Androgen-independent Prostate Cancer Models Research Articles

Co-Targeting ErbB Receptors and the PI3K/AKT Axis in Androgen-Independent Taxane-Sensitive and Taxane-Resistant Human Prostate Cancer Cells.

Simple SummaryAdvanced prostate cancer that has progressed after standard therapies such as hormone therapy and taxane-based chemotherapies is an invariably lethal disease state with limited treatment options. There remains an important need to continue to identify new treatment approaches for such patients. We used two cell culture models of prostate cancer that are resistant to hormonal therapy and chemotherapy, and which also manifest some characteristics that are often associated with advanced prostate cancer, such as neuroendocrine differentiation, to evaluate the potential anti-cancer effects of targeting the key molecules, ErbB receptors and AKT. Using several complementary approaches, we found that the concurrent targeting of ErbB receptors and AKT with specific inhibitors was more effective than targeting each of them individually, independent of the underlying molecular characteristics or relative degrees of resistance to the taxanes that defined the prostate cancer models that were studied. Enhanced anti-tumor responses occurred both in vitro and in vivo with dual targeting, with the consistent inhibition particularly of AKT occurring in both settings. These studies provide a framework to evaluate the role of signal pathway modulation as a potential therapeutic strategy in treatment-refractory prostate cancer.Using two representative models of androgen-independent prostate cancer (PCa), PC3 and DU145, and their respective paclitaxel- and docetaxel-resistant derivatives, we explored the anti-tumor activity of targeting the ErbB receptors and AKT using small-molecule kinase inhibitors. These cells manifest varying degrees of neuroendocrine differentiation characteristics and differ in their expression of functional PTEN. Although the specific downstream signaling events post the ErbB receptor and AKT co-targeting varied between the PC3- and DU145-lineage cells, synergistic anti-proliferative and enhanced pro-apoptotic responses occurred across the wild-type and the taxane-resistant cells, independent of their basal AKT activation state, their degree of paclitaxel- or docetaxel-resistance, or whether this resistance was mediated by the ATP Binding Cassette transport proteins. Dual targeting also led to enhanced anti-tumor responses in vivo, although there was pharmacodynamic discordance between the PCa cells in culture versus the tumor xenografts in terms of the relative activation and inhibition states of AKT and ERK under basal conditions and upon AKT and/or ErbB targeting. The consistent inhibition, particularly of AKT, occurred both in vitro and in vivo, independent of the underlying PTEN status. Thus, co-targeting AKT with ErbB, and possibly other partners, may be a useful strategy to explore further for potential therapeutic effect in advanced PCa.

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

Abstract Metastatic castration resistant prostate cancer (mCRPC) is an advanced form of prostate cancer (PC) associated with poor prognosis. Approximately 20 - 40% of mCRPCs are androgen-independent and do not respond to treatment with abiraterone or enzalutamide, drugs that suppress androgen synthesis or directly target androgen ligand binding domains (LBD) respectively. Patients initially responding to these drugs eventually relapse, highlighting the need for alternative therapies. Resistance is attributed to the emergence of constitutively active AR splice variants lacking C-terminal LBDs such as AR variant 7. Although ARv7 activity cannot be mitigated by current therapeutic approaches, it is known that AR mRNA is exported from the nucleus by eIF4E and exportin-1 (XPO1). Selinexor and KPT-8602 are orally bioavailable SINE (Selective Inhibitor of Nuclear Export) compounds that specifically target XPO1. The purpose of this study is to provide mechanistic evidence for using SINE compounds as novel therapies for androgen-independent mCRPC. Methods: In vitro, selinexor or KPT-8602 were tested on androgen-independent PC cell line 22Rv1. RNA and protein were analyzed by qPCR and immunoblot. Cell viability was examined using the Celltiter-Glo assay. In vivo, immune deficient SCID mice were injected subcutaneously with 22Rv1 cells. Tumors were grown to ~150 mm3 before treatment with selinexor (10 mg/kg qodx3) or KPT-8602 (15 mg/kg reduce to 10 mg/kg qdx5). Tumor growth and animal weights were monitored to determine tumor growth inhibition (TGI) and tolerability to treatment. Tumors were analyzed using immunohistochemistry (IHC). Results: We found that expression of AR, ARv7 and prostate specific antigen (PSA; transcriptionally regulated by AR) proteins are reduced following 24-hour treatment with SINE compounds. Nuclear vs. cytoplasmic fractionation of RNA revealed that ARv7 and PSA mRNA localization was increased in the nucleus (4-fold and 3-fold, respectively) and reduced in the cytoplasm (5-fold and 3-fold, respectively). Moreover, KPT-8602 potently inhibited cell viability (IC50: 100 nM), while enzalutamide and abiraterone had no effect. Finally, mice bearing 22Rv1 xenografts, treated with selinexor or KPT-8602 exhibited a complete reduction in tumor volume (95% and 94% TGI, respectively), which coincided with prolonged overall survival. IHC analysis showed a reduction of proliferation markers and a concomitant increase in cell death makers in selinexor and KPT-8602 treated tumors. Conclusion: SINE compounds show strong anti-tumor activity in androgen-independent prostate cancer models in vitro and in vivo by reducing AR, ARv7, and PSA expression. These findings highlight the promise of SINE compounds as treatment options for androgen-independent mCRPCs resistant to first line therapies. Citation Format: Christian Argueta, Boris Klebanov, Trinayan Kashyap, Hua Chang, Sharon Friedlander, Erkan Baloglu, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. Disruption of nuclear export with selinexor or KPT-8602 reduces androgen receptor expression and leads to potent anti-tumor activity in preclinical models of androgen-independent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1587. doi:10.1158/1538-7445.AM2017-1587

