Altered metastatic behavior of human breast cancer cells after experimental manipulation of matrix metalloproteinase 8 gene expression.

Previous work in our laboratory led to the cloning, from the same parent tumor cell line (MDA-MB-435), of two human breast cancer cell lines (M-4A4 and NM-2C5) with opposite metastatic phenotypes. Additional investigations revealed that the nonmetastatic cell line NM-2C5 overexpressed the neutrophil collagenase, matrix metalloproteinase (MMP)-8, relative to its partner. Because other studies have implicated the MMP family in promoting tumor metastasis, we investigated the apparently paradoxical expression of MMP-8 in these cell lines. By genetic engineering, we inverted its relative levels of expression in the two partners and studied the effects on the behavior of the tumors that they generated in athymic mice. Knock-down of expression in NM-2C5 cells by transduction with a sequence encoding a specific ribozyme and overexpression of MMP-8 in M-4A4 cells by retroviral transduction both strikingly changed metastatic performance in opposite directions, indicating that this gene plays a role in the regulation of tumor metastasis.

Expression of matrix metalloproteinase 8 (MMP-8) and tyrosinase-related protein-1 (TYRP-1) correlates with the absence of metastasis in an isogenic human breast cancer model.

The multi-step nature of metastasis poses difficulties in both design and interpretation of experiments to unveil the mechanisms causing the process. In order to facilitate such studies, we have previously derived a pair of breast tumor cell lines that originate from the same breast tumor but which have diametrically opposite metastatic capabilities. In this system, the monoclonal cell line M-4A4 is metastatic to the lungs of athymic mice, whereas clone NM-2C5 is equally tumorigenic but non-metastatic. Here, we report that representational difference analysis (RDA) of cDNA obtained from the two clonal populations revealed an increased expression of tyrosinase-related protein-1 (TYRP-1) and the matrix metalloproteinase-8 (MMP-8) genes in the non-metastatic cell line. RNA and protein analyses in cultured cells and in primary xenograft tissues confirmed that the non-metastatic cell line expresses TYRP-1 and MMP-8 at levels that are at least 20-fold higher than the metastatic counterpart. Other members of the MMP family (MMP-9 and MMP-2) and the tissue inhibitor of metalloproteinase-2 (TIMP-2) were found to be expressed at similar levels in both populations. The effects of MMP-8 and TYRP-1 on in vitro invasion and migration were assessed in cells whose expression of these genes was altered by stable transduction with sense and antisense constructs. Specific down-regulation of MMP-8 in non-metastatic NM-2C5 cells resulted in a 2.5-fold increased capacity to invade through Matrigel. Unlike other members of the matrix metalloproteinase family, MMP-8 has not previously been implicated in the processes of tumorigenesis or metastasis. The successful identification of two proteins that are differentially expressed in these matched clonal cell lines and the tumors that they produce demonstrates the feasibility of using this approach to search for genes that are associated with aberrant differentiation toward metastatic behavior.

Contrasting expression of thrombospondin-1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis.

Knowledge of the molecular mechanisms involved in metastatic spread is needed to facilitate advances in prognostic evaluation for individual patients and in the design of therapeutic interventions to inhibit the process. In an effort to establish a methodological framework for analysis of molecules and mechanisms involved in this complex multistep process, we have developed a well defined experimental system, in which the role of candidate genes can be screened and tested. By serial dilution cloning of the MDA-MB-435 breast tumor cell line and screening by orthotopic implantation into the mammary fat pad of athymic mice, we have derived a pair of breast tumor cell lines (M-4A4 and NM-2C5) that originate from the same breast tumor but have diametrically opposite metastatic capabilities. In 74% of inoculated athymic mice, clone M-4A4 metastasized consistently to the lungs, mimicking a major dissemination route of human breast cancer. Conversely, although equally tumorigenic, clone NM-2C5 did not metastasize to any distal site. We have confirmed that the cell lines originate from a single genetic source by spectral karyotyping and evaluated the expression of a number of proteins previously implicated in cellular transformation and metastasis. The ability of M-4A4 to metastasize was not associated with increased angiogenesis, as measured by immunohistochemical microvessel density analysis. However, RNA and protein analyses revealed that two secreted proteins were differentially expressed: osteopontin expression was increased approximately 30-fold in clone M-4A4 and thrombospondin-1 expression was increased approximately 15-fold in clone NM-2C5. These cell lines constitute a stable and accessible model for the identification of genes involved in the multistep process of breast tumor metastasis. Manipulation of candidate genes in these cells will permit evaluation of their functional significance in the geometric progression of breast cancer.