Article in press - uncorrected proof Horm Mol Biol Clin Invest 2011;5(2):79–89
2011 by Walter de Gruyter • Berlin • New York. DOI 10.1515/HMBCI.2010.045

The thyroid hormone receptors as tumor suppressors

Lidia Ruiz-Llorente, Olaia Martı´nez-Iglesias, Susana Garcı´a-Silva, Stephan Tenbaum, Javier Regadera and Ana Aranda* Instituto de Investigaciones Biome´dicas ‘‘Alberto Sols’’, Consejo Superior de Investigaciones Cientı´ficas and Universidad Auto´noma de Madrid, Spain

Abstract In addition to the well-known role of the thyroid hormone receptors (TRs) in growth, development and metabolism, there is increasing evidence that they have profound effects on cell proliferation and malignant transformation. TRs repress transcriptional induction of cyclin D1 by the ras oncogene and block transformation and tumor formation by Ras-transformed fibroblasts in nude mice. Mutant receptors that do not bind coactivators are able to display these actions, whereas receptors defective in corepressors binding are unable to antagonize the responses to the ras oncogene. Furthermore, expression of TRb1 in hepatocarcinoma and breast cancer cells abolishes anchorage-independent growth and migration, blocks responses to growth factors and represses expression of prometastatic genes, reducing tumor growth and strongly inhibiting invasiveness, extravasation and metastasis formation in euthyroid mice. By contrast, when cells are inoculated into hypothyroid host, tumor growth is retarded, but tumors are more invasive and metastatic growth is enhanced. Increased aggressiveness and tumor growth retardation was also observed with parental cells that do not express TRs, showing that changes secondary to hypothyroidism can modulate tumor progression and metastatic growth independently of the presence of TRs on the tumor cells. Finally, increased malignancy of skin tumors is found in mice lacking TRs, further demonstrating the role of these receptors as inhibitors of tumor progression and suggesting that they represent a potential therapeutic target in cancer. Keywords: cell proliferation; hypothyroidism; thyroid hormone receptors; tumor progression.

Transcriptional regulation by the thyroid hormone receptors The thyroid hormones (thyroxine, T4 and triiodothyronine, T3) are important regulators of growth, development and *Corresponding author: Ana Aranda, Instituto de Investigaciones Biome´dicas, CSIC-UAM, Arturo 4, 28029 Madrid, Spain Phone: q34-91-5854453, Fax: q34-91-5854401, E-mail: [email protected] Received September 6, 2010; accepted September 8, 2010; previously published online March 4, 2011

metabolism in higher animals and man. The actions of these hormones are initiated by binding to nuclear thyroid hormone receptors (TRs), the cellular counterparts of the retroviral v-erbA oncogene, which act as ligand-dependent transcription factors (1). TRs are encoded by two genes, TRa and TRb, located on human chromosomes 17 and 3, respectively. Primary transcripts of these genes undergo alternative processing generating several protein isoforms, among which TRa1, TRb1 and TRb2 are the main hormone-binding isoforms. TRs share a common structure with other nuclear receptors, displaying a modular structure with several regions: an N-terminal region (A/B), a conserved DNA binding domain (DBD) or region C composed of two zinc fingers responsible for DNA binding and sequence-specific recognition, a hinge region D that contains residues essential for interaction with corepressors, and a conserved E region containing the ligand binding domain (LBD). Transcriptional regulation by the receptors is achieved through autonomous activation functions (AFs): a constitutive N-terminal AF-1 in the A/B region and a ligand-dependent AF-2 located in the receptor C-terminal a helix (helix 12) of the LBD (2). TRs normally regulate gene expression by binding, preferentially as heterodimers with the retinoid X receptors, to specific DNA sequences (thyroid hormone response elements or TREs) located in regulatory regions of target genes (3) (Figure 1). The actions of the nuclear receptors on transcription are mediated by the recruitment of coregulators, coactivators and corepressors. Unliganded TRs can act as strong constitutive repressors when bound to TREs, owing to the binding of corepressors such as NCoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoic and thyroid receptor). NCoR and SMRT are large modular proteins that serve as platforms for the formation of multicomponent repressor complexes that contain histone deacetylases and cause chromatin compactation (4, 5). Ligand binding induces a conformational change in the receptor that leads to the release of corepressors and enables the receptor to recruit in a sequential manner multiple coactivator complexes. The main change induced by hormone binding is the repositioning of helix 12 of the LBD that contains the AF-2 domain. Residues in this helix together with residues in H3, 4 and 5 form a hydrophobic groove that accommodates the coactivator receptor interacting domain (6). A conserved glutamic acid residue in H12 and an invariable lysine residue in H3 contact directly with the coactivator and form a charge clamp that stabilizes binding. Accordingly, mutations in these residues render a receptor unable to recruit coactivators and to mediate T3-dependent transcriptional activation. Indeed, some patients suffer a thyroid hormone resistance syndrome (THR) as a consequence of a point mutation in H12 in TRb (7). Some coactivators are chromatin remodeling factors or possess histone modifying activity such as acetylation or arginine methylation, whereas others interact with the basic

