Melanoma Metastasis Inhibition by Eristostatin

描述（由申请人提供）：转移（即肿瘤扩散）是癌症治疗的主要障碍。转移过程包括许多步骤。如果一连串的事件在任何一步被打断，转移就不会发生。蛇毒蛋白是蛇毒蛋白分解素家族的一员，在免疫缺陷小鼠体内被发现可以抑制黑色素瘤的转移。迄今为止，蛇毒抑素抗转移作用的基础仍不清楚。本研究的长期目标是确定抑制黑色素瘤转移的分子机制。本文提出的具体目标是通过追求两个特定目标来研究蛇毒蛋白是如何抑制实验性转移的：(i)表征蛇毒蛋白及其突变体与四种具有明确定义的表面受体不同组合的黑色素瘤细胞系的相互作用。（ii）确定对其抗转移能力最关键的eristostatin的结构序列。重组蛇毒抑素突变将通过丙氨酸扫描诱变产生，并与谷胱甘肽- s转移酶制成细菌融合蛋白。这些纯化的突变体将用于四种人类转移性黑色素瘤细胞系的一系列功能测定。我们将通过交联和免疫学技术确定与蛇毒抑素相互作用的特定分子。为了确定蛇毒抑制素的哪些残基负责其细胞相互作用，将在细胞测定中将每个突变体与野生型蛇毒抑制素的活性进行比较。那些在细胞相互作用中表现出最大差异的突变体将用于实验转移分析。静脉注射带有黑素瘤细胞和变异蛇抑素的免疫缺陷小鼠后，观察四周，然后解剖。转移潜力将通过计算肺转移来评估。肺冷冻切片将使用免疫组织化学染色进行评估。综上所述，这些研究将深入了解一种自然产生的蛋白质是如何“合适”地结合黑色素瘤细胞并阻止其转移能力的。这些信息反过来又会导致针对这些细胞的治疗剂的合理设计。

DESCRIPTION (provided by applicant): Metastasis (i.e., tumor spread) is the major obstacle to cancer cure. The metastatic process consists of many steps. If the cascade of events is interrupted at any step, metastasis will not occur. Eristostatin, a member of the disintegrin family of viper venom proteins, has been found to inhibit melanoma metastasis in vivo using immunodeficient mice. To date, the basis for eristostatin's anti-metastatic effect remains unknown. The long term objective of this research is to identify molecular mechanisms by which melanoma metastasis may be inhibited. The specific goal proposed here is to investigate how eristostatin inhibits experimental metastasis by pursuing two specific aims: (i) Characterize the interactions of eristostatin and its mutants with four melanoma cell lines possessing distinct combinations of well-defined surface receptors. (ii) Identify the structural sequence within eristostatin which is most critical for its anti-metastatic ability. Recombinant eristostatin mutations will be created by alanine scanning mutagenesis, and made as bacterial fusion proteins with glutathione-S-transferase. These purified mutants will be used in a series of functional assays with four human metastatic melanoma cells lines. We will identify the specific molecule with which eristostatin interacts on each cell through crosslinking and immunological techniques. To determine which residue(s) of eristostatin are responsible for its cellular interactions, each mutant will be compared with wildtype eristostatin's activity in cellular assays. Those mutants which show the greatest difference in cellular interaction will be used in experimental metastasis assays. After i.v. injection of immunodeficient mice with melanoma cells and mutated eristostatin, mice will be observed for four weeks, and then necropsied. Metastatic potential will be assessed by counting lung metastases. Cryosections of lungs will be evaluated using immunohistochemical stains. Taken together, these studies will provide insights into how one naturally occurring protein possesses the "right fit" to bind melanoma cells and block their metastatic ability. This information will, in turn, lead to a rational design of therapeutic agents which would target these cells.