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cDNA微阵列研究表明存在两种主要亚型的转移性黑色素瘤，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微阵列分析已经鉴定了大量基因，其表达水平可用于区分黑色素瘤进展中的已知标志，包括在NF-κ B和WNT-2信号通路中起作用的基因。在本申请中，我们建议扩展这些观察结果：目的1：验证在一个大的黑色素瘤患者队列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。我们将使用cDNA微阵列分析，在一个更大的，前瞻性确定的转移性黑色素瘤患者队列中，证实基因表达特征对预后的影响。目标二：使用基于质粒的siRNA、核酶和cDNA的系统性递送来表征从我们对小鼠黑色素瘤转移进展的阵列研究中鉴定的四个基因的功能作用。我们将描述NF-κ B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siRNA和核酶靶向NCOA 3，PHIP，WNT 2和WIF 1 cDNA，以验证这些基因中的每一个在黑色素瘤转移进展中的重要性。目标3：检测大型人类黑色素瘤组织库中两种候选黑色素瘤进展基因的表达水平，并将这些基因的表达与黑色素瘤结局相关联。我们将评估NCOA 3和WNT 2的蛋白产物是否提供了新的分子标记物，以通过使用广泛的原发性人类黑色素瘤库对常规切片和组织阵列进行免疫组织化学分析来识别转移风险高的人类黑色素瘤患者。这些研究将有助于验证通过表达谱分析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.