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2006;66:6553-6562. Cancer Res
Abigael C.J. Polley, Francis Mulholland, Carmen Pin, et al.
Patients with Colorectal Neoplasia
Expression in the Morphologically Normal Mucosa of
Proteomic Analysis Reveals Field-Wide Changes in Protein
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Proteomic Analysis Reveals Field-Wide Changes in Protein
Expression in the Morphologically Normal Mucosa of
Patients with Colorectal Neoplasia
Abigael C.J. Polley,
1
Francis Mulholland,
1
Carmen Pin,
1
Elizabeth A. Williams,
2
D. Mike Bradburn,
3
Sarah J. Mills,
3
John C. Mathers,
2
and Ian T. Johnson
1
1
Institute of Food Research, Norwich Research Park, Norwich, United Kingdom;
2
Human Nutrition Research Centre, School of Clinical
Medical Sciences, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, United Kingdom; and
3
Wansbeck Hospital, Ashington, Northumberland, United Kingdom
Abstract
Models for the pathogenesis of colorectal cancer tend to focus
on the localized lesion, with less attention paid to changes in
normal-appearing mucosa. Here we used two-dimensional gel
electrophoresis and mass spectrometry to define patterns of
protein expression in morphologically normal colonic mucosa
from 13 healthy subjects, 9 patients with adenomatous polyps,
and 9 with cancer. Tumor samples were also compared with
the normal mucosa. Systematic gel comparisons identified a
total of 839 spots that differed significantly between one
or more groups (P < 0.05). Principle component analysis
indicated that the first three components accounted for
f37% of the total variation and provided clear evidence that
flat mucosa from healthy subjects differed significantly from
that of patients with polyps or cancer. Sixty-one proteins
differed significantly between mucosa from healthy subjects
and all other tissue types, and 206 differed significantly
between healthy mucosa and polyp mucosa. Several of the
proteins showing significant underexpression in tumor tissue
were cytokeratins and other cytoskeletal components. In
contrast, cytokeratins, including several isoforms of cytoke-
ratin 8, were overexpressed in apparently normal mucosa
from polyp and cancer patients compared with mucosa from
healthy subjects. These findings indicate that protein expres-
sion in the apparently normal colonic mucosal field is modi-
fied in individuals with neoplastic lesions at sites distant from
the lesion. Recognition and further characterization of this
field effect at the molecular level may provide protein bio-
markers of susceptibility to colorectal cancer and facilitate
development of hypotheses for the role of diet and other
environmental factors in its causation. (Cancer Res 2006;
66(13): 6553-62)
Introduction
The central paradigm for the pathogenesis of colorectal cancer is
the adenoma-carcinoma sequence, a complex stepwise series of
changes in cellular proliferation and differentiation, driven by a
progressive accumulation of genetic abnormalities, leading to
malignancy via adenomatous polyps (1). It is increasingly recog-
nized that this model is probably inadequate in that other
molecular abnormalities, including modified epigenetic marks, also
contribute and may define alternative pathways to neoplasia (2).
However, most current mechanistic models focus almost exclusively
on the localized lesion, with much less attention paid to pathologic
changes occurring in the normal-appearing mucosa from which
such lesions emerge. Physiologic anomalies in the flat mucosa,
including abnormal cell proliferation (3, 4), apoptosis (5), and gene
expression (6), have previously been reported but the nature, dura-
tion, and causes of such putative field effects are poorly defined.
The physiologic state of a complex tissue is reflected in the full
complement of proteins expressed by its constituent cells. The
pattern of expressed proteins thus constitutes a ‘‘library’’ of
information about the functional status and health of the tissue.
The development of new methods for protein extraction, display,
and analysis has led to the emergence of a new field of clinical
proteomics, in which these techniques are harnessed to identify
biomarkers of cancer and other diseases (7), but there are few
studies on the differential expression of proteins during early
colorectal carcinogenesis.
Tracy et al. (8) described reproducible patterns of protein
expression associated with the normal mucosa and specific
differences associated with tumors. Similarly, Anderson et al. (9)
compared hepatic and colorectal tumors with their normal host
tissues, and with each other, and identified characteristic patterns
of protein expression that could be used to distinguish primary and
secondary tumors from their adjacent uninvolved tissues. Several
other groups have reported similar findings and the development
of proteomic technology has enabled a more comprehensive
analysis of protein expression (10, 11). Among the most recent of
these studies is that of Friedman et al. (12), who employed two-
dimensional difference gel electrophoresis to compare tumor
samples and macroscopically normal mucosa. Using this approach,
Friedman’s group observed 52 proteins for which statistically
significant differences in abundance were detectable within the
mucosa/tumor pairs from the same patients.
Although many different techniques for the collection and
fractionation of tissues have been employed, virtually all previous
studies have compared tumors with flat mucosa from the same
individual. Whereas this approach provides a comparison of ana-
tomically normal and neoplastic tissues against the same genetic
background, it does not address the possibility that the apparently
normal mucosa of healthy individuals differs from that of
those with neoplastic lesions. The possibility of precancerous field
changes that render the mucosa more vulnerable to the emergence
of localized lesions has long been recognized but seldom explored.
In the present study, we used proteomic techniques to test the
Note: Present address for E. A Williams: Centre for Human Nutrition, Northern
General Hospital, University of Sheffield, Sheffield S5 7AU, United Kingdom.
Requests for reprints: Ian T. Johnson, Institute of Food Research, Norwich
Research Park, Norwich, NR4 7UA, United Kingdom. Phone: 01603-255330; Fax: 01603-
255288; E-mail: ian.johnson@bbsrc.ac.uk.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-0534
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hypothesis that colonic mucosa from disease-free patients would
exhibit patterns of protein expression distinct from the mucosa of
patients with adenomatous polyps or cancer. Tumors from the
cancer patients were also compared with each group of normal
mucosal samples.
