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Molecular basis of epithelial thyroid tumorigenesis
C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528 © 2000 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés S0764446900001700/REV
Concise review paper / Point sur
Molecular basis of epithelial thyroid tumorigenesis Horacio Guillermo Suareza* a
Laboratoire de génétique moléculaire UPR 42 CNRS, Institut de recherches sur le cancer – IFR Y 1221, 7, rue Guy-Môquet, BP 8, 94801 Villejuif cedex, France
Received 7 January 2000; accepted 27 March 2000 Communicated by Editorial board
Abstract – The results of experiments carried out in different laboratories (including ours) during the last 10 years have enabled us to propose the hypothesis that there are different initiators able to start the epithelial thyroid tumorigenic process via different pathways: – gsp and TSHR genes: at the origin of hyperfunctioning tumours (toxic nodules and adenomas); – ras and probably gsp genes (in a minority of samples): via a vesicular adenoma progressing eventually to a vesicular carcinoma. This could be also the case for ret but only in radiation-associated tumours; – ras, ret, trk and probably gsp and met: starting from small papillary lesions (‘spontaneous’ or radiation-induced) and progressing to a clinically evident papillary carcinoma; – the p53 gene playing a role only in the final dedifferentiation process. Simultaneous alteration in the same sample of combinations of ras, gsp, ret, trk and TSHR was found in only a minority of the approximately 150 tumours studied. These data suggest an interchangeable role for these genes in the initiation of ‘spontaneous’ or radiation-associated epithelial thyroid tumorigenesis. The requirement of one of the genes cited above to interact with other genes must not be neglected. Ras is the most frequently altered gene in ‘spontaneous’ thyroid tumours and ret in radiation-associated thyroid tumours. © 2000 Académie des sciences/ Éditions scientifiques et médicales Elsevier SAS thyroid / tumorigenic process / genetic alterations Résumé – Bases moléculaires de la tumorigenèse épithéliale thyroïdienne. Les résultats des expériences effectuées dans différents laboratoires (dont le nôtre) au cours des 10 dernières années ont conduit à l’hypothèse qu’il existe plusieurs initiateurs capables de déclencher la tumorigenèse épithéliale thyroïdienne à travers des voies différentes : 1o les gènes gsp et TSHR: à l’origine des tumeurs hyperfonctionnelles (nodules et adénomes toxiques) ; 2o les gènes ras et probablement gsp (dans une infime minorité de cas) : via un adénome vésiculaire progressant éventuellement vers un cancer de la même morphologie (cela pourrait aussi être le cas pour ret seulement dans les tumeurs associées aux radiations) ; 3o les gènes ras, ret, trk et probablement gsp et met : à partir de petites lésions papillaires « spontanées » ou radio-induites (« carcinome papillaire occulte ») progressant vers un cancer papillaire cliniquement évident ; 4o le gène p53 jouant un rôle dans les étapes finales de la dédifférentiation. Sur environ 150 tumeurs « spontanées » ou associées aux radiations étudiées, seulement une minorité présente deux ou plusieurs de ces gènes altérés simultanément. Ce fait plaide en faveur d’un rôle d’initiateur alternatif de ras, gsp, TSHR, ret ou trk dans la tumorigenèse épithéliale thyroïdienne et montre qu’il est possible de trouver pendant ce processus, dans des cas très rares, plus d’un gène altéré dans la même tumeur. La possiblité d’une coopération de ces gènes entre eux et/ou avec d’autres gènes non-identifiés, ne peut pas être négligée. Ras est le gène le plus fréquemment altéré dans les tumeurs « spontanées »
* Correspondence and reprints: [email protected]
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et ret dans celles associées aux radiations. © 2000 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS thyroïde / tumorigenèse / altérations génétiques
Version abrégée 1. Activation des gènes de la famille RAS Les trois petites protéines G appartenant à la famille ras (H-, K- et N-ras) (p21) représentent un switch moléculaire oscillant entre un état basal à GDP et un état actif à GTP. Une mutation affectant les codons 12/13 ou 61 active la protéine en supprimant sa capacité GTPasique. Ces protéines H-, K- et N-ras assurent la transmission des signaux de divers récepteurs à activité TK et couplés aux protéines G vers des kinases à activité sérine/thréonine. Les voies empruntées par ras doivent être d’une importance capitale pour la cellule vésiculaire, car une mutation activatrice de l’un de ces gènes a été retrouvée par nous dans près de 30 % des tumeurs vésiculaires bénignes aussi bien que malignes. Les mutations activatrices de ras ont été observées avec une fréquence similaire dans les adénomes folliculaires et les carcinomes soit de type papillaire soit de type folliculaire. Les mutations sont aussi bien des transitions que des transversions. Nous avons recherché aussi dans 39 tumeurs de la thyroïde provenant de malades ayant reçu à un moment donné de leur vie, une irradiation à visée médicale du cou, de la tête ou du thorax, la présence de mutations activatrices des oncogènes ras. La seule différence que nous avons trouvée entre les tumeurs thyroïdiennes radio-induites et celles « spontanées », réside dans le mécanisme de mutation des gènes ras. En effet, tandis que dans les tumeurs « spontanées » nous avons trouvé des transversions et des transitions avec une fréquence similaire, dans les tumeurs radio-induites nous n’avons détecté que des transversions, notamment des substitutions d’un G par un T (environ 70 % des cas). Ces lésions pourraient être la conséquence d’un mécanisme radicalaire d’oxydation induit par les radiations ionisantes et produisant des 8-oxo-dG, lesquelles peuvent s’apparier avec des adénines pendant la réplication de l’ADN.
2. Activation de la voie de l’AMPc dans les tumeurs hyperfonctionnelles thyroïdiennes La voie de l’AMPc induit à la fois la prolifération et la différenciation de certains tissus endocriniens comme
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ceux de la thyroïde et de l’hypophyse. Dans les tumeurs hyperfonctionnelles que développent parfois les deux glandes, le fait que la voie de l’AMPc soit maintenue dans un état d’activation permanente entraîne la prolifération excessive (normalement très réduite) et l’hyperfonctionnement des cellules endocrines concernées. Dans le cas de la thyroïde, les tumeurs hyperfonctionnelles (non malignes, en général) présentent un taux de base élevé de l’activité adényl cyclase (AAC) ne répondant plus ou très peu, à la stimulation par la TSH (thyroid stimulating hormone). On dit qu’elles ont un phénotype AAC+/TSH–. Nous, et d’autres groupes, avons montré que le phénotype AAC+/TSH– était dans environ 50 % des tumeurs examinées, la conséquence de : a) mutations situées sur les codons 201 ou 227 de la sous-unité a de la protéine Gs, entraînant son activation constitutive (oncogène gsp) (∼25 % des tumeurs) et b) mutations du gène du récepteur à sept domaines transmenbranaires de la TSH (TSHR) (∼25 % des tumeurs). L’effet de ces mutations sur l’activité biologique des protéines respectives, a été montré in vivo et in vitro.
