Androgen-sensitive Human Prostate Cancer Research Articles

Enhanced Chemoprevention of Prostate Cancer by Combining Arctigenin with Green Tea and Quercetin in Prostate-Specific Phosphatase and Tensin Homolog Knockout Mice.

The low bioavailability of most phytochemicals limits their anticancer effects in humans. The present study was designed to test whether combining arctigenin (Arc), a lignan mainly from the seed of Arctium lappa, with green tea (GT) and quercetin (Q) enhances the chemopreventive effect on prostate cancer. We performed in vitro proliferation studies on different cell lines. We observed a strong synergistic anti-proliferative effect of GT+Q+Arc in exposing androgen-sensitive human prostate cancer LNCaP cells. The pre-malignant WPE1-NA22 cell line was more sensitive to this combination. No cytotoxicity was observed in normal prostate epithelial PrEC cells. For an in vivo study, 3-week-old, prostate-specific PTEN (phosphatase and tensin homolog) knockout mice were treated with GT+Q, Arc, GT+Q+Arc, or the control daily until 16 weeks of age. In vivo imaging using prostate-specific membrane antigen (PSMA) probes demonstrated that the prostate tumorigenesis was significantly inhibited by 40% (GT+Q), 60% (Arc at 30 mg/kg bw), and 90% (GT+Q+Arc) compared to the control. A pathological examination showed that all control mice developed invasive prostate adenocarcinoma. In contrast, the primary lesion in the GT+Q and Arc alone groups was high-grade prostatic intraepithelial neoplasia (PIN), with low-grade PIN in the GT+Q+Arc group. The combined effect of GT+Q+Arc was associated with an increased inhibition of the androgen receptor, the PI3K/Akt pathway, Ki67 expression, and angiogenesis. This study demonstrates that combining Arc with GT and Q was highly effective in prostate cancer chemoprevention. These results warrant clinical trials to confirm the efficacy of this combination in humans.

Electrochemical Aptamer-Based Biosensor Developed to Monitor PCA3 and PSA Released by Prostate Cancer Cells

1. Introduction Detection of prostate cancer (PCa) in the early stage of the disease is fundamental for the prevention of metastases and for the initiation of treatment, which may bring greater chance of patient cured; so several research groups have sought the manufacture of a simple and non-invasive methodology [1]. The early diagnosis relies on prostate specific antigen (PSA) serum test, even if it showed clear limits [2]. Recently, prostate cancer gene 3 (PCA3), a non-coding mRNA which is overexpressed in tumor cells when compared with noncancerous prostate tissue [3]. Here, we report a new method for detection of prostate cancer-specific antigen 3 (PCA3) and prostate specific antigen (PSA) released by cancer cells using an aptasensor. Electrochemical impedance spectroscopy (EIS), SEM and EDS were used to evaluate biosensor surface. 2. Experimental Part The aptamers were modified through Biosearch Technologies at 5′-terminus in C6-disulfide linker to have it bond to the gold electrode; and at 3′-end in methylene blue (MB) (as redox probe) to perform the electrochemical analysis, which was followed by the HPLC purification process to remove truncated-synthesis products and to enrich the purity of the aptamer sequences. Aptamer immobilization was done by incubation of 1 μM thiolated aptamer on the top of the gold-coated silicon electrode overnight, in the dark, at 4°C. Subsequently, the electrodes were washed with PBS and a backfilling consisted of 3 mM of MCH aqueous solution for 30 minutes was done to displace the nonspecifically adsorbed aptamer molecules and to passivate the electrode surface [26]. Finally, the electrodes were once more rinsed with PBS to remove MCH excesses. The electrochemical characterization was done in a potentiostat μAutolabIII (Metrohn, NL), using a three-electrode system, which consisted of Ag/AgCl (3M KCl), as reference electrode; Pt wire, as a counter electrode; and gold-coated silicon wafer, as a working electrode. The prostate cell lines used were normal human prostate cells (RWPE-1; ATCC® CRL-11™), androgen-sensitive human prostate cancer cells (LNCaP; ATCC® CRL-1740™), and androgen-independent human prostate cancer cells (PC-3; ATCC® CRL-1435™). The three lineages were chosen to better understand PCA3 and PSA production patterns by different cell types. 3. Results and Discussion The gold-coated silicon wafe electrode was analyzed by SEM and EDS, which acts as working electrode and substrate to anchor the thiolated aptamer on the surface. By using square wave voltammetry, the present aptasensor was able of monitoring the PCA3 and PSA released from prostate cells (normal and cancerous). The PSA and PCA3 detection limits were 1 ng/mL and 9.2 ng/mL, respectively, at linear range up to 150 ng/mL, and it was also able to detect both biomarker even in presence of several interfering compounds (UA, AA, BSA and MUC1). In addition, the herein developed aptasensor was able to verify, at accurate and robust response, the patterns of the PCA3 and PSA released by different cell lines (RWPE-1, LNCaP and PC3). The detection of PSA and PCA3 by aptasensor agree with the cell lineage type, the LNCaP is an androgen-dependent lineage. In this way, an overexpressed both biomarkres was release was verified [49,50] and it can be compared to early-stage patients. On the other hand, the PC3 cells PSA and PCA3 releasing was nearly background signal, demonstrating almost absent production of studied targets, the PC3 lineage is androgen-independent which can be compared to advanced-disease patients. Funding This work has been supported by a grant from FAPESP-Brazil, CNPq and Capes.

