Pierre P Roger

Wilma C G Van Staveren

Katia Coulonval

IRIBHM Jacques E Dumont
email: jedumont@ulb.ac.be

Carine Maenhaut

SIGNAL TRANSDUCTION IN THE HUMAN THYROCYTE AND ITS PERVERSION IN THYROID TUMORS

Keywords: signal transduction, thyroid carcinomas, TSH pathways, growth factor pathway, models, caveats

The study of normal signal transduction pathways regulating the proliferation and differentiation of a cell type allows to predict and to understand the perversions of these pathways which lead to tumorigenesis. In the case of the human thyroid cell, three cascades are mostly involved in tumorigenesis: the TSH receptor-cyclic AMP cascade, the constitutive activation of which leads to autonomous adenomas and congenital hyperthyroidism.

the growth factor-Ras-MAP kinase pathway, whose constitutive activation causes follicular and papillary carcinomas.

the PI-3Kinase-PKB pathway, the activity of which is necessary for both pathways and the corresponding tumors.

The pathways and genetic events affecting them are described. Caveats in the use of models and the interpretation of results are formulated and the still pending questions are outlined.

A c c e p t e d M a n u s c r i p t 3

I. Introduction

Tumor cells are deviant descendants of normal cells which have gained a proliferation phenotype independent of normal physiological controls. Cancer cells have also lost, in part or totally, their differentiation status and gained a capacity to invade other tissues by migration or through the vascular or lymphatic systems. Knowing the properties of these cells is a condition for understanding their biology, and thus for proposing diagnostic signatures and therapeutic targets.

Tumors and their models

In this short review, we shall describe our knowledge of signal transduction in normal thyroid cells and show how this allowed us twenty years ago to predict how the perversion of these regulations could lead to tumors of various types and thus to anticipate the oncogenes that were discovered later [START_REF] Dumont | The cyclic AMP-mediated stimulation of cell proliferation[END_REF]Dumont et al., 1992a). The striking similitude of the conditions we defined as necessary for thyroid cell growth i.e. the necessary stimulation of both the TSH-cAMP or the EGF-MAPK pathway and of the IGF-I/insulin-PI-3Kinase pathway [START_REF] Roger | Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin[END_REF][START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF][START_REF] Vandeput | Role of the different mitogen-activated protein kinase subfamilies in the stimulation of dog and human thyroid epithelial cell proliferation by cyclic adenosine 5'-monophosphate and growth factors[END_REF] to the conditions of thyroid carcinoma induction in mice [START_REF] Miller | Oncogenic Kras requires simultaneous PI3K signaling to induce ERK activation and transform thyroid epithelial cells in vivo[END_REF] illustrates a posteriori the power of this approach.

Many properties of tumor cells can be defined from the clinical evolution of the tumors and from their morphological, gene expression, epigenetic and genetic pathology.

However such knowledge applies to the fully developed tumor and not necessarily to the cells which initiated and developed it. Hence the need of models to understand the development of tumors.

Various models are used to study human tumor cells, from the closer to the in vivo situation (eg tumor cells in culture or as xenotransplants in animal) to the cancer cell lines, which, through long evolution in vitro, have diverged very much from their precursors [START_REF] Van Staveren | Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells?[END_REF], and also to animal cell lines expressing human oncogenes. Another approach is to study the regulation of the normal cell and the mechanisms that have to be subverted in tumorigenesis.

1) Caveats on the use of data of in vivo human tumors. However three caveats must be kept in mind :

1.

The tumor, as removed, represents a given late stage in its evolution and therefore many findings (eg mutation, etc…) may be late irrelevant passenger events.

The comparison of several tumors, by allowing to sort out the presumably important general common findings and thus identify a signature of anterior steps, might partially obviate this.

2. Tumors are composed of a mix of tumor cells, fibroblasts, inflammatory cells and endothelial and other vascular cell types. Tumor cells may even be the minority in the tumor. This should be taken into account in analyzing results obtained on whole tumor tissue (e.g. gene/protein expression, etc) [START_REF] Delys | Gene expression and the biological phenotype of papillary thyroid carcinomas[END_REF].

3.

The tumor cells themselves are heterogeneous, and might contain a minor population of tumor driving cells e.g. the so called cancer stem cells (CSC), the properties of which could be diluted by less competitive cells in the whole population.

One might however hope that many of the properties of the driving cells will still be present in the derived cells.

All these caveats also apply to models in vivo (xenotransplants) or in vitro (direct cultures of cancer cells) of the tumors.

2) Caveats on the use of other models.

a) Primary cultures of normal human and dog thyrocytes Human thyroid cells in primary cultures, treated with either TSH + insulin or EGF + serum + insulin, demonstrate a remarkable congruence of the patterns of gene expression in cells stimulated for one or two days, and those of autonomous adenomas and papillary carcinomas respectively [START_REF] Van Staveren | Gene expression in human thyrocytes and autonomous adenomas reveals suppression of negative feedbacks in tumorigenesis[END_REF][START_REF] Hebrant | Long-term EGF/serum-treated human thyrocytes mimic papillary thyroid carcinomas with regard to gene expression[END_REF]. However there are important differences. For instance, while the activation of the TSH-cAMP pathway in vitro induces several negative feedbacks at transcriptional and enzyme activity levels, several of these feedbacks are not regulated or downregulated in autonomous adenomas [START_REF] Van Staveren | Gene expression in human thyrocytes and autonomous adenomas reveals suppression of negative feedbacks in tumorigenesis[END_REF]. This suggests that tumorigenesis not only requires oncogenic events but also the suppression of negative feedbacks. This, together with the only partial congruence of results,

shows that this model also has its limits. Nevertheless, investigation in human and dog

A c c e p t e d M a n u s c r i p t 5 thyroid cells has allowed to predict the tumorigenic potential of deregulated TSH-cAMP cascade, as well as the necessity of both prolonged and marked stimulation of the Raf-MAPK and the PI-3K pathway in the generation of dedifferentiated carcinomas. The in vitro studies have also shown that general regulation schemes appearing in the literature, especially in reviews, should be considered with much caution and not be interpreted literally (Dumont et al., 2001;[START_REF] Dumont | Cross signaling, cell specificity, and physiology[END_REF].

b) Other models are even more distant from the human tumor in vivo. In vivo models of thyroid tumors in mice offer the advantages of enabling a host environment for the thyroid cells. Moreover the use of inducible tissue-specific knock-down or oncogene expression systems should allow a clearer dissection of carcinogenic pathways in vivo. However, transgenic mice expressing an oncogene in the thyroid (eg RET/PTC3) develop tumors whose morphology, gene expression and evolution are partly different from their human counterpart [START_REF] Burniat | Gene expression in RET/PTC3 and E7 transgenic mouse thyroids: RET/PTC3 but not E7 tumors are partial and transient models of human papillary thyroid cancers[END_REF][START_REF] Jin | Human thyroid tumours, the puzzling lessons from E7 and RET/PTC3 transgenic mice[END_REF].

Normal thyroid cell lines, such as the best rat cell line PC Cl3, differ from the rat thyroid cells in vivo and even more from human cells in vivo [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]. FRTL-5, WRT and PC Cl3 cells have been widely used to demonstrate the activity of transfected thyroid oncogenes on signaling cascades, proliferation and differentiation (see below).

However the mechanistic logics of cell cycle stimulation by cAMP diverge, sometime profoundly, from the situation observed in normal human and canine thyrocytes [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]Paternot et al., 2006b). Moreover, effects of oncogenic Ras on differentiation expression are different in human thyroid primary cultures [START_REF] Gire | RAS oncogene activation induces proliferation in normal human thyroid epithelial cells without loss of differentiation[END_REF][START_REF] Jones | Evidence for a telomereindependent "clock" limiting RAS oncogene-driven proliferation of human thyroid epithelial cells[END_REF] versus rat thyroid cell lines [START_REF] Fusco | Oneand two-step transformations of rat thyroid epithelial cells by retroviral oncogenes[END_REF][START_REF] Avvedimento | Reversible inhibition of a thyroid-specific trans-acting factor by Ras[END_REF][START_REF] Kupperman | Ras inhibits thyroglobulin expression but not cyclic adenosine monophosphate-mediated signaling in Wistar rat thyrocytes[END_REF][START_REF] De Vita | Dose-dependent inhibition of thyroid differentiation by RAS oncogenes[END_REF], and, even among the latter, downstream mechanisms may vary [START_REF] Cass | Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation[END_REF][START_REF] Miller | RalGDS functions in Ras-and cAMP-mediated growth stimulation[END_REF][START_REF] Cobellis | Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation[END_REF][START_REF] Gire | Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras[END_REF]. Illegitimate coupling of tested oncogenes to downsteam effector pathways may result from their overexpression. c) Finally human cancer cell lines retain the activated oncogenes of the cancers from which they originate, thus allowing investigation of their signaling cascades [START_REF] Meireles | Molecular and genotypic characterization of human thyroid follicular cell carcinoma-derived cell lines[END_REF][START_REF] Rocha | Cyclic AMP inhibits the proliferation of thyroid carcinoma cell lines through regulation of CDK4 phosphorylation[END_REF]. However they are the result of a long Darwinian evolution in vitro.

Gene expression wise, they are much more similar to each other than to the corresponding in A c c e p t e d M a n u s c r i p t 6 vivo tumors [START_REF] Van Staveren | Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells?[END_REF][START_REF] Van Staveren | Human thyroid tumor cell lines A c c e p t e d M a n u s c r i p t 52 derived from different tumor types present a common dedifferentiated phenotype[END_REF]. As a result of this evolution, under very restricted and standard conditions these cells can simplify their signal transduction and are more apt to display oncogene addiction, i.e. the reliance on one major oncogenic pathway. This may explain in part why so many successful treatments of cell lines in vitro fail on the more diversified in vivo cancers [START_REF] Couto | How molecular pathology is changing and will change the therapeutics of patients with follicular cell-derived thyroid cancer[END_REF][START_REF] Van Staveren | Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells?[END_REF]. On the other hand, if the activation of several pathways is needed for tumor growth,

the inhibition of each one should be sufficient to impair it. Since many widely used "thyroid" cancer cell lines have been found to derive from non-thyroid cancers [START_REF] Schweppe | Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification[END_REF], researchers are urged to confirm the identity of the cell lines they are using [START_REF] Ringel | Thyroid cancer" cell line misidentification: a time for proactive change[END_REF].

II. Our present knowledge of signal transduction pathways controlling differentiation

and proliferation in the normal dog and human thyroid cells.

1. Caveats on the use of general signal transduction schemes as they are presented in reviews and commercial literature.

The main caveats are : a) Most of the schemes integrate in one map data obtained in several species, organs and models. They therefore represent a catalogue of possibilities, few of which apply to any given type at all [START_REF] Dumont | Crosstalk and specificity in signalling. Are we crosstalking ourselves into general confusion?[END_REF]. Each cell type of each species is characterized by its well defined specialized regulations. Our figures (Fig. 1A,B) are based on data obtained in dog thyroid cells in cultures; many of them have been validated in human cell cultures.

b) The schemes do not take into account kinetic and levels of activation differences.

