Androgen Receptor Reactivation Research Articles

Abstract 1859: Discerning the molecular basis of DNA-PK pro-tumorigenic functions and translational capacity as a therapeutic target in prostate cancer

Abstract Prostatic adenocarcinoma (PCa) is dependent on androgen receptor (AR) signaling at all stages of disease, as AR activation induces both cell proliferation and survival. Though organ-confined disease can be treated, the response is transient and reactivation of AR results in incurable state of disease (castration resistant prostate cancer, CRPC). Thus, there is a significant need to discern the mechanisms by which aberrant AR activity arises, and to develop new means to clinically target advanced disease. Recently, AR activation was found to promote tumor cell survival and proliferation through a feed-forward loop involving the repair factor DNA-dependent protein kinase (DNA-PK). Emerging new data correlates elevated DNA-PK expression with poor prognosis and identifies DNA-PK as a driver of metastatic signaling. Although the role of DNA-PK activity in DNA repair is well characterized, the underpinning mechanisms and consequences of DNA-PK in transcriptional regulation are understudied. Data to be discussed will build on our new preliminary DNA-PK interactome data to identify sequence-specific transcription factors found in complex with DNA-PK. In order to molecularly dissect DNA-PK transcriptional events, the DNA-PK cistrome and transcriptome will be defined using clinically relevant models, resulting in a detailed understanding of DNA-PK mediated transcriptional regulation. The correlation of high expression of DNA-PK with poor prognosis in advanced PCa, as well as the known roles of DNA-PK in cancer-relevant pathways make DNA-PK a viable therapeutic target for PCa. Despite the current clinical assessment of DNA-PK inhibitors in multiple tumor types, there are no existing trials targeting PCa. Additional studies that assess the efficacy of current DNA-PK inhibitors in PCa will be presented and used to determine the best clinical space for their use. Citation Format: Emanuela Dylgjeri, Jonathan Goodwin, Karen Knudsen. Discerning the molecular basis of DNA-PK pro-tumorigenic functions and translational capacity as a therapeutic target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1859. doi:10.1158/1538-7445.AM2015-1859

The Deubiquitinating Enzyme USP7 Regulates Androgen Receptor Activity by Modulating Its Binding to Chromatin

The androgen receptor (AR), a nuclear receptor superfamily transcription factor, plays a key role in prostate cancer. AR signaling is the principal target for prostate cancer treatment, but current androgen-deprivation therapies cannot completely abolish AR signaling because of the heterogeneity of prostate cancers. Therefore, unraveling the mechanism of AR reactivation in androgen-depleted conditions can identify effective prostate cancer therapeutic targets. Increasing evidence indicates that AR activity is mediated by the interplay of modifying/demodifying enzymatic co-regulators. To better understand the mechanism of AR transcriptional activity regulation, we used antibodies against AR for affinity purification and identified the deubiquitinating enzyme ubiquitin-specific protease 7, USP7 as a novel AR co-regulator in prostate cancer cells. We showed that USP7 associates with AR in an androgen-dependent manner and mediates AR deubiquitination. Sequential ChIP assays indicated that USP7 forms a complex with AR on androgen-responsive elements of target genes upon stimulation with the androgen 5α-dihydrotestosterone. Further investigation indicated that USP7 is necessary to facilitate androgen-activated AR binding to chromatin. Transcriptome profile analysis of USP7-knockdown LNCaP cells also revealed the essential role of USP7 in the expression of a subset of androgen-responsive genes. Hence, inhibition of USP7 represents a compelling therapeutic strategy for the treatment of prostate cancer.

Recepteur d'origine nantais (RON), more than a kinase: Role in castrate-resistant prostate cancer.

