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Tissue Microarray Analysis of Hepatocyte Growth Factor/Met Pathway Components Reveals a Role for Met, Matriptase, and Hepatocyte Growth Factor Activator Inhibitor 1 in the Progression of Node-negative Breast Cancer¹

Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520 [J. Y. K., M. D-F., I. T. O., D. L. R., R. L. C.], and Lombardi Cancer Center, Departments of Oncology and Pathology, Georgetown University School of Medicine, Washington, DC 20007 [B. S., C-Y. L., R. B. D.]

	ABSTRACT

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Numerous studies have demonstrated that overexpression of Met, the hepatocyte growth factor(HGF) receptor, plays an important role in tumorigenesis. Met activation can either occur through ligand-independent or -dependent mechanisms, both of which are mediated by a series of proteases and modulators. We studied the protein expression of several components of the HGF/Met pathway on a cohort of 330 node-negative breast carcinomas using a tissue microarray annotated with 30-year, disease-specific patient follow-up data. We examined HGF, matriptase (an activator of HGF expressed on mammary epithelial cell surfaces), HAI-I (the cognate inhibitor of matriptase), and the Met receptor itself. Our studies demonstrate tight correlation between the expression of HGF, matriptase, and Met in breast carcinoma. High-level expression of Met, matriptase, and HAI-I were associated with poor patient outcome. Met and HAI-I showed independent prognostic value when compared with traditional breast markers in a multivariate analysis. Intriguingly, antibodies against the intracellular but not the extracellular domain of Met were prognostic, suggesting that overexpression of the cytoplasmic-tail of Met, perhaps through cleavage or truncating mutation, may play an important role in breast cancer progression.

	INTRODUCTION

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Many studies have demonstrated the importance of the HGF³ pathway in carcinogenesis (1) . HGF is produced both by tumor cells as well as by surrounding stromal elements, and can act in either a paracrine or autocrine fashion (2, 3, 4, 5) . HGF is secreted as an inactive propeptide, which must be cleaved to become biologically active. One enzyme responsible for this cleavage is matriptase, an epithelial-localized transmembrane serine protease (6, 7, 8) . Matriptase is, in turn, regulated by a naturally occurring inhibitor, HAI-1 (9 , 10) . When cleaved, HGF can bind to its receptor, Met, thereby stimulating multiple downstream pathways, leading to mitogenesis, motogenesis, and morphogenesis (11) .

Several studies have analyzed individual components of the HGF pathway for their association with tumor aggression and/or patient survival. Early biochemical studies demonstrated that overall levels of HGF in breast cancers correlated with worse patient outcome (2 , 3 , 5) . Whether HGF production by tumors and/or surrounding stroma is an important prognostic feature is unclear; although tumor cells themselves are a major producer of HGF (4 , 5 , 12) . The use of matriptase and HAI-1 as prognostic markers in breast cancer has not been reported previously. However, recently, one study demonstrated that high matriptase and low HAI-1 levels were associated with advanced-stage ovarian tumors (13) . Another report demonstrated that the glycosylation of matriptase stabilized and enhanced its proteolytic activity, and promoted tumor aggression (14) .

The expression of Met has been more extensively studied. Met overexpression associates with poor prognosis in a variety of tumors (1) . Whether such expression is ligand- (HGF) dependent or independent is unclear; however, the constitutive activation of Met, via several ligand-independent mechanisms, is established. These mechanisms include activating point mutations (15, 16, 17, 18, 19) , chromosomal translocations (20 , 21) , and truncations of the cytoplasmic domain (22 , 23) . In addition, dysregulation of Met-associated phosphatases may also lead to Met activation (24) .

We have now studied several elements of the HGF pathway including HGF, matriptase, HAI-1, and Met in a single cohort of node-negative breast cancer patients with 30-year follow-up, correlating the expression of each element and determining their prognostic value. This study was facilitated by the use of tissue microarrays: arrays of hundreds of patient histological samples on a single glass slide. Our study demonstrates a significant correlation between members of the HGF pathway and shows that several members have independent prognostic value in determining patient outcome.

	MATERIALS AND METHODS

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Cohort Design and Tissue Microarray Construction.
Tissue microarrays were constructed, as described previously, and reviewed recently (25 , 26) . Three hundred and thirty cases of formalin-fixed, paraffin-embedded, node-negative breast carcinoma were obtained from the archives of the Department of Pathology, Yale University. Cases were taken sequentially, as available, from 1962 to 1980, with a median survival time of 15.6 years. Complete treatment history is not available from this cohort, but the vast majority of patients in this era were not treated with chemotherapy. Representative tumor regions were selected for coring by a pathologist (R. L. C.). Because prior studies have demonstrated that a single core adequately represents the staining pattern of an entire slide, all of the studies were performed using a single sample of each tumor (27 , 28) . Our previous study has also demonstrated the durability of antigens from archival specimens as old as 70 years (27) . In the present study, all of the tumors demonstrated some degree of staining with one or more of the antibodies tested, demonstrating that no cases were antigenically "dead" because of fixation artifacts or tissue age.

