Patient-derived Prostate Cancer Research Articles

Abstract A009: Evaluation of hormonal therapy in a panel of prostate patient-derived xenograft models and comparison to standard cell line models

Abstract Background: Prostate cancer is the second most common cancer worldwide for males, and the fourth most common cancer overall, with more than 1 million new cases diagnosed. However, progress toward understanding the biology of prostate cancer and the development of new therapies has been hampered by the lack of in vivo models that adequately represent the spectrum of benign, latent, aggressive, and metastatic forms of the human disease. Here we report the validation of a panel of patient-derived xenograft (PDX) models and their utilization in preclinical studies alongside cell line models of prostate cancer that are routinely used. Methods: Prostate cancer samples obtained from patients undergoing surgery were collected with ethical consent, disaggregated, and established subcutaneously in Rag-2 knockout mice (The Jackson Laboratory) to generate PDX models. Tumor material was diagnosed on the basis of operative histology and immunohistochemistry (IHC for PSA, androgen receptor expression). Subcutaneous tumor growth of prostate models was evaluated 3 times a week by measuring the tumor in two dimensions using electronic calipers for the duration of the study, and tumor volumes were estimated. For orthotopic PC3M, tumor growth was checked once weekly by bioluminescent imaging (BLI). Briefly, the mice were injected (s.c.) with 150mg/kg D-Luciferin prior to imaging, anesthetized following administration of D-Luciferin, and placed into the imaging chamber (Spectrum CT) and imaged for luminescence (ventral view). Results: We have established a bank of transplantable prostate cancer PDX. Two models were reported to be hormone sensitive and 2 models represented castrate-resistant prostate cancer (CRPC), one of which has a TMPRSS-ETS fusion. In comparison, cell-derived LNCaP requires testosterone to grow whereas the bioluminescent PC3M CRPC orthotopic model develops metastasis in the liver, lungs, lumbar lymph nodes, fore limbs, and hind limbs, representing the late stage of cancer. The tumor response to standard-of-care agents such as docetaxel, abiraterone, and enzalutamide will be reported. Conclusions: We have characterized both cell- and patient-derived prostate cancer xenograft models that will provide a clinically relevant platform spanning the different stages of the disease for preclinical drug evaluation. Citation Format: Nektaria Papadopoulou, Louise Woolley, Louise Wainwright, Lucy Harris, Jane Wrigley, Jason King, Anne Collins, Rajendra Kumari. Evaluation of hormonal therapy in a panel of prostate patient-derived xenograft models and comparison to standard cell line models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A009.

Spontaneous development of Epstein-Barr Virus associated human lymphomas in a prostate cancer xenograft program.

Prostate cancer research is hampered by the lack of in vivo preclinical models that accurately reflect patient tumour biology and the clinical heterogeneity of human prostate cancer. To overcome these limitations we propagated and characterised a new collection of patient-derived prostate cancer xenografts. Tumour fragments from 147 unsupervised, surgical prostate samples were implanted subcutaneously into immunodeficient Rag2-/-γC-/- mice within 24 hours of surgery. Histologic and molecular characterisation of xenografts was compared with patient characteristics, including androgen-deprivation therapy, and exome sequencing. Xenografts were established from 47 of 147 (32%) implanted primary prostate cancers. Only 14% passaged successfully resulting in 20 stable lines; derived from 20 independent patient samples. Surprisingly, only three of the 20 lines (15%) were confirmed as prostate cancer; one line comprised of mouse stroma, and 16 were verified as human donor-derived lymphoid neoplasms. PCR for Epstein-Barr Virus (EBV) nuclear antigen, together with exome sequencing revealed that the lymphomas were exclusively EBV-associated. Genomic analysis determined that 14 of the 16 EBV+ lines had unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements, confirming their B-cell origin. We conclude that the generation of xenografts from tumour fragments can commonly result in B-cell lymphoma from patients carrying latent EBV. We recommend routine screening, of primary outgrowths, for latent EBV to avoid this phenomenon.

Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation

Background:Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear.Methods:PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays.Results:PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation.Conclusions:A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.

Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

ABSTRACTAdvancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies.

In Vivo Hemin Conditioning Targets the Vascular and Immunologic Compartments and Restrains Prostate Tumor Development.

