Abstract
Androgen ablation inhibits androgen receptor (AR) activity and is as an effective treatment for advanced prostate cancer (PCa). Invariably, PCa relapses in a form resistant to further hormonal manipulations. Although this stage of the disease is androgen-refractory, or androgen depletion-independent (ADI), most tumors remain AR-dependent through aberrant mechanisms of AR activation. We employed the LNCaP/C4-2 model of PCa progression to study AR activity in androgen-dependent and ADI PCa cells. In this report, we show that the AR is transcriptionally inactive in androgen-dependent LNCaP cells in the absence of androgens. However, in ADI C4-2 cells, the AR displays a high level of constitutive, androgen-independent transcriptional activity. To study the mechanisms of ligand-dependent and ligand-independent AR activation in these AR-expressing cells, we generated a reporter system based on swapping the DNA binding domain of the AR with the DNA binding domain of the yeast Gal4 transcription factor. In androgen-dependent PCa cells, the well characterized C-terminal AR activation function-2 (AF-2) domain was critical for strong, ligand-dependent activity. Conversely, in ADI PCa cells, constitutive, ligand-independent AR activity was AF-2-independent but instead dependent on N-terminal AR domains. Importantly, the ligand- and AF-2-independent mode of AR activation observed in ADI PCa cells was completely resistant to the antiandrogen, bicalutamide. Our data thus demonstrate that the AR can inappropriately activate transcription in ADI PCa cells via mechanisms that are resistant to castration and AR antagonism, the two modes of androgen ablation used to treat advanced PCa.
Highlights
Targeted androgen receptor (AR) inhibition decreases prostate specific antigen (PSA) expression, cell proliferation, and survival in various cell-based models of androgen depletion-independent (ADI) prostate cancer (PCa) (9 –12). These findings suggest that ADI PCa cells continue to proliferate and survive through aberrant mechanisms of AR activation, and the AR remains a valid target for therapy
We observed a decrease in PSA mRNA expression when we compared C4-2 cells transfected with AR-targeted siRNA to cells transfected with non-targeted siRNA (Fig. 1B)
We designed a novel Gal4-based reporter system, which we demonstrated was completely autonomous of endogenous AR
Summary
Relieves this negative regulation on AR AF-1 and allows the AR AF-2 domain to adopt a structure that can bind FXXLF or LXXLF motifs present in AR coactivators such as ARA70 and members of the p160 coactivator family [14, 15] Both the AF-1 and AF-2 modules are able to recruit coregulatory proteins to genomic androgen response elements (AREs) encoded in the promoter and enhancer regions of target genes [16]. We identified transcriptionally active domains in the AR NTD and found that transactivation unit (TAU)-1 was important for full ligand-dependent as well as ligand-independent AR activity in androgen-dependent and ADI cells Together, these findings suggest that therapies targeted to the AR CTD may lack long term effectiveness due to an inability to inhibit AF-2-independent AR activity. AR NTD modules such as TAU-1 may be more important and relevant targets for the development of novel anti-AR strategies in androgen-dependent and ADI PCa
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