Increased Membrane Type 1 Matrix Metalloproteinase Expression from Adenoma to Colon Cancer A Possible Mechanism of Neoplastic Progression Sandeep Malhotra, M.D.,* Elliot Newman, M.D.,* David Eisenberg, M.D.,* John Scholes, M.D.,† Rosemary Wieczorek, M.D.,† Paolo Mignatti, M.D.,* Peter Shamamian, M.D.* From the Departments of *Surgery, S. Arthur Localio Laboratory for Surgical Research, and †Pathology, New York University School of Medicine, New York, New York lon cancer: a possible mechanism of neoplastic progression. Dis Colon Rectum 2002;45:537–543.

PURPOSE: Membrane type 1 matrix metalloproteinase is a membrane-associated matrix metalloproteinase central to the degradation of basement membrane components via the activation of matrix metalloproteinase-2. Although membrane type 1 matrix metalloproteinase is overexpressed in invasive colon cancer, its expression in colonic polyps and carcinoma in situ has not been defined. In addition, the association of membrane type 1 matrix metalloproteinase expression by a primary tumor and recurrence of colon cancers has not been examined. METHODS: Immunoperoxidase staining was performed on randomly selected specimens containing adenoma (n ⫽ 17), carcinoma in situ (n ⫽ 9), or metastatic colon carcinoma (n ⫽ 8) with mouse monoclonal antibody to human membrane type 1 matrix metalloproteinase. Similar staining was also performed on randomly selected node-negative colon cancers that recurred within five years of resection (n ⫽ 17), matched for age, gender, stage, grade, and vascular, lymphatic, and perineural invasion, and node-negative colon cancers that did not recur within five years of resection (n ⫽ 17). Staining for membrane type 1 matrix metalloproteinase was graded. Mean scores for the groups were compared by Wilcoxon test. RESULTS: We found a progressive and significant increase in the mean score of membrane type 1 matrix metalloproteinase from normal mucosa to adenoma (P ⬍ 0.001), carcinoma in situ (P ⬍ 0.006), and invasive cancer (P ⬍ 0.009). However, there was no difference in membrane type 1 matrix metalloproteinase expression between the recurrent and nonrecurrent groups of nodenegative colon cancer (P ⫽ not significant). CONCLUSIONS: These data suggest that membrane type 1 matrix metalloproteinase expression increases with progression from normal mucosa to invasive adenocarcinoma; however, it cannot be used as a prognostic indicator on which adjuvant therapy is based in node-negative colon cancer because of its failure to predict recurrence in this patient group. [Key words: Membrane type 1 matrix metalloproteinase; Metalloproteinase; Colon cancer; Colonic polyps]

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umor cell invasion through the basement membrane is a critical step in the metastatic cascade.1 To disseminate to secondary organs, metastatic cells must cross basement membranes as they leave the primary tumor and invade the blood vessels.1 This process involves tumor-cell attachment to and local degradation of the basement membrane components by proteolytic enzymes and cell migration through the digested matrix. Among the various proteinases involved in this process, the matrix metalloproteinases (MMPs) are a family of zinc-dependent and calcium-dependent enzymes that degrade components of the extracellular matrix.2 The MMPs are divided into four major subclasses: interstitial collagenases, gelatinases, stromelysins, and membrane-type MMPs.2 The membranetype MMPs (MT-MMPs) are a subclass of MMPs that are membrane bound. MT1-MMP, the first MT-MMP described,3 can directly degrade several extracellular matrix components, such as collagen types I and III, gelatin, fibronectin, laminin-1, aggrecan, and tenascin.4,5 Perhaps a more important function of MT1-MMP is its ability to activate MMPs (2 and 13). The two enzymes (MT1-MMP and MMP-2) are often colocalized,3,6–8 and recent studies suggest that MT1-MMP may be a critical activator of MMP-2 in tumor tissues.9–12 Increased MMP-2 expression is associated with higher metastatic potential in a variety of systems and likely plays a central role in tumor invasion.13 MT1-MMP contains a C-terminal transmembrane domain that allows it to localize to the cell surface.14,15 It functions by binding tissue inhibitor of

