Download Colorectal Cancer Cell Lines Are Representative

Published OnlineFirst April 22, 2014; DOI: 10.1158/0008-5472.CAN-14-0013
Cancer
Research
Molecular and Cellular Pathobiology
Colorectal Cancer Cell Lines Are Representative Models of
the 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
Abstract
Human 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 diversity
of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and
DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression
was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers
in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in
colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included
the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair
and DNA polymerase e proofreading deficiency, along with concordant mutation profiles in the broadly
altered WNT, MAPK, PI3K, TGFb, and p53 pathways. Furthermore, we documented mutations enriched in
genes 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 in
nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines
derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico
simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In
conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes
of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer
biology and drug responses. Cancer Res; 74(12); 1–10. 2014 AACR.
Introduction
Colorectal cancer is a leading cause of cancer-related
morbidity and mortality (1). Human colorectal cancer cell
lines are an important, commonly used preclinical model
system for studying this disease, and have provided essential
insights into tumor molecular and cell biology. Cell lines are
a fundamental tool used in the discovery of new antitumor
compounds and for the discovery of drug sensitivity, resisAuthors' Affiliations: 1Ludwig Institute for Cancer Research; 2Systems
Biology 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, University
of Melbourne, Parkville; 5VLSCI Life Sciences Computation Centre, a
collaboration between Melbourne, Monash and LaTrobe Universities,
c/o The University of Melbourne, Carlton; 6Oncogenic Transcription Laboratory, Ludwig Institute for Cancer Research, Austin, VIC, Australia;
7
Cancer and Population Studies Group, Queensland Institute of Medical
Research, Herston, QLD, Australia; 8Group of Molecular Oncology, CIBBIM-Nanomedicine, Vall d'Hebron University Hospital, Research Institute
noma de Barcelona, Passeig Vall d'Hebron, 119(VHIR), Universitat Auto
129, 08035 Barcelona; 9CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN); Spain; 10Ludwig Collaborative Laboratory for
Cancer Biology and Therapy, Department of Neurosurgery, Johns Hopkins
University School of Medicine, Baltimore, Maryland; 11Ludwig Institute for
Cancer Research Ltd., New York, New York; 12Cancer and Immunogenetics Laboratory, Weatherall Institute of Molecular Medicine (WIMM),
John Radcliffe Hospital, University of Oxford; and 13Molecular and Pop-
tance, and toxicity biomarkers, with molecular markers of
response to conventional chemotherapies and targeted
agents showing clinical utility in patients (2–7). Examples
include the relationship between tumor microsatellite instability-high (MSI-H) status and lack of 5-fluorouracil (5FU)
response (2), and mutations in KRAS, BRAF, and PIK3CA
exon 20 and resistance to anti-EGFR antibody therapy (3).
However, to what extent colorectal cancer cell lines
ulation Genetics Laboratory, Wellcome Trust Centre for Human Genetics,
Oxford, United Kingdom
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: John Mariadason, Oncogenic Transcription
Laboratory, 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: 61-3-94965334. E-mail: [email protected]; and Oliver Sieber, Colorectal Cancer Genetics Laboratory, Systems Biology and Personalised
Medicine Division, Walter and Eliza Hall Institute of Medial Research, 1G
Royal 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 primary
cancers remains controversial.
More than the past two decades, major cancer genes and
pathways central to colorectal cancer development have been
delineated, including the WNT, MAPK, PI3K, TGFb, and p53
pathways. Two broad molecular subtypes of colorectal cancer
have emerged, characterized by MSI-H (15%) or chromosomal instability (CIN, 60%; refs. 8–10). MSI-H colorectal
cancers occur predominantly in the proximal colon, and often
show poor differentiation, mucinous histology, and increased
peritumoral lymphocytic infiltration (11). These tumors exhibit hypermutation caused by defective DNA mismatch repair
(MMR), tend to be near-diploid and to have a CpG island
methylator phenotype (CIMP), and harbor mutations in a
distinct set of driver genes, including BRAF, PTEN, and TGFBR2
(12–14). In contrast, chromosomally unstable tumors are more
common in the distal colorectum and tend to develop along
the classical genetic pathway of colorectal tumorigenesis, with
mutations in APC, KRAS, SMAD4, and TP53 (15).
