Molecular Mechanisms of Metastasis Suppression by NM23

描述(申请人提供)：尽管近年来调控肿瘤发生的机制已基本阐明，但对肿瘤细胞转移的机制知之甚少，肿瘤细胞转移是癌症死亡的最终原因。转移抑制基因在控制这一过程中起着关键作用，但目前只发现了少数几个。NM23(转移阴性)基因是第一个被描述的转移抑制基因，在黑色素瘤、乳腺癌和胃癌中被认为具有抗转移活性。本项目的总体目标是系统地剖析NM23转移抑制活性的分子机制和基因组途径。我们最近发现NM23-H1亚型是一种3‘-5’外切酶(3‘-5’EXO)，这种活性通常与基因组完整性、细胞凋亡和染色质重塑有关。虽然这些过程与恶性进展有明显的潜在相关性，但我们也将测量NM23-H1的核苷二磷酸激酶(NDPK)和组氨酸激酶(Hisk)活性对其转移抑制特性的贡献程度。在特定的目标1中，我们将完成详细的结构-功能分析，以描绘3‘5’EXO的活性部位，并鉴定一组含有选择性破坏NM23-H1分子的3‘5’EXO、NDPK和HISK功能的突变体。这些突变体将被用作分子工具，利用细胞培养和体内模型来测量致瘤和转移潜力，以确定每个突变体在抑制人类黑色素瘤细胞转移表型(特定目标2)方面的相对贡献。由NM23-H1变异体的表达引起的基因表达变化将通过微阵列分析和实时荧光聚合酶链式反应(特异性目标3)来确定。相关分析将确定NM23调控的基因，其表达与转移表型和转移进展密切相关。已识别基因的功能相关性将通过病毒介导的过度表达和敲除方法进行验证。在具体目标4中，我们将直接在哺乳动物和酵母细胞模型中检测NM23-H1的DMA修复活性，并鉴定相关的酶功能。这些研究将首次对NM23蛋白在多种癌症类型中对抗转移的途径进行系统分析，因此可能会提出新的策略来对抗这种疾病的最后和棘手的阶段。

DESCRIPTION (provided by applicant): While mechanisms regulating oncogenesis have been elucidated substantially in recent years, comparatively little is yet known about the mechanisms that underlie metastasis of tumor cells, the ultimate cause of death in cancer. Metastasis suppressor genes play pivotal roles in controlling this process, but only a handful have been identified. NM23 ("negative in metastasis") genes represent the first described metastasis suppressors and are recognized for their antimetastatic activity in melanoma, breast and gastric carcinoma. The overall goal of this project to systematically dissect the molecular mechanisms and genomic pathways underlying the metastasis suppressor activity of NM23. We recently found that the NM23-H1 isoform is a 3'-5' exonuclease (3'5' EXO), an activity often associated with genome integrity, apoptosis and chromatin remodeling. Although there is clear potential relevance of these processes to malignant progression, we will also measure the extent to which the nucleoside diphosphate kinase (NDPK) and histidine kinase (hisK) activities of NM23-H1 contribute to its metastasis suppressor properties. In Specific Aim 1, we will complete a detailed structure-function analysis to delineate the active site of the 3'5 EXO, and characterize a panel of mutants harboring lesions that selectively disrupt the 3'5' EXO, NDPK and hisK functions of the NM23-H1 molecule. The mutants will be used as molecular tools to determine the relative contributions of each to suppression of the metastatic phenotype (Specific Aim 2) in human melanoma cells, using cell culture and in vivo models to measure tumorigenic and metastatic potential. Gene expression changes elicited by expression of NM23-H1 variants will be determined by microarray analysis and real-time PCR (Specific Aim 3). Correlational analysis will identify NM23-regulated genes whose expression tracks with metastatic phenotype and metastatic progression. Functional relevance of identified genes will be validated by viral-mediated overexpression and knockdown approaches. In Specific Aim 4, we will directly test the DMA repair activity of NM23-H1 in mammalian and yeast cell models, and identify the relevant enzymatic function. These studies should provide the first systematic analysis of pathways through which NM23 proteins oppose metastasis in multiple cancer types and, therefore could suggest novel strategies to combat this final and intractable stage of the disease.