ret-familial medullary thyroid carcinoma mutants y791f …that stat3 activation is mediated by a...

RET-Familial Medullary Thyroid Carcinoma Mutants Y791F

and S891A Activate a Src/JAK/STAT3 Pathway, Independent

of Glial Cell Line–Derived Neurotrophic Factor

Ivan Plaza Menacho,1Roelof Koster,

1Almer M. van der Sloot,

2Wim J. Quax,

2Jan Osinga,

1

Tineke van der Sluis,3Harry Hollema,

3Grzegorz M. Burzynski,

1Oliver Gimm,

5

Charles H.C.M. Buys,1Bart J.L. Eggen,

4and Robert M.W. Hofstra

1

Departments of 1Medical Genetics, 2Pharmaceutical Biology, 3Pathology, and 4Developmental Genetics, University of Groningen,Groningen, the Netherlands; 5Department of Surgery, Ernst-Grube-Strasse, Martin-Luther-University, Halle-Wittenberg, Germany

Abstract

The RET proto-oncogene encodes a receptor tyrosine kinasewhose dysfunction plays a crucial role in the development ofseveral neural crest disorders. Distinct activating RET muta-tions cause multiple endocrine neoplasia type 2A (MEN2A),type 2B (MEN2B), and familial medullary thyroid carcinoma(FMTC). Despite clear correlations between the mutationsfound in these cancer syndromes and their phenotypes, themolecular mechanisms connecting the mutated receptor tothe different disease phenotypes are far from completelyunderstood. Luciferase reporter assays in combination withimmunoprecipitations, and Western and immunohistochem-istry analyses, were done in order to characterize the signalingproperties of two FMTC-associated RET mutations, Y791F andS891A, respectively, both affecting the tyrosine kinase domainof the receptor. We show that these RET-FMTC mutants aremonomeric receptors which are autophosphorylated andactivated independently of glial cell line–derived neurotrophicfactor. Moreover, we show that the dysfunctional signalingproperties of these mutants, when compared with wild-typeRET, involve constitutive activation of signal transducer andactivator of transcription 3 (STAT3). Furthermore, we showthat STAT3 activation is mediated by a signaling pathwayinvolving Src, JAK1, and JAK2, differing from STAT3 activationpromoted by RETC634R which was previously found to beindependent of Src and JAKs. Three-dimensional modeling ofthe RET catalytic domain suggested that the structuralchanges promoted by the respective amino acids substitutionslead to a more accessible substrate and ATP-binding mono-meric conformation. Finally, immunohistochemical analysis ofFMTC tumor samples support the in vitro data, becausenuclear localized, Y705-phosphorylated STAT3 as well as ahigh degree of RET expression at the plasma membrane wasobserved. (Cancer Res 2005; 65(5): 1729-37)

Introduction

The RET proto-oncogene encodes a receptor tyrosine kinasewhich is expressed in neural crest–derived cells, including entericand sympathetic neurons, adrenal chromaffin cells, and parafollic-ular C cells of the thyroid gland (1). Ligands of the RET receptor are

members of the glial cell line–derived neurotrophic factor (GDNF)family: GDNF, neurturin, persephin, and artemin. They are able toactivate RET in the presence of GFR-a(1-4) glycosyl phosphatidyl-inositol anchored coreceptors (2–7).Activation of RET by its ligand results in transphosphorylation of

multiple tyrosine residues that, in turn, act as docking sites andinteract with specific adaptor proteins to trigger downstreamsignaling pathways crucial for the survival and differentiationof neural crest–derived lineages, as well as kidney organogenesis(8–10). Phosphotyrosine residues 905, 981, 1,015, 1,062, and 1,096serve as docking sites for Grb7/10, c-Src, phospholipase C-g, Shc/Enigma/Frs2/IRS1-2/Dok4-5, and Gbr2, respectively (11–15).In general, the signaling pathways activated by RET include Ras-mitogen-activated protein kinases, PI3K, c-Jun NH2-terminal kinase,p38, ERK-5, phospholipase C-g, and signal transducer and activatorof transcription 3 (STAT3) (16–18). Additionally, RET can activatemembers of the Rho family of GTPases, including Rho, Rac, andCdc42, that are involved in the reorganization of the cytoskeletonand are responsible for cell motility and morphology (19, 20).Tyrosine residues located at the catalytic domains such as Y687,Y806, Y809, Y900, Y826, and Y1029 have also been shown to bephosphorylated in response to RET activation but their down-stream signaling pathways have not yet been elucidated (21, 22).Specific germ line missense mutations in the RETproto-oncogene

