Colorectal Cancer Cell Lines Are Representative Models of ... · Colorectal Cancer Cell Lines Are...

Molecular and Cellular Pathobiology

Colorectal Cancer Cell Lines Are Representative Models ofthe Main Molecular Subtypes of Primary Cancer

Dmitri Mouradov1,2,4, Clare Sloggett5, Robert N. Jorissen1,2,4, Christopher G. Love1,2,4, Shan Li1,2,Antony W. Burgess3,4, Diego Arango8,9, Robert L. Strausberg10,11, Daniel Buchanan7, Samuel Wormald2,4,Liam O'Connor2,4, Jennifer L. Wilding12, David Bicknell12, Ian P.M. Tomlinson13, Walter F. Bodmer12,John M. Mariadason6, and Oliver M. Sieber1,2,4

AbstractHuman colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and

biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversityof primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations andDNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expressionwas defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancersin The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra incolorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities includedthe presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repairand DNA polymerase e proofreading deficiency, along with concordant mutation profiles in the broadlyaltered WNT, MAPK, PI3K, TGFb, and p53 pathways. Furthermore, we documented mutations enriched ingenes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation(ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent innonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell linesderived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silicosimulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. Inconclusion, our results establish that human colorectal cancer lines are representative of the main subtypesof primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancerbiology and drug responses. Cancer Res; 74(12); 3238–47. �2014 AACR.

IntroductionColorectal cancer is a leading cause of cancer-related

morbidity and mortality (1). Human colorectal cancer celllines are an important, commonly used preclinical modelsystem for studying this disease, and have provided essentialinsights into tumor molecular and cell biology. Cell lines area fundamental tool used in the discovery of new antitumorcompounds and for the discovery of drug sensitivity, resis-

tance, and toxicity biomarkers, with molecular markers ofresponse to conventional chemotherapies and targetedagents showing clinical utility in patients (2–7). Examplesinclude the relationship between tumor microsatellite insta-bility-high (MSI-H) status and lack of 5-fluorouracil (5FU)response (2), and mutations in KRAS, BRAF, and PIK3CAexon 20 and resistance to anti-EGFR antibody therapy (3).However, to what extent colorectal cancer cell lines

Authors' Affiliations: 1Ludwig Institute for Cancer Research; 2SystemsBiology and Personalised Medicine Division; 3Structural Biology Division;Walter and Eliza Hall Institute of Medical Research; 4Faculty of Medicine,Dentistry and Health Sciences, Department of Medical Biology, Universityof Melbourne, Parkville; 5VLSCI Life Sciences Computation Centre, acollaboration between Melbourne, Monash and LaTrobe Universities,c/o The University of Melbourne, Carlton; 6Oncogenic Transcription Lab-oratory, Ludwig Institute for Cancer Research, Austin, VIC, Australia;7Cancer and Population Studies Group, Queensland Institute of MedicalResearch, Herston, QLD, Australia; 8Group of Molecular Oncology, CIB-BIM-Nanomedicine, Vall d'Hebron University Hospital, Research Institute(VHIR), Universitat Aut�onoma de Barcelona, Passeig Vall d'Hebron, 119-129, 08035 Barcelona; 9CIBER de Bioingeniería, Biomateriales y Nano-medicina (CIBER-BBN); Spain; 10Ludwig Collaborative Laboratory forCancer Biology and Therapy, Department of Neurosurgery, JohnsHopkinsUniversity School of Medicine, Baltimore, Maryland; 11Ludwig Institute forCancer Research Ltd., New York, New York; 12Cancer and Immunoge-netics Laboratory, Weatherall Institute of Molecular Medicine (WIMM),John Radcliffe Hospital, University of Oxford; and 13Molecular and Pop-

ulation Genetics Laboratory, Wellcome Trust Centre for Human Genetics,Oxford, United Kingdom

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

Corresponding Authors: John Mariadason, Oncogenic TranscriptionLaboratory, Ludwig Institute for Cancer Research Ltd., Austin Hospital,Level 5, Olivia Newton-John Cancer & Wellness Centre, Studley Road,Heidelberg, VIC 3084, Australia. Phone: 61-3-9496-3068; Fax: 61-3-9496-5334. E-mail: [email protected]; and Oliver Sieber, Colo-rectal Cancer Genetics Laboratory, Systems Biology and PersonalisedMedicine Division, Walter and Eliza Hall Institute of Medial Research, 1GRoyal Parade, Parkville, VIC 3052, Australia. Phone: 61-3-9345-2885; Fax:61-3-9347-0852; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0013

�2014 American Association for Cancer Research.

