JOURNAL OF CELLULAR PHYSIOLOGY 195:168–186 (2003)

RET and NTRK1 Proto-Oncogenes in Human Diseases

LUISELLA ALBERTI, CRISTIANA CARNITI, CLAUDIA MIRANDA,EMANUELA ROCCATO, AND MARCO A. PIEROTTI*

Operative Unit ‘‘Molecular Mechanisms of Tumor Growth and Progression,’’Department of Experimental Oncology, Istituto Nazionale Tumori,

Milan, Italy

RET andNTRK1 are receptor tyrosine kinase (RTK) proteins which play a role in thedevelopment and maturation of specific component of the nervous system. Theiralterations have been associated to several human diseases, including some formsof cancer and developmental abnormalities. These features have contributed to theconcept that one gene can be responsible for more than one disease. Moreover,both genes encoding for the two RTKs show genetic alterations that belong toeither ‘‘gain of function’’ or ‘‘loss of function’’ class of mutations. In fact, receptorrearrangements or point mutations convert RET and NTRK1 in dominantly actingtransforming genes leading to thyroid tumors, whereas inactivating mutations,associated with Hirschsprung’s disease (HSCR) and congenital insensitivity to painwith anhidrosis (CIPA), impair RET and NTRK1 functions, respectively. In thisreview we have summarized the main features of the two receptors, theirphysiological and pathological roles. In addition, we attempted to identify thecorrelations between the different genetic alterations and the related pathogeneticmechanisms. J. Cell. Physiol. 195: 168–186, 2003. � 2003 Wiley-Liss, Inc.

Protein kinases, through the reversible phosphoryla-tion of specific substrata, regulate most of the processeslinked to cell growth and cell death. Functionally, theseenzymes translate signals into biological events, thanksto a complex and sometimes redundant network ofinteractions.

On the other side of this complexity, it is not surprisingthat their disfunction can be related to different diseasesincluding neoplastic growth. Several human tumorsare, in fact, associated to ‘‘gain of function’’ mutations ofgenes coding for both tyrosine and serine/threonine-specific kinases. Moreover, some other diseases, mostlyassociated with abnormal development, appear due to a‘‘loss of function’’ of these genes. In this context, oneaspect related to the phenomenon of the genetic heter-ogeneity, the concept of ‘‘one gene, many diseases,’’seems to find a paradigm in the example provided byRET and NTRK1 tyrosine kinase (TK) receptor geneswhose alterations have been determined as pathogenicboth in some neoplastic and abnormal developmentdisorders.

In this review we have described the main features ofthese TK receptors and discussed their involvement inhuman diseases. In particular, we have analyzed thedifferent mechanisms which lead to their activation orinactivation and tried to depict the molecular conse-quences of the latter. Finally, genotype–phenotyperelationships are proposed for the different geneticalterations of RET and NTRK1 found in the differentpathologies.

RET PROTO-ONCOGENE AND ITSPHYSIOLOGICAL FUNCTIONS

In 1985, a new oncogene was identified after transfec-tion into NIH3T3 cells of DNA derived from a human T

cell lymphoma (Takahashi et al., 1985). The chimericprotein comprised an N-terminal region with a di-merizing motif fused to a new TK domain that wasfound afterwards to belong to a transmembrane re-ceptor named RET (rearranged during transfection)(Takahashi et al., 1988).

RET gene lies on chromosome band 10q11.2 andcomprises 21 exons. The gene encodes a protein similarto other TK receptors (Ishizaka et al., 1989). The RETligand was unknown until 1996 when the glial derivedneurotrophic factor (GDNF) was discovered (Durbecet al., 1996; Trupp et al., 1996). This was the first of, atpresent, four ligands: GDNF, neurturin (NTN), perse-phin (PSP), and Artemin (Airaksinen et al., 1999). TheRET signal starts from a multimeric complex composedof the RET kinase and one of four different high-affinityglycosyl-phosphatidylinositol (GPI)-linked coreceptorsdesignated as GFRa 1, 2, 3, and 4 (Baloh et al., 2000).It has been demonstrated that the four RET ligandsGDNF, NTN, PSP, and Artemin interact preferentiallywith GFRa 1, 2, 3, and 4, respectively.

� 2003 WILEY-LISS, INC.

The authors have contributed equally to this work; specificallyLuisella Alberti and Cristiana Carniti for RET and ClaudiaMiranda and Emanuela Roccato for NTRK1 analysis.

*Correspondence to: Marco A. Pierotti, Department of Experi-mental Oncology—Istituto Nazionale Tumori, Via G. Venezian,1 20133, Milan, Italy. E-mail: marco.pierotti@istitutotumori.mi.it

Received 18 October 2002; Accepted 11 December 2002

DOI: 10.1002/jcp.10252

RET protein is characterized by an N-terminal signalpeptide, a cadherin-like motif and a cysteine rich regionin the extracellular domain; a transmembrane domainand a TK domain, interrupted by 27 amino acids ofkinase insert, in the intracellular portion (Itoh et al.,1992; Schneider, 1992). The C-terminal tail, startingfrom aa 1063, shows three different splicing variants:the long isoform of 1114 aa (ISO 51), the middle isoformof 1106 aa (ISO 43), and the short isoform of 1072 aa(ISO 9) (Tahira et al., 1990; Myers et al., 1995).

In situ hybridization showed that RET is present inperipheral enteric, sympathetic and sensory neurons.RET staining is also observed in central motor, dopa-mine and noradrenaline neurons localized in the ventralhalf of the spinal cord, in the neuroretina and in theolfattory epithelium (Pachnis et al., 1993; Attie-Bitachet al., 1998; Manie et al., 2001). In these districts RETactivation can promote neuronal cell survival anddifferentiation. Outside the nervous system, RET isinvolved in renal ontogenesis, in particular in meso-nephric ducts and branching ureteric bud development,where a chemotactic role for GDNF has been demon-strated (Taraviras and Pachnis, 1999). These observa-tions are supported by analysis of either RET- andcoreceptor-null mice, or transgenic mice with a defectiveRET TK domain, which show severe defects of theinnervation of the hindgut and branching of the uretericbud (Airaksinen et al., 1999; Baloh et al., 2000).

In mammals, c-RET is alternatively spliced to produceat least two major isoforms, RET-ISO9 and RET-ISO51,that differ only in the amino acid sequence of the C-terminal tail. The two RET isoforms are highly con-served between species, suggesting that these regionsmay have important functions that are evolutionaryconserved. Although the two isoforms behave similarlyin a number of in vitro assays, several observations havesuggested that they have different and tissue-specificeffects on embryogenesis and tumorigenesis. Monoiso-formic mouse strains that express only RET-ISO9 orRET-ISO51, in place of the normal complement of c-RETgene products, demonstrated that signaling by RET-ISO9 is critically important for kidney morphogenesisand enteric nervous system development and post-natal life, and signaling by RET-ISO51 alone, in theabsence of RET-ISO9 resulted in characteristic defectsin the development of the excretory and enteric nervoussystems (de Graaff et al., 2001) indicating that RET-ISO51 is dispensable during embryogenesis. Further-more, transgenic overexpression of RET-ISO51 onlypartially compensates for the loss of RET-ISO9 in kidneyand enteric nervous system development. RET-ISO51,but not RET-ISO9, is required for the metabolism andgrowth of mature sympathetic neurons (Tsui-Pierchalaet al., 2002b). Moreover, Tsui-Pierchala et al. (2002a)provided evidence that RET-ISO9 and RET-ISO51 arenot only functionally distinct, but also activate dif-ferent assortment of signaling pathways in neurons.In addition they are not able to associate with eachother after GDNF stimulation probably due to a dif-ferent subcellular localization of the two isoforms.The alternative splicing of RET might account for anevolutionary conserved mechanism to expand the num-ber of activities regulated by a receptor tyrosine kinase(RTK).

Signaling by RTKs requires ligand-induced receptoroligomerization, which results in the tyrosine autophos-phorylation of receptor intracellular portion. Phos-phorylated tyrosine residues mediate specific bindingto various scaffold, anchoring, adaptor proteins andenzymes that possess Src homology-2 (SH2) or proteintyrosine-binding (PTB) domains. Since the RET ligandswere unknown until 1996, extensive studies on RETsignaling have been performed using chimeric or on-cogenic version of the transmembrane RET, that displayconstitutive TK activity. RET C-terminal tail comprisesfive different phospho-tyrosine residues (Y687, Y826,Y1015, Y1029, and Y1062) out of the TK. In addition, thelong isoform displays two extra tyrosine residues: Y1090and Y1096; the latter is phosphorylated in RET/2A butnot in RET/2B (Liu et al., 1996). In particular, Y1062 is amultidocking site interacting with a number of trans-duction molecules: SHC, FRS2, IRS1/2, DOK proteinsand Enigma. Moreover, RET signals also through PLCg,that binds Y1015 and through GRB2 that binds directlyonly the long isoform at Y1096 (reviewed in Jhiang,2000).

