targeting the ret pathway in thyroid cancer

Molecular Pathways

Targeting the RET Pathway in Thyroid Cancer

Samuel A. Wells, Jr.1 and Massimo Santoro2

Abstract The RET (rearranged during transfection) protooncogene encodes a single pass trans-

membrane receptor that is expressed in cells derived from the neural crest and the uro-

genital tract. As part of a cell-surface complex, RET binds glial derived neurotrophic

factor (GDNF) ligands in conjunction with GDNF-family α co-receptors (GFRα).Ligand-induced activation induces dimerization and tyrosine phosphorylation of the

RET receptor with downstream activation of several signal transduction pathways. Ac-

tivating germline RET mutations play a central role in the development of the multiple

endocrine neoplasia (MEN) syndromes MEN2A, MEN2B, and familial medullary thyroid

carcinoma (FMTC) and also in the development of the congenital abnormality Hirsch-

sprung's disease. Approximately 50% of patients with sporadic MTC have somatic RET

mutations, and a significant portion of papillary thyroid carcinomas result from chro-

mosomal inversions or translocations, which activate RET (RET/PTC oncogenes). The

RET protooncogene has a significant place in cancer prevention and treatment. Timely

thyroidectomy in kindred members who have inherited a mutated RET allele, charac-

teristic of MEN2A, MEN2B, or FMTC, can prevent MTC, the most common cause of

death in these syndromes. Also, recently developed molecular therapeutics that target

the RET pathway have shown activity in clinical trials of patients with advanced MTC, a

disease for which there has been no effective therapy. (Clin Cancer Res 2009;15(23):

7119–23)

Background

Medullary thyroid carcinomas (MTC) are derived from theneural crest C cells, not from the follicular cells, as are papillarythyroid carcinoma (PTC) and follicular thyroid carcinoma(FTC). The malignancy presents either sporadically (75% ofcases) or in a hereditary pattern (25% of cases) as either multi-ple endocrine neoplasia (MEN) type 2A, MEN2B, or familialMTC (FMTC). Clinically, the hereditary forms of MTC are char-acterized by complete penetrance but variable expressivity, suchthat the MTC occurs in virtually all patients, however, only 50%of patients with MEN2A and MEN2B develop pheochromocy-tomas, and 30% of patients with MEN2A develop parathyroidhyperplasia. Patients with FMTC only develop MTC (1).The RET (rearranged during transfection) protooncogene

(Fig. 1) is located in the pericentromeric region of chromosome10q11.2 and spans 21 exons (2). RET encodes a receptor tyro-

sine kinase, which is expressed in neuroendocrine cells (includ-ing thyroid C cells and adrenal medullary cells), neural cells(including parasympathetic and sympathetic ganglion cells),urogenital tract cells, and testis germcells. RETprotein is structuredwith an extracellular portion (which contains four cadherin-like repeats, a calcium binding site, and a cysteine-rich region),a transmembrane portion, and an intracellular portion, whichcontains two tyrosine kinase subdomains (TK1 and TK2) thatare involved in the activation of several intracellular signal trans-duction pathways. Alternate splicing of RET produces three iso-forms with either 9, 43, or 51 amino acids at the C terminus,referred to as RET9, RET43, or RET51 (3).A tripartite cell-surface complex is necessary for RET signaling.

One of four glial derived neurotrophic factor (GDNF) family li-gands (GFL), GDNF, neurturin, artemin, or persephin, binds RETin conjunction with one of four glycosylphosphatidylinositol-anchored co-receptors, designated GDNF-family α receptors(GFRα): GFRα1, GFRα2, GFRα3, and GFRα4 (4–8). GDNF pri-marily associates with GFRα1, whereas neurturin, artemin, andpersephin preferentially bind GFRα2, GFRα3, or GFRα4, respec-tively. Mice lacking GFRα4 have defects in calcitonin production,highlighting a role for the persephin receptor in regulating thyroidC cell function (9). Ligand stimulation leads to activation of theRET receptorwith dimerization and subsequent autophosphoryla-tion of intracellular tyrosine residues, which serve as docking sitesfor various adaptor proteins (10, 11).RET is a versatile kinase, which is capable of directly phos-

phorylating multiple downstream targets (Fig. 1). Y905 is abinding site for Grb7/10 adaptors, however, the function of

Authors' Affiliations: 1Center for Cancer Research, National Cancer

Institute, National Institutes of Health, Bethesda, Maryland and2Dipartimento di Biologia e Patologia, Cellulare e Molecolare, Universita'

di Napoli “Federico II”, Naples, Italy

Received 6/19/09; revised 8/27/09; accepted 8/27/09; published OnlineFirst

11/24/09.

