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REVIEWEndocrine-Related Cancer (2007) 14 957–977

New insights in thyroid follicular cellbiology and its impact in thyroid cancertherapy

Garcilaso Riesco-Eizaguirre1,2 and Pilar Santisteban1

1Instituto de Investigaciones Biomedicas ‘Alberto Sols’, Consejo Superior de Investigaciones Cientıficas y Universidad Autonoma de

Madrid (CSIC-UAM), 28029 Madrid, Spain2Servicio de Endocrinologıa y Nutricion, Hospital Universitario La Paz, Madrid, Spain

(Correspondence should be addressed to P Santisteban; Email: [email protected])

Abstract

Well-differentiated thyroid cancer has in general terms a very good outcome. It has a very slowgrowth rate and, although it metastasises at a relatively high frequency, it has very high survivalrates. Whereas the prevalence of nodular thyroid disease worldwide is high, malignant conversionfrom benign thyroid nodules is rare. Treatment of thyroid cancer is usually successful, but we stilldo not have effective therapies for patients with invasive or metastatic thyroid cancer if the diseasedoes not concentrate radioiodine and it is not surgically resectable. On the other hand, from thesame thyroid cell, one of the most aggressive human tumours can arise – undifferentiated oranaplastic thyroid carcinoma – leading to death in a few months. What features of this malignancyaccount for such paradoxical behaviour? The most common type of thyroid cancer – papillarythyroid carcinoma – stands out among solid tumours because many of the tumour-initiating eventshave been identified. All of them function in a single pathway – the RTK/RAS/RAF/MAPK pathway– and obey an ‘exclusivity principle’: one and only one component of the pathway is mutated in asingle tumour. This highlights the requirement of this signal transduction pathway for thetransformation to thyroid cancer and paves the way to targeted therapies against a tumour with amutation in a known gene or any gene upstream of the target. However, it is also interesting tounderscore the differences among the tumours arising from the different mutations. Studies in vitroand in vivo, including genomic profiling and genetically engineered mouse models, have clearlyshown that each oncoprotein exerts its own oncogenic drive, conferring a distinct biologicalbehaviour on thyroid tumours. In this review, we attempt to summarise the most recent advancesin thyroid follicular cell-derived cancers research and their potential clinical impact that maychange the management of thyroid cancer in the near future.

Endocrine-Related Cancer (2007) 14 957–977

The disease

Thyroid cancer incidence has increased significantly

during the past decades (Davies & Welch 2006) and it

has become one of the ten leading cancer types in

females, accounting for 22 590 new cases per year in

the United States; it is more frequent than ovarian,

urinary bladder or pancreas cancer (Jemal et al. 2007).

As diagnostic techniques for thyroid cancer have

become more sensitive, particularly with the advent

of ultrasound and fine needle aspiration (FNAS), the

increasing incidence of thyroid cancer is predomi-

nantly due to the increased detection of small papillary

cancers or microcarcinomas. Additionally, the 5-year


1351–0088/07/014–957 q 2007 Society for Endocrinology Printed in Great

relative survival rates for thyroid cancer have increased

significantly from 93% in 1983–1985 to 97% in

1995–2001, probably due to the same reason (Jemal

et al. 2007). In this review, we will focus mainly on the

thyroid follicular cell-derived cancers.

Thyroid cancer management has not changed

substantially during the past decades. Treatment is

based on total thyroidectomy, ablative doses of

radioiodine and suppressive treatment. The follow-up

is based on measuring serum thyroglobulin (Tg) and

imaging with radioiodine scans. Since among clini-

cians there is a tendency to overtreating patients with

thyroid cancer, recently new proposals to change this

Britain

DOI:10.1677/ERC-07-0085

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G Riesco-Eizaguirre and P Santisteban: New insights in thyroid cancer

algorithm have arisen (Pacini et al. 2006). Most likely,

the 2.4-fold increase in incidence of papillary

microcarcinoma has also something to do with this. It

is a fact that a more accurate prognostic stratification

that improves thyroid cancer management is lacking

(Udelsman & Shaha 2005).

There are four main critical challenges that remain

unsolved in thyroid cancer management concerning

diagnosis, prognosis and therapeutic management.

First, 15–20% of the FNAS are inconclusive or cannot

discriminate between follicular adenoma and carci-

noma. This means that the patient needs to undergo

partial or total thyroidectomy only for diagnostic

purposes. Moreover, a sequential thyroidectomy has

to be performed in the case of follicular thyroid

carcinoma (FTC). Second, it has been estimated that

about 20% of the patients with well-differentiated

thyroid carcinoma will develop a local or distant

recurrence and 1% will die (Schlumberger & Pacini

2003). Identifying these high-risk patients at the time

of diagnosis through well-established prognostic

factors can help to ascertain the most appropriate

treatment and follow-up for these patients. Although

several prognostic scoring systems, like the tumour

node metastasis classification (AJCC 2002), have been

developed for thyroid cancer, unfortunately there is

still a debate on the definition and best management of

low- versus high-risk patients, and each centre has its

own protocols (Schlumberger & Pacini 2003). Third,

there is still no treatment for one of the most intriguing

situations that an endocrinologist may have to face:

patients with elevated serum Tg and negative 131I

scans. This is challenging for thyroid cancer manage-

ment, as anatomical localisation of recurrences cannot

be assessed and treatment with ablative doses of 131I is

not effective, predicting a poorer outcome. Fourth,

anaplastic thyroid carcinoma, although rare, is extre-

mely aggressive, leading to death in 100% of the cases

in a few months.

It is worth pointing out that about 500–600 million

people suffer nodular goitre worldwide, and 4–6% of

American adults are goitrous despite normal iodine

intake (Freitas 2000). Thyrocyte proliferation leading

to single nodule or multinodular goitre is diagnosed

every day, even in children, and thyroid nodules are

detected in more than 50% of autopsies (Derwahl &

Studer 2000). These figures are quite high and,

although malignancies are only present in 5% of all

the thyroid nodules, there is a need for accurate and

sensitive techniques that will help clinicians to

discriminate among the vast amount of thyroid nodules

diagnosed every day.

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Beyond this clinical pathological picture, thyroid

cancer biology has unique characteristics compared

with other human tumours that we must bear in mind.

First, there is a surprisingly low frequency of malignant

conversions, given the high incidence of benign

tumours in the normal population. Moreover, no

premalignant carcinomas have been identified for the

most frequent type of thyroid cancer, papillary thyroid

carcinoma (PTC). Second, the very slow growth rates

of well-differentiated thyroid carcinomas indepen-

dently of the evaluation method used, contrasts with

the much faster growth rates of well-differentiated

breast, lung, colon and stomach adenocarcinomas

(Soares & Sobrinho-Simoes 1994, Katoh et al. 1995,

Sreelekha et al. 2000). Third, well-differentiated

thyroid carcinomas show little or no apoptosis

(Moore et al. 1998, Yoshida et al. 1999, Sreelekha

et al. 2000). Fourth, even though survival rates are

high, well-differentiated thyroid carcinomas metasta-

sise at a relatively high frequency (Schlumberger &

Pacini 2003). The purpose of this review is to

summarise the most relevant advances in thyroid

cancer research and how these new insights will

impact on therapeutic options in the near future.

The genetics: cancer genes and thepathways they control

Cancer is, in essence, a genetic disease. Many genes that

have a causal role in cancer have been discovered and the

delineation of the pathways through which they act

characterised (Vogelstein & Kinzler 2004). Focusing in

pathways rather than individual genes is based on the fact

that there are always a variety of genes that, when altered,

lead to similar phenotypes. This concept is very familiar

to genetists and as there are many fewer pathways than

cancer genes, application of this concept to cancer has

been established in the past years by elucidation of

biochemical functions of the altered cancer genes, either

in cell culture systems or in mice.

The RTK /RAS /RAF /MAPK pathway

The most studied pathway involved in thyroid tumor-

igenesis is the RTK/RAS/BRAF/MAP kinase pathway,

which seems to be essential for the development of PTC

(Fig. 1). On the contrary, this pathway seems to play a

more limited role in FTC. Accordingly, the main genetic

events discovered so far related to PTC play important

roles in this pathway and, as inmany other cancers, these

genetic events do not overlap, which highlights the

requirement of this signalling system for transformation

to PTC. However, although all the aforementioned

www.endocrinology-journals.org


Figure 1 Thyroid cancer genes (black) and the pathways they control. Mutually exclusive mutations of either RET/PTC, RAS orBRAF provide compelling genetic evidence for the critical role of the MAPK pathway. Additionally, recent studies point to animportant role of the PI3K/AKT pathway in thyroid tumorigenesis. Adapted from Fagin 2004.


genetic events activate this pathway, notable differences

can be found among them.

MAPKs regulate critical cellular functions required

for homeostasis such as the expression of cytokines

and proteases, cell cycle progression, cell adherence,

motility and metabolism. MAPKs therefore influence

cell proliferation, differentiation, survival, apoptosis

and development; and not surprisingly, they also

control the growth and survival of a broad spectrum

of human tumours. Most studies exploring the effects

of oncogenes have been performed in cells in whose

growth is negatively regulated by cAMP. Thyroid cells

are an exception, since they are dependent on the

presence of thyrotrophin (TSH) for growth, primarily

through activation of adenylyl cyclase, cAMP gener-

ation and stimulation of protein kinase A activity

(Medina & Santisteban 2000). Furthermore, in contrast

with other cancers (Dorsam & Gutkind 2007),

activating mutations of the main G-protein-coupled

receptor of the thyroid, the TSH receptor or its Gsa

subunit, do not lead to malignancy, but to toxic benign


nodules. This confers a unique trait on thyroid cells

that, as we will see below, may serve as a protective

mechanism for tumour initiation in the thyroid.

RAS family genes

RAS oncogenes were the first to be associated with

thyroid cancer. TheRAS protooncogenes encode 21 kDa

G-proteins, which transduce signals from a wide variety

of growth factor receptors, particularly those of the

tyrosine kinase family. Point mutations affecting the

guanosine triphosphate (GTP)-binding domain

(codon 12/13) or the GTPase domain (codon 61) result

in the replacement of specific amino acid residues that

lock p21RAS in the active form, resulting in constitutive

activation of the protein and tumour development.

About 30% of all human tumours contain a mutation

in a RAS allele, making this one of the most widely

mutated human protooncogenes (Bos 1989, McCormick

& Wittinghofer 1996).

Oncogenic mutations of H-RAS, K-RAS and

N-RAS were among the first genetic changes to be

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identified in tumours originating from the thyroid

follicular epithelium, and numerous reports have

documented their occurrence in many different types

of thyroid tumours. Mutations of all three RAS genes

are found in benign and malignant follicular neoplasms

and in follicular variant PTC, and are believed to be

one of the early steps in thyroid tumour formation

(Lemoine et al. 1988, Namba et al. 1990). There are

significant discrepancies related to the overall fre-

quency of RAS mutations (ranging from 7 to 62%) and

their prevalence in specific thyroid tumours (Wright

et al. 1989, Suarez et al. 1990, Challeton et al. 1995).

No consistent relationship between tumour histotype or

biological behaviour and a particular pattern of RAS

activation can be inferred from a review of the

literature. In the largest series so far analysed, RAS

mutations were not very common in follicular

adenomas or in well-differentiated carcinomas (follicu-

lar or papillary) ranging from 5 to 10%. However, RAS

mutations are more prevalent in poorly differentiated

(55%) and anaplastic carcinoma (52%; Garcia-Rostan

et al. 2003). Moreover, this group found a significant

association between RAS mutations and poor survival,

proposing RAS mutations as a marker for aggressive

thyroid cancer behaviour.

As mutations in RAS are thought to be among the

initiating molecular events in thyroid tumorigenesis, an

inducible expression system in rat thyroid cells

represents an excellent model to investigate the early

biological events that take place after aberrant

activation of RAS. Remarkably, conditional expression

of H-RASV12 in rat thyroid cells induces strong

apoptosis. This is surprising, because RAS inhibits

apoptosis as part of its role in promoting expansion of

the neoplastic clone in epithelial (Rak et al. 1995,

Khwaja et al. 1997) and myeloid cells (Kinoshita et al.

1997), whereas in T lymphocytes and fibroblasts

constitutive RAS activity can induce cell death (Chen

et al. 1998). However, an important consideration

should be taken into account. In the presence of TSH,

H-RASV12 induces a transient increase in cell

proliferation but later inhibits TSH-mediated cell

growth via initiation of programmed cell death.

These effects are mediated via the ERK and JNK

signal transduction pathways and are only observed

with concomitant stimulation of the cAMP signalling

cascade (Shirokawa et al. 2000). By contrast, in the

absence of TSH, acute expression of H-RASV12

does inhibit apoptosis and, thus, accelerates cell

proliferation (TSH-independent growth). This means

that the fate of thyrocytes within the first cell cycles

after expression of oncogenic RAS is dependent on

TSH levels. Those cells that loose TSH responsiveness

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and/or inactivate the apoptotic cascade through

secondary events will undergo clonal expansion.

RAS activation also displays evidence of DNA

damage, manifesting as chromosome misalignment in

mitosis, micronuclei formation and centrosome

amplification (Saavedra et al. 2000). Interestingly,

the same authors also demonstrated that DNA damage

is not the only one responsible for activating apoptosis

after acute RAS expression. Finally, RAS also induces

dedifferentiation in a dose-dependent manner, and this

is an early phenomenon. Although TSH-independent

growth appears to be induced in the presence of both

low and high levels of oncogenic RAS expression, only

high levels of RAS expression are able to inhibit the

activity of thyroid-specific genes, including TTF1 and

PAX8, two transcription factors essential for mainten-

ance of the thyroid differentiated state (De Vita et al.

2005). Additionally, the RAF /MEK / ERK cascade

may act in concert with an as yet uncharacterised

signalling pathway to repress TTF1 function and

ultimately to inhibit thyroid cell differentiation.

Studies in vivo with transgenic mice have also given

valuable information. A transgenic mouse line in which

a human N-RAS (Gln61Lys) oncogene was expressed

in thyroid follicular cells under control of the Tg

promoter (Tg-N-RAS) was developed by Santoro’s

group. Significantly, Tg-N-RAS mice developed

thyroid follicular neoplasms; 11% developed follicular

adenomas and w40% developed invasive follicular

carcinomas, in some cases with a mixed papillary/folli-

cular morphology. About 25% of the Tg-N-RAS

carcinomas displayed large, poorly differentiated areas,

featuring vascular invasion and forming distant metas-

tases in lung, bone or liver (Vitagliano et al. 2006).

In conclusion, RAS mutations are not restricted to a

specific thyroid tumour type, and are present in follicular

adenoma, follicular carcinoma, follicular variant of PTC

and at a high frequency in poorly differentiated and

anaplastic carcinomas. RAS mediates TSH-independent

growth, TSH-dependent apoptosis, dedifferentiation and

DNA damage leading to genomic instability, mainly

through the RAF/MEK/ERK pathway. Other pathways

may contribute to apoptosis, like the SAPK / JUNK

pathway, and other unknown pathways in RAS-induced

dedifferentiation (Table 1).

RET/PTC rearrangements

The RET gene encodes a transmembrane tyrosine

kinase (TK) receptor whose expression and function is

normally restricted to a subset of cells derived from the

neural crest. In thyroid follicular cells, RET activation

occurs through chromosomal recombination resulting



Table 1 Tumour-initiating events in thyroid cancer

RAS RET/PTC BRAF

Early biological events

Apoptosis C C C

TSH-independent growth C K K

Dedifferentiation C C C

Genomic instability C K CClinicopathological features

Aggressiveness C K C

Present in PDC/AC C K CHistologic type FA, FTC, PTCfv FA, PTC (RET1), PTCsv (RET3) PTC and PTCtc

FA, follicular adenoma; FTC, follicular thyroid carcinoma; PTC, papillary thyroid carcinoma; PTCfv, follicular variant of PTC; PTCsv,solid variant of PTC; PTCtc, tall cell variant of PTC.


in expression of a fusion protein consisting of the

intracellular TK domain of RET coupled to the

N-terminal fragment of a heterologous gene. Several

forms have been identified that differ according to the

5 0 partner gene involved in the rearrangement.

