Rute B. Marques

Androgen receptor modifications in prostate cancer cells upon long-termandrogen ablation and antiandrogen treatment.

To study the mechanisms whereby androgen‐dependent tumors relapse in patients undergoing androgen blockade, we developed a novel progression model for prostate cancer. The PC346C cell line, established from a transurethral resection of a primary tumor, expresses wild‐type (wt) androgen receptor (AR) and secretes prostate‐specific antigen (PSA). Optimal proliferation of PC346C requires androgens and is inhibited by the antiandrogen hydroxyflutamide. Orthotopic injection in the dorsal‐lateral prostate of castrated athymic nude mice did not produce tumors, whereas fast tumor growth occurred in sham‐operated males. Three androgen‐independent sublines were derived from PC346C upon long‐term in vitro androgen deprivation: PC346DCC, PC346Flu1 and PC346Flu2. PC346DCC exhibited androgen‐insensitive growth, which was not inhibited by flutamide. AR and PSA were detected at very low levels, coinciding with background AR activity in a reporter assay, which suggests that these cells have bypassed the AR pathway. PC346Flu1 and PC346Flu2 were derived by culture in steroid‐stripped medium supplemented with hydroxyflutamide. PC346Flu1 strongly upregulated AR expression and showed 10‐fold higher AR activation than the parental PC346C. PC346Flu1 proliferation was inhibited in vitro by R1881 at 0.1 nM concentration, consistent with a slower tumor growth rate in intact males than in castrated mice. PC346Flu2 carries the well‐known T877A AR mutation, causing the receptor to become activated by diverse nonandrogenic ligands including hydroxyflutamide. Array‐based comparative genomic hybridization revealed little change between the various PC346 lines. The common alterations include gain of chromosomes 1, 7 and 8q and loss of 13q, which are frequently found in prostate cancer. In conclusion, by in vitro hormone manipulations of a unique androgen‐dependent cell line expressing wtAR, we successfully reproduced common AR modifications observed in hormone‐refractory prostate cancer: downregulation, overexpression and mutation.

Bypass mechanisms of the androgen receptor pathway in therapy-resistant prostate cancer cell models.

Background Prostate cancer is initially dependent on androgens for survival and growth, making hormonal therapy the cornerstone treatment for late-stage tumors. However, despite initial remission, the cancer will inevitably recur. The present study was designed to investigate how androgen-dependent prostate cancer cells eventually survive and resume growth under androgen-deprived and antiandrogen supplemented conditions. As model system, we used the androgen-responsive PC346C cell line and its therapy-resistant sublines: PC346DCC, PC346Flu1 and PC346Flu2. Methodology/Principal Findings Microarray technology was used to analyze differences in gene expression between the androgen-responsive and therapy-resistant PC346 cell lines. Microarray analysis revealed 487 transcripts differentially-expressed between the androgen-responsive and the therapy-resistant cell lines. Most of these genes were common to all three therapy-resistant sublines and only a minority (∼5%) was androgen-regulated. Pathway analysis revealed enrichment in functions involving cellular movement, cell growth and cell death, as well as association with cancer and reproductive system disease. PC346DCC expressed residual levels of androgen receptor (AR) and showed significant down-regulation of androgen-regulated genes (p-valueu200a=u200a10−7). Up-regulation of VAV3 and TWIST1 oncogenes and repression of the DKK3 tumor-suppressor was observed in PC346DCC, suggesting a potential AR bypass mechanism. Subsequent validation of these three genes in patient samples confirmed that expression was deregulated during prostate cancer progression. Conclusions/Significance Therapy-resistant growth may result from adaptations in the AR pathway, but androgen-independence may also be achieved by alternative survival mechanisms. Here we identified TWIST1, VAV3 and DKK3 as potential players in the bypassing of the AR pathway, making them good candidates as biomarkers and novel therapeutical targets.

