Best Practice & Research Clinical Endocrinology & Metabolism

Vol. 22, No. 6, pp. 10231038, 2008

doi:10.1016/j.beem.2008.09.012
available online at http://www.sciencedirect.com

9
Receptor tyrosine kinase inhibitors in thyroid
cancer
Maria Domenica Castellone

MD, PhD

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

Francesca Carlomagno

MD, PhD

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, PhD

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

Massimo Santoro *

MD, PhD

Professor 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 tyrosine
kinases (RTKs) or their downstream signalling molecules. Hence, there is a strong biological rationale for assessing the efficacy of RTK blockade to treat patients who are resistant to or not
candidates for treatment with radioactive iodine. The first results of clinical trials based on the
use of RTK inhibitors in thyroid cancer patients have recently been published. Here we discuss
targeting of specific RTKs as a potential therapeutic strategy for the treatment of thyroid cancer.
Key words: kinase; thyroid; monoclonal antibody; small-molecule inhibitor.

* Corresponding author. Tel.: 39-081-7463056; Fax: 39-081-7463037.

E-mail address: masantor@unina.it (M. Santoro).
1521-690X/$ - see front matter 2008 Elsevier Ltd. All rights reserved.

1024 M. D. Castellone et al

CANCER GENES CODING FOR RECEPTOR TYROSINE KINASES

The protein kinase complement of the human genome (the kinome) contains 518
protein kinase genes, including 58 receptor tyrosine kinases (RTKs) and 32 nonRTKs (www.kinase.com/human/kinome). In high-throughput genome-sequencing projects, approximately 120 genes encoding protein kinases were found to be mutated
and 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 juxtamembrane
segment (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 point
mutations, cancers may feature illicit expression of the RTK or its cognate growth
factor and RTK gene rearrangements.35 Thyroid cancer, as discussed below, is often
associated to the oncogenic conversion of genes coding for RTKs or their signalling
effectors.6,7
MECHANISMS OF RTK ACTIVATION
Upon growth factor binding, RTKs undergo a dimerization process that activates
the enzymatic function.3 All kinase domains are divided into an amino-terminal

Figure 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 domain (JMR), and tyrosine kinase domain (TK), is illustrated. Blue boxes summarize major biological outcomes
of the activation of specific signalling components.

RTK inhibitors in thyroid cancer 1025

(N-terminal) and a carboxy-terminal (C-terminal) lobe. The active site is located in the
cleft between the N- and C-terminal lobes. Mg-ATP binds to the base of the cleft, the
non-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 site
can also bind small-molecule inhibitors (see below).4,5 In the inactive kinase, the active
site is closed by the activation segment of the C-terminal lobe. Growth-factor-binding
causes RTK dimerization, followed by extensive movements of the activation segment
and the JMR domain; this process allows ATP binding and catalysis.
Oncogenic mutations disrupt normal regulatory mechanisms and lead to constitutive activation of the kinase. Point mutations or rearrangements in the extracellular
domain mimic ligand-binding, thereby causing constitutive dimerization. Mutations in
the 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 recruits
intracellular proteins endowed with phosphotyrosine binding domains.35 Proximal
targets of the RTKs invoke the intracellular signalling cascades RAS-RAF-MAPK (the
ERK pathway) and the phosphatidylinositol 3-kinase (PI3K) AKT that ultimately
lead to diverse biological responses: i.e. mitogenesis, survival, differentiation and motility (Figure 1).35
Genes coding for proteins working in the RTK-initiated signal transduction pathways are frequently mutated in cancer. For instance, systematic DNA sequencing of
the best-annotated protein-coding genes in breast and colorectal cancer revealed
frequent mutations in the PI3K and nuclear factor kB (NFkB) pathways, both of which
are involved in RTK signalling (Figure 1).8 In the case of thyroid cancer, paradigmatic
examples of this concept are mutations in BRAF, a RAF family serine/threonine kinase,
in papillary thyroid carcinoma (PTC) (2969%) and PTC-derived anaplastic thyroid carcinoma (ATC) (1035%), and in the RAS small GTPase that is mutated in the follicular
variant 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 PI3K
catalytic subunit, is associated with ATC and FTC, and loss of expression of PTEN, the
major phosphatase antagonist of PI3K function, is frequent in ATC.9 Finally, functional
activation of NFkB has been described in ATC.10 NFkB is crucial for RET and BRAF
oncogene signalling in thyroid cancer cells11,12, and the proteasome inhibitor bortezomib (PS-341, Velcade, Millennium Pharmaceuticals), whose complex mechanism of
action 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 targeting a feasible therapeutic strategy for cancer.4,5 Monoclonal antibodies (mAbs) can
block the growth factor or the RTK itself. Some humanized mAbs such as trastuzumab (herceptin; Genentech/Roche) against the HER2/ErbB2 receptor, cetuximab (erbitux; ImClone) against the epidermal growth factor receptor (EGFR/HER1/ErbB1),
and bevacizumab (avastin; Genentech/Roche) against vascular endothelial growth factor (VEGF) are now part of the treatment regimen for specific tumours.4,5 The antitumour activity of RTK-directed mAbs has been ascribed to different mechanisms: (1)