Purpose: Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, we assessed how hemin, a pharmacologic inducer of heme oxygenase-1 (HO-1), has an impact on prostate cancer development in an in vivo conditioning model.Experimental Design: The stroma of C57BL/6 mice was conditioned by subcutaneous administration of hemin prior to TRAMP-C1 tumor challenge. Complementary in vitro and in vivo assays were performed to evaluate hemin effect on both angiogenesis and the immune response. To gain clinical insight, we used prostate cancer patient-derived samples in our studies to assess the expression of HO-1 and other relevant genes.Results: Conditioning resulted in increased tumor latency and decreased initial growth rate. Histologic analysis of tumors grown in conditioned mice revealed impaired vascularization. Hemin-treated human umbilical vein endothelial cells (HUVEC) exhibited decreased tubulogenesis in vitro only in the presence of TRAMP-C1-conditioned media. Subcutaneous hemin conditioning hindered tumor-associated neovascularization in an in vivo Matrigel plug assay. In addition, hemin boosted CD8+ T-cell proliferation and degranulation in vitro and antigen-specific cytotoxicity in vivo A significant systemic increase in CD8+ T-cell frequency was observed in preconditioned tumor-bearing mice. Tumors from hemin-conditioned mice showed reduced expression of galectin-1 (Gal-1), key modulator of tumor angiogenesis and immunity, evidencing persistent remodeling of the microenvironment. We also found a subset of prostate cancer patient-derived xenografts and prostate cancer patient samples with mild HO-1 and low Gal-1 expression levels.Conclusions: These results highlight a novel function of a human-used drug as a means of boosting the antitumor response. Clin Cancer Res; 23(17); 5135-48. ©2017 AACR.

Inhibition of ERG Activity in Patient-derived Prostate Cancer Xenografts by YK-4-279.

The aim of the current study was to determine the effects of the ERG small-molecule inhibitor YK-4-279 on ERG+ prostate cancer patient-derived xenografts (PDX). ERG activity was blocked using YK-4-279 in three subcutaneously-implanted ERG+ (LuCaP 23.1, 86.2 and 35) and one ERG- (LuCaP 96) PDX. Treated animals tumor volume (TV), body weight (BW) and serum prostate-specific antigen (PSA) were compared to vehicle-treated control animals. Gene expression, proliferation, apoptosis, microvessel density and ERG expression were also assessed. Administration of YK-4-279 decreased TV (p=0.026), proliferation (p=0.0038) and PSA (p=0.022) in Severe Combined Immunodeficiency (SCID) mice bearing LuCaP 23.1 tumors. LuCaP 86.2, LuCaP 35 and LuCaP 96 showed no significant changes in TV, or PSA. Mineralocorticoid receptor (MR) and MR-direct target genes were up-regulated in treatment-resistant LuCaP 86.2 and LuCaP 35 PDX. YK-4-279 decreased ERG+ LuCaP 23.1 tumor growth, but not LuCaP 86.2 and LuCaP 35 ERG+ tumor growth.

Abstract 4420: Elevated glycolytic gene signature in patient-derived neuroendocrine prostate cancer xenograft models and its clinical relevance

Abstract Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PCa) that is becoming increasingly common in the clinic. While the vast majority of PCa presents as androgen-dependent adenocarcinoma, recent uses of increasingly potent therapeutics targeting the androgen receptor signaling axis has resulted in the promotion of NEPC transdifferentiation as a mechanism of treatment resistance. Unfortunately, there is currently no effective treatment option for NEPC. Altered cancer metabolism is now recognized as a hallmark of cancer and a crucial factor for promoting tumour growth and spread. In particular, altered glucose metabolism and the resultant acidification of the tumor microenvironment via increased lactic acid production has been shown to play an important role in multiple cancer-promoting processes, including tissue invasion/metastasis, angiogenesis, and suppression of local anticancer immunity. While increased glycolysis is not generally considered a phenomenon relevant to primary treatment-naive PCa, we have recently demonstrated its relavance to castration-resistant prostate cancer (CRPC) and thus suspect that it is also relevant to the more aggressive NEPC. Our laboratory has also developed a number of unique serially transplantable patient-derived xenograft (PDX) models of NEPC that are histologically highly similar to the donor tissues and retain important genetic and epigenetic features. In particular, we have developed the first spontaneous NEPC transdifferentiation model in the field (LTL331/331R). The gene expression profiles of these NEPC PDX models were compared to that of PCa adenocarcinoma PDX models. To determine whether certain metabolic pathway alterations were specific to NEPC, genes from a number of key metabolic pathways were compiled and overall pathway scores were generated using average expression z-scores. Furthermore, publically available gene expression data from NEPC patient tumors were used to validate our findings. From our analysis, we found that genes in the glycolysis pathway were signficiantlly upregulatied in both our PDX models and also in patient NEPC samples. Of particular interest is the upregulation of genes involved in the production and secretion of lactic acid, such as LDHA and MCT4. As such, our results suggest that elevated glycolysis and production of lactic acid could be a clinically important NEPC phenotype. Furthermore, the inhibition of glycolysis and particularly the inhibition of lactic acid secretion via MCT4 could be a potentially viable therapeutic strategy for NEPC. Citation Format: Stephen Y. Choi, Susan L. Ettinger, Dong Lin, Hui Xue, Robert H. Bell, Fan Mo, Michael Pollak, Colin C. Collins, Yuzhuo Wang. Elevated glycolytic gene signature in patient-derived neuroendocrine prostate cancer xenograft models and its clinical relevance [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 4420. doi:10.1158/1538-7445.AM2017-4420

Abstract LB-145: Study of prostate cancer & endosteal niche adhesive interactions can aid understanding of metastatic behavior

