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Cancer Research 68, 4183, June 1, 2008. doi: 10.1158/0008-5472.CAN-08-0413
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

RET/PTC-Induced Cell Growth Is Mediated in Part by Epidermal Growth Factor Receptor (EGFR) Activation: Evidence for Molecular and Functional Interactions between RET and EGFR

Michelle Croyle1, Nagako Akeno1, Jeffrey A. Knauf1,2, Doriano Fabbro3, Xu Chen2, Jacqueline E. Baumgartner2, Heidi A. Lane3 and James A. Fagin1,2

1 Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Cincinnati, Ohio; 2 Department of Medicine and Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York; and 3 Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland

Requests for reprints: James A. Fagin, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 646-888-2136; Fax: 646-422-0675.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
RET/PTC rearrangements are one of the genetic hallmarks of papillary thyroid carcinomas. RET/PTC oncoproteins lack extracellular or transmembrane domains, and activation takes place through constitutive dimerization mediated through coiled-coil motifs in the NH2 terminus of the chimeric protein. Based on the observation that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 decreased RET/PTC kinase autophosphorylation and activation of downstream effectors in thyroid cells, despite lacking activity on the purified RET kinase, we proceeded to examine possible functional interactions between RET/PTC and EGFR. Conditional activation of RET/PTC oncoproteins in thyroid PCCL3 cells markedly induced expression and phosphorylation of EGFR, which was mediated in part through mitogen-activated protein kinase signaling. RET and EGFR were found to coimmunoprecipitate. The ability of RET to form a complex with EGFR was not dependent on recruitment of Shc or on their respective kinase activities. Ligand-induced activation of EGFR resulted in phosphorylation of a kinase-dead RET, an effect that was entirely blocked by PKI166. These effects were biologically relevant, as the EGFR kinase inhibitors PKI166, gefitinib, and AEE788 inhibited cell growth induced by various constitutively active mutants of RET in thyroid cancer cells as well as NIH3T3 cells. These data indicate that EGFR contributes to RET kinase activation, signaling, and growth stimulation and may therefore be an attractive therapeutic target in RET-induced neoplasms. [Cancer Res 2008;68(11):4183–91]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The RET gene encodes the signaling subunit of a receptor complex for ligands of the glial-derived neurotrophic factor family (1). Germ-line point mutations of RET confer predisposition to multiple endocrine neoplasia type 2, familial medullary thyroid carcinoma, and Hirschsprung's disease. RET is normally expressed at very low levels in thyroid follicular cells. Chromosomal rearrangements linking the promoter and NH2-terminal domains of unrelated gene/s to the COOH-terminal fragment of RET result in the aberrant production of a chimeric form of the receptor in thyroid cells that is constitutively active (2). Several forms have been identified that differ according to the 5' partner gene involved in the rearrangement, with RET/PTC1 and RET/PTC3 being the most common. Multiple lines of evidence point to RET/PTC as one of the key first steps in papillary thyroid cancer (PTC) pathogenesis, particularly in those arising after radiation exposure and in pediatric PTC (reviewed in ref. 3). Besides RET, recombinational activation of the NTRK tyrosine kinase receptor is present in a small fraction of PTCs.

Dimerization is required for the constitutive activation of the RET/PTC oncoproteins, a property conferred by the partners of RET in the respective fusion proteins. For RET/PTC1, a leucine zipper region within the NH2 terminus of H4 mediates dimerization (4), whereas in RET/PTC2 this is likely dependent on domains in the NH2 terminus of RI{alpha} cyclic AMP (cAMP)-dependent protein kinase A (5). In the case of RET/PTC3, this has not been formally tested, but there is a coiled-coil motif within ELE1 (6). This results in constitutive activation of the tyrosine kinase function of RET, autophosphorylation at selected tyrosine residues, and initiation of intracellular signaling by engagement with effectors through specific tyrosine-phosphorylated domains of the receptor (2).

In addition to RET and NTRK, which are activated by genetic recombination, other tyrosine kinase receptors may play a role in the development and progression of thyroid neoplasms. The epidermal growth factor receptor (EGFR) has been reported to be overexpressed in various types of thyroid carcinomas by some (7–10) but not all (11) groups, and EGFR overexpression may correlate with poor prognosis (12, 13). Increased expression of EGF or transforming growth factor-{alpha} has also been found in PTC and anaplastic thyroid cancer. In addition, coexpression of EGF and EGFR is associated with bone metastasis of follicular thyroid cancer (14). The mechanisms responsible for EGFR overexpression in thyroid cancer are not known; however, it does not seem to be due to gene amplification (15). EGFR activity may contribute to thyroid cancer growth because anti-EGFR antibodies (16) or small-molecule EGFR kinase inhibitors such as AG 1478, gefitinib, and AEE788 (17, 18) have shown antiproliferative effects against thyroid carcinoma cell lines in vitro and in vivo. A recent study challenged these observations and failed to observe inhibition of growth of thyroid cancer cell lines using a range of concentrations of AEE788 within the IC50 for the EGFR kinase (11).

