Human Castration-resistant Prostate Cancer Research Articles

Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells.

Standard treatment for primary prostate cancer includes systemic exposure to chemotherapeutic drugs that target androgen receptor or antihormone therapy (chemical castration); however, drug-resistant cancer cells generally emerge during treatment, limiting the continued use of systemic chemotherapy. Patients are then treated with more toxic standard therapies. Therefore, there is an urgent need for novel and more effective treatments for prostate cancer. The cholesterol biosynthetic pathway is an attractive therapeutic target for treating endocrine-dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as the metabolic precursor of endogenous steroid hormones. In this study, we have examined the effects of RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate; Roche Pharmaceuticals internal reference: RO0488071) (RO), which is an inhibitor of 2, 3-oxidosqualene cyclase (a key enzyme in the cholesterol biosynthetic pathway), on prostate cancer cells. Exposure of both hormone-dependent and castration-resistant human prostate cancer cells to RO reduced prostate cancer cell viability and induced apoptosis in vitro. RO treatment reduced androgen receptor protein expression in hormone-dependent prostate cancer cells and increased estrogen receptor β (ERβ) protein expression in both hormone-dependent and castration-resistant prostate cancer cell lines. Combining RO with an ERβ agonist increased its ability to reduce castration-resistant prostate cancer cell viability. In addition, RO effectively suppressed the growth of aggressive castration-resistant human prostate cancer cell xenografts in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells in vitro. Our study is the first to demonstrate that the cholesterol biosynthesis inhibitor RO effectively suppresses growth of human prostate cancer cells. Our findings suggest that cholesterol biosynthesis inhibitors such as RO, when used in combination with commonly used chemotherapeutic drugs or ERβ specific ligands, could represent a novel therapeutic approach to prevent the growth of prostate cancer tumors.

ROR-γ drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer.

The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoid acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits coactivators SRC-1 and -3 to an AR-RORE to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR gene network. Lastly, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing but not AR-negative xenograft PCa models, and effectively sensitized CRPC tumors to enzalutamide, without overt toxicity in mice. Together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and a potential therapeutic target for advanced PCa.

Co-targeting hexokinase 2-mediated Warburg effect and ULK1-dependent autophagy suppresses tumor growth of PTEN- and TP53-deficiency-driven castration-resistant prostate cancer

Currently, no therapeutic options exist for castration-resistant prostate cancer (CRPC) patients who have developed resistance to the second generation anti-androgen receptor (AR) axis therapy. Here we report that co-deletion of Pten and p53 in murine prostate epithelium, often observed in human CRPC, leads to AR-independent CRPC and thus confers de novo resistance to second generation androgen deprivation therapy (ADT) in multiple independent yet complementary preclinical mouse models. In contrast, mechanism-driven co-targeting hexokinase 2 (HK2)-mediated Warburg effect with 2-deoxyglucose (2-DG) and ULK1-dependent autophagy with chloroquine (CQ) selectively kills cancer cells through intrinsic apoptosis to cause tumor regression in xenograft, leads to a near-complete tumor suppression and remarkably extends survival in Pten−/p53-deficiency-driven CRPC mouse model. Mechanistically, 2-DG causes AMPK phosphorylation, which in turn inhibits mTORC1-S6K1 translation signaling to preferentially block anti-apoptotic protein MCL-l synthesis to prime mitochondria-dependent apoptosis while simultaneously activates ULK1-driven autophagy for cell survival to counteract the apoptotic action of anti-Warburg effect. Accordingly, inhibition of autophagy with CQ sensitizes cancer cells to apoptosis upon 2-DG challenge. Given that 2-DG is recommended for phase II clinical trials for prostate cancer and CQ has been clinically used as an anti-malaria drug for many decades, the preclinical results from our proof-of-principle studies in vivo are imminently translatable to clinical trials to evaluate the therapeutic efficacy by the combination modality for a subset of currently incurable CRPC harboring PTEN and TP53 mutations.

Mécanismes de résistance à la castration : l’hypoxie intratumorale stimule l’expression du récepteur aux androgènes

Tumor hypoxia and its biological consequence lead to microenvironment adaptation of tumor initiation, promotion and progression. The aim of the study was to observe the influence of hypoxia on the expression of the androgen receptor (AR), using an original model of multicellular spheroids obtained from castration-resistant prostate tumor cells. Two human castration-resistant prostate cancer cell lines have been used to generate multicellular tumor spheroids (MTS). The conditions and duration of incubation modulated the final size of the MTS and the intrinsic hypoxia gradient. The expression of AR was studied by immunohistochemistry (IHC) and secretion of PSA measured in the culture medium. The IHC expression of AR was characterized by a decreasing gradient from the periphery to the center of MTS (less intense in central hypoxic zone), corresponding to a nuclear translocation of activated AR. This result was proportionally correlated with the duration of hypoxic incubation period. Hypoxia caused significant increase in AR expression at 6h of oxygen deprivation. This activation of AR was correlated with transcriptional activity increase of target genes, including increased secretion of PSA. This demonstration of activation, increased expression and increased transcriptional activity of AR by hypoxia is the first to have been made with an original model of hypoxia, closer to reality than previous models, i.e. close to tissue hypoxia observed in primary prostate cancer. 4.