3. Altérations activant des gènes récepteurs de facteurs de croissance de la famille des tyrosines kinases 3.1. Oncogène ret Le proto-oncogène ret est un récepteur à activité tyrosine kinase (TK) qui est activé, comme d’autres gènes de cette famille, non par mutation ponctuelle, mais par un remaniement du gène. Les ligands de ret sont le GDNF (Glial cell line derived neurotrophic factor) et la neurturine. La protéine normale comporte une partie N-terminale extracellulaire qui, en l’absence du ligand, semble inhiber l’activité du domaine TK intracellulaire. À la suite d’une cassure du gène ret, suivie d’une translocation ou d’une inversion chromosomique, le domaine TK de la protéine est séparé de la région inhibitrice en amont. Celle-ci est remplacée par la partie N-terminale d’une protéine « neutre », et le domaine TK se trouve donc libéré du contrôle inhibiteur normal. Dans la thyroïde, on a trouvé des réarrangements activateurs de ret avec un gène de fonction inconnue, nommé H4, plus récemment avec le gène de la sous-unité RIa de la protéine kinase A et enfin, avec deux gènes non encore caractérisés appelés ELE-1 et RFG5 (ret fused gene 5). Les cinq gènes chimères
H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
produits par la fusion de ret avec ces quatre gènes étrangers s’appellent respectivement ret/PTC1 à ret/ PTC5. Ces réarrangements ont été trouvés avec une fréquence de 10-15 % exclusivement dans les carcinomes « spontanés » de type papillaire de la thyroïde. Les résultats des études effectuées par nous (Saïd et al., 1994) et dans d’autres laboratoires (Fugazzola et coll., 1995 ; Ito et al., 1994 ; Klugbauer et al. 1995 ; Nikiforov et al., 1997), sur les tumeurs de type carcinome papillaire de la thyroïde apparues après l’accident de Tchernobyl, ont montré une fréquence très élevée de réarrangements activateurs du gène ret de type ret/PTC. Cette fréquence était d’environ 60–80 % contre 5–30 % (dépendant des études) dans les tumeurs « spontanées ». Ces observations nous ont amenés à étendre nos recherches à des tumeurs thyroïdiennes bénignes et malignes, apparues chez des patients ayant subi une irradiation à visée médicale de la tête, du cou et/ou du thorax (39 tumeurs). Nos résultats ont montré que la fréquence absolue de réarrangements ret/PTC dans ces tumeurs était de 64 %. Le gène chimère le plus fréquemment trouvé dans nos séries était ret/PTC1 (∼75 %). Les tumeurs porteuses d’un gène ret/PTC3 étaient minoritaires et ret/PTC2 n’a jamais été mis en évidence. Ces derniers résultats sont en contradiction avec les observations effectuées dans les tumeurs postTchernobyl où le type de réarrangement prédominant était ret/PTC3 (∼70 %), suivi dans l’ordre de ret/PTC1 et ret/PTC2. Notre étude a montré aussi, pour la première fois, la présence d’un gène ret/PTC1 dans 45 % des adénomes folliculaires associés aux radiations thérapeutiques. 3.2. Oncogène trk Dans les tumeurs thyroïdiennes, trk est associé d’une manière similaire à ret à trois gènes activants, codant pour des protéines structurales : la tropomyosine nonmusculaire, tpr (translocated promoter region) et TAG (trk activating gene). Comme ret, le gène trk a été trouvé activé par réarrangement exclusivement dans les carcinomes papillaires « spontanés » (fréquence 5–15 %). Outre cet effet qualitatif, le nouveau gène hybride acquiert le taux d’expression du partenaire non oncogénique, ce qui entraîne généralement sa surexpression.
1. Introduction The roles of somatic mutations, gene rearrangement(s) and level of gene expression in carcinogenesis are now well established. Several techniques can be used to detect
Nous avons recherché aussi dans nos tumeurs associées aux radiations, la présence d’un proto-oncogène trk activé. Nos études ont montré que : a) la présence de réarrangements activateurs, était similaire dans les tumeurs « spontanées » ou associées aux radiations (∼8 %) ; b) l’oncogène TRK jouerait un rôle dans le développement de seulement une minorité des carcinomes papillaires associés aux radiations thérapeutiques et c) contrairement aux résultats obtenus avec ret, nous n’avons pas observé de réarrangements activateurs de trk dans les tumeurs bénignes de la thyroïde associées aux radiations (adénomes folliculaires). Les réarrangements activateurs du proto-oncogène TRK étaient, dans nos tumeurs radio-induites, uniquement de type intrachromosomique (ch.1) avec le gène de la tropomyosine non-musculaire (TPM3), donnant lieu à la formation du gène chimère trk-a.
4. Étude combinée des gènes ras, gsp, ret et trk dans une même tumeur Sur plus de 150 tumeurs « spontanées » ou associées aux radiations étudiées, dans seulement dix cas, deux ou plusieurs de ces gènes ont été trouvés altérés simultanément dans la même tumeur. Ce fait plaide en faveur d’un rôle d’initiateur alternatif de ras, gsp, ret ou trk dans la tumorigenèse épithéliale thyroïdienne et montre qu’il est possible de trouver pendant ce processus, dans des cas très rares, plus d’un gène altéré dans la même tumeur. La possibilité d’une coopération de ces gènes entre eux et/ou avec d’autres gènes nonidentifiés ne peut pas être négligé. Ras est le gène le plus fréquemment altéré dans les tumeurs « spontanées » et ret dans celles associées aux radiations.
5. Gènes suppresseurs de tumeurs Les mutations ponctuelles inactivatrices du gène p53, ont été les seules altérations génétiques concernant un gène suppresseur de tumeurs, observées dans les tumeurs folliculaires tyroïdiennes « spontanées » ou radio-induites. Elles ont été mises en évidence exclusivement dans les carcinomes anaplasiques, suggérant que p53 pourrait jouer un rôle dans la dédifférentiation et l’agressivité tumorale.
such genetic alterations in human tumours [1]. The application of these techniques to thyroid tumours has focused particular attention on the role of point mutations activating (or inhibiting) the genes for the TSH receptor (TSHR) [2–4], ras [5–8], gsp [9, 10] and p53 [11], specific rearrangements of the oncogenes ret and trk [12–16] and
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Table I. Frequency of ras mutations in ‘spontaneous’ human thyroid tumours in different studies.
Country Canada (19) UK (20) Hungary (19) Italy (12) Italy (14) USA (21) USA (22) France (23) Overall
PC
FC
AC
AD (cold)
HN
0 (0/10) 21 (4/19) 0 (0/12) 13 (2/16) 0 (0/20) 21 (3/14) 6 (1/15) 45 (16/35) 18 (26/141)
10 (1/10) 52 (11/21) 50 (3/6) nd
nd
nd
55 (6/11) nd nd
20 (2/10) 27 (8/29) 92 (12/13) nd
nd
nd
nd
nd
0 (0/3) 14 (2/14) 35 (7/20) 32 (24/74)
nd
25 (6/24) 0 (0/9) 40 (12/30) 35 (40/115)
nd
nd 100 (1/1) 58 (7/12)
nd nd nd
nd 7 (2/28) 7 (2/28)
PC: papillary carcinoma; FC: follicular carcinoma; AC: anaplasic carcinoma; AD: cold adenoma; HN: hot nodule. Results are given as % frequency of activation with (positive tumours/studied tumours) in parentheses; nd: not done.
alterations in the pattern of expression of the oncogene met [17, 18]. In this review we will present data concerning the activation of ras, gsp, TSHR, ret and trk and discuss whether they play an alternative or a complementary role in human epithelial thyroid ‘spontaneous’ or radiation-induced tumorigenesis (the most frequent radiation-induced tumorigenic process in human). We will also discuss the role of tumour-supressor genes in this process.
