Olaia Martínez-Iglesias

Thyroid Hormone Receptor β1 Acts as a Potent Suppressor of Tumor Invasiveness and Metastasis

Loss of thyroid hormone receptors (TR) is a common feature in some tumors, although their role in tumor progression is currently unknown. We show here that expression of TRbeta1 in hepatocarcinoma and breast cancer cells reduces tumor growth, causes partial mesenchymal-to-epithelial cell transition, and has a striking inhibitory effect on invasiveness, extravasation, and metastasis formation in mice. In cultured cells, TRbeta1 abolishes anchorage-independent growth and migration, blocks responses to epidermal growth factor, insulin-like growth factor-I, and transforming growth factor beta, and regulates expression of genes that play a key role in tumorigenicity and metastatic growth. The receptor disrupts the mitogenic action of growth factors by suppressing activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling pathways that are crucial for cell proliferation and invasiveness. Furthermore, increased aggressiveness of skin tumors is found in genetically modified mice lacking TRs, further demonstrating the role of these receptors as inhibitors of tumor progression. These results define a novel role for the thyroid hormone receptor as a metastasis suppressor gene, providing a starting point for the development of novel therapeutic strategies for the treatment of human cancer.

A Permissive Retinoid X Receptor/Thyroid Hormone Receptor Heterodimer Allows Stimulation of Prolactin Gene Transcription by Thyroid Hormone and 9-cis-Retinoic Acid

ABSTRACT Heterodimers of the retinoid X receptor (RXR) with the thyroid hormone receptor (TR) are considered to be nonpermissive. It is believed that within these complexes RXR acts as a “silent partner.” We demonstrate here that a permissive heterodimer mediates stimulation of prolactin expression by the thyroid hormone T3 and by 9-cis retinoic acid (9-cis-RA). A response element located in the prolactin distal enhancer mediates transactivation by both ligands in pituitary cells, and RXR recruits coactivators when bound to this element as a heterodimer with TR. Furthermore, transcription by the RXR agonist can be obtained in CV-1 cells only after overexpression of coactivators, and overexpression of corepressors inhibits the response in pituitary cells. Thus, cell type-specific differences in coregulator recruitment can determine the cellular response to both ligands. Coactivator recruitment by 9-cis-RA requires the ligand-dependent transactivation domains (AF-2) of both heterodimeric partners. Interestingly, the presence of the RXR ligand can overcome the deleterious effect of the AF-2 mutation E401Q on association with coactivators and transactivation. These results demonstrate an unexpected role for RXR in TR signaling and show that in particular cellular environments this receptor can act as a “nonsilent” partner of TR, allowing stimulation by RXR agonists.

Hypothyroidism Enhances Tumor Invasiveness and Metastasis Development

Background Whereas there is increasing evidence that loss of expression and/or function of the thyroid hormone receptors (TRs) could result in a selective advantage for tumor development, the relationship between thyroid hormone levels and human cancer is a controversial issue. It has been reported that hypothyroidism might be a possible risk factor for liver and breast cancer in humans, but a lower incidence of breast carcinoma has been also reported in hypothyroid patients Methodology/Principal Findings In this work we have analyzed the influence of hypothyroidism on tumor progression and metastasis development using xenografts of parental and TRβ1–expressing human hepatocarcinoma (SK-hep1) and breast cancer cells (MDA-MB-468). In agreement with our previous observations tumor invasiveness and metastasis formation was strongly repressed when TRβ–expressing cells were injected into euthyroid nude mice. Whereas tumor growth was retarded when cells were inoculated into hypothyroid hosts, tumors had a more mesenchymal phenotype, were more invasive and metastatic growth was enhanced. Increased aggressiveness and tumor growth retardation was also observed with parental cells that do not express TRs. Conclusions/Significance These results show that changes in the stromal cells secondary to host hypothyroidism can modulate tumor progression and metastatic growth independently of the presence of TRs on the tumor cells. On the other hand, the finding that hypothyroidism can affect differentially tumor growth and invasiveness can contribute to the explanation of the confounding reports on the influence of thyroidal status in human cancer.