Abstract Introduction: Prostate cancer (PCa) is the second leading cause of cancer related death in men in the US and remains incurable in the metastatic stage. Recent reports have shown that the adhesion molecule N-cadherin, a mesenchymal cadherin associated with epithelial-to-mesenchymal transition (EMT), is expressed in a large percentage of castration resistant prostate cancer tumors, allows the interaction between N-cadherin expressing osteoblasts (OSB) at the endosteal niche and prostate cancer cells via homophilic binding, and can promote osteoclastogenesis and osteolytic disease state. There is also evidence that direct adhesive interactions between tumor cells and the bone marrow stroma are responsible for the development of drug resistance and relapse. This phenomenon termed "cell adhesion-mediated drug resistance" (CAM-DR) is thought to be one of the major mechanisms by which PCa, as well as other tumors that reside at the endosteal niche, escape the cytotoxic effects of therapeutic agents. In this study we evaluated the adhesive interaction of patient-derived primary PCa cells (i.e., conditionally reprogrammed PCa cells (Liu, X et al., Am J Pathol, 2012)) with the bone metastatic microenvironment as a first step to: 1) identify potential differences in patient response to therapy using physical cues (i.e., adhesion to the tumor microenvironment) as “physical biomarkers” of putative response to treatment, and 2) potentially target these interactions therapeutically. Materials and Methods: The human OSB cell line hFOB 1.19 was used to model the endosteal niche. Conditionally reprogrammed patient-derived PCa cells: 1) PP8T; A Gleason Score 8 CRC line from a primary site tumor and, 2) PCA3-CRC cells, which we derived from a metastatic lymph node organoid (a kind gift from Drs Charles Sawyers and Yo Chen (Gao, D, et al., Cell, 2014)), were co-cultured in a microfluidic device, layered with OSB to monitor PCa-OSB interactions using time lapse fluorescence microscopy. In separate experiments, PCa cells were labeled with the cell proliferation dye eFluor® 670 to quantify and compare proliferation of PCa as a function of their adhesive interaction with OSB. Finally, N-cadherin (CDH2) and cadherin-11 (CDH11) protein expressions were also quantified for each of the PCa lines studied as well as the OSB line utilized. Results and Conclusions: Real-time imaging showed that PCA3 cells (i.e., metastatic line) preferentially adhered to OSB whereas PP8T cells (from primary site) presented with a more invasive, yet not adherent, behavior. In addition, PP8T cells proliferated at a significantly faster rate than PCA3 cells when co-cultured with OSB. Concomitantly with these results we also found that PCA3 cells expressed significantly higher levels of N-cadherin and cadherin-11 than PP8T cells, corroborating the presence of EMT in the metastatic line. These data suggest that examination of the PCa-OSB physical interface maybe useful in determining the mechanisms by which PCa cells interact with the bone niche. Our studies represent a potential first step to predicting and targeting these interactions, prior to the development of CAM-DR. Citation Format: Jenny Zilberberg, Leah Dziopa, Eugenia Dziopa, Maia Iyer, Qiaoling Sun, Saba Choudhary, Woo Y. Lee, Lucas Tricoli, Shain Assefnia, Christopher Albanese. Study of prostate cancer & endosteal niche adhesive interactions can aid understanding of metastatic behavior [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 LB-145. doi:10.1158/1538-7445.AM2017-LB-145

Abstract 2824: Validation of a panel of patient-derived xenograft models for prostate caner and cell line models for preclinical drug evaluation

Abstract Background: Prostate cancer is the second most common cancer worldwide for males, and the fourth most common cancer overall, with more than a million new cases diagnosed. However, progress toward understanding the biology of prostate cancer and the development of new therapies has been hampered by the lack of in vivo models that adequately represent the spectrum of benign, latent, aggressive, and metastatic forms of the human disease. Here we report the validation of a panel of patient-derived xenograft (PDX) models and their utilization in preclinical studies alongside cell line models of prostate cancer. Methods: Prostate cancer samples obtained from patients undergoing surgery were collected with ethical consent, disaggregated and established subcutaneously in Rag2-/-γC-/- mice (The Jackson Laboratory) to generate PDX models. Tumor material was diagnosed on the basis of operative histology and immunohistochemistry (IHC for PSA, androgen receptor expression). Tumor growth in both Rag2-/-γC-/- and NSG mice was evaluated in comparison to cell line models such LNCaP and PC3M. Briefly tumours were measured 3 times a week and tumour volumes were estimated using the formula 0.5 (LxW2) by measuring the tumour in two dimensions using electronic callipers for the duration of the study. For orthotopic PC3M, tumour growth was checked once weekly by bioluminescent imaging (BLI). Briefly, the mice were injected (s.c.) with 150mg/kg D-Luciferin prior to imaging, anaesthetised following administration of D-Luciferin and placed into the imaging chamber (Spectrum CT) and imaged for luminescence (ventral view). Results: We have established a bank of transplantable prostate cancer PDX and stocks are maintained in liquid nitrogen which can be resuscitated in in both Rag2-/-γC-/- and NSG mice. Two models were reported to be hormone sensitive and 2 models represented castrate resistant prostate cancer (CRPC) one of which has a TMPRSS-ETS fusion. In comparison cell-derived LNCaP requires testosterone to grow whereas the bioluminescent PC3M CRPC orthotopic model develops metastasis in the liver, lungs, lumbar lymph nodes, fore limbs and hind limbs representing the late stage of cancer. Conclusions: We have characterised both cell and patient-derived prostate cancer xenograft models which will provide a clinically relevant platform spanning the different stages of the disease for preclinical drug evaluation. Citation Format: Nektaria Papadopoulou, Jane Wrigley, Andrew McKenzie, Jason King, Louise Wainwright, Kelly Jones, Anne Collins, Rajendra Kumari. Validation of a panel of patient-derived xenograft models for prostate caner and cell line models for preclinical drug evaluation [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 2824. doi:10.1158/1538-7445.AM2017-2824

