Valentina Guarino

Maria Domenica Castellone

Elvira Avilla

Rosa Marina Melillo

Thyroid cancer and inflammation

Keywords: thyroid cancer, inflammatory cell, chemokine

Some cancer types are strongly associated with chronic inflammatory or infectious diseases whereas others are not, but an inflammatory component is present in most human neoplastic lesions. This review focuses on various aspects of thyroid cancer and inflammation. The incidence of thyroid cancer, in particular of well-differentiated papillary thyroid carcinomas (PTCs), is increased in autoimmune thyroid diseases such as Hashimoto's thyroiditis. Thyroid cancer often has an inflammatory cell infiltrate, which includes lymphocytes, macrophages, dendritic cells and mast cells, whose role in thyroid cancer is still not completely understood. However, most experimental evidence suggests these cells exert a pro-tumorigenic function. Moreover, oncoproteins typically expressed in human PTCs, such as RET/PTC, RAS, and BRAF, trigger a pro-inflammatory programme in thyreocytes. These data suggest that inflammatory molecules are promising targets for thyroid cancer therapy.

Thyroid cancer, although a rare disease, is the most frequent endocrine neoplasia, and its incidence is rapidly increasing. The papillary histotype is the most frequent. Thyroid cancer can derive from both the follicular and the parafollicular component of the thyroid gland. Three malignant lesions may derive from follicular cells: well-differentiated (WDTC), poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas. WDTCs are either papillary (PTC) or follicular (FTC). These two types are characterized by specific histological features, and by the maintenance of some differentiating features [START_REF] Kondo | Pathogenetic mechanisms in thyroid follicular-cell neoplasia[END_REF]. WDTCs have a favorable prognosis thanks to the efficacy of combined surgical and radioiodine-based therapy. Nevertheless, 5-10% of WDTCs progress to radioactive iodine refractory-disease. PDTCs and ATCs, which are thought to derive from pre-existing differentiated tumors, do not accumulate radioiodine and respond poorly to medical and surgical treatment [START_REF] Rosai | Atlas of Tumor Pathology -Tumors of the thyroid gland. 3 rd series[END_REF]. Medullary thyroid carcinoma (MTC), which arises from the parafollicular C-cells of the thyroid, is generally treatable by surgery. However, novel chemotherapeutic agents like vandetanib, which target growth factor receptor, are showing promise in the treatment of MTC. This tumor can be inherited in the context of autosomal dominant MEN 2 (multiple endocrine neoplasia type 2) syndromes (MEN 2A, MEN 2B and familial medullary thyroid carcinoma [FMTC]) [START_REF] Roman | Medullary thyroid cancer: early detection and novel treatments[END_REF].

Non overlapping mutations of the RET, TRKA, RAS and BRAF genes are found in about 70% of PTCs. These genes encode activators of the mitogen-activated protein kinase (MAPK) cascade.

RET encodes the tyrosine kinase receptor of growth factors belonging to the glial-derived neurotrophic factor (GDNF) family [START_REF] Manie | The RET receptor: function in development and dysfunction in congenital malformation[END_REF]. RET/PTC rearrangements are thought to be early events in thyroid tumorigenesis since they are very frequent in clinically-silent small PTC (Fusco et al., 2002). Similar rearrangements of the high affinity receptor (TRKA) for nerve growth factor (NGF) are also found, albeit at a low prevalence, in human PTC [START_REF] Alberti | RET and NTRK1 protooncogenes in human diseases[END_REF].

Activating point-mutations in RAS small GTPases are found mainly in the follicular variant of PTC (PTC-FV) [START_REF] Zhu | Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations[END_REF]. Point-mutations in BRAF are the most common genetic lesion A c c e p t e d M a n u s c r i p t 4 in these tumors [START_REF] Kimura | High Prevalence of BRAF Mutations in Thyroid Cancer: Genetic Evidence for Constitutive Activation of the RET/PTC-RAS-BRAF Signaling Pathway in Papillary Thyroid Carcinoma[END_REF][START_REF] Xu | High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines[END_REF][START_REF] Soares | BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC[END_REF][START_REF] Cohen | BRAF mutation in papillary thyroid carcinoma[END_REF].

BRAF is a member of the RAF family of serine/threonine kinases, which includes ARAF and RAF1, and it is a component of the RAF-MEK-ERK signaling module.

FTCs are characterized by RAS point-mutations [START_REF] Nikiforova | Prevalence of RET/PTC rearrangements in Hashimoto's thyroiditis and papillary thyroid carcinomas[END_REF] or by the PAX8/PPAR rearrangement [START_REF] Kroll | PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma[END_REF]. PDTC and ATC can derive from pre-existing WDTCs. RAS point-mutations and the V600E BRAF mutation are prevalent in PDTC and ATC (Garcia-Rostan et al., 2003;[START_REF] Nikiforova | RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma[END_REF][START_REF] Soares | BRAF mutations typical of papillary thyroid carcinoma are more frequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas[END_REF][START_REF] Begum | BRAF mutations in anaplastic thyroid carcinoma: implications for tumor origin, diagnosis and treatment[END_REF]. Finally, p53 mutations are often found in ATC [START_REF] Donghi | Gene p53 mutations are restricted to poorly differentiated and undifferentiated carcinomas of the thyroid gland[END_REF]Fagin et al., 1993).

MTC can be sporadic or it could be one of the lesions that characterize the autosomal dominant MEN 2 syndromes (MEN 2A,MEN 2B and FMTC). MEN 2 syndromes are caused by germline point mutations that convert RET into a dominant oncogene [START_REF] Santoro | RET/PTC activation in human thyroid carcinomas[END_REF]. In all MEN 2A cases, mutations target extracellular cysteine residues in RET. In more than 80% of cases, MEN 2B is caused by the Met918Thr substitution in the kinase domain of the receptor. Roughly 40% of sporadic MTC cases harbor point mutations in RET [START_REF] Leboulleux | Medullary thyroid carcinoma[END_REF].

Chronic inflammation and cancer

Inflammation is a physiological, protective process used by the organism in response to tissue damage. Several chemical signals initiate and sustain the inflammatory response whose aim is to fight pathogens and to repair tissue damage. Several cell types migrate to the sites of tissue damage. This migration is mediated by chemotactic and adhesion proteins, including members of the integrin and selectin family [START_REF] Coussens | Inflammation and cancer[END_REF]. The first migrating cells are neutrophils, macrophages and mast cells, all of which secrete reactive oxygen species (ROS), vasoactive molecules, such as histamine and leukotrienes, and cytokines, chemokines and proteases that remodel the extracellular matrix [START_REF] Visser | Paradoxical roles of the immune system during cancer development[END_REF]. Inflammation is an auto-limiting process; however, abnormal persistence of the stimuli that induced the inflammatory response or the failure A c c e p t e d M a n u s c r i p t 5 of the mechanisms that make it end, result in chronic inflammation [START_REF] Coussens | Inflammation and cancer[END_REF].

A functional relationship between chronic inflammation and cancer was first proposed by

Virchow in 1863, and has been sustained by clinical and epidemiological evidence. The most compelling evidence is the association between: a) intestinal chronic inflammatory diseases (Crohn's disease and ulcerative rectocolitis) and adenocarcinoma of the colon; b) chronic HBV or HCV hepatitis and liver carcinoma; c) Helicobacter pylori-induced chronic gastritis and gastric carcinoma; d) asbestosis and mesothelioma; e) chronic obstructive pulmonary disease (COPD) and lung cancer; and f) chronic esophagitis and carcinoma of the esophagus [START_REF] Balkwill | Inflammation and cancer: back to Virchow[END_REF]. Moreover, a 40-50% reduction in the incidence of colorectal cancer is associated with the regular use of non-steroidal anti-inflammatory drugs, the COX enzymes that catalyze the synthesis of pro-inflammatory mediators, such as prostaglandins (Baron and Sadler, 2000;[START_REF] Williams | The role of cyclooxygenases in inflammation, cancer, and development[END_REF].

Further support for the concept that inflammation can cause cancer comes from the observation that polymorphisms in genes encoding proinflammatory chemokines are associated with an increased risk of some forms of human cancer. For instance, IL1-polymorphisms are associated with an increased incidence of gastric cancer (Gianfagna et al., 2008). Moreover, gastric-specific expression of this cytokine in transgenic mice can induce gastric inflammation and cancer [START_REF] Tu | Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice[END_REF].

Various infiltrating cells have been identified in tumors, namely, tumor-associated lymphocytes, tumor-associated macrophages (TAM), immature dendritic cells, mast cells and myeloid-derived suppressor cells. The presence of specific inflammatory-immune cells such as macrophages and mast cells in tumor site has been associated with a poor prognosis of the disease (Fujimoto et al., 2009;[START_REF] Maltby | Mast cells in tumor growth: Angiogenesis, tissue remodelling and immune-modulation[END_REF]. Pollard and colleagues have shown that transgenic mice prone to breast cancer develop less proliferating and invasive tumors when crossed with mice deficient for CSF-1, a macrophage chemotactic factor [START_REF] Lin | Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy[END_REF]. Other cell populations also contribute, via distinct mechanisms, to tumor growth. Indeed, mice deficient in mast cells or CD4+

A c c e p t e d M a n u s c r i p t 6 T cells display a reduction in tumor incidence and progression in models of mice carcinogenesis [START_REF] Daniel | Immune enhancement of skin carcinogenesis by CD4+ T cells[END_REF][START_REF] Soucek | Mast cells are required for angiogenesis and macroscopic expansion of Myc-induced pancreatic islet tumors[END_REF]. Among the cell populations recruited into tumor sites by cancer cells, a peculiar role is played by bone marrow-derived precursors. These include myeloidderived suppressor cells that contribute to tumor-growth through tumor-induced immune tolerance [START_REF] Marigo | Tumor-induced tolerance and immune suppression by myeloid derived suppressor cells[END_REF], and bone marrow-derived stem cells, which are thought to be recruited from the bloodstream and to repopulate areas of epithelial destruction. Indeed, these cells constitute all the metaplastic, dysplastic and neoplastic areas in the Helicobacter-induced gastric cancer model in mice. Again, IL1-plays an important role in this processindeed it seems to be required for both recruitment and activation of bone marrow-derived stem cells [START_REF] Houghton | Gastric cancer originating from bone marrow-derived cells[END_REF][START_REF] Tu | Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice[END_REF].