Multinucleation and Mesenchymal-to-Epithelial Transition Alleviate Resistance to Combined Cabazitaxel and Antiandrogen Therapy in Advanced Prostate Cancer.

Patients with metastatic castration-resistant prostate cancer (CRPC) frequently develop therapeutic resistance to taxane chemotherapy and antiandrogens. Cabazitaxel is a second-line taxane chemotherapeutic agent that provides additional survival benefits to patients with advanced disease. In this study, we sought to identify the mechanism of action of combined cabazitaxel and androgen receptor (AR) targeting in preclinical models of advanced prostate cancer. We found that cabazitaxel induced mitotic spindle collapse and multinucleation by targeting the microtubule depolymerizing kinesins and inhibiting AR. In androgen-responsive tumors, treatment with the AR inhibitor, enzalutamide, overcame resistance to cabazitaxel. Combination treatment of human CRPC xenografts with cabazitaxel and enzalutamide reversed epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) and led to multinucleation, while retaining nuclear AR. In a transgenic mouse model of androgen-responsive prostate cancer, cabazitaxel treatment induced MET, glandular redifferentiation, and AR nuclear localization that was inhibited by androgen deprivation. Collectively, our preclinical studies demonstrate that prostate tumor resistance to cabazitaxel can be overcome by antiandrogen-mediated EMT-MET cycling in androgen-sensitive tumors but not in CRPC. Moreover, AR splice variants may preclude patients with advanced disease from responding to cabazitaxel chemotherapy and antiandrogen combination therapy. This evidence enables a significant insight into therapeutic cross-resistance to taxane chemotherapy and androgen deprivation therapy in advanced prostate cancer.

Mechanistic Support for Combined MET and AR Blockade in Castration-Resistant Prostate Cancer

A recent phase III trial of the MET kinase inhibitor cabozantinib in men with castration-resistant prostate cancer (CRPC) failed to meet its primary survival end point; however, most men with CRPC have intact androgen receptor (AR) signaling. As previous work supports negative regulation of MET by AR signaling, we hypothesized that intact AR signaling may have limited the efficacy of cabozantinib in some of these patients. To assess the role of AR signaling on MET inhibition, we first performed an in silico analysis of human CRPC tissue samples stratified by AR signaling status (+ or −), which identified MET expression as markedly increased in AR− samples. In vitro, AR signaling inhibition in AR+ CRPC models increased MET expression and resulted in susceptibility to ligand (HGF) activation. Likewise, MET inhibition was only effective in blocking cancer phenotypes in cells with MET overexpression. Using multiple AR+ CRPC in vitro and in vivo models, we showed that combined cabozantinib and enzalutamide (AR antagonist) treatment was more efficacious than either inhibitor alone. These data provide a compelling rationale to combine AR and MET inhibition in CRPC and may explain the negative results of the phase III cabozantinib study in CRPC. Similarly, the expression of MET in AR− disease, whether due to AR inhibition or loss of AR signaling, suggests potential utility for MET inhibition in select patients with AR therapy resistance and in AR− prostate cancer.

Quercetin-loaded nanomicelles to circumvent human castration-resistant prostate cancer in vitro and in vivo.

Prostate cancer is highly prevalent and has become the second leading cause of cancer-related death in men. Its treatment remains a challenge in the clinic, particularly in patients who have advanced to "castration-resistant prostate cancer" (CRPC). Thus, more effective therapeutic strategies are required. Quercetin (QCT) is a natural flavonoid compound that has attracted increasing interest due to its anticancer activity. However, the clinical application of quercetin is largely hampered by its poor water solubility and low bioavailability. The objective of this study was to evaluate the therapeutic potential of novel QCT-loaded nanomicelles (M-QCTs) assembled from DSPE-PEG2000 for prostate cancer treatment. Our results indicated that QCT was efficiently encapsulated into micelles up to 1 mg mL(-1), which corresponds to a 450-fold increase of its water solubility. In vitro studies showed that the half-maximal inhibitory concentration (IC50) value (20.2 μM) of M-QCTs was much lower than free QCT (>200 μM). Thus, M-QCTs were considerably more effective than free QCT in proliferation inhibition and apoptosis induction of human androgen-independent PC-3 cells. Furthermore, M-QCTs showed superior antitumor efficacy and the tumor proliferation rate reduced by 52.03% compared to the control group in the PC-3 xenograft mouse model, possibly due to increased accumulation of M-QCTs at the tumor site by the enhanced permeability and retention (EPR) effect. Collectively, our studies demonstrated that M-QCTs significantly increase drug accumulation at the tumor site and exhibit superior anticancer activity in prostate cancer. Thus, our nanomicelle-based drug delivery system constitutes a promising and effective therapeutic strategy for clinical treatment.

PDLIM2 suppression efficiently reduces tumor growth and invasiveness of human castration-resistant prostate cancer-like cells.

