9Francesca Carlomagno MD, PhD

targeting of specific RTKs as a potential therapeutic strategy for the treatment of thyroid cancer.

1521-690X/$ - see front matter 2008 Elsevier Ltd. All rights reserved.

Best Practice & Research Clinical Endocrinology & MetabolismVol. 22, No. 6, pp. 10231038, 2008

doi:10.1016/j.beem.2008.09.012available online at http://www.sciencedirect.com* Corresponding author. Tel.: 39-081-7463056; Fax: 39-081-7463037.E-mail address: masantor@unina.it (M. Santoro).Key words: kinase; thyroid; monoclonal antibody; small-molecule inhibitor.Research Associate

Istituto di Endocrinologia ed Oncologia Sperimentale CNR, 80131 Naples, Italy c/o Dipartimento di Biologia

e Patologia Cellulare e Molecolare, Universita Federico II, 80131 Naples, Italy

Giuliana Salvatore MD, PhDAssociate Professor

Dipartimento di Studi delle Istituzioni e dei Sistemi Territoriali, Universita Parthenope, 80133 Naples, Italy

Massimo Santoro* MD, PhDProfessor of Pathology

Dipartimento di Biologia e Patologia Cellulare e Molecolare, L. Califano, Universita` Federico II, via S. Pansini 5,

80131 Naples, Italy

Thyroid cancer is frequently associated with the oncogenic conversion of receptor tyrosinekinases (RTKs) or their downstream signalling molecules. Hence, there is a strong biological ra-tionale for assessing the efficacy of RTK blockade to treat patients who are resistant to or notcandidates for treatment with radioactive iodine. The first results of clinical trials based on theuse of RTK inhibitors in thyroid cancer patients have recently been published. Here we discussReceptor tyrosine kinase inhibitors in thyroid

cancer

Maria Domenica Castellone MD, PhDResearch Associate

Istituto di Endocrinologia ed Oncologia Sperimentale CNR, 80131 Naples, Italy c/o Dipartimento di Biologia

e Patologia Cellulare e Molecolare, Universita Federico II, 80131 Naples, Italy

CANCER GENES CODING FOR RECEPTOR TYROSINE KINASES

The protein kinase complement of the human genome (the kinome) contains 518protein kinase genes, including 58 receptor tyrosine kinases (RTKs) and 32 non-RTKs (www.kinase.com/human/kinome). In high-throughput genome-sequencing pro-jects, approximately 120 genes encoding protein kinases were found to be mutatedand causally implicated in cancer development.1,2

RTKs are transmembrane proteins that have an extracellular ligand-binding domain,a transmembrane segment, and an intracellular domain containing the juxtamembranesegment (JMR), the tyrosine kinase catalytic domain (TK) and a carboxy-terminal tail(Figure 1). RTKs are often involved in cancer development.1,2 Besides RTK pointmutations, cancers may feature illicit expression of the RTK or its cognate growthfactor and RTK gene rearrangements.35 Thyroid cancer, as discussed below, is oftenassociated to the oncogenic conversion of genes coding for RTKs or their signallingeffectors.6,7

MECHANISMS OF RTK ACTIVATION

Upon growth factor binding, RTKs undergo a dimerization process that activatesthe enzymatic function.3 All kinase domains are divided into an amino-terminal

1024 M. D. Castellone et alFigure 1. Overview of the signalling mechanism of receptor tyrosine kinases (RTKs). The general structure

of a prototypic RTK, with the extracellular domain (EC), transmembrane domain (TM), juxtamembrane do-

main (JMR), and tyrosine kinase domain (TK), is illustrated. Blue boxes summarize major biological outcomes

of the activation of specific signalling components.

(N-terminal) and a carboxy-terminal (C-terminal) lobe. The active site is located in thecleft between the N- and C-terminal lobes. Mg-ATP binds to the base of the cleft, the

RTK inhibitors in thyroid cancer 1025non-transferable b-phosphate of Mg-ATP binds to the P-loop of the N-terminal lobe,and the peptide substrate binds along the surface of the cleft.3,4 The ATP-binding sitecan also bind small-molecule inhibitors (see below).4,5 In the inactive kinase, the activesite is closed by the activation segment of the C-terminal lobe. Growth-factor-bindingcauses RTK dimerization, followed by extensive movements of the activation segmentand the JMR domain; this process allows ATP binding and catalysis.

