22Rv1 Cell Lines Research Articles

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.

Lipocalin 2 over-expression facilitates progress of castration-resistant prostate cancer via improving androgen receptor transcriptional activity.

BackgroundCastration-resistant prostate cancer (CRPC) is the lethal phenotype of prostate cancer. Lipocalin 2 (LCN2) is aberrantly expressed in many cancers including primary prostate cancer (PCa), but its role in CRPC has not been reported.ResultsLCN2 expression was upregulated in human primary PCa and CRPC tissues. Overexpression of LCN2 promoted C4-2B and 22RV1 cell proliferation while knockdown of LCN2 markedly inhibited C4-2B and 22RV1 cell growth. LCN2 overexpression led to increased AR downstream gene SLC45A3 without upregulating AR expression. In the xenograft model, overexpression of LCN2 significantly promoted tumor growth.MethodsLCN2 expression was detected in primary PCa and CRPC tissues and cell lines C4-2B and 22RV1 using immunohistochemistry and western blotting, respectively. Serum LCN2 level was detected vi ELISA. Lentiviruses-mediated over-expression of LCN2 and LCN2 knockdown were performed in CRPC cell lines. Expressions of androgen receptor (AR) downstream genes was examined in cell lines, in CRPC tissues, and in animal models.ConclusionLCN2 could facilitate cell proliferation of CRPC via AR transcriptional activity. LCN2 could be a novel target in CRPC.

Positive expression of NR6A1/CT150 as a predictor of biochemical recurrence-free survival in prostate cancer patients.

NR6A1/CT150, as an orphan receptor, is a novel member of the cancer-testis (CT) antigen family. Here, we investigated the expression and function of NR6A1 and its underlying mechanisms in prostate cancer (PCa) patients who underwent radical prostatectomy. A total of 303 cases of prostate cancer after radical prostatectomy were analysed in a tissue microarray (TMA) for NR6A1 immunohistochemistry-based protein expression. Kaplan–Meier/log-rank analysis and Cox regression analysis were used to investigate the relationship between NR6A1 expression and clinicopathological factors in PCa. NR6A1 mRNA expression was examined by reversing transcriptase-polymerase chain reaction (RT-PCR). Knockdown of NR6A1 by small interfering RNA mediated gene silencing and overexpression of NR6A1 through lentivirus were utilized to investigate its potential role in prostate cancer cells. NR6A1 protein expression was 29.7% (90/303) and mRNA expression was 28.1%(9/32) in PCa patients. NR6A1 expression was significantly associated with Gleason score (GS) (P=0.003) and tumor stage (P=0.042). The patients with positive NR6A1 expression have a shorter biochemical recurrence-free survival. NR6A1 predicted biochemical recurrence in univariate (P=0.0159) and multivariate models (P=0.0317). In addition, gene silencing of NR6A1 resulted in G0/G1 phase cell cycle arrest, and decreased metastatic and invasive potential of prostate cancer cells DU145 and PC3. In contrast, overexpression of NR6A1 reduced G0/G1 phase cell cycle arrest, and promoted metastatic and invasive potential of prostate cancer cells 22RV1. And overexpression of NR6A1 significantly promoted tumor growth in vivo. What's more, down regulation of NR6A1 could reverse epithelial-to-mesenchymal transition (EMT) process in DU145 and PC3 cell lines, and the overexpression could enhance EMT process in 22RV1 cell line. NR6A1 played a prominent role in migration and invasion of PCa cells, and it is indicated that NR6A1 may act as a novel marker for biochemical recurrence after radical prostatectomy.

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.