Materials and Methods
Patients and biopsies. Volunteers were either patients with previously
diagnosed colorectal cancer or outpatients with no known major pathology,
typically presenting for investigation of symptoms, including abnormal
bowel habit or rectal bleeding, and undergoing flexible sigmoidoscopy or
colonoscopy as a diagnostic procedure. All patients were recruited from
the gastroenterology outpatient and surgical lists of the Wansbeck General
Hospital, Ashington, Northumberland, United Kingdom. Ethical approval
for the project was received from the Northumberland Local Research
Ethics Committee (project reference NLREC2/2001). Patients were con-
tacted in advance and sent an information leaflet, and those patients
consenting to the study were advised to attend endoscopy or theatre as
expected. Experimental biopsies were collected from the rectum of the
endoscopy patients, in addition to those obtained for diagnostic purposes.
For the cancer patients, samples of normal rectal mucosa (>10 cm from
tumor margin) and tumor tissue were collected at surgery. All samples
were immediately snap frozen in liquid nitrogen and transferred to a 80jC
freezer. Medical notes for each volunteer were reviewed 6 to 8 weeks after
the procedure and the findings of the pathology report and the conclusions
of the responsible consultant were recorded. The final groups were
composed of 13 ‘‘normal’’ individuals [9 females (mean age, 54.8 years;
SD, 8.4 years) and 4 males (mean age, 68 years; SD, 3.6 years)] showing no
evidence of neoplasia; 9 ‘‘polyp’’ patients [5 females (mean age, 66.6 years;
SD, 15.4 years) and 4 males (mean age, 55.8 years; SD, 4.3 years)] in whom
adenomatous polyps were detected at endoscopy; and 10 ‘‘cancer’’ patients.
The latter group was composed of six females (mean age, 64.5 years; SD,
8.4 years) and four males (mean age, 66.5 years; SD, 11.6 years), most of
whom had moderately differentiated adenocarcinomas of the recto-
sigmoidal region (Table 1). Two-dimensional gel electrophoresis was done
on 41 samples; one mucosal biopsy from a cancer patient was lost.
Protein extraction. Mucosal biopsies and samples of tumor were
thawed, weighed, and extracted without further manipulation. Resected
mucosa was allowed to thaw and subsamples of mucosal tissue (10-15 mg)
were scraped from the underlying muscle layers using a glass microscope
slide. The tissue was extracted using Bio-Rad ReadyPrep Sequential
Extraction Kit (Bio-Rad, Hemel Hempstead, United Kingdom) with the
following additions to Reagent 1 just before use: MgCl
2
(3 mmol/L),
protease inhibitor cocktail (Sigma, Poole, United Kingdom; 2.5 AL/mL),
DNase I (RNase-free; 5 units/mL), and RNase A (5 Ag/mL). Modified
Reagent 1 (25 AL) was added and each tissue sample was hand homo-
genized. A further 175 AL of Reagent 1 were added and the whole volume
was sonicated for 10 minutes at room temperature, centrifuged to obtain a
pellet, and the supernatant was removed. The volume equivalent to 100 Ag
protein was determined using a Bio-Rad Protein Assay according to the
instructions of the manufacturer, with bovine g globulin as standard. The
extraction supernatants were stored at 80jC.
Two-dimensional gel electrophoresis. For isoelectric focusing in the
first dimension, a sample volume equivalent to 100 Ag protein was added to
a rehydration mix, containing urea (7 mol/L), thiourea (2 mol/L), CHAPS
(2%), bromophenol blue, DTT (18.2 mmol/L), and IPG buffer (0.5%, pH 4-7;
GE Healthcare, Little Chalfont, United Kingdom) to make a final volume of
450 AL. The whole volume was transferred into a well of the Immobiline
DryStrip re-swelling tray and IPG strips (24 cm, pH 4-7; GE Healthcare) were
rehydrated overnight at 20jC. Each strip was then transferred to a ceramic
strip-holder and submerged in DryStrip Cover Fluid (f3.5 mL). The iso-
electric focusing was run on an Ettan IPGphor bed (GE Healthcare) with
a gradient of 500 V for 1 hour (500 V-h), 4,000 V for 1.5 hours (6,000 V-h),
and a ‘‘step-n-hold’’ of 8,000 V for 6.75 hours (51,600 V-h). After completion
of isoelectric focusing, the strips were stored at 80jC.
The second-dimension protein separation was carried out on 1-mm-thick
10% gels, prepared in 28
23-cm gel-plate cassettes. Focused strips were
rinsed free of excess mineral oil, conditioned in modified Tris Acetate
Equilibration Buffer (Genomic Solutions, Huntingdon, United Kingdom),
treated first with 8 mg/mL DTT in equilibration buffer (9 mL; 30 minutes
with gentle shaking), transferred to 25 mg/mL iodoacetamide in equilibration
buffer (9 mL; 30 minutes with gentle shaking), and placed in a gel cassette
before transfer to the gel tank. The top reservoir contained the cathode
buffer (200 mmol/L Tris base, 200 mmol/L Tricine, 14 mmol/L SDS; Sigma)
and the bottom reservoir contained the anode buffer (25 mmol/L Tris-acetate
buffer, pH 8.3). Electrophoresis conditions were set to give an upper voltage of
500 V, power of 20,000 mW/gel, and a total run time of f3.5 hours.
Gel imaging and analysis. After electrophoresis, the gels were fixed and
stained using SYPRO Ruby Protein Gel Stain (Bio-Rad) and imaged using
ProXPRESS Proteomics Imaging System and Perkin-Elmer imaging
software. Images were saved as TIF files and analysis was carried out
using ProteomWeaver analysis software (Definiens, Munich, Germany).