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represent and maintain the genetic diversity of primarycancers remains controversial.More than the past two decades, major cancer genes and

pathways central to colorectal cancer development have beendelineated, including the WNT, MAPK, PI3K, TGFb, and p53pathways. Two broad molecular subtypes of colorectal cancerhave emerged, characterized by MSI-H (�15%) or chromo-somal instability (CIN, �60%; refs. 8–10). MSI-H colorectalcancers occur predominantly in the proximal colon, and oftenshow poor differentiation, mucinous histology, and increasedperitumoral lymphocytic infiltration (11). These tumors exhib-it hypermutation caused by defective DNA mismatch repair(MMR), tend to be near-diploid and to have a CpG islandmethylator phenotype (CIMP), and harbor mutations in adistinct set of driver genes, including BRAF, PTEN, and TGFBR2(12–14). In contrast, chromosomally unstable tumors aremorecommon in the distal colorectum and tend to develop alongthe classical genetic pathway of colorectal tumorigenesis, withmutations in APC, KRAS, SMAD4, and TP53 (15).Recent cancer genome sequencing studies have revealed

additional details of the genetic profiles of human colorectalcancer, highlighting their diversity. An initial whole-exomesequencing study on 11 microsatellite stable (MSS) colorectalcancers demonstrated that such tumors harbor �80 codingsequencemutations with a small number of commonly mutat-ed driver genes and a large number of "private" mutations (16).Subsequently, The Cancer Genome Atlas (TCGA) Networkreported comprehensive data on 223 unselected sporadiccolorectal cancers (17). Hypermutation was identified in�15% of carcinomas, with three quarters of these displayingthe expected MSI-high (MSI-H) phenotype associated withepigenetic silencing and/or mutation of MMR genes. However,one-quarter of hypermutated tumors did not show MSI-H andseemed to be associated with DNA polymerase e (POLE)mutations, perhaps representing a distinct colorectal cancersubtype. Twenty-four genes were identified as significantlymutated in colorectal cancer, including several novel candi-date genes such asATM,ARID1A, TCF7L2, SOX9, and FAM123B.A number of recurrent DNA copy-number alterations werereported comprising potentially drug-targetable amplifica-tions of ERBB2 and IGF2. A similar study on 74 pairs of primaryhuman colon tumors reported highly concordant results, andin addition identified low frequency fusion transcripts involv-ing R-spondin family members RSPO2 and RSPO3 (18).Here, we report the first comprehensive whole-exome and

DNA copy-number analyses of 70 of the most widely usedcolorectal cancer cell lines, and undertake a detailed compar-ison of genetic alterations with those reported for TCGA-analyzed primary cancers. We demonstrate that the spectraof mutations and DNA copy-number aberrations in colorectalcancer cell lines are representative of primary tumors, includ-ing hypermutation phenotypes and targeting of major signal-ing pathways. Our data further highlight novel aspects ofcolorectal cancer biology, including significant enrichment ofmutated genes involved in chromatin remodeling and histonemethylation or acetylation. Our results verify established colo-rectal cancer cell lines as a useful preclinicalmodel system, andprovide a comprehensive genomic data resource enabling

informed choices when selecting cell lines for functional andpharmacogenomics research.

Materials and MethodsColorectal cancer cell lines and TCGA-analyzed primarycancers

The 70 colorectal cancer cell lines studied were obtainedfrom a range of sources, listed below, over a period spanningseveral years (Supplementary Table S1). C10, C106, C125, C135,C32, C70, C80, C84, andC99were generated by theW.F. Bodmerlaboratory, CACO2, COLO201, COLO205, COLO320-DM, DLD1,HCC2998, HCT116, HCT15, HCT8, HT29, LOVO, LS174T, LS180,LS411, LS513, NCI-H716, NCI-H747, RKO, SKCO-1, SNU-C1,SNU-C2B, SW1116, SW1417, SW1463, SW403, SW48, SW480,SW620, SW837, SW948, and T84 were obtained from theAmerican Type Culture Collection, KM12 was obtained fromDCTD, COLO678, and CX-1 were obtained from DSMZ, Gp2D,Gp5D, HT115, and HT55 were obtained from ECACC, CCK81,and CoCM-1 were obtained from HSRRB, VACO10, andVACO4S were provided by Dr. J.A. McBain (University ofWisconsin School of Medicine, Madison, WI), SNU-175, SNU-283, and SNU-C4were obtained fromKCLB, LIM1215, LIM1899,LIM2099, LIM2405, and LIM2551 were obtained from theLudwig Institute for Cancer Research, SW1222 were providedby Prof. M. Herlyn (The Wistar Institute, Philadelphia, PA),HDC101, HDC54, HDC82, HDC87, andHDC90were provided byProf. M, Schwab (DKFZ, Germany), HCA46, HCA7, and HRA19were providedbyDr. S.C. Kirkland (Royal PostgraduateMedicalSchool, United Kingdom), and RW2982 and RW7213 wereprovided by Dr. P. Calabresi (Roger Williams General Hospital,Providence, RI). Cells were cultured with Dulbecco's ModifiedEagle Medium and 10% FBS at 37�C and 5% CO2. Cell lineauthentication was performed using the Promega StemElite IDSystemat theQueensland Institute ofMedical Research (QMIR,Queensland, Australia) DNA Sequencing and Fragment Anal-ysis Facility (January 2013). Cells were tested for mycoplasmaby the MycoAlert Mycoplasma Detection Kit (Lonza). Pub-lished exome-capture sequencing data on 223 patients withcolorectal cancer were retrieved from the TCGA (19). SNP arraydata were available for 213 of these patients. Mutation datawere filtered for exons and at splice sites (�3 bp).

