Colorectal carcinogenesis is associated with stromal expression of COL11A1 and COL5A2
Heléne Fischer,
Roger Stenling,2,
Carlos Rubio,1 and
Annika Lindblom,3
Department of Molecular Medicine and
1 Department of Pathology, Karolinska Institute, S 171 76 Stockholm, and
2 Department of Pathology, Umeå University Hospital, S 901 85 Umeå, Sweden
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Abstract
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Collagen is the major component of the interstitial extracellular matrix (ECM). ECM is known to play an active role in numerous biological processes such as cell shape, proliferation, migration, differentiation, apoptosis as well as carcinogenesis. We used mRNA differential display RTPCR to study differentially expressed genes in tissue samples from 24 colorectal cancers and four normal colon epithelia. Twenty of the 24 tumours showed expression of a gene COL11A1, not expressed in the normal samples. This gene is not normally expressed in adult colon tissue, but was here found to be expressed in 27 out of a total of 34 (79%) colorectal carcinomas. An analysis of other collagens showed that COL5A2 was not expressed in normal colon but was co-expressed with COL11A1 in the tumours. Our results suggest that stromal expression of COL11A1 and COL5A2 is associated with malignancy in colorectal cancer.
Abbreviations: ECM, extracellular matrix; FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal cancer; mRNA DDPCR, mRNA differential displayPCR.
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Introduction
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The majority of colorectal tumours are sporadic, while a minority of colorectal tumours represent an inherited form (1,2). Colorectal cancer development commonly involves an early inactivation of the APC tumour suppressor gene, which is mutated in germline in familial adenomatous polyposis (FAP) and somatically in the majority of sporadic colorectal cancers (35). In hereditary nonpolyposis colorectal cancer (HNPCC), tumour progression is evoked by mutations in various cancer genes, which mutate somatically as a result of an inherited deficiency in one of the DNA mismatch repair genes (6,7). In addition to the genes involved in the APC pathway and the DNA mismatch repair genes, genetic alterations in other genes are suggested to be of importance in colorectal carcinogenesis. These alterations involve genes such as KRAS (8), TGFR2 (9), TP53 (10), SMAD4 (11), SRC (12) and the PPAR-
(13).
Some genes have been suggested to be of importance in tumorigenesis through modulating a response in stromal cells. Interactions between cancer cells and the surrounding stroma is one of the key aspects in the mechanism of tumour cell invasion (14). Metalloproteinases and cadherins are discussed in relation to their involvement in the spread of metastatic cells (15,16). Growth factors contribute to establish microvessels in the stroma surrounding the cancer cells and are considered a prerequisite for tumour growth (17). Tumours invading beyond the muscularis mucosae characteristically have abundant stroma rich in collagen, although the significance of this desmoplastic response is unknown.
In an attempt to identify additional genes involved in the developmental pathways of colorectal cancer, we compared four normal colon epithelium samples and 24 colorectal tumours using mRNA differential displayPCR (mRNA DDPCR) (18,19).
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Materials and methods
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Tumours
The mRNA DDPCR was run on a panel of 24 fresh frozen colorectal tumours and four normal tissue samples collected at the Umeå hospital between 1987 and 1991. All tumours were at Dukes' stage B. For the expression study we used the same tumours as above as well as five additional normal colon tissue samples, three HNPCC tumours collected through the Cancer Family Clinic, Karolinska Hospital, Stockholm, and five adenomas and three sporadic carcinomas collected at Umeå Hospital between 1987 and 1991. The RNA in situ study used samples from the same tissues. All samples were obtained immediately after surgery and frozen at 70°C.
RNA and DNA preparation
Total RNA was extracted from frozen tissue by homogenizing with a power homogenizer (IKA Labortechnik, Staufen, Germany) in Trizol Reagent according to the manufacturer's protocol (Life Technologies, Gaithersburg, MD) and used for DDPCR, RTPCR and northern blotting. RNA was treated with deoxyribonuclease I (Ambion, Austin, TX).
mRNA DDPCR
mRNA differential display RTPCR (mRNA DDPCR) was performed using the method previously described by Liang and Pardee (18,19) using the one base anchoring. Primers were synthesized by Genset, Paris, France. The anchoring primer for COL11A1 was AAGCT11G and the arbitrary primer was AAG CTT ATA CAG G. The band was cut out and re-amplified with the same primers and then cloned without purification into a TA vector (PCR II, Invitrogen, Carlsbad, CA). Clones were then selected by size followed by inverse hybridization (20). Positive clones were sequenced with Thermo Sequenase Radiolabelled Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Homology searches were performed using Blast search in the GenBank database.
RTPCR
Primers specific for the isolated fragments were used on template from tumours and normal tissue for comparison of expression. In a volume of 40 µl, 2 µg total RNA was reverse transcribed under the following conditions: 5 mM MgCl2, 50 mM KCl, 10 mM TrisHCl, pH 8.3, 1 mM dNTPs, 1 U RNase inhibitor (Perkin-Elmer, Foster City, CA) and 2.5 U MuLV reverse transcriptase (Perkin-Elmer).
