143

REVIEW

Molecular genetics of medullary thyroid carcinoma: the quest for novel therapeutic targets

Aniello Cerrato, Valentina De Falco and Massimo Santoro Istituto di Endocrinologia ed Oncologia Sperimentale CNR c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, ‘L. Califano’, Universita` Federico II di Napoli, via S. Pansini 5, 80131 Naples, Italy

(Correspondence should be addressed to M Santoro; Email: [email protected])

Abstract

Medullary thyroid carcinoma (MTC) is a rare tumour arising from neural crest-derived parafollicular C-cells. Metastatic MTC patients are incurable because the cancer does not respond to radiotherapy or chemotherapy. The REarranged during Transfection (RET) proto- plays a key role in the development of MTC. However, one-half of the sporadic MTC do not carry RET mutations. Mice models and early evidence obtained in human samples suggest that other , including those encoding components of the RB1 (retinoblastoma) and TP53 tumour-suppressor pathways, may be involved in MTC formation. Here, we review the data on the involvement of genes acting in the RET and RB1/TP53 pathways in MTC. Understanding genetic lesions that occur in MTC is a prerequisite to identifying molecular therapeutic targets in MTC and in improving the efficacy of RET-targeted therapies. Journal of Molecular Endocrinology (2009) 43, 143–155

Medullary thyroid carcinoma: a genetic Finally, about 50% of sporadic MTC do not carry RET overview mutations. Whether another frequently mutated or multiple low-frequency mutated genes occur in RET Medullary thyroid carcinoma (MTC) arises from wild-type MTC samples is unknown. calcitonin-producing neural crest-derived parafollicular In MEN 2, MTC is associated in about 50% of cases to (C) cells of the thyroid. MTC accounts for 5–8% of all pheochromocytoma (MEN 2A and 2B), and in 10–35% thyroid cancers (Matias-Guiu et al. 2004, Schlumberger of cases to parathyroid hyperplasia or adenoma (MEN et al. 2008). MTC is sporadic in about 75% of the cases; 2A; Gagel & Marx 2003). This suggests that MTC shares in the remaining cases, it occurs as a component of the pathogenetic mechanisms with pheochromocytomas autosomal dominant familial multiple endocrine neo- and parathyroid tumours. However, MTC is not a plasia type 2 (MEN 2) syndrome. MEN 2, first described phenotype of non-MEN 2 familial forms of parathyroid by Sipple (1961), includes three disorders: MEN 2A; tumours or pheochromocytomas (Gagel & Marx 2003, MEN 2B; and familial MTC (FMTC; Marx 2005, Elisei Marx 2005, Zbuk & Eng 2007). Accordingly, Prad1/ et al. 2007, Zbuk & Eng 2007). MEN 2-associated MTC is Ccnd1, Men1 and Hprt2 genes (associated to non-MEN 2 bilateral and multicentric, and it is usually preceded by forms of parathyroid tumours; Ferris & Simental 2004) multifocal C-cell hyperplasia (CCH; Gagel & Marx and Vhl, Nf1 and succinate dehydrogenase subunits B, C 2003). REarranged during Transfection (RET)is and D (SdhB, SdhC and SdhD; associated to non-MEN 2 mutated in roughly 50% of sporadic MTC and in forms of pheochromocytomas; Maher & Eng 2002, more than 95% of MEN 2 families. Kaelin 2008) do not seem to be mutated in MTC. SdhB, In FMTC, although the germ line RET mutation is C and D were not found to be mutated in sporadic present in all somatic cells of the affected individual, MTC (Lima et al. 2003, Cascon et al. 2005, Montani tumours are monoclonal, which suggests that other et al. 2005), although germ line SdhB and SdhD variants genetic alterations must occur at the somatic level and were over-represented in MTC samples with respect to act in concert with RET mutations for the tumour to healthy individuals (Sobrinho-Simo˜es et al. 2008). One develop (Gagel & Marx 2003). Moreover, a few MEN 2 mutation and three monoallelic deletions were found families negative for RET mutations have been in the Vhl gene in five familial RET mutant MTC described, suggesting the existence of additional loci samples, suggesting cooperation of Ret gain with Vhl loss predisposing to MEN 2 (Montero-Conde et al. 2007). in MTC formation (Koch et al. 2006). Intriguingly, Vhl,

Journal of Molecular Endocrinology (2009) 43, 143–155 DOI: 10.1677/JME-09-0024 0952–5041/09/043–143 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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Nf1 and Sdh gene products collaborate with RET in a common signalling pathway involved in controlling EglN3 prolyl hydroxylase-mediated neuronal cell apop- tosis. In this pathway, RET (gain-of-function) and NF1 and VHL (loss-of-function) mutations lead to increased JunB , which, in turn, blunts the expression of EglN3, thereby leading to inappropriate cell survival and tumourigenesis (Kaelin 2008). Loss of SDH activity results in higher succinate levels; this in turn triggers the survival pathway because EglN3 is feedback-inhibited by