Paul S. Rennie

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin‐dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone‐dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Intermittent androgen suppression delays progression to androgen-independent regulation of prostate-specific antigen gene in the LNCaP prostate tumour model

In most patients with prostate cancer, continuous androgen suppression (CAS) therapy causes tumour regression and an accompanying decrease in serum prostate specific antigen (PSA). However, with tumour progression, regulation of both tumour growth and PSA gene expression becomes androgen-independent. Because androgen resistance develops, in part, from adaptive cell survival mechanisms activated by androgen withdrawal, we hypothesize that intermittent re-exposure to androgens may prolong time to androgen-independent progression. The objective of this study was to determine whether intermittent androgen suppression (IAS) could delay the onset of androgen-independent PSA gene regulation in LNCaP prostate tumour model when compared to CAS. Five or six cycles of IAS were possible before progression developed. IAS prolonged time to androgen-independent PSA gene regulation from an average of 26 days in CAS to 77 days in IAS. Serum PSA increased above pre-castrate levels in all mice treated with CAS by 28 days post-castration, but remained below pre-castrate levels in 75% of IAS-treated mice by 60 days post-castration. By 15 weeks post-castration, serum PSA levels increased 7-fold above pre-castrate levels in CAS-treated mice compared to 1.9-fold increase in IAS-treated mice. PSA mRNA expression levels highly correlated with serum PSA levels in both groups. Maintenance of androgen dependency through IAS may be due to androgen-induced differentiation and/or down-regulation of androgen-suppressed gene expression.

Androgenic Induction of Prostate-specific Antigen Gene Is Repressed by Protein-Protein Interaction between the Androgen Receptor and AP-1/c-Jun in the Human Prostate Cancer Cell Line LNCaP

In exploring the possible mechanisms of androgen independence of prostate-specific antigen (PSA) gene expression, we investigated the effect of elevating AP-1 by both 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment and transfection of the c-Jun expression vector in LNCaP cells. Transcription of PSA is initiated when ligand-activated androgen receptor (AR) binds to a region in the PSA promoter that contains an androgen-responsive element (ARE). It was found that TPA inhibited androgen-induced PSA gene expression by a mechanism that did not alter nuclear levels of AR protein. Overexpression of AP-1 (junand fos proteins) also inhibited androgen-induced PSA promoter activity. These observations were apparently related to the disruption of AR·ARE complexes as demonstrated by the results of electrophoretic mobility shift assays. Specifically, c-Jun inhibited the formation of AR·ARE complexes and conversely that AR-glutathioneS-transferase proteins inhibited the formation of c-Jun·TPA-responsive element (TRE) complexes. Consistent with the inhibitory effect of both proteins, anti-c-Jun antibody blocked the inhibition of AR·ARE complex formation by c-Jun. A similar, but less marked, effect was obtained when anti-AR antibody was used to prevent AR inhibition of c-Jun·TRE complex formation. These findings together with results obtained from co-immunoprecipitation experiments strongly suggest that mutual repression of DNA binding activity is due to direct interaction between the two proteins and that the degree of repression may be determined by the ratio of AR to c-Jun. The mechanism of repression studied in mutant analysis experiments yielded evidence of an interaction between the DNA- and ligand-binding domains of AR and the leucine zipper region of c-Jun. Thus, the AR is similar to other nuclear receptors in its ability to interact with AP-1. This association provides a link between AP-1 and AR signal transduction pathways and may play a role in the regulation of the androgen-responsive PSA gene.

Cooperative interactions between androgen receptor (AR) and heat-shock protein 27 facilitate AR transcriptional activity.

Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser(78) and Ser(82) residues in an AR- and p38 kinase-dependent manner. After this androgen-induced, non-nuclear phospho-activation, Hsp27 displaces Hsp90 from a complex with AR to chaperone AR into the nucleus and interact with its response elements to enhance its genomic activity. Inhibition of Hsp27 phosphorylation, or knockdown using the antisense drug OGX-427, shifted the association of AR with Hsp90 to MDM2, increased proteasome-mediated AR degradation, decreased AR transcriptional activity, and increased prostate cancer LNCaP cell apoptotic rates. OGX-427 treatment of mice bearing LNCaP xenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased bioluminescence and serum PSA levels as pharmacodynamic readouts of AR activity, as well as AR, Hsp27, and Hsp90 protein levels in LNCaP tumor tissue. These data identify novel nongenomic mechanisms involving androgen, AR, and Hsp27 activation that cooperatively interact to regulate the genomic activity of AR and justify further investigation of Hsp27 knockdown as an AR disrupting therapeutic strategy in prostate cancer.

Short Hairpin RNA Knockdown of the Androgen Receptor Attenuates Ligand-Independent Activation and Delays Tumor Progression

Progression to androgen independence is the lethal end stage of prostate cancer. We used expression of androgen receptor (AR)-targeted short hairpin RNAs (shRNA) to directly test the requirement for AR in ligand-independent activation of androgen-regulated genes and hormone-independent tumor progression. Transient transfection of LNCaP human prostate cancer cells showed that AR shRNA decreased R1881 induction of the prostate-specific antigen (PSA)-luciferase reporter by 96%, whereas activation by forskolin, interleukin-6, or epidermal growth factor was inhibited 48% to 75%. Whereas the antiandrogen bicalutamide provided no further suppression, treatment with the mitogen-activated protein kinase (MAPK) inhibitor U0126 completely abrogated the residual activity, indicating a MAPK-dependent, AR-independent pathway for regulating the PSA promoter. Expression of doxycycline-inducible AR shRNA expression in LNCaP cells resulted in decreased levels of AR and PSA as well as reduced proliferation in vitro. When these cells were grown as xenografts in immunocompromised mice, induction of AR shRNA decreased serum PSA to below castration nadir levels and significantly retarded tumor growth over the entire 55-day experimental period. This is the first demonstration that, by inducibly suppressing AR expression in vivo, there is an extensive delay in progression to androgen independence as well as a dramatic inhibition of tumor growth and decrease in serum PSA, which exceeds that seen with castration alone. Based on these findings, we propose that suppressing AR expression may provide superior therapeutic benefit in reducing tumor growth rate than castration and may additionally be very effective in delaying progression to androgen independence.

