Estrogen and Its Role in Thyroid Cancer

M Derwahl and D Nicula Estrogen in thyroid cancer 21:5 T273–T283 Thematic Review

Estrogen and its role in thyroid cancer

Correspondence Michael Derwahl and Diana Nicula should be addressed to M Derwahl Department of Medicine, St Hedwig Hospital and Charite, University Medicine Berlin, Grosse Hamburger Straße Email 5-11, 10115 Berlin, Germany [email protected] or [email protected]

Abstract

Proliferative thyroid diseases are more prevalent in females than in males. Upon the onset of Key Words puberty, the incidence of thyroid cancer increases in females only and declines again after " thyroid menopause. Estrogen is a potent growth factor both for benign and malignant thyroid cells " estrogen that may explain the sex difference in the prevalence of thyroid nodules and thyroid cancer. " cell signaling It exerts its growth-promoting effect through a classical genomic and a non-genomic pathway, " growth factor receptor mediated via a membrane-bound estrogen receptor. This receptor is linked to the tyrosine " neoplasia kinase signaling pathways MAPK and PI3K. In papillary thyroid carcinomas, these pathways may be activated either by a chromosomal rearrangement of the tyrosine receptor kinase TRKA, by RET/PTC genes, or by a BRAF mutation and, in addition, in females they may be stimulated by high levels of estrogen. Furthermore, estrogen is involved in the regulation of angiogenesis and metastasis that are critical for the outcome of thyroid cancer. In contrast to other

carcinomas, however, detailed knowledge on this regulation is still missing for thyroid cancer. Endocrine-Related Cancer Endocrine-Related Cancer (2014) 21, T273–T283

Introduction

Both benign and malignant thyroid tumors are 3–4 times (Rossing et al. 2000, Sakoda & Horn-Ross 2002, Cornet more prevalent in females than in males (Dean & Gharib 2013). However, in a recent retrospective cohort study of 2008, Li et al. 2013). Whereas in prepubertal girls and boys 145 007 postmenopausal women, no association between (rare), thyroid cancer is roughly equally represented, with the risk of thyroid cancer and hormonal or reproductive the onset of puberty the incidence increases in females by factors such as age at menarche or menopause, parity, and up to 14 times (Farahati et al. 1997). After menopause, the bilateral oophorectomy was observed (Kabat et al. 2012). incidence decreases again (Li et al. 2013). Only women who had a ﬁrst live birth between the age In several studies, pregnancy was associated with a of 20 and 24 years had an increased risk of papillary higher risk of thyroid cancer (Galanti et al. 1995, Rossing thyroid cancer (PTC; Kabat et al. 2012). et al. 2000, Sakoda & Horn-Ross 2002, Horn-Ross et al. Genetic factors do not seem to confer a higher risk 2011). In contrast, low dose of estrogen in contraceptives of thyroid cancer to females. In a case–control study of 344 and in replacement therapy in postmenopausal women PTCs and 452 controls, the association between PTC and does not contribute to a higher risk of thyroid cancer (Kabat 1151 tag single-nucleotide polymorphisms (SNPs) in 58 et al. 2012). Some studies even found an inverse association candidate gene regions involved in sex hormone synthesis between the use of contraceptives and the risk of thyroid and metabolism did not show a signiﬁcant correlation cancer, which may be explained by the low concentrations between the investigated SNPs and the risk of PTC

of 17b-estradiol (E2) in currently used contraceptives (Schonfeld et al. 2012).

http://erc.endocrinology-journals.org q 2014 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/ERC-14-0053 Printed in Great Britain This paper forms part of a special thematic review section on New Concepts and Challenges in Thyroid Cancer. The guestDownloaded editor forfrom this Bioscientifica.com section was Laura at 09/30/2021 Ward, 05:56:17AM Universidade Estadual de Campinas, Brazil. via free access Thematic Review M Derwahl and D Nicula Estrogen in thyroid cancer 21:5 T274