Immunohistochemistry.
Briefly, 5-µm tissue microarray slides were deparaffinized with xylene and ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0) pressure-cooking (29) . Primary antibodies were incubated overnight at 4°C, with the exception of antibodies to ERs, PRs, and Her2, which were incubated at room temperature for 1 h. Monoclonal anti-matriptase and anti-HAI-1 antibodies were prepared as described previously (9 , 30) . Commercially acquired antibodies included: polyclonal (goat) anti-HGF antibody (R&D Systems, Minneapolis, MN); monoclonal antibody to the extracellular domain of Met (DO-24; Upstate Biotechnology, Lake Placid, NY); and monoclonal antibody to the intracellular domain of Met (3D4; Zymed, South San Francisco, CA). The specificities of all of the antibodies used were verified using immunoprecipitation and Western blotting. Antibodies to ER, PR, and HER-2/neu were obtained from DAKO (Carpinteria, CA) and used according to the manufacturer’s specifications. Antibodies were either detected using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) for anti-HAI-1 or the DAKO Envision TM + System (DAKO) for the others. Signal from the HGF antibody was amplified using biotin-tyramide signal amplification followed by a streptavidin-horseradish peroxidase conjugate (TSA kit; Perkin-Elmer Life Sciences, Boston, MA). Staining was visualized using diaminobenzidine and counterstained with acidified hematoxylin. Slides were also stained in the absence of primary antibody to evaluate nonspecific secondary antibody reactions.

Evaluation of Immunostaining.
Immunostaining was scored on a scale of 0 to 3+ (negative/weak/moderate/intense staining). Distinctions between membrane and cytoplasmic staining were impractical given the diffuse staining of the antigens (visualized using the chromogenic substrate, diaminobenzidine). Therefore, scores represent the combined staining intensity of membranous and cytoplasmic staining. Histospots with <10% of their area covered by tumor were excluded from analysis. Scoring was performed by two independent observers (J. Y. K. and M. D-F.), and histocores with discrepant scores were re-examined by both observers to achieve a consensus score. Cases with scores of 2+ or 3+ were designated as "high," whereas cases with scores of 0 or 1+ were designated as "low."

Statistical Analysis.
All of the analyses were completed using Statview 5.0.1 (SAS Institute Inc., Cary, NC). Correlations between markers were performed using a ² test. Prognostic significance was assessed using both univariate and multivariate Cox proportional hazards models with 30-year survival as an end point. Survival curves were calculated using the Kaplan-Meier method, with significance evaluated using the Manel-Cox log rank test.

	RESULTS

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We analyzed the expression of several components of the Met pathway including HGF, matriptase, HAI-1, and Met itself (using both intracellular- and extracellular-specific antibodies; Fig. 1 ). The number of tumors expressing high levels of these antigens ranged from 18 to 46% (Table 1) . Although tissue microarrays have been shown to adequately represent tumor antigen expression (27) , they most likely do not fully represent the expression of stromal markers. Therefore, we limited our analysis to the expression of markers by tumor cells and not stroma. In the case of HGF, this resulted in our selective study of autocrine (tumor) expression.

View larger version (150K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Immunohistochemical staining of breast cancer tissue microarrays for four members of the HGF/Met pathway. Each histospot shows a representative positive case (3+) for matriptase (A), HAI-1 (B), HGF (C), and Met (D), at low (x40) and high (x200, insets) magnification. Matriptase, HAI-1, and Met show a membrano-cytoplasmic localization, whereas HGF is predominantly cytoplasmic. The cellular localization of Met using antibodies to the intracellular and extracellular domains was comparable (D, left and right insets, respectively). Representative histospots stained using an antibody to the intracellular domain of Met and scored as 0, 1, 2, or 3+ are shown in E–H, respectively.

View this table: [in this window] [in a new window]   Table 2 Met pathway associations: 2 analysisa

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Kaplan-Meier analysis of disease-related survival demonstrates that Met (intracellular domain), matriptase, and HAI-1 show long-term prognostic benefit. Statistical significance was assessed using the log rank test.

We then determined the independent predictive power of the Met pathway. First, we limited our analysis solely to the Met pathway components. Using multivariate analysis, both the cytoplasmic tail of Met and HAI-1 retained independent predictive power (Table 4) . When we included these two markers with traditional breast cancer markers, they retained their independence. Tumor size was the only other independent predictor of poor outcome (Table 5) .

