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Molecular Biology, Pathobiology, and Genetics |
Departments of 1 Medicine and 2 Cancer Biology, 3 Breast Cancer Research Program, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee and Departments of 4 Medicine and 5 Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, New York
Requests for reprints: Carlos L. Arteaga, Division of Oncology, Vanderbilt University Medical Center, 2220 Pierce Avenue, 777 PRB, Nashville, TN 37232-6307. Phone: 615-936-3524/1919; Fax 615-936-1790; E-mail: carlos.arteaga{at}vanderbilt.edu.
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Abstract |
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Introduction |
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In addition to amplification of signal transduction by the EGFR, maintenance of the active state of receptor as a result of impaired desensitization has been shown to contribute to oncogenicity (5, 6). Once activated by stimulation, EGFR initiates both positive and negative processes that convey signals to downstream pathways via the interaction with adaptor proteins (7) and remove receptors from the cell surface (8). In the latter case, the Cbl family of ubiquitin ligases plays a major role in ligand-dependent ubiquitination of many receptor tyrosine kinases (RTK; ref. 9). For the EGFR, Cbl ubiquitinates activated receptors, and this post-translational modification carries both internalization and degradation signals that control endocytosis and sorting of receptors for destruction in the lysosome (10). Cbl also promotes EGFR internalization via its interaction with the adaptor protein CIN85 (11), which constitutively interacts with endophilins, the regulatory component of clathrin-coated pits. Mutations that prevent interaction between Cbl and CIN85 have been shown to block receptor endocytosis and degradation without affecting Cbl-mediated active EGFR ubiquitination (11). Therefore, recognition and modification of activated EGFR by Cbl-CIN85 complex controls receptor levels, their intracellular trafficking, and the intensity of signaling output.
EGFR gene mutations have recently been identified in specific subsets of patients with NSCLC (12–14). The mutations are clinically relevant, as they are linked to tumor sensitivity to ATP-competitive, small-molecule EGFR inhibitors, such as gefitinib and erlotinib. These are localized in exons 18, 19, 20, and 21 of the EGFR gene, which encode the kinase domain. Although a secondary mutation responsible for drug resistance has also been predicted (15) and reported (16), the molecular mechanism of signaling by drug-sensitive and drug-resistant EGFRs is not entirely defined. As EGFRs prefer homodimerization or heterodimerization with other ErbB family members, it is not easy to address the sole and intrinsic function of these EGFR mutants. Therefore, to minimize background signaling caused by other ErbB receptors and ligands, we developed a "clean system" with 32D mouse hematopoietic cells. These are interleukin-3 (IL-3)–dependent cells, which do not express any of the ErbB coreceptors or ligands and have been used previously to study EGFR signaling (17–21) as well as that of other oncogenic tyrosine kinases (22–24). Thus, we used 32D cells transfected with EGFR mutant vectors and human lung cancer cell lines harboring EGFR gene mutations in the activation loop. We report herein that EGFR mutants escaped ligand-induced receptor down-regulation (LIRD) and exhibited constitutive association with HSP90 and Cbl. The receptor mutants preferred the unphosphorylated form of Cbl and failed to recruit CIN85 to the endocytic complex on the addition of ligand. Inhibition of HSP90 permitted down-regulation of EGFR mutants in response to ligand. Taken together, these findings highlight a novel mechanism of increased signaling involving impaired receptor desensitization and maintenance of an active conformation as a result of binding to HSP90.
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Materials and Methods |
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(TGF-) and geldanamycin were from R&D Systems (Minneapolis, MN) and Calbiochem (San Diego, CA), respectively. Cell viability assay. Growth and viability of 32D cells (transfected with EGFRs) was analyzed with the trypan blue dye exclusion assay. At the indicated times, cells were harvested and resuspended in a trypan blue solution and counted using a hemocytometer. The cells with and without blue dye staining inside were recorded as dead and alive, respectively.
