Expression of Matrix Metalloproteinase (MMP)-2 and MMP-9 in Breast Cancer with a Special Reference to Activator Protein-2, HER2, and Prognosis

Vol. 10, 7621–7628, November 15, 2004 Clinical Cancer Research 7621

Johanna M. Pellikainen,1,2,3 Kirsi M. Ropponen,2 positive disease. In the univariate survival analysis, positive Vesa V. Kataja,3 Jari K. Kellokoski,4 stromal MMP-9 predicted shorter recurrence-free survival and breast cancer-related survival (0.0389 ؍ RFS; P) 1,2,3,6 5 ؉ ؍ Matti J. Eskelinen, and Veli-Matti Kosma 1 (BCRS; P 0.0081) in ER disease, especially in the sub- ؍ > Department of Pathology and Forensic Medicine, University of ؉ 2 3 group of ER tumors of 2 cm in diameter (T1; P 0.0031 ؍ ,Kuopio, Kuopio, Finland; Departments of Pathology, Oncology 4Otorhinolaryngology, Oral and Maxillofacial Unit, and 5Surgery, for RFS, and P 0.0089 for BCRS). High MMP-9 expres- in the (0.0351 ؍ Kuopio University Hospital, Kuopio, Finland; and 6Department of sion in cancer cells predicted longer RFS (P Pathology, Centre for Laboratory Medicine, University of Tampere whole patient group. In the multivariate analysis of the and Tampere University Hospital, Tampere, Finland whole patient group, the independent predictors of shorter RFS were reduced MMP-9 expression in carcinoma cells ؍ ؍ ABSTRACT (P 0.0248), HER2 overexpression (P 0.0001), and ad- -Shorter BCRS was pre .(0.0002 ؍ vanced-stage disease (P Purpose: In the present study, we investigated the ex- dicted by advanced-stage disease (P < 0.0001). pression and prognostic value of matrix metalloproteinase Conclusions: Expression of MMP-2 and MMP-9 in (MMP)-2 and MMP-9 in breast cancer as well as their breast cancer seems to be partly related to expression of relation to transcription factor activator protein (AP)-2 AP-2 and HER2. Positive stromal MMP-9 expression pre- and HER2 oncogene. The role of invasion and metastasis- dicts poor survival in the hormone-responsive small tumors, promoting MMPs and their potential regulators, AP-2 and whereas MMP-9 expression in carcinoma cells favors sur- HER2, is currently still unclear in breast cancer. vival. Evaluation of MMP-9 expression seems to add valua- Experimental Design: MMP-2 and MMP-9 expressions ble information on breast cancer prognosis. were analyzed immunohistochemically in a large prospective series of 421 breast cancer patients diagnosed and treated between 1990 and 1995 at Kuopio University Hos- INTRODUCTION pital (Kuopio, Finland). The relation of MMP-2 and MMP-9 Matrix metalloproteinase (MMP)-2 and MMP-9 (gelatin- expressions to AP-2, HER2, clinicopathological data, and ase A and B) are zinc-dependent endopeptidases of a large survival was investigated. MMP family (1, 2). MMP-2 and MMP-9 are related to tumor Results: Both MMP-2 and MMP-9 were expressed in invasion and metastasis by their capacity for tissue remodeling the cytoplasm of malignant and stromal cells. High expres- via extracellular matrix as well as basement membrane degra- sion of MMPs in carcinoma cells was related to small tumors dation and induction of angiogenesis (1, 2). These gelatinases are secreted as zymogens and cleaved to the active form, and (T1, stage I), whereas positive stromal expression of MMPs was associated with aggressive factors. High expression of their function is tightly regulated by several different mecha- MMP-2 and MMP-9 in carcinoma cells, but not in stromal nisms (1, 2). In breast cancer, both gelatinases seem to be cells, was related to high AP-2 expression. Positive stromal expressed in cancer tissue, although the results have not been MMP-2 expression was associated with HER2 overexpres- consistent (3–6). To date, only a few studies have investigated sion in the whole patient group and in the node-negative the prognostic value of MMP-2 and MMP-9 in breast cancer patient subgroup. Positive stromal MMP-9 expression was (5, 7–10). related to HER2 overexpression in estrogen receptor (ER)- Aberrant expressions of transcription factor activator protein (AP)-2 and HER2 oncogene are related to disease progression and increased invasive capacity in breast cancer (11–13), which may be due in part to increased MMP activity (14, 15). Received 