AR Signaling Research Articles

The dark proteome: translation from noncanonical open reading frames.

Omics-based technologies have revolutionized our understanding of the coding potential of the genome. In particular, these studies revealed widespread unannotated open reading frames (ORFs) throughout genomes and that these regions have the potential to encode novel functional (micro-)proteins and/or hold regulatory roles. However, despite their genomic prevalence, relatively few of these noncanonical ORFs have been functionally characterized, likely in part due to their under-recognition by the broader scientific community. The few that have been investigated in detail have demonstrated their essentiality in critical and divergent biological processes. As such, here we aim to discuss recent advances in understanding the diversity of noncanonical ORFs and their roles, as well as detail biologically important examples within the context of the mammalian genome.

Germline BRCA2, ATM and CHEK2 alterations shape somatic mutation landscapes in prostate cancer.

148 Background: Prostate cancer (PCa) is a highly heritable malignancy. Germline mutations in homologous recombination repair (HRR) genes, especially in in ATM, BRCA2 and CHEK2, are common in men with PCa, but how these influence somatic mutations is unknown. Characterizing the somatic landscape of tumors from germline HRR-altered PCa patients may provide insights into further therapeutic strategies and identify individuals most likely to benefit from existing therapies. Methods: We retrospectively identified 118 men with germline HRR-altered PCa from Johns Hopkins with available somatic mutation data (g ATM, n = 30; g BRCA2, n = 58; g CHEK2, n = 30). In these patients, somatic NGS analysis were done primarily using prostatic (rather than metastatic) tissues. The proportion of patients in each group with somatic biallelic inactivation was determined. We then compared the somatic mutation landscapes of the 3 groups, focusing on key PCa-relevant genes including AR, TMPRSS2-ERG, PTEN, TP53, BRCA2, ATM, CDK12, APC, FOXA1, AKT1, PIK3CA, and KDM6A among others. Somatic mutation data from primary prostate cancer specimens (n = 3965) found in pooled studies (n = 23) from cBioPortal were included as a reference standard for comparison. Results: Biallelic somatic inactivation was detected in 59% (34/58) of g BRCA2-altered, 47% (14/30) of g ATM-altered, and 0% (0/30) of g CHEK2-altered patients. In all 118 germline HRR-altered individuals, the most common somatic mutations were in BRCA2 (31%), FOXA1 (26%), TMPRSS2-ERG (20%), TP53 (16%), PTEN (15%), and ATM (15%). g BRCA2-altered patients had a higher prevalence of somatic mutations in AR (9% vs 3% in cBioPortal, P < 0.05) and FOXA1 (29% vs 13% in cBioPortal, P > 0.05), suggesting a greater dependency on AR signaling, but a lower prevalence of TMPRSS2-ERG fusions (17% vs 33% in cBioPortal, P < 0.05). g ATM-altered patients had a strikingly low prevalence of somatic TP53 mutations (3% vs 25% in cBioPortal, P < 0.01), indicating mutual exclusivity between these genes. g CHEK2-altered patients had a higher prevalence of somatic CDK12 mutations (10% vs 0% in gATM/gBRCA2 patients, P < 0.05), implying an interaction between CHEK2 and CDK12. No differences were seen between the three groups with respect to alterations in the PI3K/PTEN pathway, WNT pathway, or chromatin-related genes. Conclusions: Biallelic inactivation was frequently observed in PCa patients harboring g ATM and g BRCA2 mutations, but not in those with g CHEK2 mutations. g BRCA2-altered patients were enriched for somatic AR (and FOXA1) mutations and were depleted for TMPRSS2-ERG fusions. g ATM-altered patients were depleted for somatic TP53 mutations, while g CHEK2-altered patients were enriched for somatic CDK12 mutations. Thus, germline mutations appear to impose selective pressures that influence somatic mutation landscapes in PCa.

Patient derived tumor xenograft study with CDK4/6 inhibitor plus AKT inhibitor for management of metastatic castrate resistant prostate cancer.

