Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921
Cancer
Research
Tumor and Stem Cell Biology
Somatic Ephrin Receptor Mutations Are
Associated with Metastasis in Primary
Colorectal Cancer
€ m1, Jessica Svedlund2, Lotte Moens1,
Lucy Mathot1, Snehangshu Kundu1, Viktor Ljungstro
1
1
1
€ rqvist ,Vero
nica Rendo , Claudia Bellomo3, Markus Mayrhofer4,
Tom Adlerteg , Elin Falk-So
5
1
€ m , Patrick Micke1, Johan Botling1, Anders Isaksson4,
Carme Cortina , Magnus Sundstro
3
Aristidis Moustakas , Eduard Batlle5, Helgi Birgisson6, Bengt Glimelius1, Mats Nilsson1,2,
€ blom1
and Tobias Sjo
Abstract
The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. To find mutations involved in
the colorectal cancer metastatic process, we performed deep
mutational analysis of 676 genes in 107 stages II to IV primary
colorectal cancer, of which half had metastasized. The mutation
prevalence in the ephrin (EPH) family of tyrosine kinase receptors
was 10-fold higher in primary tumors of metastatic colorectal
than in nonmetastatic cases and preferentially occurred in stage III
and IV tumors. Mutational analyses in situ confirmed expression
of mutant EPH receptors. To enable functional studies of EPHB1
mutations, we demonstrated that DLD-1 colorectal cancer cells
expressing EPHB1 form aggregates upon coculture with ephrin
B1 expressing cells. When mutations in the fibronectin type III
and kinase domains of EPHB1 were compared with wild-type
EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin
B1–induced compartmentalization. These observations provide a
mechanistic link between EPHB receptor mutations and metastasis in colorectal cancer. Cancer Res; 77(7); 1730–40. 2017 AACR.
Introduction
colorectal cancer patients into those at a higher risk of developing metastatic disease is of great clinical importance, as the
decision to give adjuvant therapy following resection of the
primary tumor is difficult (4). Many patients who may never
develop metastatic disease receive chemotherapy resulting in
unnecessary side effects, and a number of patients who would
benefit from elimination of micrometastases following radical
resection remain unidentified. Discovery of mutations that can
predict development of metastatic disease would help to reduce
the number of patients who are overtreated, and could help to
increase the overall survival for higher risk patients.
Although several features of cancer genomes have been
associated with the dissemination of colorectal cancer, specific gene mutations related to metastatic processes have yet to
be confirmed. For example, loss of 1p36 has consistently been
associated with metastatic disease (5, 6) and mutations in the
tumor suppressor FBXW7 did not occur with distant metastases (7, 8). If metastasis-causing mutations arise late in
tumor development, they may be subclonal and therefore
present at a low frequency in the primary tumor (9). For this
reason, and as the vast majority of genetic events known to
contribute to colorectal cancer development are base-level
somatic mutations, we applied targeted deep sequencing to
determine the mutational spectrum of pathways and systems
associated with colorectal cancer development. Our aim was
to analyze tumor samples from metastatic and nonmetastatic
patients in order to identify less frequently mutated candidate
cancer genes with high sensitivity to gain insight into the
mutational spectrum of these tumors and discover novel
mechanisms of metastasis.
Colorectal cancer develops due to a series of well-characterized mutations that arise in a particular order and affect both
oncogenes and tumor suppressor genes (TSG; ref. 1). The
progression of colorectal cancer has been extensively studied
and characterized on a genetic level, due to the availability of
biopsies from various stages in disease progression, that is, from
benign polyps to advanced carcinomas (2). However, while
much is known about frequent mutations causing colorectal
cancer, the genetic basis of metastasis is unclear (3). Stratifying
1
Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Sweden. 2Science for Life Laboratory, Department of
Biochemistry and Biophysics, Stockholm University, Solna, Sweden. 3Department of Medical Biochemistry and Microbiology, Ludwig Cancer Research,
Science for Life Laboratory, Uppsala University, Sweden. 4Science for Life
Laboratory, Department of Medical Sciences, Uppsala University, Sweden.
5
Oncology Programme, Institute for Research in Biomedicine (IRB Barcelona),
The Barcelona Institute of Science and Technology, Barcelona, Spain. 6Department of Surgical Sciences, Colorectal Surgery, Uppsala University, Sweden.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
L. Mathot and S. Kundu contributed equally to this work.
€blom, Uppsala University, IGP, Dag HamCorresponding Author: Tobias Sjo
€ld v 20, SE-751 85 Uppsala, Sweden. Phone: 46184715036; Fax:
marskjo
46184714808; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-16-1921
2017 American Association for Cancer Research.
1730 Cancer Res; 77(7) April 1, 2017
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Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921
Eph Receptor Mutations in Colorectal Cancer Are Associated with Metastasis
Table 1. Summary of patients and sequencing
Instability phenotype
Distant metastases
Stage
Cases
Postoperative adjuvant
therapy
Median no. of mutations
per sample (range)
Point mutations
InDels
No
CIN (n ¼ 83)
II
15
1
III
16
11
Yes, during follow-up
II
III
7
20
1
7
10
(2–13)
111
21
9.5
(6–16)
146
12
9
(3–17)
55
10
9
(1–36)
181
25
MSI (n ¼ 24)
Yes, at
diagnosis
IV
25
—
II
8
0
III
10
8
8
(3–33)
221
30
83
(7–122)
330
201
69.5
(10–161)
530
228
No
Yes, during follow-up
II
III
2
2
0
0
81.5
(61–102)
99
64
87
(82–92)
112
62
Yes, at
diagnosis
IV
2
—
128.5
(87–170)
169
88
NOTE: The protein coding regions of 676 genes in 107 T/N pairs, representing 100 colon cancer and 7 rectal cancer cases, were enriched and sequenced by Illumina
sequencing to >1,000-fold average sequence depth followed by mutational analysis. Tumors were considered MSI if 1 of five Bethesda markers showed instability.
