MECHANISMS OF DIETARY MODULATION OF MELANOMA INVASION

恶性黑色素瘤的发病率持续增加，并且通常与转移有关。一旦发生转移，几乎是无法治愈的。这个项目的目标是发现黑色素瘤转移发生的机制，这样我们就可以破坏这个致命的过程。我们已经证明，在饮食中限制酪氨酸（Tyr）和苯丙氨酸（Phe）可以显著抑制转移，使转移性B16BL6黑色素瘤小鼠的存活时间增加一倍以上，并改变B16BL6黑色素瘤细胞的侵袭性和转移性表型。在缺乏Tyr/ ph的条件下，体外培养的细胞可以复制对侵袭和转移的抑制。我们建议研究抑制入侵的机制。初步数据表明，Tr/Phe限制诱导G0/G1细胞周期阻滞，减少对一组被称为硫酸肝素蛋白多糖（HSPG）的细胞基质成分的附着，减少通过重建的细胞外基质（Matrigel）的侵袭，生长因子减少黑色素瘤细胞中的Matrigel。同时，这种剥夺也降低了一些功能蛋白的表达，如1)局灶黏着激酶（FAK）的表达和磷酸化，2)Ras和c-Raf-1的表达，3)cyclin D1的表达，4)组织纤溶酶原激活物（tPa）、尿激酶纤溶酶原激活物（uPA）和金属蛋白酶（MMPs） 2和9的分泌。我们假设FAK表达和激活的抑制以及Ras和c-Raf-1信号通路的减少可以解释Tyr/Phe限制在黑色素瘤细胞中的抗侵袭作用。生化和分子方法将用于检查以下具体目标：1)确定FAK蛋白在黑色素瘤细胞附着于HSPG和通过HSPG侵袭的过程中所起的作用（使用体外技术，将缺乏特定氨基酸的细胞与在完整培养基中生长的细胞进行比较）；2)确定黑色素瘤细胞中氨基酸剥夺对FAK Tyr磷酸化的抑制是否具有Src Try激酶特异性；3)确定Ras/Raf/Erk信号通路中Ras在黑色素瘤细胞中受氨基酸剥夺影响的纤溶酶原激活物（Pas）和MMPs的合成和分泌中的作用；4)确定抗ras和抗cyclin D1治疗对侵袭的影响。本研究将增强对Tyr/Phe限制抗侵袭活性机制的认识。了解Tyr/Phe剥夺的抗粘附和抗信号效应之间的联系，将为设计新的治疗方法来减缓或阻止高度侵袭性和转移性黑色素瘤的进展提供合理的理论依据。

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.