Fig. 7

HER2-overexpressing cell lines are more sensitive to HIF-2α inhibition. a Western blot showing siRNAs knock-down of HIF-2α in MCF7-HER2 in normoxia. SiRNa knock-down was performed with 25 μM of four different siRNAs as well as 5–100 μM of SMARTpool, combined siRNAs. Protein level was reduced to < 10% of that in cells treated with an equivalent concentration of non-targeting siRNA up to 96 h after treatment. SiRNAs #4 and SMARTpool (10 μM) were chosen for the following experiments as HIF-2α was convincingly reduced and HIF-1α levels were not affected (data not shown). b Pre-treatment with either siRNA but not controls was effective at stopping the hypoxic upregulation of HIF-2α in MCF7-HER2 cells. This resulted in undetectable levels of HIF-2α protein after 24, 48 and 72 h hypoxia (0.5% oxygen). c MCF7 and MCF7-HER2 cells were treated with HIF-2α siRNAs and grown on 96-well plates for 5 days in either normoxia or hypoxia. Cellular density was assessed by SRB assay. Bars represent OD values relative to the non-targeting control (error bars = SEM, n = 12 repeats from n = 2 individual experiments). Differences between targeting and non-targeting siRNAs were assessed in each cell line using ANOVA with Dunnett’s multiple comparison test. In normoxia and hypoxia, MCF7-HER2 cellular growth was significantly reduced by both siRNA when compared to non-targeting siRNA (P < 0.0001 in all cases). MCF7 growth was reduced significantly by just one of the siRNAs in normoxia (P < 0.0001), and this was to a lesser extent than MCF7-HER2. In hypoxia, MCF7 was unaffected by either siRNA. MCF7-HER2 growth was inhibited to a significantly greater degree when compared to wild-type MCF7 for both siRNAs in normoxia (P = 0.0421 and P = 0.0004) and hypoxia (P < 0.0001 in both cases). d Breast cancer cell lines, including ER+/HER2- (MCF7/T47D/ZR751), HER2+ (MDAMB361, SKBR3, BT474) and triple-negative (MDAMB231 and HBL100) cells were treated for 5 days with C76 a specific inhibitor of HIF-2α translation. A range of concentrations were tested and an IC₅₀ value for each cell line was established (e). All HER2+ cell lines had lower IC₅₀ values for this compound, with no difference between ER+/HER2− and triple-negative lines