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Expression of Extracellular Matrix Metalloproteases Inducer on Micrometastatic and Primary Mammary Carcinoma Cells

¹ Institute of Tumor Biology, ² Department of Gynecology, ³ Department of Gynecopathology, and ⁴ Institute of Mathematics and Computer Science in Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany, and ⁵ Institute of Pathology, University of Basel, Basel, Switzerland

	ABSTRACT

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Purpose: EMMPRIN (extracellular matrix metalloprotease inducer) is a glycosylated member of the immunoglobulin superfamily known to stimulate the production of matrix metalloproteases (MMPs) 1, 2, and 3 and MT1-MMP in peritumoral fibroblasts. We here evaluated whether EMMPRIN expression is related to tumor progression in human breast cancer.

Experimental Design: An immunohistochemical study using high-density tissue microarrays (n = 2222 breast cancer samples) and EMMPRIN-specific antibodies HIM6 and MEM-M6/1 was performed, and staining results were statistically correlated with various clinicopathological parameters. To analyze the putative association between EMMPRIN expression and bone marrow (BM) micrometastasis, an additional set of 55 breast tumors from patients with or without micrometastatic cells as determined with anti-cytokeratin antibody A45-B/B3 were included in our study. Cytokeratin-positive cells in BM were costained with EMMPRIN-specific antibody 1G6.2.

Results: Positive EMMPRIN staining correlated significantly with various histopathological risk factors (higher tumor grade, increased tumor size, negative estrogen receptor status and progesterone receptor status, and higher mitotic index) as well as decreased tumor-specific survival (log-rank, P = 0.0027). In particular, in patients > 50 years (i.e., postmenopausal women), EMMPRIN expression was an independent prognosticator as shown by Cox regression analysis (relative risk = 1.7, 95% confidence interval 1.4–4.3, P = 0.036). An involvement of EMMPRIN in tumor progression was also supported by the fact that it was expressed on 90% of micrometastatic cells in BM.

Conclusions: EMMPRIN expression in primary tumor predicts an unfavorable prognosis in breast cancer, suggesting a crucial role of EMMPRIN in progression of human mammary carcinomas.

	INTRODUCTION

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Breast cancer is the leading cause of women cancer mortality in Western industrial countries. Although only <10% of women with primary breast cancer have clinicopathological signs of overt metastases, metastatic relapse occurs in about half of the cases with apparently localized tumors within 5 years after surgery. Understanding the molecular mechanisms underlying the spread of cancer cells to distant organs therefore is a prerequisite for the development of novel cancer therapies.

The degradation of extracellular matrix (ECM) surrounding both the primary tumor and tumors at distant secondary sites is a critical step in invasion and metastasis of epithelial tumor cells (1) . The degradative process mainly is catalyzed by zinc-dependent endopeptidases, the matrix metalloproteases (MMPs; Ref. 2 ). The major source of MMP activity in a tumor are surrounding stromal cells, and an increasing body of evidence suggests that cancer cells are able to stimulate MMP production by fibroblasts in a paracrine fashion. Indeed, several tumor-derived factors known to enhance MMP production have been identified, one of which is EMMPRIN (ECM metalloprotease inducer), also known as TCSF, CD147, M6, and Basigin (3 , 4) . It has been shown that native and recombinant EMMPRIN induces expression of MMP-1, MMP-2, MMP-3, and MT1-MMP in fibroblast in vitro (5 , 6) . This highly glycosylated 58-kDa type I transmembrane protein harbors two immunoglobulin-folds within its extracellular domain, indicating that it is a member of the immunoglobulin superfamily. Several molecular interaction partners of EMMPRIN have been identified thus far, including the ₃ß₁- and ₆ß₁-integrins, which are known to mediate cell-matrix interactions (7) . Recent data suggest that EMMPRIN itself may act as a counterpart receptor on the fibroblast cell surface, indicating that EMMPRIN also participates in heterotypic and homotypic cell-cell adhesion through homophilic interactions (8) . Taken together, these observations strongly suggest a role of EMMPRIN in invasion and metastasis by remodeling of the ECM through activation of MMP production and modulation of cell-substrate and cell-cell adhesion processes (9) .