176 Background: Despite androgen deprivation therapy, prostate cancer will develop resistance known as castrate resistant prostate cancer (CRPC). Androgen signaling is associated with pathways that influence cell proliferation and survival via cell cycle regulation. AR signaling results in inactivation of Rb tumor suppressor by way of AKT and reduction in intrinsic CDK4/6 inhibitors. Since PTEN loss is found in 40% of mCRPC and AKT pathway is a mechanism of CDK4/6 inhibitor resistance, we hypothesize adding an AKT inhibitor to a CDK4/6 inhibitor will have synergistic activity while delaying CDK4/6 inhibitor resistance. Preclinical xenografts have been shown to recapitulate patient response, therefore, can be used to guide clinical trials using novel combinations. Methods: We have established patient derived tumor xenograft models (GUR-017M, TMA-027 and PRJ-88T) from patients with mCRPC with each having a unique genomic alteration profile. In the clinical setting, GUR-017M was resistant to abiraterone, enzalutamide, cabazitaxel and had exposure to docetaxel. TMA-027 and PRJ-88T were resistant to enzalutamide. We combined a CDK4/6 inhibitor with an AKT inhibitor and treated each of these patient tumors in NOD-SCID mice for a total duration of 40 days. Mice were implanted and separated into 4 groups (placebo, CDK4/6 inhibitor, AKT inhibitor and CDK4/6 + AKT inhibitor) with 10-13 mice in each group. Tumor volumes were measured twice a week. For GUR-017M, abemaciclib (50 mg/kg) was combined with MK2206 (120 mg/kg) and was compared to each placebo and each single agent alone. For TMA-027, palbociclib (50 mg/kg) was combined with ipatasertib (50 mg/kg). After 40 days, tumors were harvested for immunohistochemistry analysis. PRJ-88T was grown as 3D spheroids in ultra-low attachment plates and treated with palbociclib + ipatasertib (10 uM-100uM) to evaluate for synergy. Results: GUR-017M and TMA-027 showed significant delayed tumor growth compared to placebo or either single agent alone. In the GUR-017M group, the final mean tumor volume for the combination group was 149 mm3, compared to 377 mm3 (abemaciclib), 358 mm3 (MK2206) and 527 mm3 placebo group, representing a percent decrease compared to control by 72%, 29%, and 32%, respectively. For TMA-027, the final mean tumor volume for the combination group was 102 mm3, compared to 259 mm3 (palbociclib), 416 mm3 (ipatasertib), and 402 mm3 placebo group, representing a percent decrease compared to control by 75%, 36%, and 4%, respectively. PRJ-88T 3D spheroids showed synergy with palbociclib + ipatasertib. In vivo experiments using PRJ-88T are in progress. Conclusions: The preclinical results show combining CDK4/6 inhibitors with AKT inhibitors is a promising treatment strategy and provides the basis for a clinical trial in patients with mCRPC.

Phase I/II trial of pembrolizumab and AR signaling inhibitor +/- 225Ac-J591 for chemo-naive metastatic castration-resistant prostate cancer (mCRPC).

TPS216 Background: The role of immune checkpoint inhibition (ICI) in prostate cancer remains undefined outside of the subset with mismatch repair. Several studies have suggested that ICI combined with androgen receptor signaling inhibitors (ARSI) or kinase inhibitors may result in improved and/or more durable response in a proportion of men with mCRPC. While not yet proven, addition of external beam radiation to ICI may improve outcomes (for instance Kwon et al Lancet Oncol 2014, Fizazi K et al, Eur Urol 2020). PSMA targeted therapy with 177Lu-PSMA-617 improves survival in men with CRPC, has been combined with ICI in early phase studies, and we have previously demonstrated the benefit of PSMA targeted therapy using radiolabeled mAb J591. PSMA-targeted alpha-emitters have a very high potency and the potential to generate immune response. Based upon i) ARSI may increase PSMA expression, ii) ARSI may radiosensitize tumors, iii) ARSI resistance may lead to increased PD-L1 expression, and iv) alpha emitters may generate an immune response, we hypothesize that the addition of an alpha-emitting radionuclide (225Ac) targeting prostate cancer (i.e PSMA+ tumors targeted with J591) will lead to double-stranded DNA breaks, cell death, and subsequent release of neoantigens, and thus will increase the response proportion to pembrolizumab plus ARSI resulting in more durable response. Methods: Key eligibility criteria include progressive mCRPC by PCWG3 on at least 1 prior AR pathway inhibitor and no prior chemotherapy for mCRPC. A phase I dose-finding study will first test safety of the triplet combination of pembrolizumab, an ARSI of physician choice, and 2 different doses of 225Ac-J591 (one with minimal and one with moderate single-agent toxicity). Following determination of the optimal dose, a randomized phase II trial will treat subjects with a fixed dose of pembrolizumab 400 mg every 6 weeks (for up to 2 years) plus a standard ARSI (until progression or intolerance) with or without 225Ac-J591. The primary endpoint of the study will test the hypothesis that the addition of a PSMA-targeted alpha emitter increases the composite of RECIST measurable disease, PSA, and CTC count response to immuno-hormonal therapy with 90% power. Key secondary clinical endpoints include 1-year progression-free survival, duration of response, and overall survival. Exploratory objectives include assessment of immunogenic cell death, immune serologic and host microbiome changes, plasma ctDNA, serial PSMA PET, and patient reported outcomes (FACT-P, BPI, EQ-5D-5L). The phase I portion of this DOD-funded study was activated in summer 2021 with the randomized phase II portion expected to open at PCCTC sites in 2022. Clinical trial information: NCT04946370.