Recent cancer genome sequencing studies have revealed
additional details of the genetic profiles of human colorectal
cancer, highlighting their diversity. An initial whole-exome
sequencing study on 11 microsatellite stable (MSS) colorectal
cancers demonstrated that such tumors harbor 80 coding
sequence mutations with a small number of commonly mutated driver genes and a large number of "private" mutations (16).
Subsequently, The Cancer Genome Atlas (TCGA) Network
reported comprehensive data on 223 unselected sporadic
colorectal cancers (17). Hypermutation was identified in
15% of carcinomas, with three quarters of these displaying
the expected MSI-high (MSI-H) phenotype associated with
epigenetic silencing and/or mutation of MMR genes. However,
one-quarter of hypermutated tumors did not show MSI-H and
seemed to be associated with DNA polymerase e (POLE)
mutations, perhaps representing a distinct colorectal cancer
subtype. Twenty-four genes were identified as significantly
mutated in colorectal cancer, including several novel candidate genes such as ATM, ARID1A, TCF7L2, SOX9, and FAM123B.
A number of recurrent DNA copy-number alterations were
reported comprising potentially drug-targetable amplifications of ERBB2 and IGF2. A similar study on 74 pairs of primary
human colon tumors reported highly concordant results, and
in addition identified low frequency fusion transcripts involving 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 used
colorectal cancer cell lines, and undertake a detailed comparison of genetic alterations with those reported for TCGAanalyzed primary cancers. We demonstrate that the spectra
of mutations and DNA copy-number aberrations in colorectal
cancer cell lines are representative of primary tumors, including hypermutation phenotypes and targeting of major signaling pathways. Our data further highlight novel aspects of
colorectal cancer biology, including significant enrichment of
mutated genes involved in chromatin remodeling and histone
methylation or acetylation. Our results verify established colorectal cancer cell lines as a useful preclinical model system, and
provide a comprehensive genomic data resource enabling
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informed choices when selecting cell lines for functional and
pharmacogenomics research.
Materials and Methods
Colorectal cancer cell lines and TCGA-analyzed primary
cancers
The 70 colorectal cancer cell lines studied were obtained
from a range of sources, listed below, over a period spanning
several years (Supplementary Table S1). C10, C106, C125, C135,
C32, C70, C80, C84, and C99 were generated by the W.F. Bodmer
laboratory, 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 the
American Type Culture Collection, KM12 was obtained from
DCTD, 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, and
VACO4S were provided by Dr. J.A. McBain (University of
Wisconsin School of Medicine, Madison, WI), SNU-175, SNU283, and SNU-C4 were obtained from KCLB, LIM1215, LIM1899,
LIM2099, LIM2405, and LIM2551 were obtained from the
Ludwig Institute for Cancer Research, SW1222 were provided
by Prof. M. Herlyn (The Wistar Institute, Philadelphia, PA),
HDC101, HDC54, HDC82, HDC87, and HDC90 were provided by
Prof. M, Schwab (DKFZ, Germany), HCA46, HCA7, and HRA19
were provided by Dr. S.C. Kirkland (Royal Postgraduate Medical
School, United Kingdom), and RW2982 and RW7213 were
provided by Dr. P. Calabresi (Roger Williams General Hospital,
Providence, RI). Cells were cultured with Dulbecco's Modified
Eagle Medium and 10% FBS at 37 C and 5% CO2. Cell line
authentication was performed using the Promega StemElite ID
System at the Queensland Institute of Medical Research (QMIR,
Queensland, Australia) DNA Sequencing and Fragment Analysis Facility (January 2013). Cells were tested for mycoplasma
by the MycoAlert Mycoplasma Detection Kit (Lonza). Published exome-capture sequencing data on 223 patients with
colorectal cancer were retrieved from the TCGA (19). SNP array
data were available for 213 of these patients. Mutation data
were filtered for exons and at splice sites (3 bp).