EGFR activity is elevated in many human solid tumors, and in many cases, this is associated with progression and poor prognosis (19, 20). Several mechanisms are involved in aberrant EGFR signaling in cancer (reviewed in ref. 21): (a) activating mutations of EGFR, which are present in a variety of tumor types, including glioma, non–small cell lung cancer, prostate, breast, ovary, and stomach; (b) overexpression or activation of EGFR cognate ligands; (c) overexpression of wild-type EGFR resulting from gene amplification, increased transcription, translation, or other posttranscriptional mechanisms; (d) heterodimerization with other Erb family members (i.e., ErbB2, ErbB3, or ErbB4) leading to signal amplification; and (e) transactivation by other tyrosine kinase, cytokine, and G protein–coupled receptors.

Ligand activation of the insulin-like growth factor-I (IGF-I) receptor and platelet-derived growth factor receptor (PDGFR), respectively, transactivates EGFR, albeit through different mechanisms. Thus, phosphorylation of Shc and activation of the mitogen-activated protein kinase (MAPK) pathway by IGF-I in Cos-7 cells are due primarily to transactivation of EGFR through metalloproteinase activation and release of heparin-bound EGF (HB-EGF; ref. 22). By contrast, the PDGFβR and EGFR form heterodimers basally, and PDGF-induced MAPK activation is impaired when these heterodimers are disrupted (23).

Signaling activated by RET/PTC oncoproteins is initiated by formation of homodimers through coiled-coil motifs in the NH2 terminus of the respective fusion proteins, leading to autophosphorylation of at least 12 tyrosine residues, which then serve as docking sites for intracellular signaling molecules. Our interest in exploring possible interactions between oncogenic RET and EGFR activity was prompted by our observation that RET-induced signaling and cell growth were inhibited by EGFR kinase inhibitors. Interactions between RET and other transmembrane receptors have, to our knowledge, not been previously identified. Here, we show that RET/PTC induces EGFR gene expression and kinase activity and forms a complex with EGFR in a kinase-independent manner. EGFR in turn stimulates RET phosphorylation. Moreover, inhibition of EGFR kinase activity markedly impairs cell growth induced by a spectrum of activated RET mutants in different cellular contexts, supporting the functional relevance of these observations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
DNA constructs. The wild-type and kinase-dead (K721M) EGFR expression plasmids were a gift from Dr. Gibbes R. Johnson (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD). The RET/PTC1 and RET/PTC3 cDNAs were gifts from Sissy Jhiang (Ohio State University, Columbus, OH) and Dr. Massimo Santoro (University of Naples, Naples, Italy), respectively. pUG10-3 RET/PTC3 and pUG10-3 RET/PTC1 have been described previously (24). RET/PTC2-PDZ cDNA, in which nucleotides encoding the 22 COOH-terminal amino acids (including Y1062) were replaced with the Enigma PDZ domain (provided by Susan Taylor, University of California, San Diego, CA; ref. 25), was subcloned into pUG10-3. pEGFRpr-Luc, which contains nucleotides –1,180 to +29 of the mouse EGFR promoter, was cloned upstream of the firefly luciferase gene in the pGL2 basic plasmid and was a gift from Elaine Fuchs (Rockfeller University, New York, NY; ref. 26). To generate epitope-tagged RET/PTC3, the RET/PTC3 SmaI/XbaI fragment was excised from pUG10-3 and ligated in frame into EcoRV/XbaI-digested pcDNA4HisMax vector C (Invitrogen). To generate the kinase-defective tagged RET/PTC3S765P, wild-type RET/PTC3 in pUG10-3 was digested with BlpI and a double-stranded oligo (Table 1 ) containing the S765P substitution was annealed and ligated into the BlpI site. The RET/PTC3S765P SmaI/XbaI fragment was excised from pUG10-3 and ligated into pcDNA4HisMax vector C. All constructs were confirmed by DNA sequencing.


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  		Table 1. Primer pairs used