Although PDLIM2 gene may have a context-dependent role in various human malignancies and can be a potential therapeutic target, only a limited number of in vitro studies addressed the molecular functions of PDLIM2 in prostate cancer. Here, we aimed to explore the role of PDLIM2 and the effect of the PDLIM2 gene suppression on oncogenic phenotypes of human castration-resistant prostate cancer (CRPC)-like cells. We used human CRPC-like cell lines (PC3, DU145, and C4-2B) for our experiments. Transcription levels of PDLIM2 and relevant genes were measured by real time-PCR and protein expression was analyzed by western blot. Cell viability, proliferation, clonogenic growth, and tumor sphere formation were examined after a specific inhibition of PDLIM2 using RNA interference. Flow cytometry was used to examine apoptotic cell death and cell cycle disturbances. Wound healing and transwell migration assays were performed to investigate the invasion capabilities of CRPC-like cells. Additionally, key oncogenic signaling pathways were examined using western blot. Lastly, we evaluated the in vivo efficacy of PDLIM2 suppression on tumor growth of human CRPC xenografts in mice. We observed a significant enhancement of PDLIM2 expression in human CRPC-like cell lines, while a specific inhibition of PDLIM2 reduced cell viability and proliferation due to apoptotic cell death. Conversely, PDLIM2 overexpression significantly reduced cell proliferation compared to the negative control in androgen-sensitive LNCaP cells. Moreover, PDLIM2 suppression led to a decrease of clonogenic growth and tumor sphere formation in three-dimensional cultures with the G2/M cell cycle arrest in human CRPC-like cells. PDLIM2 inhibition also attenuated cellular migration and invasion capabilities of human CRPC-like cells, and reduced the expression of mesenchymal marker. Among several oncogenic signaling pathways, only the MAPK/ERK signaling cascade was decreased by PDLIM2 inhibition and reciprocally, ERK inhibition down-regulated PDLIM2 expression. Importantly, PDLIM2 inhibition remarkably compromised tumor growth in a human CRPC xenograft model. In summary, the suppression of PDLIM2 significantly reduced such oncogenic phenotypes as proliferation, clonogenicity, invasiveness, and tumor cell growth in human CRPC-like cells both in vitro and in vivo, indicating that PDLIM2 may be considered a novel therapeutic target gene for treating human CRPC.

LncRNA MALAT1 enhances oncogenic activities of EZH2 in castration-resistant prostate cancer

The Polycomb protein enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in advanced human prostate cancer (PCa), especially in lethal castration-resistant prostate cancer (CRPC). However, the signaling pathways that regulate EZH2 functions in PCa remain incompletely defined. Using EZH2 antibody-based RNA immunoprecipitation-coupled high throughput sequencing (RIP-seq), we demonstrated that EZH2 binds to MALAT1, a long non-coding RNA (lncRNA) that is overexpressed during PCa progression. GST pull-down and RIP assays demonstrated that the 3' end of MALAT1 interacts with the N-terminal of EZH2. Knockdown of MALAT1 impaired EZH2 recruitment to its target loci and upregulated expression of EZH2 repressed genes. Further studies indicated that MALAT1 plays a vital role in EZH2-enhanced migration and invasion in CRPC cell lines. Meta-analysis and RT-qPCR of patient specimens demonstrated a positive correlation between MALAT1 and EZH2 expression in human CRPC tissues. Finally, we showed that MALAT1 enhances expression of PRC2-independent target genes of EZH2 in CRPC cells in culture and patient-derived xenografts. Together, these data indicate that MALAT1 may be a crucial RNA cofactor of EZH2 and that the EZH2-MALAT1 association may provide a new avenue for development new strategies for treatment of CRPC.

Abstract 1848: PPARγ function is attenuated by full length androgen receptor and the AR-V7 variant in human prostate cancer cells

Abstract The peroxisome proliferator activated receptor gamma (PPARγ) is a ligand-activated transcription factor that regulates in vitro and in vivo growth of castration-resistant human prostate cancer (PCa) cells. However, the factors that control expression of PPARγ within human PCa have not been characterized. We have previously shown that the androgen dihydrotestosterone decreases PPARγ levels and transcriptional activity in human PCa cells. Dihydrotestosterone serves as a high affinity ligand for the 110 kD form of the androgen receptor (AR-FL). Recent studies have shown the development of castration-resistant forms of prostate cancer is not only due to reactivation of AR-FL but also results from the expression of androgen receptor splice variants (ARVs) that lack the ligand-binding domain of the receptor. The goal of this study is to define the extent to which AR-FL and an ARV found in castration-resistant prostate cancer, AR-V7, regulate PPARγ expression and function. We first examined the role of AR-FL in the AR-positive C4-2 cell line. siRNA-mediated knockdown of endogenous AR-FL within C4-2 cells increased PPARγ protein and transcriptional activity. We next used reporter assays to further define the role of AR-FL and AR-V7 in PPARγ function. In these experiments, we first transfected plasmid vectors that express the AR-V7 or AR-FL into the AR null PC-3 prostate cancer cell line. A luciferase-based reporter assay was then used to measure alterations in PPARγ transcriptional activity within transfected cells. The addition of AR-FL to PC-3 cells resulted in reduced basal PPARγ luciferase activity. The ability of the ligand rosiglitazone to increase PPARγ activation was also suppressed in AR-FL positive PC-3 cells. A comparable decrease in ligand-induced PPARγ activation was detected in PC-3 cells that expressed AR-V7. The reduction in PPARγ activity produced by AR-FL and AR-V7 was likely not due to alterations in PPAR gamma expression, for we did not detect a consistent reduction in PPARγ mRNA and protein in transfected cells. Overall these data suggest that both AR-FL and AR-V7 are negative regulators of PPARγ function. Moreover, the AR-V7 stimulated reduction in PPARγ activity does not require the presence of AR-FL. Studies currently are underway to define the effect of endogenous AR-V7 on PPARγ expression and activity. This project was supported by NIGMS RISE grant (2R25GM059994-13), a NCI Mentored Career Development Award (K01CA114253), and the Vanderbilt CTSA grant UL1 RR024975-01 from NCRR/NIH. Citation Format: Emuejevoke Olokpa, Lamonica V. Stewart. PPARγ function is attenuated by full length androgen receptor and the AR-V7 variant in human prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1848. doi:10.1158/1538-7445.AM2015-1848