Exome-capture sequencingExome-capture was performed using the TruSeq Exome

Enrichment Kit (Illumina) and 100 bp paired-end readsequencing performed on an Illumina HiSeq 2000 at theAustralian Genome Research Facility (AGRF). Variant detec-tion was implemented using a modified GATK Best Practiceprotocol; variants were filtered against known germlinevariations and those detected in 114 in-house normal colo-rectal tissues (Supplementary Methods). Regions of knowngermline chromosomal segmental duplications were exclud-ed (20).

Transcriptome sequencingRNA-Seq analysis was performed at the AGRFon an Illumina

HiSeq2000 to a depth of >100 million paired reads. Alignmentto the human reference genome (hg19) was achieved using

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TopHat (21), and RPKM values calculated. Absence of geneexpression was defined as a RPKM value of <1 (SupplementaryMethods).

SNP microarray analysisSNP assays were performed by the AGRF using Illumina

Human610-Quad BeadChips (GSE55832) and processed usingGenomeStudio (Illumina). SNPs showing germline alterations,based on 637 normal samples, were excluded. DNA copy-number segmentation was performed using OncoSNPv2.18(22). Regions of significantly altered genome were identifiedusing GISTIC2.0 (23).

Microsatellite instability analysisMSI analysis was performed for the National Cancer Insti-

tute recommendedmicrosatellitemarker panel BAT25, BAT26,D2S123, D5S346, and D17S250 using fluorescently labeledprimers on a 3130xl Genetic Analyzer (Applied Biosystems;ref. 24). MSI-H was diagnosed if instability was evident at 2 ormore markers.

CIMP marker and MLH1 promoter methylation analysisMethyLight real-time PCR was performed for the Weisen-

berger and colleagues CIMP marker panel (IGF2, SOCS1,RUNX3, CACNA1G, NGN1), MLH1, and the reference ALU(C-4; ref. 25). The percentage of methylated reference (PMR)was calculated for GENE:ALU ratio of template amount in asample against GENE:ALU ratio of template amount in meth-ylated reference DNA. Samples with a PMR greater than 10 for�3 CIMP markers were classified as CIMP-positive.

Statistical analysisStatistical analyses were conducted using the R statistical

computing software (http://www.R-project.org/). Differencesbetween groups were assessed using Fisher exact test or x2 testfor categorical variables and the Student t test for continuousvariables. Correlation between RNA-Seq and microarray geneexpression data were assessed using Spearman rank correla-tion coefficient. Overlap of gene lists was assessed using ahypergeometric test. GISTIC2.0 analysis for significantlyaltered focal regions of chromosomal deletion or gain/ampli-ficationwere adjusted formultiple testing, implementing aBH-FDR adjusted Q-value cut-off of 0.05. All statistical analyseswere 2-sided and considered significant when P < 0.05.

ResultsExomemutationprofiles in 70 colorectal cancer cell lines

Seventy commonly used colorectal cancer cell lines, com-prising 63 unique lines and 7 duplicate lines, were analyzed formutations in protein-coding exons and exon–intron bound-aries by whole-exome capture sequencing (SupplementaryTable S2). In all cases, >20-fold coverage of >80% of targetedexons was achieved. As matched normal tissue for these celllines was not available, putative somatic mutations wereidentified by annotation against databases of known humangermline variants. Regions of known germline chromosomalsegmental duplications were excluded to reduce the possibilityof false-positive variants caused by read mismapping. Mean

pipeline sensitivity and specificity for nonsilent variants wereshown to be 79.2% and 98.6% in an analysis of 10 primarycolorectal cancers with and without paired normal tissue, withthe majority (93.4%) of false-negative calls resulting fromsomatic mutations mimicking annotated germline variants(Supplementary Data). Accuracy of mutation calling was ver-ified by Sanger resequencing for 12 selected genes (APC,CTNNB1, KRAS, BRAF, NRAS, PIK3CA, PTEN, SMAD2, SMAD3,SMAD4, FBXW7, and TP53) on 43 cell lines, with validation of97.6% (165/169) of mutations (Supplementary Table S3).