succinate. Finally, the recently discovered KIF1B tumour suppressor, which maps in a chromosomal region (1p36) frequently deleted in MTC (see below), is required for EglN3 pro-apoptotic activity (Kaelin 2008). Thus, even if not frequently mutated, these should be functionally analyzed in Figure 2 Schematic representation of RB and signalling pathways leading to cell proliferation arrest and apoptosis. relation to MTC formation. Potential oncoproteins are in white, whereas tumour suppressors In the following sections we focus on genes acting in are in grey, kinases. the RET (Fig. 1) and RB/TP53 (Fig. 2) pathways. will summarize a few key points. The RET product is a single pass transmembrane with RET signalling pathway in MTC an intracellular domain (RTKZ ) that binds glial-derived The RET gene in FMTC and sporadic MTC neurotrophic factor (GDNF) ligands. Ret was initially described as a bona fide proto-oncogene because it is The RET gene has been extensively reviewed elsewhere activated by chromosomal aberrations in papillary (Kodama et al. 2005, Kondo et al. 2006, Santoro & thyroid carcinoma (PTC; Kondo et al. 2006, Santoro & Carlomagno 2006, Asai et al. 2006b). Therefore, here we Carlomagno 2006). Subsequently, it was found that the germ line point mutations in Ret causes MEN 2 syndrome, and similar mutations at the somatic level are the most common genetic alterations identified so far in sporadic MTC (Gagel & Marx 2003, Kouvaraki et al. 2005, Marx 2005, Zbuk & Eng 2007). Most MEN 2B patients (95% of cases) carry the M918T-mutation in RET; the remaining fraction harbours the A883F substitution or other rare mutations. In 98% of MEN 2A, the mutations affect one of the five cysteines in the extracellular cysteine-rich domain of RET. In FMTC, mutations affect either the extracellular cysteines or the intracellular domain of RET (Niccoli-Sire et al. 2001, Gagel & Marx 2003, Kouvaraki et al. 2005, Marx 2005, Elisei et al. 2007, Zbuk & Eng 2007). The genotype– phenotype correlation between the type of Ret mutation and penetrance and expressivity of the disease further supports the prime role exerted by Ret mutations in FMTC (Machens & Dralle 2007). Thanks to this close correlation between a specific genetic lesion and cancer Figure 1 Schematic representation of the RTK (receptor tyrosine occurrence, MEN 2 is the best example in oncology of kinase) signalling pathways. Potential oncoproteins are in white, the efficacy of molecular diagnosis in mainstream whereas tumour suppressors are in grey, kinases. It should be noted that only some signalling effectors are represented. RAS clinical management. In fact, early thyroidectomy in and AKT proteins have several effectors (like RALGDS for RAS) in Ret mutation carriers significantly improved their addition to those represented in the figure. In addition, only some prognosis (Brandi et al. 2001, Gagel & Marx 2003, of the interactions that occur among the various proteins are Skinner et al. 2005, Machens & Dralle 2007). Sporadic represented. For instance, RTKs like RET are known to directly phosphorylate beta-catenin and STAT, besides activating them MTC, particularly more aggressive cases, also frequently through RAS and AKT. Moreover, AKT may directly phosphor- (30–50% of cases) feature the M918T-RET mutation ylate IKK proteins. (Elisei et al.2007, 2008). Finally, susceptibility to

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Downloaded from Bioscientifica.com at 09/29/2021 01:28:29PM via free access Molecular genetics of MTC . A CERRATO and others 145 sporadic MTC could be influenced by the RET RET signalling cascade polymorphisms G691S/S904S (Robledo et al. 2003, Genetic screenings in model organisms have shown Elisei et al. 2004, Cebrian et al. 2005, Lesueur et al. 2006), that the same phenotype can arise from alterations however, these findings require confirmation on larger in any of several genes acting epistatically in casistics (Weber & Eng 2005). common signalling cascades. Similarly, although the As discussed above, secondary genetic alterations at number of potential cancer driver genes is large, this somatic level must act in concert with mutations in Ret probably reflects changes in only a few pathways. For for MTC to develop (Gagel & Marx 2003). Probably, instance, a systematic cancer genome analysis only a small number of secondary genetic events are recently revealed that many mutations in colon and required in MEN 2B mutation carriers because, in these breast cancer cluster in genes acting in few signalling patients, the disease develops in the first few months of cascades (Wood et al. 2007, Sjo¨blom 2008). Thus, if their life. A secondary genetic hit may target the Ret gain-of-function RET-mutations are associated with gene itself, either through duplication of the mutant human MTC, it is equally plausible that mutations in allele or