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. 2012 Aug;14(8):670-7.
doi: 10.1593/neo.12986.

Wild-type EGFR is stabilized by direct interaction with HSP90 in cancer cells and tumors

Aarif Ahsan  1 , Susmita G Ramanand, Christopher Whitehead, Susan M Hiniker, Alnawaz Rehemtulla, William B Pratt, Shruti Jolly, Christopher Gouveia, Kristy Truong, Carter Van Waes, Dipankar Ray, Theodore S Lawrence, Mukesh K Nyati
Affiliations

Wild-type EGFR is stabilized by direct interaction with HSP90 in cancer cells and tumors

Aarif Ahsan et al. Neoplasia. 2012 Aug.

Abstract

The epidermal growth factor receptor (EGFR) has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90) is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT) WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA) and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.

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Figures

Figure 1
Figure 1
Interaction of WT-EGFR with HSP90. Immunoprecipitation using anti-HSP90 antibody revealed minimum EGFR in the complex; therefore, ammonium molybdate (20 mM), which is known to stabilize the interaction of HSP90 with its client proteins, was added to the lysis buffer. (A) IP results showed an increase in EGFR-HSP90 interaction with increasing concentrations of ammonium molybdate. Densitometric analysis of the films was performed using ImageJ software, and relative intensity is shown. Two EGFR antibodies were used to confirm these results. (B) Interaction between HSP90 and EGFR was assessed in five cell lines, representing normal EGFR-expressing cells (MRC5), EGFR-negative (SW620), ErbB2-driven, EGFR-independent (BT474), erlotinib-resistant (T790M-EGFR; NCIH1975), and EGFR-amplified (UMSCC1) tumor cells. To address the issue of cross-reactivity, multiple EGFR antibodies were used, and their pattern was compared with ErbB2. One EGFR and one ErbB2 antibody were selected for the IP experiments. UMSCC1 and NCI-H1975 cell lines showed maximum interaction between EGFR with HSP90, whereas BT474 and MRC5 cells showed minimal interaction. EGFR-HSP90 interaction was absent in EGFR null SW620 cells. Colocalization of EGFR and HSP90 in tumor cells, xenografts, head and neck cancer patient's tumor was assessed by dual immunostaining for EGFR and HSP90. (C) UMSCC1 cells showed modest colocalization of these two proteins, which was also confirmed in (D) xenograft and (E) patient tumor specimen.
Figure 2
Figure 2
Localization, specificity, and nature of EGFR and HSP90 interaction. (A) To analyze whether the mature form of EGFR interacts with HSP90, subcellular fractionation followed by IP was carried out in UMSCC1 cells. EGFR-HSP90 interaction was confirmed in cytosolic, nuclear, and membrane fractions of UMSCC1 cells. (B) The specificity of this interaction was confirmed by expression of FLAG-tagged HSP90 in UMSCC11B cells, incubated with or without GA or (C) full-length EGFR in EGFR-negative CHO cells. (D) Direct interaction between EGFR and HSP90 was confirmed in vitro by GST pull-down assay.
Figure 3
Figure 3
Effects of HSP90 inhibition on WT-EGFR and ErbB2. To assess EGFR degradation relative to ErbB2, UMSCC1, and UMSCC29B cell lines harboring WT-EGFR were treated with either GA (30 and 100 nM) or AT13387 (30 and 100 nM) for 12 hours. Immunoblot analysis was carried out to detect the effect on EGFR and ErbB2 levels. At 12 hours, loss of EGFR and ErbB2 in response to GA or AT13387 was comparable.
Figure 4
Figure 4
Effects of HSP90 inhibition on EGFR stability. To assess whether inhibition of HSP90 affects stability of EGFR, UMSCC1 cells were treated with AT13387 (30 nM) or DMSO for 12 hours, followed by CHX (100 µg/ml) to block the new protein synthesis. (A) The level of EGFR was assessed at multiple time points using immunoblot analysis, and half-life was calculated. Inhibition of HSP90 reduced the half-life of EGFR from 8 to 3.7 hours. (B) Representative blots of EGFR are shown.
Figure 5
Figure 5
Effect of HSP90 inhibitor AT13387 on UMSCC1 tumor growth. (A) SCID mice were inoculated on day 0 with UMSCC1 cells and then randomized and treated on day 7. Mice received 55 mg/kg AT13387 dissolved in cyclodextrin solution or vehicle alone. Animals were treated through the intraperitoneal route on two consecutive days per week (Monday and Tuesday, indicated by the arrows), for a total of 3 weeks. Mice were euthanized when moribund beginning on day 18, as shown in B by the Kaplan-Meier plot. Student's t test revealed a significant difference (P < .05) in tumor volume between AT13387 treated and control animals during the treatment. (B) Effect of AT13387 on the survival of mice. AT13387 treatment improved the survival of UMSCC1-bearing mice. (C) Vehicle- (n = 2) and AT13387- (n = 3) treated mice were euthanized on day 16, tumors were harvested, and the effect of AT13387 on EGFR, ErbB2, and HSP70 was analyzed by immunoblot analysis. (D) Effect on EGFR expression by AT13387 was further confirmed by immunostaining.
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