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Keywords:

  • colon carcinoma;
  • hereditary nonpolyposis colorectal cancer;
  • tumor stroma;
  • metalloproteinases

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Hereditary nonpolyposis colorectal cancers (HNPCCs) are an important subgroup of colorectal carcinomas. Compared to sporadic variants, they present several particular features, the most important of which are less invasive and metastatic properties linked to a more favorable prognosis. This contrasts to the generally poor differentiation of the epithelial tumor component. Since matrix-degrading proteases secreted by stromal fibroblasts contribute significantly to tumor invasion, we analyzed the stromal expression of 2 matrix metalloproteinases (MMP-1 and -9) and of one of their regulators, the Ets 1 transcription factor, by both immunohistochemistry and in situ hybridization in sporadic colorectal carcinomas and HNPCC tumors. We found that MMP-1 and -9 as well as Ets 1 are upregulated in the fibroblastic stroma during the development from sporadic adenomas to invasive carcinomas. HNPCC tumors exhibited a significantly lower expression of Ets 1, MMP-1 and -9. These findings on the basis of lower matrix-degrading properties of the fibroblastic tumor stroma in HNPCC tumors might help to explain why, in spite of their less differentiated phenotype, HNPCC tumors have a less invasive and metastatic potential compared to sporadic cancers. © 2003 Wiley-Liss, Inc.

Colorectal carcinoma is among the most common malignancies in industrialized nations. While most cases are sporadic, up to 5% occur as part of hereditary syndromes, among which familiar adenomatous polyposis (FAP) and the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome are the most common.1, 2, 3 Defects of different mismatch repair genes, particularly MSH2 and MLH1, have been identified in recent years to be associated with HNPCC.4, 5, 6 Compared to sporadic colorectal cancers, HNPCC tumors present several particular features, the most important of which are less invasive and metastatic properties linked to a more favorable prognosis.7, 8, 9 These less invasive and metastatic properties are observed in spite of the less differentiated morphologic phenotype with a lower tendency toward a glandular differentiation in most HNPCC tumors compared to sporadic colorectal cancers. The reasons for these contradictory features of HNPCC tumors are not known. While invasive properties have long been attributed exclusively to the epithelial tumor cells (particularly to poorly differentiated ones), it has become evident that tumor stroma plays a major role for this critical process as well. Stromal fibroblasts or myofibroblasts are a major source of matrix-degrading metalloproteinases (MMPs) necessary for tumor cell invasion. Stromal expression of MMP-2, MMP-9 and stromelysin 1 has been shown to be upregulated during the development of invasive sporadic colorectal cancers10 and high levels of MMP-1 expression have been directly linked to poor prognosis.11 In general, the activity of matrix-degrading proteases is tightly regulated at several levels. They include an induction by different cytokines and growth factors (such as EGF, bFGF or TNFα), the activation of secreted MMP-proenzymes by a proteolytic cleavage and inhibition by several naturally occurring inhibitors such as TIMP1 and -2.12, 13, 14 One of the transcription factors considered important for this regulation is Ets 1, which is encoded by the ets 1 protooncogene. According to cotransfection assays and mutational analysis, the genes encoding MMP-1, MMP-9, stromelysin 1 (MMP-3) and uPA are among the target genes of Ets 1.15, 16, 17, 18 Several of these enzymes are coexpressed with Ets 1 in fibroblastic stromal cells of invasive sporadic colon carcinomas.19 To find out whether a different regulation of matrix-degrading proteases exists in the fibroblastic stroma of HNPCC tumors, we compared in the present study the expression of Ets 1 and of 2 of its target genes, MMP-1 and -9, between HNPCC and sporadic colorectal cancers.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We analyzed 10 resection specimens with sporadic and 10 with HNPCC carcinomas that had been sent to the Institute of Pathology, University of Bonn, for diagnostic purposes (Table I). HNPCC cancers presented with germline mutations of either MLH1 or MSH2 genes (see Results and Table I). We also included 18 adenomas from non-HNPCC patients with low-grade (n = 9) or high-grade (n = 9) dysplasias.

