Castration-resistant Prostate Cancer Models Research Articles

Abstract 4700: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer

Abstract Background: While treatment options for patients with castration-resistant prostate cancer (CRPC) are expanding, one American man is still predicted to die every 19 minutes from this disease this year. Moreover, more widespread use of novel and more potent AR-targeting agents has led to increased clinical frequency of virulent androgen and AR-independent CRPC subsets(Small, Huang et al. 2015). Currently, there are limited treatment options for men with CRPC who are resistant to these AR-targeting agents, clearly demonstrating an urgent need to develop more effective therapies for CRPC patients. Recent published reports demonstrate an important role for BET bromodomain chromatin reader proteins in prostate cancer models that are androgen or AR-dependent (Gao, Schwartzman et al. 2013; Wyce, Degenhardt et al. 2013; Asangani, Dommeti et al. 2014; Chan, Selth et al. 2015). However, there was limited information about the anti-tumor activity of this class of drugs in androgen-independent, enzalutamide-resistant, or AR-independent CRPC models. The studies reported herein were designed to address that deficit. Methods: We treated a panel of CRPC cell lines with dose escalation of the BET bromodomain inhibitor JQ1 and measured cell viability. To clarify molecular mechanisms that contribute to the anti-tumor effect, we treated CRPC cell lines with JQ1 and measured gene expression changes with RNA-sequencing. Finally, to determine the anti-tumor activity of JQ1 in vivo, we implanted enzalutamide resistant or AR-null CRPC xenografts in immunocompromised mice and treated them with JQ1. Results: All cell lines were sensitive to JQ1 with similar GI50 values (all < 1μM). Our analysis of RNA-seq data after JQ1 treatment identified a BET bromodomain response signature that was conserved across the population of cell lines. Further, we applied Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to identify novel Master Regulator transcription factors that mediate response to JQ1(Aytes, Mitrofanova et al. 2014). Finally, JQ1 treatment of enzalutamide resistant or AR-null CRPC xenografts implanted in vivo suppressed tumor growth without appreciable toxicity. Conclusions: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal CRPC. Our work using a broad panel of CRPC models sheds further light on pharmacodynamic markers of response and target proteins whose function is impacted by BET bromodomain inhibition. These findings may have implications for the design of BET bromodomain inhibitor clinical trials in men with CRPC and interpretation of on-target effects in those trials. Citation Format: Lina Gao, Daniel J. Coleman, Carly J. King, Jacob Schwartzman, Nicholas Wang, Amanda Esch, Joshua Urrutia, Archana Sehrawat, Laura M. Heiser, Joshi J. Alumkal. BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4700.

Abstract 4963: A distinct AMP-activated protein kinase phosphorylation site is associated with metastasis and castration-resistance in prostate cancer

Abstract AMP-activated protein kinase (AMPK) is a critical regulator of cellular metabolism and plays important role in the development and progression of several human diseases including cancer. We have previously shown that AMPKα activity is significantly inhibited by Ser-485/491 phosphorylation in cell culture and in vivo models of metastatic and castration-resistant prostate cancer. To determine potential translatability, we investigated AMPKα-Ser-485/491 phosphorylation status in clinical prostate cancer specimens and its correlation with metastasis and progression to castration-resistance. Metastasis and matched primary tumor specimens from 45 metastatic prostate cancer patients, 31.1% of which were post-androgen deprivation therapy failure (castration-resistant prostate cancer), and primary tumor specimens from 30 non-metastatic, hormone-dependent prostate cancer patients with undetectable PSA for a mean of 97.1 ± 17.5 months were obtained from the University Hospitals Case Medical Center Pathology Archive. Slides were cut from paraffin blocks, immunostained for p-Ser-485/491 AMPKα and total AMPKα, analyzed by a pathologist, and statistical significance determined by T-test, Kruskal-Wallis test, Fisher's exact test, or Chi-Square test. Mean pre-diagnostic biopsy PSA was 6.5 ng/mL (range 1.20-25.3 ng/mL) in non-metastatic patients and 501.9 ng/mL (range 1.82-3200 ng/mL) in metastatic patients (p < 0.0001). Mean total Gleason score of primary tumors was 6.5 in the non-metastatic group and 8.2 in the metastatic group (p < 0.0001); primary prostate tumor volume (mean ± S.D.) was 22.1 ± 19.5% in the non-metastatic group and 68.1 ± 28.7% in the metastatic group (p < 0.0001). The intensity of overall p-Ser-485/491 AMPKα staining and the number of p-Ser-485/491 AMPKα positively-stained cells was significantly increased in primary prostate tumors (p = 0.0003 and p = 0.0008, respectively) and metastases (p = 0.0011 and p = 0.0062, respectively) of metastatic prostate cancer patients when compared to staining of primary tumor specimens from non-metastatic prostate cancer patients. Further, the intensity of overall p-Ser-485/491 AMPKα staining increased 2.21-fold (p = 0.0050) and the number of p-Ser-485/491 AMPKα positively-stained cells increased 1.77-fold (p = 0.0041) in castration-resistant metastatic prostate cancer specimens compared to androgen-dependent prostate cancer specimens. Therefore, AMPKα Ser-485/491 phosphorylation is associated with metastasis and castration-resistance in clinical prostate cancer and may be a novel target for treatment. Citation Format: Melissa A. Babcook, Mahmut Akgul, Seunghee Margevicius, Gregory T. MacLennan, Pingfu Fu, Robert Abouassaly, Sanjay Gupta. A distinct AMP-activated protein kinase phosphorylation site is associated with metastasis and castration-resistance in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4963.