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Figure 1 Transcriptional regulation by the thyroid hormone receptors. Thyroid hormones, thyroxine (T4) and triiodothyronine (T3) enter the cell through transporter proteins. Although the major form of thyroid hormone in the blood is T4, it is converted to the more active T3 within cells by 59-deiodinases. T3 binds to nuclear thyroid hormone receptors (TRs) that regulate transcription by binding, generally as heterodimers with the retinoid X receptor (RXR), to thyroid hormone response elements (TREs) located in regulatory regions of target genes. Activity is regulated by an exchange of corepressor (CoR) and coactivator (CoA) complexes. TRs can also transrepress the activity of genes that do not contain a TRE through ‘‘crosstalk’’ with other transcription factors (TFs) that stimulate target gene expression.

transcriptional machinery and can recruit the RNA Polymerase II to the target promoter. Recruitment of coactivators causes chromatin decompactation and transcriptional stimulation. The nuclear receptors can also regulate the expression of genes that do not contain a hormone response element by positive or negative interference with the activity of other transcription factors or signaling pathways, a mechanism referred to as transcriptional crosstalk (2). In this case, the receptors do not bind directly to the DNA recognition motifs for those transcription factors, but can be tethered to these elements of the target promoter via protein-to-protein interactions (Figure 1). Thus, we have shown that TRs can antagonize AP-1 (8, 9), cyclic AMP (cAMP) response element-binding protein (CREB) (10, 11) or NF-kB-mediated transcription (12, 13). Functional crosstalk between nuclear receptors and these transcription factors has been reported for various classes of receptors (14) and has been shown to be crucial for regulation of many cellular functions (15, 16).

The thyroid hormone receptor antagonizes induction of proliferation and cyclin D1 transcription by the ras oncogene Members of the ras family encode small GTP-binding proteins that play a key role in normal and malignant cell growth. Oncogenic mutations in ras result in a constitutively active protein that is present in at least 30% of human tumors and can efficiently transform most immortalized rodent cell

lines such as the NIH-3T3 fibroblasts (17). Several downstream pathways are initiated after Ras activation, among them the phosphatidylinositol-3-OH kinase (PI3K) pathway, involved in cell survival, and the Ras/mitogen-activated protein kinase (MAPK) signaling pathway, an important mediator for mitogenic signaling and Ras-induced transformation (18, 19). In the MAPK pathway, activation of the MAPK extracellular signal-regulated kinase 1/2 (Erk1/2) permits its translocation to the nucleus, where it regulates gene expression via the direct phosphorylation of transcription factors, for instance, factors or the Ets family, or by activation of downstream kinases such as Rsk or Msk (20, 21), which then phosphorylate and activate, among other substrates, b-Zip transcription factors of the CREB/activation transcription factor 2 (ATF-2) family. These transcription factors contain a kinase-inducible domain necessary for activation in response to external stimuli. A serine residue in this domain can be phosphorylated in response to multiple kinases, which are activated in response to different signaling pathways (22). Cyclin D1 plays an important role on cell cycle progression and is one of the main targets for the proliferative and transforming effects of ras oncogene. It has been shown that ras-induced tumorigenesis depends on signaling pathways that act preferentially through Cyclin D1, and multiple effector pathways and promoter elements can contribute to cyclin D1 transcription in response to Ras activation (23). Our group has demonstrated the existence of a crosstalk between TRs and ras signaling pathways. In N2a neuroblastoma cells, which express TRb1 (N2a-b cells), T3 blocks