leading to a constitutive activation of the receptor cause a dominantinherited cancer syndrome called multiple endocrine neoplasia type2 (MEN2) and a familial form of medullary thyroid cancer (FMTC).Depending on the affected tissues, two different clinical subtypes ofMEN2 can be recognized. MEN2A is characterized by MTC,pheochromocytoma, and hyperplasia of the parathyroid. MEN2Bis characterized by MTC, pheochromocytoma, but instead ofhyperplasia of the parathyroid, patients develop neuromas intongue, lips, and eyelids, as well as intestinal ganglioneuromas.In FMTC, only the C cells of the thyroid become malignant (23).Mutations located in the cysteine-rich domain of RET (codons

609, 611, 618, 620, 630, and 634) give rise to MEN2A and FMTC.Distinct mutations found in the tyrosine kinase domain of thereceptor can give rise to FMTC (codons 768, 790, 791, 804, and 891)or to MEN2B (codons 883 and 918; ref. 24). Interestingly, mutationsin the cysteine-rich domain (codons 609, 611, 618, and 620) are notonly found in families with MEN2A/FMTC but also in patients withHirschsprung’s disease, a congenital malformation characterized byan absence of enteric ganglia cells in the distal part of the colon, orpatients having a combination of MEN2 and Hirschsprung’sdisease (25, 26).As mentioned, FMTC mutations can be localized both in the

cysteine-rich motif as well as in the catalytic domain of the protein. In

Requests for reprints: Robert M.W. Hofstra, Department of Medical GeneticsUniversity of Groningen, Antonius Deusinglaan 4, 9713 AW Groningen,the Netherlands. Phone: 31-50-363-2925; Fax: 31-50-363-2947; E-mail:[email protected].

I2005 American Association for Cancer Research.

www.aacrjournals.org 1729 Cancer Res 2005; 65: (5). March 1, 2005

Research Article

Research. on January 26, 2020. © 2005 American Association for Cancercancerres.aacrjournals.org Downloaded from

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this report, we investigate the activation and downstream signalingroutes triggered by two RET-FMTC mutants, Y791F (27), and S891A(28), both mutations are localized in the tyrosine kinase domain of thereceptor.

Materials and Methods

Cell Lines and Cell Culture Reagents. SK-N-SH, COS-7, HepG2,

HEK293, and MZ-CRC-1 (29) cells were grown in DMEM supplemented

with 10% FCS (Life Technologies, Rockville, MD), 100 Ag/mL penicillin,

100 Ag/mL streptomycin and 2 mmol L-glutamine. MTC-TT cells (30) were

grown in RPMI supplemented with 15% FCS (Life Technologies), 100 Ag/mL

penicillin, 100 Ag/mL streptomycin, and 2 mmol L-glutamine.

Cells were stimulated with 50 or 100 ng/mL GDNF where indicated(Prepotech, Rocky Hill, NJ). Different inhibitors were employed: U0126

(Promega, Madison, WI), AG1296, AG490, and PP2 were purchased from

Calbiochem (San Diego, CA).Expression and Reporter Plasmids. The pRC-cytomegalovirus-RETwt

(RETWT) plasmid encoding the short form of the human RET proto-

oncogene cDNA was used to create the RET S891A (RETS891A) and

RETY791F (RETY791F) mutants by site-directed mutagenesis according tothe manufacturer’s instructions (Stratagene, La Jolla, USA) using

the following forward and reverse primers: RETS891A 5V-GGA-AGATGAAGATTGCGGATTTCGGCTTGTCCC-3V and RETS891A 3V-GGG-ACAAGCCGAAATCCGCAATCTTCATCTTCC-5V, RETY791F 5V-CCACATGTCATCAAATTGTTTGGGGCCTGCAGCCAGCATGGCCC-3, and

RETY791F 3V-GGGCCATGCTGGCTGCAGGCCCCAAACAATTTGATGA-ACATGTGG-5, respectively. Following mutagenesis, all constructs were

fully sequenced.

The Mercury Pathway Profiling Luciferase System #K2049-1 (Clontech,

Palo Alto, CA) containing several pathway-specific reporter constructs was

used. We also tested the pIRE-ti-Luc (containing two copies of the

interleukin-6 response element of the intercellular adhesion molecule-1

promoter) and pIREmut-ti-Luc reporters (17). The dominant-negative (DN)

JAK1 and JAK2 constructs were kindly provided by Dr. Lu-Hai Wang

(Department of Microbiology, Mount Sinai School of Medicine, New York,

NY) and were described previously (31). The DN-Src plasmid was a gift of

Dr. Yung H. Wong (Department of Biochemistry, Hong Kong University of

Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China) and

was described previously (32).