Western blot and immunoprecipitation. Cells were washed with PBS once, disrupted on ice for 30 minutes in NP-40 lysis buffer, and cleared by centrifugation. Protein concentration was determined with BCA reagent (Pierce Chemical Corp., Rockford, IL). Equal amounts of protein (20 µg) in cell lysates were separated by SDS-PAGE, transferred to membranes, immunoblotted with specific primary and secondary antibodies, and detected by chemiluminescence with the enhanced chemiluminescence detection reagents (Amersham Biosciences, Piscataway, NJ). We used the following antibodies: phosphorylated EGFR (Tyr1068), AKT, and phosphorylated AKT (Ser473; Cell Signaling Technology, Beverly, MA); Cbl, p-Cbl, and ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA); CIN85 (Calbiochem); HSP90 (Stressgen, Victoria, British Columbia Canada); actin (Sigma, St. Louis, MO); and phosphotyrosine (P-Tyr; 4G10; Molecular Recognition Core Laboratory, Vanderbilt). For immunoprecipitation, cell lysate protein (500-1,000 µg) was incubated overnight at 4°C with the designated antibody; protein G-Sepharose (Amersham Biosciences) was added for 3 hours while rocking. Precipitates were washed thrice with lysis buffer and once with PBS, resuspended in 2x Laemmli buffer, and resolved by SDS-PAGE followed by immunoblot analysis.
Plasmid construct and stable transfection. The pMSCVpuro-IRES-EGFP vector is derived from pMSCVpuro and pIRES2-EGFP vector (Clonetech Laboratories, Inc., Mountain Veiw, CA). A 1.6-kb XhoI/HpaI IRES-EGFP fragment from pIRES2-EGFP was cloned into pMSCVpuro vector (EcoRI site deleted) at same sites. A synthetic DNA fragment containing unique cloning sites was cloned into the resultant vector to generate pMSCVpuro-IRES-EGFP. Unique cloning sites in it were BglII, XhoI, PmeI, BstBI, MluI, EcoRI, SwaI, NotI, and BamHI (in order). EGFR wild-type (WT) cDNA sequence (accession no. NM_005228) was introduced into pcDNA3.1 vector (Invitrogen, Carlsbad, CA) to add myc and His tags at the COOH terminal. Mutations were introduced in the EGFR sequence using QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and using the following primers: for L858R (t2573g) sense, 5'-CACAGATTTTGGGCGGGCCAAACTGCTGGG-3', and L858R (t2573g) antisense, 5'-CCCAGCAGTTTGGCCCGCCCAAAATCTGTG-3'; for L861Q (t2582a) sense, 5'-GGCTGGCCAAACAGCTGGGTGCGGAA-3', and L861Q (t2582a) antisense, 5'-TTCCGCACCCAGCTGTTTGGCCAGCC-3'. Mutations were confirmed by sequencing (Vanderbilt DNA Sequencing Core Laboratory). WT EGFR and mutants were cut from pcDNA3.1 with PmeI and ligated into the pMSCVpuro-IRES-EGFP retrovirus vector. cDNA insert and correct orientation was confirmed by digestion with BglII. All constructs, including null vector, were stably transfected into 32D cells and sorted by fluorescence-activated cell sorting (FACS) based on green fluorescence expression. The expression of these receptors was regularly measured by Western blotting and FACS.
Xenograft studies. Six-week-old athymic BALB/c female mice [National Cancer Institute (NCI)-Frederick Cancer Center, Frederick, MD] were maintained in pressurized ventilated cages. Experiments were carried out under an Institutional Animal Care and Use Committee approved protocol, and institutional guidelines for the humane use of animals in research were followed. To generate xenograft tumor bearing mice, 5 x 106 H1975 cells were mixed with Matrigel (Collaborative Research, Bedford, MA) and inoculated s.c. in the right flank via a 22-gauge needle. Mice were treated with 17-allylaminogeldanamycin (17-AAG) or the egg phospholipid vehicle only as control. 17-AAG was given thrice weekly using one of two schedules (Monday, Wednesday, and Friday) or (Monday, Tuesday, and Wednesday). Mice were weighed, and tumor volumes were calculated with the formula: 
/ 6 x larger diameter x (smaller diameter)2. To analyze cellular markers, mice with established tumors were treated with one dose 17-AAG (50, 75, or 100 mg/kg) or vehicle only as control and then sacrificed 6 hours later. For immunoblotting, tumor tissue was homogenized in 2% SDS lysis buffer (pH 7.4).
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Results |
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was more sensitive to the EGFR inhibitor gefitinib than that of 32D-EGFR-WT (Fig. 1A
). We next examined the effect of the gefitinib and erlotinib, which reversibly inhibit ATP binding to the EGFR tyrosine kinase (26). In ligand-stimulated cells, the phosphorylation on tyrosine residues of both mutant receptors was markedly more sensitive to gefitinib than WT EGFR (Fig. 1B).
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in the presence or absence of receptor inhibitors. Cetuximab partially reduced ligand-stimulated EGFR-WT but not mutant receptor phosphorylation. Monovalent Fab fragments of cetuximab had no effect. Gefitinib and erlotinib inhibited phosphorylation of all EGFRs regardless of TGF- stimulation (Fig. 1C).
EGFR mutants generate ligand-independent cell survival. In the absence of serum and IL-3, 32D cells rapidly undergo apoptosis. Therefore, we examined if mutant EGFRs will abrogate cell death under these conditions. Both 32D-EGFR-L861Q and 32D-EGFR-L858R cells but not cells expressing WT receptor proliferated for the first >24 hours following serum and IL-3 deprivation. However, all cells exhibited low viability after 3 days in culture (Fig. 2A
), suggesting that survival signals generated by mutant EGFRs do not completely replace those provided by serum and IL-3. This difference in viability was more marked in serum-starved cells treated with TGF- (Fig. 2A), suggesting that the mutants are hyperresponsive to receptor ligands. In both 32D-EGFR-L861Q and 32D-EGFR-L858R cells, EGFR phosphorylation at Tyr1068 and AKT phosphorylation in Ser473 were detected in the presence or absence of ligand, whereas, in cells expressing EGFR-WT, these were phosphorylated only in the presence of TGF- (Fig. 2B).
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for 1 to 6 hours. In 32D-EGFR-WT cells, receptor levels were markedly reduced 2 hours after ligand stimulation. However, 32D-EGFR-L861Q and 32D-EGFR-L858R cells maintained receptor levels on ligand stimulation (Fig. 3A
). Similar results to these were observed in H3255 and H1975 human lung cancer cells. H3255 cells express L858R EGFR, and H1975 cells contain a receptor with two missense mutations, L858R and T790M (Fig. 3A, bottom).
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These data suggest that mutant receptors prefer binding of unphosphorylated Cbl, whereas WT receptor recruits phosphorylated Cbl in response to the ligand. As COOH-terminal residues of Cbl, including Tyr774, link to binding of CIN85, we hypothesized that dephosphorylation of Cbl abrogates CIN85 binding to the EGFR-Cbl complex. Therefore, we next examined the association of CIN85 to the EGFR-Cbl complex. In contrast to EGFR-WT-Cbl-CIN85 complex formation by ligand stimulation, EGFR-L861Q and EGFR-L858R showed lack of association with CIN85 even in the presence of TGF- (Fig. 3C). These data suggest that the loss of phosphorylation of Cbl results in failure of CIN85 recruitment to the mutant EGFR-Cbl complex.
HSP90 protects EGFR mutants from LIRD. Chaperone molecules, such as HSP90, contribute to the stability of oncogenic kinases. HSP90 helps the folding of nascent polypeptides as well as stabilizes mature kinases, such as HER-2 (ErbB2), Raf, and Src-family kinases. Thus, we proposed that stabilization by HSP90 may contribute to the resistance of EGFR mutants to LIRD. To test this speculation, we examined the association of receptors with HSP90. HSP90 was detected in immunoprecipitates of EGFR from 32D-EGFR-L861Q cells but not from 32D-EGFR-WT cells (Fig. 4A ). This observation was similar to a recent report using lung cancer cells harboring EGFR mutations (25). To determine the dependence of the EGFR mutants on its association with the chaperone, we examined the effect of the HSP90 inhibitor geldanamycin on receptor stability. Geldanamycin binds to an NH2-terminal ATP-binding pocket of HSP90, thus inhibiting its function by preventing the folding of HSP90-dependent client proteins. On the addition of geldanamycin, receptor depletion was markedly faster in 32D-EGFR-L861Q and 32D-EGFR-L858R cells than 32D-EGFR-WT cells (Fig. 4B and C).
