Drug Target In Prostate Cancer Research Articles

Abstract 7486: Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay

Abstract Background Prostate cancer drug targets and biomarkers may be investigated non-invasively on circulating tumor cells (CTCs) obtained via liquid biopsies. PSMA is an important drug target in prostate cancer, and ARv7 is a splice variant that confers resistance to second-generation androgen receptor inhibitors. In this study, we describe the development and analytic validation of a novel assay to quantify ARv7 and PSMA protein expression on CTCs. The assay was validated with blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. To our knowledge, a PSMA/ARv7 dual protein assay for CTCs has not previously been reported. Methods LNCaP (PSMA positive), PC3 (PSMA negative), 22Rv1 (ARv7 positive), and BT474 (ARv7 negative) cells were spiked into healthy donor blood to generate model CTCs. Nucleated cells were collected from these spike-in samples using the AccuCyte® sample preparation kit and spread onto microscope slides. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude white blood cells (WBCs), and with PSMA and ARv7 to evaluate protein expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers based on machine learning algorithms and quantitatively measures biomarker signal mean fluorescence intensity (MFI). Results For ARv7/PSMA expression classification of the model CTCs, the dual biomarker assay showed analytic sensitivity of 83%/97% and specificity 98%/99%, yielding accuracy of 90.2%/97.9%, respectively. The dynamic range of MFI for both biomarkers was approximately three orders of magnitude. Inter-run reproducibility CV was 13.6% (ARv7) and 6.9% (PSMA). Slide repeatability mean CV was 11.1% (ARv7) and 12.0% (PSMA). Clinical feasibility of the assay was investigated in 9 progressing Stage IV prostate cancer patient blood samples. CTCs were identified in all clinical samples (median 4, range 1 - 384). Thresholds for biomarker positivity were established based on MFI measurements of positive and negative control cell lines. ARv7 positivity in clinical samples ranged from 33% to 100%, and PSMA positivity ranged from 0% to 75%. Conclusions We have developed a dual ARv7/PSMA assay that simultaneously measures protein expression of both biomarkers on individual CTCs. The assay is accurate and reproducible for the assessment of ARv7 and PSMA expression by liquid biopsy in clinical studies. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Eric Kaldjian, Arturo Ramirez. Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7486.

Abstract A037: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2

Abstract Background: Treatment-related neuroendocrine prostate cancer (NEPC) is an aggressive subset of castration-resistant prostate cancer (CRPC), found in ~20% of lethal CRPC. The mechanisms underlying the progression of prostate cancer to NEPC are largely unclear, and new drug targets are desperately needed. NEPC is known to be highly vascularized. Elevated expression of NE markers and increased angiogenesis are two prominent phenotypes of NEPC, and thus are expected to be linked. However, direct molecular links between these two phenotypes are still elusive, whose elucidation will substantially expand our knowledge in NEPC and enable the development of effective treatments for NEPC. Through RNAi & cDNA screening and functional validations, we previously discovered that GPCR-kinase 3 (GRK3) is essential preferentially for highly metastatic cancer cells as compared to lowly metastatic cancer cells. The mechanisms of GRK3 in prostate cancer progression were mostly unknown. Methods: We assessed GRK3’s expression in patient samples through IHC staining on TMA and data mining on public RNA-seq datasets with large cohorts. Using molecular and cell biology methods, we determined the impacts of GRK3 and HDAC2 overexpression and silencing on NE marker expression and angiogenesis in prostate cancer cells. Through biochemistry, we investigated the nature of GRK3-HDAC2 relation. By ChIP-PCR, we measured the impacts of GRK3 genetic and pharmacological modulations on HDAC2’s epigenetic activity. Through compound library screening and biochemical characterization, we searched for novel GRK3 inhibitors to study GRK3 biology and to evaluate whether GRK3 is a suitable drug target for prostate cancer. Results: GRK3 is significantly overexpressed in metastatic prostate tumors from patients, especially in NEPC. GRK3 promotes both angiogenesis and neuroendocrine differentiation in prostate cancer cells, indicating that it is a key missing link for these two phenotypes. Mechanistically, GRK3 enhances the epigenetic repressor activity of histone deacetylase 2 (HDAC2) to suppress key repressors of angiogenesis or NE phenotype. We have identified several compounds that block kinase activity of GRK3 much more potently than that of GRK2, the closest-related kinase to GRK3. Of note, our GRK3 inhibitors could substantially reduce angiogenesis and NE marker expression, as well as significantly inhibit NEPC cell growth in culture and in mouse xenografts. Conclusion: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression. Its mechanism of actions is at least in part through enhancing HDAC2’s epigenetic activity. Results based on our novel GRK3 inhibitors suggest that GRK3 is a valuable new drug target for aggressive prostate cancer. Citation Format: Samira Naderinezhad, Zheng Wang, Guoliang Zhang, Michael Ittmann, Martin Gleave, Wenliang Li. Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2 [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A037.

Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain and Modulation via Direct Interactions with DNA Abasic Sites: Understanding the Mechanisms Involved in Castration-Resistant Prostate Cancer.

The androgen receptor (AR) is an important drug target in prostate cancer and a driver of castration-resistant prostate cancer (CRPC). A significant challenge in designing effective drugs lies in targeting constitutively active AR variants and, most importantly, nearly all AR variants lacking the ligand-binding domain (LBD). Recent findings show that an AR's constitutive activity may occur in the presence of somatic DNA mutations within non-coding regions, but the role of these mutations remains elusive. The discovery of new drugs targeting CRPC is hampered by the limited molecular understanding of how AR binds mutated DNA sequences, frequently observed in prostate cancer, and how mutations within the protein and DNA regulate AR-DNA interactions. Using atomistic molecular dynamics (MD) simulations and quantum mechanical calculations, we focused our efforts on (i) rationalising the role of several activating DBD mutations linked to prostate cancer, and (ii) DBD interactions in the presence of abasic DNA lesions, which frequently occur in CRPC. Our results elucidate the role of mutations within DBD through their modulation of the intrinsic dynamics of the DBD-DNA ternary complex. Furthermore, our results indicate that the DNA apurinic lesions occurring in the androgen-responsive element (ARE) enhance direct AR-DNA interactions and stabilise the DBD homodimerisation interface. Moreover, our results strongly suggest that those abasic lesions may form reversible covalent crosslinks between DNA and lysine residues of an AR via a Schiff base. In addition to providing an atomistic model explaining how protein mutations within the AR DNA-binding domain affect AR dimerisation and AR-DNA interactions, our findings provide insight into how somatic mutations occurring in DNA non-coding regions may activate ARs. These mutations are frequently observed in prostate cancer and may contribute to disease progression by enhancing direct AR-DNA interactions.

Molecular dynamics simulations reveal the plausible agonism/antagonism mechanism by steroids on androgen receptor mutations

Androgen receptors (AR) are the primary drug target in prostate cancer (PCa). There are several drugs developed against its activity for prostate cancer treatment, but cancer cells revive AR signaling against those drugs by using alternative steroids such as glucocorticoids. In addition, antagonists become agonists due to emergence of mutations in AR gene. The mechanism by which antagonists are converted into agonists and how AR signaling is recovered by other steroids has yet to be fully elucidated. In this study, we interrogated the role of bicalutamide conformation in its antagonist function and how glucocorticoids such as prednisolone and dexamethasone revive AR signaling at the molecular level by means of molecular dynamics. We found that the ‘‘closed’’ conformation of bicalutamide is essential for its antagonist function and W741 residue is forcing it into this conformation. Moreover, we show that prednisolone and dexamethasone behave like natural agonist DHT which confirm the experimental results that show their role in the reviving AR signaling in the case of ARL701H mutation.

