Androgen Receptor Isoforms Research Articles

The combined effect of USP7 inhibitors and PARP inhibitors in hormone-sensitive and castration-resistant prostate cancer cells.

Purpose of the studyReduced levels of the tumor suppressor protein CCDC6 sensitize cancer cells to the treatment with PARP-inhibitors. The turnover of CCDC6 protein is regulated by the de-ubiquitinase USP7, which also controls the androgen receptor (AR) stability. Here, we correlated the expression levels of CCDC6 and USP7 proteins in primary prostate cancers (PC). Moreover, we tested the efficacy of the USP7 inhibitors, in combination with PARP-inhibitors as a novel therapeutic option in advanced prostate cancer.Experimental techniquesPC cells were exposed to USP7 inhibitor, P5091, together with cycloheximide, to investigate the turnover of the USP7 substrates, AR and CCDC6. As outcome of the AR downregulation, transcription targets of AR and its variant V7 were examined by qPCR. As a result of CCDC6 degradation, the induction of PARP inhibitors sensitivity was evaluated by analyzing PC cells viability and foci formation. We scored and correlated CCDC6 and USP7 expression levels in a prostate cancer tissue microarray (TMA).ResultsP5091 accelerated the degradation of AR and V7 isoform affecting PSA, UBE2C, CDC20 transcription and PC cells proliferation. Moreover, P5091 accelerated the degradation of CCDC6 sensitizing the cells to PARP-inhibitors, that acted sinergistically with genotoxic agents. The immunohistochemical analysis of both CCDC6 and USP7 proteins exhibited significant correlation for the intensity of staining (p ≤ 0.05).Data interpretationThus, CCDC6 and USP7 represent predictive markers for the combined treatment of the USP7-inhibitors and PARP-inhibitors in advanced prostate cancer.

Detection of AR-V7 using cell-free RNA (cfRNA) in prostate cancer.

e16613Background: In prostate cancer (PC), the androgen-receptor isoform that is encoded by the splice variant 7 (AR-V7) is characterized by an absence of the ligand-binding domain that is the targ...

Abstract B18: Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent

Abstract African American (AA) men exhibit a nearly 2-fold higher incidence and 3-fold higher mortality rate from prostate cancer compared to Caucasian American (CA) men and disparities in tumor aggressiveness remain after controlling for social determinants of health. Previous work from our laboratory and others has revealed differences in gene expression that contribute to prostate cancer health disparities among AAs. Oncogenic signaling pathways exhibiting up-regulation more often in AA prostate cancer include androgen receptor (AR) and epidermal growth factor receptor (EGFR). Thus, novel therapeutic strategies capable of driving production of inhibitory AR and EGFR isoforms and capable of limiting aberrant constitutively active AR isoforms are urgently needed. Such strategies will increase our understanding of these molecular mechanisms underlying prostate cancer in AA men. In addition, such strategies have the potential to ultimately result in novel specific approaches for treatment that will help reduce prostate cancer disparities for AAs and will improve treatment of advanced stage disease in all men with aggressive disease driven by these mechanisms. Splice-switching oligonucleotides (SSOs) represent a novel therapeutic strategy to combat prostate cancer. Unlike the strategy of using RNA interference to inhibit the expression of a gene, SSOs simultaneously limit the production of pathogenic proteins and induce the expression of protein variants with therapeutic value. SSOs modulate pre-mRNA splicing by binding to target pre-mRNAs and blocking access of the splicing machinery to a particular splice site, and can be used to produce novel splice variants or correct aberrant splicing. Herein we have designed and synthesized novel SSOs targeting AR and EGFR pre-mRNAs to produce inhibitory splice variants and correct aberrant splicing as follows. We have synthesized a SSO to correct the aberrant splicing that leads to production of the constitutively active AR-V7 variant leading to restoration of ligand dependency. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the cryptic exon included in AR-V7, decreases AR-V7 RNA. In addition, we have synthesized a SSO to drive production of a naturally occurring dominant-negative AR variant (AR45) leading to interference with wild type AR transactivation activity. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the first exon of wild type AR and leads to inclusion of an alternative first exon encoding the unique N-terminal extension characterizing AR45, increases AR45 RNA. Furthermore, we have synthesized SSOs targeting exons in the transmembrane domain and the tyrosine kinase domain of EGFR to drive production of a soluble, naturally expressed inhibitory EGFR isoform and a dominant-negative EGFR isoform, respectively. Transfection of prostate cancer cells with these chemically modified SSOs increase RNA corresponding to these isoforms. Studies are underway to examine the effects of these SSOs on AR and EGFR signaling, respectively, and the effects of these SSOs on prostate tumor cell biology. These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs have the potential to further our understanding of the contribution of the targeted molecular mechanisms to AA prostate cancer and have the potential to yield novel therapeutic modalities to combat prostate cancer in AA men as well as men of all races with aggressive disease driven by these mechanisms. Citation Format: Jennifer A. Freedman, Timothy J. Robinson, Bonnie LaCroix, Brendon M. Patierno, Daniel J. George, Bruce A. Sullenger, Steven R. Patierno. Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B18.