The strongest support for estrogen as a risk factor of In addition, a novel membrane-bound ER, referred differentiated thyroid cancer comes from experimental to as ERx, was detected in the brain and the uterus

studies that have clearly demonstrated that E2, the main (Toran-Allerand et al. 2002). The existence of this receptor female sex hormone, is a potent stimulator of both human in the thyroid is still unknown. benign and malignant thyroid cells (Manole et al. 2001, et al et al et al Zeng . 2007, Chen . 2008, Kumar . 2010, ER expression in benign and et al Rajoria . 2010). Interestingly, E2 was equally potent malignant thyroid tissues in its growth-promoting effect on human thyroid cells derived from male and female thyroid glands, which First indirect evidence for the presence of ERs in benign underlines the relevance of the hormone rather than of and malignant thyroid tissues (Table 1) came from ligand other sex-speciﬁc factors (Manole et al. 2001). studies (Molteni et al. 1981). Later ER immunoreactivity In summary, epidemiological, clinical, and experi- was detected in normal thyroid tissue, thyroid adenomas, mental data are indicative of a role of estrogen in the goiters, and differentiated and undifferentiated thyroid pathogenesis of proliferative thyroid diseases. carcinomas (Diaz et al. 1991, Mizukami et al. 1991, Hiasa et al. 1993). However, widely varying and, in part, contradictory results were reported. Some authors did Estrogen and its receptors not ﬁnd ERs by immunohistochemical staining in normal thyroid tissue and adenomas or goiters (Jaklic et al. 1995, Estrogens are steroid hormones that play a key role in Arain et al. 2003), while others detected ER staining in the regulation of not only growth, differentiation, and approximately 10% to more than 50% of benign thyroid function of the reproductive organs but also bone, lesions (Diaz et al. 1991, Mizukami et al. 1991, Hiasa et al. and the cardiovascular and immune systems in males 1993, Tavangar et al. 2007). and females (Heldring et al. 2007). Estrogens that are A similar picture emerged when comparing immu-

effective in humans include estrone, estriol, and E2,of nostaining of thyroid cancer samples. Although ER

which E2 has the highest afﬁnity to the estrogen receptors immunostaining was described in less than 10% to over (ERs) and the highest potency (Kuiper et al. 1997). The 60% of the samples in the majority of investigations action of estrogen occurs via a classical genomic and a (Haruta et al. 1990, Diaz et al. 1991, Mizukami et al. 1991, non-genomic pathway. The genomic mode of action is Hiasa et al. 1993, Lewy-Trenda 1998, Tavangar et al. 2007), Endocrine-Related Cancer mediated through ERa and ERb, which are members of a no ER immunoreactivity was observed in other studies large family of nuclear transcription factors (Mangelsdorf (Jaklic et al. 1995, Arain et al. 2003). These widely varying et al. 1995). For activation, estrogen has to enter the cell results were described for both papillary carcinoma and where it binds to its receptors ERa and ERb, which is follicular thyroid carcinoma (FTC) samples. The discre- followed by a nuclear translocation, homo- or hetero- pancies may be explained by technical factors such as the dimerization of the estrogen–ER complex, and its binding use of different monoclonal or polyclonal antibodies or to an estrogen-responsive element (ERE) in the promoter various staining procedures. region of different target genes (Nilsson et al. 2001). For the However, using considerably more sensitive reverse transactivation of genes, a process of recruitment and transcription PCR, detection of ER mRNAs in malignant binding of different coactivators is mandatory. thyroid tissues also varied between less than 20% and up In contrast to the slow process of transactivation of to 100% of samples (Yane et al. 1994, Dalla Valle et al. target genes, the non-genomic action of estrogen, which 1998). Therefore, it is likely that these discrepancies may is mediated by a membrane-associated estrogen receptor be also due to epigenetic modiﬁcations of ER expression (mER), is very fast (Moriarty et al. 2006). Both the genomic rather than only the result of technical factors. Epigenetic and the non-genomic actions of estrogen are operative in regulation of ER expression was demonstrated in different benign and malignant thyroid cells (Manole et al. 2001). tumors including breast cancer (Hervouet et al. 2013). Alternatively, rapid signal transduction of estrogen may Moreover, both isoforms, ERa and ERb, were detected be mediated by a novel G protein-coupled receptor, in goiter tissues, thyroid carcinomas, and different thyroid designated GPR30 (Revankar et al.2005). This receptor has carcinoma cells (Manole et al. 2001, Santin & Furlanetto been detected in some thyroid carcinoma cell lines that lack 2011). Most of the thyroid cancer cell lines that are widely classical ERs and stimulate growth via GPR30 but not in used in thyroid research express both ER isoforms (Manole benign and malignant thyroid tissues (Vivacqua et al.2006). et al. 2001, Santin & Furlanetto 2011). However, similarly,