Abstract 5080: BET inhibitors INCB054329 and INCB057643 display significant activity in androgen-independent prostate cancer models

Abstract Prostate cancer is a leading cause of cancer death worldwide. Deregulated transcriptional factors and epigenetic effectors contribute to prostate cancer progression and castration resistance providing ideal targets for developing new therapeutic strategies. Bromodomain and extra-terminal (BET) proteins act transcriptional co-activators interacting with multiple co-regulatory molecules at gene promoters and enhancers. BET inhibitors (BETi) disrupt transcriptional regulatory complexes and have broad anticancer activity. INCB054329 and INCB057643 are BETi with proven pre-clinical activity in hematological and solid tumors. Both compounds are currently in clinical trials (NCT02431260 and NCT02711137) in advanced cancer patients. In this study we explored for the first time the activity of these two BETi in prostate cancer, comparing their effects in androgen-dependent and independent models in vitro and in vivo. We assessed the effect of INCB054329 and INCB057643 on cell proliferation, colony formation and tumor-sphere assays in androgen-dependent (LNCaP and VCaP) and androgen-independent (DU145, PC3, 22Rv1) cells. The in vivo antitumor activity was evaluated in mice with 22Rv1 derived subcutaneous xenografts with drugs administered orally (BID, daily for 3 weeks). INCB054329 and INCB057643 inhibited proliferation of prostate cancer cell lines. In short-term cell proliferation assays the BETi appeared more effective against androgen-dependent (VCaP and LNCaP) than androgen-independent (DU145 and PC3) cells (GI50 of ≤100 nM and ≥500 nM, respectively). 22Rv1 cells, which express androgen-independent AR splice variants, exhibited an intermediate level of sensitivity to both compounds (GI50: 150-250 nM). Interestingly, in colony and tumor-sphere forming assays all the cell lines showed substantially greater sensitivity to the BETi than in short-term assays. Notably, androgen-independent 22Rv1 cells were highly responsive with GI50 values ≤100 nM similar to those seen in androgen-sensitive LNCaP cells. Treatment of mice bearing 22Rv1 tumor xenografts with INCB054329 (50 mg/kg) and INCB057643 (3 mg/kg) led to significant inhibition of tumor growth (T/C%: 42 and 45%, respectively) and consistent reduction of tumor weight relative to vehicle-treated mice. These results provide evidence of activity of INCB054329 and INCB057643 in prostate cancer cell lines. Although short term assays suggest a preferential anti-proliferative effect in androgen sensitive prostate cancer cells, both compounds also exhibit significant activity in an androgen-independent model both in vitro and in vivo suggesting that they might represent a valid therapeutic option for treatment of castration-resistant prostate cancer. Citation Format: Ramiro Vázquez, Gianluca Civenni, Martina Marchetti, Sabrina Zadic, Phillip Liu, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. BET inhibitors INCB054329 and INCB057643 display significant activity in androgen-independent prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2017-5080

Canine REIC/Dkk-3 interacts with SGTA and restores androgen receptor signalling in androgen-independent prostate cancer cell lines