5/31/04; revised 8/8/04; accepted 8/13/04. Indeed, AP-2 has been shown to be critically responsible for Grant support: EVO funding of Kuopio and Tampere University MMP-2 transcription (16–22) and to enhance MMP-9 promoter Hospitals, Kuopio University Funding, Research Foundation of Orion activity (23, 24). In melanoma cells, inactivation of AP-2 leads Corporation, The Culture Fund of Finland (North Savo Fund), and The to increased invasiveness via increased MMP-2 expression and Finnish Medical Society Duodecim. activity (14), whereas in breast cancer, the relationship between The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked AP-2 and gelatinase expression has not been studied. The effect advertisement in accordance with 18 U.S.C. Section 1734 solely to of HER2 overexpression on the invasion capacity of tumor cells indicate this fact. is related, at least in part, to the up-regulation of MMP-2 and Requests for reprints: Veli-Matti Kosma, Department of Pathology MMP-9 expression and proteolytic activity (15, 25–28). Fur- and Forensic Medicine, University of Kuopio, P. O. Box 1627, FIN- 70211 Kuopio, Finland. Phone: 358-17-162-750, Fax: 358-17-162-753; thermore, c-ErbB ligands are able to up-regulate several MMPs E-mail: [email protected]. (29, 30). To the best of our knowledge, this is the first study to ©2004 American Association for Cancer Research. investigate the relation of MMP-2 and MMP-9 to AP-2 and

HER2 expression in a large prospective clinical breast cancer Table 1 Clinicopathological data study. Characteristic n (%) No. of patients 421 (100) Age (y) PATIENTS AND METHODS Mean (range) 59.2 (23.3–91.6) Patients. The present study is part of the Kuopio Breast Tumor size Cancer Project, a prospective long-term clinical study involving T1 221 (53) T 166 (39) 520 breast cancer patients (31–34) diagnosed among the 2,500 2 T 24 (6) women who were referred to Kuopio University Hospital be- 3 T4 10 (2) cause of a clinical breast lump, suspicious mammographic find- Lymph node status ing, or a breast symptom (e.g., pain, nipple discharge) between Negative 245 (58) April 1990 and December 1995. Women willing to participate in Positive 168 (40) Unknown 8 (2) the project were interviewed and examined by a trained study Stage nurse before any diagnostic procedures. The participation rate of I 162 (39) patients with diagnosed breast cancer was 98%. Thus, the pa- II 212 (50) tient series represents unselected typical breast cancer cases III 39 (9) from the University Hospital catchment area. Altogether, 479 Unknown 8 (2) Histological type invasive and 41 noninvasive carcinomas were diagnosed. After Ductal 272 (64) surgical treatment, the patients were offered adjuvant chemo- Lobular 67 (16) therapy and/or hormonal therapy and radiotherapy, depending Other 82 (20) on the mode of the surgery, the patient’s menopausal status, and Histological grade I 110 (26) the stage of the disease, according to the national guidelines II 192 (46) (35). In brief, postoperative radiotherapy was given to all pa- III 119 (28) tients treated with breast-conserving surgery and to all patients ER status with axillary node-positive (pNϩ) status irrespective of the Positive 326 (78) mode of surgery. All premenopausal patients with pNϩ status Negative 94 (22) Data not available 1 (Ͻ1) and some with axillary node-negative (pNϪ) status presenting PR status with other adverse prognostic factors, such as estrogen receptor Positive 261 (62) (ER)/progesterone receptor (PR) negative or poorly differenti- Negative 158 (38) Ͻ ated tumor, were given adjuvant chemotherapy (intravenous Data not available 2 ( 1) Menopausal status cyclophosphamide, mitoxantrone, methotrexate, and 5-fluorou- Premenopausal 132 (31) racil) for six cycles, which was the standard therapy at the time. Postmenopausal 289 (69) All postmenopausal women with ER- and/or PR-positive tumors Recurrence at 5 y were adjuvantly treated with either tamoxifen or toremifene for No 346 (82) 3 years within a clinical study protocol. Thus, within a stage, the Yes 75 (18) Cause of death at 5 y postoperative treatment was rather uniform, with only a few Alive 335 (79) exceptions due to concurrent conditions. Stage was assessed