Abstract 4327: Dietary tomato reduces castration-resistant prostate cancer progression in TRAMP mice

Abstract Epidemiological data support the hypothesis that consumption of diets rich in tomato products are associated with a reduced risk of prostate cancer. Castration-resistant prostate cancer (CRPC) represents the late and lethal phase of human prostate carcinogenesis, and is defined as cancer progression after surgical castration or pharmacologic reduction of serum testosterone through androgen deprivation therapy. Mechanisms involved in the transition to CRPC include cancer cells’ acquired capacity for androgen synthesis or activation of ligand-independent signaling. We have previously shown that dietary tomato can inhibit prostatic androgen signaling and expression of androgen biosynthetic genes, as well as reduce primary cancer incidence in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Therefore, we examined whether dietary tomato might be an effective inhibitor of CRPC progression in mice. We hypothesized that lifelong dietary intake of tomato, as well as dietary tomato intervention following castration, would reduce tumor burden and growth rate in a mouse model of CRPC. TRAMP mice were fed an AIN-93G diet (CON) for one week and then randomized to the CON diet (n = 28) or a similar diet with 10% w/w lyophilized tomato paste (TP; n = 27) from 4 wks of age until euthanization. A third group, modeling dietary intervention, consumed CON from 4 to 12 wks of age, and the 10% w/w lyophilized tomato paste diet from wk 12 until euthanization (TP-I; n = 25). All animals were castrated at 12 wks of age. Beginning at 10 wks of age, mice were monitored longitudinally with ultrasound for tumor detection, tumor volume estimation, and calculation of tumor growth rate. Serial 2D image slices were used to generate a 3D volume estimate. Mice were euthanized after 5 consecutive tumor scans, or if no tumor had been detected by 30 weeks of age. Tumor growth area under the curve (AUC) was reduced approximately 46% by tomato intervention (TP-I) and 27% by lifelong tomato consumption (TP). At euthanization, TP-I reduced tumor weight by approximately 26%. Additionally, incidence of gross metastases to distant organs (lung, liver, kidney) was strongly inhibited by TP-I, compared to CON (0% and 26%, respectively). TP diet reduced incidence of distant organ gross metastasis to <10%. The consumption of tomato products following castration inhibits CRPC progression in the TRAMP model. Our findings support the development of human studies of dietary tomato interventions in combination with anti-androgen therapy for men with advanced prostate cancer. Citation Format: Joshua W. Smith, Joe L. Rowles, Rita J. Miller, Steven K. Clinton, William D. O’Brien, John W. Erdman. Dietary tomato reduces castration-resistant prostate cancer progression in TRAMP mice. [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 4327.