1026 M. D. Castellone et al

blockade of ligand-binding; (2) blockade of RTK homo- or hetero-dimerization; (3) interference with the active-like RTK conformation; (4) down-regulation of the receptor
from the cell surface (receptor internalization); (5) shedding of the extracellular
domain 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 obstruct kinase activity by binding to the ATP pocket of the kinase in competition with
cellular ATP. The effectiveness of imatinib (imatinib mesylate/gleevec/glivec, STI571; Novartis), an inhibitor of ABL, KITand platelet-derived growth factor receptor (PDGFR), in
BCR-ABL-positive chronic myelogenous leukaemia (CML) and KITor PDGFR-a mutant gastrointestinal stromal tumours (GISTs) has illustrated the power of this approach.4 The
ATP binding site is highly conserved across the kinome. Thus, at best, TKIs may be selective but not specific and affect more than one RTK. As most cancers are the result
of a number of mutations, it is reasonable to suppose that TKIs able to target multiple
kinases or a rational combination of TKIs will be clinically more effective than agents
blocking a single kinase. As discussed in the next paragraph, multi-targeting TKIs or
combination 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 secondary mutations of the target kinase. These mutations either prevent the kinase from
adopting the conformation to which the compound binds or alter the compound contact 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 to
be highly dependent on specific genes and related pathways. This dependence can
be exploited therapeutically by appropriate targeting. Thus, the use of TKIs should
be restricted to those tumours that are addicted to the kinase that is targeted by
that specific agent. Finally, compensatory cross-talk between RTKs may attenuate
TKI efficacy. Tumour cells become resistant to an EGFR TKI by an adaptation mechanism involving activation of alternative RTKs, such as MET or PDGFR.14 Another study
showed that tumour cells are resistant to EGFR TKI up front, because they simultaneously activate the MET, EGFR and PDGFR RTKs.15 Cocktails of drugs or multitargeting 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 studies based on the use of TKIs for thyroid cancer patients (www.clinicaltrials.gov). These
are summarized in Table 1, and include imatinib mesylate1618, gefitinib (ZD1839/Iressa; 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 The
RTKs targeted by these agents are illustrated in Figure 2, and their involvement in
thyroid 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)

RTK inhibitors in thyroid cancer 1027

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

Name
Imatinib mesylate
(Novartis)
Gefitinib
(AstraZeneca)
Axitinib
(Pfizer)
Vandetanib
(AstraZeneca)
Sunitinib (Pfizer)

Sorafenib
(Bayer)
Motesanib
diphosphate
(Amgen)
XL184 (Exelixis)
Pazopanib
(GlaxoSmithKline)