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Table 1 Detection of estrogen receptors (ERs) a and b in benign and malignant thyroid tissues (ER-positive samples/total number of samples)

Benign lesions Carcinoma

Isoform Goiter Adenoma Papillary Follicular Anaplastic Methodology

Molteni et al. (1981) ER 2/4 SDG Hampl et al. (1985) ER 0/5 RBA Haruta et al. (1990) ER 30/52 0/12 IHC Mizukami et al. (1991) ER 4/8 47/62 IHC Diaz et al. (1991) ER 20/30 23/30 11/20 IHC Hiasa et al. (1993) ER 23/39 44/130 19/115 7/23 0/6 IHC Yane et al. (1994) ER 5/27 (in all neoplastic RT-PCR lesions) Jaklic et al. (1995) ER 0/5 0/1 0/4 IHC Dalla Valle et al. (1998) ER 6/7 12/12 26/26 1/1 1/1 RT-PCR Lewy-Trenda et al. (1998) ER 0/20 2/19 4/8 3/5 0/4 IHC Arain et al. (2003) ER 0/8 0/9 0/19 0/10 IHC Ceresini et al. (2006) ERa 0/17 0/17 IHC ERb 17/17 14/17 Tavangar et al. (2007) ER 31/130 8/37 37/119 2/18 0/12 IHC Vaiman et al. (2010a,b)ERa 0/150 0/34 0/90 0/6 0/5 IHC ERb 126/150 30/34 60/90 4/6 3/5 Huang et al. (2014)a ERa 2/30 30/50 IHC ERb1 28/30 48/50

IHC, immunohistochemical assay; RBA, radioligand-binding assay; SDG, sucrose density gradient assay. aTissues from female patients of reproductive age.

the patterns of ERa and ERb expression in thyroid postulated by Chen and coworkers for thyroid cancer (Chen carcinoma tissues are widely varying. Some authors did et al.2008, Sotoca et al.2008).This hypothesis states that not observe ERa immunostaining in thyroid adenomas, higher expression of ERa enhances cell proliferation, goiters, and papillary, follicular, and anaplastic thyroid whereas higher expression of ERb promotes differentiation carcinomas, but ERb immunoreactivity in all benign and and induces cell apoptosis. Evidence for this concept came Endocrine-Related Cancer malignant thyroid samples (Ceresini et al. 2006, Vaiman from studies with specific agonists, which showed that et al. 2010a,b). In contrast, others reported an increased selective stimulation of ERa increased thyroid carcinoma expression of ERa in PTC tissues in comparison to low or growth and expression of anti-apoptotic BCL2 protein, absent expression in normal thyroid tissues (Chen et al. while selective stimulation of ERb decreased proliferation 2008, Kansakar et al. 2009). Again, other immunohisto- rate and enhanced expression of apoptotic BAX protein chemical studies showed similar ERa staining in normal (Zeng et al.2007). Silencing with selective siRNAs of either thyroid and tumor tissues (Hampl et al. 1985, Vaiman et al. ERa or ERb showed contrary effects and thus confirmed 2010a,b). Recently, in papillary thyroid carcinomas, over- the stimulation experiments. Very recently, a study of 89 expression of ERa but lack of ERa expression in the females with PTC, which correlated ER expression with surrounding tissue and absence of ERb expression in the tumor size and proliferation marker Ki-67, reaffirmed the tumor samples have been described (Di Vito et al. 2011). role of ERa in growth stimulation of these tumors and, on the These authors used the sensitive technique of laser capture other hand, an inhibitory effect of ERb (Huang et al.2014). microdissection to isolate homogeneous cell population from PTC surgical samples to analyze ERa and ERb expression. Effect of estrogen on benign and In this context, it is of special interest that undiffer- malignant thyroid cells entiated thyroid stem and progenitor cells that are widely Estrogen and growth of benign and believed to be the origin of thyroid tumors predominantly malignant thyroid cells express ERa with eightfold higher expression levels than differentiated thyroid cells (Xu et al. 2013). A direct growth-stimulatory effect of estrogen was first As suggested for breast cancer, an imbalance between demonstrated in the differentiated FRTL-5 rat thyroid cell ERa and ERb expression with overexpression of ERa was also line (Furlanetto et al. 1999). These cells express functional