Abiraterone Acetate, in Combination with Apigenin, Attenuates the Survival of Human Castration-Sensitive Prostate Cancer Cells.

Abiraterone acetate (AA) is a selective inhibitor of CYP17 α-hydroxylase, which is crucial for androgen biosynthesis. Apigenin (Api) is a natural plant-derived flavonoid with potent antiproliferative and antimigration effects. We aimed to investigate the possible role of Api in combination with the androgen receptor inhibitor AA in the treatment of androgen-sensitive human prostate cancer LNCaP cells. The cells were either exposed to 10 μM AA, 25 μM Api, or in combination for 48 hours, then the viability rate was determined by the MTT test, whilst apoptosis and cell cycle phases were assessed by image-based cytometry. The expression of selected mRNA and proteins were evaluated by RT-qPCR and Western blot, respectively. The combination of AA and Api significantly inhibited LNCaP as well as androgen-insensitive PC3 cell survival in a manner more marked than observed with either single treatment. Co-administration of Api with AA triggered apoptosis. This effect was demonstrated by Hoechst staining, and up-regulation of Bax, cytochrome c, caspase -3, and - 8 and down-regulation of Bcl-2 expression confirmed the effect. AA and Api each individually arrested the cell cycle in the G1 phase, with dual applications, leading to no further increase in the effect produced. The expression of NF-κB p105/p50 and the phosphorylation of AKT markedly decreased after apigenin treatment, with combination treatment leading to a favourable effect in terms of further augmenting the reduction. The co-administration of Api with AA strongly enhanced the efficacy of AA therapy in the treatment of prostate cancer cells. These data suggested that the combination of AA and Api would be a potential chemotherapeutic strategy against prostate cancer.

Abstract LB087: Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models

Abstract TMEFF2, a transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is primarily restricted in expression to healthy brain and prostate and is enriched in prostate cancer. JNJ-70218902 (JNJ-902) is a bispecific antibody that engages TMEFF2-expressing tumor cells and CD3 on T cells causing exposure dependent pro-inflammatory responses and target tumor cell lysis. Here we report the preclinical characterization of JNJ-902 using in vitro and in vivo prostate cancer models. Expression of TMEFF2 transcript and protein in brain and prostate was confirmed in healthy human tissue, as was its elevated expression in advanced prostate cancer tumors, including in 41/45 (91%) metastatic castration-resistant prostate cancer (mCRPC) samples. JNJ-902 bound to TMEFF2-positive LNCaP-AR cells (androgen-sensitive human prostate cancer cell line) in a concentration-dependent manner (EC50 = 9.6 nM); no binding to TMEFF2-negative DU145 prostate cancer cells was observed. The binding affinity (KD) of JNJ-902 for CD3 on primary human T cells was determined to be ~151 nM. T cell-mediated killing, as measured by caspase-3 activity, was induced upon incubation of JNJ-902 with healthy human donor T cells (n = 5) and LNCaP-AR cells at a 3:1 effector to target ratio (EC50 = 1.4 nM). No cytotoxicity was detected using DU145 cells or with control antibodies. Corresponding T cell activation was confirmed by concentration-dependent increases in cell surface CD8+CD25+ expression and proinflammatory cytokine production (eg, GM-CSF, IFN-γ, IL-10, TNF-α). Antitumor activity of JNJ-902 at increasing concentrations up to 5 mg/kg was observed in T cell humanized NSG mice bearing LNCaP xenografts (tumor growth inhibition [∆%TGI] of 75-122%) and in LuCaP 86.2 patient-derived xenografts (∆%TGI of 72-88%) as compared with control-treated mice. JNJ-902-dependent intratumoral infiltration of CD8+ T cells was detected in both models with increases in frequency of CD8+granzymeB+ effector cells. The data summarized here are the first report on the pharmacological activity of a novel TMEFF2 × CD3 bispecific antibody, supporting further investigation of TMEFF2 in mCRPC given its expression in advanced prostate cancer. Targeting TMEFF2 with the TMEFF2 × CD3 bispecific antibody JNJ-902 elicited robust T cell-mediated responses in both prostate cancer cells and xenograft models. Based on these data, a phase 1 dose escalation trial of JNJ-902 in patients with mCRPC is currently ongoing (NCT04397276). Citation Format: Subhasree Basu, Mike Russell, Katrin Sproesser, Bethany Mattson, Cassandra L. Lowenstein, Sathya Venkataramani, Maura Diamond, Robin E. Ernst, Stephen Jarantow, Jackson Wong, Gerald Chu, Kathryn Packman, Shaozhou Ken Tian, Scott R. Brodeur, Margaret Yu, Brent A. Rupnow. Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB087.