Table I. Microsatellite Instability and Mutations of Mismatch Repair Genes MLH1 and MSH2 in 10 HNPCC, and 10 Sporadic Colorectal Cancers
Age (years)Loci with MSI/loci investigatedLoss of MMR proteinGermline mutation
285/7MSH2MSH2; c.942+3a[RIGHTWARDS ARROW]t
444/6MSH2MSH2; c.1226delAG
532/2MSH2MSH2; c.187delG
484/5MLH1MLH1; c.1640 T[RIGHTWARDS ARROW]AL547X
44Not investigatedMLH1MLH1; c.2T[RIGHTWARDS ARROW]A
237/10MLH1MLH1; c.184C[RIGHTWARDS ARROW]T;Q62X
385/6MLH1MLH1; c.791delATCG
524/6MLH1MLH1; c.1489insC
535/7MLH1MLH1; c.1622delC
41Not investigatedNoneMLH1; c.1068-1075delTGGGGAGA
650/5NoneSporadic carcinoma
630/5NoneSporadic carcinoma
750/5NoneSporadic carcinoma
750/5NoneSporadic carcinoma
790/5NoneSporadic carcinoma
570/5NoneSporadic carcinoma
680/5NoneSporadic carcinoma
850/5NoneSporadic carcinoma
380/5NoneSporadic carcinoma
580/5NoneSporadic carcinoma

Tissue preparation

Specimens were fixed immediately in 4% paraformaldehyde for 24 hr at 4°C for in situ hybridization and immunohistochemistry and then embedded routinely into paraffin. Sections were cut from the paraffin blocks at 4 μm, mounted on positively charged slides (Superfrost) and air-dried in an incubator at 42°C overnight.

In situ hybridization

In situ hybridization of ets 1 mRNA was performed according to our previously published method.19, 20, 21 All experiments were done twice in the same run. Briefly, sections were deparaffinized in xylene, rehydrated in graded alcohols, subjected to gentle proteinase K digestion (1 μg/ml proteinase K in 0.1 M Tris HCl, pH 8.0, 0.05 M EDTA and 0.1% SDS, 5 min at 37°C), postfixed in 4% PFA (15 min at room temperature, RT), acetylated (10 min at RT) with 0.1 M triethanolamin (pH 8.0) and 25% acetic acid anhydride and finally dehydrated in graded alcohols. 35S-labeled ets 1 antisense and sense riboprobes (negative control) were synthesized by in vitro transcription from an 825 bp human ets 1 cDNA template (nucleotides 260–1086), which had been cloned into a pSP64 plasmid vector. SP6 RNA-polymerase (Promega) was used for transcription (1 hr at 37°C). Transcription was followed by thorough DNAse I digestion of the template (1 U DNAseI/μl; Promega; 15 min at 37°C), RNA extraction with TRIzol (Gibco) and riboprobe purification by phenol-chloroform extraction and Sephadex G-50 columns (Boehringer). Hybridization was carried out for 16 hr at 53°C (riboprobe activity 2 × 104 cpm/μl; hybridization cocktail: 8 mM Tris HCl, pH 8.0, 0.12 M NaCl, 2 mM EDTA, 0.5 M DTT, 50% deionized formamide). Slides were stringently washed in 20 mM Tris HCl, pH 8.0, 0.15 M NaCl, 5 mM EDTA, 0.1 M DTT and 50% deionized formamide for 30 min at 64°C. RNAse A digestion (0.02 mg/ml) was then carried out for 45 min at 37°C. After dehydration in graded alcohols, sections were subjected to autoradiography for 10–14 days (photoemulsion NTB2, Kodak). Positive signals were visualized by dark-field illumination of the slides after fluorescent counterstaining of the nuclei with Hoechst 33258.