ERs and enhance DNA synthesis and proliferation in Receptor E2 tyrosine E2 kinase response to E2 stimulation. Similar results were obtained RET/PTC in thyroid cells derived from goiter nodules and in human mER RAS TRK thyroid carcinoma cells (Manole et al. 2001, Zeng et al. mutation c-Src Adaptor RAS PI3K 2007, Kumar et al. 2010, Rajoria et al. 2010). E2-stimulated BRAF mutation cell growth was associated with increased expression of PTEN BRAF α β cyclin D1 protein, which plays a key role in the regulation ER ER MAPK PI3K–AKT pathway AKT pathway of the G1/S transition in the cell cycle (Motokura & Arnold MEK 1993, Manole et al. 2001). CCND1, whose overexpression mTOR has been repeatedly reported in thyroid cancer, has an ERE ERK

in the promoter region of its gene (Altucci et al. 1996, Farid β ER P 1996). TFF ERE β TF ERα α In benign and malignant thyroid cells, E2 via its ER membrane-bound receptor mER also stimulated activation of the MAP kinase signaling pathway through phosphorylation of extracellular receptor kinases 1 and 2, whose Figure 1 Estrogen-dependent genomic and non-genomic signaling pathways. activity in thyroid carcinoma cells is mainly controlled The genomic mode of action is mediated through ERa and ERb, which are by growth factors (Manole et al. 2001, Kumar et al. members of a large family of nuclear transcription factors. For activation, estrogen has to enter the cell where it binds to its receptors ERa and ERb, 2010, Rajoria et al. 2010). In addition, through mER, E2 which is followed by nuclear translocation, homo- or hetero-dimerization activated the phosphatidylinositol 3-kinase (PI3K) signal- of the estrogen–ER complex, and its binding to an estrogen-responsive ing pathway (Kumar et al. 2010, Saji & Ringel 2010). element (ERE) in the promoter region of different target genes (Nilsson Both the MAPK and the PI3K pathways are critical et al. 2001). For the transactivation of genes, a process of recruitment and binding of different coactivators is mandatory (left). The non-genomic for proliferation and propagation of thyroid cancer. signaling of E2 occurs via the membrane-bound receptor mER, which Inhibition of both signaling pathways prevents estrogen- stimulates activation of the MAP kinase and the PI3 kinase signaling induced mitogenesis. pathways, whose activity in thyroid carcinoma cells is mainly controlled by growth factors (right). In PTC with aberrant activation of these tyrosine kinase pathways by chromosomal rearrangement of the tyrosine kinase receptor TRKA,byRET/PTC genes, or by a BRAF mutation (or a RAS Estrogen upregulates ERa expression mutation in FTCs), E2 synergistically stimulates these pathways (see text). Endocrine-Related Cancer In addition, in thyroid cancer, the PI3K/Akt pathway may also be activated For the promotion of thyroid adenomas, goiter, and by mutational inactivation of PTEN, a tumor-suppressor gene, or decreased/ thyroid cancer in females, the effect of estrogen on its absent expression of its mRNA or protein (Bruni et al. 2000). receptors is of clinical relevance. Indeed, it is well documented in benign and thyroid carcinoma cells that Thus, estrogen-dependent signaling targets the mitogenic

