The major objective of this proposal is the identification and functional analysis of prostate cancer biomarkers that predict disease progression and outcome.  The specific aims for this project are:

1. Establishment of comprehensive collections of biological specimens and clinical data for prostate cancer biomarker research in four European populations.

In order to identify associations between inherited genetic variation and clinical sub-forms of prostate cancer, a large collection of patient samples along with detailed and accurate diagnostic and clinical information is needed.  We will focus on four European populations, each with its own distinct epidemiology and clinical relevance.  The outcome of this part of the project is a collection of samples and clinical information from over 8,000 prostate cancer cases from different parts of Europe.  We will have established the first biospecimen repository for prostate cancer in Romania and greatly added to three existing sample collections.  This resource will be used to assess the utility of inherited genetic variants as biomarkers of disease progression and outcomes.

2. Testing of the utility of genetic prostate cancer risk variants as biomarkers of disease severity, progression and outcomes.

We have combined results from our genome-wide prostate cancer association study with publicly available data to search for variants that show different frequencies in prostate cancer cases with aggressive disease (Gleason score >6, stage T3 or T4, and/or metastatic disease) versus cases with an early stage disease (Gleason<6, stage T1a-T2b).  The purpose of this analysis is to try to identify genetic variants that are directly associated with a more aggressive disease and less favorable outcome.  Approximately 3,000 of these variants will be selected for replication in this study.  Furthermore, we will select 20 previously reported variants in candidate genes in order to test their association to disease phenotype.

We will genotype over 8,000 prostate cancer cases from four different regions in Europe and a large number of controls for these variants and put the results into context of clinical parameters.  Major emphasis will be put on testing if the risk variants associate with any of the following: i)  stage and grade of disease, ii) age at diagnosis, iii)  recurrence, iv) survival (disease-free and overall), v)  family history of the disease.

3. Elucidation of the biological mechanisms that cause increased prostate cancer risk.

We will apply state-of-the-art molecular and cellular approaches to characterize the functional aspects of variants associated with different clinical forms of the disease, using exclusively cells and tissues from prostate cancer patients and individuals with benign prostate hyperplasia.  Currently, one variant on Chr8q24 has been identified that reproducibly is associated with high Gleason score but the other variants remain to be tested in this respect.  We will focus our functional studies on two variants, the Chr8q24 variant and the variant in the TCF2 transcription factor.  As a first step, we will create primary cultures of non-neoplastic human prostate epithelial cells from 20 individuals.  The cells will be genotyped to identify carriers of the variants and immortalized to provide relevant cell line models for functional studies.  To elucidate the function of the Chr8q24 variant, we will over-express and down-regulate selected transcripts (identified previously by RACE and through an ongoing microarray project), using lenti-viral vectors and stable shRNA, respectively.  A comparison of global gene expression and pathway alterations in cells over and under-expressing the candidate genes will be performed to identify the relevant pathways involved.  For TCF2 we will seek to identify binding sites for the different TCF2 variants in prostate cell-lines.  By pairing this with expression array profiling we hope to uncover pathways regulated by TCF2 and potentially predictive of disease or recurrence.