In Vitro characterization of the endocrine disrupting effects of per- and poly-fluoroalkyl substances (PFASs) on the human androgen receptor

Per- and poly-fluoroalkyl substances (PFASs) are used extensively in a broad range of industrial applications and consumer products. While a few legacy PFASs have been voluntarily phased out, over 5000 PFASs have been produced as replacements for their predecessors. The potential endocrine disrupting hazards of most emerging PFASs have not been comprehensively investigated. In silico molecular docking to the human androgen receptor (hAR) combined with machine learning techniques were previously applied to 5206 PFASs and predicted 23 PFASs bind the hAR. Herein, the in silico results were validated in vitro for the five candidate AR ligands that were commercially available. Three manufactured PFASs namely (9-(nonafluorobutyl)− 2,3,6,7-tetrahydro-1 H,5 H,11 H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (NON), 2-(heptafluoropropyl)− 3-phenylquinoxaline (HEP), and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl)pentanamide (NNN) elicited significant antiandrogenic effects at relatively low concentrations. We further investigated the mechanism of AR inhibition and found that all three PFASs inhibited AR transactivation induced by testosterone through a competitive binding mechanism. We then examined the antiandrogenic effects of these PFASs on AR expression and its responsive genes. Consistently, these PFASs significantly decreased the expression of PSA and FKBP5 and increased the expression of AR, similar to the effects elicited by a known competitive AR inhibitor, hydroxyflutamide. This suggests they are competitive antagonists of AR activity and western blot analysis revealed these PFASs decreased intracellular AR protein in androgen sensitive human prostate cancer cells. Hence, the findings presented here corroborate our published in silico approach and indicate these emerging PFASs may adversely affect the human endocrine system.

Metformin and Androgen Receptor-Axis-Targeted (ARAT) Agents Induce Two PARP-1-Dependent Cell Death Pathways in Androgen-Sensitive Human Prostate Cancer Cells.

Simple SummaryIn the present study, we sought to determine whether a commonly used oral drug to treat adult-onset diabetes, metformin, which has a longstanding clinical history and known safety and tolerability profile, can improve the anti-cancer effects of two well-established oral agents currently in use to treat advanced prostate cancer, abiraterone and enzalutamide. We used androgen-sensitive cell culture models of human prostate cancer to test our hypothesis. We found that metformin and the oral anti-prostate cancer agents together are more effective in inhibiting prostate cancer cell growth and inducing prostate cancer cell death than when used alone. We identified new pathways by which the enhanced anti-cancer effects occur with the combination treatments. The present work suggests that incorporating metformin with abiraterone or enzalutamide may improve treatment outcomes in hormone sensitive prostate cancer.We explored whether the anti-prostate cancer (PC) activity of the androgen receptor-axis-targeted agents (ARATs) abiraterone and enzalutamide is enhanced by metformin. Using complementary biological and molecular approaches, we determined the associated underlying mechanisms in pre-clinical androgen-sensitive PC models. ARATs increased androgren receptors (ARs) in LNCaP and AR/ARv7 (AR variant) in VCaP cells, inhibited cell proliferation in both, and induced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage and death in VCaP but not LNCaP cells. Metformin decreased AR and ARv7 expression and induced cleaved PARP-1-associated death in both cell lines. Metformin with abiraterone or enzalutamide decreased AR and ARv7 expression showed greater inhibition of cell proliferation and greater induction of cell death than single agent treatments. Combination treatments led to increased cleaved PARP-1 and enhanced PARP-1 activity manifested by increases in poly(ADP-ribose) (PAR) and nuclear accumulation of apoptosis inducing factor (AIF). Enhanced annexin V staining occurred in LNCaP cells only with metformin/ARAT combinations, but no caspase 3 recruitment occurred in either cell line. Finally, metformin and metformin/ARAT combinations increased lysosomal permeability resulting in cathepsin G-mediated PARP-1 cleavage and cell death. In conclusion, metformin enhances the efficacy of abiraterone and enzalutamide via two PARP-1-dependent, caspase 3-independent pathways, providing a rationale to evaluate these combinations in castration-sensitive PC.

Phosphodiesterase Type-5 Inhibitor Tadalafil Modulates Steroid Hormones Signaling in a Prostate Cancer Cell Line.

Background: The androgen receptor (AR) plays a key role in normal prostate homeostasis and in prostate cancer (PCa) development, while the role of aromatase (Cyp19a1) is still unclear. We evaluated the effects of a treatment with Tadalafil (TAD) on both these proteins. Methods: Androgen-sensitive human PCa cell line (LnCAP) was incubated with/without TAD (10−6 M) and bicalutamide (BCT) (10−4 M) to evaluate a potential modulation on cell proliferation, protein and mRNA expression of Cyp19a, AR and estrogen receptor-β (ERβ), respectively. Results: TAD increased early AR nuclear translocation (p < 0.05, after 15 min of exposure), and increased AR transcriptional activity (p < 0.05) and protein expression (p < 0.05) after 24 h. Moreover, after 24 h this treatment upregulated Cyp19a1 and ERβ mRNA (p < 0.05 and p < 0.005 respectively) and led to an increase in protein expression of both after 48 h (p < 0.05). Interestingly, TAD counteracted Cyp19a1 stimulation induced by BCT (p < 0.05) but did not alter the effect induced by BCT on the AR protein expression. Conclusion: We demonstrate for the first time that TAD can significantly modulate AR expression and activity, Cyp19a1 and ERβ expression in PCa cells, suggesting a specific effect of these proteins. In addition, TAD potentiates the antiproliferative activity of BCT, opening a new clinical scenario in the treatment of PCa.

Leelamine suppresses cMyc expression in prostate cancer cells in vitro and inhibits prostate carcinogenesis in vivo.