Immunohistochemistry

Prior to immunohistochemistry, fresh paraffin sections were deparaffinized in xylene, rehydrated in graded alcohols and washed in TRIS buffer. Monoclonal antihuman mouse antibodies were used for the detection of the mismatch repair enzymes MSH2 and MLH1 (Pharmingen). Antibodies were generated using the full-length recombinant protein. Heat-induced epitope retrieval (HIER, 600 W microwave treatment for 2 × 15 min in prewarmed 10 mM sodium citrate buffer, pH 6) was employed prior to MSH2 and MLH1 staining. Primary antibodies were then added (dilution: MSH2 1:50, MLH1 1:75) and slides incubated overnight at 4°C. Slides were then processed on an immunostainer (TechMate 500, Dako, Hamburg, Germany). For the detection of Ets 1 protein, both a monoclonal mouse antibody (Dianova, against amino acids 122–288) and a polyclonal rabbit antiserum were utilized (Santa Cruz, against amino acids 422–441), dilution of both antibodies 1:500. Collagenases I (MMP-1) and IV (MMP-9) were demonstrated likewise by monoclonal mouse antibodies (Chemicon, dilution 1:25). Heat-induced epitope retrieval (HIER; 400 W microwave treatment for 8 min) was employed prior to staining of both MMP-1 and MMP-9. In order to block endogenous peroxidase activity, slides were incubated in 1% hydrogen peroxide diluted in methanol (30 min at room temperature). Slides were then washed in phosphate-buffered saline (PBS). This was followed by incubation in blocking solution (PBS with 5% nonfat dry milk and 2% normal rabbit serum, 30 min at RT) and application of the avidin/biotin blocking kit (Vector, 2 × 15 min). Solution was removed from the slides using a filter paper. Primary antibodies were then added and slides incubated overnight at 4°C. Washings were performed with PBS and PBS containing 0.1% Triton ×100.

In all cases, bound antibody was detected using the ABC method (Dako) with 3-amino-9-ethylcarbazol (AEC) as a visualizing reagent. Sections were finally slightly counterstained with hematoxylin, mounted in aqueous mounting media and analyzed by standard light microscopy. Replacement of the first antibody by phosphate-buffered saline was used as a negative control to assess specificity of the antibodies. Staining of adjacent normal colon mucosa, stromal cells and/or lymphocytes served as internal positive control for the detection of the mismatch repair enzymes MSH2 and MLH1. A semiquantitative scoring system (number of positive cells × staining intensity) was used to evaluate Ets 1 and MMP-1 and -9 expression. The number of positive cells was graded as negative (0), < 10% (1), < 50% (2), < 80% (3) and > 80% (4). The staining intensity was graded as negative (0), weak (1), medium (2) and strong (3). Three randomly chosen microscopic fields were semiquantitatively evaluated by 2 independent pathologists.

Microsatellite analysis

Microsatellite analysis in sporadic tumors was performed with a set of 5 markers (BAT25, BAT26, D2S123, D5S346, D17S250) according to Lamberti et al.22 using fluorescence-labeled primers and fragment analysis on an ABI 377 sequencer. In the 10 HNPCC tumors, microsatellite instability and mutations have been determined previously.22

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The age of all 20 patients as well as microsatellite instability (MSI) and mutations of mismatch repair genes MLH1 and MSH2 in the 10 HNPCC colorectal cancers are presented in Table I. We obtained the following findings.

Sporadic colorectal carcinomas and adenomas

None of the sporadic colorectal cancers had a family history of mutational changes suggestive of HNPCC. Age of patients ranged from 38 to 85 years (average, 66 years). The tumors were located in the descending and sigmoid colon and rectum and exhibited moderate to poor differentiation. In 7 of 10 cases, the tumor reached the pericolonic fat tissue (pT3). Lymph node metastases were present in 7 cases. Distant metastases to the liver were present in one case. None of the carcinomas had MSI or lack of expression of MLH1 or MSH2 proteins.

Expression of Ets 1, MMP-1 and -9 was compared between normal colon mucosa, adenomas with different degrees of dysplasias and sporadic colorectal cancers (Table II). The medium score values of the cases are given in brackets. No or only weak signals were detected for either Ets 1 [1, 2] or MMP-1 [1, 2] and MMP-9 [1] in stromal cells and capillary endothelia of normal mucosa (Fig. 1a–c). In contrast, about 20–30% of fibroblastic stromal cells and of stromal capillaries expressed ets 1 mRNA and Ets 1 protein [3, 2] as well as MMP-1 [3, 4] and -9 [3] in adenomas (Fig. 1d–f). Ets 1 protein exhibited cytoplasmatic and nuclear distribution. No relation was noted between the degree of dysplasia and expression of Ets 1, MMP-1 and -9 in the adenomas. Further significant upregulation of ets 1 transcripts, Ets 1 protein [9, 2] and of both MMP-1 [8, 6] and -9 [8, 8] was found in the stroma of invasive sporadic cancers, which demonstrated expression in about 70–80% of fibroblasts and endothelial cells (Fig. 2a–c). Expression of Ets 1 and MMP-1 and -9 was homogeneous in the entire tissue section.