Northern blot analysis
Total RNA (10 µg) was separated by electrophoresis on a 1.5% denaturing agarose gel. RNA was transferred to a nylon plus membrane (Qiabrane; Qiagen, Hilden, Germany). The membrane was UV cross-linked, prehybridization and hybridization was performed according to a standard protocol (21). A 1800 bp gel-purified COL11A1 PCR fragment generated with primer col11a1.210 (5'-TCT GGG ATT TCA CCG TAA CAAC-3') and col11a1.1020 (5'-TGG GTC CCT CTG TTA CAC-3') was 32P-labelled using a random primer labelling kit (Redi Prime DNA Labelling System; Amersham Pharmacia Biotech, Uppsala, Sweden) and used as a probe. The blot was reprobed with radiolabelled ß-actin cDNA (Clontech, Palo Alto, CA) as loading control. Signals were visualized by autoradiography using a PhosphorImager (Fudjix 1000).
Semiquantitative PCR
PCR reaction volume was 25 µl containing 250 ng each primer, 250 nM dNTPs and 2.5 U ampliTaq (Gibco Life Technologies, Gaithersburg, MD). cDNA was diluted 1:20 in water and 10 µl was used for all samples and controls. The primers used are listed in Table I
. Thermocycler (PTC 225; MJ Research, Watertown, MA) was used with an initial denaturation step of 94°C for 5 min, followed by cycles of 94°C for 45 s, 59°C for 30 s, 72°C for 30 s and a final elongation step of 72°C for 5 min. PCR products amplified after 25, 30 and 35 cycles were separated on a 1.5% agarose gel containing ethidium bromide for comparison between samples and controls. The experiment using the number of PCR cycles that made the best PCR product for each primer was repeated three to five times for the same cDNA. For quantification, the intensity of the PCR bands was estimated with Image Gauge V3.41 software (Fuji Film Science, Japan).
In situ hybridization
Tissue from two normal colonic mucosa tissues, one sporadic colonic cancer, one sporadic rectal cancer and one HNPCC cancer were cut in 14 µm sections in a cryostat (Jung CM 3000, Leica Instrument) at 18°C. In situ mRNA hybridization was performed as previously described (22,23). Oligonucleotide probes with sequence complementary to mRNA encoding COL11A1 (nucleotides 1415714203, antisense and 1599716039, sense) were synthesized (Genset, Paris, France).
Statistical analysis
The difference between normal samples and adenomas and carcinomas regarding expression was tested by a non-parametric method, KruskalWallis. No assumption was made about the underlying distribution. Post hoc pairwise comparisons were made by MannWhitney U-test. Correlation between COL11A1 and expression of RNA from other genes was studied by linear correlations and estimated by Pearson's correlation coefficient. Analyses were made twice after synthesizing new cDNA from the same RNA.
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Results
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COL11A1 is expressed in colorectal cancer, but not in normal colonic tissue
The mRNA differential display RTPCR was used to compare colonic cancers with colonic normal epithelium. Several differentially expressed transcripts were obtained. One band of approximately 200 bp was expressed in 20 out of 24 of the tumours but not in the normal samples used in the DDPCR experiment. The band was cut out and re-amplified using the same primers and cloned. Clones were then selected by size, and shown to contain the same DNA sequence by inverse northern hybridization. Sequence analysis and sequence homology search (EMBL/GenBank) showed that this differentially expressed gene was identical to the sequence of the
-chain of collagen XI. This result was confirmed by PCR on newly synthesized cDNA from our original tumours in the DDPCR experiment and by northern blot (Figure 1
). The number of samples used for northern blot analysis was small and was dependent on the limited amounts of RNA from the samples. We extended the experiment and also included five sporadic colonic adenomas and four carcinomas from HNPCC patients. Using a semiquantitative RTPCR, we found that most of the carcinomas, but none of the normal or the adenoma samples, expressed COL11A1. In total, 13 out of 15 (87%) rectal carcinomas, 10 out of 15 (67%) colonic carcinomas and 4 out of 4 (100%) HNPCC carcinomas expressed COL11A1. There was no apparent expression in any of nine normal samples or five sporadic adenomas. Image Gauge V3.41 software was used to compare the intensity of the PCR generated bands for COL11A1 and a statistically significant difference was found between the expression in 30 carcinomas compared with eight samples from normal epithelium and five adenomas (P < 0.001) (Figure 2
). There was no statistically significant difference between the expression in normal cells and adenomas (P = 0.14); however, the number of samples was small.

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Fig. 1. Northern blot demonstrating COL11A1 in tumours but not in normal samples. Lanes from left to right, two normal colon tissues, two HNPCC and one colon cancer were separated by electrophoresis on 1.5% denaturing agarose gel.