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proliferation and induces morphological differentiation. We demonstrated that T3 coordinately regulates the expression of several genes that play a key role in cell cycle control (24). An element in the region responsible for premature termination of transcription mediates a rapid repression of c-myc gene expression by T3 (25). The hormone also causes a decrease of Cyclin D1 and a strong and sustained increase of the levels of the cyclin kinase inhibitors p27Kip1 and p21Cip is found in T3-treated cells (26). The increased levels of p27Kip1 lead to a marked inhibition of the kinase activity of the cyclin/CDK2 complexes. As a consequence of these changes, retinoblastoma proteins are hypophosphorylated in T3-treated N2a-b cells and progression through the restriction point in the cell cycle is blocked. In N2a-b cells expression of oncogenic Ras increases proliferation and incubation with T3 inhibits the proliferative response to ras. The oncogene causes a significant increase of Cyclin D1 levels and incubation with T3 markedly inhibits induction of Cyclin D1 expression by oncogenic ras. Furthermore, overexpression of Cyclin D1 reverses to a significant extent the inhibitory effect of T3, demonstrating that the reduction of Cyclin D1 levels by T3 is an important component of the mechanism by which the hormone antagonizes ras-mediated proliferation (27). Transcriptional activation of the cyclin D1 gene by mitogenic signals and oncogenes such as ras and v-src can be mediated by multiple cis elements, including AP-1, Sp-1 and CRE sites (28). Our results show that a CRE located at the proximal cyclin D1 promoter is the main acceptor for Cyclin D1 induction by oncogenic ras in neuroblastoma and hepatocarcinoma cells (27, 29). This element binds CREB and ATF-2, and their transcriptional activity is markedly enhanced upon expression of Ha-rasval12. Incubation with T3 strongly antagonizes activation of the cyclin D1 promoter by oncogenic ras N2a-b cells. The hormone also blocks induction of cyclin D1 promoter activity by ras in human hepatocarcinoma cells or murine fibroblasts transfected with TRs, and in rat pituitary cells which express high endogenous TR levels, indicating that T3-dependent repression or Ras-mediated transcription is not a specific effect on neuroblastoma cells and can be extended to other cell types. Src also stimulates transcription of this gene in a Ras-dependent manner and T3 also antagonizes the transcriptional response to this oncoprotein. T3 represses expression of the cyclin D1 gene in response to the ras oncogene through promoter sequences that do not contain a TRE by interference with the activity of the MAPK pathway and CRE-mediated transcription. In addition to antagonization of the MAPK pathway and the activation of downstream kinases such as Rsk2 or Msk, TRs can interact directly with ATF-2 and with CREB inhibiting its phosphorylation (10). The CRE constitutively binds b-Zip factors and, accordingly, neither Ras nor the receptor alters the abundance of the factors that bind this motif. However, transcriptional activation of CREB and ATF-2 by ras is blocked by the TR in a T3-dependent manner (Figure 2). As described by the Cheng group (30), deregulation of Cyclin D1 expression by TRs also appears to play an important role in the development of thyrotropin-secreting pituitary

Figure 2 Model of transrepression of the cyclin D1 gene by the thyroid hormone receptors. Oncogenic Ras stimulates the MAPK pathway that leads to phosphorylation of b-Zip transcription factors such as CREB or ATF-2 that bind to a cyclic AMP response element (CRE) located in the proximal cyclin D1 promoter. The liganded thyroid hormone receptor antagonizes activation of the MAPK pathway and interacts with the b-Zip factors blocking their activation by Ras.

adenomas (TSHomas) in a knock-in mouse model harboring a C-terminal 14 amino acid frameshift mutation (PV mutation) of TRb. This mutant receptor that was initially identified in a patient with THR syndrome has lost T3-binding and transcriptional activity and acts as a strong dominant-negative of the native receptor. TRbPV/PV mice spontaneously develop follicular thyroid carcinoma and pituitary tumors (31). In these tumors, Cyclin D1 is overexpressed and the retinoblastoma protein (Rb) is hyperphosphorylated. Liganded TRb represses cyclin D1 transcription in the pituitaries through the CRE and is recruited to the promoter via interaction with CREB transcription factors. This repressive effect is lost in TRbPV, thereby resulting in constitutive activation of Cyclin D1 expression and leading to aberrant proliferation of thyrotropes in the mutant mice. To analyze the receptor domains responsible for the antagonism of ras-induced transcription, we examined the effect of mutations in TRb1 on cyclin D1 promoter activity in hepatocarcinoma HepG2 cells and murine NIH-3T3 fibroblasts (Figure 3). Interestingly, mutants E457Q in H12 or K288I in H3 still presented a significant activity to antagonize the stimulation of the cyclin D1 promoter by rasval12 or to reduce Cyclin D1 protein levels. This indicates the dispensability of the charge clamp between H3 and H12 for inhibition of Ras responses by the receptor, although the clamp is crucial for coactivators recruitment and liganddependent transactivation. The finding that association with classical coactivators appears to be dispensable for the antagonism of Ras-mediated responses by TRb1 supports a model wherein receptor-mediated antagonism is not intrinsically linked to activation and that both can be separated mechanistically. TRb1 mutants in the hinge domain were used to analyze the role of corepressors on this antagonism. A receptor containing a triple point mutation AHT-GGA in the so-called CoR box located in H1 that abolishes interaction

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Figure 3 Thyroid hormone receptor domains involved in blocking the responses to the ras oncogene. The upper part of the figure presents a scheme of the TRb1 isoform showing the receptor functional domains and different mutations. These mutants were used to analyze T3dependent in vitro recruitment of coactivators and corepressors in ‘‘pull-down’’ assays, transactivation of a reporter plasmid containing a consensus TRE, transrepression of the response of the cyclin D1 promoter to Ha-rasval12, and inhibition of formation of transformation foci in NIH-3T3 fibroblasts transfected with the oncogene.