Standard Treatments Induce Antigen-Specific Immune Responses in Prostate Cancer

Purpose: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses. Experimental Design: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment. Results: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2. Conclusion: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

In vivo knockdown of the androgen receptor results in growth inhibition and regression of well-established, castration-resistant prostate tumors.

Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm3. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

Intermittent Androgen Suppression for Prostate Cancer: Rationale and Clinical Experience

The rationale behind intermittent androgen suppression (IAS) is based on: (1) observations that androgen ablation is palliative, not curative, in most patients with prostate cancer, and that quality of life must be considered; (2) the assumption that immediate androgen ablation is superior to delayed therapy in improving survival; (3) the hypothesis that if tumour cells surviving androgen withdrawal are forced into a normal pathway of differentiation by androgen replacement, then apoptotic potential might be restored and progression to androgen independence delayed. Several centres have now tested the feasibility of IAS therapy in non-randomized groups of prostate cancer patients using serum of prostate-specific antigen levels as trigger points. Clinical data suggest that prostate cancer is amenable to control by IAS and offers clinicians an opportunity to improve patients’ quality of life by balancing the benefits of immediate androgen ablation (delayed progression and prolonged survival) while reducing treatment-related side effects and expense. Whether time to progression and survival is affected in a beneficial or adverse way is being studied in randomized, prospective protocols.

Targeting the Binding Function 3 (BF3) Site of the Human Androgen Receptor Through Virtual Screening

The androgen receptor (AR) is the best studied drug target for the treatment of prostate cancer. While there are a number of drugs that target the AR, they all work through the same mechanism of action and are prone to the development of drug resistance. There is a large unmet need for novel AR inhibitors which work through alternative mechanism(s). Recent studies have identified a novel site on the AR called binding function 3 (BF3) that is involved into AR transcriptional activity. In order to identify inhibitors that target the BF3 site, we have conducted a large-scale in silico screen followed by experimental evaluation. A number of compounds were identified that effectively inhibited the AR transcriptional activity with no obvious cytotoxicity. The mechanism of action of these compounds was validated by biochemical assays and X-ray crystallography. These findings lay a foundation for the development of alternative or supplementary therapies capable of combating prostate cancer even in its antiandrogen resistant forms.

Functional localization and competition between the androgen receptor and T-cell factor for nuclear β -catenin: a means for inhibition of the Tcf signaling axis

Recent reports suggest that the β-catenin-T-cell factor (Tcf) (BCT) signaling pathway is important in the progression of prostate cancer. Evidence suggests that the androgen receptor (AR) can repress BCT-mediated transcription both in prostate cancer and colon cancer cells (Chesire and Isaacs, 2002). In this study, we validate such findings and show that repression of BCT signaling is facilitated by competition between the AR and Tcf. Measurements of the Tcf transcriptional reporter (TOPFLASH) indicated that AR+DHT-mediated repression can inhibit BCT transcription in the presence of WT and exogenous activating β-catenin (Δ1–130 bp). Transient transfections in SW480 cells (APCmut/mut) showed that this mode of repression is functionally independent of APC-mediated β-catenin ubiquitination. Using a recently developed red flourescent protein (HcRed), we demonstrate novel observations about the nuclear distribution of Tcf. Furthermore, with the use of red (HcRed-AR and HcRed-Tcf) and green fusion proteins (β-catenin-EGFP), we provide morphological evidence of a reciprocal balance of nuclear β-catenin-EGFP (BC-EGFP). By cotransfecting in LNCaP prostate tumor cells and using quantitative imaging software, we demonstrated a 62.0% colocalization of HcRed-AR and BC-EGFP in the presence of DHT and 63.3% colocalization of HcRed-Tcf/BC-EGFP in the absence of DHT. Costaining for activated RNA Pol II (phosphoserine 2) and HcRed-Tcf suggested that Tcf foci contain transcriptional ‘hotspots’ validating that these sites have the capacity for transcriptional activity. Given this apparent androgen-dependent competition for nuclear BC-EGFP, we chose to assess our hypothesis by in vivo and in vitro binding assays. SW480 cells transiently transfected with an AR expression construct, treated with DHT and immunoprecipitated for Tcf showed less associated β-catenin when compared to Tcf precipitates from untreated cells. Furthermore, by treating cells with DHT+Casodex, we were able to abrogate the androgen-sensitive AR/β-catenin interaction, in addition to relieving transcriptional repression of the TOPFLASH reporter. In vitro binding assays, with increasing amounts of ARS35, resulted in decreased TcfS35 association with immunoprecipitated recombinant β-catenin-HIS. These data suggest that in steady-state conditions, AR has the ability to compete out Tcf binding for β-catenin. Finally, using SW480 cells, we show that AR-mediated repression of the BCT pathway has implications for cell cycle progression and in vitro growth. Using FACs analysis, we observed a 26.1% increase in accumulation of cells in the G1 phase of the cell cycle, while in vitro growth assays showed a 35% reduction in viable cells transfected with AR+DHT treatment. Together, our data strongly suggest that a reciprocal balance of nuclear β-catenin facilitates AR-mediated repression of BCT-driven transcription and cell growth.