Constitutive activation of the androgen receptor by a point mutation in the hinge region: a new mechanism for androgen-independent growth in prostate cancer

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.     

Identification of steroid derivatives that function as potent antiandrogens

We have hypothesized that some steroid derivatives bind to the androgen receptor (AR) with very low androgenic activity and therefore potentially function as better AR antagonists than clinically used antiandrogens, such as flutamide. Indeed, we previously found such a compound, 3beta-acetoxyandrosta-1,5-diene-17-one ethylene ketal (ADEK), with some estrogenic activity. Here we report the identification of 2 additional steroid derivatives, 3beta-hydroxyandrosta-5,16-diene (HAD) and androsta-1,4-diene-3,17-dione-17-ethylene ketal (OAK), as new potent antiandrogens. Like ADEK, HAD and OAK could interrupt androgen binding to the AR and suppress both dihydrotestosterone- and androstenediol-induced transactivations of wild-type and mutant ARs in prostate cancer cells. These 2 compounds also inhibited prostate-specific antigen expression in LNCaP as well as growth of different AR-positive prostate cancer cell lines stimulated by androgen. Significantly, HAD and OAK had only marginal agonist effects, as compared to hydroxyflutamide. More importantly, in contrast to ADEK, OAK was shown to possess marginal estrogenic activity. These results strengthen our hypothesis and suggest that selective steroid derivatives could be potent antiandrogenic drugs with less unfavorable effects for the treatment of prostate cancer.     

Modulation of androgen receptor transactivation by gelsolin: a newly identified androgen receptor coregulator

The partial agonist effect of antiandrogens has been well documented, and such effect is amplified by derived mutant androgen receptors (ARs) in prostate cancer cells. Here we report the identification of gelsolin (GSN) as an AR-associated protein. Hydroxyflutamide (HF), as well as androgens, can promote the interaction between AR and GSN in a dose-dependent manner. GSN interacts with AR DNA-binding domain and ligand-binding domain via its COOH-terminal domain. Immunolocalization studies show that GSN interacts with AR during nuclear translocation. Functional analyses additionally demonstrate that GSN enhances AR activity in the presence of either androgen or HF. Two peptides representing partial regions of the AR DNA-binding domain and the ligand-binding domain can block the GSN-enhanced AR activity. The expression of GSN is enhanced in LNCaP cells, LNCaP xenografts, and human prostate tumors after androgen depletion. Increasing expression of GSN enhances the AR activity in the presence of HF. Together, these data suggest that the weak androgenic effect of HF may be amplified by increasing the amount of GSN after androgen ablation treatment. Therefore, blockage of the interaction between AR and GSN could become a potential therapeutic target for the treatment of prostate cancer.     

Activation of mitogen-activated protein kinase pathway by the antiandrogen hydroxyflutamide in androgen receptor-negative prostate cancer cells

Whereas hydroxyflutamide (HF) has been used as an antiandrogen to block androgen-stimulated prostate tumor growth, the antiandrogen withdrawal syndrome that allows antiandrogens to stimulate prostate tumor growth still occurs in many patients treated with androgen ablation therapy. This was previously explained by mutations in the androgen receptor (AR) and/or modulation from AR coregulators, so that HF becomes an AR agonist. Using immunohistochemical analysis, we analyzed four prostate cancer patients undergoing androgen ablation therapy with flutamide and compared their phospho-extracellular signal-regulated kinase 1/2 levels in prostate cancer biopsies before receiving HF and after experiencing disease progression while taking HF. We found a significant increase of activated mitogen-activated protein (MAP) kinase in prostate tumors from patients receiving HF during androgen ablation therapy. In vitro studies showed that HF induced a rapid activation of the Ras/MAP kinase pathway in human prostate cancer DU145 cells which lack the AR, as well as in PC-3AR2 and CWR22 cells which express the AR. Cycloheximide failed to inhibit this activation, but both AG1478, an inhibitor of the epidermal growth factor receptor (EGF-R), and an EGF-R-neutralizing antibody blocked this HF-mediated activation of MAP kinase, which suggests that the activation of Ras/MAP kinase by HF is a membrane-initiated, non-AR-mediated, and nongenomic action. The consequence of this activation may result in increasing cell proliferation and cyclin D1 expression. This raises a concern for using HF in the complete-androgen-ablation therapy in prostate cancer treatment and provides a possible pathway that might contribute to the HF withdrawal syndrome.     