Cancer cells secrete several cytokines and chemokines, thus sustaining cancer cell growth and recruiting leukocytes into tumor sites. Leukocytes physiologically secrete ROS and reactive nitrogen species to eliminate pathogens. However, these highly reactive metabolites induce the production of peroxynitrite and other mutagenic agents; therefore, they can induce "DNA damage", i.e. mutations in proliferating cells [START_REF] Coussens | Inflammation and cancer[END_REF]. Thus, in the case of persistent tissue damage, the O 2 and N highly reactive metabolites secreted by inflammatory cells induce point mutations, rearrangements and double-strand breaks in the DNA [START_REF] Colotta | Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability[END_REF]. This results in a higher probability of oncogene activation or of tumor suppressor loss of function.

Proinflammatory cytokines produced in tumor sites are pivotal in cancer progression. The production of these cytokines by inflammatory and epithelial cancer cells depends mainly on the NFkB transcription factor. Through conditional deletion of the NFkB upstream activator IKKb, in myeloid and epithelial cells, it has been shown that NFkB plays a major protumorigenic role in an experimental colitis-associated cancer (CAC) model [START_REF] Greten | IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer[END_REF]). Among the cytokines induced by NFkB, IL6 has been identified as one of the most relevant myeloid-derived factors that promote tumorigenesis. Moreover, activation of the IL6 pathway, in particular of the STAT3 transcription factor, which is a downstream mediator of IL6 activity, is necessary for CAC initiation

Thyroid cancer and autoimmune thyroid diseases

A link between thyroid cancer, in particular the PTC histotype, and autoimmune thyroid diseases (AITD) has long been recognized, although the precise relationship between the two diseases is still debated. This group of diseases includes Hashimoto's thyroiditis and Grave's disease. Hashimoto's thyroiditis (HT) is an autoimmune disorder in which the immune system reacts against a variety of thyroid antigens. The overriding feature of HT is the progressive depletion of thyroid epithelial cells, which are gradually replaced by mononuclear cell infiltration and fibrosis. Multiple immunologic mechanisms may contribute to the death of thyrocytes.

Sensitization of autoreactive CD4+ T-helper cells to thyroid antigens appears to be the initiating event. Hashimoto's thyroiditis is characterized by proliferating nodules as well as by cytological alterations and nuclear modifications similar to those of papillary carcinomas [START_REF] Weetman | Cellular immune responses in autoimmune thyroid disease[END_REF]).

An epidemiological association has been identified between Hashimoto's thyroiditis and thyroid cancer [START_REF] Di Pasquale | Pathologic features of Hashimoto's-associated papillary thyroid carcinoma[END_REF]Wirtschafter.et al., 1997;[START_REF] Mechler | Papillary thyroid carcinoma: 6 cases from 2 families with associated lymphocytic thyroiditis harbouring RET/PTC rearrangements[END_REF][START_REF] Segal | Hashimoto's thyroiditis and carcinoma of the thyroid gland[END_REF]Eisemberg et al., 1989;[START_REF] Ott | The incidence of thyroid carcinoma in Hashimoto's thyroiditis[END_REF][START_REF] Sclafani | Hashimoto's thyroiditis and carcinoma of the thyroid: optimal management[END_REF][START_REF] Pisanu | Coexisting Hashimoto's thyroiditis with differentiated thyroid cancer and benign thyroid disease: indications for thyroidectomy[END_REF]. The increased incidence of carcinomas in patients with thyroiditis suggests thyroiditis might be a precancerous condition. Most thyroiditis-associated carcinomas are papillary; however, also follicular, anaplastic, medullary and squamous carcinomas have been reported in cases of thyroiditis. Thyroid follicular cells may have chromosomal defects, such as the RET/PTC rearrangement, which is the hallmark of many PTCs. Several authors found RET/PTC rearrangements in non neoplastic thyroid lesions, such as HT [START_REF] Wirtschafter | Expression of the RET/PTC fusion gene as a marker for papillary carcinoma in Hashimoto's thyroiditis[END_REF][START_REF] Sheils | ret/PTC-1 Activation in Hashimoto Thyroiditis[END_REF][START_REF] Elisei | RET/PTC rearrangements in thyroid nodules: studies in irradiated and not irradiated, malignant and benign thyroid lesions in children and adults[END_REF]. Additional evidence implicating RET/PTC in the association between thyroiditis and cancer comes from the finding that patients exposed to radiation from the Chernobyl nuclear power plant disaster often develop not only RET/PTC-induced papillary tumors but also autoimmune thyroiditis [START_REF] Williams | Cancer after nuclear fallout: lessons from the Chernobyl accident[END_REF]. Accordingly, transgenic mice engineered to express Two models can be hypothesized to explain the association between Hashimoto's thyroiditis and the RET/PTC rearrangement. The first suggests that inflammation might facilitate the rearrangement. According to this hypothesis, production of free radicals, cytokine secretion, cellular proliferation and other phenomena correlated with inflammation might predispose to the rearrangement of RET/PTC in follicular cells. In support of this hypothesis, it has been observed that the mutational rate in inflamed tissues is much higher than in normal tissues [START_REF] Colotta | Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability[END_REF]. It is also possible that the inflammatory microenvironment supports thyreocyte survival in stress conditions. It is generally assumed that normal human epithelial cells do not tolerate It has recently been suggested that tissue stem cells are the targets of malignant transformation. In the setting of chronic inflammation, the pool of resident or bone marrow-derived stem cells is thought to expand [START_REF] Li | Stem cells and cancer: evidence for bone marrow stem cells in epithelial cancers[END_REF]. If this applies also to thyroid cancer, AITDmediated inflammation could cause the expansion of stem cells in which a RET/PTC rearrangement has occurred.

An alternative hypothesis, which does not exclude the previous hypothesis, is that the RET/PTC rearrangement itself, and the consequent activation of the downstream signaling pathways can induce thyroid inflammation. Accordingly, RET/PTC induces the synthesis of many inflammatory proteins in epithelial thyroid cells (Table 1) and a severe inflammatory response is observed in TG-RET/PTC transgenic mice in which RET/PTC expression is confined to the thyroid gland (Powell et 2009). Indeed, RET/PTC transgenic mice develop PTCs and chronic thyroiditis. However, RET/PTC itself is not sufficient to induce complete Hashimoto's thyroiditis in these mice since this disease is characterized not only by lymphocytic infiltration, but also by a humoral autoimmune reaction that results in the production of autoantibodies against thyroid antigens, and by the formation of lymphoid follicles in the thyroid parenchyma. These features are not present in TG-RET/PTC transgenic animals. In conclusion, we favor the hypothesis that AITD creates a protumorigenic microenvironment in which the RET/PTC rearrangement is tolerated. The rearrangement itself then contributes to maintaining the inflammatory reaction.

Thyroid cancer and immune-inflammatory infiltrate

There are various reports of immune-inflammatory cell infiltrates in thyroid cancer. Not only are PTCs frequent in the context of Hashimoto's thyroiditis, but these carcinomas are often present with a remarkable lymphocytic infiltrate in the absence of the typical signs of autoimmune thyroiditis, such as autoantibody production and tertiary follicle formation. The prevalence of lymphocytic A c c e p t e d M a n u s c r i p t 10 infiltrate is generally significantly higher in patients with PTC than in patients with benign thyroid lesions [START_REF] Okayasu | The Relationship of Lymphocytic Thyroiditis to the Development of Thyroid Carcinoma[END_REF], which indicates that these cells might favor cancer development.

However, the presence of chronic lymphocytic thyroiditis in patients with PTC correlates with an improved prognosis [START_REF] Kebebew | Coexisting chronic lymphocytic thyroiditis and papillary thyroid cancer revisited[END_REF]. Moreover, thyroid carcinomas with a poor prognosis, such as PDTCs and ATCs, are characterized by a strongly reduced lymphocyte cell infiltrate compared with PTCs, thus suggesting that these cells may play a protective role in thyroid cancer [START_REF] Ugolini | Lymphocyte and immature dendritic cell infiltrates in differentiated, poorly differentiated, and undifferentiated thyroid carcinoma[END_REF]. Macrophages and dendritic cells, mainly characterized by an immature phenotype, have been identified in human PTCs [START_REF] Scarpino | Papillary carcinoma of the thyroid: hepatocyte growth factor (HGF) stimulates tumor cells to release chemokines active in recruiting dendritic cells[END_REF]. Again, PDTCs and ATCs are characterized by a greatly reduced dendritic cell infiltrate with respect to PTCs, thus suggesting that these cell types also may play a protective role in thyroid cancer [START_REF] Ugolini | Lymphocyte and immature dendritic cell infiltrates in differentiated, poorly differentiated, and undifferentiated thyroid carcinoma[END_REF]. Tumorassociated macrophages are generally considered protumorigenic [START_REF] Sica | Cancer related inflammation: the macrophage connection[END_REF]. Ryder and colleagues found that the density of the TAM infiltrate was higher in PDTCs and ATCs than in PTCs and FTCs. Moreover, increased TAM infiltration in PDTCs was found to be positively correlated with capsule invasion, extrathyroidal extension and poor prognosis [START_REF] Ryder | Increased density of tumor-associated macrophages is associated with decreased survival in advanced thyroid cancer[END_REF].

