Tumor cell metastasis is a complex, multi-step process that is a major cause of death and morbidity amongst cancer patients. Rather than being a totally random process, tumors display a preferential spread to particular organs. In breast cancer, bone metastasis occurs frequently and causes significant problems including pain, fracture, immobility, and even spinal cord compression leading to paralysis. Although bone metastasis represents a major clinical concern, the molecular mechanisms responsible for breast cancer predilection to metastasize to bone are not well understood, and few effective therapies currently exist. The major goal of this proposal is to identify novel molecular modulators of breast-bone metastasis that will ultimately result in the definition of new molecular targets to which novel therapeutics can be developed. To achieve this goal, we have utilized the intra-cardiac nude mouse model and identified sublines and subclones of the human breast cancer cell line MDA-MB-231 that display differential bone metastatic profiles. Through the use of gene array technology we have molecularly profiled and compared these sublines/subclones and identified a number of genes whose expression consistently correlates either positively or negatively with bone metastasis. We propose to define the role of two of these genes in breast-bone metastasis, one of which positively correlates with bone metastasis and the other that has been found to negatively correlate. To determine whether the genes function to enhance or reduce bone metastasis, we will employ standard molecular biology to increase and reduce the expression of the target genes in MDA-MB-231 sublines that have either a high (MDA-231Tx-SARFP) or low (MDA-231Tx-PRFP) bone metastatic phenotype (Aim 1). These sublines have previously been stably transfected with a construct that expresses red fluorescent protein (RFP) to allow full fluorescent body imaging of metastases in the in vivo bone metastatic models. Cells generated in AIM 1 will be examined for their ability to cause bone metastasis in in vivo models (intra-cardiac and intra-tibial inoculation) as well as their ability to grow at the orthotopic site (mammary-fat pad inoculation) (Aim 2). We will further examine the functional roles of the genes through a number of in vitro assays (adhesion dependent and independent proliferation, cell migration, cell invasion, cell adhesion, osteoclastogenesis assays) designed to examine the invasive and metastatic potential of cells (Aim 3). In addition we will examine a number of signal transduction pathways (EGF, IGF-I/II, TGFbeta) that have been show to be important in breast cancer progression (Aim 3). Analysis of the protein sequence of the targets reveals that each contains a number of TPR domains that are involved in protein-protein interactions. We therefore propose to identify proteins that interact with the targets to determine the possible involvement of other molecules and complexes in breast-bone metastasis (Aim 4). To determine the significance of the genes in human breast cancer we will examine their expression in archival primary and secondary breast tumors via immunohistochemistry and/or in situ hybridization to elucidate whether they may predict bone metastasis and/or survival (Aim 5). This proposal represents a unique opportunity to identify novel modulators of breast-bone metastasis.
