Prostate Cancer Cell Lines 22Rv1 Research Articles

Abstract 1587: Disruption of nuclear export with selinexor or KPT-8602 reduces androgen receptor expression and leads to potent anti-tumor activity in preclinical models of androgen-independent prostate cancer

Abstract Metastatic castration resistant prostate cancer (mCRPC) is an advanced form of prostate cancer (PC) associated with poor prognosis. Approximately 20 - 40% of mCRPCs are androgen-independent and do not respond to treatment with abiraterone or enzalutamide, drugs that suppress androgen synthesis or directly target androgen ligand binding domains (LBD) respectively. Patients initially responding to these drugs eventually relapse, highlighting the need for alternative therapies. Resistance is attributed to the emergence of constitutively active AR splice variants lacking C-terminal LBDs such as AR variant 7. Although ARv7 activity cannot be mitigated by current therapeutic approaches, it is known that AR mRNA is exported from the nucleus by eIF4E and exportin-1 (XPO1). Selinexor and KPT-8602 are orally bioavailable SINE (Selective Inhibitor of Nuclear Export) compounds that specifically target XPO1. The purpose of this study is to provide mechanistic evidence for using SINE compounds as novel therapies for androgen-independent mCRPC. Methods: In vitro, selinexor or KPT-8602 were tested on androgen-independent PC cell line 22Rv1. RNA and protein were analyzed by qPCR and immunoblot. Cell viability was examined using the Celltiter-Glo assay. In vivo, immune deficient SCID mice were injected subcutaneously with 22Rv1 cells. Tumors were grown to ~150 mm3 before treatment with selinexor (10 mg/kg qodx3) or KPT-8602 (15 mg/kg reduce to 10 mg/kg qdx5). Tumor growth and animal weights were monitored to determine tumor growth inhibition (TGI) and tolerability to treatment. Tumors were analyzed using immunohistochemistry (IHC). Results: We found that expression of AR, ARv7 and prostate specific antigen (PSA; transcriptionally regulated by AR) proteins are reduced following 24-hour treatment with SINE compounds. Nuclear vs. cytoplasmic fractionation of RNA revealed that ARv7 and PSA mRNA localization was increased in the nucleus (4-fold and 3-fold, respectively) and reduced in the cytoplasm (5-fold and 3-fold, respectively). Moreover, KPT-8602 potently inhibited cell viability (IC50: 100 nM), while enzalutamide and abiraterone had no effect. Finally, mice bearing 22Rv1 xenografts, treated with selinexor or KPT-8602 exhibited a complete reduction in tumor volume (95% and 94% TGI, respectively), which coincided with prolonged overall survival. IHC analysis showed a reduction of proliferation markers and a concomitant increase in cell death makers in selinexor and KPT-8602 treated tumors. Conclusion: SINE compounds show strong anti-tumor activity in androgen-independent prostate cancer models in vitro and in vivo by reducing AR, ARv7, and PSA expression. These findings highlight the promise of SINE compounds as treatment options for androgen-independent mCRPCs resistant to first line therapies. Citation Format: Christian Argueta, Boris Klebanov, Trinayan Kashyap, Hua Chang, Sharon Friedlander, Erkan Baloglu, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. Disruption of nuclear export with selinexor or KPT-8602 reduces androgen receptor expression and leads to potent anti-tumor activity in preclinical models of androgen-independent prostate cancer [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 1587. doi:10.1158/1538-7445.AM2017-1587

Oncolytic alphavirus SFV-VA7 efficiently eradicates subcutaneous and orthotopic human prostate tumours in mice

Background:Despite recent therapeutic and diagnostic advances, prostate cancer remains the second leading cause of cancer-related deaths among men in the Western world. Oncolytic viruses that replicate selectively in tumour cells represent a novel treatment candidate for these malignancies.Methods:We analysed infectivity of avirulent Semliki Firest virus SFV-VA7 in human prostate cancer cell lines VCaP, LNCaP and 22Rv1 and in nonmalignant prostate epithelial cell line RWPE-1. Therapeutic potency of SFV-VA7 was evaluated in subcutaneous and orthotopic mouse LNCaP xenograft models.Results:SFV-VA7 infected and killed the tested human prostate cancer cell lines irrespective of their hormone response status, while the nonmalignant prostate epithelial cell line RWPE-1 proved highly virus resistant. Notably, a single peritoneal dose of SFV-VA7 was sufficient to eradicate all subcutaneous and orthotopic LNCaP tumours.Conclusions:Our results indicate that SFV-VA7 is a novel, promising therapeutic virus against prostate cancer warranting further testing in early clinical trials.

Taxane-Grafted Metal-Oxide Nanoparticles as a New Theranostic Tool against Cancer: The Promising Example of Docetaxel-Functionalized Titanate Nanotubes on Prostate Tumors.