Materials and Methods
Study design
We aimed to collect 20 to 25 cases each of stages II and III
colorectal cancers with and without distant metastases, as well as
25 stage IV cases (6, 10). Fresh frozen tissue and whole blood
samples from 112 colorectal cancer patients [224 matched tumor/
normal (T/N) paired samples] were collected (Table 1). Sequencing libraries prepared from gDNA extracted from all patients
(tumor and matched normal tissue or blood) was sequenced on
an Illumina HiSeq platform and a somatic mutation analysis was
performed. All data were included if the T/N pairs were correctly
matched, a read depth >250-fold was obtained for >90% of the
sequencing region of interest and the tumor was >40%.
Patient samples
DNA was extracted from 196 frozen tissue samples (3 10-mmthick sections) on a liquid handling workstation (Tecan Evo 150
MCA LiHa RoMa; ref. 11). DNA from 28 EDTA whole blood
samples was extracted using a QIAamp DNA Blood Midi Kit
(Qiagen). DNA was quantified using the Qubit HS dsDNA Kit
(Invitrogen by Life Technologies). For the validation cohort of
metastatic colorectal cancer samples used for BMPR2 mutation
detection, DNA from 19 microsatellite unstable (MSI) FFPE
tumor sections was extracted using the QIAamp DNA FFPE
Tissue Kit (Qiagen).
Tumor tissue purity, determination of genomic stability,
and T/N matching
Affymetrix SNP 6.0 microarrays were used to assess tumor
purity and to confirm that samples had 40% TCC as initially
assessed histologically. Microsatellite instability status was determined using MSI Analysis System, version 1.2 (ProMega) with
6 ng genomic DNA and analysis of five mononucleotide repeat
Bethesda markers (BAT25, BAT26, NR-21, NR-24, and MONO27) on a 3130xl genetic analyzer (Applied Biosystems). T/N pair
matching was performed using an MLGA based genotyping
method described in ref. 12.
Target enrichment by HaloPlex and Illumina sequencing
Genomic DNA (225 ng) from tumor and normal tissue or
blood was used in HaloPlex target enrichment (Agilent) for the
enrichment of the coding regions of 676 genes. The quality and
molarity of the sequencing libraries was assessed on a Bioanalyzer
instrument using a High Sensitivity DNA Kit (Agilent). The
enriched and barcoded targets were then deep sequenced on a
next-generation sequencing HiSeq platform (Illumina). The frac-
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tion coverage of the region of interest was required to be greater
than 90% and the mean read depth >600-fold. Samples not
passing these criteria on the first attempt were resequenced and
sequencing data from both runs was merged. Samples not exceeding >600-fold mean read depth thereafter were accepted if the
mean read depth was greater than 250-fold (three T/N pairs).
Statistical analyses
Illumina sequencing adaptors were removed by cutAdapt version 0.9.5 (13) and the trimmed reads were subsequently aligned
to the reference genome (hg19, March 2009 assembly) using
MosaikAligner version 2.1.33 allowing for a maximum of 5%
mismatches in a read. A software for somatic mutation analysis in
deep sequence datasets, ConfIdent (Adlerteg and colleagues;
manuscript in preparation), was used for somatic mutation
detection. We assessed which genes were significantly mutated
in CIN and MSI tumors using a combination of MutSigCV (14),
number of nonsynonymous mutations per Mb and the nonsynonymous to synonymous ratio (NS: S). We used the following
cut-offs for CIN tumors: Q-value of <0.1 for MutSigCV output, >1
mutation per Mb, and >2:1 NS: S ratio. Differences in the average
number of mutations and in mutation prevalence between metastatic and nonmetastatic groups were assessed using Welch t test
and Fisher exact test. The Bonferroni method was used to correct
for multiple comparisons.
In situ mutation analysis with padlock probes
Fresh frozen 4 mm sections of colon tumors with EPH receptor
mutations were mounted on Superfrost Plus slides (ThermoFisher
Scientific). Because of limited sample availability, a total of 15
EPH receptor mutations in 12 tumors were assessed by in situ
mutation detection (two tumor sections contained two separate
sections, called 1 and 2). Probes specific for EPH receptor mutations were designed with one general and one specific detection
oligo sequence (Supplementary Table S1). Probe performance
was first tested in T47D and U251 cell lines with known ephrin
receptor mRNA expression levels (Supplementary Table S2). The
in situ detection method was performed as outlined in Grundberg
and colleagues (15). Briefly, tissue sections and cells were fixed in
3.7% PFA for 45 or 20 minutes, respectively. Next, mRNA was
reverse transcribed using specific LNA primers (Supplementary
Table S3) amplifying wild-type and mutated regions of the tumorspecific EPH receptor mutations. Single-stranded cDNA was created through Rnase H cleavage and padlock probes were hybridized and ligated, followed by rolling circle amplification. Rolling
circle products (RCP) were identified through fluorophorelabeled detection oligos (Supplementary Table S4) and the nuclei
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1731
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Mathot et al.
