of viable cells upontransduction with siCRY2 dropped more rapidly than in the control cells. B, downregulation of CRY2 expression enhances the proapoptotic effects ofoxaliplatin on DLD-1 and SW480 cell line. Binding of Annexin V and uptake of PI were analyzed by flow cytometry. The mean of three datasets wastaken and calculated from the corresponding quadrants (bar graphs, right). Error bars represent 95% confidence intervals. Treatment of 4-mmol/Loxaliplatin dramatically increased the portion of Annexin VþPIþ/Annexin VþPI� in DLD-1 cells and SW480 transduced with siCRY2. C, CRY2 knockdownleads to increased PARP cleavage. DLD-1 cells transfected with siCRY2 or control siRNA were treated with or without 4-mmol/L oxaliplatin for 48 hours.Cell lysates were immunoblotted with indicated antibodies. CRY2 overexpression reduced cleavage of PARP. DLD-1 cells transfected with myc-CRY2 or control vector were treated with or without 4-mmol/L oxaliplatin for 48 hours. Cell lysates were immunoblotted with indicated antibodies.D, TUNEL assay of DLD-1 and SW480 cell line. Cells transfected with control or SiCRY2 were assayed for TUNEL signals. TUNEL-positive cells weremeasured and presented as bar graphs (P < 0.01; n ¼ 3 experiments).






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Figure 3.FBXW7 interacts with CRY2. A, FBXW7 negatively regulates the steady-state expression of CRY2. DLD-1 cells were transfected with indicated plasmidsand increasing amounts of FBXW7. Equal amounts of cell lysates were immunoblotted with the indicated antibodies. B, interaction of FBXW7 withCRY2. Equal amounts of DLD-1 cell lysates transfected with Flag-FBXW7 were immunoprecipitated with anti-CRY2 and immunoblotted with anti-Flag. FBXW7interacts with CRY2 in vitro. Flag-FBXW7 and Myc-CRY2 cDNAs were transcribed and translated in vitro (TNT). FBXW7 and CRY2 proteins wereincubated overnight and immunoprecipitated with anti-Myc followed by immunoblotting with anti-Flag. (Continued on the following page.)

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binding interface (Fig. 3D). The degron motif of CRY2 binds tothe narrow face WD40 domain of FBXW7. Residues T300 andP301 (in the TPXXS motif) of CRY2 make hydrogen bondswith R465 and R479 of FBXW7 (Fig. 3D). Besides the interac-tions involving in the TPXXS motif, some additional interac-tions are also observed from this CRY2–FBXW7 complex mod-el. Some electrostatic interactions are made between thecharged residues R178, K183, K184, and K296 of CRY2 andthe charged residues D381, D399, D440, and R689 of FBXW7(Fig. 3D). This complex structural model reveals the interac-tions between CRY2 and WD40 domain of FBXW7.

FBXW7 regulates CRY2 via enhancing ubiquitin-mediateddegradation

To investigate the impact of FBXW7 on CRY2 stability, weexamined the turnover rate of CRY2 in the presence of de novoprotein synthesis inhibitor cycloheximide in HCT116 andHCT116 FBXW7�/� cells, and found that HCT116 FBXW7�/�

cells had a decelerated turnover rate of endogenous CRY2(Fig. 4A). On the other hand, we transfected FBXW7 and per-formed the cycloheximide experiments. The data showedthat CRY2 had a faster turnover rate in the presence of FBXW7(Fig. 4A). FBXW7-mediated CRY2 downregulation was

(Continued.) C, the consensus FBXW7-binding motif is highlighted. Sequences of CRY2 and other known FBXW7 substrates are shown for comparison(left). Sequence alignment of CRY2 containing FBXW7-binding motifs from different species is shown (right). Equal amounts of cell lysates from DLD-1cotransfected with WT myc-CRY2 or myc-CRY2-T300A and Flag-FBXW7 plasmids were immunoprecipitated with anti-Flag and immunoblotted withindicated antibodies. D, ribbon presentation of the CRY2–FBXW7 complex model. The loop of CRY2 degron motif (residues T300–S304) interacts withthe WD40 domain of FBXW7. Close-up view of the interface in the CRY2–FBXW7 complex. The interaction residues involving in the interface ofCRY2–FBXW7 molecules are shown in stick.