Abstract 4981: Extracellular spacer and co-stimulatory domains define target sensitivity and persistence of CAR T cells for the treatment of PSCA+ bone metastatic prostate cancer

Abstract Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer should be amenable to CAR-based immunotherapy given that several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. The selectivity of CARs for solid cancers is crucial, however, due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that optimized co-stimulatory signaling and extracellular spacer domains are essential in defining a CAR’s selectivity towards tumor cells that over-express the target antigen. The 4-1BB co-stimulatory domain in PSCA-CARs confers enhanced tumor selectivity with dampened yet dose-responsive cytokine production, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory domain. CARs containing a short extracellular spacer demonstrate the most selective killing of high PSCA-expressing tumors but are unable to produce the inflammatory cytokines important for a complete anti-tumor response. We show that longer extracellular spacers in PSCA-CARs are necessary for both tumor killing and cytokine production. PSCA-CARs exhibit robust in vivo anti-tumor activity in subcutaneous and orthotopic bone-metastatic patient-derived prostate cancer xenograft models, and 4-1BB-containing CARs show superior persistence in controlling metastatic disease compared with CD28-containing CARs. Our study demonstrates the critical impact of CAR components in defining an optimized tumor-selective CAR T cell, and also highlights the importance of clinically relevant animal models in developing effective solid cancer CAR T cell therapies. Citation Format: Kelly Kennewick, Dileshni Tilakawardane, Ethan Gerdts, John Murad, Anthony Park, Brook Jeang, Yukiko Yamaguchi, Stephen J. Forman, Saul Priceman. Extracellular spacer and co-stimulatory domains define target sensitivity and persistence of CAR T cells for the treatment of PSCA+ bone metastatic 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 4981. doi:10.1158/1538-7445.AM2017-4981

Abstract 20: Xentuzumab, a humanized IGF-1 and IGF-2 ligand neutralizing antibody, improves the antitumor efficacy of enzalutamide in preclinical models of prostate cancer

Abstract Background: The proliferative and pro-survival signals driven by the insulin-like growth factor (IGF) ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 activates the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. A large body of preclinical evidence suggests that IGF signaling plays a key role in cancer by driving therapy resistance, due to cross-talk with other signaling networks such as androgen receptor signaling. The aim of this study was to explore the potential of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[1]), to enhance the anti-tumor activity of enzalutamide in prostate cancer cell lines and in a patient-derived prostate cancer xenograft model. Methods: Effects of enzalutamide, xentuzumab and combinations thereof on in vitro proliferation, survival, cell cycle and signaling were evaluated using the prostate cancer cell lines VCaP, DuCaP, MDA PCa 2b, and LNCaP. The in vivo efficacy of enzalutamide, alone and in combination with xentuzumab was investigated using LuCaP 96CR, a patient-derived xenograft model of castration-resistant prostate cancer. Tumors were implanted s.c. into castrate SCID mice. When tumors exceeded 150mm3 animals were randomized into groups: 1) Control; 2) enzalutamide (50 mg/kg QD po), 3) xentuzumab (BI 836845[1], 200 mg/kg QW ip) in combination with enzalutamide. Results: Cell viability was more effectively reduced by the combination of enzalutamide and xentuzumab than either drug alone in three of four cell lines expressing the IGF-1R and the androgen receptor (AR). In VCaP cells, prolonged inhibition of IGF pathway signaling and enhanced blockade of proliferation as well as induction of apoptosis was observed after combination treatment. In vivo, enzalutamide monotherapy did not show significant antitumor efficacy in the LuCaP 96CR model, however, combined treatment with xentuzumab significantly inhibited progression of LuCaP 96CR tumor growth (p<0.001 vs. enzalutamide alone). Reduced serum PSA levels were observed after enzalutamide and combination treatment. Enzalutamide plus xentuzumab inhibited tumor growth at tolerated doses and resulted in significant improvements in survival. Conclusions: These studies demonstrated that addition of the IGF-1/-2 neutralizing antibody xentuzumab to enzalutamide results in improved anti-neoplastic activity in a subset of prostate cancer cell lines in vitro, and to re-sensitization to enzalutamide in a patient-derived xenograft model of CRPC. Reference: [1] Friedbichler K et al. (2014). Mol Cancer Ther 13(2):399-409. Citation Format: Ulrike Weyer-Czernilofsky, Marco H. Hofmann, Paul J. Adam, Flavio Solca, Katrin Friedbichler, Norbert Kraut, Eva Corey, Thomas Bogenrieder. Xentuzumab, a humanized IGF-1 and IGF-2 ligand neutralizing antibody, improves the antitumor efficacy of enzalutamide in preclinical models of 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 20. doi:10.1158/1538-7445.AM2017-20