High Efficacy of Combination Therapy Using PI3K/AKT Inhibitors with Androgen Deprivation in Prostate Cancer Preclinical Models

BACKGROUNDnThe phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT pathway is frequently activated during prostate cancer (PCa) progression through loss or mutation of the phosphatase and tensin homolog (PTEN) gene. Following the androgen receptor (AR) pathway, it is the second major driver of PCa growth.nnnOBJECTIVEnTo assess efficacy of novel PI3K/AKT-targeted therapies in PCa models, as a single agent and in combination with androgen deprivation.nnnDESIGN, SETTING, AND PARTICIPANTSnTwelve human PCa cell lines were tested in vitro for sensitivity to the AKT inhibitor AZD5363 and the PI3K beta/delta inhibitor AZD8186. The combination of AZD5363 and AZD8186 with castration was evaluated in vivo in PTEN-negative versus PTEN-positive patient-derived xenografts. Tumors and plasma were collected for biomarker analysis.nnnOUTCOME MEASUREMENTS AND STATISTICAL ANALYSISnIn vitro growth inhibition was determined by methylthiazolyldiphenyl-tetrazolium bromide assay. In vivo efficacy was monitored by caliper measurements of subcutaneous tumor volume. PI3K/AKT and AR pathway activity was analyzed by Western blot, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction.nnnRESULTS AND LIMITATIONSnAZD5363 and AZD8186 inhibited in vitro growth of 10 of 12 and 7 of 12 PCa cell lines, respectively, with increased sensitivity under androgen depletion. In vivo, AZD5363 and AZD8186 as single agents significantly inhibited growth of PTEN-negative PC346C xenografts compared to placebo by 60% and 66%, respectively. Importantly, combination of either agent with castration resulted in long-lasting tumor regression, which persisted after treatment cessation. Expression of AR-target genes kallikrein-related peptidase 3 (KLK3, also known as PSA); transmembrane protease, serine 2 (TMPRSS2); and FK506 binding protein 5 (FKBP5) was upregulated after PI3K/AKT inhibition. Neither compound inhibited tumor growth in the PTEN-positive PC310 model.nnnCONCLUSIONSnCombination with hormonal therapy improved efficacy of PI3K/AKT-targeted agents in PTEN-negative PCa models. Upregulation of AR-target genes upon PI3K/AKT inhibition suggests a compensatory crosstalk between the PI3K-AR pathways. These data strongly advocate for further clinical evaluation.nnnPATIENT SUMMARYnInactivation of the PTEN gene is a common event promoting prostate cancer (PCa) progression. This preclinical study illustrates the potent anticancer activity of novel PTEN-targeted drugs on PCa models, particularly in combination with hormonal therapy.

Movember GAP1 PDX project: An international collection of serially transplantable prostate cancer patient-derived xenograft (PDX) models.

While it has been challenging to establish prostate cancer patient‐derived xenografts (PDXs), with a take rate of 10‐40% and long latency time, multiple groups throughout the world have developed methods for the successful establishment of serially transplantable human prostate cancer PDXs using a variety of immune deficient mice. In 2014, the Movember Foundation launched a Global Action Plan 1 (GAP1) project to support an international collaborative prostate cancer PDX program involving eleven groups. Between these Movember consortium members, a total of 98 authenticated human prostate cancer PDXs were available for characterization. Eighty three of these were derived directly from patient material, and 15 were derived as variants of patient‐derived material via serial passage in androgen deprived hosts. A major goal of the Movember GAP1 PDX project was to provide the prostate cancer research community with a summary of both the basic characteristics of the 98 available authenticated serially transplantable human prostate cancer PDX models and the appropriate contact information for collaborations. Herein, we report a summary of these PDX models.

Tumor heterogeneity, aggressiveness, and immune cell composition in a novel syngeneic PSA-targeted Pten knockout mouse prostate cancer (MuCaP) model

Prostate cancer is recognized as a heterogeneous disease demanding appropriate preclinical models that reflect tumor complexity. Previously, we established the PSA‐Cre;PtenLoxP/LoxP genetic engineered mouse model (GEMM) for prostate cancer reflecting the various stages of tumor development. Prostate tumors in this Pten KO model slowly develop, requiring more than 10 months. In order to enhance its practical utility, we established a syngeneic panel of cell lines derived from PSA‐Cre targeted Pten KO tumors, designated the mouse prostate cancer (MuCap) model.