Oncogenic mutations disrupt normal regulatory mechanisms and lead to constitu-tive activation of the kinase. Point mutations or rearrangements in the extracellulardomain mimic ligand-binding, thereby causing constitutive dimerization. Mutations inthe intracellular domain target regulatory domains of the kinase, such as the P-loop,the activation segment, or the JMR, thereby disrupting auto-inhibitory mechanisms.3

MECHANISMS OF RTK SIGNALLING

Mutual trans-phosphorylation of tyrosine residues within active RTK dimers recruitsintracellular proteins endowed with phosphotyrosine binding domains.35 Proximaltargets of the RTKs invoke the intracellular signalling cascades RAS-RAF-MAPK (theERK pathway) and the phosphatidylinositol 3-kinase (PI3K) AKT that ultimatelylead to diverse biological responses: i.e. mitogenesis, survival, differentiation and mo-tility (Figure 1).35

Genes coding for proteins working in the RTK-initiated signal transduction path-ways are frequently mutated in cancer. For instance, systematic DNA sequencing ofthe best-annotated protein-coding genes in breast and colorectal cancer revealedfrequent mutations in the PI3K and nuclear factor kB (NFkB) pathways, both of whichare involved in RTK signalling (Figure 1).8 In the case of thyroid cancer, paradigmaticexamples of this concept are mutations in BRAF, a RAF family serine/threonine kinase,in papillary thyroid carcinoma (PTC) (2969%) and PTC-derived anaplastic thyroid car-cinoma (ATC) (1035%), and in the RAS small GTPase that is mutated in the follicularvariant of PTC (up to 40%), in follicular thyroid carcinoma (FTC) (4053%) and in ATC(2060%).7 Moreover, mutations or amplification of PIK3CA, which codes for the PI3Kcatalytic subunit, is associated with ATC and FTC, and loss of expression of PTEN, themajor phosphatase antagonist of PI3K function, is frequent in ATC.9 Finally, functionalactivation of NFkB has been described in ATC.10 NFkB is crucial for RET and BRAFoncogene signalling in thyroid cancer cells11,12, and the proteasome inhibitor bortezo-mib (PS-341, Velcade, Millennium Pharmaceuticals), whose complex mechanism ofaction includes NFkB blockade, was effective in thyroid carcinoma cell lines.13

RTKs AS MOLECULAR TARGETS FOR CANCER TREATMENT

The advent of small-molecule drugs and monoclonal antibodies have made RTK target-ing a feasible therapeutic strategy for cancer.4,5 Monoclonal antibodies (mAbs) canblock the growth factor or the RTK itself. Some humanized mAbs such as trastuzu-mab (herceptin; Genentech/Roche) against the HER2/ErbB2 receptor, cetuximab (er-bitux; ImClone) against the epidermal growth factor receptor (EGFR/HER1/ErbB1),and bevacizumab (avastin; Genentech/Roche) against vascular endothelial growth fac-tor (VEGF) are now part of the treatment regimen for specific tumours.4,5 The anti-tumour activity of RTK-directed mAbs has been ascribed to different mechanisms: (1)

blockade of ligand-binding; (2) blockade of RTK homo- or hetero-dimerization; (3) in-terference with the active-like RTK conformation; (4) down-regulation of the receptor

1026 M. D. Castellone et alfrom the cell surface (receptor internalization); (5) shedding of the extracellulardomain of the receptor; or (6) antibody-dependent cell-mediated cytotoxicity(ADCC).4,5

Most RTK-directed small-molecule drugs are tyrosine kinase inhibitors (TKIs) and ob-struct kinase activity by binding to the ATP pocket of the kinase in competition withcellular ATP. The effectiveness of imatinib (imatinib mesylate/gleevec/glivec, STI571; No-vartis), an inhibitor of ABL, KITand platelet-derived growth factor receptor (PDGFR), inBCR-ABL-positive chronicmyelogenous leukaemia (CML) andKITor PDGFR-amutant gas-trointestinal stromal tumours (GISTs) has illustrated the power of this approach.4 TheATP binding site is highly conserved across the kinome. Thus, at best, TKIs may be se-lective but not specific and affect more than one RTK. As most cancers are the resultof a number of mutations, it is reasonable to suppose that TKIs able to target multiplekinases or a rational combination of TKIs will be clinically more effective than agentsblocking a single kinase. As discussed in the next paragraph, multi-targeting TKIs orcombination therapies may also attenuate resistance formation.