For instance in thyroid cells, both EGF and insulin/IGF-I activate both the Raf-MAPK and the PI-3K cascades. One would therefore wonder why the two signals are necessary for

proliferation ! The answer is that the effect of EGF on MAPK pathway is strong and persistent (hours) while the effect on PI-3K is weak and transient. The converse is true for insulin/IGF-I. HGF which activates strongly and persistently both pathways in dog thyroid cells can act without insulin [START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF][START_REF] Vandeput | Role of the different mitogen-activated protein kinase subfamilies in the stimulation of dog and human thyroid epithelial cell proliferation by cyclic adenosine 5'-monophosphate and growth factors[END_REF] (Fig. 2). c) Although the physiological control of thyroid cells as well as other cells is generally similar in different species, the signal transduction mechanisms used to perform these controls vary much. For instance, while TSH activates H 2 O 2 generation and thyroid hormone synthesis in all the investigated vertebrate species, it does so through the Gq-PLC d) Signal transduction has been defined in primary cultures of thyroid cells. Therefore the results apply to thyrocytes themselves but do not consider the role of the microenvironment or the interaction in thyroid with other cell types.

2. Normal controls exerted on the human thyroid cell.

Studies using primary cultures of dog and human thyrocytes have demonstrated the unique coexistence in thyroid of two distinct mitogenic modes elicited by (i) TSH acting through its receptor coupled to Gs and adenylyl cyclase activation (Fig. 1A), and (ii) growth factors (such as EGF [START_REF] Roger | Epidermal growth factor controls the proliferation and the expression of differentiation in canine thyroid cells in primary culture[END_REF], FGF [START_REF] Roger | Factors controlling proliferation and differentiation of canine thyroid cells cultured in reduced serum conditions: effects of thyrotropin, cyclic AMP and growth factors[END_REF] and HGF [START_REF] Dremier | Mitogenic, dedifferentiating, and scattering effects of hepatocyte growth factor on dog thyroid cells[END_REF] in dog cells and EGF in human thyrocytes [START_REF] Errick | Growth and differentiation in cultured human thyroid cells: effects of epidermal growth factor and thyrotropin[END_REF])) acting through their receptor-tyrosine protein kinase pathways (Fig. 1B). TSH and cAMP induce differentiation expression, whereas growth factors and more potently EGF reversibly inhibit the expression of all differentiation markers while inducing a morphological transformation analogous to an epithelium/mesenchyme transition (EMT) [START_REF] Roger | Factors controlling proliferation and differentiation of canine thyroid cells cultured in reduced serum conditions: effects of thyrotropin, cyclic AMP and growth factors[END_REF][START_REF] Pohl | Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells[END_REF][START_REF] Roger | Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells[END_REF][START_REF] Coclet | Intermediate filaments in normal thyrocytes: modulation of vimentin expression in primary cultures[END_REF][START_REF] Hebrant | Long-term EGF/serum-treated human thyrocytes mimic papillary thyroid carcinomas with regard to gene expression[END_REF]. Both TSH and EGF mitogenic stimulations are permitted or facilitated by the comitogenic activity of IGF -I (reproduced in vitro by high insulin concentrations) acting through IGF-I tyrosine kinase receptors [START_REF] Roger | Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin[END_REF][START_REF] Burikhanov | Thyrotropin via cyclic AMP induces insulin receptor expression and insulin co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells[END_REF]. By themselves, IGF-I and insulin only weakly stimulate proliferation (Roger et al., 1988a). In dog and human thyrocytes in primary cultures, after delayed induction of insulin receptors by TSH, physiological concentrations of insulin also permit the proliferative action of TSH [START_REF] Burikhanov | Thyrotropin via cyclic AMP induces insulin receptor expression and insulin co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells[END_REF]Van Keymeulen et al., 2000a). In dog thyrocytes, in the absence of insulin/IGF-I, acetylcholine [START_REF] Van Keymeulen | Respective roles of carbamylcholine and cyclic AMP in their synergistic regulation of cell cycle in thyroid primary cultures[END_REF] and phorbol myristate ester are also permissive for TSH-cAMP mitogenic stimulation action, presumably by the stimulation of protein kinase C and consequently activation of Ras (Van Keymeulen et al., 2000b), MAP kinase and increased cyclin D expression [START_REF] Van Keymeulen | Respective roles of carbamylcholine and cyclic AMP in their synergistic regulation of cell cycle in thyroid primary cultures[END_REF]. Thus the IGF-I-PI-3K pathway may complement the TSH-cAMP and the EGF-MAPK cascades by at least partially different mechanisms.

All the proliferation effects of TSH are mimicked by nonspecific modulators of the cAMP cascade, that is, cholera toxin and forskolin (which stimulate adenylyl cyclase), cAMP

A c c e p t e d M a n u s c r i p t 8 analogs (which activate the cAMP-dependent protein kinases (PKA)), and even synergistic pairs of cAMP analogues acting on the different sites of these kinases [START_REF] Roger | Stimulation by thyrotropin and cyclic AMP of the proliferation of quiescent canine thyroid cells cultured in a defined medium containing insulin[END_REF][START_REF] Van Sande | Pairs of cyclic AMP analogs, that are specifically synergistic for type I and type II cAMPdependent protein kinases, mimic thyrotropin effects on the function, differentiation expression and mitogenesis of dog thyroid cells[END_REF]Roger et al., 1988b;[START_REF] Dremier | Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases, but not exchange proteins directly activated by cAMP (Epac), mediate thyrotropin/cAMPdependent regulation of thyroid cells[END_REF]. They are reproduced in vitro and in vivo by expression of the adenosine A 2 receptor, which is constitutively activated by endogenous adenosine [START_REF] Maenhaut | RDC8 codes for an adenosine A2 43 receptor with physiological constitutive activity[END_REF][START_REF] Ledent | The TSH cyclic AMP cascade in the control of thyroid cell proliferation: the story of a concept[END_REF][START_REF] Ledent | Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism[END_REF], and by constitutively active Gsα and cholera toxin [START_REF] Zeiger | Thyroid-specific expression of cholera toxin A1 subunit causes thyroid hyperplasia and hyperthyroidism in transgenic mice[END_REF]. They are inhibited by microinjected antibodies blocking G s [START_REF] Meinkoth | Inhibition of thyrotropin-induced DNA synthesis in thyroid follicular cells by microinjection of an antibody to the stimulatory G protein of adenylate cyclase, Gs[END_REF]. Inhibition of PKA inhibits the proliferation and differentiation effects of cAMP [START_REF] Kupperman | Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase[END_REF][START_REF] Dremier | Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells[END_REF]. Moreover, stimulation of PKA by selective cAMP analogs that do not activate EPAC proteins is sufficient to fully mimic mitogenic effects of TSH and forskolin in dog thyrocytes [START_REF] Dremier | Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases, but not exchange proteins directly activated by cAMP (Epac), mediate thyrotropin/cAMPdependent regulation of thyroid cells[END_REF]. There is, therefore, no doubt that the mitogenic and differentiating effects of TSH are mainly and probably entirely mediated by PKAs. A complementary role of the Rap1 guanyl nucleotide exchange factor EPAC and of Rap1 has been proposed in cell lines and in mice [START_REF] Ribeiro-Neto | On the mitogenic properties of Rap1b: cAMP-induced G(1)/S entry requires activated and phosphorylated Rap1b[END_REF][START_REF] Hochbaum | Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis[END_REF][START_REF] Hong | A novel Epac-Rap-PP2A signaling module controls cAMP-dependent Akt regulation[END_REF] but it is not observed in canine thyroid primary cultures [START_REF] Dremier | Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases, but not exchange proteins directly activated by cAMP (Epac), mediate thyrotropin/cAMPdependent regulation of thyroid cells[END_REF].

Unfortunately the role and mechanism of action of other pathways such as JAK/STAT, Wnt-FZZL, NOTCH and Sonic Hedgehog in normal human or dog thyroid cells have been little studied so far. There are indications that STATs and catenin (Wnt pathway) play a role in human thyroid cancers. Moreover lithium stimulates human thyrocyte proliferation by probably activating the Wnt/ catenin signaling [START_REF] Rao | Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/beta-catenin signalling[END_REF]. The fact that the higher frequency of thyroid tumors in women versus men is limited to the period between puberty and menopause (B. Jarzab, personal communication) suggests a proliferative effect of estrogens on human thyroid cells. One study thoroughly investigated the impact of sex steroids on proliferation in rat thyroid primary cultures, showing that testosterone and estradiol differentially regulate TSH-induced proliferation in cells from immature and adult rats in a gender-specific manner [START_REF] Banu | Testosterone and estradiol differentially regulate thyroid growth in Wistar rats from immature to adult age[END_REF]. Similar effects remain to be studied in normal human cells, nevertheless estrogen has been reported to increase proliferation in thyroid cancer cell lines [START_REF] Zeng | Oestrogen mediates the growth of human thyroid carcinoma cells via an oestrogen receptor-ERK pathway[END_REF].

3. Biochemical aspects of the proliferative response. EGF and phorbol esters (TPA) [START_REF] Contor | Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester[END_REF]. EGF, HGF and phorbol ester (Roger et al., 1986) actions rapidly converge on the activation of Ras (Van Keymeulen et al., 2000b) and the resulting activation of ERK1/2 and p90 RSK [START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF][START_REF] Lamy | Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway[END_REF][START_REF] Vandeput | Role of the different mitogen-activated protein kinase subfamilies in the stimulation of dog and human thyroid epithelial cell proliferation by cyclic adenosine 5'-monophosphate and growth factors[END_REF]. PI-3-kinase and its effector enzyme PKB are activated for several hours only by insulin and IGF-I, the effect of EGF being weak and short lived [START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF]. This activity is therefore the one specific feature of insulin/IGF-I action and presumably the mechanism of its facilitative effect on mitogenesis (Fig. 2). In dog thyrocytes, only HGF can trigger cell proliferation in the absence of insulin/IGF-I; this is explained by the fact that only this factor strongly activates both PI-3K and Ras-ERK cascades [START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF] (Fig. 2). By contrast, TSH and cAMP are very unique as mitogens, as they do not activate Ras, the PI-3K-PKB pathway, or any of different classes of MAP kinases in dog thyrocytes [START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF][START_REF] Lamy | Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway[END_REF][START_REF] Vandeput | Role of the different mitogen-activated protein kinase subfamilies in the stimulation of dog and human thyroid epithelial cell proliferation by cyclic adenosine 5'-monophosphate and growth factors[END_REF]Van Keymeulen et al., 2000b)(Fig. 2). TSH and cAMP also do not activate MAP kinases in human thyrocytes [START_REF] Vandeput | Role of the different mitogen-activated protein kinase subfamilies in the stimulation of dog and human thyroid epithelial cell proliferation by cyclic adenosine 5'-monophosphate and growth factors[END_REF]. The activation of the mTOR cascade (as initially indicated by p70 S6K phosphorylation and activation [START_REF] Cass | Differential effects of cyclic adenosine 3',5'-monophosphate on p70 ribosomal S6 kinase[END_REF][START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF]) apparently constitutes the only early convergence point of growth factor and cAMP-dependent mitogenic cascades (Blancquaert et al, submitted). A recent study has demonstrated the crucial role of this cascade for TSH-elicited thyroid follicular hyperplasia in vivo in mice [START_REF] Brewer | Thyroid-stimulating hormone initiated proliferative signals converge in vivo on the mTOR kinase without activating AKT[END_REF]. Indeed, as found in dog thyroid primary cultures [START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF] and PC Cl3 cells (Blancquaert et al, submitted), TSH stimulates in mice the mTOR-p70 S6K axis without activating PKB, and a rapamycin derivative abrogates the hyperplastic (but, interestingly, not the hypertrophic) responses to TSH [START_REF] Brewer | Thyroid-stimulating hormone initiated proliferative signals converge in vivo on the mTOR kinase without activating AKT[END_REF]. Growth factors and IGF-I most likely activate the mTOR-raptor complex (mTORC1) through various inactivating phosphorylations of the tumor suppressor TSC2 (tuberin) [START_REF] Ma | Molecular mechanisms of mTOR-mediated translational control[END_REF], whereas both PKB and PKA (activated by IGF-I/insulin and TSH, respectively) stably activate mTORC1 by phosphorylating PRAS40 at [START_REF] Uyttersprot | Requirement for cAMPresponse element (CRE) binding protein/CRE modulator transcription factors in thyrotropininduced proliferation of dog thyroid cells in primary culture[END_REF]. The role of the possibly important Ral pathway downstream of Ras has been little studied. It was necessary for the transformation of human thyrocytes in vitro [START_REF] Bounacer | Mutant ras-induced proliferation of human thyroid epithelial cells requires three effector pathways[END_REF].