Taken together, these data support the concept that TAM may favor the malignant progression of thyroid cancer.

We compared the density of tryptase-positive mast cells in 96 PTCs versus normal thyroid tissue from 14 healthy individuals. Mast cell density was higher in PTCs than in control tissue.

Mast cell infiltrate correlated with capsule invasion. We also observed that mast cells promoted proliferation, survival and the invasive ability of thyroid cancer cells, thereby contributing to thyroid carcinoma growth and invasiveness (Melillo et

al., unpublished observations).

The presence of inflammatory cells has been evaluated in transgenic mice expressing RET/PTC3 in the thyroid gland by means of a tissue-specific promoter. In this system, mice develop PTC-like lesions, characterized by a leukocytic infiltrate, mainly constituted by macrophages [START_REF] Russell | Proinflammatory mediators and genetic background in oncogene mediated tumor progression[END_REF]. This effect seemed to be RET-specific because a similar oncogene, TRK-T1, which also derives from a rearrangement of a tyrosine kinase receptor with a A c c e p t e d M a n u s c r i p t 11 heterologous gene, did not have the same effect. Interestingly, the tumor incidence and burden in RET/PTC3 mice were influenced by the genetic background of the animals. In fact, tumors were significantly larger in C57BL/6 mice expressing RET/PTC3 than in C3H/HeJ animals. Cytokine expression was much higher in large tumors, suggesting that these molecules play a role in tumor growth. This effect could be due to the different polarization of TCD4+ cells and consequently to the distinct cytokine environment mounted by the C57BL/6 mice with respect to the C3H/HeJ animals. Taken together, these data demonstrate that the RET/PTC3 oncoprotein can induce recruitment of immune cells into tumor sites, and that cytokines produced in a tumor site play an important role in tumor progression.

In another set of experiments, Pufnock and Rothstein investigated whether RET/PTC3 is involved in the recruitment of other immune cell populations into tumor sites [START_REF] Pufnock | Oncoprotein signaling mediates tumor-specific inflammation and enhances tumor progression[END_REF]. In that study, RET/PTC3 and its mutant isoform (RET/PTC3 Y1062F), which is defective in the activation of the most relevant RET-mediated signaling pathways, were transduced into a mouse fibrosarcoma cell line. Tumors were induced by injecting RET/PTC3-and RET/PTC3 Y1062F-expressing cells in syngeneic mice. RET/PTC3 tumors were significantly larger than RET/PTC3 mutant tumors. CD4+ and CD8+ T cell infiltrate density was comparable in both tumor groups. However, RET/PTC3, but not RET/PTC3 Y1062F tumors, displayed a remarkable leukocytic infiltrate characterized by CD11b+, Gr1+ myeloid cells, previously described as innate suppressive inflammatory cells [START_REF] Marigo | Tumor-induced tolerance and immune suppression by myeloid derived suppressor cells[END_REF]Gabrilovich et al., 2009). These data suggest that RET/PTC3-positive thyroid cancer, like other cancer types, can induce the recruitment of CD11b+, Gr1+ cells. These cells can mediate tumor escape from the immune response; this phenomenon is dependent on the integrity of the Y1062 residue of RET. Whether other mechanisms of immune evasion also operate in thyroid cancer is unknown. Interestingly, the BRAF-MAPK signaling pathway has been shown to induce the synthesis and secretion of immunosuppressive cytokines in melanomas [START_REF] Sumimoto | The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells[END_REF]. These factors include IL-10, VEGF and IL6, which are cytokines that are produced also by thyroid cancer cells [START_REF] Stassi | Thyroid cancer resistance to chemotherapeutic drugs via autocrine production of interleukin-4 and interleukin-10[END_REF]

Oncoprotein signaling in thyroid cancer and inflammation

The activation of RET in PTC derives from a genetic rearrangement between the RET tyrosinekinase domain and heterologous genes. The consequence of these rearrangements is the constitutive activation of RET due to constitutive dimerization-oligomerization of the oncoprotein induced by the different RET-fused genes. In MEN2A and FMTC, RET activation is achieved by constitutive dimerization of RET through the replacement of an extracellular cysteine and the formation of disulfide bonds [START_REF] Santoro | Dysfunction of the RET receptor in human cancer[END_REF]. Differently, mutations that affect the intracellular domain induce kinase activation in the absence of dimerization, presumably through a modification of the kinase structure. For instance, the MEN2B M918T point mutation induces a change in substrate specificity with respect to MEN2A mutants, which is reflected in the different capability of MEN2B mutants to phosphorylate endogenous tyrosines and signaling adaptors. These differences can account, at least in part, for the phenotypic differences between the two syndromes. Indeed, although both MEN2A and MEN2B are characterized by the presence of pheochromocytomas and MTCs, MEN2B MTCs occur earlier and are more aggressive. Furthermore, MEN2B patients also present skeletal abnormalities and ganglioneuromas [START_REF] Hansford | Multiple endocrine neoplasia type 2 and RET: from neoplasia to neurogenesis[END_REF].

Both point mutations and genetic rearrangements cause constitutive activation of the tyrosine kinase activity of RET in the absence of ligands. Several studies conducted in different laboratories have shown that activation of RET, either by physiological ligands or by oncogenic conversion, results in the phosphorylation of intracellular tyrosine residues that serve as docking sites for the recruitment of signaling adapters. Among the tyrosines phosphorylated after RET activation, Y1062 has been shown to be important for RET-mediated biological activity in thyroid cells. Y1062 is a multi-docking site, being able to bind several PTB-containing adapters, namely, ShcA, FRS2, IRS1, Several groups demonstrated that RET-induced transcriptional activity depends almost entirely on the integrity of the Y1062 residue and on activation of the RAS/BRAF/MAPK pathway. Russel and colleagues reported that the expression of the RET/PTC3 isoform in a rat thyroid cell line (PC Cl3) induced an increase in NFkB DNA-binding activity and a consequent increase in proinflammatory cytokine secretion [START_REF] Russell | Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors[END_REF]. CXCL1/ Gro , CCL2/mcp-1 and GM-CSF were up-regulated upon RET/PTC3 expression, and this increase depended on the integrity of residue 1062 of RET.

GDNF stimulation of the neuroectodermal tumor cell line SK-N-MC, ectopically expressing the human wild-type RET, induced the production of high levels of IL8. This cytokine has also been found in the TT human medullary thyroid carcinoma cell line carrying the RET/MEN2A oncogene and in the TPC1 human PTC cell line [START_REF] Iwahashi | Activation of RET tyrosine kinase regulates interleukin-8 production by multiple signaling pathways[END_REF]. IL8 is a pro-inflammatory, mitogenic and proangiogenic chemokine that contributes to several human cancers (Waug and Wilson, 2008). Russel and colleagues found that RET/PTC3 caused the expression of the proinflammatory proteins IL-1 , IL1 , IL6 and IL24 in thyroid cancer cells [START_REF] Russell | Proinflammatory mediators and genetic background in oncogene mediated tumor progression[END_REF][START_REF] Shinohara | Interleukin 24 is induced by the RET/PTC3 oncoprotein and is an autocrine growth factor for epithelial cells[END_REF]. Similar results were obtained by Puxeddu and colleagues, who demonstrated that prostaglandin E2 (PGE2), microsomal prostaglandin E synthase1 (mPGES1), cyclooxygenase2 (COX2) and several other genes involved in immune response and inflammation were induced by RET/PTC3 in PC Cl3 cells [START_REF] Puxeddu | Microsomal prostaglandin E2 synthase-1 is induced by conditional expression of RET/PTC in thyroid PCCL3 cells through the activation of the MEK-ERK pathway[END_REF][START_REF] Puxeddu | RET/PTC-induced gene expression in thyroid PCCL3 cells reveals early activation of genes involved in regulation of the immune response[END_REF].

To study the transcriptional changes associated with thyroid transformation, we used an oligonucleotide-based DNA microarray (Affymetrix) on PC Cl3 cells engineered to stably express wild-type and mutant isoforms of the oncogenic RET/PTC3 protein, or the HRAS (V12) or A c c e p t e d M a n u s c r i p t 14 BRAF(V600E) oncoproteins, respectively. When transfected cells were compared with the parental cells, it was evident that RET/PTC3 induced a complex pattern of gene expression that depended entirely on the integrity of the Y1062 residue. The HRAS (V12) and BRAF(V600E) oncoproteins each modified the expression of several genes, and 50% of these genes were common between the three oncoproteins. Furthermore, by using siRNA and pharmacological inhibitors, we showed that most of these gene changes depended on the ERK pathway [START_REF] Melillo | The RET/PTC-RAS-BRAF linear signalling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells[END_REF]. Our gene expression profiling studies also revealed the induction of cytokine and chemokines and of their respective receptors in PC RET/PTC3 cells (Fig. 1). Indeed, we found that RET/PTC3 cells expressed high levels of osteopontin (OPN), VEGFA, CCL2, CXCL1 and CXCL10 [START_REF] Melillo | The RET/PTC-RAS-BRAF linear signalling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells[END_REF]. Interestingly, CD44, CXCR2 and CXCR3, the receptors for OPN, CXCL1 and CXCL10, respectively, were also expressed by transformed cells. We also found the expression of CXCR4, the chemokine receptor for the chemokine CXCL12, in transformed cells with respect to the normal counterparts (Castellone et al., 2004b). These results were confirmed by Borrello and coworkers in human primary thyroid cells transduced with the RET/PTC oncogene [START_REF] Borrello | Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1 oncogene[END_REF].