with corepressors (32) and ligand-independent repression on a TRE was unable to repress cyclin D1 promoter induction by Ha-rasval12, suggesting that binding of corepressors to TRb1 is required for transcriptional antagonism. In addition, mutation P214R in a residue preceding H1, homologous to that present in a v-erbA transformation deficient mutant (td359) (33), also blocks interaction with corepressors (34) and lacks the ability to antagonize Ras-mediated transcription in a T3-dependent manner (Figure 3). The hinge TRb1 mutants that were unable to mediate transrepression of the cyclin D1 promoter also lost the ability of antagonizing ATF2 activation by the oncogene, favoring again the hypothesis that corepressors play a role in the transrepressing effects of the receptor. This is supported by the finding that overexpression of NCoR or SMRT further increases T3-dependent transrepression of cyclin D1 transcription (29). The finding that both corepressor mutants are still able to mediate TREdependent transcription again demonstrated that Ras antagonism could be mechanistically separated from classical ligand-dependent transactivation.

transforming activity of the receptor, we also examined the influence of overexpression of this protein on foci formation. The results obtained demonstrated that the antitransforming effect of TRb1 was lost in cells transfected with cyclin D1, indicating its involvement not only in the antiproliferative but also in the antitransforming effects of the receptor (27).

The thyroid hormone receptors antagonize transformation and tumorigenesis by oncogenic Ras Because TRs display a strong repressive activity of ras-mediated proliferation and transcriptional responses, we also explored the possibility that these receptors could inhibit rasmediated cellular transformation. To prove this hypothesis, we performed foci formation assays with NIH-3T3 fibroblasts transfected with Ha-rasval12 in the presence or absence of TRs. The results obtained showed that the transforming ability of the ras oncogene is greatly reduced in cells coexpressing TRs, although TRb1 appears to have a stronger antitransforming effect than the a1 isoform (Figure 4A). TRs are also able to block fibroblast transformation by v-src (27). To analyze whether Cyclin D1 levels can modulate the anti-

Figure 4 The thyroid hormone receptor antagonizes transformation and tumorigenesis by the ras oncogene. (A) Transformation assays in NIH-3T3 cells transfected with Ha-rasval12 alone or in combination with TRa1 or TRb1. Formation of transformation foci was scored in cells treated for 14 days in the absence and presence of T3. (B) Immunodeficient nude mice were injected into both flanks with NIH-3T3 fibroblasts expressing Ha-rasval12 and TRa1 or TRb1 in a stable manner. Photographs were taken 25 days after inoculation.

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Foci formation assays in NIH-3T3 fibroblasts showed a strict parallelism between repression of Ha-rasval12-induced transcription and inhibition of transformation by the TRb1 mutants. The number of transformation foci was greatly reduced by T3 in fibroblasts coexpressing the oncogene and the native receptor (Figure 4A), whereas the AHT and P214R receptors that do not bind corepressors were unable to mediate ligand-dependent antitransforming effects. Furthermore, the E457 and K288 mutants that bind T3 but do not recruit coactivators can antagonize Ras-mediated transcription and were able to repress fibroblast transformation by Ha-rasval12 (Figure 3). In addition, the antitransforming effects of T3 are more notorious after cotransfection of TRb1 with expression vectors for SMRT or NCoR, suggesting again that these corepressors participate in the inhibition of the responses to Ha-rasval12 by the receptor. The relevant role of endogenous SMRT and NCoR on the antitransforming effects of TRb1 was demonstrated with siRNA experiments that revealed that corepressor downregulation counteracted to a significant extent the inhibitory effect of T3 on cellular transformation. Furthermore, NCoR knockdown increased foci formation by Ras in the absence of the transfected receptor, indicating that corepressors act to inhibit fibroblast transformation (29). These results are in agreement with the increasing evidence that corepressors influence cellular transformation and tumorigenesis, and that NCoR could be considered as a novel tumor suppressor in the model of metastastic thyroid carcinoma caused by the PV mutant TRb1 in mice (35). By contrast, the oncogenic TR mutants such as v-erbA or PV that act as dominant-negatives blocking the antiproliferative effects of the wild-type receptors do not suppress Ras function although they bind corepressors constitutively, indicating that ligand binding is also required for this receptor action. This is further suggested by the finding that another dominant-negative receptor, a mutant TRa in three conserved lysine residues in the carboxy-terminal extension of the DBD that, unexpectedly, displays a strongly reduced ligand binding affinity, is unable to antagonize transformation by the ras oncogene in a T3-dependent manner (36). To analyze the role of TRs as suppressors of tumor formation by the ras oncogene in vivo, we prepared NIH-3T3 fibroblasts expressing in a stable manner oncogenic Ha-rasval12 alone or in combination with TRa1 or TRb1 and the different transfectants were injected into the flanks of immunodeficient mice (Figure 4B). Whereas large tumors developed in mice injected with fibroblasts expressing Harasval12 alone, no tumors were detected in mice injected with fibroblasts coexpressing the oncoprotein and TRb1. By contrast, coexpression of Ha-rasval12 with TRa1 caused a substantial delay in the appearance of tumors (27). Moreover, although all tumors were aggressive fibrosarcomas, histological analysis demonstrated that the presence of TRa1 conferred a relatively higher degree of tumor differentiation, as shown by an increased presence of collagen and a more fusiform morphology (27). Therefore, TRs could play a relevant role as suppressors of ras-dependent tumors, and although both isoforms suppress tumor growth TRb appears to have stronger antitumorigenic effects in vivo.

