DestabilizationoftheEpidermalGrowthFactorReceptor (EGFR ... The epidermal growth factor receptor (EGFR)2

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  • Destabilization of the Epidermal Growth Factor Receptor (EGFR) by a Peptide That Inhibits EGFR Binding to Heat Shock Protein 90 and Receptor Dimerization*� Received for publication, June 7, 2013, and in revised form, July 9, 2013 Published, JBC Papers in Press, July 29, 2013, DOI 10.1074/jbc.M113.492280

    Aarif Ahsan‡, Dipankar Ray‡, Susmita G. Ramanand‡, Ashok Hegde‡, Christopher Whitehead§, Alnawaz Rehemtulla‡, Yoshihiro Morishima¶, William B. Pratt¶, Yoichi Osawa¶, Theodore S. Lawrence‡, and Mukesh K. Nyati‡1

    From the Departments of ‡Radiation Oncology, §Radiology, and ¶Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109

    Background: An eight-amino acid segment lying within the �C-�4 loop region of many protein kinases determines sensi- tivity to Hsp90 inhibitors. Results:Apeptide comprised of this segment of the EGFR inhibits bothHsp90 binding andEGF-dependent EGFRdimerization. Conclusion: The peptide selectively degrades EGFR versus other Hsp90 clients. Significance: This peptide represents a unique approach to the therapy of EGFR-driven tumors.

    An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two pep- tides were synthesized as follows: one containing the wild- type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degra- dation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is spe- cific for EGFR versus other Hsp90 client proteins. In the pres- ence of EGF, Disruptin, but not the scrambled peptide, inhib- its EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Dis- ruptin inhibits cancer cell growth by a nonapoptotic mecha- nism. Disruptin provides proof of concept for the develop- ment of a new class of anti-tumor drugs that specifically cause EGFR degradation.

    The epidermal growth factor receptor (EGFR)2 is a member of the ErbB family of transmembrane tyrosine kinases (EGFR/ ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), which play critical roles in regulating cell proliferation, differ- entiation, and migration (1). The EGFR is activated by ligand- induced dimerization (2), and abnormal activation is associated with a variety of human cancers (1), making the EGFR a target of considerable interest for anti-cancer drug development. The major clinically approved approaches have been to develop EGFR kinase inhibitors, such as gefitinib or erlotinib, and the production of monoclonal anti-EGFR antibodies like cetux- imab. An experimental approach has been to target the EGFR for degradation by using inhibitors of Hsp90, which cycles with the EGFR monomer to stabilize it (3). The EGFR is activated by forming an asymmetric homodimer

    with itself (4, 5)orby formingaheterodimerwithanothermember of the ErbB family. ErbB2 (HER2) functions as a ligandless core- ceptor that heterodimerizes with other members of the ErbB family to amplify signaling. The kinase domain of ErbB2 forms a stable complex with Hsp90, and Hsp90 regulates ErbB2 func- tion by limiting heterodimer formation (6). In contrast, the EGFR undergoes dynamic cycling with Hsp90 and is less strin- gently regulated by the chaperone machinery (7). Upon Hsp90 inhibition by geldanamycin, ErbB2 is polyubiquitinated and rapidly degraded, whereas the EGFR is modestly ubiquitinated andmore slowly degraded (8, 9). This difference ingeldanamycin sensitivity is accounted for by a short eight-amino acid segment within the highly homologous kinase domains (6, 10). Swapping the eight-amino acid segments between the EGFR and ErbB2 yields the appropriate exchange of dynamic versus stable cycling withHsp90 and the corresponding change in geldanamycin sensi- tivity (6). This eight-amino acid segment lies within the �C-�4 loop

    region of many protein kinases, and it is proposed to define a

    * This work was supported, in whole or in part, by National Institutes of Health Grants R01CA131290 (to M. K. N.), R01CA160981 (to D. R.), GM077430 (to Y. O.), P50CA097248 (project IV to M. K. N.), P50CA097248 (Career Devel- opment award to A. A.), and Cancer Center Support Grant P30CA46592. This work was also supported by the Michigan Institute for Clinical and Health Research (to M. K. N.), the James Stuart and Barbara Padnos Funds for Cancer Research (to M. K. N.), and an Alfred Taubman Scholarship (to T. S. L.). This project is a component of the University of Michigan Medical School’s Fast Forward Strategic Research Initiative.

    �This article was selected as a Paper of the Week. 1 To whom correspondence should be addressed: Dept. of Radiation Oncol-

    ogy, University of Michigan Medical School, Medical Science Bldg. I, Ann Arbor, MI 48109-2026. Tel.: 734-936-9162; Fax: 734-763-1581; E-mail: nyati@umich.edu.

    2 The abbreviations used are: EGFR, EGF receptor; Hsp, heat shock protein; PARP, poly(ADP ribose) polymerase; BisTris, 2-[bis(2-hydroxyethyl)amino]- 2-(hydroxymethyl)propane-1,3-diol; GR, glucocorticoid receptor.

    THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 37, pp. 26879 –26886, September 13, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

    SEPTEMBER 13, 2013 • VOLUME 288 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 26879

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  • common surfacewithwhichHsp90 interacts (11). The�Chelix is a region that regulates kinase activity (12), and it forms part of the dimerization interface that interactswith activator kinase in the asymmetric EGFR dimer (4, 5). Although the function and turnover of a wide variety of signaling proteins are regulated by Hsp90 (13), there is no specific motif that determines interac- tion with the chaperone, and the eight-amino acid segments of the EGFR andErbB2 are unrelated to a seven-amino acid region of the glucocorticoid receptor that similarly determines its sta- ble versus dynamic cycling with Hsp90 (14). There is consider- able evidence supporting the proposal that Hsp90 interacts with proteins in the region where their ligand binding clefts open onto the protein surface (reviewed in Ref. 7). Inasmuch as the eight-amino acid segment lies in close asso-

    ciation with the EGFR ATP binding cleft (12) and the receiver dimerization face (4, 5) as well as controllingHsp90 binding (6), we explore here the role of the segment in determining EGFR stability.We show first that scrambling the first six amino acids of the wild-type segment markedly reduces EGFR stability and function, which was assessed by EGF-dependent dimerization and phosphorylation. We then synthesized two peptides, one with the wild-type sequence, which we call Disruptin, and one with the scrambled sequence. Treatment of EGFR-dependent cancer cell lineswithDisruptin inhibits EGFRbinding toHsp90 and destabilizes the receptor. The effect is specific for the EGFR and does not pertain to other Hsp90 client proteins. Disruptin, but not the scrambled peptide, also inhibits EGF-dependent dimerization of the EGFR and cell growth. These observations suggest a model in which Disruptin interacts directly with the EGFR to inhibit an intermolecular or intramolecular protein interaction to inhibit both Hsp90 binding and dimerization. They also provide a basis for the development of unique drugs that will specifically target EGFR-driven tumors.

    EXPERIMENTAL PROCEDURES

    Materials

    Geldanamycin was acquired from Enzo Life Sciences, Farm- ingdale, NY. EGFR (sc-03) antibody was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for glyceral- dehyde-3-phosphate dehydrogenase (GAPDH),Hsp70, cleaved PARP, Src, and Akt were purchased from Cell Signaling Tech- nology (Danvers, MA), and antibodies to detect ErbB2 and Hsp90 were purchased from Neomarkers (Kalamazoo, MI), and Pharmingen, respectively. Cycloheximide and the cross-linking agentdisuccinimidyl suberatewereobtained fromSigma.Peptides were synthesized by Peptide 2.0 (Chantilly, VA) and American Peptide Co. (Sunnyvale, CA). The peptide transfection reagent Chariot was purchased fromActiveMotif (Carlsbad, CA).

    Methods

    Cell Culture—EGFR-null CHO cells were purchased from the American Type Culture Collection. The human head and neck squamous cell carcinoma cell line UMSCC1 was kindly provided by Dr. Thomas Carey (University of Michigan, Ann Arbor, MI). The lung cancer cell line NCI-H1975 was provided by Dr. J. A. Engelman (Massachusetts General Hospital, Bos- ton). All cell lines were grown in RPMI 1640 medium supple- mented with 10% cosmic calf serum. For all in vitro experi-

    ments, cells were released from flasks using PBS containing 0.25% trypsin and 0.2 mM EDTA, and cells were plated onto culture dishes 2 days prior to any treatment. Immunoblotting—Cells were scraped into PBS containing a

    sodium orthovanadate and protease inhibitor mixture (Roche Diagnostics). Cells were incubated for 15min on ice in Laemmli buffer (63 mM Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, and 0.005% (w/v) bromphenol blue) containing 100 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 �g/ml aprotinin. Afte