Androgen Receptor Activity In Prostate Cancer Research Articles

Characterization of the Peroxisomal Proteome and Redox Balance in Human Prostate Cancer Cell Lines.

Prostate cancer (PCa) is associated with disruptions in cellular redox balance. Given the intricate role of peroxisomes in redox metabolism, we conducted comprehensive proteomics analyses to compare peroxisomal and redox protein profiles between benign (RWPE-1) and malignant (22Rv1, LNCaP, and PC3) prostate cell lines. Our analyses revealed significant enrichment of the "peroxisome" pathway among proteins notably upregulated in androgen receptor (AR)-positive cell lines. In addition, catalase (CAT) activity was consistently higher in these malignant cell lines compared to RWPE-1, which contrasts with previous studies reporting lower CAT levels and increased H2O2 levels in PCa tissues compared to adjacent normal tissues. To mimic this clinical scenario, we used RNA interference to knock down CAT expression. Our results show that reduced CAT levels enhanced 22Rv1 and LNCaP cell proliferation. R1881-induced activation of AR, a key driver of PCa, increased expression of the H2O2-producing peroxisomal β-oxidation enzymes acyl-coenzyme A oxidase 1 and 3, reduced CAT expression and activity, and elevated peroxisomal H2O2 levels. Considering these changes and other antioxidant enzyme profile alterations, we propose that enhanced AR activity in PCa reduces CAT function, leading to increased peroxisomal H2O2 levels that trigger adaptive stress responses to promote cell survival, growth, and proliferation.

Androgen receptor monomers and dimers regulate opposing biological processes in prostate cancer cells

Most prostate cancers express the androgen receptor (AR), and tumor growth and progression are facilitated by exceptionally low levels of systemic or intratumorally produced androgens. Thus, absolute inhibition of the androgen signaling axis remains the goal of current therapeutic approaches to treat prostate cancer (PCa). Paradoxically, high dose androgens also exhibit considerable efficacy as a treatment modality in patients with late-stage metastatic PCa. Here we show that low levels of androgens, functioning through an AR monomer, facilitate a non-genomic activation of the mTOR signaling pathway to drive proliferation. Conversely, high dose androgens facilitate the formation of AR dimers/oligomers to suppress c-MYC expression, inhibit proliferation and drive a transcriptional program associated with a differentiated phenotype. These findings highlight the inherent liabilities in current approaches used to inhibit AR action in PCa and are instructive as to strategies that can be used to develop new therapeutics for this disease and other androgenopathies.

Epigenetic Coregulation of Androgen Receptor Signaling.

The androgen receptor (AR) is a ligand-activated transcription factor belonging to the nuclear receptor (NR) superfamily. As with other members of the NR family, transcriptional activity of the AR is regulated by interactions with coregulatory proteins, which either enhance (coactivators) or repress (corepressors) its transcriptional activity. AR associated coregulators are functionally diverse, but a large fraction are epigenetic histone and chromatin modifiers. Epigenetic coregulators are recruited to gene regulatory regions as part of multi-protein complexes, often acting in a dynamic and inter-dependent manner to remodel chromatin, thereby allowing or inhibiting the access of AR-associated transcriptional machinery to target genes; functional consequences being regulation of transcriptional output. Epigenetic modifiers, including those that function as AR coregulators, are frequently mutated or aberrantly expressed in prostate cancer and are implicated in disease progression. Some of these modifiers are being investigated as therapeutic targets in several cancer types and could potentially be used to modulate aberrant AR activity in prostate cancer. In this chapter we will summarise the functional role of epigenetic coregulators in AR signalling, their dysregulation during prostate cancer progression and the current status of drugs targeting these enzymes.

A glutaminase isoform switch drives therapeutic resistance and disease progression of prostate cancer

Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to hormonal therapy resistance and disease progression in prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) up-regulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), an androgen-independent GLS1 isoform with more potent enzymatic activity, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyperproliferation, and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GAC shows better treatment effect for castration-resistant PCa than for hormone-sensitive PCa in vitro and in vivo. In summary, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is one of the key mechanisms in therapeutic resistance and disease progression.