Leelamine (LLM) inhibits growth of human prostate cancer cells but the underlying mechanism is not fully understood. The present study was undertaken to determine the effect of LLM on cMyc, which is overexpressed in a subset of human prostate cancers. The effect of LLM on cMyc expression and activity was determined by western blotting/confocal microscopy and luciferase reporter assay, respectively. A transgenic mouse model of prostate cancer (Hi-Myc) was used to determine chemopreventive efficacy of LLM. Exposure of androgen sensitive (LNCaP) and castration-resistant (22Rv1) human prostate cancer cells to LLM resulted in downregulation of protein and mRNA levels of cMyc. Overexpression of cMyc partially attenuated LLM-mediated inhibition of colony formation, cell viability, and cell migration in 22Rv1 and/or PC-3 cells. LLM treatment decreased protein levels of cMyc targets (e.g., lactate dehydrogenase), however, overexpression of cMyc did not attenuate these effects. A trend for a decrease in expression level of cMyc protein was discernible in 22Rv1 xenografts from LLM-treated mice compared with control mice. The LLM treatment (10 mg/kg body weight, 5 times/week) was well-tolerated by Hi-Myc transgenic mice. The incidence of high-grade prostatic intraepithelial neoplasia, adenocarcinoma in situ, and microinvasion was lower in LLM-treated Hi-Myc mice but the difference was not statistically significant. The present study reveals that LLM inhibits cMyc expression in human prostate cancer cells in vitro but concentrations higher than 10 mg/kg may be required to achieve chemoprevention of prostate cancer.

Abstract B089: Kaiso as a novel therapeutic target in castration-resistant prostate cancer

Abstract Androgen receptor (AR) is a critical driver in the progression of prostate cancer (PCa). However, the PCa develops to castration resistant prostate cancer (CRPC) in almost 53% of patients after 18-32 month’s therapy. Metastatic CRPC has poor prognosis and mean survival time is fewer than 2 years. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous works from our lab have demonstrated that transcription factor Kaiso was upregulated in the progression of PCa. Therefore, our objective is to explore the interrelationship between Kaiso and AR, further to clarify Kaiso as a potential therapeutic target in PCa progression and CRPC. To investigate molecular mechanisms underlying how aberrant expression of Kaiso contributes to CRPC, the androgen sensitive human prostate cancer LNCaP cells were treated with 10μM anti-androgen Enzalutamide (MDV3100). Kaiso expression levels were increased as detected by RT-PCR and immunofluorescence. To systematically investigate Kaiso targets in PCa, we performed Kaiso ChIP-Seq assay using prostate cancer cell lines LNCaP, C4-2B and PC3. The Kaiso ChIP-Seq peaks indicate androgen receptor motif enrichment in AR expressing LNCaP and C4-2B cells but not in AR-negative PC3 cells, indicating Kaiso could cooperate with AR as co-regulator and potentially control transcription of a subset of its target genes. The interaction of them was confirmed by co-immunoprecipitation of AR using Kaiso antibody in LNCaP cells. To identify Kaiso target genes that interacted with AR pathway, Kaiso knockdown and overexpression stable cell lines were established in LNCaP cells and C42B cells. RT2 Profiler PCR array of AR pathway were performed using LNCaP scramble (LNCaP-Scr) cells and shKaiso cells. The results showed multiple genes were upregulated and downregulated, such as FOLH1, PTEN, RAC3 and IGF1. More importantly, LNCaP Kaiso overexpression cells were more resistant to MDV3100 treatment compared to resistance observed in LNCaP-Scr cells. There were significant negative correlations between Kaiso expression levels and castration-sensitivity. Our results suggest that Kaiso is a novel AR-interacting protein. Kaiso regulates the genes in AR pathway. Targeting Kaiso presents a potential therapeutic strategy for disrupting AR signaling in CRPC and may prevent CRPC development. Citation Format: Huixian Lin, Honghe Wang, Jason White, Balasubramanyam Karanam, Anghesom Ghebremedhin, Benjaminal Adu-Addai, William Grizzle, Clayton Yates. Kaiso as a novel therapeutic target in castration-resistant prostate cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B089.

4,4′-Diisothiocyanatostilbene-2,2′-disulfonate modulates voltage-gated K+ current and influences cell cycle arrest in androgen sensitive and insensitive human prostate cancer cell lines

The stilbene derivative, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), an anion channel blocker is used in the present study to evaluate its modulatory effect on voltage-gated K+ current (IK) in human prostate cancer cell lines (LNCaP and PC-3). Voltage-gated K+ (KV) channels in the plasma membrane are critically involved in the proliferation of tumor cells. Therefore, KV channels are considered as a novel potential target for cancer treatment. The results of the present study show that the external perfusion of DIDS activates IK in a concentration-dependent manner, although the known K+ channel blocker TEA failed to block the DIDS activated IK in PC-3 cells. Whereas, in LNCaP cells, the higher concentration of DIDS blocked IK, though this effect was not completely recovered after washout. The difference in function of DIDS might be due to the expression of different Kv channel isoforms in LNCaP and PC-3 cells. Further, the anticancer studies show that treatment of DIDS significantly induced G2/M phase cell cycle arrest and induced moderate and low level of cell death in LNCaP and PC-3 cells respectively. This finding reveals that DIDS modulates IK and exerts cell cycle arrest and cell death in LNCaP and PC-3 cells.