BackgroundThe pathological condition of canine prostate cancer resembles that of human androgen-independent prostate cancer. Both canine and human androgen receptor (AR) signalling are inhibited by overexpression of the dimerized co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), which is considered to cause the development of androgen-independency. Reduced expression in immortalised cells (REIC/Dkk-3) interferes with SGTA dimerization and rescues AR signalling. This study aimed to assess the effects of REIC/Dkk-3 and SGTA interactions on AR signalling in the canine androgen-independent prostate cancer cell line CHP-1.ResultsMammalian two-hybrid and Halo-tagged pull-down assays showed that canine REIC/Dkk-3 interacted with SGTA and interfered with SGTA dimerization. Additionally, reporter assays revealed that canine REIC/Dkk-3 restored AR signalling in both human and canine androgen-independent prostate cancer cells. Therefore, we confirmed the interaction between canine SGTA and REIC/Dkk-3, as well as their role in AR signalling.ConclusionsOur results suggest that this interaction might contribute to the development of a novel strategy for androgen-independent prostate cancer treatment. Moreover, we established the canine androgen-independent prostate cancer model as a suitable animal model for the study of this type of treatment-refractory human cancer.

Paired box 2 upregulates androgen receptor gene expression in androgen-independent prostate cancer.

Androgen-independent prostate cancer is known as a hormone-refractory disease. Although the androgen receptor (AR) is considered to be a key regulator of androgen-independent prostate cancer progression, the mechanism through which AR gene expression is regulated is not well understood. In the present study, we showed that the AR gene was upregulated by paired box 2 (PAX2) in androgen-independent prostate cancer. When PAX2 upregulated AR gene expression, a decrease in DNA methylation of the AR gene locus was also observed. PAX2 was highly expressed and promoted cell growth in an androgen-independent prostate cancer cell line (22Rv1). The cell growth inhibition by PAX2 knockdown was rescued by AR overexpression in 22Rv1 cells. In a mouse xenograft model of androgen-independent prostate cancer, PAX2 knockdown inhibited tumor growth and AR gene expression and also increased DNA methylation of the AR gene. Consistent with this, AR and PAX2 expression levels were positively correlated in prostate cancer patients. These findings suggested that PAX2 promoted cancer cell growth in androgen-independent prostate cancer by regulating AR gene expression through an epigenetic mechanism.

GnRH-Gemcitabine Conjugates for the Treatment of Androgen-Independent Prostate Cancer: Pharmacokinetic Enhancements Combined with Targeted Drug Delivery

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone-Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake, and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely, GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG showed a significant advantage in tumor growth inhibition when compared to gemcitabine.

Abstract 2126: Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of androgen independent prostate cancer progression.

Abstract Introduction: Prostate cancer (PCa) is the second most common cause of death from cancer and the leading cancer diagnosed in the US men. Approximately 1 in 6 American men will be diagnosed with prostate cancer during his lifetime. Estimated new cases and deaths from prostate cancer in the United States for 2012 include: 241,740 new cases and 28,710 deaths. Initial treatment for metastatic PCa involves androgen ablation therapy, which causes regression of androgen-dependent tumors. However they all eventually relapse resulting in recurrent androgen independent prostate cancer (AIPC). Methods: This study aims at assessing the role of Hedgehog (Hh) and Androgen receptor (AR) signaling in the development of AIPC. Quantitative RT-PCR analysis was performed for AIPC cells in vitro and for AIPC xenograft after castration, to assess the levels of Hh and AR signaling components. Hh inhibitors, LDE225 (Novartis) and Cyclopamine, and Androgen receptor inhibitor Pyrvinium pamoate were tested individually and in combination to assess the suppression of AIPC progression both in vitro and in vivo. AR-responsive and Gli-responsive promoter-luciferase assay and MTT assay were used for in vitro studies. Intraperitoneal administration of drugs and bioluminescence imaging for monitoring orthotopic tumors in mice were used to study the effect of drugs on the growth of AIPC in vivo. Results: We demonstrate that androgen deprivation leads to increase in Hedgehog (Hh) signaling components in the AIPC cells in vitro and in vivo. AIPC cells also show increased sensitivity to low concentrations of androgen, suggesting increased androgen receptor expression. Together, these results suggest that increased Hh signaling and androgen receptor expression in the absence of androgen agonist plays an important role in the development and progression of AIPC. Our results show that inhibiting either Hh or androgen receptor signaling pathway individually was not effective in preventing AIPC cell proliferation in vitro as well as in inhibiting xenograft tumor growth in mice. In contrast, combination of an Hh pathway inhibitor with an androgen receptor inhibitor synergistically suppressed the growth of AIPC cells in vitro as assessed by MTT assay and reduced tumor burden in a mouse xenograft model of AIPC as compared to individual treatments.Conclusion: Upregulated Hh signaling and AR expression mediates development and progression of AIPC. Combined inhibition of Hh and androgen receptor signaling pathways may be a novel effective therapeutic strategy for the treatment of androgen independent prostate cancer. Citation Format: pramod gowda, Jianhong Deng, Sweta Mishra, LuZhe Sun. Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of androgen independent prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2126. doi:10.1158/1538-7445.AM2013-2126

