Androgen Deprivation Therapy Resistance Research Articles

Abstract IA3: The Co-Clinical Trial Project

Abstract Herein we report the first application of a “Co-Clinical” approach, and assess the effectiveness of this integrated platform to investigate the genetic basis of resistance to androgen deprivation therapy (ADT) in prostate cancer (CaP). Surprisingly, we find that physical castration counteracts tumour progression in a Pten-loss driven mouse model of CaP through the induction of a potent apoptotic response and proliferation block. We took advantage of these findings to identify “co-clinically” new determinants of ADT resistance. We show that the tumour suppressive response to ADT is completely bypassed as a consequence of concomitant deletion of either Trp53 or Lrf together with Pten, leading to the development of castration resistant prostate cancer (CRPC). Mechanistically, we find that compound loss of Pten with Lrf or Trp53 triggers the down-regulation of the pro-apoptotic factor Xaf1, along with the up-regulation of the steroid 5-alpha-reductase enzyme Srd5a1, hence promoting CaP cell survival in low androgen conditions. Integrated acquisition of data from mouse and human CaP identifies p53/LRF/XAF1/SRD5A1 as a novel predictive signature for castration resistance. Importantly, we further show both in vitro and in vivo that combined inhibition of XAF1/XIAP and AR pathways overcomes castration resistance. Thus these results provide a proof of principle for the “Co-Clinical” approach as a means to facilitate the stratification of human patients and the development of tailored therapeutic treatments. Citation Format: Pier Paolo Pandolfi. The Co-Clinical Trial Project. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA3.

Abstract 2112: AR splice variants ARv7 and ARv567 utilize different mechanisms of cytoplasmic trafficking and nuclear translocation: Therapeutic implications for PC

Abstract Prostate cancer (PC) is one of the most prevalent cancers among men worldwide. While androgen deprivation therapy (ADT) is initially effective in the treatment of PC, resistance emerges resulting in castration-resistant prostate cancer (CRPC) disease, which is driven by continuous signaling from the androgen receptor (AR). Several new drugs designed to inhibit AR signaling (abiraterone and enzalutamide) have been recently approved for PC treatment. However resistance to these next-generation AR inhibitors inevitably occurs and is mediated by adaptive mechanisms that restore AR function. Overexpression of AR splice variants (ARv567 and ARv7), which lack the ligand-binding domain (LBD), was shown to mediate resistance to abiraterone and enzalutamide. Following ADT resistance, CRPC patients are treated with the taxanes Docetaxel (DTX) and Cabazitaxel as first and second line chemotherapy. Our recent work showed that AR binds to microtubules (MTs) utilizing them as tracks for its nuclear translocation, and that taxanes inhibit AR signaling by sequesting AR in the cytoplasm downstream of MT stabilization. We showed that MT binding is mediated by the Hinge region of AR, which is present in ARv567 but missing in ARv7 variant. In contrast to ARv567, ARv7 does not bind MTs and its nuclear localization is not affected by taxane treatment and its expression confers taxane resistance in vitro and in vivo. Since the main nuclear localization signal (NLS) of AR is in the hinge region we hypothesized that ARv7 has a different nuclear translocation mechanism than Arv567 or AR-WT. Co-immunoprecipitation experiments revealed that AR-WT and ARv567 interact with importin-α, and that DTX treatment inhibits this interaction. In contrast, ARv7 does not interact with importin-α and is insensitive to DTX. To monitor the dynamics of AR variant nuclear accumulation we performed live cell confocal microscopy using PC3 cells microinjected with GFP-AR-WT, -ARv7 or -ARv567. Our results showed that both AR-WT and AR-v567 were sensitive to inhibition of the importin pathway, using the importin-β inhibitor importazole, while ARv7 was unaffected. FRAP experiments of M12 cells stably expressing GFP-tagged AR-WT, or ARvs revealed that the recovery after photobleaching of ARv7 nuclear fraction was much faster than that of ARv567 or AR-WT, suggesting enhanced motility of ARv7. FRAP experiments in DTX or importazole treated cells further confirmed the insensitivity of ARv7 to perturbations of the MT network and importin pathway. Taken together our results show that AR-WT and ARv567 utilize MTs and, the importin-α/β pathway for nuclear translocation; while ARv7 is MTs and importin independent. Elucidation of the mechanisms of cytoplasmic trafficking and nuclear translocation of ARv7 variant will allow us to identify new druggable targets in order to revert resistance to taxanes and next generation AR targeting drugs. Citation Format: Luigi Portella, Paraskevi Giannakakou. AR splice variants ARv7 and ARv567 utilize different mechanisms of cytoplasmic trafficking and nuclear translocation: Therapeutic implications for PC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2014-2112

MicroRNA expressions associated with progression of prostate cancer cells to antiandrogen therapy resistance

