Androgen Receptor Transcriptional Activity Research Articles

Abstract 2015: A transcriptionally enhanced biosensor to detect and monitor biopsy-dissociated primary prostate cancer single cell drug responses by bioluminescence microscopy

Abstract Background: In the past decade, the therapeutic landscape of metastatic castration resistant prostate cancer (mCRPC) has been transformed with the introduction of novel pharmaceutical agents including novel antiandrogens (nAA) such as darolutamide, enzalutamide or apalutamide. Currently, the management of mCRPC is mainly based on biomarkers obtained from the patient's blood or bulk tumor samples, which do not take into account for prostate cancer (PCa) cell heterogeneity. Objectives: To develop a bioluminescence imaging technology enabling: 1) the detection of PCa single cells from dissociated biopsies and 2) single cell nAA sensitivity assessment. Methods: The PCA3 promoter (PCa specific) and PSEBC promoter (androgen receptor-driven to monitor response to nAA therapy) have been incorporated in the non-replicating adenoviral multi-promoter integrated two-step transcriptional amplification system (MP-ITSTA) that allows imaging of complementary activities of two different promoters by a single output reporter gene. Thus, PCA3/PSEBC-ITSTA system was designed to monitor androgen receptor (AR) activity specifically within each single PCa cell by bioluminescence microscopy. Prostatic biopsies were dissociated in the presence of collagenase II and DNase for 18h. PCa cell lines or fresh biopsies-dissociated cancer cells were transduced for 72h and then covered with an extracellular matrix gel. Single cell bioluminescence microscopy images were done before and after exposure to DHT or DHT+AA to specifically monitoring therapy response. Changes in cell number and luminescence intensity after AA were normalized to the DHT group to exclude non-specific cell-death due to infection or time spent in culture. Results: PCA3/PSEBC-ITSTA was specific to PCa cell lines. By determining the ratio of AR active cells before and after DHT or DHT+AA treatment in culture, PCA3-Cre-PSEBC-ITSTA was able to determine the AA sensitivity of LNCaP (sensitive), LAPC4 (moderately resistant) and 22Rv1 (resistant) PCa cell lines. Also, our method could detect and monitor AA sensitivity of primary PCa cells harvested from eight radical prostatectomy specimens. PCA3/PSEBC-ITSTA could dynamically quantify AR transcriptional activity during enzalutamide or bicalutamide treatments, in a dose dependent manner, and could unveil tumor AA response heterogeneity. As expected, inhibition of AR activity was stronger with enzalutamide than with bicalutamide. Conclusions: Our bioluminescence microscopy-based biosensor could detect primary PCa cells in culture and it allows dynamic evaluation of AA sensitivity at a single-cell level in a heterogeneous cell population harvested from radical prostatectomy biopsies. We believe our approach opens to a novel field of treatment response predictive tools for cancer treatment decision-making. Citation Format: Audrey Champagne, Pallavi Jain, Bertrand Neveu, Frédéric Pouliot. A transcriptionally enhanced biosensor to detect and monitor biopsy-dissociated primary prostate cancer single cell drug responses by bioluminescence microscopy [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 2015.

Ralaniten Sensitizes Enzalutamide-Resistant Prostate Cancer to Ionizing Radiation in Prostate Cancer Cells that Express Androgen Receptor Splice Variants

Blocking androgen receptor (AR) transcriptional activity by androgen deprivation therapy (ADT) improves the response to radiotherapy for intermediate and high risk prostate cancer. Unfortunately, ADT, antiandrogens, and abiraterone increase expression of constitutively active splice variants of AR (AR-Vs) which regulate DNA damage repair leading to resistance to radiotherapy. Here we investigate whether blocking the transcriptional activities of full-length AR and AR-Vs with ralaniten leads to enhanced sensitivity to radiotherapy. Combination therapies using ralaniten with ionizing radiation were evaluated for effects on proliferation, colony formation, cell cycle, DNA damage, and Western blot analyses in human prostate cancer cells that express both full-length AR and AR-Vs. Ralaniten and a potent next-generation analog (EPI-7170) decreased expression of DNA repair genes whereas enzalutamide had no effect. FACS analysis revealed a dose-dependent decrease of BrdU incorporation with increased accumulation of γH2AX with a combination of ionizing radiation with ralaniten. An additive inhibitory effect on proliferation of enzalutamide-resistant cells was achieved with a combination of ralaniten compounds with ionizing radiation. Ralaniten and EPI-7170 sensitized prostate cancer cells that express full-length AR and AR-Vs to radiotherapy whereas enzalutamide had no added benefit.