Abstract 5744: Preclincial therapy of androgen independent prostate cancer using combinationLu-177-BB2r targeting peptides and docetaxel

Abstract Introduction: The bombesin receptor subtype 2 (BB2r) is an attractive target for the selective delivery of peptide receptor targeted radiation therapy for androgen independent prostate cancer (AIPC). Earlier work from our group has focused on the use of the BB2r agonist 177Lu-177-DOTA-8-AOC-BBN(7-14)NH2 administered concurrently with docetaxel to achieve tumor control in flank xenograft models of AIPC. We now present a direct comparison of 177Lu-BB2r agonist verses 177Lu-BB2r antagonist demonstrating the superiority of employing the BB2r antagonist, RM2, for the treatment of AIPC using a SCID mouse model. Methods: DOTA-8-AOC-BBN(7-14)NH2 was synthesized in house. RM2 was commercially synthesized. 177Lu radiolabeled peptides were prepared and purified using analytical HPLC resulting in a high specific activity preparation. The AIPC tumor bearing mice were created following bilateral flank inoculation of 1X106 PC-3 cells. Pharmacokinetic analysis and Micro-SPECT imaging of 177Lu-RM2 was conducted out to 14 days post administration. Survival study design compared a 177Lu-BB2r agonist /docetaxel arm with a 177Lu-BB2r antagonist /docetaxel arm including a non-treated tumor bearing control arm. Multi-modal therapy consisting of docetaxel (8mg/kg; administered weekly) and 177Lu-BB2r peptide therapy (37MBq/25g; administered weekly) was delivered for six consecutive weeks. Subjects were removed from study based on evidence of morbidity and body condition score. Results: Pharmacokinetic analysis and Micro-SPECT imaging of 177Lu-RM2 demonstrated prolonged tumor retention with minimal non-target tissue accumulation observed within 24 hours post administration. Survival analysis at Day 100 demonstrated that 67% of the mice in the multi-modal BB2r antagonist therapy arm (chemo + targeted 177Lu radiation therapy) were responding to treatment as compared to a 17% survival of the combination BB2r agonist/chemotherapy arm only arm at the same time point. 177Lu-RM2 antagonist therapy demonstrated tumor regression and complete control whereas 177Lu agonist therapy tumor regression was short lived with complete regrowth observed 45 days following last treatment. Conclusions: Our preclinical data suggests that 177Lu-BB2r antagonist therapy is superior to 177Lu-BB2r agonist therapy when used concurrently with docetaxel employing a preclinical model of AIPC. The use of 177Lu-BB2r antagonists permits accurate SPECT imaging of therapeutic agent localization at prolonged times post injection. More extensive evaluation of 177Lu-BB2r antagonist therapy for AIPC are warranted to exploit the potential of these agents for clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5744. doi:1538-7445.AM2012-5744

Differential effects of PXD101 (belinostat) on androgen-dependent and androgen-independent prostate cancer models