Expression of Fibroblast Activation Protein Is Enriched in Neuroendocrine Prostate Cancer and Predicts Worse Survival.

Background: Advanced prostate cancer (PC) may accumulate genomic alterations that hallmark lineage plasticity and transdifferentiation to a neuroendocrine (NE) phenotype. Fibroblast activation protein (FAP) is a key player in epithelial-to-mesenchymal transition (EMT). However, its clinical value and role in NE differentiation in advanced PC has not been fully investigated. Methods: Two hundred and eight patients from a multicenter, prospective cohort of patients with metastatic castration-resistant prostate cancer (CRPC) with available RNA sequencing data were analyzed for tumor FAP mRNA expression, and its association with overall survival (OS) and NE tumor features was investigated. Results: Twenty-one patients (10%) were found to have high FAP mRNA expression. Compared to the rest, this subset had a proportionally higher exposure to taxanes and AR signaling inhibitors (abiraterone or enzalutamide) and was characterized by active NE signaling, evidenced by high NEPC- and low AR-gene expression scores. These patients with high tumor mRNA FAP expression had a more aggressive clinical course and significantly shorter survival (12 months) compared to those without altered FAP expression (28 months, log-rank p = 0.016). Conclusions: FAP expression may serve as a valuable NE marker indicating a worse prognosis in patients with metastatic CRPC.

Comparison between Heat-Clearing Medicine and Antirheumatic Medicine in Treatment of Gastric Cancer Based on Network Pharmacology, Molecular Docking, and Tumor Immune Infiltration Analysis.

Background Clinical research found that TCM is therapeutic in treating gastric cancer. Clearing heat is the most common method, while some antirheumatic medicines are widely used in treatment as well. To explore the pharmacological mechanism, we researched the comparison between heat-clearing medicine and antirheumatic medicine in treating gastric cancer. Methods First, related ingredients and targets were searched, respectively, and are shown in an active ingredient-target network. Combining the relevant targets of gastric cancer, we constructed a PPI network and MCODE network. Then, GO and KEGG enrichment analyses were conducted. Molecular docking experiments were performed to verify the affinity of targets and ligands. Finally, we analyzed the tumor immune infiltration on gene expression, somatic CNA, and clinical outcome. Results A total of 31 ingredients and 90 targets of heat-clearing medicine, 31 ingredients and 186 targets of antirheumatic medicine, and 12,155 targets of gastric cancer were collected. Antirheumatic medicine ranked the top in all the enrichment analyses. In the KEGG pathway, both types of medicines were related to pathways in cancer. In the KEGG map, AR, MMP2, ERBB2, and TP53 were the most crucial targets. Key targets and ligands were docked with low binding energy. Analysis of tumor immune infiltration showed that the expressions of AR and ERBB2 were correlated with the abundance of immune infiltration and made a difference in clinical outcomes. Conclusions Quercetin is an important ingredient in both heat-clearing medicine and antirheumatic medicine. AR signaling pathway exists in both types of medicines. The mechanism of the antitumor effect in antirheumatic medicine was similar to trastuzumab, a targeted drug aimed at ERBB2. Both types of medicines were significant in tumor immune infiltration. The immunology of gastric tumor deserves further research.