MiR-100-5p inhibition induces apoptosis in dormant prostate cancer cells and prevents the emergence of castration-resistant prostate cancer

Carcinoma of the prostate is the most common cancer in men. Treatment of aggressive prostate cancer involves a regiment of radical prostectomy, radiation therapy, chemotherapy and hormonal therapy. Despite significant improvements in the last decade, the treatment of prostate cancer remains unsatisfactory, because a significant fraction of prostate cancers develop resistance to multiple treatments and become incurable. This prompts an urgent need to investigate the molecular mechanisms underlying the evolution of therapy-induced resistance of prostate cancer either in the form of castration-resistant prostate cancer (CRPC) or transdifferentiated neuroendocrine prostate cancer (NEPC). By analyzing micro-RNA expression profiles in a set of patient-derived prostate cancer xenograft tumor lines, we identified miR-100-5p as one of the key molecular components in the initiation and evolution of androgen ablation therapy resistance in prostate cancer. In vitro results showed that miR-100-5p is required for hormone-independent survival and proliferation of prostate cancer cells post androgen ablation. In Silico target predictions revealed that miR-100-5p target genes are involved in key aspects of cancer progression, and are associated with clinical outcome. Our results suggest that mir-100-5p is a possible therapeutic target involved in prostate cancer progression and relapse post androgen ablation therapy.

MP83-08 COMBINATION OF METFORMIN AND SODIUM VALPROATE FOR PROSTATE CANCER: A RAPID APPROACH FROM BENCH TO CLINICAL TRIAL.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-08 COMBINATION OF METFORMIN AND SODIUM VALPROATE FOR PROSTATE CANCER: A RAPID APPROACH FROM BENCH TO CLINICAL TRIAL. Linh N.K. Tran, Ganessan Kichenadasse, Rebecca J. Ormsby, Katherine L. Morel, Lisa M. Butler, Margaret M. Centenera, and Pamela J. Sykes Linh N.K. TranLinh N.K. Tran More articles by this author , Ganessan KichenadasseGanessan Kichenadasse More articles by this author , Rebecca J. OrmsbyRebecca J. Ormsby More articles by this author , Katherine L. MorelKatherine L. Morel More articles by this author , Lisa M. ButlerLisa M. Butler More articles by this author , Margaret M. CenteneraMargaret M. Centenera More articles by this author , and Pamela J. SykesPamela J. Sykes More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2576AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The anti-diabetic drug metformin (MET) and the anti-epileptic drug sodium valproate (VPA), when used alone, have shown anti-cancer effects in prostate cancer (PCa). However high doses are required which results in unacceptable toxicity. Here, we aimed to determine if the combination of MET and VPA (MET+VPA) at clinically relevant doses would (1) induce a synergistic anti-tumor effect in human PCa cell lines and patient-derived PCa explants, (2) cause minimal toxicity to normal tissues in vivo, (3) identify prognostic molecular markers for MET+VPA responses and (4) lead to rapid clinical application. METHODS Human PCa cell lines (PC-3 and LNCaP) and radical prostatectomy explants from 8 patients were analyzed for response to MET+VPA. Ki67, cleaved caspase-3, the androgen receptor (AR), and p53 expression in explants were analyzed using immunohistochemistry. The combinatorial effects of MET+VPA were calculated using the Drug Combination Index (CI). The role of p53 and AR in response to MET+VPA was determined using TP53 gene knock-down in LNCaP and TP53 ectopic expression in PC3, and AR was chemically inhibited using Enzalutamide in LNCaP. The toxicity of MET+VPA was determined using histological scoring systems for kidney and liver toxicity in nude mice. RESULTS MET+VPA synergistically inhibited proliferation in both PC-3 and LNCaP and synergistically induced apoptosis in LNCaP cells (CI<0.9). All PCa explants (pathological stage pT2C-pT3A and Gleason score 7-9) demonstrated a significant reduction (90%, p<0.001) in proliferation and a significant dose-dependent increase in apoptosis (300%, p<0.001) compared to vehicle. The p53 protein plays a role in MET+VPA as depletion of TP53 in LNCaP significantly reduced the apoptotic response by 58% (p=0.001) and the ectopic expression of TP53 in PC3 significantly increased the apoptotic response by 15% (p=0.047). The AR signaling pathway was also confirmed to play an important role in the apoptotic response to MET+VPA as Enzalutamide significantly reduced apoptosis by 28.5% (p=0.029) in LNCaP cells. No kidney or liver toxicity was detected in nude mice after 8 weeks administration of MET+VPA. These results support evaluation in a Phase I clinical trial which will start early in 2017 (Trial ID: ACTRN12616001021460p). CONCLUSIONS Our study has only taken 2 years from the first experiment to a phase I clinical trial. Although MET+VPA with an AR inhibitor may not show benefit, MET+VPA has potential to control clinically localized or metastatic PCa, particularly in tumors with functional p53 and/or AR signaling. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1108-e1109 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Linh N.K. Tran More articles by this author Ganessan Kichenadasse More articles by this author Rebecca J. Ormsby More articles by this author Katherine L. Morel More articles by this author Lisa M. Butler More articles by this author Margaret M. Centenera More articles by this author Pamela J. Sykes More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

BMI-1 Targeting Interferes with Patient-Derived Tumor-Initiating Cell Survival and Tumor Growth in Prostate Cancer.

Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach. We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1. We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell-like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor-directed therapies, without toxic effects on normal tissues. Our data offer a paradigm for targeting TICs and support the development of BMI-1-targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176-91. ©2016 AACR.

Abstract 243: Investigating microtubule growth dynamics in patient-derived metastatic prostate cancer cells

Abstract Advanced prostate cancer (PC) is treated primarily by means of chemotherapy with one of the clinically approved microtubule (MT)-targeting agents (MTAs): paclitaxel, docetaxel and cabazitaxel. The mechanism of action of the MTAs on the cellular level is to bind to MTs and inhibit MT-dependent intracellular pathways, ultimately leading to cell death. In PC, MTAs are the only chemotherapy class shown to improve survival, however, there is high variability of response to MTA chemotherapy among patients as well as emergence of resistance to drug action which hampers the clinical efficacy. Clinicians need additional information to match individual patient tumors to the most effective drugs or refrain from treating patients that will not respond. However, the precise mechanism of action or resistance development to MTAs is not understood, which represents a critical gap in our knowledge to select the best treatment. Our hypothesis is that there are inherent differences in tumor MT dynamics among individual patients which dictates their response to treatment with specific MTAs. To this end, we envision that a systematic characterization of PC MT dynamics and their response to MTAs will allow chemotherapy customization and prolong survival. Specifically, our approach consists of the analysis of metastatic castrate resistant PC (mCRPC) organoids (Gao D. et al, 2014) derived from bone (acetabulum, vertebrae), soft tissue (salvage prostatectomy, lymph nodes), pleural effusion and circulating tumor cells biopsy samples obtained from metastatic patients who have received hormonal therapy and the MTA docetaxel or hormonal therapy alone or no treatment. We apply a quantitative single-cell image analysis algorithm (ClusterTrack) to measure a panel of twelve distinct MT dynamics parameters (a MT dynamics signature). Preliminary analysis suggests differences between different mCRPC organoids. MT tracking results indicate a correlation of decreased MT growth rate with increased Op18/stathmin expression. In this context, we hypothesize that differential expression of MT regulating genes affects specific parameters of MT dynamics, thus determining the sensitivity to MTA treatment. Citation Format: Alexandre Matov, Johan de Rooij, Jay Gatlin, Julia Rohrberg, Nikta Ahmad, Rahul Aggarwal, Jeff Simko, Andrei Goga, Charles Ryan, Torsten Wittmann. Investigating microtubule growth dynamics in patient-derived metastatic prostate cancer cells. [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 243.

Abstract 3074: Addition of ARN509 and docetaxel, alone or in combination, to castration demonstrates improved efficacy and heterogeneity of molecular response in prostate cancer patient-derived xenografts

Abstract Results from clinical and preclinical investigations suggest potential survival benefits from more aggressive early treatment of some patients. Our objective was to evaluate in vivo the efficacy of adding docetaxel (DOC) or ARN509, alone or in combination, to primary androgen ablation and to investigate the associated molecular response and resistance. We used hormone-sensitive LuCaP patient-derived prostate cancer xenografts (PDXs) models 35, 77, and 147 that all express AR but differ significantly in responses to castration (CX) and exhibit diverse molecular characteristics. Tumors were grown in intact mice and when established, mice were randomized for CX alone or CX with DOC or ARN-509, or their combination. Tumor volume (TV), and serum PSA responses were monitored. Tumors were harvested 5 days post-treatment (d5) and at end of study (EOS) for analyses. Our results show differential efficacy of the treatments, mimicking the heterogeneity of clinical response. LuCaP 77 progression was inhibited by CX and the additional treatments (TXs) further inhibited TV, with the most decreased TV and PSA detected in the combination group. When compared to CX, CX combined with ARN509 demonstrated the most significantly improved survival benefit (10w vs 16.5w median survival; HR = 0.2, p&amp;lt;0.001), while CX combined with DOC resulted in a smaller survival benefit (10w vs 12.5w, median survival HR = 0.5 p = 0.036). The combination TX did not result in improved survival due to negative health side effects that resulted in early sacrifice. LuCaP 147 exhibited similar responses to CX as LuCaP 77 but only small additional inhibition of TV with the combination TXs and no significant survival improvements over CX alone. LuCaP 35 was the most responsive to CX and the additional treatments did not enhance the TV inhibition, decreases in serum PSA or survival. Preliminary IHC analyses of the early response (d5 tumors) did not show significant decrease in AR or GR in any of the PDXs but decrease in PSA indicated the efficacy of androgen suppression. Decreased proliferation (Ki67) was detected in LuCaP 77 and LuCaP 147 after CX and more pronounced decreases in the combination vs CX groups in LuCaP 77 and ARN509 and the combination vs CX in LuCaP 147 with no difference detected in LuCaP 35. Analyses of early gene expression responses at d5 by qPCR revealed changes specifically associated with single and combination treatment with ARN509 in LuCaP 77 for both AR-regulated genes (FKBP5, PCNA, and AKR1C3) and proliferation-associated genes (UBE2C3 and NUSAP1). Further analyses of the responses and alteration of genes and proteins associated with AR signaling, proliferation and drug resistance at d5 and EOS are ongoing. Citation Format: Ryan McMullin, Himanshu Gupta, Yashoda Rajpurohit, Holly Nguyen, Lisha Brown, Daniel Sondheim, Gormley Michael, Deborah Ricci, Shibu Thomas, Corey Eva. Addition of ARN509 and docetaxel, alone or in combination, to castration demonstrates improved efficacy and heterogeneity of molecular response in prostate cancer patient-derived xenografts. [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 3074.