RET / PTC1 is formed by a paracentric inversion of

the long arm of chromosome 10, leading to fusion of

RET with a gene named H4 /D10S170 (Grieco et al.

1990). RET / PTC2 is formed by a reciprocal transloca-

tion between chromosomes 10 and 17, resulting in the

juxtaposition of the TK domain of c-RET with a

portion of the regulatory subunit of RIacAMP-

dependent protein kinase A (Bongarzone et al. 1993).

RET / PTC3 is also a result of an intrachromosomal

rearrangement and is formed by fusion with the

RFG / ELE1 gene (Santoro et al. 1994). Recently,

several additional variants of RET/PTC have been

observed in papillary carcinomas arising in children

exposed to radiation after Chernobyl (Klugbauer et al.

1996, 1998). The fusion proteins dimerise in a ligand-

independent manner due to motifs present in the

N-terminal domains. This results in constitutive

activation of the TK function of RET, autophos-

phorylation at selected tyrosine residues and initiation

of intracellular signalling by engagement with effectors

through specific tyrosine phosphorylated domains of

the receptor. Three sites of tyrosine phosphorylation

have been shown to function as docking sites for

signalling molecules (Fig. 1): pY905 mediates the

recruitment of the SH2 domain-containing proteins

Grb7 and Grb10; pY1015 mediates the association

between phospholipase Cg (PLC) and RET (Borrello

et al. 1996) and Shc and Frs2 interact with pY1062

mediating RAS/RAF/MAPK activation (Arighi et al.

1997, Melillo et al. 2001).

RET / PTC rearrangements were initially associated

with PTC (Santoro et al. 1992), radiation exposure

(Fugazzola et al. 1995, Klugbauer et al. 1995,

Bounacer et al. 1997, Nikiforov et al. 1997) and young


age (Ito et al. 1994, Bongarzone et al. 1996, Kumagai

et al. 2004, Lima et al. 2004, Penko et al. 2005, Powell

et al. 2005). However, RET mutations are not

restricted to a malignant phenotype as they have also

been found in sporadic follicular adenomas (Ishizaka et

al. 1991), benign thyroid nodules (Bounacer et al.

1997, Elisei et al. 2001) and Hashimoto’s thyroiditis

(Nikiforova et al. 2002a). Moreover, RET / PTCs are

also frequently present in non-radiated thyroid

tumours and in adults (Williams et al. 1996, Motomura

et al. 1998, Smida et al. 1999, Elisei et al. 2001).

Unknown ionising radiation exposure may explain

these findings and/or other factors may act indepen-

dently or in cooperation with radiation to trigger DNA

damage leading to protooncogene activation. Reported

frequencies of RET / PTC rearrangements in sporadic

PTC vary widely among different countries, ranging

from as low as 2.5% in Saudi Arabia to 59% in the

United Kingdom. Several reasons have been advocated

for such broad variability: ethnicity or genetic back-

ground in the occurrence of RET rearrangements

(See review by Nikiforov 2002), the use of different

detection methods, the genetic heterogeneity of the

tumours (Zhu et al. 2006) and finally, we cannot rule

out unknown environmental exposure to ionising

radiations or to other mutagenic factors. Two different

groups have reported a high frequency of RET / PTC

rearrangements in papillary microcarcinomas: 42.3%

in an Italian series (Viglietto et al. 1995) and 77% in a

Canadian series (Sugg et al. 1998). Finally, there is

some evidence that different types of RET / PTC

may be associated with distinct subtypes of PTC.

RET / PTC1 tends to be more common in tumours

with typical papillary growth and microcarcinomas

and to have a more benign clinical course, whereas in

some populations RET / PTC3 shows a strong corre-

lation with the solid variant of papillary carcinoma

(Nikiforov 2002). However, the presence of the

mutation does not seem to influence the biological

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behaviour of the tumour or its response to conventional

treatment modalities (Pacini et al. 2000).

Like RAS mutations, RET/PTC rearrangements are

thought to be tumour-initiating events. The early

biological consequences of RET/PTC activation, as

observed using an inducible system in rat thyroid cells,

are an induction of apoptosis by both RET/PTC1 and

RET/PTC3. However, in contrast to RAS mutations,

none of the RET/PTCs bestowed cells with the ability

to grow in the absence of TSH and no genomic

instability was observed (Wang et al. 2003). The same

group also demonstrated that the Y1062 phosphoryl-

ation site mediates RET/PTC-induced thyroid cell

dedifferentiation and DNA synthesis through Shc,

which activates the RAS/RAF/MEK/MAPK pathway

(Knauf et al. 2003). Additionally, acute expression of

the oncoprotein decreased TSH-mediated growth

stimulation due to interference with TSH signalling

by RET/PTC at multiple levels. Taken together, these

data indicate that RET/PTC is a weak tumour-initiating

event and that TSH action is disrupted by this

oncoprotein at several points, and also predict that

secondary genetic or epigenetic changes are required

for clonal expansion. Finally, exposure of cell lines to

ionising radiation results in induction of expression of

RET/PTC within hours (Ito et al. 1993), supporting a

direct role for radiation in the recombination of RET.

Thyroid-specific overexpression of either

RET/PTC1 (Jhiang et al. 1996, Santoro et al. 1996)

or RET/PTC3 (Powell et al. 1998) in transgenic mice

leads to development of tumours with histological

features consistent with PTC. RET/PTC1-transgenic

mice develop thyroid tumours displaying thyroid

hyperplasia and the main histological features of

PTC. These tumours are slowly progressive and do

not cause metastasis (Santoro et al. 1996). Transgenic

mice expressing human RET/PTC3 develop thyroid

hyperplasia and solid tumour variants of PTC that

metastasise to regional lymph nodes (Powell et al.

1998). Buckwalter et al. (2002) investigated the

contribution of the three signalling pathways triggered

by the specific phosphorylated tyrosine domains of the

receptor by characterising transgenic mice expressing

thyroid-targeted RET/PTC1 mutants with phenyl-

alanine substitutions at either Y905, Y1015 or

Y1062. Tumour formation was significantly decreased

in all of the mutants, but, in particular, in RET/PTC1-

Y905F double mutants. This points to significant

contributions by all of these pathways to RET/PTC-

induced thyroid cell transformation. However, as

tumours are still able to form in some transgenic

mice from all three single mutant transgenic groups,

the authors speculate that the signalling pathways

962

involving any one of these three phosphorylation sites

can be compensated by alternative redundant or

complementary pathways and lead to thyroid tumour

formation in vivo. This assumption has been confirmed

partially in vitro in PCCl3 cells, where neither Y1015

nor Y1062 alone are required for the RET/PTC-

induced effects on growth and apoptosis. By contrast,

in the same in vitro model there is an absolute

requirement of Y1062 for RET/PTC-induced dediffer-

entiation, as determined by decreased expression of

thyroid-specific gene products such as the sodium

iodide symporter (NIS), Tg or PAX8. RET/PTC-

mediated dedifferentiation requires activation of

Shc/RAS/RAF/MAP kinase (Knauf et al. 2003).

Overall, RET mutations are present in sporadic PTC,

in PTC arising after radiation exposure and in children.

They are highly prevalent in microcarcinomas, have

not been described in poorly differentiated and

anaplastic carcinomas and are not related to poor

prognosis. RET/PTC1 tends to be more common in

tumours with typical papillary growth and microcarci-

nomas and tends to have a more benign clinical course,

whereas RET/PTC3 shows a correlation with the solid

variant of PTC and more aggressive tumour behaviour.

Early biological events after RET/PTC activation are

consistent with apoptosis and dedifferentiation, but

RET/PTC is not able to induce TSH-independent

growth or genomic instability, exerting a weak

oncogenic drive. None of the RET/PTC tyrosine

residues alone is absolutely required for tumour

formation, and all appear to contribute to the ultimate

phenotype to some extent.

