The incidence of malignant melanoma continues to increase, and often is associated with metastasis. Once metastasis occurs, it is virtually incurable. The goal of this project is to discover the mechanism by which melanoma metastasis occurs so that we can disrupt this fatal process. We already demonstrated that restricting tyrosine (Tyr) and phenylalanine (Phe) in the diet dramatically inhibits metastasis, more than doubles survival time of mice with metastatic B16BL6 melanoma, and alters the invasive and metastatic phenotype of B16BL6 melanoma cells. Inhibition of invasion and metastasis can be replicated after culture of cells in vitro under Tyr/Phe-deprived conditions. We propose to examine the mechanism underlying the inhibition of invasion. Preliminary data indicate that Tr/Phe restriction induces G0/G1 cell cycle arrest, decreases attachment to a group of constituents of the cell matrix known collectively as heparan sulfate proteoglycans (HSPG), and decreases invasion through reconstituted extracellular matrix (Matrigel), and growth factor reduced Matrigel in melanoma cells. Meanwhile, this deprivation also decreases some functional proteins, such as 1) focal adhesion kinase (FAK) expression and phosphorylation, 2) Ras and c-Raf-1 expression, 3) cyclin D1 expression, and 4) secretion of tissue plasminogen activation (tPa), urokinase plasminogen activator (uPA) and metalloproteases )(MMPs) 2 and 9. We hypothesize that the inhibition of FAK expression and activation along with decreased Ras and c-Raf-1 signaling pathways accounts for the anti-invasive effect of Tyr/Phe restriction in melanoma cells. Biochemical and molecular approaches will be used to examine the following specific aims: 1) Determine what role(s) FAK protein plays in the attachment of melanoma cells to HSPG and invasion through HSPG (using in vitro techniques, comparing cells deprived of specific amino acids with cells grown in complete media); 2) Determine whether the inhibition of Tyr phosphorylation of FAK by amino acid deprivation in melanoma cells is Src Try kinase specific; 3) Determine the role of the Ras of the Ras/Raf/Erk signaling plays in synthesis and secretion of plasminogen activators (Pas) and MMPs in melanoma cells as influenced by amino acid deprivation; and 4) Determine the effect of anti-Ras and anti- cyclin D1 treatment on invasion. This study will enhance knowledge of the mechanisms underlying the anti-invasion activity of Tyr/Phe restriction. Understanding the connection between anti-adhesion and anti-signaling effects by Tyr/Phe deprivation will provide a sound rationale for designing new therapeutic approaches to slow or block progression of highly invasive and metastatic melanoma.