E2 stimulates growth and ampliﬁes its growth-promoting pathways that are critical for growth regulation of thyroid effect by upregulation of ERa (Manole et al. 2001, Vivacqua cancer (Fig. 1). et al. 2006). By contrast, ERb expression is not affected by E stimulation. 2 Estrogen and thyroid stem cells

There is increasing evidence that mutated thyroid stem Estrogen and other mitogenic pathways of the thyroid cells or progenitor cells rather than primary thyroid cells In the thyroid gland, there are 3 distinct mitogenic are the origin of thyroid cancer (Derwahl 2011). Support for pathways operative: i) the hormone receptor–Gs–adenylyl this concept comes from the ﬁnding that normal differ- cyclase–cAMP-dependent protein kinase system, ii) the entiated thyrocytes divide only ﬁve times during adult hormone receptor–tyrosine protein kinase pathways, and lifetime with a mean turnover rate of 8.5 years (Dumont iii) the hormone receptor–Gq–phospholipase C cascade et al. 1992). Cell division is, however, a prerequisite for (Dumont et al. 1992). In thyroid papillary cancer, the the accumulation of mutations and/or other genetic and tyrosine kinase signaling pathways, Ras/Raf/MAPK and epigenetic changes and in turn the generation of a tumor. PI3K–Akt, which are also stimulated by estrogen via mER, Indeed, cancer occurs more frequently in organs with a are activated by chromosomal rearrangements of the higher cellular turnover such as the colon. tyrosine kinase receptor TRKA, by RET/PTC genes, and As an alternative source for the generation of tumors by a BRAFV600E mutation (Ciampi & Nikiforov 2007). including thyroid cancer, stem and progenitor cells have

been proposed (Davies et al. 2011, Derwahl 2011). Stem uptake (Furlanetto et al.1999, 2001). In contrast, cells are undifferentiated cells that possess the capacity of expression of thyroglobulin was enhanced in response to

self-renewal and the generation of more differentiated E2 stimulation (del Senno et al. 1989).

progenitor cells (Davies et al. 2011). These cells that reside In thyroid-progenitor-cell-derived thyroid nodules, E2 in any organ and tissue for the lifetime of the organism decreased the expression of TSH-induced differentiation have been detected in the human thyroid gland and in markers such as TPO, TSHR, and NIS mRNAs (Xu et al.

goiters and cancer stem cells in thyroid cancer cell lines 2013). The most pronounced effect of E2 was observed on and thyroid cancer tissues (Thomas et al. 2006, Lan et al. NIS expression. This result is in accordance with the report 2007, Mitsutake et al. 2007, Fierabracci et al. 2008, Todaro on primary FRTL-5 cells mentioned above. Thyroglobulin