b) Induction of early mitogenic response transcription factors.

As in other types of cells, EGF and TPA first enhance c-fos and c-myc mRNA and protein levels in dog and human thyrocytes [START_REF] Reuse | Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression[END_REF][START_REF] Baptist | Intercellular heterogeneity of early mitogenic events: cAMP generalizes the EGF effect on c-Fos protein appearance but not on MAP kinase phosphorylation and nuclear translocation in dog thyroid epithelial cells[END_REF]Pirson et al., 1996;[START_REF] Heinrich | The protein kinase A pathway inhibits c-jun and c-fos protooncogene expression induced by the protein kinase C and tyrosine kinase pathways in cultured human thyroid follicles[END_REF]. On the other hand, TSH and forskolin only very transiently enhance c-myc mRNA expression [START_REF] Pirson | c-Myc expression is controlled by the mitogenic cAMP-cascade in thyrocytes[END_REF]. In fact, cAMP even decreases c-myc expression stimulated by insulin and growth factors [START_REF] Reuse | Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression[END_REF][START_REF] Pirson | c-Myc expression is controlled by the mitogenic cAMP-cascade in thyrocytes[END_REF]. This second phenomenon is akin to what has been observed in fibroblasts, in which cAMP negatively regulates growth. As in fibroblasts, EGF and TPA enhance c-jun, and egr1 mRNA expression. However, as in fibroblasts, activators of the cAMP cascade had a minimal effect on c-fos and decreased c-jun and egr1 mRNA expression [START_REF] Reuse | Differential regulation of protooncogenes c-jun and jun D expressions by protein tyrosine kinase, protein kinase C, and cyclic-AMP mitogenic pathways in dog primary thyrocytes: TSH and cyclic-AMP induce proliferation but downregulate C-jun expression[END_REF]Deleu et al., 1999a;Deleu et al., 1999b;[START_REF] Heinrich | The protein kinase A pathway inhibits c-jun and c-fos protooncogene expression induced by the protein kinase C and tyrosine kinase pathways in cultured human thyroid follicles[END_REF][START_REF] Hebrant | Long-term EGF/serum-treated human thyrocytes mimic papillary thyroid carcinomas with regard to gene expression[END_REF].

c) Cell cycle regulatory proteins (Fig. 3).

The investigation of the pattern of proteins synthesized in response to the various proliferation stimuli has suggested very early that the proliferation of dog thyroid cells is controlled during G1 phase by at least two largely distinct, cAMP-dependent or cAMPindependent, pathways [START_REF] Lamy | Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways[END_REF]. Recent microarray analyses have confirmed and extended this concept in human thyrocytes [START_REF] Van Staveren | Gene expression in human thyrocytes and autonomous adenomas reveals suppression of negative feedbacks in tumorigenesis[END_REF][START_REF] Hebrant | Long-term EGF/serum-treated human thyrocytes mimic papillary thyroid carcinomas with regard to gene expression[END_REF].

Nevertheless, the different mitogenic cascades are expected to finally modulate the level and activity of proteins that are the common primary regulators of the cell cycle machinery. As generally considered, mitogenic signals regulate mammalian cell cycle by stimulating the accumulation of D-type cyclins and their assembly through a ill-defined mechanism with their partners, the cyclin-dependent kinases (CDK) 4 and 6. These complexes operate in mid-tolate G1 phase to promote progression through the restriction point, and thus commit cells to replicate their genome [START_REF] Bartek | The retinoblastoma protein pathway and the restriction point[END_REF][START_REF] Sherr | CDK inhibitors: positive and negative regulators of G1-phase progression[END_REF]Bockstaele et al., 2006a).

In the current model, this key decision depends on the initiation by cyclin D-CDK4/6

A c c e p t e d M a n u s c r i p t 11 complexes of the phosphorylation of the growth/tumor suppressor protein pRb (Fig. 3), which triggers the activation of transcription factors, including those of the E2F family, the synthesis of cyclin E and then cyclin A, and CDK2 activation by these cyclins. Activated CDK2 in turn further phosphorylates pRb and other substrates and initiates and organizes the progression through the DNA synthesis phase [START_REF] Sherr | CDK inhibitors: positive and negative regulators of G1-phase progression[END_REF]. The down regulation of p27 kip1 , a CDK inhibitor of the CIP/KIP family, by mitogenic factors and/or its sequestration by cyclin D-CDK complexes participate to CDK2 activation, but the proposed role of p27 kip1 and p21 cip1 as adaptors and/or nuclear anchors for cyclin D-CDK complexes suggests positive influences on cell cycle progression as well [START_REF] Sherr | CDK inhibitors: positive and negative regulators of G1-phase progression[END_REF]Bockstaele et al., 2006a).

These mechanisms have been well studied in dog thyroid cells (Bockstaele et al., 2006a). The different mitogenic stimulations (TSH, cAMP, growth factors) require the activity of CDK4 [START_REF] Lukas | Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled G1 checkpoint[END_REF], and converge on the inactivating phosphorylation of pRb and related proteins p107 and p130 [START_REF] Coulonval | Phosphorylation of the three Rb protein family members is a common step of the cAMP-, the growth factor, and the phorbol ester-mitogenic cascades but is not necessary for the hypertrophy induced by insulin[END_REF][START_REF] Van Keymeulen | Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture[END_REF], on the phosphorylation and nuclear translocation of CDK2, and on the induction of cyclin A and CDK1 [START_REF] Baptist | Expression and subcellular localization of CDK2 and cdc2 kinases and their common partner cyclin A in thyroid epithelial cells: comparison of cyclic AMP-dependent and -independent cell cycles[END_REF]. These effects are dependent on insulin [START_REF] Coulonval | Phosphorylation of the three Rb protein family members is a common step of the cAMP-, the growth factor, and the phorbol ester-mitogenic cascades but is not necessary for the hypertrophy induced by insulin[END_REF][START_REF] Van Keymeulen | Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture[END_REF]. What is strikingly different between the cascades is the mechanism of D-type cyclin-CDK4 activation. TSH, unlike all the other known mitogenic factors, does not induce the accumulation of cyclins D, but it paradoxically stimulates the expression of p27 kip1 [START_REF] Depoortere | Paradoxical accumulation of the cyclindependent kinase inhibitor p27kip1 during the cAMP-dependent mitogenic stimulation of thyroid epithelial cells[END_REF]. However the predominant cyclin D3 is required for the proliferation stimulated by TSH, but not in the proliferation of dog thyrocytes stimulated by EGF or HGF which induce cyclins D1 and D2 [START_REF] Depoortere | A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes[END_REF]Paternot et al., 2006a). In dog thyrocytes, the formation and the nuclear translocation of essential cyclin D3-CDK4 complexes depend on the synergistic interaction of TSH and insulin. The presence of the required cyclin D3 depends on insulin/IGF-I (Van [START_REF] Van Keymeulen | Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture[END_REF], whereas TSH stimulates the assembly of the cyclin D3-CDK4 complexes and their import into nuclei [START_REF] Depoortere | A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes[END_REF][START_REF] Van Keymeulen | Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture[END_REF] where these complexes are anchored by their association with p27 kip1 [START_REF] Depoortere | Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1)[END_REF][START_REF] Coulonval | The cyclin D3-CDK4-p27kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis[END_REF]. This also sequesters p27 away of CDK2 complexes [START_REF] Depoortere | Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1)[END_REF], thus contributing to CDK2 activation. Moreover, cAMP exerts an additional crucial function in very late G1 phase [START_REF] Roger | Nature of the critical labile event that controls RB phosphorylation in the cyclic AMP-dependent cell cycle of thyrocytes in primary culture[END_REF][START_REF] Van Keymeulen | Respective roles of carbamylcholine and cyclic AMP in their synergistic regulation of cell cycle in thyroid primary cultures[END_REF] to stimulate the enzymatic activity of cyclin D3-CDK4-p27 complexes, which involves the stimulation of the activating Thr172-phosphorylation of CDK4 (Bockstaele et al., 2006b;[START_REF] Paternot | Cyclic AMP-dependent phosphorylation of cyclin D3-bound CDK4 determines the passage through the cell cycle restriction point in thyroid epithelial cells[END_REF].

A c c e p t e d M a n u s c r i p t 12

The nature of the regulated CDK4-activating kinase complex, and whether it contains CDK7 as the catalytic subunit, remains to be defined (Bockstaele et al., 2006b;Bockstaele et al., 2006a;[START_REF] Bockstaele | Differential regulation of CDK4 and CDK6, evidence that CDK4 might not be activated by CDK7, and design of a CDK6 activating mutation[END_REF][START_REF] Paternot | Rb inactivation in cell cycle and cancer : The puzzle of highly regulated activating phosphorylation of CDK4 versus constitutively active CDK-activating kinase[END_REF] (Fig. 3). Importantly, TSH-stimulated CDK4 phosphorylation and activity are partly inhibited by rapamycin, indicating the involvement of the mTOR-raptor complex activated by TSH (Blancquaert et al, submitted).