Abstract 267: Combining androgen deprivation with immune checkpoint blockade delays the development of castration resistance in a murine model of prostate cancer

Abstract Androgen deprivation therapy induces immune cell infiltration in human prostate cancer. These findings suggest that immunotherapy may be most efficacious when administered concurrently with androgen deprivation, early in disease progression. We used a subcutaneous allograft model of murine prostate cancer (Myc-Cap), which mimics the development of human castration-resistant prostate cancer (CRPC) progression to study the anti-tumor effects of concurrent hormonal/immunotherapy. Implanted Myc-Cap tumors initially respond to androgen deprivation (degarelix acetate or bilateral orchiectomy), but mice eventually progress with CRPC. To test the hypothesis that the combination of androgen deprivation and immune checkpoint blockade could mediate pre-clinical benefit, we treated mice with either anti-PD-1, a depleting anti-CTLA-4 antibody (IgG2A), a non-depleting anti-CTLA-4 antibody (IgG1 D265A) or antibody combinations in the peri-castration period, then followed mice for the development of castration-resistant disease. Interestingly, the depleting anti-CTLA-4 antibody with/without anti-PD-1 antibody was strikingly effective in preventing the emergence of castration-resistant disease. The median castration-resistance free survival was 22 days in mice treated with androgen deprivation alone versus 32 days in mice treated with androgen deprivation and depleting anti-CTLA-4 antibody (P<0.05, compared to androgen deprivation alone) versus 30 days in mice treated with androgen deprivation, depleting anti-CTLA-4 and anti-PD-1 (P<0.05, compared to androgen deprivation alone; non-significant, compared to androgen deprivation and depleting anti-CTLA-4 antibody). Immunologically, we found that castration increases intratumoral infiltration of helper, cytotoxic and regulatory T cells (Tregs), natural killer cells, and macrophages, as well as effector cytokine production of T cells. We also found up-regulated expression of CTLA-4 and PD-1 as well as their respective ligands. Mechanistic studies showed that androgen deprivation combined with depleting anti-CTLA-4 /anti-PD-1 significantly reduces intratumoral Tregs and increases interferon-γ- or tumor necrosis factor-α- producing T cells in the tumor and its draining lymph node. In conclusion, while androgen deprivation renders the tumor microenvironment more immunogenic; the combination of androgen deprivation and depleting anti-CTLA-4 antibody with/without anti-PD-1 can significantly delay the development of CRPC. Citation Format: Ying-Chun Shen, Christina Kochel, Brian J. Francica, Angela Alme, Christopher Nirschl, Thomas Nirschl, Zoila Areli Lopez Bujanda, Maria A. Carrera H, Mark Selby, Alan Korman, Charles G. Drake. Combining androgen deprivation with immune checkpoint blockade delays the development of castration resistance in a murine model of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 267. doi:10.1158/1538-7445.AM2015-267

Repurposing of phentolamine as a potential anticancer agent against human castration-resistant prostate cancer: A central role on microtubule stabilization and mitochondrial apoptosis pathway.

