(Verbatim from the Applicant): The mechanism underlying the preferential metastasis of breast cancer to bone is poorly understood. We propose that gap junctional intercellular communication (GJIC) contributes to breast cancer metastasis to bone by facilitating heterotypic cell-cell interactions between breast cancer cells and osteoblastic cells. These interactions contribute to breast cancer cell transosteoblastic migration, which is a critical step in bone metastasis. The long-term goals of this project are to characterize gap junction expression and function in breast cancer cells (metastasizing, metastasis suppressed and non-metastatic) and how this contributes, through activation of cytosolic Ca2+ mobilization and myosin light chain kinase (MLCK) activity, to breast cancer cell transosteoblastic migration and bone metastasis. These goals will be accomplished through the completion of five Specific Aims: (1) examine homotypic GJIC among breast cancer cell lines and heterotypic GJIC between breast cancer cells and osteoblastic cells; (2) quantify the effect of breast cancer cell contact, in the presence and absence of inhibitors of GJTC and cytosolic Ca2+ mobilization, on cytosolic Ca2+ concentration ([Ca +i]) in osteoblastic hFOB 1.9 cells; (3) quantify MLCK activity in osteoblastic cells following contact with breast cancer cells; (4) quantify transcellular migration of breast cancer cells through osteoblastic monolayers; and (5) genetically alter expression of connexins in breast cancer cells and assess metastatic potential in an in vivo model of bone metastasis. As breast cancer cell lines, we will use MDA-MB-23 I and MDA-MB-435, these cells expressing the recently identified metastasis suppressing gene BRMS1, these cells expressing green fluorescent protein (GFP) and appropriate vector controls. We will also examine non-metastasic MDA-MB-468 and neol 1/435 breast cancer cells. GJIC will be quantified by dye transfer techniques, [Ca2+], by microspectrofluorometry, MLCK activity by in vitro phosphorylation assays, and transcellular migration by quantification of cell migration through Matrigel, utilizing confocal microscopy. Bone metastasis will be assessed fluorometrically utilizing GFP tagged breast cancer cells. The results of this project will provide insights into the mechanism by which breast cancer cells preferentially metastasize to bone, an important step in developing approaches to inhibit bone metastasis.
