Tissue distribution of major matrix metalloproteinases and their transcripts in human breast carcinomas

Abstract

The key event of invasive growth of malignant epithelial tumors is the dissolution of the peritumoral basement, membrane (BM). Accordingly, numerous immunohistochemical studies have shown that particularly in breast carcinomas there is an almost complete loss of the BM, even in well-differentiated carcinomas. In order to find out the significance of tumor-associated BM-degradation we localized major matrix metalloproteinases (MMPs) as the most important proteolytic enzymes for connective tissue dissolution. As a prerequisite, we had to identify antibodies reacting specifically on paraffin-embedded tissue material. Extensive pretesting of MMP-2, -3 and -9 antibodies of various sources provided evidence that only a small proportion of the antibodies analyzed showed a specific, positive staining result, as most of the commercial antibodies did not react on the paraffin material or revealed non-specific staining results. Using the specifically reacting antibodies, we analyzed material from 65 cases of invasive ductal breast cancer by immunohistochemistry for the localization of MMP-2, -3 and -9 and by the non-radioactive in-situ hybridization technique for the localization of the MMP-3-mRNA. The specificity of the in-situ hybridization was analyzed using the sense control. We observed a distinct positive immunoreaction for MMP-2, -3 and -9 over both invasive, as well as non-invasive tumor cells, without apparent differences in the staining intensity. Remarkably, there was a significant staining of tumor cell complexes undergoing lymphangiotic dissemination. In addition to this tumor cell staining pattern, a positive immunoreaction, although to reduced proportion, was observed over peritumoral fibroblastic and endothelial stroma cells. Normal breast tissue also revealed a positive immunostaining of epithelial and stromal cells. Using in-situ hybridization, we observed mRNA expression for MMP-3 both in tumor and stroma cells, comparable to the protein data. Normal breast epithelia reacted weakly positive for MMP-3-mRNA. Our data indicate that there is a major active expression of important MMPs in invasive breast carcinomas as the possible cause for the matrix dissolution. These MMPs are synthesized both by tumor and peritumoral stroma cells which may interact with each other. However, the de-novo synthesis and the amount of immunoreactive enzyme protein does not seem to be significantly enhanced in invasive versus noninvasive tumor areas or normal breast epithelia, indicating that other mechanisms, such as enzyme activation and/or differences in the levels of proteinase inhibitors may be biologically essential factors.