The proinflammatory properties of RET/PTC in the thyroid might have a dual effect: on one hand, molecules such as OPN, CXCL1, CXCL10, CCL2 and GM-CSF can influence the immune response to the tumor by recruiting and functionally regulating immune cells. For instance, transplantation of RET/PTC3-expressing thyreocytes into mice in vivo induced intense macrophage infiltrate and neovascularization followed by cell death [START_REF] Russell | Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors[END_REF]. On the other hand, secreted cytokines and chemokines, such as OPN, CXCL1, CXCL10 and IL24, can act as autocrine growth and survival factors for thyroid tumor cells that express the cognate receptors on their plasma membrane [START_REF] Shinohara | Interleukin 24 is induced by the RET/PTC3 oncoprotein and is an autocrine growth factor for epithelial cells[END_REF]Castellone et al., 2004a;[START_REF] Melillo | The RET/PTC-RAS-BRAF linear signalling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells[END_REF]. These data are corroborated by studies conducted on human thyroid tumors. Indeed, it has been shown that human thyroid samples express most inflammatory genes (CXCR4, CD44, OPN, CXCL1, CXCL10 Other studies support the concept that thyroid cancer epithelial cells can produce inflammatory factors, and that these inflammatory factors can sustain the resistance of thyroid cancer cells to various apoptotic stimuli. Stassi and colleagues looked for cytokines involved in the resistance of thyroid cancer cells to chemotherapeutic agents and identified IL4 and IL10. These cytokines are typically secreted by T CD4+ cells polarized toward a T H 2 phenotype. T H 2 cytokines induce humoral immunity and promote thyreocyte survival in AITD by up-regulating levels of antiapoptotic proteins such as Bcl2 and Bcl-XL [START_REF] Stassi | Thyroid cancer resistance to chemotherapeutic drugs via autocrine production of interleukin-4 and interleukin-10[END_REF]. Moreover, the authors demonstrated that IL4 and IL10 also induce resistance of thyroid cancer cells to FAS/FASLmediated apoptosis. This effect is mediated by IL4 and IL10-induction of two anti-apoptotic proteins, namely cFLIP and PED/PEA15 [START_REF] Todaro | Autocrine production of interleukin-4 and interleukin-10 is required for survival and growth of thyroid cancer cells[END_REF].

Conclusions

The relationship between inflammation and thyroid cancer is complex and still not completely understood. Like other cancer types, thyroid cancer is influenced by and modulates inflammation.

Epidemiological and histological data indicate that thyroid cancer frequently occurs in the context of one of the most common AITDs, Hashimoto's thyroiditis, and that thyroid cancer is frequently infiltrated by inflammatory-immune cells.

The role of these cells is complex, and several studies indicate that, depending on the specific cell population, the effect can be either pro-or anti-tumorigenic. Specifically, the presence of lymphocytes, which belong to the adaptive branch of immunity, is significantly higher in neoplastic than in non neoplastic lesions. However, lymphocytic infiltration seems to confer protection against cancer progression. On the other hand, the presence of cells belonging to innate immunity, such as macrophages and mast cells, enhances tumor progression and is associated with an unfavorable prognosis. By activating the MAPK cascade, the oncogenes activated in thyroid carcinomas (i.e., RET/PTC, RAS and BRAF) can activate a cell-autonomous pro-inflammatory transcriptional A c c e p t e d M a n u s c r i p t 16 program that involves mainly cytokines, chemokines and their receptors (Table 1). These molecules exert two main effects in thyroid cancer. First, by acting in an autocrine fashion, they sustain most of the malignant phenotypic features of thyroid cancer cells namely, proliferation, survival and invasiveness. Second, by acting in a paracrine and, possibly, in an endocrine manner, they induce remodeling of the tumoral stroma by recruiting inflammatory, immune, endothelial and bone marrow-derived cells. In this way, cytokines can further enhance tumor progression through the release of mediators from infiltrating cells, by stimulating angiogenesis, and by inducing subversion of the anti-tumoral immune response. Based on these observations, we favor the concept that, at least in the full blown phase, thyroid cancer growth and progression are positively influenced by two major inflammatory components, one dependent on the cells that are present in the cancer stroma, the other dependent on activation, in epithelial cancer cells, of specific oncoprotein-mediated signaling. As a consequence, not only oncoproteins, but also inflammatory molecules are promising targets for novel thyroid cancer therapeutical strategies. In support of this, we have shown that the blockade of CXCR4 chemokine receptor inhibits the growth of ATC cell xenografts in immunodeficient mice [START_REF] De Falco | Biological role and potential therapeutic targeting of the chemokine receptor CXCR4 in undifferentiated thyroid cancer[END_REF]. Curr. Opin. Genet. Dev. 1, 25-31. Fagin, J.A., Matsuo, K., Karmakar, A., Chen, D.L., Tang, S.H., Koeffler, H.P., 1993 High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J. Clin. Invest. 91, 179-84. Fujimoto, H., Sangai, T., Ishii, G., Ikehara, A., Nagashima, T., Miyazaki, M., Ochiai, A., 2009. Stromal MCP-1 in mammary tumors induces tumor-associated macrophage infiltration and contributes to tumor progression. Int. J. Cancer. Feb 18. Fusco, A., Chiappetta, G., Hui, P., Garcia-Rostan, G., Golden, L., Kinder, B.K., Dillon, D.A., Giuliano, A., Cirafici, A.M., Santoro, M., Rosai, J., Tallini, G., 2002. Assessment of RET/PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer. Am. J Pathol. 160, 2157-67.

Gabrilovich, D.I., Nagaraj, S., 2009. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 9, 162-74. Gianfagna, F., De Feo, E., van Duijn, C.M., Ricciardi, G., Boccia, S., 2008. A systematic review of meta-analyses on gene polymorphisms and gastric cancer risk. Curr.

Genomics. 9, 361-74. Cytokine that functions in inflammation and the maturation of B cells. The protein is primarily produced at sites of acute and chronic inflammation, where it is [START_REF] Russell | Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors[END_REF][START_REF] Russell | Proinflammatory mediators and genetic background in oncogene mediated tumor progression[END_REF][START_REF] Puxeddu | RET/PTC-induced gene expression in thyroid PCCL3 cells reveals early activation of genes involved in regulation of the immune response[END_REF]; [START_REF] Russell | Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors[END_REF][START_REF] Russell | Proinflammatory mediators and genetic background in oncogene mediated tumor progression[END_REF][START_REF] Melillo | The RET/PTC-RAS-BRAF linear signalling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells[END_REF] Small cytokine belonging to the [START_REF] Puxeddu | Microsomal prostaglandin E2 synthase-1 is induced by conditional expression of RET/PTC in thyroid PCCL3 cells through the activation of the MEK-ERK pathway[END_REF]

  Grivenikov et al., 2009. Bollrath et al., 2009).

  develop papillary carcinomas and chronic thyroiditis[START_REF] Powell | The RET/PTC3 oncogene: metastatic solid-type papillary carcinomas in murine thyroids[END_REF]). Finally, Wirtschafter and colleagues detected RET/PTC expression in about 90% of the cases of Hashimoto's thyroiditis they analyzed[START_REF] Wirtschafter | Expression of the RET/PTC fusion gene as a marker for papillary carcinoma in Hashimoto's thyroiditis[END_REF]. However, these data partially contrast with other reports. Nikiforova and colleagues detected RET/PTC rearrangements only in PTCs not associated with Hashimoto's disease[START_REF] Nikiforova | Prevalence of RET/PTC rearrangements in Hashimoto's thyroiditis and papillary thyroid carcinomas[END_REF].Rhoden et al. detected only a few follicular cells expressing very low levels of RET/PTC in Hashimoto's thyroiditis, which suggests that RET/PTC expression does not necessarily predict the development of PTC in patients with thyroiditis[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].

  oncogene expression because excessive growth signals induce DNA replication stress, which, in turn, induces oncogene-mediated senescence or apoptosis (Lowe and Sherr, 2003; Halazonetis et al., 2008; Evan and D'adda di Fagagna, 2009). Thus, evasion from apoptosis is required for neoplastic transformation and can occur through additional genetic lesions that lead to activation of anti-apoptotic pathways, such as those mediated by phosphatydilinositole 3-kinase. This concept is supported by the finding that the ectopic expression of the RET/PTC oncogene in a continuous rat thyroid cell line (PC Cl3) induces apoptosis (Castellone et al., 2003; Wang et al., 2003). This effect might be due to the strong RET-mediated mitogenic stimuli. It is possible that cytokines and chemokines released by the inflammatory tumoral stroma sustain the survival of those thyroid cells in which RET/PTC rearrangements randomly occur, thereby allowing the selection of clones that A c c e p t e d M a n u s c r i p t 9 acquire additional genetic lesions and thus become resistant to oncogene-induced apoptosis.

  . Substitution of the Y1062 residue of Ret with a phenylalanine, both in the context of a wild-type and of an oncogenically activated receptor, abrogates Ret-mediated signal transduction and biological activities[START_REF] Takahashi | The GDNF/RET signaling pathway and human diseases[END_REF]. We and others have shown that Ret can activate the MAPK pathway in a Y1062-RAS-and BRAF-dependent manner[START_REF] Kimura | High Prevalence of BRAF Mutations in Thyroid Cancer: Genetic Evidence for Constitutive Activation of the RET/PTC-RAS-BRAF Signaling Pathway in Papillary Thyroid Carcinoma[END_REF] Melillo et al., 2005).

  and SDF-1)(Castellone et al., 2004; Guarino et al., 2005; De Falco et al., 2007; Melillo et al., 2005; Borrello et al., 2005).