Here, we show for the first time that expression of EMMPRIN was strongly associated with poor prognosis in human breast carcinomas. Moreover, we observed relationships between EMMPRIN expression and grade of differentiation, histological type, negative estrogen receptor (ER) and progesterone receptor status, and tumor size. We also showed that EMMPRIN is expressed on 90% of micrometastatic tumor cells isolated from breast cancer patients without overt metastasis, indicating a possible EMMPRIN-dependent selection mechanism for these cells.

	MATERIALS AND METHODS

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Patients and Tissue Microarrays (TMAs).
Collection of tissue cores was carried out under highly controlled conditions. Areas of nonnecrotic vital-appearing cancer tissue were selected for arraying by an experienced pathologist (G. Sauter). The quality of tissues was demonstrated by cytokeratin (CK) staining of the TMAs. In total, <5% of the spots lacked complete staining with all of the three antibodies against CK8, CK18, and CK19 (data not shown). Processing and staining of tissue cores was carried out under highly standardized conditions. All TMA sections were simultaneously stained with in the same reagents on 1 day. A high-density breast cancer prognosis TMA containing a total of 2222 formalin-fixed, paraffin-embedded breast cancer tissues was generated as described previously (10) . The median age of patients was 63 years (range, 26–101 years). The series included 1551 ductal, 312 lobular, 337 other known breast cancer entities, and 22 nonclassified mammary tumors. The average follow-up period was 63 months for all patients (range, 1–176 months).

For the correlation between EMMPRIN expression and the existence of micrometastatic cancer cells in bone marrow (BM), paraffin-embedded tissue specimens of additional 55 breast cancer patients were evaluated immunohistochemically for EMMPRIN expression. The patients underwent surgical treatment at the Department of Gynecology and Obstetrics, University Hospital Eppendorf (Hamburg, Germany) and the Department of Gynecology and Obstetrics, University Hospital (Kiel, Germany) between May 1999 and July 2000. Written informed consent was obtained from all patients of this study.

Immunohistochemistry.
Formalin-fixed, paraffin-embedded tumor arrays or primary tumor tissue samples were deparaffinized and subsequently subjected to a 20 min microwave pretreatment in citrate buffer (10 mM, pH 6.0). To visualize EMMPRIN expression a combination of two EMMPRIN-specific monoclonal antibodies (mAbs) (clone MEM-M6/1, Biogenesis, Kingston, NH, at 10 µg/ml; and clone HIM6, RDI, Flanders, NJ, at 5 µg/ml) was used. The preparation and specificity of these antibodies has been described in detail elsewhere (11 , 12) . Automatic immunohistochemical staining was performed using the DAKO Autostainer and a DAKO ChemMate Detection kit, peroxidase/3,3'-diaminobenzidine (DAKO, Glostrup, Denmark) according to the manufacturer’s instructions. Finally, sections were counterstained with Mayer’s hemalaun solution (Merck, Darmstadt, Germany). MOPC21 (Sigma, Deisenhofen, Germany, at 15 µg/ml), an unrelated mouse myeloma immunoglobulin, served as the IgG1 isotype control. Staining results were judged by a board-certified pathologist. For each tissue sample, the fraction of immunostained tumor cells was recorded, and the staining intensity was estimated on a four-step scale (0, 1, 2, 3). Tumors were then initially categorized according to arbitrarily predefined criteria into four groups, including completely negative, strongly positive, and two intermediate groups similarly as described previously (13) . The exact criteria for these groups were as follows: negative (no staining at all); weak (1+ staining regardless of positive cell percentages or 2+ staining of 30% of cells); moderate (2+ staining of >30% of cells or 3+ staining of 50% of cells); and strong (3+ staining of >50% of cells). However, as the statistical analyses did not show differences in the associations between the different levels of EMMPRIN expression and tumor phenotype and prognosis, tumors with weak, moderate, and positive immunostaining were eventually combined into one group as EMMPRIN positive.

Immunocytochemical Detection of CK-Positive Cells in BM.
BM cytospin samples of mononucleated interphase cells were analyzed for the presence of CK-positive cells using mAb A45-B/B3 (Micromet, Martinsried, Germany, at 2 µg/ml) directed against a common epitope of CK-polypeptides, including the CK heterodimers 8-18 and 8-19 as described in detail elsewhere (14) . For each patient, a total of 2 x 10⁶ mononucleated interphase cells was analyzed with the Automated Cellular Imaging System (ChromaVision Medical Systems, Inc., San Juan Capistrano, CA; Ref. 15 ). A negative staining control was accomplished by using an appropriate dilution of the mAb MOPC21 (DAKO). The breast carcinoma cell line MCF-7 served as a positive control for CK immunostaining.

