AR Regulation Research Articles

The Androgen Receptor Does Not Directly Regulate the Transcription of DNA Damage Response Genes.

Our data indicate that the clinical benefit of combining ADT with RT is not due to direct AR regulation of DDR gene transcription, and that the field needs to consider alternative mechanisms for this clinical benefit.

Analysis of a novel flux adjustable axial flux permanent magnet eddy current coupler

Permanent magnet eddy current couplers have been widely used in large fan and pump loads to achieve the purpose of speed regulation and energy saving. In order to reduce the coupler axial volume and simplify the actuator, a novel flux adjustable axial flux permanent magnet eddy current coupler (FAAF-PMECC) is proposed in this paper. The permanent magnet rotor is divided into three parts: the inner, middle and outer parts, where the permanent magnets are embedded in the core and the adjacent poles are magnetised in opposite directions. The middle part is the adjusting permanent magnet ring (AR), which can rotate around the shaft. The inner part and outer part are fixed permanent magnet rings (FR) that are fixed with the shaft, and the output torque can be controlled by adjusting the relative angle of AR and FR. The structure and working principle of FAAF-PMECC are described in detail, and the output torque analytical model of the whole regulating process is established based on the magnetic equivalent circuit method. The AR regulation process is simulated by the variable reluctance model. The validation results show that the proposed structure can achieve a good speed regulation effect, and the output torque calculated by the analytical model in a certain slip speed range matches well with the output torque obtained using the 3D finite element method and experimental measurements. The sensitivity analysis of the structure parameters is also carried out. The analysis shows that the proposed coupler can achieve a wide speed range.

Novel AR/AR-V7 and Mnk1/2 Degrader, VNPP433-3β: Molecular Mechanisms of Action and Efficacy in AR-Overexpressing Castration Resistant Prostate Cancer In Vitro and In Vivo Models.

Prostate cancer (PCa) relies in part on AR-signaling for disease development and progression. Earlier, we developed drug candidate galeterone, which advanced through phase 2-clinical trials in treating castration-resistant PCa (CRPC). Subsequently, we designed, synthesized, and evaluated next-generation galeterone-analogs including VNPP433-3β which is potently efficacious against pre-clinical models of PCa. This study describes the mechanism of action of VNPP433-3β that promotes degradation of full-length AR (fAR) and its splice variant AR-V7 besides depleting MNK1/2 in in vitro and in vivo CRPC models that stably overexpresses fAR. VNPP433-3β directly engages AR within the cell and promotes proteasomal degradation of fAR and its splice variant AR-V7 by enhancing the interaction of AR with E3 ligases MDM2/CHIP but disrupting AR-HSP90 binding. Next, VNPP433-3β decreases phosphorylation of 4EBP1 and abates binding of eIF4E and eIF4G to 5′ cap of mRNA by depleting MNK1/2 with consequent depletion of phosphorylated eIF4E. Finally, RNA-seq demonstrates modulation of multiple pathways that synergistically contribute to PCa inhibition. Therefore, VNPP433-3β exerts its antitumor effect by imposing 1) transcriptional regulation of AR and AR-responsive oncogenes 2) translational regulation by disrupting mRNA-5′cap-dependent translation initiation, 3) reducing AR half-life through enhanced proteasomal degradation in vitro and AR-overexpressing tumor xenografts in vivo.

Ribonuclease H2 Subunit A Preserves Genomic Integrity and Promotes Prostate Cancer Progression.

RNASEH2A was demonstrated to be highly upregulated in aggressive prostate cancer to degrade R-loop accumulation and preserve genomic stability for tumor growth, suggesting that RNase H2 activity could be a promising therapeutic target.

Mechanistic Insights into the Selective Dual BET and PLK1 Inhibitory Activity of a Novel Benzamide Compound in Castration-Resistant Prostrate Cancer.