Oncogenic role of PinX1 in prostate cancer cells through androgen receptor dependent and independent mechanisms

Coregulators play an important role in prostate cancer (PCa), modulating androgen receptor (AR) action and representing a possible cause of androgen deprivation therapy failure. Pin2-interacting protein X1 (PinX1) is a nucleolar protein described as a steroid hormone receptor coregulator in breast cancer cell lines. In this work, we studied the effect of PinX1 on AR action in PCa. Our results demonstrate that PinX1 acts as an AR coactivator, increasing its transcriptional activity and target gene expression, as well as proliferation, migration and colony formation in PCa cell lines. These effects are observed in the presence and absence of AR agonist and antagonists, suggesting a possible androgen independent pathway for PinX1. We present the first oncogenic roles described for PinX1, acting as a coactivator of the AR.

A Glutaminase Isoform Switch Drives Therapeutic Resistance and Disease Progression of Prostate Cancer

Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to the hormonal therapy resistance and disease progression of prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) upregulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), a shorter splicing isoform of GLS1that is upregulated by MYC (c-MYC and N-Myc) with more potent in glutaminolysis, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyper-proliferation and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GLS1 isoforms differentially suppresses the growth of hormone sensitive prostate cancer and castration resistant prostate cancer in vitro and in vivo. Therefore, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is a key mechanism in therapeutic resistance and disease progression.

Androgen receptor-modulatory microRNAs provide insight into therapy resistance and therapeutic targets in advanced prostate cancer

Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced ‘castrate-resistant’ disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition. Corresponding miRs increased AR activity through a novel and anti-dogmatic mechanism of direct association with AR 6.9 kb 3′UTR and transcript stabilisation. In addition, miR-346 and miR-361-3p modulation altered levels of constitutively active AR variants, and inhibited variant-driven PC cell proliferation, so may contribute to persistent AR signalling in CRPC in the absence of circulating androgens. Pathway analysis of AGO-PAR-CLIP-identified miR targets revealed roles in DNA replication and repair, cell cycle, signal transduction and immune function. Silencing these targets, including tumour suppressors ARHGDIA and TAGLN2, phenocopied miR effects, demonstrating physiological relevance. MiR-346 additionally upregulated the oncogene, YWHAZ, which correlated with grade, biochemical relapse and metastasis in patients. These AR-modulatory miRs and targets correlated with AR activity in patient biopsies, and were elevated in response to long-term enzalutamide treatment of patient-derived CRPC xenografts. In summary, we identified miRs that modulate AR activity in PC and CRPC, via novel mechanisms, and may represent novel PC therapeutic targets.

A Standardized Wedelia chinensis Extract Overcomes the Feedback Activation of HER2/3 Signaling upon Androgen-Ablation in Prostate Cancer

Crosstalk between the androgen receptor (AR) and other signaling pathways in prostate cancer (PCa) severely affects the therapeutic outcome of hormonal therapy. Although anti-androgen therapy prolongs overall survival in PCa patients, resistance rapidly develops and is often associated with increased AR expression and upregulation of the HER2/3-AKT signaling pathway. However, single agent therapy targeting AR, HER2/3 or AKT usually fails due to the reciprocal feedback loop. Previously, we reported that wedelolactone, apigenin, and luteolin are the active compounds in Wedelia chinensis herbal extract, and act synergistically to inhibit the AR activity in PCa. Here, we further demonstrated that an herbal extract of W. chinensis (WCE) effectively disrupted the AR, HER2/3, and AKT signaling networks and therefore enhanced the therapeutic efficacy of androgen ablation in PCa. Furthermore, WCE remained effective in suppressing AR and HER2/3 signaling in an in vivo adapted castration-resistant PCa (CRPC) LNCaP cell model that was insensitive to androgen withdrawal and second-line antiandrogen, enzalutamide. This study provides preclinical evidence that the use of a defined, single plant-derived extract can augment the therapeutic efficacy of castration with significantly prolonged progression-free survival. These data also establish a solid basis for using WCE as a candidate agent in clinical studies.

Development of Bag-1L as a therapeutic target in androgen receptor-dependent prostate cancer.

Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth. Clinically, Bag-1L protein levels increase with progression to castration-resistant PCa (CRPC) and high levels of Bag-1L in primary PCa associate with a reduced clinical benefit from abiraterone when these tumors progress. Intriguingly, residues in Bag-1L important for its interaction with the AR AF-1 are within a potentially druggable pocket, implicating Bag-1L as a potential therapeutic target in PCa.