For the 63 unique cell lines, the total number of putativesomatic mutations ranged from 219 to 8657 (mean ¼ 1498.3).Similar to the primary colorectal cancers reported by TheTCGA (17), the prevalence of mutations varied substantially,ranging from 6.6 to 260.0 per 106 bases, with evidence fornonhypermutated and hypermutated groups (Fig. 1). Cell lineswith a mutation prevalence of >25 per 106 bases were desig-nated as hypermutated. Hypermutation was observed in 34.9%(22/63) of cell lines as compared with 15.7% (35/223) of theTCGA cancers (P < 0.001). 86.3% (19/22) of hypermutated celllines exhibited MSI-H, similar to the proportion found incancers (80.0%, 28/35, P ¼ 0.725). MSI-H status in cell lineswas associated with MLH1 hypermethylation, mutations inMMRgenes and presence of CIMP (P< 0.009; Fig. 1). Consistentwith the established association between MSI-H and proximaltumor location in colorectal cancer, all MSI-H cell lines withavailable site details originated from the proximal colon (Sup-plementary Table S1). There was no evidence of imbalance insite distribution between cell lines and cancers (P ¼ 0.937).

Mutation spectra in hypermutated colorectal cancer celllines

Comparedwith nonhypermutated cell lines, MSI-H cell linesdisplayed a higher number of InDels (41.1-fold; P < 0.001) andSNVs (9.7-fold; P < 0.001) as observed in the TCGA cancers

Figure 1. Mutation frequencies in 70 human colorectal cancer celllines. Cell lines were separated into distinct hypermutated andnonhypermutated cases. MSI-H, microsatellite instability-high; CIMP,CpG island methylator phenotype; MLH1 meth., MLH1 promoterhypermethylation, MMR mut., DNA mismatch repair gene mutation;inset, mutations in MMR genes among the hypermutated samples, withcases in the same order as in the main graph.

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(InDels: 18.3-fold, SNVs: 10.8-fold; P < 0.001; Fig. 2A and B). Asexpected, InDels inMSI-H cases tended to occur at nucleotide-repeat (�N5 or �NN3) sequences (MSI-H vs. nonhypermu-tated: cell lines 82.7% vs. 17.9%, cancers 73.4% vs. 38.4%; P <0.001), a bias not observed for the SNVs (cell lines 2.9% vs. 3.7%,cancers 2.3% vs. 2.9%; Supplementary Table S2). The SNVspectrum differed significantly between MSI-H and nonhyper-mutated cases, with an increased proportion of A>G/T>Ctransitions (cell lines 1.5-fold, cancers 1.8-fold; P < 0.011) anddecreased C>G/G>C transversions (cell lines �4.5-fold, can-cers �5.2-fold; P < 0.001; Fig. 2C).A second type of hypermutated tumor was observed among

cell lines and the TCGA cancers. These cases were MSS andcomparedwith nonhypermutated cases exhibited a nucleotide-substitution hypermutator phenotype (NSHP) characterized bya substantial increase of SNVs (cell lines 16.8-fold, cancers 56.8-fold; P < 0.005; Fig. 2A and B). The SNV spectrum in 2 of 3 NSHPcell lines (HT115, HCC2998) and 7 of 7 NSHP cancers furtherdisplayed increased proportions of C>A/G>T transversions(MSI-H vs. nonhypermutated: cell lines 1.5-fold, cancers 2.4-fold) and A>C/T>G transversions (cell line 3.8-fold, cancers 2.0-fold), as well as decreased C>G/G>C transversions (cell lines�13.6-fold,cancers�22.3-fold;Fig.2C).Combiningcell linesandcancers, this mutator phenotype was significantly associatedwiththepresenceofPOLEmutations (NSHP:90.0%,9/10vs.MSI-H: 29.8%, 14/47 vs. nonhypermutated: 1.3%, 3/229; P < 0.001). Inaddition, all nine NSHP caseswith POLEmutation had at least 1missense mutation in the POLE exonuclease domain (EDM), ascomparedwithonly1of17non-NSHPcaseswithPOLEmutation(P < 0.001; Supplementary Table S4). 82.4% (14/17) of POLEmutated non-NSHP samples were MSI-H, and there was noevidence that the non-EDM POLE mutations in these cases

modified the MSI-H phenotype with similar SNV frequenciesand transition/transversion spectra compared with MSI-Hcancers without POLEmutation (P > 0.05 for all comparisons).