Prostate cancer (PCA) is the second leading cause of cancer-related deaths in men in the United States. It is natural for a hormone-driven malignancy such as prostate cancer that androgen deprivation therapy (ADT) would be the preferred treatment for clinical disease management. However, after initial treatment response a vast majority of patients develop metastatic castrate-resistant prostate cancer (CRPC), which is fatal. While great headway has been made to understand the possible mechanisms that drive castrate-resistant disease, a bonafide cure remains elusive. Reactivation of androgen receptor (AR) signaling partly contributes to the emergence of CRPC. Here we briefly examine some of the known mechanisms of AR reactivation including intratumoral synthesis of androgens, modulation of AR coregulators, and AR variants with constitutive activity as well as activation of receptor tyrosine kinases. We primarily focus on the emerging dual function of the receptor tyrosine kinase (recepteur d'origine nantais; RON) as a traditional tyrosine kinase and transcription factor. We further discuss activation of RON as an alternate mechanism in the development of CRPC and available therapeutic approaches for clinical management of CRPC by combined inhibition of RON and AR.

Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

Implications of ubiquitin ligases in castration-resistant prostate cancer.

Significant advances have been made in the study of ubiquitination-mediated regulation of androgen receptor (AR). This review will highlight the latest developments in the mechanisms by which E3 ubiquitin ligases control AR activity, with implications in castration-resistant prostate cancer (CRPC). Several ubiquitin ligases have been identified to interact with and ubiquitinate AR, and consequently regulate the AR transcriptional programme. Different ubiquitin ligases can use distinct mechanisms to modulate the expression of AR target genes, including local turnover of AR chromatin complex, recruitment of AR coactivators and global AR stability. The expression or activity of ubiquitin ligases can be altered in prostate cancer and thus contribute to the growth of androgen-insensitive prostate cancer cells by modulating the AR transcriptional activity. Understanding the regulation of AR transcriptional activity by ubiquitin ligases will contribute to the elucidation of mechanisms underlying AR reactivation that is believed to drive the development of CRPC. Ubiquitin ligases could potentially serve as promising targets for developing therapeutics in the treatment of advanced prostate cancers.

Targeting the adaptive molecular landscape of castration-resistant prostate cancer.

Castration and androgen receptor (AR) pathway inhibitors induce profound and sustained responses in advanced prostate cancer. However, the inevitable recurrence is associated with reactivation of the AR and progression to a more aggressive phenotype termed castration-resistant prostate cancer (CRPC). AR reactivation can occur directly through genomic modification of the AR gene, or indirectly via co-factor and co-chaperone deregulation. This mechanistic heterogeneity is further complicated by the stress-driven induction of a myriad of overlapping cellular survival pathways. In this review, we describe the heterogeneous and evolvable molecular landscape of CRPC and explore recent successes and failures of therapeutic strategies designed to target AR reactivation and adaptive survival pathways. We also discuss exciting areas of burgeoning anti-tumour research, and their potential to improve the survival and management of patients with CRPC.

315 Biological characterization of TAS3681, a new type of androgen receptor antagonist with androgen receptor downregulating activity

Background: Recent evidence from both preclinical and clinical studies is consistent with the importance of reactivation of androgen receptor (AR) signaling in a majority of castration-resistant prostate cancer (CRPC). This knowledge has driven the clinical development of new inhibitors of androgen production (e.g., abiraterone) and AR signaling (e.g., enzalutamide). However, resistance to these new therapies has already been reported. Reactivation of AR signaling occurs by several mechanisms in CRPC. In the present study, we describe the biological characterization of TAS3681, which is a new AR antagonist with AR downregulation activity, and propose this concept as a potential new approach for the treatment of CRPC. Methods: For cell growth assay, prostate cancer cells were treated with androgen and TAS3681 in steroid depleted media for 3 days, and the living cells were determined by using wst-8 reagent. For assay of AR transactivation, COS-7 cells were transiently transfected with androgenresponsive reporter gene construct and expression vectors encoding wt or mu ARs. The transfected cells were treated with TAS3681 and androgen in steroid depleted media for 24 h, and luciferase activity was measured. For an in vivo pharmacodynamic (PD) assay to confirm AR downregulation in tumor, TAS3681 was orally dosed in CRPC tumor xenograft model. Tumor was isolated and AR level in tumor was determined by Western blot. Results: In cell growth assay, TAS3681 suppressed the growth of AR positive prostate cancer cells but did not affect that of AR-negative DU145 prostate cancer cells, indicating a dependency on AR for efficacy. TAS3681 did not stimulate AR nuclear translocation and suppressed wt and mu ARs (including F876L) transactivation in cells, indicative of its pure AR antagonist profile. In contrast to enzalutamide and bicalutamide, TAS3681 effectively suppressed androgen-independent AR transactivation by growth factors and cytokines via AR downregulation activity. Interestingly, in prostate cancer cells which express full-length and splice variant AR, TAS3681 reduced the expression of both ARs. Moreover, TAS3681 treatment effectively decreased AR level in CRPC tumors in vivo. Conclusion: Our findings suggest that TAS3681, a new type of AR antagonist with AR downregulation activity, has the potential to circumvent resistance to current and 2nd-generation therapies targeting AR signaling and could provide a new therapeutic strategy in the treatment of CRPC.