by Breast cancer 49 (12) using the UICC classification (36). Patients with noninvasive Other 37 (9) carcinomas, a previous history of breast cancer, metastatic disease (stage IV), or insufficient tumor material were excluded from the present study. Thus, 421 patients with sufficient primary tumors and complete clinical histories were available for four original sections of the primary tumor. All tumors were MMP-2 analyses, 415 patients were available for MMP-9 anal- simultaneously reevaluated for histologic type and grade by two yses, 417 patients were available for AP-2 analyses, and 419 senior pathologists, who were unaware of the clinical data. The patients were available for HER2 analyses. The mean age of the most representative blocks were selected for cutting into new 421 patients was 59.2 years (median, 56.8 years; range, 23.3– 5-␮m–thick sections for immunohistochemical analyses. 91.6 years). The mean follow-up time was 55.0 months (median, Immunohistochemistry. The MMP-2 and MMP-9 stain- 57.5 months; range, 1.2–115.1 months). During the first 5 years ings were demonstrated using Sequenza Immunostaining Center of follow-up, 75 of the 421 patients (18%) had a recurrence, 49 (Shandon Scientific Limited, Astmoor, United Kingdom). In patients (12%) died of breast cancer, and 37 patients (9%) died brief, the sections were deparaffinized in xylene, rehydrated in of other causes. The 5-year recurrence-free survival (RFS) rate EtOH, and washed twice with distilled water. For better antigen of the patients was 79%, the breast cancer-related survival retrieval, the samples were boiled three times for 5 minutes in a (BCRS) was 86%, and the overall 5-year survival rate was 77%. microwave oven in a citrate buffer (pH 6.0). Endogenous per- The 5-year survival of excluded stage IV patients (n ϭ 19) was oxidases were blocked by 5% hydrogen peroxidase treatment 21%. None of the patients were lost to follow-up. The clinico- for 5 minutes. The samples were washed with PBS (pH 7.2) and pathological data of the patients are summarized in Table 1. incubated in 1.5% normal horse serum for 35 minutes to prevent Histology. The tumor samples were fixed in 10% neutral nonspecific antigen binding. The primary antibody for MMP-2 buffered formalin, processed routinely, and embedded in paraf- was a mouse monoclonal antibody toward a human proform of fin. The histologic diagnosis was confirmed by reviewing one to MMP-2 (Ab-1; clone CA-4001; Neomarkers, Fremont, CA),

used at a working dilution of 1:25. The primary antibody used MMP-9 immunohistochemistry, AP-2 and HER2 expressions, for detection of MMP-9 was a mouse monoclonal antibody, and clinicopathological parameters were tested with contin- which recognizes both the pro- and active forms of human gency tables and a ␹2-test. The univariate survival analyses were MMP-9 (MAB3309; clone 56-2A4; Chemicon, Temecula, CA), performed using Kaplan-Meier’s log-rank analysis, and the in- used at a dilution of 1:2,500. The samples were incubated with dependent prognostic value of variables was further examined the primary antibody overnight at 4°C. Before applying the with Cox’s regression analysis. Probability values of Յ0.05 secondary antibody, the samples were washed twice with PBS. were considered significant in the analyses. In Cox’s multiva- The slides were incubated for 45 minutes with the biotinylated riate analysis, the Enter method was used with an additional secondary antibody, followed by a wash and a 50-minute incu- removal limit of P Ͻ 0.10. Both BCRS and RFS were examined. bation in an avidin-biotinylated peroxidase complex reagent The recurrence-free time was defined as the time between (Vectastain Rabbit ABC Elite Kit; Vector Laboratories, Burl- diagnosis and the date of first local recurrence or a distant ingame, CA). Expressions were visualized with a 5-minute metastasis, whichever appeared first. Patients who remained diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) healthy or died without breast cancer during the follow-up treatment. The slides were counterstained with Mayer’s hema- period were censored at the time of the last follow-up visit or toxylin, dehydrated, and mounted with DePex (BDH Ltd, Poole, death. United Kingdom). A routinely processed breast cancer section without the primary antibody served as a negative control in each staining series. Placenta and known positive breast cancer RESULTS samples served as positive controls. Control sections stained as Expression of Matrix Metalloproteinase-2 and Matrix expected. Metalloproteinase-9. The expression patterns of MMP-2 and Activator Protein-2 and HER2 Immunohistochemistry. MMP-9 were cytoplasmic both in tumor cells and in stromal The immunohistochemical procedures for AP-2 (12) and HER2 fibroblasts and inflammatory cells. In addition, benign breast (37) have been reported previously. In brief, a rabbit polyclonal epithelium and vascular endothelium stained positively for antibody for human AP-2 (C-18, specific for AP-2␣, AP-2␤, MMPs. The general expressions of MMP-2 and MMP-9 in the and AP-2␥; Santa Cruz Biotechnology, Santa Cruz, CA) was tumor cells were high, with median values of 80% for MMP-2 used as a primary antibody at a working dilution of 1:2,000. An and 85% for MMP-9 (range, 0–100%). Fifty four percent and AP-2–positive melanoma was used as a positive control. HER2 52% of tumors expressed high levels of MMP-2 and MMP-9, immunohistochemistry was performed at Kuopio University respectively. Stromal MMP-2 positivity was observed in 46% of Hospital according to a routine staining method using a Tech- the cases, and MMP-9 positivity was observed in 38% of the Mate 500 staining automat (DAKO, Glostrup, Denmark). The cases. High MMP-2 expression in carcinoma cells was posi- primary antibody used was NCL-CB11 (Novocastra Laborato- tively associated with high stromal MMP-2 expression (␹2 ϭ ries Ltd., Newcastle-upon-Tyne, United Kingdom), diluted 6.9; P ϭ 0.009), whereas MMP-9 expression in cancer cells and 1:100. Expression was visualized by the labeled streptavidin- MMP-9 expression in stromal cells were not associated with biotin system (LSAB, ChemMate Detection Kit; DAKO). A each other. known 3ϩ stained, amplified breast cancer sample served as a Expression of Activator Protein-2 and HER2. The ex- positive control. pression of nuclear AP-2 (range, 0–100%) was high (Ն80%) in Scoring of Immunoreactivity. The specimens were an- 50% of the carcinomas, and cytoplasmic AP-2 expression was alyzed by two observers (J. M. P. and K. M. R.), who were seen in 47% of the cases. Pathological membranous HER2 unaware of the patients’ clinical outcome. Discrepancies be- overexpression (i.e., 2–3ϩ) was observed in 13% of the carci- tween the observers were found in Ͻ10% of the slides exam- nomas. ined, and consensus was reached on further review. Expression Relation of Matrix Metalloproteinase-2 and Matrix of both MMP-2 and MMP-9 was cytoplasmic and was recorded Metalloproteinase-9 Expressions to Activator Protein-2. separately for tumor and stromal cells. The median value (80% High MMP-2 expression in carcinoma cells was associated with for MMP-2; 85% for MMP-9) was used as a cutoff for MMP high nuclear AP-2 expression (␹2 ϭ 9.6; P ϭ 0.002) and high expression in the tumor cells. The categories for MMP-2 were cytoplasmic AP-2 expression (␹2 ϭ 22.0; P Ͻ 0.001) in the reduced (Ͻ80%) and high (Ն80%), and the categories for whole patient group, as well as in the nodal subgroups (Table 2). MMP-9 were reduced (Յ85%) and high (Ͼ85%). Stromal ex- Stromal MMP-2 expression was not associated with AP-2 pression of MMPs was divided into positive and negative ex- (Table 2). pression groups according a 20% cutoff for positively stained Similarly, high MMP-9 expression in malignant cells was stromal cells. HER2 staining was analyzed according to a scor- associated with high nuclear AP-2 expression (␹2 ϭ 8.5; P ϭ ing proposed by the HercepTest (DAKO), and samples with a 0.004) in the whole patient group and in the node-negative score of 2ϩ or 3ϩ were considered HER2 positive (37). For patient group (␹2 ϭ 5.4; P ϭ 0.020). Stromal MMP-9 expres- nuclear AP-2 expression, the median value (80%) was used as a sion and AP-2 were not statistically related. Results are sum- cutoff value (12). If Ͼ10% of the cells in the tumor area were marized in Table 2. stained (12), cytoplasmic AP-2 expression was considered pos- Relation of Matrix Metalloproteinase-2 and Matrix itive. Metalloproteinase-9 Expressions to HER2. Stromal MMP-2 Statistical Analyses. The statistical analyses were car- positivity was associated with HER2 overexpression in the ried out by using the SPSS for Windows 9.0 program (SPSS whole patient group (␹2 ϭ 5.8; P ϭ 0.016) and in the node- Inc., Chicago, IL). The associations between MMP-2 and negative patient group (␹2 ϭ 7.7; P ϭ 0.005; Table 2). Expres-

