Molecular Mechanisms of Metastasis Suppression by NM23

描述（由申请人提供）：虽然近年来已经基本阐明了调节肿瘤发生的机制，但对肿瘤细胞转移（癌症死亡的最终原因）的基础机制知之甚少。转移抑制基因在控制这一过程中起着关键作用，但只有少数已被确定。NM 23（“转移阴性”）基因代表了第一个描述的转移抑制因子，并因其在黑素瘤、乳腺癌和胃癌中的抗转移活性而被认可。本项目的总体目标是系统地剖析NM 23的转移抑制活性的分子机制和基因组途径。我们最近发现，NM 23-H1亚型是一种3 '5'核酸外切酶（3 '5' EXO），其活性通常与基因组完整性、细胞凋亡和染色质重塑相关。虽然这些过程与恶性进展有明确的潜在相关性，但我们也将测量NM 23-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。在具体目标1中，我们将完成详细的结构-功能分析，以描绘3 '5 EXO的活性位点，并表征一组携带病变的突变体，这些病变选择性地破坏NM 23-H1分子的3' 5 ' EXO、NDPK和hisK功能。突变体将用作分子工具，以确定每种突变体对抑制人黑色素瘤细胞中转移表型（特异性目的2）的相对贡献，使用细胞培养和体内模型测量致瘤性和转移潜力。通过微阵列分析和实时PCR（特异性目的3）确定由NM 23-H1变体表达引起的基因表达变化。相关性分析将确定NM 23调节的基因，其表达与转移表型和转移进展有关。将通过病毒介导的过表达和敲低方法验证鉴定的基因的功能相关性。在具体目标4中，我们将在哺乳动物和酵母细胞模型中直接测试NM 23-H1的DNA修复活性，并鉴定相关的酶功能。这些研究应该提供第一个系统分析的途径，通过这些途径，NM 23蛋白对抗多种癌症类型的转移，因此可以提出新的策略来对抗这种疾病的最终和棘手的阶段。

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.