Synthesis and crystal structures of D-annulated pentacyclic steroids: looking within and beyond AR signalling in prostate cancer

Crystal structures of D-annulated steroids were used for docking studies against the human androgen receptor. The combination of the selected steroid with bicalutamide was found to exhibit significant antiproliferative effects in 22Rv1 cells.

Tomato and Olive Bioactive Compounds: A Natural Shield against the Cellular Effects Induced by β-Hexachlorocyclohexane-Activated Signaling Pathways.

The β-isomer of hexachlorocyclohexane (β-HCH) is a globally widespread pollutant that embodies all the physicochemical characteristics of organochlorine pesticides, constituting an environmental risk factor for a wide range of noncommunicable diseases. Previous in vitro studies from our group disclosed the carcinogenic potential of β-HCH, which contributes to neoplastic transformation by means of multifaceted intracellular mechanisms. Considering the positive evidence regarding the protective role of natural bioactive compounds against pollution-induced toxicity, micronutrients from olive and tomato endowed with the capability of modulating β-HCH cellular targets were tested. For this purpose, the solution obtained from a patented food supplement (No. EP2851080A1), referred to as Tomato and Olive Bioactive Compounds (TOBC), was administered to the androgen-sensitive prostate cancer cells LNCaP and different biochemical and cellular assays were performed to evaluate its efficiency. TOBC shows a dose-dependent significant chemoprotection by contrasting β-HCH-induced intracellular responses such as STAT3 and AhR activation, disruption of AR signaling, antiapoptotic and proliferative activity, and increase in ROS production and DNA damage. These experimental outcomes identified TOBC as a suitable functional food to be included in a diet regimen aimed at defending cells from β-HCH negative effects, recommending the development of tailored enriched formulations for exposed individuals.

G3BP1 inhibits Cul3SPOP to amplify AR signaling and promote prostate cancer

SPOP, an E3 ubiquitin ligase, acts as a prostate-specific tumor suppressor with several key substrates mediating oncogenic function. However, the mechanisms underlying SPOP regulation are largely unknown. Here, we have identified G3BP1 as an interactor of SPOP and functions as a competitive inhibitor of Cul3SPOP, suggesting a distinctive mode of Cul3SPOP inactivation in prostate cancer (PCa). Transcriptomic analysis and functional studies reveal a G3BP1-SPOP ubiquitin signaling axis that promotes PCa progression through activating AR signaling. Moreover, AR directly upregulates G3BP1 transcription to further amplify G3BP1-SPOP signaling in a feed-forward manner. Our study supports a fundamental role of G3BP1 in disabling the tumor suppressive Cul3SPOP, thus defining a PCa cohort independent of SPOP mutation. Therefore, there are significantly more PCa that are defective for SPOP ubiquitin ligase than previously appreciated, and these G3BP1high PCa are more susceptible to AR-targeted therapy.

Large Multicohort Study Reveals a Prostate Cancer Susceptibility Allele at 5p15 Regulating TERT via Androgen Signaling-Orchestrated Chromatin Binding of E2F1 and MYC.

Aberrant telomerase reverse transcriptase (TERT) expression is crucial for tumor survival and cancer cells escaping apoptosis. Multiple TERT-locus variants at 5p15 have been discovered in association with cancer risk, yet the underlying mechanisms and clinical impacts remain unclear. Here, our association studies showed that the TERT promoter variant rs2853669 confers a risk of prostate cancer (PCa) in different ethnic groups. Further functional investigation revealed that the allele-specific binding of MYC and E2F1 at TERT promoter variant rs2853669 associates with elevated level of TERT in PCa. Mechanistically, androgen stimulations promoted the binding of MYC to allele T of rs2853669, thereby activating TERT, whereas hormone deprivations enhanced E2F1 binding at allele C of rs2853669, thus upregulating TERT expression. Notably, E2F1 could cooperate with AR signaling to regulate MYC expression. Clinical data demonstrated synergistic effects of MYC/E2F1/TERT expression or with the TT and CC genotype of rs2853669 on PCa prognosis and severity. Strikingly, single-nucleotide editing assays showed that the CC genotype of rs2853669 obviously promotes epithelial–mesenchymal transition (EMT) and the development of castration-resistant PCa (CRPC), confirmed by unbiased global transcriptome profiling. Our findings thus provided compelling evidence for understanding the roles of noncoding variations coordinated with androgen signaling and oncogenic transcription factors in mis-regulating TERT expression and driving PCa.