Requests for reprints: Samuel A. Wells, Jr., Center for Cancer Research,

National Cancer Institute, National Institutes of Health, Building 10, Room

3-2571, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-435-7864;

Fax: 301-496-9020; E-mail: [email protected].

F 2009 American Association for Cancer Research.

doi:10.1158/1078-0432.CCR-08-2742

7119 Clin Cancer Res 2009;15(23) December 1, 2009www.aacrjournals.org

Cancer Research. on October 16, 2015. © 2009 American Association forclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 24, 2009; DOI: 10.1158/1078-0432.CCR-08-2742

http://clincancerres.aacrjournals.org/

the adaptors has not been completely elucidated. The transcrip-tion factor STAT3 is activated through phosphorylation of Y752and Y928. Janus kinases (JAK), c-Src, and the Ras/ERK pathwayhave also been involved in STAT3 activation by different RET-derived oncoproteins (12). Phosphorylated Y981 is the majorbinding site for Src, which is essential for neuronal survival.Src is also necessary for RET signaling through focal adhesionkinase (FAK), an important mediator of tumor cell migrationand metastases (13). RET 981 is also involved in AKT activation(14). RET phospholipase Cγ interacts with activated RET bybinding Y1015, thereby activating protein kinase C enzymes,

which in turn are key regulators of receptor tyrosine kinases(15). Mice lacking RET 1015 have defects in kidney develop-ment (16). Nonphosphotyrosine dependent mechanisms areinvolved in RET signaling as well. Phosphorylation of RETS697 is critical for activation of the RAC1/JUN NH(2)-terminalkinase (JNK) pathway and mice lacking S697 exhibit entericnervous system defects (17). The C-tail of RET9 but notRET51 binds the PDZ-containing scaffold protein Shank3, con-tributing to sustained ERK and PI3K signaling (18).Finally, Y1062 is a highly important multidocking binding

site for such proteins as DOK1/4/5, Enigma, FRS2, IRS1/2,

Fig. 1. The RET structure and signalingnetwork. The structure of the RETprotein and the signaling network,showing docking sites and targets.ART, artemin; NTN, neurturin; PSP,persephin; Ca++, calcium ion.

7120Clin Cancer Res 2009;15(23) December 1, 2009 www.aacrjournals.org

Molecular Pathways




Shc, and ShcC. Phosphorylation of 1062 also results in activa-tion of major intracellular pathways, including RAS/ERK, PI3K/AKT, nuclear factor κB (NFκB), and JNK (10, 12, 19–25). Thewell-characterized RAS > RAF > MEK > ERK pathway plays animportant role in thyroid carcinoma as RAS mutations arefound in FTC and in the follicular variant of PTC. BRAF muta-tions are found in PTC and anaplastic thyroid carcinoma. Therole of RAS mutations in MTC is controversial (26, 27). ThePI3K > AKT pathway plays a key role in RET signaling, however,to date there has been little genetic analysis of this pathwayin MTC.Recently, the biochemical characterization and structure of

the human RET tyrosine kinase domain was reported toshow that both the phosphorylated and nonphosphorylatedforms adopt the same active kinase conformation necessaryto bind ATP and substrate and have a pre-organized activa-tion loop conformation that is independent of phosphoryla-tion status (28).

Clinical-Translational Advances

In 1990 Grieco and associates reported that some PTCs werecaused by a transforming oncogene, resulting from the rear-rangement of a heterologous amino terminal sequence to thetyrosine kinase domain of RET (29). It was subsequently shownthat this hybrid oncogene, termed RET/PTC, was present in ap-proximately 5 to 40% of PTCs and resulted from double-strandedDNA breaks with erroneous rearrangements involving theC-terminal portion of RET and the promoter and coding regionof the N terminus of a constitutively expressed unrelated genefrom chromosome 10 or a different chromosome (30). Thesefusion oncogenes were found to be particularly common inpatients previously exposed to radiation, such as the childrenliving near the Chernobyl nuclear accident, in patients exposedto external irradiation, or in victims of the atomic bomb explo-sion in Japan during World War II (31–33). Spatial proximityof RET and fusion partner's loci involved in the recombinationin interphase chromatin has been shown as a mechanism ofradiation-induced recombination (34). Most PTCs, however,are caused by an activating mutation in the B isoform of Rafkinase, BRAFV600E (35). PTCs caused by BRAF mutations, com-pared with those resulting from RET/PTC translocations, arelarger, have a higher incidence of local tissue invasion and lymphnode metastases, and have a poorer clinical prognosis (36).In 1993 and 1994 it was shown that point mutations in the