Exome-capture sequencing
Exome-capture was performed using the TruSeq Exome
Enrichment Kit (Illumina) and 100 bp paired-end read
sequencing performed on an Illumina HiSeq 2000 at the
Australian Genome Research Facility (AGRF). Variant detection was implemented using a modified GATK Best Practice
protocol; variants were filtered against known germline
variations and those detected in 114 in-house normal colorectal tissues (Supplementary Methods). Regions of known
germline chromosomal segmental duplications were excluded (20).
Transcriptome sequencing
RNA-Seq analysis was performed at the AGRF on an Illumina
HiSeq2000 to a depth of >100 million paired reads. Alignment
to the human reference genome (hg19) was achieved using
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Genomic Landscape of Colorectal Cancer Cell Lines
TopHat (21), and RPKM values calculated. Absence of gene
expression was defined as a RPKM value of <1 (Supplementary
Methods).
SNP microarray analysis
SNP assays were performed by the AGRF using Illumina
Human610-Quad BeadChips (GSE55832) and processed using
GenomeStudio (Illumina). SNPs showing germline alterations,
based on 637 normal samples, were excluded. DNA copynumber segmentation was performed using OncoSNPv2.18
(22). Regions of significantly altered genome were identified
using GISTIC2.0 (23).
Microsatellite instability analysis
MSI analysis was performed for the National Cancer Institute recommended microsatellite marker panel BAT25, BAT26,
D2S123, D5S346, and D17S250 using fluorescently labeled
primers on a 3130xl Genetic Analyzer (Applied Biosystems;
ref. 24). MSI-H was diagnosed if instability was evident at 2 or
more markers.
CIMP marker and MLH1 promoter methylation analysis
MethyLight real-time PCR was performed for the Weisenberger 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 a
sample against GENE:ALU ratio of template amount in methylated reference DNA. Samples with a PMR greater than 10 for
3 CIMP markers were classified as CIMP-positive.
Statistical analysis
Statistical analyses were conducted using the R statistical
computing software (http://www.R-project.org/). Differences
between groups were assessed using Fisher exact test or x2 test
for categorical variables and the Student t test for continuous
variables. Correlation between RNA-Seq and microarray gene
expression data were assessed using Spearman rank correlation coefficient. Overlap of gene lists was assessed using a
hypergeometric test. GISTIC2.0 analysis for significantly
altered focal regions of chromosomal deletion or gain/amplification were adjusted for multiple testing, implementing a BHFDR adjusted Q-value cut-off of 0.05. All statistical analyses
were 2-sided and considered significant when P < 0.05.
pipeline sensitivity and specificity for nonsilent variants were
shown to be 79.2% and 98.6% in an analysis of 10 primary
colorectal cancers with and without paired normal tissue, with
the majority (93.4%) of false-negative calls resulting from
somatic mutations mimicking annotated germline variants
(Supplementary Data). Accuracy of mutation calling was verified 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 of
97.6% (165/169) of mutations (Supplementary Table S3).
For the 63 unique cell lines, the total number of putative
somatic mutations ranged from 219 to 8657 (mean ¼ 1498.3).
Similar to the primary colorectal cancers reported by The
TCGA (17), the prevalence of mutations varied substantially,
ranging from 6.6 to 260.0 per 106 bases, with evidence for
nonhypermutated and hypermutated groups (Fig. 1). Cell lines
with a mutation prevalence of >25 per 106 bases were designated as hypermutated. Hypermutation was observed in 34.9%
(22/63) of cell lines as compared with 15.7% (35/223) of the
TCGA cancers (P < 0.001). 86.3% (19/22) of hypermutated cell
lines exhibited MSI-H, similar to the proportion found in
cancers (80.0%, 28/35, P ¼ 0.725). MSI-H status in cell lines
was associated with MLH1 hypermethylation, mutations in
MMR genes and presence of CIMP (P < 0.009; Fig. 1). Consistent
with the established association between MSI-H and proximal
tumor location in colorectal cancer, all MSI-H cell lines with
available site details originated from the proximal colon (Supplementary Table S1). There was no evidence of imbalance in
site distribution between cell lines and cancers (P ¼ 0.937).