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Figure 4.FBXW7 regulates CRY2 stability viaubiquitination. A, CRY2 turnover rateand polyubiquitination are regulatedby FBXW7. HCT116 and FBXW7�/�

HCT116 cells were treated withcycloheximide (CHX; 100 mg/mL) forthe indicated times. After transfectionwith the FBXW7 plasmid, DLD-1 cellswere treated with cycloheximide (100mg/mL) for the indicated times. Equalamounts of cell lysates wereimmunoblotted with the antibodies.DLD-1 cells were cotransfected withFBXW7 and His-Ubi. MG132 wasadded 6 hours before harvesting cellswith guanidine-HCl–containing buffer.The cell lysateswere pulled downwithnickel beads and immunoblotted byanti-CRY2 antibody. B, DLD-1 cellswere transfected with the FBXW7plasmids. Cells were treated with MG132 for 6 hours before harvesting.Equal amounts of cell lysates wereimmunoblotted with the antibodies.C, DLD-1 cells were transfected withmyc-CRY2 or myc-CRY2 (T300A).After treatment with cycloheximidefor indicated times, cells wereharvested. Equal amounts of celllysates were immunoblotted with theantibodies. D, DLD-1 cell weretransfected with myc-CRY2 or myc-CRY2 (T300A). MG132 was added 6hours before harvesting cells withguanidine-HCl–containing buffer. Thecell lysates were pulled down withnickel beads and immunoblotted byanti-myc antibody.






suppressed by MG132, a proteasome inhibitor, suggesting theinvolvement of the 26S proteasome (Fig. 4B). Indeed, wefound that FBXW7 increased the ubiquitination level of CRY2(Fig. 4A). We then identified that CRY2 T300A mutant had aslower turnover rate than WT CRY2 even in the presence ofFBXW7 (Fig. 4C). Accordingly, CRY2 T300A mutant is resistantto FBXW7-mediated ubiquitination (Fig. 4D). These results sug-gest that FBXW7 activity negatively regulates CRY2 stabilityvia enhancing the levels of CRY2 ubiquitination, thereby increas-ing CRY2 turnover rate.

FBXW7 enhances chemosensitivity of colorectal cancer cellline

We examined whether FBXW7 expression had an impact onchemoresistance of colon cancer cells and found that DLD-1 cellswith overexpressed FBXW7 were more sensitive to oxaliplatincompared with control cells (Supplementary Fig. S1A). Accord-

ingly, overexpressed FBXW7 resulted inmore oxaliplatin-inducedPARP cleavage than the control cells (Fig. 5A). As expected,FBXW7 overexpression led to downregulation of CRY2 duringthis process (Fig. 5A). Another line of evidence showed that in thepresence of 4-mmol/L oxaliplatin, overexpressed FBXW7 signifi-cantly potentiated the drug-mediated apoptosis, as evident byAnnexin V staining (Fig. 5B and Supplementary Fig. S1B). Thesedata suggest that the FBXW7–CRY2 axis is critical forchemosensitivity.

LowFBXW7expression correlateswith highCRY2 expression incolorectal cancer and manifests poor survival

To determine the clinical relevance, we subjected a tissuemicroarray containing 289 human colorectal cancer specimensto IHC staining for CRY2 and FBXW7. The colorectal cancersamples had high CRY2 expression, which correlated withlow FBXW7 expression (representative case 1). Accordingly,