SFRP1 increases TMPRSS2-ERG expression promoting neoplastic features in prostate cancer in vitro and in vivo

BackgroundProstate cancer (PCa) is the second cause of cancer related death in North American men. Androgens play an important role in its progression by regulating the expression of several genes including fusion ones that results from structural chromosome rearrangements. TMPRSS2-ERG is a fusion gene commonly observed in over 50% of PCa tumors, and its expression can be transcriptionally regulated by the androgen receptor (AR) given its androgen responsive elements. TMPRSS2-ERG could be involved in epithelial–mesenchymal transition (EMT) during tumor development. ERG has been reported as a key transcriptional factor in the AR-ERG-WNT network where five SFRP proteins, structurally similar to WNT ligands and considered to be WNT pathway antagonists, can regulate signaling in the extracellular space by binding to WNT proteins or Frizzled receptors. It has been shown that over-expression of SFRP1 protein can regulate the transcriptional activity of AR and inhibits the formation of colonies in LNCaP cells. However, the effect of SFRP1 has been controversial since differential effects have been observed depending on its concentration and tissue location. In this study, we explored the role of exogenous SFRP1 protein in cells expressing the TMPRSS2-ERG fusion.MethodsTo evaluate the effect of exogenous SFRP1 protein on PCa cells expressing TMPRSS2-ERG, we performed in silico analysis from TCGA cohort, expression assays by RT-qPCR and Western blot, cell viability and cell cycle measurements by cytometry, migration and invasion assays by xCELLigance system and murine xenografts.ResultsWe demonstrated that SFRP1 protein increased ERG expression by promoting cellular migration in vitro and increasing tumor growth in vivo in PCa cells with the TMPRSS2-ERG fusion.ConclusionsThese results suggest the possible role of exogenous SFRP1 protein as a modulator of AR-ERG-WNT signaling network in cells positive to TMPRSS2-ERG. Further, investigation is needed to determine if SFRP1 protein could be a target in against this type of PCa.

AR phosphorylation and CHK2 kinase activity regulates IR-stabilized AR-CHK2 interaction and prostate cancer survival.

We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR-CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR-CHK2 interactions. The destabilization of AR - CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage.

Shaping Chromatin States in Prostate Cancer by Pioneer Transcription Factors.

The androgen receptor (AR) is a critical therapeutic target in prostate cancer that responds to antagonists in primary disease, but inevitably becomes reactivated, signaling onset of the lethal castration-resistant prostate cancer (CRPC) stage. Epigenomic investigation of the chromatin environment and interacting partners required for AR transcriptional activity has uncovered three pioneer factors that open up chromatin and facilitate AR-driven transcriptional programs. FOXA1, HOXB13, and GATA2 are required for normal AR transcription in prostate epithelial development and for oncogenic AR transcription during prostate carcinogenesis. AR signaling is dependent upon these three pioneer factors both before and after the clinical transition from treatable androgen-dependent disease to untreatable CRPC. Agents targeting their respective DNA binding or downstream chromatin-remodeling events have shown promise in preclinical studies of CRPC. AR-independent functions of FOXA1, HOXB13, and GATA2 are emerging as well. While all three pioneer factors exert effects that promote carcinogenesis, some of their functions may inhibit certain stages of prostate cancer progression. In all, these pioneer factors represent some of the most promising potential therapeutic targets to emerge thus far from the study of the prostate cancer epigenome.

Mithramycin suppresses DNA damage repair via targeting androgen receptor in prostate cancer.

The dependency of prostate cancer (PCa) growth on androgen receptor (AR) signaling has been harnessed to develop first-line therapies for high-risk localized and metastatic PCa treatment. However, the occurrence of aberrant expression, mutated or splice variants of AR confers resistance to androgen ablation therapy (ADT), radiotherapy or chemotherapy in AR-positive PCa. Therapeutic strategies that effectively inhibit the expression and/or transcriptional activity of full-length AR, mutated AR and AR splice variants have remained elusive. In this study, we report that mithramycin (MTM), an antineoplastic antibiotic, suppresses cell proliferation and exhibits dual inhibitory effects on expression and transcriptional activity of AR and AR splice variants. MTM blocks AR recruitment to its genomic targets by occupying AR enhancers and causes downregulation of AR target genes, which includes key DNA repair factors in DNA damage repair (DDR). We show that MTM significantly impairs DDR and enhances the effectiveness of ionizing radiation or the radiomimetic agent Bleomycin in PCa. Thus, the combination of MTM treatment with RT or radiomimetic agents, such as bleomycin, may present a novel effective therapeutic strategy for patients with high-risk, clinically localized PCa.

