Functional analysis of TSPY and its role in prostate carcinogenesis

Abstract

Testis specific protein Y chromosome encoded (TSPY) is a multicopy gene located on the human Y chromosome. It has been reported that the human genome harbours between 20 to 40 copies of the gene. Mammalian homologues of human TSPY were also found to be repetitive. The gene is expressed in human foetal and adult testis. The precise function of the expressed TSPY gene product is not fully defined, but it has been postulated to regulate proliferation of testicular spermatogonia. Up-regulation of TSPY expression has been detected in gonadoblastoma, testicular cancer and prostate cancer. The contribution of abnormal expression of TSPY to prostate carcinogenesis has not been investigated. Prostate cancer (CaP) and benign prostate hyperplasia (BPH) are the most common diseases of the human prostate. Prostate cancer is a disease of the elderly and is the second most common cancer and second most common cause of death from cancer among men in UK. In this thesis, the role of TSPY in prostate carcinogenesis was studied in four related aspects. First, TSPY protein and transcript expression pattern in CaP compared to Benign Prostate Hyperplasia (BPH) was studied using a combination of immunohistochemistry and mRNA in situ hybridisation. Second, the ability of TSPY to regulate cellular proliferation was investigated by transfection experiments of the prostate cancer LNCaP cell line. Third, TSPY genomic copy number in prostate cancer was characterised and compared to BPH. Finally, the downstream genes regulated by TSPY were investigated using high density microarray gene profiling method. An anti-human TSPY polyclonal antibody was developed for immunodetection of TSPY expression level in resected prostate tissues. In total, 72 cases of patients with prostate cancer and 20 cases of patients affected with BPH were studied by immunohistochemistry. TSPY was predominantly detected in the prostatic epithelium. In the benign gland, TSPY expression was limited to the basal cells compartment. TSPY expression was significantly up-regulated in prostate cancer when compared to BPH (P<0.0001). Furthermore, increased TSPY expression level was associated with aggressive disease (tumour with high Gleason score; P<0.02) and the presence of bone metastasis at the time of diagnosis (P<0.028). To address the functional role of 3 TSPY in prostate cancer, FLAG-TSPY was cloned and stably transfected into LNCaP cells. The presence of transfected TSPY increased LNCaP proliferation by two fold compared to empty vector control, consistent with a mitogenic function in CaP (P<0.0001). An absolute quantitative real time PCR based on Taqman assay was established and validated. TSPY genomic copy number was determined from comparing 161 samples: CaP-serum (n=47), resected tumour (n=31); BPH-serum (n=27), resected prostate (n=13) and control-serum (n=45). Of the clinical samples analysed, interpersonal variability of TSPY copy numbers was observed with the majority of cases containing between 20 to 50 TSPY copies per genome. Although, there were variability in TSPY copy numbers among individuals, there was no statistically significant correlation between TSPY copy number (serum and prostatic tissue) and the development of prostate cancer. Studying LNCaP stably transfected with TSPY and empty vector control, the key genes mediating the functional effect of TSPY were identified using Affymetrix oligonucleotide microarray method. In total, 332 genes have been altered 1.5 to 90 fold due to the effect of TSPY over-expression. Ten genes were selected and the gene expression levels were confirmed using semi-quantitative RT-PCR method. Gene clustering analysis has indicated changes in genes that regulate cellular differentiation (NRDG1, NF2, C-MAF and BMP11), apoptotic gene (BAX), cell cycle gene (cyclin G), detoxification gene (GST-2A) and genes linked to adhesion (PCDH7a, PLOD2 and IRS 1). In summary, TSPY is over-expressed in prostate cancer. This abnormal expression is linked to prostate cancer progression and metastasis. The up-regulation of TSPY expression is unlikely to be due to increased copy number. Expression of exogenous TSPY in prostate cancer cells enhanced cellular proliferation. The gene meditates its effect by down-regulating the expression pattern of selected differentiation, apoptosis and adhesion genes. Hence, over-expression of TSPY contributes to prostate carcinogenesis.