et al. 2010, Zheng et al. 2010, Malaguarnera et al. 2011). expression was not affected by E2 stimulation in thyroid Proliferation of stem cells is regulated by niches, progenitor cells. signals from the surrounding microenvironment, which prevent the undifferentiated cells from undergoing ERs and hypoxia and inflammation uncontrolled cell division (Rezza et al.2014). Under certain conditions, e.g., induction of apoptosis, and Owing to insufficient neovascularization, necrosis is intense stimulation with hormones and growth factors, frequently observed in solid tumors. The resulting hypoxia the strict control of niche may be overcome, resulting in is critical for the progress of the tumor (Bertout et al. 2008). proliferation of stem cells. Owing to their longevity, stem Under this condition, the hypoxia-inducible factor 1 cells are prone to acquiring mutations and other molecu- (HIF1), a transcription factor, induces the transcription lar aberrations (Dick 2008). of several genes involved in angiogenesis (e.g., vascular In differentiated thyroid cells and thyroid cancer cells, endothelial growth factor (VEGFA)), glucose metabolism, it has been demonstrated that apoptotic stimuli can apoptosis resistance, inflammation, invasion, and metas- overcome the control of niches, which in vitro in response tasis (Semenza 2012). to intense stimulation with growth factors results in the HIF1 is overexpressed in thyroid cancer with the outgrowth of thyroid stem and progenitor cells as highest expression in therapy-resistant, undifferentiated spheroids, designated thyrospheres, in vitro (Lan et al. thyroid carcinomas (Burrows et al. 2010). Interestingly, 2007, Fierabracci et al. 2008, Zheng et al. 2010). HIF1a also regulates ERa expression (Tafani et al. 2011). Very recently, it has been reported that thyroid stem Lymphocytic infiltrations are frequently observed in Endocrine-Related Cancer and progenitor cells are also a target of estrogen action (Xu thyroid cancer (Guarino et al. 2010). Both HIT1a and et al. 2013). Thyroid stem and progenitor cells derived nuclear factor kB(NFkB), a transcription factor that from thyroid nodules express ERa and ERb with eight coordinates immune and inflammation processes, have times higher expression levels of ERa compared with very recently been demonstrated to upregulate pro- normal thyroid cells (Xu et al. 2013). This high ERa inflammatory genes that play a key role in the progression expression in undifferentiated stem/progenitor cells is of transformed thyroid cells to a malignant phenotype reminiscent of the above-mentioned overexpression of (Tafani et al. 2013). Based on the links between ERa, ERa in some papillary thyroid carcinomas (Di Vito et al. hypoxia (HIF1a activation), and inflammation (NFkB 2011). High expression levels of ERa in thyroid stem/pro- activation), a concept has very recently been proposed genitor cells were further upregulated by stimulation with that postulates a major role for the interaction between

E2, which is in accordance with ﬁndings for human benign ERa and the two transcription factors in the progression of and malignant thyroid cells (Manole et al. 2001, Xu et al. thyroid cancer (Tafani et al. 2013). 2013). A predominant expression of ERa was also reported in stem and progenitor cells derived from other tissues ER expression and outcome of thyroid cancer such as prostate (Hu et al. 2011). Clinical proof for the concept that ERa promotes proliferation while ERb mediates differentiation came Estrogen and thyroid function from studies of breast cancer, ovarian cancer, and lung There is little known about the effect of estrogen on cancer, which demonstrated that low ERb expression is

thyroid function. In the FRTL-5 rat thyroid cell line, E2 associated with a poor outcome of these tumors (Omoto downregulated expression of the sodium–iodide sympor- et al. 2001, Mauro et al. 2010, Halon et al. 2011). In general, ter (NIS) and consecutively inhibited radioactive iodine ERb is believed to play a protective role in carcinogenesis