The pSG5-STAT3a and pGS5-STAT3h expression plasmids were a gift

from Dr. R. de Groot (Department of Pulmonary Diseases, University

Medical Center, Utrecht, The Netherlands).

Luciferase Reporter Assays. Cells were transfected using the calcium

phosphate precipitation method as described previously (17, 33).

Western Blotting and Immunoprecipitation. The following antibodies

(1:1,000) were used in protein analyses: phosphotyrosine 4G10 (Upstate,

New York, NY), RET (H-300), phospho-Y1062RET, STAT3 (C-20) (Santa Cruz

Biotechnology, Santa Cruz, CA), phospho-Y981RET (15), phospho-ERK1/2,

ERK1/2, and phospho-STAT3 (Tyr705; Cell Signaling Technology, New

England Biolabs, United Kingdom). Western blotting and immunoprecipi-

tations were done as described previously (17).Blue Native SDS-PAGE. Cells were lysed on ice with lysis buffer

[10 mmol Tris-Cl (pH 7.4), 150 mmol NaCl, 2 mmol EDTA, 1% Nonidet P40,

1 mmol sodium vanadate, 10 Ag/mL aprotinin, 2 Ag/mL leupeptin, 0.2 mmol

phenylmethylsulfonyl fluoride, 10% glycerol] and blue native-PAGE was

done as described previously (34).

Immunohistochemistry. From the tumor cell lines, MTC-TT and MZ-

CRC-1, f2 � 106 cells were fixed with buffered formalin (pH 7.4) for

Figure 1. Signaling properties of RETWT,RETY791F, and RETS891A. (A, B and D )SK-N-SH cells were transfected with eitherRETWT, RETY791F, or RETS891A incombination with different reporterplasmids and stimulated with GDNF asindicated. Columns, mean fold inductionof normalized reporter activity; bars,F SD of triplicates. C, COS-7 cells weretransfected with RETWT, RETY791F, andRETS891A as indicated. Cell lysates weresubjected to RET immunoprecipitation(IP) and immunoblotting (IB) withantiphosphotyrosine and RET antibodies,respectively. COS-7 cell were transfectedwith RETWT, RETC634R, RETY791F, andRETS891A, respectively. Cell lysates wereanalyzed using blue native PAGE followedby Western analysis using an anti-RETantibody.

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Cancer Res 2005; 65: (5). March 1, 2005 1730 www.aacrjournals.org



1 hour at 4jC. After washing with PBS, 150 AL plasma (Sigma

coagulation control level 1; C7916) was added, followed by 50 AL

thromboplastine/Ca2+ (Sigma, St. Louis, MO; T7280). This was incubated

for 10 minutes at 37jC in a water bath. The gel was embedded in

paraffin. Immunohistochemistry was done on five tumor samples from

independent patients all carrying the Y791F-RET mutation, and it was

done as described previously (35).

Structural Modeling of RET Cytoplasmic Tyrosine Kinase Domain.The crystal structure of the human fibroblast growth factor receptor 1

(PDB accession code 1FGK) determined at 2.0 A resolution (36) was

selected as the template to construct a model of the RET tyrosine kinase

domain. The tyrosine kinase domains of fibroblast growth factor receptor1 and RET (RETK) share 50% amino acid sequence identity. The homology

models of the wild-type and mutant RETKs were constructed using

MODELLER (37) version 7, as implemented in DS Modeling 1.1 (Accelrys,

Inc., San Diego, CA) using standard settings. The quality of the modelswas evaluated using WHAT IF (38) and Procheck (39). Quality parameters

of the models, such as structure Z scores and G factors, were comparable

to those of the template structure. A short energy minimization usingconstraints on the backbone atoms was done using the CHARMM (40)

module of DS Modeling 1.1.

Results

Glial Cell Line–Derived Neurotropic Factor–Dependency ofRETWT, RETY791F, and RETS891A Signaling. In order to determinethe signaling properties of the wild-type RET receptor in response toGDNF, SK-N-SH cells, which are GFRa-1 positive (data not shown),were transfected with an RETWT expression plasmid in combinationwith different luciferase reporter constructs. GDNF treatment (100

ng/mL) resulted in activation of the SRE, NF-nB and, to a lesserextent, the IRE reporters (Fig. 1A), where the HSE, activator proteinand GRE reporters were not activated. When SK-N-SH cells weretransiently transfected with either RETY791F or RETS891A expressionplasmids in combination with the different reporters, activation ofthe SRE, NF-B, and IRE reporters was observed. The level of IREreporter activation was however clearly higher than that observedwith RETWT (Fig. 1B and C). GDNF did not further enhance reporteractivation by RETY791F and RETS891A, indicating that in these mutantRET receptors, signaling is independent of GDNF. To validate theresponsiveness of the reporter constructs used, the HSE, AP1, andIRE reporters were treated with a heat shock (43jC, 30 minutes), UV(45minutes), or cotransfected with v-Src, respectively. An increase inreporter activity in response to these treatments was observed (HSE,20-fold; activator protein, 3-fold; and IRE, 12-fold; data not shown).To determine whether the observed GDNF-independent signaling

of these FMTC mutants is reflected by constitutive receptorautophosphorylation, COS-7 cells were transfected with RETWT,RETY791F, and RETS891, respectively. Cell lysates were subjected toanti-RET immunoprecipitations followed by Western analysis usinganti-phosphotyrosine and anti-RET antibodies. Whereas tyrosinephosphorylation was not observed in the wild-type RET receptor;strong GDNF-independent phosphorylation was observed in bothFMTCmutants. Tyrosine phosphorylation of both mutant receptorswas not further increased by GDNF stimulation (data not shown).These results supported the findings obtained in the reporter

assays where these mutants activated reporter activity in theabsence of GDNF.

Figure 2. RETY791F and RETS891A induceconstitutive STAT3 transactivation.SK-N-SH (A ), HepG2, and COS-7 cells(B and D ) were transfected with IRE andIRE-mut reporter constructs in combinationwith STAT3a, STAT3h, RETY791F, andRETS891A expression plasmids asindicated. C, HEK293 cells transfected withRETWT, RETY791F, and RETS891A, andtreated or untreated with GDNF,respectively. Cell lysates were Westernanalyzed using antibodies againstphospho-Y705-STAT3, STAT3, and RET,respectively.

Activation of STAT3 by RET in FMTC




Monomeric and Dimeric RET Proteins. Iwashita et al. (41)showed that RETS891A functions as a monomeric oncoprotein.

Whether the Y791F mutant signals as a monomer or as a dimer

is at present unknown (24). In order to determine the

monomeric/dimeric state of RETY791F, COS-7 cells were trans-fected with wild-type and mutant RET expression plasmids, after

which cell lysates were analyzed using blue native gel

electrophoresis and Western blotting. RETC634R, a known dimer,RETS891A (monomeric) and RETWT (monomeric) were included

as controls. From these studies, we concluded that, like the

S891A mutant, the Y791A mutant functions as a monomericreceptor (Fig. 1C).STAT3 Activation by RETY791F and RETS891A. GDNF-

independent activation of the IRE reporter by RETY791F and RETS891A

was observed. As the IRE reporter contains specific binding sites formembers of the STAT family of transcription factors (42) andbecause it has previously been shown that the RET receptor containstwo STAT3-specific docking sites (Y752 and Y928; ref. 17), wedetermined whether the observed IRE transactivation by RETY791F

and RETS891A was mediated by STAT3. For this purpose, SK-N-SHcells were transiently transfected with an IRE-Luc reporter incombination with STAT3a and RETS891A. In case the IRE reporter

was cotransfected with RETS891A, a 5-fold increase in IREreporter activity was observed. When STAT3a was also included,IRE reporter activation increased to 20-fold. Overexpression ofSTAT3a alone had no effect on reporter activity (Fig. 2A). In caseSTAT3h, a splice variant of STAT3a which lacks the COOH-terminaltail of the molecule, was cotransfected, RETS891A induced activationof the IRE luciferase reporter progressively decreased whenincreasing amounts of STAT3h were used (Fig. 2A). These resultsindicate that activation of the IRE reporter by RETS891A is mediatedby STAT3. Furthermore, when an IRE reporter was used in which theSTAT3 binding sites are mutated (IRE-mut-Luc), no reporteractivation was observed on cotransfection with RETS891A. To confirmthat these results are not cell type–dependent, we did similarexperiments using HepG2 and COS-7 cells. In both cell lines,potentiation of IRE activity in response to RETS891A by STAT3a andinhibition of IRE-Luc activation by STAT3h was again observed(Fig. 2B). When RETY791F was used, similar results as observedwith RETS891A were obtained (Fig. 2D).To further substantiate that FMTC-RET mutants can activate

STAT3, the effect of these mutants on STAT3 Y-705 phosphorylation

was determined. HEK293 cells were transiently transfected with

RETWT, RETY791F, and RETS891A expression plasmids, treated in the

Figure 3. RETY791Fand RETS891A activate STAT3 through a Src-, JAK1-, and JAK2-dependent pathway. A, SK-N-SH cells were transiently transfected with theIRE-Luc reporter together with RETS891A. Twenty-four hours before harvesting, cells were treated over night with 10 Amol/L U0126 (mitogen-activated protein/ERKkinase inhibitor), 20 and 40 Amol/L AG1296 (tyrosine kinase receptor inhibitor), 15 and 30 Amol/L AG490 (JAK2 inhibitor), and 30 nmol/L PP2 (Src inhibitor) as indicated.Western analyses of HEK293 cells expressing RETS891A and treated with AG1296. HEK293 expressing RETC634R, RETY791F, and RETS891A were Western analyzedusing antibodies against phospho-Y981RET, phospho-Y1062RET, and RET, respectively. B and D, HepG2 cells were transiently transfected with a IRE-Luc reporterand both RETY791F or RETS891A in combination with STAT3a, dominant-negative Src (DN-Src) and dominant-negative JAK1 or JAK2 (DN-JAK1 or DN-JAK2)expression plasmids (1 and 2 Ag, respectively) as indicated. C, HEK293 cell were transiently transfected with both a RETC634R expression plasmid and an IRE reporter,and cells were either treated with PP2 (30 nmol/L) and AG490 (15 and 30 Amol/L), or transfected with 1 Ag of DN-Src and DN-JAKs, respectively.

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presence (50 ng/mL) and in the absence of GDNF, and cell lysateswere Western analyzed. Both FMTC mutants (RETY791F and

RETS891A) induced an increase in STAT3-Y705 phosphorylation

levels where overexpression of RETWT did not result in STAT3phosphorylation. STAT3 tyrosine phosphorylation induced by both

FMTC mutants was not further increased by GDNF stimulation

(Fig. 2C).RETY791Fand RETS891A Activate STAT3 through a Src-, JAK1-,

and JAK2-Dependent Pathway. To determine whether the

activation of STAT3 by RETY791F and RETS891A was mediated bythe tyrosine kinase domain of RETexclusively (17) or whether other

cytoplasmic kinases contribute to this process (43, 44), different

inhibitors of known STAT3 activators were used in SK-N-SH cellstransiently expressing RETS891A in combination with the IRE-Luc

reporter (Fig. 3A). First, we showed that the tyrosine kinase inhibitor

AG1296 (20 and 40 Amol/L) reduced IRE reporter activity in a dose-dependent manner suggesting that the kinase domain of RET is

indeed involved in STAT3 activation. To confirm that AG1296 was

indeed inhibiting RETactivity, cells expressing RETS891A were treatedwith increasing amounts of AG1296 (1, 5, 10, 20, and 40 Amol/L,

respectively) and they were Western analyzed using a phospho-

RETY981 antibody. A clear reduction of RET tyrosine 981

phosphorylation, at concentrations of 20 and 40 Amol/L, wasobserved, demonstrating that indeed AG1296 was inhibiting RET

activity/phosphorylation. This effect, reduction of phosphorylation,

was also observed in downstream RET-dependent signaling proteinpathways such as STAT3 and ERK1/2 (Fig. 3A).When cells were treated with JAK inhibitor AG490 (15 and

20 Amol/L), a dose-dependent decrease in IRE reporter activity was

observed, suggesting that besides RET, JAK is also involved in the

activation of STAT3. Moreover, the treatment of cells with Srcinhibitor PP2 (30 nmol/L) led to a reduction of IRE reporter activity

as well. Furthermore, the mitogen-activated protein/extracellularsignal-regulated kinase inhibitor U0126 (10 Amol/L) partially

inhibited IRE reporter activity, suggesting the involvement of amitogen-activated protein/extracellular signal-regulated kinase-mitogen-activated protein kinase pathway in regulating the

transcriptional activity of STAT3 through phosphorylation of serine727 (45). The same experiment was done with STAT3a over-

expression, and the same results were obtained (data not shown).However, none of the inhibitors completely abolished IRE reporter

activation by FMTC-RET, suggesting that there is interplay betweenthese molecules during signaling. To further confirm the involve-

ment of Src and JAKs, we determined the effect of dominant-negative forms of Src (32), JAK1 (containing a three-amino acidchange in the conserved region VIII of the kinase domain) and

JAK2 (containing a lysine to alanine mutation in the ATP bindingsite; ref. 31) on IRE reporter activation by RETY791F and RETS891A.