Histone deacetylase inhibitors (HDACi) are promising epigenetic cancer chemotherapeutics rapidly approaching clinical use. In this study, we tested using in vitro and in vivo models the differential biological effects of a novel HDAC inhibitor [belinostat (PXD101)], in a wide panel of androgen-sensitive and androgen-independent tumor cells. Belinostat significantly increased acetylation of histones H3 and H4. Belinostat potently inhibited the growth of prostate cancer cell lines (IC50 range from 0.5 to 2.5 µM) with cytotoxic activity preferentially against tumor cells. This agent induced G2/M arrest and increased significantly the percentage of apoptosis mainly in androgen-sensitive tumor cells confirming its growth-inhibitory effects. The cell death mechanisms were studied in three different prostate cancer cell lines with different androgen dependence and expression of androgen receptor; LAPC-4 and 22rv1 (androgen-dependent and expressing androgen receptor) and PC3 (androgen-independent not expressing androgen receptor). Belinostat induced the expression of p21 and p27, acetylation of p53 and G2/M arrest associated with Bcl2 and Bcl-Xl downmodulation and significant reduction of survivin, IAPs and Akt/pAkt and increased caspase-8 and -9 expression/activity. Belinostat effectiveness was dependent on the androgen receptor (AR), since the stable transfection of AR greatly increased the efficacy of this HDAC inhibitor. These observations were correlated using in vivo models. We demonstrated that belinostat preferentially resulted in antitumor effect in androgen-dependent tumor cells expressing AR. Our findings provide evidence that belinostat may be a promising anticancer drug for prostate cancer expressing AR, supporting its clinical role in prostate cancer.

Xenotropic murine leukemia virus-related virus (XMRV) in prostate cancer cells likely represents a laboratory artifact

The prevalence of xenotropic murine leukemia virus-related virus (XMRV) in human population and its involvement in prostate cancer are subjects of ongoing research and debate. 22Rv1, which is a human cell line that serves as a common model of androgen-independent prostate cancer, was recently reported to carry infectious copies of XMRV. 22Rv1 was derived from a prostate cancer xenograft CWR22 that was serially passaged in immunodeficient mice. Based on the analysis of the DNA from CWR22 and 22Rv1, we present evidence against the presence of XMRV in CWR22 and, by inference, the tumor, from which CWR22 and 22Rv1 were established. While the presence of XMRV in 22Rv1 is likely to be an artifact, it may be a significant factor in determining the biological properties of this cell line. This consideration warrants additional caution for the interpretation of the relevance of the studies, which utilize this popular cell line as a model. It also invites a closer look at the sources of viral contamination in xenografts and cultured cells, as well as in the experiments that allege the presence of this virus in human cells and populations.

Evaluation of Lu-177 RM2 as a targeted radiopharmaceutical in a PC-3 xenograft model of androgen-independent prostate cancer.

e13522 Background: Prostate cancer is the most common cancer in American men (after nonmelanoma skin cancers). In 2010 more than 200,000 cases were diagnosed and more than 30,000 men died of prostate cancer. One in six men will be diagnosed with prostate cancer during his lifetime and one in thirty six men will die of prostate cancer. Human prostate cancer has been shown to widely express the bombesin receptor (BB2R). Lutetium-177 (177 Lu) radiolabeled BB2r agonist analogs are currently being investigated preclinically for their utility as targeted radiation therapy agents. (Maddalena, J Nucl Med, 50:2017, 2009 and Johnson, Can Biother & Radiopharm, 21(2):155, 2006) Recently, Maecke and co-workers identified a high affinity BB2r antagonist (Ga-68 and In-111-RM2) for potential use as a PET/SPECT diagnostic imaging agent. (Mansi, Clin Can Res, 15(16):5240, 2009 and Mansi, Eur J Nucl Med Mol Imaging, May 2010) The purpose of this work was to investigate the biodistribution of Lu-177-RM2 for potential as a radiotherapeutic agent using a PC-3 xenografted SCID mouse model. Methods: Male SCID mice were inoculated subcutaneously with 5 million PC-3 cells/site in 100uL. At 4 weeks post-inoculation, pharmacokinetic studies of 177Lu-VA RM2 were conducted at 1, 4, 24, 48, 72, and 96 hours post-injection. Micro-SPECT/CT imaging studies were done at 24, 48, 72, and 96 hrs. Results: See table; shown in %ID/g. Conclusions: Our study demonstrates that Lu-177-RM2 shows promising pharmacokinetic properties with high affinity for the BB2 receptor and excellent tumor to blood and tumor to organ ratios. The prostate tumor uptake of Lu-177-RM2 is equal or greater than any previously identified BB2r targeting radiotherapeutic agent. Lu-177-RM2 exhibits rapid washout from the pancreas and other non-target organs making it an ideal candidate for tumor targeted therapy research. 1 HR 4 HRS 24 HRS 48 HRS 72 HRS 96 HRS Blood 0.36 +/- 0.15 0.02 +/- 0.01 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 Pancreas 17.53 +/- 1.97 1.59 +/- 0.7 0.26 +/- 0.03 0.18 +/- 0.04 0.19 +/- 0.03 0.03 +/- 0.00 Tumor 9.25 +/- 3.35 11.46 +/- 5.38 5.56 +/- 1.59 5.28 +/- 1.42 4.03 +/- 0.92 3.14 +/- 0.33