BRAF

There are three isoforms of the serine–threonine kinase

RAF in mammalian cells: ARAF, BRAF, and CRAF or

RAF1. CRAF is expressed ubiquitously, whereas

BRAF is expressed at higher levels in hematopoietic

cells, neurons and testes (Daum et al. 1994). BRAF has

a higher affinity for MEK1 and MEK2 and is more

efficient in phosphorylating MEKs than other RAF

isoforms (Peyssonnaux & Eychene 2001). The

majority of the oncogenic mutations of BRAF

destabilise the inactive BRAF structure, thereby

promoting an active conformation and leading to a

constitutive catalytic activation (Wan et al. 2004,

Moretti et al. 2006). The V600E mutant of BRAF is

one of the most prevalent somatic genetic events in

human cancer and possesses the hallmarks of a

conventional oncogene (Davies et al. 2002). The

kinase activity of this mutant protein is greatly

elevated; it constitutively stimulates ERK activity




in vivo independent of RAS and potently transforms

NIH3T3 cells.

The BRAFV600E mutation is the most common

genetic change in PTC, present in about 29–83%

of cases (see review by Xing 2005). Unlike RAS or

RET/PTC rearrangements, BRAF mutations are unique

to PTC and are not found in any other form of

well-differentiated follicular neoplasm arising from the

same cell type. BRAF mutations can occur early in the

development of PTC, based on evidence that they are

present in microscopic PTC (Nikiforova et al. 2003b).

The tall cell (TC) variant papillary thyroid cancers,

widely regarded as more aggressive, have a particu-

larly high prevalence of BRAF mutations (Nikiforova

et al. 2003b). Undifferentiated or anaplastic carci-

nomas arising from pre-existing papillary thyroid

cancers also have a significant prevalence of BRAF

mutations, whereas those arising from pre-existing

follicular carcinoma do not (Namba et al. 2003,

Nikiforova et al. 2003b, Begum et al. 2004, Soares

et al. 2004, Xing et al. 2004b, Quiros et al. 2005).

Some studies, but not all, have found BRAF to be

associated with aggressive clinicopathological features

such as advanced clinical stages and extrathyroidal

extension (Namba et al. 2003, Nikiforova et al. 2003b,

Xing et al. 2005, Riesco-Eizaguirre et al. 2006).

Moreover, we have demonstrated, along with another

group, that BRAFV600E is associated with a high

recurrence rate during the early follow-up of the

patients and that the majority of these recurrences have

no avidity for radioiodine (131I) and, thus, are

unresponsive to 131I treatment, and point out that this

genetic event is a new biological marker that predicts

poor prognosis and resistance to treatment of thyroid

cancer (Xing et al. 2005, Riesco-Eizaguirre et al.

2006). We further confirmed that these data in studies

in vitro in which assessment of NIS expression by

immunohistochemistry in human tumour samples and

transfection experiments in rat thyroid cells demon-

strated that BRAFV600E sharply impairs both NIS

expression and NIS trafficking to the membrane.

Like RAS mutations and RET/PTC rearrangements,

BRAF mutations are thought to be a tumour-initiating

event. The early biological consequences of BRAF

activation show that BRAF induces apoptosis and does

not confer on cells the ability to grow in the absence of

TSH, because of concomitant stimulation of both DNA

synthesis and apoptosis, resulting in no net growth in

the cell population (Mitsutake et al. 2005). BRAF-

induced apoptosis was also seen in the presence of

TSH. Acute BRAFV600E expression in PCCL3 cells

induces dedifferentiation and genomic instability

(Mitsutake et al. 2005). These data indicate that


BRAFV600E expression confers little growth advantage

on thyroid cells because of concomitant activation of

DNA synthesis and apoptosis. However, in contrast to

RET/PTC, and similarly to RAS, BRAFV600E may

facilitate the acquisition of secondary genetic events

(or primary epigenetic events) through induction of

genomic instability, which may account for its

aggressive properties (Mitsutake et al. 2005). Both

RET/PTC and BRAF decrease expression of the TSH

receptor (TSHR). However, the mechanisms by which

RET/PTC and BRAF interfere with TSH action distal

to the receptor differ in important aspects. In contrast to

RET/PTC, BRAF activation does not impair key

activation steps distal to the TSHR, such as forsko-

lin-induced adenylyl cyclase activity or cyclic AMP-

induced DNA synthesis (Mitsutake et al. 2005).

Thyroid-specific overexpression of the mutant

BRAF in transgenic mice leads to development of

tumours with histological features consistent with

invasive PTC, which exhibit foci of classic features,

foci of TC features and foci of poorly differentiated

carcinomas. These mice had a 30% decrease in

survival at 5 months (Knauf et al. 2005). This closely

recapitulates the phenotype of BRAF-positive PTCs in

humans. These data indicate that BRAFV600E is present

exclusively in PTC, particularly in the classic and TC

variant, and in poorly and anaplastic carcinomas that

most likely arise from PTC. It may be an alternative

tumour-initiating event in PTC, and tumours with this

genotype may carry a less favourable prognosis.

Overview

There are two kinds of evidence demonstrating that the

genetic events mentioned above are tumour-initiating

events. One of them is that these mutations are present in

microcarcinomas. The other one arises from studies on

transgenic mice, where the specific activation of these

oncogenes in the thyroid provokes a tumour phenotype

very similar to the one seen in humans. Assuming that

these genetic events are tumour-initiating events, the

study of the early biological events using inducible

systems represents an excellent model to provide new

insights into thyroid tumorigenesis. Oncogene-induced

apoptosis is an early and common biological event

present in the three oncogenic phenotypes. This

phenomenon usually does not happen in other epithelial

cells, where the oncogene itself triggersmechanisms that

are able to override apoptosis. Dedifferentiation is also

widely seen after early oncogene induction, although the

mechanisms and pathways involved differ among them.

TSH-independent growth is seen only after RAS

induction. This trait is only achieved by BRAF and

963



RET after stably transfecting the cell, indicating that

TSH-independent growth occurs in a second step during

the tumorigenesis in BRAF- and RET-positive tumours.

Finally, BRAF and RAS induce genomic instability,

while RET does not. Whether this genomic instability

induces secondary genetic events that confer more

aggressiveness to both tumour phenotypes is something

that needs to be elucidated. It should be noted that other

signalling pathways and molecular targets, although not

directly activated through genetic mutations, may prove

to be crucial for thyroid cancer progression and thus

appropriate for targeted inhibition (Table 1).

The phosphatidylinositol 3-kinase (PI3K)/Akt

pathway

Activation of Akt plays a pivotal role in fundamental

cellular functions such as cell proliferation and

survival by regulating the function of many cellular

proteins. It has been reported that alterations to the

PI3K/Akt signalling pathway are frequent in human

cancer. Constitutive activation of the PI3K/Akt

pathway occurs due to mutations or amplification of

the PIK3CA gene encoding PI3K or the Akt gene, or as

a result of inactivating mutations in components of the

pathway, for example PTEN (phosphatase and tensin

homologue deleted on chromosome 10), which inhibit

the activation of Akt.

The first evidence of the implication of this pathway

in thyroid tumours was provided by a syndrome called

Cowden disease. Germline mutations of the PTEN

gene confer predisposition to Cowden disease, a

condition characterised by development of hamarto-

mas in multiple organs, benign thyroid disorders such

as multinodular goitre and adenoma, and increased risk

of thyroid (mostly of the follicular type), breast and

other cancers (Eng 1998). However, in sporadic

thyroid neoplasm, a role for PTEN has not been firmly

established. Loss of heterozygosity (LOH) was found

in 27% of follicular carcinomas and 7% of follicular

adenomas, one of which was a small hemizygous

deletion (Halachmi et al. 1998), and loss or reduction

of PTEN expression as well as inappropriate sub-

cellular compartmentalisation has been reported in

thyroid tumours (Bruni et al. 2000, Gimm et al. 2000,

Vasko et al. 2004). The absence of somatic PTEN

mutations has lead to the screening for other genes

involved in the PI3K/Akt pathway. The potential

relevance of this pathway is also supported by the

observation that Akt expression and activation is

increased in thyroid cancers, particularly in FTC

(Ringel et al. 2001, Vasko et al. 2004). Additionally,

PTEN loss in transgenic mice and subsequent

964

activation of the PI3K/Akt pathway causes goitre and

follicular adenomas but is not sufficient for malignant

transformation of thyroid cells (Yeager et al. 2007).