NTRK1 PROTO-ONCOGENE AND ITSPHYSIOLOGICAL FUNCTIONS

NTRK1 (also known as TrkA) is a TK receptor for thenerve growth factor (NGF) which primarily regulatesgrowth, differentiation, and programmed cell death ofneurons in both the peripheral and central nervoussystem. NGF action occurs also through p75LNGFR

cognate receptor which does not possess kinase activityand belongs to the TNF receptor family. The combinedaction of these two receptors gives rise to NGF signaling(Kaplan and Miller, 2000).

NTRK1 was originally discovered as an oncogene inone case of human colon carcinoma, and named TRK(tropomyosin rearranged kinase) (Martin-Zanca et al.,1986). Cloning of the full length gene (Martin-Zancaet al., 1989) and identification of the NGF as a ligandoccurred few years later (Kaplan et al., 1991; Klein et al.,1991). The NTRK1 gene is located on chromosome1q21–22 (Weier et al., 1995) and consists of 17 exonsdistributed within a 25-kb region (Greco et al., 1998).The NTRK1 receptor is a glycosylated protein of140 kDa, comprising an extracellular portion, includingIg-like and Leucine rich domains for ligand binding,a single transmembrane region, a juxta-membrane do-main, a TK domain and a C-terminal tail. FollowingNGF binding, NTRK1 undergoes dimerization andautophosphorylation of five tyrosine residues (Y490,Y670, Y674, Y675, and Y785). Activated receptor ini-tiates several signal transduction cascades, includingthe mitogen activated protein kinase (MAPK), thephosphatidylinositol 3-kinase (PI3K) and the PLC-gpathways. These signaling cascades culminate in theactivation of transcription factors that alter gene ex-pression (Kaplan and Miller, 2000).

NTRK1 activity is highly regulated. In particular,NGF/NTRK1 signaling supports survival and differen-tiation of sympathetic and sensory neurons responsiveto temperature and pain. In the mouse embryo, NTRK1is expressed in sensory ganglia of neural crest origin.In adult animals the gene was found expressed in thetrigeminal, dorsal root and sympathetic ganglia of the

PROTO-ONCOGENES IN HUMAN DISEASES 169

peripheral nervous system as well as in a small subset ofcholinergic neurons in the caudatoputamen and thebasal forebrain of the central nervous system (Martin-Zanca et al., 1990). In addition to its neurotrophicfunctions, NGF also stimulates proliferation of anumber of cell types such as lymphocytes, keratinocytes,and prostate cells (Otten et al., 1989; Djakiew et al.,1991; Di Marco et al., 1993).

The Trk family of receptors consists of three mem-bers named NTRK1, NTRK2, and NTRK3 (or TrkA, B,and C, respectively), displaying binding specificity fordifferent neurothrophins (Barbacid, 1995). In particu-lar, NTRK1 is preferentially activated by NGF, but canalso bind with lower affinity to NT-3 and -4. NTRK2 isthe receptor for both BDNF and NT-4, while NTRK3responds only to NT-3. All the neurothrophins bind p75receptor with low affinity. Recently, several evidenceshave shown that Trk and p75 exist in a paradoxicalrelationship, each acting to suppress or enhance theother’s actions (reviewed in Kaplan and Miller, 2000).

Although both involved in development and matura-tion of the nervous system, as summarized in Table 1,it is interesting to note the different strategy of RETand NTRK1 receptors to exert their different physiolo-gical effects. In fact, NTRK1 belongs to a family of highlyrelated receptors with defined ligand specificity, allsharing only one cognate receptor. The expressionpattern of all these receptors confers the broad rangeof action of the NT/RTK family. At variance, RET is theonly component of its family; however it may accomplishdistinct functions by using different isoforms andinteracting with different coreceptors.

RET AND NTRK1 ONCOGENES INPAPILLARY THYROID CANCER

Chromosomal rearrangements producing chimericoncogenes are frequently associated with human cancerand several lines of evidence suggest that they areinvolved in the pathogenesis of tumors. Among solidtumors, papillary thyroid carcinoma (PTC) provides aunique model of a frequent generation of chimericoncogenes through chromosomal rearrangements.Transfection of DNA from PTCs into NIH3T3 cells, ledto the identification of rearrangements of RET andNTRK1 TK receptors with foreign sequences. In fact,PTCs are characterized by the generation of fusion

proteins, made of the N-terminus derived from dif-ferent partners and the C-terminus of one of the tworeceptors, carrying the TK domain (Pierotti et al., 1996)(Fig. 1).

In 1987, in collaboration with the group of Vecchioand Fusco of the University of Naples, we have isolatedthe first activated version of the RET oncogene, namedRET/PTC (papillary thyroid cancer) (Fusco et al., 1987).Afterwards, a number of different RET/PTCs have beenisolated, where RET TK domain was found fused todifferent partner genes (Fig. 1).

RET/PTC1 originates by chromosome 10 inversion,inv(10)(q11.2q21.2), and results from the fusion of theRET-TK domain and H4 (D10S170) gene, whose func-tion is still unknown. The H4/RET fusion incorporates101 amino acids of H4, predicted to encode a leucinezipper domain responsible for RET/PTC1 oligomeriza-tion and constitutive TK activity (Tong et al., 1997). Anovel rearrangement, containing the N-terminal 150residues of H4, creates an oncoprotein named RET/PTC1L able to transform NIH3T3 cells with fivefoldlower efficiency than RET/PTC1. Its low transformingability may explain its low frequency in human thyroidcarcinomas (Giannini et al., 2000). Recently H4 hasbeen found fused to the platelet-derived growth factorreceptor beta gene in atypical chronic myeloid leukemia(t(5; 10)(q33;q22) (Schwaller et al., 2001). InterestinglyRET/PTC1 rearrangement has been found to be asso-ciated with post-Chernobyl PTC of long latency(see below).

In RET/PTC2, RET-TK is fused to the type I alpharegulatory subunit of protein kinase A (RI alpha) and isgenerated by a reciprocal and balanced chromosometranslocation (Lanzi et al., 1992; Bongarzone et al.,1993). The resulting 596-aa protein contains the firsttwo-thirds of RIa. The wild-type RIa subunit dimerizesin an antiparallel orientation between Cys-16 and -37(Bubis et al., 1987). RET/PTC2 deletion mutants showedthat the RIa dimerization domain is the only portion ofRIa required for RET/PTC2 mitogenic activity, thussuggesting that RET TK is activated in RET/PTC2 viathe dimerization domain of RIa (Durick et al., 1995).

Both RET/PTC3 and RET/PTC4 oncogenes are gen-erated by an intrachomosomal rearrangement withELE1a/ARA70 gene. RET/PTC3 contains the first 238amino acids of the androgen receptor-associated protein

TABLE 1. RET and NTRK1 TK receptors in physiology

RETTK receptor for GDNF, NTN, PSP, ArteminExpressed in

Neural crestUrogenital precursor cells

Required forKidney morphogenesisMaturation of peripheral nervous system lineages (enteric, autonomic, and sensory neurons)Differentiation of spermatogonia

NTRK1TK receptor for nerve growth factorExpressed in

Sensory ganglia of neural crest origin (mouse embryo)Trigeminal dorsal root and sympathetic ganglia of the peripheral nervous system (adult animals)Small subset of cholinergic neurons of central nervous system (adult animals)

Required forGrowthDifferentiation programmed cell death in PNS and CNS

170 ALBERTI ET AL.

70 (Santoro et al., 1994). Bongarzone et al. (1997)identified a short homology sequence (3–7 bp) in thetwo rearranging genes and a break cluster region (Bcr)in ELE1, in AþT rich regions. The N-terminal coiled-coil domain of ELE1a/ARA70 mediates oligomeriza-tion, RET kinase activation and transforming ability.In fact, expression of RET/PTC3 mutants lacking theN-terminal coiled-coil domain does not lead to fociformation in NIH3T3 cells. Moreover the same domainmediates the interaction between RET/PTC3 andELE1a/ARA70, causing the oncoprotein re-localizationto the plasma membrane (Monaco et al., 2001). Intra-chromosomal rearrangements involving RET and theadjacentH4orELE1a/ARA70geneonchromosome10arevery frequent events (58%) in thyroid cancer of childrenof the Chernobyl-contaminated zone (Klugbauer et al.,1995; Nikiforov et al., 1997). In addition RET/PTC3

rearrangement is strongly associated with PTC of shortlatency and connected with the solid-follicular variant(Thomas et al., 1999). Consistent with this, micecarrying RET/PTC3 display an aggressive tumor pheno-type, including competence for lymph node metastases(Powell et al., 1998). In contrast, RET/PTC1 transgenicmice develop follicular hyperlasia and carcinoma, butnot invasive cancer (Santoro et al., 1996). In RET/PTC3transgenic mice, solid-type PTCs were found with highfrequency, similarly to what was observed in PTC3 posi-tive patients from the Chernobyl-contaminated area(Ahmed et al., 1997). This suggests that RET/PTC3 geneis critical for the development of the solid subtype ofPTC. Recently Basolo et al. (2002) associated RET/PTC3rearrangement also with the Toll-Cell Variant of PTCs.The finding that RET/PTC3 is present in the aggressivehistological PTC subtypes (Solid PTC and TCV) could

Fig. 1. Mechanisms of chromosomal rearrangements generating fusion transforming genes. RET andTRK thyroid oncogenes are represented below. Activating genes are indicated; SP, signal peptide; TM,transmembrane domain; TK, tyrosine kinase domain; C, coiled-coil domain.

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depend on the more efficient in vitro mitogenic abilityand MAPK activation of this RET rearrangement incomparison to RET/PTC1.

In the case of RET/PTC4, in spite of the presence ofthe same RET/PTC3 breakpoint in exon 5 of ELE1a/ARA70 gene, the sequence of the rearranged genomicDNA showed different intra-exonic breakpoint in theRET proto-oncogene. Moreover, it has been demon-strated that the exon 5 of ELE1a/ARA70 joined to exon11 instead of to exon 12 of RET gene; as a consequence,the RET/PTC4 cDNA sequence is 93 nucleotides largerthan the RET/PTC3 one (Fugazzola et al., 1996).

After the Chernobyl power plant explosion, an un-usual number of thyroid cancers were noted in Belarusand Ukraine, between 10 and 30 fold higher than in therest of Europe. The analysis of the PTCs derived fromthe contaminated zones led to the identification of otherrearranged forms of RET, where RET-TK is fused toseven different donor genes. For instance, RET/PTC5fusion partner protein is GOLGA5, a coiled-coil proteinexpressed on the Golgi surface (Klugbauer et al., 1998).RET/PTC6 and RET/PTC7 display rearrangementswith the transcriptional intermediary factor 1-alphaand gamma, respectively (Klugbauer and Rabes, 1999).This protein family is able to bind to the ligand-dependent activation function (AF2)-activating domainof the estrogen receptor, RARs, RXRs, vitamin D3 re-ceptor and regulate transcription. Kinectin is the RET/PTC8 partner (Salassidis et al., 2000), whereas RFG9, aputative cytoplasmic protein that might be involved inintracellular transport processes, is rearranged withRET to form RET/PTC9 (Klugbauer et al., 2000). InRET/PCM-1 the activating sequences belong to a genecoding for a centrosomal protein that displays distinctcell cycle distribution (Corvi et al., 2000). The last RETrearrangement found is ELKS/RET. The ELKS mRNAis ubiquitously expressed with the highest expression inheart, placenta, pancreas, thyroid, and testis but thefunction of the ELKS protein is still unknown (Nakataet al., 1999).

As mentioned above, NTRK1 is the other RTK activat-ed in PTCs, although with a lower frequency comparedto RET. The 30 end of NTRK1 gene was found fused tothe 50 end of three different genes, forming four fusiongene variants. The NTRK1 gene joins to TPM3 (Martin-Zanca et al., 1986), TPR (Greco et al., 1992) or TFG(Greco et al., 1995) generating chimeric genes designedTRK, TRK-T1, TRK-T2, and TRK-T3, respectively.TRK-T1 and TRK-T2 are two fusion variants of TPR-NTRK1 rearrangement (Greco et al., 1997). NTRK1locus spans a 25 Kb region, however all the rearrange-ments occur in an NTRK1 genomic region of 2.9 kb,showing a GC content of 58.8% (Greco et al., 1993a).Differently from the RET oncogenes, all known TRKoncogenes, except TRK-T1, also contain the NTRK1transmembrane domain (Fig. 1).

As anticipated, NTRK1 was originally detected asan oncogene in a human colon carcinoma. In thisactivated version of the gene, named TRK, the NTRK1TK domain is fused to sequences of one isoform ofthe non-muscle tropomyosin gene, TPM3, localized onchromosome 1q22–q23 (Wilton et al., 1995). Afterwards,molecular analysis detected the activation of NTRK1 byTPM3 sequences, resulting from an intrachromosomal

inversion inv(1) (q21-22q22-23), in human PTCs. TRKoncogene consists of 619 amino acids, 221 of whichderived from the first seven exons of TPM3. Molecularanalysis of TRK positive PTCs revealed the presence ofeither the oncogenic rearrangement TPM3-NTRK1 orthe reciprocal event NTRK1-TPM3, indicating intra-chromosomal balanced reciprocal inversions (Buttiet al., 1995).

TRK-T1 oncogene contains sequences belonging to thetranslocated promoter region (TPR) gene, encoding afilamentous protein of the nuclear pore complex impli-cated in nuclear protein transport (Byrd et al., 1994) andoriginally identified as part of MET oncogene (Parket al., 1986). TPR locus mapped on chromosome 1q25(Miranda et al., 1994) and the mechanism leading to theformation of TRK-T1 oncogene is an intrachromosomalinversion inv(1) (q21–22q25). TRK-T1 encodes a 55-kDaprotein, consisting of 192 amino acids of TPR precedingthe NTRK1 portion. TPR sequences in TRK-T1 containleucine repeat motifs predicted to form coiled-coildomains, which are responsible for protein dimeriza-tion. It has been demonstrated that TRK-T1 oncogene isable to induce differentiation of PC12 cells similar toNGF treatment (Greco et al., 1993b) and to induceNIH3T3 foci formation (Bongarzone et al., 1989).Transgenic mice containing the human TRK-T1 trans-gene expressed under the control of the bovine thyro-globulin promoter, developed follicular hyperplasia orpapillary carcinomas (Russell et al., 2000). This indi-cates that TRK-T1 is oncogenic in vivo and contributes tothe neoplastic transformation of the thyroid.

In addition, TPR is involved in generating TRK-T2oncogene, which contains a TPR portion longer by 737amino acids than in TRK-T1 (Greco et al., 1997). TRK-T2encodes a 150-kDa protein and, differently from TRK-T1, maintains the NTRK1 transmembrane domain inits structure.

The TRK-T3 oncogene is activated by TRK fusedgene (TFG), a novel gene localized on chromosome 3(3q11–q12), encoding a protein of yet unknown func-tions (Greco et al., 1995; Mencinger et al., 1997). Dif-ferently from the TRK oncogenes described above, inthis case the rearrangement is a translocation involvingchromosomes 1 and 3 t(1;3)(q21–22;q11–12). The TRK-T3 protein has a molecular weight of 68 kDa and is madeof 592 amino acids, 193 of which are encoded by TFG.TFG gene contains a coiled-coil domain that mediatesthe capability of TRK-T3 to form complexes and there-fore is essential for its oncogenic activation. However,the TFG N-terminal portion preceding the coiled-coildomain has been shown to be required for TRK-T3 fullytransforming activity, suggesting that other regions arealso important for oncogenic activation (Greco et al.,1998). In this context it is worth noting that in additionto the coiled-coil domain, the TFG portion in TRK-T3contains putative consensus sequences for SH2- andSH3-binding motifs. Several of these sites are conservedin TFG proteins from different species, indicating thatthis protein might be involved in basic cellular processes(Mencinger and Aman, 1999).

Very recently, two fusion partner genes of NTRK1,TFG and TPM3, were reported to fuse to ALK in ana-plastic large cell lymphoma (Lamant et al., 1999;Hernandez et al., 2002). It has been demonstrated that

172 ALBERTI ET AL.

three different variants of TFG-ALK involving differentbreakpoints in TFG gene, have in vitro transformingpotential and bind to signal transduction pathwayproteins such as Grb2, Shc, and PLCg (Hernandezet al., 2002). Interestingly, the TFG portion presentin TFG-ALKL chimeric gene was the same as in theTFG-NTRK1 translocation occurring in thyroid carci-nomas (Hernandez et al., 1999, 2002).

Some features of the different activating genes fusedto RET and NTRK1 TK domains are summarized inTable 2.

Following chromosomal rearrangements, includinginversions or translocations, a common mechanism ofactivation for RET and TRK oncogenes could be pro-posed, taking into account the features shared by theactivating gene products (Fig. 1). In fact, all the fusionpartner proteins are ubiquitously expressed, displaycytoplasmic localization, and contain different protein–protein interaction motifs, including coiled-coil do-mains. These latter often represent the dimerization/oligomerization domains present in the activating genes(Lupas et al., 1991; Lupas, 1996).

As anticipated, the rearranged oncoproteins sharethree different features. The capability of self-associa-tion, mediated by the oligomerization domain, triggersthe constitutive transautophosphorylation of the TKdomain, thus mimicking the receptor dimerization uponligand binding (Fig. 2). The importance of promoting anoligomerization, which in turn leads to the constitutiveactivation, has been demonstrated in several cases in-volving different oncogenes (i.e., RET/PTC1, RET/PTC2, RET/PTC3, TRK-T3, and also TPR/MET, Bcr/Abl, PLM/RAR) (McWhirter et al., 1993; Rodrigues andPark, 1993; Durick et al., 1995; Tong et al., 1997; Grecoet al., 1998; Grignani et al., 1999; Monaco et al., 2001).A second common feature of these oncoproteins is adifferent subcellular localization with respect to the wildtype receptors, which physiologically act on the cellsurface. In fact the chimeras are localized in the cytosoland a specific sub-localization could be driven by theactivating portion. In the case of RET/PTC2, differentlocalization for the two isoforms was suggested (Borrelloet al., 2002). Finally, the rearrangements lead to theectopic expression of the kinases in the epithelialfollicular cells, although it has been recently demon-

strated that RET could be physiologically active inthyrocytes (Bunone et al., 2000).

All the chromosomal rearrangements are balancedreciprocal events. The rearrangements involve one ofthe two alleles of the activating gene. The non-rear-ranged allele is still present but the encoded proteinscould have a different subcellular localization or theirexpression level could be decreased, as demonstratedfor RET/PCM1 (Corvi et al., 2000). This could be eitherdue to the allelic inactivation or to the coexistence ofthe wild type and the rearranged forms that couldaffect protein function or stability. The haploin-sufficency could be directly responsible for tumori-genesis if the proteins involved in the oncogeneactivation are important for physiological cell activityas it is RI alpha for RET/PTC2, TPM3 for TRK.

CLINICAL FEATURES OF PTC EXPRESSINGRET OR TRK ONCOGENES

Analysis of the clinical characteristics of sporadicand radiation-induced thyroid tumors indicates theexistence of correlation between RET and NTRK1 posi-tivity and young age of patients. In fact it has beenreported that in papillary thyroid neoplasias, thefrequency of RET and NTRK1 activation is significantlyhigher in patients under the age of 30 (Bongarzoneet al., 1996). In addition, children are much more sensi-tive to the tumorigenic effect of external irradiation,because of the high degree of replication of thyroid cells.This could amplify the possibility to fix and propagatemutational changes. More recently, controversial data(Elisei et al., 2001) suggested that RET/PTCs rear-rangements in thyroid tumor are not restricted to themalignant phenotype, are not higher in radiation in-duced tumors compared with those occurring natur-ally, are not different after exposure to radioiodineor external radiation, and are not dependent onyoung age.

However, the Chernobyl accident has increased therisk of childhood thyroid cancer. These tumors aremore aggressive than sporadic tumors and have an un-usually short latency period between the exposure andthe disease. Moreover several studies indicate a lowerfemale to male ratio than sporadic thyroid tumors inchildren (Moysich et al., 2002).

TABLE 2. RET and NTRK1 oncogenes in papillary thyroid carcinomas

Oncogene Activating gene Activating gene function Chromosome

PTC1a H4/D10S170 Unknown 10q21PTC2 RI a (PKA) PKA regulatory subunit 17q23–q24PTC3a-PTC4a ELE1 Androgen receptor-associated protein

(co-transcription factor)10q11.2

PTC5a GOLGA5 Golgi auto antigen 14qPTC6a hTIF1a Transcription intermediary factor 7q32–q34PTC7a hTIF1 g RFG7 Transcription intermediary factor 1p13PTC8a Kinectin Vesicle membrane anchored protein 14q22.1PTC9a RFG9 Putative intracellular transport protein 18q21–22RET/PCM1 PCM1 Centrosomal protein 8p22–p21.3Elks-RET ELKS Unknown 12p13TRKa TPM3 Stabilization of cytoskeleton actin filaments 1q22–q23TRK-T1; TRK-T2a TPR Nuclear protein transport 1q25TRK-T3 TFG Unknown 3q11–q12

aFrom patient exposed to Chernobyl radiations.

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Since many authors described RET and TRK rear-rangements in occult PTCs (Viglietto et al., 1995) andpapillary microcarcinomas (Tallini et al., 1998; Nasiret al., 2000; Corvi et al., 2001), the oncogene activation isconsidered an early event in thyroid carcinogenesis.This hypothesis is also supported by the evidence thattransgenic mouse models of RET/PTC1, RET/PTC3, andTRK-T1, driven by a thyroglobulin promoter, developmultifocal thyroid tumors at an early age, and thetumors are histologically very similar to human PTCs,with ground glass nuclei, grooves and inclusions(Santoro et al., 1996; Russell et al., 2000). However,TRK-T1 and RET/PTC1 transgenic mice infrequentlydeveloped solid-type carcinomas, differently from whatobserved in RET/PTC3 transgenic mice (Sagartz et al.,1997; Powell et al., 1998; Russell et al., 2000). Inaddition, the infection of short-term cultures of normalhuman thyroid cells with a retroviral vector expres-sing RET/PTC1 or TRK-T3 alters nuclear morphologywith similar or identical changes diagnostic of PTC(Fischer et al., 1998; Fischer personal communication).Interestingly, the failure to identify RET/PTC in poorlydifferentiated and anaplastic thyroid carcinomas(Soares et al., 1998), the apparent inability of papillarytumors harboring RET/PTC rearrangements to pro-gress to less differentiated form (Tallini et al., 1998)and, on the contrary, the discovery of RET oncogene intransformed cells with apparent limited growth poten-tial (Bond et al., 1994), point to a limited role for RET/PTC in the development of aggressive forms of thyroid

cancer. Tallini et al. (1998), analyzing 316 thyroidtumors concluded that only PTCs with alterations otherthan RET, es: NTRK1 gene, can progress to less dif-ferentiated, more malignant thyroid cancer. Recently,the same authors using immunohistochemical tech-niques, identify RET rearrangement also in a lowpercentage (12.9%) of poorly differentiated thyroidcarcinomas (Santoro et al., 2002) but the proportionraises to 20% among poorly differentiated thyroid carci-nomas (PDC) with evidence of evolution from a PTC.This finding may suggest that PDC could derive fromdedifferentiation of PTC but do not support a role forRET/PTC in the development of the more malignantphenotype.

RESTRICTION OF RET AND NTRK1 ONCOGENICREARRANGEMENTS TO PTCs

Thyroid carcinoma was the first adult epithelialmalignancy where specific chromosomal rearrange-ments have been identified. Thyroid cancer is a raremalignant disease of the endocrine system and has beenclearly linked to external ionizing radiation exposure.It has been demonstrated that Chernobyl-related ioniz-ing radiation exposure was directly related to the in-creased risk of PTC development in children fromaffected areas. Gene rearrangements involving theRET proto-oncogene, and less frequently NTRK1, havebeen shown to be causative events specific for PTC(Wajjwalku et al., 1992; Bongarzone et al., 1996;Beimfohr et al., 1999; Elisei et al., 2001). However, in

Fig. 2. Mechanism of activation of the physiological receptor and its oncogenic version. L, ligand; TK,tyrosine kinase domain; Y, tyrosine residue; Y*, phosphorylated tyrosine residue; D, dimerizationdomain.

174 ALBERTI ET AL.

experimental models, transforming ability of the RET/PTCs oncogenes is not restricted to the thyroid epithe-lium. In fact, transgenic mice carrying RET/PTC1 geneunder the control of the H4 promoter, developedmammary adenocarcinomas, hyperplasia of sebaceousglands, and pilomatrixomas (Portella et al., 1996). Thespecificity of these oncogenic rearrangements as pecu-liar feature for thyrocytes has been related to the higherfrequency of proximity RET and H4 loci in interphasenuclei of human thyroid cells, compared to nuclei ofperipheral blood lymphocytes and mammary epithe-lial cells. Spatial contiguity of RET and H4 suggestsa structural basis for generation of RET/PTC1 rear-rangement, because a single event could produce adouble-strand break in each gene at the same site in thenucleus (Nikiforova et al., 2000a,b). This tendency isalso proposed from the same authors for RET/PTC3rearrangement (Nikiforov et al., 1999). Moreover RET/PTC activation can be induced in irradiated humanthyroid tissue implanted in mice (Mizuno et al., 2000)but not in irradiated mice thyroid. This may be due todifferent interphase architecture of chromosome carry-ing RET locus in thyroid cells of mice compared tohuman. This finding could explain the high level ofRET rearrangements in patients irradiated for benigndiseases or in children exposed to radiation afterChernobyl accident, where the incidence of RET/PTCactivation ranges from 60–70% versus the 5–30% ofthe spontaneous papillary carcinomas. On the otherhand Yang et al. (1997) observed that thyroid cells withDNA damage induced by exposure to ionizing radiationwere resistant to apoptosis. In this context, the inducedexpression of wild type p53 might play an important rolein promoting DNA end-jointing enzymatic activity inthyroid cells.

Follicular adenomas and carcinomas arise through anoncogenic pathway distinct from that of papillarycarcinomas, characterized by a higher prevalence ofactivating mutations of all the three RAS genes and agreater predisposition to develop DNA copy abnormal-ities. In follicular carcinomas, a fusion oncogene involv-ing PAX8 has been recently described, which encodesa paired domain transcription factor essential forthyroid development, and the peroxisome proliferator-activated receptor PPARg. Such rearrangement was notfound either in follicular adenomas or in PTCs. Theoncogene resulted from a chromosomal translocationt(2;3)(q13;p25) in follicular tumors and displayed do-minant negative suppression of wild type PPARgactivities (Kroll et al., 2000).

In conclusion, a single thyrocyte can give rise to twodifferent types of well-differentiated carcinomas, thepapillary and the follicular. In this context the specificgenetic alteration becomes crucial to drive the transfor-mation of the thyrocytes towards the papillary (RET andNTRK1 rearrangements) or the follicular phenotype(PPARg rearrangements or RAS point mutations). It ispossible that papillary carcinomas lacking RET or TRKactivation may carry rearrangements of other TK re-ceptors rather than defects in genes involved in RET andNTRK1 signaling pathways.

Finally, it is worth mentioning that a few cases offamilial papillary thyroid carcinomas (FPTCs) havebeen recently described. Nevertheless no linkage to RET

and NTRK1 has been found in these tumors (Lesueuret al., 1999).

NTRK1 DEREGULATION IN OTHERNEOPLASTIC DISEASES

Although oncogenic activation of NTRK1 plays amajor role in PTCs, its involvement has been demons-trated in other malignancies.

Molecular analysis identified a mutation in theNTRK1 gene in a patient with acute myeloid leukemia.This deletion removes 75 amino acids in the extracel-lular domain of the NTRK1 receptor. The resultingmutated protein displayed in vitro transforming activ-ity, it constitutively phosphorylates and stimulateddownstream signaling pathways. This is the first de-monstration indicating that an NTRK1 mutation maycontribute to leukemogenesis and the first example ofa deletion within NTRK1 involved in human cancer(Reuther et al., 2000).

A novel recurrent rearrangement that fuses ETV6(TEL) gene to NTRK3 (TRKC) gene was identified(Knezevich et al., 1998) in congenital fibrosarcoma(CFS). The translocation t(12;15)(p13;q25) creates achimeric oncoprotein ETV6-NTRK3 (EN) containing thehelix-loop-helix dimerization domain (HLH) of ETV6fused to the TK domain of NTRK3. ETV6-NTRK3displays transforming activity in NIH3T3 cells andrequires the intact HLH dimerization domain and thefunctional TK domain for transformation (Wai et al.,2000).

NTRK1 was also shown to play a role in regulatingneurogenic neoplasms such as neuroblastoma (NBL).High levels of NTRK1 are expressed in NBLs with goodprognosis which often showed spontaneous regression.A limited amount of NGF, supplied by stromal cells,accounts for regulation of differentiation and apoptosisof the NBL cells (Nakagawara et al., 1993). On thecontrary, NTRK1 expression is highly down-regulatedin NBL with aggressive behavior, disturbing down-stream signaling (Nakagawara et al., 1992; Borrelloet al., 1993).

Mutations in the RET proto-oncogene have beenfound in familiar and sporadic medullary carcinomas(MTC, see below), however it has also been reportedthat changes in TRK receptor family expression appearto be involved in both preneoplastic thyroid C cellhyperplasia and later tumor progression. In particular,NTRK1, not expressed in normal C cells, is up-regulatedin C cell hyperplasia and MTC, although the correlationwith aggressive disease is not as prominent as seen forNTRK3 (TrkC) (McGregor et al., 1999).

Deregulation of NGF/NTRK1 pathway, may also leadto prostate and breast cancer. In particular, normalprostate epithelial cells express NTRK1 and its cognatereceptor p75, but do not express NGF or any neuro-trophin (Weeraratna et al., 2000). A paracrine model hasbeen proposed by Djakiaew, in which NGF, produced bystromal cells, diffuses and binds to the NTRK1 and p75receptors expressed in the epithelial cells. However, thefunction of the paracrine NGF/NTRK1/p75 loop innormal prostate cells is not clear. It has been hypothe-sized that such paracrine loop may have a generaltrophic effect within the prostate (Djakiew et al., 1991).Prostate carcinogenesis gives rise to alteration of

PROTO-ONCOGENES IN HUMAN DISEASES 175

the NGF/NTRK1/p75 axis. In fact, prostate cancer cellslack the expression of p75 and become able to produceNGF. All these events lead to a switch from a para-crine to an autocrine loop, which enhances growthand survival of malignant cells (Dalal and Djakiew,1997). In particular, prostate cancer cells acquire adependence on functional NGF/NTRK1 pathway fortheir survival. Inhibition of TRK kinase activity resultsin in vivo tumor regression by increasing the apoptosisof the cancerous but not normal cells. A TRK inhibitor(CEP-701) has entered clinical development and iscurrently undergoing Phase II evaluation in hormone-refractory prostate cancer patients (Weeraratna et al.,2001).

NGF has also been demonstrated to stimulate theproliferation of breast cancer but not normal cell lines,via activation of the MAPK cascade (Descamps et al.,1998). Moreover, NGF stimulates survival of these cells,through a distinct mechanism involving p75 and NFkB(Descamps et al., 2001). Recently it has been shown thatTamoxifen is able to inhibit NGF-induced proliferationand NTRK1 phosphorylation in a human breast cancercell line (Chiarenza et al., 2001).

In conclusion, NTRK1 activation in human malig-nancies occurs by several mechanisms, mainly struc-tural rearrangements and altered expression. It isinteresting to outline that, differently from other RTKssuch as Met, FGFR, Kit and RET, NTRK1 pointmutations have never been found responsible for humancancer.

RET ACTIVATION IN INHERITEDAND SPORADIC MEDULLARY

THYROID CARCINOMAS

The C cells of the thyroid are derived from the neuralcrest and are believed to be the precursors from whichmedullary thyroid carcinoma (MTC) arise. As many as75% of all MTCs are sporadic; the remainding heredi-tary forms of MTC are associated with multiple endo-crine neoplasia type 2 (MEN2). This was the first of theinherited endocrine neoplasia syndromes to be eluci-dated at the genetic level with the discovery in 1993 thatgermline mutations in the RET proto-oncogene werepresent in affected individuals (Mulligan et al., 1993).Unlike other cancer syndromes, which are associatedwith inactivation of tumor suppressor genes, MEN2arises as a result of activating mutations of the RETproto-oncogene.

Germline point mutations of RET are responsible forthe inheritance of MEN2 cancer syndromes which areusually divided into three different clinical subtypes:MEN2A, MEN2B, and FMTC. MEN2A, MEN2B, andFMTC are autosomal dominant cancer syndromes.

The MEN2A subtype is characterized by MTC, pheo-chromocytoma, and parathyroid hyperplasia (Mulliganet al., 1995; Eng et al., 1996) whereas MTC, pheochro-mocytoma, ganglioneuromas of the intestinal tract andskeletal and ocular abnormalities characterize MEN2B.MEN2B is the most aggressive of the three sub-types,often displaying an earlier age of onset.

MTC is the only feature of FMTC and it usuallydevelops at a later stage of life. The course of MTC inFMTC families is more benign and prognosis is good(Famdon et al., 1986).

MULTIPLE ENDOCRINE NEOPLASIATYPE 2A (MEN2A)

In virtually all MEN2A, mutations affecting RETcysteine-rich extracellular domain, each convertinga cysteine to another amino acid, at codons 630, 634(exon 11) or codons 609, 611, 618, 620, (exon 10). Thesemutations account for 98% of all mutations associatedwith MEN2A; the most common mutation, accountingfor over 80% of all mutations associated with MEN2A,affects codon 634 and converts a cysteine into an argi-nine. Rarer mutations associated with MEN2A havebeen described such as an in-frame germline duplicationof 12 and 9 bp in exon 11 (Hoppner and Ritter, 1997;Hoppner et al., 1998) and de novo cases of MEN2A havebeen associated with two new germline mutations(at both codon 634 and 640) on the same RET allele(Tessitore et al., 1999) or at codon 624 (Aguild, 1999).RETMEN2A oncoproteins display constitutive kinaseactivity consequent to ligand-independent dimeriza-tion. The substitution of one cysteine residue leads toconstitutive receptor dimerization and hence activationas the loss of an intramolecular disulfide bond resultsin an unpaired cysteine residue available to take part inan intermolecular disulfide bond between two mutantRET receptors (Fig. 3) (Asai et al., 1995; Borrello et al.,1995; Santoro et al., 1995). Consistent with this,transgenic mice in which the C634R mutation in theret gene coding for the short isoform was expressedunder the control of the human calcitonin promoter orunder the MoMuLv LTR, develop C-cell tumors resem-bling human MTC (Michiels et al., 1997; Kawai et al.,2000). Interestingly, transgenic mice expressing thesame mutation in ret long isoform, developed both MTCsand PTC (Reynolds et al., 2001).

MULTIPLE ENDOCRINE NEOPLASIATYPE 2B (MEN2B)

Most MEN2B cases (95%) are caused by the M918Tmutation (exon 16) that is frequently a de novo mutationlocated on the allele inherited from the patient’s father(Carlson et al., 1994a). Other rarer (5%) intracellularmutations involve codon 883 (exon 15) in the RETTK domain. The M918T substitution is also found insporadic MTC, with M918T mutation-positive tumorsoften displaying a more aggressive phenotype. Recently,infrequent germline missense mutations have beenreported in MEN2B de novo cases: in exon 16 at codons912 and 922 (Carlson et al., 1994b). Moreover, a doublemutation at codons 804 and 806 has been found in aJapanese patient that had clinical features character-istic of MEN2B (Miyauchi et al., 1999). The reason whymore than 95% of MEN2B cases are accounted for bygermline M918T and only fewer than 5% by A883F, isunknown. The M918T mutation does not cause consti-tutive dimerization although GDNF stimulation seemsto be necessary for the full activation of MEN2B mutantRET (Bongarzone et al., 1998), and the overall activationlevels of the RET kinase it induces, can hardly accountfor its high oncogenic potential (Borrello et al., 1995;Santoro et al., 1995). Thus, RETMEN2B is probablymore than simply an active RET kinase and qualitativechanges in RET kinase activity may be responsible forits specific neoplastic phenotype. RET methionine at

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codon 918 is highly conserved in RTKs and it maps in thePþ1 loop of the kinase domain that is predicted tointeract with the protein substrate. A threonine is foundat the equivalent position in cytosolic TKs, and the twokinase classes (receptorial and cytosolic) have differentsignaling specificities (Marengere et al., 1994). Accord-ingly, the MEN2B mutation converts the substrate-binding pocket of RET to resemble that of non-RTKssuch as c-src and c-abl (Carlson et al., 1994b; Hofstraet al., 1994). The change in substrate specificity canaffect RET-mediated phosphorylation of intracellularproteins as well as the pattern of RET autophosphoryla-tion sites. Both possibilities have been experimentallyproven. The pattern of phosphorylated intracellularproteins differs in RETMEN2B- and RETMEN2A-expressing cells (Santoro et al., 1995). Moreover, phos-phopeptide mapping has shown that RETMEN2Bautophosphorylation sites differ from those of wild-typeRET and of RETMEN2A (Santoro et al., 1995; Liu et al.,1996; Murakami et al., 2002). Thus, the shift of RETautophosphorylation sites and of RET intracellularsubstrates, rather than the simple rise of RET kinaseactivity, may be crucial for the oncogenic activity ofRETMEN2B alleles. It is not known how the A883Faffects RET function. However, residue 883 is located ina subdomain of RET that defines substrate preference(Smith et al., 1997) thus suggesting that the alteration ofsubstrate specificity may be the common etiologic threadthat underlies the pathogenesis of MEN2B. The produc-tion of a mouse model of MEN2B by introduction ofthe corresponding mutation into the ret gene demons-trated that heterozygous mutant mice displayed several

features of the human disease, including C-cell hyper-plasia progressing to pheochromocytoma, while homo-zygous displayed more severe thyroid adrenal diseaseas well as male infertility. Only homozygous mice diddevelop ganglioneuromas of the adrenal medulla andenlargement of the associated sympathetic ganglia(Smith-Hicks et al., 2000).

FAMILIAL MEDULLARY THYROIDCARCINOMA (FMTC)

FMTC mutations can be found either in the extra-cellular or in the TK domain of RET. The ones occurringin the extracellular RET domain are usually a set ofsubstitutions of cysteines 609, 611, 618, 620 (exon 10)and 630 and 634 which are also found associated withMEN2A, whereas in the TK domain mutations occurat residues 768, 790, 791 (exon 13), 804, 844 (exon 14)or 891 (exon 15). Rare mutations have been recentlyreported, such as a 9-base pair duplication in exon 8 inan FMTC family (Pigny et al., 1999) or mutations atcodons 804 and 778 on the same RET allele which areassociated with both FMTC and prominent cornealnerves (Kasprzak et al., 2001). FMTC mutations, occur-ring in the intracellular RET domain, were thought tobe infrequent and only a small number of families bear-ing a RET mutation within exons 13, 14, and 15 havebeen described. However, in the past 2 years thefrequency of detection of these mutations has increased(Niccoli-Sire et al., 2001) due to more accurate analysisand screening. Of note, mutations in exon 13 at codons790 and 791 and in exon 14 at codon 804, until recentlyassociated with the FMTC phenotype, are also found in

Fig. 3. Mechanism of disulfide bond-linked RET dimerization. SS, disulfide bond; TK, tyrosine kinasedomain; Y, tyrosine residue; Y*, phosphorylated tyrosine residue.

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MEN2A. Cysteine mutations in the RET extracellulardomain sometimes induce the FMTC phenotype and incontrast with what happens for the MEN2A families,the substitution at codon 634 occurs only in 30% of thecases whereas mutations at codons 609, 611, 618, 620,630, occurred in 60% of the FMTC families (Ponder andSmith, 1996). The transforming activity of RET with asubstitution in cysteine 609, 611, 618, 620, or 630 wassignificantly low compared with that of RET with thecysteine substitution at codon 634. This low transform-ing activity could then predispose to the development ofFMTC rather than MEN2A.

No data are yet available on the mechanisms ofactivation of FMTC mutations occurring in RET TKdomain. Patients with RET mutations in exons 13, 14,and 15 exhibit a mild C cell disease phenotype (Berndtet al., 1998; Fattoruso et al., 1998) confirmed by in vitrostudies. RET expressing E768, V804, and S891 RETmutations display lower transforming activity (Pasiniet al., 1997; Iwashita et al., 1999) compared with RETsubstitutions at codons 634, 918, or 883 stronglyassociated with MEN2A and MEN2B, respectively.Computer modeling has suggested that the E768Dsubstitution modifies the kinase activity of the re-ceptor by altering the substrate specificity or the ATP-binding capacity (Pasini et al., 1997). As for its location,also the substitution at position 804 may exert anactivating effect by altering the kinetics of interactionswith normal cellular substrates or by modifying therange of substrates that are phosphorylated (Bolinoet al., 1995; Eng et al., 1995; Pasini et al., 1997; Iwashitaet al., 1999).

SPORADIC MTC

Somatic RET mutations are found in a fraction ofsporadic MTCs and, rarely, in pheochromocytomas.Mutation at the codon 918 or at the cysteine codons609, 611, 618, 620, 630, 634 appear in a significantamount of sporadic MTC cases and recently threenew somatic missense mutations (at codons 639, 641,and 922) of the RET proto-oncogene associated withsporadic MTC have been described (Kalimin et al.,2001).

Recent studies (Feldman et al., 2000; Brauckhoffet al., 2002) have reported cases of patients harbor-ing RET germline mutations in exons 14 and 15 (atcodons 790, 791, 804) resulting in papillary microcarci-noma. Moreover, Rey et al. (2001) also described the caseof a kindred in which a novel single point germline RETmutation (K603E in exon 10) cosegregates with medul-lary and PTCs. Despite the low number, these observa-tions suggest that there might be a correlation betweenthe occurrence of PTC and RET germline mutations inexons 13 and 14 that might play a role in pathogenesisof PTC. Of note, PTC seems to be present just in pa-tients with low penetrance RET germline mutations. Itremains an open question whether the simultaneousoccurrence of inherited MTC and PTC is coincidental orthe result of partly common pathogenic pathways.Reynolds et al. (2001) found the co-existence of MTCand PTC in transgenic mice expressing the long isoformof MEN2A RET and suggested that this might be dueto the possible existence of an ultimobranchial stemcell of endodermal origin, which gives rise to a subset

of both thyroid follicular cells and C-cells (Kovacset al., 1994).