Drug repurposing of phentolamine, an α-adrenoceptor antagonist, as an anticancer agent has been studied in human castration-resistant prostate cancer (CRPC). Cell proliferation was examined by sulforhodamine B and CFSE staining assays. Cell cycle progression and mitochondrial membrane potential (ΔΨm) were detected by flow cytometric analysis. Protein expression was detected by Western blotting. Effect on tubulin/microtubule was determined using confocal immunofluorescence microscopic examination, microtubule assembly detection, tubulin turbidity assay, and binding assay. Several assessments were used to characterize apoptotic signaling pathways and combinatory effect. Phentolamine induced anti-proliferative effect in PC-3 and DU-145, two CRPC cell lines, and P-glycoprotein (P-gp) overexpressing cells. This effect was not significantly reduced in paclitaxel-resistant cells. Rhodamine 123 efflux assay showed that phentolamine was not a P-gp substrate. Phentolamine induced mitotic arrest of the cell cycle and formation of hyperdiploid cells, followed by an increase of apoptosis. Mitotic arrest was confirmed by cyclin B1 up-regulation, Cdk1 activation, and a dramatic increase of mitotic protein phosphorylation. Both in vitro and cellular identification demonstrated that phentolamine, similar to paclitaxel, induced tubulin polymerization and formation of multiple nuclei. Besides, it did not compete with paclitaxel binding on tubulin. Phentolamine induced the phosphorylation and degradation of Bcl-2 and Bcl-xL, two anti-apoptotic Bcl-2 family members, and the loss of ΔΨm indicating the induction of mitochondrial damage. It ultimately induced the activation of caspase-9, -8, and -3 and apoptotic cell death. Moreover, combination treatment with phentolamine and paclitaxel caused a synergistic apoptosis. The data suggest that phentolamine is a potential anticancer agent. In contrast to a wide variety of microtubule disrupting agents, phentolamine induces microtubule assembly, leading to mitotic arrest of the cell cycle which "in turn" induces subsequent mitochondrial damage and activation of related apoptotic signaling pathways in CRPC cells. Furthermore, combination between phentolamine and paclitaxel induces a synergistic apoptotic cell death. Phentolamine has a simple chemical structure and is not a P-gp substrate. Optimization of phentolamine structure may also be a potential approach for further development.

The AhR Ligand, TCDD, Regulates Androgen Receptor Activity Differently in Androgen-Sensitive versus Castration-Resistant Human Prostate Cancer Cells.

The reported biological effects of TCDD include induction of drug metabolizing enzymes, wasting syndrome and tumor promotion. TCDD elicits most of its effects through binding the aryl hydrocarbon receptor (AhR). TCDD induced degradation of AhR has been widely reported and requires ubiquitination of the protein. The rapid depletion of AhR following TCDD activation serves as a mechanism to modulate AhR mediated gene induction. In addition to inducing AhR degradation, TCDD has been reported to induce degradation of hormone receptors. The studies reported here, evaluate the effect of TCDD exposure on androgen receptor (AR) expression and activity in androgen-sensitive LNCaP and castration-resistant C4-2 prostate cancer cells. Our results show that TCDD exposure does not induce AhR or AR degradation in C4-2 cells. However, both AhR and AR are degraded in LNCaP cells following TCDD exposure. In addition, TCDD enhances AR phosphorylation and induces expression of AR responsive genes in LNCaP cells. Our data reveals that TCDD effect on AR expression and activity differs in androgen-sensitive and castration-resistant prostate cancer cell models.

Novel Imidazopyridine Derivatives Possess Anti-Tumor Effect on Human Castration-Resistant Prostate Cancer Cells.

Prostate cancer (PCa) is the second leading cause of cancer-related death afflicting United States males. Most treatments to-date for metastatic PCa include androgen-deprivation therapy and second-generation anti-androgens such as abiraterone acetate and enzalutamide. However, a majority of patients eventually develop resistance to these therapies and relapse into the lethal, castration-resistant form of PCa to which no adequate treatment option remains. Hence, there is an immediate need to develop effective therapeutic agents toward this patient population. Imidazopyridines have recently been shown to possess Akt kinase inhibitory activity; thus in this study, we investigated the inhibitory effect of novel imidazopyridine derivatives HIMP, M-MeI, OMP, and EtOP on different human castration-resistant PCa cells. Among these compounds, HIMP and M-MeI were found to possess selective dose- and time-dependent growth inhibition: they reduced castration-resistant PCa cell proliferation and spared benign prostate epithelial cells. Using LNCaP C-81 cells as the model system, these compounds also reduced colony formation as well as cell adhesion and migration, and M-MeI was the most potent in all studies. Further investigation revealed that while HIMP primarily inhibits PCa cell growth via suppression of PI3K/Akt signaling pathway, M-MeI can inhibit both PI3K/Akt and androgen receptor pathways and arrest cell growth in the G2 phase. Thus, our results indicate the novel compound M-MeI to be a promising candidate for castration-resistant PCa therapy, and future studies investigating the mechanism of imidazopyridine inhibition may aid to the development of effective anti-PCa agents.

Enhanced Sensitivity to Sunitinib by Inhibition of Akt1 Expression in Human Castration-resistant Prostate Cancer PC3 Cells Both In Vitro and In Vivo

To investigate whether antitumor activity of sunitinib is enhanced by silencing Akt1 in a human castration-resistant prostate cancer PC3 model. We initially established PC3 in which the expression vector containing a short hairpin ribonucleic acid targeting Akt1 was introduced (PC3/sh-Akt1). Changes in various phenotypes of PC3/sh-Akt1 after treatment with sunitinib were compared with those of PC3 transfected with control vector alone (PC3/C) both in vitro and in vivo. When cultured in the standard medium, in vitro growth of PC3/sh-Akt1 was almost similar to that of PC3/C. However, compared with PC3/C, PC3/sh-Akt1 showed a significantly higher sensitivity to sunitinib, accompanying impaired phosphorylation of p44/42 mitogen-activated protein kinase, downregulation of Bcl-2, and upregulation of Bax. In addition, treatment with sunitinib significantly suppressed the migration ability of PC3/sh-Akt1 compared with that of PC3/C. In vivo, administration of sunitinib induced the significantly marked growth inhibition of PC3/sh-Akt1 compared with that of PC3/C, and apoptotic index in PC3/sh-Akt1 tumor in mice treated with sunitinib was significantly greater than that in PC3/C tumor. Combined treatment with Akt1 inhibitor and sunitinib could be a promising therapeutic approach for men with castration-resistant prostate cancer.