 
Cell lines. All PCCL3 cell lines were maintained in H4 complete medium consisting of Coon's medium/F12 high zinc supplemented with 5% fetal bovine serum (FBS), 0.3 mg/mL L-glutamine, 1 mIU/mL thyrotropin (TSH), 10 µg/mL insulin, 5 µg/mL apo-transferrin, 10 nmol/L hydrocortisone, and penicillin/streptomycin. H3 complete medium was identical to H4 but without the addition of TSH. Doxycycline-inducible cell lines expressing RET/PTC3, RET/PTC1, RET/PTC2-PDZ (24), BRAFV600E (27), reverse tetracycline transactivator (rTTA), and HRASV12 (28) have been described previously. The TT cell line (a gift from Robert Gagel, University of Texas M. D. Anderson Cancer Center, Houston, TX), which was derived from a human medullary thyroid carcinoma expressing RETC634W (MEN2A), was grown in Ham's F12 medium with 10% FBS. NIH3T3 cells expressing RETC634W were a gift from Dr. Massimo Santoro. NIH3T3 cells expressing RET/PTC3 or RETM918T (MEN2B) were generated by stable transfection of pcDNA3.1-RET/PTC3 or pBABE puro RETM918T, respectively, and G418-resistant clones were isolated and expression of the respective proteins was determined by Western blotting. All NIH3T3-derived cell lines were grown in DMEM containing 10% calf serum and 1.0 µg/mL puromycin (Sigma) unless indicated. Cos-7 cells were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Antibodies and reagents. The goat anti-EGFR, goat anti-RET, anti-protein kinase C{alpha} (PKC{alpha}), anti-phospholipase C{gamma} (PLC{gamma}), anti-phospho-Y783 PLC{gamma}, anti-MET, and the species-specific horseradish peroxidase (HRP)-conjugated IgGs were purchased from Santa Cruz Biotechnology, Inc. The anti-phosphotyrosine and rabbit anti-EGFR were obtained from Upstate Biotechnology. Anti-phospho-Y783 PLC{gamma} and anti-phospho-Y905 RET were purchased from Cell Signaling Technology. Anti-phospho-Y1173 EGFR was from BIOMOL Research Laboratories. Mouse anti-EGFR was from Sigma. The mouse anti-RET was a kind gift from Dr. Yuri Nikiforov (University of Cincinnati, Cincinnati, OH). The pan-matrix metalloproteinase (MMP) inhibitor GM6001 was from Chemicon International; indomethacin and U0126 were from Calbiochem.

Transient transfection and immunoprecipitation. Cos-7 cells were transfected using Lipofectamine 2000 as directed by the manufacturer (Invitrogen). Ten hours after transfection, the medium was replaced with serum-free medium and cells were incubated for 10 to 16 h. Cells were then washed with ice-cold PBS containing 0.2 mmol/L sodium orthovanadate and lysed by incubating with ice-cold radioimmunoprecipitation assay buffer [20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1.0% NP40, 1.0% Tween 20, 20 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1 mmol/L EGTA, 5 mmol/L EGTA, 0.2 mmol/L phenylmethylsulfonyl fluoride (Sigma)] for 20 min and passage through a 26-gauge needle. Lysates were centrifuged for 30 min at 4°C, the supernatant was collected, and the protein concentration was determined using Coomassie Plus (Pierce) as directed by the manufacturer. Equal amounts of protein from lysates were incubated with anti-EGFR (Upstate Biotechnology) for 2 h at 4°C and then with prewashed protein A/G agarose (Santa Cruz Biotechnology) for 1 h at 4°C. The immunoprecipitate was washed thrice with washing buffer [50 mmol/L HEPES (pH 7.2), 20 mmol/L MnCl2, 5 mmol/L MgCl2], incubated with SDS-PAGE loading buffer for 10 min at 95°C, and subjected to SDS-PAGE. Proteins were transferred to nitrocellulose or polyvinylidene difluoride (PVDF) membranes and Western blotted with the indicated antibody.

Western blotting. Cell lysates were collected by centrifugation at 4°C for 20 min. Protein concentrations were determined using Coomassie Plus and 100 µg of total protein were subjected to SDS-PAGE and transferred to nitrocellulose or PVDF membranes, and membranes were probed with the indicated antibody. Bands were detected by incubating with species-specific HRP-conjugated IgGs and then with enhanced chemiluminescence reagent (Amersham Biosciences Corp.). Images were captured using the Kodak Image Station 440CF. Band densities were quantitated using Kodak 1D image analysis software.

Quantitative reverse transcription-PCR. The indicated cell lines were grown until confluent and incubated in H3 medium for 5 d before being incubated with H3 or H4 medium with or without doxycycline for 48 h. RNA was then isolated using TRI reagent as directed by the manufacturer (Molecular Research Center, Inc.). Total RNA (2 µg) was reverse transcribed with 200 units of SuperScript III First-Strand Synthesis System (Invitrogen) in the presence of 2.5 µmol/L of random 9-mer primers and 20 µmol/L deoxynucleotide triphosphate for 60 min at 50°C. Quantitative PCR amplifications were performed using the QuantiTect SYBR Green PCR kit as directed by the manufacturer (Qiagen, Inc.). The amplification conditions were optimized for the LightCycler instrument (Cepheid) and shown to result in a single PCR product by melting curve and electrophoretic analysis. Primer pairs for each gene (Table 1) were designed with the Primer3 software (Whitehead Institute for Biomedical Research). PCR primer pairs were designed to span a large intron whose location was determined by BLAST analysis of the cDNA sequence of EGFR, ErbB2, ErbB3, cMET, or β-actin against the National Center for Biotechnology Information rat genome database. This was further verified by the lack of a signal when reactions were performed in the absence of reverse transcriptase. The CT value, which was determined using the second derivative, was used to calculate the β-actin normalized expression of the different mRNAs using the Q-Gene program (29). Reactions were performed in triplicate.