2. Activation of ras family genes The products of members of this oncogene family (HaKi- and N-ras) are 21-kD proteins (p21) with nucleotide binding activity, involved in the transduction of information from the cell surface to the nucleus. These genes are all oncogenically activated by a single amino acid substitution in codon 12 or 61 and sometimes in codon 13 or 59 [9]. The activation of the ras oncogenes by point mutation was found in about 30 % of ‘spontaneous’ epithelial thyroid tumours studied in different laboratories and was the most frequent genetic alteration in this type of tumour (table I). The mutations, which in ‘spontaneous’ thyroid tumours are transitions as well as transversions, are randomly distributed between the three ras genes with similar frequencies (11–15 %), without predominance of one of the critical codons or their constituting bases. The mutations occur in follicular adenomas, papillary and follicular carcinomas, at approximately the same frequency (table I). The thyroid gland is, along with the colon, a tissue in which ras mutations are observed in benign tumours [1]. Compared to other studies, we found a higher frequency of ras mutations in ‘spontaneous’ papillary carcinomas (table I). This discrepancy may be the consequence of a
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difference in iodine intake between the different populations [19–23]. Concerning thyroid tumours from patients with a history of external radiation for benign or malignant conditions, activating mutations in the ras genes have been described, and their overall frequency (about 30 %) (table II) was similar to that observed in ‘spontaneous’ benign or maligant tumours [20, 24]. In tumours arising in children after the Chernobyl accident, Nikiforov et al. [25] observed ras point mutations in follicular carcinomas and adenomas but not in papillary carcinomas (table II). It has been concluded that ras does not appear to be important in the development of the Chernobyl papillary carcinomas. Challeton et al. [24] showed that all three ras genes were mutated with similar frequencies in malignant radiation-associated tumours, as was the case in ‘spontaneous’ tumours (see above). However, the mechanism by which ras mutations occurs seems to be different between ‘spontaneous’ and radiation-associated tumours. Whereas in ‘spontaneous’ thyroid tumours, as previously mentioned, transversions as well as transitions were detected in the ras genes [3, 9, 23], in radiation-associated benign and malignant tumours, only transversions (mainly G→T) were present [24]. It can be postulated that the mutations in the radiation-induced tumours could have arisen through an ionizing radiation-associated oxidative lesion, producing 8-OXO-dG which can pair with A during DNA replication. Mutations of ras which, as stated, can be found in ‘spontaneous’ and radiation-associated adenomas or carcinomas at roughly the same frequency, can be proposed as an early event in the thyroid tumorigenic process. Support for this hypothesis has therefore been obtained from experiments in which mutant ras has been introduced into normal follicular cells either in vitro or in vivo
H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
Table II. Frequency of ras and gsp mutations in human radiation-associated thyroid tumours.
Country
UK* (20) France* (24) Belarus** (25) Overall
RAS
RAS
PC
FC
AC
AD (cold)
PC
FC
AC
AD (cold)
50 (1/2) 40 (6/15) 0 (0/33) 14 (7/50)
60 (3/5) 0 (0/2) 100 (1/1) 50 (4/8)
0 (0/1) nd
0 (0/4) 25 (4/16) 43 (3/7) 25 (7/27)
nd
nd
nd
nd
7 (1/15) nd
50 (1/2) nd
nd
0 (0/16) nd
7 (1/15)
50 (1/2)
nd 0 (0/1)
nd nd
0 (0/16)
Abbreviations are as in table I. Results are given as overall frequency (%) with (positive tumours/studied tumours) in parentheses. * Tumours from patients irradiated for benign or malignant conditions; ** post-Chernobyl tumours.
[1]. Finally, activated ras protein has also been shown to stimulate the growth and inhibit differentiation of thyroid epithelial cells [3, 9, 26].
3. Genetic alterations activating the cAMP pathway in hyperfunctioning tumours Increasing evidence demonstrates that constitutive activation of adenylate cyclase (AC) induced by acquired or inherited mutations of Gs protein and G-protein-coupled receptors plays a major role in human disease [1]. In thyroid tumours, the cAMP signalling cascade is rarely involved in malignant transformation, even if activating mutations of gsp (the α subunit of Gs protein) and TSH receptor (TSHR) genes have been described in a subset of differentiated thyroid carcinomas with high basal adenylate cyclase activity [5, 10, 23]. On the contrary, it has been firmly established that constitutive activation of the cAMP cascade through the mutation of gsp or TSHR, is responsible for the hyperfunction and growth of a majority of thyroid follicular toxic adenomas [6, 7, 27–30]. Our experiments, studying the entire coding sequence of both genes in the same hyperfunctioning tumours, showed that both the Gαs protein and TSHR alterations
may contribute with a similar frequency (about 25–30 %) to the constitutive activation of AC in this type of tumour [5, 6, 7, 23]. However, in a subgroup of hyperfunctioning adenomas (40–50 %), no mutations were identified in the genes studied. It may be hypothesized that in gsp- and TSHR-negative tumours, alterations in another gene participating in the cAMP pathway (e.g. AC or protein kinase A (PKA)) may be responsible for the phenotype. The mutations detected in gsp and TSHR genes are concentrated in domains of the protein that are important for their normal biological activity. In the case of gsp the two ‘criticial’ codons are 201, related to the GTPase activity of the protein, and 227, the equivalent of ras codon 61 and located in the GTP binding domain [4, 23, 28]. For TSHR the mutations are predominantly present in an area of the receptor concerned in the interaction with the Gs protein and including the III intracellular loop and the adjacent VI transmembrane region [7, 30]. A role for gsp activating mutations as an early event in the development of toxic adenomas has been shown using transgenic mice [31]. The gsp oncogene was also studied by Challeton et al. [24] in 33 radiation-associated thyroid tumours (table II). The comparison of these results with data obtained in other studies of ‘spontaneous’ hypofunctional tumours ([23] and table III) showed that the overall frequency (5–10 %) and the spectrum of mutations in the gsp oncogene were similar in both types of tumour.
Table III. gsp oncogene in ‘spontaneous’ human thyroid tumours in different studies.
Country France (10) France + Italy (6) UK (28) USA (4) Overall
PC
FC
AC
AD (cold)
HN
9 (3/35) nd
10 (2/20) nd
0 (0/1) nd
7 (2/30) nd
0 (5/5) 0 (0/14) 15 (8/54)
0 (0/3) 0 (0/3) 8 (2/26)
nd
0 (0/16) 0 (0/12) 3 (2/58)
32 (9/28) 25 (9/37) 38 (5/13) 7 (1/14) 26 (24/92)
nd 0 (0/1)
Abbreviations are as in table I. Results are given as overall frequency (%) with (positive tumours/studied tumours) in parentheses.
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4. Gene alterations activating genes from the tyrosine protein kinase receptor family
It has been reported that in humans external radiation as a consequence of an atomic accident (e.g. Chernobyl) or as a treatment for benign or malignant conditions may be a direct inducer of activating ret rearrangements at relatively high frequency [13, 39–41].
Since the discovery of tyrosine kinases over a decade ago, this gene superfamily has been steadily growing to reach its current size of almost 50 members [32]. C-ret and the members of the trk family (trk, trkB, trkC and trkD), which plays a key role in epithelial thyroid tumorigenesis, belong to this superfamily.
Concerning Chernobyl tumours, different studies [39–41] found ret/PTC rearrangements in 55–85 % of the tumours, which were all papillary carcinomas. Ret/PTC3 was the most frequent chimeric gene in each of the studies, the other rearrangements being of the PTC1 and PTC2 types (figure 2), in contrast to what is observed in ‘spontaneous’ papillary carcinomas where the frequency of PTC1 and PTC3 is more or less the same (48 and 45 %, respectively).