TGF selectively inhibits the cAMP-dependent proliferation of dog thyrocytes by preventing the association of the cyclin D3-CDK4 complex with nuclear p27 kip1 and the consequent activating phosphorylation of CDK4 [START_REF] Depoortere | Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1)[END_REF][START_REF] Coulonval | The cyclin D3-CDK4-p27kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis[END_REF].

The investigation of cell cycle regulatory proteins has thus clearly established that both CDK4 activation and pRb phosphorylation result from distinct but complementary actions of TSH and insulin, rather than from their interaction at an earlier step of the signalling cascades [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF][START_REF] Van Keymeulen | Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture[END_REF][START_REF] Van Keymeulen | Respective roles of carbamylcholine and cyclic AMP in their synergistic regulation of cell cycle in thyroid primary cultures[END_REF] (Fig. 1A and3). Together with the fact that the necessary increase of cell mass before division depends on insulin/IGF-I but not TSH (Deleu et al., 1999b), these observations provide a molecular basis for the well established physiological concept that in the regulation of normal thyroid cell proliferation, TSH is the "decisional" mitotic trigger, while locally produced IGF-I and/or circulating insulin are supporting "permissive" factors. Of note, in all these experiments, the facilitative action of insulin on TSH action but not on the EGF action can be replaced by activation of the Gq/PLC cascade by carbamylcholine (Van Keymeulen et al., 2001).

In the "dedifferentiating" proliferation mode induced by EGF and serum in dog thyrocytes, cyclin D1 and p21 cip1 are transiently induced, and p21 (instead of p27 which is down regulated by growth factors) supports the nuclear translocation of cyclin D1-CDK4 complexes, whose activity also depends on regulated CDK4 phosphorylation [START_REF] Depoortere | A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes[END_REF]Paternot et al., 2006b) (Fig. 3). The main differences between the differentiationassociated mitogenic stimulation by TSH and cAMP and dedifferentiating mitogenic mode of EGF have been confirmed in human cells. Indeed, in primary cultures of normal human thyrocytes, EGF+serum increases cyclin D1 and p21 accumulation, and it stimulates the assembly and activity of cyclin D1-CDK4-p21 (Paternot et al., 2006a) (Fig. 4). By contrast, TSH (cAMP) represses cyclin D1 and p21, but stimulates the activating phosphorylation of CDK4 and the pRb-kinase activity of preexisting cyclin D3-CDK4 complexes (Paternot et al., 2006a) (Fig. 4). Cyclin D1 or cyclin D3 are thus differentially used in the distinct mitogenic Homeostasis in tissues implies at all levels a dynamic control with negative feedbacks.

In the thyroid, the main physiological feedbacks involve the thyroid hormone inhibition of TSH synthesis in, and release by, the thyrotrophs of the pituitary and the dampening by substrate availability (i.e. iodide) of the thyroid response to TSH. These feedbacks are functional, thus do not play any role in dedifferentiated tumors. The pituitary thyroid hormone feedback does not play a role either in thyroid cells, which thanks to a constitutive activation of a protein of the TSH-cAMP cascade (TSH or Gs ), are no longer dependent on TSH.

There are also negative feedback lopps within the thyrocytes themselves. Some have been well defined for the TSH-cAMP pathway and involve direct effects on enzyme activity such as the activation of cyclic AMP phosphodiesterases by phosphorylations and induction of negative effectors of the cascade such as GRK (inhibiting the receptor), RGS (inhibiting Gs), phosphodiesterases, and ICER (inhibiting the action of CREB). The conjunction of these feedback loops makes very unlikely a rapid second division of a stimulated cell. It is therefore not surprising that a suppression of some of these feedback loops is observed in autonomous adenomas which led us to suggest this is a necessary condition of tumorigenesis [START_REF] Van Staveren | Gene expression in human thyrocytes and autonomous adenomas reveals suppression of negative feedbacks in tumorigenesis[END_REF]. The suppression of negative intracellular feedback loops in tumors resulting from other cascades, has also been observed later but not yet investigated in thyroid tumors.

Proliferation and differentiation.

Different mechanisms might contribute to explain the very different proliferation/differentiation relationship in the distinct mitogenic modes elicited by TSH and growth factors. Activation of Ras and ERK1/2, induction of c-jun, sustained expression of cmyc, induction of cyclin D1 and down regulation of p27 kip1 , all have been shown to be causatively associated not only with proliferation, but also with loss of differentiation in a large variety of systems, sometimes independently of the proliferation effects. The effects of the cAMP cascade are in striking contrast with this general concept. Indeed, TSH and cAMP A c c e p t e d M a n u s c r i p t 14 induce the proliferation of dog thyrocytes while maintaining differentiation expression; both proliferation and differentiation programs can be triggered by TSH in the same cells at the same time [START_REF] Pohl | Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells[END_REF][START_REF] Pohl | Thyroperoxidase mRNA in quiescent and proliferating thyroid epithelial cells: expression and subcellular localization studied by in situ hybridization[END_REF]. It is tempting to relate this apparent paradox to the unique characteristics of the cAMP-dependent mitogenic pathway, such as the lack of activation (or even the inhibition) of the Ras-ERK-c-jun-cyclin D1 cascade, as demonstrated in dog and human thyrocytes. The fact that c-myc is even repressed by cAMP (after a first very transient induction) also likely prevents its dedifferentiating impact observed in a variety of cell types. The specific involvement of cyclin D3 in the cAMP-dependent mitogenic stimulation of dog and human thyrocytes is also interesting in this context [START_REF] Depoortere | A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes[END_REF]Paternot et al., 2006a). We have recently shown that the differential use of cyclin D1 or cyclin D3 affects quantitatively and qualitatively the phosphorylation site specificity of the pRb-kinase CDK4, including in dog and human thyrocytes (Paternot et al., 2006a;Paternot et al., 2006b) (Fig. 4). In addition to inhibiting E2F-dependent gene transcription related to cell cycle progression, pRb plays positive roles in the induction of tissue-specific gene expression by directly interacting with a variety of transcription factors, including Pax8 in thyroid cells [START_REF] Miccadei | Retinoblastoma protein acts as Pax 8 transcriptional coactivator[END_REF]. Whether, the selective use of cyclin D3 in the TSH cascade, associated with a more restricted pRb-kinase activity, could allow the preservation of some differentiation-related functions of pRb thus remains to be examined. Indeed, unlike cyclins D1 and D2, cyclin D3 is highly expressed in several quiescent tissues in vivo, and its expression is not only stimulated by mitogenic factors but also induced during several differentiation processes associated with a repression of cyclin D1 [START_REF] Bartkova | Cyclin D3: requirement for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation[END_REF].

We now consider the distinct cAMP-dependent mitogenic pathway, which appears to be superimposed to the more general mechanisms used by growth factors, as pertaining to the specialized differentiation program of thyroid cells and some other organs (eg pituitary) [START_REF] Roger | Multiple facets of the modulation of growth by cAMP[END_REF][START_REF] Lyons | Transforming growth factors and the regulation of cell proliferation[END_REF]Lyons et al., 1990). In dog thyrocytes, the proliferation in response to serum or growth factors specifically extincts their capacity to respond to TSH/cAMP as a mitogenic stimulus [START_REF] Roger | A mechanism generating heterogeneity in thyroid epithelial cells: suppression of the thyrotropin/cAMP-dependent mitogenic pathway after cell division induced by cAMP-independent factors[END_REF]. Similarly, in less differentiated thyroid cancers generated by the subversion of growth factor mechanisms, the TSH-dependence of growth is generally found to be lost, and in various cell lines derived from thyroid carcinomas, cAMP and PKA activation even inhibit CDK4 activity and cell cycle progression [START_REF] Rocha | Cyclic AMP inhibits the proliferation of thyroid carcinoma cell lines through regulation of CDK4 phosphorylation[END_REF].

Validity of these concepts in vivo.