Association of ARV7 expression with molecular and clinical characteristics in prostate cancer.

109 Background: Prostate cancer (PC) is the most common cancer in men over the age of 60 and the second leading cause of cancer mortality in the United States. Clinicopathological parameters such as Gleason score, tumor volume, surgical margins, prostate-specific antigen (PSA), Ki-67 index and clinical stage are used as prognostic markers for clinical outcomes. Identification of novel molecular markers could improve our understanding of the clinical behavior of this disease. Androgen receptor isoforms, in particular variant 7 (ARV7 or AR3) have been recently studied for elucidating their potential role in PC progression, associated with epithelial-mesenchymal transition (EMT), disease aggressiveness, increased proliferation and therapeutic resistance. Our study is analyzing the association of ARV7 mRNA expression to clinical characteristics and is analyzing the genomic data to identify differentially altered genes by ARV expression status, summarized as a potential functional network. Methods: We obtained the TCGA public dataset of prostate adenocarcinoma tumors (N=499) that included the clinical data, gene and isoform expression and mutation data. Cases were categorized into ARV7 over-expressing (ARV+) and normal or low expression (ARV –/N) by using a cut-off of upper 25th percentile of the background genomic expression. Analysis was performed in R and Perl by using custom-made scripts. Differentially altered genes and pathways were identified and were summarized as potential functional networks. Results: We categorized 30 out of the 499 tumors as ARV+. ARV7 over-expression was found to be significantly associated with older age at diagnosis (>70), advanced clinical stage, nodal involvement, high Gleason score and a poor therapeutic response. We also observed a trend towards shorter disease-free survival among ARV+ tumors. In addition, ARV+ tumors showed significantly higher number of mutations in 20 key regulatory pathways including Jak-STAT signaling, homologous recombination, ErbB and Wnt signaling pathways. Conclusions: ARV7 overexpression is associated with genomic alterations in key regulatory pathways and poorer clinical outcome in PC patients.

Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model.

Age- and Sex-Dependent Changes in Androgen Receptor Expression in the Developing Mouse Cortex and Hippocampus.

During the perinatal period, male mice are exposed to higher levels of testosterone (T) than females, which promotes sexual dimorphism in their brain structures and behaviors. In addition to acting via estrogen receptors after being locally converted into estradiol by aromatase, T also acts directly through androgen receptor (AR) in the brain. Therefore, we hypothesized that AR expression in the developing mouse cortex and hippocampus was sexually dimorphic. To test our hypothesis, we measured and determined AR mRNA and protein levels in mouse cortex/hippocampus collected on the day of birth (PN0) and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. We demonstrated that, as age advanced, AR mRNA levels increased in the cortex/hippocampus of both sexes but showed no sex difference. Two AR proteins, the full-length (110 kDa) and a smaller isoform (70 kDa), were detected in the developing mouse cortex/hippocampus with an age-dependent increase in protein levels of both AR isoforms at PN21 and a transient masculine increase in expression of the full-length AR protein on PN7. Thus, we conclude that the postnatal age and sex differences in AR protein expression in combination with the sex differences in circulating T may cause sexual differentiation of the mouse cortex/hippocampus.