EGFR promoter assay. PC-PTC3 cells were grown until confluent and incubated in H3 medium for 3 d. Cells were transfected with cytomegalovirus (CMV)-Renilla and pEGFRpr-Luc or pGL3-Basic using Fugene6 (Roche) and then incubated with H3 or H4 medium with or without doxycycline for 48 h. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System directed by the manufacturer (Promega). Fold induction between cells incubated with or without doxycycline was calculated after normalizing to CMV-Renilla and subtracting luciferase activity in pGL3-Basic–transfected cells.

Preparation of glutathione S-transferase-RET and RET kinase assays. The activity profile of PKI166 has been reported previously (30). To explore its effects on RET kinase, a glutathione S-transferase (GST)-fused RET kinase domain was expressed in baculovirus and purified over glutathione-Sepharose. Kinase activity was tested by measuring the phosphorylation of a synthetic substrate [poly(Glu, Tyr)] by purified GST fusion kinase domains of the respective protein kinase in the presence of radiolabeled ATP; ATP concentrations used were optimized within the Km range for the individual kinases. Briefly, each kinase was incubated under optimized buffer conditions in 20 mmol/L Tris-HCl (pH 7.5), 1 to 3 mmol/L MnCl2, 3 to 10 mmol/L MgCl2, 10 µmol/L Na3VO4, 1 mmol/L DTT, 0.2 µCi [{gamma}-33P]ATP, 1 to 8 µmol/L ATP, 3 to 8 µg/mL poly(Glu:Tyr) (4:1), and 1% DMSO in a total volume of 30 µL in the presence or absence of NVP-AST487 for 10 min at ambient temperature. Reactions were terminated by adding 10 µL of 250 mmol/L EDTA, and the reaction mixture was transferred onto an Immobilon PVDF membrane (Millipore). Filters were washed (0.5% H3PO4), soaked in ethanol, dried, and counted in a liquid scintillation counter. IC50s for PKI166 were calculated by linear regression analysis of the percentage inhibition.

Growth curves. The various cell lines were seeded in multiple six-well plates, and 24 h later, the number of attached cells in a representative plate was determined by counting the trypsinized cells with a Z1 Coulter counter (Beckman Coulter). The remaining plates were incubated in the indicated experimental condition and the cells were counted as described above. All experiments were performed in triplicate and medium was replaced every 48 h.

Statistical evaluation. A single-sample t test was used to compare experimental samples where control values were normalized to 1. In all other cases, a standard two-tailed t test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Induction of EFGR by RET/PTC1 and RET/PTC3 in PCCL3 cells. We first examined factors controlling EGFR gene expression in thyroid cells. TSH induced EGFR mRNA by >3-fold in PCCL3 cells expressing the reverse rTTA as the sole heterologous gene. These served as controls for the other cell lines modified to conditionally express the indicated oncoproteins. Similar effects of TSH were seen in all other PCCL3-derived cell lines when grown without doxycycline (Fig. 1A ). Accordingly, TSH also increased EGFR abundance as determined by Western blotting (Fig. 1B). Doxycycline-inducible expression of RET/PTC1 or RET/PTC3 increased EGFR mRNA levels by ~3- to 4-fold in TSH-deprived cells. The combined effects of TSH and RET/PTC1 or RET/PTC3 on EGFR mRNA (Fig. 1A) and protein (Fig. 1B) were at least additive. In addition, RET/PTC induced EGFR phosphorylation as determined by Western blotting with an anti-EGFR pY1173 antibody, consistent with activation of the receptor. The phosphorylated EGFR to total EGFR ratio was not significantly increased by RET/PTC3 (Fig. 1C), indicating that RET-induced EGFR phosphorylation could primarily be due to overexpression of the receptor. To determine whether RET/PTC increases EGFR transcription, PC-PTC3 cells were transiently transfected with a reporter construct consisting of –1,210 bp (–1,180 to 29) of the mouse EGFR promoter. RET/PTC activation was associated with 3.2- and 32.6-fold induction in EGFR promoter activity in the absence and presence of TSH, respectively (Fig. 1D). To explore whether other factors may also have contributed to RET-induced EGFR activation, we first explored whether RET-induced release of HB-EGF may have participated in this process. For this, PC-PTC3 cells were grown with or without TSH for 3 days and then incubated with doxycycline in the presence or absence of 20 µmol/L GM6001, a pan-MMP inhibitor that prevents release of HB-EGF. Treatment with GM6001 did not alter EGFR phosphorylation in either basal conditions or after stimulation with TSH- or doxycycline-induced RET/PTC (data not shown). Similarly, RET-induced prostaglandin biosynthesis was not involved because EGFR phosphorylation was not prevented by a 24-h treatment with 50 µmol/L indomethacin (data not shown).


Figure 1
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  		Figure 1. Effects of TSH and/or RET/PTC on EGFR, MET, ErbB2, and ErbB3 mRNA abundance in PCCL3 cells. A, PCCL3 cell lines expressing only rTTA or doxycycline (Dox)-inducible RET/PTC1 (PTC1) or RET/PTC3 (PTC3) were grown to confluence and placed in H3 medium (no TSH) for 4 to 6 d before switching to the indicated conditions for 48 h. Quantitative reverse transcription-PCR (RT-PCR) was performed in triplicate using β-actin as the internal control. Data are expressed relative to cells grown in the absence of TSH and doxycycline. Columns, mean of at least two different experiments; bars, SD. *, P < 0.05 versus control. B, conditions were as described in A. Cell lysates containing 100 µg protein was separated on a 4% to 20% gradient polyacrylamide denaturing gel. The membrane was immunoblotted sequentially with antibodies to EGFR, MET, and RET. Protein loading was determined by immunoblotting with PKC{alpha}. C, a second set of cell lysates containing 100 µg protein was separated on a 4% to 20% gradient polyacrylamide denaturing gel. The membrane was immunoblotted sequentially with antibodies to EGFR (pY1173), EGFR, and RET. D, PC-PTC3 cells were transfected with pEGFRpr-Luc and incubated with or without doxycycline for 48 h. Columns, mean fold increase in luciferase activity versus doxycycline; bars, SE. *, P < 0.02.

 
By contrast to the effects on EGFR, neither TSH nor oncogenic RET altered expression of other members of the Erb family (i.e., ErbB2 or ErbB3; Fig. 1A). RET/PTC oncoproteins, but not TSH, did induce expression of the tyrosine kinase receptor MET, which is often overexpressed in thyroid cancers (31).

RET/PTC signaling via RAS-RAF-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK has been implicated in thyroid cell transformation toward a papillary lineage. We explored the contribution of this pathway to RET/PTC-induced EGFR overexpression. Doxycycline-inducible expression of oncogenic mutants of HRAS and BRAF (HRASG12V or BRAFV600E, respectively) also increased EGFR mRNA abundance (Fig. 2A ), although to a lesser extent than activated RET. Neither HRASG12V nor BRAFV600E significantly affected MET mRNA levels. The RET/PTC-induced increase in EGFR mRNA showed a partial requirement for MEK/ERK, as treatment of cells with the MEK inhibitor U0126 at the time doxycycline was added markedly blunted RET induction of EGFR (Fig. 2B).


Figure 2
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  		Figure 2. Effects of oncogenic HRAS or BRAF on EGFR and MET mRNA abundance in PCCL3 cells. A, PCCL3 cell lines with doxycycline-inducible expression of HRASG12V (HRAS) or BRAFV600E (BRAF) were grown as described in Fig. 1A. RNA was isolated and quantitative RT-PCR for MET or EGFR was performed in triplicate using β-actin as the internal control. B, PCCL3 cells with doxycycline-inducible expression of RET/PTC3 were treated with or without 10 µmol/L U0126 added simultaneously with doxycycline. RNA was prepared as described in Materials and Methods and quantitative RT-PCR for EFGR was performed. Data are expressed relative to cells grown in the absence of TSH and doxycycline. Columns, mean of at least two different experiments; bars, SD. *, P < 0.05.

 
Coimmunoprecipitation of EGFR and RET/PTC3. As will be shown in greater detail further below, we began to entertain the hypothesis that RET and EGFR may reciprocally transactivate each other because PKI166, a potent EGFR kinase antagonist, inhibited RET/PTC phosphorylation in PCCL3 cell extracts despite lacking inhibitory activity on a purified GST-RET kinase. To determine if RET/PTC was present in a complex with EGFR, extracts prepared from PC-PTC1 or PC-PTC3 cells treated with or without doxycycline were immunoprecipitated with rabbit anti-EGFR and blotted with mouse anti-RET. Both RET/PTC1 and RET/PTC3 coimmunoprecipitated with EGFR (Fig. 3A ). The RET/PTC oncoproteins were not detected in extracts prepared from cells not treated with doxycycline or when extracts were immunoprecipitated with a normal rabbit IgG (Fig. 3A). None of the commercially available RET antisera immunoprecipitated RET in our hands, so we were unable to confirm the association using a reciprocal approach. The EGFR-RET association does not require the extracellular domain of RET (which is absent in RET/PTC chimeric proteins) or RET-Y1062, as the PDZ-RET/PTC fusion protein, which lacks the COOH-terminal 22 amino acids including Y1062, also coimmunoprecipitated with EGFR (Fig. 3B). As this fusion protein lacks Y1062, it cannot recruit Shc, thus indicating that signaling via Shc-Grb2-RAS is not required for the interaction between these two proteins. Moreover, coimmunoprecipitation with EGFR is not dependent on the specific NH2-terminal domains of the respective RET/PTC oncoproteins because RET/PTC1, RET/PTC2, and RET/PTC3 associate with EGFR.