Resistance development is a critical issue in the use of TKIs in cancer treatment.Resistance is primarily mediated by the clonal expansion of cancer cells carrying sec-ondary mutations of the target kinase. These mutations either prevent the kinase fromadopting the conformation to which the compound binds or alter the compound con-tact point.4,5 Another important point in the clinical use of TKIs is target selection.Although cancer cells frequently contain mutations in multiple genes, they appear tobe highly dependent on specific genes and related pathways. This dependence canbe exploited therapeutically by appropriate targeting. Thus, the use of TKIs shouldbe restricted to those tumours that are addicted to the kinase that is targeted bythat specific agent. Finally, compensatory cross-talk between RTKs may attenuateTKI efficacy. Tumour cells become resistant to an EGFR TKI by an adaptation mecha-nism involving activation of alternative RTKs, such as METor PDGFR.14 Another studyshowed that tumour cells are resistant to EGFR TKI up front, because they simulta-neously activate the MET, EGFR and PDGFR RTKs.15 Cocktails of drugs or multi-targeting TKIs may be used to overcome these mechanisms.

RTK INHIBITORS IN THYROID CANCER

The last few months have witnessed the publication of the first results of clinical stud-ies based on the use of TKIs for thyroid cancer patients (www.clinicaltrials.gov). Theseare summarized in Table 1, and include imatinib mesylate1618, gefitinib (ZD1839/Ire-ssa; Astra Zeneca)19, axitinib (AG-013736; Pfizer)20, sorafenib (nexavar/BAY 43-9006;Bayer)21,22, and motesanib (motesanib diphosphate/AMG706; Amgen).23 Moreover,promising early results have emerged from studies with vandetanib (ZD6474/zactima;AstraZeneca)24, sunitinib (sutent/SU11248; Pfizer)2527, and XL184 (Exelixis).28 TheRTKs targeted by these agents are illustrated in Figure 2, and their involvement inthyroid cancer is discussed hereafter.

RET (GLIAL-DERIVED GROWTH FACTOR RECEPTOR)

RET (REarranged during Transfection) has been extensively reviewed elsewhere(Figure 2).6,7,29 RET is the receptor of glial-derived neurotrophic factor (GDNF)

Table 1. Receptor tyrosine kinase inhibitors in clinical trials in thyroid cancer.

RTK inhibitors in thyroid cancer 1027Name Other names Tyrosine kinase targets

(only those IC50< 1 mM)

Clinical development

Imatinib mesylate

(Novartis)

Gleevec, glivec,

STI571

ABL, KIT, PDGFR Approved for

CML, GIST

Gefitinib

(AstraZeneca)

ZD1839, iressa EGFR Approved in some

countries for NSCLC

Axitinib

(Pfizer)

AG-013736 VEGFR-1, -2, -3, PDGFR, KIT Investigational

Vandetanib

(AstraZeneca)

Zactima,

ZD6474

EGFR, RET, VEGFR-2, -3 Investigational

Sunitinib (Pfizer) Sutent,

SU11248

VEGFR-2, PDGFR, KIT, FLT3,

RET, FGFR-1, CSF-1R

Approved for metastatic

renal carcinoma and

imatinib-resistant GIST

Sorafenib

(Bayer)

Nexavar,

BAY 43-9006

RAF, BRAF, P38, VEGFR-1, -2, -3,

PDGFR, FLT3, RET, KIT, FGFR-1

Approved for metastatic

renal carcinoma

Motesanib

diphosphate

(Amgen)

AMG 706 VEGFR-1, -2, -3, KIT, RET,

PDGFR, FLT3

Investigational

XL184 (Exelixis) e RET, VEGFR-2, MET Investigational

Pazopanib GW-786034 VEGFR-1, -2, -3, PDGFR, Investigationalligands. In PTC, chromosomal inversions or translocations cause the fusion of the RETkinase-encoding domain with the 50-end of heterologous genes. The resulting chimericsequences are called RET/PTC.6,7 Germline point mutations in RET cause MEN-2(multiple endocrine neoplasia type 2) syndromes that predisposes to MTC. Somaticmutations of RET are also found in sporadic MTC.29 Transgenic mouse models dem-onstrated that REToncogenes are able to drive PTC and MTC formation.6 Moreover,RET knock-down by dominant-negative mutants or RNAi impairs proliferation ofMTC and PTC cell lines harbouring RET-derived oncogenes.30,31 In this scenarioRET appears to be a promising target for the molecular therapy of PTC and MTC.However, the heterogeneous distribution of RET mutations in some tumour sampleshas challenged the notion that RET alteration is the driving lesion in those individualpatients.32,33

Some of the TKIs in clinical experimentation in thyroid cancer patients have anti-RET activity (Table 1 and Figure 2). Most of them e.g. vandetanib34, sunitinib35, sor-afenib36, XL-18428, and motesanib37, inhibit both RETand VEGFRs. Thus, potentiallythese drugs can simultaneously attack both neoplastic and endothelial cells. X-raystructural analysis has demonstrated that vandetanib binds the ATP pocket of thekinase and is therefore an ATP-competitive RET TK inhibitor.38

(GlaxoSmithKline) KIT, FGFR-1

ABL, Abelson murine leukaemia viral (v-abl) oncogene homologue; PDGFR, platelet-derived growth fac-

tor receptor; KIT, stem-cell factor receptor; EGFR, epidermal growth factor receptor; VEGFR, vascular

endothelial growth factor receptor; FLT3, fms-related tyrosine kinase; RET, rearranged during transfec-

tion; FGFR, fibroblast growth factor receptor; MET, hepatocyte growth factor receptor; CSF-1R, colony-

stimulating factor receptor.

**CML chronic myelogenous leukaemia, GIST gastro intestinal stromal tumours; NSCLC nonsmall cell lung carcinoma.

1028 M. D. Castellone et alNTRK1 (NERVE GROWTH FACTOR RECEPTOR)

NTRK1 (also known as TRKA) belongs to the RTK neurotrophin receptor family thatincludes NTRK2 (TRKB) and NTRK3 (TRKC) (Figure 2).39 NTRK1 is the high-affinityreceptor for nerve growth factor. In 513% of PTC cases, the NTRK1 TK-encodingdomain is illegitimately recombined with heterologous sequences, thereby generatingTRK-T oncogenes.7,39 As yet there is no evidence that other members of the NTRKfamily are mutated in thyroid carcinoma.40 However, changes in their expressionwere involved in thyroid C-cell transformation, with NTRK2 being reduced andNTRK3 being up-regulated in MTC.41 The CEP-751 TKI, which is active againstboth RET and NTRK, had a cytostatic effect in MTC cells.42

MET (HEPATOCYTE GROWTH FACTOR RECEPTOR)

MET is a cell-surface receptor for hepatocyte growth factor (HGF, also known as scat-ter factor) (Figure 2).43 The MET homologue, RON, is the receptor for macrophage-stimulating protein (MSP). Amplification of the MET gene has been reported in severalhuman tumours; more rarely, MET carries activating point mutations. Most often, MET

Figure 2. Schematic representation of some receptor tyrosine kinases (RTKs). Specific domains of the ex-

tracellular portion are indicated: immunoglobulin-like, plexin and transcription factor domain (IPT); sema-

phorin domain (Sema); plexins, semaphorins, and integrins domain (Psi). Some RTKs have a tyrosine

kinase domain (TK) domain split by a peptide insert. Examples of agents monoclonal antibodies (mAbs)

or tyrosine kinase inhibitors (TKIs) able to obstruct RTK activity are indicated at the bottom (see text

for details).

is transcriptionally up-regulated in carcinomas. Since HGF is ubiquitously expressed,particularly in tumour stroma, this is believed to lead to constitutive MET activation.43

RTK inhibitors in thyroid cancer 1029Once activated, MET stimulates cell scattering, invasion, protection from apoptosisand anoikis, and angiogenesis.43

Approximately 70% of PTCs over-express MET, whereas normal thyrocytes do notexpress MET. HGF is locally produced by PTC stromal fibroblasts.44,45 METwas iden-tified as one of the most consistently up-regulated markers of thyroid cancer, partic-ularly in BRAF mutant PTC cases and in PTCs of the aggressive tall-cell variant.4649

Moreover, MET and HGF are co-expressed in a subset of MTCs.50 MET gene copygains have been identified in approximately 10% of ATCs and less frequently inFTCs,51 MET mutations are rare in thyroid cancer; a missense mutation, T1010I,was found in 6% of thyroid carcinomas and in germline DNA.52 Importantly, HGFwas identified as one of the most potent growth factors for cultured thyrocytes.53

The RON RTK was reported to be over-expressed in PTC and FTC, and its levels cor-related with advanced clinical stage.54

Monoclonal antibodies against HGF or the extracellular MET domain and METATP-competitive TKIs have been described.43 The TKI PF2341066 (Pfizer) is undergoingphase-I/II clinical trials, and the related compound, PHA665752, was effective againstPTC cells in vitro.55 Another two TKIs, XL880 and XL184 (Exelixis), are undergoingclinical experimentation. It is noteworthy that XL184, which is also a RET inhibitor, isbeing studied also in thyroid cancer patients (Table 1, Figure 2).28

EGFR (EPIDERMAL GROWTH FACTOR RECEPTOR)

The epidermal growth factor (EGF) receptor (EGFR, also named ErbB1 or HER1) be-longs to the ErbB/HER family of RTKs, which in addition includes ErbB2/Neu/HER2,ErbB3/HER3 and ErbB4/HER4.56,57 The four ErbBs share an overall structure of twocysteine-rich regions in their extracellular region and an intracellular TK domain(Figure 2). ErbB2, which does not have a direct ligand, and ErbB3, which is devoidof intrinsic kinase activity, are non-autonomous receptors and require heterodimeri-zation with other ErbB family members for activation and signalling. ErbB2 is themost potent oncoprotein in the ErbB family and it is the preferred heterodimeric part-ner of the other ErbBs. ErbB3 is a potent PI3K activator.56 The EGF family includesvarious ligands: EGF, transforming growth factor-a (TGF-a) and amphiregulin(AREG), which bind specifically to EGFR; b-cellulin (BTC), heparin-binding EGF(HB-EGF) and epiregulin (EREG), which bind EGFR and ErbB4; and neuregulins(NRGs), which bind either ErbB3/ErbB4 heterodimers or ErbB4 homodimers.56,57

Gene amplification of EGFR is often found in human cancers. In many tumours, EGF-related growth factors are produced either by the tumour cells or by stromal cells.56

In gliomas, EGFR amplification is often accompanied by structural rearrangements. So-matic mutations in the TK domain of EGFR are present in non-small-cell lung cancers(NSCLCs). Amplification of ErbB2 occurs in breast and other carcinomas. Mutations inthe kinase domain of ErbB2 occur in a small number of NSCLCs.4,5,56,57

Structural alterations in the EGFR gene are uncommon in thyroid cancer. No EGFRmutation (exons 1821) was found in 31 ATCs58, and only two mutations were de-tected in 62 thyroid cancers and 11 cell lines.59 In another study, no EGFR mutationwas found in 51 ATC and 64 FTC samples; however, EGFR gene copy gains were fre-quent in these tissues.51 Over-expression of EGFR in thyroid cancer is controversial:some studies report up-regulation in thyroid carcinomas, particularly in ATC58,60,61,whereas others report expression levels similar to those observed in normal

tissue.59,62 Growth factors of the EGF family (TGF-a, AREG, EREG)63 as well as ErbB2,ErbB3 and ErbB4 were found to be up-regulated in PTC61,64 and ATC.58 Importantly,

1030 M. D. Castellone et alEGF is mitogenic for thyrocytes, and long-term treatment with EGF induced gene ex-pression profiles similar to those of PTC samples.65 It was recently reported that RET/PTC oncogenes induce EGFR expression, and that EGFR and RET/PTC proteins forma complex that mediates EGFR-dependent RET phosphorylation.66

Four different anti-EGFR agents are approved in several countries for NSCLC andfor colorectal, pancreatic and head and neck carcinoma (Figure 2). These include twomAbs, cetuximab (a mouse/human chimeric mAb) and panitumumab (vectibix; Abge-nix) (a fully human mAb), and two TKIs, gefitinib and erlotinib (tarceva; Genentech).57

Trastuzumab (the humanized mAb targeting HER2) and lapatinib (GW572016/Tykerb;GlaxoSmithKline), a pan-ErbB TKI, have been approved for the treatment of HER2-over-expressing breast cancers.4,5,57

In preclinical studies, micromolar doses of gefitinib60,67 or NVP-AEE788 (Novartis),an EGFR and VEGFRTKI with additional activity against RET59,68,69, were effective in cul-tured ATC cells. NVP-AEE788 and cetuximab reduced the secretion of VEGF from thy-roid cancer cells, but only NVP-AEE788 dose-dependently inhibited proliferation.70 Incells expressing activated REToncogenes, NVP-AEE788 and PKI166 (Novartis), anotherEGFRTKI, were active at submicromolar concentrations, secondary to interaction be-tween EGFR and RET.66 No objective response was obtained in a phase-II study of gefi-tinib in patients with locally advanced or metastatic thyroid cancer, although a fraction ofpatients achieved prolonged stable disease and there was a reduction in tumour size andplasma thyroglobulin concentration. Therefore, EGFR inhibition is not sufficient, at leastby itself, to induce a major clinical response in thyroid cancer patients.19

FGFR (FIBROBLAST GROWTH FACTOR RECEPTOR)

The fibroblast growth factor (FGF) family currently includes more than 20 membersthat are important regulators of angiogenesis and tumourigenesis. FGFs signal throughfour RTKs (FGFR-1, -2, -3 and -4).7 Each receptor has two or three immunoglobulin-like extracellular domains, a transmembrane domain, and an intracellular TK(Figure 2).

Thus far, no mutations or rearrangements involving FGFRs have been identified inthyroid cancer.7 Expression of FGF1 and FGF2 (also known as basic FGF, a potent an-giogenic factor) is increased in thyroid cancer.7173 Increased expression of FGFR-1, -3and -4 has been observed in benign and malignant thyroid tumours.71,74 FGFR-2 ex-pression, instead, was down-regulated in thyroid cancer, and its re-expression in thy-roid carcinoma cells interrupted signalling upstream of BRAF and MAPK and reducedcell growth.75 FGFR-4 is expressed predominantly in aggressive thyroid tumour typesand MTC cells. Molecular targeting of FGFR-4 with the ATP-competitive inhibitorPD173074 (Pfizer) inhibited growth and reduced tumourigenesis of MTC cells.76 Itis noteworthy that sorafenib, sunitinib and pazopanib, which are undergoing clinical ex-perimentation in thyroid cancer patients, exert anti-FGFR activity (Table 1 andFigure 2).

IGF-1R (INSULIN-LIKE GROWTH FACTOR RECEPTOR 1)

IGF-1R (insulin-like growth factor receptor 1) is a ubiquitous transmembrane tyrosinekinase structurally similar to the insulin receptor (IR). IGF-1R is composed of two

extracellular a-subunits and two intracellular b-subunits (Figure 2). The a-subunitsbind ligands (IGF-I, IGF-II, and insulin at supraphysiological doses), whereas b-subunits

77

well as in endothelial cells.

RTK inhibitors in thyroid cancer 1031VEGF targeting is a promising anti-cancer therapeutic approach. VEGF-targetedtherapy acts through various mechanisms: inhibition of new vessel formation, apopto-sis of pre-existing vessels, blockade of endothelial cell progenitors, and vessel constric-tion (with reduced blood flow and ischaemia).81,82 There are currently more than 20different VEGF-targeted agents undergoing clinical evaluation (Figure 2). These includeneutralizing mAbs against VEGF or VEGFRs. Bevacizumab is a humanized mAb againstVEGF currently registered for colorectal cancer, breast cancer and NSCLC.4,5,81 Sev-eral multi-targeting TKIs that block VEGFRs have shown promising clinical activityagainst various solid tumours, and two of them (sorafenib and sunitinib) are registeredas anti-cancer agents.4,5,81 Unfortunately, in most cases the effects of anti-angiogenictreatment are only transitory. Various mechanisms have been evoked to explain thisphenomenon: production by cancer cells of angiogenic growth factors other thanVEGF (FGF2, PDGF, ephrins, angiopoietin, IL-8), recruitment of angiogeniccontain the TK domain. Most of the effects of IGF-I are mediated by IGF-1R. Theeffects of IGF-II may be mediated by both IGF-IR and an alternatively spliced variantof IR.

IGF-II and IGF-1R are over-expressed in many cancer types. High circulating levelsof IGF-I have been indicated as risk factors for various tumour types. Moreover, IGF-1R up-regulation was found to mediate resistance to TKIs in different types of cancercells. IGF-I and IGF-1R are over-expressed in thyroid cancer, particularly in the mostaggressive variants.78 Importantly, IGF-I and insulin are essential for the mitogenic ac-tion of TSH and EGF in thyroid follicular cells.79 Both anti-IGF-R mAbs and TKIs arebeing developed (Figure 2). The TKI NVP-ADW742 (Novartis) is cytotoxic for FTC-and MTC-derived cancer cells.80 IMC-A12 (ImClone) is a fully human mAb that targetsthe human IGF-1R. IMC-A12 was effective in treating an orthotopic mouse model ofATC.78

VEGFR (VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR)