Involvement of the Androgen and Glucocorticoid Receptors in Bladder Cancer.

Bladder cancer is encountered worldwide having been associated with a host of environmental and lifestyle risk factors. The disease has a male to female prevalence of 3 : 1. This disparity has raised the possibility of the androgen receptor (AR) pathway being involved in the genesis of the disease; indeed, research has shown that AR is involved in and is likely a driver of bladder cancer. Similarly, an inflammatory response has been implicated as a major player in bladder carcinogenesis. Consistent with this concept, recent work on anti-inflammatory glucocorticoid signaling points to a pathway that may impact bladder cancer. The glucocorticoid receptor- (GR-) α isoform has an important role in suppressing inflammatory processes, which may be attenuated by AR in the development of bladder cancer. In addition, a GR isoform that is inhibitory to GRα, GRβ, is proinflammatory and has been shown to induce cancer growth. In this paper, we review the evidence of inflammatory mediators and the relationship of AR and GR isoforms as they relate to the propensity for bladder cancer.

Prostate cancer characteristics associated with response to pre-receptor targeting of the androgen axis.

BackgroundFactors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth.MethodsWe characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy.ResultsIn LuCaP35 tumors (intra-tumoral T:DHT ratio 2∶1) dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone (337 vs.152 days, HR 2.8, p = 0.0015). In LuCaP96 tumors (T:DHT 10∶1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels (5-fold higher SRD5A1, 41 fold higher, 99-fold higher RL-HSD, p<0.0001 for both), reconstitution of intra-tumoral DHT (to ∼30% of untreated tumors), and ∼2 fold increased expression of full length AR. In contrast, LuCaP96 demonstrated higher levels of steroid catabolizing enzymes (6.9-fold higher AKR1C2, 3000-fold higher UGT2B15, p = 0.002 and p<0.0001 respectively), persistent suppression of intra-tumoral DHT, and 6–8 fold induction of full length AR and the ligand independent V7 AR splice variant. Human metastases demonstrated bio-active androgen levels and AR full length and AR splice-variant expression consistent with the range observed in xenografts.ConclusionsIntrinsic differences in basal steroidogenesis, as well as variable expression of full length and splice-variant AR, associate with response and resistance to pre-receptor AR ligand suppression. Expression of steroidogenic enzymes and AR isoforms may serve as potential biomarkers of sensitivity to potent AR-axis inhibition and should be validated in additional models.

AR-V7 and Resistance to Enzalutamide and Abiraterone in Prostate Cancer

BackgroundThe androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone.MethodsWe used a quantitative reverse-transcriptase–polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression–free survival), clinical or radiographic progression–free survival, and overall survival.ResultsA total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7–positive patients had lower PSA response rates than AR-V7–negative patients (0% vs. 53%, P=0.004) and shorter PSA progression–free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression–free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7–positive patients had lower PSA response rates than AR-V7–negative patients (0% vs. 68%, P=0.004) and shorter PSA progression–free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression–free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA.ConclusionsDetection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.)

New Emerging Androgenic Actions in the Regulation of Sperm Production and Function

Androgenic actions are determinant for sperm production and function and, thus, for male fertility. Androgens exert their effects by interaction with the androgen receptor (AR), a transcription factor that modulates gene expression in target-cells and tissues. Variants of AR protein have been identified in the testis, revealing a new complexity in androgen signaling pathways. In addition, androgens may evoke responses by controlling intracellular calcium (Ca2+) levels and/or activating Ca2+-dependent pathways. However, until recently the knowledge about the role of androgens controlling testicular expression and activity of membrane and intracellular Ca2+ regulatory proteins was very limited or inexistent. Also the function of Ca2+ in sperm maturation in the epididymis only recently started to be known. This review describes recent advances identifying new AR isoforms in the testis, as well as the novel actions of androgens as modulators of Ca2+ homeostasis in reproductive tract discussing the consequent impact for male fertility.

