MECHANISMS OF DIETARY MODULATION OF MELANOMA INVASION

恶性黑色素瘤的发病率持续增加，而且常常与转移有关。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，以便我们能够干扰这一致命的过程。我们已经证明，限制饮食中的酪氨酸(Tyr)和苯丙氨酸(Phe)可以显著抑制转移，使转移性B16BL6黑色素瘤小鼠的生存时间增加一倍以上，并改变B16BL6黑色素瘤细胞的侵袭和转移表型。体外培养的细胞在缺乏Tyr/Phe的条件下，可以抑制侵袭和转移。我们建议研究抑制侵袭的机制。初步数据表明，Tr/Phe限制可诱导黑色素瘤细胞G0/G1期停滞，减少与一组细胞基质成分的粘附性，并通过重组的细胞外基质(Matrigel)减少侵袭，生长因子降低Matrigel。同时，这种剥夺也降低了一些功能蛋白，如1)粘着斑激酶(FAK)的表达和磷酸化，2)RAS和c-Raf-1的表达，3)细胞周期蛋白D1的表达，4)组织纤溶酶原激活物(TPA)、尿激酶型纤溶酶原激活物(UPA)和金属蛋白酶(MMPs)2和9的分泌。我们推测，抑制FAK的表达和激活以及减少RAS和c-Raf-1的信号通路是Tyr/Phe限制黑色素瘤细胞抗侵袭作用的原因。我们将用生物化学和分子生物学的方法来研究以下特殊目的：1)确定(S)FAK蛋白在黑色素瘤细胞与HSPG的黏附和侵袭中所起的作用(使用体外技术，比较缺乏特定氨基酸的细胞和完全培养的细胞)；2)确定氨基酸剥夺对黑色素瘤细胞FAK酪氨酸磷酸化的抑制是否具有Src try激酶特异性；3)确定Ras/Raf/Erk信号通路的RAS在氨基酸剥夺影响黑色素瘤细胞合成和分泌纤溶酶原激活物(Pas)和基质金属蛋白酶(MMPs)中的作用；4)检测抗RAS和抗细胞周期蛋白D1对肿瘤侵袭能力的影响。这项研究将加强对Tyr/Phe限制的抗侵袭活性的潜在机制的了解。了解Tyr/Phe缺失所产生的抗黏附和抗信号作用之间的联系，将为设计新的治疗方法以减缓或阻止高侵袭性和转移性黑色素瘤的进展提供可靠的理论基础。

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.