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;
[email protected] 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
