by As genomic profiling of PCa patients becomes increasingly feasible, the developed AR functional encyclopedia could provide decision-makers with a tool to guide the treatment choice for PCa patients based on their AR mutation status. cancer genomics portal data base [18,19], we found that the frequency of AR mutants can vary between patient cohorts and can reach up to 15% in metastatic CRPC [4,20]. is common in prostate cancer (PCa), and the gain-of-function mutations in human androgen receptor (AR) represent one of the most dominant drivers of progression to resistance to AR pathway inhibitors (ARPI). Previously, we evaluated the in vitro response of 24 AR mutations, identified in men with castration-resistant PCa, to five AR antagonists. In the current work, we evaluated 44 additional PCa-associated AR mutants, reported in the literature, and thus SB 525334 expanded the study SB 525334 of the effect of darolutamide to a total of 68 AR mutants. Unlike other AR antagonists, we demonstrate that darolutamide exhibits consistent efficiency against all characterized gain-of-function mutations in a full-length AR. Additionally, the response of the AR mutants to clinically used bicalutamide and enzalutamide, as well as to major endogenous steroids (DHT, estradiol, progesterone and hydrocortisone), was also investigated. As genomic profiling of PCa patients becomes increasingly feasible, the developed AR functional encyclopedia could provide decision-makers with a tool to guide the treatment choice for PCa patients based on their AR mutation status. cancer genomics portal data base [18,19], we found that the frequency of AR mutants can vary between patient cohorts and can reach up to 15% in metastatic CRPC SB 525334 [4,20]. We also reported the results of functional characterization of 24 AR mutants identified in liquid biopsies from CRPC patients or reported in the literature, and demonstrated that all these mutants exhibited resistance to at least one of four available AR antagonists, including hydroxyflutamide, bicalutamide, enzalutamide and apalutamide [13]. The remarkable plasticity of the AR under selective pressure of AR pathway inhibition (ARPI), coupled with the marked heterogeneity and negative prognostic significance of its cfDNA mutants, indicates that there is no one size fits all treatment for PCa patients. Furthermore, the results of our initial functional characterization of clinically observed AR mutants clearly indicate the need for novel AR antagonist(s) capable of inhibiting all forms of AR mutants. Darolutamide, a structurally distinct AR antagonist compared to ABS antagonists hydroxyflutamide, bicalutamide, enzalutamide and apalutamide (Figure 1), showed complete inhibition of several documented AR-resistant mutants [21] and might provide broader antagonist activity with emergent AR mutants. Hence, we evaluated the inhibition of 44 PCa-associated AR mutants identified in the literature and public databases by darolutamide. Additionally, the response of the AR mutants to most clinically used bicalutamide and enzalutamide, as well as to major endogenous steroids (DHT, estradiol, progesterone and hydrocortisone), was investigated. Open in a separate window Figure 1 Chemical structures of clinically used AR antagonists. 2. Materials and Methods 2.1. Constructs Full-length human AR (WT-AR) was encoded on a pcDNA3.1 expression plasmid (Life Technologies, Carlsbad, CA, USA). The AR point mutations were generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) as per manufacturers instructions using WT-AR as the template. The mutagenic oligonucleotide primers were designed individually with the desired mutation in the middle of the primer with ~10C15 bases of correct sequence on both sides (the sequences of the used primers are presented in Table S1). 2.2. Steroid Activation Assay PC3 cells lacking the AR and authenticated by Genetica using STR profiling were maintained in RPMI 1640 media (Life Technologies) and 5% FBS (Hyclone Thermo Fisher Scientific, Waltham, MA, USA) at 37 C and 5% CO2. Cultures were routinely monitored for mycoplasma contamination. For the steroid activation assay, IkB alpha antibody cells were seeded in 96-well plates (5000 cells/well) in RPMI 1640 medium with 5% charcoal-stripped serum (CSS) (Hyclone). After 24 h, cells were co-transfected with 25 ng of wild-type or mutated AR and 25 ng of the reporter plasmid pARR3-tk-luciferase using TransIT20/20 transfection reagent (3 L/g of DNA) (Mirus Bio LLC, Madison, WI, USA) in Opti-MEM serum-free media (Life Technologies) SB 525334 for 48 h according to manufacturers suggested protocol. Cells were stimulated with increasing concentrations of DHT, estradiol, SB 525334 progesterone or hydrocortisone in 100% ethanol (0 to 500 nM). Control cells were treated with 100% ethanol alone. At 24 h after treatment, the medium was aspirated off and the cells were lysed by adding 60 L of 1 1.