loss of the wild-type allele (Huang et al. 2003). the genes encoding co-receptors/ligands that trigger Additional hits may involve deletion and RET activation or signalling effectors that mediate amplification events, such as the deletion in chromo- RET intracellular effects play a role in MTC. An some lp (Mathew et al. 1987, Khosla et al. 1991, Mulligan important proof of this concept was provided by et al. 1993, Marsh et al. 2003, Ye et al. 2008). genetic analysis of PTC, another thyroid tumour type, MTC-associated Ret-mutations convert Ret into a in which RET is implicated. Indeed, it was found that dominantly transforming oncogene. Extracellular most PTC cases that are negative for RET (RET/ cysteine MEN 2A/FMTC RET-mutants exert constitu- PTC) rearrangements harbour either mutations of tive kinase activity consequent to -independent BRAF, an effector of the RET-initiated extracellular- homodimerization. In the case of mutation M918T, signal regulated kinase (ERK) signalling cascade or, constitutive RET activation probably results from less frequently, in NTRK1, another disruption of an auto-inhibited head-to-tail RET TK receptor (Fig. 1; Fagin 2005, Kondo et al. 2006, homodimer (Knowles et al. 2006). Pierotti & Greco 2006). Transgenic mouse models demonstrated that RET RET is activated through the binding of four GDNF are able to drive MTC formation. Mice family ligands (GFL) GDNF, neurturin (NRTN), artemin expressing RET-C634R or RET-M918T, but not wild-type (ARTN), persephin (PSPN) together with the four RET, under the control of the calcitonin gene promoter corresponding membrane co-receptors (GFRa1, 2, 3 developed MTC (Michiels et al. 1997, Acton et al. 2000). and 4; Airaksinen & Saarma 2002). GFRa4, in particular, Also transgenic mice carrying RET-C634R under the is expressed in normal C-cells and the corresponding control of a ubiquitous viral promoter developed MTC, ligand, PSPN, is required for calcitonin production by suggesting that murine C-cells are highly susceptible to C-cells (Lindahl et al. 2001, Lindfors et al. 2006). No RET-mediated transformation (Kawai et al. 2000). somatic mutation in any of the GFL/GFRA encoding However, the knock-in of the M918T mutation into genes has been reported in MTC (Marsh et al.1997, mouse endogenous Ret gene caused CCH but not MTC, Borrego et al. 1998), although GFL/GFRA genes map in suggesting that, in the background of a normally chromosomal regions where allelic imbalances were expressed Ret-mutant allele, the accumulation of detected in MTC (Marsh et al. 2003). Polymorphic secondary genetic alterations is required for develop- variants of GFL/GFRA genes, particularly GFRA1 in ment of MTC (Smith-Hicks et al. 2000). Genetic familial (Gimm et al.2001a, Lesueur et al. 2006)and background strongly affected the MTC phenotype in GFRA4 in sporadic (Cebrian et al.2005, Vanhorne et al. transgenic mice, with tumour penetrance varying from 2005, Ruiz-Llorente et al. 2007)MTCcases,havebeen 0% in FVB/N to 98% in CBA/ca mice, which suggests reported. that the genetic modifiers greatly affect RET-driven Once activated, RET transmits mitogenic, survival and MTC risk (Cranston & Ponder 2003). motogenic signals (Kodama et al. 2005, Asai et al. 2006b, RET knock-down by dominant-negative mutants, Santoro & Carlomagno 2006). Two major signalling ribozymes or RNAi impaired proliferation of RET-mutant cascades, namely RAS and phosphatidylinositol 3-kinase MTC cell lines (Parthasarathy et al. 1999, Drosten (PI3K), are triggered by RET (Fig. 1). In turn, RAS and et al.2004). Taken together, these studies strongly PI3K contribute to the activation of many signalling implicated RET in the formation and maintenance of a effectors and, as described below, they concur to the subset of MTC, and provided the conceptual framework activation of nuclear factor-kB (NF-kB), signal transdu- for the use of RET kinase inhibitory compounds in MTC cer and activator of transcription (STAT) and beta- clinical trials (Wells & Nevins 2004, Castellone et al. 2008, catenin. Other signalling effectors, namely SRC Schlumberger et al.2008, Sherman 2008). (Encinas et al. 2004, Iavarone et al. 2006), phospholipase www.endocrinology-journals.org Journal of Molecular Endocrinology (2009) 43, 143–155