Abstract 5219: Kaiso as a novel therapeutic target in castration-resistant prostate cancer (CRPC)

Abstract Androgen receptor (AR) is a critical driver in the progression of prostate cancer (PCa). Androgen deprivation therapy(ADT) has been a standard treatment of PCa. However, the PCa develops to castration resistant prostate cancer (CRPC) in almost 53% of patients after 18-32 month’s therapy. Metastatic CRPC has poor prognosis and mean survival time is fewer than 2 years. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous works from our lab have demonstrated that transcription factor Kaiso was upregulated in the progression of PCa. Therefore, our objective is to explore the interrelationship between Kaiso and AR, further to clarify Kaiso as a potential therapeutic target in PCa progression and CRPC. To investigate molecular mechanisms underlying how aberrant expression of Kaiso contributes to CRPC, the androgen sensitive human prostate cancer LNCaP cells were treated with 10µM anti-androgen Enzalutamide (MDV3100). Kaiso expression levels were increased as detected by RT-PCR and immunofluorescence. To systematically investigate Kaiso targets in PCas, we performed Kaiso ChIP-Seq assay using prostate cancer cell lines LNCaP, C4-2B and PC3. The Kaiso ChIP-Seq peaks indicate androgen receptor motif enrichment in AR expressing LNCaP and C4-2B cells but not in AR-negative PC3 cells, indicating Kaiso could cooperate with AR as co-regulator and potentially control transcription of a subset of its target genes. The interaction of them was confirmed by co-immunoprecipitation of AR using Kaiso antibody in LNCaP cells. To identify Kaiso target genes that interacted with AR pathway, Kaiso knockdown and overexpression stable cell lines were established in LNCaP cells and C42B cells. RT2 Profiler PCR array of AR pathway were performed using LNCaP scramble (LNCaP-Scr) cells and shKaiso cells. The results showed multiple genes were upregulated and downregulated, such as FOLH1, PTEN, RAC3 and IGF1. More importantly, LNCaP Kaiso overexpression cells were more resistant to MDV3100 treatment compared to resistance observed in LNCaP-Scr cells. There were significant negative correlations between Kaiso expression levels and castration-sensitivity. Our results suggest that Kaiso is a novel AR-interacting protein. Kaiso regulates the genes in AR pathway. Targeting Kaiso presents a potential therapeutic strategy for disrupting AR signaling in CRPC and may prevent CRPC development. These studies were supported by grants U54MD007585, (NIH/RCMI) [CY], U54CA118623-01 (NIH/NCI) [CY] Citation Format: Hui-Xian Lin, Honghe Wang, Jason White, Balasubramanyam Karanam, Anghesom Ghebremedhin, Benjamin Adu Addai, William E. Grizzle, Clayton Yates. Kaiso as a novel therapeutic target in castration-resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5219.

Piperine blocks voltage gated K+ current and inhibits proliferation in androgen sensitive and insensitive human prostate cancer cell lines

Piperine is an attractive therapeutic alkaloid from black pepper that exhibits a broad spectrum of pharmacological properties over various pathological disorders including cancer. Voltage gated K+ (KV) channels play an important role in regulating cancer cell proliferation and are considered as potential target for cancer treatment. However, the implication of piperine in KV associated anticancer activities on human prostate cancer cells LNCaP and PC-3 remains unrevealed. The electrophysiological and pharmacological data identifies that both androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cells typically expressed voltage gated K+ current (IK). This current was significantly blocked by piperine in a concentration-dependent manner with an IC50 value 39.91 μM in LNCaP and 49.45 μM in PC-3 cells. Analysis of voltage-dependence of activation kinetics showed that piperine induces a positive shift in the relative activation curve in both the cells. Piperine also depolarized the resting membrane potential by an average of 10.2 mV and 8.3 mV in LNCaP and PC-3 cells, respectively. The anticancer studies showed that, treatment with piperine concentration dependently induced G1 phase cell cycle arrest and apoptosis in LNCaP and PC-3 cells. These results unravel that the IK inhibition might be responsible for the anticancer effect of piperine on androgen sensitive and insensitive human prostate cancer cells.

Pirfenidone, an Anti-Fibrotic Drug, Suppresses the Growth of Human Prostate Cancer Cells by Inducing G1 Cell Cycle Arrest

Pirfenidone (PFD) is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis by inducing G1 cell cycle arrest in fibroblasts. We hypothesize that PFD can induce G1 cell cycle arrest in different types of cells, including cancer cells. To investigate the effects of PFD treatment on the growth of human prostate cancer (PCa) cells, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines (androgen-low-sensitive E9 and F10 cells and androgen-insensitive AIDL cells), as well as an androgen-insensitive human PCa cell line (PC-3). PFD treatment suppressed the growth of all PCa cells. Transforming growth factor β1 secretion was significantly increased in PFD-treated PCa cells. In both LNCaP and PC-3 cells, PFD treatment increased the population of cells in the G0/G1 phase, which was accompanied by a decrease in the S/G2 cell population. CDK2 protein expression was clearly decreased in PFD-treated LNCaP and PC-3 cells, whereas p21 protein expression was increased in only PFD-treated LNCaP cells. In conclusion, PFD may serve as a novel therapeutic drug that induces G1 cell cycle arrest in human PCa cells independently of androgen sensitivity. Thus, in the tumor microenvironment, PFD might target not only fibroblasts, but also heterogeneous PCa cells of varying androgen-sensitivity levels.