A single NSHP cell line, HT55, exhibited a distinct bias toA>T/T>A transversions (49.0% vs. nonhypermutated mean4.6%) and was wild type for POLE, but a similar case was notpresent among the TCGA cancers (Fig. 2C). This cell lineharbored a heterozygous truncating mutation in the MMRgene PMS1, which may be related to this mutator phenotype,although Pms1-deficient mice have been reported to show noevidence of tumor predisposition or hypermutation (26).

Chromosomal and subchromosomal aberrationsDNA copy-number alterations in cell lines were profiled

using Illumina Human 610-Quad BeadChips. As anticipated,DNA copy-number profiles were similar between cell lines andprimary cancers (Fig. 3A). Hypermutated MSI-H and NSHPcases showed stable profiles with a modal chromosome copy-number of 2n. In contrast, nonhypermutated cases tended toexhibit unstable profiles with modal chromosome copy num-bers ranging from 2n to 4n in cell lines and 2n to 6n in cancers.For nonhypermutated groups, the most common deletedchromosome arms were 8p, 17p (including TP53), and 18q(including SMAD4), and themost common gained regionswerechromosome 7, 8q (including MYC), 13, and 20q. Some differ-ences were apparent between cell lines and primary cancers,including more frequent chromosome 4 deletions in nonhy-permutated and chromosome 7 gains and 18q deletions inhypermutated cell lines.

Focal regions significantly targeted by DNA copy-numberalterations in both cell lines and cancers were deduced usingGISTIC2.0 software (23). Eleven and 4 significant focal regions

Figure 2. Types of mutations for 70 human colorectal cancer cell lines and 223 TCGA-analyzed primary cancers. A–C, counts of SNVs and InDels (A and B),and proportions of nucleotide transitions and transversions (C). Paired cell lines originating from the same tumor are indicated by identical colored stars. MSI-H, microsatellite instability-high; NSHP, nucleotide-substitution hypermutator phenotype; POLE EDM,missensemutation in the POLE exonuclease domain.






were found to overlap between nonhypermutated and hyper-mutated groups, respectively (Fig. 3B and C and Supplemen-tary Table S5). For nonhypermutated cases, overlappingregions included gain/amplification of MYC, a known keymediator of colorectal tumorigenesis (27), and deletion ofthe candidate cancer genes PARK2 and WRN, detected in29.5% and 36.3% of cell lines and 21.9% and 31.6% of primarycancers, respectively. The 4 recurrent regions identified inhypermutated cases—which were also present in the nonhy-permutated group—were all known fragile sites (includingFHIT, A2BP1, WWOX, or MACROD2), the functional relevanceof which is uncertain.

Cancer genes and pathwaysGene mutation profiles, excluding silent mutations, were

compared between colorectal cancer cell lines and primary

cancers. Only genes with well-defined expression in cell lines[reads per kilobase per million reads mapped (RPKM) >1 in atleast 3/13 colorectal cancer cell lines analyzed by RNA-Seq]were considered (n ¼ 20,702 genes; Supplementary Table S6).NSHP cases were excluded from this comparison because ofthe small sample size.

Mutation landscapes of cell lines markedly resembled thoseof primary cancers. In both cohorts, nonhypermutated casesdisplayed a small number of distinctmutation peaks, withAPC,TP53, and KRAS being the most common targets, whereashypermutated MSI-H cases showed frequent mutations inmultiple genes, with the anticipated bias to genes comprisingnucleotide repeats (SupplementaryTables S7). Significant over-lap was observed for the top 5% ofmutated genes (based on theproportion of affected samples), with 54 and 62 genes inter-secting for nonhypermutated and MSI-H groups, respectively

Figure 3. Genome-wide DNA copy-number aberrations in 63 uniquecolorectal cancer cell lines and 213TCGA primary cancers stratifiedinto nonhypermutated andhypermutated cases. A, absoluteDNA copy-number gains andlosses relative to diploid status (2n)deduced using oncoSNP. B and C,minimal significant regions ofchromosomal loss (blue) and gain/amplification (red) deduced usingGISTIC2.0. Selected candidategenes in overlapping regions areindicated and total numbers ofgenes are shown in brackets.

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(P < 0.001; Supplementary Tables S8). Overlapping genesincluded the expected members of the WNT, MAPK, PI3K,TGFb, and p53 pathways. For nonhypermutated cases, thesecomprisedAPC,CTNNB1,FBXW7,KRAS,BRAF,PIK3CA, SMAD4,and TP53, as well as the candidate cancer genes, AXIN2, BCL9L,FAT1, SOX9, ERBB3, PIK3C2B, TIAM1, and ATM. For MSI-Hcases these encompassed APC, FBXW7, PIK3CA, and TGFBR2,along with the candidates CREBBP, TCF7L2, RNF43, andACVR2A. Mutation distributions across colorectal cancer-associated signaling pathways were also similar betweennonhypermutated cell lines and cancers, with the samepathway members tending to show the highest mutationfrequencies (Fig. 4). Greater variability in mutation frequen-cies between pathway members was observed for MSI-Hcases, at least partly related to the higher mutation back-ground and smaller sample size. A notable difference forMSI-H cases was a differential prevalence of CTNNB1 muta-tions, which were frequent in cell lines (47%) but notreported in primary cancers (P < 0.001).In addition to the established colorectal cancer-associated