Modulation of androgen receptor DNA binding activity through direct interaction with the ETS transcription factor ERG

The androgen receptor (AR) is a type I nuclear hormone receptor and the primary drug target in prostate cancer due to its role as a lineage survival factor in prostate luminal epithelium. In prostate cancer, the AR cistrome is reprogrammed relative to normal prostate epithelium and particularly in cancers driven by oncogenic ETS fusion genes. The molecular basis for this change has remained elusive. Using purified proteins, we report a minimal cell-free system that demonstrates interdomain cooperativity between the ligand (LBD) and DNA binding domains (DBD) of AR, and its autoinhibition by the N terminus of AR. Furthermore, we identify ERG as a cofactor that activates AR's ability to bind DNA in both high and lower affinity contexts through direct interaction within a newly identified AR-interacting motif (AIM) in the ETS domain, independent of ERG's own DNA binding ability. Finally, we present evidence that this interaction is conserved among ETS factors whose expression is altered in prostate cancer. Our work highlights, at a biochemical level, how tumor-initiating ETS translocations result in reprogramming of the AR cistrome.

Vemurafenib Inhibits Active PTK6 inPTEN-null Prostate Tumor Cells

Protein tyrosine kinase 6 (PTK6, also called BRK) is overexpressed and activated in human prostate cancer. Loss of the tumor suppressor PTEN, a frequent event in prostate cancer, leads to PTK6 activation at the plasma membrane and its oncogenic signaling. The small molecule inhibitor vemurafenib, also known as PLX4032, and its tool analog PLX4720 were designed to inhibit constitutively active BRAF V600E, yet they also have potent effects against PTK6. Vemurafenib is used in the treatment of metastatic melanoma, but its efficacy in prostate cancer has not been assessed. When activated at the plasma membrane, PTK6 promotes signaling through FAK, EGFR, and ERK1/2, and we show this can be blocked by vemurafenib. In addition, PTK6-mediated cell growth, migration, and invasion are inhibited upon vemurafenib administration. Using a flank xenograft model, vemurafenib treatment reduced tumor burden. Using saturation transfer difference NMR and molecular docking, we demonstrate that vemurafenib binds in the active site of PTK6, inhibiting its activation. These structural studies provide insight into the PTK6-vemurafenib complex, which can be utilized for further refinement chemistry, whereas functional studies demonstrate that active PTK6 is a viable drug target in prostate cancer.

Abstract A056: Is non-mutated GPR89A protein a potential drug target in prostate cancer?

Abstract Mutated genes as drug targets to treat cancer are a widely known concept; however, the effect is moderate. Targeting nonmutated genes is an alternative and probably has potential to benefit patients. GPR89A, as a nonmutated protein in prostate cancer, was evaluated as a drug target in this project. Data showed that GPR89A knockdown could induce high cancer cell death rate. Combination use of GPR89A siRNA and docetaxel triggered synergism effect, which implicates a possibility that GPR89A inhibitors could be used together with docetaxel. The high-throughput screening discovered 39 drug candidates that could reduce cancer cell viability and GPR89A expression simultaneously. These findings highlight the importance of nonmutated genes in cancer progression. Targeting nonmutated genes, e.g., GPR89A, should be evaluated further to find significant association between these genes/proteins and cancers. Citation Format: Yuanjun Ma, Yanling Liu, Sten Nilsson, Chunde Li. Is non-mutated GPR89A protein a potential drug target in prostate cancer? [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A056.

