Prostate Cancer Gene Identification within Candidate Region in African Americans

 描述（由申请人提供）：在美国，非洲裔美国人（AA）和欧洲裔美国人（EA）男性之间的前列腺癌（PCa）发病率和死亡率存在充分记录的种族差异。这些差异不能完全由种族的筛查差异或治疗差异来解释，其他环境和生物风险因素尚未得到很好的表征。我们推测，种族差异的遗传风险因素的分布可能有助于观察到的差异。我们最近 在染色体7 q31 -32上发现了一个区域，该区域在统计学上与AA男性的前列腺癌显著相关，并且也有与PCa侵袭性相关的暗示性证据。所确定的区域是基因丰富的，到目前为止，我们已经确定了两个候选基因（CPED 1和FLNC）在这个区域。这些基因在文献中还没有得到很好的描述，但是在前列腺癌中可以找到它们的作用的暗示。 以前的表达研究。在这里，我们建议使用来自多个数据库的现有样本和数据，更深入地研究染色体7 q31 -32上的区域。在目标1中，我们首先将在一个更大的独立样本集中检查该区域的SNP关联，以证实我们的原始发现（目标1.A）。然后，我们将检查可用的在线数据库，包括dbGAP，GEO和Oncomine，以进一步证明该区域的基因与PCa风险和侵袭性相关（Aim 1.B）。基于目标1.A和B的发现，我们将选择基因和区域，以密集tagSNP面板为目标，以更好地完善我们在230例AA PCa病例和230例AA对照中的目标基因。目标2将使用库存组织样本（目标2.A）和细胞系（目标2.B）探索在我们先前的工作和目标1中鉴定的前五个候选基因（包括CPED 1和FLNC）的功能意义。对于目标2.A，将在112个新鲜冷冻PCa样品中评估mRNA的表达，所述样品被选择以同样地代表EA男性、AA男性、高Gleason分级（GG，>4+3）和低GG（<3+4）。将使用相同人种/等级采样方法，在石蜡包埋的PCa样本中使用IHC评估蛋白表达。对于目标2.B，将使用代表一系列PCa侵袭性以及EA和AA来源的多个细胞系来评价这些蛋白质的蛋白质表达和生物学功能。将使用免疫印迹分析测定蛋白质表达。如果临床数据表明候选基因在前列腺癌中过表达，我们将敲除它，如果在前列腺癌中表达丢失，我们将过表达它。我们将进行细胞生长，转化，迁移和侵袭试验，以确定改变基因表达是否会调节细胞功能。我们还将进行细胞计数试验，以测量8天内的细胞生长。软琼脂和克隆形成试验将用于测定转化。Boyden小室试验将用于评估迁移和侵袭的变化。研究结果将提供深入了解PCa的遗传风险因素，这可能有助于解释发病率和死亡率的种族差异，从而为减少这些差异提供生物学目标。

 DESCRIPTION (provided by applicant): There are well documented racial disparities in prostate cancer (PCa) incidence and mortality in the United States between African American (AA) and European American (EA) men. These disparities cannot be fully explained by differences in screening or treatment differences by race, and other environmental and biological risk factors have not been well characterized. We hypothesize that racial differences in the distribution of genetic risk factors may contribute to the observed disparities. We recently identified a region on chromosome 7q31-32 that is statistically significantly associated with prostate cancer in AA men, and there is suggestive evidence for association with PCa aggressiveness as well. The identified region is gene-rich, and thus far we have identified two candidate genes (CPED1 and FLNC) in this region. These are genes that have not been well characterized in the literature, however hints of their role in prostate cancer can be found within previous expression studies. Here, we propose to examine the region on chromosome 7q31-32 in greater depth, using existing samples and data from multiple databases. In Aim 1, we first will examine SNP associations in this region in a much larger independent sample set to confirm our original findings (Aim 1.A). We will then examine available online databases, including dbGAP, GEO, and Oncomine for further evidence of association of genes in this region with PCa risk and aggressiveness (Aim 1.B). Based on the findings of Aims 1.A and B, we will select genes and regions to target with a dense tagSNP panel to better refine our genes of interest in 230 AA PCa cases and 230 AA controls. Aim 2 will explore the functional significance of the top five candidate genes identified in our prior work and in Aim 1, including CPED1 and FLNC, using banked tissue samples (Aim 2.A) and cell lines (Aim 2.B). For Aim 2.A, Expression of mRNA will be assessed in 112 fresh frozen PCa samples selected to represent EA men, AA men, high Gleason grade (GG, >4+3) and low GG (<3+4) equally. Protein expression will be assessed using IHC in paraffin embedded PCa samples, using the same race/grade sampling method. For Aim 2.B, multiple cell lines representing a range of PCa aggressiveness and both EA and AA origin will be used to evaluate protein expression and biological function of these proteins. Protein expression will be determined using immunoblot analysis. If clinical data suggest the candidate gene is overexpressed in PCa, we will knock it down, and if expression is lost in PCa we will overexpress it. We will perform cell growth, transformation, migration and invasion assays to determine if altering gene expression will regulate cellular function. We will also perform cell counting assays to measure cell growth over an eight day period. Soft agar and clonogenic assays will be used to assay transformation. Boyden chamber assays will be used to assess changes in migration and invasion. Study results will provide insight into genetic risk factors for PCa that might help explain racial disparities in incidence and mortality and thereby provide biological targets for reducing these disparities.