Abstract 4972: Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin

Abstract Prostate specific membrane antigen (PSMA), a transmembrane glycoprotein, is a clinically validated marker of prostate cancer. PSMA is overexpressed in primary and metastatic prostate cancer when compared to normal prostate tissue which makes it an attractive target for immunotherapy. Several antibodies are being considered to target PSMA for the development of an anticancer drug. The expression levels of PSMA on tumor cells may play a role in response to treatment with anti PSMA drugs. Therefore, it is important to monitor PSMA expression on tumor cells before and during treatment. We used CellSearch® CTC assay to determine the presence of PSMA on circulating tumor cells (CTCs) using anti PSMA Centyrin as a biomarker. Centyrins are a class of alternative scaffold molecules which are proteins of small size (∼ 10 kDa). They are engineered to bind to target molecules with high specificity and sensitivity. The anti PSMA Centyrin was tested for specificity and sensitivity using tissue cultured tumor cell lines spiked into normal healthy blood and then compared to anti PSMA antibody. The anti PSMA Centyrin showed 100% positivity with the high PSMA expressing cell line (LNCap), 25% positivity with the low PSMA expressing cell line (22Rv1) and 0% positivity with the PSMA negative cell line (PC3-9). The anti PSMA Centyrin was further tested with prostate patient blood samples to determine the expression levels of PSMA on CTCs. The patient samples which contain CTCs are positive for anti PSMA Centyrin and the percentage of CTCs positive for PSMA ranged from 25-100%. Thus, PSMA expression on CTCs can be determined using CellSearch® system and anti PSMA Centyrin as a biomarker in a clinical trial. Citation Format: Bhavesh Vaidya, Peter Vulfson, Renouard Sanders, Donna Klein, Shalom Goldberg, Steve Jacobs, Chandra Rao. Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin. [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 4972.

Abstract 1825: ONC201 induces cell death in androgen receptor positive prostate cancer cells and shows synergistic effect with anti-prostate cancer drugs

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer related death among men in the United States. Current therapy for advanced prostate cancer is mainly based on androgen deprivation, though in many cases tumors become androgen-independent and resistant to available treatments. Thus, accessing novel therapies for prostate cancer and resistant tumors remains an important goal in the field. ONC201 is a first-in-class small molecule anti-cancer drug, shown to selectively induce cell death in most cancer cells tested in contrast to matched normal cells. In this study we investigated the single-agent and combination efficacy of ONC201 on a panel of prostate cancer cell lines with varied androgen receptor (AR) and receptor tyrosine kinases (RTKs) expression status. Our preliminary data suggests that the pro-apoptotic effects of ONC201 correlate with expression of androgen receptor and other receptor tyrosine kinases (RTKs). Specifically, high AR and low RTK (c-MET, EGFR, HER2 and IGFR) expression demonstrated higher sensitivity to ONC201 as compared to low AR and high RTK expression as evaluated by cell viability using the Cell-Titer Glo assay. We further modeled these findings in ONC201-sensitive 22Rv1 and ONC201-resistant DU145 cell lines. ONC201 induced robust apoptosis (cleaved PARP) in ONC201-sensitive 22Rv1 cells as compared to ONC201-resistant DU145 cells In addition, ONC201-sensitive 22RV1 cells showed abrogation of total AR expression 72 hours following treatment with ONC201. Thus, AR and RTK status seem to be emerging efficacy markers for response of ONC201 in prostate cancer cells. We are further investigating the involvement of ONC201 in the signal transduction pathway of AR and other RTKs in prostate cancer. Lastly, our initial screening for treatment of cells with a combination of ONC201 and FDA-approved therapies for prostate cancer showed synergistic potential of ONC201 with everolimus and docetaxel in both AR-dependent and independent cells. As expected, synergism of anti-androgen MDV3100 (enzalutamide) with ONC201 was restricted to AR positive prostate cancer cell lines. Our results indicate that ONC201 has therapeutic potential both as a single agent and in combination therapy for prostate cancer. Our long-term goal is to integrate our pre-clinical studies and translate the findings to single agent/combination trials of ONC201 for prostate cancer patients. Citation Format: Avital Lev, Amriti R. Lulla, David T. Dicker, Wafik S. El-Deiry. ONC201 induces cell death in androgen receptor positive prostate cancer cells and shows synergistic effect with anti-prostate cancer drugs. [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 1825.

