Figure 7

Benzylguanine phosphorylates ERα at Ser167 site. Tamoxifen-resistant MCF7 breast cancer cells were treated in presence or absence of BG (140 µmol/L) and 24 h later either TAM (1 µmol/L) or ICI (1 µmol/L) was added for another 48 h after treatment and 30 min prior to harvest 17β-estradiol (10 nmol/L) was added. Western blot analysis was performed. (A) BG either alone or in combination with antiestrogen therapy (TAM/ICI) significantly increased ERα Serl67 phosphorylation. (Control (□); 4-OH TAM ; ICI ; BG ; 4-OHTAM + BG ; ICI + BG ). (B) Inhibition of MGMT by specific siRNA led to similar increase in phosphorylation of ERα Ser 167. Mice with mammary tumors were treated with BG or tamoxifen or ICI, or combination therapy (BG + TAM/ICI). The sections were immunostained for expression of phosphorylated ERα Serl 67 and phosphorylated ERα Serl 18. (NT (□); MGMT KD ). (C) BG either alone, or in combination with tamoxifen or ICI, significantly increased ERα Ser167. Alternatively, phosphorylated of ERα Ser118 was decreased significantly. This dual action of increased ERα Ser167 phosphorylation and decreased ERα Ser118 phosphorylation suggests that BG and BG combination therapies may lead to functional restoration of ERα in tamoxifen-resistant tumors. Representative samples (40×) are shown. IHC correlative quantified analysis of ERα phosphorylated Ser167 and Ser118 was done using ImageJ/ImmunoRatio plugin (C), as described in Materials and Methods. (p-ERα Ser 167 ;p-ERα Ser 118 ).