The adrenal androgen androstenediol is present in prostate cancer tissue after androgen deprivation therapy and activates mutated androgen receptor

Despite an initial response to androgen deprivation therapy, prostate cancer (PCa) progresses eventually from an androgen-dependent to an androgen-independent phenotype. One of the mechanisms of relapse is antiandrogen withdrawal phenomenon caused by mutation of 877th amino acid of androgen receptor (AR). In the present study, we established a method to measure the concentration of androstenediol (adiol) in prostate tissue. We found that adiol maintains a high concentration in PCa tissue even after androgen deprivation therapy. Furthermore, adiol is a stronger activator of mutant AR in LNCaP PCa cells and induces more cell proliferation, prostate-specific antigen (PSA) mRNA expression, and PSA promoter than dihydrotestosterone (DHT). Because antiandrogen, bicalutamide, blocked adiol activity in LNCaP cells, it was suggested that adiol effect was mediated through AR. However, high concentration of bicalutamide was necessary to block completely adiol activity. These effects were specific to LNCaP cells because adiol had less effect in PC-3 PCa cells transfected with wild-type AR than DHT and had similar effect in PC-3 cells transfected with mutant AR. The mechanism that adiol activates mutant AR in LNCaP cells did not result from the increased affinity to mutant AR or from AR's association with coactivator ARA70. However, low concentration of adiol induced more AR nuclear translocation than DHT in LNCaP cells and not PC-3 cells transfected with AR. These results indicate that adiol may cause the progression of PCa even after hormone therapy.     

Xenoestrogen action in prostate cancer: pleiotropic effects dependent on androgen receptor status

Androgen is critical for prostate development, growth, and survival. Therapies for advanced prostate cancer aim to block androgen receptor (AR) action. However, recurrent tumors ultimately arise, which harbor restored AR activity. One mechanism of such reactivation occurs through AR mutations, rendering the receptor responsive to noncanonical ligands. We have shown previously that a known xenoestrogen, bisphenol A (BPA), activates a tumor-derived AR mutant (T877A), leading to androgen-independent prostate cancer cell proliferation. Here, we show that BPA cooperates with androgen to activate AR-T877A as shown by both reporter assays and increased levels of prostate-specific antigen expression. Further investigations using both yeast and mammalian model systems revealed that multiple AR alleles are responsive to BPA, thus expanding the potential influence of xenoestrogens on prostate cancer. Moreover, in vitro radioligand binding assay revealed that BPA alters 5alpha-dihydrotestosterone binding to AR-T877A likely through noncompetitive inhibition. We also show that higher concentrations of BPA block proliferation of AR-positive, androgen-dependent prostate adenocarcinoma cells (LNCaP and LAPC-4), with a more modest inhibitory effect on androgen-independent cells (22Rv-1). By contrast, AR-negative prostate cancer cells failed to show growth inhibition after exposure to high BPA dose. Together, these data show that BPA can serve as a potential "hormone sensitizer" of the mutant ARs present in advanced prostate adenocarcinomas, thereby possibly contributing toward therapeutic relapse in advanced prostate cancer patients and supporting the notion that nonsteroidal environmental compounds can alter the function of nuclear receptor complexes.     