Abstract 1796: Novel triazole nucleoside inhibits heat shock factor 1 and androgen receptor to fight against prostate cancer

Abstract Prostate cancer is one of the most commonly diagnosed cancers and leading cause of cancer-related mortality in men in developed countries. While most patients initially respond to first-line androgen deprivation therapy (ADT), many become refractory to hormonal and other conventional therapies and eventually develop castration-resistant prostate cancer (CRPC) with time. CRPC is often characterized by the reactivation of androgen receptor (AR) and constitutes the most important therapeutic target in CRPC. It is known that AR is bound to heat shock protein (HSPs) complex to maintain its stable and mature conformation with high affinity to androgen. Targeting HSPs are therefore to result in perturbation of AR. Using a lead optimization process, we have recently identified a novel triazole nucleoside VM-1 which can down-regulate heat shock transcription factor 1 (HSF1), the dominant upstream transcriptional regulator for HSPs. The down-regulation of HSF1 by VM-1 leads to the simultaneous suppression of multiple HSPs and results in growth inhibition of various prostate cancer cells. Interestingly, VM-1 displays more potent antiproliferation activity in AR-positive LNCaP, C4-2 and 22RV-1 cells than AR-negative PC-3 cells. Our further studies have showed that VM-1 reduces the expression of AR and a panel of AR targeted genes as well as inhibits AR transactivation, which consequently leads to effective caspase-dependent apoptosis in LNCaP, C4-2 and 22RV-1 cells. Moreover, VM-1 arrests cell cycle of prostate cancer cells in AR-governed phase suggesting the potential functionality of this compound involved in the crosstalk between AR and cell cycle pathways. Taken together, the above data suggest the role of HSF1 in regulation of AR, and the inhibition on both HSF1 and AR contribute to the activity of VM-1 in prostate cancer cells. Therefore, VM-1 may serve as a novel structural motif in the search of anticancer candidates to fight against prostate cancer via inhibiting HSF1 and AR. Citation Format: Yi Xia, Eliana Beraldi, Amina Zoubeidi, Ling Peng, Martin Gleave. Novel triazole nucleoside inhibits heat shock factor 1 and androgen receptor to fight against 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 1796. doi:10.1158/1538-7445.AM2014-1796

Lyn tyrosine kinase regulates androgen receptor expression and activity in castrate-resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) progression is a complex process by which prostate cells acquire the ability to survive and proliferate in the absence or under very low levels of androgens. Most CRPC tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes owing to reactivation of AR. Protein tyrosine kinases have been implicated in supporting AR activation under castrate conditions. Here we report that Lyn tyrosine kinase expression is upregulated in CRPC human specimens compared with hormone naive or normal tissue. Lyn overexpression enhanced AR transcriptional activity both in vitro and in vivo and accelerated CRPC. Reciprocally, specific targeting of Lyn resulted in a decrease of AR transcriptional activity in vitro and in vivo and prolonged time to castration. Mechanistically, we found that targeting Lyn kinase induces AR dissociation from the molecular chaperone Hsp90, leading to its ubiquitination and proteasomal degradation. This work indicates a novel mechanism of regulation of AR stability and transcriptional activity by Lyn and justifies further investigation of the Lyn tyrosine kinase as a therapeutic target for the treatment of CRPC.