4.1. ret oncogene
The ret proto-oncogene, located on chromosome 10q11.2, encodes a protein structurally related to transmembrane receptors with a tyrosine kinase domain [13, 16], and recently, it has been shown that its putative ligands are the glial-cell-line-derived neurotrophic factor (GDNF) and the neurturine [33]. Five oncogenically activated forms of the ret proto-oncogene have been identified in epithelial thyroid tumours and designated ret/PTC1 to PTC5 [1]. All these activated forms of the proto-oncogene are the consequence of specific oncogenic rearrangements fusing the tyrosine kinase domain of ret with the 5’ domain of different genes (figure 1). The 5’ domain of the foreign genes, acting as a promoter, permanently activates the tyrosine kinase activity of ret, normally undetected in the thyroid follicular cells. ret/PTC1 is formed by an intrachromosomal rearrangement fusing the ret tyrosine kinase domain to a gene designated H4, whose function is still unknown [1, 16]. In ret/PTC2, the catalytic domain of ret is fused to the 5’ terminal sequence of the gene located on chromosome 17 encoding the RIα regulatory subunit of PKA [34], while ret/PTC3 is formed by fusion of the tyrosine kinase domain of ret with another gene located on chromosome 10, Ele1 [35]. Fugazzola et al. [36] and Klugbauer et al. [37] have described two other forms of oncogenic rearrangements between ret and Ele1 (PTC4) and ret and an unknown gene called ret fused gene 5 (RFG5): PTC5. These last chimeric genes were isolated in post-Chernobyl thyroid tumours. A role of ret/PTC as an initiator of the thyroid tumorigenic process has been shown using transgenic mice [1]. The frequency of ret activation in ‘spontaneous’ thyroid papillary carcinomas varies widely between different studies: from 2.5 to 35 % [1, 13]. It has been postulated that this variation could be the result of the different geographical origins of populations studied, the age at tumour occurrence or the sensitivity of the experimental methods used to detect the rearrangement. In ‘spontaneous’ tumour series all the rearranged ret genes were detected in papillary carcinomas. The exception is a report by Ishizaka et al. [38] who reported the detection of an activated ret oncogene in four out of sixteen follicular adenomas. As the prevalence of occult thyroid carcinomas in the Japanese population is high, it is possible that these ret/PTCpositive benign tumour samples were the consequence of the presence of an occult papillary tumour.
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In a series of 39 thyroid tumours (19 carcinomas and 20 adenomas) from patients who received external radiation for benign or malignant conditions studied in our laboratory [13], the overall frequency of ret/PTC rearrangements was not significantly different to that observed in Chernobyl tumours (about 64 %). However, interestingly, the most frequently observed chimeric gene was ret/PTC1 and not PTC3 (78 %) (figure 2). Moreover, 45 % of the radiation-associated follicular adenomas screened by us contained a ret/PTC1 gene. The reason why in Chernobyl and post-therapeutic irradiation tumours the most frequent chimeric genes detected are different is actually unknown. Results obtained in our and other laboratories [13, 41] suggest that in papillary carcinomas from Chernobyl PTC3 is associated with an aggressive behaviour and histologically with the presence of a solid structure. In contrast, most of the papillary carcinomas appearing after external irradiation, show the classical biological and histological features of ‘spontaneous’ tumours of the same type [13]. 4.2. trk oncogene
The human N-trk gene, located in chromosome 1, encodes a cell surface protein representing one of the receptors for the nerve growth factor (NGF) [1]. Following chromosomal rearrangements, the trk 5’ domain is removed and replaced by sequences provided by three activating genes [1]. The resulting chimeric genes are ubiquitously expressed and present a constitutively active tyrosine kinase domain, these features representing, presumably, the bases for its transforming activity. The rearrangements originate four chimeric genes: three are intrachromosomic rearrangements (TRK, TRK-T1 and TRK-T2) and one is an interchromosomic one (TRK-T3) [1]. In the thyroid N-trk proto-oncogene has been found to be activated only in a minority of ‘spontaneous’ and radiation-associated papillary carcinomas ([12, 23, 42]; Bounacer et al., in press) with similar frequencies (12 %). Only the TRK form of rearrangement is present in radiationassociated thyroid tumours. TRK rearrangements play the role of an initiator in the development of ‘spontaneous’ and radiation-associated epithelial thyroid tumours [1].
H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
Figure 1. Schematic structure of the product of the ret proto-oncogene and its oncogenic versions. Signal peptide (SP), cadherin-rich (Cad), cystein-rich (Cys), transmembrane (TM) and tyrosine kinase 1 (TK1) and 2 (TK2) domains are indicated.
5. Combined study of the ras, gsp, ret and trk oncogenes
associated series which harboured a second genetic alteration, argues in favour of an alternative role of these genes in the epithelial thyroid tumorigenic process.
Simultaneous alteration in the same sample of combinations of the ras, gsp, ret, trk and TSHR genes was sought in benign and malignant ‘spontaneous’ and radiationassociated thyroid tumours [5, 13, 24, 29]. Simultaneous altered oncogenes were detected in 5/114 ‘spontaneous’ tumours screened and in 5/39 radiation-associated tumours (table IV). It is not possible at present to say which of the simultaneous genetic alterations occurred first, in the ‘spontaneous’ or radiation-associated tumours, or to speculate about an eventual mechanism of co-operation between the altered genes. We cannot neglect the co-operation of the studied genes with other until now unknown genes. However, the low number of ras-, gsp-, ret-, trk- or TSHRpositive tumours in the ‘spontaneous’ or radiation-
6. Tumour suppressor genes No genomic abnormalities were found in the Rb gene in different series of thyroid tumours studied by WynfordThomas [26] or in 40 benign and malignant tumours studied in our laboratory [1]. p53 is by far the most often modified gene in human cancer, being found altered at high frequency in tumours of colon, breast and lung and in acute leukaemia [1]. In thyroid ‘spontaneous’ tumours, inactivating point mutations in the p53 gene were observed with relatively high frequency in anaplastic but not differentiated carcinomas [1, 26]. These data suggest that mutational inactivation of the p53 gene may be a key event in the progression from differentiated to anaplastic carcinoma.
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Figure 2. ret/PTC activation in thyroid radiation-induced papillary carcinomas in different studies.
Mutations of the p53 gene in radiation-associated tumours are rare, but they seem to be related to greater aggression in well-differentiated carcinomas [43].
7. Conclusions and perspectives In conclusion, the results discussed in this manuscript show or suggest: Table IV. Studies of five genes in the same ‘spontaneous’ or radiationassociated human thyroid tumour.
Oncogenes*
ras + gsp ras + TSHR ras + ret/PTC ras + TRK gsp + ret/PTC gsp + TRK gsp + TSHR ret/PTC +TRK TSHR + ret/PTC TSHR + TRK
‘Spontaneous’** (n = 114)
Radiationassociated*** (n = 39)
1 2 1 1 – – – – – –
2 1 1 – – 1 – –
* Simultaneous alterations in the same tumour (point mutation or rearrangement); ** data from [13, 23]; *** data from [13, 24] and Bounacer et al. (2000, B.J.C. in press); –: negative.
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– an interchangeable role for ras, gsp, TSHR, ret and trk genes in the initiation or progression of ‘spontaneous’ or radiation-associated thyroid epithelial tumours and, perhaps, a role for p53 in their differentiation process; – that ras activating mutations are the most frequent genetic alterations in ‘spontaneous’ benign or malignant thyroid tumours; – that the ret rearrangements resulting in ret/PTC chimeric genes play a crucial role in the development of radiation-associated benign and malignant thyroid tumours appearing after therapeutic or accidental ionizing irradiation. They are the most frequent genetic alteration in this type of tumours; – that genes other than gsp or TSHR participating in the cAMP pathway (e.g. AC and PKA) may play a role in the development of hyperfunctioning thyroid tumours; that the simultaneous activation of two genes (or more) is a rare event, but may lead to a super-additive effect. Further identification of new oncogenes or tumour suppressor genes and a better knowledge of the physiology of the normal follicular cells, in terms of proliferation, differentiation and expression of differentiated functions, are needed to open up new avenues in the biology and clinical management of thyroid tumours.
Acknowledgements. The author wishes to thank all the colleagues who participated in our experiments, allow-
H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
ing publication of this article. I am also indebted to M. Chaker for typing the manuscript. Work in our laboratory at the UPR42 of CNRS (IFR Y1221) in Villejuif was
supported by grants from CNRS, ARC, EDF, Ligue Nationale contre le Cancer, Fondation de France and Ministère de l’Education et de la Recherche.
References
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Concise review paper / Point sur
Molecular basis of epithelial thyroid tumorigenesis Horacio Guillermo Suareza* a
Laboratoire de génétique moléculaire UPR 42 CNRS, Institut de recherches sur le cancer – IFR Y 1221, 7, rue Guy-Môquet, BP 8, 94801 Villejuif cedex, France
Received 7 January 2000; accepted 27 March 2000 Communicated by Editorial board
Abstract – The results of experiments carried out in different laboratories (including ours) during the last 10 years have enabled us to propose the hypothesis that there are different initiators able to start the epithelial thyroid tumorigenic process via different pathways: – gsp and TSHR genes: at the origin of hyperfunctioning tumours (toxic nodules and adenomas); – ras and probably gsp genes (in a minority of samples): via a vesicular adenoma progressing eventually to a vesicular carcinoma. This could be also the case for ret but only in radiation-associated tumours; – ras, ret, trk and probably gsp and met: starting from small papillary lesions (‘spontaneous’ or radiation-induced) and progressing to a clinically evident papillary carcinoma; – the p53 gene playing a role only in the final dedifferentiation process. Simultaneous alteration in the same sample of combinations of ras, gsp, ret, trk and TSHR was found in only a minority of the approximately 150 tumours studied. These data suggest an interchangeable role for these genes in the initiation of ‘spontaneous’ or radiation-associated epithelial thyroid tumorigenesis. The requirement of one of the genes cited above to interact with other genes must not be neglected. Ras is the most frequently altered gene in ‘spontaneous’ thyroid tumours and ret in radiation-associated thyroid tumours. © 2000 Académie des sciences/ Éditions scientifiques et médicales Elsevier SAS thyroid / tumorigenic process / genetic alterations Résumé – Bases moléculaires de la tumorigenèse épithéliale thyroïdienne. Les résultats des expériences effectuées dans différents laboratoires (dont le nôtre) au cours des 10 dernières années ont conduit à l’hypothèse qu’il existe plusieurs initiateurs capables de déclencher la tumorigenèse épithéliale thyroïdienne à travers des voies différentes : 1o les gènes gsp et TSHR: à l’origine des tumeurs hyperfonctionnelles (nodules et adénomes toxiques) ; 2o les gènes ras et probablement gsp (dans une infime minorité de cas) : via un adénome vésiculaire progressant éventuellement vers un cancer de la même morphologie (cela pourrait aussi être le cas pour ret seulement dans les tumeurs associées aux radiations) ; 3o les gènes ras, ret, trk et probablement gsp et met : à partir de petites lésions papillaires « spontanées » ou radio-induites (« carcinome papillaire occulte ») progressant vers un cancer papillaire cliniquement évident ; 4o le gène p53 jouant un rôle dans les étapes finales de la dédifférentiation. Sur environ 150 tumeurs « spontanées » ou associées aux radiations étudiées, seulement une minorité présente deux ou plusieurs de ces gènes altérés simultanément. Ce fait plaide en faveur d’un rôle d’initiateur alternatif de ras, gsp, TSHR, ret ou trk dans la tumorigenèse épithéliale thyroïdienne et montre qu’il est possible de trouver pendant ce processus, dans des cas très rares, plus d’un gène altéré dans la même tumeur. La possiblité d’une coopération de ces gènes entre eux et/ou avec d’autres gènes non-identifiés, ne peut pas être négligée. Ras est le gène le plus fréquemment altéré dans les tumeurs « spontanées »
* Correspondence and reprints: [email protected]
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et ret dans celles associées aux radiations. © 2000 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS thyroïde / tumorigenèse / altérations génétiques
Version abrégée 1. Activation des gènes de la famille RAS Les trois petites protéines G appartenant à la famille ras (H-, K- et N-ras) (p21) représentent un switch moléculaire oscillant entre un état basal à GDP et un état actif à GTP. Une mutation affectant les codons 12/13 ou 61 active la protéine en supprimant sa capacité GTPasique. Ces protéines H-, K- et N-ras assurent la transmission des signaux de divers récepteurs à activité TK et couplés aux protéines G vers des kinases à activité sérine/thréonine. Les voies empruntées par ras doivent être d’une importance capitale pour la cellule vésiculaire, car une mutation activatrice de l’un de ces gènes a été retrouvée par nous dans près de 30 % des tumeurs vésiculaires bénignes aussi bien que malignes. Les mutations activatrices de ras ont été observées avec une fréquence similaire dans les adénomes folliculaires et les carcinomes soit de type papillaire soit de type folliculaire. Les mutations sont aussi bien des transitions que des transversions. Nous avons recherché aussi dans 39 tumeurs de la thyroïde provenant de malades ayant reçu à un moment donné de leur vie, une irradiation à visée médicale du cou, de la tête ou du thorax, la présence de mutations activatrices des oncogènes ras. La seule différence que nous avons trouvée entre les tumeurs thyroïdiennes radio-induites et celles « spontanées », réside dans le mécanisme de mutation des gènes ras. En effet, tandis que dans les tumeurs « spontanées » nous avons trouvé des transversions et des transitions avec une fréquence similaire, dans les tumeurs radio-induites nous n’avons détecté que des transversions, notamment des substitutions d’un G par un T (environ 70 % des cas). Ces lésions pourraient être la conséquence d’un mécanisme radicalaire d’oxydation induit par les radiations ionisantes et produisant des 8-oxo-dG, lesquelles peuvent s’apparier avec des adénines pendant la réplication de l’ADN.
2. Activation de la voie de l’AMPc dans les tumeurs hyperfonctionnelles thyroïdiennes La voie de l’AMPc induit à la fois la prolifération et la différenciation de certains tissus endocriniens comme
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ceux de la thyroïde et de l’hypophyse. Dans les tumeurs hyperfonctionnelles que développent parfois les deux glandes, le fait que la voie de l’AMPc soit maintenue dans un état d’activation permanente entraîne la prolifération excessive (normalement très réduite) et l’hyperfonctionnement des cellules endocrines concernées. Dans le cas de la thyroïde, les tumeurs hyperfonctionnelles (non malignes, en général) présentent un taux de base élevé de l’activité adényl cyclase (AAC) ne répondant plus ou très peu, à la stimulation par la TSH (thyroid stimulating hormone). On dit qu’elles ont un phénotype AAC+/TSH–. Nous, et d’autres groupes, avons montré que le phénotype AAC+/TSH– était dans environ 50 % des tumeurs examinées, la conséquence de : a) mutations situées sur les codons 201 ou 227 de la sous-unité a de la protéine Gs, entraînant son activation constitutive (oncogène gsp) (∼25 % des tumeurs) et b) mutations du gène du récepteur à sept domaines transmenbranaires de la TSH (TSHR) (∼25 % des tumeurs). L’effet de ces mutations sur l’activité biologique des protéines respectives, a été montré in vivo et in vitro.