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0.40.50.91Figure 5.FBXW7 regulates CRY2 expression andcolon cancer chemoresistance. A,overexpression of FBXW7 expressionleads to PARP cleavage. DLD-1 cellstransfected with FBXW7 or control vectorwere treated with or without 4 mmol/Loxaliplatin for 48 hours. Cell lysates wereimmunoblotted with indicated antibodies.Ratio of CRY2 expression was shown. B,overexpression of FBXW7 enhances theproapoptotic effects of oxaliplatin. DLD-1cells transfected with FBXW7 or controlvector were treated with or without 4mmol/L oxaliplatin for 48 hours. Binding ofAnnexin V and uptake of PI were analyzedby flow cytometry. C, representative IHCstaining for FBXW7 and CRY2 in serialsections from colon cancer patientsamples. Case 1 represents high CRY2-expressing patient. Case 2 representslow CRY2-expressing patient. Thesample used was derived from coloncancer cases. Scale bars, 50 mm.D, model of FBXW7 in modulating CRY2ubiquitination and chemosensitivity.FBXW7 binds to phosphorylatedT300 of CRY2 to ubiquitinate CRY2 andcauses subsequent degradation, whichreduces CRY20s activity.

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colorectal cancer samples with low CRY2 expression had highFBXW7 expression (representative case 2; Fig. 5C). The CRY2and FBXW7 expression levels were reversely correlated witheach other (Table 2). Importantly, the Kaplan–Meier analysisshowed that high expression of CRY2 and low expression ofFBXW7 were correlated with poor survival (Supplementary Fig.S1C). These results strongly suggest that the FBXW7–CRY2 axisis deregulated during the development of human colorectalcancer.

DiscussionAdjuvant chemotherapy with 5-FU and oxaliplatin currently

remains a standard treatment for patients with advanced colo-rectal cancer. However, chemotherapy resistance leading totreatment failure and local recurrence is still a critical problem.One of the biggest challenges is to identify the subpopulationof patients who are most likely to respond to a specific therapy.If one or more biomarkers could predict patient response tochemotherapy, one could spare the nonresponders from inef-fective treatment and direct them to alternative treatmentstrategies that could be more effective. There was some evidencethat showed that the circadian clock can modulate drug metab-olism and efficacy of antitumor therapy. Patient response tochemotherapy was closely correlated to the functional statusof the circadian genes. However, chronotherapeutic approachhas not become routine in clinical practice, perhaps in part,because of the lack of a clear mechanistic basis (49). Notice-ably, we found that circadian clock protein CRY2 is over-expressed in chemoresistant colorectal cancer and that FBXW7has unprecedented biologic activity in downregulating CRY2to mediate chemosenstivity; this demonstrates that cicadianclock proteins may play an important role in colorectal cancerchemosensitivity.

There has been evidence that the circadian clock couldmodulate multidrug resistance genes, thereby playing impor-tant roles in chemoresistance (50), as mdr1a gene can beaffected by the circadian clockwork. Also, overexpression ofthe circadian clock gene Bmal1 can increase sensitivity tooxaliplatin in colorectal cancer (51). These findings suggestthat dysregulation of the circadian clock may be associated withdrug sensitivity. Paradoxically, we have shown that high expres-sion of CRY2 was significantly associated with poor chemo-therapeutic outcomes in colorectal cancer patients whoreceived standard adjuvant chemotherapy. It is possible thatCRY2 expression is involved in DNA-damage response, therebyaffecting the chemosensitivity. It was reported that mutation ofCry could protect p53-mutant mice from early onset of cancerand extend their survival time after ionizing radiation (26),suggesting that Cry may be involved in p53-mediated DNA-damage response. However, there was no study regarding thecorrelation between CRY2 expression and p53 in patients interms of clinical outcome. Interestingly, our in vitro study didshow that repression of CRY2 in DLD-1 and SW480 cells (bothare p53 mutant) leads to decreased cell viability and increasedapoptosis in the presence of oxaliplatin, thereby resulting in anincreased sensitivity to chemotherapy drugs. Thus, previousfindings and current study indicate that CRY2 expression leveland p53 status might be factors in predicting chemoresistancein colorectal cancer patients.