Rapid Induction of Androgen Receptor Splice Variants by Androgen Deprivation in Prostate Cancer

Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer that progresses after castration (castration-resistant prostate cancer; CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear. Prostate cancer xenografts were examined to identify mechanism of progression after castration and abiraterone. AR reactivation in abiraterone-resistant VCaP xenografts was not associated with restoration of intratumoral androgens or alterations in AR coregulators. In contrast, mRNA encoding full-length AR (AR-FL) and a constitutively active splice variant (AR-V7) were increased compared with xenografts before castration, with an increase in AR-V7 relative to AR-FL. This shift toward AR-V7 was due to a feedback mechanism whereby the androgen-liganded AR stimulates expression of proteins that suppress generation of AR-V7 relative to AR-FL transcripts. However, despite the increases in AR-V7 mRNA, it remained a minor transcript (<1%) relative to AR-FL in resistant VCaP xenografts and CRPC clinical samples. AR-V7 protein expression was similarly low relative to AR-FL in castration-resistant VCaP xenografts and androgen-deprived VCaP cells, but the weak basal AR activity in these latter cells was further repressed by AR-V7 siRNA. AR-V7 at these low levels is not adequate to restore AR activity, but its rapid induction after androgen deprivation allows tumors to retain basal AR activity that may be needed for survival until more potent mechanisms emerge to activate AR. Agents targeting AR splice variants may be most effective when used very early in conjunction with therapies targeting the AR ligand-binding domain.

Synthesis and in vitro characterization of ionone-based compounds as dual inhibitors of the androgen receptor and NF-κB

Current therapeutic strategy for advanced prostate cancer is to suppress the androgen receptor (AR) signaling. However, lethal castration-resistant prostate cancer (CRPC) arises due to AR reactivation via multiple mechanisms, including mutations in the AR and cross-talk with other pathways such as NF-κB. We have previously identified two ionone-based antiandrogens (SC97 and SC245), which are full antagonists of the wild type and the clinically-relevant T877A, W741C and H874Y mutated ARs. Here, we discovered SC97 and SC245 also inhibit NF-κB. By synthesizing a series of derivatives of these two compounds, we have discovered a novel compound 3b that potently inhibits both AR and NF-κB signalling, including the AR F876L mutant. Compound 3b showed low micromolar antiproliferative activites in C4-2B and 22Rv1 cells, which express mutated ARs and are androgen-independent, as well as DU-145 and PC-3 cells, which exhibit constitutively activated NF-κB signalling. Our studies indicate 3b is effective against the CRPC cells.

Molecular Pathogenesis and Progression of Prostate Cancer

Prostate cancer (PCa) is the most commonly diagnosed noncutaneous malignancy and second leading cause of cancer-related deaths in US males. Clinically, locally confined disease is treated surgically and/or with radiation therapy. Invasive disease, however, must be treated with pharmacological inhibitors of androgen receptor (AR) activity, since disease progression is fundamentally reliant on AR activation. However, despite initially effective treatment options, recurrent castration-resistant PCa (CRPC) often occurs due to aberrant reactivation of AR. Additionally, it is appreciated that many other signaling molecules, such as transcription factors, oncogenes, and tumor suppressors, are often perturbed and significantly contribute to PCa initiation and progression to incurable disease. Understanding the interplay between AR signaling and other signaling networks altered in PCa will advance therapeutic approaches. Overall, comprehension of the molecular composition promoting neoplastic growth and formation of CRPC is paramount for developing durable treatment options.