MP55-19 SUPPRESSION OF AUTOPHAGY BY CHLOROQUINE POTENTIATES THE ANTI-CANCER EFFECT OF DOCETAXEL ON CASTRATION-RESISTANT PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2015MP55-19 SUPPRESSION OF AUTOPHAGY BY CHLOROQUINE POTENTIATES THE ANTI-CANCER EFFECT OF DOCETAXEL ON CASTRATION-RESISTANT PROSTATE CANCER CELLS Wen Song, Jun Yang, Yang Cai, Yang Luan, Li Wu, Xia Liu, Tao Wang, Shao Wang, Ji Liu, and Zhang Ye Wen SongWen Song More articles by this author , Jun YangJun Yang More articles by this author , Yang CaiYang Cai More articles by this author , Yang LuanYang Luan More articles by this author , Li WuLi Wu More articles by this author , Xia LiuXia Liu More articles by this author , Tao WangTao Wang More articles by this author , Shao WangShao Wang More articles by this author , Ji LiuJi Liu More articles by this author , and Zhang YeZhang Ye More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2062AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Autophagy has been recognized as a potential target for cancer therapy. The world wide used antimalarial drug chloroquine (CQ) is able to inhibit autophagy and therefore is being considered as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. Docetaxel is the first-line chemotherapeutic agent in prostate cancer, but in most cases, resistance to docetaxel develops through various mechanisms. This study focused on the combination of CQ as an approach to potentiate the inhibitory effect of docetaxel on human castration-resistant prostate cancer (CRPC) cells. METHODS PC3/22RV1 cells were treated with CQ and/or docetaxel, and their proliferative ability, apoptosis and autophagy induction effects, and the variation of the cell cycle were evaluated. The proliferative ability of PC3/22RV1 was analyzed by the Cell Counting Kit-8 (CCK8) assay. Apoptosis was quantified by flowcytometry after double-staining of the cells with Annexin V/PI and Western blot analysis. The cell cycle was evaluated by flowcytometry after staining of cells with PI. Autophagy was quantified by immunofluorescence of GFP-LC3 and Western blot analysis. Finally, the in vivo investigation further confirmed the advantages of such a strategy. RESULTS Docetaxel inhibited the proliferative activity of PC3/22RV1 cells, which was dependent on the arrest of the cells to the G2/M-phase and also partially on apoptosis induction, and the effect was potentiated by CQ co-treatment. The potentiation of the inhibitory effect of docetaxel by CQ was dependent on the suppression of autophagy and induction of apoptosis. PC3/22RV1 induced autophagy in response to docetaxel, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer effect of docetaxel. The result of experiment in vivo was consistent. CONCLUSIONS The combination therapy with CQ should be a novel therapeutic modality to improve efficacy of docetaxel-based chemotherapy on CRPC, possibly by inhibiting autophagy-dependent resistance to chemotherapy. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e680 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wen Song More articles by this author Jun Yang More articles by this author Yang Cai More articles by this author Yang Luan More articles by this author Li Wu More articles by this author Xia Liu More articles by this author Tao Wang More articles by this author Shao Wang More articles by this author Ji Liu More articles by this author Zhang Ye More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP37-13 ACCELERATED CELL PROLIFERATION AND ENHANCED RESISTANCE TO DOCETAXEL BY INHIBITION OF 4E-BINDING PROTEIN 1 EXPRESSION IN HUMAN CASTRATION-RESISTANT PROSTATE CANCER PC3 CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2015MP37-13 ACCELERATED CELL PROLIFERATION AND ENHANCED RESISTANCE TO DOCETAXEL BY INHIBITION OF 4E-BINDING PROTEIN 1 EXPRESSION IN HUMAN CASTRATION-RESISTANT PROSTATE CANCER PC3 CELLS Hiromoto Tei, Hideaki Miyake, and Masato Fujisawa Hiromoto TeiHiromoto Tei More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1276AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES 4E-binding protein 1 (4E-BP1) is an eukaryotic initiation factor 4E (eIF4E) binding protein that plays a critical role in the control of protein synthesis, cell growth and survival. When 4E-BP1 binds to eIF4E, this complex is shown to impede the cap-mediated translation, resulting in the induction of apoptotic cell death in a wide variety of cancer cells. The objective of this study was to clarify the functional significance of 4E-BP1 in the regulation of cell proliferation and sensitivity to proapoptotic stimuli in human castration-resistant prostate cancer (CRPC) PC3 cells. METHODS We initially established PC3, in which the expression vector containing short hairpin RNA (shRNA) targeting 4E-BP1, was introduced (PC3/sh-4E-BP1). Changes in the growth and sensitivity to docetaxel in PC3/sh-4E-BP1 were compared with those in PC3 transfected with control vector alone (PC3/C) both in vitro and in vivo. We also evaluated the expression of key molecules associated with apoptosis and signal transduction in PC3 sublines by Western blot analyses. RESULTS In vitro growth of PC3/sh-4E-BP1 was significantly faster than that in PC3/C. Expression levels of phosphorylated (p)-eIF4E, c-Myc, cyclin D1, p-Akt, p-p44-42 mitogen activated kinase (MAPK), p-mammalian target of