Abstract 5732: PI3K/Akt activity regulates androgen receptor expression and predicts poor clinical outcome in non-metastatic hormone-naïve prostate cancer

Abstract Activation of PI3K/Akt pathway is associated with adverse outcome and aggressive disease in many cancers. In prostate cancer (PCa), the activity of this pathway has been shown to promote disease progression and metastasis. However, it is still controversial how PI3K/Akt regulates androgen receptor (AR), a central signaling molecule in prostate pathophysiology, and whether it has an active role in hormone naïve non-metastatic PCa. Here, we show using immunohistochemistry (IHC) and advanced quantitative multiplexed IHC that the expression of phosphorylated-Akt(S473) and AR are highly correlated in clinical PCa, even at the cellular level. Furthermore, we found that high expression of p-Akt(S473) predicts poor clinical outcome in two independent hormone-naïve non-metastatic PCa cohorts. To study whether PI3K/Akt regulates AR expression, we performed an in vitro drug screen with 32 PI3K/Akt/mTOR inhibitors in PC346C, an AR expressing cell line derived from a hormone-naïve primary tumor of prostate. We observed a strong correlation between p-Akt(S473) and AR also in vitro in individual cells independent of the inhibitor used. Although both PI3K and Akt specific inhibition reduced cell viability, the response in nuclear expression of AR was highly dependent on the target of inhibition: Akt specific inhibition reduced AR nuclear expression and resulted in large, spindle-shaped cells, whereas PI3K specific inhibition increased AR nuclear expression and resulted in smaller, round-shaped cells. These data suggest that PI3K and Akt have different roles in sustaining AR activity in PCa as perturbations of the two components leads to differential responses in terms of AR nuclear expression and cell morphology. In conclusion, activated Akt associates with AR expression and predicts poor clinical outcome in hormone-naïve non-metastatic PCa. Furthermore, the differing roles of PI3K and Akt in AR regulation warrants for further studies as it may have implications in the design of PCa therapy targeting PI3K/Akt, especially when the inhibitors are administered in combination with anti-androgens. Citation Format: Sami Blom, Petra Mäki-Teeri, Andrew Erickson, Lassi Paavolainen, Tuomas Mirtti, Antti Rannikko, Swapnil Potdar, Päivi Östling, Wytske van Weerden, Olli Kallioniemi, Teijo Pellinen. PI3K/Akt activity regulates androgen receptor expression and predicts poor clinical outcome in non-metastatic hormone-naïve prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5732. doi:10.1158/1538-7445.AM2017-5732

CXCL13 is androgen-responsive and involved in androgen induced prostate cancer cell migration and invasion.

Androgen receptor (AR) is a key transcription factor playing a critical role in prostate cancer (PCa) initiation and progression. However, the molecular mechanisms of AR action in prostate cancer are not very clear. CXCL13, known as B cell attracting chemokine1 (BCA-1), is a member of CXC chemokine family and relevant to cancer metastasis. This study shows that CXCL13 is an androgen-responsive gene and involved in AR-induced PCa cell migration and invasion. In clinical specimens, expression of CXCL13 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of CXCL13 is up-regulated by androgen-AR axis at both mRNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen responsive elements (ARE) at CXCL13 enhancer and dual-luciferase reporter assays reveals that the ARE is highly responsive to androgen while mutations of the ARE abolish the reporter activity. Additional chromatin immunoprecipitation (ChIP) assays also identify that the ARE presents androgen responsiveness. In addition, CXCL13 promotes G2/M phase transition by increasing Cyclin B1 levels in PCa cells. Functional studies demonstrate that reducing endogenous CXCL13 expression in LNCaP cells largely weakens androgen-AR axis induced cell migration and invasion. Taken together, our study implicates for the first time that CXCL13 is an AR target gene and involved in AR-mediated cell migration and invasion in primary PCa.