Abstract 2192: Fusogenic targeted liposomes as next-generation nanomedicine for prostate cancer

Abstract Metastatic or castrate-resistant is the second-leading cause of cancer mortality in males. In the past 3 years, chemotherapies have extended survival, but efficacy is limited by dose-limiting toxicities due to suboptimal biodistribution. We address the lack of specificity and accumulation in Castrate Resistant Prostate Cancer (CRPC) by developing a unique nano-carrier for drug delivery: targeted fusogenic liposomes. We have developed a platform whereby liposomes are formulated with fusion associated small transmembrane protein p14 displaying targeting ligands. The p14 protein catalyzes mixing of the liposomal bilayer with cell membranes to deliver the cargo directly into the cytoplasm, while the targeting ligand bombesin, allows for targeting to the Gastrin-releasing Peptide (GRPR) that is overexpressed in prostate cancer. We hypothesized that this novel targeted fusogenic liposome formulation would significantly improve the biodistribution and efficacy of chemotherapy. A clinical liposomal doxorubicin formulation (DOXIL) was modified to incorporate targeted fusogenic p14 protein. We then evaluated the efficacy and biodistribution of these new formulations using in vitro and in vivo models of CRPC. In PC3 cells, compared to conventional liposomes, intracellular levels of doxorubicin are increased by 15 and 25 times when p14 or p14-bombesin liposomes are used. Additionally, the IC50 is reduced from 85mM to 2mM. In mice bearing PC3 tumors treated with targeted fusogenic liposomal doxorubicin, we observed tumor growth inhibition of 57% (vs control). This establishes a proof of concept for an innovative targeted drug delivery system that may improve the outcome of patients with CRPC by enhancing the effect of approved drugs. Citation Format: Jihane M. Mriouah, Rae Lynn Nesbitt, Deborah Sosnowski, Desmond Pink, Roy Duncan, Andries Ziljstra, John D. Lewis. Fusogenic targeted liposomes as next-generation nanomedicine for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2192.

Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD(+)-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen-dependent versus castration-resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration-resistant tumor cells expressed higher levels of UGDH, UGT2B15, and UGT2B17. However, the magnitude of androgen-activated UGDH and prostate-specific antigen (PSA) expression, as well as the androgen receptor (AR)-dependent repression of UGT2B15 and UGT2B17, was blunted several-fold in these cells. Consistent with these results, the ligand-activated binding of AR to the PSA promoter and subsequent transcriptional activation were also significantly reduced in castration-resistant cells. Analysis of the UDP-sugar pools and flux through pathways downstream of UDP-glucuronate production revealed that these glucuronidation precursor metabolites were channeled through proteoglycan and glycosaminoglycan biosynthetic pathways, leading to increased surface expression of Notch1. Knockdown of UGDH diminished Notch1 and increased glucuronide output. Overall, these results support a model in which the aberrant partitioning of UDP-glucuronate and other UDP-sugars into alternative pathways during androgen deprivation contributes to the loss of prostate tumor cell androgen sensitivity by promoting altered cell surface proteoglycan expression.

PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer

Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.

Cellular androgen content influences enzalutamide agonism of F877L mutant androgen receptor.

Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation.

Restoration of the cellular secretory milieu overrides androgen dependence of in vivo generated castration resistant prostate cancer cells overexpressing the androgen receptor

It is believed that growth of castration resistant prostate cancer (CRPC) cells is enabled by sensitization to minimal residual post-castrate androgen due to overexpression of the androgen receptor (AR). Evidence is derived from androgen-induced colony formation in the absence of cell-secreted factors or from studies involving forced AR overexpression in hormone-dependent cells. On the other hand, standard cell line models established from CRPC patient tumors (e.g., LNCaP and VCaP) are hormone-dependent and require selection pressure in castrated mice to re-emerge as CRPC cells and the resulting tumors then tend to be insensitive to the androgen antagonist enzalutamide. Therefore, we examined established CRPC model cells produced by castration of mice bearing hormone-dependent cell line xenografts including CRPC cells overexpressing full-length AR (C4-2) or co-expressing wtAR and splice-variant AR-V7 that is incapable of ligand binding (22Rv1). In standard colony formation assays, C4-2 cells were shown to be androgen-dependent and sensitive to enzalutamide whereas 22Rv1 cells were incapable of colony formation under identical conditions. However, both C4-2 and 22Rv1 cells formed colonies in conditioned media derived from the same cells or from HEK293 fibroblasts that were proven to lack androgenic activity. This effect was (i) not enhanced by androgen, (ii) insensitive to enzalutamide, (iii) dependent on AR (in C4-2) and on AR-V7 and wtAR (in 22Rv1) and (iv) sensitive to inhibitors of several signaling pathways, similar to androgen-stimulation. Therefore, during progression to CRPC in vivo, coordinate cellular changes accompanying overexpression of AR may enable cooperation between hormone-independent activity of AR and actions of cellular secretory factors to completely override androgen-dependence and sensitivity to drugs targeting hormonal factors.