Though multifactorial, BET and PLK1 proteins have been found to be key players in the oncogenic process leading to castration-resistant prostate cancer through regulation of AR and MYC-mediated transcription. Hence, dual inhibition of these proteins appears to be an auspicious approach for CRPC therapy. WNY0824 has been reported to exhibit nanomolar range inhibition as well as significant anti-proliferative activity on AR-positive CRPC cells in vitro. However, structural, and mechanistic events associated with its dual inhibitory and anti-proliferative mechanisms remain unclear. Utilizing integrative computer-assisted atomistic techniques, analyses revealed that the dual-inhibitory activity of WNY0824 against BRD4 and PLK1 proteins is mediated by conserved residues present in the binding cavities of both proteins which are shown to elicit various strong intermolecular interactions and thus favour binding affinity. Also, binding orientation of the ligand at the protein binding cavities allowed for important hydrophobic interactions which resulted in high binding free energy of -42.50 kcal/mol and -51.64 kcal/mol towards BRD4 and PLK1, respectively. While van der Waals interactions are very important to ligand binding in BRD4-WNY complex, electrostatic interactions are pertinent to PLK1-WNY complex. Intriguingly, WNY0824 triggered conformational alterations in both proteins through increased structural instability, decreased structural compactness and mitigation in exposure of residues to solvent surface area. Consequently, critical interactions peculiar to the oncogenic activities of BRD4 and PLK1 were inhibited, a phenomenon that results in an antagonism of CRPC progression. The mechanistic insights presented in this report would further assist in the structure-based design of improved inhibitors useful in CRPC therapy.

RETRACTED ARTICLE: Targeting KDM4A-AS1 represses AR/AR-Vs deubiquitination and enhances enzalutamide response in CRPC

Castration-resistant prostate cancer (CRPC) is a highly malignant type of advanced cancer resistant to androgen deprivation therapy. One of the important mechanisms for the development of CRPC is the persistent imbalanced regulation of AR and AR splice variants (AR/AR-Vs). In this study, we reported KDM4A-AS1, a recently discovered lncRNA, as a tumor promoter that was significantly increased in CRPC cell lines and cancer tissues. Depletion of KDM4A-AS1 significantly reduced cell viability, proliferation, migration in vitro, and tumor growth in vivo. We found that by binding to the NTD domain, KDM4A-AS1 enhances the stability of USP14-AR/AR-Vs complex, and promoted AR/AR-Vs deubiquitination to protect it from MDM2-mediated ubiquitin-proteasome degradation. Moreover, KDM4A-AS1 was found to enhance CRPC drug resistance to enzalutamide by repressing AR/AR-Vs degradation; antisense oligonucleotide drugs targeting KDM4A-AS1 significantly reduced the growth of tumors with enzalutamide resistance. Taken together, our results indicated that KDM4A-AS1 played an important role in the progression of CRPC and enzalutamide resistance by regulating AR/AR-Vs deubiquitination; targeting KDM4A-AS1 has broad clinical application potential.

Sex differences feed into nuclear receptor signaling along the digestive tract

Sex differences in physiology are noted in clinical and animal studies. However, mechanisms underlying these observed differences between males and females remain elusive. Nuclear receptors control a wide range of physiological pathways and are expressed in the gastrointestinal tract, including the mouth, stomach, liver and intestine. We investigated the literature pertaining to ER, AR, FXR, and PPAR regulation and highlight the sex differences in nutrient metabolism along the digestive system. We chose these nuclear receptors based on their metabolic functions, and hormonal actions. Intriguingly, we noted an overlap in target genes of ER and FXR that modulate mucosal integrity and GLP-1 secretion, whereas overlap in target genes of PPARα with ER and AR modulate lipid metabolism. Sex differences were seen not only in the basal expression of nuclear receptors, but also in activation as their endogenous ligand concentrations fluctuate depending on nutrient availability. Finally, in this review, we speculate that interactions between the nuclear receptors may influence overall metabolic decisions in the gastrointestinal tract in a sex-specific manner.