was stained with 4',6-diamidino-2-phenylindole (DAPI). The
tissues and cells were then imaged with an automated Zeiss
Axioplan II epifluorescence microscope (Zeiss) using a Z-stack
of 0.49 mm 6 and a tile overlap of 10%. Images were orthogonally processed and tiles were stitched together using ZEN
software (Zeiss). The colon sections contained features that were
highly autofluorescent; therefore, each RCP was dual-labeled (one
general stain and one specific stain) to minimize false-positive
signals. Moreover, for each RCP all fluorophore intensities were
measured and a quality score was applied to call the correct signal
(16). A quality score from 0.5 to 0.8 was used depending on the
autofluorescence in each tumor section. Images were analyzed in
Cellprofiler v.2.1.1 calling ImageJ plugins (Broad Institute, MA)
and specific signals were called using a Matlab script (v.8.5.1,
Mathworks). All tumor sections were then stained for hematoxylin and eosin (Sigma), imaged and aligned to the DAPI (nuclei)
stain of the fluorescent images.
Cell lines and cell culture
Parental DLD-1 (CCL-221) cells were purchased from ATCC, in
2010. All the DLD-1 cell lines used in this study were authenticated by short tandem repeat (STR) profiling from the ATCC cell
line authentication service in October 2016. DLD-1 cells ectopically expressed EPHB1-GFP and its four mutant versions were
generated by lentiviral transductions. DLD-1 cells ectopically
expressing GFP, RFP, EPHB2-GFP, and ephrin B1-RFP were from
ref. 17. All cell lines were maintained in DMEM (Invitrogen)
medium supplemented with 10% FBS and 1% penicillin–streptomycin (Invitrogen) at 37 C in 5% CO2.
Generation of stable ectopic expressing cell lines of wild-type
and mutant EPHB1
Lentiviral particles were purchased from Labomics. The day
before transduction, 50,000 cells were plated in each well of a 24well plate. Viruses were diluted in 250 mL of normal growth
medium with 7.5 mg/mL Sequa-Brene per well. The plating
medium was removed and 250 mL of diluted virus was added to
each well. After 24 hours of incubation at 37 C, virus containing
media were replaced with fresh medium. After 48 hours of
incubation, transduced cells were selected with Puromycin (1
mg/mL) for two to four passages to remove any Puromycinresistant cells in the cell pool. Expression levels of EPHB1 and
its four different mutated transcripts were determined by qPCR.
In vitro compartmentalization experiments
Compartmentalization experiments were performed as
described in ref. 17. Briefly, DLD-1 cells expressing GFP and RFP
were mixed in suspension at a 1:3 ratio and plated at a density of
130,000 cells/cm2 on coverslips coated with 2 mg/cm2 laminin
and incubated at 37 C in 5% CO2. Culture medium was changed
every 24 hours. After 48 hours, the coverslips were fixed in 4%
paraformaldehyde and mounted in Fluoromount G with DAPI
(SouthernBiotech). This experiment was performed twice.
Confocal image analysis and quantitation of GFP clusters by
ImageJ software
Slides from compartmentalization experiments were subjected to confocal (LSM 700) image analysis. Images were
acquired from five random fields with 20 objective and two
confocal planes in z-axis from two experiments. Cell sorting
was quantified by counting the number of cells present in each
1732 Cancer Res; 77(7) April 1, 2017
GFP-positive cluster of approximately 10 representative fields at
two different confocal planes in the z-axis and from two experimental repeats by creating an ImageJ macro. Images throughout the paper show the basal plane.
Ethical approval
This study was approved by the Regional Ethical Review Board
of Uppsala (2007/116).
Biosafety declaration
The Swedish work environment authority approved the work
with genetically modified and replication-deficient lentiviral particles (Arbetsmilj€
overket ID 202100-2932 v72). All the experiments with GMO lentiviral particles were conducted under Biosafety Level 2.
Results
Samples and mutation frequency
Tumor and normal tissues from 112 patients exhibiting stages
II, III, and IV colorectal cancer, of which approximately half (58
patients) had metastasized either at diagnosis or during follow-up
were analyzed (Table 1). Five T/N pairs were excluded due to
contamination of the normal sample with tumor tissue or a failed
sequencing library preparation. The coding regions and adjacent
splice sites of 676 genes (Supplementary Table S5) were enriched
in the remaining samples (Supplementary Table S6) using HaloPlex (Agilent) and sequenced. Genes were chosen on a pathwayoriented basis, including members of canonical colorectal cancer
pathways such as Wnt, Ras-MAPK, PI3K, p53, and TGFb as well as
families and processes with a putative role in colorectal cancer.