Detection of EMMPRIN on Micrometastatic Cancer Cells.
A double-staining method was developed for the staining of micrometastatic tumor cells in BM aspirates. The cells were centrifuged onto glass slides, dried overnight, and permeabilized in 0.5% Triton (Sigma). Cells were washed in PBS, and unspecific binding was blocked with 10% AB-serum (Biotest, Dreieich, Germany). EMMPRIN was detected with mAb 1G6.2 (Chemicon, Hofheim, Germany, at 50 µg/ml). mAb MOPC 21 (DAKO) served as an isotype control in this experiment. Slides were incubated with biotinylated rabbit-antimouse antibody (DAKO). After washing, streptavidin alkaline phosphatase-conjugated antibody (DAKO) was applied. Finally, alkaline phosphatase activity was developed with Fast Blue BB salt (Sigma). Micrometastatic cancer cells were detected using Cy-3-labeled mAb A45-B/B3 (Micromet, at 2 µg/ml). Slides were covered with Vectashield mounting medium supplemented with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). The entire slide was scanned microscopically. Screening was performed for CK-positive cells, and EMMPRIN expression was determined by two independent observers in a double-blinded fashion. A BM aspirate from a breast cancer patient with clinically detectable metastases in BM served as a positive control for the double-staining method.

Statistical Analysis.
The ² test was used to analyze the putative association between EMMPRIN expression and various clinicopathological parameters. For analysis of follow-up data, life table curves were calculated using the Kaplan-Meier method, and survival distributions were compared by log-rank statistics. The primary end point was cancer-related survival, as measured from the date of surgery to the time of the last follow-up or cancer-related death. Data of patients still alive and without evidence of disease at the end of the study were censored. The joint effects with already recognized prognostically relevant variables were examined via Cox proportional hazard analysis. Tumor size, lymph-node status, ER status, and tumor grading were entered stepwise forward into the model to test these covariables for possible prognostic joint effects with EMMPRIN expression (threshold for statistical significance was P = 0.05). For statistical analyses, we used SPSS software for PC (version 11 for Windows).

	RESULTS

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EMMPRIN Expression in Human Breast Cancer.
To analyze the overall incidence of EMMPRIN expression in human mammary carcinomas, an immunohistochemical study was undertaken using breast cancer TMAs containing tumor samples plus control tissue sections (10) . The putative association between EMMPRIN expression and various clinicopathological factors was evaluated statistically on the basis of 1816 tumor sections where interpretable stainings could be obtained. The results are summarized in Table 1 , and a representative staining of a breast cancer TMA is shown in Fig. 1 . In total, EMMPRIN expression was found in 49.8% (n = 904) of the tumors with 41.4% (n = 374) showing weak, 25.4% (n = 230) moderate, and 33.2% (n = 300) strong staining. For statistical analysis, staining results were considered positive if any staining was detected. This was because all statistical analysis showed no relevant differences in outcome and tumor phenotype between tumors of varying staining intensity. Although individual carcinomas from all histological types stained positive for EMMPRIN, marked differences in the expression of EMMPRIN between breast cancer entities were observed. EMMPRIN expression was significantly less frequent in lobular (28.6%) carcinomas than in ductal (51.8%) or other (59.2%) carcinomas (P < 0.001), and staining for EMMPRIN appeared to be inversely correlated to age of cancer patients (P = 0.003). Furthermore, EMMPRIN expression correlated with high tumor grade, with a continuous increase of staining frequency from grade 1 to grade 3 (P = 0.001). In addition, EMMPRIN staining correlated inversely with ER and progesterone receptor staining (P < 0.001, P = 0.028). Positivity was also associated with the histologically determined mitotic index (P < 0.001) and tumor size (P = 0.001).

View larger version (85K): [in this window] [in a new window]   Fig. 1. High-density breast cancer prognosis tissue microarray and CK-positive cells in bone marrow stained for extracellular matrix metalloprotease inducer (EMMPRIN). A tissue microarray (B) and two enlarged tumor samples representing negative (A) and positive (C) staining for EMMPRIN are shown. A disseminated tumor cell in a BM cytospin preparation from a breast cancer patient double-stained for EMMPRIN (D) and CK (E).