Table 2 Relation of gelatinase expression to AP-2 and HER2 (P ϭ 0.0031 for RFS, and P ϭ 0.0089 for BCRS; n ϭ 171), AP-2 in which the 5-year RFS for stromal MMP-9–negative patients was 89% compared with 70% for stromal MMP-9– High High HER2 positive patients (Fig. 2). However, stromal MMP-9 expres- Variable nuclear cytoplasmic overexpression MMP-2 expression in cancer cells All Pϭ0.0021 PϽ0.0011 PϾ0.05 Table 3 Immunohistochemical and statistically significant NϪ Pϭ0.0451 Pϭ0.0101 PϾ0.05 clinicopathological variables of BCRS and RFS in univariate survival Nϩ Pϭ0.0361 PϽ0.0011 PϾ0.05 analysis Stromal MMP-2 expression BCRS at 5 y RFS at 5 y All PϾ0.05 PϾ0.05 Pϭ0.0161 Variable n % Pn% P NϪ PϾ0.05 PϾ0.05 Pϭ0.0051 Nϩ PϾ0.05 PϾ0.05 PϾ0.05 Stage Ͻ0.0001 Ͻ0.0001 MMP-9 expression in I 162 92 162 87 cancer cells II 212 87 212 77 All Pϭ0.0041 PϾ0.05 PϾ0.05 III 39 55 39 55 NϪ Pϭ0.0201 PϾ0.05 Pϭ0.0461 Lymph node status Ͻ0.0001 Ͻ0.0001 Nϩ PϾ0.05 Pϭ0.0281 PϾ0.05 Negative 245 92 245 86 Stromal MMP-9 Positive 168 78 168 69 expression Histological grade 0.0506 0.0747 All PϾ0.05 PϾ0.05 PϾ0.05 I 110 92 110 85 NϪ PϾ0.05 PϾ0.05 PϾ0.05 II 192 85 192 78 Nϩ PϾ0.05 PϾ0.05 PϾ0.05 III 119 82 119 74 ER status Ͻ0.0001 0.0878 Abbreviation: 1, positive association. Positive 326 90 326 80 Negative 94 72 94 74 PR status 0.0009 0.1660 Positive 261 90 261 81 sion of MMP-2 in carcinoma cells was not associated with Negative 158 79 158 76 HER2 expression (Table 2). HER2 expression 0.0056 Ͻ0.0001 In the node-negative patient group, high MMP-9 expres- Negative 365 88 365 82 sion in carcinoma cells was associated with HER2 overexpres- Positive 54 71 54 56 MMP-2 in cancer cells 0.6340 0.8672 ␹2 ϭ ϭ sion ( 4.0; P 0.046; Table 2). This was not seen in the Reduced 195 86 195 79 whole patient group or in the node-positive patient subgroup High 226 85 226 79 (Table 2). Positive stromal MMP-9 expression was related to Stromal MMP-2 0.6957 0.2428 HER2 overexpression in ERϩ tumors (␹2 ϭ 4.4; P ϭ 0.036), Negative 228 86 228 76 Positive 193 85 193 83 but not in other investigated subgroups. MMP-9 in cancer cells 0.2819 0.0351 Relation of Matrix Metalloproteinase-2 and Matrix Reduced 198 84 198 74 Metalloproteinase-9 Expressions to Clinicopathological High 217 87 217 83 Data. High MMP-2 expression in carcinoma cells associated Stromal MMP-9 0.6078 0.6253 with stage I disease (␹2 ϭ 6.4; P ϭ 0.040). Stromal MMP-2 Negative 259 86 259 79 Positive 156 84 156 77 positivity, instead, was related to poor differentiation (␹2 ϭ 21.8; P Ͻ 0.001), ER negativity (␹2 ϭ 20.0; P Ͻ 0.001), and ductal or other carcinoma types (␹2 ϭ 15.7; P Ͻ 0.001). High MMP-9 expression in carcinoma cells was associated with small tumor size (␹2 ϭ 10.0; P ϭ 0.007) and lower recurrence rate (␹2 ϭ 4.4; P ϭ 0.036). Positive stromal MMP-9 expression was associated with poor differentiation (␹2 ϭ 8.8; P ϭ 0.013) and ductal carcinoma type (␹2 ϭ 8.1; P ϭ 0.018). Univariate Survival Analysis. In the univariate analysis, MMP-2 expression, in either carcinoma cells or stromal cells, possessed no prognostic value for RFS or BCRS in the whole patient group (Table 3) or in the nodal subgroups. The 5-year RFS rate for patients with high MMP-9 expression in carcinoma cells was 83%, compared with 74% for patients with low MMP-9 expression (P ϭ 0.0351; Fig. 1; Table 3). This survival advantage was not seen in the BCRS analysis (Table 3) or in the nodal subgroups. Interestingly, positive MMP-9 expression in stromal cells predicted shorter RFS and BCRS in ERϩ disease (P ϭ 0.0389 for RFS, and P ϭ 0.0081 for BCRS; n ϭ 321). A Fig. 1 RFS in the whole patient group according to MMP-9 expression ϩ ϭ similar result was obtained in a subgroup of T1,ER tumors in carcinoma cells (n 415).