The mean read depth in regions of interest was 1,063-fold (range
256–3516) in tumor samples and 1,103-fold (range 258–14,956)
in the normal samples. A conservative mutational analysis tool
(ConfIdent, see Supplementary Methods) was applied to call
somatic mutations at base positions covered by 30 reads in
both tumor and patient-matched normal sample. We identified
3,392 somatic mutations, of which 696 were synonymous single
nucleotide variations (SNV), 1,941 nonsynonymous SNVs, 717
insertion/deletion polymorphisms (InDels), and 38 splice site
mutations in the genes of interest (Supplementary Table S7). The
chromosomally unstable (CIN) tumors had an average of 9.9
nonsynonymous somatic mutations (SNV, InDels, and splicing
mutations) per patient, whereas the MSI tumors had 75.4 such
mutations in the 676 genes of interest (Fig. 1A; Supplementary
Table S7). We then assessed the genes with the highest nonsynonymous mutation density in both CIN and MSI tumors to confirm
expected mutation patterns. In CIN tumors, these were APC, TP53,
KRAS, SMAD4, PIK3CA, and BRAF. In contrast, the genes with the
highest nonsynonymous mutation density in MSI tumors were
ACVR2A, SETD1B, TGFBR2, BMPR2, BRAF, and TCF7L2 (Fig. 1B
and C). The recurrent frameshift InDels in SETD1B and BMPR2
were validated by Sanger sequencing (Supplementary Materials
and Methods). Using a separate cohort of 19 stage IV MSI-high
colorectal cancers, we sequenced the region of BMPR2 containing
this mutation by Sanger sequencing and found that 31% (6/19) of
tumors had this mutation. Using a luciferase reporter assay to
measure BMP pathway activity using 293T cell lines overexpressing either the mutant or wild-type BMPR2 protein, we observed a
downregulation of activity when overexpressing the mutant form
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Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921
Eph Receptor Mutations in Colorectal Cancer Are Associated with Metastasis
A
Mutation rate (Mutation per MB)
100
10
1
Non silent
Silent
0,1
Hypermutated
Non-hypermutated
MSI Status
Metastasis
40
15%
16%
20
17%
30
15
60%
48%
20
72%
No. of mutations
50
43%
No. of mutations
60
80%
25
70
64%
30
76%
80
C
68%
90
72%
B
10
5
10
0
0
Figure 1.
Deep targeted mutational analyses of metastatic and nonmetastatic primary colorectal cancers. A, Number of mutations per sample in 676 genes in colorectal
cancer pathways and systems. Red bars, MSI-high tumors; blue bars, MSI-low tumors; empty bars, CIN tumors. Black bars, patients metastatic at diagnosis;
orange bars, patients that developed metastases; empty bars, patients that did not develop metastases. B and C, Genes with the highest mutation prevalence in CIN
tumors (B) and MSI tumors (C). The percentage of tumors with mutation is indicated. Black bars, missense mutations; blue bars, frameshift InDels; red bars,
nonsense mutations; grey bars, splicing mutations; empty bars, silent mutations.
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Mathot et al.
of BMPR2, although this difference was not significant (Supplementary Methods and Supplementary Fig. S1).
Enrichment of Eph receptor mutations in metastatic tumors
The aim of the study was to investigate if alterations in a
particular pathway or gene group could predict the metastasis of
CIN tumors. The average number of mutations per sample did not
differ between the metastatic and nonmetastatic groups in CIN
tumors (10 vs. 9.4, P ¼ 0.71, Welch two-sample t test). We did not
find any significant differences in mutation prevalence between
the metastatic and nonmetastatic samples in any of the pathways
or large gene families included in the study (Supplementary Table
S8). To assess enrichment of biological themes in the metastatic
samples compared with the nonmetastatic, we compared the 50
genes with the highest number of nonsynonymous mutations per
Mb in each group. Genes found only in the metastatic group were
analyzed using the DAVID functional annotation tool (18),
revealing an enrichment of the Ephrin receptor family (Supplementary Methods and Supplementary Table S9). The mutation
prevalence of EPH receptor genes in CIN patients with metastases
(18/52 of stage IV and stage II and III tumors that later developed
distant metastases) versus those that did not develop metastases
(2/31 patients) was significantly different (P ¼ 0.0032, Fisher
exact test; Supplementary Table S10). There was no difference in
sequencing read depth in any of the EPH receptor genes between
the metastatic and nonmetastatic groups (Supplementary Table
S11) and 21/24 mutations were independently validated by
Sanger sequencing (Supplementary Table S10). The three mutations that could not be validated had a variant allele ratio (VAR)
0.11 to 0.13, which is at the limit of detection for Sanger sequencing. Sixty-seven percent (16/24) of mutations were located in a
functional domain, with six mutations in a kinase domain. Six
Eph receptor mutations had a low VAR of 0.1 to 0.15 despite being
found in tumors with 50% to 75% tumor cell content (TCC).
Mutations in EPH receptors seemed to predominantly affect stage
III and IV cancers (22/24 mutations). Together, this suggests that
Eph mutations in colorectal cancers are late events or part of
subclones that become increasingly more prevalent due to selection pressure as the disease progresses. As there is a high level of
sequence similarity between the Eph receptors, we used the
prediction tool Consurf to assess the degree of conservation at
the affected amino acids (19). We found that the affected amino
acids with the highest conservation scores (>7) were generally
those with the most damaging mutations as predicted by the effect
of specific point mutations using PredictProtein (20). Seventynine percent (19/24) of these alterations were predicted to have
damaging effects using the PolyPhen-2 prediction tool (Supplementary Tables S10 and S12; ref. 21). Of the two mutations in
nonmetastatic samples, one was predicted to be benign whereas
the other was predicted to have a probably damaging consequence. In total, there were 6/24 (25%) positions in a predicted
functional residue, which is greater than the fraction of functional
residues found in the EPH receptors (15%), indicating that they
are selected for. Randomly selecting 14 genes (the same number of
Eph receptor genes) from the panel 100,000 times and comparing
the number of significant differences in mutation rate between
nonmetastatic and metastatic CIN patients indicated that EPH
receptors are involved primarily in the metastatic groups (see
Supplementary Methods). EPHB1, EPHA5, and EPHA6 were the
1st, 3rd, and 7th most frequently found in groups of genes
showing significant differences. Other genes frequently found in
1734 Cancer Res; 77(7) April 1, 2017
significant groups had a lower mutation density than any of the
EPH receptor genes (Supplementary Fig. S2). Comparing the
number of mutations in EPH receptor genes in the two groups
indicated that these alterations might have clinical relevance in
predicting the development of metastatic disease (Fig. 2A). The
mutations found were distributed across the coding regions of
both EPHA and EPHB-type receptors (Fig 2B and C). Importantly,
of the two patients with EPH receptor mutations that did not
develop metastatic disease, one had adjuvant therapy after radical
resection of the tumor, which may have eradicated metastatic cells
remaining after surgery.