Table II. SEMIQUANTITATIVE IMMUNOHISTOCHEMICAL EVALUATION OF Ets 1, MMP1 AND MMP9 EXPRESSION IN NORMAL COLONIC MUCOSA, ADENOMAS, HNPCC AND SPORADIC COLORECTAL CARCINOMAS
 Ets 1MMP1MMP9
  1. The immunohistochemical staining scores (percentage of stained cells × staining intensity) for Ets 1 protein and matrix-degrading proteases (MMP1 and MMP9) are given for each case after semiquantitative evaluation by 2 independent observers. The following scores have been used. Percentage of stained cells: 0, < 10% (1), < 50% (2), < 80% (3), > 80% (4). Staining intensity: negative (0), weak (1), medium (2), strong (3).

Normal   
 11 × 11 × 11 × 1
 21 × 11 × 11 × 1
 31 × 12 × 11 × 1
 41 × 21 × 11 × 1
 51 × 11 × 11
  Average1, 21, 21
Adenoma   
 11 × 12 × 22 × 1
 23 × 22 × 22 × 2
 32 × 22 × 22 × 2
 41 × 22 × 12 × 2
 52 × 12 × 12 × 1
 62 × 12 × 21 × 2
 72 × 23 × 22 × 2
 83 × 23 × 23 × 2
 91 × 12 × 11 × 2
 102 × 12 × 12 × 1
 112 × 11 × 21 × 2
 122 × 22 × 22 × 2
 132 × 12 × 22 × 1
 142 × 22 × 22 × 1
 152 × 21 × 22 × 2
 163 × 23 × 22 × 2
 173 × 12 × 12 × 1
 182 × 11 × 22 × 1
  Average3, 23, 43
Sporadic   
 13 × 33 × 24 × 3
 24 × 24 × 23 × 2
 33 × 34 × 34 × 3
 44 × 34 × 24 × 3
 54 × 34 × 33 × 3
 63 × 23 × 24 × 2
 73 × 33 × 33 × 2
 83 × 33 × 34 × 2
 94 × 34 × 23 × 3
 102 × 33 × 33 × 2
  Average9, 28, 68, 8
HNPCC   
 12 × 12 × 22 × 1
 22 × 12 × 12 × 1
 32 × 22 × 22 × 2
 43 × 22 × 23 × 2
 53 × 23 × 23 × 2
 63 × 23 × 22 × 2
 73 × 22 × 22 × 2
 82 × 22 × 12 × 1
 92 × 23 × 23 × 2
 102 × 22 × 13 × 2
  Average4, 444, 2
thumbnail image

Figure 1. Expression of ets 1 and of MMP-1 and -9 in normal colon mucosa and colorectal adenomas. Neither ets 1 transcripts (a) nor Ets 1 (b) or MMP-1 protein (c) are present in normal colon mucosa (a, in situ hybridization; b and c, immunohistochemistry). In contrast, expressions of ets 1 (d and e) and of MMP-9 (f) are demonstrated in stromal fibroblasts of colorectal adenomas by in situ hybridization (d) and immunohistochemistry (e and f). (a) and (d) illustrate dark-field illuminations of the slides after fluorescent counterstaining of the nuclei with Hoechst 33258. Magnification, ×200 (a and d) and ×400 (b, c, e and f)

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thumbnail image

Figure 2. Expression of Ets 1 and of MMP-1 in the stroma of HNPCC and sporadic colorectal cancers. Ets 1 transcripts (a), Ets 1 protein (b) and MMP-1 (c) are strongly expressed within stromal fibroblasts of a sporadic colorectal carcinoma. In contrast, expression of ets 1 mRNA (d), Ets 1 protein (e) and MMP-1 (f) is significantly lower in HNPCC. (g) demonstrates lack of expression of MLH1 protein within the tumor cells of an HNPCC. In contrast, adjacent normal mucosa and stromal lymphocytes exhibit positive staining. (h) Ets 1in situ hybridization (sense riboprobe) shows a negative control section of a sporadic cancer. (a), (d) and (h) illustrate dark-field illuminations of the slides after in situ hybridization and fluorescent counterstaining of the nuclei with Hoechst 33258. Magnification, ×200 (a, d and h) and ×400 (b, c, eg).