A c c e p t e d M a n u s c r i p t 15 The qualitative validity of these concepts in vivo has been established by using transgenic mice models. The expression in thyroid of oncogene E7 of HPV-16, which sequestrates pRb protein, leads to thyroid growth and (partially dedifferentiated) euthyroid goiter [START_REF] Burniat | Gene expression in RET/PTC3 and E7 transgenic mouse thyroids: RET/PTC3 but not E7 tumors are partial and transient models of human papillary thyroid cancers[END_REF][START_REF] Coppee | Early occurrence of metastatic differentiated thyroid carcinomas in transgenic mice expressing the A2a adenosine receptor gene and the human papillomavirus type 16 E7 oncogene[END_REF]. Expression in the thyroid of the adenosine A 2 receptor, which behaves as a constitutive activator of adenylyl cyclase, induces thyroid growth, goitrogenesis, and hyperthyroidism [START_REF] Ledent | Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism[END_REF]. Similar, albeit weaker phenotypes are obtained in mice expressing constitutive Gs (the G protein activating adenylyl cyclase) [START_REF] Michiels | Oncogenic potential of guanine nucleotide stimulatory factor alpha subunit in thyroid glands of transgenic mice[END_REF] or cholera toxin [START_REF] Zeiger | Thyroid-specific expression of cholera toxin A1 subunit causes thyroid hyperplasia and hyperthyroidism in transgenic mice[END_REF]. A contrario, the expression in thyroid of a dominant negative CREB provokes a marked thyroid hypotrophy, suggesting the crucial role of CREB and its activating phosphorylation by PKA [START_REF] Nguyen | A dominant negative CREB (cAMP response element-binding protein) isoform inhibits thyrocyte growth, thyroid-specific gene expression, differentiation, and function[END_REF]. By contrast, transgenic mice overexpressing both human IGF-I and IGF-I receptor in their thyroid (TgIGF-I-TgIGF-IR) develop only a mild thyroid hyperplasia and respond to a goitrogenic effect of antithyroid drugs while maintaining a comparatively low serum TSH level. This indicates some autonomy of these thyroids, as in acromegalic patients, and a much greater sensitivity to endogenous TSH [START_REF] Clement | Low TSH requirement and goiter in transgenic mice overexpressing IGF-I and IGF-Ir receptor in the thyroid gland[END_REF]. Recently, thyrocyte-specific deficiency of Gq/G 11 (the G proteins activating PLC ) in mice was shown to impair not only the TSH-stimulated iodide-organification and thyroid hormone synthesis, but also TSHdependent development of goiter [START_REF] Kero | Thyrocyte-specific Gq/G11 deficiency impairs thyroid function and prevents goiter development[END_REF]. It remains to be defined whether this impaired follicular cell hyperplasia could result in part from a direct requirement, from the lack of induction of VEGF and angiogenesis [START_REF] Kero | Thyrocyte-specific Gq/G11 deficiency impairs thyroid function and prevents goiter development[END_REF] which normally accompany goitrogenesis, or from another indirect effect. Nevertheless, the phenotype of these mice suggests a role of PLC, at least permissive, in TSH-dependent goitrogenesis. PLC is activated by TSH [START_REF] Van Sande | Thyrotropin stimulates the generation of inositol 1,4,5-trisphosphate in human thyroid cells[END_REF] but even more strongly but often transiently by neurotransmitters [START_REF] Raspe | Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells[END_REF][START_REF] Raspe | ATP, bradykinin, TRH and TSH activate the Ca(2+)-phosphatidylinositol cascade of human thyrocytes in primary culture[END_REF]. Moreover, activation of Gq/PLC by carbamycholine can facilitate cAMP-dependent mitogenesis in dog thyrocytes cultured without insulin or IGF-I (Van Keymeulen et al., 2001). Noteworthy, section of inferior laryngeal nerve in rats was similarly reported to impair both thyroid function and growth stimulated by TSH [START_REF] Romeo | Effect of inferior laryngeal nerve section on thyroid function in rats[END_REF]. However the success of thyroid xenotransplants argues against a necessary role of neurotransmitters in vivo. On the other hand, expression of RET/PTC alone, which is a constitutive growth factor receptor, in papillary thyroid carcinoma (PTC), leads to growth and hypothyroidism and later cancer in mice [START_REF] Jhiang | Targeted expression of the ret/PTC1 oncogene induces papillary thyroid carcinomas[END_REF][START_REF] Powell | The RET/PTC3 oncogene: metastatic solid-type papillary carcinomas in murine thyroids[END_REF] "Immortal" rat thyroid cell lines (FRTL-5, WRT and PC Cl3 cells) are well responsive to insulin/IGF-I but, when used alone, their impact on DNA-synthesis and proliferation much vary, even in different subclones of same cell line or during repeated cell passages [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]. Fibroblast growth factors stimulate proliferation while inhibiting differentiation expression in FRTL-5 and PC Cl3 cells [START_REF] Logan | Basic fibroblast growth factor: an autocrine mitogen of rat thyroid follicular cells?[END_REF][START_REF] Isozaki | Opposite regulation of deoxyribonucleic acid synthesis and iodide uptake in rat thyroid cells by basic fibroblast growth factor: correlation with opposite regulation of c-fos and thyrotropin receptor gene expression[END_REF][START_REF] Battaglia | Mitogenic and dedifferentiating effect of the K-fgf/hst oncogene on rat thyroid PC clone 3 epithelial cells[END_REF]. By contrast, the responsiveness to EGF of FRTL-5 and WRT cells appears a characteristic developed by "aged" cells [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]. In our hands, the proliferation of PC Cl3 cells is not affected by EGF and HGF [START_REF] Kimura | Insulin and TSH promote growth in size of PC Cl3 rat thyroid cells, possibly via a pathway different from DNA synthesis: comparison with FRTL-5 cells[END_REF]. Nevertheless these cell lines have been widely used to demonstrate/confirm the impact of thyroid oncogenes on signaling cascades, proliferation, differentiation, and transcriptomes [START_REF] Saavedra | The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway[END_REF][START_REF] De Vita | Akt/protein kinase B promotes survival and hormone-independent proliferation of thyroid cells in the absence of dedifferentiating and transforming effects[END_REF][START_REF] Cobellis | Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation[END_REF][START_REF] Miyagi | Chronic expression of RET/PTC 3 enhances basal and insulin-stimulated PI3 kinase/AKT signaling and increases IRS-2 expression in FRTL-5 thyroid cells[END_REF][START_REF] Vitagliano | Regulation of p27Kip1 protein levels contributes to mitogenic effects of the RET/PTC kinase in thyroid carcinoma cells[END_REF][START_REF] Cass | Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation[END_REF][START_REF] Mesa | Conditional activation of RET/PTC3 and BRAFV600E in thyroid cells is associated with gene expression profiles that predict a preferential role of BRAF in extracellular matrix remodeling[END_REF]. Conditional expression [START_REF] Mitsutake | Conditional BRAFV600E expression induces DNA synthesis, apoptosis, dedifferentiation, and chromosomal instability in thyroid PCCL3 cells[END_REF][START_REF] Saavedra | The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway[END_REF][START_REF] Wang | Conditional expression of RET/PTC induces a weak oncogenic drive in thyroid PCCL3 cells and inhibits thyrotropin action at multiple levels[END_REF] or activity [START_REF] De Vita | Dose-dependent inhibition of thyroid differentiation by RAS oncogenes[END_REF] avoid several selection artefacts. As far as Ras-Raf-MAPK and PI3-K-PKB signal transductions are concerned, results are most often compatible with the other in vivo and in vitro models and allowed to better understand the biology of thyroid cancers. Evidence for novel potential RET/PTC effectors were obtained, such as Erk8 [START_REF] Iavarone | Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene[END_REF]), Rap1 (De Falco et al., 2007), EGF receptor [START_REF] Croyle | RET/PTC-induced cell growth is mediated in part by epidermal growth factor receptor (EGFR) activation: evidence for molecular and functional interactions between RET and EGFR[END_REF] and -catenin [START_REF] Castellone | The beta-catenin axis integrates multiple signals downstream from RET/papillary thyroid carcinoma leading to cell proliferation[END_REF].

The mechanisms of TSH/cAMP mitogenic effects have also been investigated using FRTL-5 and WRT cells [START_REF] Rivas | TSH-activated signaling pathways in thyroid tumorigenesis[END_REF][START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]. The capacity of TSH and cAMP to induce DNA synthesis and proliferation, either alone or in combination with insulin/IGF-I, much vary in different subclones of FRTL-5 cells, depending on variations in culture media, or even on the "aging" of this cell line in the same laboratory [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]. In different publications of the same group using WRT cells, TSH/cAMP was reported as a fully sufficient mitogen due to activation of multiple pathways [START_REF] Cass | Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation[END_REF]Tsygankova et al., 2000;[START_REF] Vuchak | Protein kinase A and B-Raf mediate extracellular signal-regulated kinase activation by thyrotropin[END_REF], or to only enhance the effect of serum [START_REF] Lewis | Thyrotropin and serum regulate thyroid cell proliferation through differential effects on p27 expression and localization[END_REF]. Whereas the signaling cascades involved in the action of growth factors and IGF-I are likely to be well conserved in the different thyroid systems, as generally observed in the other cell types, the mechanistic logics of cell cycle regulation by cAMP has disappointingly turned out to strongly diverge in the various thyroid in vitro models [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]. These divergences do not only reflect species differences (Dumont et al., 1992b). Among the apparently similar rat thyroid cell lines, or even among different A c c e p t e d M a n u s c r i p t 17 subclones of FRTL-5 cells, major differences have been observed [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]. For instance, the PI-3K-PKB cascade is claimed to be activated by cAMP in WRT cells (Tsygankova et al., 2001), but inhibited by cAMP in PC Cl3 cells [START_REF] Lou | cAMP inhibition of Akt is mediated by activated and phosphorylated Rap1b[END_REF]. The induction of c-jun by TSH/cAMP in FRTL-5 cells and its repression by cAMP in WRT cells (Tominaga et al., 1994) as in dog [START_REF] Reuse | Differential regulation of protooncogenes c-jun and jun D expressions by protein tyrosine kinase, protein kinase C, and cyclic-AMP mitogenic pathways in dog primary thyrocytes: TSH and cyclic-AMP induce proliferation but downregulate C-jun expression[END_REF] and human thyrocytes [START_REF] Heinrich | The protein kinase A pathway inhibits c-jun and c-fos protooncogene expression induced by the protein kinase C and tyrosine kinase pathways in cultured human thyroid follicles[END_REF] likely reflect major differences in upstream signaling cascades, and should result in divergent expression of downstream target genes, such as cyclin D1. Cyclin D1 synthesis, an accepted endpoint of mitogenic cascades, is indeed induced by cAMP in FRTL-5 and PC Cl3 cells, but rather repressed by cAMP in dog and human thyroid primary cultures [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]Paternot et al., 2006b). Conversely TSH used alone down-regulates p27 in FRTL-5 cells [START_REF] Medina | Somatostatin is expressed in FRTL-5 thyroid cells and prevents thyrotropin-mediated down-regulation of the cyclin-dependent kinase inhibitor p27kip1[END_REF], but not in WRT cells [START_REF] Lewis | Thyrotropin and serum regulate thyroid cell proliferation through differential effects on p27 expression and localization[END_REF], whereas it up-regulates it in dog and human thyrocytes [START_REF] Depoortere | Paradoxical accumulation of the cyclindependent kinase inhibitor p27kip1 during the cAMP-dependent mitogenic stimulation of thyroid epithelial cells[END_REF]Paternot et al., 2006b).

The reasons for such discrepancies are unclear. Some signaling features, when they lead to selective proliferative advantages, might have been acquired during the establishment and continuous cultures of the cell lines and stabilized by subcloning. On the other hand, both the specific cyclin D3 requirement [START_REF] Depoortere | A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes[END_REF][START_REF] Motti | Critical role of cyclin D3 in TSH-dependent growth of thyrocytes and in hyperproliferative diseases of the thyroid gland[END_REF]Paternot et al., 2006a) and the cAMP-dependent, rapamycin-sensitive, regulation of CDK4 phosphorylation have been observed in both dog thyrocytes and PC Cl3 cells (Blancquaert et al, submitted).

Many mechanisms demonstrated in the dog thyroid primary culture system so far apply to normal human thyrocytes, but much remains to be defined [START_REF] Kimura | Regulation of thyroid cell proliferation by thyrotropin and other factors : a critical evaluation of in vitro models[END_REF]Paternot et al., 2006b).

III. Genetic alterations commonly found in thyroid tumors.

Different genetic alterations are involved in thyroid tumorigenesis. Their regroupment in signalling pathways illustrates the functional approach of tumorigenesis.