Validation of mutant transcript expression in tumors by in situ
mutational analyses
EPH receptor mutations in tissue samples from 12 patients
were evaluated by in situ mutation detection using padlock
probes. This revealed both the expression of these receptors in
the tissue as well as validating the mutations present on an RNA
transcript level (Supplementary Fig. S3; Table S13). There was no
localization of any of the mutations in the tissue; rather signals
that were detected were spread throughout the section. For the
majority of mutations, the mutant: wild type ratio was greater at
the transcript level than at the genomic level, revealing a preferential expression of the mutant transcript. A number of EPH
receptors showed a low overall expression level; namely, EPHA5
and EPHA8 (Supplementary Fig. S3A) and EPHA6 (Supplementary Fig. S3H). Mutations in EPHB1 (C268T) and EPHA3
(A2399G) had a lower mutant: wild type ratio at the transcript
level compared with the corresponding VAR at the genomic level
(Supplementary Table S13), indicating that these variants may be
silenced at the transcript level. The mutations found in EPH
receptors may have various different roles in disease progression
and are therefore seen both up and downregulated in these tumors.
In vitro compartmentalization assay to study EPHB1 mutations
EphB receptor signaling is of particular importance in the
colonic crypt, where expression of Eph receptors and ephrin
ligands control the correct positioning of cells in the intestine
due to repulsive mechanisms (22). We therefore sought to establish a model system with the potential to compare mutant
phenotypes across the EPHB receptor family. It is known that
Ephrin-B1 activation of EphB2 and EphB3 receptors induces
sorting and compartmentalization of colorectal cancer cells in
vitro (17). However, this phenotype has not previously been
demonstrated for EphB1. We first assessed whether ephrin B1–
expressing cells induce in vitro compartmentalization of EPHB1
expressing colorectal cancer cells using the coculture system
described in ref. 17 complemented by DLD-1 cell lines engineered
to express wild-type or mutant EPHB1 tagged with GFP (Supplementary Fig. S4). Continuous cell contact-mediated EPHB1 and
ephrin B1 bidirectional activation (23) was achieved by coculturing these two cell populations (Fig. 3A–E). Intermingling of GFP
and RFP populations was virtually absent in EPHB1-GFP and
EphB2-GFP cocultures with ephrin B1-RFP (Fig. 3A; Supplementary Fig. S5A). Conversely, GFP and RFP cells were completely
mixed and scattered to a similar extent in the case of EPHB1-GFP
(Fig. 3F–J), EphB2-GFP, or GFP (Supplementary Fig. S5A) expressing cells cocultured with RFP-labeled control cells. Quantification of cell distribution demonstrated the presence of large
homogenous GFP clusters (>50 cells) of EPHB1-GFP (Fig. 3K)
and EphB2-GFP (Supplementary Fig. S5A and S5B) expressing
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Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921
Eph Receptor Mutations in Colorectal Cancer Are Associated with Metastasis
A
8
Number of mutations
7
6
5
4
3
2
1
0
B
EPHRIN BINDING
EPHA6
EPHA8
EPHA1
FIBRONECTIN TYPE III
EPHA5
EPHA10
EPHA2
EPHA3
EPHA4
TRANSMEMBRANE
TYROSINE KINASE
SAM
TRANSMEMBRANE
TYROSINE KINASE
SAM
EPHA7
C
EPHRIN BINDING
EPHB1
EPHB2
FIBRONECTIN TYPE III
EPHB6
Figure 2.
Mutations in protein-coding regions of Ephrin receptor tyrosine kinases are associated with metastasis of colorectal cancer. A, The number of nonsynonymous
mutations in Eph receptor genes in the two groups by disease stage is shown. Red bars, metastatic stage IV patients; black bars, metastatic stage III patients;
gray bars, metastatic stage II patients; empty bars, stage III patients that did not develop metastases. B and C, Schematic representation of the distributions of
mutations in EPHA genes (B) and EPHB genes (C). Symbols on top of the gene ideogram represent mutations in genes found in this study (no border) and in
nonhypermutated and CIN samples (black border) in refs. 7, 24, 49, and 50. All mutations are exonic and nonsynonymous.
cells in the presence of ephrin B1-RFP cells compared with the cell
distribution using RFP expressing cell alone. Thus, DLD-1 cells
expressing EphB1 display a similar clustering phenotype as EphB2
expressing cells (17) when exposed to ephrin B1. Notably, the cell
sorting phenotype of EPHB1-GFP cells was comparatively weaker
than EphB2-GFP cells in the presence of ephrin B1-RFP cells (Fig.
3A and K; Supplementary Fig. S5A and S5B).
Somatic mutations in the fibronectin type III domain and
tyrosine kinase domain compromised the
compartmentalization of EphB1-expressing cells
We found 7 somatic, nonsynonymous EPHB1 mutations in
CIN colorectal cancer cases that developed metastatic disease. Of
these, four were located in functional domains of EPHB1 and
therefore more likely to be of relevance to protein function
(Supplementary Table S10). These four mutations were also
predicted to be damaging using PolyPhen-2 (Supplementary
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Table S12) and in situ mutation detection revealed expression of
three of the mutations in the Ephrin binding and tyrosine kinase
domains (Fig. 4). We therefore engineered DLD-1 colorectal
cancer cell lines each expressing one of the four different EPHB1
mutants G251A, C268T, C1051T, and G1882T tagged with GFP
and performed compartmentalization assays by coculturing with
RFP-tagged ephrin B1-RFP ligand expressing cells (Fig 3B–E) or
RFP alone (Fig 3G–J; ref. 17). The presence of the desired mutation
was confirmed by Sanger sequencing of the constructs (Supplementary Table S14; Supplementary Fig. S6A–S6D). There were no
deleterious effects on the growth of DLD-1 cells ectopically
expressing mutated EPHB1 (Supplementary Fig. S6E) or ephrin
B1-RFP (Supplementary Fig. S6F). Cells expressing EphB1
C1051T and G1882T showed abrogated compartmentalization
capacity when cocultured with ephrin B1 ligand-expressing cells
(Fig. 3D, E, and K), whereas the mutations G251A and C268T
formed large clusters (>50 cells) to a similar extent as wild type
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Mathot et al.
B (G251A)-GFP
C (C268T)-GFP
D (C1051T)-GFP E (G1882T)-GFP
F EPHB1-GFP
G (G251A)-GFP
H (C268T)-GFP
I (C1051T)-GFP
ephrin B1-RFP
A EPHB1-GFP
RFP
J (G1882T)-GFP
400 µm
100
0–10
31–50
11–30
>50
80
60
40
EPHB1-GFP
(G251A)-GFP
(C268T)-GFP
(C1051T)-GFP
RFP
EphrinB1
-RFP
RFP
EphrinB1
-RFP
RFP
EphrinB1
-RFP
RFP
RFP
0
EphrinB1
-RFP
**
**
20
EphrinB1
-RFP
Percentage of GFP + cells in clusters
K
(G1882T)-GFP
Figure 3.
EPHB1 activity induces cell sorting and compartmentalization of colorectal cancer cells, and point mutations in the fibronectin and tyrosine kinase domains
reduced these effects in vitro. A–J, In vitro compartmentalization assay. Representative confocal images of DLD-1 cells expressing ephrin B1-RFP ligand (A–E) or RFP
alone (F–J) cocultured with DLD-1 cells expressing EPHB1-GFP alone (A and F), four different mutated versions of EPHB1, (G251A)-GFP (B and G), (C268T)-GFP
(C and H), (C1051T)-GFP (D and I), and (G1882T)-GFP (E and J). Images were captured 48 hours after plating. Arrows point to examples of large, homogeneous
GFPþ cell clusters indicative of cell sorting and compartmentalization. K, Quantitative results of the compartmentalization experiments. Cell distribution was
quantified by counting the percentage of GFPþ cells forming clusters of different sizes. In cocultures of ephrin B1 ligand with EPHB1 and its two mutated
versions, (G251A) and (C268T), a high percentage of GFPþ cells were distributed into large homogeneous clusters (>50), whereas this was significantly reduced in
coculture of ephrin B1 ligand with mutated versions (C1051T) and (G1882T) where the majority of GFPþ cells form small groups of fewer than ten cells. The
experiments were performed twice and quantitation was based on these two experimental repeats. Error bars, SD (n ¼ 10 random fields). Statistical
analysis was performed by Student t test, where P < 0.01.
1736 Cancer Res; 77(7) April 1, 2017
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Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921
Eph Receptor Mutations in Colorectal Cancer Are Associated with Metastasis
EPHRIN BINDING
T
82
T
C
G
10
18
51
8T
26
C
G
25
1A
Wild-type
Mutated
FIBRONECTIN TYPE III
TRANSMEMBRANE
TYROSINE KINASE
SAM
Figure 4.
Schematic representation of EPHB1 including the four nonsynonymous mutations studied in the compartmentalization experiments and in situ detection of wild-type
and mutant transcripts in patient samples. The number of signals in the tissue represents the expression level of the transcript in the section.
EPHB1–ephrin B1 ligand interaction (Fig. 3B, C, and K). The
fraction of GFPþ cells expressing EPHB1 with C1051T and
G1882T mutations present in large homogenous clusters of
>50 cells was more than two-fold lower (P ¼ 0.0085 and
0.0066, respectively) than the fractions of wild-type EPHB1 and
G251A and C268T mutants in such clusters (Fig. 3K). Notably, the
number of small clusters (10 cells) was more than two-fold
higher in these two mutants as compared with normal EPHB1.
Taken together, the C1051T and G1882T EPHB1 mutations yield
protein products with impaired compartmentalization ability as
compared with wild-type EPHB1.