Figure 3
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  		Figure 3. EGFR coimmunoprecipitates with RET/PTC1 and RET/PTC3 in PCCL3 cells. A, PC-PTC1 or PC-PTC3 cells were grown to near confluence and then placed in H3 medium (no TSH) for 5 to 6 d before switching to the indicated conditions for 48 h. Cell lysates containing 600 µg of total protein were immunoprecipitated (IP) with nonimmune IgG (nIgG) or anti-EGFR antibodies. The washed immunocomplexes were then separated on a gradient gel (4–20%) and subjected to Western blot analysis. The membranes were cut and immunoblotted (IB) with either anti-RET or EGFR IgGs. B, cells with doxycycline-inducible expression of RET/PTC3 (PTC3) or RET/PTC2-PDZ (PDZ) were grown in H4 medium (with TSH) until almost confluent and then treated with or without doxycycline for 48 h. Cell lysates were prepared and immunoprecipitated with nonimmune IgG or EGFR antibodies. Representative blot of four experiments is shown. The arrows mark EGFR, RET/PTC2-PDZ, and RET/PTC3. C, representative Western blot of PC-PTC3 cell extracts treated with or without 1 µmol/L doxycycline in the presence of TSH for 24 h to activate RET/PTC3, in the presence or absence of the indicated concentration of PKI166. Blot was sequentially probed with anti-phospho-Y905 RET, anti-phospho-Y783 PLC{gamma}, anti-RET, and anti-PLC{gamma}. PKI, PKI166. D, dose-dependent inhibition of RET pY905 and PLC{gamma} pY783 by PKI166. Bars, average percent decrease compared with untreated cells from three independent experiments. *, P < 0.001; **, P < 0.01 versus no PKI166.

 
Role for EFGR in activation of RET/PTC. To investigate the contribution of EGFR activity to RET/PTC-mediated signaling, cells were treated with PKI166 (30), a potent EGFR kinase inhibitor with no in vitro activity on GST-RET kinase (see Materials and Methods). PKI166 inhibited RET/PTC-induced RET-Y905 and PLC{gamma} phosphorylation in PCCL3 cells (Fig. 3C and D). These data could be explained if PKI166 effects on RET kinase were indirect and mediated via inhibition of EGFR. As shown in Fig. 4A , association of EGFR with RET/PTC was likely not dependent on their respective kinase activities, as KD-EGFR and KD-RET/PTC3S765P also coimmunoprecipitated. This is further supported by the observation that KD-RET/PTCS765P and EGFR coimmunoprecipitate in the presence of 500 nmol/L PKI166, which completely blocks tyrosine phosphorylation of both proteins (Fig. 4C). The RET/PTC association with EGFR did not result in EGF-independent EGFR phosphorylation. The trace amount of EGFR immunoreactivity observed with the phosphotyrosine antibody in cells transfected with KD-EGFR is likely the result of endogenous EGFR activation in the host cells.


Figure 4
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  		Figure 4. EGFR-dependent RET phosphorylation in Cos-7 cells. A, representative Western blot of anti-EGFR immunoprecipitates of Cos-7 cells transiently transfected with the following constructs: empty vector, EGFR, EGFRKD, EGFR + RET, EGFR + RETKD, EGFRKD + RET, or EGFRKD + RETKD. Before harvesting, cells were washed and placed in complete medium for 10 h before switching to serum-free medium for 16 h. Where indicated, 60 ng/mL EGF was added for 5 min before harvesting. Lysates were prepared and 400 µg of total protein were immunoprecipitated with anti-EGFR IgG. The washed immunocomplexes were then separated and sequentially immunoblotted with antibodies to anti-phosphotyrosine, total EGFR, and RET. B, columns, fold induction of RET phosphorylation by EGF of three different experiments; bars, SE. *, P = 0.037. C, representative Western blot of anti-EGFR immunoprecipitates of Cos-7 cells transiently transfected with the indicated constructs. Cells were treated as in A except that, where indicated, 500 nmol/L PKI166 was added 12 h before harvest.

 
By contrast, EGF-mediated activation of EGFR was associated with greater phosphorylation of kinase-defective RET/PTC (RET-KD; Fig. 4B). There was no effect of EGF on wild-type RET/PTC3 phosphorylation possibly because the baseline phosphorylation state of the overexpressed oncoprotein was very high, making additional increments hard to detect.

To confirm phosphorylation of RET/PTC by EGFR, RET-KD and EFGR were cotransfected into Cos-7 cells and cells were treated with EGF. The addition of EGF produced a marked increase in phosphorylation of EGFR and RET-KD. The EGF-induced increase in phosphorylation of both EGFR and RET-KD was completely blocked by the addition of PKI166 (Fig. 4C), consistent with a requirement of EGFR kinase activity for phosphorylation of the kinase-defective RET mutant.

Role EGFR in RET-mediated growth. Growth of NIH3T3 cells stably expressing constitutively active mutants of RET-RET-C634W (RET-MEN2A), RET-M918T (RET-MEN2B), or RET/PTC3 was markedly inhibited by 300 nmol/L PKI166, whereas growth of vector-transfected NIH3T3 cells was not (Fig. 5A and C ). Similar inhibitory effects were seen on the medullary carcinoma cell line TT. These effects were reproduced with the EGFR kinase inhibitors gefitinib (Fig. 5B) and AEE788 (Fig. 5C), which inhibited RET-mediated growth of NIH3T3 cells and of the human PTC cell line TPC1, which harbors a naturally occurring RET/PTC1 rearrangement.


Figure 5
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  		Figure 5. EFGR kinase inhibitors impair growth of cell lines expressing constitutively active RET-mutant proteins. A, the following cell lines were grown in 1% or 5% FBS in the presence or absence of 300 nmol/L PKI166: human TT and NIH3T3 cells stably transfected with empty vector, RET-MEN2A, RET-MEN2B, or RET/PTC3. Cells were plated in six-well dishes and harvested after 10 d, with medium changes every 2 d. Data represent fold change compared with vector-transfected NIH3T3 in 1% serum. *, P < 0.001, PKI166 versus vehicle. Columns, mean of two independent experiments performed in triplicate; bars, SD. B, gefitinib inhibits RET-MEN2A–induced growth of NIH3T3 cells. NIH3T3 cells stably transfected with empty vector or an expression vector for RET-MEN2A were grown in 1% or 5% FBS in the presence or absence of 1 µmol/L gefitinib for 10 d. Columns, mean of two different experiments performed in triplicate; bars, SD. *, P < 0.001, gefitinib versus vehicle. C, NIH3T3 cells stably expressing RET-MEN2A, human papillary cancer TPC1 cells, and vector-transfected NIH3T3 cells were incubated with the indicated concentration of inhibitors and counted at the indicated times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
EGFR is frequently overexpressed in cancer cells. As EGFR transduces growth and survival signals in many cell types, pharmacologic approaches to inhibit its enzymatic activity have been the subject of considerable interest for cancer therapy. EGFR signaling can be increased in cancer cells through several mechanisms: higher expression, activating mutations, heterodimerization with other members of the ErbB family, greater ligand concentrations, and alterations in signaling molecules downstream of EGFR that enhance signal output (32).

Most studies report increased EGFR expression in thyroid cancer, but the mechanisms responsible for this are not well understood. To explore factors regulating EGFR gene expression in thyroid cells, we examined the effects of TSH, RET/PTC3, and BRAF in PCCL3 cells. TSH induced a robust increase in EGFR mRNA and protein. This is consistent with the observations of Westermark and colleagues (33) who reported that ligand activation of the TSH receptor, likely acting via cAMP, increased the number of EGFR in porcine thyroid membranes. In UMR 106-01 cells, PTH and cAMP also induce EGFR gene transcription (34). Indeed, there is a cAMP-responsive element in the EGFR gene promoter, likely mediating these effects (35). RET/PTC1 and RET/PTC3 also up-regulated EGFR expression, with a magnitude of induction similar to that seen with TSH. RET/PTC induction of EGFR expression showed a partial requirement for MAPK signaling, as it was decreased by pharmacologic inhibition of MEK and recapitulated by expression of activated RAS or BRAF. The effects of TSH and RET/PTC were at least additive, suggesting that independent pathways are involved in its regulation. Taken together, these data indicate that two of the critical signaling pathways implicated in thyroid oncogenesis, TSH receptor-adenyl cyclase-cAMP-protein kinase A and RET-RAS-RAF-MEK, cooperate to evoke a major increase in EGFR abundance in thyroid cells.

Besides increasing EGFR expression, activation of RET/PTC resulted in EGFR phosphorylation, but this was proportional to the increase in receptor mass. We examined whether other mechanisms of EGFR transactivation may also be contributing to this effect. Tyrosine kinase receptors and G protein–coupled receptors have been reported to stimulate metalloproteinases, which cleave and release EGF-like precursors in the extracellular surface of the plasma membrane (36). Activation of the EP1 receptor by prostaglandin E2, which is commonly elevated in a variety of cancers as a result of overexpression of cyclooxygenase (COX)-2 (37), increases phosphorylation of EGFR in cholangiocarcinoma (38) and hepatocellular carcinoma (39) cell lines. Neither of these mechanisms seemed to be involved in RET-induced EGFR phosphorylation, as the process was not prevented by incubation with MMP or COX inhibitors.