RET protooncogene cause MEN2A, MEN2B, and FMTC (seeCOSMIC catalog3 and the MEN2 database;4 refs. 37, 38).MEN2A is associated with mutations involving the extracellularcysteine codons 609, 611, 618, 620 (exon 10) 630, or 634 (ex-on 11). The mutations associated with FMTC involve a broadrange of codons including some associated with MEN2A, par-ticularly, 609, 618, and 620, as well as others: 768, 790, and791 (exon 13), 804 and 844 (exon 14), or 891 (exon 15). In95% of patients with MEN2B there is a point mutation in co-don 918 (exon 16, Met918Thr) within the intracellular domain

of RET (39). A few patients with MEN2B have a mutation incodon 883 (exon 15; ref. 40). Single MEN2B patients with dou-ble RET mutations Val804Met + Ser904Cys and Val804Met +Tyr806Cys have been reported (41, 42). In MEN2A and FMTCthe RET mutations lead to a ligand-independent homodimeri-zation and constitutive kinase activity such that RET becomes adominant oncogene. In MEN2B RETmutations activate the RETreceptor in its monomeric state, leading to phosphorylation ofY1062 and other tyrosines, and also causing a change in sub-strate specificity (9).It is likely that RET does not act alone in the genesis of he-

reditary MTC, and studies of MTC tissues and cell lines frompatients with MEN2A have shown evidence of a second hit asevidenced by trisomy 10 with duplication of the mutant RETallele, loss of the wild-type RET allele, and a tandem duplica-tion event with amplification of mutant RET (43). Almost cer-tainly additional genetic defects, such as chromosomaldeletions and amplifications will be shown in future studies.Probably, given the young age at presentation, fewer additionalgenetic alterations occur in MTC in patients with the germlineMEN2B mutation. A recent array CGH study unveiled manygene copy number alterations in MTC patients (44). Approxi-mately 50% of patients with sporadic MTC have somatic muta-tions in RET codon 918 and these tumors seem to be moreaggressive clinically compared with tumors lacking the somaticRET mutation (45).In patients with MEN2A, MEN2B, and FMTC there is a corre-

lation between genotype and phenotype, both in the clinicalexpression of the disease spectrum and the clinical aggressive-ness of MTC. Whereas, virtually all patients develop MTC,pheochromocytomas develop in approximately 50% of pa-tients with MEN2A and MEN2B. In MEN2A they occur mostoften in patients with RET mutations in codon 634 and less fre-quently in patients with mutations in codons 618, 620, or 791.Hyperparathyroidism occurs only in patients with MEN2A whohave mutations in codons 618, 630, 634, or 791. Consideringthe age of onset and clinical aggressiveness of MTC, patients areclassified as being at highest risk, higher risk, or high risk. Pa-tients with MEN2B, who have mutations in codons 918 or 883represent the highest risk group, whereas patients with muta-tions in codons 634, 630, 609, 611, 618, and 620 are in thehigher risk group. The high risk group includes patients withmutations in codon 768, 790, 791, 804, and 891 (46).Rarely, the dermatologic disorder cutaneous lichen amyloid-

osis and the congenital disorder Hirschsprung's disease (HSCR;aganglionic megacolon) may occur in association with MEN2A(46, 47). Hirschsprung's disease, the most common cause of in-testinal obstruction in the newborn, is characterized by the ab-sence of enteric ganglia in variable segments of intestine. A RETmutation has been identified in only 50% of familial and 15 to20% of sporadic cases of HSCR (48, 49). More than 100 differ-ent RETmutations have been described, and, in contrast to mu-tations in MEN2A, MEN2B, FMTC, and sporadic MTC, whichare virtually all missense mutations, they include microdele-tions, insertions, nonsense and missense, and splicing muta-tions. In some cases the entire RET gene is deleted (50). TheRET mutations associated with MEN2A, MEN2B, and FMTCsyndromes are generally gain-of-function mutations, whereasthe RET mutations occurring in HSCR are loss-of-function mu-tations, most likely associated with a haploinsufficiency ordominant negative effect.

3 http://www.sanger.ac.uk4 http://www.arup.utah.edu/database/MEN2/MEN2_welcome.php






Rarely, patients with MEN2A who have RET mutations in co-dons 609, 611, 618, or 620 (exon 10) develop HSCR (47).Functional studies have shown that cell surface expression ofthese RET mutants is lower than that associated with otherRET mutations, such as 634 (51). Thus, these four mutationslead to constitutive activation of RET and continued prolifera-tion of C cells on one hand, whereas on the other, they result ina marked reduction of RET expression at the cell membrane,and a resultant apoptosis of enteric neurons.There is no better example of the translation of genetic infor-

mation into clinical practice than that provided by detection ofRET mutations in patients with hereditary MTC. Shortly, afterthe discovery that MEN2A, MEN2B, and FMTC were causedby mutations in the RET protooncogene, it became clear thatfamily members who carried a mutated RET allele could be de-tected by direct DNA analysis. Knowing that the MTC was themost common cause of death in patients with these syndromesand that the thyroid gland was an expendable organ whosefunction could be easily replaced by the oral administrationof the drug thyroxin, several centers initiated screening pro-grams in MEN type 2 families to identify kindred members withRET mutations. The thyroid gland can be removed safely andthe operation is curative if done at a young age, before theMTC develops or is still confined to the thyroid gland (52,53). Although, there is general agreement that prophylactic thy-roidectomy is indicated in members of families with hereditaryMTC, the timing of thyroidectomy depends on the site of theRET codon mutation. Thyroidectomy is advised at a very earlyage (even in the first months of life) in patients at highest risk(mutations in codon 918 or 883), at 5 years of age in patients athigher risk (mutations in codons 611, 618, 620, and 634), andbetween 5 and 10 years of age in patients at high risk (muta-tions in codons 609, 768, 790, 791, 804, or 891; refs. 54, 55).The finding that the molecular targeted therapeutic (MTT)

STI571 (imatinib mesylate, Novartis AG) induced remissionsin patients with chronic myelogenous leukemia has had aprofound effect on clinical medicine and has changed the land-scape of cancer therapy (56). The anilinoquinazoline vandeta-nib (AstraZeneca Limited) selectively inhibits the kinase insert

domain-containing receptor [KDR/vascular endothelial growthfactor receptor (VEGFR2) tyrosine kinase activity (IC50 ∼40 nM)]. The compound also has activity against VEFGR3;IC50 = 110 nM and epidermal growth factor receptor (EGFR/HER1; IC50 = 500 nM; ref. 57). In 2002 Carlomagno and associ-ates showed that ZD6474 is a potent inhibitor (IC50 = 100 nM)of RET oncoproteins. These investigators also found thatZD6474 has a marked inhibitory effect on the growth of thy-roid cancer cell lines with spontaneous RET/PTC rearrange-ments and also the growth of NIH-RET/PTC xenografts (58).Phase I studies showed that ZD6474 administration is associat-ed with minimal toxicity and subsequent phase II clinical trialsshowed that ZD6474 has activity in patients with locally ad-vanced or metastatic hereditary MTC. Approximately 20% ofpatients experienced a partial remission, as shown by RECIST(response evaluation criteria in solid tumors) criteria and an-other 60% experienced stable disease for a disease control rateof 80% (59). Following treatment, there was reduction in se-rum levels of calcitonin and carcinoembryonic antigen, tumormarkers secreted by the MTC cells. Subsequently, it was shownthat vandetanib induced confirmed partial remissions in 85%of children with advanced MEN2B (60). Phase II clinical trialsof other MTTs have also shown activity in patients with ad-vanced MTC (61, 62).The molecular analysis of tumor tissue in patients whose

chronic myeloid leukemia recurred following initial treatmentwith imatinib mesylate was fundamental to the design of suc-cessful rescue therapy and to the development of combinatorialregimens to prevent disease recurrence following primary treat-ment (63). Unfortunately, there has been a paucity of suchstudies in clinical trials of MTTs in advanced thyroid cancer, pri-marily because tissue acquisition has been infrequent or non-existent. This issue must be corrected if we are to improve thetreatment of patients with this disease.

Disclosure of Potential Conflicts of Interest

S.A. Wells, Jr., consultant, AstraZeneca. M. Santoro, commercial

research grant, AstraZeneca.

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2009;15:7119-7123. Published OnlineFirst November 24, 2009.Clin Cancer Res Samuel A. Wells, Jr. and Massimo Santoro

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