On the Origins of the Androgen Receptor Low Molecular Weight Species

Prostate cancer (CaP), a commonly diagnosed malignancy, is readily treated by androgen ablation. This treatment temporarily halts the disease, but castration-resistant neoplasms that are refractory to current therapies emerge. While these neoplasms are no longer dependent on physiological levels of androgens, they remain reliant on the expression of the androgen receptor (AR). There are multiple mechanisms by which CaP cells circumvent androgen ablation therapies. These include AR mutations that broaden ligand specificity, AR overexpression, AR activation by growth factors and cytokines, overexpression of AR co-activators, altered steroid metabolism, and a locus-wide histone transcriptional activation of some AR targets. This review focuses on a more recently described mechanism: the expression of low molecular weight AR species that are missing the ligand-binding domain and function independently of ligand to drive proliferation. The etiology, biological activity, unique features, predictive value, and therapeutic implication of these androgen receptor isoforms are discussed in depth.

Abstract 4772: Efficacy of VT-464, a novel selective inhibitor of cytochrome P450 17,20-lyase, in castrate-resistant prostate cancer models.

Abstract Cytochrome P450 17α-hydroxylase /17,20-lyase (CYP17) is a validated treatment target for metastatic castrate-resistant prostate cancer (mCRPC). Abiraterone acetate (AA; Zytiga) inhibits 17α-hydroxylase and 17,20-lyase reactions catalyzed by CYP17 and thus reduces androgen biosynthesis. However, co-administration of prednisone is required to suppress the mineralocorticoid excess from 17α-hydroxylase inhibitions. VT-464, a non-steroidal small molecule inhibits androgen production without mineralocorticoid excess or cortisol depletion by selective inhibition of CYP17 17,20-lyase. We determined the impact of VT-464 on tumor growth of a mCRPC xenograft, MDA-PCa-133, in vivo, and on androgen signaling in C4-2B prostate cancer cells in vitro. The MDA-PCa-133 xenograft is derived from a clinical CRPC bone metastasis. Subcutaneous MDA-PCa-133 tumor expresses PSA, full-length androgen receptor (AR) and AR-V7 isoform. We determined the effect of VT464 and AA on MDA-PCa-133 growing in tumor-bearing castrated male mice: randomization into three groups; oral treatment with vehicle only, VT-464, (100 mg/kg bid), or AA (100 mg/kg bid) for 25 days. Both VT-464 and AA reduced tumor volume (&amp;gt;two fold compared to vehicle; p&amp;lt;0.05). These results indicate that selective VT-464 CYP17 lyase inhibition is as effective as AA CYP17 inhibition in this model. We determined the effects of VT-464 on AR-dependent luciferase reporter activity and expression of AR signaling genes, PSA and NKX3.1, in cultured C4-2B prostate cancer cells with or without VT-464 or AA treatment. VT-464 or AA highly reduced AR-dependent reporter activity and specific expression of PSA and NKX3.1. AR-dependent signaling in the presence of VT-464 or AA was restored however by 10 nM DHT, consistent with VT-464- or AA-mediated depletion of androgen in C4-2B cells. The AR-dependent reporter activity was also measured following overexpression of full-length AR or AR-V7 isoform in C4-2B cells. Full-length AR-dependent reporter activity was (&amp;gt;80%) inhibited; AR-V7-dependent reporter activity was inhibited by 50% by 1 μM VT-464 or AA. We conclude that VT-464 inhibited AR signaling as effectively as AA in C4-2B prostate cancer cells. The findings suggest that AR signaling in cells, which express high levels of AR isoform (AR-V7) are more resistant to androgen biosynthesis inhibitors than those with wild type AR. Preliminary observations suggest C4-2B cells with AR-V7 are less sensitive to AA than VT- 464. Ongoing clinical and co-clinical studies will determine if the observed differences in steroid profile and relative efficacy of VT-464 compared to AA are clinically and biologically meaningful. Citation Format: Sankar N. Maity, Guanglin Wu, Jing-fang Lu, Joel R. Eisner, Stephen W. Rafferty, Robert J. Schotzinger, William R. Moore, Christopher J. Logothetis, John C. Araujo, Eleni Efstathiou. Efficacy of VT-464, a novel selective inhibitor of cytochrome P450 17,20-lyase, in castrate-resistant prostate cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2013-4772