Mutation spectra in hypermutated colorectal cancer cell
lines
Compared with nonhypermutated cell lines, MSI-H cell lines
displayed a higher number of InDels (41.1-fold; P < 0.001) and
SNVs (9.7-fold; P < 0.001) as observed in the TCGA cancers
Results
Exome mutation profiles in 70 colorectal cancer cell lines
Seventy commonly used colorectal cancer cell lines, comprising 63 unique lines and 7 duplicate lines, were analyzed for
mutations in protein-coding exons and exon–intron boundaries by whole-exome capture sequencing (Supplementary
Table S2). In all cases, >20-fold coverage of >80% of targeted
exons was achieved. As matched normal tissue for these cell
lines was not available, putative somatic mutations were
identified by annotation against databases of known human
germline variants. Regions of known germline chromosomal
segmental duplications were excluded to reduce the possibility
of false-positive variants caused by read mismapping. Mean
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Figure 1. Mutation frequencies in 70 human colorectal cancer cell
lines. Cell lines were separated into distinct hypermutated and
nonhypermutated cases. MSI-H, microsatellite instability-high; CIMP,
CpG island methylator phenotype; MLH1 meth., MLH1 promoter
hypermethylation, MMR mut., DNA mismatch repair gene mutation;
inset, mutations in MMR genes among the hypermutated samples, with
cases in the same order as in the main graph.
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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. MSIH, microsatellite instability-high; NSHP, nucleotide-substitution hypermutator phenotype; POLE EDM, missense mutation in the POLE exonuclease domain.
(InDels: 18.3-fold, SNVs: 10.8-fold; P < 0.001; Fig. 2A and B). As
expected, InDels in MSI-H cases tended to occur at nucleotiderepeat (N5 or NN3) sequences (MSI-H vs. nonhypermutated: 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 SNV
spectrum differed significantly between MSI-H and nonhypermutated cases, with an increased proportion of A>G/T>C
transitions (cell lines 1.5-fold, cancers 1.8-fold; P < 0.011) and
decreased C>G/G>C transversions (cell lines 4.5-fold, cancers 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 and
compared with nonhypermutated cases exhibited a nucleotidesubstitution hypermutator phenotype (NSHP) characterized by
a substantial increase of SNVs (cell lines 16.8-fold, cancers 56.8fold; P < 0.005; Fig. 2A and B). The SNV spectrum in 2 of 3 NSHP
cell lines (HT115, HCC2998) and 7 of 7 NSHP cancers further
displayed increased proportions of C>A/G>T transversions
(MSI-H vs. nonhypermutated: cell lines 1.5-fold, cancers 2.4fold) and A>C/T>G transversions (cell line 3.8-fold, cancers 2.0fold), as well as decreased C>G/G>C transversions (cell lines
13.6-fold, cancers 22.3-fold; Fig. 2C). Combining cell lines and
cancers, this mutator phenotype was significantly associated
with the presence of POLE mutations (NSHP: 90.0%, 9/10 vs. MSIH: 29.8%, 14/47 vs. nonhypermutated: 1.3%, 3/229; P < 0.001). In
addition, all nine NSHP cases with POLE mutation had at least 1
missense mutation in the POLE exonuclease domain (EDM), as
compared with only 1 of 17 non-NSHP cases with POLE mutation
(P < 0.001; Supplementary Table S4). 82.4% (14/17) of POLE
mutated non-NSHP samples were MSI-H, and there was no
evidence that the non-EDM POLE mutations in these cases
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modified the MSI-H phenotype with similar SNV frequencies
and transition/transversion spectra compared with MSI-H
cancers without POLE mutation (P > 0.05 for all comparisons).
A single NSHP cell line, HT55, exhibited a distinct bias to
A>T/T>A transversions (49.0% vs. nonhypermutated mean
4.6%) and was wild type for POLE, but a similar case was not
present among the TCGA cancers (Fig. 2C). This cell line
harbored a heterozygous truncating mutation in the MMR
gene PMS1, which may be related to this mutator phenotype,
although Pms1-deficient mice have been reported to show no
evidence of tumor predisposition or hypermutation (26).
Chromosomal and subchromosomal aberrations
DNA 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 and
primary cancers (Fig. 3A). Hypermutated MSI-H and NSHP
cases showed stable profiles with a modal chromosome copynumber of 2n. In contrast, nonhypermutated cases tended to
exhibit unstable profiles with modal chromosome copy numbers ranging from 2n to 4n in cell lines and 2n to 6n in cancers.