The combination of anticancer drugs and metal oxide nanoparticles is of great interest in cancer nanomedicine. Here, the development of a new nanohybrid, titanate nanotube-docetaxel (TiONts-DTX) is reported, the two parts of which are conjugated by covalent linkages. Unlike most nanoparticles currently being developed for biomedical purposes, TiONts present a needle-shaped morphology. The surface of TiONts is linked with 3-aminopropyl triethoxysilane and with a hetero-bifunctional polymer (polyethylene glycol) to create well-dispersed and biocompatible nanovectors. The prefunctionalized surface of this scaffold has valuable attachments to graft therapeutic agents (DTX in our case) as well as chelating agents (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to monitor the nanohybrids. To evaluate drug efficacy, in vitro tests have demonstrated that the association between TiONts and DTX shows cytotoxic activity against a hormone-refractory prostate cancer cell line (22Rv1) whereas TiONts without DTX do not. Finally, the first in vivo tests with intratumoral injections show that more than 70% of TiONts nanovectors are retained within the tumor for at least 7 d. Moreover, tumor growth in mice receiving TiONts-DTX is significantly slower than that in mice receiving free DTX. This nanohybrid can thus become a promising new tool in biomedicine to fight against prostate cancer.

Ca2+ Selective Host Rotaxane Is Highly Toxic Against Prostate Cancer Cells.

New therapies are needed to eradicate androgen resistant, prostate cancer. Prostate cancer usually metastasizes to bone where the concentration of calcium is high, making Ca2+ a promising toxin. Ionophores can deliver metal cations into cells, but are currently too toxic for human use. We synthesized a new rotaxane (CEHR2) that contains a benzyl 15-crown-5 ether as a blocking group to efficiently bind Ca2+. CEHR2 transfers Ca2+ from an aqueous solution into CHCl3 to greater extent than alkali metal cations and Mg2+. It also transfers Ca2+ to a greater extent than CEHR1, which is a rotaxane with an 18-crown-6 ether as a blocking group. CEHR2 was more toxic against the prostate cancer cell lines PC-3, 22Rv1, and C4-2 than CEHR1. This project demonstrates that crown ether rotaxanes can be designed to bind a targeted metal cation, and this selective cation association can result in enhanced toxicity.

RK-33 Radiosensitizes Prostate Cancer Cells by Blocking the RNA Helicase DDX3.

Despite advances in diagnosis and treatment, prostate cancer is the most prevalent cancer in males and the second highest cause of cancer-related mortality. We identified an RNA helicase gene, DDX3 (DDX3X), which is overexpressed in prostate cancers, and whose expression is directly correlated with high Gleason scores. Knockdown of DDX3 in the aggressive prostate cancer cell lines DU145 and 22Rv1 resulted in significantly reduced clonogenicity. To target DDX3, we rationally designed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 preferentially bound to DDX3 and perturbed its activity. RK-33 treatment of prostate cancer cell lines DU145, 22Rv1, and LNCaP (which have high DDX3 levels) decreased proliferation and induced a G1 phase cell-cycle arrest. Conversely, the low DDX3-expressing cell line, PC3, exhibited few changes following RK-33 treatment. Importantly, combination studies using RK-33 and radiation exhibited synergistic effects both in vitro and in a xenograft model of prostate cancer demonstrating the role of RK-33 as a radiosensitizer. Taken together, these results indicate that blocking DDX3 by RK-33 in combination with radiation treatment is a viable option for treating locally advanced prostate cancer. Cancer Res; 76(21); 6340-50. ©2016 AACR.

Marine compound rhizochalinin shows high in vitro and in vivo efficacy in castration resistant prostate cancer.

Development of drug resistance is an inevitable phenomenon in castration-resistant prostate cancer (CRPC) cells requiring novel therapeutic approaches. In this study, efficacy and toxicity of Rhizochalinin (Rhiz) – a novel sphingolipid-like marine compound – was evaluated in prostate cancer models, resistant to currently approved standard therapies. In vitro activity and mechanism of action of Rhiz were examined in the human prostate cancer cell lines PC-3, DU145, LNCaP, 22Rv1, and VCaP. Rhiz significantly reduced cell viability at low micromolar concentrations showing most pronounced effects in enzalutamide and abiraterone resistant AR-V7 positive cells. Caspase-dependent apoptosis, inhibition of pro-survival autophagy, downregulation of AR-V7, PSA and IGF-1 expression as well as inhibition of voltage-gated potassium channels were identified as mechanisms of action. Remarkably, Rhiz re-sensitized AR-V7 positive cells to enzalutamide and increased efficacy of taxanes.In vivo activity and toxicity were evaluated in PC-3 and 22Rv1 NOD SCID mouse xenograft models using i.p. administration. Rhiz significantly reduced growth of PC-3 and 22Rv1 tumor xenografts by 27.0% (p = 0.0156) and 46.8% (p = 0.047) compared with controls with an increased fraction of tumor cells showing apoptosis secondary to Rhiz exposure. In line with the in vitro data, Rhiz was most active in AR-V7 positive xenografts in vivo. In animals, no severe side effects were observed.In conclusion, Rhiz is a promising novel marine-derived compound characterized by a unique combination of anticancer properties. Its further clinical development is of high impact for patients suffering from drug resistant prostate cancer especially those harboring AR-V7 mediated resistance to enzalutamide and abiraterone.