Characterization of the bioactive compounds with efficacy against enteritis in Polygonum hydropiper L. by UHPLC‐Q‐Orbitrap HRMS combined with network pharmacological analysis**

AbstractPolygonum hydropiper L. (PL) is widely used in treating enteritis in China. This study interpreted the active ingredient and mechanism of PL against enteritis through UHPLC‐Q‐Orbitrap HRMS, network pharmacology and molecular docking. UHPLC‐Q‐Orbitrap HRMS of PL manifested 68 compounds. In addition, the primary candidate genes and potential active components were identified by topological analysis of the single‐component disease gene interaction network. The interaction between the active ingredient, whose drug‐likeness properties were confirmed by Lipinski's rule, and the therapeutic gene was confirmed by molecular docking analysis. AutoDock Vina in AutoDock Tools was used to conduct molecular docking between significant components and critical genes. The advantage of this experiment is that the UHPLC‐Q‐Orbitrap HRMS method increases the total chemical composition of PL. Secondly, we obtained active compounds through network pharmacology and found that PL mainly acts on multiple inflammatory pathways in the treatment of enteritis. 61 potential genes of PL for the treatment of enteritis were obtained. The genes were mainly involved in biological processes such as response to oxidative stress, inhibition of inflammatory factors and inflammatory pathways, and role in treating enteritis by participating in AR, NOS2, MMP9, MMP2, SRC, and other signaling pathways.

82P Correlations of androgen receptor (AR) and transforming growth factor beta receptor type 2 (TGFbR2) expression in prostate cancer (PCa)

Transforming growth factor beta (TGFb) is a potent inducer of epithelial-mesenchymal transition. Of all members of the TGFb receptor family only TGFbR2 is capable of direct binding to ligand. In experimental conditions interactions were shown between TGFbR2 and AR signaling pathways, often leading to negative feedback loops. However, data on expression levels of both receptors in clinical cancer samples are scarce.

Activity of New Synthetic (2-Chloroethylthio)-1,4-naphthoquinones in Prostate Cancer Cells.

Development of resistance to currently available standard therapies in advanced prostate cancer (PCa) emphasizes the need for novel therapeutic options. Here, we report the synthesis of new hybrid molecules consisting of 2-chloroethylthio and 1,4-naphthoquinone pharmacophores and describe their activity in PCa. In screening analyses, the introduction of one 2-chloroethylthio group improved the anticancer properties of 1,4-naphthoquinones, whereas the introduction of a second 2-chloroethylthio moiety rather decreased activity. Two most promising of the synthesized compounds, 30 and 32, were highly active in different human PCa cell lines harboring varying resistance profiles at nanomolar concentrations. The generated data suggest that the compounds are capable of mitochondria targeting, cytotoxic ROS induction, and DNA damage, which resulted in apoptosis presumably executed in a caspase-dependent manner. The substances synergized with the clinically approved PARP inhibitor olaparib and resensitized AR-V7-expressing PCa cells to antiandrogen enzalutamide, as well as to a combination of enzalutamide and an AKT inhibitor. This was at least in part exerted via down-regulation of AR-V7 expression and inhibition of AR signaling. Mild antagonism was observed in combination with platinum- or taxane-based chemotherapy, which was putatively related to treatment-induced activation of p38, JNK1/2, ERK1/2, MEK1/2, and AKT, functioning as potential pro-survival factors. Thus, the synthesized (2-chloroethylthio)-1,4-naphthoquinone derivatives exhibit promising anticancer properties in vitro, suggesting their further development as potential therapeutics for the treatment of castration-resistant PCa.