Overexpression of DN-Src resulted in decreased IRE reporteractivity by both FMTC mutations in a dose-dependent manner

(Fig. 3B). IRE reporter activity was also markedly decreased byoverexpression of either DN-JAK1 or DN-JAK2 (Fig. 3D). Theseresults suggest that RETY791F and RETS891A activate STAT3 through

a pathway involving Src, JAK1, and JAK2.Similar experiments were done with RETC634R. In contrast to the

results obtained for the FMTC mutant, the Src inhibitor PP2 does

not have any effect on IRE reporter activation by RETC634R (Fig. 3C).

The same results were obtained using a dominant-negative Src

Figure 4. Immunohistochemistrydetection of RET in FMTC tumors sectionsand tumor cell lines. A-C, three differentMTC (�400) from patients carrying a germline FMTC RETY791F mutation showinghigh degree of RET expression on theplasma membrane. D, tumor cell lineMTC-TT (�400) showing high degreeof RET expression on the plasmamembrane as well.





construct (Fig. 3C). We determined the phosphorylation status of

tyrosine 981, the tyrosine residue involved in Src activation. We

show that Tyr981 is more strongly phosphorylated in both FMTC

mutants than in the case of RETC634R, when compared with thelevel of tyrosine 1062 phosphorylation, which proved to be morephosphorylated in the case of the RETC634R mutant compared withboth FMTC mutants. It has to be noticed that the higher levels ofphospho-Y981 displayed by RETS891A when compared with theRETY791F are supported by the dominant-negative data, where theS891A mutant showed a higher sensitivity for DN-Src. Thesefindings suggest that tyrosine 981 in the FMTC mutants plays a keyrole in Src activation (Fig. 3A).The JAK inhibitor AG490 shows a slight inhibition of the IRE

reporter activation by C634R (Fig. 3C), but to a lesser extent thanFMTC-mediated activation, which was almost completely inhibited.Using DN-JAK1 and DN-JAK2 (data not shown), we were able toobserve that this slight inhibition could be JAK1-dependent as noeffect on the IRE reporter was observed for DNJAK2, whereasDNJAK1 was slightly inhibited. From these experiments,we concluded that indeed, RETC634R activates STAT3 througha pathway independent of Src and JAK2.

Immunohistochemical Analysis of RET in Familial MedullaryThyroid Carcinoma Tumor Samples and Tumor Cell Lines.Analysis of tumor sections from five patients carrying germ lineRETY791F mutation with an anti-RET antibody displayed a highdegree of RET staining at the plasma membrane (Fig. 4A-C).Paraffin-embedded tumor cell lines MTC-TT (MEN2A) and MZ-

CRC-1 (MEN2B) were also analyzed for RET expression as positivecontrols, showing a high degree of the RET receptor at the plasmamembrane as well (Fig. 4D). MZ-CRC-1 cells as well as all tumortissues of FMTC patients carrying the RETY791F mutation alsoshow cytoplasmic RET staining, aside from the previouslymentioned positive RET staining at the plasma membrane. Incase of the MTC-TT (MEN2A) cell line, RET staining was mainlyobserved at the plasma membrane.Immunohistochemical Analysis of Phospho-Y705-STAT3 and

STAT3 in Familiar Medullary Thyroid Carcinoma TumorSamples and Tumor Cell Lines. As mentioned in the previousparagraph, staining of the same tumor samples and cell lines withanti-STAT3 and anti–phospho-Y705-STAT3 showed high levels ofphosphorylated STAT3 in the nucleus in all samples (Fig. 5A-C),whereas STAT3 was detected both in the cytoplasm and thenucleus (Fig. 5D). These results indicate strong activation of STAT3in MTCs.Structural Modeling of RET Cytoplasmic Tyrosine Kinase

Domain. Structural models of wild-type and mutant RETKswere obtained by comparative modeling using the tyrosinekinase domain of fibroblast growth factor receptor 1 astemplate. The models show the characteristic bilobate structureof tyrosine kinase catalytic domains. Like the inactivatedtyrosine kinase domains of fibroblast growth factor receptors1 and 2, the model of wild-type RETK shows a relatively openATP binding site (Fig. 6A). Residue Ser891 is located at thebeginning of the activation loop in the cleft between the

Figure 5. Immunohistochemistrydetection of phospho-Y705-STAT3 andSTAT3 in FMTC tumors sections andtumor cell lines. A-C, staining with aphospho-Y705-STAT3 antibody (Tyr705)displayed a clear nuclear localization of theactive transcription factor in medullarythyroid carcinomas (A and B ) and in thetumor cell line MTC-TT (�400; C ). D,immunohistochemistry done with STAT3antibody show a clear cytoplasmic andnuclear signal. Bar, 100 Am.