Abstract 1537: The biological role of BAT-1 in prostate cancer

Abstract Prostate cancer is the most common cancer in men in the United States and is the second cause of cancer death in men. In Puerto Rico, prostate cancer is the most common type of cancer and the leading cause of cancer death in men. The current available biomarkers are unable to predict malignant outcomes such as recurrence. Thus, there is a critical demand for the development of innovative diagnostic and prognostic tools for the management of prostate cancer. In the current study, we evaluated the biological role of BAT-1 in prostate cancer. Preliminary data from patients who had prostate cancer recurrence identified that HLA-B associated transcript 1 (BAT-1) was down-regulated in patients with prostate cancer recurrence when compared with non-recurrent patients. We down-regulated BAT-1 in PC3 and 22RV1 prostate cancer cell lines using small interfering RNA (siRNA). In vitro assays were performed to measure de-differentiation, proliferation, apoptosis, migration and invasion. Flow cytometry analysis using the cell Viability/Annexin V cell staining kit showed that down-regulation of BAT-1 significantly increased the proliferation of PC3 cells (P<0.05) but decreased apoptosis. Down-regulation of BAT-1 showed no significant change in the expression of vimentin, desmin, and alpha-smooth muscle actin (α-SMA). Wound healing and Boyden assays showed that cells treated with BAT-1 siRNA significantly increased migration and motility at 12h and 24h of PC3 cells when compared to control (P<0.05). Our results showed that BAT-1 down-regulation increased migration and cell motility. Cells also tend to proliferate more, but have reduced apoptosis. These results were expected due to its down-regulation in recurrent prostate cancer patient samples, suggesting that BAT-1 down-regulation promotes aggressiveness in prostate cancer recurrence. A possible mechanism of action for BAT1 is the modulation of cell proliferation and migration. Citation Format: Aileen M. Garcia-Vargas, Maylein C. Juan-Rivera, Milaris M. Sánchez-Cordero, Maria Sánchez-Vázquez, Krizia Rohena, Magaly Martínez-Ferrer. The biological role of BAT-1 in prostate cancer. [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 1537.

Abstract 1824: Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes

Abstract Background: Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells. Androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Cancer cell escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. Methods: Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. Results: A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen and AR dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial EP156T-AR cells, but did not exert a positive feedback on endogenous AR expression. Mesenchymal type prostate EPT3-AR cells with exogenous AR expression, in contrast to epithelial type EP156T-AR cells, were androgen non-responsive and were unable to produce detectable PSA in the culture supernatants even with higher levels of exogenous AR protein than in EP156T-AR and LNCaP cells for up to 2 weeks in androgen containing growth medium. The restricted PSA expression in the mesenchymal context suggests that if ADT increases the pool of mesenchymal type prostate cancer cells, then this might go undetected during PSA monitoring of disease progression. Citation Format: Jan Roger Olsen, Waqas Azeem, Margrete R. Hellem, Kristo Marvyin, Yaping Hua, Yi Qu, Lisha Li, Biaoyang Lin, XiSong Ke, Anne Margrete Oyan, Karl-Henning Kalland. Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes. [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 1824.

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes.

BackgroundExpression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable.MethodsImmortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.ResultsA variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s).ConclusionsRegulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2453-4) contains supplementary material, which is available to authorized users.

Carnosic acid promotes degradation of the androgen receptor and is regulated by the unfolded protein response pathway in vitro and in vivo.