BackgroundDevelopment of resistance to androgen deprivation therapy (ADT) is a major obstacle for the management of advanced prostate cancer. Therapies with androgen receptor (AR) antagonists and androgen withdrawal initially regress tumors but development of compensatory mechanisms including AR bypass signaling leads to re-growth of tumors. MicroRNAs (miRNAs) are small regulatory RNAs that are involved in maintenance of cell homeostasis but are often altered in tumor cells.ResultsIn this study, we determined the association of genome wide miRNA expression (1113 unique miRNAs) with development of resistance to ADT. We used androgen sensitive prostate cancer cells that progressed to ADT and AR antagonist Casodex (CDX) resistance upon androgen withdrawal and treatment with CDX. Validation of expression of a subset of 100 miRNAs led to identification of 43 miRNAs that are significantly altered during progression of cells to treatment resistance. We also show a correlation of altered expression of 10 proteins targeted by some of these miRNAs in these cells.ConclusionsWe conclude that dynamic alterations in miRNA expression occur early on during androgen deprivation therapy, and androgen receptor blockade. The cumulative effect of these altered miRNA expression profiles is the temporal modulation of multiple signaling pathways promoting survival and acquisition of resistance. These early events are driving the transition to castration resistance and cannot be studied in already developed CRPC cell lines or tissues. Furthermore our results can be used a prognostic marker of cancers with a potential to be resistant to ADT.

PI3K-AKT-mTOR signaling in prostate cancer progression and androgen deprivation therapy resistance.

Prostate cancer (PCa) is the second most common malignancy among men in the world. Castration-resistant prostate cancer (CRPC) is the lethal form of the disease, which develops upon resistance to first line androgen deprivation therapy (ADT). Emerging evidence demonstrates a key role for the PI3K-AKT-mTOR signaling axis in the development and maintenance of CRPC. This pathway, which is deregulated in the majority of advanced PCas, serves as a critical nexus for the integration of growth signals with downstream cellular processes such as protein synthesis, proliferation, survival, metabolism and differentiation, thus providing mechanisms for cancer cells to overcome the stress associated with androgen deprivation. Furthermore, preclinical studies have elucidated a direct connection between the PI3K-AKT-mTOR and androgen receptor (AR) signaling axes, revealing a dynamic interplay between these pathways during the development of ADT resistance. Thus, there is a clear rationale for the continued clinical development of a number of novel inhibitors of the PI3K pathway, which offer the potential of blocking CRPC growth and survival. In this review, we will explore the relevance of the PI3K-AKT-mTOR pathway in PCa progression and castration resistance in order to inform the clinical development of specific pathway inhibitors in advanced PCa. In addition, we will highlight current deficiencies in our clinical knowledge, most notably the need for biomarkers that can accurately predict for response to PI3K pathway inhibitors.

The HIF1A functional genetic polymorphism at locus +1772 associates with progression to metastatic prostate cancer and refractoriness to hormonal castration

The hypoxia inducible factor 1 alpha (HIF1a) is a key regulator of tumour cell response to hypoxia, orchestrating mechanisms known to be involved in cancer aggressiveness and metastatic behaviour. In this study we sought to evaluate the association of a functional genetic polymorphism in HIF1A with overall and metastatic prostate cancer (PCa) risk and with response to androgen deprivation therapy (ADT).The HIF1A +1772 C>T (rs11549465) polymorphism was genotyped, using DNA isolated from peripheral blood, in 1490 male subjects (754 with prostate cancer and 736 controls cancer-free) through Real-Time PCR. A nested group of cancer patients who were eligible for androgen deprivation therapy was followed up. Univariate and multivariate models were used to analyse the response to hormonal treatment and the risk for developing distant metastasis. Age-adjusted odds ratios were calculated to evaluate prostate cancer risk.Our results showed that patients under ADT carrying the HIF1A +1772 T-allele have increased risk for developing distant metastasis (OR, 2.0; 95%CI, 1.1–3.9) and an independent 6-fold increased risk for resistance to ADT after multivariate analysis (OR, 6.0; 95%CI, 2.2–16.8). This polymorphism was not associated with increased risk for being diagnosed with prostate cancer (OR, 0.9; 95%CI, 0.7–1.2).The HIF1A +1772 genetic polymorphism predicts a more aggressive prostate cancer behaviour, supporting the involvement of HIF1a in prostate cancer biological progression and ADT resistance. Molecular profiles using hypoxia markers may help predict clinically relevant prostate cancer and response to ADT.

Castration-resistant prostate cancer: a strategy to enhance response to androgen deprivation

A new research study evaluated the mechanisms of prostate cancer (PCa) resistance to androgen deprivation therapy (ADT) and progression to castrate-resistant disease. Using a coclinical approach, the investigators were able to identify key genetic determinants of ADT resistance, gain insight into the molecular pathways that play a key role in this transition and propose a potential management strategy to overcome ADT resistance in select cancers. This represents a step forward towards personalized medicine guided by specific molecular markers of prostate cancer enabling more effective therapies targeted towards altered metabolic pathways.

GEMMs Shine a Light on Resistance to Androgen Deprivation Therapy for Prostate Cancer

Androgen deprivation therapy (ADT) for advanced prostate cancer inexorably leads to resistance, and clinically useful biomarkers are lacking. The value of genetically engineered mice for coclinical studies isclearly demonstrated in a recent publication that reveals XAF1, XIAP, and SRD5A1 as novel predictive biomarkers and therapeutic targets for ADT resistance.