in that it mediates anti-proliferative and pro-apoptotic believed to promote growth of benign and malignant signals and thus counteracts ERa-induced cell prolifer- nodules (Kimura et al. 1990). However, there are only a ation (Gustafsson & Warner 2000, Satake et al. 2006, few studies that have assessed the outcome of thyroid Rajoria et al. 2010).There is some evidence that loss of cancer, the overall survival, and the DTC-related death ERb expression is a common step in estrogen-dependent in pregnancy (recently reviewed by Alves et al. 2011). tumor progression (Bardin et al. 2004). Remarkably, no differences in survival, recurrence, or Although for thyroid tumors, as mentioned above, data death were observed between pregnancy-associated on expression of ER isoforms are, at least in part, contra- thyroid cancer and cancer in aged-matched non- dictory, some recent studies have been in accordance with pregnant women (Herzon et al. 1994, Moosa & Mazza- the findings for other carcinomas that low or even loss of ferri 1997, Yasmeen et al. 2005). ERb expression is also a hallmark of thyroid carcinomas By contrast, a more recent study has found a with a poor prognosis. In a very recent study with follicular significantly higher rate of recurrent or persistent disease thyroid adenomas (FTAs) and FTCs, ERb expression was in pregnant women (Vannucchi et al. 2010). These authors significantly lower in FTCs than in FTAs (Heikkila et al. demonstrated a higher expression of ERa immuno- 2013). This lower level of expression was correlated with a reactivity in tumor samples from pregnant women than b poor clinical outcome. Interestingly, ER expression was a in those from controls. Two recent studies have confirmed stronger prognostic marker than expression of the Ki-67 the clinical outcome of this study but not the increased proliferation marker routinely used in the histological ERa expression: Shindo et al. (2014) reported on the diagnosis of thyroid cancer (Tallini et al. 1999). Moreover, enlargement of a papillary thyroid microcarcinoma in four the transition from FTAs to FTCs seemed to go along with out of nine pregnant patients (44%). However, in all three the loss of ESR2 expression (Heikkila et al. 2013). This fits patients who underwent surgery ER expression was not well with the lack of ERb expression in papillary carcinomas detectable. In the other study, which included 38 and the finding of ERb negativity in some small differ- pregnant patients, DTC persistence and recurrence were entiated thyroid carcinomas with a more aggressive significantly higher in pregnant than in non-pregnant phenotype (Di Vito et al. 2011, Magri et al. 2012). Lower women (Messuti et al. 2014). But, no differences among expression of ERb was also described in undifferentiated the groups were observed regarding ER expression thyroid stem and progenitor cells when compared with patterns, NIS expression, and BRAF mutations. The results differentiated human thyrocytes (Xu et al. 2013). Thus, Endocrine-Related Cancer of the two studies do not indicate a role of ER expression lower or even loss of ERb expression that maintains cell in thyroid cancer during pregnancy. differentiation and the epithelial phenotype may also be a hallmark of dedifferentiation in thyroid cancer as in other carcinomas (Thomas & Gustafsson 2011). ERs and epigenetic regulation in thyroid cancer The considerably differing expression patterns of ERa The reasons for the absence of ERa expression in some make it difficult to define its role in the pathogenesis of thyroid cancer. However, there is no doubt that estrogen is a immunohistochemical studies of differentiated and undif- potent growth factor for benign and malignant thyroid cells ferentiated thyroid cancers or its restriction to few samples and that proliferation is mediated via ERa-dependent remain unclear. signaling (Manole et al. 2001, Lee et al.2005, Chen et al.2008). A most likely explanation is that ER and ER-targeted This assumption is also supported by the findings that genes in thyroid tumors are also controlled by epi- ERa positivity was associated with a more aggressive genetic pathways, as has demonstrated for other cancers phenotype of differentiated thyroid cancer and of an eight including breast cancer (recently reviewed by Hervouet to ten times higher expression of ERa in undifferentiated, et al. 2013). Epigenetic pathways regulate gene trans- growth-prone thyroid stem and progenitor cells than in cription by DNA methylation and post-translational differentiated thyrocytes (Magri et al.2012, Xu et al.2013). modification of histones. DNA methylation may silence the promoter of the ESR1 gene that encodes ERa, which occurs in the pathogenesis of different cancers (Yao et al. ER expression and outcome of thyroid cancer in 2010, Wei et al. 2012). In breast cancer, DNA hyper- pregnancy methylation is a major reason for the loss of ERa During pregnancy, iodine deficiency, the TSH receptor- expression and confers a poor prognosis to this tumor stimulating effect of hCG and high estrogen levels are (Ramos et al. 2010).