Predicting cancer drug TARGETS - TreAtment Response Generalized Elastic-neT Signatures

We are now in an era of molecular medicine, where specific DNA alterations can be used to identify patients who will respond to specific drugs. However, there are only a handful of clinically used predictive biomarkers in oncology. Herein, we describe an approach utilizing in vitro DNA and RNA sequencing and drug response data to create TreAtment Response Generalized Elastic-neT Signatures (TARGETS). We trained TARGETS drug response models using Elastic-Net regression in the publicly available Genomics of Drug Sensitivity in Cancer (GDSC) database. Models were then validated on additional in-vitro data from the Cancer Cell Line Encyclopedia (CCLE), and on clinical samples from The Cancer Genome Atlas (TCGA) and Stand Up to Cancer/Prostate Cancer Foundation West Coast Prostate Cancer Dream Team (WCDT). First, we demonstrated that all TARGETS models successfully predicted treatment response in the separate in-vitro CCLE treatment response dataset. Next, we evaluated all FDA-approved biomarker-based cancer drug indications in TCGA and demonstrated that TARGETS predictions were concordant with established clinical indications. Finally, we performed independent clinical validation in the WCDT and found that the TARGETS AR signaling inhibitors (ARSI) signature successfully predicted clinical treatment response in metastatic castration-resistant prostate cancer with a statistically significant interaction between the TARGETS score and PSA response (p = 0.0252). TARGETS represents a pan-cancer, platform-independent approach to predict response to oncologic therapies and could be used as a tool to better select patients for existing therapies as well as identify new indications for testing in prospective clinical trials.

PD44-08 NOVEL DUAL TARGETING AKR1C3 AND AR/AR VARIANTS INHIBITS ANDROGEN SIGNALING AND OVERCOMES RESISTANCE TO ANTIANDROGEN TREATMENT

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (PD44)1 Sep 2021PD44-08 NOVEL DUAL TARGETING AKR1C3 AND AR/AR VARIANTS INHIBITS ANDROGEN SIGNALING AND OVERCOMES RESISTANCE TO ANTIANDROGEN TREATMENT Cameron Armstrong, Shu Ning, Wei Lou, Jinge Zhao, Christopher Evans, Pui-Kai Li, and Allen Gao Cameron ArmstrongCameron Armstrong More articles by this author , Shu NingShu Ning More articles by this author , Wei LouWei Lou More articles by this author , Jinge ZhaoJinge Zhao More articles by this author , Christopher EvansChristopher Evans More articles by this author , Pui-Kai LiPui-Kai Li More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002058.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: AR/AR-V7 and AKR1C3 play key roles in prostate cancer progression and drive resistance to current therapies. Thus far, there are no clinically available therapies to specifically target either AR-V7 or AKR1C3, nor are there dual inhibitors for AR/AR-V7 and AKR1C3. We have developed a number of novel small molecules (LX) that are dual inhibitors of AR/AR-V7 and AKR1C3. METHODS: We designed and synthesized a library of novel compounds according to the structure based computer modeling. Fourteen were selected for initial study. Cell growth assays were used to assess their efficacy at inhibiting CRPC growth. The effects of the LXs on AR/AR variants and AKR1C3 expression were determined by Western blot. PSA-luciferase assays were used to determine effects on AR signaling activity. RNA-seq was performed on selected LXs (LX-1 and LX-5) based on their improved efficacy over the other LX compounds. Resistant cell sublines generated from C4-2B cells resistant to enzalutamide (MDVR), apalutamide (ApalR), darolutamide (DaroR), or abiraterone (AbiR) were treated with LX-1 or their respective antiandrogen and cell number was determined. Mice bearing VCaP xenograft tumors and LuCaP35CR PDX tumors were treated with LX-1 and the effects on tumor growth were determined. RESULTS: Of the 14 LX compounds, LX-1 had the greatest effect at reducing cell number, AR/AR variant expression, and AKR1C3 activity. LX-5 was the next most effective compound. PSA-luciferase activity was greatly reduced by both LX-1 and LX-5. RNA-seq analysis demonstrated a robust reduction in AR and AR-V7 signaling gene expression by both LX-1 and LX-5. MDVR, ApalR, DaroR, and AbiR cells all showed a reduction in cell number when treated with LX-1. LX-1 inhibited conversion of the testosterone precursor androstenedione into testosterone in AKR1C3 overexpressing C4-2B and LNCaP cells. Additionally, LX-1 treatment reduced testosterone production by LUCaP35CR tumor cells which express high levels of AKR1C3 in the presence of androstenedione in a dose-dependent manner ex vivo. In addition, treatment with LX-1 reduced tumor growth in both VCaP and LuCaP35CR PDX models. Furthermore, LX-1 treatment reduced intratumoral testosterone. CONCLUSIONS: We generated novel small molecule inhibitors that dual target AKR1C3 and AR/AR variants. These compounds, specifically LX-1, effectively reduce CRPC growth in vitro and in vivo, suggesting a clinical potential for treating advanced prostate cancer. Source of Funding: NIH/NCI CA 225836 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e740-e740 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cameron Armstrong More articles by this author Shu Ning More articles by this author Wei Lou More articles by this author Jinge Zhao More articles by this author Christopher Evans More articles by this author Pui-Kai Li More articles by this author Allen Gao More articles by this author Expand All Advertisement Loading ...