The vascular endothelial growth factor (VEGF) family consists of five ligands: VEGFA,VEGFB, VEGFC, VEGFD and PGF (placenta growth factor).81 The best characterized isVEGFA (commonly referred to as VEGF). VEGFA and VEGFB are angiogenic proteins,whereas VEGFC and VEGFD are primarily lymphangiogenic proteins.81,82 The VEGFreceptors are VEGFR-1 (FLT-1), VEGFR-2 (KDR) and VEGFR-3 (FLT-4) (Figure 2).VEGFR-2 binds VEGFA and is expressed primarily on blood vessel endothelium.VEGFR-1 binds VEGFA, VEGFB and PGF and is expressed mainly in the vasculatureand also in other cell types. VEGFR-3 is the receptor for VEGFC and VEGFD and isexpressed primarily on lymphatic endothelium.81,82 However, it has been recentlydemonstrated that VEGFR-3 is also expressed in tumour blood vessels where it con-tributes to angiogenic sprouting, and simultaneous blockade of VEGFR-3 and VEGFR-2resulted in potent inhibition of tumour angiogenesis.83

Over-expression of VEGFA has been reported in thyroid carcinoma tissue73,8486

and plasma.87 In most85,87,88 but not all73,89 reports this was correlated with stage, tu-mour size and metastasis. Over-expression of VEGFC in PTC has been reported inthree studies85,90,91, whereas over-expression of VEGFC in MTC is controversial.85,91

Finally, expression of VEGFR-192 and VEGFR-285,93 has been detected in thyrocytes as

bone-marrow precursors, survival of blood vessels covered by pericytes, and migra-tion of cancer cells outside the primary tumour to co-opt pre-existing vessels.91

1032 M. D. Castellone et alImportantly, clinical trials with VEGFR-blocking multi-target TKIs (sorafenib,axitinib, motesanib, sunitinib, vandetanib and XL-184) have yielded promising resultsin thyroid cancer patients in the last few months (Table 1).2028 In addition, pazopanib(GW-786034; GlaxoSmithKline), a multi-targeted pan-VEGFR inhibitor, is undergoingclinical experimentation in thyroid cancer patients (www.clinicaltrials.gov).

Preclinical studies with NVP-AEE788, a dual VEGFR and EGFR inhibitor, in FTC andATC cells6870, and with PTK787/ZK222584 (Novartis and Schering), a pan-VEGFRTKI, in thyroid carcinoma mouse xenografts93 showed promising results. Cediranib(AZD2171; AstraZeneca), another pan-VEGFRTKI, inhibited tumour growth and pro-longed animal survival in an orthotopic nude mouse model of ATC.92 Finally, VEGFmAbs, including the murine version of bevacizumab, inhibited the growth of mousexenografts of thyroid cancer cells.94

PDGFR (PLATELED-DERIVED GROWTH FACTOR RECEPTOR)

The plateled-derived growth factor (PDGF) family consists of polypeptides PDGF-A, -B,-C and D that form homo- or more rarely hetero-dimers. PDGFs act via two RTKs(PDGFR-a and PDGFR-b) with common structures, including extracellular immuno-globulin (Ig) loops and a split intracellular TK domain (Figure 2).95 PDGF-AA andPDGF-CC ligands act via PDGFR-a, while PDGF-BB and probably PDGF-DD act viaPDGFR-b. PDGF-B is expressed mainly in vascular endothelial cells, megakaryocytesand neurons. PDGF-A and PDGF-C are expressed in epithelial cells, muscle, and neu-ronal progenitors. PDGFR-b is expressed in vascular smooth cells and pericytes,whereas PDGFR-a is expressed in mesenchymal cells.95,96

Amplification of PDGFs or PDGFRs genes is a frequent finding in glial tumours.A subset of GIST carries activating point mutations in PDGFR-a. Translocations ofthe PDGFR genes have been reported in myeloid disorders and leukaemias. Autocrineor paracrine loops involving PDGFs and their receptors are commonly observed insolid tumours.95,96 Autocrine PDGF loops are involved in tumours that originatefrom PDGFR-positive cells, such as tumours of glial origin and sarcomas. Autocrinesignalling may also play a role in carcinomas in conjunction with ectopic PDGFRexpression. Moreover, a paracrine PDGF loop is commonly observed in epithelialcancers. In fact, PDGF is expressed in the neoplastic component whereas PDGFR isexpressed in the stromal compartment. Tumour stroma contains a vascular part con-sisting of endothelial cells and associated mural cells (PDGFR-b-positive pericytes) anda fibrous part consisting of mesenchymal cells (PDGFR-a-positive tumour fibroblasts).Thus PDGF enhances pericyte recruitment to tumour vessels and recruits fibroblaststhat secrete angiogenic and tumour growth factors.95,96 PDGFR-b signalling could alsomediate the increase in interstitial fluid pressure that reduces drug uptake.96