Recently, Garcia-Rostan et al. (2005) reported in a

European series that somatic mutations within the

PI3K catalytic subunit, PIK3CA, is present in 23% of

anaplastic carcinomas and in 8% of FTC and is likely

to function as an oncogene in anaplastic thyroid cancer

(ATC) and less frequently in well-differentiated

thyroid carcinomas. The authors also argued for a

role of PIK3CA targeting in the treatment of ATC

patients. This PIK3CA mutant has also been observed

in colorectal, gastric, breast and ovarian cancers, and in

high-grade brain tumours (25–40%). However, Wu

et al. (2005) did not find any PIK3CA gene mutations

in an American series of thyroid tumours. Instead, they

observed PIK3CA gene amplification in 4 of 34 (12%)

benign thyroid adenomas, 3 of 59 (5%) PTC, 5 of 21

(24%) FTC, none of 14 (0%) medullary thyroid cancers

and in 5 of 7 (71%) thyroid tumour cell lines.

In an attempt to investigate the overall occurrence of

genetic alterations in the PI3K/Akt pathway in

sporadic thyroid cancer, Hou et al. (2007) investigated

the presence of PIK3CA copy number gain and

mutation, RAS mutation and PTEN mutation in a

large series of thyroid tumours. However, RET/PTC

rearrangements, which also activate the PI3K pathway,

were not included. The occurrence of any of these

genetic alterations was found in 25 of 81 (31%) benign

thyroid adenomas, 47 of 86 (55%) follicular thyroid

cancers, 21 of 86 (24%) papillary thyroid cancers and

29 of 50 (58%) ATCs, with FTC and ATC most

frequently harbouring these genetic alterations.

PIK3CA copy gain was associated with increased

PIK3CA protein expression. Although Hou et al.

describe a mutual exclusivity of these alterations in

well-differentiated carcinomas and adenomas, they did

not observe this in anaplastic carcinomas, arguing for a

role in the progression of FTC to ATC as the genetic

alterations of this pathway accumulate.

More studies in vitro and in vivo are needed to

elucidate the role this aberrant activation of PI3K/Akt

pathway plays in thyroid tumorigenesis, particularly in

FTC and ATC. Several small molecules designed to

specifically target PI3K/Akt have been developed and

induced cell cycle arrest or apoptosis in human cancer

cells in vitro and in vivo. Moreover, the combination of

an inhibitor with various cytotoxic agents enhances the

anti-tumour efficacy (Osaki et al. 2004). Therefore,

specific inhibition of the activation of Akt may be a

valid approach to treating thyroid malignancies,

particularly FTC and ATC.




The p53 pathway

Inactivating point mutations of the p53 tumour

suppressor gene are highly prevalent in anaplastic

and poorly differentiated thyroid tumours, but not in

well-differentiated papillary or follicular carcinoma

(Ito et al. 1992, Fagin et al. 1993). These data imply

p53 inactivation as an important step in late stage

progression of thyroid cancer. Thyroid cells carrying a

mutated p53 gene did not form colonies in soft agar or

tumours in athymic mice, suggesting that a mutation of

the p53 gene is not sufficient for the induction of the

malignant phenotype, and probably cooperation with

other oncogenes is necessary to accomplish full

malignancy. However, a mutated p53 gene results in

a marked loss of the differentiated phenotype in the rat

thyroid cell line PCCl3, including inhibition of the

expression of the thyroid-specific transcription factor

PAX8 (Battista et al. 1995). Conversely, re-expression

of wt-p53 activity in undifferentiated thyroid carci-

noma cell lines inhibits cell proliferation and restores

differentiation (Fagin et al. 1996, Moretti et al. 1997).

The PAX8/PPARg rearrangement

This translocation fuses the thyroid-specific transcrip-

tion factor PAX8 gene with the PPARg gene, a

ubiquitously expressed transcription factor that has

been shown to play an important role in regulating

genes involved in adipocyte differentiation and lipid

metabolism. It involves a chromosome 3p25 and 2q13

translocation (Kroll et al. 2000) creating a fusion gene,

encompassing the promoter and proximal 50 coding

sequence of the thyroid-specific transcription factor

PAX8 gene and most of the coding sequence of the

PPARg gene. PAX8/PPARg rearrangement has been

identified in a significant proportion of FTC (36–45%),

follicular adenoma (FA; 4–33%), follicular variant of

PTC (37.5%) or Hurthle cell carcinoma (Kroll et al.

2000, Marques et al. 2002, Nikiforova et al. 2002b,

2003a, French et al. 2003, Castro et al. 2006).

The mechanism of transformation induced by

PAX8/PPARg is still unclear. It has been shown to

have a dominant negative effect on thiazolidinedione-

induced transactivation by PPARg (Kroll et al. 2000)

and overexpression of wild-type PPARg1 in thyroid

cancer cell lines inhibits cell growth, an effect that is

further enhanced by PPAR agonist (Martelli et al.

2002). However, another group did not find this

dominant negative effect in the rat thyroid cells

FRTL5 nor in immortalised human thyroid cells,

raising the question whether the transforming proper-

ties of PAX8/PPARg can be attributed only to

inhibition of PPARg function (Au et al. 2006).


Epigenetics of thyroid cancer

Epigenetic modifications, such as histone deacetylation

and DNA methylation, play an important role in the

regulation of gene expression and are likely to be

involved in carcinogenesis. DNA methylation is a

covalent modification of cytosine residues that occurs

at the dinucleotide sequence CpG in vertebrates.

Nearly half of all human genes have CpG islands

associated with transcriptional start sites. Unmethy-

lated CpG islands are seen in highly transcribed genes,

whereas heavily methylated CpG islands result in

heritable inhibition of gene transcription (Baylin

1997). Although overall DNA methylation is often

decreased in cancers, CpG islands in critical gene

promoter regions can become hypermethylated, result-

ing in heritable inhibition of gene expression.

Abnormal patterns of DNA methylation are observed

consistently in human tumours, including benign and

malignant human thyroid tumours (Matsuo et al.

1993), usually occurring as an early event (Counts &

Goodman 1995).

Aberrant methylation affecting thyroid-specific

genes in benign and malignant thyroid tumours have

been described by several groups, demonstrating a role

for this mechanism in thyroid dedifferentiation.

Affected genes include the TSHR gene (Xing et al.

2003b), the Pendred syndrome gene SLC26A4 (Xing

et al. 2003a) and the NIS gene (Venkataraman et al.

1999, Neumann et al. 2004). The observation of

epigenetic alterations of the NIS promoter has opened

new approaches to recovering NIS expression.

Demethylating agents and inhibitors of histone

deacetylase have been used successfully in vitro to

restore iodine uptake in thyroid cancer cell lines (see

review Riesco-Eizaguirre & Santisteban 2006).

Recently, several tumour suppressor genes and

candidate tumour suppressor genes have been shown to

be silenced due to aberrantmethylation in thyroid cancer.

Examples of these genes include RASSF1A (Schagdar-

surengin et al. 2002, Xing et al. 2004a), genes encoding

the cyclin-dependent kinase inhibitors p15INK4b and

p16INK4a (Elisei et al. 1998, Boltze et al. 2003), the

tissue inhibitor of metalloproteinase-3 (TIMP3; Hu et al.

2006), SLC5A8 (also called sodium monocarboxylate

transporter (SMCT; Hu et al. 2006), death-associated

protein kinase (DAPK; Li et al. 2003), retinoic acid

receptor b-2 (RARb-2; Hoque et al. 2005) and PTEN

(Alvarez-Nunez et al. 2006). Methylation of some of

these genes (TIMP3, SLC5A8 and DAPK) was

significantly associated with several aggressive features

of PTC, and may have a role as potential prognostic

markers (Hu et al. 2006).However, there is still a lot to be

965



learned about all these silencing events and their potential

pathogenic significance is still uncertain. For instance,

although restoration of RASSF1A expression inhibits

tumorigenicity in vitro and in vivo in lung cancer, the

biological function of RASSF1 and its isoforms (A, B, C,

D, E, F and G) is still under investigation. Interestingly,

mutational inactivation of this gene is very rare (2%), and

the main mechanism of its inactivation is through

promotermethylation andLOH.Future studies focussing

on the mechanisms underlying these epigenetic altera-

tions need to be done in order to have a better view of the

pathogenic significance in thyroid tumorigenesis.

The genomics of thyroid cancer

Tumour gene expression profiling by DNA microarrays

has brought new important clues to our understanding of

cancer pathophysiology and simultaneously has pro-

vided clinically valuable information onmanymalignant

neoplasms. Taking advantage of this new approach,

several studies have given hints about the molecular

pathways involved in thyroid tumorigenesis and may

provide newbiomarkers for clinical use.Gene expression

studies are beginning to be applied to thyroid cancer

(Huang et al. 2001,Barden et al. 2003,Aldred et al. 2004,

Chevillard et al. 2004, Finley et al. 2004, Frattini et al.

2004, Mazzanti et al. 2004, Wreesmann et al. 2004,

Giordano et al. 2005, Jarzab et al. 2005) and should yield

significant improvements in thyroid cancer diagnosis,

prognosis and treatment.

In the first study performed using this methodology

(Huang et al. 2001), results fromeight PTC tumourswere

comparedwith normal thyroid tissue from the same eight

individuals. Although PTC is clinically heterogeneous,

the authors concluded that PTC is characterised by

consistent and specific global expression patterns. They

detected differential expression of genes that were

previously known to be altered in PTC, validating the

feasibility of their experimental approach, such as MET,

LGALS3 (galectin 3), KRT19, DPP4,MDK, TIMP1 and

FN1 (fibronectin).

They also reported numerous additional genes to be

differentially expressed, proposing some of them as

candidate clinical markers, since they were signi-

ficantly upregulated in the great majority of more than

40 PTCs. Finally, these findings gave clues about the

molecular pathways involved in PTC. Genes related

with molecular adhesion were clearly overexpressed in

PTC versus normal thyroid, whereas genes under-

expressed in PTC included tumour suppressors, thyroid

function-related proteins and fatty acid binding

proteins. In a similar study, Wasenius et al. confirmed

in a larger series much of the data seen by Huang et al.

966

but focused on different candidate genes that could

serve as biological markers for PTC.

Using more advanced bioinformatics methods that

were not used before addressing thyroid cancer by

other groups, Jarzab et al. (2005) demonstrated that the

difference between tumour and normal samples was

the major source of variability in the gene expression

pattern of thyroid tissues, confirming the results

obtained by Huang et al. (2001) However, despite

very distinct changes in expression, none of the genes

proved to be an ideal single marker of PTC in an

independent set of PTCs analysed by Q-PCR. This

study confirmed the overexpression of cell adhesion

genes that was indicated by the aforementioned

microarray study. This group of genes, possibly related

to invasion and metastasis processes, constituted the

most numerous gene ontology class. Concerning signal

transduction-related genes, the very distinct upregula-

tion of MET is a consistent feature also found in other

genomic studies of PTC (Wasenius et al. 2003, Finley

et al. 2004). The authors also emphasised that among

the five transcription factors upregulated, RXR-gwas a

novel gene, which could have implications for the

response to retinoids in thyroid cancer. However,

genomic profiling provides a very partial view of

functionality and a wide spectrum of descriptive data

that it is not easy to interpret. Also, one of the major

drawbacks of this kind of approach is its lack of

reproducibility.

Another study made a comparative analysis of

microarray expression data for PTC and FTC, the

two most common forms of non-medullary thyroid

carcinoma. Although there were some similarities

between the two tumour types among underexpressed

genes, distinct expression patterns could be observed.

Five genes (CITED1, CAV1, CAV2, IGFBP6 and

CLDN10) were identified that collectively distinguish

PTCs from FTCs (Aldred et al. 2004). Although PTC is

characterised by consistent and specific global gene

expression patterns, in a very interesting study,

Giordano et al. (2005) observed a relationship between

morphologic subtype of PTC and gene expression. The

follicular variants (FV) were tightly grouped in the

PCA plot, and the TC variants were loosely grouped

among the remaining classic types (CT). Interestingly,

the authors further observed that incorporating the

BRAF, RET/PTC and RAS mutational status revealed

a strong relationship between mutation and gene

expression, with PTCs closely grouped for each

mutation type. Interestingly, tumours with BRAF

mutations displayed either TC or classical variant

morphology; tumours with RET/PTC rearrangements

displayed predominantly the classical morphology and




tumours with RAS mutations exclusively displayed the

follicular variant morphology. Tumours with no

apparent mutation predominantly had the follicular

variant morphology. Also, the analysis revealed that

mutation was more strongly correlated with gene

expression than with morphology. Frattini et al. (2004)

performed a similar though smaller profiling study and,

although they showed no strong differences in global

gene expression in PTC, they found some expression

differences between tumours with RET or NTRK1

rearrangements and tumours with BRAF mutations.

The striking relationship between gene expression

and genotype indicates that mutations affecting the

RET/RAS/BRAF/MAPK pathway are the predominant

source of gene expression variation and suggests that

they represent the earliest mutational events occurring

in PTC, resulting in persistent and distinct patterns of

abnormal gene expression. In addition, the absence of

premalignant lesions in well-differentiated PTC, in

contrast to other epithelial tumour types such as colon

carcinoma, suggests that PTCs are the morphological

manifestation of single dominant activating mutations.

This finding suggests that these mutations are able to

signal through alternative pathways and may confer

discrete mutation-specific phenotypical and biological

features on tumours. For instance, the authors observed

that among the mutation-specific differentially

expressed genes in PTC, some have roles in signal

transduction such as VAV3, a member of the VAV

oncogene family involved in PI3K signalling and

subsequent AKT activation. Its preferential expression

in PTCs with RET/PTC and RAS mutations may

provide evidence that these PTCs signal more through

PI3K than MAPK pathways. The identification of

TM7SF4 as one of the most preferentially expressed

genes in PTCs with BRAF mutations has implications

for the immunological aspects of PTC. TM7SF4 is a

transmembrane protein expressed in dendritic cells and

has a role in antigen processing and initiation of the

immune response. Its expression profile lead to the

authors to speculate that BRAF mutation has a unique

role in initiating an immune response in PTC.

As mentioned before, genomic profiling has its

limitations and provides a very partial view of

functionality. For instance, despite the variability in

the genes mentioned above, the expression of genes

related to DNA replication, cell cycle, mRNA splicing

and protein biosynthesis showed much less variation

than could be expected (Jarzab et al. 2005). We should

also bear in mind that the functions of selected genes

may be related to the stromal component of the tumour.

Thyroid tumours consist of neoplastic cells inter-

mingled irregularly with normal (connective tissue and


vessels) and reactive (stromal and immune) cells (Kroll

2002). Quantitative relations between these com-

ponents may vary between patients and even inside

one tumour. Most microarray studies include tumour

fragments containing more than 80–90% of tumour

cells and some authors recommend investigation of

microdissected cells (Jarzab et al. 2005).

Apart from all this genomic profiles made in human

tumour samples, several in vitro studies have tried to

define the functional role of selected genes in tumoral

transformation, particularly in metastasis progression.

In thyroid cell lines, the activation of unique patterns of

gene expression exerted by the three main oncopro-

teins have also been demonstrated. Melillo et al. (2005)

examined the transcriptional profile of rat thyroid

PCCL3 cells stably expressing RET/PTC, RAS or

BRAF. A large fraction of the genes activated by

RET/PTC3 were also activated by RAS or BRAF,

consistent with the primary role of the RET/RAS/

BRAF/MAPK pathway in transformation. This was

confirmed for a subset of genes by use of RNA

interference (RNAi) for BRAF or by MEK inhibitors.

However, there were relatively large sets of genes

specifically modulated by each oncogene. Interest-

ingly, they also showed a critical role of this pathway

in stimulating cell motility, mediated in part by

induction of the chemokines CXCL1 and CXCL10

(Melillo et al. 2005). In a slightly different model,

Mesa et al. (2006) examined the pattern of gene

expression after conditional activation of these

oncoproteins in thyroid PCCL3 using an inducible

system. Metalloproteinases were preferentially

induced by BRAF, particularly matrix metalloprotei-

nase 3 (MMP3), MMP9 and MMP13. Enhanced

production of MMPs in the tumour environment, either

by stromal or by tumour cells, is believed to be an

important determinant of tumour invasion. As BRAF

was associated with markedly increased invasion into

Matrigel compared with cells expressing RET/PTC3,

the preferential induction of MMPs by BRAF could

explain in part the more invasive behaviour of thyroid

cancers with BRAF mutations (Mesa et al. 2006).