Transcriptional profiling identifies an androgen receptor activity-low, stemness program associated with enzalutamide resistance

The androgen receptor (AR) antagonist enzalutamide is one of the principal treatments for men with castration-resistant prostate cancer (CRPC). However, not all patients respond, and resistance mechanisms are largely unknown. We hypothesized that genomic and transcriptional features from metastatic CRPC biopsies prior to treatment would be predictive of de novo treatment resistance. To this end, we conducted a phase II trial of enzalutamide treatment (160 mg/d) in 36 men with metastatic CRPC. Thirty-four patients were evaluable for the primary end point of a prostate-specific antigen (PSA)50 response (PSA decline ≥50% at 12 wk vs. baseline). Nine patients were classified as nonresponders (PSA decline <50%), and 25 patients were classified as responders (PSA decline ≥50%). Failure to achieve a PSA50 was associated with shorter progression-free survival, time on treatment, and overall survival, demonstrating PSA50's utility. Targeted DNA-sequencing was performed on 26 of 36 biopsies, and RNA-sequencing was performed on 25 of 36 biopsies that contained sufficient material. Using computational methods, we measured AR transcriptional function and performed gene set enrichment analysis (GSEA) to identify pathways whose activity state correlated with de novo resistance. TP53 gene alterations were more common in nonresponders, although this did not reach statistical significance (P = 0.055). AR gene alterations and AR expression were similar between groups. Importantly, however, transcriptional measurements demonstrated that specific gene sets-including those linked to low AR transcriptional activity and a stemness program-were activated in nonresponders. Our results suggest that patients whose tumors harbor this program should be considered for clinical trials testing rational agents to overcome de novo enzalutamide resistance.

Extragonadal Steroids Contribute Significantly to Androgen Receptor Activity and Development of Castration Resistance in Recurrent Prostate Cancer after Primary Therapy.

Beyond testosterone, several steroids contribute to the activation of the androgen receptor pathway, but their relative contributions to the activation of the androgen receptor signaling axis in patients with castrated prostate cancer remain unknown. Serum levels of 9 steroids were measured by mass spectrometry from continuously castrated patients of the PR.7 study (219) and from the PCA24 cohort (116). For each steroid standard curves for dose dependent prostate specific antigen promoter activation were built in castration sensitive (LAPC4) and resistant (VCaP) prostate cancer models. Standard curves were used to determine the androgen receptor activation potency for each steroid measurement from patients in these trials. In LAPC4 and VCaP cells testosterone, dihydrotestosterone and androstenedione induced androgen receptor transcriptional activity, while dehydroepiandrosterone, 5alpha-androstan-3beta,17beta-diol, androstenediol and androsterone stimulated androgen receptor only in VCaP cells. Extragonadal steroids were responsible for 34% (LAPC4) and 88% (VCaP) of the serum total androgen receptor transcriptional activity found in castrated cases. The total androgen receptor transcriptional activity secondary to testosterone, dihydrotestosterone and androstenedione was associated with time to castration resistance in patients from the PR.7 study (HR 2.17, 95% CI 1.12-4.23, p=0.02) in multivariate analysis using the castration sensitive model (LAPC4). Androgen receptor transcriptional activity of extragonadal androstenedione was the only steroid statistically associated with time to castration resistance in univariate analysis (HR 1.89, 95% CI 1.04-3.44, p=0.036). Extragonadal steroids contribute significantly to the androgen receptor axis activation at castration levels of testosterone in recurrent nonmetastatic prostate cancer and these sustain the development of castration resistance after primary local treatment.

Androgen receptor targeted therapy sensitizes tumor bearing mice to effective immunotherapy

Abstract Sex hormones impact both the innate and adaptive immune response, contributing to differences in autoimmune diseases, response to infection, and cancer incidence between males and females. Androgens are known to exert a general immunosuppressive effect. Historically, immune checkpoint blockade (ICB) has not proven to be efficacious in patients with prostate cancer despite standard of care consisting of androgen deprivation therapy (ADT). Upon disease progression, men will be treated with second line ADT therapy; some of which can prevent androgen receptor transcriptional activity. One such drug is enzalutamide. Given T cells express AR, we hypothesized that enzalutamide treatment would relieve the immunosuppressive effects of androgens on T cells, rendering them more responsive to immunotherapy. In the studies presented, we sensitize castration resistant prostate cancer tumor bearing mice to effective ICB with a combination of castration and enzalutamide. This combination therapy leads to ~40% tumor rejection and long-term survival; an effect that is dependent on CD8 T cells. These tumor infiltrating CD8 T cells made 6-fold more IFNγ within the tumor in the triple therapy group compared to control group. Surprisingly, this combination therapy showed efficacy in the androgen independent sarcoma tumor model MCA-205. Using in vitro and in vivo assays, we show that enzalutamide has a direct effect on T cell function. Taken together, our data suggests that ADT, and specifically enzalutamide treatment, can sensitize tumor bearing animals to effective immune checkpoint blockade and allow for tumor control by T cells.

