Critical role of androgen receptor level in prostate cancer cell resistance to new generation antiandrogen enzalutamide.
ABSTRACT: Enzalutamide is an androgen receptor (AR) inhibitor approved for therapy of metastatic castration resistant prostate cancer. However, clinical application revealed that 30 to 40% of patients acquire resistance after a short period of treatment. Currently, the molecular mechanisms underlying such resistances are not completely understood, partly due to a lack of model systems. In the present study we established three different cellular models of enzalutamide resistance including a cell line with wild type AR (LAPC4), DuCaP cells which overexpress wild-type AR, as well as a cell which has been adapted to long term androgen ablation (LNCaP Abl) and harbors the AR T878A mutation. After 10 months of cultivation, sustained growth in the presence of enzalutamide was achieved. When compared to controls, resistant cells exhibit significantly decreased sensitivity to enzalutamide as measured with 3[H]thymidine incorporation and WST assay. Moreover, these cell models exhibit partly re-activated AR signaling despite presence of enzalutamide. In addition, we show that enzalutamide resistant cells are insensitive to bicalutamide but retain considerable sensitivity to abiraterone. Mechanistically, enzalutamide resistance was accompanied by increased AR and AR-V7 mRNA and protein expression as well as AR gene amplification, while no additional AR mutations have been identified.
Project description:Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool.
Project description:Despite the introduction of novel therapies that maximally decrease androgen-receptor (AR) signaling activity, metastatic castration-resistant prostate cancer (mCRPC) remains a lethal disease. Even though abiraterone and enzalutamide represent breakthroughs in the treatment of mCRPC and have demonstrated significant survival benefits, a significant proportion of patients have primary resistance to these agents and virtually all patients develop secondary resistance. While the mechanisms of resistance to these agents are not fully understood, many hypotheses of AR-dependent and AR-independent mechanisms are emerging, including upregulation of AR and cytochrome P450 17?-hydroxylase/17,20-lyase (CYP17), induction of AR splice variants, AR point mutations, upregulation of glucocorticoid receptor, activation of alternative oncogenic signaling pathways, neuroendocrine transformation, and immune evasion via programmed death-ligand 1 upregulation. The aim of this review is to summarize the most clinically relevant mechanisms of resistance to novel androgen-directed agents, focusing on escape from enzalutamide and abiraterone.
Project description:Patients with metastatic castration-resistant prostate cancer (CRPC) frequently develop therapeutic resistance to taxane chemotherapy and antiandrogens. Cabazitaxel is a second-line taxane chemotherapeutic agent that provides additional survival benefits to patients with advanced disease. In this study, we sought to identify the mechanism of action of combined cabazitaxel and androgen receptor (AR) targeting in preclinical models of advanced prostate cancer. We found that cabazitaxel induced mitotic spindle collapse and multinucleation by targeting the microtubule depolymerizing kinesins and inhibiting AR. In androgen-responsive tumors, treatment with the AR inhibitor, enzalutamide, overcame resistance to cabazitaxel. Combination treatment of human CRPC xenografts with cabazitaxel and enzalutamide reversed epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) and led to multinucleation, while retaining nuclear AR. In a transgenic mouse model of androgen-responsive prostate cancer, cabazitaxel treatment induced MET, glandular redifferentiation, and AR nuclear localization that was inhibited by androgen deprivation. Collectively, our preclinical studies demonstrate that prostate tumor resistance to cabazitaxel can be overcome by antiandrogen-mediated EMT-MET cycling in androgen-sensitive tumors but not in CRPC. Moreover, AR splice variants may preclude patients with advanced disease from responding to cabazitaxel chemotherapy and antiandrogen combination therapy. This evidence enables a significant insight into therapeutic cross-resistance to taxane chemotherapy and androgen deprivation therapy in advanced prostate cancer.
Project description:Molecular mechanisms underlying resistance to androgen deprivation therapy (ADT) and, in particular, to antiandrogen Enzalutamide, in treating castration-resistant prostate cancer (CRPC), remain incompletely understood. Through screening >120 CRPC patient samples, we observed 3 expression patterns of androgen receptor (AR) protein: primarily nuclear (nuc-AR), mixed nuclear/cytoplasmic expression (nuc/cyto-AR), and low/no expression (AR-/lo). Xenograft CRPC modeling in 4 models (i.e., LNCaP, VCaP, LAPC4, and LAPC9) recapitulated the 3 AR expression patterns in castration-resistant tumors developed from parental androgen-dependent tumors. Strikingly, although the 3 CRPC models that retained AR expression (LNCaP, VCaP, and LAPC4) responded, to different levels and in different kinetics, to Enzalutamide, the AR-/lo LAPC9 CRPC was completely refractory to Enzalutamide. By combining whole-genome RNA-Seq and biochemical analyses together with experimental combinatorial therapy in the LNCaP and LAPC9 models, we identified BCL-2 as a critical therapeutic target in both AR+/hi and AR-/lo, Enzalutamide-resistant CRPC models. Overall design: 1) In LNCaP model, total RNA profiles of 4 pairs of LNCaP AD, LNCaP Primary CRPC and LNCaP Secondary CRPC tumors were generated by deep sequencing. 2) In LAPC9 model, total RNA profiles of 5 paris of LAPC9 AD and LAPC9 CRPC tumors were generated by deep sequencing.