Abstract 3574: Studies of castrate resistant 22Rv1 cells identifies AR regulated interrelated networks of transcription factors, co-regulators, chromatin, and nuclear scaffolding proteins.

Abstract Prostate cancer development, progression and acquisition of castrate resistance are reliant on the activity of the androgen receptor (AR), a transcription factor that governs the proliferation of prostate cancer cells. Multiple studies have focused on the principle components that are critical for cell cycle transversal and these studies have identified members of the E2F/RB family, c-Myc, cyclins and cyclin dependent kinase inhibitors as key proteins that are instrumental in prostate cell proliferation and development of castrate resistance. But the mechanism by which the AR dominates this process is unclear. To identify the link between the AR and cell cycle control we used 22Rv1 cells which are castrate resistant due to the expression of low molecular weight AR forms that are missing the ligand binding domain. However, a siRNA mediated decrease in AR expression induces a growth arrest confirming that proliferation is AR dependent. RNA-seq analysis of gene expression at castrate levels of androgen and following AR ablation identified transcripts that were AR dependent. This methodology reduced the number of AR regulated transcripts, by excluding transcripts regulated by super-physiological levels of androgens. The AR regulated transcripts included previously identified genes (PMEPA1, C1orf116, PCDH7, APP) including NFAT a gene shown to be regulated by a low molecular weight AR isoform. The analysis also revealed that interrelated networks of transcription factors and co-factors were AR transactivated or repressed. These included proteins such as the LBR, LMNB, TMPO and SATB1 that do not directly promote transcription, but rather link chromatin to the nuclear membrane or scaffolding, suggesting that the AR may modulate gene expression through epigenetics mechanisms. Network analysis showed connections between these proteins and cell cycle components including E2Fs and Myc where a number of the regulated transcripts are known E2F or Myc targets. In addition, this analysis identified a number of non-coding RNAs; some that have been previously described and some that have yet to be annotated. qPCR studies found that a number of the transcripts regulated in 22Rv1 cells are also AR regulated in LNCaP cells. Coupled with ChIP-seq data, the study characterizes the link between the AR and the cell cycle machinery and chromatin modulating proteins. Citation Format: Maria Mudryj, Stephen J. Libertini, Alan P. Lombard. Studies of castrate resistant 22Rv1 cells identifies AR regulated interrelated networks of transcription factors, co-regulators, chromatin, and nuclear scaffolding proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2013-3574

Modeling Truncated AR Expression in a Natural Androgen Responsive Environment and Identification of RHOB as a Direct Transcriptional Target

Recent studies identifying putative truncated androgen receptor isoforms with ligand-independent activity have shed new light on the acquisition of androgen depletion independent (ADI) growth of prostate cancer. In this study, we present a model system in which a C-terminally truncated variant of androgen receptor (TC-AR) is inducibly expressed in LNCaP, an androgen-dependent cell line, which expresses little truncated receptor. We observed that when TC-AR is overexpressed, the endogenous full length receptor (FL-AR) is transcriptionally downmodulated. This in essence allows us to “replace” FL-AR with TC-AR and compare their individual properties in exactly the same genetic and cellular background, which has not been performed before. We show that the TC-AR translocates to the nucleus, activates transcription of AR target genes in the absence of DHT and is sufficient to confer ADI growth to the normally androgen dependent LNCaP line. We also show that while there is significant overlap in the genes regulated by FL- and TC-AR there are also differences in the respective suites of target genes with each AR form regulating genes that the other does not. Among the genes uniquely activated by TC-AR is RHOB which is shown to be involved in the increased migration and morphological changes observed in LN/TC-AR, suggesting a role of RHOB in the regulation of androgen-independent behavior of prostate cancer cells.