The antiandrogen bicalutamide activates the androgen receptor (AR) with a mutation in codon 741 through the mitogen activated protein kinase (MARK) pathway in human prostate cancer PC3 cells

The objective of this study was to assess the effect of antiandrogen on the activation of mutated androgen receptor (AR) and its signaling pathway in prostate cancer. We transfected the AR gene with a point mutation at codon 741 (tryptophan to leucine; W741L) into human androgen-independent prostate cancer PC3 cells lacking the expression of AR, and established PC3 cells overexpressing mutant type AR (PC3/W741L). Changes in the phenotype in these cells were compared to those in PC3 cells transfected with wild- type AR (PC3/Wild) and control vector alone (PC3/Co). There was no significant differences in the growth among PC3/Co, PC3/Wild and PC3/W741L cells. A transactivation assay using these cells showed that bicalutamide activated W741L mutant type AR, but not wild-type AR, while hydroxyflutamide failed to activate either type of ARs. Treatment with specific inhibitors of the MAPK or STST3 pathway (UO126 or AG490, respectively), in contrast to treatment with the Akt pathway inhibitor LY294002, significantly inhibited the dihydrotestosterone-induced activation of both wild-type and mutant ARs; however, activation of W741L mutant AR by bicalutamide was significantly inhibited by treatment with UO126, in contrast to treatment with AG490 or LY294002. Furthermore, treatment of PC3/W741L with bicalutamide, in contrast to treatment with hydroxyflutamide, resulted in significant upregulation of phosphorylated p44/42 MAPK. These findings suggest that the MAPK pathway might be involved in the activation of the AR with a point mutation at codon 741 induced by treatment with the antiandrogen bicalutamide.     

Interactions of abiraterone, eplerenone, and prednisolone with wild-type and mutant androgen receptor: a rationale for increasing abiraterone exposure or combining with MDV3100

Prostate cancer progression can be associated with androgen receptor (AR) mutations acquired following treatment with castration and/or an antiandrogen. Abiraterone, a rationally designed inhibitor of CYP17A1 recently approved for the treatment of docetaxel-treated castration-resistant prostate cancer (CRPC), is often effective, but requires coadministration with glucocorticoids to curtail side effects. Here, we hypothesized that progressive disease on abiraterone may occur secondary to glucocorticoid-induced activation of mutated AR. We found that prednisolone plasma levels in patients with CRPC were sufficiently high to activate mutant AR. Mineralocorticoid receptor antagonists, such as spironolactone and eplerenone that are used to treat side effects related to mineralocorticoid excess, can also bind to and activate signaling through wild-type or mutant AR. Abiraterone inhibited in vitro proliferation and AR-regulated gene expression of AR-positive prostate cancer cells, which could be explained by AR antagonism in addition to inhibition of steroidogenesis. In fact, activation of mutant AR by eplerenone was inhibited by MDV3100, bicalutamide, or greater concentrations of abiraterone. Therefore, an increase in abiraterone exposure could reverse resistance secondary to activation of AR by residual ligands or coadministered drugs. Together, our findings provide a strong rationale for clinical evaluation of combined CYP17A1 inhibition and AR antagonism.     

Collocation of androgen receptor gene mutations in prostate cancer.

Consistent with both the development of the normal prostate gland and prostate tumorigenesis being dependent on testicular androgens, targeting the androgen-signaling axis (i.e., androgen ablation therapy) remains the predominant treatment regime for patients with metastatic prostate cancer. Although there is a very good initial response to androgen ablation, these treatments are essentially palliative. Recent evidence suggests that treatment failure may not result from a loss of androgen signaling but, rather, from the acquisition of genetic changes that lead to aberrant activation of the androgen-signaling axis. A consistent finding is that androgen receptor (AR) gene mutations, present in metastatic prostate cancer and in human prostate cancer cell lines as well as in xenograft and other animal models, result in decreased specificity of ligand-binding and inappropriate receptor activation by estrogens, progestins, adrenal androgens, glucocorticoids and/or AR antagonists. Because a significant proportion of missense mutations in the AR gene reported in prostate cancer collocate to the signature sequence and AF-2, two discrete regions of the ligand-binding domain critical for androgen signaling, we recently proposed that collocation of mutations identified in prostate cancer would identify additional regions of the AR important in receptor function. This approach led to the identification of a four-amino acid region at the boundary of the hinge and ligand-binding domains of the receptor that forms half of a potential protein-protein binding site. AR gene mutations have also been identified that collocate to areas in the DNA-binding domain, to the NH(2)-terminal transactivation domain, and to the hinge region in prostate tumors. In nearly every case, missense mutations in the AR gene identified in prostate cancer that collocate to discrete regions of the receptor contribute to altered androgen signaling and provide a potential mechanism to explain the reemergence of tumor growth during the course of hormone ablation therapies.     