To examine the TRb1 domains responsible for the repression of tumor formation by Ha-rasval12, NIH-3T3 cells expressing the oncoprotein were transfected in a stable manner with wild-type TRb1 or with the AHT and E457Q mutants. Tumor formation was strongly repressed in mice injected with fibroblasts coexpressing Ha-rasval12 in combination with either the native TRb1 or the E457 mutant, whereas tumor development was not inhibited by the AHT mutant. These results demonstrate the importance of corepressors and the dispensability of coactivators recruitment not only for the antitransforming effects of the receptor but also for inhibiting tumor formation in mice (Figure 5).

TRb1 acts as a potent suppressor of tumor invasiveness and metastasis Reduced expression of TRs, as well as alterations in TR genes, is a common event in human cancer. These alterations, reviewed in (37, 38), include loss of heterozygosity, gene rearrangements, promoter methylation, aberrant splicing and point mutations. In particular, TRb mutations are frequent in breast cancers, biallelic inactivation of this gene by promoter methylation is found in early stage breast cancer and altered expression and anomalous subcellular localization of both TRa and TRb has been found in these tumors. Aberrant TRs have also been found in more than 70% of human hepatocellular carcinomas and these mutant receptors have been shown to act in general as dominant-negative inhibitors of wild-type TR activity (39) and to display an altered target gene repertoire (40). The tendency for TRb expression to disappear as malignancies progress suggests that TRb can act as a tumor suppressor in human cancers and that therefore loss of expression and/or function of this receptor could result in a selective advantage for cell transformation and tumor development. To analyze the role of TRs in tumor progression and metastatic growth, we reexpressed TRb1 in hepatocarcinoma SK-hep1 (SK) cells and breast cancer MDA-MB-468 (MDA) cells which have lost

Figure 5 TRb1 domains involved in the repression of tumorigenesis by the ras oncogene. Tumor formation was analyzed in nude mice 30 days after inoculation of NIH-3T3 fibroblasts expressing Ha-rasval12 alone or in combination with native TRb1 or with the E457Q or AHT mutants. The results are expressed as the percentage of injections that caused a tumor.

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receptor expression. We generated cells that stably express this receptor (SK-TRb and MDA-TRb, respectively) and these cells, as well as cells infected with the empty vector, were inoculated into nude mice. It was observed that tumor volume was significantly reduced in mice injected with cells that express TRb1 and that these tumors show enhanced expression of epithelial markers, such as keratin 8/18 or bcatenin, and diminished expression of the mesenchymal marker vimentin (41). Therefore, TRb1 seems to facilitate mesenchymal-to-epithelial transition. This could retard tumor progression because epithelial-mesenchymal transition plays a key role in tumor invasion by disrupting intercellular contacts and enhancing motility and migration of tumor cells to the surrounding tissues. In addition, TRb1 increased the necrotic area of the tumors and significantly reduced angiogenesis, also compatible with reduced tumor growth (41). TRb1 also reduced tumor invasiveness. Whereas tumors formed by the parental hepatocarcinoma and breast cancer cells present a diffuse highly infiltrative growth pattern, TRb1-expressing cells caused the appearance of tumors with a more compact structure and surrounded by a pseudocapsule of collagen and inflammatory cells. These tumors do not infiltrate the adjacent tissues such as muscle or lymph and blood vessels (Figure 6A) and they do not originate distant nodular metastasis. To analyze the effect of the receptor in formation of experimental metastasis, parental and TRb1expressing cells were injected into the tail vein of nude mice. Examination of the lungs at necropsy showed that TRb1 had a potent inhibitory effect on metastasis formation (Figure 6B). Whereas most mice inoculated with MDA or SK cells developed metastasis, only 20% of mice injected with TRb1expressing cells had metastatic lesions. Furthermore, large nodular metastases were found in lungs of mice injected with