Novel androgen receptor antagonist identified by structure-based virtual screening, structural optimization, and biological evaluation

Androgen receptor (AR) plays important roles in the development of prostate cancer (PCa), and therefore it has been regarded as the most important therapeutic target for both hormone-sensitive prostate cancer (HSPC) and advanced PCa. In this study, a novel hit (C18) with IC50 of 2.4 μM against AR transcriptional activity in LNCaP cell was identified through structure-based virtual screening based on molecular docking and free energy calculations. The structure-activity relationship analysis and structural optimization of C18 resulted in the discovery of a structural analogue (AT2), a more potent AR antagonist with 16-fold improved anti-AR potency. Further assays indicated that AT2 was capable of effectively inhibiting the transcriptional function of AR and blocking the nuclear translocation of AR like the second-generation AR antagonists. The antagonists discovered in this study may be served as the promising lead compounds for the development of AR-driven PCa therapeutics.

Contribution of extragonadal steroids to the androgen receptor activity and to the castration-resistance development in recurrent prostate cancers after primary therapy.

148 Background: Beyond testosterone, several steroids contribute to the activation of the androgen receptor (AR) pathway, but their relative contributions in castrated prostate cancer (PCa) patients remain unknown. Methods: Serum levels of nine steroids were measured by mass spectrometry from continuously castrated patients of the PR.7 study (n=219) and from the PCA24 cohort (n=116). For each steroid, standard curves for dose-dependent prostate-specific antigen promoter activation were built in castration-sensitive (LAPC4) and resistant (VCaP) PCa models. Standard curves were used to determine the AR activation potency for each steroid measurement from patients. Results: In LAPC4 and VCaP cells, testosterone, dihydrotestosterone and androstenedione induced AR transcriptional activity, while dehydroepiandrosterone, 5alpha-androstan-3beta,17beta-diol, androstenediol, androsterone stimulated AR only in VCaP cells. Extragonadal steroids were responsible for 34% (LAPC4) and 88% (VCaP) of the serum total AR transcriptional activity found in castrated patients. The total AR transcriptional activity secondary to testosterone, dihydrotestosterone and androstenedione was associated with time to castration resistance in patients from the PR.7 study (HR=2.17; 95% CI: 1.12-4.23; p=0.02) in multivariate analysis using the castration-sensitive model (LAPC4). AR transcriptional activity of extragonadal androstenedione was the only steroid statistically associated with time to castration resistance in univariate analysis (HR=1.89; 95% CI: 1.04-3.44; p=0.036). Conclusions: Extragonadal steroids contribute significantly to the AR axis activation at testosterone castration levels in recurrent non-metastatic PCa and these sustain the development of castration resistance after primary local treatment.

Abstract P4-08-04: Downregulation of DYRK2 contributes to tumor cell proliferation by enhancing CDK14 expression in breast cancer

Abstract Introduction: Tumor progression is the main cause of death in patients with breast cancer. Accumulating evidence suggests that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. However, little is known about the mechanisms of transcriptional regulation by DYRK2 in cancer progression, particularly with respect to cancer proliferation and invasion. Here, using a comprehensive expression profiling approach, we show that cyclin-dependent kinase 14 (CDK14) is a target of DYRK2. The aim of this study is to clarify whether DYRK2 suppresses the proliferation of breast cancer cells through CDK14 expression. Methods: Cell lines: We established stable DYRK2-depleted cells. MCF-7 cells were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) along with puromycin to isolate stable cell lines. We generated stable DYRK2-overexpressing MDA-MB-231 cells using GFP-DYRK2 (GFP-DYRK2 cells). A GFP vector was transfected as a vehicle control (GFP control cells) with G418 to isolate stable cell lines. Moreover, we established both stable DYRK2- and CDK14-depleted cells. The shDYRK2 cells were transfected with the pSuper-neo vector (shDYRK2-pSuper control) or pSuper-neo CDK14 shRNAs (shDYRK2-shCDK14). The pSuper control cells were transfected with the pSuper-neo vector (pSuper control-pSuper control cells) as a vehicle control with puromycin and G418 to isolate stable cell lines. These manipulated cells were compared with the control cells by various assays. Immunohistological staining: Sixty samples from surgically treated breast cancer patients were obtained from the Surgery Department at the Jikei University Hospital between 2001 and 2002. The Jikei University School of Medicine Ethics Review Committee approved the study protocol, and informed consent was obtained. Results: We analyzed cancer cell proliferation by MTS and colony formation assays. We investigated the effects of CDK14 in DYRK2-depleted cells in a breast tumor xenograft model. We found that reduced DYRK2 expression increased CDK14 expression to promote cancer cell proliferation in vitro and tumorigenicity in vivo. We further identified androgen receptor (AR) as a candidate of DYRK2-dependent transcription factors to control CDK14. Real-time RT-PCR and western blotting revealed that inhibition of AR activity using an AR inhibitor MDV3100 decreased both mRNA and protein levels of CDK14 in shDYRK2 and GFP control cells. In contrast, knocking down DYRK2 induced AR transcription activity in MCF-7 cells. shDYRK2 cells were more sensitive to MDV3100 compared with pSuper control cells. Immunohistological staining of sixty samples from surgically treated breast cancer patients showed an inverse correlation between DYRK2 and CDK14 expression. Conclusion: These findings demonstrate that reduced DYRK2 expression in breast cancer promotes tumor cell proliferation by modulating CDK14 expression via AR. Citation Format: Yoshimi Imawari, Rei Mimoto, Noriko Yamaguchi, Makiko Kamio, Hiroko Nogi, Ken Uchida, Hiroshi Takeyama, Kiyotsugu Yoshida. Downregulation of DYRK2 contributes to tumor cell proliferation by enhancing CDK14 expression in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-04.