Abstract 5566: Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer

Abstract Introduction: Androgen receptor (AR) stability has been shown to be regulated by MDM2 E3 ubiquitin ligase. Our data suggest a role for PMEPA1 and NEDD4-1 in the regulation of AR protein in prostate cancer cells. The AR inducible PMEPA1 recruits NEDD4-1, an E3 ubiquitin ligase, to degrade AR. Decreased or absent PMPEA1 was observed in more than 65% of prostate tumors. PMEPA1 depleted transfectant is established in LNCaP cells with a shRNA lentivirus system and shown elevated AR protein levels, AR signaling and increased cell growth. In the current study, we investigated the growth and AR signaling of PMEPA1 depleted prostate cancer cells in the presence of AR inhibitors. Also we investigated the role of PMEPA1 silencing by DNA methylation in the regulation of AR. Methods: PMEPA1 depleted LNCaP cells were treated with AR inhibitors (MDV3100 or bicalutamide) in the presence or absence of the synthetic androgen R1881. Cell growth was monitored by cell counting, BrdU incorporation, colony plating and soft agar assays. Cell cycle was analyzed with propidium iodide staining based flowcytometry. AR, PMEPA1, PSA proteins were assessed by immunoblot assay. Methylation of the PMEPA1 gene was evaluated in LCM-enriched tumor samples by combing precipitation of methylated-DNA and methylation-sensitive restriction enzyme digestion (COMPARE-MS). The transcript levels of PMEPA1 gene was assessed by qRT-PCR. Results: PMEPA1 depleted LNCaP cells are more resistance to growth inhibition by MDV3100 or bicalutamide in both hormone and hormone-depleted conditions, which demonstrated stronger BrdU incorporation abilities, cell plating efficiency and colony formation capacities in soft agar under MDV3100 or bicalutamid treatment compared to controls. The half life of AR was increased, and more cells in S-phase and less apoptotic cells in PMEPA1 depleted LNCaP cells. Methylation of PMEPA1 gene and mRNA levels showed significant correlation in prostate tumors (n=77, P=0.001). The higher frequency of PMEPA1 gene methylation was noticed in Caucasian American patients. Conclusions: PMEPA1 depleted prostate cancer cells develop increased resistance to AR inhibitors through enhanced AR signaling. Silencing of PMEPA1 may contribute to AR activation in prostate cancer. These data highlight that PMEPA1 deficiency may confer resistance to AR inhibitors during prostate cancer progression. This study is supported by the NIH grant (R01CA106653) to SS. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Michael Haffner, Daniel Heidenberg, Srinivasan Yegnasubramanian, Yongmei Chen, Ahmed Mohamed, Alagarsamy Srinivasan, Gyorgy Petrovics, Shiv Srivastava. Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5566. doi:10.1158/1538-7445.AM2014-5566

Posttranslational Modification of the Androgen Receptor in Prostate Cancer

The androgen receptor (AR) is important in the development of the prostate by regulating transcription, cellular proliferation, and apoptosis. AR undergoes posttranslational modifications that alter its transcription activity, translocation to the nucleus and stability. The posttranslational modifications that regulate these events are of utmost importance to understand the functional role of AR and its activity. The majority of these modifications occur in the activation function-1 (AF1) region of the AR, which contains the transcriptional activation unit 1 (TAU1) and 5 (TAU5). Identification of the modifications that occur to these regions may increase our understanding of AR activation in prostate cancer and the role of AR in the progression from androgen-dependent to castration-resistant prostate cancer (CRPC). Most of the posttranslational modifications identified to date have been determined using the full-length AR in androgen dependent cells. Further investigations into the role of posttranslational modifications in androgen-independent activation of full-length AR and constitutively active splicing variants are warranted, findings from which may provide new therapeutic options for CRPC.

MED1 Coactivation of Androgen Receptor‐dependent Transcription is Mediated Through a Newly Discovered Noncanonical Binding Motif

The androgen receptor (AR) is uncommon among Nuclear receptor (NR) because of its intramolecular interaction(termed the N/C interaction) and thus coactivators can alternatively bind to surfaces in the AR amino terminal. The Mediator complex plays a key coregulatory role in steroid hormone dependent transcription and is chiefly targeted to NRs via the LxxLL‐containing MED1 subunit. However, the mechanisms by which AR recruits MED1 have remained unclear. Here we show that MED1 binds to a distinct AR amino‐terminal region termed transactivation unit‐1 (Tau‐1) via two newly discovered noncanonical α‐helical motifs of MED1. The two MED1 LxxLL motifs are not required for AR binding whereas loss of the intramolecular AR N/C interaction decreases MED1 binding. We further demonstrate that mitogen‐activated protein kinase phosphorylation of MED1 enhances the AR‐MED1 interaction in prostate cancer cells. In sum, our findings reveal a novel AR‐coactivator binding mechanism that may have clinical implications for AR activity in prostate cancer.