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Cg (Borrello et al. 1996, Jain et al. 2006), and RAC1/JUN findings excluded that RAS/BRAF gene mutations exert NH(2)-terminal kinase ( JNK; Chiariello et al. 1998, a prominent role in MTC formation. However, a recent Fukuda et al. 2002, Asai et al. 2006a) are activated by RET study led to a different conclusion by showing 41% (Fig. 1). In principle, gain-of-function of these pathways KRAS-mutations and 68% BRAF-mutations in MTC may contribute to MTC. Moreover, negative regulators samples (Goutas et al. 2008). of RET signalling have also been identified and, in The degree and duration of activation dictate principle, their loss-of-function may contribute to MTC the final biological outcome of RAS signalling. formation (Fig. 1). For example, in PC12 pheochromocytoma cells, transient Hereafter, we focus on the RET pathways that have RAS activation stimulates proliferation, whereas been more extensively studied in MTC. Components of sustained RAS activation induces differentiation these pathways may be exploited as molecular targets (Schubbert et al. 2007). Similarly, oncogenic HRAS for MTC treatment. and CRAF or RAF1 alleles decreased MTC cell proliferation and increased calcitonin gene expression (Nakagawa et al.1987, Carson-Walter et al. RAS pathway 1998). Such a pro-differentiating effect of constitutive Growth-factor binding to cell surface RTKs creates RAS–RAF signalling may explain why mutations in docking sites for adaptor molecules that activate these genes are unlikely to occur in MTC. In this guanine nucleotide-exchange factors, which in turn context, NRAS exerted a protective effect against MTC favours GTP binding to RAS small G-proteins (KRAS, formation as shown by the finding that NRAS deletion HRAS and NRAS; Schubbert et al. 2007). Intrinsic RAS- increased MTC formation in RB1-knock-out mice (see GTPase activity terminates signalling, a reaction that is also below; Takahashi et al. 2006). However, the role of accelerated thousands of fold by GTPase-activating RAS signalling in MTC cells is probably complex and proteins such as neurofibromin (NF1; Fig. 1; Schubbert different components of the RAS family may exert et al. 2007). Once activated, RAS stimulates numerous different effects. In fact, when targeted to C-cells, intracellular transducers, including RAF, PI3K and Ral an oncogenic HRAS mutant caused MTC in transgenic guanine nucleotide-dissociation stimulator, to regulate mice (Johnston et al. 1998). Similarly, Moloney murine proliferation, survival and differentiation (Fig. 1; sarcoma virus oncogene (MOS) another oncogene that Halilovic & Solit 2008). The RAS–RAF–MEK–ERK potently activates ERK induced MTC and pheochro- cascade is the best characterized RAS-effector pathway. mocytoma in transgenic mice (Schulz et al. 1992). With There are three RAF serine/threonine kinases (ARAF, the caveat that findings obtained in artificial animal BRAF and CRAF) that activate the MEK (MAP2K1/ models should be interpreted with caution, it is MAP2K2)–ERK (MAPK3/MAPK1) kinase cascade. conceivable that RAS signalling along the ERK cascade ERK, in turn, stimulates gene transcription by directly is involved in mitogenic signalling in MTC cells. In this phosphorylating transcription factors or by targeting context, it is noteworthy that inhibition of the ERK intracellular kinases like p90RSK (Fig. 1; Schubbert pathway reduced proliferation of a RET-mutant MTC cell et al. 2007). Negative regulators attenuate RAS line (Zatelli et al.2005). Inhibitors of MEK are currently signalling at various levels of the signalling cascade undergoing clinical experimentation in thyroid cancer (Fig. 1; see below). patients (Sherman 2008). RAS genes are most commonly activated by point mutations in cancer. Alternatively, the RAS pathway PI3K pathway can be triggered indirectly by loss of the negative regulator NF1, by upstream activation of cell surface Class I PI3K are constituted by a regulatory (p85a, RTKs or PTPN11 (which encodes the Src homology-2- p55a, p50a, p85b, p55g) and a catalytic (p110a, p110b, containing protein tyrosine phosphatases-2 (SHP-2)) p110d) subunit. Upon recruitment to the plasma or by downstream activation of RAS signalling effectors membrane by activated RTK or RAS, class I PI3K (Fig. 1; Wellbrock et al. 2004, Halilovic & Solit 2008). phosphorylates phosphatidylinositol-4, 5-bisphosphate This paradigm applies to thyroid carcinoma of to generate phosphatidylinositol-3, 4, 5-triphosphate follicular cell lineage, where RET gene rearrangements (PIP3; Fig. 1). PIP3, in turn, activates downstream are prevalent in PTC, RAS-mutations in follicular molecules such as the RAC small GTPase, carcinoma and in follicular-variant PTC, and BRAF- 3-phosphoinositide-dependent protein kinase 1 mutations in PTC and anaplastic carcinoma (Kondo (PDK1), and the AKT (also known as PKB) serine/ et al. 2006). Sequencing analysis of all three RAS family treonine kinase (Yuan & Cantley 2008). The lipid members did not reveal any mutation in about 30 MTC phosphatase, phosphatase and tensin homologue samples (Moley et al. 1991, Horie et al. 1995, Bockhorn deleted on chromosome 10 (PTEN) antagonizes this et al. 2000). Similarly, no BRAF-mutation was found in cascade by dephosphorylating PIP3 (Salmena et al. 65 MTC samples (Xing 2005). Taken together, these 2008). Besides, buffering the PI3K pathway, PTEN also