Malhotra S, Newman E, Eisenberg D, Scholes J, Wieczorek R, Mignatti P, Shamamian P. Increased membrane type 1 matrix metalloproteinase expression from adenoma to coPresented at the meeting of the Society for Surgery of the Alimentary Tract, San Diego, California, May 20 to 24, 2000. Address reprint requests to Dr. Newman: 530 First Avenue, Suite 6C, New York, New York 10016.

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MMP-2 (TIMP-2) and the N-terminal end of MMP-2, which results in a 105-kd trimer that effects the cleavage of pro-MMP-2 to its active (64/62 kd) form.16 MT1-MMP binding of pro-MMP-2 (72 kd) alone does not efficiently activate it; however, overexpression of MT1-MMP results in the generation of the intermediate form of MMP-2 (68/66 kd). Other mechanisms, including autocatalysis, contribute to MMP-2 cleavage to its active (64/62 kd) form. Neutrophil-derived serine proteinases (elastase, cathepsin B, and proteinase 3) activate pro-MMP-2 and enhance MT1-MMP– dependent tumor-cell invasion.17,18 MT1-MMP expression has been correlated with tumor invasiveness in a variety of tumor types, including breast, liver, lung, gastric, and cervical cancers.9– 11,13,19–26 A correlation of increased MT1-MMP mRNA with advanced tumor stage has been demonstrated in colon cancer.27 However, little is known about the expression of this enzyme in preinvasive colon lesions or whether there is any variation in MT1-MMP expression in node-negative (TxN0) colon cancer patients who have experienced recurrence within five years compared with those who have not. Because there is a clear association between MT1-MMP overexpression and increased invasiveness of tumor cells in experimental in vitro models, the purpose of the present study was to determine whether there is a progressive increase in the expression of MT1-MMP in humans when normal mucosal tissue is compared with adenoma, carcinoma in situ, and invasive cancer. The present study also attempted to determine whether an increased level of expression of MT1MMP in node-negative colon cancer patients was associated with a higher recurrence rate after surgical resection.

METHODS Patient Selection Tissue samples from either endoscopic biopsy or surgical resections were selected at random, and only those with good-quality paraffin blocks were used for the study. MT1-MMP expression was evaluated in patients with adenoma (n ⫽ 17), carcinoma in situ (n ⫽ 9), and node-positive (TxN1–3) carcinomas (n ⫽ 8). Adjacent normal mucosa in 12 specimens from these 34 patients was examined to represent baseline control values. To characterize MT1-MMP expression in patients with node-negative (TxN0) colon cancers and assess

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whether its level predicted recurrence, a separate group of 17 patients who underwent curative resection between 1989 and 1994 were selected randomly. These patients were pathologically staged as node negative by hematoxylin and eosin staining at the time of resection but developed histologically proven recurrences within five years of follow-up. MT1-MMP expression in this recurrent group was then compared with that in 17 patients matched for T-stage, age, gender, grade, and vascular and lymphatic invasion who underwent resection during the same time period but were without evidence of recurrence at five years’ follow-up.