No mutation was found in PDGFR-a gene in ATC; however, ATC, and to a lesserextent FTC, featured frequent PDGFR-b and PDGFR-a gene copy gains.51 Normalthyroid cells lack PDGFR, whereas FTC, PTC and ATC cancer cells over-expressPDGFR-b.58,97

Several PDGFRs TKIs have been developed. Imatinib, which inhibits both PDGFR-b and PDGFR-a, has been approved for the treatment of GIST carrying activatingmutations in PDGFR-a.4,5 Imatinib was poorly effective in cultured ATC and MTCcells,98,99 and had practically no effect in MTC patients, suggesting that at least inthis thyroid cancer type PDGFR inhibition is not sufficient to achieve a clinical

response.1618 Notably, some VEGFR-targeted TKIs inhibit the activity of PDGFRs(Table 1 and Figure 2), raising the possibility that these compounds, some of which

renchyma, have given promising results in thyroid cancer patients. It may be envis-aged that a more detailed understanding of the mechanism of action of thoseprotein kinases, such as RET, that already have a strong track record of involvementin thyroid oncogenesis, together with the systematic analysis of other genes of theRTK family by way of such post-genomic techniques as high-throughput sequencingand copy number analysis, as well as functional screens such as silencing by kinomeRNAi (RNA interference) libraries, will foster more research on RTK targeting forthe treatment of thyroid cancer. Meanwhile, clinical trials are of paramount impor-tance to address the efficacy of the RTK inhibitors and possible side-effects. Whencoupled with the genotyping of patients (identifying a mutation in the targeted ki-nase, such as RET for example) and measurement of surrogate markers of targetinhibition (for instance, RET phosphorylation in pre- and post-treatment tumour bi-opsies), clinical trials will also help to validate the RTK as a target for therapeuticintervention.

Research agenda

preclinical studies in appropriate cell system models are needed to character-

RTK inhibitors in thyroid cancer 1033ACKNOWLEDGEMENTS

We gratefully acknowledge members of our laboratory for continuous support. Weare grateful to Jean Ann Gilder for text editing and StudioCiotola for art-work. Wethank ISO (Istituto Superiore di Oncologia) and Nogec (Naples Oncogenomic Center)for support.

ize the activity of RTK inhibitors and to validate RTK targets preclinical studies in appropriate animal models (tissue-specific transgenic miceor nude mice xenografts) are needed to validate RTK targets and the activity ofthe compounds of interest

patients enrolled in clinical studies need to be genotyped for the compound(s)target(s) (example: RET mutation in patients treated with anti-RET agents)

studies, both preclinical and clinical, are needed to identify surrogate markers(for example, phosphorylation of the receptor by immunoblot or enzyme-linked immunosorbent assay; shedding of the receptor ectodomain) for targetinhibition

combinations of agents against different targets should be investigatedare undergoing clinical experimentation in patients affected by thyroid cancer, maymount a dual attack (VEGFR against endothelial cells and PDGFR against pericytes)on the tumour vasculature.

SUMMARY

Clinical trials with several agents that target RTKs, in either tumour stroma or pa-

CONFLICT OF INTEREST STATEMENT

1034 M. D. Castellone et alM. Santoro has received research funding support from AstraZeneca, Bayer andAmgen.

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1038 M. D. Castellone et al

Receptor tyrosine kinase inhibitors in thyroid cancerCancer genes coding for receptor tyrosine kinasesMechanisms of RTK activationMechanisms of RTK signallingRTKs as molecular targets for cancer treatmentRTK inhibitors in thyroid cancerRET (glial-derived growth factor receptor)NTRK1 (nerve growth factor receptor)MET (hepatocyte growth factor receptor)EGFR (epidermal growth factor receptor)FGFR (fibroblast growth factor receptor)IGF-1R (insulin-like growth factor receptor 1)VEGFR (vascular endothelial growth factor receptor)PDGFR (Plateled-derived growth factor receptor)SummaryAcknowledgementsConflict of interest statementReferences