3. Altérations activant des gènes récepteurs de facteurs de croissance de la famille des tyrosines kinases 3.1. Oncogène ret Le proto-oncogène ret est un récepteur à activité tyrosine kinase (TK) qui est activé, comme d’autres gènes de cette famille, non par mutation ponctuelle, mais par un remaniement du gène. Les ligands de ret sont le GDNF (Glial cell line derived neurotrophic factor) et la neurturine. La protéine normale comporte une partie N-terminale extracellulaire qui, en l’absence du ligand, semble inhiber l’activité du domaine TK intracellulaire. À la suite d’une cassure du gène ret, suivie d’une translocation ou d’une inversion chromosomique, le domaine TK de la protéine est séparé de la région inhibitrice en amont. Celle-ci est remplacée par la partie N-terminale d’une protéine « neutre », et le domaine TK se trouve donc libéré du contrôle inhibiteur normal. Dans la thyroïde, on a trouvé des réarrangements activateurs de ret avec un gène de fonction inconnue, nommé H4, plus récemment avec le gène de la sous-unité RIa de la protéine kinase A et enfin, avec deux gènes non encore caractérisés appelés ELE-1 et RFG5 (ret fused gene 5). Les cinq gènes chimères
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produits par la fusion de ret avec ces quatre gènes étrangers s’appellent respectivement ret/PTC1 à ret/ PTC5. Ces réarrangements ont été trouvés avec une fréquence de 10-15 % exclusivement dans les carcinomes « spontanés » de type papillaire de la thyroïde. Les résultats des études effectuées par nous (Saïd et al., 1994) et dans d’autres laboratoires (Fugazzola et coll., 1995 ; Ito et al., 1994 ; Klugbauer et al. 1995 ; Nikiforov et al., 1997), sur les tumeurs de type carcinome papillaire de la thyroïde apparues après l’accident de Tchernobyl, ont montré une fréquence très élevée de réarrangements activateurs du gène ret de type ret/PTC. Cette fréquence était d’environ 60–80 % contre 5–30 % (dépendant des études) dans les tumeurs « spontanées ». Ces observations nous ont amenés à étendre nos recherches à des tumeurs thyroïdiennes bénignes et malignes, apparues chez des patients ayant subi une irradiation à visée médicale de la tête, du cou et/ou du thorax (39 tumeurs). Nos résultats ont montré que la fréquence absolue de réarrangements ret/PTC dans ces tumeurs était de 64 %. Le gène chimère le plus fréquemment trouvé dans nos séries était ret/PTC1 (∼75 %). Les tumeurs porteuses d’un gène ret/PTC3 étaient minoritaires et ret/PTC2 n’a jamais été mis en évidence. Ces derniers résultats sont en contradiction avec les observations effectuées dans les tumeurs postTchernobyl où le type de réarrangement prédominant était ret/PTC3 (∼70 %), suivi dans l’ordre de ret/PTC1 et ret/PTC2. Notre étude a montré aussi, pour la première fois, la présence d’un gène ret/PTC1 dans 45 % des adénomes folliculaires associés aux radiations thérapeutiques. 3.2. Oncogène trk Dans les tumeurs thyroïdiennes, trk est associé d’une manière similaire à ret à trois gènes activants, codant pour des protéines structurales : la tropomyosine nonmusculaire, tpr (translocated promoter region) et TAG (trk activating gene). Comme ret, le gène trk a été trouvé activé par réarrangement exclusivement dans les carcinomes papillaires « spontanés » (fréquence 5–15 %). Outre cet effet qualitatif, le nouveau gène hybride acquiert le taux d’expression du partenaire non oncogénique, ce qui entraîne généralement sa surexpression.
1. Introduction The roles of somatic mutations, gene rearrangement(s) and level of gene expression in carcinogenesis are now well established. Several techniques can be used to detect
Nous avons recherché aussi dans nos tumeurs associées aux radiations, la présence d’un proto-oncogène trk activé. Nos études ont montré que : a) la présence de réarrangements activateurs, était similaire dans les tumeurs « spontanées » ou associées aux radiations (∼8 %) ; b) l’oncogène TRK jouerait un rôle dans le développement de seulement une minorité des carcinomes papillaires associés aux radiations thérapeutiques et c) contrairement aux résultats obtenus avec ret, nous n’avons pas observé de réarrangements activateurs de trk dans les tumeurs bénignes de la thyroïde associées aux radiations (adénomes folliculaires). Les réarrangements activateurs du proto-oncogène TRK étaient, dans nos tumeurs radio-induites, uniquement de type intrachromosomique (ch.1) avec le gène de la tropomyosine non-musculaire (TPM3), donnant lieu à la formation du gène chimère trk-a.
4. Étude combinée des gènes ras, gsp, ret et trk dans une même tumeur Sur plus de 150 tumeurs « spontanées » ou associées aux radiations étudiées, dans seulement dix cas, deux ou plusieurs de ces gènes ont été trouvés altérés simultanément dans la même tumeur. Ce fait plaide en faveur d’un rôle d’initiateur alternatif de ras, gsp, ret ou trk dans la tumorigenèse épithéliale thyroïdienne et montre qu’il est possible de trouver pendant ce processus, dans des cas très rares, plus d’un gène altéré dans la même tumeur. La possibilité d’une coopération de ces gènes entre eux et/ou avec d’autres gènes nonidentifiés ne peut pas être négligé. Ras est le gène le plus fréquemment altéré dans les tumeurs « spontanées » et ret dans celles associées aux radiations.
5. Gènes suppresseurs de tumeurs Les mutations ponctuelles inactivatrices du gène p53, ont été les seules altérations génétiques concernant un gène suppresseur de tumeurs, observées dans les tumeurs folliculaires tyroïdiennes « spontanées » ou radio-induites. Elles ont été mises en évidence exclusivement dans les carcinomes anaplasiques, suggérant que p53 pourrait jouer un rôle dans la dédifférentiation et l’agressivité tumorale.
such genetic alterations in human tumours [1]. The application of these techniques to thyroid tumours has focused particular attention on the role of point mutations activating (or inhibiting) the genes for the TSH receptor (TSHR) [2–4], ras [5–8], gsp [9, 10] and p53 [11], specific rearrangements of the oncogenes ret and trk [12–16] and
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Table I. Frequency of ras mutations in ‘spontaneous’ human thyroid tumours in different studies.
Country Canada (19) UK (20) Hungary (19) Italy (12) Italy (14) USA (21) USA (22) France (23) Overall
PC
FC
AC
AD (cold)
HN
0 (0/10) 21 (4/19) 0 (0/12) 13 (2/16) 0 (0/20) 21 (3/14) 6 (1/15) 45 (16/35) 18 (26/141)
10 (1/10) 52 (11/21) 50 (3/6) nd
nd
nd
55 (6/11) nd nd
20 (2/10) 27 (8/29) 92 (12/13) nd
nd
nd
nd
nd
0 (0/3) 14 (2/14) 35 (7/20) 32 (24/74)
nd
25 (6/24) 0 (0/9) 40 (12/30) 35 (40/115)
nd
nd 100 (1/1) 58 (7/12)
nd nd nd
nd 7 (2/28) 7 (2/28)
PC: papillary carcinoma; FC: follicular carcinoma; AC: anaplasic carcinoma; AD: cold adenoma; HN: hot nodule. Results are given as % frequency of activation with (positive tumours/studied tumours) in parentheses; nd: not done.
alterations in the pattern of expression of the oncogene met [17, 18]. In this review we will present data concerning the activation of ras, gsp, TSHR, ret and trk and discuss whether they play an alternative or a complementary role in human epithelial thyroid ‘spontaneous’ or radiation-induced tumorigenesis (the most frequent radiation-induced tumorigenic process in human). We will also discuss the role of tumour-supressor genes in this process.