Androgen regulation of pulmonary AR, TMPRSS2 and ACE2 with implications for sex-discordant COVID-19 outcomes

The sex discordance in COVID-19 outcomes has been widely recognized, with males generally faring worse than females and a potential link to sex steroids. A plausible mechanism is androgen-induced expression of TMPRSS2 and/or ACE2 in pulmonary tissues that may increase susceptibility or severity in males. This hypothesis is the subject of several clinical trials of anti-androgen therapies around the world. Here, we investigated the sex-associated TMPRSS2 and ACE2 expression in human and mouse lungs and interrogated the possibility of pharmacologic modification of their expression with anti-androgens. We found no evidence for increased TMPRSS2 expression in the lungs of males compared to females in humans or mice. Furthermore, in male mice, treatment with the androgen receptor antagonist enzalutamide did not decrease pulmonary TMPRSS2. On the other hand, ACE2 and AR expression was sexually dimorphic and higher in males than females. ACE2 was moderately suppressible with enzalutamide administration. Our work suggests that sex differences in COVID-19 outcomes attributable to viral entry are independent of TMPRSS2. Modest changes in ACE2 could account for some of the sex discordance.

Study on the mechanism of anti-cancer action of rosa roxbughii tratt based on network pharmacology

The active chemical components and targets of rosa roxbughii tratt were screened by the method of network pharmacology, and the molecular mechanism of its action was discussed. By consulting relevant literature reports, we established drug-disease targets by using databases such as TCMSP, TCMIP, Genecards and OMIM. Using STRING database to construct the protein interaction network between rosa roxbughii tratt and cancer target; and Cytoscape software was used to construct the interactive network diagram of “active ingredient-target-disease”. The function enrichment analysis of Gene Ontology (GO) and pathway enrichment analysis based on Kyoto Encyclopedia of genes and genomes (KEGG) were carried out by using biological information annotation database (David). We obtained 11 active components and 80 drug-disease targets were obtained, DAVID database was used to carry out GO function enrichment analysis and KEGG pathway enrichment analysis on 80 potential targets, and 303 biological processes and 114 signaling pathways were screened out to participate in the anti-cancer effect of rosa roxbughii tratt. The Pathway in cancer has the strongest correlation with cancer, which is related to the regulation of IL6, AR, CASP3 and other key genes. These key genes are related to myricetin, quercetin, kaempferol and other compounds in rosa roxbughii tratt. The anti-cancer compounds of rosa roxbughii tratt might were myricetin, quercetin, kaempferol and other compounds. The anti-cancer effect of rosa roxbughii tratt may regulation of key genes, indirectly affecting the sustained angiogenesis, developing apoptosis and promotion, so as to promote or inhibit the production of cancer.