Thyroid receptor: roles in cancer

The thyroid hormone receptors, encoded by the TRalpha and TRbeta genes, are ligand-dependent transcription factors that belong to the nuclear receptor superfamily. In addition to the role of these receptors in growth, development and metabolism, there is increasing evidence that they also inhibit transformation and act as tumor suppressors. Aberrant TR action, as well as receptor silencing, are common events in human cancer, and TRs also have an important role in tumor progression in experimental animal models, suggesting that these receptors constitute a novel therapeutic target in cancer. This review highlights recent studies on mechanisms by which loss of expression and/or function of these receptors results in a selective advantage for cellular transformation, tumor development and metastatic growth.

Anti-estrogenic actions of histone deacetylase inhibitors in MCF-7 breast cancer cells

Anti-estrogens are the current endocrine therapy of choice in the treatment of estrogen receptor (ER)-positive breast cancers. Histone deacetylase inhibitors (HDACi) also constitute a promising treatment for therapy, and combination of anti-estrogens with HDACi may improve efficacy while reducing side effects. We have examined the effect of the HDACi sodium butyrate and suberoylanilide hydroxamic acid (SAHA), alone and in combination with 17beta-estradiol (E2) and the pure anti-estrogen ICI 182.780 (ICI) in human MCF-7 breast cancer cells. HDACi caused a sustained increase of histone H3 acetylation and caused cell death as shown by flow cytometry analysis. In surviving cells, both inhibitors were even stronger than ICI in depleting cyclin D1 levels, inducing expression of the cyclin kinase inhibitor p21Waf1/Cip1, blocking phosphorylation of the retinoblastoma protein, or inhibiting cell growth. No additive effects of ICI with either butyrate or SAHA were found. In addition, these drugs were able to antagonize the effects of E2 on expression of cell cycle proteins, cell growth, and transcription of ER-dependent genes. The anti-estrogenic effects of HDACi appear to be related to a strong downregulation of the expression of ERalpha that appears to be secondary to both transcriptional and post-transcriptional regulation. ERalpha phosphorylation is involved in estrogen signaling, and HDACi also prevented receptor phosphorylation in Ser-118 both in the absence and presence of ER ligands. These results provide further support for the use of deacetylase inhibitors as chemotherapeutic agents in the treatment of breast cancer tumors.

Thyroid hormone receptor β1 domains responsible for the antagonism with the ras oncogene: role of corepressors

The thyroid hormone receptor (TR) is a suppressor of ras-mediated responses. To characterize the receptor domains involved in this function, we analyzed a panel of TRβ1 mutants for their ability to interfere with ras-driven cyclin D1 activation, formation of transformation foci and tumor growth in nude mice. Our results show that the domains and mechanisms responsible for the anti-transforming and anti-tumorigenic actions of the receptor are divergent from those operating in classical T3-dependent transcriptional activation. TRβ1 mutants that do not bind coactivators and do not transactivate retained the capacity of suppressing cellular transformation and tumor growth, whereas selective mutations in the hinge region affecting corepressors recruitment abolished these actions, while preserving ligand-dependent transcription. There was a strict parallelism between anti-transforming activity of the various mutants and their ability to antagonize cyclin D1 stimulation by ras, indicating that transrepression mechanisms may have an important function in suppression of the transforming effects of the oncogene by TRβ1. The inhibitory action of T3 on transformation was further enhanced after over-expression of corepressors, while corepressor depletion by means of small-interference RNA reversed significantly hormonal action. This shows an important functional role of endogenous corepressors in suppression of ras-mediated transformation and tumorigenesis by TRβ1

Autoregulatory loop of nuclear corepressor 1 expression controls invasion, tumor growth, and metastasis.

