Homologous cell systems for the study of progression of androgen-dependent prostate cancer to castration-resistant prostate cancer

2013-08-22Despite immense research progress made in recent years, the recurrence of castration-resistant prostate cancer (CRPC) still remains poorly understood. Androgen receptor (AR), an essential player during the differentiation and maintenance of normal prostate as well as the initiation of prostate cancer, has been widely accepted to be the "Partner-in-Crime" even in the stage of CRPC, mediating and being regulated by numerous factors and signaling pathways. Therefore, to decipher the role of AR and its regulation in contributing to the occurrence of CRPC has become a priority topic in the prostate cancer (PCa) research. ❧ This dissertation describes establishment of a study system composed of murine PCa cell lines derived from tumors of the conditional Pten deletion mouse model of prostate adenocarcinoma obtained at two distinct phases of the disease, namely androgen-dependent (AD) growth phase and the castration-resistant (CR) growth phase. We have characterized five mouse PCa cell lines - E2/E4 and E8 from two separate AD tumors and cE1/cE2 from one CR tumor. All the cell lines manifest biallelic deletion of the Pten gene, corresponding to epithelial origin since the Cre/LoxP system used in the modeling is targeted to prostate epithelium. Analyses of various molecular expressions as well as morphology of these cells suggest a degree of epithelial-mesenchymal transition (EMT) in E2/E4 but not much in E8, which possesses increased epithelial phenotype. The cell lines from the CRPC tumor generally display epithelial characteristics and demonstrate significantly better growth capacity as compared to E-series in the absence of androgen, although sensitivity to supplemented androgen is retained. All the cell lines established are able to induce in vivo tumor growth in immunodeficient mice. However, while E8 and cE1/cE2 give rise to adenocarcinomas, E2 and E4 yields tumors that are akin to sarcomatoid carcinoma expressing protein makers of both epithelial and stromal cells. In this regard, there is a fairly good correlation between the in vitro and in vivo properties. ❧ Among the multiple mechanisms by which AR activity may be aberrantly regulated, we have focused our studies of the cell lines to determine the status of the androgen receptor splice variants (AR-Vs). We have identified a set of novel mouse AR-Vs (termed as mAR-Vabc) in these homologous mouse PCa cell systems. Their expression is present in all five cell lines and is generally up-regulated in response to androgen ablation. The relative proportion of these mAR-Vs varies with respect to the stage of the disease from which the cell lines originated. In cE-series, mAR-Vb and mAR-Vc are more abundant than that in E-series. However, mAR-Va displays an opposite profile of expression. The mRNA of these mAR-Vs is detected in the normal prostates; however, their abundance relative to the full-length AR (mAR-FL) is significantly increased in the tumor tissues. Interestingly, among the normal prostates, aged prostates harbor the highest levels. These findings implicate a potential role of these mAR-Vs in both etiological and normal biological processes. Structurally, mAR-Va is similar to mAR-FL except lacking the ligand binding domain, and mAR-Vb and AR-Vc, differing in C-terminal sequences, basically harbor the N-terminal transcription activation domain (NTD) of mAR-FL. Functionally, as tested via reporter gene transcription assays in non-prostate heterologous COS-1 cells, all of these variants appear to lack the ability to activate AR response elements whether androgen is supplemented or not. However, each one, to a variable extent, can suppress the activity of AR-FL in co-transfection assays and in the presence of a low level of androgen. These variants appear to serve like transduced NTD "decoy" molecules that were reported before. ❧ Based on these findings, it is speculated that AR, that is necessary for both AD and CR prostatic tumors may be negatively regulated by production of the AR natural decoys to maintain a balance in overall AR functions in the activated and reactivated states. These decoys may also have a similar biological role in suppressing or down-regulating AR function in the ageing process. These are exciting clues and deserve to be further investigated. A critically important topic then would be to determine the mechanisms by which abnormal splice variants are formed. Once this mechanism is unveiled, it might be possible to design small molecular inducers for increased expression of these decoys for potential therapy or prevention of prostate cancer, in a unique but still natural manner