Other names

Tyrosine kinase targets

(only those IC50 < 1 mM)
ABL, KIT, PDGFR

Clinical development

Gleevec, glivec,
STI571
ZD1839, iressa

EGFR

AG-013736

VEGFR-1, -2, -3, PDGFR, KIT

Approved for
CML, GIST
Approved in some
countries for NSCLC
Investigational

Zactima,
ZD6474
Sutent,
SU11248

EGFR, RET, VEGFR-2, -3

Investigational

VEGFR-2, PDGFR, KIT, FLT3,

RET, FGFR-1, CSF-1R

Nexavar,
BAY 43-9006
AMG 706

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

PDGFR, FLT3, RET, KIT, FGFR-1
VEGFR-1, -2, -3, KIT, RET,
PDGFR, FLT3

Approved for metastatic

renal carcinoma and
imatinib-resistant GIST
Approved for metastatic
renal carcinoma
Investigational

e
GW-786034

RET, VEGFR-2, MET

VEGFR-1, -2, -3, PDGFR,
KIT, FGFR-1

Investigational
Investigational

ABL, Abelson murine leukaemia viral (v-abl) oncogene homologue; PDGFR, platelet-derived growth factor 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 transfection; FGFR, fibroblast growth factor receptor; MET, hepatocyte growth factor receptor; CSF-1R, colonystimulating factor receptor.
**CML chronic myelogenous leukaemia, GIST gastro intestinal stromal tumours; NSCLC non
small cell lung carcinoma.

ligands. In PTC, chromosomal inversions or translocations cause the fusion of the RET
kinase-encoding domain with the 50 -end of heterologous genes. The resulting chimeric
sequences are called RET/PTC.6,7 Germline point mutations in RET cause MEN-2
(multiple endocrine neoplasia type 2) syndromes that predisposes to MTC. Somatic
mutations of RET are also found in sporadic MTC.29 Transgenic mouse models demonstrated that RET oncogenes are able to drive PTC and MTC formation.6 Moreover,
RET knock-down by dominant-negative mutants or RNAi impairs proliferation of
MTC and PTC cell lines harbouring RET-derived oncogenes.30,31 In this scenario
RET appears to be a promising target for the molecular therapy of PTC and MTC.
However, the heterogeneous distribution of RET mutations in some tumour samples
has challenged the notion that RET alteration is the driving lesion in those individual
patients.32,33
Some of the TKIs in clinical experimentation in thyroid cancer patients have antiRET activity (Table 1 and Figure 2). Most of them e.g. vandetanib34, sunitinib35, sorafenib36, XL-18428, and motesanib37, inhibit both RET and VEGFRs. Thus, potentially
these drugs can simultaneously attack both neoplastic and endothelial cells. X-ray
structural analysis has demonstrated that vandetanib binds the ATP pocket of the
kinase and is therefore an ATP-competitive RET TK inhibitor.38

1028 M. D. Castellone et al

Figure 2. Schematic representation of some receptor tyrosine kinases (RTKs). Specific domains of the extracellular portion are indicated: immunoglobulin-like, plexin and transcription factor domain (IPT); semaphorin 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).

NTRK1 (NERVE GROWTH FACTOR RECEPTOR)

NTRK1 (also known as TRKA) belongs to the RTK neurotrophin receptor family that
includes NTRK2 (TRKB) and NTRK3 (TRKC) (Figure 2).39 NTRK1 is the high-affinity
receptor for nerve growth factor. In 513% of PTC cases, the NTRK1 TK-encoding
domain is illegitimately recombined with heterologous sequences, thereby generating
TRK-T oncogenes.7,39 As yet there is no evidence that other members of the NTRK
family are mutated in thyroid carcinoma.40 However, changes in their expression
were involved in thyroid C-cell transformation, with NTRK2 being reduced and
NTRK3 being up-regulated in MTC.41 The CEP-751 TKI, which is active against
both 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 scatter factor) (Figure 2).43 The MET homologue, RON, is the receptor for macrophagestimulating protein (MSP). Amplification of the MET gene has been reported in several
human tumours; more rarely, MET carries activating point mutations. Most often, MET

