BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

Abstract Histidine acid phosphatases are characterized by the presence of a conserved RHGXRXP motif. One medically important acid phosphatase is the Prostatic Acid Phosphatase (PAP). PAP has been associated with prostate cancer for a long time and has been used as a marker to stage prostate carcinoma. Yet, there is no clear understanding on the functioning of the enzyme in vivo. This thesis work focuses on the characterization of putative ligands and elucidation of the reaction mechanism of PAP using computational methods. The ligand-enzyme complexes were generated by docking and molecular dynamics simulations. The complexes showed that the conserved arginines of RHGXRXP motif are important for binding the highly negatively charged phosphate group. The complexes also highlighted that the active site aspartate (Asp258) should be neutral in the complex and is involved as a general acid-base in the reaction. The studies support that PAP could dephosphorylate the growth factor receptors EGFR and ErbB-2. The studies also found that the majority of tyrosine phosphorylated peptides from these growth factor receptors could bind to PAP. The affinities were assessed based on theoretical calculations and were further confirmed by experimental measurements in the feasible cases. To clearly understand the mechanism of PAP, quantum mechanical methods were employed. The enzymatic reaction involves two steps. In the first step, the phosphate moiety is transferred from the ligand to the conserved histidine. The calculations on the first step of the reaction involved generating the transition state (TS) structures and estimating the respective barriers. The calculations clearly support that Asp258 becomes neutral by picking up the proton from the monoanionic ligand entering the binding site. The proton from neutral Asp258 is later transferred to the leaving group via a water bridge, restoring the negative state of Asp258. The second step involves the hydrolysis of phosphohistidine enzyme intermediate. Using hybrid quantum mechanics/molecular mechanics calculations, it was found that the Asp258 accepts a proton from the nucleophilic water only after the TS is crossed. This proton is possibly then transferred to the free phosphate while it leaves the binding site, restoring the enzyme to its free state. The study highlights the importance of active site arginines in the binding as well as the stabilization of TS. Further, the analysis of TS structures in both the steps showed an associative mechanism, based on the distance of the nucleophilic and the leaving atoms to the phosphate atom. These distances are much smaller than what has been found in other well studied nonmetallo-phopshatases. Thus, the study finds a novel mechanism of enzymic phosphotransfer in PAP mediated catalysis.

Abstract Human prostatic acid phosphatase (hPAP) was the first laboratory parameter used for prostate cancer diagnosis, whereas the mechanisms behind the androgen regulation and tissue-specific expression of this prostate epithelium-specific differentiation antigen are not yet clear. In this study, a transient transfection model and transgenic animal model have been set up for functional analysis of the promoter and first intron region of the hPAP gene. The promoter constructs covering the region-734/+467 of the gene were functional in both prostatic and nonprostatic cells. Although hPAP constructs included two putative AREs with in vitro AR-binding ability at -178 and +336, androgen treatment had little effect on the promoter activity of the gene in transiently transfected cells. The hPAP fragment -734/+467 could trigger the expression of the CAT reporter gene and restrict the expression mainly in the prostates of transgenic mice. The DNA-binding site with the sequence GAAAATATGATA of a regulatory protein involved in prostate-specific and androgen receptor-dependent gene expression was identified from rPB promoter. The exact same 12 bp sequence was found in the first intron +1144/+1155 of the hPAP gene. Five homologous sequence, A, B, C, D and E, were located in the -734/+467 region of the hPAP gene, where site C and E could bind the regulatory protein in EMSA. Deletion of site C decreased the transcriptional activities significantly compared to those of corresponding wild-type constructs in LNCaP cells when androgens were present. Deletion of site E or both sites D and E increased the promoter activity in LNCaP when androgens were absent. In conclusion, androgens could not directly regulate hPAP expression via receptor-binding to the AREs in LNCaP cells. The promoter and first intron fragment -734/+467 of the hPAP gene could direct and restrict the gene expression mainly in prostate epithelium. A prostatic regulatory protein binds to multiple sites with the GAAAATATGATA or homologous sequences along the regulatory areas of the hPAP gene with different affinities, modulating the prostate-specific expression of the gene in a bidirectional manner, depending on the hormone status.