Prognostic Significance of EMMPRIN Expression in Human Breast Carcinomas.
The prognostic significance of EMMPRIN staining was then evaluated on the basis of 774 samples from breast cancer patients, where clinical follow-up data and interpretable EMMPRIN staining were available. Survival analysis of cancer-related death was performed by comparing samples from patients showing EMMPRIN staining of tumor cells (n = 439) with those exhibiting no detectable expression of EMMPRIN (n = 335). Comparison of survival distributions by statistical analysis using the log-rank test showed that EMMPRIN expression was significantly associated with shorter tumor-specific survival in breast cancer (P = 0.003; Fig. 2A ). However, in multivariate analysis, including the clinicopathological parameters local tumor extension, lymph-node status, ER receptor status, and histological grade, EMMPRIN expression was not an independent prognostic factor (P = 0.275).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Extracellular matrix metalloprotease inducer (EMMPRIN) protein expression is significantly associated with shorter tumor-specific survival in breast cancer. Survival analysis was performed by evaluating samples where EMMPRIN staining yielded interpretable results, and clinical follow-up information on tumor-specific survival was available (n = 774; A). Separate Kaplan-Meier analyses were performed for patients older or younger than 50 years (n = 772, age of 2 patients was unknown; B and C).

EMMPRIN Expression on Autologous Primary and Micrometastatic Tumor Cells.
To analyze whether an increased EMMPRIN expression on primary tumors is associated with the existence of micrometastatic tumor cells in BM, BM aspirates from 55 patients without overt skeletal metastases were taken and screened for the presence of micrometastatic cancer cells using the CK-specific mAb A45-B/B3. CK-positive tumor cells were found in 36% (n = 20) of cytospin preparations of the breast cancer patients analyzed. Immunohistochemical staining of the corresponding primary tumors revealed EMMPRIN expression in 55% (n = 11) of tissue samples from patients with micrometastatic cancer cells in BM as compared with 54% (n = 19) of patients without detectable CK-positive cells in this organ. Despite the relatively small number of samples analyzed, these results suggest that an elevated expression of EMMPRIN in the primary tumor is not a predictor for early hematogenous spread of mammary carcinoma cells to BM.

However, staining intensity within primary tumors generally was highly variable, possibly reflecting the known genetic and morphological heterogeneity of cancer cell populations in solid tumors. Because it is still controversial as to whether metastasis arises from preexisting metastatic subclones of a tumor or whether metastasis represents a late event in the progression of tumors (16 , 17) , we could not exclude the possibility that EMMPRIN is still involved in early hematogenous dissemination of breast cancer cells to BM. To address this question, we also analyzed the expression of EMMPRIN in disseminated mammary carcinoma cells in BM cytospin preparations of a second cohort consisting of 31 patients staged clinically M0. A double-staining method was developed to detect individual micrometastatic cancer cells using CK-specific mAb A45-B/B3 and to assess EMMPRIN expression in these cells with mAb 1G6.2 recognizing EMMPRIN (Fig. 1, D and E) . Staining for EMMPRIN was not restricted to tumor cells alone but was also observed less intensively in mononucleated interphase cells present in the cytospin preparations, indicating that EMMPRIN is a widely distributed cell surface molecule. A BM aspirate from a breast cancer patient with clinically detectable metastases in BM served as a positive control in this experiment and yielded 190 CK-positive cancer cells/2 x 10⁶ mononucleated interphase cells, 169 (89%) of which also expressed EMMPRIN. Consistent with the results obtained with the first series of breast cancer patients, in 11 of 31 (35%) BM cytospin preparations, CK-positive cells could be detected. In total, 38 disseminated CK-positive cancer cells were found in these 11 samples, 34 (89%) of which also stained positive for EMMPRIN. However, only 3 of 7 primary tumors available from patients with micrometastatic cancer cells in BM also stained positive for EMMPRIN. Taken together, our results suggest that EMMPRIN is frequently expressed in micrometastatic cancer cells in BM independent of the EMMPRIN expression in the primary tumor.

	DISCUSSION

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Tumor cell metastasis represents an extremely complex process consisting of multiple sequential steps. The metastatic cell must exhibit a complex phenotype that includes the capability to escape from the primary tumor mass, invade the surrounding normal tissue, penetrate into the circulation (bloodstream or lymphatics), implant into the capillary beds of distant organs, extravasate the target tissue, and finally proliferate in the parenchyma of specific organs to produce clinically relevant metastases. An increasing body of evidence suggests that EMMPRIN, a multifunctional molecule on the surface of tumor cells, modulates key steps of the metastatic cascade and therefore might play a crucial role in the progression of carcinomas (6, 7, 8, 9) .