Four gel groups were established representing the anatomically normal
mucosa from 13 healthy patients (‘‘healthy mucosa’’), 9 polyp patients
(‘‘polyp mucosa’’), and 9 cancer patients (‘‘cancer mucosa’’), and samples
of tumor from 10 cancer patients (‘‘tumor tissue’’). Each gel underwent
automatic spot detection and manual editing before automatic spot
matching both within and between groups. Manual matching was done
where necessary. Base-paired normalization was done on all the gels before
examination of spot volume data. Average gels were constructed using spots
that were detectable in a minimum of 50% of the gels in each group.
Selected spots were picked from a gel using the ProPick spot-picking robot
(Genomic Solutions) and gel plugs were transferred to a modified 96-well
microtitre plate.
Protein analysis. In-gel trypsin digestion was carried out using a
ProGest Protein Digester (Genomic Solutions). After preincubation, the
digestions were carried out at 37jC for 3 hours using 50 ng of sequencing
Table 1. Characteristics of cancer patients from whom mucosal and tumor biopsies were obtained in the present study
Patient no. Sex Age (y) Tumor site Duke’s stage Histology
1 F 51 Rectum C Poorly differentiated adenocarcinoma
2 F 66 Hepatic flexure B Histopathology report incomplete
3 F 60 Hepatic flexure B Moderately differentiated adenocarcinomas
4 F 72 Sigmoid colon C1 Moderately differentiated adenocarcinoma
5 F 64 Sigmoid colon/rectum B Moderately differentiated adenocarcinoma
6 F 74 Hepatic flexure B Moderately differentiated adenocarcinoma
7 M 66 Ascending colon B Moderately differentiated adenocarcinoma
8 M 60 Sigmoid colon/rectum C1 Moderately differentiated adenocarcinoma
9 M 83 Sigmoid colon/rectum A Moderately differentiated adenocarcinoma
10 M 57 Sigmoid colon/rectum B Moderately differentiated adenocarcinoma
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grade porcine trypsin (5 AL/well; Promega, Southampton, United Kingdom).
The digests were analyzed using a Reflex III MALDI/ToF (Bruker Ltd.,
Coventry, United Kingdom) with Scout 384 ion source, fitted with a nitrogen
laser (wavelength, 337 nm) to desorb/ionize the matrix/analyte material
from the sample substrate. All spectra were acquired in a positive-ion
reflector set at the following variables: 25 kV acceleration voltage, 28.7 kV
reflection voltage, 20.9 kV ion source acceleration voltage, and 1.65 kV
reflector-detector voltage. Calibration was carried out using a set of peptide
standards having an approximate concentration of 1 pmol/AL from spots
adjacent to the samples. In some cases, quadrupole time-of-flight mass
spectrometry was done using a Micromass quadrupole time-of-flight
electrospray fitted with a Waters Cap LC system.
Peptide masses obtained from the matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry were searched (on the basis of
mass) against the MSDB protein database using the Mascot peptide mass
fingerprint program from Matrix Science.
4
The search parameters were as
Figure 1. Two-dimensional protein map
based on the average gel for the healthy
mucosa. A, arrows, positions and spot
numbers for all the identified proteins
listed in Table 2. B, a similar map of
cancer mucosa is shown together with
spot numbers and positions for proteins
identified as cytokeratins. C, coordinates
for each tissue sample on the first
three principal components, following
principal component analysis carried
out on 839 proteins that differed
significantly across the 41 samples of
tissue analyzed. Each sample is
numbered to indicate its origin from
healthy mucosa (1), polyp mucosa (2 ),
cancer mucosa (3), or tumor tissue (4 ).
4
http://www.matrixscience.com.
Proteomic Analysis of Colorectal Mucosa
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follows: (a) tryptic digest was assumed to have a maximum number of one
missed cleavage; (b) peptide masses were stated to be monoisotopic; (c)
methionine residues were assumed to be partially oxidized; (d) the
carbamidomethylation of cysteine residues was considered; (e) the mass
tolerance was kept at 75 ppm; and ( f ) the taxonomy group searched was
Homo sapiens. The results give a Probability Based Mowse Score (13), equal
to 10
Log(P), where P is the probability that the observed match is a
random event. Protein scores >63 are considered statistically significant
(P < 0.05) under the above parameters.
Statistical analysis. Gels were normalized in accordance with
ProteomWeaver software protocols using base-paired normalization. The
spot-density data were transferred to Excel spreadsheets for statistical
analysis using a nonparametric inference approach. Statistical filtering
retained only those spots for which average spot-density was >0.07
(arbitrary units), the within-group frequency was >50%, or which were
present in more than 6 of the 41 gels. To test the null hypothesis of equal
distribution of protein expression between groups, a nonparametric one-
way ANOVA (Kruskal-Wallis) was done on the ranked data (14). The null
hypothesis was rejected for any protein where the rank differed significantly
(P < 0.05) in at least one of the four groups. For all proteins showing
evidence of unequal expression between groups, principal component
analysis was carried out on the correlation matrix of the ranks of the 41
independent samples. F tests were used to assess the significance of
differences between groups and combinations of groups (15). Models were
set up to determine whether a particular protein differed significantly
between independent subsets of the four groups of observations. Differ-
ences were considered significant when the P value associated with the F
statistic was <0.05.
Results
Figure 1A is a two-dimensional protein map based on the
average gel for the healthy mucosa. The equivalent map for cancer
mucosa is shown in Fig. 1B. Gel-analysis software identified a total
of 6,494 unique spots across all the 41 gels and statistical filtering
yielded 1,910 spots of potential interest. Among these, the Kruskal-
Wallis test identified a total of 839 spots for which the average
density in one set of gels differed significantly (P < 0.05) from that
in at least one other set. For these proteins, principle component
analysis was carried out on the correlation matrix of the ranks of
the 41 independent samples; the first three principal components
were found to account for 37% of the total variability. Principal
component 1, which was highly correlated with 166 proteins
(correlation coefficient > 0.6) and represented f21% of the total
variation, indicated that the greatest differences in the ranks of
proteins occurred between the tumor tissues and the three sets of
morphologically normal mucosa. A total of 72 of the 839 proteins
Table 2. Characteristics of 26 identified proteins differentially expressed in tumor tissue compared with healthy mucosa, polyp
mucosa, and cancer mucosa
Spot no. Protein name Score Accession
no.