Dietary Tomato Reduces Castration‐Resistant Prostate Cancer Burden in the TRAMP Model

Epidemiological evidence continues to support the hypothesis that consumption of diets rich in tomato products are associated with a reduced risk of prostate cancer, a relationship supported by studies in rodent models. Castration‐resistant prostate cancer (CRPC) represents the late and lethal phase of human prostate carcinogenesis, and is defined as cancer progression after surgical castration or pharmacologic reduction of serum testosterone through androgen deprivation therapy (ADT). One mechanism involved in the transition to CRPC is cancer cells’ acquired capacity for androgen synthesis. We have previously shown that dietary tomato can reduce prostatic expression of androgen biosynthetic genes. Therefore, we questioned whether dietary tomato might be effective in preventing or controlling the progression of CRPC. We hypothesized that lifelong dietary intake of tomato, as well as dietary tomato intervention following castration, would reduce tumor burden and growth rate in a mouse model of CRPC. TRAMP mice (3 weeks of age, n=79) were acclimated to a powdered, AIN‐93G diet (CON) for one week and then randomized to consume CON (n=28) or 10% w/w lyophilized tomato paste (TP; n=27) from 4 weeks of age until euthanization. A third group, modeling adjuvant dietary intervention, consumed CON from 4 weeks of age until 12 weeks of age, and then 10% w/w lyophilized tomato paste from week 12 until euthanization (TP‐I; n=25). All animals were castrated at 12 weeks of age. Beginning at 10 weeks of age, mice were monitored longitudinally with biweekly ultrasound scans for tumor detection. For each ultrasound scan, serial 2D image slices were used to generate a 3D volume measurement. Upon tumor detection, mice were switched from biweekly to weekly scans and imaged 4 additional times for volumetric tumor measurement and determination of tumor growth rate. Two criteria were implemented for euthanasia: after 5 tumor scans (detection + 4 weekly), or if no tumor had been detected by 30 weeks of age. Longitudinal tumor area under the curve (AUC), as measured by ultrasound, was reduced nearly 50% by tomato intervention (TP‐I) and approximately 25% by lifelong tomato consumption (TP). At euthanization, TP‐I, but not TP, reduced tumor weight approximately 30%. Both measures of tumor burden (tumor growth AUC and tumor weight at euthanization) were reduced by the adjuvant TP‐I, while only tumor growth AUC was decreased by lifelong TP. Observational studies have shown that the lycopene content of the tomato diets in the present study, in human equivalent dose, is both achievable and protective against prostate cancer. Previously, we have shown that lifelong tomato consumption reduces cancer incidence in TRAMP models of primary prostate cancer; current results suggest that pathways regulating primary and castration‐resistant prostatic carcinogenesis in the TRAMP model may be differentially impacted by lifelong tomato feeding. Future work should address efficacy of adjuvant dietary tomato with pharmacologic ADT to assess the potential for this approach to translate into clinical benefits for men with CRPC.Support or Funding InformationThis work is supported by the USDA NIFA Hatch project ILLU‐971‐334 and NIH R37 EB002641.

Peptide-Drug Conjugate GnRH-Sunitinib Targets Angiogenesis Selectively at the Site of Action to Inhibit Tumor Growth.

The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [d-Lys(6)]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R-expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphorylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide-drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site (∼4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation.

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.

Abstract A150: Anti-BIRC6 antisense oligonucleotide inhibits enzalutamide-resistant castration-resistant prostate cancer growth in patient-derived xenograft model by suppressing multiple signaling pathways

