) Metastasis in particular to the bone marrow and to the bone is a major cause of morbidity and mortality and is present in 60 percent of the case at diagnosis. Matrix degrading proteases such as matrix metalloproteases (MMP) and plasmin/plasminogen activators (PA) play an active role in metastasis and we have begun to explore their role in metastasis. In a recently completed analysis of the expression of proteases and protease inhibitors in primary neuroblastoma tumors we have observed that MMP-9 (gelatinase B) and PA inhibitor-1 (PAI-1) are increasingly expressed in primary neuroblastoma tumors derived from metastatic stage and that they are present not in malignant cells but in stromal cells. The overall goal of this project is to understand the role of MMP-9 and PAI- in neuroblastoma metastasis with a special emphasis on examining the interaction between tumor cells and stromal cells. We hypothesize that, in metastatic neuroblastoma, tumor cells induce the expression of MP-9 and PAI-1 by stromal cells which enhances tumor cell dissemination.
In Specific Aim 1, we will explore the mechanisms responsible for MMP-9 over-expression in stromal cells. Using a gain (over-expression of MMP-9 in neuroblastoma cells) and loss (MMP-9 deficient mice) of function approach, we will determine whether MMP-9 is causally involved in metastasis and/or angiogenesis. We will test the ant- tumor and anti-metastatic effect of a synthetic MMP inhibitor (AG3340) in experimental and orthotopic neuroblastoma tumor models developed in our laboratory.
In Specific Aim 2, we will determine whether bFGF and VEGF produced by neuroblastoma cells are responsible for up-regulating PAI-1 expression in capillary endothelial cells and investigate whether Pai-1 plays a positive role in angiogenesis by promoting endothelial cell detachment from vitronectin and migration towards fibronectin. We will then determine whether PAI-1 is actively involved in metastasis by testing the metastatic potential of neuroblastoma cell lines in PAI-1 deficient mice.
In Specific Aim 3, we will study the mechanisms of bone metastasis in vitro and in vivo. Using a bone organ culture model we will investigate whether neuroblastoma cells up- regulate bone resorption by osteoclasts. We will test positive cell lines for their ability to form osteolytic bone metastasis in scid mice. This model will then be used in preclinical studies to test synthetic inhibitors of MMP's and inhibitors of osteoclasts such as biophosphonates. We anticipate that these studies will improve our understanding of the role of matrix degrading proteases and their inhibitors in neuroblastoma metastasis and will lead to the understanding of new approaches to treat and prevent metastasis in this type of cancer.
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