Abstract 1278: Elucidating the mechanistic role of ERG in prostate cancer

Abstract Introduction: The transcription factor ERG is important for the development of angiogenesis, blood vessel integrity, and maintenance of hematopoietic stem cells. However, aberrant overexpression of ERG has been shown to be associated with various cancers. For example, ERG is recurrently involved in a translocation with TMPRSS2, which shifts it under AR regulation, and is thus subjected to overexpression in about 50% of prostate cancers in Caucasian males. While the clinical prognostic value of the TMPRSS2:ERG fusion is still debated, mechanistic studies of ERG-positive prostate cancers have demonstrated that ERG promotes cell invasion and metastasis. However, the molecular and cellular mechanisms of oncogenic ERG function in prostate cancer remain largely unclear. Methods/Results: To more comprehensively study the molecular and phenotypic roles of the TMPRSS2:ERG fusion product in prostate cancer, we developed an inducible ERG overexpression model in multiple cell lines as well as a CRISPR-Cas9-mediated TMPRSS2:ERG fusion translocation model. Using these models, we first performed RNA-seq and observed that ERG overexpression induced a global transcriptional program that increased the expression of genes related to epithelial-mesenchymal transition and decreased the expression of genes related to cell-cycle promotion. Further, using Boyden Chamber Assays as well as IncuCyte, we showed that overexpression of ERG promoted cell invasion but suppressed cell proliferation. We then used flow cytometric analysis to show that cells overexpressing ERG undergo a prolonged S phase. Interestingly, ERG antagonization through peptidomimetic inhibition partially rescued the proliferation rates of these ERG-overexpressing cells. Finally, using genome-wide CRISPR/Cas9 screening and NGS of the integrated sgRNA expression cassettes in the inducible ERG model, we identified panels of genes mediating ERG functions. Discussion: Unlike oncogenic kinases such as BCR-ABL in Chronic Myeloid Leukemia, transcription factors such as ERG have been more challenging to directly target in cancer. Thus, elucidation of the mechanisms of ERG-mediated transcriptional reprogramming, ERG binding partners, as well as the regulation of ERG, may result in the development of new strategies to target ERG by selectively antagonizing ERG oncogenic functions. Using our models, we are able to gain a better understanding of the role of ERG in cancer development and progression and thus provide insight for the development of new strategies for the prevention, detection, and treatment of prostate cancer. Citation Format: Caleb Cheng, Lanbo Xiao, Jean Tien, Cynthia Wang, Jesse Cheng, Eileen Shiau, Xiaoju Wang, Arul M. Chinnaiyan. Elucidating the mechanistic role of ERG in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1278.

SUN-002 Control of Metastasis by DAX-1 in Prostate Cancer Cells, an Androgen-Induced Orphan Nuclear Receptor

The ability of cancer cells to metastasize is the pivotal step that allows a primary tumor to become a life-threatening malignancy. This mechanism is coordinated by transcription factors driving cancer cells to acquire a mesenchymal state through a process called epithelial-mesenchymal transition (EMT). The study of potential therapeutic markers that can prevent or even reverse this phenomenon in metastatic cancers is crucial. To this effort, we have focused on studying the role of DAX-1 (Dosage-sensitive, sex-reversal, adrenal hypoplasia congenita, on the X chromosome, gene 1) as a transcriptional repressor of genes involved in EMT. DAX-1 is a member of the nuclear hormone receptor superfamily with key roles in adrenal and gonadal development. However, recent studies have focused on studying DAX-1 transcriptional regulation of genes involved in cancer progression. We hypothesize that DAX-1 is able to repress the transcription of genes involved in EMT leading to a reduction of metastatic rates in prostate cancer cells. We found that introducing DAX-1 into a prostate cancer cell line that lacks endogenous expression of DAX-1 was sufficient to repress the transcription of EMT markers and lead to a drastic reduction of metastatic rates. Parallel to these findings, we used a CRISPR KO model of DAX-1 to study its role in hormone-dependent prostate cancer. We have previously shown that treatment of hormone-dependent prostate cell lines with a non-aromatizable androgen leads to up-regulation of DAX-1 expression. Here, we use the same mechanism to analyze DAX-1 and AR regulation of metastatic markers in an androgen induced DAX-1 model in parallel to an androgen stimulated DAX-1 KO model. Results have shown that DAX-1 is sufficient to repress the expression of metastatic markers and reduce metastatic rates in prostate cancer cells. These results shed light into the convoluted web of biological processes coordinating prostate cancer progression and could reveal new therapeutics for the treatment of prostate cancer.