RTK inhibitors in thyroid cancer 1029

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
Once activated, MET stimulates cell scattering, invasion, protection from apoptosis
and anoikis, and angiogenesis.43
Approximately 70% of PTCs over-express MET, whereas normal thyrocytes do not
express MET. HGF is locally produced by PTC stromal fibroblasts.44,45 MET was identified as one of the most consistently up-regulated markers of thyroid cancer, particularly 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 copy
gains have been identified in approximately 10% of ATCs and less frequently in
FTCs,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, HGF
was 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 correlated with advanced clinical stage.54
Monoclonal antibodies against HGF or the extracellular MET domain and MET ATPcompetitive TKIs have been described.43 The TKI PF2341066 (Pfizer) is undergoing
phase-I/II clinical trials, and the related compound, PHA665752, was effective against
PTC cells in vitro.55 Another two TKIs, XL880 and XL184 (Exelixis), are undergoing
clinical experimentation. It is noteworthy that XL184, which is also a RET inhibitor, is
being 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) belongs 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 two
cysteine-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 devoid
of intrinsic kinase activity, are non-autonomous receptors and require heterodimerization with other ErbB family members for activation and signalling. ErbB2 is the
most potent oncoprotein in the ErbB family and it is the preferred heterodimeric partner of the other ErbBs. ErbB3 is a potent PI3K activator.56 The EGF family includes
various 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, EGFrelated growth factors are produced either by the tumour cells or by stromal cells.56
In gliomas, EGFR amplification is often accompanied by structural rearrangements. Somatic 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 in
the 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 EGFR
mutation (exons 1821) was found in 31 ATCs58, and only two mutations were detected in 62 thyroid cancers and 11 cell lines.59 In another study, no EGFR mutation
was found in 51 ATC and 64 FTC samples; however, EGFR gene copy gains were frequent 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

1030 M. D. Castellone et al

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,
EGF is mitogenic for thyrocytes, and long-term treatment with EGF induced gene expression 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 form
a complex that mediates EGFR-dependent RET phosphorylation.66
Four different anti-EGFR agents are approved in several countries for NSCLC and
for colorectal, pancreatic and head and neck carcinoma (Figure 2). These include two
mAbs, cetuximab (a mouse/human chimeric mAb) and panitumumab (vectibix; Abgenix) (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 HER2over-expressing breast cancers.4,5,57
In preclinical studies, micromolar doses of gefitinib60,67 or NVP-AEE788 (Novartis),
an EGFR and VEGFR TKI with additional activity against RET59,68,69, were effective in cultured ATC cells. NVP-AEE788 and cetuximab reduced the secretion of VEGF from thyroid cancer cells, but only NVP-AEE788 dose-dependently inhibited proliferation.70 In
cells expressing activated REToncogenes, NVP-AEE788 and PKI166 (Novartis), another
EGFR TKI, were active at submicromolar concentrations, secondary to interaction between EGFR and RET.66 No objective response was obtained in a phase-II study of gefitinib in patients with locally advanced or metastatic thyroid cancer, although a fraction of
patients achieved prolonged stable disease and there was a reduction in tumour size and
plasma thyroglobulin concentration. Therefore, EGFR inhibition is not sufficient, at least
by 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 members
that are important regulators of angiogenesis and tumourigenesis. FGFs signal through
four RTKs (FGFR-1, -2, -3 and -4).7 Each receptor has two or three immunoglobulinlike extracellular domains, a transmembrane domain, and an intracellular TK
(Figure 2).
Thus far, no mutations or rearrangements involving FGFRs have been identified in
thyroid cancer.7 Expression of FGF1 and FGF2 (also known as basic FGF, a potent angiogenic factor) is increased in thyroid cancer.7173 Increased expression of FGFR-1, -3
and -4 has been observed in benign and malignant thyroid tumours.71,74 FGFR-2 expression, instead, was down-regulated in thyroid cancer, and its re-expression in thyroid carcinoma cells interrupted signalling upstream of BRAF and MAPK and reduced
cell growth.75 FGFR-4 is expressed predominantly in aggressive thyroid tumour types
and MTC cells. Molecular targeting of FGFR-4 with the ATP-competitive inhibitor
PD173074 (Pfizer) inhibited growth and reduced tumourigenesis of MTC cells.76 It
is noteworthy that sorafenib, sunitinib and pazopanib, which are undergoing clinical experimentation in thyroid cancer patients, exert anti-FGFR activity (Table 1 and
Figure 2).
IGF-1R (INSULIN-LIKE GROWTH FACTOR RECEPTOR 1)
IGF-1R (insulin-like growth factor receptor 1) is a ubiquitous transmembrane tyrosine
kinase structurally similar to the insulin receptor (IR). IGF-1R is composed of two