Significance Inactivating nuclear corepressor 1 (NCoR) mutations have been found in human tumors. Here, we report that NCoR has a tumor-suppressive role, inhibiting extravasation, metastasis formation, and tumor growth in mice. These changes are related to repressed transcription of genes associated with increased metastasis and poor prognosis in cancer patients. An autoregulatory loop maintains NCoR gene expression, suggesting that NCoR loss can be propagated, contributing to tumor progression in the absence of NCoR gene mutations. The nuclear receptor thyroid hormone receptor β1 increases NCoR expression, and this induction is essential in mediating its tumor-suppressive actions. Both are reduced in human hepatocarcinoma and aggressive breast cancer tumors, identifying NCoR as a potential molecular target for the development of novel therapies. Nuclear corepressor 1 (NCoR) associates with nuclear receptors and other transcription factors leading to transcriptional repression. We show here that NCoR depletion enhances cancer cell invasion and increases tumor growth and metastatic potential in nude mice. These changes are related to repressed transcription of genes associated with increased metastasis and poor prognosis in patients. Strikingly, transient NCoR silencing leads to heterochromatinization and stable silencing of the NCoR gene, suggesting that NCoR loss can be propagated, contributing to tumor progression even in the absence of NCoR gene mutations. Down-regulation of the thyroid hormone receptor β1 (TRβ) appears to be associated with cancer onset and progression. We found that expression of TRβ increases NCoR levels and that this induction is essential in mediating inhibition of tumor growth and metastasis by this receptor. Moreover, NCoR is down-regulated in human hepatocarcinomas and in the more aggressive breast cancer tumors, and its expression correlates positively with that of TRβ. These data provide a molecular basis for the anticancer actions of this corepressor and identify NCoR as a potential molecular target for development of novel cancer therapies.

Residues K128, 132, and 134 in the Thyroid Hormone Receptor-α Are Essential for Receptor Acetylation and Activity

The thyroid hormone receptor (TR)-alpha is a nuclear receptor that mediates both transrepression and ligand-dependent transactivation. Here we show that TRalpha is posttranslationally modified by acetylation in response to its own ligand (T(3)). Acetylation increases binding to DNA. Using mutagenesis, we identified three conserved lysine residues in the carboxi-terminal extension (CTE) of the DNA binding domain that are targets of the cAMP-response element-binding protein acetyltransferase. Substitution of these lysines by arginines in TRalpha decreased ligand binding affinity and precluded ligand-dependent release of corepressors and recruitment of coactivators. The acetylation TRalpha mutant lost the ability to transactivate even at high T(3) concentrations and acts as a dominant-negative inhibitor of wild-type TR activity. In addition, whereas native TRalpha interferes with AP-1 function, the mutant is unable to mediate transrepression. Finally, TRalpha suppresses NIH-3T3 fibroblast transformation by the Ras oncogene both in a ligand-dependent and -independent manner, but the CTE mutant is unable to mediate ligand-dependent repression of transformation. These results reveal a key role for the CTE region on acetylation, ligand affinity, transactivation, transrepression, and antitransforming properties of TRalpha.

Thyroid hormones inhibit TGF-β signaling and attenuate fibrotic responses

Significance We show here that binding of the thyroid hormone triiodothyronine to the thyroid hormone receptors (TRs) antagonizes TGF-β/SMAD (mothers against decapentaplegic)-dependent transcription. Transcriptionally inactive TR mutants that do not bind coactivators retained most of the capacity of suppressing transactivation by TGF-β/SMAD, whereas selective mutations in the DNA binding domain abolished this action. TGF-β is a major profibrogenic cytokine, and through this transcriptional mechanism, the hormone-bound TRs act as an endogenous barrier to moderate liver and skin fibrosis. These antagonistic actions on TGF-β/SMAD transcription suggest that TR ligands might be used to block the progression of fibrotic diseases. The natural hormone cannot be used clinically because of severe adverse effects, but novel synthetic ligands with fewer effects might be potentially developed and used. TGF-β, the most potent profibrogenic factor, acts by activating SMAD (mothers against decapentaplegic) transcription factors, which bind to SMAD-binding elements in target genes. Here, we show that the thyroid hormone triiodothyronine (T3), through binding to its nuclear receptors (TRs), is able to antagonize transcriptional activation by TGF-β/SMAD. This antagonism involves reduced phosphorylation of SMADs and a direct interaction of the receptors with SMAD3 and SMAD4 that is independent of T3-mediated transcriptional activity but requires residues in the receptor DNA binding domain. T3 reduces occupancy of SMAD-binding elements in response to TGF-β, reducing histone acetylation and inhibiting transcription. In agreement with this transcriptional cross-talk, T3 is able to antagonize fibrotic processes in vivo. Liver fibrosis induced by carbon tetrachloride is attenuated by thyroid hormone administration to mice, whereas aged TR knockout mice spontaneously accumulate collagen. Furthermore, skin fibrosis induced by bleomycin administration is also reduced by the thyroid hormones. These findings define an important function of the thyroid hormone receptors and suggest TR ligands could have beneficial effects to block the progression of fibrotic diseases.

The thyroid hormone receptors as tumor suppressors.

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 TRβ1 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.