Androgen receptor protein is down-regulated by basic fibroblast growth factor in prostate cancer cells

Interactions between polypeptide growth factors and the androgen receptor (AR) are important for regulation of cellular events in carcinoma of the prostate. Basic fibroblast growth factor (bFGF), the prototype of heparin-binding growth factors, and the AR are commonly expressed in prostate cancer. bFGF diminished prostate-specific antigen protein in the supernatants of androgen-stimulated human prostate cancer cells LNCaP by 80%. In the present study, we asked whether the bFGF effect on prostate-specific antigen is preceded by action on AR expression. LNCaP cells were treated with bFGF and AR protein expression was determined by immunoblotting and ligand binding assay. bFGF down-regulated AR protein in a dose-dependent manner showing a maximal effect at 50 ng ml−1both in the presence or absence of dihydrotestosterone. Down-regulation of AR protein expression occurred already after 8 h of bFGF treatment and a maximal inhibition was observed 24 h after addition of bFGF to culture media. As AR expression can be reduced by an increase in intracellular calcium levels, we investigated whether the bFGF effect on AR protein is mediated by this mechanism. Calcium release from intracellular stores and store-operated calcium influx after treatment with either bFGF or calcium ionophore A 23187 were measured by single cell fluorescence technique. The ionophore A 23187 was able to induce calcium influx and an increase in cytoplasmic calcium concentration in LNCaP cells. In contrast, bFGF was incapable of eliciting a similar effect. In contrast to AR protein, AR mRNA levels were not affected by bFGF as shown by semiquantitative reverse transcription polymerase chain reaction. In summary, these studies show that bFGF is a potent negative regulator of AR protein expression in the human prostate cancer cell line LNCaP. © 2000 Cancer Research Campaig

Secreted Frizzled-related protein-1 is a negative regulator of androgen receptor activity in prostate cancer

Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/β-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised β-catenin or β-catenin shRNA, suggesting it does not involve Wnt/β-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca2+ and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of β-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens

<p>Abstract</p> <p>Background</p> <p>A crucial event in Prostate Cancer progression is the conversion from a hormone-sensitive to a hormone-refractory disease state. Correlating with this transition, androgen receptor (AR) amplification and mutations are often observed in patients failing hormonal ablation therapies. β-Catenin, an essential component of the canonical Wnt signaling pathway, was shown to be a coactivator of the AR signaling in the presence of androgens. However, it is not yet clear what effect the increased levels of the AR could have on the Wnt signaling pathway in these hormone-refractory prostate cells.</p> <p>Results</p> <p>Transient transfections of several human prostate cancer cell lines with the AR and multiple components of the Wnt signaling pathway demonstrate that the AR overexpression can potentiate the transcriptional activities of Wnt/β-Catenin signaling. In addition, the simultaneous activation of the Wnt signaling pathway and overexpression of the AR promote prostate cancer cell growth and transformation at castration levels of androgens. Interestingly, the presence of physiological levels of androgen or other AR agonists inhibits these effects. These observations are consistent with the nuclear co-localization of the AR and β-Catenin shown by immunohistochemistry in human prostate cancer samples. Furthermore, chromatin immunoprecipitation assays showed that Wnt3A can recruit the AR to the promoter regions of Myc and Cyclin D1, which are well-characterized downstream targets of the Wnt signalling pathway. The same assays demonstrated that the AR and β-Catenin can be recruited to the promoter and enhancer regions of a known AR target gene PSA upon Wnt signaling. These results suggest that the AR is promoting Wnt signaling at the chromatin level.</p> <p>Conclusion</p> <p>Our findings suggest that the AR signaling through the Wnt/β-Catenin pathway should be added to the well established functional interactions between both pathways. Moreover, our data show that via this interaction the AR could promote prostate cell malignancy in a ligand-independent manner.</p

Switch from antagonist to agonist of the androgen receptor blocker bicalutamide is associated with prostate tumour progression in a new model system

Advanced prostate cancer is treated by androgen ablation and/or androgen receptor (AR) antagonists. In order to investigate the mechanisms relevant to the development of therapy-resistant tumours, we established a new tumour model which closely resembles the situation in patients who receive androgen ablation therapy. Androgen-sensitive LNCaP cells were kept in androgen-depleted medium for 87 passages. The new LNCaP cell subline established in this manner, LNCaP-abl, displayed a hypersensitive biphasic proliferative response to androgen until passage 75. Maximal proliferation of LNCaP-abl cells was achieved at 0.001 nM of the synthetic androgen methyltrienolone (R1881), whereas 0.01 nM of this compound induced the same effect in parental cells. At later passages (> 75), androgen exerted an inhibitory effect on growth of LNCaP-abl cells. The non-steroidal anti-androgen bicalutamide stimulated proliferation of LNCaP-abl cells. AR protein expression in LNCaP-abl cells increased approximately fourfold. The basal AR transcriptional activity was 30-fold higher in LNCaP-abl than in LNCaP cells. R1881 stimulated reporter gene activity in LNCaP-abl cells even at 0.01 nM, whereas 0.1 nM of R1881 was needed for induction of the same level of reporter gene activity in LNCaP cells. Bicalutamide that acts as a pure antagonist in parental LNCaP cells showed agonistic effects on AR transactivation activity in LNCaP-abl cells and was not able to block the effects of androgen in these cells. The non-steroidal AR blocker hydroxyflutamide exerted stimulatory effects on AR activity in both LNCaP and LNCaP-abl cells; however, the induction of reporter gene activity by hydroxyflutamide was 2.4- to 4-fold higher in the LNCaP-abl subline. The changes in AR activity were associated neither with a new alteration in AR cDNA sequence nor with amplification of the AR gene. Growth of LNCaP-abl xenografts in nude mice was stimulated by bicalutamide and repressed by testosterone. In conclusion, our results show for the first time that the non-steroidal anti-androgen bicalutamide acquires agonistic properties during long-term androgen ablation. These findings may have repercussions on the natural course of prostate cancer with androgen deprivation and on strategies of therapeutic intervention. © 1999 Cancer Research Campaig

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies