Resistance due to the stress response surpasses the contribution of P-glycoprotein mediated resistance to HSP90-targeted therapy

A186 HSP90 is critical in the folding, stabilization, and trafficking of many client proteins involved in proliferation and antiapoptotic signaling necessary for the malignant process. HSP90-targeted therapy has been introduced into the clinic with the expectation that with degradation of many client proteins, a significant antitumor response would be observed. However, single agent clinical trials to date have shown only limited and anecdotal activity of this multitargeted kinase approach, even though stress response proteins such as HSP27 and HSP70 are induced in patients after treatment with HSP90-directed agents. P-glycoprotein (P-gp) has been implicated in the resistance to HSP90-targeted therapy. We and others have shown that expression of HSP27 and HSP70 also contribute to such resistance. To further delineate the contribution of these factors to HSP90-targeted therapy resistance observed in patients, we undertook in vitro studies of parent A549 cells and those selected for resistance (A549GARS) to geldanamycin (GA). A549GARS have been selected by exposure to increasing concentrations of GA in a stepwise fashion, and are 10-fold more resistant to GA and the clinical analogs 17-allylaminogeldanamycin (17AAG) and (dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) than parent A549 cells. These cells overexpress P-gp and HSP27. We found that treatment of A549 cells with 1 μM GA, 17AAG, or DMAG for 24 hours induced P-gp expression and increased HSP70 and HSP27 levels, indicating that P-gp, like HSP27 and HSP70, is regulated by the stress response during treatment with HSP90-targeted agents. We treated KB3-1 parent cells and KB-T10 cells, which were selected for resistance to colchicine and overexpress P-gp , with 17AAG and found that the KB-T10 cells were 6-fold more resistant to 17AAG compared to the KB3-1 cells (IC50: 218+/-43 nM vs 36+/-19.8 nM, respectively). However, simultaneous treatment of the A549GARS cells with verapamil (VP) and 17-AAG did not affect the 17-AAG IC50 (1048+/-121 nM with VP vs 957+/-123 nM without VP). Despite the overexpression of P-gp in the A549GARS, 17AAG caused dissociation of p23 from HSP90, an indicator of GA binding to HSP90, regardless of whether cells were treated with VP. In addition, HSP27 and HSP70 levels were increased after 17AAG treatment in the A549GARS with or without VP treatment. Knockdown of HSP27 and/or HSP70 reversed the resistance of the A549GARS resulting in an IC50 similar to that of A549 parent cells (228+/-22.3 nM [HSP27 siRNA], 235+/-42.7 nM [HSP70 siRNA] and 179+/-67.6 nM [both siRNAs]). Our data suggests that 17AAG, GA and DMAG can acutely induce P-gp expression. This may cause increased efflux of chemotherapeutic agents which are known substrates of P-gp. In cells chronically exposed to HSP90-targeted agents, the resistance induced by the stress response surpasses P-gp-mediated resistance to HSP90-targeted therapy. These results indicate that selective targeting of HSP90 alone may have limited clinical efficacy. On the other hand, a combined approach that affects the stress response by targeting other stress-related proteins such as HSP27 and HSP70 in addition to HSP90 may overcome this self-induced resistance. Supported in part by CA90390 and CA15083.