For nonhypermutated groups, the most common deleted
chromosome arms were 8p, 17p (including TP53), and 18q
(including SMAD4), and the most common gained regions were
chromosome 7, 8q (including MYC), 13, and 20q. Some differences were apparent between cell lines and primary cancers,
including more frequent chromosome 4 deletions in nonhypermutated and chromosome 7 gains and 18q deletions in
hypermutated cell lines.
Focal regions significantly targeted by DNA copy-number
alterations in both cell lines and cancers were deduced using
GISTIC2.0 software (23). Eleven and 4 significant focal regions
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Genomic Landscape of Colorectal Cancer Cell Lines
Figure 3. Genome-wide DNA copynumber aberrations in 63 unique
colorectal cancer cell lines and 213
TCGA primary cancers stratified
into nonhypermutated and
hypermutated cases. A, absolute
DNA copy-number gains and
losses relative to diploid status (2n)
deduced using oncoSNP. B and C,
minimal significant regions of
chromosomal loss (blue) and gain/
amplification (red) deduced using
GISTIC2.0. Selected candidate
genes in overlapping regions are
indicated and total numbers of
genes are shown in brackets.
were found to overlap between nonhypermutated and hypermutated groups, respectively (Fig. 3B and C and Supplementary Table S5). For nonhypermutated cases, overlapping
regions included gain/amplification of MYC, a known key
mediator of colorectal tumorigenesis (27), and deletion of
the candidate cancer genes PARK2 and WRN, detected in
29.5% and 36.3% of cell lines and 21.9% and 31.6% of primary
cancers, respectively. The 4 recurrent regions identified in
hypermutated cases—which were also present in the nonhypermutated group—were all known fragile sites (including
FHIT, A2BP1, WWOX, or MACROD2), the functional relevance
of which is uncertain.
Cancer genes and pathways
Gene mutation profiles, excluding silent mutations, were
compared between colorectal cancer cell lines and primary
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cancers. Only genes with well-defined expression in cell lines
[reads per kilobase per million reads mapped (RPKM) >1 in at
least 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 of
the small sample size.
Mutation landscapes of cell lines markedly resembled those
of primary cancers. In both cohorts, nonhypermutated cases
displayed a small number of distinct mutation peaks, with APC,
TP53, and KRAS being the most common targets, whereas
hypermutated MSI-H cases showed frequent mutations in
multiple genes, with the anticipated bias to genes comprising
nucleotide repeats (Supplementary Tables S7). Significant overlap was observed for the top 5% of mutated genes (based on the
proportion of affected samples), with 54 and 62 genes intersecting for nonhypermutated and MSI-H groups, respectively
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Integrating mutation and DNA copy-number data across
cell lines and primary cancers showed the anticipated tumor
suppressor signatures for APC, TP53, SMAD4, and SOX9 with a
significant overrepresentation of 2 mutational hits (2þ mutations or 1 mutation and loss of heterozygosity; P < 0.023). There
was further an association of mutation in BRAF, ERBB3,
PIK3CA, and KRAS with copy-number gain of 5 at the
respective chromosomal regions (P < 0.018). Trends to mutual
exclusivity between pathway member mutations were
observed for KRAS and BRAF, TP53, and ATM (P < 0.005).
Figure 4. Mutation frequencies for WNT, MAPK, PI3K, TGFb, and p53
pathway members as well as chromatin regulators identified among the
top 5% of mutated genes overlapping between colorectal cancer cell
lines and TCGA-analyzed primary cancers for nonhypermutated or
hypermutated MSI-H samples.
(P < 0.001; Supplementary Tables S8). Overlapping genes
included the expected members of the WNT, MAPK, PI3K,
TGFb, and p53 pathways. For nonhypermutated cases, these
comprised APC, 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-H
cases these encompassed APC, FBXW7, PIK3CA, and TGFBR2,
along with the candidates CREBBP, TCF7L2, RNF43, and
ACVR2A. Mutation distributions across colorectal cancerassociated signaling pathways were also similar between
nonhypermutated cell lines and cancers, with the same
pathway members tending to show the highest mutation
frequencies (Fig. 4). Greater variability in mutation frequencies between pathway members was observed for MSI-H
cases, at least partly related to the higher mutation background and smaller sample size. A notable difference for
MSI-H cases was a differential prevalence of CTNNB1 mutations, which were frequent in cell lines (47%) but not
reported in primary cancers (P < 0.001).