2. Activation of ras family genes The products of members of this oncogene family (HaKi- and N-ras) are 21-kD proteins (p21) with nucleotide binding activity, involved in the transduction of information from the cell surface to the nucleus. These genes are all oncogenically activated by a single amino acid substitution in codon 12 or 61 and sometimes in codon 13 or 59 [9]. The activation of the ras oncogenes by point mutation was found in about 30 % of ‘spontaneous’ epithelial thyroid tumours studied in different laboratories and was the most frequent genetic alteration in this type of tumour (table I). The mutations, which in ‘spontaneous’ thyroid tumours are transitions as well as transversions, are randomly distributed between the three ras genes with similar frequencies (11–15 %), without predominance of one of the critical codons or their constituting bases. The mutations occur in follicular adenomas, papillary and follicular carcinomas, at approximately the same frequency (table I). The thyroid gland is, along with the colon, a tissue in which ras mutations are observed in benign tumours [1]. Compared to other studies, we found a higher frequency of ras mutations in ‘spontaneous’ papillary carcinomas (table I). This discrepancy may be the consequence of a
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difference in iodine intake between the different populations [19–23]. Concerning thyroid tumours from patients with a history of external radiation for benign or malignant conditions, activating mutations in the ras genes have been described, and their overall frequency (about 30 %) (table II) was similar to that observed in ‘spontaneous’ benign or maligant tumours [20, 24]. In tumours arising in children after the Chernobyl accident, Nikiforov et al. [25] observed ras point mutations in follicular carcinomas and adenomas but not in papillary carcinomas (table II). It has been concluded that ras does not appear to be important in the development of the Chernobyl papillary carcinomas. Challeton et al. [24] showed that all three ras genes were mutated with similar frequencies in malignant radiation-associated tumours, as was the case in ‘spontaneous’ tumours (see above). However, the mechanism by which ras mutations occurs seems to be different between ‘spontaneous’ and radiation-associated tumours. Whereas in ‘spontaneous’ thyroid tumours, as previously mentioned, transversions as well as transitions were detected in the ras genes [3, 9, 23], in radiation-associated benign and malignant tumours, only transversions (mainly G→T) were present [24]. It can be postulated that the mutations in the radiation-induced tumours could have arisen through an ionizing radiation-associated oxidative lesion, producing 8-OXO-dG which can pair with A during DNA replication. Mutations of ras which, as stated, can be found in ‘spontaneous’ and radiation-associated adenomas or carcinomas at roughly the same frequency, can be proposed as an early event in the thyroid tumorigenic process. Support for this hypothesis has therefore been obtained from experiments in which mutant ras has been introduced into normal follicular cells either in vitro or in vivo
H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
Table II. Frequency of ras and gsp mutations in human radiation-associated thyroid tumours.
Country
UK* (20) France* (24) Belarus** (25) Overall
RAS
RAS
PC
FC
AC
AD (cold)
PC
FC
AC
AD (cold)
50 (1/2) 40 (6/15) 0 (0/33) 14 (7/50)
60 (3/5) 0 (0/2) 100 (1/1) 50 (4/8)
0 (0/1) nd
0 (0/4) 25 (4/16) 43 (3/7) 25 (7/27)
nd
nd
nd
nd
7 (1/15) nd
50 (1/2) nd
nd
0 (0/16) nd
7 (1/15)
50 (1/2)
nd 0 (0/1)
nd nd
0 (0/16)
Abbreviations are as in table I. Results are given as overall frequency (%) with (positive tumours/studied tumours) in parentheses. * Tumours from patients irradiated for benign or malignant conditions; ** post-Chernobyl tumours.
[1]. Finally, activated ras protein has also been shown to stimulate the growth and inhibit differentiation of thyroid epithelial cells [3, 9, 26].
3. Genetic alterations activating the cAMP pathway in hyperfunctioning tumours Increasing evidence demonstrates that constitutive activation of adenylate cyclase (AC) induced by acquired or inherited mutations of Gs protein and G-protein-coupled receptors plays a major role in human disease [1]. In thyroid tumours, the cAMP signalling cascade is rarely involved in malignant transformation, even if activating mutations of gsp (the α subunit of Gs protein) and TSH receptor (TSHR) genes have been described in a subset of differentiated thyroid carcinomas with high basal adenylate cyclase activity [5, 10, 23]. On the contrary, it has been firmly established that constitutive activation of the cAMP cascade through the mutation of gsp or TSHR, is responsible for the hyperfunction and growth of a majority of thyroid follicular toxic adenomas [6, 7, 27–30]. Our experiments, studying the entire coding sequence of both genes in the same hyperfunctioning tumours, showed that both the Gαs protein and TSHR alterations
may contribute with a similar frequency (about 25–30 %) to the constitutive activation of AC in this type of tumour [5, 6, 7, 23]. However, in a subgroup of hyperfunctioning adenomas (40–50 %), no mutations were identified in the genes studied. It may be hypothesized that in gsp- and TSHR-negative tumours, alterations in another gene participating in the cAMP pathway (e.g. AC or protein kinase A (PKA)) may be responsible for the phenotype. The mutations detected in gsp and TSHR genes are concentrated in domains of the protein that are important for their normal biological activity. In the case of gsp the two ‘criticial’ codons are 201, related to the GTPase activity of the protein, and 227, the equivalent of ras codon 61 and located in the GTP binding domain [4, 23, 28]. For TSHR the mutations are predominantly present in an area of the receptor concerned in the interaction with the Gs protein and including the III intracellular loop and the adjacent VI transmembrane region [7, 30]. A role for gsp activating mutations as an early event in the development of toxic adenomas has been shown using transgenic mice [31]. The gsp oncogene was also studied by Challeton et al. [24] in 33 radiation-associated thyroid tumours (table II). The comparison of these results with data obtained in other studies of ‘spontaneous’ hypofunctional tumours ([23] and table III) showed that the overall frequency (5–10 %) and the spectrum of mutations in the gsp oncogene were similar in both types of tumour.
Table III. gsp oncogene in ‘spontaneous’ human thyroid tumours in different studies.
Country France (10) France + Italy (6) UK (28) USA (4) Overall
PC
FC
AC
AD (cold)
HN
9 (3/35) nd
10 (2/20) nd
0 (0/1) nd
7 (2/30) nd
0 (5/5) 0 (0/14) 15 (8/54)
0 (0/3) 0 (0/3) 8 (2/26)
nd
0 (0/16) 0 (0/12) 3 (2/58)
32 (9/28) 25 (9/37) 38 (5/13) 7 (1/14) 26 (24/92)
nd 0 (0/1)
Abbreviations are as in table I. Results are given as overall frequency (%) with (positive tumours/studied tumours) in parentheses.
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H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
4. Gene alterations activating genes from the tyrosine protein kinase receptor family
It has been reported that in humans external radiation as a consequence of an atomic accident (e.g. Chernobyl) or as a treatment for benign or malignant conditions may be a direct inducer of activating ret rearrangements at relatively high frequency [13, 39–41].
Since the discovery of tyrosine kinases over a decade ago, this gene superfamily has been steadily growing to reach its current size of almost 50 members [32]. C-ret and the members of the trk family (trk, trkB, trkC and trkD), which plays a key role in epithelial thyroid tumorigenesis, belong to this superfamily.
Concerning Chernobyl tumours, different studies [39–41] found ret/PTC rearrangements in 55–85 % of the tumours, which were all papillary carcinomas. Ret/PTC3 was the most frequent chimeric gene in each of the studies, the other rearrangements being of the PTC1 and PTC2 types (figure 2), in contrast to what is observed in ‘spontaneous’ papillary carcinomas where the frequency of PTC1 and PTC3 is more or less the same (48 and 45 %, respectively).
