In this study, we have found that mature WT-EGFR interacts with HSP90 in both tumor and normal cells. We detected this interaction using immunoadsorption of endogenous or ectopically expressed HSP90 or WT-EGFR and confirmed the direct interaction between HSP90 and EGFR by in vitro GST pull-down experiment. The degradation of EGFR on HSP90 inhibition is due to a decrease in the protein stability of mature EGFR, indicating that WT-EGFR stability is critically dependent on HSP90's chaperone function. The finding that HSP90 inhibition by AT13387 degrades EGFR and suppresses growth of WT-EGFR-driven HNSCC tumors underscores the biologic and potential clinical significance of these observations.
Although the major focus of research related to EGFR-targeted therapy has been development of agents to block EGFR phosphorylation [27
], we and others have found that the physical presence of EGFR is critical for cell survival. Small interfering RNA, chemotherapy- or radiotherapy-induced degradation of EGFR causes cell death in EGFR-driven tumor cells [1,3,5,6,28
]. Blockade of HSP90 activity is known to induce EGFR degradation in cells that harbor erlotinib-resistant T790M or the ligand-independent truncated form of EGFR (EGFRvIII) [16,17
]. Overall, these results suggest that HSP90 inhibitors may have a role in overcoming erlotinib resistance.
Although it is known that HSP90 inhibitors cause overall EGFR levels to decrease over time [29
], this has been attributed to an effect only on the nascent EGFR, which is a client of HSP90 [30
]. More recently, the stability of mutant [15,16
] and truncated forms of EGFR (EGFRvIII) was shown to be regulated by HSP90 [17
]. However, only minimal interactions between mature EGFR and HSP90 have been reported [31,32
], and none of these reports has indicated that WT-EGFR and HSP90 interact directly. This apparent lack of interaction was unexpected because other EGFR family members such as ErbB2 [32,33
] and ErbB3 [34
] are known to interact directly with HSP90, and the stability of both nascent and mutant forms of EGFR seems to depend on the HSP90 activity [17
]. There are a number of possible reasons why WT-EGFR and HSP90 interaction was previously not detected. First, the studies that have investigated the interaction between nascent EGFR and HSP90 have focused on COS7 cells in an overexpression system [23,30
]. It is possible that tumor cells, which tend to contain much higher levels and a more active form of HSP90, would be more likely to reveal HSP90-EGFR binding. This may also explain why only a small amount of EGFR is immunoadsorbed with HSP90 from MRC5 cells (normal fibroblasts), which express only a moderate amount of EGFR. The second, and perhaps more likely possibility, is related to the dynamic nature of the interaction between EGFR and HSP90. At any given time, the amount of EGFR interacting with HSP90 may be minimal compared with other clients such as ErbB2, which is known to form a more stable interaction with HSP90 [31
]. Our data would be consistent with this idea because stabilization of the HSP90 clients using ammonium molybdate caused the amount of EGFR immunoadsorbed with HSP90 to be enhanced several fold.
HSP90 expression in tumors is known to be elevated relative to that in normal tissue [35,36
]. We also observed a high expression of HSP90 and EGFR in HNSCC patient tumor, similar to UMSCC1 xenografts (). This high expression of HSP90 in tumors most likely provides stability to many oncogenic kinases that are either overexpressed or activated through mutations. Previous studies have demonstrated that only nascent or mutated EGFR binds to HSP90 [17
], but in this study using subcellular fractions, we found that not only the cytoplasmic but also the membrane-bound mature EGFR coimmunoprecipiates with HSP90 (). Treatment with HSP90 inhibitors led to a rapid loss of total EGFR ( and ), indicating a critical role of HSP90 in regulation of EGFR stability. These findings were confirmed by a proof-of-principle in vivo
therapy experiment where inhibition of HSP90 activity by AT13387 treatment caused growth delay of a WT-EGFR-driven head and neck carcinoma, which correlated with a decreased expression of EGFR.
Our data confirm that blocking the chaperone function of HSP90 with HSP90 ATPase inhibitors, leading to EGFR degradation, is an attractive approach for treatment of EGFR-dependent tumors. However, given the essential proteins for which HSP90 functions as a chaperone [37
], an approach targeting the specific interaction between EGFR and HSP90 could result in more selective cancer cell killing. Although we have demonstrated that mature WT-EGFR is an HSP90 client protein, the details of the interaction between EGFR and HSP90 still need to be determined. Indeed, an in-depth knowledge of HSP90 interaction with EGFR would provide an opportunity to develop an agent that would selectively disrupt EGFR-HSP90 interactions and cause EGFR degradation without affecting HSP90's other chaperone functions. The contact surface through which one EGFR family member, ErbB2, interacts with HSP90 has been shown to be in the M5 domain [32
], which could be the starting point for designing a more targeted approach to disrupt the interaction between EGFR and HSP90. Studies focusing on identifying this region and developing specific methods to block the interaction between EGFR and HSP90 are currently underway in our laboratory.