Theo. pI Theo. MW Act. pI Act. MW Expression
in tumor
117903 h-Actin 157 P60709 5.55 40,536 5.12 42,093 1.104
17654 a1-Antitrypsin precursor 235 P01009 5.37 46,878 4.98 59,532 0.788
3385 Apolipoprotein A-I precursor 162 P02647 5.56 30,759 5.24 24,471 3.031
9315 Calgizzarin 70 P31949 6.56 11,847 5.86 12,048 2.032
9669 Creatine kinase 261 P12277 5.34 42,902 5.43 42,655 2.571
12691 Cytokeratin 9 76 P35527 5.14 62,178 4.81 50,192 0.07
18566 Cytokeratin 19 268 P08727 5.04 44,065 4.87 45,588 0.298
17043 Cytokeratin 19 260 P08727 5.04 44,065 4.82 45,484 0.275
16185 Cytokeratin 19 223 P08727 5.04 44,065 4.78 45,640 0.449
64231 Cytokeratin 19 66 P08727 5.04 44,065 4.92 44,917 1.012
7046 Glycyl-tRNA synthetase 148 P41250 6.61 83,828 5.95 77,814 0.597
17391 IgG Fc binding protein 59 O95784 5.56 81,017 5.31 132,499 0.147
16796 IgG Fc binding protein 52 O95784 5.56 81,017 5.22 135,256 0.113
86052 Maspin 76 P36952 5.72 42,586 5.75 42,325 0.395
17865 Nucleoside diphosphate kinase A 164 P15531 5.42 17,309 5.57 19,758 1.154
114132 Protein disulfide isomerase 46 P07237 4.76 57,480 4.77 58,722 0.301
145857 Serum albumin, human 147 1AO6_A 5.73 68,126 5.45 52,323 0.207
13756 Serum albumin, human 91 1A06A 5.63 67,690 5.43 41,401 0.539
16253 Human serum albumin
complexed with myristic acid
131 1BJ5 5.73 68,126 5.77 24,841 0.447
5764 Stathmin 107 P16949 5.77 17,161 5.64 18,761 0.412
10280 Transthyretin (prealbumin)
complex with thyroxine (T4)
93 2ROX_A 5.33 12,996 5.48 16,838 1.144
8115 Tropomyosin a4 chain
(tropomyosin 4; TM30-pl)
63 P67936 4.67 28,487 4.68 31,786 1.928
9264 Ubiquitin thiolesterase 125 P15374 4.48 26,337 4.79 28,835 0.414
17404 Vinculin (metavinculin) 165 P18206 5.51 124,161 5.56 127,631 0.104
18975 14-3-3 h 120 P31946 4.76 28,179 4.76 28,296 4.11
17784 21K tumor protein 93 P13693 4.84 19,697 4.84 25,485 1.723
Abbreviations: Theo, theoretical data based on whole sequence in database; Act, actual gel pI and MW data from calibrated gel; Score, probability-based
Mowse score (see ref. 15); pI, isoelectric point.
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differed significantly between tumor tissue and all three sets of
mucosal tissues. The second principal component accounted for
f10% of the total variation and was highly correlated with 56
proteins, 35 of which differed significantly among the three sets of
normal mucosal samples. The third component accounted for
about 6% of the total variation and was most highly correlated with
the polyp mucosa. Figure 1C illustrates the coordinates for each
tissue sample on the first three principal components. Each sample
is numbered to indicate its origin from healthy mucosa (1), polyp
mucosa (2), cancer mucosa (3), or tumor tissue (4). The separation
of the tumor samples and the three sources of mucosa into discreet
clusters is clearly apparent, with some evidence of a further
segregation within group 3.
Differential expression of proteins in tumor tissue com-
pared with flat mucosa. Use of the F test for between-group
comparisons indicated differential expression of 588 proteins in
tumor tissue compared with healthy mucosa, 536 compared with
polyp mucosa, and 520 compared with cancer mucosa. A total of
291 proteins differed significantly between tumor tissue and all
three groups of flat mucosal samples; of these, 90 were underex-
pressed in tumor tissue and 201 were overexpressed. A large
proportion of these proteins were so weakly expressed that analysis
was impractical, but 26 were positively identified by mass
spectrometry and are listed in Table 2.
Many of the spots showing differential expression among the four
groups of tissues were identified as cytoskeletal proteins. Among
these, we noted two separate spots identified as h-actin. This
protein is widely regarded as a ‘‘housekeeping gene,’’ characterized
by stable expression across a range of cell types, although variations
in expression in colorectal cancer (10) and other tissues (16) have
previously been reported. Most of the variation in h-actin
expression was associated with a weakly expressed spot (117903)
which was more prominent in tumor tissue (Table 2). Figure 2
indicates that the only detectable difference between spots 3476
and 117903 was the absence in the latter of the peptide of mass
1,516.75 Da. This corresponds to the peptide sequence QEYDESG-
PSIVHR, found at the COOH-terminal of the protein, which was the
fourth highest intensity peptide of the 14 used to derive the identity.
As all the other tryptic peptides were detected, it seems probable
that it was genuinely absent from spot 117903 and that a limited
COOH-terminal clipping accounted for the small shifts in molecular
mass and charge observed on the gel.
