Purpose

Src is over-expressed or hyper-activated in a variety of human cancers including thyroid carcinoma. Src is a central mediator in multiple signaling pathways that are important in oncogenesis and cancer progression. In this study, we evaluated the effects of a Src inhibitor, SKI-606 (bosutinib), in a spontaneous metastatic thyroid cancer model with constitutively activated Src (ThrbPV/PVPten+/− mice).

Experimental Design

ThrbPV/PVPten+/− mice were treated with SKI-606 or vehicle controls, beginning at 6 weeks of age until the mice succumbed to thyroid cancer. We assessed the effects of SKI-606 on thyroid cancer progression and analyzed the impact of SKI-606 on aberrant Src-mediated signaling.

Results

SKI-606 effectively inhibited aberrant activation of Src and its downstream targets to markedly inhibit the growth of thyroid tumor, thereby prolonging the survival of treated mice. While Src inhibition did not induce cell apoptosis, it decreased cell proliferation by affecting the expression of key regulators of cell cycle progression. Importantly, SKI-606 dramatically prevented de-differentiation, vascular invasion, and lung metastasis of thyroid cancer cells. These responses were meditated by down-regulation of mitogen-activated protein kinase pathways and inhibition of the epithelial-mesenchymal transition.

Conclusions

Our findings suggest that Src is critical in the progression of thyroid cancer, making oral SKI-606 a promising treatment strategy for refractory thyroid cancer.

Purpose of review

Thyroid hormone (3,3′,5-triiodo-l-thyronine) plays an important role in thermogenesis and maintenance of lipid homeostasis. The present article reviews the evidence that 3,3′,5-triiodo-l-thyronine regulates lipid metabolism via thyroid hormone receptors, focusing particularly on in-vivo findings using genetically engineered mice.

Recent findings

That lipid metabolism is regulated via thyroid hormone receptor isoforms in a tissue-dependent manner was recently uncovered by using knockin mutant mice harboring an identical mutation in the Thra gene (Thra1PV mouse) or the Thrb gene (ThrbPV mouse). The mutation in the Thra gene dramatically decreases the mass of both white adipose tissue and liver. In contrast, the mutation in the Thrb gene markedly increases the mass of liver with an excess depot of lipids, but no significant abnormality is observed in white adipose tissue. Molecular studies show that the expression of lipogenic genes is decreased in white adipose tissue of Thra1PV mice, but not in ThrbPV mice. Markedly increased lipogenic enzyme expression, and decreased fatty acid beta-oxidation activity contribute to the adipogenic steatosis and lipid accumulation in the liver of ThrbPV mice. In contrast, reduced expression of genes critical for lipogenesis mediates decreased liver mass with lipid scarcity in Thra1PV mice.

Summary

Studies using Thra1PV and ThrbPV mice indicate that apo-thyroid hormone receptor-beta and apo-thyroid hormone receptor-alpha-1 mediate distinct deleterious effects on lipid metabolism. Thus, both thyroid hormone receptor isoforms contribute to the pathogenesis of lipid abnormalities in hypothyroidism, but in a target tissue-dependent manner. These studies suggest that thyroid hormone receptor isoform-specific ligands could be designed as therapeutic targets for lipid abnormalities.

Accumulated evidence suggests that thyroid hormone receptor β (TRβ) could function as a tumor suppressor, but the detailed mechanisms by which TRβ inhibits tumorigenesis are not fully understood. The present studies explored the mechanisms by which TRβ acted to inhibit thyroid tumor development mediated by simian virus-40 (SV40). In mouse xenograft models, SV40 large T antigen (SV40Tag)-immortalized human thyroid epithelial (HTori) cells rapidly induced tumors, but the tumor development was totally blocked by TRβ stably expressed in HTori cells. Previous studies showed that the SV40Tag oncoprotein binds to and inactivates tumor suppressors p53 and retinoblastoma protein (Rb), thereby inducing tumorigenesis. Here we showed that one of the mechanisms by which TRβ suppressed tumor development was by competing with p53 and Rb for binding to SV40Tag. The interaction of TRβ with SV40Tag led to reactivation of Rb to inhibit cell cycle progression. TRβ- SV40Tag interaction also resulted in reactivating p53 to increase the expression of Pten, thus attenuating PI3K-AKT signaling to decrease cell proliferation and to induce apoptosis. The present study uncovered a novel action of TRβ as a tumor suppressor initiated via interfering with the recruitment of Rb and p53 by SV40Tag oncoprotein through protein-protein interaction, thereby acting to block tumor development.

