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    Awarded Grants
    Stromal-Derived MMPs Activate PAR1 Promoting Breast Cancer Cell Migration and Invasion.

    Scientific Abstract:
    Much of the morbidity and mortality associated with cancer is due to the metastatic phase of the disease. Metastasis is marked by the cellular events of migration and invasion. The molecular mechanisms underlying these cellular processes remain poorly understood. Indeed, metastasis has been described as “the last great frontier for exploratory cancer research” (Hanahan and Weinberg 2000). Work in our lab suggests that the receptor PAR1 plays an integral role in the processes of breast cancer cell migration and invasion. PAR1 is a seven-transmembrane G-protein-coupled receptor activated through proteolytic cleavage of the extracellular N-terminal domain. The highly invasive human breast cancer cell line, MDA-MB-231, endogenously expresses high levels of PAR1. We found that inhibition of PAR1 expression by siRNA transfection inhibited both cellular migration and invasion in this cell line. Pharmacologic inhibition of PAR1 signaling also prevented migration of invasive breast cancer cells. Conversely, transfection of PAR1 into the poorly invasive MCF-7 cell line promoted migration and invasion. Expression of a non-activatable PAR1 mutant did not increase cell migration or invasion. Together, these data support the hypothesis that PAR1 activation is both necessary and sufficient to promote migration on breast cancer cells. Because the PAR1 receptor is activated by proteases, we further hypothesized that a secreted protease was responsible for PAR1 activation. We found that MDA-MB-231 cells prefer to migrate toward conditioned medium derived from stromal cells such as NIH3T3 and CRL2076 fibroblasts rather than toward their own conditioned medium. Preliminary characterization of the stromal-derived conditioned medium revealed a PAR1-activating protease sensitive to MMP inhibitors. With these data in hand, the first objective of this proposal is to identify the stromal-derived MMP responsible for PAR1 cleavage and activation. The second aim of this proposal is to characterize interaction of PAR1 and MMPs at the molecular level. MMPs represent a new class of PAR1-activating proteases, and it is therefore of great interest to describe the enzymatic parameters for this receptor activation. In light of the importance of MMPs in breast cancer metastasis, it is of great therapeutic interest to define its physiologically relevant substrates. To this end, we propose in vivo studies of breast cancer metastasis employing both MMP inhibitors and PAR1 inhibitors. These data will significantly advance the understanding of breast cancer metastasis and will lay the foundation for improved design of breast cancer treatments.

    Lay Abstract:
    Much of the morbidity and mortality associated with cancer is due to the metastatic phase of the disease. Metastasis is marked by the cellular events of migration and invasion. The molecular mechanisms underlying these cellular processes remain poorly understood. Indeed, metastasis has been described as “the last great frontier for exploratory cancer research” (Hanahan and Weinberg 2000). Work in our lab suggests that the protein PAR1 plays a key role in the processes of breast cancer metastasis. Using in vitro models of human breast cancer, we found that highly invasive human breast cancer cells express PAR1. We found that this expression is important for breast cancer metastasis. We also found that PAR1 needs to be activated in order to promote metastasis. PAR1 activation has been previously studied. Enzymes which cut protein called proteases are the agents that activate PAR1. Because the PAR1 receptor is activated by proteases, we postulated that a protease was responsible for PAR1 activation in breast cancer metastasis. We wanted to find the protease responsible for PAR1 activation. Because breast cancer cells prefer to migrate toward other nearby cells that are not cancerous, we examined proteases from these normal neighbor cells. We have begun to identify the protease responsible for PAR1 activation. We found that the protease belongs to the family of enzymes called MMPs, or matrix metalloproteases. MMPs have been implicated in breast cancer metastasis, but are still incompletely understood. The first objective of this grant proposal is to definitively identify the protease (an MMP) responsible for PAR1 activation. The second aim of this proposal is to characterize interaction of PAR1 and MMPs at the molecular level. MMPs represent a new class of PAR1-activating proteases, and it is therefore of great interest to describe the parameters for this interaction. In light of the importance of MMPs in breast cancer metastasis, it is of great therapeutic interest to define its physiologically relevant targets. To this end, we propose in vivo studies of breast cancer metastasis employing both MMP inhibitors and PAR1 inhibitors. Together, these data will significantly advance the understanding of breast cancer metastasis and will lay the foundation for improved design of breast cancer treatments.