rapamycin, p-S6K, Bcl-2, Bcl-xL and Mcl-1 in PC3/sh-4E-BP1 were significantly upregulated compared with those in PC3/C. Furthermore, PC3/sh-4E-BP1 showed a significantly higher sensitivity to docetaxel; that is, the IC50 of docetaxel in PC3/sh-4E-BP1 was approximately 10-times as high as that in PC3/C. Sensitivities of both PC3 sublines to docetaxel became similar by additional treatment with a specific inhibitor of c-Myc, Akt or MAPK. In vivo, the growth of PC3/sh-4E-BP1 tumors in nude mice was significantly faster than that of PC3/C tumors. Administration of docetaxel induced the growth inhibition of both PC3/C and PC3/sh-4E-BP1 tumors; however, the growth inhibitory effect of docetaxel on PC3/C tumors was more marked than that on PC3/sh-4E-BP1 tumors. CONCLUSIONS These findings suggest that 4E-BP1 plays an important role in the growth as well as resistance to proapoptotic therapeutic stimuli in CRPC cells through the regulation of major molecules mediating the signal transduction and apoptosis. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e442-e443 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiromoto Tei More articles by this author Hideaki Miyake More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP55-06 GALECTIN-3 IS A THERAPEUTIC TARGET FOR CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2015MP55-06 GALECTIN-3 IS A THERAPEUTIC TARGET FOR CASTRATION-RESISTANT PROSTATE CANCER Tomoharu Fukumori, Tsogt-Ochir Dondoo, Kei Daizumoto, Tomoya Fukawa, Yasuyo Yamamoto, Kunihisa Yamaguchi, Masayuki Takahashi, and Hiro-omi Kanayama Tomoharu FukumoriTomoharu Fukumori More articles by this author , Tsogt-Ochir DondooTsogt-Ochir Dondoo More articles by this author , Kei DaizumotoKei Daizumoto More articles by this author , Tomoya FukawaTomoya Fukawa More articles by this author , Yasuyo YamamotoYasuyo Yamamoto More articles by this author , Kunihisa YamaguchiKunihisa Yamaguchi More articles by this author , Masayuki TakahashiMasayuki Takahashi More articles by this author , and Hiro-omi KanayamaHiro-omi Kanayama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2049AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Castration-resistant prostate cancer (CRPC) has been common and CRPC-related death has been increasing. Therefore, to clarify the mechanism of tumor progression and resistance to anti-androgen drug is useful for the strategy of appropriate treatment for CRPC. Galectin-3 has been shown to be correlated with tumor progression and metastasis in a variety of cancer cells through the regulation of tumor proliferation, angiogenesis, and apoptosis. Here, we investigate the effects of galectin-3 on the tumor progression and resistance to anti-androgen drug in CRPC. METHODS Endogenous galectin-3 expression was examined by immunohistochemical analysis in human CRPC specimen, and serum galectin-3 was measured by ELISA. We generated continuous galectin-3-overexpressed LNCaP cells and galectin-3-downregulated PC-3 cells using siRNA. The invasion and migration assays in LNCaP and PC-3 cells with or without galectin-3 were performed by the xCELLigence system. Androgen receptor (AR)-dependent gene PSA expression was measured by RT-PCR in LNCaP cells with or without galectin-3 cultured in androgen-depleted media and treated for 24 hours with or without DHT (1 nM) combined with mock, enzalutamide (10 µM), and bicalutamide (10 µM). Transcriptional activity of AR were measured by luciferase reporter assay for the PSA promoter. RESULTS The CRPC highly expressed galectin-3 in both cytoplasm and nuclei, and mean concentration of human serum galectin-3 adjusted by prostate volume in CRPC was higher than that of non-malignancy control (6178 vs. 1671 pg/ml, p<0.001) and hormone-naive prostate cancer (6178 vs. 3021 pg/ml, p=0.008). Overexpression of galectin-3 in LNCaP cells promoted invasion (cell index; 0.39 vs. 0.07) and migration of tumor cells (cell index; 0.30 vs. 0.02) comparing with control LNCaP cells. Downregulation of galectin-3 in PC-3 cells suppressed invasion (cell index; 1.64 vs. 0.89) and migration (cell index; 0.84 vs. 0.42) comparing with control PC-3 cells. Galectin-3 significantly suppressed anti-androgen effect induced by enzalutamide (PSA expression ratio; 0.47 vs. 0.15, p=0.005) or bicalutamide (PSA expression ratio; 0.49 vs. 0.26, p=0.001) in LNCaP comparing with controls by activating androgen transcriptional activity of AR. CONCLUSIONS These data indicate that galectin-3 is involved in the tumor progression and anti-androgen drug resistance of CRPC by regulating invasion, migration, and transcriptional activity of AR. These results suggest that galectin-3 is one of the target molecules for future treatments in CRPC. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e675 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomoharu Fukumori More articles by this author Tsogt-Ochir Dondoo More articles by this author Kei Daizumoto More articles by this author Tomoya Fukawa More articles by this author Yasuyo Yamamoto More articles by this author Kunihisa Yamaguchi More articles by this author Masayuki Takahashi More articles by this author Hiro-omi Kanayama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP46-16 CASTRATION-INDUCED ACCELERATION OF BONE METASTASIS PREVENTED BY RANK INHIBITOR OSTEOPROTEGERIN IN MURINE CASTRATION-RESISTANT PROSTATE CANCER MODEL