Polymorphic Cytosine-Adenine-Guanine Repeat Length of Androgen Receptor Gene and Gender Incongruence in Trans Women: A Systematic Review and Meta-Analysis of Case-Control Studies

IntroductionIt has been hypothesized that gender incongruence in transgender women could result from an antenatal impaired androgen activity on the developing brain. As the length of polymorphic cytosine-adenine-guanine (CAG) repeat sequences in the androgen receptor (AR) gene is inversely correlated with AR transcriptional activity, some studies explored a possible association between long CAG repeats and gender incongruence in trangender women. Yet results remain inconclusive. AimTo systematically evaluate whether a difference exists in the length of AR CAG repeat sequences between trans women and men without gender incongruence. MethodsA thorough search of Medline, Scopus, Cochrane Library, Web of Science, and CINAHL databases was carried out to identify suitable case-control studies. Methodological quality of the included articles was assessed using the Newcastle-Ottawa Scale. In the absence of between-studies heterogeneity, as assessed by the Cochrane's Q and I2 tests, standardized mean differences (SMDs) in the length of AR CAG repeats were combined using a fixed effect model. Funnel plot and trim-and-fill analysis were used to assess publication bias. Main Outcome MeasureThe association of gender incongruence in transgender women with longer length of AR CAG repeat sequences was evaluated by calculating pooled standardized mean difference with 95% confidence interval (CI). Results5 studies included in the quantitative analysis collectively provided information on 795 trans women and 1,355 control men. At the overall estimate, the MtF group exhibited a significantly longer length of AR CAG repeat sequences (pooled standardized mean difference: 0.13, 95% CI: 0.04 to 0.22; P = 0.005; I2 = 0%, Pfor heterogeneity = 0.51). Sensitivity analysis demonstrated the high stability of the result. Funnel plot revealed a possible publication bias, and the trim-and-fill test detected 2 putative missing studies. Nevertheless, the significant association persisted even when pooled estimate was adjusted for publication bias. Clinical ImplicationsThese findings could suggest a contribution of a genetically mediated impairment in androgen signaling in development of gender incongruence for transgender women. Strength & LimitationsThis is the first meta-analysis exploring the relationship between AR CAG repeat polymorphism and gender incongruence. However, interactions with other functional genetic variants were not explored, and caution should be exercised when generalizing these results because of the possible variability in the distribution of CAG repeats among different populations and ethnic groups. ConclusionTrans woman population exhibits significantly longer polymorphic CAG repeat sequences in the AR gene. Further studies are warranted to elucidate whether, how and to what extent multiple functional variants in sex hormone signaling genes could be associated with gender incongruence/dysphoria.D'Andrea S, Pallotti F, Senofonte G, et al. Polymorphic Cytosine-Adenine-Guanine Repeat Length of Androgen Receptor Gene and Gender Incongruence in Trans Women: A Systematic Review and Meta-Analysis of Case-Control Studies. J Sex Med 2020;17:543–550.

A Novel Small Molecule Targets Androgen Receptor and Its Splice Variants in Castration-Resistant Prostate Cancer.

Reactivation of androgen receptor (AR) appears to be the major mechanism driving the resistance of castration-resistant prostate cancer (CRPC) to second-generation antiandrogens and involves AR overexpression, AR mutation, and/or expression of AR splice variants lacking ligand-binding domain. There is a need for novel small molecules targeting AR, particularly those also targeting AR splice variants such as ARv7. A high-throughput/high-content screen was previously reported that led to the discovery of a novel lead compound, 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(4-(2,3-dimethylphenyl)piperazin-1-yl)ethan-1-one (IMTPPE), capable of inhibiting nuclear AR level and activity in CRPC cells, including those resistant to enzalutamide. A novel analogue of IMTPPE, JJ-450, has been investigated with evidence for its direct and specific inhibition of AR transcriptional activity via a pulldown assay and RNA-sequencing analysis, PSA-based luciferase, qPCR, and chromatin immunoprecipitation assays, and xenograft tumor model 22Rv1. JJ-450 blocks AR recruitment to androgen-responsive elements and suppresses AR target gene expression. JJ-450 also inhibits ARv7 transcriptional activity and its target gene expression. Importantly, JJ-450 suppresses the growth of CRPC tumor xenografts, including ARv7-expressing 22Rv1. Collectively, these findings suggest JJ-450 represents a new class of AR antagonists with therapeutic potential for CRPC, including those resistant to enzalutamide.