Other cytoskeletal proteins showing evidence of differential
expression in tumor tissue included cytokeratin 9 (CK9), four
isoforms of cytokeratin 19 (CK19), and vinculin. The latter is involved
in the attachment of actin-based microfilaments to the inner surface
of the plasma membrane (17). Conversely, h-actin and tropomyosin
(TM30-pl) both showed strong evidence of overexpression in tumor
tissue. Other proteins showing evidence of significant under-
expression included a1-antitrypsin precursor, creatine kinase,
protein disulfide isomerase, immunoglobulin G (IgG) Fc binding
protein, and the putative plasma proteins transthyretin, human
Table 2. Characteristics of 26 identified proteins differentially expressed in tumor tissue compared with healthy mucosa, polyp
mucosa, and cancer mucosa (Cont’d)
Expression in
healthy mucosa
Fold change Expression in
polyp mucosa
Fold change Expression in
tumor mucosa
Fold change P
0.141 8 0.125 9 0.12 9 9.7
10
5
1.853 -2.3 1.692 -2.1 2.997 -3.8 8.8
10
4
6.876 -2.3 5.381 -1.8 7.257 -2.4 2.3
10
3
0.594 3.4 0.56 3.6 0.765 2.7 5.5
10
7
9.158 -3.6 14.158 -5.51 8.004 -3.1 7.47
10
5
0.365 -5.2 0.809 -11.6 0.322 -4.6 1.9
10
3
1.45 -4.9 2.005 -6.7 1.76 -5.9 1.99
10
5
1.408 -5.1 2.064 -7.5 1.42 -5.2 6.24
10
5
1.467 -3.3 1.868 -4.2 1.03 -2.3 1.08
10
3
1.869 -1.8 2.24 -2.2 2.469 -2.4 8.05
10
3
0.226 2.6 0.188 3.2 0.199 3 6.8
10
5
0.667 -4.5 0.952 -6.5 0.547 -3.7 4.47
10
6
0.467 -4.1 0.649 -5.7 0.339 -3 1.6
10
6
0.058 6.8 0.09 4.4 0.188 2.1 2.3
10
4
0.462 2.5 0.424 2.7 0.494 2.3 5.3
10
7
0.738 -2.5 0.806 -2.7 0.551 -1.8 1.0
10
3
0.821 -4 0.568 -2.7 0.639 -3.1 7.9
10
3
1.888 -3.5 1.613 -3 1.336 -2.5 4.63
10
4
1.285 -2.9 1.054 -2.4 1.083 -2.4 2.2
10
3
0.135 3.1 0.143 2.9 0.181 2.3 2.1
10
3
1.839 -1.6 1.661 -1.5 2.677 -2.3 1.6
10
3
1.127 1.7 0.991 1.9 1.024 1.9 4.3
10
4
0.241 1.7 0.252 1.6 0.217 1.9 3.1
10
4
0.68 -6.5 0.516 -5 0.621 -6 1.37
10
6
2.628 1.6 2.658 1.5 2.568 1.6 1.3
10
3
0.867 2 0.873 2 1.044 1.7 1.3
10
5
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serum albumin and APO A1. S100A11 (calgizzarin), stathmin, 14-3-3
h, glycyl-tRNA synthetase, and ubiquitin thiolesterase all showed
evidence of increased expression in tumor compared with
morphologically normal mucosa.
Differential expression of proteins in mucosa of patients
with and without neoplasia. The expression of 61 proteins
differed significantly between healthy mucosa and all of the other
tissue types (polyp mucosa, cancer mucosa, and tumor tissue).
Moreover, a total of 206 proteins differed significantly between
healthy mucosa and polyp mucosa. Sixteen positively identified
proteins with expression in polyp mucosa differing significantly
compared with healthy mucosa are listed in Table 3. In contrast
with tumor tissue, in which there was reduced expression of
cytokeratins, three proteins identified as CK8 and two identified
as CK9 were overexpressed in polyp mucosa compared with
healthy mucosa. Two isoforms of a1-antitrypsin precursor were
identified; one was markedly increased in expression and the other
was significantly reduced, an effect which may reflect differing post-
translational modification of this protein in polyp mucosa. Of the
seven identified proteins that were underexpressed in polyp mucosa
compared with healthy mucosa (Table 3), two proteins, desmin
and transgelin, are cytoskeleton-associated proteins. Other proteins
showing evidence of underexpression in polyp mucosa included
calvasculin, 14-3-3 e, and 70K thyroid antigen fragment (Ku protein).
There was further evidence of increased expression of cytoker-
atins in cancer mucosa compared with healthy mucosa (Fig. 1B;
Table 4). Seven proteins positively identified as cytokeratins (CK8,
CK9, and CK20) were overexpressed but a2-actin and other
proteins associated with the cytoskeleton (desmin, transgelin, and
vimentin) were all underexpressed. As in polyp mucosa, there
was evidence for modified expression of members of the serpin
superfamily of protease inhibitors. One isoform of a1-antitrypsin
precursor (24831) was increased by 4.7-fold, but again this was
associated with a reduction in a second isoform of the same
protein (163130). There was increased expression of the related
proteins, elastase inhibitor and maspin. Other proteins listed in
Table 4 are associated with the regulation of a variety of cellular
functions including proliferation and apoptosis.
Discussion
ThemultivariateanalysisillustratedinFig.1C provides
confirmation that colorectal tumor tissue can be distinguished
from morphologically normal mucosa on the basis of protein
expression. This is not unexpected, given the many histologic,
metabolic, and cytokinetic differences between normal and
neoplastic tissues, and our findings are consistent with a number
of other proteomics studies in which tumor tissue has been
Figure 2. Mass spectrometric data (mass and intensity) and sequences identified by peptide mass fingerprinting for two spots (117903 and 3476) identified as
h-actin. The relative positions and intensities of the spots are shown in detail from Fig. 1A . The two spots apparently differ because of a single peptide (highlighted in
3476) that is absent in 117903.
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compared with flat mucosa from the same patients (9, 11, 12).