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Downloaded from Bioscientifica.com at 09/29/2021 01:28:29PM via free access Molecular genetics of MTC . A CERRATO and others 147 exerts phosphatase-independent nuclear functions that (sirolimus), mTOR is one of the most appealing may contribute to the potent oncogenic effect resulting therapeutic targets in this pathway (Bjornsti & from its inactivation in tumours (Salmena et al. 2008). Houghton 2004). AKT phosphorylates and inactivates pro-apoptotic transcription factors of the forkhead-box class O (FOXO) family, the cell-cycle inhibitor CDKN1B NF-kB (p27Kip1), and the GSK3B kinase, thereby releasing The NF-kB family includes five transcription factors beta-catenin from the inhibitory effects of GSK3B named NF-kB1 (p50), NF-kB2 (p52), Rel, RelA (p65) (see below; Fig. 1; Yuan & Cantley 2008). In addition, and RelB. NF-kB activates transcription of genes AKT stimulates the serine/threonine kinase mamma- associated with cell proliferation, angiogenesis, metas- lian target of rapamycin (mTOR; Fig. 1). mTOR is tasis, and inflammation and suppression of apoptosis associated with two complexes: the rapamycin-sensitive (Baud & Karin 2009). NF-kB proteins are rendered TORC1 complex (that phosphorylates S6K to regulate inactive in non-stimulated cells through binding to protein translation); and the rapamycin-insensitive inhibitors, known as the IkB(IkB a, b, 3) proteins. TORC2 (which is the PDK2 activity that controls serine Activation of most forms of NF-kB, especially the most 473 phosphorylation of AKT itself; Bjornsti & common form (the p50/RelA dimer), depends on k Houghton 2004). TORC1 also contributes to NF- B phosphorylation-induced ubiquitination of IkB that is activation (see below; Fig. 1). mediated by the IkB kinase (IKK) complex (CHUK, The PI3K–AKT–mTOR cascade is important in IKBKB, IKBKG or NEMO; Baud & Karin 2009). Thus, tumourigenesis because of its ability to promote growth NF-kB is activated by different membrane receptors as (cell size) and proliferation (cell number) and to well as by BRAF that directly associates with IKK prevent cell death. Mutations in major nodes of this (Encinas et al. 2008) and by PI3K/AKT that mediates cascade are prevalent in human cancer and include an mTOR/IKK interaction (Dan et al. 2008; Fig. 1). RET gain-of-function mutations and amplification of the stimulates IKK phosphorylation and NF-kB activation, genes encoding the catalytic subunit p110a of PI3K thus contributing to MTC cell survival (Ludwig et al. (PIK3CA) and AKT (Zbuk & Eng 2007, Yuan & Cantley 2001, Encinas et al. 2008). 2008). Mutations in this pathway are very frequent, for Recent studies have found mutations that directly instance, in breast and colon cancer (Wood et al. 2007). target NF-kB pathway components in human cancer Germ line inactivating mutations of PTEN cause (Wood et al. 2007). A genetic analysis of the NF-kB autosomal dominant hamartoma syndromes, and pathway in MTC has not yet been reported. Histochem- somatic PTEN inactivation by deletion is very frequent ical analysis of MTC tissue samples revealed that many (up to 30–50%) in sporadic tumours (Zbuk & Eng 2007, proteins of the NF-kB family, particularly p65, p52 and Paes & Ringel 2008). Many studies have demonstrated c-Rel, are localized in the nucleus (Gallel et al. 2008). that the PI3K–AKT system plays a key role in RET NF-kB inhibitors, particularly IKK inhibitors, are being signalling (Segouffin-Cariou & Billaud 2000, Kodama exploited in cancer therapy (Baud & Karin 2009). et al. 2005, Asai et al. 2006b). However, no systematic Moreover, inhibitors of the 26S proteasome, such as genetic analysis of PI3K pathway components has been bortezomib (Velcade), that prevent IkB degradation reported so far in MTC. PIK3CA gene amplification, and NF-kB nuclear translocation, exerted cytotoxic which frequently occurs in aggressive tumours of effects in MTC cells (Mitsiades et al. 2006b). thyroid cells of follicular lineage, was not detected in 13 MTC samples (Wu et al. 2005). PTEN analysis in MTC has so far been limited to promoter methylation assessment, and no methylation was detected in a small Beta-catenin (CTNNB1) MTC set (Schagdarsurengin et al. 2006). However, CCH and MTC occur in PTEN heterozygous mice, particularly Beta-catenin, which is encoded by the CTNNB1 gene, when crossed with mice knocked-out for CDKN2C plays an important role in cellular adhesion by (encoding the p18Ink4c cell-cycle inhibitor; see below; associating with E-cadherin and alpha-catenin. Upon Bai et al. 2006). Thus, as discussed for RAS, the PI3K– disassembling of the membrane complex, beta-catenin AKT cascade, even though infrequently mutated, may migrates into the nucleus where it acts as a co-activator play a role in MTC. Accordingly, in vitro chemical PI3K of TCF/LEF (T-cell factor/lymphoid-enhancing factor) inhibition reduced MTC cell proliferation and survival, transcriptional factors (Brembeck et al. 2006). RET which indicates that this pathway could be a molecular stimulates beta-catenin activation via direct phosphoryl- target in MTC treatment (Kunnimalaiyaan et al. 2006a). ation on Y654 and via PI3K/AKT- and RAS/ERK- Given its central role in PI3K–AKT signalling, and mediated inhibition of GSK3B (Fig. 1; Cassinelli et al. the availability of potent and selective inhibitors (ever- 2009, Gujral et al. 2008, Castellone et al. 2009). Although olimus, temsirolimus) derived from rapamycin an analysis of the CTNNB1 gene in MTC has not yet www.endocrinology-journals.org Journal of Molecular Endocrinology (2009) 43, 143–155