The activity of the androgen receptor variant AR‐V7 is regulated by FOXO1 in a PTEN‐PI3K‐AKT‐dependent way

The androgen receptor (AR) AR-V7 splice isoform is a constitutively active outlaw transcription factor. Transition of prostate cancer (PC) to the castration-resistant phenotype correlates with AR-V7 accumulation, suggesting that PC progression in patients refractory to conventional therapy is due to the activity of this AR isoform. The mechanism of AR-V7 constitutive activation is not known. We analyzed potential signaling pathways associated with AR-V7 constitutive activation in PTEN (-) PC-3 and LNCaP cells. We used transient and stable transfection, reporter gene assay, RNAi technology together with a number of kinase inhibitors to determine if AR-V7 activation is linked to a kinase-dependent signaling pathway. In these cell lines, AR-V7 transcriptional activity was inhibited by LY294002, Wortmanin, and AKT inhibitor II. Analysis of the contributing mechanisms demonstrated the involvement of the Phosphatidylinositol 3-kinase (PI3K)-AKT-FOXO1 signaling pathway, and a significant reduction of AR-V7 constitutive activity under conditions of PTEN reactivation. Our study identifies a pathway regulating AR-V7 constitutive activity and potential therapeutic targets for the treatment of castration-resistant PC.

Abstract 2924: Constitutively active androgen receptor splice variants function independently of full-length androgen receptor in a cell-based model of castrate-resistant prostate cancer

Abstract Recent studies suggest that progression to castrate-resistant prostate cancer (CRPC) is due to generation of androgen receptor (AR) splice variants, lacking the ligand binding domain. These AR variants originally identified in cell-based models of CRPC, have also been detected in benign and malignant human prostate tissue, with the highest levels observed in late state CRPC. The diversity and complexity of AR variants is well appreciated but not fully understood. Using RT-PCR we assessed the expression of a number of AR variants in cell-based models of both castrate-resistant and androgen-dependent prostate cancer. Several AR variants were expressed in both 22Rv1 and VCaP cells, but not in LNCaP cells. Coding sequences for AR variants were cloned into expression vectors and constitutive transcriptional activity was observed using a luciferase reporter assay. Augmented transcriptional activity of AR variants was observed in cells that express full-length AR, when full-length AR was silenced using shRNA targeting exons 7/8. This observation suggests the constitutive activity of AR variants may be independent of full-length AR. We profiled the AR variant transcriptome in 22Rv1 cells, using the Illumina HumanHT12 v4 whole-genome expression array platform. shRNA targeting exon 1 was used to silence all AR isoforms and shRNA targeting exons 7/8 was used to silence full-length AR. 5,256 genes were modulated by knocking down all AR isoforms and 5,086 genes were modulated by knocking down full-length AR. A total of 3,714 genes were common to both groups, leaving 1542 genes modulated by AR variants and 1372 modulated by full-length AR (p&amp;lt;0.05). The represents a subset of genes differentially regulated by AR variants independently of full length AR. Aggregate function of multiple AR variants may confer a CRPC phenotype independent of full-length AR. Pathway analysis of these genes differentially regulated by AR variants may reveal potential targets for therapeutic intervention in patients who progress despite androgen deprivation or anti-androgen therapy. (Supported by NIH grants CA125747, CA091956 and CA121277, T.J. Martell Foundation and the Mayo Clinic Prostate Cancer SPORE). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2924. doi:1538-7445.AM2012-2924