However, it is also clear that the three sources of morphologically
normal mucosa do not form a single coherent group; of particular
interest is the fact that the cluster of samples derived from patients
without polyps or cancer is clearly separated from both sets of
patients with neoplastic lesions. This observation contradicts
the conventional, but largely unexamined, assumption that the
morphologically normal mucosa of cancer patients is also
functionally normal. However, individuals with a history of multiple
colorectal polyps or cancer are well known to be at a greater risk
of developing another cancer at sites remote from the original
lesion. This implies that the whole mucosal field of such individuals
has undergone functional changes that make it persistently
vulnerable to neoplasia. Chen et al. (6) established that, for a
panel of genes, the expression profiles measured in polyp-free
mucosa differed significantly between APC
min
and wild-type mice
and between human patients with and without colorectal cancer.
A later study from the same group reported consistent differences
in gene expression in subjects with a family history of cancer
compared with those without (18). Our present results are
consistent with these findings and show that the changes in gene
expression associated with increased vulnerability to colorectal
cancer encompass a larger number of genes than previously
reported and extend to the level of protein expression. Although it
was not our primary intention to conduct a comprehensive
analysis of all the proteins showing differential expression in polyp
and cancer mucosa, some of our observations may shed light on
the functional changes taking place in the mucosal field of
individuals at increased risk of bowel cancer.
Epithelial cells contain 20 different cytokeratins, numbered 1 to
8 (type II CKs) and 9 to 20 (type I CKs); cytokeratins 1 to 6 are
characteristic of squamous epithelia whereas the columnar
epithelia of the gastrointestinal tract typically contain CKs 7, 8,
and 18 to 20 (19). In the present study, we observed differential
expression of seven isoforms of CK8, four of CK19, and one each of
CK9 and CK20. All four isoforms of CK19 were underexpressed in
tumor tissue compared with morphologically normal mucosa,
regardless of source, and one of the spots identified as CK9 was
absent from tumor samples (Table 2). In contrast, four CK8
isoforms and both CK9s were overexpressed in polyp mucosa
relative to normal mucosa (Table 3) and seven CK8 isoforms, one of
CK9 and one of CK20, were overexpressed in cancer mucosa
relative to healthy mucosa (Table 4). The increased abundance of
cytokeratins in cancer mucosa is highlighted in Fig. 1B. Overall,
these findings indicate that expression of CK8 increases in the
morphologically normal mucosa as the adenoma-carcinoma
sequence progresses, with multiple isoforms differing slightly in
mass and charge.
In this complex situation, the increased abundance of certain
isoforms may be balanced by reductions in others so that total
amounts of protein remain the same. A full characterization of the
subtle changes in peptide structure and abundance that we have
identified presents a considerable technical challenge, which is
beyond the scope of the present study, although further work to
identify their structure and cellular localization is certainly
warranted.
Apart from the maintenance of normal epithelial architecture,
cytokeratins have functional roles in epithelial physiology. CK8-null
mice develop chronic inflammation of the colon (20) and have
impaired electrolyte and fluid transport (21). Mutations affecting
the structure of CK8 in humans also lead to epithelial fragility in
the gut and may play a role in some types of inflammatory bowel
disease (22). The colitis of CK8-null mice is due to a primary
epithelial defect rather than an immune deficiency, and colonic
bacteria are required for its development (23). Thus, CK-8 may be
directly involved in the defense of the mucosal epithelium against
proinflammatory stimuli that contribute to neoplastic change (24).
a1-Antitrypsin [a1 proteinase inhibitor (API)] is a member of
the serine-protease superfamily (25) and an anti-inflammatory
protein, the main function of which is to prevent degradation of
connective tissues by opposing the activity of endogenous
proteases (26). In the present study, four distinct spots were
Table 3. Characteristics of 15 proteins differentially expressed in polyp mucosa compared with healthy mucosa
Spot no. Protein name Score Accession
no.
Theo.
pI
Theo.
MW
Act.
pI
Act.
MW
Expression
in healthy
mucosa
Expression
in polyp
mucosa
Fold
change
P
3476 h-Actin 178 P60709 5.29 42,052 5.25 42,893 51.2 71.73 1.4 1.3
10
2
24831 a1-antitrypsin precursor 85 P01009 5.37 46,878 5.02 58,483 0.629 2.04 3.1 2.1
10
3
163130 a1-Antitrypsin precursor 168 P01009 5.37 46,878 5.03 58,483 2.045 0.596 3.5 6.6
10
5
18889 Calcyclin 88 P06703 5.33 10,230 5.05 9,671 6.29 8.32 1.3 2.2
10
2
12155 Calvasculin 49 P26447 5.85 11,949 5.19 10,727 0.208 0.103 23.1
10
2
68702 Cytokeratin 8 75 P05787 5.52 53,510 5.04 51,491 0.167 0.534 3.2 9.1
10
3
64844 Cytokeratin 8 116 P05787 5.52 53,510 5.10 52,913 0.195 0.686 3.5 1.9
10
3
65803 Cytokeratin 9 82 P35527 5.14 62,178 5.06 62,008 0.026 0.135 5.2 1.7
10
2
12691 Cytokeratin 9 76 P35527 5.14 62,178 4.81 50,192 0.365 0.81 2.2 4.5
10
2
63453 Desmin 70 P17661 5.21 53,429 4.86 48,541 0.794 0 — 3.2
10
10
2887 Fibrinogen g chain,
isoform g-A precursor
79 P02679 5.70 49,465 5.63 49,356 0.553 0.36 1.5 3.8
10
2
17391 IgG Fc binding protein 59 O95784 5.56 81,017 5.31 132,499 0.667 0.952 1.4 2.1
10
2
17076 Transgelin-2
(SM22-a homologue)
59 P37802 8.45 22,417 5.22 18,242 0.205 0.067 31.6
10
2
19586 14-3-3 e 53 P62258 4.63 29,326 4.68 30,803 0.158 0.072 2.2 7.0
10
3
72917 70K thyroid
antigen fragment
66 P12956 6.23 69,953 5.14 32,121 0.169 0 — 2.1
10
5
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positively identified as API precursor, which is a 52-kDa poly-
peptide that subsequently becomes glycosylated to form the
55-kDa secreted protein. One spot (24831) was significantly
overexpressed in both polyp mucosa and cancer mucosa relative
to healthy mucosa, but expression in tumor tissue was not
significantly different from healthy mucosa. A second isoform
(17654) was overexpressed in cancer mucosa only and two others
were unchanged or underexpressed. Overall, the data suggest that
modified expression of API isoforms in the morphologically normal
mucosal field occurs during development of neoplasia. API has
previously been shown to be synthesized by hepatocytes, macro-
phages, and intestinal epithelial cells (27), and gastric cancer has
been reported to be associated with high levels of API in the gastric
juice (28). It is also detectable in the feces of both healthy subjects
and patients with inflammatory bowel disease (29), and in vitro
evidence suggests that it is secreted in response to certain
proinflammatory cytokines (30). There are also reports suggesting
that high levels of API expression in sporadic colorectal tumors are
associated with poor prognosis (31). A second serpin, elastase
inhibitor, also showed evidence of increased expression in cancer
mucosa compared with healthy mucosa. BLAST searches con-
firmed that both proteins were members of the serpin superfamily
but a comparison of their sequences established that they were
products of different genes, with 31.4% homology across the amino
acid sequence.