In conclusion, the genotype/phenotype associations inMEN2 reflect differences in behavior and functionamong the RET mutant forms. The above data suggesta model in which there are tissue specific differencesin sensitivity to RET activation. All RET activatingmutations in fact, are sufficient to induce tumorigenesisin the thyroid glands and MTC is associated with allMEN2 subtypes. On the other hand, pheochromocytomaand hyperparathyroidism are only found in associationwith the most penetrant of RET mutations affectingcysteine 634 suggesting that a high transformingactivity is required to induce abnormal growth in thesetissues.

DIAGNOSIS AND MANAGEMENT OF MEN2

The prognosis for MEN2 patients is very good withearly diagnosis and intervention thus implying thatadequate testing is required to screen subjects at riskfor MTC. Currently, early genetic screening for RETmutations is considered the standard care for MEN2 aspatients having a MEN2/FMTC-specific germline muta-tion have a high risk of developing MTC. Prophylac-tic thyroidectomy is recommended before the age of6 years (reviewed in Gimm et al., 2001) as every singleC-cell inherits the genetically determined potential tobecome neoplastic. Current clinical research concen-trates on finding an existing genotype-phenotype cor-relation, mutations which are associated with a late ageof onset, that would allow to postpone surgery, thusrestricting the extent of surgery. Several mutationssuch as E768D and V804M seem to qualify for suchrecommendation (Gimm et al., 2001). MEN2B-asso-ciated MTC is the most aggressive and early diagnosis isrequired and total thyroidectomy is recommended asearly as 1 year. Given that even low penetrance RETmutations, particularly codon 804 mutations, mightexist in MEN2, it is standard clinical practice for allindividuals who present with MTC to undergo RETtesting. The discovery of an occult germline RET muta-tion in an apparently sporadic MTC case means that theindividual has MEN2. In order to simplify the detectionof RET missense mutations, a method for the rapidmutation analysis of gene sequences has been proposedin a very recent paper (Kim et al., 2002) using oligonu-cleotide microarrays instead of the commonly usedanalysis of RET mutations by single-strand conforma-tional polymorphism or direct sequencing. This mightrepresent an effective diagnostic genetic tool to test forthe presence of MEN2 mutations at early stages.

As MTCs respond very poorly to chemotherapeuticagents and total thyroidectomy is the only way to treatthese tumors, the availability of inhibitors specific forRET oncoproteins could help in developing new ther-apeutic strategies for RET-associated diseases. Carlo-magno et al. (1996) have very recently demonstratedthat the pyrazolo-pyrimidine PP1 blocks tumorigenesisinduced by RET/PTC cytoplasmic oncogenes as it caninhibit RET enzymatic activity and its transformingeffects. Our results also suggest that PP1 treatmentwould represent a promising new strategy to selectivelytarget RET oncogenic products to destruction holdingpromise for MTC therapy.

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RET GERMLINE INACTIVATING MUTATIONSIN HIRSCHPRUNG’S DISEASE

Hirschsprung’s disease (HSCR), or colonic aganglio-nosis, is a common congenital disorder (one in 5000 livebirths) leading to intestinal obstruction or chronic cons-tipation (Parisi and Kapur, 2000). Most HSCR casesare sporadic, however 15–20% are familial forms andgenetic analysis identified mutations in pathways relat-ed to RET, endothelin and in two transcription factors,Sox 1 and the SMAD-interacting protein-1 (SIP1) (Parisiand Kapur, 2000; Cacheux et al., 2001; Wakamatsuet al., 2001). Heterozygous mutations of GDNF andNTRN have also been identified in a small fraction ofHSCR patients (Parisi and Kapur, 2000) but no muta-tions have been described for RET coreceptors (Angristet al., 1998; Myers et al., 1999; Onochie et al., 2000;Vanhorne et al., 2001) although Gfraa1 knockout miceare phenotypically very similar to RET and GDNF�/�mice (Cacalano et al., 1998; Enomoto et al., 1998).However, RET seems to be the major gene involved inHSCR as even when the major mutation is in the endo-thelin receptor B (EDNRB) gene (Puffenberger et al.,1994), RET variants make some contribution to suscept-ibility and homozygous RET-null mice have full sex-independent penetrance of aganglionosis (Schuchardtet al., 1994). Moreover, even the mutations affectingRET ligands are not sufficient by themselves to inducethe HSCR phenotype but contribute to the disease whenassociated with other mutations in RET (Parisi andKapur, 2000).

Inactivating mutations of one allele of the RET proto-oncogene have been detected in half of the dominantlyinherited cases of HSCR that displays incompletepenetrance, and in one third of HSCR sporadic cases(Eng, 1996; Eng and Mulligan, 1997). RET mutationsare spread throughout the coding sequence and includedeletion, insertion, frameshift, nonsense and missensemutations (Eng and Mulligan, 1997; Parisi and Kapur,2000). Most of these mutations impair RET function(Parisi and Kapur, 2000; Iwashita et al., 2001) thussuggesting that HSCR results from RET haploinsuf-ficiency but the allelic heterogeneity at the RET locus inHSCR is associated with various mechanisms of actionall leading to RET dysfunction (Pelet et al., 1998;Iwashita et al., 2001). Mutations located within theextracytoplasmic domain impair RET maturation andits translocation at the plasma membrane (Carlomagnoet al., 1996; Iwashita et al., 1996). This probably resultsin a decrease in the amount of RET protein level at thecell surface that is insufficient to trigger RET signalingin enteric neuroblasts during embryogenesis. HSCRmutations in RET TK domain, affect the catalytic activ-ity to different extent. Some of them completely abolishthe kinase activity of the receptor, others interfere withthe binding of components of RET signaling such asPLC-g for the E762Q substitution (Iwashita et al., 2001)Shc, IRS-2 or FRS2 (Geneste et al., 1999; Bordeaux et al.,2000; Melillo et al., 2001a,b). Recently, Bordeaux et al.(2000) suggested that RET can induce caspase depen-dent apoptosis, in the absence of its ligand. HSCR maythen result from apoptosis of RET-expressing entericneuroblasts as HSCR mutations render this proapopto-tic activity ligand insensitive.

COSEGREGATION OF MEN2A/FMTC AND HSCR

In rare families, MEN2A/FMTC and HSCR co-segregate (Mulligan et al., 1994; Decker et al., 1998;Takahashi et al., 1999) and affected individuals carry asingle substitution at one of the four cysteines in theextracellular RET domain (codons 609, 611, 618, and620). The reason why a single mutation can displayantagonistic effects is still under current studies. It ispossible to speculate that this is due to the differencesin the timing of RET expression in each particular tissuethat might differently affect neural-crest derived cellstissue development. These mutations result in fact, inuncontrolled cellular proliferation in endocrine tissuesand on the other hand, result in lack of neural growth inthe enteric system (Takahashi et al., 1999). Genetic orenvironmental factors might also influence the clinicalexpression of the enteric phenotype. In endocrinetissues, these cysteine substitutions have a dominanteffect whereas it is not yet clear if in the enteric systemthe loss or inactivation of the wild type RET allele isassociated with the HSCR phenotype.

Experimental data demonstrated that RET MEN2A/HSCR mutations markedly decrease the expression ofRET at the cell surface (Ito et al., 1997) resembling theeffect of the typical HSCR mutations occurring in RETextracellular domain. Interestingly, both transfectionexperiments and biochemical analysis have shown thatthe cysteine codon substitutions were also able to causeligand independent dimerization, activation, and trans-formation as the classical MEN2A cysteine substitu-tions do (Asai et al., 1995; Borrello et al., 1995; Santoroet al., 1995), although to different extents. As thesemutations trigger the development of MTC as well as ofpheochromocytoma, the mislocalization of the receptorsaffect pathways not involved in cell proliferation butprobably controlling other pathways such as cell move-ment migration, probably essential for neuronal devel-opment and survival.

NTRK1 GERMLINE INACTIVATINGMUTATIONS IN CONGENITAL INSENSITIVITY

TO PAIN WITH ANHIDROSIS (CIPA)

Although several evidences show that NTRK1 can beactivated as an oncogene, no naturally occurring germ-line activating point mutations in NTRK1 have beenfound associated to malignancies. Conversely, develop-mental abnormalities have been found associated withNTRK1 inactivation.