In addition to the established colorectal cancer-associated
pathways, significant enrichment was observed for mutations in chromatin-state regulators (P < 0.001; gene ontology
enrichment analysis). These comprised ASH1L, CHD6,
PRDM2, and MLL3 in nonhypermutated and ARID1A, EP300,
EP400, MLL2, CHD6, PRDM2, TRRAP, and SRCAP in MSI-H
cases (Supplementary Table S8). Approximately 49% and
19% of nonhypermutated and 100% and 93% of MSI-H cell
lines and primary cancers carried mutations across these
candidates, respectively.
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Mutation and DNA copy-number differences in paired
colorectal 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, and
DLD1/HCT8/HCT15), and 1 pair derived from a primary tumor
and subsequent lymph node metastasis (SW480/SW620).
LS174T and LS180 were established by trypsin treatment and
scraping of primary cultures from the same tumor, respectively
(28), and have been shown to differ with respect to E-cadherin
expression and cell adhesion, with LS174T displaying complete
loss of E-cadherin protein (2, 29).
Assessment of the overlap between mutations detected in
paired cell lines identified substantial discrepancies, with 63
and 356 mutational differences in the nonhypermutated pairs
COLO201/COLO205 and CX-1/HT29, and 2,763, 480, 6,503,
5,377, and 6,369 mutational differences in the MSI-H pairs
Gp2D/Gp5D, LS174T/LS180, DLD1/HCT8, DLD1/HCT15, and
HCT8/HCT15 (Fig. 5). Eight hundred and forty-nine mutations
differed in the nonhypermutated primary/metastasis pair
SW480/SW620. Nonsilent and silent mutations contributed in
similar proportions to these discrepancies (54.4% vs. 56.6%),
suggesting no selection for these differential alterations.
DNA copy-number profiles showed multiple differences for
nonhypermutated pairs, with 41.1% and 53.8% of the genome
varying for COLO201/COLO205 and CX-1/HT29 (Fig. 5). In
contrast, discrepancies were limited in MSI-H pairs with 0.2%,
0.4%, 6.6%, 2.9%, and 4.3% of the genome differing between
Gp2D/Gp5D, LS174T/LS180, DLD1/HCT8, DLD1/HCT15, and
HCT8/HCT15, respectively. 47.5% of the genome differed in the
nonhypermutated primary/metastasis pair SW480/SW620.
Notably, mutation differences between paired cell lines did
not obscure known driver genes. For the established colorectal
cancer genes APC, TP53, SMAD4, PIK3CA, KRAS, and BRAF, 29 of
33 (87.9%) nonsilent mutations were concordant between cell
line pairs.
Discussion
In this study we show that the mutation and DNA copynumber landscapes determined for 70 colorectal cancer cell
lines closely resemble those of primary tumors, underscoring
the utility of cell lines as an appropriate model system for this
malignancy. The 3 molecular subtypes of colorectal cancer
recently defined by the TCGA, nonhypermutated, hypermutated with microsatellite instability, and hypermutated without microsatellite instability were faithfully captured in the
cell line panel. As expected, MSI-H cell lines exhibited
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Genomic Landscape of Colorectal Cancer Cell Lines
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.
hypermethylation of the MLH1 promoter and/or mutations in
MMR genes, whereas hypermutated lines without microsatellite instability were instead characterized by mutations in the
exonuclease domain of POLE. Consistent with defective DNA
POLE proofreading function, the latter tumors showed a
nucleotide substitution hypermutator phenotype with a bias
to C>A/G>T and A>C/T>G transversions. Germline missense
mutations in the POLE exonuclease domain have recently been
associated with familial predisposition to colorectal cancer
(30), and elevated tumor predisposition and base-substitution
mutations observed in Pole exonuclease-mutant mice (31).