Discussion
Somatic mutations in the exomes of colorectal cancers have
previously been identified in a limited sample set by Sanger
sequencing and hybridization capture enrichment coupled to
Illumina sequencing to 20-fold coverage (7, 24). Although the
number of samples analyzed limited the first approach, the
second was limited in coverage by the sequence depth. Other
groups have performed targeted deep sequencing of colorectal
cancer with panels of genes implicated in the disease, showing a
high degree of concordance between primary and metastatic
lesions from the same patient (25). Here, we have performed
pathway-oriented targeted deep sequencing for the further characterization of colorectal cancer genomes. In contrast to other
targeted sequencing studies, we included genes with both known
and putative roles in colorectal cancer development and progression to uncover novel mechanisms for metastatic disease development. We sequenced these genes to approximately 1,000-fold
coverage in normal and tumor samples to validate our approach
to mutation detection, confirm known patterns of mutations in
colorectal cancer and extend the compendium of potential cancer
genes in these pathways. The expected frequencies and types of
mutations were observed in known colorectal cancer genes such as
APC, KRAS, and TP53, confirming the sensitivity and specificity of
mutation detection (7). Interestingly, we uncovered recurring
mutations in repeat sequences in BMPR2 and SETD1B in MSI
tumors. The frameshift mutation N583fs in the A (7) microsatellite repeat sequence of BMPR2 observed in 56% of MSI tumors
www.aacrjournals.org
has been reported in both gastric cancer and colorectal cancer
(frequency of 5–13%; refs. 26, 27). Functional studies including
those reported here revealed an impaired expression of BMPR2 in
MSI colorectal cancers (28) and downregulation of the BMP
pathway (Supplementary Fig. S1), indicating a potential role of
this mutation in colorectal cancer. In addition, it was recently
reported that 35% of Lynch syndrome colorectal cancers had the
same mutation seen in this study (29). Similarly, the frameshift
InDel seen here in 72% of MSI tumors in SETD1B (H8fs) has been
reported as a confirmed somatic mutation in colorectal cancer,
albeit at a mutation rate of 6% (7). The mutation prevalence of
TCF7L2, thought to have a tumor suppressor role in colorectal
cancer (30, 31), was also higher than expected in MSI tumors
(48% vs. 27% in hypermutated tumors; ref. 7). We hypothesize
that the higher mutation rate in microsatellites shown here
compared with similar studies is due to improved sensitivity of
our mutation calling in these repeat sequences, due to the ability
of ConfIdent to filter out artefactual InDels in the corresponding
normal sample that are due to enzyme slippage introduced by the
sequencing technique.
In this study, we aimed to assess mutational differences
between patients that developed metastases and those that did
not. It has been proposed that FBXW7 mutations may be protective for the development of metastatic disease (7). Although we
did not find a statistically significant correlation between mutations in FBXW7 and the development of metastatic disease (P ¼
0.23, Fisher exact test), 79% (11 out of 14) of mutations in FBXW7
occurred in patients that did not develop metastases. We next
examined mutations in Ephrin receptor tyrosine kinases as we
noted an enrichment of this gene family in tumors giving rise to
metastasis. A major challenge in understanding Eph receptor
mutations observed in cancers is the combinatorial nature and
complexity of Ephrin signaling. In the intestine, TCF and b-catenin inversely control the expression of EphB genes, and the
expression of EphB genes is required for the stabilization of the
correct positioning of epithelial cells along the intestinal crypt
(22). Eph receptors have previously been associated with metastatic disease development due to their role in tumor growth,
invasiveness, angiogenesis, and metastasis in vivo (32). However,
no mutational evidence has yet been presented to explain this
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Mathot et al.
association. The Ephrin receptors have a complex role in tumor
progression and have been found both up- and downregulated in
several cancer types. In colorectal cancer, EphA1-3, EphA8, and
EphB4 have upregulated expression, whereas EphA6, EphA7,
EphB1, and EphB2 have downregulated expression (33). A tumor
suppressor role for Ephrin B receptors has been suggested based
on animal models and transcriptional downregulation in human
colorectal cancers (34). EphB2, EphB3, and EphB4 were silenced
in metastatic intestinal tumors (35–37) and EphB2 expression
and silencing of EphB2 was inversely correlated with patient
survival in colorectal cancer (35, 38).
Here, EPHB1, EPHA5, and EPHA6 were the most frequently
mutated Eph receptors, accounting for 63% of Eph mutations.
EPHA5 has been associated with metastases in breast cancer,
where promoter hypermethylation results in downregulation of
expression (39, 40). Although EphA6 expression was found
strongly downregulated in colorectal cancer compared with normal colon, mutations have not previously been reported in
CIN tumors (41). Reduced EPHB1 expression in colon cancer
is associated with poor differentiation and increased invasive
capacity (42). In our in situ mutation detection assay, we demonstrate that the expression of wild type EPHA6 and EPHB1 is
generally reduced compared with the mutant transcript, indicating preferential expression of mutant transcripts in a number of
samples (Supplementary Fig. S3; Supplementary Table S13). In
addition, four nonsynonymous mutations (Supplementary Table
S10; Fig. 3) in protein coding functional domains of EPHB1 were
assessed using an in vitro compartmentalization assay. To our
knowledge, this is the first time the interaction of EPHB1 with
ephrin B1 ligand in vitro has been demonstrated. As EphB receptors play an important role in controlling the position of different
cell types in the crypt–villus axis of the epithelium (22), we
hypothesized that dysregulation of EphB receptor activity by
somatic mutations could be linked to metastases by abrogating
restrictive forces maintained between cells under normal conditions. It was found that the mutations C1051T and G1882T in the
fibronectin and tyrosine kinase domains respectively, abrogated
the cell sorting and compartmentalization capacity more than
two-fold when compared with wild-type EPHB1 (Fig. 3K). In
contrast, mutations in the ephrin-binding domain (G251A and
C268T) had no such effect (Fig. 3K). The lack of phenotypes of
these mutations can either mean that they are passenger mutations or that the compartmentalization assay is a suboptimal tool
to study their phenotype. Interestingly, in situ mutation detection
revealed that the mutant allele ratio for C268T was decreased
compared with the VAR found by DNA sequencing, indicating
that expression of this mutation may be downregulated at a
transcript level. The opposite was true for the G251A and
G1882T mutations (Supplementary Table S13). The fibronectin
domain is responsible for the interactions of many extracellular
matrix proteins such as integrins (43, 44) and plays an important
role in metastasis and invasion (45). Bidirectional downstream
signaling of EPHB1 is transduced by phosphorylation of the
tyrosine kinase domain upon binding with ephrin B1 (46, 47).
