The Different Expression And Function Of Androgen Receptor Isoforms In Prostate Cancer

BackgroundProstate cancer (PCa) is one of the most common malignant tumor in men. AR plays important role in the development and progression of prostate cancer. A new AR isoform protein, identified as an NH2-terminally truncated protein of AR-B was first described in human genital skin fibroblasts in 1993 by Carol. The AR-A, migrates with an apparent mass of 87 kDa, appears to derive from internal translation initiation at methionine-188 in the AR open-reading frame, and usually constitutes 20% or less of the immunoreactive AR present. The B form migrates with an apparent mass of 110 kDa and constitutes more than 80% of the immunoreactive receptor in most cell types. Within nuclear receptor superfamily, the two different isoforms of the AR bear the most striking similarity to the A and B forms of the PR. AR and PR appear to be unique in that a single gene gives rise to two distinct isoforms which differ in the N-terminal amino acids. In case of PR, PR-A and PR-B are different in transgenic functions as previous report. There are also reports that high expression of PR-A are related to human breast cancers and endometrial cancer.Researches on AR isoforms are far less explored than PR isoform till now. Catalano discovered that neoplastic colon tissue shows a characteristic loss of expression of the AR-B isoform, while AR-A expression remains unaltered, compared to healthy colon mucosa, either at mRNA or protein level. Liegibel's study demonstrated that AR-A lacks the ability to stimulate cell proliferation in cultured human bone cells and genial skin fibroblasts possibly due to reduced binding of AR co-activating proteins to the truncated N-terminal transactivation domain rather than due to impaired stability or low expression of the AR-A isoform.Despite the importance of AR in normal prostate development and prostate cancer, it is still unknown whether androgen actions in prostate are mediated by AR-A and/or AR-B. Given the different transcriptional activities of the two AR isoforms in cultured human bone cells and genial skin fibroblasts, analysis of their expression in different prostate tissues and different function in prostate cancer cell is crucial to understand the roles of AR isoforms in prostate cancer.Objective This study was designed to investigate AR isoforms protein and AR mRNA expression in normal prostate tissue, BPH and PCa by means of western blotting, Dual immunofluorescent staining and Realtime-PCR, and further investigate the different function in prostate cancer cell by means of transfection of AR-A vector to find out whether the AR isoforms was associated with the development and the progress of the PCa.Methods1. Western blotting, Realtime-PCR and Dual immunofluorescent staining were used to dectecte the expression of AR isoforms protein and mRNA in normal prostate, BPH and PCa tissues.2. The AR-A expression plasmid were recombinated from pEGFP-AR. AR-defective prostate cancer PC3 cells was chosen as the platform for transfection. The expression of the AR-A and AR-B expression levels were detected by Western blotting. They were divided into three groups:control group, AR-A group and AR group. In the control group PC3 cells were untreated. In the blank group PC3 cells were transfected with blank pEGFP vector. In the test group PC3 cells were ransfected with pEGFP-AR-A and pEGFP-AR. Transwell and CCK8 proliferation were done to test the different fuction of AR-A and AR-B.ResultsIn normal prostate and BPH tissues, AR-A and AR-B were co-expressed within the same cells at a stable proportion, implicating both isoforms involved in androgen action. And the expression in these two kinds of tissues got no statistically difference. In PCa, however, there was an increased expression of AR-A,2.8～2.38 times as compared to BPH and normal prostate, and AR-B increased by 1.47～1.37 times in PCa. Thus the AR-A/AR-B ratio increased by 1.92～1.75 times. Statistically we found no relationship between the expression level of AR-A expression and the patients'age. However Gleason score are related to higher expression level of AR-A, AR-B and AR-A/AR-B proportion (p<0.05).Dual immunofluorescent staining was also applied in sections of different prostate tissues to exploring the expression of AR isoforms. AR-A expression were higher in PCa than BPH and normal prostate. Clinic stage and pathological stage are also related to higher expression level of AR-A. These data were well consistent with the result from western blotting. The differential expression of AR-mRNA was also checked by means of Realtime-PCR. The expression of AR-A in PCa was increased by 10.65～11.93 times as compared to normal prostate and BPH. Statistically we found no relationship between the expression level of AR-A expression and the patients' age. However Gleason score are related to higher expression level of AR mRNA.Occasionally we separated the nuclei and cytosol protein from normal prostate tissue and LNCaP cancer cells and noticed that both AR-A and AR-B were present in cytosol, while only AR-B, but not AR-A was present in cell nuclear. To further investigate this phenomenon, we culture the LNCaP cells starving from androgen for 5 days and regain androgen for 2h, the AR-A isoform appeared in cell nuclei upon hormone stimulation. This phenomenon of differential distribution and translocation activity demonstrated that AR-A should be a biologically active androgen receptor and different from AR-B. We also did co-immunoprecipitation and western blotting of prostate tissues demonstrated that AR-B and AR-A can not form heterodimers. This could validate the different distribution and translocation activity of AR-A from AR-B. If AR-A and AR-B can form heterodimers upon androgen activation, their distribution would be alike, rather than different.For further study the biological activity of AR-A, we manufacture pEGFP-AR and pEGFP-AR-A vector which could translate AR and AR-A. PC3 cells were transfected with the pEGFP-AR-A vector and pEGFP-AR. Tumor Invasion Assay outcomes demonstrated that AR-A and AR decrease the migration and invasion of PC3 in vitro. Cell proliferation experiment showed that AR-A decreased proliferation ratio and promoted the apoptosis of PC3 cells. The results suggested that AR-A can effectively inhibit the growth and proliferation PC3 cells in vitro.Conclusion:AR-A may play some role in the development and progress of the PCa. The over expression of the AR-A in PCa and the decrease of migration and invasion might be some kind of self-protect in PCa progression. So increasing the activity of AR-A might be a new therapeutic strategy for the PCa. It has potential value in targeting gene therapy of prostate cancer.