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Metastatic prostate carcinoma is a significant cause of morbidity and mortality in men world-wide. The aim of our study was to (i) identify novel metastasis associated genes using a cell line model for metastatic prostate cancer (ii) determine the clinical relevance of any candidate proteins. Using Proteomic two-dimensional (2D) gel electrophoresis, we compared the protein profile of the human prostate cancer cell line LNCaP with its metastatic variants (LNCaP-LN3 and LNCaP-Pro5). Protein identification was performed by Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF-MS), and Electrospray Ionisation tandem mass spectrometry (ESI-MS/ MS). The expression pattern of any candidate protein(s) was assessed by immunohistochemisty in matched benign and malignant prostate tissues (n= 60) arranged on a tissue micro-array. Promoter hyper-methylation was assessed by bisulfite modified DNA sequencing. The LNCaP-LN3 cell line was treated with the demethylating agent 5’ azacytidine and mRNA expression status was determined. Analysis of silver stained gels showed the absence of a major protein in LNCaP-LN3 and LNCaP-Pro5, but was present in the parental LNCaP cell line. MALDI-TOF-MS and ESI-MS/ MS analyses identified this protein as Lactate Dehydrogenase B (LDH-B), and its identity was further confirmed by 2D Western blotting. Furthermore, enzyme kinetic assays showed a higher activity of LDH in the LNCaP cell line compared with LNCaP-LN3 and LNCaP-Pro5. Immunostaining showed high expression of LDH-B in the basal cells of benign epithelium with weak/ absent expression in luminal cells. In prostate cancer, absent staining was seen in 70% of cases while the remaining 30% of cases showed LDH-B expression. Significantly, LDH-B promoter hyper-methylation was seen in 8/17 cases (47%) of adenocarcinoma and in none of the 10 cases of benign epithelium. In addition promoter hyper-methylation was seen in LNCaP-LN3, but not in the LNCaP cell line. By RT-PCR, LNCaP-LN3 cells showed near absent levels of LDH-B mRNA, however treatment of these cells with 5’ azacytidine caused re-expression of LDH-B mRNA. Our results suggest that loss of LDH-B expression is frequent in prostate cancer. DNA sequencing of bisulfite modified DNA suggests that the underlying mechanism may involve promoter hyper-methylation. Enzyme kinetic assays on the cell lines suggest that loss of LDH-B protein leads to a reduction in LDH enzyme activity. The finding of loss of LDH-B expression in a high frequency of prostate cancers but its expression in benign tissue suggests that loss of LDH-B is associated with prostate cancer development.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]