Thyroid hormone receptors (TRs) mediate the critical activities of the thyroid hormone (T3) in growth, development, and differentiation. Decreased expression and/or somatic mutations of TRs have been shown to be associated with several types of human cancers including liver, breast, lung, and thyroid. A direct demonstration that TRβ mutants could function as oncogenes is evidenced by the spontaneous development of follicular thyroid carcinoma similar to human cancer in a knockin mouse model harboring a mutated TRβ (denoted as PV; ThrbPV/PV mice). PV is a dominant negative mutation identified in a patient with resistance to thyroid hormone. Analysis of altered gene expression and molecular studies of thyroid carcinogenesis in ThrbPV/PV mice show that the oncogenic activity of PV is mediated by both nucleus-initiated transcription and extranuclear actions to alter gene expression and signaling transduction activity. This article focuses on recent findings of novel extranuclear actions of PV that affect signaling cascades and thereby the invasiveness, migration, and motility of thyroid tumor cells. These findings have led to identification of potential molecular targets for treatment of metastatic thyroid cancer.

Therapeutic choices are limited for undifferentiated metastatic thyroid carcinomas. Although implanted subcutaneous thyroid tumors are standard preclinical models to examine the efficacy of new therapeutic agents, these xenograft models frequently fail to predict the outcomes of clinical trials in patients with metastatic thyroid carcinomas. Genetically engineered mouse models with alterations similar to human cancers in their pathological progression and in an immunocompetent environment offer unparalleled opportunities for evaluating novel potential molecular targets. We review recent advances in the modeling of follicular thyroid carcinoma with distant metastasis and in the use of these mouse models in preclinical studies, emphasizing the significance of genetically engineered mouse models in clinical applications.

Thyroid hormone (T3) is critical in growth, development, differentiation, and maintenance of metabolic homeostasis. Recent studies suggest that thyroid hormone receptors (TRs) not only mediate the biological activities of T3 via nucleus-initiated transcription, but also could act via nongenomic pathways. The striking phenotype of thyroid cancer exhibited by a knockin mutant mouse that harbors a dominant negative TRβ mutant (TRβPV/PV mouse) allows the elucidation of novel oncogenic activity of a TRβ mutant (PV) via extra-nuclear actions. PV physically interacts with the regulatory p85α subunit of phosphatidylinositol 3-kinase (PI3K) to activate the downstream AKT-mammalian target of rapamycin (mTOR) and p70S6K and PI3K-integrin-linked kinase-matrix metalloproteinase-2 signaling pathways. The PV-mediated PI3K activation results in increased cell proliferation, motility, migration, and metastasis. Remarkably, a nuclear receptor corepressor (NCoR) was found to regulate the PV-activated PI3K signaling by competing with PV for binding to the C-terminal SH2 domain of p85α. Overexpression of NCoR in thyroid tumor cells of TRβPV/PV mice reduces AKT-mTOR- p70S6K signaling. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells leads to over-activated PI3K-AKT signaling to increase cell proliferation and motility. Furthermore, NCoR protein levels are significantly lower in thyroid tumor cells than in wild type thyrocytes, allowing more effective binding of PV to p85α to activate PI3K signaling, thereby contributing to tumor progression. Thus, PV, an apo-TRβ, could act via direct protein-protein interaction to mediate critical oncogenic actions. These studies also uncovered a novel extra-nuclear role of NCoR in modulating the nongenomic actions of a mutated TRβ in controlling thyroid carcinogenesis.

Metastasis is the major cause of thyroid cancer-related death. However, little is known about the genes involved in the metastatic spread of thyroid carcinomas. We have created a mouse that spontaneously develops metastatic follicular thyroid carcinoma (FTC). This mouse harbors a targeted mutation (denoted TRβPV) in the thyroid hormone receptor β gene (ThrbPV/PV mice). Our recent studies show that the highly elevated level of thyroid stimulating hormone (TSH) in ThrbPV/PV mice promotes proliferation of thyroid tumor cells, but requires the collaboration of the oncogenic action of TRβPV to empower the tumor cells to undergo distant metastasis. To uncover genes destined to drive the metastatic process, we used cDNA microarrays to compare the genomic expression profile of laser capture microdissected thyroid tumor lesions of ThrbPV/PV mice with that of hyperplastic thyroid cells of wild-type mice having elevated TSH induced by treatment with the anti-thyroid drug propylthiouracil (WT-PTU mice). Analyses of microarray data indicated that the expressions of 150 genes were significantly altered between ThrbPV/PV and WT-PTU mice (87 genes had higher expression and 63 genes had lower expression in ThrbPV/PV mice than in WT-PTU mice). Thirty-six percent of genes with altered expression function as key regulators in metastasis. The remaining genes were involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/extracellular matrix signaling. The present studies have uncovered novel genes responsible for the metastatic spread of FTC and, furthermore, have shown that the metastatic process of thyroid cancer requires effective collaboration among genes with diverse cellular functions. Importantly, the present studies indicate that the tumor cells in the primary lesions are endowed with the genes destined to promote metastasis. Thus, our study has provided new insights into the understanding of the metastatic spread of human thyroid cancer.

