Search

The overall goal of our project is to identify novel treatments for breast cancer invasion and metastasis. We view invasion and metastasis as a breakdown of mechanisms that specify tissue organization. We postulated that metastasis is initiated by molecular cues that improperly stimulate cancer cell motility. Therefore, our approach is to block metastasis by understanding, and then interfering with, molecular and cellular mechanisms that regulate cell motility. We found that, in the mammary gland, several of these mechanisms revolve around the interaction of matrix metalloproteinases (MMP) with laminin-5 (Ln-5), an extracellular matrix macromolecule of the breast gland basal lamina. Our major findings are as follows: I) identification of the Ln-5 site onto which cells adhere and migrate; 2) mapping, relative to this cell adhesion site, of a docking site for antibodies that block cell motility; 3) mapping of Ln-5 sites that are cleaved by MMPs; 4) identifying the composition of the Ln-5 fragments resulting from this proteolytic MMP activity. This information should aid the design of reagents for in vivo animal experiments in which to test the ability of antibodies to Ln-5, or Ln-5 fragments, to hopefully block breast cancer cell motility and metastasis.

The goal of our project is to find novel treatments for breast cancer metastasis. We view metastasis as a breakdown of mechanisms that govern tissue organization. We postulated that metastasis is initiated by molecular cues that improperly stimulate cancer cell motility. Therefore, our approach is to block metastasis by understanding, and then interfering with, molecular and cellular mechanisms that regulate cell motility. We found that, in the mammary gland, several of these mechanisms revolve around the interaction of matrix metalloproteases (MMP) with laminin-5 (Ln-5), an extracellular matrix protein of the breast gland basement membrane. One major finding in this past year was the identification of the Ln-5 site onto which cells adhere and migrate. We also defined the position, relative to this site, of the docking site for antibodies that block cell motility. A second important finding is the location of Ln-5 sites that are cleaved by MMPs, and the compositon of the Ln-5 fragments resulting from this proteolytic activity. This information is critical to design in vivo animal experiments in which we will test the ability of antibodies to Ln-5, or Ln-5 fragments, to block tumor cell motility and hopefully metastasis.

The subject of this research is breast cancer metastasis. Our long-term goal is to find cancer treatments based on targeting directly the metastasis process. An Important element of innovation in our approach is that we visualize metastasis as a problem of breakdown in tissue organization. Consequently, for the purposes of cancer treatment, our target is the mammary gland as a tissue, rather than the individual cells. By taking a strictly reductionist approach, we are investigating the actual molecular mechanisms that keep breast epithelial cells segregated on the luminal side of the basal lamina. These epithelial cells are the ones from which invasive breast cancer arises. In our previous work, we identified a molecular mechanism (1, 2) that determines whether normal or cancer breast cells may or may not cross the basal lamina. This mechanism relies on the interaction of laminin-5, a major extracellular matrix molecule of basal lamina, with matrix metalloproteases and integrins. The specific challenge of this proposal is to determine how pervasive this mechanism is in regulating migratory versus stationary behavior of breast epithelial cells. If such mechanism is an important one, we will be one step away from entering a discovery phase for novel drugs or treatments that prevent or block breast cancer invasion.

Cell migration is crucial to embryonic development, tissue remodeling and cancer invasion. To migrate properly, cells must integrate multiple incoming signals. Once committed to migration, they coordinately regulate, both spatially and temporally, surface receptors and cytoskeleton, in order to generate traction and movement (Lauffenburger and Horwitz, 1996). Migration usually occurs over extracellular matrix (ECM), and is accompanied by characteristic morphological changes. Cell protrusions, e.g., filopodia or lamellipodia, are sites where adherence contacts for traction generally are formed. To accomplish forward movement, there must be a balance between the establishment of plasma membrane-ECM adherence contacts at the cell leading edge, and their coordinated, asymmetric release at the cell trailing edge (Huttenlocher et al., 1995). While significant advances have been made in identifying molecules involved in cell migration, molecular mechanisms are poorly defined.

We are attempting to identify integrin-specific signalling events that may contribute to malignant transformation in mammary epithelium. We are focusing on the extracellular matrix protein Iaminin-5r, which is located in mammary epithelial basement membrane. We hypothesize that binding of integrins to laminin-5r elicits downstream signalling events that may help define the normal phenotype of mammary epithelial cells; disruption of these signals may contribute to the development of malignancy. To date, we have established that normal and malignant cell lines plated on laminin-5r differ in at least three ways: normal and malignant cells use different integrin receptors to bind laminin-5, malignant cells migrate while normal cells do not, and normal and malignant cells exhibit different patterns of tyrosine phosphate-containing proteins following binding to laminin-5. The integrins that mediate these responses have been identified in two cell types, and we hypothesize that in these two cells, the conversion from normal to malignant phenotype involves the activation of the a3Beta1 integrin. Our goals for the upcoming year are to further elucidate the signalling pathways associated with the alpha 3Beta1 integrin following binding of laminin-5r, and to identify functionally active regions of laminin-5r responsible for eliciting these effects.

The expression of different cytoplasmic domains could influence the function of the alpha 6 beta l laminin receptor integrin by affecting the ligand binding characteristics of the integrin or by altering the intracellular interactions between alpha 6 and cytosolic components (i.e.

The purpose of this study is to identify integrin-specific signalling events that may be responsible for development of the malignant pheno type in mammary epithelium. Our laboratory has focused on a novel ligand for epithelial integrins, laminin-5r, which is located in mammary epithelial basement membrane. We hypothesize that binding of integrins to laminin-5r elicits downstream signalling events that may help define the normal phenotype of mammary epithelial cells; disruption of these signals may contribute to the development of malignancy. To date we have succeeded in raising six monoclonal anti-bodies against laminin-5r, and have used them to define distinct structural conformations of laminin-5r using a combination of immuno-precipitation, western blot, and elisa assays. Also, we have used these antibodies to affinity purify laminin-5r from conditioned medium of a rat epithelial cell line, 804G. Finally, we have shown that one of these antibodies blocks adhesion of a variety of cells, including mammary epithelial cells, to purified laminin-5r., Original contains color plates: All DTIC/NTIS reproductions will be in black and white.