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NH2- and COOH-terminal lobes and just proximal to theconserved DFG motif (residues 892-894). Comparison betweenthe inactivated (46) and activated (47) structures of the insulinreceptor kinase domain shows that at the position before theDFG motif, the activation loop conformations begin to diverge.The activation loop in the inactive conformation blocks accessof peptide substrate to the active site and aspartic acid 892 inRET is not properly positioned for interaction with the h-phosphate group of ATP through coordination with Mg2.Mutation of Ser891 in RET to the smaller hydrophobic alaninepossibly permits the activation loop to change to a conforma-tion compatible with peptide substrate binding, as well as aproper interaction with the ATP, in the monomeric state(Fig. 6B). The other FMTC mutation, Y791F, is located in theh4 strand of the NH2-terminal lobe and is spatially located nearthe conserved WE motif and is partly solvent exposed.Phosphorylation of this residue has not been shown (21). TheY791F mutation probably disrupts interactions with surroundingGlu805 and Lys789 residues, resulting in a conformational changeof the loop connecting the aC helix and the h4 strand.Through the subsequent spatial rearrangement of the aC helix,the conformation of the activation loop changes into the activestate by interaction of aC helix residues with phenylalanine 893of the DFG motif (Fig. 6C).

Discussion

Many groups have investigated how mutations in the RET proto-

oncogene can result in constitutive active receptors and which

substrates and signaling routes are being activated by these aberrant

proteins. Finding mutation-specific alterations in RET signaling

properties might help us to understand how different RET-

associated cancer syndromes develop. Nevertheless, the oncogenic

activation and downstream signaling pathways activated by FMTC

mutations located within the catalytic domain of RET have been just

partially uncovered (41, 48).We now show that RETY791F is active as a monomeric protein and

that both RETY791F and RETS891A are phosphorylated and activatedindependently of GDNF. Results similar to the observations reportedfor RETS891A (41). Signaling by the monomeric RETY791F andRETS891A proteins is GDNF-independent and strongly targets theSTAT3 signaling pathway. Although there is evidence that RET-induced signaling is cell type–specific (24), our results indicate thatthe activation of STAT3 by both RETY791F and RETS891A mutants wasnot cell type–dependent as four different cell lines (HepG2, HEK293,COS-7, and SK-N-SH) gave similar results. Previously, it was reportedthat the oncogenic transactivation of STAT3 by RETC634R

(a mutation found in MEN2A patients) was required for cellulartransformation, and this process was mediated by the intrinsic

Figure 6. Structural modeling of the RETtyrosine kinase domain affected bymutations Y791F and S891A. Schematicrepresentation of modeled RET-TKshowing the NH2-terminal lobe at theleft-hand side and the COOH-terminal lobeat the right-hand side. The activation loop isdepicted in dark blue and the loopconnecting the aC helix and the h4 strandin yellow (A). Activation loop conformationof inactive RET-TK (dark blue ) andactivated RET-TK (dark gray ) diverge atresidue Ser891. Activation loop in theinactive conformation blocks access ofpeptide substrate to the active site andAsp892 is not properly positioned forinteraction with the h-phosphate group ofATP through coordination with Mg2+.Backbone of modeled RET-TK in lightgray , activation loop in blue , and aC helixin red . The superimposed activation loop ofRET-TK in active conformation wasmodeled using activated insulin tyrosinereceptor kinase as template structure, ATPanalogue AMP-PNP and tyrosine peptidesubstrate (purple ) coordinates were alsotaken from this structure (B ; ref. 47). Localenvironment around tyrosine 791:NH2-terminal lobe backbone residues inlight gray and backbone residues of theDFG motif in dark gray , aC helix in red ,and loop connecting the aC helix and theh4-strand in yellow (C ).





tyrosine kinase domain of RET, independently of JAKs and Src (17),data we largely confirm (see Results). Here, we show, from the useof either specific inhibitors or dominant-negative forms of Src, JAK1,and JAK2, that Src and JAKs are implicated in STAT3 activationby RETY791F and RETS891A.Our results suggest that different disease phenotype–associated