A preclinical therapeutic schedule optimizing docetaxel plus estramustine administration in prostate cancer

Androgen-dependent and castration-resistant prostate cancer (PC) is usually sensitive to docetaxel chemotherapy. Nevertheless, docetaxel resistance frequently appears after several cycles of treatment, raising the problem of salvage treatment for docetaxel-resistant PC patients. Although the combination of docetaxel and estramustine prolongs metastasis-free and overall survival of patients with androgen-independent PC, the use of this modality remains limited in elderly patients or patients with several comorbidities, especially vascular disease or gastrointestinal toxicity, because of unacceptable toxicity including venous thrombosis. The aims of this study were therefore (i) to evaluate the in-vivo efficacy of estramustine combined with docetaxel since initial tumor growth and following the appearance of docetaxel resistance in the androgen-dependent human PC xenograft PAC120, and (ii) to evaluate the efficacy of estramustine in six human androgen-independent PC models derived from PAC120. In docetaxel-resistant tumor-bearing mice, estramustine alone induced a TGD2 of 18 days, whereas the combination of docetaxel and estramustine induced a TGD2 of 50 days (P<0.05) with no significantly different overall survival of mice treated by docetaxel and estramustine since day 1 or since the onset of resistance to docetaxel. Among the six human androgen-independent tumors treated with estramustine alone, two highly sensitive models, two intermediate responding tumors, and two resistant models were observed. Altogether, these results suggest that estramustine should be combined with docetaxel in PC patients, but the use of this treatment could be limited, particularly in elderly patients, to docetaxel-resistant cases.

Ozarelix, a fourth generation GnRH antagonist, induces apoptosis in hormone refractory androgen receptor negative prostate cancer cells modulating expression and activity of death receptors

Antagonistic or agonistic analogues of gonadotropin-releasing hormone are extensively used for the treatment of advanced hormone-dependent prostate cancer. However, the majority of recurrent prostate tumors is androgen independent. This study explored the in vitro effects on DU145 and PC3 cell lines, two models of androgen-independent prostate cancer, of a fourth generation GnRH antagonist (Ozarelix). Ozarelix was added to cultures and toxicity, cell cycle modifications, cell viability and caspase activity were investigated. Ozarelix showed antiproliferative effects and produced an accumulation of cells in G2/M cell cycle phase. Apoptosis was related with caspase-8-dependent caspase 3 activation with down-regulation of c-FLIP (L) and a sensitization to TRAIL-induced apoptosis linked also to increased expression and activity of death receptors DR4/5 and Fas. TRAIL-resistant cancer cells can be sensitized to TRAIL by Ozarelix. This effect may be achieved by the activation of apoptotic pathway improving the therapeutic effects in androgen independent tumor cell lines. However, a better understanding of molecular mechanisms by which GnRH antagonists may act in androgen independent models is necessary.

Correction for Gao et al., Combinatorial activities of Akt and B-Raf/Erk signaling in a mouse model of androgen-independent prostate cancer

Correction for Gao et al., Combinatorial activities of Akt and B-Raf/Erk signaling in a mouse model of androgen-independent prostate cancer

Inhibitory Effects of Castration in an Orthotopic Model of Androgen-Independent Prostate Cancer Can Be Mimicked and Enhanced by Angiogenesis Inhibition

