Androgen receptor (AR) mutations that modify both the ligand binding and the transactivation capacities of the AR represent one of the mechanisms involved in the transition of prostate cancer (PCa) from androgen-dependent to androgen-independent growth. We use a yeast-based functional assay to detect and analyze mutant ARs in PCa. We report the detection of 2 different mutant ARs within the same metastatic tumour sample harvested in a patient with advanced PCa who had escaped androgen deprivation. Concomitantly to the widely described T877A mutant AR, we identified an additional double mutant AR harboring the nonsense mutation Q640Stop just downstream the DNA binding domain together with the T877A point mutation. This type of mutation, which leads to a c-terminal truncated AR, has not been described yet in PCa. Using luciferase reporter assays we demonstrated that this truncated AR exhibited constitutive transactivation properties. In conclusion, our data suggest that mutation-induced constitutive activation of the AR could be a mechanism used by PCa cells to escape androgen deprivation. © 2003 Wiley-Liss, Inc. Key words: androgen receptor mutations; prostate cancer; androgen independence; constitutive transactivationProstate cancer (PCa) is the most frequently diagnosed malignancy in men and the second leading cause of cancer deaths in Western countries. As prostate cancer growth depends on androgens, the standard first line therapy for advanced PCa consists of decreasing the plasma concentration of testicular testosterone by medical or surgical castration. 1,2 This androgen ablation therapy is eventually combined to anti-androgens to further block the effects of androgens produced from adrenal precursors. The response to such androgen ablation is high and provides subjective relief to more than 80% of patients. Unfortunately, the benefit of this therapy is only transitory lasting some months or years, with a median duration of 12-18 months, and all patients will eventually relapse with tumour cells whose growth escapes the androgen deprivation. Although these relapsed tumours are clinically androgen-independent, many lines of evidence indicate that the androgen receptor (AR) signaling pathway remains active and may contribute to the escape to androgen ablation. 3 In this regard, the amplification and the overexpression of the AR gene or the stabilization of the AR may allow PCa cells to continue androgendependent growth even in very low levels of androgens left in serum after castration. 4 -6 Also, the cross-talk with the mitogenactivated protein kinase (MAPK) or the Akt/protein kinase B phosphorylation pathways 7 and the altered expression of AR coactivator proteins 8 may lead to the activation of AR in a ligandindependent manner. Finally, mutations in the ligand binding domain of the AR that expand the specificity or the affinity of the receptor to other hormones may allow it to respond to different steroids as well as anti-androgens. 9 We have described previously a yeast-based functional assay to simultane...

Advanced prostate cancer (PCa) has emerged as a public health concern due to population aging. Although androgen deprivation has proven efficacy in this condition, most advanced PCa patients will have to face failure of androgen deprivation as a treatment. Mutations in the androgen receptor (AR) from tumor cells have been shown to induce androgen independency both in PCa cell lines and in the clinic. We have investigated the molecular events leading to androgen independency in the 22Rv1 cell line, a commonly used preclinical model of PCa. Besides AR mutants that have been described so far, including nonsense mutations, recent data have focused on AR pre-mRNA aberrant splicing as a new mechanism leading to constitutively active truncated AR variants. In this article, we describe two novel variants arising from aberrant splicing of AR pre-mRNA, characterized by long mRNA transcripts that encode truncated, constitutively active proteins. We also describe several new nonsense mutants that share ligand independency and transcriptional activity. Finally, we show that alongside these mutants, 22Rv1 cells also express a mutant AR lacking exon 3 tandem duplication, a major feature of this cell line. By describing unreported AR mutants in the 22Rv1 cell line, our data emphasize the complexity and heterogeneity of molecular events that occur in preclinical models, and supposedly in the clinic. Future work on the 22Rv1 cell line should take into account the concomitant expression of various AR mutants.

The androgen receptor (AR) is a ligand-activated transcription factor that displays genomic actions characterized by binding to androgen-response elements in the promoter of target genes as well as nongenomic actions that do not require nuclear translocation and DNA binding. In this study, we report exclusive cytoplasmic actions of a splicing variant of the AR detected in a metastatic prostate cancer. This AR variant, named AR23, results from an aberrant splicing of intron 2, wherein the last 69 nucleotides of the intronic sequence are retained, leading to the insertion of 23 amino acids between the two zinc fingers in the DNA-binding domain. We show that the nuclear entry of AR23 upon dihydrotestosterone (DHT) stimulation is impaired. Alternatively, DHT-activated AR23 forms cytoplasmic and perinuclear aggregates that partially colocalize with the endoplasmic reticulum and are devoid of genomic actions. However, in LNCaP cells, this cytoplasmic DHT-activated AR23 remains partially active as evidenced by the activation of transcription from androgen-responsive promoters, the stimulation of NF-kappaB transcriptional activity and by the decrease of AP-1 transcriptional activity. Our data reveal novel cytoplasmic actions for this splicing AR variant, suggesting a contribution in prostate cancer progression.

Androgen receptor (AR) signaling pathway remains the foremost target of novel therapeutics for castration-resistant prostate cancer (CRPC). However, the expression of constitutively active AR variants lacking the carboxy-terminal region in CRPC may lead to therapy inefficacy. These AR variants are supposed to support PCa cell growth in an androgen-depleted environment, but their mode of action still remains unresolved. Moreover, recent studies indicate that constitutively active AR variants are expressed in primary prostate tumors and may contribute to tumor progression. The aim of this study was to investigate the impact of constitutively active AR variants on the expression of tumor progression markers. N-cadherin expression was analyzed in LNCaP cells overexpressing the wild type AR or a constitutively active AR variant by qRT-PCR, Western blot and immunofluorescence. We showed here for the first time that N-cadherin expression was increased in the presence of constitutively active AR variants. These results were confirmed in C4-2B cells overexpressing these AR variants. Although N-cadherin expression is often associated with a downregulation of E-cadherin, this phenomenon was not observed in our model. Nevertheless, in addition to the increased expression of N-cadherin, an upregulation of other mesenchymal markers expression such as VIMENTIN, SNAIL and ZEB1 was observed in the presence of constitutively active variants. In conclusion, our findings highlight novel consequences of constitutively active AR variants on the regulation of mesenchymal markers in prostate cancer.