Abstract 2142: Plumbagin, a medicinal plant-derived naphthoquinone, inhibits the growth of docetaxel resistant prostate cancer cells via inhibition of the expression of P-glycoprotein and NF-kB activation

Abstract Prostate cancer (PCa) is one of the leading causes of death in men across the world. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy manifests by progression to castration resistant prostate cancer (CRPC), which is the end stage that accounts for the majority of PCa patient deaths. Docetaxel (DTX) is an approved drug for the treatment of CRPC. However, development of DTX resistance and toxicity are the major limitations in the use of DTX in the treatment of CRPC. Now we investigated to determine whether these limitations may be overcome by combining DTX with plumbagin (PL). PL is a quinoid constituent isolated from the root of Plumbago zeylanica L. The roots of Plumbago zeylanica L. have been used in Indian medicine for more than 2500 years for treatment of various ailments. We were the first to report that PL inhibits the growth and PCa metastasis. Now we present that PL: 1) sensitizes CRPC cells to DTX and thus reduces its effective therapeutic dose to prevent DTX resistance and toxicity, and 2) inhibits the viability and growth of DTX resistant cells (22Rv1R). In combination therapeutic experiments, the effects of different concentrations of PL (0, 1.0, 2.0, and 5.0 μM) alone or in combination with DTX (0.25 nM and 0.5 nM) on colony formation in 22Rv1 and C4-2 prostate cancer cells, was determined. Combined treatment of PL and DTX has more than additive inhibitory effects on colony formation. This synergism of PL and DTX on colony formation can be explained on the basis of unique mechanism of action of PL and DTX. PL-induced inhibition of PCa growth and metastasis involves inhibition of the expression of multiple targets including PKCϵ, which correlates with the aggressiveness of PCa and plays roles in both androgen independent androgen receptor (AR) activation and promotion of PCa cell survival. DTX targets microtubules, impairs AR signaling, and inhibits cell division. To determine the effects of PL on DTX resistant PCa cells, we established DTX-resistant 22Rv1 cell line (22Rv1R). 22Rv1R overexpresses multidrug resistant protein 1 (MDR1, also known as P-glycoprotein). MDR1 is overexpressed in many tumors and thus causing intrinsic drug resistant. Transcription factor NF-KB, which is linked to cell proliferation and survival, is also activated in 22Rv1R. PL inhibited the colony forming efficiency of 22Rv1R cells which accompanied inhibition of MDR1 expression and NF-KB activation. In summary, the results presented here and the ones reported by us previously with preclinical mouse models (TRAMP and PTEN knockout mice) of PCa indicate that PL has the potential to prevent as well as treat PCa. PL is a potential natural agent worthy of investigation for prevention and treatment of human PCa. (Support: NIH grant CA102431). Citation Format: Ashok Singh, Anupama Singh, Satyamedha Bathula, Saivenkateshkomal Bathula, Ajit K. Verma. Plumbagin, a medicinal plant-derived naphthoquinone, inhibits the growth of docetaxel resistant prostate cancer cells via inhibition of the expression of P-glycoprotein and NF-kB activation. [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 2142.

Metformin Elicits Antitumor Effects and Downregulates the Histone Methyltransferase Multiple Myeloma SET Domain (MMSET) in Prostate Cancer Cells.

This study explored the biological effects of metformin on prostate cancer (PCa) cells and determined molecular pathways and epigenetic regulators implicated in its mechanism of action. We performed mRNA expression profiling in 22Rv1 cells following 2.5 mM and 5 mM metformin treatment. Genes significantly modified by metformin treatment were ranked based on altered expression, involvement with cancer-related processes, and reported dysregulation in PCa. The effects of the top ranked gene, MMSET, on the proliferative and invasive capabilities of PCa cells were investigated via siRNA knockdown alone and also combined with metformin treatment. Metformin treatment decreased cell growth of PCa cell line 22Rv1 and stalled cells at the G1/S checkpoint in a time- and dose-dependent manner, resulting in increased cells in G1 (P < 0.05) and decreased cells in S (P < 0.05) phase. Metformin activated the AMPK/mTOR signaling pathway as shown by increased p-AMPK and decreased p-p70S6K. mRNA expression profiling following metformin treatment identified significant changes in 136 chromatin-modifying genes. The top ranked gene, multiple myeloma SET domain (MMSET) showed increased expression in PCa cell lines (22Rv1 and DU145) when compared to the benign prostate epithelium-derived cell-line RWPE-1, and its expression was decreased upon metformin treatment. siRNA-mediated knockdown of MMSET showed decreased cellular migration and invasion in DU-145 cells. MMSET knockdown in combination with metformin treatment resulted in further reduction in the capacity of PCa cells to migrate and invade. These data suggest MMSET may play a role in the inhibitory effect of metformin on PCa and could serve as a potential novel therapeutic target for PCa. Prostate 76:1507-1518, 2016. © 2016 Wiley Periodicals, Inc.

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.

Combined AKT and MEK Pathway Blockade in Pre-Clinical Models of Enzalutamide-Resistant Prostate Cancer.

Despite recent improvements in patient outcomes using newer androgen receptor (AR) pathway inhibitors, treatment resistance in castrate resistant prostate cancer (CRPC) continues to remain a clinical problem. Co-targeting alternate resistance pathways are of significant interest to treat CRPC and delay the onset of resistance. Both the AKT and MEK signaling pathways become activated as prostate cancer develops resistance to AR-targeted therapies. This pre-clinical study explores co-targeting these pathways in AR-positive prostate cancer models. Using various in vitro models of prostate cancer disease states including androgen dependent (LNCaP), CRPC (V16D and 22RV1) and ENZ-resistant prostate cancer (MR49C and MR49F), we evaluate the relevance of targeting both AKT and MEK pathways. Our data reveal that AKT inhibition induces apoptosis and inhibits cell growth in PTEN null cell lines independently of their sensitivity to hormone therapy; however, AKT inhibition had no effect on the PTEN positive 22RV1 cell line. Interestingly, we found that MEK inhibition had greater effect on 22RV1 cells compared to LNCaP, V16D or ENZ-resistant cells MR49C and MR49F cells. In vitro, combination AKT and MEK blockade had evidence of synergy observed in some cell lines and assays, but this was not consistent across all results. In vivo, the combination of AKT and MEK inhibition resulted in more consistent tumor growth inhibition of MR49F xenografts and longer disease specific survival compared to AKT inhibitor monotherapy. As in our in vitro study, 22RV1 xenografts were more resistant to AKT inhibition while they were more sensitive to MEK inhibition. Our results suggest that targeting AKT and MEK in combination may be a valuable strategy in prostate cancer when both pathways are activated and further support the importance of characterizing the dominant oncogenic pathway in each patient’s tumor in order to select optimal therapy.

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.

PD32-01 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 ...

MP62-01 TARGETING THE DNA BINDING DOMAIN OF THE ANDROGEN RECEPTOR: EFFICACY OF PROSTATE CANCER COMPOUND WITH NOVEL MECHANISM OF ACTION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2016MP62-01 TARGETING THE DNA BINDING DOMAIN OF THE ANDROGEN RECEPTOR: EFFICACY OF PROSTATE CANCER COMPOUND WITH NOVEL MECHANISM OF ACTION Hendrik Borgmann, Kush Dalal, Eliana Beraldi, Artem Cherkasov, Paul Rennie, and Martin Gleave Hendrik BorgmannHendrik Borgmann More articles by this author , Kush DalalKush Dalal More articles by this author , Eliana BeraldiEliana Beraldi More articles by this author , Artem CherkasovArtem Cherkasov More articles by this author , Paul RenniePaul Rennie More articles by this author , and Martin GleaveMartin Gleave More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.909AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In castration-resistant prostate cancer, resistance to conventional treatments targeting the ligand binding domain of the androgen receptor (AR) occur inevitably, prompting the need to develop small molecules with a different binding location on the AR and novel mechanism of action. Recently, we identified a pocket on the AR-DNA binding domain (DBD) protein surface with potential as an alternative drug-target site. Here, we present preclinical data on an AR-DBD specific compound and its inhibitory effects across multiple prostate cancer cell lines in vitro and in vivo. METHODS We tested the effect of VPC-14449 on cell growth of the hormone sensitive cell line LNCaP, the Enzalutamide resistant cell line MR49C and the AR Variant driven cell line 22Rv1 by MTS assay and compared to enzalutamide. Moreover, we assessed the effect of VPC-14449 on AR transcriptional activity of these cell lines using luciferase reporter assay. In castrated mice, change in volume of LNCaP tumour xenografts and serum PSA levels was determined after compound treatment over 4 weeks. RESULTS Treatment with VPC-14449 led to dose-dependent cell growth inhibition in all three cell lines. Enzalutamide inhibited cell growth of LNCaP, but not 22Rv1 and MR49C cells for which an agonistic effect of Enzalutamide was observed at high compound concentrations. VPC-14449 blocked transcriptional activity of the AR in LNCaP, MR49C and 22Rv1 cell lines. Finally, the compound suppressed the growth of LNCaP tumor xenografts by 68% (p<0.01) and serum PSA levels by 81% (p<0.01) in mice at week 4. CONCLUSIONS The developed AR-DBD inhibiting compound showed excellent efficacy in preclinical studies across multiple prostate cancer cell lines in vitro and in vivo. AR-DBD inhibiting compounds may complement the armamentarium of drugs as effective non-cross-resistant treatment of castration-resistant prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e812 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hendrik Borgmann More articles by this author Kush Dalal More articles by this author Eliana Beraldi More articles by this author Artem Cherkasov More articles by this author Paul Rennie More articles by this author Martin Gleave 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.