parental cells, whereas cells that express the receptor at most gave rise to micrometastasis with a pattern of interstitial cells. The number of lesions was also strongly reduced by TRb1 and quantification of parenchymal involvement showed that TRb1 drastically decreased the area of the lungs affected by metastatic growth. When extravasion was analyzed, it could be observed that it was reduced in TRb1-expressing cells, indicating that the receptor has antimetastatic activity by blocking not only the ability of cancer cells to proliferate and colonize the lung parenchyma but also by limiting cancer cell extravasation (41). The ability of cancer cells to survive and proliferate in the absence of a solid substrate is an important component of the acquisition of an invasive and metastatic phenotype. TRb1 blocked colony formation by MDA and SK cells in soft agar and prevented their ability to grow in suspension under rocking conditions. In addition, migration assays through Matrigel showed that receptor expression markedly impaired in vitro invasiveness (41). The inhibitory effect of TRb1 on tumor invasiveness observed in vivo in the mice is compatible with this reduced migration observed in the cultured cells. This could restrain their entry into the circulation, as well as their exit from the bloodstream into the lung parenchyma. Furthermore, the inability to grow in the absence of a solid substrate could decrease survival of TRb1expressing cells in the bloodstream. Autocrine factors, as well as paracrine signals produced by stromal cells, influence proliferation, survival and tumor cell invasiveness. We, therefore, tested if TRb1 could affect the response of cancer cells to growth factors. Whereas EGF or IGF-I triggered proliferation of parental hepatocarcinoma and breast cancer cells, expression of TRb1 abolished this response. Our results show that TRb1 disrupted the mito-

Figure 6 TRb1 inhibits tumor invasiveness and formation of experimental metastasis. (A) MDA-MB-468 breast cancer cells were stably transfected with an empty vector or with TRb1 (MDA and MDA-TRb, respectively) and inoculated orthotopically into the fat mammary pad of nude mice. The percentage of tumors that infiltrate surrounding tissues, such as muscle or blood and lymph vessels was scored after 30 days. Representative H&E staining of tumor infiltration is also shown. (B) Mice were injected with parental and TRb1-expressing cells into the tail vein of nude mice and formation of lung metastasis was analyzed 30 days later. The percentage of animals bearing metastatic lesions, the number of lesions/lung and the area of lung parenchyma affected (mean"SE) were calculated. Representative H&E staining of lungs showing the appearance of a large nodular metastasis (delineated by a line) in mice injected with parental MDA cells.

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genic action of these factors by suppressing activation of ERK and PI3K signaling pathways that are crucial for cell proliferation and invasiveness (41). Downregulation of growth factor receptors could participate in the insensitivity of TRb1-expressing cells to EGF and IGF-I. Indeed, transcripts for IGFIR, EGFR or ErbB3, although not ErbB2, were strongly reduced by TRb1. In addition, and in accordance with our observations that TRs can repress Ras-mediated responses downstream of ERK, receptor expression abolished activation of transcription factors, such as ELK1 or ATF-2 in the cells. Furthermore, TRb1 blocks TGFbdependent proliferation in hepatocarcinoma and breast cancer cells without altering TGFbRII expression. Many actions of TGFb are mediated by SMAD activation but these factors can also induce MAPK and PI3K activation, and TRb also blocked stimulation of these pathways by TGFb (41). Taken together, these results show that TRb antagonizes stimulation of signaling pathways by growth and transforming growth factors that play a key role in tumor progression and invasiveness. Recent searches for genetic determinants of metastasis have led to the identification of gene sets or ‘‘signatures’’ for which the expression in primary tumors is associated with high risk of metastasis and poor survival in patients, and genes that are relevant for metastatic progression have been identified. Transcripts for the prostaglandin-synthesizing enzyme cyclooxygenase 2 and the transcriptional inhibitor ID1, recently identified among the genes that mediate breast cancer metastasis to the lungs, were strongly reduced by TRb. The same occurred with the transcripts for the chemokine receptors CXCR4, CCR6 and CCR1 or for c-Met, also markers of metastastic growth. This reduction could explain the reduced invasiveness and engraftment of TRb1expressing cells. Matrix-remodeling metalloproteinases (MMP) are also important for invasiveness, and MMP-1 and MMP9 transcripts were also reduced after TRb expression. Inversely, loss of caspase-1 is associated with metastasic growth and caspase-1 mRNA was increased in TRb1expressing cells (41). The finding that TRb1 coordinately downregulates the expression of prometastatic genes suggests a common molecular mechanism for this receptor action. The finding that inhibitors of MAPK or PI3K inhibit their expression in parental cells suggests that antagonism of these pathways by TRb1 could participate in their transcriptional repression (41).

Hypothyroidism enhances tumor invasiveness and metastasis development In contrast with the role of TRs as tumor suppressors, no consistent association between thyroidal status and cancer has been demonstrated. For instance, the connection between thyroid disorders and human breast cancer is a controversial issue. Beatson proposed the use of thyroid extracts for breast cancer treatment more than a century ago (42), and hypothyroidism has been described to be frequently found in cancer patients and to be associated with poor response to