Immunostaining Sections 8 ␮m thick were immunostained with a Techmate 500 Immunostainer (Ventana, Tucson, AZ). After they underwent dewaxing and rehydration, slides were brought to distilled water (DW), automation buffer (Biomeda, Foster City, CA), and finally 0.01 M citrate buffer, pH 6.0 (Sigma, St. Louis, MO). The slides were then autoclaved at 15 lb per square inch for 3 to 5 minutes and cooled for 15 minutes. After a DW/0.1 percent Triton X-100 (Fisher, Springfield, NJ) wash, they were placed back into automation buffer and loaded onto the automated immunostainer. The slides were subjected to avidin/biotin blocking (Vector, Burlingame, CA) and then protein blocking (Research Genetics, Huntsville, AL) for 2 minutes at 37°C. The slides were incubated overnight at 4°C with 0.2 ␮g/ml mouse monoclonal antibody to human MT1MMP (Calbiochem, Cambridge, MA), then with 10 ␮g/ml of biotinylated horse anti-mouse immunoglobulin G (Vector) for 15 minutes at 37°C, streptavidin peroxidase (LAB Probe, Biomeda) for 5 minutes at 37°C, and finally diaminobenzidine (Research Genetics) for 1 minute at 42°C. Three automation buffer washes were performed at room temperature between each step, except for a DW/0.1 percent Triton X wash before and after the chromogen. The slides were counterstained lightly with hematoxylin (Biomeda). As a control, a pellet of cultured human fibrosarcoma cells (HT 1080) transfected with MT1-MMP cDNA, fixed in formalin and embedded in paraffin, was cut into 8-␮m sections and immunostained as described above. One slide from each case was immunostained with nonimmune mouse serum as negative control.

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Grading of Immunostained Slides Each slide was viewed independently by two experienced pathologists blinded to the source of the tissue and the outcome of the patient. Tumor on the entire slide was assessed, and staining for MT1-MMP was graded for both intensity and distribution. A score of 0 to 3⫹ was assigned for each parameter (Table 1) compared with positive and negative controls. The final MT1-MMP score was a product of the two scores, with a range of 0 to 9. Similar systems for grading immunostained slides have been used frequently to assess estrogen receptors in breast cancer.28 All data were expressed as mean ⫾ standard error of mean. Interobserver variability was noticed in 5 percent of the slides reviewed by the pathologists. This discrepancy was resolved by a thorough review of the slides by both pathologists together at a multihead microscope.

Statistics Mean MT1-MMP scores were compared by Wilcoxon’s rank-sum test. Significance was defined as P ⬍ 0.05.

RESULTS Pattern of Staining Most of the staining represented by dark brown granules was observed on the cytoplasmic membrane of the tumor cells and some in the cytoplasm. No staining was observed in the nuclei. The staining was particularly prominent on the luminal aspect of the epithelial cells. No staining was observed in the stromal cells. There was variability in staining in different parts of the same tumor. Representative slides from Table 1. Quantification of Immunostaining for MT1-MMP Score

Intensity of Staining

Distribution of Staining (%)

0 No staining No staining 0.5/⫹ Very weak staining Rare cells stained (⬍5) 1⫹ Weak staining Focal (5–20) 2⫹ Moderate staining Focal (20 –50) 3⫹ Strong staining Diffuse (⬎50) Each immunostained slide was graded independently for intensity and distribution of staining and assigned a score of 0 to 3⫹. The final score was the product of the two scores.

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the normal mucosa, adenoma, carcinoma in situ, and invasive carcinoma groups are shown in Figure 1.

Quantification of MT1-MMP Expression Because of the variability in staining within the tumors, both the intensity of maximum staining and the extent of staining within a tumor were taken into account while MT1-MMP expression was expressed. The highest intensity of staining is relevant because the tumor behaves as aggressively as the most aggressive cells. The MT1-MMP score was 0.2 ⫾ 0.1 for normal colonic mucosa, 1.6 ⫾ 0.3 for adenoma (P ⬍ 0.001 vs. normal mucosa), 3.7 ⫾ 0.7 for carcinoma in situ (P ⬍ 0.006 vs. adenoma), and 7.9 ⫾ 0.5 for node-positive (TxN1–3) colon cancer (P ⬍ 0.009 vs. carcinoma in situ). Thus, there was a significant increase in the expression of MT1-MMP from normal colonic mucosa to adenoma, carcinoma in situ, and node-positive (TxN1–3) colon cancer. The results are summarized in Figure 2. The MT1-MMP score for tumors in the group of patients with node-negative (TxN0) colon cancer that had not recurred within five years of resection was 5.1 ⫾ 0.5. The score for the node-negative patients who had experienced recurrence within five years was 5.1 ⫾ 0.6 (P ⫽ 0.54; Fig. 3). Thus, there was no significant difference in the expression of MT1-MMP between the two groups of node-negative (TxN0) patients. However, the level of MT1-MMP expression in nodenegative colon cancer was higher than that in carcinoma in situ but less than that in node-positive colon cancer.