pathways, significant enrichment was observed for muta-tions in chromatin-state regulators (P < 0.001; gene ontologyenrichment analysis). These comprised ASH1L, CHD6,PRDM2, and MLL3 in nonhypermutated and ARID1A, EP300,EP400, MLL2, CHD6, PRDM2, TRRAP, and SRCAP in MSI-Hcases (Supplementary Table S8). Approximately 49% and19% of nonhypermutated and 100% and 93% of MSI-H celllines and primary cancers carried mutations across thesecandidates, respectively.

Integrating mutation and DNA copy-number data acrosscell lines and primary cancers showed the anticipated tumorsuppressor signatures for APC, TP53, SMAD4, and SOX9 with asignificant overrepresentation of 2 mutational hits (2þ muta-tions or 1mutation and loss of heterozygosity; P < 0.023). Therewas further an association of mutation in BRAF, ERBB3,PIK3CA, and KRAS with copy-number gain of �5 at therespective chromosomal regions (P < 0.018). Trends to mutualexclusivity between pathway member mutations wereobserved for KRAS and BRAF, TP53, and ATM (P < 0.005).

Mutation and DNA copy-number differences in pairedcolorectal cancer cell lines

Included in our colorectal cancer cell line panel were 5 pairs/triplets originally derived from the same tumor (COLO201/COLO205, CX-1/HT29, Gp2D/Gp5D, LS174T/LS180, andDLD1/HCT8/HCT15), and 1 pair derived from a primary tumorand subsequent lymph node metastasis (SW480/SW620).LS174T and LS180 were established by trypsin treatment andscraping of primary cultures from the same tumor, respectively(28), and have been shown to differ with respect to E-cadherinexpression and cell adhesion, with LS174T displaying completeloss of E-cadherin protein (2, 29).

Assessment of the overlap between mutations detected inpaired cell lines identified substantial discrepancies, with 63and 356 mutational differences in the nonhypermutated pairsCOLO201/COLO205 and CX-1/HT29, and 2,763, 480, 6,503,5,377, and 6,369 mutational differences in the MSI-H pairsGp2D/Gp5D, LS174T/LS180, DLD1/HCT8, DLD1/HCT15, andHCT8/HCT15 (Fig. 5). Eight hundred and forty-nine mutationsdiffered in the nonhypermutated primary/metastasis pairSW480/SW620. Nonsilent and silent mutations contributed insimilar proportions to these discrepancies (54.4% vs. 56.6%),suggesting no selection for these differential alterations.

DNA copy-number profiles showed multiple differences fornonhypermutated pairs, with 41.1% and 53.8% of the genomevarying for COLO201/COLO205 and CX-1/HT29 (Fig. 5). Incontrast, discrepancies were limited in MSI-H pairs with 0.2%,0.4%, 6.6%, 2.9%, and 4.3% of the genome differing betweenGp2D/Gp5D, LS174T/LS180, DLD1/HCT8, DLD1/HCT15, andHCT8/HCT15, respectively. 47.5% of the genome differed in thenonhypermutated primary/metastasis pair SW480/SW620.

Notably, mutation differences between paired cell lines didnot obscure known driver genes. For the established colorectalcancer genesAPC,TP53, SMAD4, PIK3CA,KRAS, andBRAF, 29 of33 (87.9%) nonsilent mutations were concordant between cellline pairs.

DiscussionIn this study we show that the mutation and DNA copy-

number landscapes determined for 70 colorectal cancer celllines closely resemble those of primary tumors, underscoringthe utility of cell lines as an appropriate model system for thismalignancy. The 3 molecular subtypes of colorectal cancerrecently defined by the TCGA, nonhypermutated, hypermu-tated with microsatellite instability, and hypermutated with-out microsatellite instability were faithfully captured in thecell line panel. As expected, MSI-H cell lines exhibited

Figure 4. Mutation frequencies for WNT, MAPK, PI3K, TGFb, and p53pathway members as well as chromatin regulators identified among thetop 5% of mutated genes overlapping between colorectal cancer celllines and TCGA-analyzed primary cancers for nonhypermutated orhypermutated MSI-H samples.






