The objective of this proposal is to understand the role of mammary adipocytes and adipocyte-secreted factors (such as, Type-VI collagen): i) in the growth/survival of mammary epithelial cells; and ii) in the pathogenesis of breast cancer. Mammary epithelial cells/ducts are embedded in a matrix that consists predominantly of adipocytes. However, it remains unknown what adipocyte-secreted factors are required for the survival of mammary epithelia. Similarly, these unknown adipocyte-specific factors may contribute to the pathogenesis of human breast cancers. To test this hypothesis, we will use a variety of novel in vivo approaches, such as """"""""reconstituted mammary fat pads (RFPs)"""""""" employing the transfectable 3T3-L1 model adipocyte cell line, and Type-VI collagen (-/-) null mice. The three specific aims of the project are: 1) To identify paracrine interactions between mammary epithelia and their surrounding adipocytes. We will establish co-culture and conditioned media systems using 3T3-L1 adipocytes and the human breast cancer epithelial cell line, MCF-7. We will use microarray analysis to define adipocyte-induced transcriptional changes in MCF-7 and thereby identify genes that mediate the growth and survival of MCF-7 cells. To functionally examine the role of specific factors, we will generate """"""""reconstituted fat pads (RFPs)"""""""" using stably-transfected 3T3-L1 adipocytes over-expressing a given gene product. These RFPs will be co-implanted with MCF-7 cells into nude mice to assess their contribution to tumorigenicity in vivo; 2) To determine the role of Type-VI collagen in mammary tumorigenesis. Our preliminary data demonstrate that Type-VI collagen is an abundant extracellular matrix component of mammary tissue that is secreted by adipocytes and that is up-regulated during mammary tumor progression. We will cross Type-VI collagen null mice with an established mouse model of mammary tumorigenesis, MMTV-ErbB2/Neu. We predict that loss of Type-VI collagen expression will block or diminish the development of mammary tumors; and 3) To determine if selective ablation of mammary adipocytes in the adult mouse prevents or diminishes mammary tumor development. For this purpose, we have developed a novel transgenic inducible system that allows the selective apoptotic ablation of mammary adipocytes, in a temporally controlled fashion that will be crossed to MMTV-PyMT mice. In a complementary approach, we will induce the selective-ablation of mammary epithelial cells. We have placed a ligand-activatable transgenic cassette containing pro-caspase-8 under the control of the aP2-promoter (for expression in adipocytes) and the MMTV-promoter (for expression in mammary epithelia). These studies will help define the role of mammary adipocytes in the pathogenesis of breast cancer.
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