  Metab. 86, 3211-6. Evan, G.I., d'Adda di Fagagna, F., 2009. Cellular senescence: hot or what?

  most relevant inflammatory molecules in thyroid cancer cellsThe official symbol and full name, Gene ID, and known function for each molecule are listed.References regarding their role in thyroid cancer are also reported.

Figure 1

 1 Figure1

al., 2003; Melillo et al., 2005; Puxeddu et al., 2005; Pufnock and Rothstein,

Table 1 . Inflammatory molecules in thyroid cancer cells Inflammatory molecules Official symbol

 1

			Official full name	Gene ID	Function	References
			colony stimulating		Cytokine that controls the
	M-CSF	CSF1	factor 1 (macrophage)	1435	production, differentiation, and function of macrophages.	Borrello et al., 2005
			colony stimulating		Cytokine that controls the
	G-CSF	CSF3	factor 3 (granulocyte)	1440	production, differentiation, and function of granulocytes.	Borrello et al., 2005
			colony stimulating		Cytokine that controls the
			factor 2		production, differentiation, and	Russell et al., 2003, 2004; Borrello
	GM-CSF	CSF2	(granulocyte-macrophage)	1437	function of granulocytes and macrophages.	et al., 2005
					Member of the interleukin 1
					cytokine family. Pleiotropic
					cytokine involved in various
					immune responses, inflammatory
					processes, and hematopoiesis. It is
	IL-1	IL1A	interleukin 1, alpha 3552	mainly produced by monocytes and macrophages.	Russell et al., 2003, 2004
					Member of the interleukin 1
					cytokine family. It is produced by
					activated macrophages as a
					proprotein, which is proteolytically
					processed to its active form by
					caspase 1 (CASP1/ICE). This	Russell et al., 2003, 2004; Borrello
	IL-1	IL1B	interleukin 1, beta	3553	cytokine is an important mediator of the inflammatory response.	et al., 2005
	IL-6	IL6	interleukin 6 (interferon, beta 2)	3569

Table 1 A

 1

	IL-8/CXCL8 IL-4	IL8 IL4		interleukin 8 interleukin 4	secreted into the serum and induces a transcriptional inflammatory response. Member of the CXC chemokine family. It is one of the major mediators of the inflammatory response. It is secreted by several cell types, functions as a a n u s c r i p t chemoattractant, and is also a Iwahashi et al., 2002; Borrello et potent angiogenic factor. al., 2005 Pleiotropic cytokine produced by M 3576 activated T cells. This cytokine is a Stassi et al., 2003; Todaro et al., 3565 ligand for interleukin 4 receptor. 2006
						Cytokine produced primarily by
	IL-10 IL-24	IL10 IL24	interleukin 10 interleukin 24 c c e p t e d 3586 11009	monocytes and to a lesser extent by lymphocytes. It has pleiotropic effects in immunoregulation and inflammation. It down-regulates the expression of Th1 cytokines, and enhances B cell survival, proliferation, and antibody production. Member of the IL10 family of cytokines. It can induce apoptosis selectively in various cancer cells. Member of the PDGF/VEGF growth factor family, acts on endothelial cells and mediates	Stassi et al., 2003; Todaro et al., 2006 Shinohara et al., 2004
						increased vascular permeability,
						induces angiogenesis,
				vascular		vasculogenesis and endothelial cell
				endothelial growth		growth, promotes cell migration,
	VEGFA	VEGFA	factor A	7422	and inhibits apoptosis.	Belletti et al., 1999
				secreted		Cytokine secreted by immune and	Castellone et al., 2004a; Borrello et
	OPN/IL28/SPP1	SPP1		phosphoprotein 1	6696	non immune cells. Involved in cell	al., 2005


Francesco Pacifico

Antonio Leonardi
email: leonardi@unina.it

ROLE OF NF-B IN THYROID CANCER

Keywords: NF-B, thyroid cancer, apoptosis, inflammation NF-κB, Nuclear Factor-kappa B, IB, Inhibitor of NF-κB, IKK, IκB kinase

Thyroid cancer is the most common neoplasia of the endocrine system and accounts for approximately 1% of all newly diagnosed cancer cases. Its incidence has rapidly grown over the past few decades. Although most thyroid carcinomas are of the well-differentiated papillary histology, and respond well to treatment with surgical resection followed by radioactive iodine ablation, tumors with more aggressive phenotype, such as follicular, poorly differentiated, anaplastic, and medullary cancers, lead to almost 1,500 patient deaths annually. Therefore, understanding molecular mechanisms that regulate the biology of these carcinomas could be helpful to identify new molecules acting as novel targets for therapeutic intervention.

NF-B has been recently shown to play an important role in thyroid cancer for its ability to control the proliferative and the anti-apoptotic signaling pathways of thyroid neoplastic cells.

Oncogenic proteins RET/PTC, RAS and BRAF, that are involved in many aspects of thyroid carcinogenesis, can induce NF-B activation in papillary, follicular, and medullary thyroid carcinomas, while constitutive de-regulated NF-B activity has been found in anaplastic thyroid carcinomas. A number of NF-B inhibitors have been demonstrated to induce anti-proliferative effects and/or massive apoptosis, especially in combination with radio-or chemo-therapy. The results obtained suggest that targeting NF-B could be a promising strategy for advanced thyroid cancer treatment.

Introduction

Thyroid carcinomas mainly comprise four types of tumors: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), both of which may be summarized as differentiated thyroid carcinoma, medullary thyroid carcinoma (MTC), and undifferentiated anaplastic thyroid carcinoma (ATC). PTC, FTC and ATC derive from the thyroid follicular epithelial cells, while MTC derives from the parafollicular C-cells [START_REF] Sherman | Thyroid carcinoma[END_REF]. PTC is the most common malignant thyroid neoplasm in countries with sufficient iodine diets, and comprises up to 80% of all thyroid malignancies. FTC is more common in regions with insufficient iodine diets and represents approximately 10-20% of all thyroid malignancies [START_REF] Fagin | Molecular pathology of thyroid cancer: diagnostic and clinical implications[END_REF]. The overall 5-10 year survival rate of patients with PTC is about 80-95%, while that of patients with FTC is about 70-95% [START_REF] Gimm | Thyroid cancer[END_REF]. The incidence of MTC is not well known because epidemiologic studies are rare. Generally, it is believed that MTC comprises about 5-10% of all thyroid malignancies [START_REF] Gimm | Thyroid cancer[END_REF][START_REF] Sherman | Thyroid carcinoma[END_REF]. ATC is one of the most aggressive human malignancies, with a very poor prognosis. Although rare, accounting for up to 1-2% of clinically recognized thyroid cancers, the overall median survival is limited to months [START_REF] Smallridge | Anaplastic thyroid cancer: molecular pathogenesis and emerging therapies[END_REF]. Unfortunately, at the time of diagnosis most of ATC patients already show local and distant metastases, so that surgery, radiotherapy, and chemotherapy, based primarily on doxorubicin and cisplatin treatment, do not meaningfully improve survival of these patients. Consequently, there is a need for new diagnostic and therapeutic tools for the treatment of these tumors.

An emerging body of literature shows that NF-B plays a role in thyroid cancer [START_REF] Visconti | Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression[END_REF][START_REF] Ludwig | Nuclear factor-kappaB is constitutively active in C-cell carcinoma and required for RET-induced transformation[END_REF][START_REF] Russell | Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors[END_REF][START_REF] Vasudevan | Suppression of PTEN expression by NF-kappa B prevents apoptosis[END_REF][START_REF] Kato | PPARgamma insufficiency promotes follicular thyroid carcinogenesis via activation of the nuclear factor-kappaB signaling pathway[END_REF][START_REF] Palona | BRAFV600E promotes invasiveness of thyroid cancer cells through nuclear factor kappaB activation[END_REF][START_REF] Gombos | Characterization of microarray gene expression profiles of early stage thyroid tumours[END_REF][START_REF] Gallel | Nuclear factor-kappaB activation is associated with somatic and germ line RET mutations in medullary thyroid carcinoma[END_REF], especially of anaplastic type [START_REF] Pacifico | Oncogenic and anti-apoptotic activity of NF-kappa B in human thyroid carcinomas[END_REF]Starenki et al., 2004a;[START_REF] Pacifico | RbAp48 is a target of nuclear factor-kappaB activity in thyroid cancer[END_REF][START_REF] Iannetti | The Neutrophil Gelatinase-Associated Lipocalin (NGAL), a NF-B regulated gene, is a survival factor for thyroid neoplastic cells[END_REF][START_REF] Festa | R-roscovitine sensitizes anaplastic thyroid carcinoma cells to TRAIL-induced apoptosis via regulation of IKK/NF-kappaB pathway[END_REF][START_REF] Zhu | A small[END_REF]. This is an important issue because NF-B, and particularly the genes under its transcriptional regulation, could potentially become novel molecular targets in ATC therapy, given A c c e p t e d M a n u s c r i p t the ability of NF-B to control many aspects of thyroid cancer biology. This review will focus on these aspects, in particular on the molecular mechanisms by which NF-B exerts its role in thyroid tumors.