Furthermore, BRAF-induced expression of matrix

metalloproteinase and cell invasion into matrigel seems

to be mediated NF-kB pathway (Palona et al. 2006),

although the intrinsic molecular mechanism of BRAF-

induced metastatic progression remains to be elucidated.

An integrated perspective of thyroidcancer pathogenesis

We now have compelling evidence that indicate

that PTC requires constitutive activation of the

967



RTK/RAS/RAF/MAPK pathway for tumour initiation

and progression. Similarly, we are starting to have

more evidence that the PI3K/AKT pathway seems to

play an important role in tumour initiation and

progression of FTC. The p53 pathway appears to be

involved exclusively in tumour progression of both

phenotypes, as they are only present in ATC. One can

speculate that tumour phenotype may differ in part

according to the pre-eminence of the pathway activated

and its occurrence early or later in tumour progression.

This point of view is probably too simplistic but it is a

good starting point that, for instance, has lead to the

development of new targeted therapies aimed at

blocking these pathways. In addition to the

RTK/MAPK, PI3K/AKT and p53 pathways, there are

others that have a role in many tumour types, including

those involving the adenomatous polyposis coil (APC),

glioma-associated oncogene (GLI), hypoxia-inducible

transcription factor (HIF)-1 and SMADs. However,

their role in thyroid cancer is yet to be discovered.

The nature of the tumour-initiating event is also

important in determining the tumour phenotype. PTCs

harbouring RET/PTC variants, RAS and BRAF

mutations exert its own oncogenic drive, exhibiting

distinct pathological features, genomic profiles, epige-

netic changes and biological behaviour. Follicular

variant of PTC commonly has RAS mutations, the

classical and TC variant PTC have BRAF mutations

and solid variants of PTC harbour RET/PTC3

rearrangements. Obviously, there has been an attempt

to study the clinical significance of all these genetic

events. Unfortunately, as RAS or PAX8/PPARg is not

restricted to a malignant phenotype, they are not

useful as biological markers to discriminate between

follicular adenoma and carcinoma. On the contrary,

BRAF mutations are common and specific for PTC.

FNAC is routinely used in the preoperative diagnosis

of thyroid nodules and about 15–20% of FNAC are

undeterminate. A BRAF mutation can be reliably

detected in cells aspirated from a FNAC of a thyroid

nodule and it has been shown to establish the diagnosis

of PTC in 16% of carcinomas within the indeterminate

group (Cohen et al. 2004, Xing et al. 2004c). Besides,

the prognostic impact of BRAF and its role in

discriminating between low- and high-risk patients is

still a controversial matter, pointing to the need of

prospective randomised studies in large series of

patients. The observation that BRAF-positive tumours

have lower levels of NIS expression and are associated

with recurrences that have lost the ability to

concentrate radioiodine may pave the way to elucidate

the mechanisms underlying the dedifferentiation

process that affects some thyroid cancer metastasis.

968

Indeed, new strategies are needed to treat the

challenging situation of elevated Tg and negative

radioiodine scans in patients with thyroid cancer.

Targeted therapies in thyroid cancer

Since thyroid follicular cancer conserves a certain degree

of differentiation, one logical therapeutic approach is to

redifferentiate the cells and reinduce endogenous NIS

expression so that radioiodine treatment canbeperformed.

Before targeted therapy became feasible, many groups

focused on this strategy and several compounds, also

known to have tumour-inhibitory effects, have partially

succeeded in reaching this goal. Among them, the most

well known has been retinoic acid (RA). Several clinical

trials have been done using RA in order to increase

radioiodide uptake and improve clinical outcome of

patients with recurrent thyroid cancer (see review by

Dohan et al. 2003). In general terms, radioiodide uptake

was improved in 20–42% of the cases, but tumour

shrinkage was observed in very few cases after 131I

treatment. The largest study was done on 50 iodide scan-

negative patients: 26% had a significant increase in

radioiodide uptake, but only 16% had reduced tumour

volume (Simon et al. 2002). Other compounds such as

Troglitazone, HDAC inhibitors and demethylating agents

are currently being tested with promising results (see

review by Riesco-Eizaguirre & Santisteban 2006).

The prevalence of activating BRAF mutations,

RET/PTC rearrangements and RAS mutations and

consistent downstream activation of ERK suggests that

activation of the MAP kinase signalling cascade may

be an obligatory step in the transformation of

thyrocytes. Allegedly, such dependency may represent

a point of therapeutic attack (Table 2). However, we

should bear in mind that, as we have explained before,

deregulation of other pathways and/or secondary

genetic events may be playing important roles in

thyroid tumorigenesis. Several lines of experimental

evidence affirm the notion that targeting RAF activity

can be a good starting point. First, depletion of BRAF,

by small interfering RNA, in BRAF-positive thyroid

cancer cell lines inhibits both ERK activation and

proliferation while abrogating tumour xenograft

formation (Salvatore et al. 2006). Secondly and most

interestingly, RAF activity seems to be required for the

transforming effects of the non-overlapping mutations

of the other oncogenes signalling through the MAPK

pathway. Indeed, RAF binds to and is a direct effector

for RAS (Troppmair et al. 1994), and selective knock-

down of BRAF (but not CRAF) abrogates RET/PTC3-

induced ERK phosphorylation in thyroid cells (Melillo

et al. 2005, Mitsutake et al. 2006). These data suggest



Table 2 Targeted therapies in thyroid cancer

Agent Target Additional targets Structure IC50 (nM) Observations

BAY 43-9006a

(Sorafenib)

RAF VEGFR, PDGFR Biaryl urea 12 Clinical study phase I/II

AAL881b RAF C-ABL, KDR Isoquinoline 100

LBT613b RAF RET, C-ABL, KDR Isoquinoline 100

AMG 706 VEGFR KIT, PDGFR Carboxamide 10 Clinical study phase I/II

ZD6474c RET VEGFR, EGFR Quinazoline 100

PP1 RET SRC Pyrazolopyrimidine 80

PP2 RET SRC Pyrazolopyrimidine 100

CEP-701 RET NTRK Indolocarbazole !100 Medullary thyroid carcinoma

CEP-751 RET NTRK Indolocarbazole !100 Medullary thyroid carcinoma

Plitidespind Unknown – Marine derivative 10 Anaplastic thyroid carcinoma

PS-341e

(Borte-

zomib)

NF-kB – Boronic acid dipeptide 5–20 Anaplastic and medullary

aBayer/Onyx; bNovartis; cAstra Zeneca; dPharmaMar; eJanssen-Cilag.


that blocking RAF activity may be a logical approach

to interfere with the effects of RET/PTC, RAS and

BRAF oncoproteins.

Given the prominence of this ‘oncogene addiction’

phenotype involving the MAP kinase pathway in

thyroid and other cancer types, several small molecules

that target this pathway are currently being tested

in vitro and in vivo. Sorafenib (BAY 43-9006) was one

of the first compounds to be evaluated in clinical trials.

This biaryl urea exhibits in vitro inhibition of both

wild-type and mutant BRAF activities in the 20–40 nM

range (Ahmad & Eisen 2004). Sorafenib is a multi-

kinase inhibitor that also inhibits CRAF and VEGFR3

at low concentrations, while inhibiting platelet-derived

growth factor receptor (PDGFR), VEGFR-2, c-KIT

and FLT3 at higher concentrations in vitro. Despite this

robust effect in vitro, the potency of this compound

against MAPK activity in whole cells has proved more

variable. Salvatore et al. (2006) reported half-maximal

inhibitory concentrations (IC50) values for sorafenib

against BRAF-positive thyroid cancer cell lines of

1 mM to reduce proliferation, and 5 mM to virtually

arrest growth (50- to 100-fold higher than the required

concentration for in vitro kinase inhibition. In

xenografts, daily administration of 60 mg/kg sorafenib

attenuated tumour growth, almost completely inhibited

phospho-MEK activity and reduced Ki67/MIB-1

immunolocalisation (a proliferation marker). It also

diminished the number of blood vessels in the

xenograft, demonstrating an antiangiogenic effect

(Salvatore et al. 2006). Carlomagno et al. (2006)

obtained similar results using sorafenib against onco-

genic RET/PTC-positive thyroid cancer cell lines. In

this case, the IC50 was significantly lower (50 nM) and


the same oral doses in xenografts (60 mg/kg per day)

reduced tumour growth significantly.