Abstract Background: Enzalutamide (ENZ) is the latest androgen receptor antagonist approved for treating castration-resistant prostate cancer (CRPC) and improves patient survival. However, increasing reports showing the development of ENZ-resistant tumor in patients and no known therapies were shown to be effective to-date. Our group has identified that BIRC6, an Inhibitor of Apoptosis (IAP) member, was elevated in CRPC. We developed antisense oligonucleotides (ASOs) that specifically target BIRC6 and demonstrated a significant inhibitory effect towards various CRPC models in vitro and in vivo. Most recently, we observed that BIRC6 expression was also elevated in an ENZ-resistant CRPC patient-derived xenograft (PDX) model. Thus, we hypothesize that anti-BIRC6 ASO may inhibit Enz-resistant CRPC. Materials and Methods: PDX model LTL313BR was a castration relapsed tumor line developed after androgen ablation in animal bearing LTL313B, a patient derived prostate cancer tumor line (androgen sensitive, AR+). LTL313BR was a typical CRPC (adenocarcinoma, AR+) and was resistant to Bicalutamide and Enzalutamide. The model was used to study the effect of anti-BIRC6 ASO on the growth of ENZ-resistant CRPC. Mice bearing LTL313BR tumors were randomized into scrambled control (Scrb) ASO or anti-BIRC6 ASO groups for 21-day treatment (n = 30 per group). Tumors were harvested 1 week after the end of treatment. Tumor volume and serum PSA was measured and the effect on tumor apoptosis was examined. Gene expression profiling was performed to investigate the mechanism of action of anti-BIRC6 ASO. Results: We demonstrated that anti-BIRC6 ASO significantly impeded the growth of Enz- resistant CRPC LTL313BR. Anti-BIRC6 ASO treated group showed median tumor volume of 385mm3 comparing to 521 mm3 in control ASO group, i.e. a 37% reduction. The marked tumor suppression was also coupled with significant reduction in serum PSA and induction of tumor apoptosis. Pathway enrichment analysis of gene expression profile indicates that anti-BIRC6 ASO altered gene expressions that inhibit pathways in mitogenic signalling, proliferation, cell migration, neutrophils chemotaxis and increased T-cell recruitment. Conclusion: Current study provides proof-of-principle data that anti-BIRC6 ASO may represent as novel therapeutic agent against ENZ-resistant CRPC. Citation Format: Iris Sze Ue Luk, Raunak Shrestha, Hui Xue, Yuwei Wang, Peter Gout, Colin Collins, Martin Gleave, Yuzhuo Wang. Anti-BIRC6 antisense oligonucleotide inhibits enzalutamide-resistant castration-resistant prostate cancer growth in patient-derived xenograft model by suppressing multiple signaling pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A150.

SiRNA Lipid Nanoparticle Potently Silences Clusterin and Delays Progression When Combined with Androgen Receptor Cotargeting in Enzalutamide-Resistant Prostate Cancer.

Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signaling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model. Gene silencing of a luciferase reporter gene in the PC-3M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models. LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. LNP CLU-siRNA potently suppressed CLU and AR ASO-induced CLU and AKT and ERK phosphorylation in ENZ-R LNCaP cells in vitro, more potently inhibiting ENZ-R cell growth rates and increased apoptosis when compared with AR-ASO monotherapy. In subcutaneous ENZ-R LNCaP xenografts, combinatory treatment of LNP CLU-siRNA plus AR-ASO significantly suppressed tumor growth and serum PSA levels compared with LNP LUC-siRNA (control) and AR-ASO. LNP siRNA can silence target genes in vivo and enable inhibition of traditionally non-druggable genes like CLU and other promising cotargeting approaches in ENZ-R CRPC therapeutics.

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.

A robust blood gene expression-based prognostic model for castration-resistant prostate cancer

BackgroundCastration-resistant prostate cancer (CRPC) is associated with wide variations in survival. Recent studies of whole blood mRNA expression-based biomarkers strongly predicted survival but the genes used in these biomarker models were non-overlapping and their relationship was unknown. We developed a biomarker model for CRPC that is robust, but also captures underlying biological processes that drive prostate cancer lethality.MethodsUsing three independent cohorts of CRPC patients, we developed an integrative genomic approach for understanding the biological processes underlying genes associated with cancer progression, constructed a novel four-gene model that captured these changes, and compared the performance of the new model with existing gene models and other clinical parameters.ResultsOur analysis revealed striking patterns of myeloid- and lymphoid-specific distribution of genes that were differentially expressed in whole blood mRNA profiles: up-regulated genes in patients with worse survival were overexpressed in myeloid cells, whereas down-regulated genes were noted in lymphocytes. A resulting novel four-gene model showed significant prognostic power independent of known clinical predictors in two independent datasets totaling 90 patients with CRPC, and was superior to the two existing gene models.ConclusionsWhole blood mRNA profiling provides clinically relevant information in patients with CRPC. Integrative genomic analysis revealed patterns of differential mRNA expression with changes in gene expression in immune cell components which robustly predicted the survival of CRPC patients. The next step would be validation in a cohort of suitable size to quantify the prognostic improvement by the gene score upon the standard set of clinical parameters.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-015-0442-0) contains supplementary material, which is available to authorized users.

