MECHANISMS OF DIETARY MODULATION OF MELANOMA INVASION

恶性黑色素瘤的发病率不断增加，且常伴有转移。一旦发生转移，几乎无法治愈。该项目的目标是发现黑色素瘤转移发生的机制，以便我们能够破坏这一致命过程。我们已经证明，限制饮食中的酪氨酸（Tyr）和苯丙氨酸（Phe）可显著抑制转移，使转移性B16 BL 6黑色素瘤小鼠的存活时间增加一倍以上，并改变B16 BL 6黑色素瘤细胞的侵袭性和转移性表型。在Tyr/Phe剥夺条件下体外培养细胞后，可以复制对侵袭和转移的抑制。我们建议检查的机制抑制入侵。初步数据表明，Tr/Phe限制诱导G 0/G1细胞周期停滞，减少附着的一组成分的细胞基质统称为硫酸乙酰肝素蛋白聚糖（HSPG），并减少入侵通过重建的细胞外基质（基质胶），生长因子减少基质胶在黑色素瘤细胞。 同时，这种剥夺也降低了一些功能蛋白，如1）粘着斑激酶（FAK）表达和磷酸化，2）Ras和c-Raf-1表达，3）细胞周期蛋白D1表达，以及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。我们推测FAK表达和活化的抑制沿着Ras和c-Raf-1信号通路的减少是Tyr/Phe限制在黑素瘤细胞中的抗侵袭作用的原因。本研究将采用生物化学和分子生物学的方法，对以下几个方面进行研究：1）确定FAK蛋白在黑色素瘤细胞与HSPG的粘附和通过HSPG的侵袭中的作用（使用体外技术，将缺乏特定氨基酸的细胞与在完全培养基中生长的细胞进行比较）; 2）确定在黑素瘤细胞中通过氨基酸剥夺对FAK的Tyr磷酸化的抑制是否是Src Try激酶特异性的; 3）确定Ras/Raf/Erk信号传导中的Ras在受氨基酸剥夺影响的黑素瘤细胞中纤溶酶原激活物（Pas）和MMP的合成和分泌中的作用;和4）确定抗Ras和抗细胞周期蛋白D1处理对侵袭的影响。本研究将有助于进一步了解Tyr/Phe限制性酶抗侵袭活性的机制。了解Tyr/Phe剥夺的抗粘附和抗信号传导作用之间的联系将为设计新的治疗方法以减缓或阻断高度侵袭性和转移性黑色素瘤的进展提供合理的理论基础。

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.