RET mutations activate STAT3 through different signaling routes.Nevertheless, it is still unclear if the interaction between c-Src, JAKsand both RETY791F and RETS891A is promoted by direct phosphor-ylation through the catalytic domain of the receptor itself, or byanother parallel mechanism of signaling. Overexpression of JAK1,JAK2, and c-Src in combination with both RETY791F and RETS891A inthe absence of STAT3 did not increase IRE reporter activitysignificantly; but expression of v-Src did enhance it (data notshown), suggesting that aberrant signaling through STAT3 alsorequires the catalytic domain of the receptor ‘‘per se’’, as it ismediated by a Src/JAK-dependent mechanism that could not bedirectly linked to the RET receptor itself.The oncogenic mechanism of activation promoted by the

investigated monomeric FMTC-RET mutations located within thecatalytic domain differs from the MEN2A/FMTC mutations locatedat the cysteine-rich motif. The extracellular mutations lead to theformation of covalent dimers due to intermolecular disulfide boundsbetween RET monomers. Hence, the receptor is activated constitu-tively and independently of GDNF (49). FMTC mutations affectingthe catalytic domain of RET, however, seem to have an effect similarto the MEN2B mutations. These mutations result in monomericoncoproteins, altering both the catalytic activity and substratespecificity of the receptor due to structural changes of the bindingpocket of the tyrosine kinase domain. They lead to an aberrantphosphorylation of substrates preferred by cytoplasmic tyrosinekinases such as c-Src and c-abl (50). We have seen such an effect inRETY791F and RETS891A mutant proteins. However, RETM918T

(MEN2B) has been also shown to be GDNF-responsive, suggestingthat differences in the mechanism of receptor activation combinedwith differences in GDNF-responsiveness of these receptors, as wellas tissue-specific GDNF expression (or related ligands), could giverise to different disease phenotypes (24).STATs are a family of latent transcription factors that become

activated in response to cytokines and growth factors (43). IFN,granulocyte colony-stimulating factor, and cytokines activateSTAT3 through their receptors in a JAK-dependent manner, whereasgrowth factor receptors can phosphorylate STAT3 through theirintrinsic tyrosine kinase domains (43, 44). STAT3 has beenimplicated in the oncogenesis of many types of tumors, such asleukemia, lymphomas, multiple myeloma, breast cancer, and head

and neck cancers. In all of these cases, constitutive, ligand-independent activation of STAT3 has been detected (51). In thisstudy, we show that STAT3 plays an important role in thedevelopment of MTC, not only based on in vitro data, but alsobased on the immunohistochemistry analysis of MTCs. Tumorsfrom patients carrying the RETY791F germ line mutation showed aclear nuclear localization of activated STAT3 (phospho-Tyr705) incontrast to normal cells where STAT3 is not phosphorylated andmainly cytoplasmic localized. Therefore, we hypothesize thataberrant STAT3 activation by both FMTC mutants could promotethe dysregulation of the transcriptional control of genes playing animportant role in tumorigenesis, contributing to the malignanttransformation of the C cells resulting in MTC. Immunohistochem-ical analysis of tumor tissues also revealed high levels of RETproteinat the plasma membrane, similar to those observed in MTC-derivedcell lines, MTC-TT and MZ-CRC-1. Interestingly in FMTC tumortissues, cytoplasmic localized RET was also observed. We assume,therefore, that the antibodies also recognize the immature form ofthe receptor located in the cytoplasm. It remains unclear if theimmature form of RET could also be active inside the endoplasmicreticulum while it is being processed and transported to the plasmamembrane.How RET, which is normally activated upon dimerization of

two proteins, can be active as a monomer is yet unclear. Wetherefore modeled the RET catalytic domain using the tyrosinekinase domain of the fibroblast growth factor receptor 1 as atemplate. Our modeling studies suggest that the mutationsresult in a modification of the tertiary structure of the catalyticdomain giving rise to a protein with a more accessible substrateand ATP conformation.In summary, these results show that FMTC-RET mutants Y791F

and S891A constitutively activate STAT3 in vitro , a findingsupported by observations in tumor material from FMTC patients.Constitutive ligand-independent signaling of these mutant recep-tors through STAT3 might contribute to the development ofmedullary thyroid carcinomas.

Acknowledgments

Received 7/22/2004; revised 12/14/2004; accepted 12/30/2004.Grant support: Supported by grants from the Ubbo Emmius Foundation (RUG)

and GUIDE.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We would like to thank Hein Schepers, Jan Jacob Schuringa, Bart Jan Wieringa, andLoes Drenth–Diephuis, as well as Liesbeth Veenhoff for technical and materialsupport.

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2005;65:1729-1737. Cancer Res Ivan Plaza Menacho, Roelof Koster, Almer M. van der Sloot, et al.

Derived Neurotrophic Factor−of Glial Cell Line and S891A Activate a Src/JAK/STAT3 Pathway, Independent

RET-Familial Medullary Thyroid Carcinoma Mutants Y791F

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