NF-B

NF-B is a family of transcription factors that plays a central role in the regulation of apoptosis, inflammation and immune response (Ghosh et al., 1998;[START_REF] Karger Karin | Phosphorylation meets ubiquitination: the control of NF [kappa]B activity[END_REF][START_REF] Tak | NF-kappaB: a key role in inflammatory diseases[END_REF]. In mammals, the NF-B family is composed of five members: RelA(p65), RelB, c-Rel, NF-B1 (p50 and its precursor p100), NF-B2 (p52 and its precursor p105) [START_REF] Vallabhapurapu | Regulation and function of NF-kappaB transcription factors in the immune system[END_REF]). These proteins form homodimers and heterodimers and their activity is regulated by two major pathways. The first pathwayknown as the canonical pathway-regulates the activity of NF-B dimers composed of RelA(p65), c-Rel and p50. These dimers are held in the cytoplasm in an inactive form bound to specific inhibitors known as inhibitor of B (IB). Following different stimuli, such as cytokines, pathogens and pathogen-related factors, IB is phosphorylated on specific serine residues, ubiquitinated and degraded through a proteasome dependent pathway. NF-B dimers are then free to move to the nucleus and to transcribe target genes [START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF].

The second pathway affects NF-B2, which preferentially dimerizes with RelB. Following triggering of some members of the TNF receptor superfamily, such as LT-R and BAFF-R, NF-B2 is phosphorylated, ubiquitinated, and processed to form the mature form p52. RelB-p52 dimers then move to the nucleus, where they control transcription of genes involved in secondary lymphoid organ development and lymphocytes survival [START_REF] Claudio | BAFF-induced NEMOindependent processing of NF-kappa B2 in maturing B cells[END_REF][START_REF] Kayagaki | BAFF/BLyS receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface loop and promotes processing of NF-kappaB2[END_REF][START_REF] Dejardin | The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-kappaB pathways[END_REF]. The phosphorylation of the IB and NF-B2 is mediated by a high molecular weight complex, the IB Kinase (IKK) complex. This complex is composed by two catalytic subunits, IKK and IKK, and a regulatory subunit IKK/NEMO [START_REF] Ghosh | Missing pieces in the NF-kappaB puzzle. Cell 109, S81-S96. conserved mediators of immune responses[END_REF]. IKK and IKK have distinct substrates. IKK directly phosphorylates IB, then activating the canonical pathway, while et al., 2001;Senftleben et al., 2001). IKK/NEMO does not have any enzymatic activity but its presence in the complex is absolutely necessary for a correct activation of the IKK complex, very likely because it serves to connect upstream signal mediators to the IKK [START_REF] Yamaoka | Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation[END_REF][START_REF] Rothwarf | IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex[END_REF]. Once in the nucleus, NF-B transcribes different genes controlling different aspects of cell physiology. Since many of these genes fall in the six categories characterizing a neoplastic cell (1. self-sufficiency in growth, 2. insensitivity to growth-inhibitory signals, 3. evasion of apoptosis, 4. limitless replicative potential, 5. sustained angiogenesis and 6. tissue invasion and metastasis) [START_REF] Hanahan | The hallmarks of cancer[END_REF], it is not surprising that the NF-B pathway is involved in cell transformation (Fig. 1).

No information is available about the role played by NF-B in thyroid gland development, and very little is known about the influence of NF-B on the regulation of thyroid functions, as well as on the expression of thyroid specific genes, such as thyroglobulin, thyroid stimulating hormonereceptor, and thyroperoxidase. It has been reported that the murine Pax8 gene contains B sites, however no experimental evidence confirmed that theses sites are functional (Okladova et al. 1997).

Role of NF-B in thyroid cancer

Several lines of evidence show that NF-B plays a role in cancer. Its activity has been found constitutively elevated in many types of human tumors from either haematological or solid origin [START_REF] Pacifico | NF-kappaB in solid tumors[END_REF]. The role of NF-B in solid tumors has been well documented in several studies performed on primary tumors, and neoplastic cell lines derived from different human tissues. These studies show that the inhibition of constitutive NF-B activity blocks the oncogenic potential of neoplastic cells by different ways: by sensitizing tumor cells to chemotherapeutic drug-induced apoptosis, by decreasing the highly proliferative rate which characterizes transformed cells, and by inhibiting tissue invasiveness and metastatic potential of A c c e p t e d M a n u s c r i p t highly malignant cells [START_REF] Karin | NF-B in cancer : from innocent bystander to major culprit[END_REF]. In addition, since it is now generally accepted that chronic inflammation contributes to the genesis of many solid tumors, such as gastric, colon and hepatic carcinomas, activation of NF-B by the classical IKK-dependent pathway, is a crucial mediator of inflammation-induced tumor growth and progression in animal models of inflammation-associated cancer [START_REF] Greten | The IKK/NF-kappaB activation pathway-a target for prevention and treatment of cancer[END_REF][START_REF] Pikarsky | NF-B functions as a tumour promoter in inflammation-associated cancer[END_REF].

The first evidence of NF-B constitutive activation in thyroid carcinomas was reported by Visconti et al. in 1997, who described the presence of a strong de-regulated NF-B activity in the nuclei of different thyroid cancer-derived cell lines. In this work the authors pointed out the requirement of NF-B for the expression of neoplastic phenotype of thyroid carcinoma cell lines.

Successively, Ludwig and co-workers demonstrated that NF-B was strongly activated in tissue specimens from parafollicular C-cell carcinomas, and that RET oncogene needs NF-B to exert its transforming activity in the MTC-derived TT cell line. Since then, a number of papers reported that different signaling pathways leading to the neoplastic transformation of thyroid cells, merge into NF-B activation: the anti-apoptotic behaviour and invasiveness of thyroid cancer cells transformed by oncogenic BRAF is mediated by NF-B activation [START_REF] Palona | BRAFV600E promotes invasiveness of thyroid cancer cells through nuclear factor kappaB activation[END_REF]; blocking PPAR tumor suppressor promotes follicular thyroid carcinogenesis via NF-B [START_REF] Kato | PPARgamma insufficiency promotes follicular thyroid carcinogenesis via activation of the nuclear factor-kappaB signaling pathway[END_REF]; constitutive activation of the PI3K/Akt pathway due to PTEN inactivation, accelerates thyroid cancer progression following increase of NF-B activity [START_REF] Guigon | PTEN deficiency accelerates tumour progression in a mouse model of thyroid cancer[END_REF]. It has also been reported that FOXO3 activity is negatively regulated by the PI3K/Akt pathway, suggesting that FOXO3 inactivation might cooperates with NF-B in controlling survival of thyroid cancer cells [START_REF] Guigon | PTEN deficiency accelerates tumour progression in a mouse model of thyroid cancer[END_REF], Karger et al. 2009).

In addition to cell lines, additional studies have shown that primary human thyroid carcinomas exhibit high basal activity of NF-B, especially in tissue specimens from ATC (Fig. 2) [START_REF] Pacifico | Oncogenic and anti-apoptotic activity of NF-kappa B in human thyroid carcinomas[END_REF][START_REF] Mitsiades | Antitumor effects of the proteasome inhibitor bortezomib in medullary and anaplastic thyroid carcinoma cells in vitro[END_REF]. The increased NF-B activity detected in both cell lines and primary tumors, is not a simple epiphenomenon of the neoplastic transformation, but A c c e p t e d M a n u s c r i p t rather one of the causes that contributes to the establishment of the neoplasia. In fact, inhibition of NF-B by ectopic expression of a superepressor form of IB in the ATC-derived cell line FRO, determines an increased susceptibility to chemotherapeutic drug-induced apoptosis, and the block of the oncogenic activity [START_REF] Pacifico | Oncogenic and anti-apoptotic activity of NF-kappa B in human thyroid carcinomas[END_REF]. The specific antisense oligonucleotides against RelA reduces the growth rate of different thyroid cell lines [START_REF] Visconti | Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression[END_REF]. Several NF-B inhibitory reagents have been demonstrated to promote thyroid cell death alone, or in combination with chemotherapy or radiation exposure (Starenky et al. 2004a;Starenky et al. 2004b;[START_REF] Mitsiades | Antitumor effects of the proteasome inhibitor bortezomib in medullary and anaplastic thyroid carcinoma cells in vitro[END_REF]). These works indicate that NF-B plays a central role in thyroid cancer being important to maintain the transformed phenotype, and to confer resistance to apoptosis. Much of the studies present in the literature, point to an exclusive role of the canonical pathway in thyroid tumorigenesis. However, constitutive processing of p100 is associated with the development of various lymphomas/leukemia, and with some solid cancer such as breast cancer and pancreatic cancer, then the possibility that the alternative pathway may also be involved in thyroid cancer needs to be explored (Cogswell et al. 2000[START_REF] Chandler | Increased expression of NF-kappa B subunits in human pancreatic cancer cells[END_REF], Baud and Karin 2008).

NF-B and apoptosis in thyroid cancer

Cancer cells have the feature to circumvent apoptosis by up-regulating and/or downregulating a number of genes involved in the control of programmed cell death. This property allows neoplastic cells to survive beyond their normal lifespan, providing protection against hypoxia as tumor mass expands, promoting angiogenesis and invasiveness during tumor progression, and allowing tumor cells to become resistant to radio-and chemo-therapy. Thus, deregulated apoptosis is a fundamental aspect of the biology of cancer.

