Identification of new biomarkers for clinical trials of hsp90 inhibitors

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The geldanamycin derivative, 17-allyamino-17-demethoxygeldanamycin (17-AAG), is a selective HSP90 inhibitor that is now under clinical investigation. The biomarkers that have been used in 17-AAG clinical trials to date are HSP70, Raf-1 and CDK4. HSP70 was induced during 17-AAG treatment, whereas Raf-1 and CDK4 were repressed. All three biomarkers have to be analyzed by western blot of cellular samples, either from tumor biopsy or PBMC cells isolated from patient blood. This analytical method is time-consuming and laborious. We have identified two new biomarkers, IGFBP2 and HER-2 ECD (HER-2 extracellular domain), both of which can be readily detected in patient sera by ELISA. IGFBP-2 is an Insulin-like Growth Factor binding protein that modulates the activity and facilitates the transportation of IGF and is regulated via the PI3K/AKT pathway. Inhibition of HSP90 causes AKT degradation and thus should in turn attenuate secretion of IGF-BP2. HER-2 ECD is a shed form of HER-2 that circulates in the bloodstream. Since HER-2 is a sensitive HSP90 client protein, circulating HER-2 ECD might be a sensitive serum marker of HSP90 inhibitor activity. In vitro experiments in BT474 cells with a panel of HSP90 inhibitors revealed that secretion of both IGFBP-2 and HER-2 ECD into culture medium was decreased in a dose-dependent manner. Moreover, both IGFBP-2 and HER-2 ECD exhibited greater sensitivity to HSP90 inhibitors than did previously identified biomarkers, and the repressive effect lasted up to 48 hours after drug was removed. Structurally-related control compounds with no HSP90 inhibitory activity had no effect on either protein. In sera from BT474 tumor-bearing mice, both IGFBP-2 and HER-2 ECD were down-regulated by 17-AAG and other HSP90 inhibitors, and that correlated with the degradation of HER-2 and attenuation of AKT activity in the tumors. In addition, we also found that both IGFBP-2 and HER-2 ECD levels are elevated in patient sera from several cancer types, suggesting that these novel secreted biomarkers could be valuable pharmacodynamic tools in clinical trials of HSP90 inhibitors.