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Fig. 2. Box plot illustrating the difference between expression of COL11A1 in carcinomas compared with normal and adenoma samples. The boxes mark the interval between the 25th and 75th percentiles. The whiskers denote the interval between the 10th and 90th percentiles. An observation that lies more than 1.5 times the height of a box from the box is defined as outlier and when an observation lies more than three times the height of a box from the box, it is defined as an extreme value.
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mRNA in situ expression in stromal cells
RNA in situ hybridization was used to determine which cells expressed COL11A1. Tissue from two normal colonic mucosa tissues, one sporadic colonic cancer, one sporadic rectal cancer and one HNPCC cancer were tested. No signal was detected in normal tissues but a clear positive signal for COL11A1 was found in all tumours (Figure 3B
). As expected the signal was expressed in and around the stromal cells. There was no positive signal in the epithelial cells, which looked similar to background (Figure 3A
). The epithelial cells give an auto-fluorescent signal in all figures, while the staining with antisense probe is seen as small bright dots.


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Fig. 3. RNA in situ demonstrating collagen expression in (A) normal sample and (B) tumour sample, stromal cells express COL11A1, while epithelial cells show no signal different from background.
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Expression of other collagens
Collagens normally form trimers using different
-chains. We therefore tested the expression of the
2-chain of collagen 11 by RTPCR. There was no expression. However, the
2-chain of collagen 5 has been reported to be expressed in association with COL11A1 (24). RTPCR experiments showed that COL5A2 was not expressed in normal epithelium or adenomas but up-regulated together with COL11A1 (P < 0.001) in carcinomas (Figure 4
). To explore if this up-regulation of the two types of fibrillar collagens was specific or involved other collagens, the mRNA expression of two other collagens, the fibrillar COL3A1 and the basal membrane COL4A1, were tested. In contrast to COL11A1 and COL5A2, COL3A and COL4A were all expressed in normal colonic epithelium as well as in adenomas and carcinomas, and the expression was not correlated to the expression of COL11A1 (Table II
).

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Fig. 4. Scatter plot showing the correlation between expression of COL11A1 and COL5A2 (correlation coefficient, 0.464; P level = 0.002).
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Discussion
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Collagen is composed of three polypeptide chains built of repeated Gly-X-Y triplets that fold into typical triple-helical domains. Minor-fibrillar collagen types V and XI play a critical role in formation of the type I and type II fibrillar network in cartilage and non-cartilagenous tissues, respectively (24). They can also give rise to crosstype trimers consisting of one
-2 (V) and two
-1 (XI) chains in skeletal muscles and in developing bone, fetal brain and other tissues (25). The structure and biological properties of V and XI seem to be closely related. They are usually buried within the major collagen fibrils and suggested to be of crucial importance in physiological processes such as development and wound healing. It has become evident that several molecules are in fact heterotypic associations of chains from both collagen types V and XI, demonstrating that these two collagens are not distinct types but rather a single type which can be called collagen V/XI (24). The fact that these two collagens are up-regulated together in colorectal cancer is likely to be of significance. The type XI or V collagens have not been found to be expressed in adult colon. The expression of pro-
-2 (V) collagen was found in human fetal gut (25) suggesting an involvement of this collagen in development of the gut. The
-1 (XI) chain seems to participate in a stage- and tissue-specific manner in intestine and many other tissues in mouse development (26). Since the normal adult bowel does not express COL5A2 and COL11A1, it seems that in carcinogenesis some carcinomas are associated with an embryological expression pattern in the stromal tissue.
In normal colon mucosa, the membrane functions as a barrier that prevents the invasion of the non-hematogenous origin. Its main structural component is type IV collagen, whereas the main component of the stroma are the classical fibril forming type I and III collagens. Both the epithelial membrane and the collagenous matrix immediately beneath it are degraded in malignant tissue, due to the up-regulation of several degradative enzymes or an altered expression of collagen subtypes in tumour (27,28). We found a specific up-regulation of COL5A2 and COL11A1 in stromal cells in colon carcinoma. An increase of type V collagen expression has also been observed in mouse skin tumours (29) and human breast carcinoma (30).
The up-regulation of mRNA expression of some collagens in stromal cells could be a downstream effect of tumorigenic changes in colonic epithelial cells in colorectal cancer. Alternatively, the expression of COL11A1 and COL5A2 could be the primary change giving rise to a tumorigenic response in epithelial cells.
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Notes
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3 To whom correspondence should be addressed E-mail: annika.lindblom{at}cmm.ki.se 
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Acknowledgments
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This work was supported by The Swedish Cancer Foundation, The Cancer Foundation in Stockholm, Åke Wiberg Foundation and Lion's Cancer Research Foundation in Umeå. The authors would like to thank Susanne Petersson for valuable advice.
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Received June 8, 2000;
revised March 1, 2001;
accepted March 2, 2001.