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2015MP46-16 CASTRATION-INDUCED ACCELERATION OF BONE METASTASIS PREVENTED BY RANK INHIBITOR OSTEOPROTEGERIN IN MURINE CASTRATION-RESISTANT PROSTATE CANCER MODEL Koichiro Takayama, Takamitsu Inoue, Shintaro Narita, Mingguo Huang, Yoko Mitobe, Hiroshi Tsuruta, Susumu Akihama, Mitsuru Saito, Norihiko Tsuchiya, and Tomonori Habuchi Koichiro TakayamaKoichiro Takayama More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Mingguo HuangMingguo Huang More articles by this author , Yoko MitobeYoko Mitobe More articles by this author , Hiroshi TsurutaHiroshi Tsuruta More articles by this author , Susumu AkihamaSusumu Akihama More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Norihiko TsuchiyaNorihiko Tsuchiya More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1579AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen deprivation therapy (ADT) decreases bone mineral density by stimulating RANK signaling in osteoclasts. It has been reported that the RANK inhibitor osteoprotegerin (OPG) prevents bone metastases in a murine prostate cancer model. However, the possibility of castration-induced acceleration of bone metastasis in castration-resistant prostate cancer (CRPC) has not been examined. Here, the effect of castration on bone mineral density and bone metastases was investigated using a murine CRPC model. METHODS Male Balb/c nude mice were divided into three groups: the noncastration group, the castration group, and the castration + OPG group. OPG was intravenously injected (2 mg/kg) twice a week in the castration + OPG group. A human CRPC cell line (PC3M–luc–C6), which is prone to the formation of bone metastases and has luciferase activity, was injected into the left ventricle of mice. Bone mineral density was measured by microcomputed tomography twice a week. Three weeks after injection into the ventricle, the number of mice with bone metastasis, the mean number of bone metastases, and mean photon counts of bone metastases were measured using the IVIS imaging system§ (Xenogen, Alameda, CA, USA). RESULTS Two weeks after castration, bone mineral density in the castration group was significantly lower than that in the noncastration group and the castration + OPG group (p = 0.034 and p = 0.010, respectively). The number of mice with bone metastases in the castration group was significantly higher than that in the noncastration group and the castration + OPG group (p = 0.024 and p = 0.028, respectively). The mean number of bone metastases and the mean photon counts of bone metastases in the castration group was significantly higher than those in the castration + OPG group (p = 0.020 and p = 0.041, respectively). CONCLUSIONS Castration reduced bone mineral density and accelerated bone metastasis in the murine CRPC model. OPG prevented both bone mineral loss and bone metastasis. The castration-induced acceleration of bone metastasis in CRPC could be caused by activation of osteoclasts due to RANK signaling. These results suggested that it is necessary to inhibit RANK signaling from the beginning of ADT to prevent bone metastasis in prostate cancer patients. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e549-e550 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Koichiro Takayama More articles by this author Takamitsu Inoue More articles by this author Shintaro Narita More articles by this author Mingguo Huang More articles by this author Yoko Mitobe More articles by this author Hiroshi Tsuruta More articles by this author Susumu Akihama More articles by this author Mitsuru Saito More articles by this author Norihiko Tsuchiya More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Establishment of a syngeneic orthotopic model of prostate cancer in immunocompetent rats.

We previously established 3 cell lines (PLS10, PLS20 and PLS30) from a chemically-induced prostate carcinoma in F344 rats, and demonstrated high potential for metastasis in nude mice. In the present study, we investigated the feasibility of establishing an orthotopic model using the 3 rat prostate cancer cell lines in immunocompetent rats with the aim of resolving species-mismatch problems and defects of immune systems. The PLS10, PLS20 and PLS30 cell lines were injected into the ventral prostates of 6-week-old rats, which were then sacrificed at experimental weeks 4 and 8. Tumor mass formation was found in rats with PLS10, but not in those with PLS20 or PLS30. Additionally, metastatic carcinomas could be detected in lymph nodes and lungs of PLS10-inoculated rats. Genetic analysis demonstrated K-ras gene mutations in PLS10 and PLS20, but not in PLS30 cells. There were no mutations in p53 and KLF6. In conclusion, we established a syngeneic orthotopic model for prostate cancer in immunocompetent rats simulating human castration-resistant prostate cancer (CRPC), which should prove useful for development and validation of therapeutic agents, especially with immunotherapy.