1. The cAMP pathway: hyperfunctioning autonomous adenomas and congenital non autoimmune hyperthyroidism (Fig. 1A)

In Europe and in Japan, one of the main mechanisms responsible for the hyperfunction and growth of thyroid autonomous adenomas is the constitutive activation of the cAMPdependent mitogenic cascade, through mutations conferring constitutive activity of the TSH receptor (50 to 80%) or an activating mutation of G s (8%) (Lyons et al., 1990;[START_REF] Parma | Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas[END_REF] A c c e p t e d M a n u s c r i p t 18 1993; [START_REF] Fuhrer | Identification of a new thyrotropin receptor germline mutation (Leu629Phe) in a family with neonatal onset of autosomal dominant nonautoimmune hyperthyroidism[END_REF][START_REF] Russo | Activating mutations of the TSH receptor in differentiated thyroid carcinomas[END_REF]Tonacchera et al., 1999;[START_REF] Van Sande | Somatic and germline mutations of the TSH receptor gene in thyroid diseases[END_REF][START_REF] Vanvooren | Oncogenic mutations in the thyrotropin receptor of autonomously functioning thyroid nodules in the Japanese population[END_REF][START_REF] Hebrant | Long-term EGF/serum-treated human thyrocytes mimic papillary thyroid carcinomas with regard to gene expression[END_REF][START_REF] Ledent | The TSH cyclic AMP cascade in the control of thyroid cell proliferation: the story of a concept[END_REF][START_REF] Ledent | Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism[END_REF][START_REF] Hebrant | Thyroid gene expression in familial nonautoimmune hyperthyroidism shows common characteristics with hyperfunctioning autonomous adenomas[END_REF]. Similar mutations account for hyperfunctioning nodules in multinodular goiters (Tonacchera et al., 1999) . Autonomous adenomas are thus a well-defined example for human thyroid tissue in vivo of the predicted results of long-term stimulation by the physiological TSH receptor/cAMP dependent cascade [START_REF] Dumont | The cyclic AMP-mediated stimulation of cell proliferation[END_REF]. Besides affecting thyroid cells, activating mutations in partners of the cAMP pathway also induce uncontrolled growth of other cells or tissues. Gain-of-function mutations of GNAS1, encoding G s , are found in different sporadic endocrine tumors (e.g. growth hormone-secreting pituitary adenomas, Leydig cell tumors) and diverse tissues of patients with McCune-Albright syndrome, in which the mutations occur early during embryogenesis [START_REF] Lania | G protein mutations in endocrine diseases[END_REF]. Inactivating mutations in the gene encoding PKA type 1-alpha regulatory subunit (PRKAR1A), have been identified in Carney Complex, a familial multiple neoplasia syndrome (Kirschner et al, 2000). The Carney Complex has features overlapping those of the McCune-Albright syndrome, and both have thyroid pathologies as part of their phenotype. Dominant activating mutations of the TSH receptor are also the cause of familial non autoimmune hyperthyroidism (FNAH) and of sporadic congenital non autoimmune hyperthyroidism (SCNAH) [START_REF] Kopp | Brief report: congenital hyperthyroidism caused by a mutation in the thyrotropin-receptor gene[END_REF][START_REF] Lania | G protein mutations in endocrine diseases[END_REF][START_REF] Polak | Congenital hyperthyroidism: the fetus as a patient[END_REF][START_REF] Hebrant | Thyroid gene expression in familial nonautoimmune hyperthyroidism shows common characteristics with hyperfunctioning autonomous adenomas[END_REF]. In both cases, the whole gland is affected since embryogenesis, whereas in autonomous adenomas, the mutation is somatic and thus affects only one cell later in the adulthood.

In the case of autonomous adenomas, the TSH receptor mutation strongly activates the cAMP cascade in one cell which initiates the adenoma. In the case of FNAH the mutations are milder but affect all the cells. Very active mutations in all the thyroid cells would probably be lethal while mild mutations in one cell would have only a local effect. The SCNAH cases were caused by strong mutations (as in adenoma) and had to be treated energetically after birth to save the patients. We have proposed to regroup all these syndromes as one disease, genetic hyperthyroidism [START_REF] Hebrant | Thyroid gene expression in familial nonautoimmune hyperthyroidism shows common characteristics with hyperfunctioning autonomous adenomas[END_REF]. Of course hyperthyroidism secondary to increased TSH secretion of pituitary origin or to thyroid stimulating antibodies (Graves disease) leads to a similar thyroid phenotype.

Animal models include transgenic mice expressing in the thyroid the adenosine A 2 receptor [START_REF] Ledent | Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism[END_REF]. Constitutively active Gs [START_REF] Michiels | Oncogenic potential of guanine nucleotide stimulatory factor alpha subunit in thyroid glands of transgenic mice[END_REF] or cholera toxin [START_REF] Zeiger | Thyroid-specific expression of cholera toxin A1 subunit causes thyroid hyperplasia and hyperthyroidism in transgenic mice[END_REF]. Moreover thyroid tumors have been observed as a consequence of both [START_REF] Kirschner | A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues[END_REF].

2. The Ras-MAPK pathway (Fig. 1B): Thyroid carcinomas The distinct forms of thyroid carcinomas are characterized by different genetic alterations in signalling pathways. These genetic alterations are generally mutually exclusive. Differentiated carcinomas (PTC, FTC) are essentially characterized by constitutive activation of the MAPK signalling pathway, resulting from mutations or rearrangements in different effectors of this pathway, essentially RET, BRAF or RAS. It should be noted that the constitutive activation of these pathways in the carcinomas is only partial but chronic contrary to the drastic effects of EGF in vitro. Animal models include transgenic mice expressing in their thyroid the RET/PTC1, RET/PTC3, and mutated B-Raf and Ras [START_REF] Jhiang | Targeted expression of the ret/PTC1 oncogene induces papillary thyroid carcinomas[END_REF][START_REF] Powell | The RET/PTC3 oncogene: metastatic solid-type papillary carcinomas in murine thyroids[END_REF][START_REF] Knauf | Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation[END_REF][START_REF] Santoro | Development of thyroid papillary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice[END_REF][START_REF] Rochefort | Thyroid pathologies in transgenic mice expressing a human activated Ras gene driven by a thyroglobulin promoter[END_REF][START_REF] Santelli | Thyroid specific expression of the Ki-ras oncogene in transgenic mice[END_REF]. A doxycycline-inducible mouse model of thyroid targeted RET/PTC1 oncogene has been created [START_REF] Knostman | Creation and characterization of a doxycycline-inducible mouse model of thyroid-targeted RET/PTC1 oncogene and luciferase reporter gene coexpression[END_REF].

In addition, genetic alterations involving proteins along the PI-3K/PKB signalling pathway have been described, particularly in follicular (FTC) and anaplastic (ATC) thyroid cancers. These include genomic copy number gain and activating mutations of PIK3CA, inactivating mutations and deletions of PTEN, and RAS activating mutations (Mitsutake et al., 2006;[START_REF] Garcia-Rostan | Mutation of the PIK3CA gene in anaplastic thyroid cancer[END_REF]. PTEN deletion in mouse thyroid causes causes goiter and follicular adenomas [START_REF] Yeager | Pten loss in the mouse thyroid causes goiter and follicular adenomas: insights into thyroid function and Cowden disease pathogenesis[END_REF]. and RET/PTC3 are the most common combined forms of RET. RET/PTC1 is formed by a paracentric inversion of the long arm of chromosome 10 leading to fusion with a gene named H4/D10S170 [START_REF] Grieco | PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas[END_REF]. RET/PTC3 is formed by fusion with the RFG/ELE1 gene [START_REF] Minoletti | The two genes generating RET/PTC3 are localized in chromosomal band 10q11.2[END_REF][START_REF] Santoro | Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma[END_REF]. Many other variants have been identified, mainly in children exposed to radiation after the Chernobyl power plant explosion (Ciampi et al., 2007;[START_REF] Alberti | RET and NTRK1 proto-oncogenes in human diseases[END_REF].

The prevalence of RET/PTC rearrangements varies widely among studies (8% to 85%) but most of them report a prevalence of 20-40% in adult sporadic PTC. The wide variability is due to different factors such as geographic variation but also the different procedures to identify the rearrangements (Zhu et al., 2006;[START_REF] Rhoden | RET/papillary thyroid cancer rearrangement in nonneoplastic thyrocytes: follicular cells of Hashimoto's thyroiditis share low-level recombination events with a subset of papillary carcinoma[END_REF] and the genetic heterogeneity of PTC. The prevalence of RET/PTC is significantly higher in pediatric PTC [START_REF] Bongarzone | Age-related activation of the tyrosine kinase receptor protooncogenes RET and NTRK1 in papillary thyroid carcinoma[END_REF][START_REF] Nikiforov | Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children[END_REF] and in cancers from children exposed to radiation after the Chernobyl nuclear accident. In the last ones, RET/PTC was found in up to 80% of tumors removed 5-8 years after the accident and in 50-60% of those removed 7-11 years after exposure (Ciampi and Nikiforov, 2007). Many studies have shown that short latency tumors are usually associated with the solid variant, a very aggressive form with a higher prevalence of RET/PTC3 rearrangement. By contrast, longer latency tumors are generally less aggressive, and associated with the classical variant and with the RET/PTC1 rearrangement (Unger et al., 2004;[START_REF] Williams | Thyroid carcinoma after Chernobyl latent period, morphology and aggressiveness[END_REF].

b) Rearrangements involving TRK

The neurotrophic tyrosine kinase receptor (NTRK)-1 or TRK is similar in many ways to RET in that it encodes a transmembrane TK receptor for neuronal growth factor, which is normally restricted to neuronal crest-derived cells.

Similar to RET/PTC rearrangements, the 3' TK domain of TRK can be fused with the 5' promoter region of an ubiquitously expressed fusion gene resulting in a chimeric fusion protein with constitutively active tyrosine kinase activity. Different 5' fusion partners have (TPR) and TRK Fused Gene (TFG) [START_REF] Alberti | RET and NTRK1 proto-oncogenes in human diseases[END_REF]. To date, TRK rearrangements have exclusively been identified in papillary thyroid carcinomas, with a prevalence of 5-13 %.

c) The AKAP9-BRAF fusion AKAP9-BRAF is another chromosomal rearrangement reported in about 11% of post-Chernobyl, radiation-associated, PTC. Only 1% of sporadic PTC display this mutation.

AKAP9-BRAF results from a paracentric inversion of the long arm of the chromosome 7 and leads to the fusion of the first 8 exons of the A-kinase anchor protein 9 (AKAP9) gene with the C-terminal coding region of the BRAF protooncogene. This fusion leads to the constitutive activity of the serine-threonine kinase [START_REF] Ciampi | Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer[END_REF].

Point mutations

a) The B-Raf mutations B-Raf, a member of the Raf family, encodes a cytoplasmic serine/threonine kinase, acting along the MAPK signaling pathway. Activating point mutations in the kinase region mimic phosphorylation of the protein, leading to constitutive, Ras-independent, kinase activity [START_REF] Davies | Mutations of the BRAF gene in human cancer[END_REF]. The most common of these mutations is a T1799A missense mutation that results in a valine to glutamic acid substitution at amino acid 600 (V600E).

Mutations in the B-Raf gene have been described in a variety of human neoplasms, with its highest incidence in melanoma and nevi (about 70%) [START_REF] Davies | Mutations of the BRAF gene in human cancer[END_REF].

In differentiated thyroid tumors, B-Raf mutations are found in PTC, with a prevalence of about 40%, which constitutes the more common molecular defect in this type of tumor [START_REF] Nikiforov | Genetic alterations involved in the transition from well-differentiated to poorly differentiated and anaplastic thyroid carcinomas[END_REF]. Although the V600E substitution is the more frequent mutation, other, less frequent, alterations leading to constitutive B-Raf have been described : K601E substitution has been detected in the follicular variant of PTC (Trovisco et al., 2004), and small in-frame insertions or deletions around V600 have been reported [START_REF] Carta | Genotyping of an Italian papillary thyroid carcinoma cohort revealed high prevalence of BRAF mutations, absence of RAS mutations and allowed the detection of a new mutation of BRAF oncoprotein (BRAF(V599lns))[END_REF]Hou et al., 2007a).