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We therefore examined depletion of receptors using TGF- in combination with the HSP90 inhibitor. On the addition of TGF-, 32D-EGFR-WT cells exhibited receptor down-regulation, whereas EGFR levels in both 32D-EGFR-L861Q and 32D-EGFR-L858R cells were stable. A low dose of geldanamycin (0.1 µmol/L) had no appreciable effect against WT or the EGFR mutants. However, geldanamycin, in combination with TGF- (20 ng/mL), enhanced down-regulation of both WT and the mutant receptors at 3 hours (Fig. 5A, top
). Similar to these results in transfected 32D cells, combination treatment induced receptor down-regulation in both H1975 and H3255 NSCLC lines, whereas each single treatment showed little effect on EGFR protein steady-state levels (Fig. 5A, middle and bottom). These data suggest that geldanamycin-mediated inhibition of HSP90 function restores the sensitivity of EGFR mutants to LIRD. This was associated with restoration of ligand-induced receptor ubiquitination. TGF- induced EGFR ubiquitination in 32D-EGFR-WT but not in 32D cells or NSCLC lines expressing mutant receptors. However, 32D-EGFR-L861Q, 32D-EGFR-L858R, H1975, and H3255 cells displayed EGFR ubiquitination only in response to geldanamycin alone or cotreatment of geldanamycin and TGF- (Fig. 5B), implying that EGFR mutants interact with HSP90 not only for stabilization of their mature structure but also for prevention of ubiquitination in response to ligand.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EGFR mutants displayed ligand-independent phosphorylation at the Tyr1045, the Cbl-binding site, stable association with Cbl, and impaired ligand-mediated receptor ubiquitination and down-regulation. Previously reported mechanisms of impaired degradation of RTKs have mainly involved the sequestration of Cbl from activated EGFR (37, 38) rather than the constitutive association between both molecules. Thus, our data would imply an impairment in the processes that follow Cbl binding to mutant EGFRs. Eps15, Endophilin-2, CALM, Rabaptin-5, CIN85, and Hip1 are among several adaptor proteins involved in receptor down-regulation (39). CIN85 is recruited to the EGFR endocytic complex by Cbl (11, 40). In this study, we discovered that mutant receptors failed to recruit CIN85 to the EGFR-Cbl complex in response to TGF- (Fig. 3C). This is the first report of impaired down-regulation of activated EGFR that is persistently associated with Cbl. The EGFR mutant-associated Cbl is mainly unphosphorylated, whereas Cbl bound to ligand-stimulated WT EGFR is tyrosine phosphorylated (Fig. 3B and C). Because the tyrosine-rich tail of Cbl plays a role in the recruitment of CIN85 to activated receptors (5), the preference of the mutants to bind unphosphorylated forms of Cbl may explain the defective recruitment of CIN85. The lack of receptor ubiquitination in TGF--stimulated 32D-EGFR-L861Q and 32D-EGFR-L858R cells suggests a mechanism inhibiting Cbl-mediated ubiquitination of the mutants. Several mechanisms have been proposed to explain negative regulation of Cbl function leading to receptor stabilization, such as dephosphorylation of Cbl by the SHP1 phosphatase, degradation of Cbl by AIP4, and sequestration of Cbl from activated receptors (30). Our data herein do not support any of these mechanisms.