Androgen deprivation therapy in prostate cancer is extremely effective; however, due to the continuous expression and/or mutagenesis of androgen receptor (AR), the resistance to antihormonal therapy is a natural progression. Consequently, targeting the AR for degradation offers an alternate approach to overcome this resistance in prostate cancer. In this study, we demonstrate that carnosic acid, a benzenediol diterpene, binds the ligand-binding domain of the AR and degrades the AR via endoplasmic reticulum (ER) stress-mediated proteasomal degradative pathway. In vitro, carnosic acid treatment induced degradation of AR and decreased expression of prostate-specific antigen in human prostate cancer cell lines LNCaP and 22Rv1. Carnosic acid also promoted the expression of ER proteins including BiP and CHOP in a dose-dependent manner. Downregulation of CHOP by small interfering RNA somewhat restored expression of AR suggesting that AR degradation is dependent on ER stress pathway. Future studies will need to evaluate other aspects of the unfolded protein response pathway to characterize the regulation of AR degradation. Furthermore, cotreating cells individually with carnosic acid and proteasome inhibitor (MG-132) and carnosic acid and an ER stress modulator (salubrinal) restored protein levels of AR, suggesting that AR degradation is mediated by ER stress-dependent proteasomal degradation pathway. Degradation of AR and induction of CHOP protein were also evident in vivo along with a 53% reduction in growth of xenograft prostate cancer tumors. In addition, carnosic acid-induced ER stress in prostate cancer cells but not in normal prostate epithelial cells procured from patient biopsies. In conclusion, these data suggest that molecules such as carnosic acid could be further evaluated and optimized as a potential therapeutic alternative to target AR in prostate cancer.

Abstract B31: Characterization of a novel model of prostate cancer metastasis resulting from androgen receptor suppression

Abstract Background: Prostate cancer (PC) is the second leading cause of male cancer-related mortality: 30,000 men die each year from metastatic disease. The Androgen Receptor (AR) may be the earliest known example of a lineage oncogene – a master regulator of prostate cancer cell survival and growth. The AR protein is detectable at all stages of prostate cancer progression and is the primary target of pharmacological agents capable of suppressing AR signaling to extend survival in men with metastatic prostate cancer (mPC). The CYP17 inhibitor Abiraterone, and the 3rd generation AR antagonist, MDV3100 prolong the survival of men with mPC, though cures remain exceeding rare using these agents individually. In order to develop additional therapies to be used in combination with current AR inhibitors, preclinical models that mimic PC progression need to be established. Tissue microarray studies from hormone-naïve, nodal positive, prostate cancer patients revealed a significant down-regulation of AR protein level in matched nodal metastases versus primary tumors. These results suggest that loss of AR or AR activity could play a role in the progression to metastatic disease. Methods: We used a short-hairpin RNA targeting exon 1 of the AR to reduce AR protein in vivo using a doxycycline-inducible system in the non-metastatic AR-positive human prostate cancer cell line 22Rv1. This cell line model, derived from a primary tumor, is castration-resistant and enzalutamide/MDV3100-resistant. We generated xenografts using 22Rv1shARex1 and evaluated tumor growth and propensity for metastatic spread. We profiled transcriptional changes in the context of AR suppression and compared gene expression changes between grafted tumors and sites of metastatic spread. Results: Xenograft studies in NOD-SCID mice revealed that reduction of the AR protein decreased tumor proliferation rates and promoted cancer metastasis. Tumor-bearing mice exposed to continual reduction of the AR developed large liver metastases and had significantly increased disseminated tumor cells in their bone marrow. Furthermore, intratibial and intracardiac injection of 22Rv1shARex1 followed by AR reduction, resulted in an increased formation of metastases. Transcriptional analysis revealed unique signatures between untreated parental 22Rv1 xenografts, tumors with AR reduction, and liver metastases. Genes up-regulated in xenografts following AR reduction, were also up-regulated in patient metastases versus primary tumors including transcripts encoding EGF, HGF, and ZEB2. Discussion: The AR is the primary therapeutic target for mPC. However, a cure for advanced cancer and metastatic disease remains elusive. Reduction of the AR in the 22Rv1 in vivo model mimics disease progression seen in patients. Molecular changes observed during the transformation of localized cancer to metastatic disease in this model may identify new targets to be used in combination with current therapies intent on ablating AR signaling. Citation Format: Clinton K. Matson, Peter S. Nelson. Characterization of a novel model of prostate cancer metastasis resulting from androgen receptor suppression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B31.