DISCUSSION The development of invasive cancers from precursor lesions, e.g., polyps, is well documented.29,30 The cellular events that are responsible for these phenotypic changes are the subject of intense investigation and likely multifactorial in nature. In the present study, the expression of MT1-MMP, an enzyme involved in the degradation of extracellular matrix, was investigated ex vivo by immunohistochemical staining of tissue samples containing normal colonic mucosa, adenoma, carcinoma in situ, or metastatic (TxN1–3) colon carcinoma. Because there is a clear association between MT1-MMP overexpression and increased invasiveness of tumor cells in experimental models, we sought to determine whether there is clinical evidence of this by assessing the level of MT1-MMP expression

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Figure 1. Immunohistochemical staining for membrane type 1 matrix metalloproteinase (MT1-MMP) in normal colon mucosa, tubulovillous adenoma, carcinoma in situ, and invasive colon cancer (TxN1–3). Paraffin sections (8 ␮m) were stained with a mouse monoclonal antibody to human MT1-MMP. Brown staining in the tumor cells represents MT1-MMP. Note that there is no staining in the stromal cells. Original magnification ⫻200 (all illustrations).

Figure 2. Mean membrane type 1 matrix metalloproteinase (MT1-MMP) score ⫾ standard error of the mean for normal colon mucosa, tubulovillous adenoma, carcinoma in situ, and TxN1–3 colon cancer. *P ⬍ 0.001 vs. normal mucosa, **P ⬍ 0.006 vs. tubulovillous adenoma, #P ⬍ 0.009 vs. carcinoma in situ.

in adenomatous polyps, carcinoma in situ, and invasive cancer in human colonic tissue. In addition, we assessed whether the level of expression can be predictive of recurrence in node-negative colon cancer patients. We found increased MT1-MMP expression from adenoma to carcinoma in situ and colon cancer. MT1MMP was expressed on the cytoplasmic membrane and cytoplasm of the tumor cell in colon cancers. MT1-MMP expression was not detected in the stroma

Figure 3. Mean membrane type 1 matrix metalloproteinase (MT1-MMP) score ⫾ standard error of the mean for node-negative (TxN0) colon cancers that had (Recurrence ⫹) or had not (Recurrence ⫺) recurred within five years of surgical resection. *P ⫽ not significant.

of the tumors, and there was variability in expression in different areas of the same tumor. In addition, there was no difference in the level of MT1-MMP expression in node-negative (TxN0) colon cancer patients who did or did not have a recurrence within five years of surgical resection of the primary tumor. Our data demonstrate that normal colonic mucosa expresses small amounts of MT1-MMP. The stepwise increase in MT1-MMP expression from adenoma to carcinoma in situ and invasive cancer is relevant when one considers that MT1-MMP is necessary for