A novel androgen receptor antagonist JJ-450 inhibits enzalutamide-resistant mutant ARF876L nuclear import and function.

Castration-resistant prostate cancer can develop resistance to enzalutamide because of androgen receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation ARF876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate cancer (CRPC). We employed several assays to determine the impact of JJ-450 and enzalutamide on prostate cancer cell lines expressing green fluorescent protein (GFP)-ARF876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate cancer cell proliferation. As expected, enzalutamide inhibited wild-type (WT) AR but not ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines. JJ-450 inhibits enzalutamide-resistant ARF876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.

Abstract B124: ERG mediated transcriptional regulation of DLX1 homeobox gene represents a novel mechanism underlying prostate cancer progression

Abstract Homeobox genes are the master regulatory transcription factors involved in the embryonic development and their deregulation in homeostatic biological processes can lead to cancer initiation and progression. A member of Distal less homeobox gene family (DLX), namely DLX1 plays pivotal role in the craniofacial, jaw and GABAergic (gamma aminobutyric acid) interneuron development. Higher expression levels of DLX1 has been associated with primary and metastatic prostate tumor. Moreover, DLX1 and HOXC6 are well-established diagnostic biomarkers for the early detection of prostate cancer (PCa). However, the mechanism involved in DLX1 up-regulation and functional significance in metastatic castration-resistant prostate cancer (mCRPC) progression remains unexplored. Here, we show that benign prostate epithelial cells acquire several oncogenic properties upon ectopic overexpression of DLX1. Similarly, CRISPR/Cas9 mediated DLX1 knockout in castrate-resistant 22RV1 cells show reduced proliferation, invasion, foci formation in cell-based assays, and a remarkable reduction (~80%) in tumor burden in mouse xenograft model, confirming its role in tumor progression. Moreover, our global gene expression profiling data established the role of DLX1 in regulating critical cancer hallmarks such as epithelial-to-mesenchymal transition (EMT), apoptosis and cell cycle regulation. We also demonstrate positive association between DLX1 and the most recurrent TMPRSS2-ERG gene fusion in many independent PCa cohorts, wherein, ~96% of the TMPRSS2-ERG fusion positive patients also harbour higher level of DLX1. Our ChIP-qPCR and luciferase-reporter assay data show that ERG occupies the DLX1 promoter region, and orchestrate its transcriptional control. Nevertheless, a subset of ERG-fusion negative patients also exhibits a higher DLX1 expression, where androgen receptor (AR) and AR variant 7 (AR-V7) plays critical role in transcriptional regulation of DLX1 in an ERG-independent manner. Since, Bromodomain and extra-terminal domain inhibitors (BETi) are known to inhibit the transcriptional activity of AR, AR-V7 and ERG, we examined the effect of BETi on DLX1. Interestingly, a significant decrease in the expression of DLX1 in BETi treated PCa cell lines with a concomitant loss of their oncogenic potential was observed. Taken together, our findings reveal that DLX1 serves as an oncogene thereby regulating several oncogenic properties associated with PCa progression. We discovered a novel transcriptional regulatory network involving ERG dependent up-regulation of DLX1 in TMPRSS2-ERG fusion positive PCa, while AR and AR-V7 governed expression in ERG-independent manner in CRPC cells. Conclusively, our findings suggest that BET domain inhibitors can be used as an adjuvant therapy for DLX1-positive subset of metastatic castration resistant prostate cancer patients. Citation Format: Sakshi Goel, Vipul Bhatia, Mahendra Shyamlal Palecha, Shannon Carskadon, Nallasivam Palanisamy, Bushra Ateeq. ERG mediated transcriptional regulation of DLX1 homeobox gene represents a novel mechanism underlying prostate cancer progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B124. doi:10.1158/1535-7163.TARG-19-B124

Abstract LB-A14: Combinational therapy by targeting LSD1 and BRD4 in prostate cancer

