Research ArticleOsteopontin Overexpression Induced Tumor Progression andChemoresistance to Oxaliplatin through Induction of Stem-LikeProperties in Human Colorectal CancerLui Ng,1Timothy Wan,1Ariel Chow,1,2Deepak Iyer,1Johnny Man,1Guanghua Chen,1Thomas Chung-Cheung Yau,1,2Oswens Lo,1Chi-Chung Foo,1Jensen Tung-Chung Poon,1Ronnie Tung-Ping Poon,1,2Roberta Pang,1,2and Wai-Lun Law11Department of Surgery, Li Ka Shing Faculty of Medicine, Te University of Hong Kong, Hong Kong2Centre for Cancer Research, Li Ka Shing Faculty of Medicine, Te University of Hong Kong, Hong KongCorrespondence should be addressed to Roberta Pang; robertap@hku.hkReceived 9 March 2015; Revised 7 May 2015; Accepted 12 May 2015Academic Editor: Yupo MaCopyright 2015 Lui Ng et al. Tis is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Colorectal cancer (CRC) is one of the most common and fatal malignancies worldwide. Te poor prognosis of colorectal cancerpatients is due to development of chemoresistance and cancer metastasis. Recently osteopontin (OPN) has been associated withstem-like properties in colorectal cancer. Tis study further examined the clinicopathological signifcance of OPN in CRC and itsefect on chemoresistance and transcription of stem cell markers. We examined the transcription level of OPN in 84 CRC patientsand correlated the expression with their clinicopathological parameters. Te associations of OPNoverexpression with transcriptionof stem cell markers and response to chemotherapy in DLD1-OPN overexpressing clones and CRC patients were also investigated.Our results showed that OPN was signifcantly overexpressed in CRC, and its overexpression correlated with tumor stage andpoor prognosis. Overexpression of CRC induced OCT4 and SOX2 expression in vitro and correlated with SOX2 overexpressionin CRC patients. In addition, DLD1-OPN overexpressing cells showed enhanced ability to survive upon oxaliplatin treatment, andOPN expression was higher in CRC patients who were resistant to oxaliplatin-involved chemotherapy treatment. Tus, CRC cellsoverexpressing OPN demonstrated stem-like properties and OPN inhibition is a potential therapeutic approach to combat CRCprogression and chemoresistance.1. IntroductionColorectal cancer (CRC) is the third most common malig-nancy around the world [1]. Annually, over 1.2 million peopledevelop CRCglobally, with more than 600,000 patients dyingfromthe disease in 2008 [2]. Both the incidence and the deathrates from CRC are increasing rapidly in Asian countries[3]. Incidenceandmortality ratesforCRChavedeclinedas a result of improved tests that allow early detection ofthe cancer, when it can be more easily treated by surgeryandchemotherapy along withradiotherapy [4]. Despitethose advances in clinical treatment, the overall prognosisof CRC patients is still unsatisfactory due to developmentofchemoresistanceandcancermetastasis. Terefore, itisimportant to understand how CRC cells acquired the abilitytosurviveuponchemotherapyandmetastasizetodistantregions, inorder todevelopnewtherapeutictarget andapproach to improve the prognosis and survival rates of CRCpatients.Osteopontin (OPN),a member of the SmallIntegrin-BindingLigandN-linkedGlycoprotein(SIBLING)family,is expressed in normal mineralized tissues,epithelial cellsof some metabolically active ducts,and several neoplastictissues [5]. OPNis involved in most aspects of tumor biology.Trough its diverse reported functions related to prolifera-tion, survival, angiogenesis, escape from host defense, tumordevelopment, invasion, and metastasis, OPN covers multiplehallmarks of cancer [6]. Indeed, OPNexpression signifcantlycorrelates with tumor stage in various cancer types.Hindawi Publishing CorporationStem Cells InternationalVolume 2015, Article ID 247892, 8 pageshttp://dx.doi.org/10.1155/2015/2478922 Stem Cells InternationalIncolorectal cancer, OPNdownregulationsuppressedinvitroproliferationandinvivotumorigenicityandalsosuppressed in vitro invasion and migration capacity [7]. Wealso showed in our previous study that stable overexpressionof OPNinDLD1 cells signifcantlyinducedtheproteinexpression and secretory level of OPN, and the migrationability of DLD1 cells [8]. In addition, OPN overexpression isassociated with activation of the epithelial to mesenchymalpathway through induction of Twist and Snail and downreg-ulation of E-cadherin.Recently, OPNhasbeenassociatedwithcancerstemcell nature in colorectal cancer. OPN secreted from tumorassociated cells increased CD44v6 expression in colorectalcancer stem cells by activating the Wnt/-catenin pathway,whichpromotesmigrationandmetastasis[9]. Tisstudywill further investigate the in vitro efect of OPN overex-pression on growth response to chemotherapy. In addition,a recent study demonstrated that OPN silencing suppressedtranscriptions of key stemness transcription factors SOX2,Oct3/4, and Nanog in vitro and glioblastoma stem-like cellcharacterandtumorigenicityinvivo[10]. Tisstudywillalso study the efect of OPN overexpression on stemness ofCRC cells, by investigating its correlation with transcriptionof stem cell markers.2. Materials and Methods2.1. Patients and Specimens. Te human sample collectionprotocol has beenapprovedbythe Institutional ReviewBoard (IRB) of the University of Hong Kong, and all clinicalinvestigation has been conducted according to the principlesexpressed in the Declaration of Helsinki. Informed writtenconsent has beenobtainedfromtheparticipants. Tissuesamples were obtained from 84 patients, immediately frozenin liquid nitrogen and kept at 80C until analysis. Clinico-pathological data were obtained from the patient database ofour hospital.2.2. Cell-Lines, Tissue Culture, Transfections,and Reagents.Construction of stable OPN overexpressing or vector controlDLD1 cells was described previously [8]. DLD1-OPN stableclones and vector control were maintained in DMEM with10%fetal bovine serumand antibiotics (Invitrogen, Carlsbad,CA), 5% CO2 at 37C.2.3. RNA Extraction. Total RNA was extracted using Trizolreagent andPurelinkRNAMini Kit (Life Technologies,Carlsbad, CA) according to the manufacturers instructions.Te RNA yield and quality were analyzed by NanoDrop 2000(Termo Scientifc).2.4. cDNA Synthesis and Quantitative Real-Time PolymeraseChain Reaction. 500 ng total RNA was reversely transcribedwithPrimeScript 1st strandcDNASynthesisKit (Takara,Tokyo, Japan)inaccordancewiththeinstructionsof themanufacturer. Real-time PCRwas performedina fnalvolumeof 15 Lcontaining1.5 LRTtranscript, 0.2 Mof each primer, 1x ROX reference dye, and 7.5 L of Fast-StartUniversal SYBRGreenMaster(ROX)(RocheDiag-nostics, Switzerland, Basel). AnoRTtranscript controlwas included for each gene to ensure the signal was trulydriven by target gene amplifcation. Te primer sequenceswere as follows: OPN-Forward Primer: 5