FOXA1 regulates androgen receptor variant activity in models of castrate-resistant prostate cancer.

Retention of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the requirement for the development of more effective AR targeting therapies. A key mechanism of resistance to anti-androgens is through expression of constitutively active AR variants (AR-Vs) that are refractory to next-generation therapies, including Enzalutamide and Abiraterone. By maintaining an androgenic gene signature, AR-Vs drive tumour survival and progression in castrate conditions. Critically, however, our understanding of the mechanics of AR-V-driven transcription is limited, particularly with respect to dependency on pioneer factor function. Here we show that depletion of FOXA1 in the CWR22Rv1 CRPC cell line abrogates the oncogenic potential of AR-Vs. Gene expression profiling reveals that approximately 41% of the AR-V transcriptome requires FOXA1 and that depletion of FOXA1 attenuates AR-V binding at a sub-set of analysed co-regulated genes. Interestingly, AR-V levels are elevated in cells depleted of FOXA1 as a consequence of attenuated negative feedback on the AR gene, but is insufficient to maintain cell growth as evidenced by marked anti-proliferative effects in FOXA1 knockdown cells. In all, our data suggests that AR-Vs are dependent on FOXA1 for sustaining a pro-proliferative gene signature and agents targeting FOXA1 may represent novel therapeutic options for CRPC patients.

Abstract 1685: In vivo efficacy of PSMA ADC in combination with enzalutamide in castration-resistant prostate cancer

Abstract Despite multiple new treatment options for patients with castration-resistant prostate cancer (CRPC), the overall survival benefit still remains relatively short. Therefore, it is possible that combining these and other new agents can potentially broaden individual therapeutic windows by achieving greater efficacy and improved safety profiles. Prostate-specific membrane antigen antibody-drug conjugate (PSMA ADC), a fully human anti-PSMA antibody linked to monomethylauristatin E (MMAE), is currently in phase 2 development for the treatment of CRPC. We have previously demonstrated significant efficacy of PSMA ADC in advanced prostate cancer patient-derived xenografts and moreover have also shown synergy between PSMA ADC and enzalutamide (ENZ) in vitro. The objective of the current studies was to evaluate the efficacy of PSMA ADC in combination with ENZ to inhibit CRPC patient-derived xenografts (PDX) in a preclinical setting. First, we performed a pilot study to determine a sub-optimal dose for PSMA ADC, using LuCaP 96CR, a model of CRPC with high PSMA expression that previously showed significant regressions in response to PSMA ADC. Animals bearing subcutaneous LuCaP 96CR were treated with a single dose of PSMA ADC (1, 2, or 5 mg/kg, IP) or a non-PSMA targeted ADC (5 mg/kg) and followed for up to 10 weeks. PSMA ADC demonstrated dose-dependent efficacy, and based on the results, a single dose of PSMA ADC (2 mg/kg) was selected for combination treatment with ENZ (50 mg/kg; 5x/week, oral gavage). The effects of the PSMA ADC+ENZ combination were compared to PSMA ADC alone, ENZ alone, and placebo control. At six weeks after the beginning of treatment the changes in tumor volume were: fold increases of enrollment tumor volume in control (4.9 ± 0.40); ENZ (2.83 ± 0.37); and PSMA ADC (1.6 ± 0.43); and decrease in tumor volume in the combination group (0.28 ± 0.10). In the control and ENZ groups all tumors were progressing at 6 weeks, while in the PSMA ADC group there were 4 tumors that had decreased tumor volume, and all 11 animals in the PSMA ADC + ENZ groups had regressing tumors. The tumor growth inhibition was also reflected by changes in serum PSA. Most interestingly, a 12-week follow up and survival analysis show that all three treatments resulted in significant survival benefits but the combination therapy effects were the most pronounced resulting in ENZ vs PSMA ADC+ENZ HR = 0.09 with P = 0.0415 and with no deaths observed in the combination group. No significant negative side effects were associated with the combination treatment. Additional post-treatment analyses are underway. In summary, our results clearly indicate that the combination of PSMA ADC+ENZ possesses strong antitumor activity in the LuCaP 96CR model that is much more pronounced than the inhibitory effects of either agent alone. These results provide a strong rationale for clinical testing of PSMA ADC in combination with androgen-directed treatment strategies. Citation Format: Vincent A. DiPippo, Holly M. Nguyen, Lisha G. Brown, William C. Olson, Robert L. Vessella, Eva Corey. In vivo efficacy of PSMA ADC in combination with enzalutamide in castration-resistant 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 1685. doi:10.1158/1538-7445.AM2015-1685