Abstract 5787: Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models

Abstract Introduction: Therapeutic targeting the PI3K/AKT signaling pathway is an attractive strategy to combat solid tumors that exhibit hyper-AKT activity. Ipatasertib, a potent pan-AKT inhibitor, has exhibited significant inhibitory effects on tumor growth in preclinical studies, including models of Androgen Receptor (AR)-independent prostate cancer (PCa) that lack PTEN, a negative regulator of the PI3K/AKT pathway. It is currently being investigated in several ongoing clinical trials, including evaluating its efficacy in combination with abiraterone in castrate resistant PCa (CRPC) (NCT01485861). There is cross-talk between androgen signaling and PI3K/AKT pathway activation, however the optimal setting for AKT inhibition in PCa is not clear. Here we seek to determine the benefit of ipatasertib for androgen sensitive (AS) PCa alone or in combination with androgen deprivation (AD), as well as the benefit of ipatasertib alone or in combination with the antiandrogen enzalutamide in CRPC. Methods: In vitro studies utilized human androgen sensitive PCa cell lines LAPC, LAPC4shPTEN, and LNCAP, as well as two LNCaP-derived, castrate resistant cell lines. Cell viability was determined using CellTitre Glo assay and apoptosis by flow cytometry. Inhibitory effects of AKT signaling pathway was determined by western blot assays against AKT phosphorylation sites and downstream targets as well as detection for hormone receptor expression. In vivo studies utilized LuCaP 136 and LuCaP 136-derived castrate resistant (LuCaP 136CR) patient derived xenograft (PDX) models. Cells were injected into athymic mice and grouped into 4 arms. Intact mice bearing AS tumors were treated with vehicle, ipatasertib (100mg/Kg), AD, or ipatasertib + AD. Castrated mice bearing castrate-resistant tumors were treated with vehicle, ipatasertib, enzalutamide (10mg/Kg), or ipatasertib + enzalutamide. Tumor growth was determined twice/week via blinded caliper measurements. Results: Ipatasertib effectively inhibited AKT signaling, as indicated by significant decrease in phosphorylation of the downstream targets P70S6K and 4E-BP1. Our in vitro data indicate significant anti-proliferation effects of ipatasertib across all PCa models, both androgen sensitive and castrate resistant. Interestingly, this was irrespective of PTEN status. In addition, we observed a decrease in glucocorticoid receptor (GR) expression in enzalutamide resistant lines, although AR expression was not significantly altered. Preclinical animal studies showed significant response to tumor growth inhibition with ipatasertib monotherapy, which was enhanced with combination treatment. Conclusion: Ipatasertib is beneficial in models of androgen sensitive and castrate resistant PCa and can be effective in the treatment of PCa alone or in combination with current standard therapies targeting the androgen receptor. Citation Format: Remi M. Adelaiye-Ogala, Yen T. Nguyen, Jose A. Rodriguez-Nieves, Aian Neil Alilin, Supreet Agarwal, David Vanderweele. Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models [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 5787.

Interleukin-6 induces VEGF secretion from prostate cancer cells in a manner independent of androgen receptor activation.

The reduced androgen-sensitivity of prostate cancer (PCa) cells is an important clinical development because of its association with the cells' progression to castration-resistant prostate cancer (CRPC). During androgen deprivation therapy (ADT), stroma-derived growth factors and cytokines can activate the androgen receptor (AR). For example, IL-6 is a multifunctional cytokine that is involved in the malignancy of PCa cells through AR activation. In the present study, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines to investigate the relationship between the responsiveness of PCa cells to IL-6 treatment and the cellular AR signaling pathway. The androgen-low-sensitive F10 and E9 cells were obtained from LNCaP cells by limiting dilution method in regular culture condition. In contrast, the androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging in hormone-depleted condition. Original carcinoma-associated fibroblasts (CAFs) PCaSC-8 and PCaSC-9 cells were isolated from needle biopsy samples of PCa patients. In fibroblasts derived from PCa patients, IL-6 secretion was generally higher than that observed with normal fibroblasts. In contrast, IL-6 secretion was not detected in LNCaP and its sublines. The soluble IL-6 receptor was detected in PCa cells but not in fibroblasts. IL-6 treatment suppressed cell growth of LNCaP, F10, and E9 cells but not AIDL cells and it was accompanied with neuroendocrine-like differentiation. Induction of PSA secretion was observed in IL-6-treated LNCaP and F10 cells. VEGF secretion was strongly induced in IL-6-treated LNCaP and AIDL cells. IL-6-induced VEGF secretion was significantly suppressed by a PI3K inhibitor (LY294002) and it was accompanied by inhibited phosphorylation of Akt. Our results suggest that IL-6 might induce VEGF secretion from PCa cells in a manner independent of AR activation. To prevent IL-6-induced VEGF secretion, inhibition of the PI3K/AKT signaling pathway could be an important pharmacological goal regardless of ADT.