Abstract 3498: Regulation of AR through an FGF5 driven mechanism

Abstract Purpose To investigate the role of increasing FGF5 expression in the progression of prostate cancer and emergence of castration resistant prostate cancer. Methods Utilizing a cell line model representing different stages of prostate cancer progression, we assessed FGF5 expression and function in vitro and in vivo. We analyzed both an outcomes and progression tissue microarray (TMA) containing ~400 prostate cancer patient cores to assess FGF5 expression in a clinical setting. Results There was a significant increase in the both RNA and protein expression of FGF5 in tumorigenic cell lines compared to nontumorigenic. In TMAs that were analyzed, there was a significant increase of FGF5-positive cells across progression of prostate cancer from benign to metastatic, with the highest percentage occurring in cases of cancer. When performing gain of function experiments with recombinant FGF5 (rFGF5) or overexpression plasmids, we found that FGF5 significantly increased the proliferation of AR-positive cell lines. Conversely, in AR-negative cell lines, FGF5 inhibited proliferation. Because FGF5 is associated with stem cells and differentiation events, we next asked whether the population of FGF5 cells that increases through cancer progression could lead to molecular changes in the total population. In addition to inhibiting proliferation of AR-negative nontumorigenic BPH1 cells, rFGF5 resulted in a robust AR expression. In LNCaP, C4-2, and C4-2B rFGF5 resulted in an increase in AR expression. In C4-2 and C4-2B cells an increase in AR protein coincided with an increase in nuclear YAP1, a potent transcriptional regulator. We hypothesized that androgens could be regulating FGF5 expression in the prostate. When androgen dependent LNCaP cells are subjected to an androgen-depleted environment, FGF5 protein and RNA significantly increased within 24hrs. Additionally, when AR is stimulated using dihyrotestosterone, FGF5 protein and RNA levels significantly decreased. These effects are not observed in cell lines that do not express AR. Conclusions These results suggest that FGF5 is increased in prostate cancer progression cell lines as well as patient samples, with highest expression in aggressive cancer cores. Data also suggests that AR plays a role in the regulation of FGF5 and vice versa. These data suggest that FGF5 driven AR expression could be driven by YAP1 nuclear localization. This mechanism will be further interrogated. Citation Format: Dalton McLean. Regulation of AR through an FGF5 driven mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3498.

Abstract 5900: Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells

Abstract Background and objective: Androgen deprivation therapy (ADT) is among the most effective of all cancer therapies for prostate cancer; however, resistance to therapy leading to castrate resistant prostate cancer (CRPC) is inevitable in most patients. Recently we demonstrated that CYP3A5 expressed in prostate cancer cells facilitates the nuclear translocation of AR, directly impacting the transcriptional regulation of AR-dependent genes promoting prostate cancer cell growth. Our preliminary data show that CYP3A5 is a part of the HSP90-AR complex, known to regulate AR nuclear localization and activation. Additionally, we observed that CYP3A5 inhibition also blocks growth of prostate cancer cells lacking AR ligand binding site (22RV1). This observation suggested presence of an alternative mechanism of regulation of prostate cell growth by CYP3A5, independent of its AR activation. We hypothesize that CYP3A5 inhibition can also sensitize prostate cancer cells to therapy by downregulating expression of drug resistance genes independent of its AR regulation. Our aim is to decipher the role of CYP3A5 in therapeutic resistance, by screening for CYP3A5 regulated cancer drug resistance genes and to further understand the underlying mechanism of this regulation. Method: We used cancer drug resistance PCR array after CYP3A5 siRNA knockdown to identify CYP3A5 regulated genes involved in therapeutic resistance. Further we evaluated the role of CYP3A5 in inducing synthetic lethality in combination with DNA damage repair inhibiting drugs by imagining the γ-H2AX foci. Results: CYP3A5 siRNA knockdown downregulates 11 genes involved in cancer drug resistance: TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1 with a p≤0.005 value. Of the list of downregulated genes, the three genes TOP2A, BRCA2 and BRCA1 have the most fold change and their loss is known to impair homologous recombination (HR), leading to synthetic lethality. HR defects in CRPC, sensitizes these tumors to PARP and topoisomerase inhibitors. We tested the effect of CYP3A5 inhibition in inducing HR defects by analyzing γ-H2AX foci counts in combination with topoisomerase inhibitor etoposide. Increased number of γ-H2AX foci suggest that CYP3A5 siRNA may induce synthetic lethality in prostate cancer cells when used in combination with DNA damage repair inhibiting drugs by impairing HR and downregulating TOP2A and BRCA1/2 expression. Conclusions: CYP3A5 inhibition downregulates DNA damage repair genes (TOP2A and BRCA1/2) and cell cycle progression genes (CCNE1, CDK 2/4) and impairs HR. CYP3A5 inhibition can sensitize CRPC to PARP and topoisomerase inhibitors as it induces synthetic lethality by downregulating TOP2A and BRCA1/2 expression. CYP3A5 inhibition can be potentially exploited therapeutically in combination with PARP inhibitors as it functionally impairs HR and causes synthetic lethality. Citation Format: Priyatham Gorjala, Ranjana Mitra, Oscar Goodman. Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5900.