Highlights of This Issue

Lung cancer is one of the most prevalent and deadliest cancers worldwide. A major risk factor in the disease comes from the carcinogens produced from cigarette smoking. As such, understanding the cellular mechanisms of detoxification is of general interest. In the current study, mRNA expression analysis indicated that the detoxification enzyme glutathione S-transferase GST-M2 and connective tissue growth factor CCN2 (CTGF) were reduced in lung tumor tissue. Overexpression of GST-M2 increased CTGF expression, reduced cellular migration, and inhibited tumor growth and metastasis in animal models. Importantly, higher expression of GST-M2 and CCN2 using real-time PCR is associated with a favorable prognosis in non–small cell lung cancer (NSCLC). Overall, this study reveals the potential of GST-M2 as a novel tumor suppressor via inhibiting tumor growth and metastasis, which has been demonstrated in vitro and in animal models. The antitumor effects of GST-M2 and CCN2 activity in animal models have provoked an intense interest in the development of small-molecule activators of GST-M2 and CCN2 (and their gene therapy) to prevent NSCLCs in the future.As tumors grow, there is an increased demand for nutrients and oxygen. Therefore, intratumoral hypoxia is considered a major driving force in breast cancer progression and has been linked to metastasis. Gilkes and colleagues demonstrated that hypoxia-inducible factor 1 (HIF-1) mediated increased expression of collagen hydroxylases (PLOD1 and PLOD2), which modify type I collagen prior to its secretion into the extracellular matrix (ECM). In addition, human breast cancer specimens stratified by elevated PLOD2 expression were associated significantly with increased patient mortality. In animal models, PLOD2 knockdown resulted in a marked decrease in tumor stiffness, local tissue invasion, and metastasis to lymph nodes and lungs without affecting primary tumor growth. Taken together, HIF-1 stimulates PLOD2 expression and collagen biogenesis to promote metastasis of hypoxic breast cancer cells. Thus, specific inhibitors of HIF-1 and/or collagen hydroxylases may be of clinical benefit for preventing breast cancer metastasis.The primary treatment for advanced/metastatic prostate cancer involves androgen deprivation therapy. Unfortunately, recurrence develops due to reactivation of androgen receptor (AR) activity resulting in castration-resistance and frequent osteoblastic bone metastasis. Based on genomic analyses, Zheng and colleagues mechanistically identified WNT7B as a novel target of AR in castration-resistant prostate cancer (CRPC). Manipulation of WNT7B levels promoted CRPC cell growth under androgen-deprived conditions and activated a noncanonical WNT pathway involving protein kinase C (PKC) isozymes. Critically, WNT7B was selectively upregulated in human xenografts that induce an osteoblastic reaction when grown in bone. These results establish a connection between AR and WNT signaling and suggest a unique opportunity to develop effective therapies for advanced prostate cancer by targeting the AR-WNT7B-PKCs axis, which would inhibit CRPC growth and prevent the formation of a metastatic reservoir in bone.Considered a major driver of invasive solid tumor progression, epithelial-mesenchymal transformation (EMT) is a cellular reprogramming by which malignant epithelial cells generally lose their cell–cell adhesion properties to become highly invasive. Austin and colleagues used mesenchymally transformed breast cancer cells to demonstrate that the small molecule sarasinoside A1, at noncytotoxic doses, reversed a number of aspects of EMT. Sarasinoside A1 inhibited cell invasion and promoted cell adhesion in three-dimensional cultures. Interestingly, sarasinoside A1 also initiated a morphologic re-epithelialization and stabilized the cytoskeleton at sites of nascent cell–cell contact. Combined, these findings indicate the potential to impede tumor invasion and metastasis without the need to reinduce the expression of cell adhesion molecules, such as E-cadherin, that are often lost or silenced during mesenchymal transformation.

Abstract 4771: Regulatory role of an orphan nuclear receptor LRH-1 in castration-resistant growth of prostate cancer cells.