therapy (43–45). However, a lower incidence of primary breast carcinoma and reduced risk of developing invasive disease have also been reported in hypothyroid patients (46). By contrast, it has been reported that hypothyroidism is more prevalent in hepatocarcinoma patients with no known underlying cause of liver disease and might be a possible risk factor for liver cancer (47). Thyroid hormone administration also influences hepatocarcinoma progression in experimental animals. Thus, T3 treatment in rats, despite causing liver hyperplasia, induces a rapid regression of carcinogeninduced hepatic nodules and reduces the incidence of hepatocarcinoma and lung metastasis (48–50). We examined the effect of hypothyroidism on tumor growth, invasion and formation of metastasis in nude mice. To analyze if the changes caused by hypothyroidism are dependent on a direct effect of the hormone in the tumor cell through binding to TRs, we used both parental SK and MDA cells and cells in which TRb1 has been re-expressed. These cells were inoculated into control nude mice and into mice made hypothyroid by treatment with antithyroidal drugs, and tumor growth was followed. The results obtained demonstrated that hypothyroidism has a dual effect on tumorigenesis (51). Tumor growth is slower in hypothyroid mice but the tumors are more aggressive and invasive, and metastasis formation is strongly enhanced. Expression of TRb in euthyroid animals retarded the detection of palpable tumors and reduced tumor volume, and hypothyroidism was able to retard tumor growth in mice inoculated with both parental and TRb-expressing cells (Figure 7A). The reduced tumor volume in hypothyroid hosts correlates with a lower proliferation index (Figure 7B), reduction of Cyclin E expression and enlargement of the necrotic area in the tumors (51). Tumor development by ras-transformed fibroblasts, which do not express the receptor, was also retarded in hypothyroid animals and expression of TRa1 produced a further delay in tumor appearance, whereas expression of TRb1 abolishes tumor formation by ras-transformed cells in nude mice, even under hypothyroid conditions (27). The higher degree of tumor differentiation obtained in tumors developed in euthyroid animals after injection of fibroblasts expressing the ras oncogene and TRa1 was lost in hypothyroid animals where tumors had a morphology similar to that obtained in euthyroid animals expressing ras alone (27). In addition, tumors from both parental and TRb1-expressing SK and MDA cells inoculated into hypothyroid mice have a more mesenchymal phenotype with a strong reduction of keratin 8/18 and bcatenin and a concomitant increase in vimentin (51). Therefore, hypothyroidism appears to confer a less differentiated phenotype to the tumors independently of the presence of the receptor in the cancer cells. The more mesenchymal phenotype of the tumor cells in hypothyroidism could facilitate spreading from the primary tumor to the neighboring host tissues, a critical step that allows tumor cells to invade the extracellular matrix, enter the circulation and disseminate to distant organs. In accordance with the changes in the tumor cell phenotype, hypothyroidism increased the number of invasion fronts of the tumors formed by parental and TRb1expressing cells and strongly augmented infiltration of adja-

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Figure 7 Hypothyroidism reduces tumor growth. Parental and TRb-expressing MDA cells were inoculated into euthyroid nude mice and into mice made hypothyroid by treatment with antithyroidal drugs. Treatment started 4 weeks before inoculation and was continued for the duration of the experiment. Tumor volume was followed during 30 days (A) and the percentage of proliferating cells in the tumor was measured by staining with Ki67 (B). Data are mean"SE.

cent tissues such as muscle, blood and lymph vessels or skin. Thyroidal status also influenced the formation of long distance metastasis by hepatocarcinoma and breast cancer cells. Thus, spontaneous metastasis in bone appeared when MDA cells were injected into hypothyroid but not euthyroid hosts (Figure 8A) and in lung, liver or bone in the case of SK cells (51), reinforcing the concept that metastatic growth is dependent on both the intrinsic properties of the tumor cells and the responses of the stromal cells. Furthermore, formation of experimental metastasis in lung by direct inoculation of the cancer cells into the tail vein of the hypothyroid nude mice was also markedly enhanced with regard to

the metastatic growth observed in normal hosts again both in parental and TRb1-expressing cells (51) (Figure 8B). Therefore, hypothyroidism appears to favor a permissive tissue microenvironment for cancer metastasis. A higher aggressiveness of the tumors developed from TRb-expressing cells in hypothyroid hosts would be compatible with a reduced TR activity in the cancer cells as a result of the reduced thyroid hormone availability. However, the increased malignancy of tumors formed by the parental hepatocarcinoma and breast cancer cells that do not express TRs, and consequently do not respond to thyroid hormones, show that changes in the stromal cells, most likely secondary to the

Figure 8 Hypothyroidism enhances tumor invasiveness and formation of metastasis. (A) Representative H&E staining of the tumors formed by MDA and MDA-TRb cells in control and hypothyroid nude mice (left panels). Tumor borders are marked with a line. The number of invasion fronts of the tumors was scored and is represented as mean"SE in the right panel. (B) Representative images of lungs from euthyroid and hypothyroid mice injected into the tail vein with MDA and MDA-TRb cells. Metastases are delineated with a discontinuous line. The percentage of animals bearing metastatic lesions and the number of lesions/lung (mean"SE) was determined in the different groups.