Abstract 909: Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9

Abstract Background: Androgen receptor (AR) is the key drug target in prostate cancer. AR splice variant-7 (AR-V7) is the most relevant AR variants implicated in castration-resistant prostate cancer (CRPC). Detection of AR-V7 in both liquid biopsies and tissue biopsies predicts poorer response to novel AR-targeting drugs. However, because AR-V7 always coexists with the full-length AR (AR-FL), the functional importance of AR-V7 needs to be dissected in the context of AR-FL. Lack of relevant cell line models presented a hurdle for detailed dissection of the role for AR-V7. It is essential to develop cell lines that express endogenous AR-V7 at a level comparable to clinical specimens, with or without AR-FL, to facilitate functional studies. The goal of this study is to develop and characterize LNCaP95 lines following specific and complete knock-out of AR molecules by CRISPR-Cas9 system. Methods: Different AR isoforms were knocked-out in parental LNCaP95 cells by CRISPR-Cas9. AR-FL was knocked-out through sgRNAs targeting AR exon5 or exon6. AR isoforms with functional N-terminal domain (total AR) were knocked-out through sgRNA targeting AR exon1. Complete knockout of AR-FL and total AR was achieved in LNCaP95 cells. The on-target editing of AR genomic regions were verified by Sanger Sequencing. Results: Complete knockout of AR-FL protein and total AR protein was confirmed in two clones carrying homologous indels, ARfl-KO#1/2 and AR-KO#1/2. Antibodies against different AR domains were used to differentiate expression of different AR isoforms, by Western blot and immunohistochemistry (IHC). Cell proliferation was not affected by androgen in AR-FL or total AR knockout cells, as shown in cell growth and clonogenic assays. The expression of AR-target genes such as prostate-specific antigen (PSA) were analyzed by realtime-PCR. PSA was expressed at similar level compared to parental cells in AR-FL knockout cells, which was totally lost in total AR knockout cells. Interestingly, total AR knockout cells are more resistant to ionizing radiation and PARP inhibitor olaparib compared to parental cell line. Conclusion: We have successfully developed the first prostate cancer cell line expressing endogenous AR-V7 at a level comparable to clinical specimens without AR-FL, allowing dissection of the functions mediated by AR-FL and AR-V7. Initial functional characterization suggested AR-V7 maintained the baseline level of AR signaling activity, which was not dependent on androgen. The chromatin-binding pattern of AR-V7 in the absence of AR-FL is under investigation. We have also developed a novel total AR knockout cell model from the AR-positive parental cell line. This model suggested prostate cancer cells were able to survive in an AR-null manner, although loss of AR significantly inhibited cell proliferation. Preliminary studies have found loss of total AR confers resistance to ionizing radiation and PARP inhibition. Citation Format: Yezi Zhu, Pei Zhao, Changxue Lu, Shihua Chen, Jun Luo. Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9 [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 909.

Evaluating the effects of HIPK2 Inhibition on Castration‐Resistant Prostate Cancer Cells

Prostate cancer is the second leading cause of death in men in the United States. It depends on the signaling by the androgen receptor (AR), which is activated by testosterone. AR signaling promotes normal prostate development as well as cancer. Current treatments for prostate cancer include prostatectomy and androgen deprivation therapy. Although androgen deprivation is effective in halting prostate cancer growth, a group of men are often diagnosed with castration resistant prostate cancer (CRPC), which is refractory to hormone deprivation. Therefore, new targets are needed to stop prostate cancer growth. In this study, we investigated whether Homeodomain‐interacting protein kinase 2 (HIPK2) affects AR signaling and prostate cancer cell growth. To do this, we utilized doxycycline‐induced shRNA‐mediated silencing of HIPK2 expression in 22Rv1 and LNCaP‐95, two cell lines that are refractory to androgen deprivation, and demonstrated substantial knockdown of both HIPK2 mRNA and protein. In addition, we observed significant changes in AR target gene expression. To determine the effects of HIPK2 knockdown on AR subcellular localization, we fractionated control and doxycycline‐treated 22Rv1 and LNCaP‐95 cells. We observed modest changes in chromatin‐bound AR upon HIPK2 depletion, suggesting that HIPK2 influences AR chromatin occupancy and in turn AR‐dependent transcription. We also tested HIPK2 catalytic inhibitors by examining their effects on 22Rv1 and LNCaP‐95 cellular proliferation, and phosphorylation a HIPK2 substrate, SIAH2. We found that HIPK2 inhibitors modestly affected the proliferation and substrate phosphorylation. Thus, HIPK2 can modulate AR activity, highlighting the role of HIPK2 as a new drug target for prostate cancer.Support or Funding InformationNew York University Langone Medical CenterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional characterization of the G162R and D216H genetic variants of human CYP17A1

Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5 h, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms.