PD32-02 NOVEL PI3K/P110BETA-SPECIFIC INHIBITORS ELIMINATE ENZALUTAMIDE RESISTANCE IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-02 NOVEL PI3K/P110BETA-SPECIFIC INHIBITORS ELIMINATE ENZALUTAMIDE RESISTANCE IN PROSTATE CANCER Marcus Austenfeld, Chenchen He, Yifen Wang, Qingting Hu, Brantley Thrasher, and Benyi Li Marcus AustenfeldMarcus Austenfeld More articles by this author , Chenchen HeChenchen He More articles by this author , Yifen WangYifen Wang More articles by this author , Qingting HuQingting Hu More articles by this author , Brantley ThrasherBrantley Thrasher More articles by this author , and Benyi LiBenyi Li More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.678AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We and others have demonstrated that the class IA PI3K isoform p110beta is critical in prostate cancer development and progression, indicating p110beta as a bona fide target for prostate cancer therapy. Our recent work consistently shows that prostate cancer cell-specific delivery of nano-micellar TGX221 completely blocked prostate cancer growth in vivo, suggesting that TGX221-based therapy is feasible for further development. Unfortunately, the small chemical TGX221 is not soluble in water, hampering its clinical development. To solve this problem, we recently synthesized a panel of TGX221 analogs with increased water solubility. In this study, we tested these novel inhibitors (TGX221 analogs) in multiple enzalutamide-resistant prostate cancer cell lines for their activity in androgen receptor (AR) signaling and cancer cell growth. METHODS Prostate cancer cell lines 22RV1, VCaP and C4-2 were utilized. AR transactivation was evaluated with the ARE-LUC luciferase reporter assay and PSA mRNA level was assessed with real-time RT-PCR. Cancer cell growth was assessed with NCI-approved Sulforhodamine B-based assay. PI3K activation, AR protein levels and cell apoptosis were evaluated in Western blot assays. RESULTS Our novel TGX221 analogs, shown in Figure 1 with water solubility, are much more water soluble (47-99 mM in water) than TGX221, which can only be dissolved in organic solution. Most importantly, these analogs retained a similar inhibitory effect as TGX221 on cell proliferation, AR transactivation and PI3K/AKT activation in prostate cancer cells. Addition of our TGX221 analogs significantly enhanced the enzalutamide-induced anti-cancer effect in LNCaP and LAPC4 cells already sensitive to enzalutamide treatment, as evaluated by cell proliferation and AR transactivation assays. Moreover, these novel TGX221 analogs also sensitized resistant 22RV1 cells to enzalutamide treatment with increased apoptotic cell death. CONCLUSIONS These data strongly suggest that our novel TGX221 analogs possess great potential to be developed as feasible anti-cancer agents for castration-resistant prostate cancers. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e761 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Marcus Austenfeld More articles by this author Chenchen He More articles by this author Yifen Wang More articles by this author Qingting Hu More articles by this author Brantley Thrasher More articles by this author Benyi Li More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PD32-01 PRE-CLINICAL RATIONALE FOR COMBINATION PI3K AND BRD4 INHIBITION IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-01 PRE-CLINICAL RATIONALE FOR COMBINATION PI3K AND BRD4 INHIBITION IN ADVANCED PROSTATE CANCER Paul Toren, Jared Allman, Soojin Kim, and Amina Zoubeidi Paul TorenPaul Toren More articles by this author , Jared AllmanJared Allman More articles by this author , Soojin KimSoojin Kim More articles by this author , and Amina ZoubeidiAmina Zoubeidi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.677AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The PI3K/Akt pathway is frequently activated in aggressive and resistant prostate cancer. This study details our pre-clinical evaluation of AZD8186, a novel β- and δ- selective PI3K small molecule inhibitor in prostate cancer models. We and others have recently noted the role of the oncogene myc as a mechanism of resistance to monotherapy treatment with PI3K/Akt inhibitors. Therefore, we further evaluated co-targeting strategies against both the PI3K/Akt pathway and the epigenetic reader protein BRD4. METHODS Human prostate cancer cell lines LNCaP and 22RV1 were tested for sensitivity to AZD8186 in vitro. Caspase-3 activity and flow cytometry were used to assess apoptosis. Western blotting and RT-qPCR were used to measure AR and myc pathway genes and protein expression. Castrate resistant LNCaP xenografts were treated with 10mg/kg and 25mg/kg doses of AZD8186 given orally 4 days on, 3 off. RESULTS LNCaP cells demonstrated sensitivity to AZD8186 with decreases in cell proliferation and increases in apoptosis. In vivo, castrate resistant LNCaP xenografts demonstrated a dose-dependent decrease in tumor growth velocity with AZD8186. On-target decreases in pAkt was observed in xenograft tumour samples by western blot analysis. Further, increases in myc protein and mRNA levels were seen in xenograft samples treated with AZD8186 compared to control. Downstream increases in EGFR and IGF-IR mRNA transcripts induced by AZD8186 were also seen in vitro and in vivo. Increases in myc, EGFR and IGF-IR was also seen in prostate cancer cell lines and xenograft tumours treated with an Akt inhibitor. Addition of the BDR4 inhibitor JQ1 decreased the elevation of myc transcript levels induced by AZD8186 and also partially abrogated the increases in EGFR, IGFR transcript levels. Greater suppression of PSA expression was seen with the combination of AZD8186 and JQ1 compared to AZD8186 and enzalutamide. Similar results were seen in the 22RV1 cell line. CONCLUSIONS Inhibition of PI3K with AZD8186 inhibits growth of PTEN-negative LNCaP cells. However, feedback activation of myc and AR pathways occurs and may result in treatment resistance. We demonstrate BRD4 inhibition using JQ1 as a rationale combination strategy with PI3K inhibition. This strategy co-targets both myc and AR feedback pathways and warrants further investigation in prostate cancers with an activated PI3K/Akt pathway. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e761 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Paul Toren More articles by this author Jared Allman More articles by this author Soojin Kim More articles by this author Amina Zoubeidi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PD32-03 A PHASE 2 OPEN-LABEL TRIAL OF GTX-758 IN MEN WITH CASTRATION-RESISTANT PROSTATE CANCER, FINAL ANALYSIS OF THE PRIMARY ENDPOINT

