Latif A. Wafa

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.

Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system

The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.

Cyclin G‐associated kinase: A novel androgen receptor‐interacting transcriptional coactivator that is overexpressed in hormone refractory prostate cancer

The androgen receptor (AR), a steroid receptor family member, is a ligand‐dependent transcription factor that has an integral role in normal prostate development. Alterations in AR‐mediated activity can result in abnormal gene expression, dysregulated cell growth and prostate cancer. Coregulator proteins that interact with AR to influence activity and specificity of the AR‐response may also have an important role in prostate cancer progression. Since the NH2‐terminal domain (NTD) of AR encodes the ligand‐independent activation function (AF)‐1, this domain is incompatible with conventional yeast two‐hybrid systems. Therefore, we have used the Tup1 repressed transactivator (RTA) system, which exploits the intrinsic transactivation properties of AR.NTD, for identification of novel AR‐interacting proteins. Using this system, cyclin G‐associated kinase (GAK) was identified as an AR interacting protein, and GST pull‐down assays were used to confirm the interaction. GAK was shown to enhance the AF‐1 function of AR activity in a ligand‐dependent manner. Additionally, GAK enhanced the AR transcriptional response even at low concentrations of androgens, which is relevant to AR activity in androgen‐independent prostate cancer. Finally, neo‐adjuvant hormone therapy (NHT) tissue microarray analysis demonstrated that GAK expression increased significantly with prostate cancer progression to androgen independence, which suggests a prognostic role for GAK in advanced disease.

Androgen-regulated genes differentially modulated by the androgen receptor coactivator L-dopa decarboxylase in human prostate cancer cells

BackgroundThe androgen receptor is a ligand-induced transcriptional factor, which plays an important role in normal development of the prostate as well as in the progression of prostate cancer to a hormone refractory state. We previously reported the identification of a novel AR coactivator protein, L-dopa decarboxylase (DDC), which can act at the cytoplasmic level to enhance AR activity. We have also shown that DDC is a neuroendocrine (NE) marker of prostate cancer and that its expression is increased after hormone-ablation therapy and progression to androgen independence. In the present study, we generated tetracycline-inducible LNCaP-DDC prostate cancer stable cells to identify DDC downstream target genes by oligonucleotide microarray analysis.ResultsComparison of induced DDC overexpressing cells versus non-induced control cell lines revealed a number of changes in the expression of androgen-regulated transcripts encoding proteins with a variety of molecular functions, including signal transduction, binding and catalytic activities. There were a total of 35 differentially expressed genes, 25 up-regulated and 10 down-regulated, in the DDC overexpressing cell line. In particular, we found a well-known androgen induced gene, TMEPAI, which wasup-regulated in DDC overexpressing cells, supporting its known co-activation function. In addition, DDC also further augmented the transcriptional repression function of AR for a subset of androgen-repressed genes. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time RT-PCR.ConclusionTaken together, our results provide evidence for linking DDC action with AR signaling, which may be important for orchestrating molecular changes responsible for prostate cancer progression.

TAF1 differentially enhances androgen receptor transcriptional activity via its N-terminal kinase and ubiquitin-activating and -conjugating domains.

Aberrant expression of androgen receptor (AR) coregulators has been linked to progression of prostate cancers to castration resistance. Using the repressed transactivator yeast two-hybrid system, we found that TATA binding protein-associated factor 1 (TAF1) interacted with the AR. In tissue microarrays, TAF1 was shown to steadily increase with duration of neoadjuvant androgen withdrawal and with progression to castration resistance. Glutathione S-transferase pulldown assays established that TAF1 bound through its acetylation and ubiquitin-activating/conjugating domains (E1/E2) directly to the AR N terminus. Coimmunoprecipitation and ChIP assays revealed colocalization of TAF1 and AR on the prostate-specific antigen promoter/enhancer in prostate cancer cells. With respect to modulation of AR activity, overexpression of TAF1 enhanced AR activity severalfold, whereas small interfering RNA knockdown of TAF1 significantly decreased AR transactivation. Although full-length TAF1 showed enhancement of both AR and some generic gene transcriptional activity, selective AR coactivator activity by TAF1 was demonstrated in transactivation experiments using cloned N-terminal kinase and E1/E2 functional domains. In keeping with AR coactivation by the ubiquitin-activating and -conjugating domain, TAF1 was found to greatly increase the cellular amount of polyubiquitinated AR. In conclusion, our results indicate that increased TAF1 expression is associated with progression of human prostate cancers to the lethal castration-resistant state. Because TAF1 is a coactivator of AR that binds and enhances AR transcriptional activity, its overexpression could be part of a compensatory mechanism adapted by cancer cells to overcome reduced levels of circulating androgens.