-TGGGGG-TCACTGCAATTAG-3, OPN-Reverse Primer: 5

-TGG-GGCTAGGAGATTCTG-3

; GAPDH-Forward Primer: 5

-GTCTCCTCTGACTTCAACAGCG-3

, GAPDH-ReversePrimer: 5

-ACCACCCTGTTGCTGTAGCCAA-3

; OCT4-ForwardPrimer: 5

-GTGGAGGAAGCTGACAACAA-3,OCT4-ReversePrimer: 5

-GCCGGTTACAGAACCACA-CT-3

; SOX2-Forward Primer: 5

-GACAGTTACGCG-CACATGAA-3, SOX2-Reverse Primer: 5

-TAGGTCTGC-GAGCTGGTCAT-3

; Nanog-Forward Primer: 5

-GTG-ATTTGTGGGCCTGAAGA-3, Nanog-Reverse Primer: 5

-ACACAGCTGGGTGGAAGAGA-3

. Real-time PCR wascarriedout usingtheABI 7900HTFast Real-TimePCRSystem (Applied Biosystems, Foster, CA) at 95C for 10 min,followed by 40 cycles at 95C for 15 sec and at 56C for 1 min.Each assay was done in triplicate, the average was calculated,and the expression level of target mRNA was normalized bythe expression of GAPDH (delta Ct); that is, the higher thedelta Ct, the lower the target gene expression.2.5. Cell Viability Assay. Same number of cells were seededon 96-well plate for 24 hours and then subjected to treatmentof chemotherapeuticdrugs5 Moxaliplatinor50 M5-Fluorouracil (5FU). Te cell viability at 72 hafer drugtreatment was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide (MTT) (Invitrogen).2.6. Immunohistochemical Staining. Immunohistochemicalstainingwasperformedasdescribedpreviously[11]. Sec-tions were incubatedwiththe primary antibodies anti-OPN(OriGene Technologies, Rockville, MD) and anti-SOX2(Cell Signaling Technology, Danvers, MA) at 1 : 100 dilutionsovernight at 4Cinamoist chamber. Ascoringsystemrelatedtotheextent andintensityof immunostainingofenterocytes was used. Te intensity of positive staining wasscored as 0, negative; 1, weak; 2, moderate; 3, strong by twoindependent observers. Te extent of positive staining wasscored as 1 (50%). Te fnal scorewas determined by multiplying the intensity score and extentscore, yielding a range from 0 to 12.2.7. Statistical Analysis. Te association of OPN level withstem cell markers was tested with Pearson correlation. Stu-dents -test was applied to compare diference between twogroups. Survival rates were analyzed with log-rank test. Allof these statistical analyses were performed with SigmaPlot10.0(SystatSofwareInc., SanJose, CA, USA). Statisticalsignifcance was set at 0.05.3. Results3.1. OPN Was Overexpressed in CRC. We frst compared theexpression of OPN in CRC and the paired nontumor mucosaof 84 CRC patients. Te OPN expression was determined byStem Cells International 3Table 1: Clinicopathological correlation of OPN overexpression in CRC ( = 84).Clinicopathological features Category Number of casesOPN overexpression(Ct) (mean SEM) valueAge