The inhibition of NF-B activity in mouse xenograft models of chemo-resistant tumors, provokes tumor regression by sensitizing them to chemo-therapeutic drug treatments [START_REF] Greten | The IKK/NF-kappaB activation pathway-a target for prevention and treatment of cancer[END_REF]. Consistent with this work, NF-B inhibition in many human tumor-derived cell lines, A c c e p t e d M a n u s c r i p t including malignant Reed-Sternberg (H-RS) cells of Hodgkin's disease, colon carcinoma cells, lung adenocarcinoma cells, Ewing sarcoma cells, prostate carcinoma cells, thyroid carcinoma cells, and many others, induces spontaneous apoptosis, or/and sensitizes them to killing by TNF, TRAIL or anti-cancer drugs [START_REF] Pacifico | NF-kappaB in solid tumors[END_REF]. Mechanisms for NF-B-mediated protection from apoptosis are essentially based on its ability to activate the transcription of genes capable of suppressing cell death [START_REF] Kucharczak | To be or not to be: NF-B is the answerrole of Rel/NF-B in the regulation of apoptosis[END_REF]. Genes controlled by NF-B capable of controlling apoptosis may be classified in two major groups. 1) genes interfering with caspase activation and cytochrome c release, such as members of the mammalian Bcl-2 gene family, Bcl-xL and Bfl-1/A1, [START_REF] Boise | Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death[END_REF][START_REF] Wang | NF-kappaB induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapyinduced apoptosis[END_REF], and cellular inhibitors of apoptosis c-IAP1, c-IAP2, TRAF1 and TRAF2, the zinc-finger protein A20, c-FLIP, all of them able to suppress cell death induced by TNF, death receptors or anti-cancer drugs [START_REF] Deveraux | IAPs block apoptotic events induced by caspase 8 and cytochrome c by direct inhibition of distinct caspases[END_REF][START_REF] Yeh | Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development[END_REF][START_REF] Wang | NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation[END_REF]. 2) Genes controlling JNK activity. Belongs to this group the anti-apoptotic genes XIAP, and GADD45, both of them implicated in the suppression of the prolonged, pro-apoptotic JNK activity [START_REF] Tang | Inhibition of JNK activation through NF-kappaB target genes[END_REF][START_REF] De Smaele | Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling[END_REF]. In addition to XIAP and GADD45 a similar mechanism of action is also displayed by Ferritin Heavy Chain (FHC), the principal site of storage of intracellular iron. FHC, by controlling free iron concentration inside the cell, also controls ROS production, responsible, in turn, to mediate sustained activation of the proapoptotic JNK pathway (Pham et al., 2004). All these anti-apoptotic proteins work in a coordinated manner to interfere with apoptosis at multiple steps along the apoptotic signalling cascade (Fig. 3).

The same antipoptotic mechanisms also work in thyroid cancer cells, and contribute to the maintenance of the transformed phenotype. Numerous studies reported increased expression of the pro-survival members of the Bcl-2 gene family in both primary cancer and cell lines [START_REF] Saltman | Patterns of expression of cell cycle/apoptosis genes along the spectrum of thyroid carcinoma progression[END_REF][START_REF] Smallridge | Anaplastic thyroid cancer: molecular pathogenesis and emerging therapies[END_REF]. Indeed, Bcl-2 overexpression in thyroid carcinoma cell lines confers protection against several anticancer agents, and the use of small peptide that inhibits its antiapoptotic activity sensitizes thyroid cancer cell lines to sub-lethal concentration of A c c e p t e d M a n u s c r i p t chemotherapeutic agents [START_REF] Mitsiades | Bcl-2 overexpression in thyroid carcinoma cells increases sensitivity to Bcl-2 homology 3 domain inhibition[END_REF]. BRAF promotes its oncogenic activity in thyroid cancer cells through the NF-B-mediated induction of c-IAP1, c-IAP2 and XIAP [START_REF] Palona | BRAFV600E promotes invasiveness of thyroid cancer cells through nuclear factor kappaB activation[END_REF]. These NF-B regulated proteins are also highly expressed in ATC cell lines [START_REF] Tirrò | Altered expression of c-IAP1, survivin, and Smac contributes to chemotherapy resistance in thyroid cancer cells[END_REF]. Inactivation of PPAR or PTEN proteins in FTC leads to the repression of critical genes involved in the control of apoptosis following NF-B activation [START_REF] Kato | PPARgamma insufficiency promotes follicular thyroid carcinogenesis via activation of the nuclear factor-kappaB signaling pathway[END_REF][START_REF] Guigon | PTEN deficiency accelerates tumour progression in a mouse model of thyroid cancer[END_REF].

Also in thyroid cells, the duration of JNK activity seems to be important to determine the susceptibility of thyroid cancer cells to apoptosis. Sustained JNK activation after exposure to UV increases apoptosis, while transient activation, for example the one induced by growth factors, does not [START_REF] Shklyaev | Transient activation of c-Jun NH2-terminal kinase by growth factors influences survival but not apoptosis of human thyrocytes[END_REF][START_REF] Sautin | Ceramide-induced apoptosis of human thyroid cancer cells resistant to apoptosis by irradiation[END_REF]. Inhibition of NF-B, by stable transfection of a superrepressor form of IB, or by the use of the pharmacological inhibitor DHMED, increases JNK activity, rendering FRO cells susceptible to apoptosis. Administration of a specific JNK inhibitor blocks the apoptotic response [START_REF] Pacifico | Oncogenic and anti-apoptotic activity of NF-kappa B in human thyroid carcinomas[END_REF]Starenki et al., 2004b). This effect seems to be mediated, at least partially, by the up-regulation of GADD45, whose level paralleled the level of NF-B. There is no evidence, so far, that also FHC is mediating the antiapoptotic activity of NF-B in thyroid cancer. However, the recent finding that iron uptake mediated by NGAL, is important for survival of ATC cells, suggests that intracellular iron and ROS levels are important to control thyroid cancer cell apoptosis [START_REF] Iannetti | The Neutrophil Gelatinase-Associated Lipocalin (NGAL), a NF-B regulated gene, is a survival factor for thyroid neoplastic cells[END_REF].

NF-B links inflammation and cancer.

Inflammation is central to control our fight against pathogens, and to regulate wound healing. These are very intricate biological responses that involve complex interactions among different cell types that regulate the expression of biological mediators promoting cell chemotaxis, cell migration, and cell proliferation. If inflammation is not ordered and timely, the resulting chronic inflammation can contribute to a variety of diseases including cancer. Several lines of A c c e p t e d M a n u s c r i p t evidence, based on epidemiological and molecular studies, indicate that inflammation and cancer are closely linked: inflammatory diseases increase the risk of developing many types of cancer, including thyroid cancer; non-steroidal anti-inflammatory drugs reduce the risk of developing certain types of tumors, such as colon cancer; signaling pathways involved in inflammation act downstream to oncogenes (RAS, MYC, RET); a variety of inflammatory cells, chemokines, and cytokines are present in the microenvironment of tumors; targeting chemokines and cytokines, such as TNF or IL-1, as well as key transcription factors, such as NF-B or STAT3, decreases the spread of cancer; transfer of inflammatory cells or over-expression of chemokines and cytokines promotes tumor development [START_REF] Mantovani | Cancer-related inflammation[END_REF].

The interplay between epithelial and inflammatory cells is thought to be crucial for the genesis and the establishment of carcinomas. One of the main actors in the inflammatory process is NF-B which, by regulating the expression and the function of different cytokines and chemokines in inflammatory cells, stimulates the growth and its own activity in epithelial cells. Thus, NF-B establishes a network that, after prolonged time, can lead epithelial cells to undergo malignant transformation. Known examples are the Helicobacter pylori-induced gastric cancer [START_REF] Keates | Helicobacter pylori infection activates NFkappa B in gastric epithelial cells[END_REF], the colitis-associated cancer [START_REF] Greten | The IKK/NF-kappaB activation pathway-a target for prevention and treatment of cancer[END_REF], and the cholangitis-derived hepatocarcinoma [START_REF] Pikarsky | NF-B functions as a tumour promoter in inflammation-associated cancer[END_REF]. The data obtained suggest that the NF-B pathway has multiple actions in tumor promotion, by preventing apoptosis of cells with malignant potential, and by stimulating the production of pro-inflammatory cytokines in the inflammatory cells infiltrating the tumor mass (Fig. 4). This model is consistent with the observed correlation between the number of inflammatory cells, level of cytokines, and tumor aggressiveness and prognosis in both human and mouse. However, the transformed cells themselves can contribute to the overall level of secreted pro-inflammatory cytokines, and to maintain the NF-B pathway activated. In fact, it is possible to keep malignant cells in culture without the support of the innate immunity, and still detect constitutive activation of NF-B.

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

A possible link between inflammation and cancer has been postulated in thyroid, too [START_REF] Arif | Hashimoto's thyroiditis shares features with early papillary thyroid carcinoma[END_REF][START_REF] Memon | Epidemiological evidence for a link between postpartum thyroiditis and thyroid cancer[END_REF][START_REF] Larson | Increased incidence of well-differentiated thyroid cancer associated with Hashimoto thyroiditis and the role of the PI3k/Akt pathway[END_REF]. In their work, Arif et al. showed an overlap in the morphological features, immunohistochemical staining pattern and, most importantly, molecular profile between papillary thyroid carcinoma and Hashimoto's thyroiditis (HT). Indeed, Memon and co-workers reported that women who had experienced episodes of postpartum thyroiditis, even with no previous thyroid disease, or X-ray exposure to the neck, were at a significantly increased risk of developing thyroid cancer. In a study conducted by Larson et al., patients with HT were three times more likely to have thyroid cancer, suggesting a strong link between chronic inflammation and cancer development. Interestingly, two studies have shown RET activation in a significant number of HT cases [START_REF] Sheils | ret/PTC-1 Activation in Hashimoto Thyroiditis[END_REF][START_REF] Sheils | RET/PTC rearrangements in Hashimoto's thyroiditis[END_REF], whereas another study detected no RET rearrangements in HT [START_REF] Nikiforova | Prevalence of RET/PTC rearrangements in Hashimoto's thyroiditis and papillary thyroid carcinomas[END_REF]. Another group describes RET rearrangements in non-malignant thyroid tissue associated to lymphocytic thyroiditis (LT), whereas it was absent in the pure LT areas [START_REF] Mechler | Papillary thyroid carcinoma: 6 cases from 2 families with associated lymphocytic thyroiditis harbouring RET/PTC rearrangements[END_REF]. A very recent study shows that sixty-eight percent (15 of 22) of HT were positive by FISH analysis [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]. The hypothetical association among RET, HT and thyroid cancer let to the suggestion of a possible pro-inflammatory role of RET in thyroid cells, that could predispose them to neoplastic transformation. This hypothesis is substantiated by the evidence that exogenous over-expression of oncogenic RET in primary human thyrocytes and rat thyroid cell lines, induces the expression of a distinct set of genes involved in inflammation and tumor invasion [START_REF] Russell | Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors[END_REF][START_REF] Castellone | Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas[END_REF][START_REF] Borrello | Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1 oncogene[END_REF][START_REF] Melillo | The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and A c c e p t e d M a n u s c r i p t mitogenic phenotype of thyroid cancer cells[END_REF]). In addition, oncogenic RET enhances infiltration of lymphocytes and myeloidderived cells in a transplantable tumor system and strongly stimulates tumor growth in immunocompetent mice [START_REF] Pufnock | Oncoprotein signaling mediates tumor-specific inflammation and enhances tumor progression[END_REF].