hypermethylation of theMLH1 promoter and/or mutations inMMR genes, whereas hypermutated lines without microsatel-lite instability were instead characterized by mutations in theexonuclease domain of POLE. Consistent with defective DNAPOLE proofreading function, the latter tumors showed anucleotide substitution hypermutator phenotype with a biasto C>A/G>T and A>C/T>G transversions. Germline missensemutations in the POLE exonuclease domain have recently beenassociated with familial predisposition to colorectal cancer(30), and elevated tumor predisposition and base-substitutionmutations observed in Pole exonuclease-mutant mice (31).Similarly, somatic missense mutations in the POLE exonucle-ase domain have been reported in 7% of endometrial cancers,with good evidence of associated hypermutation (32).Although differences in prognosis and chemotherapy responsehave been extensively documented for nonhypermutated ver-sus MSI-H tumors (2–4), clinical characteristics of POLE-mutant NSHP tumors are unknown. Our identification of 2cell lines representative of this colorectal cancer subtype willfacilitate an investigation of the specific aspects of the biologyof these tumors, particularly in relation to identifying thera-peutics that may exploit their unique genomic instability.

Consistent with previous lower-resolution data (33), DNAcopy-number profiles were similar between colorectal can-cer cell lines and primary tumors, with nonhypermutatedcases tending to exhibit chromosomal instability, whereasboth MSI-H and NSHP cases had overall stable copy-numberprofiles. Patterns of whole and partial chromosome gainsand losses were largely concordant. Besides recurrent gain/

amplification of the established colorectal cancer gene MYC,recurrent regions of deletion in nonhypermutated casesincluded the candidate cancer genes PARK2 and WRN. WRNhas important roles in homologous recombination repair,MUTYH-mediated repair of oxidative DNA damage, andtelomeric recombination (34–36), and WRN germline muta-tions are associated with chromosomal instability and can-cer predisposition (37). PARK2 deletion has been previouslyreported in sporadic colorectal cancer, and Park2 deficiencyshown to accelerate intestinal adenoma development in Apcmutant mice (38).

The mutation landscapes in nonhypermutated and hyper-mutated MSI-H cases showed close resemblance in cell linesand primary tumors, with the expected alterations in theWNT,MAPK, PI3K, p53, and TGFb pathways. Besides these maincolorectal cancer–associated pathways, multiple chromatin-state regulators were enriched among the top 5% of mutatedgenes, including proteins involved in chromatin remodeling(ARID1A, CHD6, and SRCAP) and histone methylation oracetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, andTRRAP). Overall,�24%of nonhypermutated and�96%ofMSI-H cases harbored mutations in these genes. Trends for muta-tions to cluster in chromatin regulators have been reported inmultiple other cancer types, including liver cancers (39), gastricadenocarcinoma (40), and transitional cell carcinoma of thebladder (41), suggesting potential pathogenicity of thesealterations.

Paired cell lines originating from the same tumor showedconsiderable differences for both mutations and DNA copy-

Figure 5. Overlap of mutations and DNA copy-number states between paired colorectal cancer cell lines. COLO201/COLO205, CX-1/HT29, Gp2D/Gp5D,LS174T/LS180, and DLD1/HCT8/HCT15 were derived from the same tumor, SW480/SW620 from a primary tumor and subsequent lymph node metastasis.

Mouradov et al.





number profiles. Nonhypermutated pairs differed for up to356 mutations and �54% of genome copy number, whereasMSI-H pairs differed for up to 6,503 mutations and �6.6% ofgenome copy number. The two main possible reasons forthese differences are preexisting heterogeneity between thecells from the original tumor grown out to establish thesepaired lines, or acquisition of alterations as a result of long-term culture. Assuming a normal mutation rate of 10�8 (perbp per cell generation) for nonhypermutated and 100-foldelevated rate for hypermutated pairs and absence of selec-tion (42), cell lines established from tumor cells separated byan average of 328 and 66 replications would be expected toexhibit the observed mutational differences with a >99%probability, respectively (Supplementary Data and Table S9).In contrast, mutations acquired during serial passaging inculture are not anticipated to reach a detectable 10% levelbefore 9,034 and 4,266 replications, respectively (Supplemen-tary Data and Fig. 6). Although serial cell passaging may havebeen common-place at the time when these paired cell lineswere established >20 years ago, stock-keeping practices tolimit the number of replications were soon introduced.Preexisting mutation heterogeneity in the original tumortherefore seems highly likely to account for the majority ofthe detected differences. Numbers of replications may beexpected to be larger between cells from a primary tumorand subsequent metastasis, and accordingly our corre-sponding nonhypermutated cell line pair showed a �4-foldhigher number of mutational differences than the othernonhypermutated pairs. Importantly, mutational differencesacross paired cell lines did not obscure known driver genes,with �88% of nonsilent mutations in established colorectalcancer genes concordant between pairs.Despite the high level of similarity between colorectal cancer

cell lines and primary tumors, there were a number of differ-ences. These included overall higher mutation and DNA copy-number frequencies, as well as a greater proportion of detected