INTRODUCTION AND OBJECTIVES: The PI3K/Akt pathway is frequently activated in aggressive and resistant prostate cancer. This study details our pre-clinical evaluation of AZD8186, a novel band dselective PI3K small molecule inhibitor in prostate cancer models. We and others have recently noted the role of the oncogene myc as a mechanism of resistance to monotherapy treatment with PI3K/Akt inhibitors. Therefore, we further evaluated co-targeting strategies against both the PI3K/Akt pathway and the epigenetic reader protein BRD4. METHODS: Human prostate cancer cell lines LNCaP and 22RV1 were tested for sensitivity to AZD8186 in vitro. Caspase-3 activity and flow cytometry were used to assess apoptosis. Western blotting and RT-qPCRwere used tomeasure ARandmyc pathway genes and protein expression. Castrate resistant LNCaP xenografts were treated with 10mg/kg and 25mg/kg doses of AZD8186 given orally 4 days on, 3 off. RESULTS: LNCaP cells demonstrated sensitivity to AZD8186 with decreases in cell proliferation and increases in apoptosis. In vivo, castrate resistant LNCaP xenografts demonstrated a dose-dependent decrease in tumor growth velocity with AZD8186. On-target decreases in pAkt was observed in xenograft tumour samples by western blot analysis. Further, increases in myc protein and mRNA levels were seen in xenograft samples treated with AZD8186 compared to control. Downstream increases in EGFR and IGF-IR mRNA transcripts induced by AZD8186 were also seen in vitro and in vivo. Increases in myc, EGFR and IGF-IR was also seen in prostate cancer cell lines and xenograft tumours treated with an Akt inhibitor. Addition of the BDR4 inhibitor JQ1 decreased the elevation of myc transcript levels induced by AZD8186 and also partially abrogated the increases in EGFR, IGFR transcript levels. Greater suppression of PSA expression was seen with the combination of AZD8186 and JQ1 compared to AZD8186 and enzalutamide. Similar results were seen in the 22RV1 cell line. CONCLUSIONS: Inhibition of PI3K with AZD8186 inhibits growth of PTEN-negative LNCaP cells. However, feedback activation of myc and AR pathways occurs and may result in treatment resistance. We demonstrate BRD4 inhibition using JQ1 as a rationale combination strategy with PI3K inhibition. This strategy co-targets both myc and AR feedback pathways and warrants further investigation in prostate cancers with an activated PI3K/Akt pathway.

7-Substituted umbelliferone derivatives as androgen receptor antagonists for the potential treatment of prostate and breast cancer

The clinically used androgen receptor (AR) antagonists (bicalutamide, flutamide and nilutamide) bind with low affinity to AR and can induce escape mechanisms. Furthermore, under AR gene amplification or mutation conditions they demonstrate agonist activity and fail to inhibit AR, causing relapse into castration resistant prostate cancer (CRPC). Discovery of new scaffolds distinct from the 4-cyano/nitro-3-(trifluoromethyl)phenyl group common to currently used antiandrogens is urgently needed to avoid cross-resistance with these compounds. In this study, a series of twenty-nine 7-substituted umbelliferone derivatives was prepared and their antiproliferative activities were evaluated. The most active compound 7a demonstrated submicromolar inhibitory activity in the human prostate cancer cell line (22Rv1); IC50=0.93μM which represents a 50 fold improvement over the clinical antiandrogen bicalutamide (IC50=46μM) and a more than 30 fold improvement over enzalutamide (IC50=32μM). Interestingly, this compound showed even better activity against the human breast cancer cell line (MCF-7); IC50=0.47μM. Molecular modelling studies provided a plausible theoretical explanation for our findings.

Pre-clinical rationale for combination PI3K and BRD4 inhibition in advanced prostate cancer.