Differential expression and function of AR isoforms in prostate cancer

The androgen receptor (AR) plays a key role in prostate cancer (PCa). Two isoforms of AR, are AR-A and -B, which differ by a lack of the first 187 amino acids in the NH2-terminal transactivation domain of AR-A. Since little is known about the expression and basic function of the AR-A/B isoforms in prostate cancer, the aim of this study was to analyze this possible association. The AR-A, -B and AR-A/B ratio was determined in the tissues of healthy controls, benign prostate hyperplasia (BPH) and PCa by means of Western blot analysis and immunofluorescence. The elevation of AR-A, and -B, as well as the AR-A/B ratio with regard to Gleason scores, were assessed in prostate cancer compared to BPH and normal prostate. In order to further investigate the role of AR A/B isoform function, we transfected PC3 cells with an AR or AR-A expression vector. The overexpression of AR-A and -B significantly decreased the invasion and proliferation of PC3 cells. However, the overexpression of AR-A further decreased proliferation but accelerated the invasion of PC3 cells compared to AR-B. In conclusion, the elevation of AR-A and -B, and the AR-A/B ratio, is associated with prostate cancer occurrence and progression. Furthermore, AR-A could provide a new potential therapy with regard to the decrease in the invasion and proliferation of prostate cancer cells. Our study provides insight into further understanding the biological role of AR-A in its interaction with AR-B and its impact on PCa clinical treatment.

The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells

Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16INK4A and p21CIP1, but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation.

Alternatively spliced androgen receptor variants

Alternative splicing is an important mechanism for increasing functional diversity from a limited set of genes. Deregulation of this process is common in diverse pathologic conditions. The androgen receptor (AR) is a steroid receptor transcription factor with functions critical for normal male development as well as the growth and survival of normal and cancerous prostate tissue. Studies of AR function in androgen insensitivity syndrome (AIS) and prostate cancer (PCa) have demonstrated loss-of-function AR alterations in AIS and gain-of-function AR alterations in PCa. Over the past two decades, AR gene alterations have been identified in various individuals with AIS, which disrupt normal AR splicing patterns and yield dysfunctional AR protein variants. Recently, altered AR splicing patterns have been identified as a mechanism of PCa progression and resistance to androgen depletion therapy. Several studies have described the synthesis of alternatively spliced transcripts encoding truncated AR isoforms that lack the ligand-binding domain, which is the ultimate target of androgen depletion. Many of these truncated AR isoforms function as constitutively active, ligand-independent transcription factors that can support androgen-independent expression of AR target genes, as well as the androgen-independent growth of PCa cells. In this review, we will summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologic conditions of AIS and PCa.

Abstract 4556: AR intragenic rearrangement and altered androgen receptor mRNA splicing in castration-resistant prostate cancer

Abstract Androgen depletion for advanced prostate cancer (PCa) targets activity of the androgen receptor (AR), a steroid receptor transcription factor required for PCa growth. The emergence of lethal castration-resistant PCa (CRPCa) is marked by aberrant re-activation of the AR despite ongoing androgen depletion. Recently, alternative splicing has been described as a mechanism giving rise to COOH-terminally truncated, constitutively active AR isoforms that can support the CRPCa phenotype. However, the pathologic origin of these truncated AR isoforms has not been described. The goal of this study was to investigate alterations in AR expression arising in a cell-based model of PCa progression driven by truncated AR isoform activity. We demonstrate that stable, high-level expression of truncated AR isoforms in 22Rv1 CRPCa cells coincides with intragenic rearrangement of a ∼35kb AR genomic segment, which contains several alternative AR exons. Analysis of metastatic CRPCa tissues indicated that related AR copy number alterations are frequent and often occur in conjunction with high-level AR amplification. Nucleotide-level resolution of the break fusion junction in 22Rv1 cells revealed a DNA repair signature consistent with a microhomology-mediated, replication-based mechanism of origin. Together, these data provide the first report of AR intragenic rearrangements in CRPCa and a link with pathologic expression of truncated AR isoforms in a cell-based model of PCa progression. 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 4556. doi:10.1158/1538-7445.AM2011-4556