Abstract The advanced development of castration-resistant prostate cancer (CRPC) in patients upon androgen-deprivation therapy is generally believed to be mostly mediated by reactivation of androgen receptor (AR) signaling or its bypass with mechanisms involved, including clonal selection of androgen-independent or stem cell-like cell populations, AR hypersensitivity due to AR overexpression, and ligand promiscuity or independence by AR mutations, intratumoral conversion of adrenal androgens to high affinity AR ligands. Besides, the de novo steroid synthesis from cholesterol in CRPC has also been proposed. In this study, we found that the nuclear receptor liver receptor homolog-1 (LRH-1) might contribute to the CRPC growth or de novo steroid synthesis in prostate cancer cells through its positive regulation on expression of several critical enzymes in steroidogenesis, including CYP17A1, HSD3B1, HSD3B2 and StAR. Functional analyses showed that overexpression of LRH-1 could induce higher resistance to antiandrogen bicalutamide and steroid-depleted culture condition in AR-positive androgen-sensitive (LNCaP) but not in AR-negative androgen-insensitive (DU145) prostate cancer cells. In vivo tumorigenicity study showed that LNCaP-LRH-1 cells grew more aggressively in castrated or intact SCID mice, contrary to LNCaP-vector control cells that did not grow in castrated mice. Importantly, liquid chromatography-tandem mass spectrometry showed that intratumoral androgen concentrations (testosterone and DHT) were significantly higher in tumors formed by LNCaP-LRH-1 cells than those by LNCaP-vector control cells. Treatment of LNCaP-LRH-1 cells with a CYP17A1 inhibitor abiraterone could restore their sensitivity to bicalutamide and steroid-depleted culture condition. Gene knockdown of endogenous LRH-1 in AR-positive VCaP prostate cancer cells could not only suppress the mRNA expression of steroidogenic enzymes but also enhance their sensitivity to antiandrogen. Together, our results indicated that LRH-1 might play a regulatory role in advanced castration-resistant growth of prostate cancer via positive control on de novo steroid synthesis in prostate cancer cells. (This work was supported by a Direct Grant for Research 2011/2012 and a RGC-GRF grant 2009/2010) Citation Format: Lijia Xiao, Shan Yu, Wendy W.L. Hsiao, Franky Leung Chan. Regulatory role of an orphan nuclear receptor LRH-1 in castration-resistant growth of prostate cancer cells. [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 4771. doi:10.1158/1538-7445.AM2013-4771

Abstract 2986: Mechanism of androgen receptor (AR) silencing in small cell prostate carcinoma.

Abstract Small cell prostate cancer (SCPC) is an androgen receptor (AR) negative variant that often emerges in the castration-resistant progression of typical AR-positive prostate adenocarcinomas. Its presence predicts for an aggressive clinical course with poor response to hormonal therapies and a short median survival. Evidence suggests that the emergence of (SCPC) occurs through cellular transdifferentiation. Therefore, we hypothesized that loss of AR gene transcription may depend on epigenetic events. Given the central role of AR in prostate cancer, we reasoned that identifying the mechanisms involved in its downregulation could improve our understanding of how the lethal SCPC subtype arises. Here we investigated the DNA methylation and histone modifications of AR in a group of six prostate tumor xenografts developed from men with CRPC (two AR-positive [MDA PCa 170.4 and MDA PCa 180.30] and four AR-negative SCPC [MDA PCa 144.13, MDA PCa 144.4, MDA PCa 155 and MDA PCa 146.10]) and three established prostate cancer cell lines (one AR-positive [LNCaP] and two AR-negative [PC3 and DU145]). We first evaluated the methylation status of selected CpG sites in the AR promoter-associated CpG island using bisulfite-sequencing followed by pyrosequencing analysis. Except for the cancer cell lines PC-3 and DU145, all other cell lines and xenografts were unmethylated regardless of the AR expression status. Next, we used chromatin immunoprecipitation (ChIP) to evaluate marking of the AR promoter and a putative proximal enhancer by active (H3K4me3 and H3K9ac) and repressive (H3K9me2 and H3K27me3) histone modifications. Marking of the constitutively expressed genes (ACTB and GAPDH) and a repressed gene (HBB) served as control for these experiments. As expected, the only samples with AR marking by H3K4me3 and H3K9ac were the two AR-positive xenografts and LNCaP. Marking by repressive histone modifications was more variable: H3K27me3 was a universal finding in AR-negative samples except for Du-145, and was accompanied by H3K9me2 in two out of four xenografts. H3K27me3 and H3K9me2 also marked the putative enhancer region when AR was repressed. Altogether, these data indicate that promoter DNA methylation of AR is an infrequent finding in prostate cancer, while polycomb protein-based silencing is nearly universal in AR-negative variants. Also, it shows that H3K9me2 promotes reinforcement of silencing. Thus, we propose that reactivation of AR may by achieved in selected cases by inhibiting H3K27me3 alone, while concomitant inhibition of both polycomb and SET domain proteins may be necessary for double-positive H3K27/K9 methylation cases. Given the purported differentiating and tumor suppressor effects of AR, its reactivation in SCPC may be of therapeutic benefit. Citation Format: Brittany N. Kleb, Marcos RH Estecio, Guanglin Wu, Jing-Fang Lu, Christopher J. Logothetis, Sankar Maity, Ana M. Aparicio. Mechanism of androgen receptor (AR) silencing in small cell prostate carcinoma. [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 2986. doi:10.1158/1538-7445.AM2013-2986

Abstract 4081: Taxane sensitivity in prostate cancer is determined by androgen receptor splice variants.