Abstract 1516: Androgen-repressed and androgen-induced genes: challenging the traditional dogma of prostate cancer therapy

Abstract Background: Benign and malignant prostate tissues are dependent upon the activity of androgen receptor (AR). The primary function of full-length AR is as a ligand activated transcription factor to increase or repress gene expression. Many androgen-repressed genes regulate the cell cycle and proliferation. With castration, the main therapeutic approach for advanced prostate cancer (PC), these genes are believed to play a role in the initial clinical response. Current approved therapies for advanced PC and castration-resistant PC (CRPC), target the AR C-terminal ligand-binding domain (LBD), such as antiandrogens. Recent antagonists of the AR N-terminal domain (NTD) have been described with EPI-506, the prodrug of EPI-002, now in Phase 1 clinical trials. EPI-002 binds tau-5 in activation function-1 (AF-1) of the NTD that is essential for AR transcriptional activity. As expected with an AF-1 antagonist, EPI-002 is an excellent inhibitor of androgen-induced gene expression and at blocking the transcriptional activities of truncated AR splice variants lacking LBD, such as AR-V7. EPI-002 blocks expression of genes regulated by truncated AR-V7 such as UBE2C while antiandrogens have no effect. Here we reveal that the major difference in gene expression regulated by full-length AR between EPI-002 and antiandrogens is their abilities to de-repress genes that are turned off by androgen. Methods: The androgen sensitive human prostate cancer cell line LNCaP, which expresses full-length AR, was treated with antiandrogens (bicalutamide [BIC] and enzalutamide [ENZA]), EPI-002 and a control vehicle, with and without androgen. Gene expression was analysed using Affymetrix microarrays. Bioinformatical analysis was completed and a selection of androgen-repressed genes that were de-repressed with antiandrogens and/or EPI-002, were selected for validation using qRT-PCR. Results: EPI-002 de-repressed known androgen-repressed genes including SPLTLC3, ST7, PSAT1, TMEM140 and TNFRSF21. EPI-002 was as effective or better than BIC or ENZA in de-repressing a subset of androgen-repressed genes. Importantly, EPI-002 failed to de-repress expression of many androgen-repressed genes that antiandrogens de-repressed, such as SLITRK3, GPR63 and DAB1. Conclusions: EPI binds AR NTD which blocks the transcriptional activities of full-length AR and truncated AR splice variants. EPI-002 was excellent at inhibiting androgen-induced genes. However, EPI did not broadly de-repress expression of genes turned off by androgen when compared to antiandrogens. Such differences between EPI-002, a tau-5/NTD antagonist, and C-terminal LBD antiandrogens probably reflect the complexities of the mechanisms of repression of gene expression that may involve different domains of AR. Citation Format: Daniel P. Caley, Nasrin R. Mawji, Marianne Sadar. Androgen-repressed and androgen-induced genes: challenging the traditional dogma of prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1516. doi:10.1158/1538-7445.AM2017-1516

Abstract 5220: Chronic exposure to a novel AR-NTD inhibitor induces resistance via a selective metabolism pathway

Abstract Background: The androgen receptor (AR) is recognized as playing a crucial role in prostate cancer (PCa) maintenance and progression; therefore its inhibition has been the cornerstone of modern therapy for men who fail primary treatment. Current treatments are initially effective, however resistance ultimately develops and the disease progresses to a lethal form termed castration-resistant prostate cancer (CRPC). Our lab has discovered a new class of molecules (EPI) which inhibit the AR by binding to the N-terminal domain (NTD). The NTD interacts with transcriptional machinery, and its presence is vital for a transcriptionally functional receptor. We have previously shown that EPI-002 specifically inhibits both full-length AR and constitutively active AR splice variants. The efficacy of EPI-506, the prodrug of EPI-002, is currently being tested in a Phase I/II clinical trial for CRPC. Here we propose novel resistance mechanisms arising from sustained AR-NTD inhibition, with the goal of preemptively developing backup compounds. Methods: The androgen sensitive human PCa cell line LNCaP was used in all experiments. A resistant cell line (LNCaP-EPIR) was generated by passaging parental LNCaP cells weekly in media supplemented with EPI-002 beginning in September 2012. In vitro and in vivo studies using LNCaP and LNCaP-EPIR cells were employed to confirm biological resistance. A human affymetrix microarray identified possible resistance mechanisms and was validated using qRT-PCR, western blot and functional studies. LNCaP and LNCaP-EPIR cells were challenged with EPI-045, an EPI-analog predicted to remain effective in the context of EPI-002 resistance. Results: LNCaP-EPIR cells treated with 25 µM EPI-002 displayed similar growth rates to vehicle treatment, both in vitro and in vivo. Conversely, parental LNCaP cells showed significant growth inhibition in response to EPI-002. LNCaP-EPIR cells retained sensitivity to anti-androgens and AR knock-down by targeted siRNA, implying functional AR remains essential for growth. qRT-PCR data demonstrated that EPI-002 had reduced ability to block AR mediated gene transcription. Interrogation of microarray data revealed candidate genes (UGT2B family) which were specifically upregulated in the resistant line, and may function to metabolize EPI-002. Supporting this hypothesis EPI-045 which is predicted to be resistant to UGT2B metabolism, was able to significantly inhibit LNCaP-EPIR proliferation and AR transcriptional activity. Conclusions: Taken together these data suggest an EPI-specific mechanism of resistance, whereby EPI-002 is preferentially metabolized and removed from the cell. LNCaP-EPIR cells remain dependent upon AR signalling, and are sensitive to anti-androgens used clinically as well as a novel EPI-analog. This work highlights the potential for combination or sequential therapy in the context of drug-resistant CRPC. Citation Format: Jonathon K. Obst, Jun Wang, David Williams, Amy H. Tien, Nasrin R. Mawji, Yu Chi Yang, Raymond J. Andersen, Marianne D. Sadar. Chronic exposure to a novel AR-NTD inhibitor induces resistance via a selective metabolism pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2017-5220

Inhibition of telomerase potentiates enzalutamide efficiency of androgen-sensitive human prostate cancer cells.