Estrogen and angiogenesis in thyroid cancer (Todaro et al. 2010). This indicates the presence of both cancer stem cells and metastatic cancer stem cells in Neovascularization that increases the local blood supply is undifferentiated thyroid cancer (Derwahl 2011). There is a prerequisite for any growing tumor. While estrogen and some evidence that a small subpopulation of cancer stem other growth factors stimulate angiogenesis in endothelial cells is critical for metastatic colonization, the initial cells, inhibition of ER results in decreased angiogenesis expansion of cancer cells at the secondary site (Malanchi (Losordo & Isner 2001). et al. 2011). Whether or not estrogen is involved in the VEGF has the capacity to induce angiogenesis by growth regulation of metastatic cancer stem cells is still detachment of quiescent endothelial cells from their unknown. parental vessels followed by migration of the cells into the neighboring stroma (Krock et al. 2011). In vitro thyroid cancer cells secreted VEGF in response to estrogen Concluding remarks treatment (Kamat et al. 2011). Incubation of human Estrogen and its receptors play a pivotal role in the umbilical vein endothelial cells with a conditioned pathogenesis and progression of cancers in females, medium from estrogen-treated cancer cells resulted in particularly breast cancer. In contrast, in thyroid cancer, angiogenesis-associated eventssuchastubulogenesis there is still a substantial gap in understanding the and migration. signiﬁcance of estrogen and ERs in the pathogenesis and VEGF expression or its overexpression has been progression of this tumor. reported in samples from differentiated, anaplastic, and Certainly, epidemiological and clinical studies metastatic thyroid carcinomas (Viglietto et al. 1995, Klein demonstrated a three to four times higher prevalence of et al. 1999). In comparison with normal thyroid cells, differentiated thyroid cancer in females than in males and higher TSH-stimulated VEGF expression and secretion an increase in the incidence of this tumor with the onset were reported for thyroid carcinoma cells (Soh et al. 1996, of puberty in females is well documented (Farahati et al. 1997). Remarkably, in tumor tissues, the number of blood 1997, Dean & Gharib 2008, Li et al. 2013). Furthermore, vessels and the tumor size were directly related to the there is some recent evidence for the progression of VEGF concentrations (Soh et al. 1997, Fenton et al. 2000). papillary microcarcinoma during pregnancy and a higher In contrast, others described higher VEGF expression in recurrence rate of thyroid cancer due to pregnancy the surrounding tissues than in cancer tissue (Kansakar (Messuti et al. 2014, Shindo et al. 2014). Results from all Endocrine-Related Cancer et al. 2009). these studies have indicated that estrogen and its receptors are relevant to the regulation of thyroid cancer growth. Estrogen and metastasis of thyroid cancer In fact, results from in vitro stimulation experiments unanimously support this view. As outlined above, In breast cancer, it was demonstrated that the three however, in contrast to these data, there are numerous, different steps of metastasis, adhesion, invasion, and in part, contradictory results showing widely varying ERa migration, are controlled by estrogen (Malek et al. 2006, and ERb expression patterns, loss of expression of one or Planas-Silva & Waltz 2007, Baranwal & Alahari 2009). the other form or overexpression of one of them in thyroid Analogously, through downregulation of b-catenin, E2 cancer tissues. also affected the metastatic phenotype of thyroid cancer To clarify these apparent contradictions, it must be cells by enhancing their adhesion, migration, and kept in mind that the current state of expression patterns invasiveness (Rajoria et al.2010). Downregulation of of a receptor may not allow conclusions to be drawn proteins of the cadherin–catenin complexes correlated regarding its role in the pathogenesis of a tumor. This is with increased proliferation, adhesion, migration, and very well known for TSH and its receptor, whose invasion of cancer cells in vitro and in vivo (Cheng et al. expression is decreased or may even be absent in thyroid 2008, Kalluri & Weinberg 2009). cancer. Nevertheless, TSH is a major regulator of thyroid In breast cancer, estrogen expanded cancer stem cells growth and also involved in the regulation of thyroid and enhanced their potential for tumor sphere formation cancer proliferation, which justiﬁes a TSH-suppressive (Fillmore et al. 2010). Interestingly, when cancer-stem- treatment in patients with differentiated thyroid cancer. cell-enriched thyrospheres derived from an undifferen- In addition, the high complexity of ER expression and tiated, metastatic thyroid cancer were transplanted onto variants, with multiple phosphorylation sites and different nude mice, a metastatic disease was generated in the mice subcellular localizations, their probable interactions with

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Received in ﬁnal form 25 June 2014 Accepted 21 July 2014 Made available online as an Accepted Preprint 22 July 2014 Endocrine-Related Cancer