RTK inhibitors in thyroid cancer 1031

extracellular a-subunits and two intracellular b-subunits (Figure 2). The a-subunits
bind ligands (IGF-I, IGF-II, and insulin at supraphysiological doses), whereas b-subunits
contain the TK domain.77 Most of the effects of IGF-I are mediated by IGF-1R. The
effects of IGF-II may be mediated by both IGF-IR and an alternatively spliced variant
of IR.
IGF-II and IGF-1R are over-expressed in many cancer types. High circulating levels
of IGF-I have been indicated as risk factors for various tumour types. Moreover, IGF1R up-regulation was found to mediate resistance to TKIs in different types of cancer
cells. IGF-I and IGF-1R are over-expressed in thyroid cancer, particularly in the most
aggressive variants.78 Importantly, IGF-I and insulin are essential for the mitogenic action of TSH and EGF in thyroid follicular cells.79 Both anti-IGF-R mAbs and TKIs are
being developed (Figure 2). The TKI NVP-ADW742 (Novartis) is cytotoxic for FTCand MTC-derived cancer cells.80 IMC-A12 (ImClone) is a fully human mAb that targets
the human IGF-1R. IMC-A12 was effective in treating an orthotopic mouse model of
ATC.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 is
VEGFA (commonly referred to as VEGF). VEGFA and VEGFB are angiogenic proteins,
whereas VEGFC and VEGFD are primarily lymphangiogenic proteins.81,82 The VEGF
receptors 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 vasculature
and also in other cell types. VEGFR-3 is the receptor for VEGFC and VEGFD and is
expressed primarily on lymphatic endothelium.81,82 However, it has been recently
demonstrated that VEGFR-3 is also expressed in tumour blood vessels where it contributes to angiogenic sprouting, and simultaneous blockade of VEGFR-3 and VEGFR-2
resulted 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, tumour size and metastasis. Over-expression of VEGFC in PTC has been reported in
three 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
well as in endothelial cells.
VEGF targeting is a promising anti-cancer therapeutic approach. VEGF-targeted
therapy acts through various mechanisms: inhibition of new vessel formation, apoptosis of pre-existing vessels, blockade of endothelial cell progenitors, and vessel constriction (with reduced blood flow and ischaemia).81,82 There are currently more than 20
different VEGF-targeted agents undergoing clinical evaluation (Figure 2). These include
neutralizing mAbs against VEGF or VEGFRs. Bevacizumab is a humanized mAb against
VEGF currently registered for colorectal cancer, breast cancer and NSCLC.4,5,81 Several multi-targeting TKIs that block VEGFRs have shown promising clinical activity
against various solid tumours, and two of them (sorafenib and sunitinib) are registered
as anti-cancer agents.4,5,81 Unfortunately, in most cases the effects of anti-angiogenic
treatment are only transitory. Various mechanisms have been evoked to explain this
phenomenon: production by cancer cells of angiogenic growth factors other than
VEGF (FGF2, PDGF, ephrins, angiopoietin, IL-8), recruitment of angiogenic