We were prompted to explore additional interactions between RET and EGFR because of the observation that PKI166, a potent EGFR kinase inhibitor, decreased RET autophosphorylation and signaling in cell extracts despite lacking effect on kinase activity of a purified GST-RET fusion protein. As autophosphorylation of RET/PTC is ligand independent, and there is no other known intracellular mediator of RET phosphorylation, this pointed to the possibility that EGFR itself may mediate RET activation. RET/PTC was found to coimmunoprecipitate with EGFR, indicating that they were part of a common complex. RET/PTC oncoproteins differ in their NH2-terminal domains, which are encoded by the upstream gene recombination partner in the rearrangement. These domains of H4, PRKAR1A, and ELE1, which are the corresponding partners for RET/PTC1, RET/PTC2, and RET/PTC3, respectively, either have been shown (5) or are believed to encode for coiled-coil motifs, which mediate oligomerization of the fusion proteins. We considered the possibility that one of these domains could also mediate the association with EGFR. Our data suggest that this is unlikely because all three RET oncoproteins coimmunoprecipitated with EGFR. Alternatively, the RET/PTC-EGFR interaction could be mediated by adaptors or other signaling effectors recruited following activation and autophosphorylation of each of these receptors. To test this proposition, we examined whether a mutant of RET/PTC modified to prevent association with Shc retained the association with EGFR, and this was indeed the case. Moreover, a kinase-defective mutant of RET (RET/PTC3S765P) coimmunoprecipitated with EGFR. It is unlikely that the RET-EGFR complex is formed through recruitment of proteins to phosphotyrosine residues of these receptors, as RET/PTC3S765P associated with KD-EGFR and with wild-type EGFR in the presence of PKI166, which completely blocks tyrosine phosphorylation of both RET/PTC3S765P and EGFR. Src has been implicated in transactivation of EGFR following agonist-induced G protein–coupled receptor signaling (40, 41). As Src kinase activity is regulated by RET, and may mediate its mitogenic effects, it is conceivable that this tyrosine kinase may play a role in the RET-EGFR interaction. The precise nature of the association of Src and EGFR is not fully understood, but it is likely dependent on receptor autophosphorylation, as is the case with PDGFR (42). Indeed, there is evidence that Src may bind to pY891 of EGFR through its SH2 domain (42). As KD-EGFR lacks any detectable autophosphorylation yet retains its ability to associate with RET, Src is an unlikely mediator of this phenomenon. At this point, we have not further attempted to identify the components of this complex or to explore the contribution of individual proteins to the EGFR-RET association.

We did not find any evidence that the immunoprecipitated complex of RET/PTC3 with KD-EGFR was associated with increased EGFR phosphorylation. This is again consistent with our conclusion that RET-induced EGFR activation is largely due to increased expression of EGFR. By contrast, ligand-stimulated EGFR does transactivate RET, as EGF-induced EGFR activation increases phosphorylation of KD-RET/PTC, which is completely blocked by treatment with the EGFR kinase inhibitor PKI166. As the KD-RET/PTC lacks an extracellular domain, this must be mediated by EGFR or an EGFR-regulated intracellular kinase, the identity of which is unknown.

The potential significance of this observation was shown in a human thyroid cancer cell line harboring an endogenous activating RET rearrangement (PTC1, derived from a PTC) as well as NIH3T3 cells stably expressing the most common activating mutants of RET. Three multikinase inhibitors with potent effects on EGFR kinase activity were tested, PKI166, gefitinib, and AEE788, all of which exerted consistent growth-inhibitory effects on the thyroid cancer line and on RET-transfected cell lines at submicromolar concentrations. PKI166 and AEE788 are pyrrolopyrimidines (30), whereas gefitinib is a quinazoline (43). Gefitinib and PKI166 lack activity against RET, whereas AEE788 is moderately active on this kinase (IC50, 0.74 µmol/L), so we cannot exclude that some of the biological effects of the latter compound may have been exerted via RET, perhaps explaining its more potent effects. However, AEE788 inhibited RET-induced growth at concentrations below its IC50 for this kinase, indicating that other targets were involved. Based on our data, it is reasonable to postulate that the growth-inhibitory properties of these compounds were mediated via EGFR, although we cannot exclude interference with other distal effectors of RET.

This has implications for preclinical development. There is now considerable interest in developing compounds that hit more than one target, either to overcome resistance or to interfere with multiple pathways of pathogenetic significance (44). ZD6474, a lead compound showing considerable effectiveness in phase 2 trials for medullary thyroid carcinomas that harbor RET mutations, is also a potent EGFR inhibitor.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
J.A. Fagin: commercial research grant, Novartis. The University of Cincinnati and J.A. Fagin: patent on PKI166 for treating mutated RET kinase–associated diseases. H.A. Lane: employment, Novartis Pharma AG. The other authors disclosed no potential conflicts of interest.


   Acknowledgments
 
Grant support: CA50706, CA72597, and Novartis Oncology.

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 2/ 1/08. Revised 3/ 6/08. Accepted 3/12/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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