Abstract Although its pivotal role in prostate cancer care, androgen deprivation therapy (ADT) is not curative as many patients progress to a castration-resistant stage (CRPC) which remains driven by pathologic reactivation of androgen receptor (AR). Taxanes represent the only chemotherapy class that improves survival in CRPC and as such Docetaxel (DTX) and Cabazitaxel (CTX) are standard of care. Recent evidence revealed the presence of several AR splice variants lacking the ligand binding domain and thus constitutively active in CRPC. Previous data from our lab showed that taxanes impair the ligand-induced nuclear accumulation and transcriptional activity of full-length AR (AR-FL) in taxane treated human prostate cancer cells and CRPC patients. Indeed we showed that AR cytoplasmic sequestration in patient's circulating tumour cells (CTCs) was significantly correlated with clinical response to taxane chemotherapy. This is due to AR binding microtubules (MTs) and using them for nuclear accumulation supported by the dynein motor protein. However, the effect of taxane on the nuclear accumulation and activity of the AR variants is unknown. Thus, we set out to investigate the mechanism underlying AR variant nuclear accumulation and the impact of taxane treatment on AR variant function focusing on the two clinically relevant AR splice variants ARv567 and AR-V7. A microtubule co-sedimentation assay revealed that ARv567 is associated with MTs, in contrast to AR-V7. Serial AR mutagenesis demonstrated that the MT binding domain comprises a region aa 559 - 663 which is missing in AR-V7. Dynamitin overexpression inhibited the nuclear accumulation of AR-FL and ARv567 but had no effect on AR-V7, suggesting that AR-V7’s nuclear translocation is independent of dynein-based MT transport. To examine the impact of taxane treatment on variant activity we microinjected GFP-tagged AR-FL, or AR variants into the nucleus of PC3 cells and monitored the dynamics of AR nuclear translocation using live-cell confocal microscopy. Moreover we stably transfected M12 cells with AR-FL, AR-V7 and AR-v567 and assessed AR's nuclear translocation in response to R1881 and in the presence or absence of DTX. Our data showed that taxanes significantly inhibited the nuclear accumulation and activity of AR-FL and ARv567 but not that of AR-V7 and that this effect is androgen independent. We are currently elucidating the effect of taxanes on AR variant transcriptional activity using luciferase reporter and specific genes expression assays. Taken together our data revealed that functional MTs are required for AR's nuclear transport in a ligand independent manner, as in the case of ARv567. However, this does not apply to AR-V7 which is not under MT control and thus insensitive to taxane treatment. These data suggest that impairment of AR variant expression might be predictive of clinical taxane sensitivity in CRPC. Citation Format: Luigi Portella, Maria Thadani-Mulero, Alexandre Matov, David M. Nanus, Stephen R. Plymate, Paraskevi Giannakakou. Taxane sensitivity in prostate cancer is determined by androgen receptor splice variants. [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 4081. doi:10.1158/1538-7445.AM2013-4081

Novel Interaction between the Co-chaperone Cdc37 and Rho GTPase Exchange Factor Vav3 Promotes Androgen Receptor Activity and Prostate Cancer Growth*