In this study, we analyzed for the first time the overall incidence of EMMPRIN expression in human breast cancer by applying the TMA technology, which allowed us to process a large number of tissue specimens from breast cancer patients and to associate staining results with various clinicopathological parameters. Our results showed that EMMPRIN expression in breast carcinomas is associated with risk factors such as poor histological grade, the mitotic index, negative hormone receptor status, and tumor size. Positive EMMPRIN staining also strongly correlates with decreased tumor-specific survival in breast cancer patients when tumors at all stages were analyzed simultaneously. Interestingly, EMMPRIN expression is only of prognostic value for patients > 50 years but not for younger patients (50 years). As EMMPRIN expression was also inversely correlated with ER and progesterone receptor status of primary tumors, it is conceivable that the prognostic effect of EMMPRIN expression is related to postmenopausal changes in hormone levels. Indeed, recently published data demonstrate that EMMPRIN expression, glycosylation, and biological activity can be modulated by hormones (18, 19, 20) .

Taken together the importance of MMPs in cancer cell invasion and metastasis (2) , it is surprising that EMMPRIN expression is higher in ductal than in highly invasive lobular carcinomas of the breast. Moreover, expression of EMMPRIN was not associated with lymph node metastasis or early hematogenous spread to BM. A potential role of EMMPRIN in cancer cell dissemination still could not be excluded as 90% of micrometastatic cancer cells on cytospin preparations of BM aspirates from breast cancer patients without overt metastases stained positive for EMMPRIN. Our finding is also supported by the recent results of Klein et al. (21) , demonstrating a frequent expression of EMMPRIN in BM micrometastases from patients with prostate and lung cancer. These observations raise the possibility that EMMPRIN expressing cancer cells represent only a small clone in the primary tumor and that these cells are positively selected in BM. An alternative explanation is that EMMPRIN-negative cells leave the primary tumor and the specific BM microenvironment up-regulates expression of EMMPRIN in these cells.

It is important to note that EMMPRIN represents a multifunctional molecule exerting various biological effects not restricted to facilitating cancer cell invasion and metastasis by induction of MMP production in peritumoral fibroblasts and degradation of the ECM. Indeed, our data revealed that EMMPRIN expression is associated with the mitotic index and tumor size of breast cancer patients, implying that EMMPRIN might promote cancer cell proliferation. These observations are in accordance with a recently published study by Zucker et al. (9) showing that overexpression of EMMPRIN in MDA-MB-436 mammary carcinomas cells resulted in enhanced tumor growth as compared with plasmid-transfected control cells when orthopically implanted into nude mice. Although breast cancer cells transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than mock-transfected MDA-MB-436 cells, an increased capability to colonize distant organs was not observed (9) . The results presented here additionally support a growth-promoting role of EMMPRIN in human breast cancer. It is tempting to speculate that the EMMPRIN-induced production of MMPs by peritumoral fibroblasts results in the breakdown of the ECM and subsequent release of growth factors sequestered within the extracellular space. This hypothesis, however, awaits in-depth analysis.

Thus far, only one immunohistochemical study on EMMPRIN expression in breast cancer tissue has been published, analyzing a small cohort of 20 patients (22) In contrast, we conducted a comprehensive analysis based on 1816 tumor samples, which led us to conclude that EMMPRIN expression is significantly correlated with risk factors such as poor histological grade, the mitotic index, tumor size, and most importantly, tumor-specific survival in human breast cancer, suggesting a crucial role of EMMPRIN in breast cancer progression.

	ACKNOWLEDGMENTS

 
We thank Sonja Santjer, Antje Andreas, Cornelia Coith and Katrin Baack for their excellent technical assistance.

	FOOTNOTES

 
Grant support: Deutsche Forschungsgemeinschaft (Bonn, Germany) Grant PA341-11/2 (K. Pantel).

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.

Note: N. Reimers and K. Zafrakas contributed equally to this work.

Requests for reprints: Klaus Pantel, Institute of Tumor Biology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. Phone: 49-40-42803-3503; Fax: 49-40-42803-5379; E-mail: pantel{at}uke.uni-hamburg.de

Received 11/19/03; revised 1/30/04; accepted 2/10/04.

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