Validation of genomic targets in melanoma

描述（由申请人提供）：最近在我们实验室进行的cDN A微阵列研究表明存在两种主要的转移性黑色素瘤亚型，具有潜在的不同临床结果。对痣、原发性和转移性黑色素瘤的cDNA微阵列分析已经发现了大量基因，这些基因的表达水平可以用来区分黑色素瘤进展中的已知标志，包括NF-kappaB和WNT-2信号通路中的基因。在此应用中，我们建议扩展这些观察结果：目的1：验证在大量黑色素瘤患者中通过基因表达谱鉴定的转移性黑色素瘤分子亚型的预后意义。我们将利用cDNA微阵列分析在更大的前瞻性转移性黑色素瘤患者队列中确认基因表达特征对预后的影响。目的2：利用系统的、基于质粒的sirna、核酶和cdna传递来表征从我们的阵列研究中鉴定出的四个基因在小鼠黑色素瘤转移进展中的功能作用。我们将描述NF-kappaB和WNT-2信号通路中四个基因在黑色素瘤转移进展中的功能重要性。我们将使用靶向NCOA3、PHIP和WNT2的sirna和核酶，以及WIF1 cDNA来验证这些基因在黑色素瘤转移进展中的重要性。目的3：检测两种候选黑色素瘤进展基因在大型人类黑色素瘤组织库中的表达水平，并将这些基因的表达与黑色素瘤结局联系起来。我们将评估NCOA3和WNT2的蛋白产物是否提供了新的分子标记，通过对常规切片和组织阵列进行免疫组织化学分析，利用广泛的原发性人类黑色素瘤库来识别转移高风险的人类黑色素瘤患者。这些研究应该有助于验证通过表达谱鉴定的基因在黑色素瘤进展中的作用。

DESCRIPTION (provided by applicant): Recent cDN A microarray studies performed in our laboratory have indicated the presence of two dominant subtypes of metastatic melanoma, with potentially different clinical outcomes. cDNA microarray analyses of moles, primary and metastatic melanomas have identified a large number of genes whose expression level can be used to distinguish between the known landmarks in melanoma progression, including genes operating in the NF-kappaB and WNT-2 signaling pathways. In this application, we propose to extend these observations: Aim 1: To validate the prognostic significance of the molecular subtypes of metastatic melanoma identified by gene expression profiling in a large cohort of melanoma patients. We will confirm the prognostic impact of gene expression signatures using cDNA microarray analysis on a larger, prospectively identified cohort of metastatic melanoma patients. Aim 2: To use systemic, plasmid-based delivery of siRNAs, ribozymes, and cDNAs to characterize the functional roles of four genes identified from our array studies on the metastatic progression of melanoma in mice. We will characterize the functional importance of four genes in the NF-kappaB and WNT-2 signaling pathways on the metastatic progression of melanoma. We will use siRNAs and ribozymes targeting NCOA3, PHIP, and WNT2, and the WIF1 cDNA to validate the importance of each of these genes in the metastatic progression of melanoma. Aim 3: To examine expression levels of two candidate melanoma progression genes in a large tissue bank of human melanomas, and to correlate the expression of these genes with melanoma outcome. We will assess whether the protein products of NCOA3 and WNT2 provide new molecular markers to identify human melanoma patients at high risk for metastasis by performing immunohistochemical analysis of routine sections and tissue arrays, using an extensive bank of primary human melanomas. These studies should help validate the role of genes identified by expression profiling in the progression of melanoma.