Functional Genomics of Metastatic Determinants of Melanoma

描述（申请人提供）：转移是一个复杂的、多步骤的过程，尽管对驱动原发肿瘤形成的遗传和基因组改变进行了大量研究，但对转移的遗传基础知之甚少。有证据表明，转移性肿瘤的基因组图谱不同于原发肿瘤，这意味着新的、独特的基因驱动转移。虽然基因组数据提供了对肿瘤病因至关重要的改变的见解，但真正的遗传决定因素必须通过体外和体内的功能性研究来确定。为此，跨物种肿瘤基因组筛选已经确定了转移性黑色素瘤中严格过滤的上调基因列表。本研究建议将这些候选细胞用于低复杂性的体外筛选，以检测细胞对细胞表面附着丧失的抵抗性或细胞凋亡。此外，将进行体内转移筛查，以确定驱动转移的各个步骤的基因，包括体内转移、循环生存和继发性肿瘤形成。最后，所有的抗瘤基因和转移基因将被纳入深入的功能和临床病理研究。使用低贴壁板，高通量96孔格式体外斑点病筛选，以确定赋予斑点病对斑点病敏感细胞抗性的基因。由于耐药只是转移的一个方面，本研究还将在体内筛选能够驱动多步骤转移的基因组合。表达候选基因的人类细胞将被皮下注射到小鼠体内，并监测小鼠的原发性和继发性肿瘤形成和肿瘤细胞在循环中的存活情况。具有转移潜力的候选细胞将通过功能获得和功能丧失分析纳入深入的生化研究。同时，利用免疫组织化学，转移性决定因素将被检测在分期的人类转移性黑色素瘤组织中的表达，以建立临床相关性。

DESCRIPTION (provided by applicant): Metastasis is a complex, multi-step process and while much has been investigated about the genetic and genomic alterations driving primary tumor formation, little has been elucidated about the genetic basis for metastasis. Evidence shows that genomic profiles of metastatic tumors are distinct from primary tumors implying that novel, distinct genes drive metastasis. While genomic data provides insight into alterations that are critical in the etiology of tumors, true genetic determinants must be identified through functional in vitro and in vivo studies. To this end, a cross-species oncogenomic screen has identified a stringently filtered list of genes up-regulated in metastatic melanoma. This research proposes to employ these candidates in a low-complexity in vitro screen for resistance to anoikis, or apoptosis due to loss of surface attachment. Additionally, an in vivo metastasis screen will be conducted to identify genes driving various steps of metastasis including intravasation, survival while in circulation, and secondary tumor formation. Finally, all pro-anoikis resistance and pro-metastatic genes will be enlisted into in-depth functional and clinicopathological studies. Using low-adherent plates, a high-throughput 96-well format in vitro anoikis screen to identify genes that confer anoikis resistance to anoikis sensitive cells will be conducted. Since anoikis resistance is but one aspect of metastasis, this research will also screen in vivo for the combination of genes capable of driving multiple steps of metastasis. Human cells expressing candidate genes will be sub-cutaneously injected into mice and mice will be monitored for primary and secondary tumor formation and survival of tumor cells in circulation. Candidates conferring metastatic potential to non-metastatic cells will be enlisted into in-depth biochemical studies through gain-of-function and loss-of-function assays. In parallel, using immunohistochemistry, metastatic determinants will be examined for expression on staged human metastatic melanoma tissues to establish clinical relevancy. PUBLIC HEALTH RELEVANCE: Unlike many cancers, the incidence of melanoma has steadily increased world-wide. Whereas primary localized melanoma is surgically excisable, therapies for metastatic melanoma have poor clinical efficacy, resulting in only 10% of patients having greater than five year survival. Therefore, it is of utmost importance to identify the genetic determinants of metastatic melanoma and thereby generate a list of genes that can serve not only as therapeutic targets but also as prognostic biomarkers with high translational potential.