HER2, and known prognostic parameters. High gelatinase expression in carcinoma cells was associated with high AP-2 expression, and positive stromal MMP expression was related to HER2 overexpression, supporting a function for AP-2 and HER2 in MMP regulation. In addition, positive stromal MMP-9 ϩ expression predicted poor survival in ER , small T1 breast cancers, whereas high MMP-9 expression in carcinoma cells favored survival. In this study, the adjacent benign breast epithelium was positive for both MMPs. In line with our results, Soini et al. (6) detected MMP-9 mRNA as well as MMP-2 and MMP-9 proteins in benign epithelial cells. Positive protein expression of MMP-2 and MMP-9 in benign epithelium was also observed by Lebeau et al. (38). In addition, Dahlberg et al. (39) detected MMP-2 mRNA in some benign glandular cells. Indeed, in zymography studies, the expression of MMP-2 and MMP-9 has been detected in benign tissue, and enzyme activities have been Fig. 2 RFS according to stromal MMP-9 expression in the subgroup of less than that in malignant tissue (40–42). However, benign ϩ ϭ T1,ER patients (n 171). epithelial cells have also remained negative for protein and/or mRNA (5, 43), or the expression has been localized in other structures (3, 44). In the present study, the antibodies used recognized pro– sion did not have prognostic value in the whole patient group MMP-2 and both the pro- and active forms of MMP-9. The (Table 3) or in the nodal subgroups. gelatinase expressions were cytoplasmic in both carcinoma and Shorter RFS and BCRS were also predicted by advanced- stromal cells, as also described previously (3, 5, 6, 44). How- Ͻ stage disease (P 0.0001 for both), axillary lymph node pos- ever, in some studies, MMP-2 protein has been expressed Ͻ ϭ itivity (P 0.0001 for both), and HER2 overexpression (P mainly in the carcinoma cells (7–10, 45), whereas MMP-9 Ͻ 0.0056 for BCRS, and P 0.0001 for RFS). Shorter BCRS was, protein and mRNA have been mostly localized into stromal cells Ͻ in addition, predicted by ER and PR negativity (P 0.0001 for (40, 46). In addition, in several in situ hybridization studies, ϭ ER, and P 0.0009 for PR). The results are summarized in MMP-2 and MMP-9 mRNAs have been identified mainly in Table 3. stromal cells, although some has been detected in carcinoma Multivariate Analysis. There were 402 patients with cells (6, 40, 43, 46–48). Previously, it has been suggested that complete clinical data available for BCRS and 403 patients stromal fibroblasts secrete MMPs, which are stored and acti- with complete clinical data available for RFS analysis. Variables vated in carcinoma cells (4). with a prognostic value in the univariate analysis were included In the present series, high nuclear AP-2 expression was in the multivariate analysis (stage, ER and PR status, and HER2 often associated with high MMP-2 and MMP-9 expression in expression in BCRS; stage, MMP-9 expression in carcinoma carcinoma cells. This is noteworthy because MMP transcription cells, and HER2 expression in RFS). Shorter BCRS was inde- has also been shown to occur in breast carcinoma cells (6), a pendently predicted by advanced-stage disease (P Ͻ 0.0001; Table 4). The independent predictors of shorter RFS were reduced MMP-9 expression in carcinoma cells (P ϭ 0.0248), advanced-stage disease (P ϭ 0.0002), and HER2 overexpres- Table 4 Results of Cox’s multivariate analysis for BCRS and RFS sion (P ϭ 0.0001; see Table 4). Relative risk Multivariate analysis was also performed in the subgroup Category (variable) Beta (SE) (95% CI) P of ERϩ patients, in which positive stromal MMP-9 expression BCRS (P ϭ 0.0023) and advanced-stage disease (P Ͻ 0.0001) inde- Stage Ͻ0.0001 pendently predicted shorter BCRS (n ϭ 306) and HER positiv- I Reference ϭ ϭ II 0.79 (0.41) 2.21 (0.99–4.95) 0.0537 ity (P 0.0210) and advanced-stage disease (P 0.0012) III 2.07 (0.44) 7.95 (3.35–18.86) Ͻ0.0001 independently predicted shorter RFS (n