Androgen deprivation therapy (ADT) is one of the main strategies to treat prostate cancer (PCa) at various stages of its development. Androgen receptor (AR) antagonists such as enzalutamide are mainstay treatments for castration-sensitive prostate cancer. Though, a majority of patients initially respond to ADT, most will eventually progress to castrate-resistant, due to the development of different mutations on the AR. PCa cells express high telomerase activity, and there is a correlation between the total activity of telomerase and the Gleason score. Therefore, we hypothesized that the combination of enzalutamide plus a telomerase inhibitor could be more effective than enzalutamide alone in decreasing cell survival. In this study MTT test, RT-qPCR and imagebased cytometry were used to investigate cell viability, apoptosis and cell cycle progression of androgen-responsive human prostate cancer LNCaP cells. The cells were treated with 5 μM enzalutamide and 40 μM telomerase inhibitor BIBR 1532, or with their combinations for 72 hrs. Enzalutamide and BIBR 1532 alone inhibited cell proliferation in a dose-dependent manner. The combinations of the two agents could synergistically induce apoptotic and necrotic cell death. Either inhibition of telomerase by BIBR 1532 or AR blockages by enzalutamide decreased prostate-specific antigen (PSA) and the catalytic component of telomerase, hTERT, expression. These results suggest that telomerase inhibition therapy may contribute to the efficacy of enzalutamide in the androgen-sensitive PCa model.

&amp;lt;i&amp;gt;In-Vitro&amp;lt;/i&amp;gt; Antiproliferative Analysis of Metformin Hydrochloride on Androgen-Sensitive, LNCAP and Androgen-Insensitive, PC-3 Human Prostate Cancer Cell Lines

Prostate cancer is one of the diseases worldwide that causes cancer-related deaths in men. Metformin is an antidiabetic drug that has been in use for over two decades for the treatment of Type II Diabetes mellitus (DM2). The purpose of this study was to evaluate the anti-proliferative property of metformin hydrochloride on androgen-sensitive, LNCAP and androgen-insensitive, PC-3 human prostate cancer cell lines at different concentrations (μM and mM) using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Metformin hydrochloride displayed a stronger cytotoxicity on the androgen-insensitive PC-3 than on the androgen-sensitive human prostate cancer cell lines. For both cell lines, the antiproliferative activity of metformin hydrochloride was best displayed at 0.1 mM concentration with average cell death percentage of 60% after 120-hour exposure.

Abstract 4971: Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients

Abstract Background: CTCs represent a real-time, liquid biopsy for analyses of a variety of molecular biomarkers that offer clinically relevant information. In prostate cancer, the AR pathway controls tumor cell growth and survival and is an important therapeutic target. AR becomes biologically active upon translocation from the cytoplasm to the nucleus in a microtubule dependent fashion, resulting in the activation of numerous genes. Real-time analysis of AR and tubulin status in CTCs may provide insight to therapy response in mCRPC patients. Methods: Androgen sensitive human prostate cancer cells, LNCaP, were treated with and without a synthetic androgen (R1881; 10 nM, 1 hr) and docetaxel, spiked into healthy donor blood, and captured using a prostate specific microfluidic device (GEDI chip), following the same protocol that we use with patient samples. Cells were fixed and stained (N-terminal AR, CD45, CK, DAPI, and tyr-tubulin) directly on the device and imaged using high resolution multiplex confocal microscopy. AR and tubulin status were analyzed using a quantitative image analysis algorithm. Nuclear AR percentage was calculated by integrating fluorescence intensity within regions defining the entire cell and nucleus. H-Score was calculated by multiplying the nuclear AR percentage by the normalized total cell AR intensity. Tubulin status and microtubule bundling were assessed using a variety of quantitative parameters including measurements of tubulin fluorescence intensity and distribution within the cell. Results: LNCaP cells in the presence of synthetic androgen exhibited higher nuclear AR percentage (p = 0.0004) and H-score (p = 0.003). Docetaxel treatment led to lower nuclear AR percentage (p = 0.04) and lower H-Score (p = 0.03). Cells treated with docetaxel exhibited higher tubulin intensity range (p = 0.008) and standard deviation (p = 0.03), consistent with docetaxel's mechanism of action. We applied these methods of quantitative image analyses to CTCs isolated from a small cohort of mCRPC patients. Single CTC image analysis revealed heterogeneity in AR status. Quantitative evidence for microtubule bundling was observed in patients receiving docetaxel chemotherapy. Conclusions: We have developed a high throughput quantitative image analysis algorithm to interrogate mCRPC specific biomarkers, such as AR and tubulin status, in single CTCs. These methods of quantitative image analyses will be prospectively validated with CTCs from mCRPC patients followed longitudinally over the course of treatment with AR inhibitors and taxanes, yielding patient specific, clinically relevant information that can guide physicians’ strategies for the clinical management of cancer. Citation Format: Daniel Worroll, Giuseppe Galletti, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4971.