Abstract 2567: Cabazitaxel chemotherapy targets mitotic kinesins resulting in multi-nucleation of prostate cancer cells: A novel mechanism of cross-resistance with antiandrogens in advanced CRPC

Abstract Progression to castration-resistant prostate cancer (CRPC) is characterized by increased androgen receptor (AR) and activated AR signaling despite castrate levels of androgens. The FDA-approved next generation microtubule stabilizing taxane, Cabazitaxel (CBZ) and novel anti-androgen Enzalutamide (MDV) have demonstrated additional survival benefits for patients with advanced CRPC. The present study pursued the mechanism of therapeutic resistance to CBZ and antiandrogen treatment in models of CRPC. The effect CBZ and/or MDV was examined in human prostate cancer cells: androgen independent (DU145 and PC3), androgen responsive (LNCaP and VCaP) and CRPC (22Rv1 and PC3-ARv567, harboring AR variants). Cell viability assessment revealed that CRPC PC3-ARv567es and the androgen-sensitive VCaP cells exhibited resistance to CBZ; however the 22Rv1 cells responded to CBZ and MDV combination with a significant increase in growth (P<0.05). Confocal microscopy and Western blot analysis were used to establish the subcellular localization and protein expression in prostate cancer cells. Antiandrogen (MDV) treatment (24hrs;1uM) led to a cytoplasmic accumulation of AR, while treatment with CBZ (72hrs, 35 -100nM) decreased AR expression, but it did not inhibit its nuclear localization. Furthermore CBZ induced mono-astral spindle formation, centrosomal clustering and multi-nucleation, potentially via targeting the “pro-mitotic kinesins”, MCAK and HSET, regardless of androgen status. To determine the in vivo efficacy of CBZ and androgen axis targeting, we used a transgenic mouse model of prostate cancer (Dominant negative TGFβRII/TRAMP) that is driven to metastasis by epithelial-mesenchymal transition (EMT). Immunohistochemical profiling of prostate tumors from untreated control and CBZ treated mice demonstrated that combination of CBZ and castration-induced androgen depletion reduced kinesin and AR expression and reversed EMT, impairing prostate cancer progression. Furthermore, CBZ treatment restored normal glandular differentiation (by H&E), without significant effect on apoptosis in tumor epithelial cells. Cell proliferation was significantly decreased in response to castration-induced androgen depletion in combination with CBZ (2wks) (P<0.05). These findings support the ability of CBZ to target pro-mitotic kinesins, providing an insight into a new mechanism of action of this taxane that can be exploited for targeting CRPC patients with kinesin overexpression. Moreover, the in vivo evidence identified that CBZ chemotherapy has novel effects beyond suppressing tumor growth, by inducing prostate glandular differentiation. This study is of translational significance in introducing a novel mechanism to overcome cross-resistance to CBZ chemotherapy and anti-androgens in advanced CRPC. Citation Format: Sarah K. Martin, Hong Pu, Craig Horbinski, Zheng Cao, Natasha Kyprianou. Cabazitaxel chemotherapy targets mitotic kinesins resulting in multi-nucleation of prostate cancer cells: A novel mechanism of cross-resistance with antiandrogens in advanced CRPC. [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 2567. doi:10.1158/1538-7445.AM2015-2567

Abstract 1856: Preclinical effects of dual AKT/MAPK inhibition in PTEN-deficient prostate cancer