Abstract As a nuclear transcriptional factor, androgen receptor (AR) plays a significant role in prostate cancer (PCa) development. Androgen deprivation therapy (ADT) blunting the AR activity is the mainstream strategy for treating PCa. Despite the initial effectiveness of ADTs, most cases will relapse to a lethal stage termed castration-resistant prostate cancer (CRPC), where AR regains activity and drives tumor growth. This unmet clinical challenge to further target AR has led to a number of clinical investigations of targeting some of the key components in AR signaling. Epigenetic regulators are promising targets as they are critical players for rewiring the AR network during PCa progression. Lysine specific demethylase 1 (LSD1), an important epigenetic modifier that is well known for its repressor function through demethylating histone 3 lysine 4 (H3K4), can also function as an activator. In PCa cells, our epigenomic profiling study showed that LSD1 resides at active chromatin regions marked by high H3K27ac and FOXA1. Further genome-wide studies showed that inhibiting LSD1 disrupts AR cistrome and impairs its transcriptional output. Our mechanistic studies show that the activator function of LSD1 is through controlling the chromatin binding of FOXA1, which is a critical pioneer factor for AR. Importantly, at the AR-regulated enhancers, we observed that LSD1 inhibition also caused a rapid and broad decrease of H3K27ac signal, which directs Bromodomain-containing protein 4 (BRD4) chromatin binding. BRD4 belongs to BET (bromodomain and extra terminal domain) family, which plays an essential role in promoting the initiation and elongation steps in gene transcription process. The most credited function of BRD4 is its regulation of oncogene c-MYC via binding to the highly active super enhancers. Moreover, the oncogenic function of BRD4 in PCa has been well attributed to its coactivator role with AR. Multiple inhibitors targeting BRD4 have been developed and shown early promising clinical outcomes, however the drug resistance also emerges in the preclinical models. Herein, we reason that targeting LSD1 and BRD4 together could further impair the AR action and achieve better anti-tumor effect. To examine whether there is a synergistic effect, we employed inhibitors that have been tested in clinical trials: ORY-1001 and I-BET762, which target LSD1 and BRD4 respectively. We found that the combinational treatment lead to a more effective reduction of AR transcriptional activity than either single treatment. The proliferation of AR-positive LNCaP PCa cell line and CWR22RV1 CRPC cell line which expresses high levels of C-terminal truncated AR splicing variants (AR-Vs) was also more profoundly inhibited by combined therapy. Besides disrupting the full-length AR cistrome, LSD1 inhibition also impairs the activity of AR-V7, a major form of AR-Vs that drives developing ADT resistance in CRPC. However, the function of BRD4 in regulating AR-V7 activity remains to be elucidated. Therefore, we tested the combined treatment in CWR22RV1 cells under hormone-depleted condition. By targeting LSD1 and BRD4 together, AR-V7 regulated gene expression was significantly reduced. Currently, we are investigating the genome-wide effect of targeting both epigenetic regulators on AR and AR-V7. To provide clinical evidence, we are testing this combined treatment in patient-derived xenograft models, which recapitulate the CRPC by harboring high levels of AR and AR-Vs and ETS gene translocation. Citation Format: Muqing Li, Dong Han, zifeng wang, shuai gao, Changmeng Cai. Combinational therapy by targeting LSD1 and BRD4 in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A14. doi:10.1158/1535-7163.TARG-19-LB-A14

Abstract LB-A13: LSD1 activates both PI3K/AKT and AR signaling in prostate cancer cells

Abstract Background: Lysine specific demethylase 1 (LSD1/KDM1A), a specific demethylase of mono- or di-methylated histone lysine 4 (H3K4me1,2, enhancer-associated signature), was first identified as a component of REST repressor complex composed of CoREST and histone deacetylases 1, 2 (HDAC1,2). In hematopoietic malignancies, targeting LSD1 has shown very promising clinical outcomes, potentially through promoting myeloid differentiation. In solid tumors, multiple mechanisms have been proposed for the antitumor effect of inhibiting LSD1. While LSD1 is well known for its transcription repressor activity, it also activates gene transcription through demethylating repressive histone marks, such as H3K9me1,2, and possibly other non-histone proteins. In particular, LSD1 functions as a major androgen receptor (AR) coactivator in prostate cancer (PCa) cells. Indeed, we reported that LSD1 interacts and colocalizes with FOXA1, a pioneer factor of AR, at AR-mediated enhancers, and this interaction may facilitate AR transcription activity. Nonetheless, since AR signaling is critical to PCa development and progression to the lethal stage of castration-resistant PCa (CRPC), studies from us and others highly suggest that targeting LSD1 may be a potential treatment strategy for CRPC, where AR signaling is commonly restored. However, whether LSD1 regulates other major tumor-promoting pathways in PCa cells remains to be determined. Identification of suchcritical LSD1 targets will provide important insights for developing more effective therapeutic strategy for treating PCa. Experiments and Results: To address this question, we conducted RNA-seq analyses of LSD1 inhibitor treated PCa cells. We first confirmed that prolonged LSD1 inhibitor treatment in the presence of androgens broadly impaired AR transcriptional output. Importantly, we found that the LSD1 inhibitor repressed genes were also enriched for PI3K/AKT pathway even in absence of androgen stimulation. We further confirmed that LSD1 inhibition and Knock-out significantly decreased AKT phosphorylation, which interestingly is independent of AR signaling, as targeting LSD1 also decreased AKT phosphorylation in AR negative PCa cells. Through functional annotation analyses and subsequent validations, we identified the regulatory subunit of PI3K, p85α and possibly its isoform p85β, as a critical transcriptional target of LSD1 that mediates its effect on PI3K/AKT pathway activation. Using a combined analysis with ChIP-seq against LSD1 and H3K4me2, we identified a regulatory region on the PIK3R1 gene loci that may be responsible for regulating its gene transcription. We further investigated the effect of LSD1 inhibitor treatment in LuCaP35CR, a patient-derived CRPC xenograft model which is TMPRSS2-ERG positive and PTEN-negative. Using this model, we found that targeting LSD1 impaired the tumor growth and suppressed PI3K/AKT signaling. Conclusion: our study suggests that LSD1 has dual functions in promoting PCa development: 1) it enhances AR signaling through its coactivator function, 2) it activates PI3K/AKT signaling through increasing p85 gene expression. Based on these findings, it is plausible that the effectiveness of LSD1 inhibitor treatment in CRPC may be due to inhibition of both AR signaling and PI3K/AKT signaling pathways. As LSD1 inhibitors are currently being tested in phase II clinical trials of leukemia, our studies can be rapidly translated into clinical trials of CRPC. Citation Format: zifeng wang, Shuai Gao, Changmeng cai. LSD1 activates both PI3K/AKT and AR signaling in prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A13. doi:10.1158/1535-7163.TARG-19-LB-A13