Non-Genomic Actions of the Androgen Receptor in Prostate Cancer.

Androgen receptor (AR) is a validated drug target for prostate cancer based on its role in proliferation, survival, and metastases of prostate cancer cells. Unfortunately, despite recent improvements to androgen deprivation therapy and the advent of better antiandrogens with a superior affinity for the AR ligand-binding domain (LBD), most patients with recurrent disease will eventually develop lethal metastatic castration-resistant prostate cancer (CRPC). Expression of constitutively active AR splice variants that lack the LBD contribute toward therapeutic resistance by bypassing androgen blockade and antiandrogens. In the canonical pathway, binding of androgen to AR LBD triggers the release of AR from molecular chaperones which enable conformational changes and protein–protein interactions to facilitate its nuclear translocation where it regulates the expression of target genes. However, preceding AR function in the nucleus, initial binding of androgen to AR LBD in the cytoplasm may already initiate signal transduction pathways to modulate cellular proliferation and migration. In this article, we review the significance of signal transduction pathways activated by rapid, non-genomic signaling of the AR during the progression to metastatic CRPC and put into perspective the implications for current and novel therapies that target different domains of AR.

Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells.

One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies.

Kinase joins the chaperone club: Androgen-regulated kinome reveals choline kinase alpha as a potential drug target in prostate cancer

ABSTRACTTo identify clinically relevant downstream effectors of androgen signaling, the androgen-regulated kinome was defined in prostate cancer (PCa). Within this study, choline kinase α (CHKA) was identified as an androgen receptor chaperone that is both a biomarker of progression and a potential therapeutic target for PCa.

A Fluorescent, HTS‐Adaptable Coupled‐Enzyme Assay for Measurement of Human Cytosolic Sulfotransferase (SULT) 2B1b Activity and Identification of Small‐Molecule Inhibitors

Human cytosolic sulfotransferase (SULT) 2B1b, as a member in the SULT family, catalyzes the transfer of a sulfonate moiety (‐SO3) from the universal donor 3‐phopho‐adenosine 5′‐phophosulfate (PAPS) to the hydroxyl groups of various substrates, which leads to a change in water solubility and thus triggers various downstream effects. SULT2B1b highly prefers 3β‐hydroxysteroids, such as cholesterol, as its substrates. By switching the sulfonation state of its substrates, SULT2B1b is involved in the regulation of steroid synthesis, transportation and signaling. Furthermore, SULT2B1b has been suggested as a potential drug target for prostate cancer. Therefore, we aim to identify small‐molecule inhibitors from high‐throughput screening (HTS), and utilize them to study the physiological roles of SULT2B1b in both normal tissues and carcinogenesis as well as provide possible leads for drug discovery in the treatment of prostate cancer. Considering the lack of convenient and HTS‐adaptable activity assays for SULT2B1b, we have developed a fluorescent, coupled‐enzyme assay with real‐time fluorescence‐monitoring capabilities as opposed to the commonly used endpoint radioactivity assays. The new assay was adapted for HTS in 384‐well plates and after non‐trivial assay optimization, this activity assay resulted in a reliable performance (Z′‐factor=0.8 in preliminary study, Z‐factor=0.6~0.7 in the actual screening). Initial hits from HTS have been further validated in vitro and in cellulo. Our criteria for highly selective inhibitors of SULT2B1b include high potency in vitro, and success in mimicking the effects of SULT2B1b siRNA in cellular studies, including attenuating prostate cancer cell proliferation and decreasing cholesterol sulfate accumulation.Support or Funding InformationThis work is funded by a grant from the Department of Defense.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target.

Background:The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling.Methods:Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided.Results:CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts.Conclusions:CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

The association of CXCR4 expression with clinicopathological significance and potential drug target in prostate cancer: a meta-analysis and literature review.