Among PTC, B-Raf mutations appear more frequently in the classical and tall cell variants, where the prevalence of RET/PTC is usually low. BRAF mutations are also associated to older patients [START_REF] Powell | Frequency of BRAF T1796A mutation in papillary thyroid carcinoma relates to age of patient at diagnosis and not to radiation exposure[END_REF] and correlate with distant metastases and tumor aggressivity [START_REF] Nikiforova | BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas[END_REF]. In addition to PTC, about 15% of poorly differentiated A c c e p t e d M a n u s c r i p t 22 and 35% of anaplastic carcinomas [START_REF] Namba | Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers[END_REF][START_REF] Nikiforova | BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas[END_REF] harbour the B-Raf V600E mutation.

The fact that Raf mutant induced papillary carcinomas are more dedifferentiated than carcinoma of other origin, thus take up less radioiodide and are more difficult to treat, fits in well with the very efficient dedifferentiating action of the Ras-MAPK pathway in vitro.

b) RAS mutations

Activating point mutations in codon 61 of H-Ras and N-Ras are not specific of a particular type of thyroid tumors. They are found in more than 30% of follicular adenoma and FTC [START_REF] Nikiforov | Genetic alterations involved in the transition from well-differentiated to poorly differentiated and anaplastic thyroid carcinomas[END_REF], but are also identified with a lower frequency in PTC (15%), particularly in the follicular variant [START_REF] Di Cristofaro | Molecular genetic study comparing follicular variant versus classic papillary thyroid carcinomas: association of N-ras mutation in codon 61 with follicular variant[END_REF]. A subset of poorly differentiated and anaplastic carcinomas also present Ras mutations (35% and 53%, respectively).

PI-3K pathway (Fig. 1A,B) a) PI-3KCA mutations and amplifications

The PI-3K signalling pathway is an important regulator of proliferation, apoptosis, metabolism and motility. Activating mutations in the p110α catalytic subunit of PI-3K (PI-3KCA) cause expression of constitutively active PI-3K proteins, resulting in PIP3 generation and activation of the downstream signalling cascade. Most of the mutations have been localized in the helicase (exon 9) and kinase (exon 20) domains of the protein [START_REF] Vogt | Cancer-specific mutations in phosphatidylinositol 3-kinase[END_REF]. In addition to activating mutations, PI-3KCA gene amplification also contributes to increased PI-3K signalling in thyroid tumors. Among differentiated carcinomas, such alterations occur mainly in FTC (8-13% mutation, 28% amplification) and less frequently in PTC (2% mutation, 12% amplification).

Their frequency in ATC increases up to 17% for PI-3KCA mutation and to 42% for gene amplification [START_REF] Garcia-Rostan | Mutation of the PIK3CA gene in anaplastic thyroid cancer[END_REF][START_REF] Wang | High prevalence and mutual exclusivity of genetic alterations in the phosphatidylinositol-3-kinase/akt pathway in thyroid tumors[END_REF].

b) PTEN mutations and loss of expression

A c c e p t e d M a n u s c r i p t 23 Inactivating mutations in PTEN, a lipid and protein phosphatase counteracting the activity of PI-3K, occur with a relatively low frequency in thyroid cancer (2% in PTC, 7% in FTC and 14 % in ATC). Loss of PTEN expression following deletion in PTEN gene is more common : 3% in follicular adenoma, 22% in PTC, 36% in PTC and 41% in ATC (Hou et al., 2007b;[START_REF] Wang | High prevalence and mutual exclusivity of genetic alterations in the phosphatidylinositol-3-kinase/akt pathway in thyroid tumors[END_REF].

In addition to these somatic mutations and deletions in PTEN, germline mutations in the PTEN gene leads to Cowden disease, a multiple hamartoma syndrome with increased susceptibility to breast, thyroid and skin tumours (Liaw et al, 1997).

4. Overexpression of growth factors and their receptors (Fig. 1B) Quantitative or qualitative changes in the expression of local growth factors and their receptors, leading to autocrine loops and autonomous proliferation, have been often reported to be associated with thyroid carcinogenesis (Fig. 1B). They also lead to permanent activation of MAPK, PI-3K or both and their respective cascades. Examples include the overexpression of c-met/HGF receptor, sometimes in association with HGF, mainly in papillary thyroid carcinomas [START_REF] Di Renzo | Overexpression of the c-MET/HGF receptor gene in human thyroid carcinomas[END_REF]Trovato et al., 1998), of erbB/EGF receptor in association with its ligand TGF [START_REF] Aasland | Co-expression of the genes encoding transforming growth factor-alpha and its receptor in papillary carcinomas of the thyroid[END_REF]) and of nine genes coding for proteins involved in EGF cascade activation [START_REF] Delys | Gene expression and the biological phenotype of papillary thyroid carcinomas[END_REF]. IGF-I was also reported to be produced by thyroid adenomas at levels sufficient to obviate the requirement for exogenous insulin or IGF-I in cultures derived from these tumors [START_REF] Williams | Loss of dependence on IGF-1 for proliferation of human thyroid adenoma cells[END_REF][START_REF] Williams | Evidence for autocrine production of IGF-1 in human thyroid adenomas[END_REF]. In some thyroid cancers, the overexpression of insulin receptors leads to an increased formation of insulin receptor/IGF-I receptor hybrids that are activated by IGF-I [START_REF] Belfiore | Insulin/IGF-I hybrid receptors play a major role in IGF-I signaling in thyroid cancer[END_REF].

Another interesting mechanism is provided by the expression in thyroid cancers of an alternatively spliced insulin receptor isoform (insulin receptor A) which binds with high affinity IGF-II produced by the same tumor cells (Fig. 1B) [START_REF] Vella | A novel autocrine loop involving IGF-II and the insulin receptor isoform-A stimulates growth of thyroid cancer[END_REF].

Other pathways a) PAX8-PPARγ rearrangement

PAX8-PPARγ rearrangement is a chromosomal translocation t(2:3)(q13;p25) which contributes to the development of thyroid cancers [START_REF] Kroll | PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma [corrected[END_REF]. PAX8 (paired-box gene 8) encodes a transcriptional factor required for the genesis of follicular cell lineages and PAX8-PPARγ rearrangements are present in follicular thyroid adenoma (up to 30 %), follicular thyroid carcinomas (35 %), and in follicular variants of PTC [START_REF] Castro | PAX8-PPARgamma rearrangement is frequently detected in the follicular variant of papillary thyroid carcinoma[END_REF][START_REF] Marques | Expression of PAX8-PPAR gamma 1 rearrangements in both follicular thyroid carcinomas and adenomas[END_REF]. To date, it has not been detected in poorly differentiated and undifferentiated (anaplastic) carcinomas. b) p53 mutations All mutations discussed above are mainly found in differentiated thyroid cancers and are believed not to be sufficient by themselves to trigger the progression to poorly and anaplastic carcinomas. Additional alterations are required such as p53 mutations.

In thyroid tumors, p53 mutations occur in 24% and 59% of poorly differentiated and anaplastic cancers, respectively, and only in isolated cases of PTC and FTC [START_REF] Nikiforov | Genetic alterations involved in the transition from well-differentiated to poorly differentiated and anaplastic thyroid carcinomas[END_REF].

c) β-catenin mutations

In thyroid tumors, stabilizing mutations in β-catenin (CTNNB1), a cadherin-mediated adhesion regulator and a component of the Wnt signaling pathway, have been reported in 16% of poorly differentiated cancers and in 66% of undifferentiated carcinomas, but not in differentiated carcinomas [START_REF] Garcia-Rostan | Frequent mutation and nuclear localization of beta-catenin in anaplastic thyroid carcinoma[END_REF][START_REF] Nikiforov | Genetic alterations involved in the transition from well-differentiated to poorly differentiated and anaplastic thyroid carcinomas[END_REF]. This occurrence suggests that mutations in β-catenin play a direct role in the dedifferentiation of thyroid cancers, especially those that progress to undifferentiated carcinomas.

Conclusion

In conclusion, extensive reports suggest a constitutive activation of the MAPK signalling pathway in PTC by genetic alterations of its components. More recently, genetic abnormalities leading to constitutive activation of the PI-3K-PKB signalling pathway have been discovered, and were shown to play also an important role in thyroid tumorigenesis, signalling activation in thyroid cancers, and PI-3K activation might be an essential factor for tumorigenesis [START_REF] Miller | Oncogenic Kras requires simultaneous PI3K signaling to induce ERK activation and transform thyroid epithelial cells in vivo[END_REF]. However, the relative importance of the PI-3K versus the MAPK signalling pathway is not fully elucidated since PI-3K activation can in turn facilitate MAPK activation [START_REF] Miller | Oncogenic Kras requires simultaneous PI3K signaling to induce ERK activation and transform thyroid epithelial cells in vivo[END_REF]. Additional alterations are required to go further in the dedifferentiation process. Undifferentiated anaplastic thyroid carcinomas commonly display B-Raf or Ras mutations, but no RET/PTC nor PAX8-PPARγ rearrangements [START_REF] Nikiforov | Genetic alterations involved in the transition from well-differentiated to poorly differentiated and anaplastic thyroid carcinomas[END_REF]. This could be explained by the fact that B-Raf or Ras mutations may facilitate the acquisition of secondary genetic events (such as p53 and β-catenin) through induction of genomic instability [START_REF] Mitsutake | BRAF mediates RET/PTC-induced MAPK activation in thyroid cells: functional support for requirement of the RET/PTC-RAS-BRAF pathway in papillary thyroid carcinogenesis[END_REF]. The specific signal transduction role of PAX8-PPAR , and the prevalence and role of the catenin mutations remain to be defined. p53 inactivating mutations seem to be involved in the progression rather than in the initiation of thyroid cancers.

IV.

Thyroid signal transduction, oncogenes and protooncogenes.

The parallelism between what is known of thyroid mitogenic pathways and the genetic events discovered in thyroid tumors is striking even with incomplete data: the differentiating, mitogenic cAMP pathway is involved at several steps in various forms of hyperthyroidism;

the dedifferentiating mitogenic growth factor MAPK kinase pathway is involved at different levels in carcinomas. There is some evidence of the involvement of the PI-3K pathway in both differentiated and dedifferentiated tumors (Fig. 1A,B).

We had believed at one time that rather than defining the tumors by their morphology, we should classify them by their genetic origin. The results obtained so far would discourage such a classification as several different but parallel genetic events in one pathway should lead to the same phenotype.

In the case of lung cancers and glioblastomas, a systematic sequence analysis of the genome of tumors or of several hundred selected genes has shown that each individual cancer harbors about 80 different mutations with little overlap between individual cancers. Of course, among these mutations there might be many more passengers than driving mutations.

However, analysis of the mutations shows that 70% of the cancers demonstrated one 3K pathway and in each of the two antioncogenic pathways: the p53 and then pRb pathway (Ding et al., 2008;The Cancer Genome Atlas Research network, 2008). This clearly indicates, that, as generally admitted, carcinogenesis requires several rather than one oncogenic event. Such events could be genetic, epigenetic, or functional lysogenic like (i.e.

heredity based on on/off signalling) positive feedback loops (Thomas, 1978). Thus, the oncogenic events described in section III, may be initiating but are not sufficient to elicit carcinogenesis. This is further supported by the fact that expression of one oncogene (eg RET-PTC3) in the thyroid of mice elicits thyroid growth and dedifferentiation but not general carcinomas. In fact the thyroids of such mice get, gene expression wise, more and more different from human papillary carcinomas as they age [START_REF] Burniat | Gene expression in RET/PTC3 and E7 transgenic mouse thyroids: RET/PTC3 but not E7 tumors are partial and transient models of human papillary thyroid cancers[END_REF]. However they develop different localized, then extensive lesions… obviously as the result of further localized oncogenic event. In humans, we still have to define many of these events, even in tumors in which one oncogenic mutation has been discovered.