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been reported, MTC samples from human patients and Incoronato et al. 2004, Zatelli et al. 2005). Intriguingly, RET(M918T) transgenic mice showed nuclear beta- SHP-1 is involved in the cytostatic effects of somato- catenin accumulation (Gujral et al. 2008). statin in MTC cells (Zatelli et al. 2005). Activated ERKs are inactivated through dephosphor- ylation of threonine and/or tyrosine residues within the STAT activation loop. The DUSP, also called ‘MAP kinase STAT transcription factors are activated in response to phosphatases’ (MKP), carries out this function (Fig. 1; cytokines and growth factors. Cytokines activate STAT Kondoh & Nishida 2007). Intriguingly, MKPs/DUSPs through JAK tyrosine kinases, whereas RTKs can are rapidly induced upon growth factor signalling, and phosphorylate STAT directly. The JAK–STAT pathway functions as feedback regulators of the pathway (Amit has been implicated in several neoplastic diseases, et al. 2007). RET-mediated signalling increased MKP-3 particularly myeloproliferative disorders (Levine & levels (Colucci-D’Amato et al. 2000). In principle, a loss- Gilliland 2008). Oncogenic RET mutants induce serine of-function of MKPs may favour RET signalling along phosphorylation through the RAS pathway, and the ERK cascade. However, it should be noted that the pro-mitogenic and anti-mitogenic effects of MKPs/DUSPs tyrosine phosphorylation of STAT3 (Fig. 1; Plaza may vary depending on the specific complement of Menacho et al. 2005, 2007). Moreover, activated MAPK family members they dephosphorylate. For STAT3 was identified in the nucleus of cells from instance, DUSP4/MKP-2, which dephosphorylates not MTC samples (Plaza Menacho et al. 2005). only p42/44 MAPK (ERK) but also p38MAPK and JNK, exerts a positive (rather than a negative) role in RET- Negative regulators of RET signalling mediated tumourigenesis and it is upregulated in MTC samples (Hasegawa et al. 2008). Several proteins function as feedback regulators to Sprouty (SPRY) and Spred proteins are evolutionarily attenuate RTK signalling and, intriguingly, the corres- conserved inhibitors of signalling that act by blocking ponding genes are often downregulated in diverse RAS–RAF interaction and ERK activation. The tumour types (van Staveren et al. 2006, Amit et al. 2007). expression of SPRY family members is induced by Negative regulators of RET signalling have been RET, and SPRY2 blunted RET–ERK signalling (Ishida identified. However, also in this case, no systematic et al. 2007). Intriguingly, genetic ablation of SPRY2 led analysis of genetic alterations in MTC has yet been to enteric neuronal hyperplasia by promoting RET reported. Below, we briefly discuss the effects exerted signalling (Taketomi et al. 2005). Similarly, SPRY1- by tyrosine phosphatases (LAR, PTPRJ, SHP-1), ERK deficient mice had kidney defects because of RET dual-specificity phosphatases (DUSP) and RAS–BRAF hypersignalling (Basson et al.2005). SPRY/SPRED signalling inhibitors (SPRY) on RET signalling. downregulation has been reported in several human Tyrosine phosphatases dephosphorylate RET and cancers (Lo et al. 2006). Germ line loss-of-function attenuate RET signalling; theoretically, their loss could mutations in SPRED1 caused a neurofibromatosis 1-like promote MTC formation (Fig. 1). Leukocyte common syndrome (Brems et al. 2007). Thus, a loss of SPRY/ antigen-related (LAR) phosphatase (also called SPRED family members in C-cells can, in principle, ‘PTPRF’, protein tyrosine phosphatase, receptor favour MTC formation. type, F) is a receptor tyrosine phosphatase that maps on a region of chromosome 1 (1p) that is frequently lost in MTC (Mathew et al. 1987, Mulligan et al. 1993). Other growth factor receptors LAR forms stable complexes with RET and dephos- It is conceivable that other RTKs, besides RET, are phorylates RET cysteine mutants (but not RET-M918T) involved in MTC. This point is of great topical interest thereby blunting cell proliferation (Qiao et al. 2001). because tyrosine kinase inhibitors are now being tested Similarly, the receptor-protein tyrosine phosphatase J in MTC patients (Castellone et al. 2008, Sherman 