A third member of the serine protease family, maspin, was
shown to be overexpressed in tumor and cancer mucosa relative to
healthy mucosa. Maspin is an unusual serpin in that it lacks
Table 4. Characteristics of 37 identified proteins differentially expressed in cancer mucosa compared with healthy mucosa
Spot no. Protein name Score Accession
no.
Theo.
pI
Theo.
MW
Act.
pI
Act.
MW
Expression
in healthy
mucosa
Expression
in cancer
mucosa
Fold
change
P
19076 a2-Actin 104 P62736 5.23 42,381 5.30 43,815 2.997 1.46 2.1 9.9
10
3
17372 a2-Actin 167 P62736 5.23 42,381 5.22 43,570 12.4 7.3 1.7 1.1
10
2
3476 h-Actin 178 P60709 5.29 42,052 5.25 42,893 51.23 63.95 1.3 4.3
10
2
24831 a1-Antitrypsin precursor 85 P01009 5.37 46,878 5.02 58,483 0.629 2.97 4.7 3.1
10
5
163130 a1-Antitrypsin precursor 168 P01009 5.37 46,878 5.03 58,483 2.045 0.969 2.1 3.0
10
3
50128 ATPase h-chain 98 P06576 5.26 56,525 5.09 54,474 0 0.209 2.5
10
4
18889 Calcyclin 88 P06703 5.33 10,230 5.05 9,671 6.29 9.21 1.5 3.4
10
3
12155 Calvasculin 49 P26447 5.85 11,949 5.19 10,727 0.208 0.034 6.1 2.5
10
4
3013 Calpain 69 P04632 5.05 28,469 4.95 27,853 0.187 0.31 1.7 1.1
10
2
66557 Cytokeratin 8 102 P05787 5.52 53,510 5.06 48,199 0 0.357 — 8.1
10
4
68702 Cytokeratin 8 75 P05787 5.52 53,510 5.04 51,491 0.167 0.869 5.2 8.5
10
4
64844 Cytokeratin 8 116 P05787 5.52 53,510 5.10 52,913 0.195 0.888 4.6 2.0
10
3
68667 Cytokeratin 8 136 P05787 5.52 53,510 5.21 50,928 0.168 0.918 5.5 2.3
10
3
88139 Cytokeratin 8 130 P05787 5.52 53,510 5.28 52,781 0.076 0.709 9.3 2.5
10
3
162428 Cytokeratin 8 154 P05787 5.52 53,510 5.13 48,714 0.018 0.079 4.4 2.9
10
3
163121 Cytokeratin 8 101 P05787 5.52 53,510 5.06 47,359 0.322 0.743 2.3 3.0
10
2
65803 Cytokeratin 9 82 P35527 5.14 62,178 5.06 62,008 0.026 0.125 4.8 2.5
10
2
80605 Cytokeratin 20 126 P35900 5.60 48,599 5.38 49,415 0.154 0.581 3.8 3.8
10
3
63453 Desmin 70 P17661 5.21 53,429 4.86 48,541 0.794 0.274 2.9 1.1
10
5
10872 Fatty acid-binding protein 132 P07148 6.60 14,256 6.02 13,502 5.002 12.452 2.5 4.3
10
3
2887 Fibrinogen g chain,
isoform g-A precursor
79 P02679 5.70 49,465 5.63 49,356 0.553 0.219 2.5 2.2
10
4
90505 Hemopexin 61 P02790 6.55 52,385 5.65 65,289 0 0.295 — 9.4
10
4
11118 Leukocyte elastase inhibitor 153 P30740 5.90 42,829 5.94 42,750 0.289 0.847 2.9 1.0
10
3
66836 Mannose-6-phosphate isomerase 76 P34949 5.63 47,065 5.57 44,917 0.301 0.191 1.6 9.9
10
3
86052 Maspin 76 P36952 5.72 42,586 5.75 42,325 0.058 0.188 3.3 2.3
10
2
146954 Peroxiredoxin 2 (thiol-specific
antioxidant protein;
natural killer
cell-enhancing factor B)
82 P32119 5.67 21,918 5.24 24,131 0.259 2.033 7.8 7.4
10
3
11873 Rho GDi, ly 88 P52566 5.10 23,031 5.10 28,896 0.952 0.58 1.6 7.5
10
3
16534 Sarcomeric tropomyosin n;TPM1-n 67 AAT68294 4.65 32,688 4.70 40,372 2.171 1.298 1.7 0.4
10
2
18169 Secernin 2 54 AAH17317 5.44 46,989 5.54 44,512 0.692 0.347 22.3
10
3
18889 S100A6 (calcyclin) 88 P06703 5.33 10,230 5.05 9,671 6.29 9.21 1.5 3.4
10
3
12155 S100A4 (calvasculin) 94 P26447 5.85 11,949 5.19 10,727 0.208 0.034 6.1 2.5
10
4
17076 Transgelin-2 (SM22-a homologue) 59 P37802 8.45 22,417 5.22 18,242 0.205 0.016 12.8 1.8
10
4
13202 Vimentin 58 P08670 5.06 53,545 4.73 46,273 0.259 0.064 42.5
10
2
72917 70K thyroid antigen frag 66 P12956 6.23 69,953 5.14 32,121 0.169 0.056 34.2
10
3
19586 14-3-3 e 53 P62258 4.63 29,326 4.68 30,803 0.158 0 — 1.9
10
4
18349 14-3-3 protein ~/y (protein kinase C
inhibitor protein 1, KCIP-1)
199 P63104 4.73 27,899 4.72 28,504 5.42 4.68 1.2 3.1
10
2
17784 21K tumor protein 93 P13693 4.84 19,697 4.84 25,485 0.867 1.04 1.2 1.2
10
2
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protease inhibitory activity and is classified as a tumor suppressor
gene. It has been shown to induce apoptosis in tumor cells via Bax
(32) and Bcl-2 (33), but in prostate cancer the tumor suppressor
function is associated with increased cell adhesion, thereby
suppressing cell mobility and malignant behavior (34). Over-
expression of maspin in colorectal tumor tissue has previously
been described (35) and a recent report suggests that this is
particularly associated with microsatellite instability (36). The
present study seems to be the first in which the expression of
maspin in morphologically normal mucosa has been described.