Today, the most important treatment of advanced prostate cancer is castration; unfortunately, however, the long-term effect of this therapy is insufficient. Recent studies suggest that castration-induced prostate involution could be caused by primary effects in the prostate vasculature; therefore, we examined if antivascular treatments could mimic the effects of castration. Androgen-independent AT-1 prostate cancer cells were grown inside the ventral prostate in adult rats. Tumor-bearing animals were treated with an inhibitor of vascular endothelial growth factor receptor 2 and epidermal growth factor receptor signaling, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine (ZD6474, AstraZeneca, Södertälje, Sweden), and short-term effects (after 3 days) were compared with those induced by castration. Castration caused decreased vascular density in the normal tissue surrounding the tumor and consequently increased tumor hypoxia and apoptosis, and moderately decreased tumor growth. ZD6474 treatment resulted in decreased tumor vascular density accompanied by increased tumor hypoxia, apoptosis, and decreased tumor growth, suggesting that castration and antiangiogenic therapy work through similar mechanisms. Interestingly, castration or ZD6474 alone worked by reducing vascular density in the surrounding normal tissue and ZD6474 also in the tumor. Combined treatment with castration + ZD6474 was more effective than castration and ZD6474 alone in inducing tumor hypoxia, apoptosis, necrosis, and decreasing tumor vascular density. These findings show that a drug that targets the vasculature in the tumor and in the surrounding ventral prostate lobe could mimic and even enhance the effects of castration. Our present findings thus suggest that castration + ZD6474 could be a particularly effective way to treat prostate tumors.

Combined In vivo Effect of A12, a Type 1 Insulin-Like Growth Factor Receptor Antibody, and Docetaxel against Prostate Cancer Tumors

A human type 1 insulin-like growth factor receptor antibody (A12) has been shown to effectively inhibit human xenograft tumor growth, including androgen-dependent and androgen-independent prostate tumors. Docetaxel, either as a single agent or combined with others, has shown a survival benefit in prostate cancer patients. Based on these data, we investigated the combined in vivo effect of A12 and docetaxel on human androgen-independent and osseous prostate tumor growth. To study human androgen-independent prostate cancer model, LuCaP35V tumors were implanted s.c. into castrated severe combined immunodeficient mice. When tumors reached about 100 mm3, animals were treated with vehicle control docetaxel (10 or 20 mg/kg) and docetaxel in combination with A12 (40 microg/kg) for 4 weeks. To study human osseous prostate cancer model, LuCaP 23.1 tumors were implanted intratibiae. When serum prostate-specific antigen reached 5 to 10 ng/mL, treatments were initiated. A12 markedly augmented the inhibition of docetaxel on tumor growth. When docetaxel is combined with A12, the inhibition of tumor growth continued after treatment cessation, which was associated with continued apoptosis and decreased proliferation of tumor cells. Gene expression profiles indicated that the posttreatment suppression of tumor growth may be due to enhanced negative regulation of cell cycle progression- and/or cell survival-associated genes, some of which have been shown to induce resistance to docetaxel. Our findings suggest that targeting type 1 insulin-like growth factor receptor can enhance the therapeutic effect of docetaxel on advanced prostate cancer. Our findings also suggest a potential mechanism to improve the treatment efficacy of docetaxel in prostate cancer.

Dose-escalated abarelix in androgen-independent prostate cancer: a phase I study

Follicle-stimulating hormone has been shown to be a mitogen in preclinical models of androgen-independent prostate cancer and abarelix has been previously shown to significantly reduce follicle-stimulating hormone levels in patients when administered monthly. Consequently, we evaluated the safety of more frequent (biweekly) dosing of abarelix and characterized the effect of this dosing schedule on serum follicle-stimulating hormone levels in men with prostate cancer that is progressing despite luteinizing hormone-releasing hormone agonist therapy. Twenty-one patients with prostate cancer progressing on gonadotropin-releasing hormone agonist therapy discontinued the gonadotropin-releasing hormone agonist and received abarelix-depot 100 mg by intramuscular injection every 2 weeks for up to 12 weeks. Safety profile and effect on serum follicle-stimulating hormone were the primary end-points, while prostate-specific antigen response was a secondary end-point. Abarelix therapy was generally well tolerated. One patient experienced an acute immediate allergic reaction. The mean follicle-stimulating hormone serum concentration declined from 3.5 mIU/ml (95% confidence interval: 2.7-4.3) to 2.0 mIU/ml (95% confidence interval: 1.3-2.6) on day 57 and to 2.0 mIU/ml (95% confidence interval: 0.9-3.0) on day 85 (P=0.008 in a Kruskal-Wallis test), but no patient's follicle-stimulating hormone has reached the lower limit of quantitation (below 0.15 mIU/ml). No patient met criteria for prostate-specific antigen response. At the end of 12 weeks of therapy, three (14.3%) patients had no change in prostate-specific antigen levels on days 57 and 85 compared with baseline. Twelve patients (57%) had stable disease throughout treatment defined as percent change from baseline within -50 to 50% at a given time-point confirmed by a second measurement at least 4 weeks later. Treatment with biweekly abarelix in patients with androgen-independent prostate cancer is feasible with no unexpected toxicity, but fails to completely suppress serum follicle-stimulating hormone levels or produce prostate-specific antigen responses.