Recently, mutations in the NTRK1 gene have beenfound to be the genetic bases for CIPA disease (Indoet al., 1996). CIPA (also known as hereditary sensoryand autonomic neuropathy IV) is a rare recessivegenetic disease characterized by loss of pain and tem-perature sensation, defects in thermal regulation andoccasionally mental retardation. These clinical featuresare due to a lack of innervation of the eccrine sweatglands by sympathetic neurons and to the absence ofsmall diameters afferent neurons responsible for tissuedamaging stimuli in the dorsal ganglia (Axelrod andPearson, 1984). The NGF pathway has been consideredas a candidate since the phenotype of CIPA overlapsphenotype of mice lacking NTRK1 or NGF genes. Muta-tional screening, performed by different laboratories

PROTO-ONCOGENES IN HUMAN DISEASES 179

including ours, identified several NTRK1 mutations inCIPA patients from different ethnic groups. So far, 37NTRK1 mutations have been identified (Indo, 2001).Mutation types include frame shift, splice site, nonsenseand missense. The former three mutation types produceaberrant proteins leading to disruption of the NGF/NTRK1 pathway. Some of the mutations occur in theextracellular region; the majority of them occur in theintracellular region, in particular in the TK domain.The causative role in the disease has been demonstrat-ed with the analysis of the biological effects of CIPAmissense mutations. By analyzing the receptor tyro-sine phosphorylation status and the biological activity(seen as the capability of NGF receptor to transformNIH3T3 cells and to differentiate PC12 cells in the pre-sence of NGF) several mechanisms of receptor inactiva-tion leading to CIPA disease have been identified. Inaddition, 3 amino acidic substitutions (R85S, H598Y,and G607V) were classified as polymorphisms (Mardyet al., 2001; Miranda et al., 2002a).

One of the mutations analyzed, L213P, occurring inthe first Ig-like domain (Mardy et al., 1999), inter-feres with the receptor processing, producing only the110 kDa partially glycosylated form of the receptor.Immunofluorescence and biochemical analysis haveshown that the L213P mutant receptor is not able toreach the plasma membrane but it is retained in theendoplasmic reticulum, thus not responding to NGF(Miranda et al., 2002a). Another mutation, L93P (Miuraet al., 2000), analogously to L213P, produces only thenon-mature 110 kDa form of the receptor, suggesting apossible common mechanism, although the ER reten-tion has not been demonstrated for it (Mardy et al.,2001). Several mutations occurring in the intracellulardomain (G571R, R643W, G708S, and R774P) produceinactive receptors, affecting the capability of NTRK1 toautophosphorylate, to transform NIH3T3 and to differ-entiate PC12 cells in the presence of NGF (Greco et al.,1999, 2000; Miranda et al., 2002a). 3-D studies (basedon a model built by analogy with the insulin receptor)suggested that some of these mutations occur in re-sidues critical for kinase activity (G571R, R643W) or inresidues that affect the whole conformation of theenzyme although not located in the active site (G708S,R774P) (Miranda et al., 2002a). A preliminary analysisbased on the phosphorylation status of the receptorshowed that G516R and R648C mutations abrogate itstyrosine phosphorylation (Mardy et al., 2001).

A mechanism reducing the biological activity of thereceptor has been described for two different mutations,D668Y and M581V, both occurring in the TK domain(Yotsumoto et al., 1999; Miura et al., 2000). In partic-ular, D668Y mutation occurs at a highly conservedresidue located in the activation loop. Surprisingly, thismutation does not cause inactivation of the receptorsince its kinase activity is not abrogated in the presenceof NGF. However a reduced biological activity of theD668Y receptor with respect to the wild type was observ-ed. Such reduction may not be sufficient for properneuronal differentiation (Miranda et al., 2002a). Inter-estingly the same substitution has been found in thecorresponding residue of the c-kit receptor causingmastocytosis (Furitsu et al., 1993). It is interesting tonote that although occurring at conserved residue,

mutation of Asp 668 causes differential effect on NTRK1versus other RTKs, one being inactivating, the otheractivating.

Recently we have shown the effect of M581V muta-tion, associated to a mild form of CIPA. The patientscarrying such mutation are adults and retain tempera-ture sensation. The mutant receptor still retains a weak,although drastically reduced, kinase activity, and dis-plays a residual biological activity. By contrast to com-plete inactivation, such reduction may account for themilder phenotype of the CIPA patients carrying M581Vmutation (Miranda et al., 2002b).

Unfortunately, no clinical treatment is possible forCIPA patients, because survival and maintenance ofspecific neurons take place during embryogenesis.However, identification of CIPA mutation in an affectedfamily may allow prenatal diagnosis.

To date, NTRK1 represents the only gene knownresponsible for the pathogenesis of CIPA. However, asmentioned above, CIPA phenotype overlaps phenotypesof both NTRK1 and NGF knockout mice. In the nextfuture, it will be interesting to investigate whethermutations of other genes involved in the NGF pathway(such as NGF or p75) may be responsible for the diseaseof CIPA patients negative for NTRK1 mutations.

CONCLUSIONS

RET and NTRK1 represent a valuable example of thenewly developing molecular medicine where the geneticaspects of a disease are elucidated.

The two genes also provide a demonstration of thenotion that different alterations of the same geneticelement can lead to different and sometime oppositepathologic manifestations.

Moreover, they also show that even in the context ofsimilar diseases, subtle differences in the type of gene-tic alterations result in different pathogenic mechan-isms which in turn account for the diverse diseases.These genotype–phenotype relationships betweengenetic alterations of RET and NTRK1 and the conse-quent pathogenic mechanism(s) of a particular diseaseare summarized in Table 3.

Here it is shown that chromosomal rearrangementsleading to a constitutive unregulated TK enzymaticactivity are genetic alterations, affecting both genes, inPTCs which provided the first example for suchmechanisms in solid tumors. The involvement of RETin MEN2 and FMTC, which share as main pathologythe MTCs, is an outstanding evidence of how muta-tions in different domains of the same gene result indifferent although related cancer syndromes.

Moreover, the creation of disulfide bridges by dif-ferent genetic mechanisms, such as insertion, deletionor point mutations, is directly related to the abnormalconstitutive activation of RET enzymatic activity andcould provide a significant target for a molecularlyoriented possible therapy aimed to the abrogation ofthese cysteine-mediated bonds. Other outstanding ex-amples of molecular specificity are represented bymutations of RET in codon 609, 611, 618, and 620which show a co-segregation with two opposite pathol-ogic phenotypes, thyroid medullary carcinoma, a gain offunction disease, and HSCR, a loss of function develop-mental abnormality.

180 ALBERTI ET AL.

How a single mutation could results in so dramaticallyopposite effects is still unclear although, at least for thecodon 620 mutations, a mislocalization of the receptorwhich is retained in the cytoplasm could be associated toa loss of function leading to HSCR. On the other hand itsstill intact capability to promote a constitutive disulfidebridges-linked dimerization could account for its role inMEN2A phenotype.

Finally, the common outcome, loss of function ofNTRK1, the high affinity NGF receptor, in CIPAsyndrome is obtained by different genetic alterationsassociated with different pathogenic mechanisms. Anaberrant protein production can result from differentalterations including frameshift, nonsense mutations oraberrant splicing. On the other hand, the same type ofgenetic alteration, missense mutations, can produce aloss of enzymatic activity, retention of the receptor in theendoplasmic reticulum or a reduced biological activity.

This constellation of different pathogenic mechan-isms could require different therapeutic approaches. Ifwe believe, as stated by the ancient Greek and Latinmasters of medicine, that we can cure a disease onlywhen we know it, these reported progresses of ourknowledge of the molecular mechanisms underlying theRET and NTRK1 associated diseases are more than ahope that soon we will develop appropriate and effectivetools for their clinical management.

ACKNOWLEDGMENTS

The authors thank Miss Cristina Mazzadi for secre-tarial help.

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TABLE 3. Involvement of RET and NTRK1 in human pathologies

Disease Genetic alteration Pathogenic mechanism

RET

PTCs Chromosomal rearrangements Constitutive TK activityMEN2A Germline point mutations in the cysteine-rich domain Constitutive disulfide linked dimerizationMEN2B Germline point mutations in RET TK domain Constitutive TK activity altered substrate specificityFMTC Germline point mutations in:

the cysteine-rich domain Constitutive dimerizationRET TK domain Constitutive TK activity? altered substrate specificity?

HSCR Germline point, frame shift, missense mutations in:RET extracellular domain Impairment of RET cell surface expressionRET tyrosine kinase domain Partial or total loss of RET TK activityRET COOH terminus Impairment of binding of docking proteins

TRK

PTCs Chromosomal rearrangements Constitutive TK activityAcute myeloid leukemia Deletion of 75 aa Constitutive TK activityCongenital fibrosarcoma Chromosomal rearrangement Constitutive TK activityNeuroblastoma Altered expression Paracrine loopProstate cancer Altered expression Autocrine loop

Frame shift mutations Aberrant protein productionNonsense Aberrant protein production

CIPA Splice site Aberrant protein productionLoss of RTK activity

Missense mutations ER retentionReduced biological activity

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