Several other cytoplasmic proteins with diverse regulatory
functions were shown to be differentially regulated in polyp and
cancer mucosa. The S100 proteins are calcium-activated signaling
molecules involved in cell proliferation, differentiation, and
cytoskeletal dynamics. In the present study, S100A11 (calgizzarin)
was overexpressed in tumor tissue compared with healthy mucosa,
polyp mucosa, and cancer mucosa. Three S100 proteins, including
S100A11, were previously identified by Chaurand et al. (37) as
tumor-specific markers of colorectal neoplasia. Stulik et al. (11)
also identified overexpression of S100A11 in their proteomic
analysis of human colorectal tumors. We observed statistically
significant overexpression of S100A6 (calcyclin) in both polyp
mucosa and cancer mucosa compared with healthy mucosa (Tables
2 and 3) whereas S1004A (calvasculin) was underexpressed in these
tissues. Stulik et al. (11) reported a statistically significant
correlation between the development of colorectal neoplasia and
the expression of different isoforms of S1006A (38), and Bronckart
et al. (39) also reported characteristic patterns of expression of
S100 proteins, including reduced expression of S100A4, associated
with colorectal dysplasia and neoplasia. A high expression of
S100A4 has been reported to be positively associated with risk of
metastasis of colorectal tumors (40) and of many other tumor types
(41). The present study shows that S100A4 is expressed in healthy
mucosa, albeit at a low level, and that reduced expression was
associated with the development of neoplasia.
Overall, the present study provides evidence for progressive
changes in protein expression patterns in the colonic mucosal field,
associated with the development of neoplastic lesions at distant
sites. This observation has a number of important implications.
The use of proteomic techniques to characterize tumors is
becoming increasingly common and many groups have described
studies in which tumor tissue was compared with supposedly
normal mucosa obtained from the same patient. The present study
shows that this is a misleading strategy if it is assumed that the
paired mucosal sample is equivalent to the healthy mucosa of
disease-free individuals. This is well illustrated by the example of
liver fatty acid binding protein, which was reported recently by
Lawrie et al. (42) to be reduced consistently in colorectal tumor
tissue compared with adjacent normal colon. This observation is
consistent with our own study in which the level of liver fatty acid
binding protein expression in cancer mucosa was 3.9-fold higher
than in tumor tissue (P = 0.0004). However, the level in cancer
mucosa was also f2.5-fold higher compared with healthy mucosa
(P = 0.004) whereas there was no statistically significant difference
between expression levels in tumor tissue and healthy mucosa.
The existence of characteristic patterns of protein expression
associated with enhanced vulnerability to neoplasia provides
opportunities for development of biomarkers of increased risk.
Kinzler and Vogelstein (43) proposed that the adverse effects of
certain dietary factors on the risk of colorectal cancer were
attributable not to food-borne carcinogens but to hypothetical
effects they described as chronic ‘‘irritation,’’ causing the colorectal
mucosa to enter a perpetual state of tissue regeneration. Under
these conditions, the survival of cells carrying preneoplastic
mutations would be favored and the vulnerability to cancer would
increase. Since Kinzler and Vogelstein’s review, evidence for the
importance of apoptosis as a protective mechanism against
carcinogenesis has grown (5, 44), but little direct biological evidence
for the putative state of chronic tissue regeneration has emerged.
The results of the present study do not provide conclusive evidence
that the mucosal field defect involves a heightened state of tissue
repair but the observed changes in cytoskeletal proteins and serine
protease inhibitors may be consistent with this hypothesis. Some
of the differences in protein expression that we have observed may
reflect altered gene expression occurring as a consequence of
aberrant CpG island methylation (2, 45). Recognition and further
characterization of the field effect will provide a framework on
which to build hypotheses for future research on the role of diet as
an etiologic factor in the development of colorectal cancer.
Acknowledgments
Received 2/10/2006; revised 4/6/2006; accepted 4/25/2006.
Grant support: Food Standards Agency (UK) and the Biotechnology and Biological
Sciences Research Council.
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.
We thank Drs. Mike Naldrett and Andrew Bottrill for the MALDI-T of analysis and
Wendy Bal, Julie Coaker, and Catherine Lamb for technical support.
Proteomic Analysis of Colorectal Mucosa
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