Whereas, in normal tissues, RTKs achieve active conformation of their kinase domain on the binding of ligand or phosphorylation of tyrosine residue(s) in the activation loop (41), oncogenic mutations in the activation loop can trigger a conformational shift of the C-helix toward an active state of the kinase domain without ligand stimulation (35, 36). Maintenance of this active conformation is required by oncogenic kinases to be protected from proteasomal degradation, which is designed for desensitization of signaling. Oncogenic kinases have been shown to adopt various mechanisms that spare them from these negative regulatory processes. One mechanism that alleviates the conformational instability of oncogenic proteins is their association with the HSP90 chaperone (31, 42, 43). Indeed, over 100 proteins, including signal transducers and transcription factors interact with HSP90 along with cochaperones (44, 45). For example, a point mutation within the kinase domain of the EGFR-homologous HER-2 receptor alters its conformation, resulting in its dissociation from HSP90. Conversely, substitution of one amino acid within the kinase domain of EGFR, such as D746G, confers binding of HSP90 (46). Shimamura et al. reported recently the association of EGFR mutants in human lung cancer cells with HSP90 (25). Other oncogenic kinases, such as Src, B-Raf, and Met are also associated with HSP90 (27, 47).
In this report, the EGFR mutants in 32D cells showed stable association with HSP90. Treatment with the HSP90 inhibitor geldanamycin induced degradation of the mutant receptors, which was much slower for WT EGFR (Fig. 4). The constitutive association of both mutant receptors with HSP90 but not the more geldanamycin-resistant WT EGFR suggests that the degradation on treatment with geldanamycin is HSP90 specific. Of note, other small molecules that also bind the HSP90 pocket exhibit an activity against client proteins that correlates directly with their affinity to HSP90 (48). Although we cannot rule out a nonspecific effect of geldanamycin, based on these cumulative data, we surmise that the effects observed on the steady-state levels of both mutant receptors are likely to be HSP90 specific. Interestingly, EGFR-L861Q and EGFR-L858R showed constitutive association with both Cbl and HSP90 in a ligand-independent manner (Fig. 4A). Because Cbl binding to EGFR requires phosphorylation at Tyr1045 in the COOH-terminal tail (49), whereas HSP90 binds to the receptor via its kinase domain (50), our result implies that Cbl and HSP90 bind independently. Inhibition of HSP90 with geldanamycin reconstituted mutant receptor ubiquitination and receptor degradation both in vitro and in vivo. Taken together, these data support a novel function of HSP90, which endows mutant EGFRs with a stable and active conformation protected from LIRD by negatively regulating Cbl function. The results also suggest that combinations of EGFR and HSP90 inhibitors are worth exploring against NSCLC containing EGFR mutations in the activation loop.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3/21/06. Revised 5/ 1/06. Accepted 5/11/06.
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12 but not G 13 to lipid rafts. J Biol Chem 2002;277:32409–12.This article has been cited by other articles:
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G. J. Riely, M. G. Kris, B. Zhao, T. Akhurst, D. T. Milton, E. Moore, L. Tyson, W. Pao, N. A. Rizvi, L. H. Schwartz, et al. Prospective Assessment of Discontinuation and Reinitiation of Erlotinib or Gefitinib in Patients with Acquired Resistance to Erlotinib or Gefitinib Followed by the Addition of Everolimus Clin. Cancer Res., September 1, 2007; 13(17): 5150 - 5155. [Abstract] [Full Text] [PDF]
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D. Padron, M. Sato, J. W. Shay, A. F. Gazdar, J. D. Minna, and M. G. Roth Epidermal Growth Factor Receptors with Tyrosine Kinase Domain Mutations Exhibit Reduced Cbl Association, Poor Ubiquitylation, and Down-regulation but Are Efficiently Internalized Cancer Res., August 15, 2007; 67(16): 7695 - 7702. [Abstract] [Full Text] [PDF]
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200 mm3) were treated with a single variable dose of 17-AAG (50-100 mg/kg) and sacrificed 6 hours later. Lysates from tumor homogenates were separated by SDS-PAGE followed by immunoblot analysis with the indicated antibodies. Treatment with 17-AAG caused dose-dependent down-regulation of HSP90 clients, including (L858R/T790M) mutant EGFR, HER-2, and Raf-1. Down-regulation of these HSP90 clients was accompanied by down-regulation of cyclin D1 expression and active (phosphorylated) Akt and MAPK. B, 17-AAG inhibited the growth of H1975 xenografts. Daily treatment for 3 consecutive days (MTW) each week was most effective. Results compare treatment versus control tumors following 2 weeks of 17-AAG treatment. Columns, mean tumor volume relative to control from a group of six mice per treatment group; bars, SE.