Ubiquitination-mediated degradation of clock proteins hasbeen highlighted as a key regulatory process in clockwork. TheFBXL3 and FBXL21 are critical for generating the CRY expressiondynamics and behavior rhythms (39, 40, 52). Both CRY1 andCRY2 repressor proteins are regulated by ubiquitin-mediateddegradation for temporally orchestrating the transcription ofclock genes. Our studies have shown that FBXW7 can bind toand destabilize CRY2 through enhancing ubiquitin-mediateddegradation of CRY2. FBXW7 is a tumor-suppressor (53) proteinthrough recognizing and degrading several oncoproteins suchas cyclinE, c-JUN, Aurora B, andMYC (33, 35, 54).Mutant FBXW7or downregulation of FBXW7 causes tumorigenesis as it failsto decrease target oncoproteins (37, 55). Although FBXW7 hasa critical function in cancer regulation, very few circadian clockproteins are the known targets of FBXW7. Giving that three F-Boxproteins are all involved in CRY2 ubiquitination, the relationshipbetween FBXW7, FBXL3 and FBXL21 in terms of regulating CRY2stability certainly warrants further investigation. Nonetheless, itmakes sense that a tumor-suppressor protein FBXW7 is involvedin degrading CRY2 when CRY20s overexpression leads to poorclinical outcome. Indeed, we found that CRY2 overexpression isquite common in chemoresistant colorectal cancer. CRY2 over-expression was negatively correlated with FBXW7 protein expres-sion in human colon cancer samples. These findings demonstratethat FBXW7 downregulation can, at least partially, account forCRY2 overexpression in colon cancer and provide importantinsights into the mechanisms underlying CRY2 overexpressionin chemoresistant colorectal cancer. The inverse relationshipbetween FBXW7 and CRY2 is demonstrated for the first time incancer.

In summary, our mechanistic studies explain the regulatoryrelationship among CRY2, FBXW7, and chemosensitivity (Fig.5D). ThatCRY2overexpressed in chemoresistant colorectal cancerraises the possibility that inhibiting the CRY2 is an efficienttherapeutic approach in overcoming chemoresistance.

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

Authors' ContributionsConception and design: L. Fang, L. Wang, J. Wang, M.-H. LeeDevelopment of methodology: L. Fang, Z. Yang, M.-H. LeeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Fang, Z. Yang, J. Zhou, Y. Deng, M.-H. LeeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Fang, J.-Y. Tung, C.-D. Hsiao, M.-H. LeeWriting, review, and/or revisionof themanuscript: L. Fang, Z. Yang, J.-Y. Tung,C.-D. Hsiao, M.-H. LeeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Fang, Z. Yang, L. Wang, P. Wang, M.-H. LeeStudy supervision: L. Fang, J. Wang, M.-H. LeeOther (used computationalmolecular docking analysis methods to interpretthe interactions between Cry2 and Fbw7): J.-Y. Tung

Table 2. Correlation betweenCRY2 andFBXW7expression in colorectal cancerpatient samples (P < 0.001)

Low CRY2 High CRY2 Total

Low FBXW7 48 (17%) 86 (30%) 134 (46%)High FBXW7 117 (40%) 38 (13%) 155 (54%)Total 165 (57%) 124 (43%) 289 (100%)

NOTE: Fisher exact test.






AcknowledgmentsThe authors thank Joseph Munch of the Department of Scientific Publica-

tions at The University of Texas MD Anderson Cancer Center for critical readingof the article.

Grant SupportThis work was supported by the BIH through grant R01CA089266 (to

M.-H. Lee), by the Fidelity Foundation (to M.-H. Lee), by the Susan G. KomenBreast Cancer Foundation grant KG081048 (to M.-H. Lee), and by the NIHCA16672 (MDACC Core grant). This work was also supported by 973 Projects

fromMinistry of Science andTechnology ofChina 2015CB554000 (to J.Wang),the Program of Introducing Talents of Discipline to Universities B12003(to J. Wang) and the International S&T Cooperation Program of China2011DFA32570 (to J. Wang).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 10, 2015; revised March 31, 2015; accepted April 1, 2015;published OnlineFirst April 8, 2015.

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2015;14:1476-1487. Published OnlineFirst April 8, 2015.Mol Cancer Ther Lekun Fang, Zihuan Yang, Junyi Zhou, et al. Chemoresistance of Colorectal CancerCircadian Clock Gene CRY2 Degradation Is Involved in

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