CXCR4/CXCL12 axis plays an important role in tumor growth, angiogenesis, metastasis, and therapeutic resistance. The aim of this study is to perform a meta-analysis and literature review to evaluate the association of CXCR4 expression with clinicopathological significance and prognosis in patients with prostate cancer (PCa). A detailed literature search was made in Medline, EMBASE, Web of Science, and Google Scholar for related research publications. The data were extracted and assessed independently. Analysis of pooled data was performed using Review Manager 5.2. Odds ratio (OR) with corresponding confidence intervals were calculated and summarized. The meta-analysis included a total of eleven studies and 630 patients. The rate of CXCR4 protein expression in PCa was significantly higher than in nonmalignant prostate tissues (OR =35.71, P<0.00001). The expression of CXCR4 protein was not significantly associated with Gleason score (P=0.73). However, the frequency of CXCR4 protein expression was significantly higher in T3–4 stage than in T1–2 stage of PCa (OR =2.35, P=0.001). The expression of CXCR4 protein was significantly associated with the presence of lymph node and bone metastasis of PCa: for lymph node metastasis positive versus negative, OR was 5.07 and P=0.0003, and for bone metastasis positive versus negative, OR was 7.03 and P=0.003. Cancer-specific survival of patients with PCa was significantly associated with CXCR4 protein expression, and the pooled Hazard ratio was 0.24 and P=0.002. In conclusion, the high expression of CXCR4 protein is a diagnostic biomarker of PCa, and it is significantly associated with T stages. The increased expression of CXCR4 protein is significantly associated with lymph nodes or bone metastasis, and CXCR4 is a poor prognosis predictor for patients with PCa. Taken together, our findings indicate that CXCR4 could be a target not only for the development of therapeutic intervention but also for the noninvasive monitoring of PCa progression.

Abstract 4370: GSK3α/β inhibition as a drug target in prostate cancer

Abstract Despite what its namesake suggests, Glycogen Synthase Kinase 3 (GSK3) is a kinase that is implicated in a myriad of signalling pathways and has recently received considerable interest due to its conflicting roles oncogenesis. Though GSK3 has classically been regarded as a tumour suppressor due to in role in supressing WNT signalling, emerging evidence suggests that GSK3 also functions as a tumor promoter by facilitating tumor cell survival and conferring drug resistance in cancer. These tumor promoting effects have been partly linked to the expression of certain NFκB target genes, whose expression in some cancers are influenced by direct GSK3 phosphorylation of NFκB's p65 subunit. Given the complex interplay between GSK3 and the androgen receptor in prostate cancer (PCa), and the inverse correlation between the androgen receptor status and NFκB, we investigate the potential of GSK3 inhibitors to treat and overcome drug resistance in PCa. A kinase inhibitor screen for 80 compounds and additional chemical inhibitors for GSK3 was carried out using the JANUS Automated Workstation at the robotics facility at NUI Galway, Ireland. The cellular fractions for treated cells were separated and assessed for differences in expression between the cytoplasmic and nuclear compartments using western blotting. Flow cytometry and western blotting using antibodies for markers of apoptosis were used to assess for mode of cell death. Differences in gene expression of NFκB target genes were assessed using gene specific primers for SYBR green RT-PCR. RNAi using siRNA directed against each of the GSK3 isoforms, α and β, was utilised for target validation The kinase inhibitor screen demonstrated significant growth inhibition among all the tested PCa cell lines to the GSK3 inhibitor, BIO. This result was validated using two additional chemical GSK3 inhibitors, LiCl and CHIR99021. Expression analysis demonstrated aberrant NFκB and GSK3 expression in the cytoplasmic and nuclear compartments of the cell lines that varied upon treatment with GSK3 inhibitors. siRNA directed against GSK3 confirmed that the effects of the inhibitors were GSK3 specific and demonstrated that both isoforms distinguished between the effects of each of the two isoforms. Our results suggest that GSK3 has a role in the pathogenesis of PCa. This notion is supported by the findings that GSK3 inhibition reduces the viability of PCa cells via a mechanism that is consistent with apoptosis. Furthermore, both NFκB and GSK3 are shown to be active and aberrantly co-expressed in our PCa model. Collectively, these results suggest that GSK3 has an effect on NFκB that may be independent of WNT signalling. Citation Format: Husnain Ali, Amy Burke, Enda O'Connell, Frank Sullivan, Frank Giles, Sharon Glynn. GSK3α/β inhibition as a drug target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4370. doi:10.1158/1538-7445.AM2015-4370

Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Background:We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors.Methods:We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)).Results:We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA.Conclusions:In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required.

Abstract B133: Regulation of the androgen receptor function by a metabolic kinase in prostate cancer.

Abstract In a recent genome-wide study to identify androgen receptor target genes we found that the AR promotes tumor growth by enhancing the expression and activity of enzymes required for metabolic biosynthesis. This raises the possibility for effective therapeutic approaches directed towards AR target genes. Kinases are well-established drug targets. In this study we identify androgen regulated kinome and show that an important androgen regulated metabolic kinase is a drug target in prostate cancer. Our proteome study shows that this kinase interacts with the androgen receptor and regulates AR stability. Inhibition of this kinase significantly reduces AR protein levels and suppresses prostate cancer growth in cell-lines and ex vivo cultures. In clinical prostate cancer this kinase is highly over-expressed in metastatic prostate cancers and inhibition represses metastatic potential of prostate cancer cells. Our study therefore establishes this kinase for the first time as an AR target gene that regualtes AR itself as part of feedback loop and makes a case for further investigation of its inhibitors as novel AR antagonists in both localised and advanced disease. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B133. Citation Format: Mohammad asim, Charlie Massie, Heather Zecchini, Ajoeb Baridi, Nelma Gomes, Hisham Mohammed, Vincent Zecchini, Basetti Madhu, Arham Qureshi, Roslin Russell, Wiebke Hessenkemper, Rouchelle Sriranjan, Carrie Yang, Andy Lynch, Elena Gregorengko, Rory Stark, Paul Rennie, John Griffiths, Aria Baniahmad, Jasol Carroll, Ian Mills, David Neal. Regulation of the androgen receptor function by a metabolic kinase in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B133.

EGF promotes neuroendocrine-like differentiation of prostate cancer cells in the presence of LY294002 through increased ErbB2 expression independent of the phosphatidylinositol 3-kinase-AKT pathway

An increased neuroendocrine (NE) cell population in prostate cancer is associated with more aggressive disease and recurrence after androgen-deprivation therapy, although the mechanism responsible is unknown. In this study, we report that the treatment of LNCaP cells with epidermal growth factor (EGF) in the presence of LY294002, an inhibitor of the phosphoinositol 3'-kinase (PI3K)-AKT pathway, induced an increase of levels and activity of ErbB2. Under these conditions, we also observed cell survival and NE differentiation. When we treated with wortmannin, another PI3K inhibitor, or we knocked down PI3K or AKT isoforms in the presence of EGF, ErbB2 up-regulation was not observed, suggesting that the increase of ErbB2 induced by EGF plus LY294002 is not mediated by the PI3K-Akt pathway. Other targets of LY294002 were also discounted. We also show that ErbB2 up-regulation is directly involved in neuroendocine differentiation but not in cell survival as ErbB2 levels increased in parallel with NE differentiation marker levels, whereas ErbB2 knockdown reduced them; other NE differentiation inducers also increased the ErbB2 levels and the immunohistochemical analysis of prostate cancer samples showed colocalization of ErbB2 and chromogranin A. We found that, in LNCaP cells, EGF in combination with LY294002 increased ErbB2 levels by a PI3K/AKT-independent mechanism and that this increase was associated with the acquisition of a NE phenotype. These results suggest that is worth reconsidering ErbB2 as a drug target in prostate cancer and this should be kept in mind when designing new clinical schedules for the treatment of this disease.