MiR-133a-5p inhibits androgen receptor (AR)-induced proliferation in prostate cancer cells via targeting FUsed in Sarcoma (FUS) and AR

ABSTRACTAndrogens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly employed to treat advanced prostate cancer. Previously, FUsed in Sarcoma (FUS) was identified as an AR-interacting protein that enhances AR transcriptional activity. In the present study, we attempted to identify miRNAs that might target both FUS and AR to inhibit FUS and AR expression. Based on TCGA data and the online tools UALCAN, Kaplan Meier-plotter (KMplot), LncTar and miRWalk prediction, miR-133a-5p was selected. MiR-133a-5p expression was significantly downregulated in prostate cancer, and low miR-133a-5p expression was correlated with low survival probability. As predicted by LncTar and miRWalk, miR-133a-5p could bind to the 3′UTR of FUS and AR to inhibit their expression. MiR-133a-5p overexpression significantly suppressed the cell viability of the AR-positive prostate cancer cell lines VCaP and LNCaP, inhibited the expression of FUS, AR, as well as AR downstream targets IGF1R and EGFR. More importantly, miR-133a inhibition increased cancer cell proliferation as well as the expression of AR and AR downstream factors, while FUS knockdown exerted an opposite effect; the effect of miR-133a on cancer cell proliferation and AR could be significantly reversed by FUS knockdown. Moreover, IGF1R and EGFR knockdown reversed the effect of the miR-133a-5p inhibition. In summary, miR-133a-5p inhibits AR-positive prostate cancer cell proliferation by targeting FUS/AR, thus improving the resistance of prostate cancer to androgen ablation therapies, which requires further in vivo validation. We provided a novel miRNA regulation mechanism for proliferation regulation in AR-positive prostate cancer cells.

Histone Demethylase KDM4C Stimulates the Proliferation of Prostate Cancer Cells via Activation of AKT and c-Myc.

Our three-dimensional organotypic culture revealed that human histone demethylase (KDM) 4C, a histone lysine demethylase, hindered the acini morphogenesis of RWPE-1 prostate cells, suggesting its potential oncogenic role. Knockdown (KD) of KDM4C suppressed cell proliferation, soft agar colony formation, and androgen receptor (AR) transcriptional activity in PCa cells as well as reduced tumor growth of human PCa cells in zebrafish xenotransplantation assay. Micro-Western array (MWA) analysis indicated that KD of KDM4C protein decreased the phosphorylation of AKT, c-Myc, AR, mTOR, PDK1, phospho-PDK1 S241, KDM8, and proteins involved in cell cycle regulators, while it increased the expression of PTEN. Fluorescent microscopy revealed that KDM4C co-localized with AR and c-Myc in the nuclei of PCa cells. Overexpression of either AKT or c-Myc rescued the suppressive effect of KDM4C KD on PCa cell proliferation. Echoing the above findings, the mRNA and protein expression of KDM4C was higher in human prostate tumor tissues as compared to adjacent normal prostate tissues, and higher KDM4C protein expression in prostate tumors correlated to higher protein expression level of AKT and c-Myc. In conclusion, KDM4C promotes the proliferation of PCa cells via activation of c-Myc and AKT.