4.1. ret oncogene
The ret proto-oncogene, located on chromosome 10q11.2, encodes a protein structurally related to transmembrane receptors with a tyrosine kinase domain [13, 16], and recently, it has been shown that its putative ligands are the glial-cell-line-derived neurotrophic factor (GDNF) and the neurturine [33]. Five oncogenically activated forms of the ret proto-oncogene have been identified in epithelial thyroid tumours and designated ret/PTC1 to PTC5 [1]. All these activated forms of the proto-oncogene are the consequence of specific oncogenic rearrangements fusing the tyrosine kinase domain of ret with the 5’ domain of different genes (figure 1). The 5’ domain of the foreign genes, acting as a promoter, permanently activates the tyrosine kinase activity of ret, normally undetected in the thyroid follicular cells. ret/PTC1 is formed by an intrachromosomal rearrangement fusing the ret tyrosine kinase domain to a gene designated H4, whose function is still unknown [1, 16]. In ret/PTC2, the catalytic domain of ret is fused to the 5’ terminal sequence of the gene located on chromosome 17 encoding the RIα regulatory subunit of PKA [34], while ret/PTC3 is formed by fusion of the tyrosine kinase domain of ret with another gene located on chromosome 10, Ele1 [35]. Fugazzola et al. [36] and Klugbauer et al. [37] have described two other forms of oncogenic rearrangements between ret and Ele1 (PTC4) and ret and an unknown gene called ret fused gene 5 (RFG5): PTC5. These last chimeric genes were isolated in post-Chernobyl thyroid tumours. A role of ret/PTC as an initiator of the thyroid tumorigenic process has been shown using transgenic mice [1]. The frequency of ret activation in ‘spontaneous’ thyroid papillary carcinomas varies widely between different studies: from 2.5 to 35 % [1, 13]. It has been postulated that this variation could be the result of the different geographical origins of populations studied, the age at tumour occurrence or the sensitivity of the experimental methods used to detect the rearrangement. In ‘spontaneous’ tumour series all the rearranged ret genes were detected in papillary carcinomas. The exception is a report by Ishizaka et al. [38] who reported the detection of an activated ret oncogene in four out of sixteen follicular adenomas. As the prevalence of occult thyroid carcinomas in the Japanese population is high, it is possible that these ret/PTCpositive benign tumour samples were the consequence of the presence of an occult papillary tumour.
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In a series of 39 thyroid tumours (19 carcinomas and 20 adenomas) from patients who received external radiation for benign or malignant conditions studied in our laboratory [13], the overall frequency of ret/PTC rearrangements was not significantly different to that observed in Chernobyl tumours (about 64 %). However, interestingly, the most frequently observed chimeric gene was ret/PTC1 and not PTC3 (78 %) (figure 2). Moreover, 45 % of the radiation-associated follicular adenomas screened by us contained a ret/PTC1 gene. The reason why in Chernobyl and post-therapeutic irradiation tumours the most frequent chimeric genes detected are different is actually unknown. Results obtained in our and other laboratories [13, 41] suggest that in papillary carcinomas from Chernobyl PTC3 is associated with an aggressive behaviour and histologically with the presence of a solid structure. In contrast, most of the papillary carcinomas appearing after external irradiation, show the classical biological and histological features of ‘spontaneous’ tumours of the same type [13]. 4.2. trk oncogene
The human N-trk gene, located in chromosome 1, encodes a cell surface protein representing one of the receptors for the nerve growth factor (NGF) [1]. Following chromosomal rearrangements, the trk 5’ domain is removed and replaced by sequences provided by three activating genes [1]. The resulting chimeric genes are ubiquitously expressed and present a constitutively active tyrosine kinase domain, these features representing, presumably, the bases for its transforming activity. The rearrangements originate four chimeric genes: three are intrachromosomic rearrangements (TRK, TRK-T1 and TRK-T2) and one is an interchromosomic one (TRK-T3) [1]. In the thyroid N-trk proto-oncogene has been found to be activated only in a minority of ‘spontaneous’ and radiation-associated papillary carcinomas ([12, 23, 42]; Bounacer et al., in press) with similar frequencies (12 %). Only the TRK form of rearrangement is present in radiationassociated thyroid tumours. TRK rearrangements play the role of an initiator in the development of ‘spontaneous’ and radiation-associated epithelial thyroid tumours [1].
H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
Figure 1. Schematic structure of the product of the ret proto-oncogene and its oncogenic versions. Signal peptide (SP), cadherin-rich (Cad), cystein-rich (Cys), transmembrane (TM) and tyrosine kinase 1 (TK1) and 2 (TK2) domains are indicated.
5. Combined study of the ras, gsp, ret and trk oncogenes
associated series which harboured a second genetic alteration, argues in favour of an alternative role of these genes in the epithelial thyroid tumorigenic process.
Simultaneous alteration in the same sample of combinations of the ras, gsp, ret, trk and TSHR genes was sought in benign and malignant ‘spontaneous’ and radiationassociated thyroid tumours [5, 13, 24, 29]. Simultaneous altered oncogenes were detected in 5/114 ‘spontaneous’ tumours screened and in 5/39 radiation-associated tumours (table IV). It is not possible at present to say which of the simultaneous genetic alterations occurred first, in the ‘spontaneous’ or radiation-associated tumours, or to speculate about an eventual mechanism of co-operation between the altered genes. We cannot neglect the co-operation of the studied genes with other until now unknown genes. However, the low number of ras-, gsp-, ret-, trk- or TSHRpositive tumours in the ‘spontaneous’ or radiation-
6. Tumour suppressor genes No genomic abnormalities were found in the Rb gene in different series of thyroid tumours studied by WynfordThomas [26] or in 40 benign and malignant tumours studied in our laboratory [1]. p53 is by far the most often modified gene in human cancer, being found altered at high frequency in tumours of colon, breast and lung and in acute leukaemia [1]. In thyroid ‘spontaneous’ tumours, inactivating point mutations in the p53 gene were observed with relatively high frequency in anaplastic but not differentiated carcinomas [1, 26]. These data suggest that mutational inactivation of the p53 gene may be a key event in the progression from differentiated to anaplastic carcinoma.
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H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
Figure 2. ret/PTC activation in thyroid radiation-induced papillary carcinomas in different studies.
Mutations of the p53 gene in radiation-associated tumours are rare, but they seem to be related to greater aggression in well-differentiated carcinomas [43].
7. Conclusions and perspectives In conclusion, the results discussed in this manuscript show or suggest: Table IV. Studies of five genes in the same ‘spontaneous’ or radiationassociated human thyroid tumour.
Oncogenes*
ras + gsp ras + TSHR ras + ret/PTC ras + TRK gsp + ret/PTC gsp + TRK gsp + TSHR ret/PTC +TRK TSHR + ret/PTC TSHR + TRK
‘Spontaneous’** (n = 114)
Radiationassociated*** (n = 39)
1 2 1 1 – – – – – –
2 1 1 – – 1 – –
* Simultaneous alterations in the same tumour (point mutation or rearrangement); ** data from [13, 23]; *** data from [13, 24] and Bounacer et al. (2000, B.J.C. in press); –: negative.
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– an interchangeable role for ras, gsp, TSHR, ret and trk genes in the initiation or progression of ‘spontaneous’ or radiation-associated thyroid epithelial tumours and, perhaps, a role for p53 in their differentiation process; – that ras activating mutations are the most frequent genetic alterations in ‘spontaneous’ benign or malignant thyroid tumours; – that the ret rearrangements resulting in ret/PTC chimeric genes play a crucial role in the development of radiation-associated benign and malignant thyroid tumours appearing after therapeutic or accidental ionizing irradiation. They are the most frequent genetic alteration in this type of tumours; – that genes other than gsp or TSHR participating in the cAMP pathway (e.g. AC and PKA) may play a role in the development of hyperfunctioning thyroid tumours; that the simultaneous activation of two genes (or more) is a rare event, but may lead to a super-additive effect. Further identification of new oncogenes or tumour suppressor genes and a better knowledge of the physiology of the normal follicular cells, in terms of proliferation, differentiation and expression of differentiated functions, are needed to open up new avenues in the biology and clinical management of thyroid tumours.
Acknowledgements. The author wishes to thank all the colleagues who participated in our experiments, allow-
H.G. Suarez / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 519–528
ing publication of this article. I am also indebted to M. Chaker for typing the manuscript. Work in our laboratory at the UPR42 of CNRS (IFR Y1221) in Villejuif was
supported by grants from CNRS, ARC, EDF, Ligue Nationale contre le Cancer, Fondation de France and Ministère de l’Education et de la Recherche.
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