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Cornification and classical versus nonclassical androgen receptor signaling in mouse penile/preputial development

Mouse penile development is androgen-dependent. During development of male and female external genitalia, an internal ectodermal epithelial structure forms called the preputial lamina. At puberty the male preputial lamina canalizes to create the preputial space, effectively splitting into two layers: (a) the epithelial lining of the prepuce and (b) the surface epithelium of the penis. The female preputial lamina does not canalize, and instead remodels into the inverted U-shaped clitoral lamina of the adult female mouse. Androgen-dependent penile development was studied in transgenic mice with pathway-selective AR mutant transgenes through which AR signaling was activated either via the classical (AR-C) or the nonclassical pathway (AR-NC). Penile development and canalization of the preputial lamina was observed in AR-C and wild-type male mice naturally having both AR-C and AR-NC pathways. Conversely, clitoral development occurred in AR null (lacking both AR-C and AR-NC pathways) and AR-NC mice. The process of canalization of the preputial lamina seen in wild-type, AR-C and AR-C/AR-NC male mice involved cornification of the preputial lamina which involved up-regulation of keratin 10 and loricrin. Such up-regulation of these epidermal proteins was absent in the developing and adult clitoral lamina seen in wild-type female mice and AR-NC and AR null male (XY) mice. Thus, signaling through AR-C is sufficient to initiate and promote penile development and canalization of the preputial lamina, a process involving epithelial cornification.

Neuropilin-2 Promotes the Development of Neuroendocrine Prostate Cancer

Neuroendocrine prostate cancer (NEPC), a lethal subset of the disease, is characterized by loss of AR signaling and resulting resistance to AR-targeted therapy during neuroendocrine transdifferentiation, for which the molecular mechanisms remain unclear. Here, we report that neuropilin 2 (NRP2) is upregulated in both de novo and therapy-induced NEPC, which induces neuroendocrine markers, neuroendocrine cell morphology and NEPC cell aggressive behaviors. NRP2 silencing reduced or even eliminated NEPC tumor xenograft growth. Mechanistically, NRP2 engages in reciprocal crosstalk with AR, where NRP2 is a direct transcriptional target inhibited by AR, and in turn, suppresses AR signaling by downregulating the AR transcriptional program and confers resistance to enzalutamide. Moreover, NRP2 physically interacts with VEGFR2 through the intracellular SEA domain to activate STAT3 phosphorylation and subsequently SOX2, thus driving NEPC differentiation and growth. Together these results characterize NRP2 as a driver of NEPC and suggest NRP2 as a potential therapeutic target in NEPC.

