Prostate Cancer Susceptibility Gene Identification in Chromosome 5 Candidate Region

Wayne State University, Detroit MI

Investigators

Abstract

There are well documented, marked 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 5q35 that is associated with PCa in AA men. Preliminary analyses in this region have suggested two loci involved in chromatin regulation, and several genes including COL23A1, TSPAN17, and GRM6. These are genes that have not been well characterized in the literature, however hints of their role in PCa can be found within previous expression studies. Here, we propose to examine the region on chromosome 5q35 in greater depth, using both gene mapping and gene function analyses. In Aim 1, we will perform fine mapping in this region in a much larger set of AA men (2241 cases, 1808 controls) from multiple existing data sources to identify candidate susceptibility loci. We will then perform validation analyses in an independent set of 600 AA cases and 1650 controls and in a second independent sample of 700 cases and 700 controls from the NCI Ghana Prostate Study. Results will be combined across studies using meta-analysis. Aim 2 will explore the functional significance of the top candidate genes identified in our prior work and in Aim 1, including COL23A1 and TSPAN17, 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. The co-PIs have successfully collaborated on a similar project examining a different region on chromosome 7q31.