Obesity does not promote tumorigenesis of localized patient-derived prostate cancer xenografts.

There are established epidemiological links between obesity and the severity of prostate cancer. We directly tested this relationship by assessing tumorigenicity of patient-derived xenografts (PDXs) of moderate-grade localized prostate cancer in lean and obese severe combined immunodeficiency (SCID) mice. Mice were rendered obese and insulin resistant by high-fat feeding for 6 weeks prior to transplantation, and PDXs were assessed 10 weeks thereafter. Histological analysis of PDX grafts showed no differences in tumor pathology, prostate-specific antigen, androgen receptor and homeobox protein Nkx-3.1 expression, or proliferation index in lean versus obese mice. Whilst systemic obesity per se did not promote prostate tumorigenicity, we next asked whether the peri-prostatic adipose tissue (PPAT), which covers the prostate anteriorly, plays a role in prostate tumorigenesis. In vitro studies in a cellularized co-culture model of stromal and epithelial cells demonstrated that factors secreted from human PPAT are pro-tumorigenic. Accordingly, we recapitulated the prostate-PPAT spatial relationship by co-grafting human PPAT with prostate cancer in PDX grafts. PDX tissues were harvested 10 weeks after grafting, and histological analysis revealed no evidence of enhanced tumorigenesis with PPAT compared to prostate cancer grafts alone. Altogether, these data demonstrate that prostate cancer tumorigenicity is not accelerated in the setting of diet-induced obesity or in the presence of human PPAT, prompting the need for further work to define the at-risk populations of obesity-driven tumorigenesis and the biological factors linking obesity, adipose tissue and prostate cancer pathogenesis.

Patient-Derived Prostate Cancer: from Basic Science to the Clinic.

Systems that model cancer form the backbone of research discovery, and their accuracy and validity are a key determinant to ensure successful translation. In many tumour types, patient-derived specimens are an important model of choice for pre-clinical drug development. In this review, we consider why this has been such a challenge for prostate cancer, resulting in relatively few patient-derived xenografts (PDXs) of prostatic tumours compared to breast cancers, for example. Nevertheless, with only a few patient specimens and PDXs, we exemplify in three vignettes how important new clinical insights were obtained resulting in benefit for future men with prostate cancer.

Abstract B18: Intratumor lysophosphatidylinositol metabolites in a treatment naïve patient-derived bone metastatic prostate cancer xenograft