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HNPCC tumors

Age of patients with HNPCC carcinomas ranged from 23 to 53 years (average, 42 years). The tumors were located in the caecum, the ascending colon and the rectum and exhibited moderate to poor differentiation. In 5 cases, the tumors reached the pericolonic fat tissue (pT3). Lymph node metastases were seen in 3 cases. Distant metastases were present in one case. MSI was present in all cases tested. Detailed germline mutations in MLH1 and MSH2 genes are shown in Table I. Immunohistochemically, all cases of HNPCC lacked expression of one of the analyzed mismatch repair genes (MLH1 or MSH2; Fig. 2g). MLH1 was absent in 7 of 10 cases and MSH2 in 3 of 10 cases, respectively.

All HNPCC tumors also showed a significantly lower expression of both ets 1 [average score 4, 4] and of MMP-1 [4] and -9 [4, 2] in the tumor stroma compared to invasive sporadic carcinomas. Signals were only in part located in stromal fibroblasts, most of them being seen in small-vessel endothelia and in lymphocytes (Fig. 2d–f).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Sporadic colorectal cancers and HNPCC significantly differ in their biological behavior, with HNPCC patients presenting generally with fewer metastases at time of diagnosis.7, 8, 23, 24, 25, 26, 27 In view of the fact that HNPCC tumors are generally less differentiated than sporadic carcinomas, these differences remain largely unexplained. An immunologic response of intraepithelial cytotoxic T lymphocytes against tumor cells of HNPCC has been proposed as a possible explanation.28 Other mechanisms may involve tumor stroma, which carries out many functions promoting tumor progression. Also important is tumor vascularization as a prerequisite for tumor growth beyond a size of a few millimeters. Further, stromal fibroblasts participate in tumor invasion via the production of matrix-degrading proteases.29, 30 Both MMP-1 and -9, which have been investigated in the present study, and other proteases involved in matrix degradation such as uPA and stromelysin 1 have been found to be mainly expressed in the fibroblastic stroma of sporadic colorectal cancers and to be regulated via transcription by Ets 1.10, 31, 32, 33, 34, 35, 36, 37, 38, 39 In a previous investigation, we found that uPA, collagenase 1 and stromelysin 1 are topographically coexpressed with Ets 1 in the stroma of these tumors.19 In the present study, we demonstrated an upregulation of Ets 1 and of 2 of its target genes (MMP-1 and -9) during the adenoma-carcinoma sequence. Significantly lower levels of ets 1 mRNA and Ets 1 protein and of MMP-1 and -9 were present in the stromal fibroblasts of HNPCC carcinomas. Previous investigations have also implicated Ets 1 in the transcriptional regulation of tumor vascularization.19, 20, 21, 34, 40, 41 In line with a role of Ets 1 for the vascularization of colon cancers, we found no ets 1 expression in capillary endothelial cells of normal colon mucosa, an upregulation in adenomas and a maximal expression in the stromal capillaries of invasive colon cancers of both sporadic and HNPCC types. As in stromal fibroblasts, expression of Ets 1 in endothelial cells correlated to that of MMP-1 and -9, which are involved in matrix remodeling during early steps of angiogenesis.29, 42 We found no obvious differences of Ets 1 expression in stromal capillaries between sporadic and HNPCC tumors. Thus, Ets 1 probably promotes both invasion and vascularization of sporadic colorectal cancers. Its role seems to be more restricted to angiogenesis in HNPCC tumors, which express significantly lower levels of Ets 1 and its proteases encoding target genes in their fibroblastic stroma. The fact that MMP-1 expression is related to poor prognosis of sporadic colon cancer and that growth and progression are reduced by inhibitors of metalloproteinases is in line with this assumption.11, 43, 44

These findings may help to explain the lower invasive and metastatic potential of HNPCC tumors compared to sporadic cancers on the basis of lower matrix-degrading properties of the fibroblastic tumor stroma in HNPCC carcinomas. The present results in colon cancers are in line with our previously published findings relating expression of ets 1 and several proteases to invasion in other human tumors as well, such as lung and breast cancers.19

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank A. Kraus for technical assistance, G. Klemm, K. Moses and H. Schimikowski for photographic work.

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  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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