Mutation and microsatellite instability analysis of the androgen receptor gene in human prostate cancer

To investigate the mechanism for the development of human prostate cancer, examination was made of structural abnormality of the androgen receptor (AR) gene in 29 human prostate cancer. AR gene mutations from exons A to H were examined by PCR-SSCP and microsatellite instability analysis using (CAG)n and (GGN)n polymorphic markers in AR gene exon A. A point mutation was found in the exon D hormone-binding domain of AR leading to substitution of glutamine (GAG) for wild-type arginine (CGG) at codon 629 in 1 (3.4%) hormone-independent stage D2 patient. Microsatellite instability was detected in 5 of the 27 (18.5%) patients, 1 of 6 (16.7%) hormone-independent stage D2 and 4 of 21 (19.0%) hormone-dependent and non-treated prostate cancer patients. AR mutations may possibly be involved in the transition from androgen-dependent to independent stages during androgen ablation therapy.     

Two percent of Finnish prostate cancer patients have a germ-line mutation in the hormone-binding domain of the androgen receptor gene

Mutations of the androgen receptor (AR) gene have been reported in prostate cancer, usually from tumor tissue specimens from late-stage, androgen-independent cancer. Occasionally, germ-line mutations have been found, but a link between AR mutations and predisposition to human prostate cancer has not been firmly established. Recently, two independent studies reported the same germ-line mutation at codon 726 in exon E (CGC to CTC) in two apparently unrelated Finnish prostate cancer patients. This arginine to leucine substitution was reported to alter the transactivational specificity of the AR protein. In the present study, the R726L mutation was analyzed by allele-specific oligohybridization in DNA specimens from 418 consecutive prostate cancer patients who reported a negative family history (sporadic group) and from 106 patients with a positive family history (hereditary group). The population frequency of the R726L mutation in blood donors was 3 of 900 (0.33%). In contrast, eight (1.91%) mutations (odds ratio = 5.8; P = 0.006) were found in the sporadic group, and two (1.89%) mutations were found in the hereditary group (odds ratio = 5.8; P = 0.09). Suggestive evidence of the segregation of the mutation with prostate cancer was seen in these two families. The present study indicates that the R726L substitution in the AR may confer an up to 6-fold increased risk of prostate cancer and may contribute to cancer development in up to 2% of Finnish prostate cancer patients. These results warrant additional large-scale studies of the significance of rare mutations and polymorphisms in candidate genes along the androgen signaling pathway as risk factors for prostate cancer.     

Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor

This study discovered that Id-1 expression in androgen-dependent prostate cancer decreased immediately after androgen deprivation but increased after longer androgen deprivation both in vivo and in vitro. Id-1 expression in androgen-independent LNCaP cells was about 6 fold as that in their parental cells. As was the case with LNCaP cells, when androgen receptor (AR) was introduced into AR-negative PC-3 cells, dihydrotestosterone inhibited while flutamide increased Id-1 expression. Thus, Id-1 expression in androgen-dependent prostate cancer was negatively regulated by androgen in a receptor-dependent way. The re-increased Id-1 might partially contribute to the emergence of androgen-independent prostate cancer after longer androgen deprivation therapy.