Abstract 740: A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology

Abstract Introduction: OncoSignal test was developed to determine and quantify functional signal transduction pathway activity levels of 4 key oncogenic signaling pathways (ER, AR, PI3K and MAPK), based on the quantitative measurement of mRNA expression levels of the direct target genes. Here we present data of a 4 pathway qPCR test, that can be used in external labs and is able to measure simultaneously the 4 pathway activities in tumor tissue samples possibly providing important information for optimal therapy selection for treatment of breast cancer. Materials and methods: OncoSignal 4 pathway test was compared between 2 reference laboratories; the Philips service lab in The Netherlands and at Protean BioDiagnostics, a US CAP-certified CLIA service lab. mRNA was isolated from unstained FFPE tissue sections of well characterized breast tissues obtained from 65 patients (retrospective). Annotated areas with at least 50% tumor cells were used (total volume about 0.25mm3). The OncoSignal test was used to measure the signal transduction pathway activity of AR, ER, PI3K and MAPK pathways. IHC staining for ER, PR, Ki67 and HER2 was also available for all samples enabling tumor categorization into conventional breast cancer subtypes. Results: Excellent inter-lab concordance was found with correlation coefficients of 0.99, 0.99, 0.98 and 0.99 for ER, AR, PI3K and MAPK, respectively. Average absolute difference between pathway activities was 3.6 activity points on a 0-100 activity score scale. All 32 cases passed the criteria set for reproducibility. Of the 65 tested breast cancer samples, 58 could be subtyped based on IHC. Five of the 58 samples failed QC. Of interest, ER IHC positive luminal A (41%) and B (28%) breast cancer showed high variation in ER pathway activity between cases, suggesting that the ER signaling pathway is not the tumor driving pathway in a significant subset of IHC ER positive tumors. As expected the Her2-enriched (17%) and TNBC (17%) sub-groups predominantly had a very low ER pathway activity score. AR pathway activity was most prominent in the HER2 enriched tumor groups, while in TNBC the MAPK pathway was most active. Conclusion: The OncoSignal 4 pathway test is robust and can be implemented in external (local) labs as shown by the excellent correlation results. The results of the signal pathway activity analysis correlate well with breast subtype classification, however, the assay has the potential to uncover specific pathway activation independent of subtype which may have significant value for precision oncology and targeted therapy selection. The test has potential for wide utilization as it can be performed on conventional FFPE prepared breast specimens, and may be complementary to conventional mutational and IHC analysis for classifying breast cancer and selecting appropriate therapy. Citation Format: Eveline den Biezen, Saskia Vermeer, Diederick Keizer, Martijn Akse, Martijn van Zelst, Dianne van Strijp, Hannah Park, Anthony Magliocco. A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 740.

Abstract 2364: MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells

Abstract One of six deaths globally is due to cancer. In 2018, prostate cancer (PCa) represented 1.28 million cases worldwide, the second most frequent cancer diagnosis and fifth leading cause of death in men. Currently, the mortality rate of PCa has experienced a steady increase due to the progression of metastatic castration-resistant prostate cancer (mCRPC). As an androgen driven disease, androgen deprivation therapies (ADTs) have been employed for treatment. However, despite ADTs, mCRPC develops by maintaining AR signaling, which can eventually lead to progression of AR-independent neuroendocrine PCa (NEPC). NEPC is observable in 1 in 5 men with mCRPC and associated with poor outcome. Therefore, further understanding the mechanisms of progression to mCRPC and NEPC is required. The 8q24 human chromosomal region associated with Increased PCa incidence and aggressiveness contains the PVT1 (Plasmocytoma Variant Translocation 1) locus. The PVT1 locus encodes six microRNAs, including microRNA-1205 (miR-1205) in PCa. Our laboratory previously confirmed that miR-1205 is underexpressed in PCa tissue in comparison to normal prostatic tissue and in CRPC cells in comparison to non-CRPC cells. To understand the molecular mechanism of miR-1205, we performed RNA pull down and RNA sequencing analysis to identify miR-1205 molecular targets in non-tumorigenic RWPE-1 prostate epithelial cells and PCa cells (MDA PCa 2b (metastatic PCa), PC-3 (NEPC), and C4-2B (CRPC)). We identified one cut homeobox 2 (ONECUT2 or OC2) as a direct molecular target of miR-1205. ONECUT2 is a master regulator of transcriptional factors that drives NEPC by through regulating hypoxia signaling or inducing neuroendocrine markers. ONECUT2 enrichment was approximately 3-fold higher in RWPE-1 (P=0.26), 2-fold in PC-3 (P=0.06), 1-fold in C4-2B (P=0.05) and MDA PCa 2b (P=0.02) cells. The significant binding of miR-1205 to ONECUT-2 in the PC-3 NEPC cell line suggests that miR-1205 regulation of ONECUT2 may be an important molecular mechanism in NEPC. Consequently, further study of miR-1205 and ONECUT2 in NEPC is required. Citation Format: Bachelard Dieujuste, Michelle Naidoo, Olorunseun Ogunwobi. MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2364.