	DISCUSSION

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In addition to their role in the activation of HGF, matriptase and its cognate inhibitor, HAI-1, also play a role in the plasminogen activator cascade (6 , 31 , 32) . This cascade culminates in the activation of plasmin and the coactivation of matrix metalloproteinases, both of which degrade extracellular matrix components and potentiate tumor cell invasion, extravasation, and metastasis (33) . Matriptase promotes this pathway by activating latent uPA, which, in turn, activates plasmin (6 , 34) . Like matriptase, uPA can also cleave pro-HGF, providing another level of interaction between the Met/HGF pathway and plasmin cascade (35) . Given the multiple functions of matriptase, it is not surprising that aggressive breast tumors produce higher levels of this enzyme.

Likewise, HAI-1, as an inhibitor of matriptase, may help modulate both the Met/HGF and plasmin pathways. Interestingly, the expression of HAI-I was independent of the other members of the Met pathway indicating that its expression is regulated differently. Previous reports have suggested that HAI-I is down-regulated in colon carcinoma and high-grade ovarian carcinomas (13 , 36) . In contrast, our study demonstrates that HAI-1 expression is associated with aggressive breast carcinomas, being an independent predictor of poor outcome (Table 5) . Although this may be puzzling in light of the role of HAI-1 in inhibiting HGF-dependent Met activation, the coordinated expression of both matriptase and HAI-1 may be far more important in promoting tumor aggression than the unopposed production of active matriptase in the absence of its inhibitor. Coordinated regulation of another inhibitor of the uPA/plasmin cascade, PAI-1, is crucial for inducing tumor invasion (37) . Furthermore, a recent meta-analysis of the uPA/PAI-1 system in breast cancer demonstrated conclusively that overexpression of both uPA and its inhibitor PAI-1 were associated with poor outcome in breast cancer (32) . In our study, the importance of matriptase and HAI-1 coexpression is demonstrated in the elevated relative risk of patients with tumor expressing both markers.

Because of lot-to-lot inconsistencies in polyclonal Met-antibodies,⁴ we analyzed two different monoclonal antibodies, one to the extracellular and one to the intracellular domain. Comparison of Met expression as assessed by these two antibodies showed some interesting results. First, the expression of the intra- and extracellular domains of Met, although highly associated, was not coincident. Second, of cases with mixed expression of intra- and extracellular Met, overexpression of the intracellular domain was far more common, with 12.2% of all of the cases expressing solely the intracellular domain. Third, high levels of the cytoplasmic tail of Met were predictive of poor outcome, whereas expression of the extracellular portion was not. Although this result could be explained by differences in the affinity of the antibodies for Met in formalin-fixed, paraffin-embedded tissue, both antibodies gave strong staining and similar results across a range of titrations (data not shown). A more likely explanation is that the cytoplasmic tail of Met is either cleaved (e.g., after activation) or that mutations in Met lead to an overexpression of the cytoplasmic tail in some tumors. Indeed, recent studies have suggested that the cleavage of the cytoplasmic tail of Met may be important in signal transduction (1 , 22 , 23) . Whether overexpression of the Met intracellular domain relative to the extracellular domain is a ligand-independent or -dependent phenomenon is unclear. Interestingly, expression of the Met extracellular domain correlates with HGF levels but not matriptase levels, whereas the Met intracellular domain correlates with both. This observation would be expected if the binding of matriptase-potentiated HGF to Met induced a subsequent cleavage of the Met intracellular domain.

In summary, we have made use of tissue microarray technology to analyze various components of the Met-signaling pathway. Our studies provide evidence that the expression of the stimulatory members of this pathway (Met, HGF, and matriptase) is tightly correlated. High-level HAI-1 expression is an independent predictor of outcome. Furthermore, studies using antibodies to different domains of the Met receptor suggest that overexpression of the cytoplasmic domain is a strong independent predictor of outcome.

	ACKNOWLEDGMENTS

 
We thank Thomas D’Aquila and Lori Charette for their help in this effort.

	FOOTNOTES

 
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.

¹ Supported by grants from the Patrick and Catherine Weldon Donaghue Foundation for Medical Research, The Connecticut Breast Cancer Alliance, and grants from the NIH, including National Institute of Environmental Health Sciences Grant K0-8 ES11571 (to R. L. C.), National Cancer Institute Grant RO-1 GM57604 (to D. L. R.), United States Army Grant DAMD 01-000436, and NIH Breast Cancer Specialized Programs of Research Excellence 2P50CA72460.

² To whom requests for reprints should be addressed, at Department of Pathology, Yale University, School of Medicine, 310 Cedar Street, BML 122, New Haven, CT 06520. E-mail: robert.camp{at}yale.edu

³ The abbreviations used are: HGF, hepatocyte growth factor; HAI, hepatocyte growth factor activator inhibitor; ER, estrogen receptor; PR, progesterone receptor; CI, confidence interval; uPA, urokinase-type plasminogen activator; PAI, plasminogen activator inhibitor.

⁴ Unpublished observations.

Received 8/28/02. Accepted 1/ 3/03.

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