Similarly, somatic missense mutations in the POLE exonuclease domain have been reported in 7% of endometrial cancers,
with good evidence of associated hypermutation (32).
Although differences in prognosis and chemotherapy response
have been extensively documented for nonhypermutated versus MSI-H tumors (2–4), clinical characteristics of POLEmutant NSHP tumors are unknown. Our identification of 2
cell lines representative of this colorectal cancer subtype will
facilitate an investigation of the specific aspects of the biology
of these tumors, particularly in relation to identifying therapeutics that may exploit their unique genomic instability.
Consistent with previous lower-resolution data (33), DNA
copy-number profiles were similar between colorectal cancer cell lines and primary tumors, with nonhypermutated
cases tending to exhibit chromosomal instability, whereas
both MSI-H and NSHP cases had overall stable copy-number
profiles. Patterns of whole and partial chromosome gains
and losses were largely concordant. Besides recurrent gain/
www.aacrjournals.org
amplification of the established colorectal cancer gene MYC,
recurrent regions of deletion in nonhypermutated cases
included the candidate cancer genes PARK2 and WRN. WRN
has important roles in homologous recombination repair,
MUTYH-mediated repair of oxidative DNA damage, and
telomeric recombination (34–36), and WRN germline mutations are associated with chromosomal instability and cancer predisposition (37). PARK2 deletion has been previously
reported in sporadic colorectal cancer, and Park2 deficiency
shown to accelerate intestinal adenoma development in Apc
mutant mice (38).
The mutation landscapes in nonhypermutated and hypermutated MSI-H cases showed close resemblance in cell lines
and primary tumors, with the expected alterations in the WNT,
MAPK, PI3K, p53, and TGFb pathways. Besides these main
colorectal cancer–associated pathways, multiple chromatinstate regulators were enriched among the top 5% of mutated
genes, including proteins involved in chromatin remodeling
(ARID1A, CHD6, and SRCAP) and histone methylation or
acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and
TRRAP). Overall, 24% of nonhypermutated and 96% of MSIH cases harbored mutations in these genes. Trends for mutations to cluster in chromatin regulators have been reported in
multiple other cancer types, including liver cancers (39), gastric
adenocarcinoma (40), and transitional cell carcinoma of the
bladder (41), suggesting potential pathogenicity of these
alterations.
Paired cell lines originating from the same tumor showed
considerable differences for both mutations and DNA copy-
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Mouradov et al.
Figure 6. Simulation of the acquisition of mutations in cell culture. The process of serial passage was modeled with cell numbers repeatedly increasing
5
6
from 1 10 to 2 10 cells. Proportions of cells containing the most frequent mutation by passage number for five simulations, using fixed mutation
8
6
probabilities of 10 per base per cell replication for nonhypermutated (A) and 10 for hypermutated (B) MSI-H tumors. The black diagonal trendline
is 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 sequencing
mutation detection threshold of 10%.
number profiles. Nonhypermutated pairs differed for up to
356 mutations and 54% of genome copy number, whereas
MSI-H pairs differed for up to 6,503 mutations and 6.6% of
genome copy number. The two main possible reasons for
these differences are preexisting heterogeneity between the
cells from the original tumor grown out to establish these
paired lines, or acquisition of alterations as a result of longterm culture. Assuming a normal mutation rate of 108 (per
bp per cell generation) for nonhypermutated and 100-fold
elevated rate for hypermutated pairs and absence of selection (42), cell lines established from tumor cells separated by
an average of 328 and 66 replications would be expected to
exhibit the observed mutational differences with a >99%
probability, respectively (Supplementary Data and Table S9).
In contrast, mutations acquired during serial passaging in
culture are not anticipated to reach a detectable 10% level
before 9,034 and 4,266 replications, respectively (Supplementary Data and Fig. 6). Although serial cell passaging may have
been common-place at the time when these paired cell lines
were established >20 years ago, stock-keeping practices to
limit the number of replications were soon introduced.
Preexisting mutation heterogeneity in the original tumor
therefore seems highly likely to account for the majority of
the detected differences. Numbers of replications may be
expected to be larger between cells from a primary tumor
and subsequent metastasis, and accordingly our corresponding nonhypermutated cell line pair showed a 4-fold
higher number of mutational differences than the other
nonhypermutated pairs. Importantly, mutational differences
across paired cell lines did not obscure known driver genes,
with 88% of nonsilent mutations in established colorectal
cancer genes concordant between pairs.