ϭ 309). In the subgroup RFS ϩ ϭ of ER ,T1 tumors (n 152), shorter BCRS was independently Stage 0.0002 predicted by positive stromal MMP-9 expression (P ϭ 0.0301) I Reference ϭ ϭ II 0.48 (0.29) 1.62 (0.91–2.86) 0.0996 and advanced-stage disease (P 0.0096), and shorter RFS (n III 1.44 (0.35) 4.22 (2.10–8.46) Ͻ0.0001 167) was independently predicted by stromal MMP-9 positivity HER2 expression 0.0001 (P ϭ 0.0131) and HER2 overexpression (P ϭ 0.0050). Negative Reference Positive 1.01 (0.26) 2.75 (1.66–4.57) MMP-9 expression in 0.0248 DISCUSSION carcinoma cells In the present study we investigated in a large prospective Reduced 0.53 (0.24) 1.70 (1.07–2.70) series of breast cancer patients the expression and prognostic High Reference value of MMP-2 and MMP-9, as well as their relation to AP-2, Abbreviation: CI , confidence interval.

situation that may be regulated in part by AP-2 in breast carci- MMP-2 has often been related to poor outcome in breast carcinomas. Further evidence for this possible phenomenon has been nomas (7–10, 65, 66). In ovarian adenocarcinoma, high stromal obtained from in vitro studies, in which AP-2 has been an MMP-2 expression has been related to shorter disease-free sur- important factor in induction of MMP-2 transcription (16, 21). vival (67). In early-stage oral squamous cell carcinoma, MMP-9 In addition, Sivak et al. (24) have shown that AP-2␣ is required and TIMP-2 expressions, but not MMP-2, have shown prognos- for MMP-9 transcription in corneal epithelium. On the other tic value (68). MMP-2 staining results reported here represent hand, in melanoma cells, wild-type AP-2 could down-regulate pro–MMP-2 protein expression, and thus do not tell about the MMP-2 transcription, and inactivation of AP-2 could lead to enzyme activity, which may be better related to prognosis. increased invasiveness via an increase in MMP-2 (14). Based on Several other factors such as membrane-type MMP-1 and our results and those in the literature (12, 49), we hypothesize TIMP-2 take part in the activation and regulation of MMP-2 and that the role of AP-2 in MMP regulation in breast cancer may be may alter prognosis. In fact, patient outcome may depend on the tumor suppressive, by unknown mechanisms. Possibly this hap- balance between MMP-2 and TIMP-2 in breast cancer, as sug- pens via tissue inhibitor of metalloproteinase (TIMP)-1 and gested by Nakopoulou et al. (44). TIMP-2 regulation (50, 51), both of which can inhibit activated The prognostic value of stromal MMP-9 expression has not MMPs, including MMP-2 and MMP-9 (52–54). The complex been studied previously in a large clinical breast cancer series. regulation between AP-2 and MMPs and the separate roles of Interestingly, positive stromal MMP-9 expression predicted sig- different AP-2 family members in breast cancer still need fur- nificantly shorter RFS and BCRS for ERϩ patients, especially ther investigation (55–58). if the tumors were small (i.e., in a group with usually very good In the present study, positive stromal MMP-2 and MMP-9 prognosis). A possible explanation is that positive stromal expressions were related to HER2 overexpression. Previously, MMP-9 expression may represent an increase in the active high MMP-2 mRNA expression in the stromal cells of breast enzyme form that could lead to metastatic process and increased carcinoma has been associated with c-erbB2 positivity, too (48). malignancy, for example, via enhanced angiogenic activity (69– Interestingly, association was seen in early carcinogenesis (in 71). MMP-9 expression, in addition, may be affected by hor- Ϫ ␣ N , in stage I disease, and in T1 tumors for MMP-2; data not mones. It has been shown that activation of ER- by estrogen shown). Positive stromal MMP-9 was associated with HER2 has resulted in tumor progression by stimulating cell growth and overexpression in ERϩ disease, in which MMP-9 predicted invasiveness via acceleration of the expression of MMPs includ- shortened