234 Background: The PI3K/Akt pathway is frequently activated in aggressive and resistant prostate cancer. Here we detail our pre-clinical evaluation of AZD8186, a novel β and δ selective PI3K small molecule inhibitor. Further, we investigate how increased transcription of the myc oncogene may represent a mechanism of resistance to monotherapy PI3K/Akt inhibition. Therefore, we further evaluated co-targeting strategies against both the PI3K/Akt pathway and the epigenetic reader protein BRD4. Methods: Human prostate cancer cell lines LNCaP and 22RV1 were tested for sensitivity to AZD8186 in vitro. Caspase-3 activity and flow cytometry were used to assess apoptosis. Western blotting and RT-qPCR were used to measure AR and myc pathway genes and protein expression. Castrate resistant LNCaP xenografts were treated with 10mg/kg and 25mg/kg doses of AZD8186 given orally 4 days on, 3 off. Results: In vitro results demonstrated sensitivity to AZD8186 with decreases in cell proliferation and increases in apoptosis. In vivo results demonstrated a dose-dependent decrease in tumor growth velocity with AZD8186 with on-target decreases in pAkt was observed in xenograft tumour samples. Further, increases in myc protein and mRNA levels were seen in xenograft samples treated with AZD8186 compared to control. Downstream increases in EGFR and IGF-IR mRNA transcripts induced by AZD8186 were also seen in vitro and in vivo. Increases in myc, EGFR and IGF-IR was also seen in cells and tumours treated with an Akt inhibitor. Addition of the BDR4 inhibitor JQ1 decreased the increase of myc induced by AZD8186 and also partially abrogated the increases in EGFR, IGFR. Greater suppression of PSA expression was seen with the combination of AZD8186 and JQ1 compared to AZD8186 and enzalutamide. Similar results were seen in the 22RV1 cell line. Conclusions: Inhibition of PI3K with AZD8186 inhibits growth of PTEN-negative LNCaP cells. However, feedback activation of myc and AR pathways occurs. We demonstrate BRD4 inhibition using JQ1 which co-targets both myc and AR feedback pathways as a rationale combination strategy with PI3K inhibition. This strategy warrants further investigation in prostate cancers with an activated PI3K/Akt pathway.

Abstract PR11: Dysregulation of cholesterol homeostasis through loss of CYP27A1 in prostate cancer; Implications for early detection and prevention of over-treatment

Abstract Approximately 200,000 men are annually diagnosed with prostate cancer (PC), making PC the most commonly diagnosed malignancy among men in industrialized countries. On the other hand, only 30,000 men diagnosed with PC will succumb to the disease with the majority of patients dying from other causes. This makes identification of aggressive vs. indolent PCs one of the most important clinical conundrums in the modern health era. Indeed, identification of biomarkers that achieve this distinction would add a huge benefit and would help patients that have indolent disease avoid side effects associated with treatment modalities that should only be provided to patients with aggressive forms of PC. To achieve the above goal, we employed a bioinformatics based search to look for genes that dramatically change in PC relative to benign and can predict progression. Our mined data from eight different publically available clinical datasets clearly indicates that CYP27A1transcript levels (our top candidate) are down-regulated in PC relative to benign and are further downregulated during PC progression. Our bioinformatics data also shows that patients whose tumors express higher levels of CYP27A1 have longer cancer-free survival following local treatment with radical prostatectomy. To validate these findings, we analyzed expression levels of CYP27A1 protein in benign and cancerous prostate tissue microarrays and found that while 90% of benign prostate cores express CYP27A1, 78% of cancerous cores show low to undetectable expression levels. Given the high rate of CYP27A1 loss in PC, we investigated potential mechanisms using data extracted from The Cancer Genome Atlas (TCGA). We found a very strong inverse and nearly linear relationship between levels of CYP27A1 expression and degree of its promoter methylation (R2=0.79). Conversely, the rate of genetic alterations (mutation, deletion) that might explain loss of CYP27A1expression is very low (3%). Taken together, these results indicate that CYP27A1 loss is an epigenetic event caused by promoter hypermethylation. To investigate the role of CYP27A1 in PC, we reintroduced CYP27A1 in LNCaP and 22Rv1 PC cell lines that have silenced its expression via promoter methylation. Overexpressing CYP27A1 in LNCaP cells significantly retards proliferation in vitro and also slows growth of 22Rv1 subcutaneous xenograft tumors. Given that a major function of CYP27A1 is to convert cholesterol into 27hydroxycholesterol (27HC), we treated five different PC cell lines with 27HC and found that 27HC inhibits PC cells viability in vitro in all cell lines and induces cleavage of PARP, a cellular marker for apoptosis. Finally, we found that 27HC treatment reduces cholesterol levels in PC cells and supplementing PC cells with an exogenous source of cholesterol rescues the cells from 27HC mediated effects. Collectively, our results suggest that 27HC may act as an intracellular cholesterol biosensor and PC cells have developed mechanisms to accumulate more cholesterol through silencing of CYP27A1 and stopping the production of 27HC. This is key, as epidemiological and experimental evidence strongly suggests that hypercholesterolemia is associated with an increased risk of lethal PC. Our results highlight CYP27A1 loss as a promising biomarker for PC and also suggest that reduction of intratumoral cholesterol may have a preventative role in PC possibly through treatment with 27HC. Citation Format: Mahmoud A. Alfaqih, Erik R. Nelson, Rachid Safi, Jeff Jasper, Donald P. McDonnell, Stephen J. Freedland. Dysregulation of cholesterol homeostasis through loss of CYP27A1 in prostate cancer; Implications for early detection and prevention of over-treatment. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR11.