Abstract A patient-derived bone metastatic prostate cancer (PCa) xenograft (MDA-PCa-183) was used to investigate early resistance mechanisms after androgen deprivation therapy (ADT). The patient-derived xenograft (PDX) expresses wild-type androgen receptor (AR), TMPRSS2-ERG and was found to have homozygous loss of PTEN [1]. MDA-PCa-183 PDX was established as described by Li et al. [2] using a bone biopsy procured from a patient with metastatic PCa prior to ADT. Tumors were grown in intact male immunodeficient mice (n=23) with a subset of mice (n=16) castrated when they reached a size of approximately 900 mm3. At day 11 after castration tumors from 6 mice were harvested and these were considered at maximal androgen ablation (MAA). Of the remaining ten castrated mice, seven responded to androgen ablation by tumor shrinkage or detention of tumor growth (responsive tumors). Tumors of three mice demonstrated sustained growth after castration (relapsed tumors), were harvested between 30 to 40 days after castration and prostate specific antigen (PSA) blood levels were measured. The androgen signaling biomarkers, AR, cytochrome P450 17-alpha-hydroxylase/17,20-lyase (CYP17) and TMPSSR2-ERG protein expression were determined using immunohistochemistry. A biochemical profile consisting of lipid, amino acid, bioenergy, nucleotide (Metabolon, Inc.) and steroid were measured. The levels of testosterone, androstenedione, dihydrotestosterone, progesterone and 17-alpha-hydroxy-progestone were &amp;lt;0.1 pg/mg tissue in MAA and relapsed tumors. Immunostaining showed that tumors from castrate mice expressed AR and CYP17, but were negative for TMPSSR2-ERG. AR and CYP17 were localized in cytoplasm of epithelial cells. PSA levels were low for all castrate tumors (&amp;lt;35 ng/dL). Notably, the lipid profile showed that the lysolipids (LL); 1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 2-stearoylglycerophosphoinositol, were increased in MAA and relapsed tumors (P&amp;lt;0.05) and 2-oleoylglycerophosphoinositol, 1-oleoylglycerophosphoinositol, 2-arachidonoylglycerophosphoinositol levels were higher in MAA tumors versus intact control tumors (P&amp;lt;0.05). Arachidonic acid levels were also increased in MAA and relapsed tumors (P&amp;lt;0.05). In contrast, the eicosanoids (prostaglandin D2, prostaglandin E2 and 5-HETE) levels were similar in the MAA, relapse and control tumors. The long chain fatty acids, palmitate and oleate, levels were similar in MAA, relapsed and control tumors, however stearate levels decreased in MAA and relapsed tumors (P&amp;lt;0.05). Inositol 1-phosphate (I1P), a metabolite of glycerophosphoinositols (GPIs), levels are similar in MAA and control tumors but are increased in relapsed tumors versus control (P&amp;lt;0.05). Our data supports a possible mechanism of AR independent cell proliferation in relapsed MDA-PCa-183 PDX by LL activation of G protein-coupled receptor-55 (GPR55) signaling. The intracellular LL together with ABCC1 and GPR55 generate an autocrine loop that promotes epithelial cell growth [3] after ADT. LL signaling may also support endothelial cell proliferation within the PDX during ADT. I1P levels, a metabolite of LL, indicate that GPIs may act as a paracrine regulator of osteoblast differentiation. Overall, LL biosynthesis and metabolic metabolites may support early resistance to ADT.

Abstract A109: Radium-223 dichloride inhibits tumor growth and tumor-induced bone growth in osteoblastic prostate cancer models

Abstract Radium-223 dichloride (radium-223), an alpha particle-emitting calcium-mimetic, improves overall survival in prostate cancer patients with symptomatic bone metastases. We have defined radium-223 mode-of-action and efficacy in two clinically relevant prostate cancer xenograft models demonstrating PSA expression and osteoblastic growth upon intratibial inoculation of cancer cells. Immunocompromised male mice were inoculated with human LNCaP or patient-derived LuCaP 58 prostate cancer cells in the intratibial compartment and subsequently stratified into treatment groups based on lesion grade and/or serum PSA levels. Radium-223 (300 kBq/kg) or vehicle was administered intravenously, two times at 4-week intervals during the experiment. X-rays and serum samples were obtained biweekly and at sacrifice. Soft tissue tumors were examined macroscopically at sacrifice and tissue samples were collected and processed for γ-counter measurements, micro-CT, autoradiography and histology. Radium-223 treatment inhibited tumor-induced osteoblastic bone growth as indicated by reduced bone volume and surface in LNCaP and LuCaP 58 prostate cancer mouse models. In addition, radium-223 treatment suppressed metabolic activity in bone as evidenced by decreased number of osteoblasts and osteoclasts relative to bone surface and reduced levels of the bone formation marker PINP. Radium-223 resulted in lower PSA values as early as two weeks after the first dose, indicating constrained tumor growth following treatment. This phenomenon was further supported by reduced total bone lesion tissue and tumor area in LNCaP and LuCaP 58 models and increased percentage of necrotic tumor area in the LuCaP 58 model in radium-223-treated mice as compared to vehicle-treated mice. Moreover, DNA double-strand breaks were increased in cancer cells 24 hours post radium-223 treatment in the LuCaP 58 model providing further evidence of anti-tumor effects. Radium-223-treated mice exhibited less visceral metastases in the LuCaP 58 model (not significant). Based on autoradiography, radium-223 was deposited in the intratumoral bone matrix and in conjunction with osteoblasts in osteoblastic metastases. Our results demonstrate that radium-223 dichloride is successfully incorporated into the intratumoral bone matrix and inhibits tumor growth in both cell line- and patient-derived osteoblastic prostate cancer metastasis models. Given the α-particle range of 50-80 μm, potent radiation effects on the tumor microenvironment are evident whereas relevant effects on the more distant bone marrow are not expected. Taken together, radium-223 therapy exhibits a dual mode-of-action that impacts tumor growth and tumor-induced bone reaction, both important players in the destructive vicious cycle of osteoblastic bone metastasis in prostate cancer. Citation Format: Mari I. Suominen, Katja M. Fagerlund, Dominik Mumberg, Karl Ziegelbauer, Sanna-Maria Käkönen, Jussi M. Halleen, Robert L. Vessella, Arne Scholz. Radium-223 dichloride inhibits tumor growth and tumor-induced bone growth in osteoblastic prostate cancer models. [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 A109.