Ana Aranda

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.

The Thyroid Hormone Receptor Is a Suppressor of ras-Mediated Transcription, Proliferation, and Transformation

ABSTRACT The thyroid hormone triiodothyronine (T3) has a profound effect on growth, differentiation, and metabolism in higher organisms. Here we demonstrate that T3 inhibits ras-induced proliferation in neuroblastoma cells and blocks induction of cyclin D1 expression by the oncogene. The hormone, at physiological concentrations, strongly antagonizes the transcriptional response mediated by the Ras/mitogen-activated protein kinase/ribosomal-S6 subunit kinase (Rsk) signaling pathway in cells expressing thyroid hormone receptors (TRs). T3 blocks the response to the oncogenic forms of the three ras isoforms (H-, K-, and N-ras) and both TRα and TRβ can mediate this action. The main target for induction of cyclin D1 transcription by oncogenic ras in neuroblastoma cells is a cyclic AMP response element (CRE) located in proximal promoter sequences, and T3 represses the transcriptional activity of b-Zip transcription factors such as CREB (CRE-binding protein) or ATF-2 (activation transcription factor 2) that are direct targets of Rsk2 and bind to this sequence. The hormone also blocks fibroblast transformation by oncogenic ras when TR is expressed. Furthermore, TRs act as suppressors of tumor formation by the oncogene in vivo in nude mice. The TRβ isoform has stronger antitransforming properties than the α isoform and can inhibit tumorigenesis even in hypothyroid mice. These results show the existence of a previously unrecognized transcriptional cross talk between the TRs and the ras oncogene which influences relevant processes such as cell proliferation, transformation, or tumorigenesis.

Role of c-Src in Human MCF7 Breast Cancer Cell Tumorigenesis

To study the role of c-Src in breast cancer tumorigenesis, we generated a cell line derived from MCF7 carrying an inducible dominant negative c-Src (c-SrcDN: K295M/Y527F) under tetracycline control (Tet-On system). c-SrcDN expression caused phenotypic changes, relocation of c-Src, Fak, and paxillin, and loss of correct actin fiber assembly. These alterations were coupled to increased Fak-Tyr397 autophosphorylation and to inhibition of Fak-Tyr925, p130CAS, and paxillin phosphorylation. An increased association of total Src with Fak and a decreased interaction of p130CAS and p85-PI3K with Fak were also observed. SrcDN inhibited cell attachment, spreading, and migration. Serum and EGF-induced stimulation of cell proliferation and Akt phosphorylation were also significantly reduced by SrcDN, whereas p27Kip1 expression was increased. Consistently, silencing c-Src expression by siRNA in MCF7 cells significantly reduced cell migration, attachment, spreading and proliferation. Inoculation of MCF7 cells carrying inducible SrcDN to nude mice generated tumors. However, doxycycline administration to mice significantly reduced tumorigenesis, and when doxycycline treatment was installed after tumor development, a significant tumor regression was observed. In both situations, inhibition of tumorigenesis was associated with decreased Ki67 staining and increased apoptosis in tumors. These data undoubtedly demonstrate the relevance of the Src/Fak complex in breast cancer tumorigenesis.

Thyroid hormone receptors, cell growth and differentiation

BACKGROUND Tissue homeostasis depends on the balance between cell proliferation and differentiation. Thyroid hormones (THs), through binding to their nuclear receptors, can regulate the expression of many genes involved in cell cycle control and cellular differentiation. This can occur by direct transcriptional regulation or by modulation of the activity of different signaling pathways. SCOPE OF REVIEW In this review we will summarize the role of the different receptor isoforms in growth and maturation of selected tissues and organs. We will focus on mammalian tissues, and therefore we will not address the fundamental role of the THs during amphibian metamorphosis. MAJOR CONCLUSIONS The actions of THs are highly pleiotropic, affecting many tissues at different developmental stages. As a consequence, their effects on proliferation and differentiation are highly heterogeneous depending on the cell type, the cellular context, and the developmental or transformation status. Both during development and in the adult, stem cells are essential for proper organ formation, maintenance and regeneration. Recent evidence suggests that some of the actions of the thyroid hormone receptors could be secondary to regulation of stem/progenitor cell function. Here we will also include the latest knowledge on the role of these receptors in proliferation and differentiation of embryonic and adult stem cells. GENERAL SIGNIFICANCE The thyroid hormone receptors are potent regulators of proliferation and differentiation of many cell types. This can explain the important role of the thyroid hormones and their receptors in key processes such as growth, development, tissue homeostasis or cancer. This article is part of a Special Issue entitled Thyroid hormone signalling.