Combinatorial activities of Akt and B-Raf/Erk signaling in a mouse model of androgen-independent prostate cancer

Androgen independence is responsible for most prostate cancer lethality, yet currently there are no effective clinical treatments. We have been investigating the mechanisms underlying androgen-independent prostate cancer in Nkx3.1;Pten mutant mice, which display salient features of the disease, including a requirement for wild-type androgen receptor (AR) signaling. We now demonstrate that the Akt and Erk MAP kinase signaling pathways are activated in androgen-independent lesions of these mice. Forced activation of either Akt or Erk signaling in an androgen-responsive prostate cancer cell line promotes hormone-independent but AR-dependent growth in culture. Although these pathways act additively in culture, they act synergistically in vivo to promote tumorigenicity and androgen independence in the context of the prostate microenvironment. We propose that androgen independence emerges by means of epithelial-stromal competition, in which activation of Akt and Erk promotes AR activity in the prostate epithelium while counteracting antagonistic effects of the stroma.

Patupilone (epothilone B, EPO906) inhibits growth and metastasis of experimental prostate tumors in vivo

Microtubule agents appear promising for the treatment of prostate cancer. Patupilone (epothilone B), a highly potent non-taxane microtubule stabilizing agent, was evaluated in models of androgen-independent prostate cancer. Patupilone was administered to athymic mice bearing human prostate cancer xenografts (subcutaneous DU 145 and PC-3M, orthotopic PC-3M). One 4 mg/kg patupilone administration produced transient regression of DU 145 tumors, while two weekly administrations of 2.5 mg/kg produced stable disease followed by protracted regression, however with more pronounced body weight loss. Taxol (15 mg/kg every other day) weakly inhibited tumor growth, but with less body weight loss. Patupilone (5 mg/kg) produced protracted growth inhibition of subcutaneous PC-3M tumors, with transient body weight loss. In mice with orthotopic PC-3M tumors, 4 or 5 mg/kg/week patupilone impaired primary tumor growth, abrogated metastases and enhanced survival, with only transient body weight loss. These data suggest that patupilone holds promise for prostate cancer treatment.

Inhibitory Effect of Antagonists of Bombesin and Growth Hormone-Releasing Hormone on Orthotopic and Intraosseous Growth and Invasiveness of PC-3 Human Prostate Cancer in Nude Mice

To determine whether antagonists of growth hormone-releasing hormone (GHRH) and bombesin/gastrin-releasing peptide (BN/GRP) can inhibit the orthotopic and metastatic growth of PC-3 human androgen-independent prostate cancers. The effects of administration of GHRH antagonist MZ-J-7-118, BN/GRP antagonist RC-3940-II, and their combination on the growth and metastatic spread of PC-3 tumors implanted orthotopically into nude mice were evaluated. The efficacy of this treatment on PC-3 tumors implanted intratibially and s.c. was also determined. Treatment with MZ-J-7-118, RC-3940-II, or their combination significantly inhibited the growth of PC-3 tumors implanted orthotopically, intraosseously, and s.c. The combination of the two antagonists had the greatest effect, inhibiting orthotopic tumor growth by 77%, intratibially implanted tumors by 86%, and s.c. tumors by 86%. The therapy with BN/GRP and GHRH antagonists, especially in combination, also reduced the local tumor spread and distant metastases in animals bearing orthotopic tumors. Combination therapy was likewise the most effective in reducing the incidence and severity of tibial osteolytic lesions and pathologic fractures in intraosseously implanted tumors. High-affinity binding sites for BN/GRP and GHRH were found in s.c. and orthotopic PC-3 tumor samples. MZ-J-7-118, RC-3940-II, and the combination of both compounds inhibited in vitro growth of PC-3 cells. Our findings show the efficacy of BN/GRP antagonists and GHRH antagonists for the treatment of advanced prostate cancer in preclinical metastatic models. As BN/GRP antagonists are already in clinical trials and GHRH antagonists are effective in androgen-independent prostate cancer models, these analogues could be considered for the management of advanced prostate carcinoma.