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MMP-2 activation.3,16 Our findings are consistent with the hypothesis that the ability of cells to activate MMP-2 is associated with an increased invasiveness.17 Kjellman et al.31 demonstrated that MT1-MMP is expressed in both stromal and neoplastic cells of adrenocortical cancers but not in adenomas. Abundant clinical and histopathologic data suggest that most if not all malignant colonic tumors (carcinomas) arise from preexisting benign tumors (adenomas).30,32 Colonic tumors appear to arise as a result of the mutational activation of oncogenes coupled with inactivation of tumor suppressor genes.32 Mutations in four to five genes are required for the formation of a malignant tumor, including FAP, Kras, DCC, and p53.29,32 Ras gene mutations occur in 58 percent of adenomas larger than 1 cm, in 47 percent of carcinomas, and in only 9 percent of adenomas ⬍1 cm in size. MT1-MMP possesses a cytoplasmic tail, and therefore it has been hypothesized that it may interact with intracellular proteins to transduce signals across the plasma membrane. Because ras proteins participate in signal transduction, upregulation of MT1-MMP may be one of the mechanisms by which ras promotes colon tumorigenesis in the adenoma-carcinoma sequence. The role of MT1-MMP in cancer cell invasion has been demonstrated in malignancies outside the gastrointestinal tract. Although in one study, MT1-MMP mRNA expression was found in all breast carcinomas, there was no difference in the level of expression in patients who survived compared with patients who died of metastatic disease.33 Increased MT1-MMP mRNA levels were found in laryngeal carcinoma compared with surrounding normal tissue, and higher levels were associated with advanced stage, lymph node metastases, and shorter survival.34 Tokuraku et al.11 reported that MT1-MMP mRNA expression was increased in lung carcinoma compared with adjacent normal lung. Once we established that MT1-MMP expression was important to the development of the invasive phenotype, we further sought to determine whether the level of expression had a role in determining recurrent disease. We found that in this patient group, there was no difference in the level of expression of MT1-MMP in patients with TxN0 colon cancer whether they had a recurrence within five years of resection or not. Thus, although the level of MT1MMP expression suggests the invasive nature of a tumor, it cannot be used as a prognostic indicator on which adjuvant therapy is based in node-negative

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colon cancer because of its failure to predict recurrence in this patient group. However, the scoring value of 5.1 seen in both groups of node-negative patients falls between the 3.7 value for carcinoma in situ and the 7.9 value seen in node-positive patients. Although not predictive of recurrence, a high MT1MMP score (e.g., in the range of 7) in the primary tumor might prompt a more intense search for positive lymph nodes in patients with negative lymph nodes. Previous data from our laboratory have shown that treatment of HT-1080 (human fibrosarcoma) cells that overexpress MT1-MMP with neutrophil-derived serine protease results in the conversion of proMMP-2 (72 kd) to its active 62-kd form and increased invasiveness in a dose-dependent manner. In contrast, neutrophil-conditioned medium has no effect on the pro-MMP-2 secreted by HT-1080 cells transfected with an antisense cDNA for human MT1-MMP, which expressed very low levels of MT1-MMP.18 Thus, although a threshold amount of MT1-MMP appears to be necessary for cells to behave in an invasive fashion, additional factors are responsible for metastasis and recurrence after surgical resection. The distribution of MT1-MMP expression in various tumor types has been studied. Some investigators have described MT1-MMP expression predominantly in tumor cells,10 whereas others have found MT1MMP expression in both tumor and stromal cells.21,22,31,35 Okada et al.20 described expression of MT-MMP transcripts in stromal cells of human colon, breast, and head and neck carcinomas but not in cancer cells. Our results suggest that MT1-MMP protein expression can be detected on the cell surface and cytoplasm of the tumor cell in colon cancers. No MT1-MMP was observed in the stroma of the tumors.

CONCLUSIONS Our findings show that the level of MT1-MMP expression in adenoma, carcinoma in situ, and invasive colon cancers is progressively higher. This may contribute in part to the mechanism for progression of colonic adenomas to colon cancer by enhancing the ability of the tumor cells to activate pro-MMP-2 and thus invade the surrounding tissues, including the blood vessels. This observation opens the possibility of targeting MT1-MMP therapeutically in patients with premalignant lesions or at high risk of developing colon cancer. However, given the results in the set of node-negative patients studied, the level of MT1-MMP expression does not appear to predict recurrence and

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as such cannot be used as an independent prognostic indicator.

ACKNOWLEDGMENT The authors thank Dr. Kenneth Eng for his continued support for surgical research.

13.

14.

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