During thyroiditis, in particular HT, thyroid gland parenchyma is progressively lost and replaced by cells of the inflammatory infiltrate that produce chemokines, cytokines, and growth factors, the majority of which are under NF-B transcriptional control. The persistent stimulation of residual thyrocytes with such molecules could induce the activation of NF-B in follicular cells, A c c e p t e d M a n u s c r i p t thereby creating a functional network between epithelial thyroid cells and inflammatory cells. The microenvironment established by this interplay could strongly promote the onset of thyroid cancer, and could account for the progression of tumor growth. The microenvironment of primary tumor and lymph-node metastasis of patients diagnosed for PTC expresses the same inflammatory gene pattern produced by thyrocytes transfected with oncogenic RET [START_REF] Castellone | Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas[END_REF][START_REF] Borrello | Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1 oncogene[END_REF][START_REF] Melillo | The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and A c c e p t e d M a n u s c r i p t mitogenic phenotype of thyroid cancer cells[END_REF]. Since oncogenic RET activates NF-B, and the majority of proinflammatory molecules are under NF-B transcriptional control, it is reasonable that NF-B could be involved in the regulation of pro-inflammatory program of thyroid cells, and this event contributes to the onset of thyroid cancer. Interestingly, De Falco and co-workers found that ATC samples from human specimens, as well as ATC-derived cell lines, strongly express CXCR4, the chemokine receptor of stromal cell-derived factor (SDF)-1 [START_REF] De Falco | Biological role and potential therapeutic targeting of the chemokine receptor CXCR4 in undifferentiated thyroid cancer[END_REF]. Treatment of ATC cells with SDF-1 increases of the proliferative rate of cancer cells. This effect was blocked by a specific CXCR4 antagonist, and by CXCR4 RNA interference. Moreover, the CXCR4 antagonist effectively reduced tumor growth in nude mice inoculated with different ATC cells [START_REF] De Falco | Biological role and potential therapeutic targeting of the chemokine receptor CXCR4 in undifferentiated thyroid cancer[END_REF]. These results indicate that ATC cells promote their growth through the up-regulation of CXCR4/SDF-1 circuit in the tumor microenvironment. Also in this case, NF-B could play a determinant role because it is able to regulate CXCR4 transcription [START_REF] Helbig | NF-B promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4[END_REF] and, therefore, it could drive this paracrine mechanism, determining the increase of cell proliferation and, as a consequence, of tumor growth. Accordingly to this paracrine model, we recently demonstrate that the strong constitutive NF-B activity in ATC cells leads to the up-regulation of NGAL, a protein involved in inflammatory response, which is abundantly secreted in the extracellular milieu [START_REF] Iannetti | The Neutrophil Gelatinase-Associated Lipocalin (NGAL), a NF-B regulated gene, is a survival factor for thyroid neoplastic cells[END_REF]. The uptake of NGAL from culture medium allows the ATC cells to survive to serum withdrawal-induced apoptosis through an iron-dependent mechanism, thus enhancing tumor growth [START_REF] Iannetti | The Neutrophil Gelatinase-Associated Lipocalin (NGAL), a NF-B regulated gene, is a survival factor for thyroid neoplastic cells[END_REF]. In addition, these data also support the idea that the transformed cells themselves contribute to the overall level of secreted cytokines, chemokines and survival factors, and to maintain the NF-B pathway constitutively activated. While differentiated PTC and FTC have generally a good prognosis after surgery and radioiodine therapy, poorly differentiated MTC and undifferentiated ATC do not respond to neither radio-iodine treatment, nor conventional chemo-and radio-therapy. These tumors are obvious candidates for alternative therapeutic approaches, such as molecularly targeted therapy. The finding that NF-B is one of the most important tumor-promoting machinery, prompted the search for drugs able to suppress NF-B activity in order to improve chemo-and radio-therapy. It is possible to interfere with NF-B activation at different levels, targeting components of its signalling cascade, such as the IKK complex, the IB inhibitory protein, the proteosome, the RelA subunit of the transcriptionally active heterodimers. An increasing number of compounds, able to block NF-B have been tested, and have shown to suppress the growth of those cancer cells whose tumorigenicity depends on NF-B activity [START_REF] Karin | NF-B in cancer : from innocent bystander to major culprit[END_REF]Baud and Karin 2009). Some of these compounds have also been tested in ATC cells. The cell permeable peptide SN50, which blocks RelA nuclear translocation, has been found to promote cell death in vitro in combination with ionizing radiation (Starenki et al. 2004a). The dehydroxymethylepoxy quinomicin (DHMEQ) also blocks NF-B nuclear translocation, and has been demonstrated to induce apoptosis in vitro and in nude mice xenografted with FRO cells (Starenki et al. 2004b). Interestingly, in addition to block NF-B activation, DHMEQ treatment also induces sustained activation of the JNK pathway. This activation is, at least partially, responsible for the observed apoptosis, since administration of a specific JNK inhibitor suppressed the DHMEQ-induced apoptosis. These data further suggest that one of the ways used by NF-B to protect cells from apoptosis is to terminate JNK activity. The proteosome inhibitor Bortezomib [START_REF] Mitsiades | Antitumor effects of the proteasome inhibitor bortezomib in medullary and anaplastic thyroid carcinoma cells in vitro[END_REF], which is the only pharmacological inhibitor of NF-B approved for treatment of some haematological malignancies, induces apoptosis in a panel of thyroid carcinoma cell lines originating from papillary, follicular, and anaplastic [START_REF] Mitsiades | Antitumor effects of the proteasome inhibitor bortezomib in medullary and anaplastic thyroid carcinoma cells in vitro[END_REF]). However, one of the most problematic aspects of a cancer therapy based on inhibition of NF-B activity is represented by the difficulty to find compounds which block the oncogenic activity of NF-B, without interfering with its physiological roles in immunity, inflammation, and cellular homeostasis. Unfortunately, most of the drugs analyzed so far, also affect other cellular signaling pathways, thereby determining a number of highly toxic side effects. Much of the efforts are now concentrated on the identification of novel NF-B targets specifically activated in tumors, but not in normal cells. Thus, the inhibition of these targets should block the oncogenic potential of NF-B in cancer cells without affecting its role in normal tissues.

Conclusions

In the last few years an enormous amount of basic and clinical observations strongly implicate NF-B in a variety of human tumors, including thyroid cancer. The strongly deregulated NF-B activity in human ATC represents a novel finding in the understanding of the molecular biology of thyroid cancer, that joins to those of RET, BRAF, PPAR, PTEN, whose role has been well established in differentiated thyroid carcinomas. The future challenge is the comprehension of the molecular mechanisms mediating the oncogenic activity of NF-B, in order to design compounds interfering with the anti-apoptotic, or the pro-survival signalling, without affecting its physiological role in immunity, inflammation and cellular homeostasis. The interplay between epithelial and inflammatory cells is thought to be crucial for the genesis and the establishment of carcinomas. One of the main actors in inflammatory process is NF-B which, by regulating the expression and the function of different cytokines and chemokines in inflammatory cells, stimulates the growth and its own activity in epithelial cells. Thus, NF-B establishes a network that can sustain cell proliferation and resistance to apoptosis.

  as therapeutic target in thyroid cancer.

  s c r i p t carcinomas. Interestingly, Bortezomid induces apoptosis with an IC50 value within the range of clinically achievable concentration, suggesting the clinical use of Bortezomid alone, or in combination with conventional therapy

  s c r i p t molecule triptolide suppresses angiogenesis and invasion of human anaplastic thyroid carcinoma cells via down-regulation of the nuclear factor-kappa B pathway. Mol.Pharmacol. 75,[812][813][814][815][816][817][818][819]

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. NF-B constitutive activity in primary human anaplastic thyroid carcinomas

Figure 3 .

 3 Figure 3. NF-B controls transcription of different genes interfering with different steps in the

Figure 4 .

 4 Figure 4. Mechanisms by which NF-B contributes to cancer development during inflammation.

  Figure 2

NF-kB

Sustained angiogenesis Tissue invasion and metastasis

New vessels Invasion Invasion

Primary tumor

Metastasis

VEGF, MMPs, uPA, VCAM-1, ICAM-1, ELAM-1, CXCR4 Cyclin D1, CDK2, c-Myc, TNF, IL-1, IL-6, RbAp48

Death signals

Normal cell Apoptotic cell

Evading apoptosis

NF-kB

TRAF-1, TRAF-2, XIAP, cIAP-1, cIAP-2, c-FLIP Bcl-2 family, GADD45b, FHC, NGAL