Despite its promising preclinical properties, the

preliminary efficacy data for sorafenib in patients with

thyroid cancer appear more modest, yet significant. In a

recent phase I/II clinical trial, a total of 46 patients with

iodine-refractory measurable metastatic thyroid carci-

noma were given sorafenib at a dose of 400 mg/day for a

median of 12 weeks (range 3–28 weeks). The group was

heterogenous, including papillary, follicular,Hurthle and

anaplastic carcinomas. Among the 28 evaluable patients,

only 5 had progressive disease during the treatment, 12

(42.8%) had serum Tg decreased significantly and 5 out

of 11 patients (45%) in whom MRI was performed

showed an average decrease of 57% on exchange rate

thatwas concordantwithTg response (Kloos et al. 2005).

Sorafenib received FDA approval for the treatment of

metastastic renal cancer, a malignancy where BRAF

mutations have not been observed. In this case, sorafenib

is believed to have a prominent antiangiogenic effect, and

therefore its clinical efficacy may derive more from its

anti-VEGF activity than from BRAF blockade.

Other RAF inhibitors have been tested for thyroid

cancer, yet they did not show better in vitro potencies

than sorafenib. For example, AAL881 and LBT613

(Novartis,Cambridge,MA,USA) showapharmacologic

IC50 ofO100 nM against wild-type and mutant BRAF

in vitro. In thyroid carcinoma cell lines, the IC50 rose up

to 10 mM (tenfold higher than sorafenib), and some

toxicity was reported in vivo, particularly with LBT613.

Despite these pharmacokinetic concerns, both

compounds were effective growth inhibitors of poorly

differentiated thyroid cancer cell lineswith either RET or

RAF mutations (Ouyang et al. 2006). Several second

969



generation small molecule inhibitors of RAF and MEK

that exhibit in vitro potencies exceeding that of sorafenib

are currently being investigated, i.e. in BRAF-mutant

melanoma. In addition, a series of small molecules has

been shown to selectively target the V600E mutant of

BRAF, resulting in submicromolar IC50 values (http://

www.plexxicon.com). Presumably, these and other

emerging RAF inhibitors may provide a more robust

effect against MAP kinase activity in clinical trials.

Another small-molecule multikinase inhibitor, AMG

706, is a new orally bioavailable ATP-competitive

inhibitor of VEGFR1, VEGFR2 and VEGFR3. In

addition, AMG 706 inhibits Kit and platelet-derived

growth factor receptor (PDGFR), two receptor tyrosine

kinases (RTKs) related to VEGFR and implicated in the

pathogenesis of several human cancers. AMG 706

inhibits VEGF-induced cell proliferation and vascular

permeability and induces tumour regression in vivo by

selectively targeting neovascularisation in tumour cells

(Polverino et al. 2006). From a phase 1 study performed

on a large series of patients with solid tumours (Rosen

ASCO 2005; Herbst EORTC 2005), encouraging anti-

tumour activity was seen in a subset of patients with

thyroid cancer. Seven patients with iodine-refractory

metastatic thyroid cancer received this compound orally

for amedian time of 141 days. Histologies were PTC (3),

FTC (1), Hurthle cell (1), anaplastic (1) and medullary

thyroid carcinoma (MTC) (1). Three patients (one PTC,

one FTC and one MTC) showed a partial response

consistentwith reduced tumour volume and a decrease of

tumour markers. The authors concluded that AMG 706

appeared to bewell tolerated and, although the serieswas

very small, this inhibitor displayed a promising anti-

tumour activity in multiple histological subtypes of

thyroid cancer (Boughton et al. 2006).

Quinazolines are also some of the most promising

inhibitors of RTKs (Levitzki 1999). For instance,

ZD1839 (Iressa) is a potent and selective inhibitor of

the EGFR and is currently in advanced clinical

development (Ciardiello et al. 2001). Another anilino-

quinazoline, ZD6474, has been shown to be a selective

inhibitor of the VEGF receptor-2 (flk-1/KDR) tyrosine

kinase (Wedge et al. 2002). Interestingly, this last

compound has also been shown to inhibit the enzymatic

and transforming activity of RET oncoproteins and

arrests the development of RET/PTC3-induced tumours

in nude mice. This low molecular weight tyrosine kinase

inhibitor blocks the enzymatic activity of RET-derived

oncoproteins at a half maximal inhibitory concentration

of 100 nM in vitro and blocks in vivo phosphorylation and

signalling of the RET/PTC3 and RET/MEN2B oncopro-

teins. RET/PTC3-transformed cells lost proliferative

autonomy and showed morphological reversion after

970

treatment with ZD6474. This compound also prevented

the growth of two human PTC cell lines that carry

spontaneous RET/PTC1 rearrangements. The authors

concluded that targeting RET oncogenes with ZD6474

might offer a potential treatment strategy for carcinomas

sustaining oncogenic activation of RET such as PTC and

medullary thyroid carcinomas (Carlomagno et al. 2002).

The proteasome inhibitor bortezomib (PS-341,

Velcade, Janssen-Cilag, Belgium) has been approved

by the Food and Drug Administration for the treatment

of multiple myeloma. The inhibition of inhibitory-kBdegradation, which leads to inactivation of the

transcription factor nuclear factor-kB (NF-kB), seems

to be its mechanism of action. NF-kB has been

implicated in the pathophysiology of the most

aggressive forms of thyroid carcinoma, i.e. medullary

and anaplastic carcinomas (Ludwig et al. 2001,

Pacifico et al. 2004). Although no studies in xenografts

were performed, bortezomib induced apoptosis in

medullary and anaplastic cell lines, but not in papillary

or follicular cell lines, with IC(50) values well within

the range of clinically achievable concentrations,

showing a promising therapeutic target for aggressive

thyroid cancer (Mitsiades et al. 2006). Activation of

the PI3K/Akt signalling pathway appears to be an

important event in thyroid tumorigenesis and, perhaps,

in tumour progression too. Pharmacological disruption

of PI3-kinase activity, or disruption of Akt signalling

using dominant negative cDNA expression have

demonstrated beneficial effects on several cancer

models in vitro. Therefore, Akt represents an attractive

target for pharmaceutical development for a variety of

malignancies, including thyroid cancer (Kada et al.

2004).

Conclusions

Thyroid follicular cancer biology has unique charac-

teristics that make this malignancy a paradigm for the

initiation of tumour formation. Tumour-initiating events

have been identified in a high proportion of the most

frequent type of thyroid cancer, PTC, and all of

them converge in a single signalling pathway – the

RTK/RAS/BRAF/MAPK pathway. Further studies are

needed to understand the next steps in thyroid tumorigen-

esis as other cancer genes and/or pathways are starting to

emerge. As a consequence of the development of thyroid

follicular cancer genetics, genomics and epigenetics, our

increasing understanding of the biology of this type of

cancer is leading to newand promising approaches for the

development of thyroid cancer therapies.



http://www.plexxicon.com

http://www.plexxicon.com


Acknowledgements

We are grateful to the Ministerio de Educacion

y Ciencia (Grants BFU 2004-03169; SAF2007-

60164) and the Instituto de Salud Carlos III, FIS

(grants RGDM(C03/212), PI041216, PI042374, and

RD06/0020/0060) for supporting our work. G Riesco-

Eizaguirre is the recipient of an MD Training Grant

from FIS, Instituto de Salud Carlos III (Spain). We

thank Dr Ronald Hartong for his linguistic assistance.

The authors declare that there is no conflict of interest

that would prejudice the impartiality of this scientific

work.

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