Patient-Derived Xenograft Models of Prostate Cancer

Prostate cancer is the second leading cause of cancer in men in western countries. It is characterized by specific clinical features as androgen responsiveness, hence the initial efficacy of endocrine therapies. Recurrent prostate cancer inevitably progresses toward castration resistance with metastatic spread to the bone, responsible for most of the experienced morbidity. Research into the mechanisms of tumor growth and disease progression have been hampered significantly by the lack of experimental models, as a result of the rarity of spontaneous prostate cancer in other mammals and lack of expertise in growing prostate cancer tissue directly from the patient in a Petri dish or in immune-deficient mice to generate patient-derived xenograft (PDX) models. Major efforts by several research groups worldwide have resulted in the establishment of a substantial set of PDXs. This review highlights advances in the development of prostate cancer PDX models, with an overview of the contemporary sets of prostate cancer PDXs available and their use in experimental and translational research. Several approaches are outlined that aim to improve existing prostate cancer PDXs to better capture the complexity of the disease including orthotopic engraftment procedures, humanization of the immune and endocrine murine system, and efforts to replicate the biological diversity by extending the PDX collections with resistant disease stages. Finally, novel screening approaches, such as co-clinical trials and PDX clinical trials, are being developed that increase preclinical value and enhance translational power of (prostate cancer) PDXs.

Abstract B185: High efficacy of combination therapy of PI3K/AKT inhibitors with androgen deprivation in prostate cancer.

Introduction and Objectives: A current challenge in prostate cancer (PCa) research is how to treat metastatic patients that have relapsed after androgen deprivation therapy. The PI3K/AKT pathway is frequently activated during PCa progression, with PTEN loss or mutation reported in up to 60% of advanced tumors, making it an interesting target for therapy. Recent reports have suggested a potential cross-talk between PI3K/AKT and androgen receptor (AR) pathway (1, 2). The present study aimed to assess the efficacy of combination therapy in PTEN-positive and PTEN-negative PCa models in vitro and in vivo and elucidate the mechanisms of interaction between both pathways. Material and Methods: The PI3K beta/delta inhibitor AZD8186 and AKT inhibitor AZD5363 were supplied by AstraZeneca (Macclesfield, UK). Growth response was tested in PTEN-positive (DU145 and VCAP) and PTEN-negative (LNCaP, PC346C, PC346Flu1 and PC346DCC) PCa cell lines by MTT assay. As response biomarkers, PI3K/AKT downstream targets phospho-AKT, phospho-PRAS40 and phospho-GSK3 were analyzed by western blotting. AR pathway activity was assessed by quantitative RT-PCR against AR, PSA, and TMPRSS2. Efficacy of both compounds was also tested in vivo in the PTEN-negative PC346C subcutaneous xenograft in combination with surgical castration. Results: AZD8186 and AZD5363 inhibited growth of all the PTEN-negative cell lines (LNCaP, PC346C, PC346Flu1, and PC346DCC) at nanomolar concentrations, while PTEN-positive cells were less sensitive (VCAP) or resistant (DU145). A dose dependent decrease in phosphorylation of AKT and its downstream targets was observed after treatment with AZD8186, which correlated with inhibition of cell growth. AZD5363 treatment resulted in increased AKT phosphorylation, as this compound stabilizes AKT in an inactive hyperphosphorylated state. Both AZD8186 and AZD5363 significantly inhibited in vivo growth of PC346C xenograft compared to that of placebo treated controls (respectively, 66% and 60% tumor growth inhibition after 28 days of treatment). Androgen depletion strongly increased the sensitivity to the PI3K/AKT inhibitors both in vitro and in vivo, with complete abrogation of tumor growth in castrated tumor-bearing mice. Additionally, up-regulation of AR signaling was observed after inhibition of the PI3K/AKT pathway, further lending support for cross-talk between both pathways. Conclusions: In conclusion, AZD8186 and AZD5363 efficiently targeted PI3K/AKT pathway and inhibited the growth of PTEN-negative PCa models in vitro and in vivo, particularly in combination with androgen depletion. These data suggest cross-talk between PI3K/AKT and AR pathways and indicate that for optimal antitumor efficacy inhibition of both pathways is required. The functional mechanisms underlying this cross-talk are the focus of our current research. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B185. Citation Format: Rute Marques, Ashraf Aghai, Debra Stuurman, Corrina de Ridder, Agnes Boer, Sander Hoeben, Jan Trapman, Wytske M. van Weerden. High efficacy of combination therapy of PI3K/AKT inhibitors with androgen deprivation in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B185.