Abstract Targeting signal molecules with single agent monotherapy is met with limited or transient clinical benefits. Therefore, establishing effective rational combinatorial treatment strategies provides a better option for achieving improved clinical outcomes. The AKT/PI3K and MAPK signaling pathways have been identified as key compensatory signal transduction pathways in prostate cancer. We have used genetically engineered mouse models of prostate cancer to show the treatment efficacy of monotherapy with the AKT inhibitor AZD5363 and the MAP/ERK kinase (MEK) inhibitor selumetinib (AZD6244, ARRY142886). We hypothesized that a rational combinatorial approach utilizing both of these inhibitors would improve treatment efficacy compared to single agent monotherapy. To test this hypothesis, we evaluated the treatment effects of AZD5363 and selumetinib in preclinical models of prostate cancer. In vitro experiments were carried out using a panel of human prostate cancer cell lines, and mouse prostate cancer cell lines, derived from PTEN-deficient castration-naïve prostate cancer (CNPC) and castration-resistant prostate cancer (CRPC). Treatments with AZD5363 and selumetinib demonstrated a synergistic combination effect in both human and mouse prostate cancer cell lines. We evaluated the antitumor effects in vivo by comparing the effects of AZD5363 and selumetinib alone or in combination in PTEN-deficient mice. Our studies show that combination therapy of AZD5363/selumetinib was well-tolerated by the mice and demonstrated significantly improved inhibition of tumor growth compared to single agent therapy. In the CNPC model, mice showed 16.7, 10.0 and 24.9% reductions in tumor burden after treatments with AZD5363, selumetinib and AZD5363/selumetinib, respectively (P<0.001). In the CRPC model, mice showed 15.8, 3.8 and 27.0% reductions in tumor burden after treatments with AZD5363, selumetinib and AZD5363/selumetinib, respectively (P<0.001). Target validation studies confirmed the inhibition of downstream targets. Overall, our findings suggest a potential role for targeting the PI3K/AKT and MAPK signaling pathways with AZD5363 and selumetinib for treatment of PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yutaka Yamamoto, Yurie Kura, Emiko Fukushima, Naomi Ando, Barry Davies, Yuji Hatanaka, Takashi Oki, Nobutaka Shimizu, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Preclinical effects of dual AKT/MAPK inhibition in PTEN-deficient 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 1856. doi:10.1158/1538-7445.AM2015-1856

Abstract 4212: Inhibition of mouse PTEN-deficient prostate cancer with next generation antisense oligonucleotide targeting the androgen receptor

Abstract Resistance to androgen deprivation therapies is characterized by the persistent activation of the androgen receptor (AR). Traditional approaches for the development of novel androgen deprivation therapies have focused on inhibition of AR activity by blocking the production or binding of androgens to the AR. These novel treatment strategies have shown improvements, however, patients still develop castration-resistant disease. Continued AR activity in castration-resistant prostate cancer (CRPC) may be the result of ligand dependent or ligand independent mechanisms. Antisense oligonucleotides (ASO) have the ability to block specific genes inhibiting the associated proteins thus providing an alternate treatment strategy for CRPC. Here, we investigated the in vivo antitumor effects of a generation 2.5 ASO targeting the mouse AR in a PTEN-deficient mouse model of prostate cancer. Pharmacodynamic analysis demonstrated strong inhibition of target RNA by the AR-ASO in prostate tumor tissues. The AR-ASO also exhibited strong suppression of AR protein levels in prostate tumors as demonstrated by western blot analysis. Immunohistochemical analysis confirmed reduced levels of AR expression and transcriptional activity in tumor tissue. Furthermore, AR was also inhibited in normal tissues of the accessory sex glands. The treatment efficacy of the AR-ASO was evaluated in pharmacological intervention trials in mice with castration-naïve prostate cancer (CNPC) and CRPC. Treatments effects in CNPC were also compared to androgen deprivation by surgical castration. Compared to saline treated controls, tumor growth was significantly inhibited by the AR-ASO (34%) and was comparable to that of surgical castration (38%). In the CRPC model, significant suppression of tumor growth (10% vs control) was also observed in mice treated with the AR-ASO. Furthermore, strong inhibition of AR expression and activity was observed in AR-ASO-treated mice harboring either CNPC or CRPC, but not in mice treated with surgical castration. Gene expression profiling using the Fluidigm Biomark platform revealed that 40 AR-dependent target genes were significantly altered in CNPC after treatment with the AR-ASO; 34 of these genes were also significantly changed following castration. A decrease of ERK phosphorylation in was observed in AR-ASO-treated mice in both CNPC and CRPC intervention models and was associated with decreased protein levels of AR. These findings demonstrate that treatment with generation 2.5 AR-ASO is effective against preclinical mouse models of prostate cancer and may provide a promising approach to overcoming treatment resistance of CRPC. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Emiko Fukushima, Barry R. Davies, Hayley Campbell, Yuji Hatanaka, Yutaka Yamamoto, Nobutaka Shimizu, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Inhibition of mouse PTEN-deficient prostate cancer with next generation antisense oligonucleotide targeting the androgen receptor. [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 4212. doi:10.1158/1538-7445.AM2015-4212