InDels in cell lines. These discrepancies may be related todifferences in exome-capture and sequencing platforms, bio-informatics pipelines, the presence of contaminating normaltissue in primary cancers, and accuracy of assigning somaticalterations in cell lines. Cell lines further showed a higherproportion of hypermutated cases, and exhibited differences inthe prevalence of aberrations for certain genes and genomicregions such as CTNNB1 mutations in MSI-H cases. Theselatterfindingsmay be a reflection of preferential growing out ofcell lines from primary tumors (or their respective subclones)that contain these aberrations, a contention supported by theobservation that only �10% to 15% of colorectal cancers giverise to cell lines (43). Another possibility is that some of themutations have been acquired and selected for in tissueculture.

A caveat to our analysis of protein-coding genes is that wecould not report on untranslated exonic regions (UTR), as thelatter were inconsistently covered by our study and the TCGA.UTR mutations can impact on RNA splicing, stability, ortranslation as previously highlighted for MSI-H colorectalcancer (44). In the comparison of gene mutation profiles, wechose to exclude silent mutations (other than those affectingsplice sites), although some of these may similarly have func-tional consequences (45).

In conclusion, our comparative analysis of the genomiclandscapes of human colorectal cancer cell lines and TCGA-analyzed primary cancers identified cell lines representative ofthe three main mutational colorectal cancer subtypes. Withinthese molecular subtypes, although some differences wereevident, cell lines showed globally similar genetic alterationsto primary cancers, including genome-wide mutation, DNAcopy number, and driver gene mutation profiles. Accordingly,gene expression profiles of colorectal cancer cell lines havepreviously been shown to broadly represent those of primarytumors (46). Our genomic data significantly expand on cancercell line characterization efforts by the major cancer genome

Figure 6. Simulation of the acquisition of mutations in cell culture. The process of serial passage was modeled with cell numbers repeatedly increasingfrom 1 � 105 to 2 � 106 cells. Proportions of cells containing the most frequent mutation by passage number for five simulations, using fixed mutationprobabilities of 10�8 per base per cell replication for nonhypermutated (A) and 10�6 for hypermutated (B) MSI-H tumors. The black diagonal trendlineis the least squares fit on the log–log scale and the small vertical lines are the 99% prediction intervals. The horizontal red line corresponds to a sequencingmutation detection threshold of 10%.






centers, such as the Cancer Cell Line Encyclopedia project,which currently reports mutation data for 1,500 selected geneson 62 colorectal cancer cell lines (5). Our data will help toinform investigations of the molecular basis of colorectalcancer pathogenesis, inherent and acquired drug resistance,and exploration of novel treatment modalities for thismalignancy.

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

Authors' ContributionsConception and design: A.W. Burgess, R.L. Strausberg, J.M. Mariadason,O.M. SieberDevelopment of methodology: D. Mouradov, R.N. Jorissen, S. Li, D. Bicknell,O.M. SieberAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Arango, D. Buchanan, S. Wormald, D. Bicknell,J.M. Mariadason, O.M. SieberAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Mouradov, C. Sloggett, R.N. Jorissen, C.G. Love,A.W. Burgess, D. Arango, D. Buchanan, L. O'Connor, J.L. Wilding, W.F. Bodmer,J.M. Mariadason, O.M. Sieber

Writing, review, and/or revisionof themanuscript:D.Mouradov, C.G. Love,A.W. Burgess, D. Buchanan, J.L. Wilding, I.P.M. Tomlinson, W.F. Bodmer,J.M. Mariadason, O.M. SieberAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. Mouradov, S. Li, S. WormaldStudy supervision: J.M. Mariadason, O.M. Sieber

AcknowledgmentsThe authors thank the Victorian Cancer BioBank for patient specimens and

Prof. M. Schwab at the DKFZ for providing cell lines.

Grant SupportThis work was supported by Cancer Australia through a Project Grant

(APP1030098; O.M. Sieber), Ludwig Institute for Cancer Research (J.M. Maria-dason, A.W. Burgess, O.M. Sieber), NHMRC Overseas Postdoctoral Fellowship(519795; S. Wormald), and a Victorian State Government Operational Infra-structure Support grant. J.M. Mariadason holds an Australian Research CouncilFuture Fellowship.

The costs of publication of this article were defrayed in part 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 7, 2014; revised March 17, 2014; accepted April 8, 2014;published OnlineFirst April 22, 2014.

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2014;74:3238-3247. Published OnlineFirst April 22, 2014.Cancer Res Dmitri Mouradov, Clare Sloggett, Robert N. Jorissen, et al. Molecular Subtypes of Primary CancerColorectal Cancer Cell Lines Are Representative Models of the Main

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