survival. This may indicate that HER2 induces stro- ing MMP-9 (72). Other studies have confirmed the relationship mal MMP expression and/or activation in an early phase of between steroid hormones and increased gelatinase activity as breast cancer, which further increases disease progression and well (73–75). Evaluating stromal MMP-9 expression may add metastasis, as suggested in the literature (27, 28, 30, 59, 60). A valuable information on breast cancer prognosis, especially in possible explanation may be increased stimulus mediated by the early carcinogenesis. extracellular matrix metalloproteinase inducer, EMMPRIN (61, High MMP-9 expression in carcinoma cells independently 62). Differences between MMP-2 and MMP-9 expression in predicted improved RFS in the whole patient group. This is in relation to HER2 in the present study may be due to different line with the results of Scorilas et al. (5), who reported that low antibodies as well as different transcriptional regulation of the MMP-9 expression in breast carcinoma cells predicted poor gelatinases (19, 63). survival for NϪ patients (5). A similar trend was seen in a study In this study, high MMP-9 expression in carcinoma cells with a small number of breast cancer patients (65). Increased was associated with small tumors and a lower recurrence rate. MMP-9 mRNA expression, instead, has been inversely associ- Previously, Scorilas et al. (5) reported that reduced MMP-9 ated with the number of survivors in breast cancer (66), whereas expression in breast carcinoma cells was associated with poor in other studies, MMP-9 has not been associated with prognosis prognostic factors such as large tumor size, giving further at all (42, 64). Additional studies are required to assess whether strength to our results. Jones et al. (3) found high MMP-9 the results presented here may be related to a shift from inactive expression in lobular carcinoma cells but failed to demonstrate to active MMPs during tumor progression. other associations between MMP-9 in carcinoma or stromal We conclude that immunohistochemically detected posi- cells and clinicopathological parameters. High MMP-2 expres- tive stromal MMP-9 expression predicts shortened survival in

sion in carcinoma cells, instead, has been related to only a few hormone-sensitive, small T1 breast cancers, whereas MMP-9 inverse prognostic factors (9, 44) or shown to have no associ- expression in carcinoma cells offers survival advantage. HER2 ation with clinicopathological parameters in breast cancer (3, 7). overexpression may be responsible in part for MMP induction in In this material, high MMP-2 expression in carcinoma cells was early breast cancer, resulting in increasing invasiveness. The related to stage I disease. Positive stromal expression of MMP-2 association between AP-2 and MMP expression suggests a role and MMP-9, instead, was associated with clinicopathological for AP-2 in gelatinase regulation, but the exact mechanism, factors related to aggressive disease. Previously, an increase in however, remains to be investigated. Finally, evaluation of stromal MMP-2 mRNA in poorly differentiated breast tumors MMP-9 expression may add valuable information regarding has been reported (43), which is in accordance with our results. breast cancer. Positive correlations of MMP-2 and MMP-9 with tumor grade have been observed in zymography, too (40, 45). ACKNOWLEDGMENTS In our material, neither stromal nor carcinoma cell MMP-2 The authors thank Anna-Kaisa Lyytinen, Mervi Malinen, Aija expression possessed prognostic value, in line with previous Parkkinen, and Riikka Eskelinen for skillful technical assistance. The findings in breast cancer (42, 48, 64). On the other hand, statistical advice of Pirjo Halonen is also acknowledged.

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Johanna M. Pellikainen, Kirsi M. Ropponen, Vesa V. Kataja, et al.

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