CAUSEL: an epigenome- and genome-editing pipeline for establishing function of noncoding GWAS variants.

The vast majority of disease-associated single nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWAS) are located in the non-protein coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here, we describe a general pipeline in which candidate functional SNPs are first evaluated by fine-mapping, epigenomic profiling, and epigenome editing and then interrogated for causal function by using genome editing to create isogenic cell lines. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top scoring SNP. Epigenome editing confirmed that rs339331 possessed regulatory potential. Using transcription activator-like effector nuclease (TALEN)-mediated genome-editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the “T” risk allele increased transcription of the RFX6 gene, increased HOXB13 binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach to establish functional causality for non-coding sequence variants identified by GWAS.

Epigenetically altered miR-193b targets cyclin D1 in prostate cancer.

Micro-RNAs (miRNA) are important regulators of gene expression and often differentially expressed in cancer and other diseases. We have previously shown that miR-193b is hypermethylated in prostate cancer (PC) and suppresses cell growth. It has been suggested that miR-193b targets cyclin D1 in several malignancies. Here, our aim was to determine if miR-193b targets cyclin D1 in prostate cancer. Our data show that miR-193b is commonly methylated in PC samples compared to benign prostate hyperplasia. We found reduced miR-193b expression (P < 0.05) in stage pT3 tumors compared to pT2 tumors in a cohort of prostatectomy specimens. In 22Rv1 PC cells with low endogenous miR-193b expression, the overexpression of miR-193b reduced CCND1mRNA levels and cyclin D1 protein levels. In addition, the exogenous expression of miR-193b decreased the phosphorylation level of RB, a target of the cyclin D1-CDK4/6 pathway. Moreover, according to a reporter assay, miR-193b targeted the 3’UTR of CCND1 in PC cells and the CCND1 activity was rescued by expressing CCND1 lacking its 3’UTR. Immunohistochemical analysis of cyclin D1 showed that castration-resistant prostate cancers have significantly (P = 0.0237) higher expression of cyclin D1 compared to hormone-naïve cases. Furthermore, the PC cell lines 22Rv1 and VCaP, which express low levels of miR-193b and high levels of CCND1, showed significant growth retardation when treated with a CDK4/6 inhibitor. In contrast, the inhibitor had no effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Taken together, our data demonstrate that miR-193b targets cyclin D1 in prostate cancer.

SLUG is a direct transcriptional repressor of PTEN tumor suppressor.

PTEN/AKT signaling plays a key role in prostate cancer development and maintenance of prostate cancer stem cells. How other oncogenes or tumor suppressors interact with this pathway remain to be elucidated. SLUG is an zinc finger transcription factor of the Snail superfamily, and it promotes cancer metastasis and determines the mammary stem cell state. SLUG was overexpressed in cells by retroviral vector and knockdown of SLUG and PTEN was mediated by shRNAs-expressing lentiviruses. Expression level of SLUG and PTEN was examined by Western blot, RT-PCR, and qPCR analyses. PTEN promoter activity was measured by luciferase reporter assay. ChIP assay was used to measure the binding between SLUG and the PTEN promoter in vivo. We showed that overexpression of SLUG decreased expression of PTEN tumor repressor in prostate cancer cell lines 22RV1 and DU145; conversely, knockdown of SLUG expression elevated PTEN expresson at both protein and RNA level in these cells. We demonstrated that SLUG overexpression inhibits PTEN promoter activity through the proximal promoter region in prostate cancer cells. By ChIP assay, we confirmed that SLUG directly binds to the PTEN promoter region covering the E-box sites. We also showed that Slug deficiency leads to an increased expression of PTEN in mouse embryo fibroblasts and prostate tissues. Importantly, we found that overexpression of SLUG increases drug resistance of DU145 prostate cancer cell line and knockdown of SLUG by shRNA sensitizes DU145 cell line to chemotherapeutic drugs. We further demonstrated that PTEN knockdown converts drug sensitivity of DU145 cells expressing SLUG shRNA to anticancer drugs. We provide compelling evidence showing that PTEN is a direct functional target of SLUG. Our findings offer new insight in the regulation of the PTEN/AKT pathway and provide a molecular basis for potential targeted therapies of prostate cancer Prostate 75:907-916, 2015. © 2015 Wiley Periodicals, Inc.