SBF-1 bound to AR mutants and inhibited their activation
AR signaling is known to be caused in castrate-resistant prostate cancer (CRPC) by activating AR point mutations. The most frequently identified AR point mutations include T878A (Fenton et al., 1997; Taplin et al., 1999; Taplin et al., 1995; Veldscholte et al., 1990), W742C(Lallous et al., 2016; Steketee, Timmerman, Ziel-van der Made, Doesburg, Brinkmann & Trapman, 2002; Tan et al., 1997; Taplin et al., 2003; Watson, Arora & Sawyers, 2015; Yoshida et al., 2005), L702H(Lallous et al., 2016; Steketee, Timmerman, Ziel-van der Made, Doesburg, Brinkmann & Trapman, 2002; Tan et al., 1997; Taplin et al., 2003) and F876L mutations (Korpal et al., 2013). According to these findings, we covered these frequently occurring mutations in CRPC through the construction of AR mutants to examine the effect of SBF-1 on those mutants (Fig. 3). The mutants tagged with GFP tag were transfected into PC3 cells. After 36 h of transfection, the cell lysate from transfected PC3 cells were collected to examine the binding with SBF-1. MST was used to detect the binding affinity between SBF-1 and the AR mutants. As the result, SBF-1 showed a strong binding to all of the mutants with the affinity 10 µM to L702H, 610 nM to W742C, 1.9 µM to F876L, and 385 nM to T878A, respectively (Fig. 3A). Also, we examined the activity of those mutants and their wild type in the presence of DHT stimulation. We used SBF-1 and DHT alone or both to treat the cells. As the result, the stimulation of AR wild type and mutants with DHT (0.1 and 10 nM) caused an increase in the luciferase activity of AR wild type and its mutants, while in case of SBF-1, there was no change in the activity of neither the wild type nor the mutants. Surprisingly, the combined treatment of DHT and SBF-1 did not cause any change in the luciferase activity, suggesting that SBF-1 could block AR activity increased by DHT due to its binding to it (Fig. 3B). We examined the effect of SBF-1 on the phosphorylation state of the constructed ARWT and the mutants. After 36 h of transfection of GFP tagged constructs, the PC3 cells were treated with or without DHT or a combination of SBF-1 and DHT for 6 h. As the result, SBF-1 markedly reduced the phosphorylation of AR and its mutants even in the presence of DHT (Fig. 3C).