2008). (PTPRJ) binds and de-phosphorylates RET cysteine Proliferation of cultured MTC cells is stimulated by mutants and thus impairs their transforming effect insulin-like growth factor 1 (IGF1) and inhibited by (Iervolino et al. 2006). The SHP-1, SHP-2 are non- compounds targeting IGF1-R (Yang et al. 1992, Mitsiades transmembrane phosphotyrosine phosphatases et al. 2004). NTRKs, which are tyrosine kinase receptors (PTPN6 and PTPN11 respectively). While SHP-2 for growth factors of the nerve growth factor family, have functions as a positive RTK signal transducer and been studied in MTC because, like RET, they exert stimulates downstream RET signalling along the RAS neurotrophic effects and are involved in PTC (NTRK1 cascade (D’Alessio et al.2003), SHP-1 serves as a rearrangements; Pierotti & Greco 2006). Moreover, negative regulator of signalling systems. SHP-1 associ- there is functional evidence that NTRK1–RETsignalling ates with RET, restrains RET autophosphorylation, and is involved in neuronal cell survival (Tsui-Pierchala et al. inhibits MTC cell proliferation (Hennige et al. 2001, 2002, Luo et al. 2007, Pierchala et al. 2007). Although no

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Downloaded from Bioscientifica.com at 09/29/2021 01:28:29PM via free access Molecular genetics of MTC . A CERRATO and others 149 mutations have been found in NTRK1, 2 and 3 (Gimm factors, namely, stimulation of cell-cycle progression or et al. 1999, 2001b), NTRK2 expression was reduced, apoptosis (Fig. 2; Hahn & Weinberg 2002). Binding of whereas NTRK3 expression was increased in MTC the pRB protein to E2F/DP transcription factors is (McGregor et al. 1999). Moreover, NTRK2 expression high when pRB is hypophosphorylated in G1, and low impaired the tumourigenicity of MTC cells (McGregor when pRB is hyperphosphorylated in S and G2 phases. et al. 1999). Interaction between epidermal growth pRB is phosphorylated sequentially by D-, E- and A-type factor receptor (EGFR) and RET was recently found to cyclin-mediated CDK activity. In turn, CDKs are mediate EGFR-dependent RET activation (Croyle et al. negatively regulated by CDK inhibitors (CKI) of the 2008). Phosphorylated EGFR has been identified in INK4 (p16INK4A, p15INK4B, p18INK4C, p19INK4D) MTC cells (Gorla et al. 2008). It is noteworthy that and CIP/KIP (p21CIP1, p27KIP1, p57KIP2) families Vandetanib, a RET kinase inhibitor currently being (Fig. 2). Tethering of pRB to E2F target genes results in investigated in MTC patients, is also an EGFR inhibitor cell-cycle arrest (Trimarchi & Lees 2002). There are (Carlomagno et al. 2002). No mutation in EGFR was multiple interactions between the pRB and the p53 found in small MTC sample sets (Mitsiades et al. 2006a, pathways (Fig. 2). On one hand, by stimulating Cerrato & Santoro unpublished). Fibroblast growth transcription of the p21CIP1 (CDKN1A) cell-cycle factor receptor-4 (FGFR4) is expressed in aggressive inhibitor, p53 obstructs the activity of /CDK thyroid tumour types and MTC cells. Molecular complexes, thereby reducing pRB phosphorylation and, targeting of FGFR-4 with an ATP-competitive inhibitor consequently, E2F activity. On the other hand, loss- prevented the growth and reduced the tumourigenesis of-function of pRB releases not only the pro-mitogenic of MTC cells (Ezzat et al. 2005). but also pro-apoptotic activity of E2F transcription Finally, membrane receptors of families other than factors. The final outcome may depend on TP53 genetic the RTK family have been implicated in MTC. status because E2F-mediated apoptosis is dependent on NOTCH1 is a multifunctional transmembrane receptor the upregulation of (CDKN2B) that in turn that regulates cell differentiation, development, stabilizes p53. Therefore, in cancer, RB1 and TP53 are proliferation and survival. Binding of several ligands often concurrently mutated (Hahn & Weinberg 2002). promotes proteolytic cleavage events, which result in There is extensive genetic evidence in rodents that the release of the NOTCH1 intracellular domain that, the pRB and p53 pathways are involved in MTC. RB1- in turn, translocates to the nucleus and activates deficient mice developed MTC (Harrison et al. 1995). transcription of various target genes. NOTCH1 is a Conditional RB1 inactivation also induced highly negative regulator of ASH1 (achaete-scute homolog-1, aggressive MTC in mice (Kucherlapati et al. 2006). called ‘MASH1’ in rodents), which is a highly conserved Loss of TP53 further increased MTC formation in RB1- basic helix-loop-helix transcription factor that is critical deficient mice (Williams et al. 1994, Harvey et al. 1995). for C-cell development (Lanigan et al. 1998). Interest- E2F family transcription factors exerted a dual role ingly, MTC expresses ASH1 but not NOTCH1, and in MTC formation. Genetic deletion of E2F1 or E2F4 NOTCH1 expression arrested proliferation