RhoA as a Mediator of Clinically Relevant Androgen Action in Prostate Cancer Cells

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.

Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation

Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

Abstract A8: Androgen regulation of SRF target genes in prostate cancer cells is mediated in part by the small GTPase RhoA

Abstract Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen-responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate and correlate with aggressive disease. Moreover, the SRF-AR expression signature is associated more strongly with biochemical recurrence than similarly sized sets of AREdriven AR target genes. Thus, understanding the mechanism(s) by which SRF conveys androgen effects to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we explore the role of the RhoA signaling axis, a well-known activating pathway upstream of SRF, in androgen activation of SRF action. PCa cells were treated with inhibitors of RhoA signaling or transfected with siRNAs targeting RhoA, the critical SRF cofactor MAL or control siRNAs in the presence or absence of the synthetic androgen R1881. Real-time RT-PCR and western blotting was done to assess effects on expression of SRF target genes. Cell fractionation experiments evaluated the cellular localization of AR, MAL and SRF. ChIP was performed to assess recruitment of MAL to SRF target genes. Wild-type, dominant-active and dominant-negative versions of RhoA were overexpressed to determine effects on androgen regulation of SRF target genes. Immunohistochemistry was done to evaluate RhoA expression in clinical PCa samples and a tissue microarray derived from a CWR22 PCa xenograft model that mimics progression from androgen-stimulated (AS) to castration-recurrent (CR) disease. Effects on AS and CR PCa cell biology were assessed using proliferation and migration assays. Here, we show that the small GTPase RhoA mediates androgen responsiveness of more than half of SRF target genes tested. Interference with expression of RhoA, activity of the RhoA effector RhoA-associated kinase (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor MAL, all prevented full androgen-regulation of SRF target genes. Androgen treatment increased the nuclear content of MAL and led to MAL recruitment to the promoter of the SRF target gene FHL2. Moreover, in clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival following radical prostatectomy. Overexpression of RhoA markedly increased the androgen responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. Cell lines and a xenograft model that mimics the transition from AS to CR PCa indicate that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Silencing of RhoA, use of ROCK inhibitors or an inhibitor of SRF-MAL interaction blunted (androgen-regulated) cell viability and attenuated AS and CR PCa cell migration. Taken together, our work demonstrates that the RhoA signaling axis mediates clinically relevant AR action in PCa. In addition, these studies are the first to report on overexpression of RhoA in PCa and its association with aggressive disease and poor outcome. Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Lucy Schmidt, Thomas Sebo, Donald Tindall, Hannelore Heemers, Kelly Duncan, Neelu Yadav, Kevin Regan, Christine Lohse, Elena Pop, Kristopher Attwood, Gregory Wilding, James Mohler. Androgen regulation of SRF target genes in prostate cancer cells is mediated in part by the small GTPase RhoA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A8.

Regulation of Androgen Receptor-dependent Transcription by Coactivator MED1 Is Mediated through a Newly Discovered Noncanonical Binding Motif

Nuclear receptor (NR) activation by cognate ligand generally involves allosteric realignment of C-terminal α-helices thus generating a binding surface for coactivators containing canonical LXXLL α-helical motifs. The androgen receptor (AR) is uncommon among NRs in that ligand triggers an intramolecular interaction between its N- and C-terminal domains (termed the N/C interaction) and that coactivators can alternatively bind to surfaces in the AR N-terminal or hinge regions. The evolutionary conserved Mediator complex plays a key coregulatory role in steroid hormone-dependent transcription and is chiefly targeted to NRs via the LXXLL-containing MED1 subunit. Whereas MED1 has been demonstrated to serve as a key transcriptional coactivator for AR, the mechanisms by which AR recruits MED1 have remained unclear. Here we show that MED1 binds to a distinct AR N-terminal region termed transactivation unit-1 (Tau-1) via two newly discovered noncanonical α-helical motifs located between MED1 residues 505 and 537. Neither of the two MED1 LXXLL motifs is required for AR binding, whereas loss of the intramolecular AR N/C interaction decreases MED1 binding. We further demonstrate that mitogen-activated protein kinase phosphorylation of MED1 enhances the AR-MED1 interaction in prostate cancer cells. In sum, our findings reveal a novel AR-coactivator binding mechanism that may have clinical implications for AR activity in prostate cancer.