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. Vav3, a guanine nucleotide exchange factor, is up-regulated following progression to castration resistance in preclinical models and is overexpressed in a significant number of human prostate cancers. Vav3 is a novel co-activator of the AR. We sought to identify Vav3 binding partners in an effort to understand the molecular mechanisms underlying Vav3 enhancement of AR activity and to identify new therapeutic targets. The cell division cycle 37 homolog (Cdc37), a protein kinase-specific co-chaperone for Hsp90, was identified as a Vav3 interacting protein by yeast two-hybrid screening. Vav3-Cdc37 interaction was confirmed by GST pulldown and, for native proteins, by co-immunoprecipitation experiments in prostate cancer cells. Cdc37 potentiated Vav3 co-activation of AR transcriptional activity and Vav3 enhancement of AR N-terminal-C-terminal interaction, which is essential for optimal receptor transcriptional activity. Cdc37 increased prostate cancer cell proliferation selectively in Vav3-expressing cells. Cdc37 did not affect Vav3 nucleotide exchange activity, Vav3 protein levels, or subcellular localization. Disruption of Vav3-Cdc37 interaction inhibited Vav3 enhancement of AR transcriptional activity and AR N-C interaction. Diminished Vav3-Cdc37 interaction also caused decreased prostate cancer cell proliferation selectively in Vav3-expressing cells. Taken together, we identified a novel Vav3 interacting protein that enhances Vav3 co-activation of AR and prostate cancer cell proliferation. Vav3-Cdc37 interaction may provide a new therapeutic target in prostate cancer.

Fibroblast growth factor, estrogen, and prolactin receptor features in different grades of prostatic adenocarcinoma in elderly men

The objective was to characterize and associate the receptor reactivities of fibroblastic growth factor (FGF)-2, FGF-7, FGF-8, epidermal growth factor (EGF), α-actin and vimentin in relation to the androgen receptor (AR), α and β estrogen receptors (ERα and ERβ), and prolactin receptor in the prostate of elderly men showing low- and high-grade adenocarcinoma. Thirty prostatic samples were taken from 60- to 90-year-old patients without prostatic lesions and with low-grade cancer and high-grade cancer, from the University Hospital, School of Medicine, the State University of Campinas. The results showed that increased FGF-2, FGF-7, and FGF-8 receptor reactivities and decreased AR reactivity were verified in both high- and low-grade cancer. However, the FGF-8 receptor showed greater involvement at the beginning of the malignancy alterations. Increased EGF receptor (EGFR) reactivity and diminished α-actin immunohistochemistry were identified in both cancer groups. Also, increased ERα, PR, and vimentin receptors were verified in both cancer groups. To conclude, the ERα involvement in the reactive stroma activation led to a microenvironment, which was favorable to cancer progression, due to maximizing stromal imbalance. The prolactin could be related to cancer progression due to its interaction with ERα action, indicating that this hormone could be a relevant target to prevent the estrogenic effects in the prostatic lesions. Both FGF receptor (FGFR)-2 and FGFR-8 play a fundamental role in the early stages of prostate cancer, suggesting that these molecules could be a promising therapeutic target. The differential localization of the fibroblastic factors between the prostatic epithelium and stroma of elderly men, who presented prostate cancer, could indicate a favorable distinction for tumoral progression.

PAGE4 Positivity Is Associated with Attenuated AR Signaling and Predicts Patient Survival in Hormone-Naive Prostate Cancer

Aberrant activation of the androgen receptor (AR) plays a key role during prostate cancer (PCa) development and progression to castration-resistant prostate cancer (CR-PCa) after androgen deprivation therapy, the mainstay systemic treatment for PCa. New strategies to abrogate AR activity and biomarkers that predict aggressive tumor behavior are essential for improved therapeutic intervention. PCa tissue microarrays herein reveal that prostate-associated gene 4 (PAGE4), an X-linked cancer/testis antigen, is highly up-regulated in the epithelium of preneoplastic lesions compared with benign epithelium, but subsequently decreases with tumor progression. We show that AR signaling is attenuated in PAGE4-expressing cells both in vitro and in vivo, most likely via impaired androgen-induced AR nuclear translocation and subsequently reduced AR protein stabilization and phosphorylation at serines 81 and 213. Consistently, epithelial PAGE4 protein levels inversely correlated with AR activation status in hormone-naive and CR-PCa clinical specimens. Moreover, PAGE4 impaired the development of CR-PCa xenografts, and strong PAGE4 immunoreactivity independently predicted favorable patient survival in hormone-naive PCa. Collectively, these data suggest that dysregulation of epithelial PAGE4 modulates AR signaling, thereby promoting progression to advanced lethal PCa and highlight the potential value of PAGE4 as a prognostic and therapeutic target.

Therapy resistance by splicing: can the androgen receptor teach us about BRAF?

Therapy resistance by splicing: can the androgen receptor teach us about BRAF?