Transcribed ultraconserved region Uc.63+ promotes resistance to docetaxel through regulation of androgen receptor signaling in prostate cancer.

Docetaxel is the standard chemotherapy for metastatic castration-resistant prostate cancer (CRPC). However, nearly all patients ultimately become refractory due to the development of docetaxel resistance. The transcribed ultraconserved regions (T-UCRs) are a novel class of non-coding RNAs that are absolutely conserved across species and are involved in carcinogenesis including prostate cancer (PC). In this study, we investigated the transcriptional levels of 26 representative T-UCRs and determined the regions that were differentially expressed in PC. Quantitative real-time polymerase chain reaction analysis revealed that the expression of T-UCR Uc.63+ was increased in PC tissues. MTT assay and wound healing assay revealed that Uc.63+ was involved in cell growth and cell migration. miR-130b was predicted to have binding sites within the Uc.63+ sequence. The expression of miR-130b was significantly disturbed by the overexpression or knockdown of Uc.63+. We also showed that Uc.63+ regulated the expression of MMP2 via miR-130b regulation. Furthermore, overexpression of Uc.63+ increased the expression of AR and its downstream molecule PSA and promoted resistance to docetaxel through AR regulation. In patients treated with docetaxel, the expression of serum Uc.63+ in the docetaxel-resistant patients was higher than that in the docetaxel-sensitive patients (P = 0.011). Moreover, Kaplan-Meier analysis showed that the high expression of serum Uc.63+ correlated with a worse prognosis (P = 0.020). These results substantially support the important role that Uc.63+ plays in PC progression by interacting with miR-130b and indicate that Uc.63+ could potentially be a promising serum marker for deciding the best treatment for patients with CRPC.

Prostate cancer: Epigenetic AR regulation.

Prostate cancer: Epigenetic AR regulation.

Snail promotes resistance to enzalutamide through regulation of androgen receptor activity in prostate cancer.

Treatment with androgen-targeted therapies can induce upregulation of epithelial plasticity pathways. Epithelial plasticity is known to be important for metastatic dissemination and therapeutic resistance. The goal of this study is to elucidate the functional consequence of induced epithelial plasticity on AR regulation during disease progression to identify factors important for treatment-resistant and metastatic prostate cancer. We pinpoint the epithelial plasticity transcription factor, Snail, at the nexus of enzalutamide resistance and prostate cancer metastasis both in preclinical models of prostate cancer and in patients. In patients, Snail expression is associated with Gleason 9-10 high-risk disease and is strongly overexpressed in metastases as compared to localized prostate cancer. Snail expression is also elevated in enzalutamide-resistant prostate cancer cells compared to enzalutamide-sensitive cells, and downregulation of Snail re-sensitizes enzalutamide-resistant cells to enzalutamide. While activation of Snail increases migration and invasion, it is also capable of promoting enzalutamide resistance in enzalutamide-sensitive cells. This Snail-mediated enzalutamide resistance is a consequence of increased full-length AR and AR-V7 expression and nuclear localization. Downregulation of either full-length AR or AR-V7 re-sensitizes cells to enzalutamide in the presence of Snail, thus connecting Snail-induced enzalutamide resistance directly to AR biology. Finally, we demonstrate that Snail is capable of mediating-resistance through AR even in the absence of AR-V7. These findings imply that increased Snail expression during progression to metastatic disease may prime cells for resistance to AR-targeted therapies by promoting AR activity in prostate cancer.