of MTC reduced MTC formation in RB1-deficient mice cells (Kunnimalaiyaan et al. 2006b). The prolactin (Yamasaki et al. 1998, Lee et al. 2002). Instead, deletion receptor (PRLR) belongs to the cytokine receptor of E2F3 further increased the incidence and aggressive- family and activates the JAK–STAT pathway. Unexpect- ness of MTC (Ziebold et al. 2003). edly, PRLR-null mice developed MTC at a high Interestingly, MTC from RB1/TP53-deficient mice frequency, thereby suggesting that PRLR suppresses acquired somatic cysteine mutations in RET that closely MTC formation at least in mice (Kedzia et al. 2005). resemble activating mutations observed in human MTC. This suggested that murine MTC requires mutational dysregulation within both the RET and Tumour suppressors of the RB1 and TP53 nuclear tumour suppressor gene pathways (Coxon et al. pathways in MTC 1998). High grade MTC were observed in mice simultaneously lacking RB1 and CDKN1B (that codes The tumour-suppressor genes RB1 (retinoblastoma: for the p27Kip1 cell-cycle inhibitor; Park et al. 1999). pRB protein) and TP53 (p53 protein) are frequently Interestingly, germ line mutation in CDKN1B predis- mutated in human cancer, and several lines of evidence posed rats to a multiple endocrine neoplasia syndrome indicate that both pathways must be inactivated in featuring MTC formation (Pellegata et al. 2006). In cancer to overcome senescence or apoptosis (Hahn & transgenic mice, the loss of two CDKIs, CDKN1B and Weinberg 2002). RB1 is the prototypic member of the CDKN2C (coding for the p18INK4C cell-cycle class of tumour suppressors known as ‘gatekeepers’, inhibitor), led to accelerated MTC formation (Franklin which control tumour growth in a cell-autonomous et al. 2000, Joshi et al. 2007). CDKN2C deficiency also manner. This mainly depends on pRB’s ability to repress accelerated MTC formation in PTEN-deficient mice the effect exerted by the E2F/DP family of transcription (Bai et al. 2006). Finally, transgenic mice expressing www.endocrinology-journals.org Journal of Molecular Endocrinology (2009) 43, 143–155

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oncogenic RET crossed with mice lacking CDKN2C therapeutic approaches. In any event, the data acquired developed MTC at a higher incidence and sooner than in recent years about the signalling mechanisms their single mutant littermates (van Veelen et al. 2008). operating in MTC show that molecular targeting of Taken together, these studies provide robust evi- pathways like the RAS/ERK, PI3K/AKT and NF-kB dence that, in rodents, disruption of the RB1 and TP53 pathways is a plausible therapeutic approach for this pathways predisposes to MTC formation. However, cancer. mice models may not faithfully mimic the human situation, and the tumour spectrum may significantly differ in the two species. A prominent example of this Declaration of interest concept is provided by the phenotype of RB1-deficient There is no conflict of interest that could be perceived as prejudicing mice. In humans, loss of the RB1 gene is associated with the impartiality of the research reported. the development of retinoblastoma and osteosarcoma and, later in life, small-cell lung carcinoma, whereas RB1-deleted mice do not develop these types of Funding tumours, and develop retinoblastoma only when the RB1-related RBL1 gene is concurrently deleted This work was supported by the Associazione Italiana per la Ricerca (Rangarajan & Weinberg 2003). Previous studies did sul Cancro (AIRC, 2008–2010), the Italian Ministero della Salute (2008-2009), and Ministero dell’Universita’ e della Ricerca (MiUR) not find TP53-mutations in sets of 9 (Yoshimoto et al. (2008–2009). 1992) and 22 (Herfarth et al. 1997) MTC samples. More recent studies identified a high prevalence of TP53 mutations (Pavelic´ et al. 2006) and deletions in MTC Acknowledgements (Sheikh et al. 2004). Very recently, about 10% of MTCs were found to carry loss-of-function mutations in We gratefully acknowledge members of our laboratory for their CDKN2C (van Veelen et al. 2009); however, we did not continuous support. We are grateful to Jean Ann Gilder for text editing and StudioCiotola for art-work. find any CDKN2C-mutation in 15 MTC samples (Cerrato & Santoro unpublished). A systematic analysis of the genes in the RB1 and TP53 pathways in human samples will help to clarify their role in MTC formation. Given References the role played by these tumour-suppressor pathways in Acton DS, Velthuyzen D, Lips CJ & Ho¨ppener JW 2000 Multiple the response of tumours to therapy, this information endocrine neoplasia type 2B mutation in human RET oncogene might be important for the analysis of data from the induces medullary thyroid carcinoma in transgenic mice. Oncogene ongoing MTC trials involving the use of targeted agents. 19 3121–3125. 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