1032 M. D. Castellone et al

bone-marrow precursors, survival of blood vessels covered by pericytes, and migration of cancer cells outside the primary tumour to co-opt pre-existing vessels.91
Importantly, clinical trials with VEGFR-blocking multi-target TKIs (sorafenib,
axitinib, motesanib, sunitinib, vandetanib and XL-184) have yielded promising results
in thyroid cancer patients in the last few months (Table 1).2028 In addition, pazopanib
(GW-786034; GlaxoSmithKline), a multi-targeted pan-VEGFR inhibitor, is undergoing
clinical experimentation in thyroid cancer patients (www.clinicaltrials.gov).
Preclinical studies with NVP-AEE788, a dual VEGFR and EGFR inhibitor, in FTC and
ATC cells6870, and with PTK787/ZK222584 (Novartis and Schering), a pan-VEGFR
TKI, in thyroid carcinoma mouse xenografts93 showed promising results. Cediranib
(AZD2171; AstraZeneca), another pan-VEGFR TKI, inhibited tumour growth and prolonged animal survival in an orthotopic nude mouse model of ATC.92 Finally, VEGF
mAbs, including the murine version of bevacizumab, inhibited the growth of mouse
xenografts 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 immunoglobulin (Ig) loops and a split intracellular TK domain (Figure 2).95 PDGF-AA and
PDGF-CC ligands act via PDGFR-a, while PDGF-BB and probably PDGF-DD act via
PDGFR-b. PDGF-B is expressed mainly in vascular endothelial cells, megakaryocytes
and neurons. PDGF-A and PDGF-C are expressed in epithelial cells, muscle, and neuronal 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 of
the PDGFR genes have been reported in myeloid disorders and leukaemias. Autocrine
or paracrine loops involving PDGFs and their receptors are commonly observed in
solid tumours.95,96 Autocrine PDGF loops are involved in tumours that originate
from PDGFR-positive cells, such as tumours of glial origin and sarcomas. Autocrine
signalling may also play a role in carcinomas in conjunction with ectopic PDGFR
expression. Moreover, a paracrine PDGF loop is commonly observed in epithelial
cancers. In fact, PDGF is expressed in the neoplastic component whereas PDGFR is
expressed in the stromal compartment. Tumour stroma contains a vascular part consisting of endothelial cells and associated mural cells (PDGFR-b-positive pericytes) and
a fibrous part consisting of mesenchymal cells (PDGFR-a-positive tumour fibroblasts).
Thus PDGF enhances pericyte recruitment to tumour vessels and recruits fibroblasts
that secrete angiogenic and tumour growth factors.95,96 PDGFR-b signalling could also
mediate 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 lesser
extent FTC, featured frequent PDGFR-b and PDGFR-a gene copy gains.51 Normal
thyroid cells lack PDGFR, whereas FTC, PTC and ATC cancer cells over-express
PDGFR-b.58,97
Several PDGFRs TKIs have been developed. Imatinib, which inhibits both PDGFRb and PDGFR-a, has been approved for the treatment of GIST carrying activating
mutations in PDGFR-a.4,5 Imatinib was poorly effective in cultured ATC and MTC
cells,98,99 and had practically no effect in MTC patients, suggesting that at least in
this thyroid cancer type PDGFR inhibition is not sufficient to achieve a clinical

RTK inhibitors in thyroid cancer 1033

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
are undergoing clinical experimentation in patients affected by thyroid cancer, may
mount 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 parenchyma, have given promising results in thyroid cancer patients. It may be envisaged that a more detailed understanding of the mechanism of action of those
protein kinases, such as RET, that already have a strong track record of involvement
in thyroid oncogenesis, together with the systematic analysis of other genes of the
RTK family by way of such post-genomic techniques as high-throughput sequencing
and copy number analysis, as well as functional screens such as silencing by kinome
RNAi (RNA interference) libraries, will foster more research on RTK targeting for
the treatment of thyroid cancer. Meanwhile, clinical trials are of paramount importance to address the efficacy of the RTK inhibitors and possible side-effects. When
coupled with the genotyping of patients (identifying a mutation in the targeted kinase, such as RET for example) and measurement of surrogate markers of target
inhibition (for instance, RET phosphorylation in pre- and post-treatment tumour biopsies), clinical trials will also help to validate the RTK as a target for therapeutic
intervention.

Research agenda
 preclinical studies in appropriate cell system models are needed to characterize the activity of RTK inhibitors and to validate RTK targets
 preclinical studies in appropriate animal models (tissue-specific transgenic mice
or nude mice xenografts) are needed to validate RTK targets and the activity of
the 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 enzymelinked immunosorbent assay; shedding of the receptor ectodomain) for target
inhibition
 combinations of agents against different targets should be investigated

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

1034 M. D. Castellone et al

CONFLICT OF INTEREST STATEMENT

M. Santoro has received research funding support from AstraZeneca, Bayer and
Amgen.
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