V. Pending questions

The role in thyroid carcinomas of the Ral, Wnt, NOTCH and Sonic Hedgehog and TGF pathways and the action of estrogens has been little studied.

The striking correlation between the in vitro delineation of mitogenic signaling pathways and the nature of the oncogenes and antioncogenes discovered in thyroid pathway [START_REF] Coulonval | Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin[END_REF]Brewer et al., 2007)(Blancquaert et al. submitted) ? Could induction of insulin receptors making the thyrocyte sensitive to insulin [START_REF] Burikhanov | Thyrotropin via cyclic AMP induces insulin receptor expression and insulin co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells[END_REF]Van Keymeulen et al., 2000a), or an insulin receptor sensitive to IGF-II [START_REF] Vella | A novel autocrine loop involving IGF-II and the insulin receptor isoform-A stimulates growth of thyroid cancer[END_REF], provide the answer ? There is an apparent paradox in the fact that activating mutations of B-Raf induce a more severe form of papillary carcinoma than activation of RET which stimulates both the Raf-MAPK pathway and the PI-3K pathway (besides others such as Ral). This raises the question of the strength of the activation and of the nature of other complementary causal events. Indeed systematic sequencing of cancer genomes reveals, for each of most cancers, oncogenic events in the four major pathways: Raf-ERK, PI-3K pathway, p53 and pRb pathways (Ding et al., 2008;The Cancer Genome Atlas Research network, 2008). Moreover, even the bivalent acting RET, when expressed in the thyroid of transgenic mice, is insufficient to cause a real cancer [START_REF] Burniat | Gene expression in RET/PTC3 and E7 transgenic mouse thyroids: RET/PTC3 but not E7 tumors are partial and transient models of human papillary thyroid cancers[END_REF]. Constitutively activated RET, in C cells, leads to a well differentiated medullary carcinoma which suggests only a mild stimulation of the Raf-MAPK cascade.

How do cytosolic rearranged RET-PTC link to membrane bound Ras ? Could high concentrations be the answer ?

How does the level of dietary iodide influence the type of thyroid carcinoma appearing in the population ?

VI. Conclusion

The detailed knowledge of the normal and pathological signal transduction pathways in normal and tumor human cells is necessary for a complete understanding of the perversions which are at the basis of tumorigenesis. This knowledge in normal human thyroid cells from studies in human and other species such as dogs, led directly to the discovery of the events causing genetic hyperthyroidism. In the case of the carcinomas, although the knowledge could have led to their initiating cause, the first causing oncogene (RET/PTC) was discovered by a non a priori approach [START_REF] Grieco | PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas[END_REF].

The interpretation of the role of proposed causal events also benefits from this knowledge with some intesting diagnostic and therapeutic consequences.

The generally postulated transitions from adenoma to follicular carcinoma and from papillary and follicular to anaplastic thyroid carcinoma is supported by histological sections of human tumors. The finding of B-Raf and Ras mutations, pathognomonic of papillary, papillary and follicular carcinomas, respectively in 25% and 50% of anaplastic carcinomas [START_REF] Couto | How molecular pathology is changing and will change the therapeutics of patients with follicular cell-derived thyroid cancer[END_REF]) also supports it [START_REF] Nikiforova | BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas[END_REF]. The validity of this very plausible hypothesis would certainly confer a special interest to thyroid cancers as general models but it remains to be proved.

The fact that constitutive activation of the MAPK pathway at whatever level leads to papillary carcinomas suggests that carcinomas could be diagnosed on the basis of the activation cascades rather than the initial genetic events. The phenotypic signature (eg morphologic or gene expression) is the true reflect of the pathological process involved. In some cases, eg the presence of B-Raf mutations, the genetic signature may signal the severity of the disease [START_REF] Xing | BRAF mutation testing of thyroid fine-needle aspiration biopsy specimens for preoperative risk stratification in papillary thyroid cancer[END_REF].

For the choice of treatment, among a myriad of possibilities, the knowledge of the pathways involved, could also orient the selection of probably useful agents. Moreover, knowing the level involved will allow predicting the outcome. A priori, it is no use to inhibit a pathway upstream of its constitutively active actor (eg the EGF receptor in case of a Raf 2009). Most of the drugs in clinical trials act on the receptors [START_REF] Couto | How molecular pathology is changing and will change the therapeutics of patients with follicular cell-derived thyroid cancer[END_REF].

Also comprehensive knowledge could reveal metabolic steps that, although not causal, could be required for tumor development (eg angiogenesis, inflammation, etc..).

The understanding of the pathogenesis and physiopathology of carcinomas as of other diseases will result from the constant confrontation of clinical and pathological data in patients and data obtained on models, all of which have different limitations. or inhibitions (-). Redrawn from (Bockstaele et al., 2006a).

  human tumors (e.g. gene expression) certainly represent the real tumor.

  , the cAMP cascade in dogs, and through both in rats or mice(Massart, submitted).

  of the proliferative response occurring at sequential times of the prereplicative phase have been considered in our characterization of mechanisms of cell cycle regulation by TSH (Fig. 1A) and growth factors (Fig. 1B). a) Protein kinase cascades. The pattern of protein phosphorylation induced within minutes by TSH is reproduced by forskolin and cAMP analogues. It totally diverges from the phosphorylations induced by

  et al, submitted). The cAMP-dependent mitogenesis and gene expression also appears to require the phosphorylation by PKA and activity of CREB/CREM transcription factors

  A c c e p t e d M a n u s c r i p t 13 stimulations by growth factors and TSH, and potentially in hyperproliferative diseases generated by the overactivation of their respective signaling pathways. 4. Negative feedback loops.

  different regulations in rat thyroid cell lines.

  ablation in mice of one allele of the PRKAR1A gene, as a model of the Carney complex syndrome

2. 1

 1 Chromosomal rearrangements a) RET/PTC rearrangements RET/PTC rearrangements have been known as one of the most common molecular alterations in thyroid cancers, especially those of the papillary type. RET encodes a membrane tyrosine kinase receptor for a family of ligands, the prototype of which is glial cellderived neurotrophic growth factor. The RET protooncogene is involved in the regulation of growth, survival, differentiation, and migration of cells of neural crest origin. The RET gene is not expressed in thyroid follicular cells but is present in parafollicular C cells.

  types of RET rearrangements found in PTC, formed by the fusion of the intracellular tyrosine kinase domain of the protein with different 5' heterologous gene fragments which are ubiquitously expressed and possess a dimerization domain. This fusion results in the constitutive activation of the truncated tyrosine kinase portion of RET by autophosphorylation thanks to the dimerization domain of the heterologous gene. RET/PTC1

  including tropomyosin 3 gene (TPM3), Translocated Promotor Region gene

  -specific gene expression, whereas PPARγ (peroxisome proliferatoractivated receptor-γ) is a member of the steroid nuclear hormone receptor superfamily. The chimeric protein contains the DNA binding domain of PAX8 and functions as a dominant negative on the transcriptional activity of wild-type PPARγ.

  and ATC. In addition, RET/PTC or Ras are also responsible for PI-3K

  of the two major oncogenic pathways: the Raf-MAPK pathway and the PI-

  carcinomas should not lead us to believe that we understand fully what happens in the carcinomas. There are several unexplained discrepancies. Differences between observations and expectations should take into account the simplifications of our schemes of signal transduction: 1) the strength and duration of the activation, for instance the dedifferentiating action of Ras may be dose dependent[START_REF] De Vita | Dose-dependent inhibition of thyroid differentiation by RAS oncogenes[END_REF]; 2) the existence at each step of the pathways of more or less different isoforms (eg Ras, Raf, PI-3K etc); 3) the involvement of non studied pathways Ral, JAK, STAT, Wnt/catenin, NOTCH, sonic hedgehog, estrogen, retinoic acid, T3 receptors, etc 4) the existence of unknown positive and negative feedback loops; 5) late secondary induction or repression of signalling proteins; 6) the possible role of cancer associated endothelial, inflammatory cells and fibroblasts.

  mutations of the TSHR, or Gs succeed to lead to tumorigenesis without acting on the IGF-I-PI-3K pathway ? Is it due to the fact that both TSH-cAMP-PKA and IGF-I-PI-3K-PKB converge on the activation of the required mTOR-raptor (mTORC1)

  recent reports of new genetic changes in subsets of carcinoma tissues (Unger et al., 2008) raise the question of the evolution of the carcinogenetic process with time and in different parts of a tumor.

  this has been shown to be the case in colorectal cancers(Sartore-Bianchi et al.,

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Kinetics of ERK1/2 activation (upper panel), PI(3,4,5)P3 generation (middle panel), and PKB activation (bottom panel) in response to various (co)mitogenic stimuli in dog thyroid primary cultures.

Figure 3 .

 3 Figure 3. CDK4 regulation: the example of canine thyroid primary cultures.

Figure 4 .

 4 Figure 4. Presence and activity of D-type cyclin-CDK4 complexes in normal human thyrocytes stimulated by TSH or growth factors in the presence of high insulin concentrations. The addition of EGF and 10% serum (EGF+S) increases both the accumulation of cyclin D1 and even more the pRb-kinase activity of immunoprecipitated (IP) cyclin D1-CDK4. By contrast, TSH represses cyclin D1 but increases the activity of cyclin D3-CDK4 complexes.

  Figure 1A

Figure 3

 3 Figure 3

Acknowledgments

The work performed in the authors'laboratories was supported by grants from the Belgian Federation against Cancer, the Communauté française de Belgique -Actions de Recherches Concertées, the Belgian Fund for Scientific Medical Research (FRSM), the National Fund for Scientific Research (FRS-FNRS, Belgium), Les Amis de l'Institut Bordet, Télévie and EU Genrisk Contract. P.P. Roger, and W.C.G. van Staveren and K. Coulonval are respectively Senior Research Associate and Scientific Research Workers of the FRS-FNRS.

 . Mol. Cell Biol. 21, 1921-1929. Unger, K., Malisch, E., Thomas, G., Braselmann, H., Walch, A., Jackl, G., Lewis, P., Lengfelder, E., Bogdanova, T., Wienberg, J., Zitzelsberger, H., 2008. Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC. Oncogene 27, 4592-4602. Unger, K., Zitzelsberger, H., Salvatore, G., Santoro, M., Bogdanova, T., Braselmann, H., Kastner, P., Zurnadzhy, L., Tronko, N., Hutzler, P., Thomas, G., 2004. Heterogeneity in the