DCE and DW MRI in monitoring response to androgen deprivation therapy in patients with prostate cancer: A feasibility study

Androgen deprivation therapy (ADT) is a key primary treatment for advanced and metastatic prostate cancer and is an important neoadjuvant before radiotherapy. We evaluated 3.0 T dynamic contrast-enhanced MRI and diffusion-weighted (DW) MRI in monitoring ADT response. Twenty-three consecutive patients with prostate cancer treated by primary ADT were included. Imaging was performed at baseline and 3 months posttreatment with ADT. After 3 months therapy there was a significant reduction in all dynamic contrast-enhanced MRI parameters measured in tumor regions of interest (K(trans), k(ep), v(p), IAUGC-90); P < 0.001. Areas of normal-appearing peripheral zone showed no significant change; P = 0.285-0.879. Post-ADT, there was no significant change in apparent diffusion coefficient values in tumors, whilst apparent diffusion coefficient values significantly decreased in areas of normal-appearing peripheral zone, from 1.786 × 10(-3) mm(2) /s to 1.561 × 10(-3) mm(2) /s; P = 0.007. As expected the median Prostate-Specific Antigen (PSA) significantly reduced from 30 ng/mL to 1.5 ng/mL posttreatment, and median prostate volume dropped from 47.6 cm(3) to 24.9 cm(3) ; P < 0.001. These results suggest that dynamic contrast-enhanced MRI and diffusion-weighted MRI offer different information but that both could prove useful adjuncts to the anatomical information provided by T2-weighted imaging. dynamic contrast-enhanced as a marker of angiogenesis may help demonstrate ADT resistance and diffusion-weighted imaging may be more accurate in determining presence of tumor cell death versus residual tumor.

Abstract 5376: Antisense oligonucleotide targeting insulin-like growth factor-1 receptor (IGF-1R) enhances paclitaxel sensitivity in a castrate-resistant and paclitaxel-resistant prostate cancer model

Abstract While prostate cancer (PCa) initially responds to androgen deprivation therapy (ADT), castrate-resistant disease (CRPC) relapse is inevitable. Altered expression of insulin-like growth factor (IGF) axis components have been consistently found in PCa and associated with adaptive growth and survival signaling implicated in CRPC progression. These activities are mediated by activation of the IGF-I receptor (IGF-IR). While CRPC patients treated with taxanes (paclitaxel or docetaxel) alone or in combination with estramustine phosphate or predonisone show a survival benefit, development of chemotherapeutic resistance eventually occurs. Therefore novel strategies targeting the molecular basis of ADT and chemotherapy resistance are required. In this study, we assess the potency and anti-cancer activity of a 2′-MOE-modified antisense oligonucleotide (ASO) targeting human IGF-IR, ATL1101, on the CRPC cell line, PC3 and a paclitaxel-resistant androgen-independent PC3-derived cell line, PtxR PC3, in vitro and in vivo. PtxR PC3 cells were established by culturing in step-wise increased drug concentrations. IGF-IR mRNA and protein expression in ATL1101- and control oligonucleotides (ODN)-treated PtxR PC3 cells were measured by QT-PCR and immunoblotting. The effect of IGF-1R ASO and paclitaxel combination therapy on parental and PtxR PC3 cell growth and apoptosis in vitro was examined by crystal violet assay and flow cytometry. In vitro combination index (CI) was calculated by Calcusyn® softoware. For xenograft studies, parental and PtxR PC-3 cells were inoculated s.c. in the flanks of athymic male nude mice and tumor volume kinetics were compared for mice treated with paclitaxel injected i.v. and either ATL1101 or control ODN injected i.p. In vitro, the IC50 for paclitaxel in PtxR PC3 was 20-fold higher than that in parental cells. PtxR PC3 also show significant cross-resistance to docetaxel and mitoxantrone. We observed equivalent dose- and sequence-specific suppression of IGF-IR mRNA and protein expression, and comparable decreased proliferation and increased apoptosis in ATL1101-treated parental and PtxR PC3 cells in vitro. Combination of ATL1101 with paclitaxel showed CI values below 1 at the IC50, IC75, IC90, suggesting the drug interactions resulted in synergy. In vivo, ATL1101 significantly increased sensitivity of PtxR PC3 tumors to combination therapy with paclitaxel as compared to control ODN in murine xenografts. This study reports the first preclinical proof-of-principle data that this novel IGF-IR ASO suppresses growth of paclitaxel resistant PC tumors, synergistically enhancing paclitaxel sensitivity in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5376.

A Role of Androgen Receptor Protein in Cell Growth of an Androgen-Independent Prostate Cancer Cell Line

Prostate cancer, which develops due to androgen and is initially responsive to androgen deprivation therapy, often comes to acquire androgen deprivation therapy resistance in short order. We investigated the role of androgen receptor (AR) protein in an androgen-independent prostate cancer cell line using AR ligands and AR siRNA. Although the androgen-independent cell line scarcely responded to AR ligands, their growth was attenuated by ablation of AR protein by siRNA.