Despite the high level of similarity between colorectal cancer
cell lines and primary tumors, there were a number of differences. These included overall higher mutation and DNA copynumber frequencies, as well as a greater proportion of detected
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Cancer Res; 74(12) June 15, 2014
InDels in cell lines. These discrepancies may be related to
differences in exome-capture and sequencing platforms, bioinformatics pipelines, the presence of contaminating normal
tissue in primary cancers, and accuracy of assigning somatic
alterations in cell lines. Cell lines further showed a higher
proportion of hypermutated cases, and exhibited differences in
the prevalence of aberrations for certain genes and genomic
regions such as CTNNB1 mutations in MSI-H cases. These
latter findings may be a reflection of preferential growing out of
cell lines from primary tumors (or their respective subclones)
that contain these aberrations, a contention supported by the
observation that only 10% to 15% of colorectal cancers give
rise to cell lines (43). Another possibility is that some of the
mutations have been acquired and selected for in tissue
culture.
A caveat to our analysis of protein-coding genes is that we
could not report on untranslated exonic regions (UTR), as the
latter were inconsistently covered by our study and the TCGA.
UTR mutations can impact on RNA splicing, stability, or
translation as previously highlighted for MSI-H colorectal
cancer (44). In the comparison of gene mutation profiles, we
chose to exclude silent mutations (other than those affecting
splice sites), although some of these may similarly have functional consequences (45).
In conclusion, our comparative analysis of the genomic
landscapes of human colorectal cancer cell lines and TCGAanalyzed primary cancers identified cell lines representative of
the three main mutational colorectal cancer subtypes. Within
these molecular subtypes, although some differences were
evident, cell lines showed globally similar genetic alterations
to primary cancers, including genome-wide mutation, DNA
copy number, and driver gene mutation profiles. Accordingly,
gene expression profiles of colorectal cancer cell lines have
previously been shown to broadly represent those of primary
tumors (46). Our genomic data significantly expand on cancer
cell line characterization efforts by the major cancer genome
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Published OnlineFirst April 22, 2014; DOI: 10.1158/0008-5472.CAN-14-0013
Genomic Landscape of Colorectal Cancer Cell Lines
centers, such as the Cancer Cell Line Encyclopedia project,
which currently reports mutation data for 1,500 selected genes
on 62 colorectal cancer cell lines (5). Our data will help to
inform investigations of the molecular basis of colorectal
cancer pathogenesis, inherent and acquired drug resistance,
and exploration of novel treatment modalities for this
malignancy.
Writing, review, and/or revision of the manuscript: 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. Sieber
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Mouradov, S. Li, S. Wormald
Study supervision: J.M. Mariadason, O.M. Sieber
Acknowledgments
The authors thank the Victorian Cancer BioBank for patient specimens and
Prof. M. Schwab at the DKFZ for providing cell lines.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
Authors' Contributions
Conception and design: A.W. Burgess, R.L. Strausberg, J.M. Mariadason,
O.M. Sieber
Development of methodology: D. Mouradov, R.N. Jorissen, S. Li, D. Bicknell,
O.M. Sieber
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): D. Arango, D. Buchanan, S. Wormald, D. Bicknell,
J.M. Mariadason, O.M. Sieber
Analysis 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
This work was supported by Cancer Australia through a Project Grant
(APP1030098; O.M. Sieber), Ludwig Institute for Cancer Research (J.M. Mariadason, A.W. Burgess, O.M. Sieber), NHMRC Overseas Postdoctoral Fellowship
(519795; S. Wormald), and a Victorian State Government Operational Infrastructure Support grant. J.M. Mariadason holds an Australian Research Council
Future Fellowship.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance 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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Colorectal Cancer Cell Lines Are Representative Models of
the Main Molecular Subtypes of Primary Cancer
Dmitri Mouradov, Clare Sloggett, Robert N. Jorissen, et al.
Cancer Res Published OnlineFirst April 22, 2014.
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