Abstract B166: A RNA helicase siRNA screen to identify potential therapeutic targets in castration-resistant prostate cancer

Abstract Castration-resistant prostate cancers (CRPC) are resistant to androgen-deprivation therapies commonly used to treat carcinoma of the prostate, resulting in death from this disease. CRPC can remain AR-driven through upregulation of the expression of wild-type, mutated or alternatively spliced constitutively active AR. Targeting both full-length AR and AR splice variants may overcome endocrine resistance. Since RNA helicases play crucial roles in various aspects of RNA metabolism including transcription, pre-mRNA splicing, translation, RNA export and RNA decay, we evaluated potentially druggable RNA helicases implicated in the regulation of AR and AR-V7 expression. We performed an siRNA screen targeting RNA helicases in which the readout was the expression of AR and AR-V7 mRNA as determined by quantitative RT-PCR. Out of the 75 human RNA helicases silenced, we identified 11 that induced a 50% or greater decrease in both AR and AR-V7 mRNA expression. These included 3 RNA helicases that have no known described function, 2 that regulate ribosomal RNA transcription, 3 required in nuclear export of spliced and unspliced mRNA, 2 in translation initiation and the DEAD-box helicase EIF4A3 involved in nonsense-mediated decay. Among these, only EIF4A3 reduced cell numbers by 40% in addition to decreasing AR and AR-V7 expression. Silencing EIF4A3 resulted in a 50-80% inhibition of cell proliferation and between 2- to 11-fold induction of apoptosis in a panel of 6 prostate cancer cell lines. Inhibiting EIF4A3 in CRPC may block the expression of important oncogenes including AR and counteract the emergence of splice variants involved in resistance mechanisms. Citation Format: Hannah Wang, Emmanuel de Billy, Johann de Bono, Paul Workman. A RNA helicase siRNA screen to identify potential therapeutic targets in castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B166.

The role of HOXC6 in prostate cancer development

Homeobox (HOX) genes, which are involved in organ development and homeostasis, have been shown to be involved in normal prostate- and PCa development. In this study, we investigate the expression levels of the HOX A-D genes in PCa. The functional relevance and potential of HOX gene as biomarkers are explored. We evaluated HOX gene expression in prostate tissues of different grade and stage and related the outcome to clinical parameters. We analyzed AR regulation and function of HOXC6 in PCa cell lines. We developed a urine-based HOXC6 mRNA assay for diagnostic purposes. HOXC6 was one of the most upregulated HOX genes in all primary, metastasized, and castration-resistant PCa. HOXC6 upregulation was specific to the epithelial component of PCa, and HOXC6 was shown to be involved in epithelial cell proliferation. HOXC6 expression was not influenced by androgens nor by treatments targeting the AR signaling pathway. HOXC6 expression was not related to a prognosis after radical prostatectomy, that is, biochemical or local recurrence. We successfully developed an assay for HOXC6 mRNA detection in urine and confirmed that HOXC6 levels are higher in PCa patients. HOXC6 has a role in all PCa stages, particularly in PCa cell proliferation. Due to its stable expression, HOXC6 is a novel candidate biomarker for PCa not only in early detection but also for monitoring of progression or response to therapy.

Skp2 regulates androgen receptor through ubiquitin‐mediated degradation independent of Akt/mTOR pathways in prostate cancer

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment. Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins. Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR. The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling.

β-Adrenergic Agonists Mediate Enhancement of β1-Adrenergic Receptor N-terminal Cleavage and Stabilization In Vivo and In Vitro

The β(1)-adrenergic receptor (β(1)AR) is the predominant βAR in the heart and is the main target for β-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) β(1)AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in β(1)AR regulation, focusing on the effects of long-term treatment with β-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293(i) cells. The key findings were verified using the transiently transfected hβ(1)AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the β(1)AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of β-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of β(1)AR levels. This phenomenon may contribute to the exceptional resistance of β(1)ARs to downregulation and tendency toward upregulation following long-term ligand treatments.