Therefore, mutations in these domains may disrupt normal
function of the protein and enhance tumor progression as well
as metastatic capacity of cells bearing these mutations by reducing
the repulsive interactions between cells. To better understand
which aspects of Eph signaling the compartmentalization assay
measures and due to the fact that kinase independent functions of
Eph receptor signaling were found to be mediated by PI3K (48),
1738 Cancer Res; 77(7) April 1, 2017
we performed the assay with and without kinase inhibitors to EPH
kinase, Abl and PI3K (Supplementary Fig. S7). We found that a
strong compartmentalization phenotype was dependent on Eph
kinase activity but not Abl or PI3K activity, indicating that kinase
independent phenotypes may not be captured effectively by this
assay and the use of alternative assays could be beneficial to
convey the function of these mutations.
The pattern of mutations seen in this study and others (7,
25, 49, 50) suggest a metastasis suppressor role for Eph genes
as the mutations are widely distributed over the coding region,
both inside and outside functional domains (Fig. 2B and C),
and some mutations result in loss of function (51–53). Stimulating Eph forward signaling by activating Eph receptors
using soluble ephrin ligands has a tumor suppressive function
by promoting contact-dependent growth inhibition and reducing cell motility and invasive capacity (32). Therefore, it is
reasonable to hypothesize that mutations abrogating forward
signaling through Eph receptors are tumor suppressive. However, the mutations seen here do not appear to have the
characteristics of classical tumor suppressors (requiring inactivation of both alleles), perhaps due to a dominant negative
effect, heterodimerization effects and/or crosstalk with other
signaling pathways (32, 52). In fact, it has been shown that
depending on the ratio of mutant:wild type Eph receptor
expressed in a cell, inactivating mutations of one Eph receptor
can exert a dominant negative effect on a different wild type
receptor (54). Interestingly, three patients had mutations in
more than one Eph receptor gene, suggesting that in some
cases, several of these genes work together to suppress tumorigenesis, and the cumulative effect of several mutations is
required to promote metastasis (52). Concurrent expression
of >1 Eph receptor mutation is shown in Supplementary Fig.
S3 for two of these patients (panels A and J). The finding that
some of the mutations found in EPHB1 may contribute to an
increased invasive capacity of cancers with Eph receptor mutations is novel, and is potentially of great clinical importance to
identify patients who require close monitoring to detect recurrence and to stratify patients that would benefit most from
adjuvant treatments.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: L. Moens, C. Cortina, M. Sundstr€
om, J. Botling,
E. Batlle, B. Glimelius, M. Nilsson, T. Sj€
oblom
Development of methodology: S. Kundu, T. Adlerteg, C. Cortina, A. Moustakas,
E. Batlle, B. Glimelius, M. Nilsson
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): L. Mathot, S. Kundu, J. Svedlund, V. Rendo,
C. Bellomo, M. Sundstr€
om, P. Micke, J. Botling, A. Moustakas, H. Birgisson,
B. Glimelius, M. Nilsson
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): L. Mathot, S. Kundu, V. Ljungstr€
om, J. Svedlund,
L. Moens, T. Adlerteg, E. Falk-S€
orqvist, C. Bellomo, M. Mayrhofer, A. Isaksson,
A. Moustakas, B. Glimelius, M. Nilsson
Writing, review, and/or revision of the manuscript: L. Mathot, S. Kundu,
V. Ljungstr€
om, J. Svedlund, T. Adlerteg, E. Falk-S€
orqvist, V. Rendo, C. Bellomo,
M. Sundstr€
om, J. Botling, A. Isaksson, A. Moustakas, H. Birgisson, B. Glimelius,
M. Nilsson, T. Sj€
oblom
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): T. Adlerteg, M. Nilsson
Study supervision: B. Glimelius, M. Nilsson, T. Sj€
oblom
Cancer Research
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Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921
Eph Receptor Mutations in Colorectal Cancer Are Associated with Metastasis
Acknowledgments
We thank Uppsala Genome Center, Jeremy Adler, and Simin Tahmasebpoor
for expert technical assistance. Imaging was performed with support of the
Science for Life Lab BioVis Platform, Uppsala, Sweden.
Grant Support
This study was supported by the research grants awarded to T. Sj€
oblom from
the Swedish Cancer Foundation (2006/2154, 2007/775, and 2012/834), the
Uppsala-Umea Comprehensive Cancer Consortium (U-CAN), the European
Union's Seventh Framework Programme (FP7/2007-2013) under grant agree-
ment no. 601939 (MERIT) and the Swedish Foundation for Strategic Research
(F06-0050), and by grants awarded to M Nilsson from VINNOVA (Companion
diagnostic initiative) and the Innovative Medicines Initiative (IMI) Joint Undertaking under grant agreement no. 115234 (OncoTrack).
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 July 20, 2016; revised December 12, 2016; accepted December 29,
2016; published OnlineFirst January 20, 2017.
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Somatic Ephrin Receptor Mutations Are Associated with Metastasis
in Primary Colorectal Cancer
Lucy Mathot, Snehangshu Kundu, Viktor Ljungström, et al.
Cancer Res 2017;77:1730-1740. Published OnlineFirst January 20, 2017.
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