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 ...

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.

The Expression of Ubiquitous Mitochondrial Creatine Kinase Is Downregulated as Prostate Cancer Progression.

Background: Mitochondria play crucial roles in cell signaling events, interorganellar communication, aging, cell proliferation and apoptosis, and mitochondrial impairment has been shown to accelerate or modulate cancer progression. Ubiquitous mitochondrial creatine kinase (uMtCK) is predominantly localized in the intermembrane space of mitochondria and catalyzes the reversible exchange of high-energy phosphate between adenosine tri-phosphate (ATP) and phosphocreatine. However, little is known about its expression and function in human prostate cancer progression.Method: We investigated the expression of uMtCK in 148 prostate carcinoma tissues and matched normal tissue by immunohistochemistry. The expression and localization of uMtCK and hexokinase II, a marker of glycolysis, were examined in prostate carcinoma cell lines using western blot and immunofluorescence.Results: MtCK expression was significantly lower in high Gleason grade carcinoma compared with normal prostate or low grade carcinoma. Western blot further revealed that uMtCK was highly expressed in LNCaP and 22Rv1 cell lines, as well as in the normal prostate cell line RWPE-1. However, uMtCK expression was almost absent in PC3 and DU145 cell lines, in correlation with absent or mutant p53 expression, respectively. In contrast, hexokinase II was overexpressed in PC3 cells. Moreover, in the low uMtCK expressing cell lines, glycolytic ATP production was increased, whereas mitochondrial ATP production was decreased.Conclusions: These data suggest that uMtCK is downregulated as prostate cancer progresses in correlation with a metabolic switch in ATP usage.

Reciprocal feedback inhibition of the androgen receptor and PI3K as a novel therapy for castrate-sensitive and -resistant prostate cancer.

Gain-of-function of the androgen receptor (AR) and activation of PI3K/AKT/mTOR pathway have been demonstrated to correlate with progression to castration-resistant prostate cancer (CRPC). However, inhibition of AR or PI3K/mTOR alone results in a reciprocal feedback activation. Therefore, we hypothesized that dual inhibition of the AR and PI3K/mTOR pathway might lead to a synergistic inhibition of cell growth and overcome drug resistance in CRPC. Here, we reported that androgen-depletion increased AR protein level and Akt phosphorylation at Ser473 and Thr308 in LNCaP cells. Moreover, we developed resistance cell lines of LNCaP to Enzalutamide (or MDV3100), an AR inhibitor (named as LNCaP 'MDV-R') and PF-04691502, a PI3K/mTOR inhibitor (named as LNCaP 'PF-R'). MTS analysis showed that LNCaP 'PF-R' was strongly resistant to Enzalutamide treatment, and on the other hand, LNCaP 'MDV-R' was 6-fold resistant to PF-04691502 treatment. Mechanistically, LNCaP 'MDV-R' cells had significantly reduced AR, loss of PSA and increase Akt activity in contrast with LNCaP 'PF-R' cells. Combined inhibition of PI3K/mTOR and AR pathways with a variety of small molecular inhibitors led to a synergistic suppression of cell proliferation and a profound increase of apoptosis and cell cycle arrest in both androgen-dependent LNCaP and independent CRPC 22Rv1 cell lines. In conclusion, this study provides preclinical proof-of-concept that the combination of a PI3K/mTOR inhibitor with an AR inhibitor results in a synergistic anti-tumor response in non-CRPC and CRPC models.