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important metabolic changes associated with hypothyroidism, rather than a direct effect of the hormone on the cancer cells appear to be responsible for the increased invasiveness and metastatic activity observed in hypothyroid mice. In summary, our data point to an important role of the thyroidal status in tumor progression. Normal thyroid hormone levels appear to favor growth of primary xenografts, but they also block tumor cell dissemination and metastasis formation. These divergent effects could help to explain the confounding reports on the influence of hypothyroidism in human tumors. Furthermore, because our results show that similar effects are observed independently of the presence or absence of TR in the cancer cells, it would be expected that thyroidal status could impact tumor progression even in tumors in which TRs are deleted or mutated, a common event in human cancer.

Role of endogenous TRs on skin carcinogenesis in mice The protocol of two-stage chemical skin carcinogenesis in mice provides an excellent model to analyze the role of TRs on tumor progression. With this protocol, benign and malignant neoplasms can be induced on the backs of mice after exposure to an initiating carcinogen, such as 7,12-dimethylbenzwaxanthracene (DMBA), and subsequent chronic regenerative epidermal hyperplasia caused by topical application of a promoting agent, such as 12-O-tetradecanoyl-phorbol13-acetate (TPA). Supporting the notion that TRb could act as a suppressor of malignant transformation, TRb expression is lost as aggressiveness of skin tumors progresses. Thus, strong TRb expression can be detected by immunohistochemistry in normal skin, as well as in hyperplasic epidermis, obtained after topical application of TPA for 4 days. However, receptor expression was strongly reduced in the papillomas and was totally lost in squamous cell carcinomas (SCCs) (41). To test if endogenous TRs can suppress epithelial tumor formation and malignancy, we compared the growth of skin tumors during 30 weeks in wild-type mice and in double knockout (KO) mice lacking both TRa and TRb. Tumor number increased with time, reaching a plateau at 20 weeks after DMBA treatment in both groups. Unexpectedly, TRa –/– /TRb –/– double KO mice developed a significantly lower number of tumors than controls. However, after 20 weeks, tumor growth was significantly faster in animals devoid of the receptors. The higher proliferation rate of tumors from mice lacking TRs was reflected by an increase in the percentage of bromodeoxyuridine-positive cells in the basal layer of epithelium at necropsy (41). Interestingly, skin tumors from TRa –/– /TRb –/– mice had a more malignant phenotype. Analysis by nuclear magnetic resonance showed that the tumors had a different morphology (Figure 9). Nonaggressive papillomas were formed in the wild-type animals, but rapidly growing tumors disrupting the subcutaneous fat layer were observed in the TRa –/– /TRb –/– KO mice. This was confirmed by histological classification which demon-

Figure 9 Increased malignancy of skin tumors in mice lacking thyroid hormone receptors. Control mice and TRa –/– /TRb –/– knockout mice were subjected to a protocol of experimental twostage chemical skin carcinogenesis. Representative photographs of the tumors in both groups of mice were taken at 30 weeks of treatment. The corresponding nuclear magnetic resonance images show a benign papilloma in a control animal and a tumor invading the subcutaneous fat layer, histologically classified as a squamous cell carcinoma, in a TRa –/– /TRb –/– mouse.

strated that most tumors found in wild-type mice were typical well-differentiated papillomas, whereas in KO mice half of the tumors were diagnosed as in situ carcinoma or SCCs. Therefore, TR deficiency seems to inhibit benign tumor formation at early stages of skin carcinogenesis and increases malignant progression at later stages in this model of epithelial carcinogenesis.

Conclusions We have obtained evidence that thyroid hormone receptors are important regulators of cell growth and malignant transformation. TRs can suppresses transformation by the ras oncogene in cultured cells and tumor progression and metastasis formation in experimental animals. These results together with the fact that these receptors are often inactivated in human cancer and that a dominant-negative TR causes cancer in mice suggest their role as tumor suppressors, indicating that these receptors could constitute a novel therapeutic target in cancer. Although multiple mechanisms for the anti-oncogenic actions of TRs have already been identified, many studies will be needed to fully understand their role in cancer. By contrast, hypothyroidism can differentially affect tumor growth and invasiveness independently of the presence of functional TRs in the tumor cells. Therefore, changes in the stromal cells secondary to the thyroidal status can modulate tumor progression and metastatic growth. Furthermore, TRs are required for normal growth and cell proliferation in different tissues and they could play a dual role in carcinogenesis, because they have been described to act as growth-promoting factors in certain types

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of cancer cells. Because TR actions are complex and tissueand time-specific, aberrant functions of the various TR isoforms might have divergent effects in different tumor cells or at different stages of tumor development. Clarification of the mechanisms by which TRs can influence tumor progression and elucidation of their role in metastatic growth, which causes most deaths from solid tumors, should lead to a better understanding of the biology of cancer and its susceptibility to treatment in humans.

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Acknowledgments Research in this laboratory has been supported by grant BFU200762402 from the Ministerio de Ciencia e Innovacio´n, grant RD06/ 0020/0036 from the Fondo de Investigaciones Sanitarias and from the European Project CRESCENDO (grant FP6-018652). The authors have no conflict of interest to declare.

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Copyright of Hormone Molecular Biology & Clinical Investigation is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.