Carbidopa abrogates L-dopa decarboxylase coactivation of the androgen receptor and delays prostate tumor progression

The androgen receptor (AR) plays a central role in prostate cancer progression to the castration‐resistant (CR) lethal state. L‐Dopa decarboxylase (DDC) is an AR coactivator that increases in expression with disease progression and is coexpressed with the receptor in prostate adenocarcinoma cells, where it may enhance AR activity. Here, we hypothesize that the DDC enzymatic inhibitor, carbidopa, can suppress DDC‐coactivation of AR and retard prostate tumor growth. Treating LNCaP prostate cancer cells with carbidopa in transcriptional assays suppressed the enhanced AR transactivation seen with DDC overexpression and decreased prostate‐specific antigen (PSA) mRNA levels. Carbidopa dose‐dependently inhibited cell growth and decreased survival in LNCaP cell proliferation and apoptosis assays. The inhibitory effect of carbidopa on DDC‐coactivation of AR and cell growth/survival was also observed in PC3 prostate cancer cells (stably expressing AR). In vivo studies demonstrated that serum PSA velocity and tumor growth rates elevated ∼2‐fold in LNCaP xenografts, inducibly overexpressing DDC, were reverted to control levels with carbidopa administration. In castrated mice, treating LNCaP tumors, expressing endogenous DDC, with carbidopa delayed progression to the CR state from 6 to 10 weeks, while serum PSA and tumor growth decreased 4.3‐fold and 5.4‐fold, respectively. Our study is a first time demonstration that carbidopa can abrogate DDC‐coactivation of AR in prostate cancer cells and tumors, decrease serum PSA, reduce tumor growth and delay CR progression. Since carbidopa is clinically approved, it may be readily used as a novel therapeutic strategy to suppress aberrant AR activity and delay prostate cancer progression.

Carbidopa enhances antitumoral activity of bicalutamide on the androgen receptor‐axis in castration‐resistant prostate tumors

Response to bicalutamide after castration failure is not durable and treatment options at this stage are limited. Carbidopa, an L‐dopa decarboxylase (AR‐coactivator) inhibitor, has been shown to retard prostate tumor growth/PSA production in xenografts. Here, we hypothesize that pharmacological targeting of the AR‐axis by combination treatment with bicalutamide plus carbidopa significantly enhances antitumoral activity in vitro and in vivo compared to monotherapy with either drug.

Androgen Receptor Coregulators and Their Role in Prostate Cancer

The limiting factor in the survival of a patient with prostate cancer is the rate of progression to the noncurable androgen-independent (AI) stage of disease. The androgen receptor (AR) is a critical regulator of prostate cancer development and is involved in AI progression. Coregulators are proteins that interact directly with AR to enhance (coactivators) or reduce (corepressors) its transcriptional activity. Currently, over 165 AR coregulators have been discovered. In this chapter, we focus on a subset of the most well-characterized AR coregulators that are associated with prostate cancer. The first part of our review discusses the mechanisms by which classical type I and nonclassical type II AR coactivators/corepressors regulate AR transcriptional activity. The second section focuses on the role of coregulators in prostate cancer, including their expression profile in prostate cancer patients, tumor cell growth effects, and potential as therapeutic targets. In view of their involvement in prostate cancer progression, it is anticipated that further study of AR coregulators will provide more treatment options for increasing survival of patients with AI prostate cancer.