Project description:Prostate cancer (PCa) is the most common malignancy, and the third leading cancer-related cause of death among men of the Western world. Upon PCa progression into metastatic disease, androgen deprivation therapy is applied as the first-line treatment, and has been shown to be effective in most patients, leading to a decrease in serum prostate-specific antigen and relief of disease-related symptoms. However, advanced PCa almost inevitably progresses to a castration-resistant state, and is currently regarded as incurable. The large body of evidence indicates that PCa cells remain dependent on androgen receptor (AR) signaling even in an androgen-deprived environment. As such, development of drugs that target AR and AR signaling pathways have become one of the major milestones in treatment of castration-resistant PCa (CRPC). Nevertheless, currently available therapies that target AR signaling are still regarded as palliative and more potent therapies are in great need. Over the past few years, a wide range of novel therapies has entered clinical trial for treatment of CRPC, including androgen synthesis inhibitors (abiraterone acetate), chemotherapeutic agents (docetaxel and cabazitaxel), and immunotherapies (sipuleucel-T). In this context, enzalutamide (previously referred to as MDV3100) is a novel second generation antiandrogen that has been demonstrated to significantly improve survival in men with metastatic CRPC in several clinical trials. In this paper we summarize recently completed and ongoing clinical trials of enzalutamide, and briefly discuss the efficacy of the novel antiandrogen therapy and its limitations for treatment of CRPC.
Project description:Genomic amplification of the androgen receptor (<i>AR</i>) is an established mechanism of antiandrogen resistance in prostate cancer. Here, we show that the magnitude of <i>AR</i> signaling output, independent of <i>AR</i> genomic alteration or expression level, also contributes to antiandrogen resistance, through upregulation of the coactivator <i>GREB1</i>. We demonstrate 100-fold heterogeneity in <i>AR</i> output within human prostate cancer cell lines and show that cells with high <i>AR</i> output have reduced sensitivity to enzalutamide. Through transcriptomic and shRNA knockdown studies, together with analysis of clinical datasets, we identify <i>GREB1</i> as a gene responsible for high <i>AR</i> output. We show that <i>GREB1</i> is an <i>AR</i> target gene that amplifies <i>AR</i> output by enhancing <i>AR</i> DNA binding and promoting <i>EP300</i> recruitment. <i>GREB1</i> knockdown in high <i>AR</i> output cells restores enzalutamide sensitivity <i>in vivo</i>. Thus, <i>GREB1</i> is a candidate driver of enzalutamide resistance through a novel feed forward mechanism.
Project description:Prostate cancer (PCa) is the most common malignancy among Western men and the second leading-cause of cancer related deaths. For men who develop metastatic castration resistant PCa (mCRPC), survival is limited, making the identification of novel therapies for mCRPC critical. We have found that deficient lipid oxidation via carnitine palmitoyltransferase (CPT1) results in decreased growth and invasion, underscoring the role of lipid oxidation to fuel PCa growth. Using immunohistochemistry we have found that the CPT1A isoform is abundant in PCa compared to benign tissue (n=39, p<0.001) especially in those with high-grade tumors. Since lipid oxidation is stimulated by androgens, we have evaluated the synergistic effects of combining CPT1A inhibition and anti-androgen therapy. Mechanistically, we have found that decreased CPT1A expression is associated with decreased AKT content and activation, likely driven by a breakdown of membrane phospholipids and activation of the INPP5K phosphatase. This results in increased androgen receptor (AR) action and increased sensitivity to the anti-androgen enzalutamide. To better understand the clinical implications of these findings, we have evaluated fat oxidation inhibitors (etomoxir, ranolazine and perhexiline) in combination with enzalutamide in PCa cell models. We have observed a robust growth inhibitory effect of the combinations, including in enzalutamide-resistant cells and mouse TRAMPC1 cells, a more neuroendocrine PCa model. Lastly, using a xenograft mouse model, we have observed decreased tumor growth with a systemic combination treatment of enzalutamide and ranolazine. In conclusion, our results show that improved anti-cancer efficacy can be achieved by co-targeting the AR axis and fat oxidation via CPT1A, which may have clinical implications, especially in the mCRPC setting.
Project description:Despite enzalutamide's efficacy in delaying the progression of metastatic castration-resistant prostate cancer (CRPC), resistance to this anti-androgen inevitably occurs. Several studies have revealed that the signal transducer and activator of transcription (STAT) 5 plays a role in tumour progression and development of drug resistance such as enzalutamide. Data mining revealed heterogeneous expression of STAT5 in enzalutamide-treated mCRPC patients and enzalutamide-resistant prostate cancer (PCa). Isobologram analysis revealed that the STAT5 inhibitor pimozide combined with enzalutamide has? additive and synergistic inhibitory effects on cell viability in the used models. Functional analysis with siRNA-mediated STAT5 knockdown yielded divergent results. The LNCaP-derived cell line MR49F could be resensitised to enzalutamide by siRNA-mediated STAT5b-knock-down. In contrast, neither STAT5a nor STAT5b knockdown resensitised enzalutamide-resistant LAPC4-EnzaR cells to enzalutamide. In conclusion, our results indicate that STAT5 may be a possible target in a subgroup of enzalutamide-resistant PCa. However, based on the data presented here, a general role of STAT5 in enzalutamide-resistance and its potential as a therapeutic target could not be shown.
Project description: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.