Freshly prepared sulfo-NHS-SS-biotin was added to the final concentration of 0.5 mg/ml in PBS. family members, in cell-based assays they are effective at inhibiting both EGFR and HER2 and equally effective at suppressing the growth of EGFR and HER2 driven tumor cells 15C19. WZ4002 They are also effective at inhibiting EGFR and HER2 phosphorylation in patients tissues and tumors 5C8. But these brokers show very limited clinical anti-tumor activity 1C5. Their clinical development to this point has been driven largely by the detection of modest delays in tumor progression. The failure to reverse malignancy progression despite an apparent inhibition of HER kinase function has created an enigma in the concept of TKI Rabbit Polyclonal to DYR1A therapy of malignancy that we have been exploring. It is through heterodimerization and transphosphorylation that this HER family performs its signaling functions. Importantly, downstream PI3K/Akt pathway signaling is usually predominantly mediated through the transphosphorylation of the kinase-inactive member HER3 9,10. We have previously reported that sensitivity to HER family TKI therapy correlates with the inhibition of PI3K/Akt pathway signaling 15,20. We as well as others have also reported that failure to inhibit PI3K/Akt signaling prospects to WZ4002 TK inhibitor resistance 20C22. WZ4002 In contrast to reports from models, Akt activity is not inhibited in most patients on HER TKI therapy 5,6,8. This discordancy has led us to look more closely at the inhibition of PI3K/Akt signaling. To investigate this discrepancy, we analyzed the durability of Akt inhibition by TKI with amazing results. Although as previously reported, gefitinib inhibits Akt signaling in HER2-driven malignancy cells, this inhibition is not durable. Akt signaling resumes after a transient inhibition despite continued drug therapy (figures 1A,B). In light of this finding, we looked at the broader HER family signaling activities over a period of 96 hours following continuous exposure of BT474 breast malignancy cells to gefitinib at concentrations that nonselectively inhibit EGFR and HER2. TKI treatment effects a sustained inhibition of EGFR and HER2 phosphorylation and a durable inhibition of downstream MAPK and JNK pathway signaling (physique 1A). However phosphorylation of the kinase-inactive family member HER3 is merely transient. HER3 signaling resumes and persists despite continued drug exposure and effective suppression of EGFR and HER2 (physique 1A,B). The reactivation of HER3 signaling explains the reactivation of Akt signaling since HER3 is the principal HER family member that binds PI3K and drives Akt signaling 9,10. TKI-refractory Akt signaling remains sensitive to PI3K inhibitors as expected (not shown). These time-dependent findings are not due to drug degradation since the drug is usually replenished daily in these studies and HER3/Akt signaling resumes despite repeatedly refreshing drug supply up to and beyond the point of resumption of Akt signaling (not shown). There is no significant expression of HER4 before or after drug treatment in these cells (data not shown). These findings are not unique to BT474 and SkBr3 cells and have been confirmed in other HER2 overexpressing breast malignancy cells including MDA-453, AU565, MDA-361, HCC1954 (supplementary physique 1). These findings are not unique to gefitinib and are seen with other HER TKIs including brokers with selectivity profiles favoring EGFR or HER2, such as erlotinib or AG825 (physique 1C,D). These findings are not artifacts of the models either. Treatment of mice bearing numerous HER2-driven xenograft tumors with gefitinib similarly fails to durably supress HER3 and Akt signaling, despite a transient suppression (physique WZ4002 1E, and supplementary physique 2). This is not due to WZ4002 ineffective drug biodistribution, since in these models gefitinib was dosed three times higher than doses known to accomplish sustained xenograft tumor concentrations above 2C4uM and averaging 6C10uM 23. Since we had previously established that inactivation of PI3K/Akt signaling is usually mechanistically linked to HER family TK inhibitor sensitivity in HER family driven cancers, we felt that this failure of these drugs to durably.