SOD Mimetics: A Novel Class of Androgen Receptor Inhibitors That Suppresses Castration-Resistant Growth of Prostate Cancer

Advanced prostate cancer is the second leading cause of cancer-related deaths among American men. The androgen receptor (AR) is vital for prostate cancer progression, even in the face of castrate levels of serum testosterone following androgen ablation therapy, a mainstay therapy for advanced prostate cancer. Downregulation of superoxide dismutase 2 (SOD2), a major intracellular antioxidant enzyme, occurs progressively during prostate cancer progression to advanced states and is known to promote AR activity in prostate cancer. Therefore, this study investigated the effects of SOD mimetics on AR expression and function in AR-dependent LNCaP, CWR22Rv1, and LAPC-4AD prostate cancer cells. Treatment with Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a SOD mimetic, not only lowered cellular superoxide levels but also concomitantly attenuated AR transcriptional activity and AR target gene expression in a dose- and time-dependent manner, in the presence and absence of dihydrotestosterone, the major endogenous AR agonist. Inhibition of AR by Tempol was mediated, in large part, by its ability to decrease AR protein via increased degradation, in the absence of any inhibitory effects on other nuclear receptors. Inhibitory effects of Tempol on AR were also reproducible with other SOD mimetics, MnTBAP and MnTMPyP. Importantly, effects of Tempol on AR function were accompanied by significant in vitro and in vivo reduction in castration-resistant prostate cancer (CRPC) survival and growth. Collectively, this study has shown for the first time that SOD mimetics, by virtue of their ability to suppress AR function, may be beneficial in treating the currently incurable CRPC, in which SOD2 expression is highly suppressed.

Abstract 2163: Microtubule-dependent regulation of a constitutively active androgen receptor splice variant

Abstract Prostate cancer progression is dependent on androgens and can be suppressed by androgen ablation. Androgen deprivation provides palliation but not cure in men with prostate cancer, and the disease progresses to a castrate-resistant stage (CRPC), which is often accompanied by molecular alterations in the androgen receptor (AR). Recently, an AR splice variant lacking the ligand binding domain was found in human prostate tissue. This AR variant (ARv567) was not expressed in normal prostate epithelium, suggesting that the presence of a constitutively active splice variant of the AR arises following castration and plays a role in the progression of prostate cancer. Work from our lab shows that taxanes, used in CRPC treatment, impair the ligand-induced nuclear accumulation of full-length AR (AR-FL) and hence its transcriptional activity. We also show that AR trafficking towards the nucleus occurs along microtubules (MTs) with the aid of the dynein motor protein using a variety of assays including co-immunoprecipitation, immunofluorescence and microtubule co-sedimentation. In order to identify the minimum AR domain that mediates MT binding we have generated deletion mutants of the AR, and are testing their MT binding ability both in cells and in vitro. We have extended these studies to the clinically relevant AR splice variant ARv567, and showed that it binds MTs as evidenced by microtubule co-sedimentation assays and live cell imaging. Next we sought to determine the dynamics of AR-FL and variant nuclear accumulation in relation to taxane-induced MT stabilization. To do so we microinjected GFP-tagged AR-FL or GFP-tagged ARv567 into the nucleus of PC3 cells stably expressing mCherry tubulin and monitored the effect of taxane treatment on MT dynamics and AR nuclear translocation using live-cell confocal microscopy. This study revealed rapid AR-FL nuclear accumulation within 30 min of R1881 treatment, while it did not induce further nuclear accumulation of the variant, consistent with the lack of ligand binding domain in ARv567. On the other hand, taxane pre-treatment attenuated the nuclear accumulation of both AR-FL and ARv567, suggesting that functional MTs are required for AR's nuclear transport in a ligand independent manner. Interestingly, we observed that the presence of the variant sensitizes cells to taxane treatment as seen by greater decreases in tumor volumes in response to taxanes in xenografts expressing variant vs those with AR-FL. Taken together, these data, reveal a new mechanism of regulation of AR activity in prostate cancer. Further elucidation of this mechanism may help us better understand the molecular basis of clinical response to taxane treatment in CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2163. doi:10.1158/1538-7445.AM2011-2163