An element in the region responsible for premature termination of transcription mediates repression of c-myc gene expression by thyroid hormone in neuroblastoma cells

The thyroid hormone (T3) blocks proliferation and induces differentiation of neuroblastoma N2a-β cells that express the thyroid hormone receptor (TR) β1 isoform. c-Myc is required for cell cycle progression, and this study shows that T3-induced neuronal differentiation is preceded by a rapid decrease of c-mycgene expression. A negative T3 responsive element (TRE), arranged as an inverted palindrome spaced by three nucleotides, has been identified within the first exon between nucleotides +237 and +268. The TRE is adjacent to the binding site for the transcriptional repressor CCCTC binding factor and maps precisely within the region of RNA polymerase II pausing and release, suggesting a direct implication of TR on premature termination of transcription. Furthermore, the TRE confers repression by T3 to an heterologous promoter only when inserted downstream of the transcription initiation site. Binding of CCCTC binding factor and TR to their cognate sites in the region of transcriptional attenuation, as well as direct interactions between both factors, could facilitate the formation of a repressor complex and the inhibition of c-myc gene expression. These studies provide insight into mechanisms by which TR mediate transcriptional repression and contribute to the understanding of the important effects of thyroid hormones on growth and differentiation of neuronal cells.

Combined effects of retinoic acid and histone deacetylase inhibitors on human neuroblastoma SH-SY5Y cells

All-trans retinoic acid (RA) causes differentiation of neuroblastoma cells, and retinoids have been used in clinical trials in children with advanced neuroblastoma. Combination of RA with histone deacetylase inhibitors (HDACi) could result in improved antitumorigenic activity. We have examined the effect of the HDACi trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA), alone and in combination with RA in human neuroblastoma SH-SY5Y cells. At concentrations that cause sustained increase of histone H3 acetylation, HDACi produced extensive apoptotic cell death as shown by flow cytometry analysis and induction of poly(ADP-ribose) polymerase proteolysis. HDACi inhibited SH-SY5Y cell growth at a much larger extent than RA. This compound did not cause apoptosis and did not further increase HDACi-mediated cell death. In contrast, both types of drugs cooperated to inhibit cell growth, although synergistic effects were not found. In surviving cells, HDACi repressed cyclin D1 expression and increased the cyclin kinase inhibitors (CKI) p21Waf1/Cip1 and p27Kip1. Cyclin D1 was not affected by RA, but this retinoid also increased CKI levels. Induction of p21Waf1/Cip1 and p27Kip1 by HDACi was further enhanced in the presence of RA. This effect seems to be at least partially due to transcriptional stimulation of CKI gene expression because both types of drugs cooperated to increase CKI mRNA levels and to activate the CKI promoters in transient transfection assays. These results show the strong antitumorigenic effects of HDACi in neuroblastoma cells and reinforce the idea that combination therapy could be useful to inhibit tumor growth. [Mol Cancer Ther 2007;6(4):1425–32]

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.

The thyroid hormone receptor antagonizes CREB-mediated transcription

Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein–protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)‐containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein–protein interactions. In turn, expression of CREB reduces TR‐dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand‐dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross‐talk between CREB and TR signalling pathways, which can have important functional consequences.

Comparison of the effects of forskolin and dibutyryl cyclic AMP in neuroblastoma cells: evidence that some of the actions of dibutyryl cyclic AMP are mediated by butyrate.

Abstract: We have compared the effects of forskolin, N6,2′‐O‐dibutyryladenosine 3′:5′‐cyclic monophosphate (dibutyryl cyclic AMP, Bt2‐cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2‐cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose‐dependent inhibition of cell proliferation, but the effect of Bt2‐cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2‐cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2‐cAMP than in untreated cells or in forskolin‐treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2‐cAMP produced histone acetylation, a well‐known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2‐cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H1°, a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H1° in the cells treated with Bt2‐cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2‐cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cyclic nucleotide itself.

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.