Thyroid hormone receptors (TRs) are critical in regulating gene expression in normal physiological processes. Decreased expression and/or somatic mutations of TRs have been shown to be associated several types of human cancers including liver, breast, lung, and thyroid. To understand the molecular mechanisms by which mutated TRs promote carcinogenesis, an animal model of follicular thyroid carcinoma (FTC) (ThrbPV/PV mice) was used in the present study. The ThrbPV/PV mouse harbors a knockin dominant negative PV mutation, identified in a patient with resistance to thyroid hormone. To understand whether oncogenic actions of PV involve not only the loss of normal TR functions but also gain-of-function activities, we compared the gene expression profiles of thyroid lesions in ThrbPV/PV mice and Thra1−/−Thrb−/− mice that also spontaneously develop FTC, but with less severe malignancy. Analysis of the cDNA microarray data derived from microdissected thyroid tumor cells of these two mice showed contrasting global gene expression profiles. With stringent selection using 2.5-fold change (p<0.01) in cDNA microarray analysis, 241 genes with altered gene expression were identified. Nearly half of the genes (n=103: 42.7% of total) with altered gene expression in thyroid tumor cells of ThrbPV/PV mice were associated with tumorigenesis and metastasis; some of these genes function as oncogenes in human thyroid cancers. The remaining genes were found to function in transcriptional regulation, RNA processing, cell proliferation, apoptosis, angiogenesis, and cytoskeleton modification. These results indicate that the more aggressive thyroid tumor progression in ThrbPV/PV mice was not due simply to the loss of tumor suppressor functions of TR via mutation but also, importantly, to gain-of-function in the oncogenic activities of PV to drive thyroid carcinogenesis. Thus, the present study identifies a novel mechanism by which a mutated TRβ evolves with an oncogenic advantage to promote thyroid carcinogenesis.

Thyroid hormone receptors (TRs) are critical in regulating gene expression in normal physiological processes. Decreased expression and/or somatic mutations of TRs have been shown to be associated several types of human cancers including liver, breast, lung, and thyroid. To understand the molecular mechanisms by which mutated TRs promote carcinogenesis, an animal model of follicular thyroid carcinoma (FTC) (Thrbpv/pv mice) was used in the present study. The Thrbpv/pv mouse harbors a knockin dominant negative PV mutation, identified in a patient with resistance to thyroid hormone. To understand whether oncogenic actions of PV involve not only the loss of normal TR functions but also gain-of-function activities, we compared the gene expression profiles of thyroid lesions in Thrbpv/pv mice and Thra1-/- Thrb-/- mice that also spontaneously develop FTC, but with less severe malignancy. Analysis of the cDNA microarray data derived from microdissected thyroid tumor cells of these two mice showed contrasting global gene expression profiles. With stringent selection using 2.5-fold change (p<0.01) in cDNA microarray analysis, 241 genes with altered gene expression were identified. Nearly half of the genes (n=103: 42.7% of total) with altered gene expression in thyroid tumor cells of Thrbpv/pv mice were associated with tumorigenesis and metastasis; some of these genes function as oncogenes in human thyroid cancers. The remaining genes were found to function in transcriptional regulation, RNA processing, cell proliferation, apoptosis, angiogenesis, and cytoskeleton modification. These results indicate that the more aggressive thyroid tumor progression in Thrbpv/pv mice was not due simply to the loss of tumor suppressor functions of TR via mutation but also, importantly, to gain-of-function in the oncogenic activities of PV to drive thyroid carcinogenesis. Thus, the present study identifies a novel mechanism by which a mutated TRβ evolves with an oncogenic advantage to promote thyroid carcinogenesis.

Metastasis is the major cause of thyroid cancer-related death. However, little is known about the genes involved in the metastatic spread of thyroid carcinomas. We have created a mouse that spontaneously develops metastatic follicular thyroid carcinoma (FTC). This mouse harbors a targeted mutation (denoted TRβPV) in the thyroid hormone receptor β gene (ThrbPV/PV mice). Our recent studies show that the highly elevated level of thyroid stimulating hormone (TSH) in ThrbPV/PV mice promotes proliferation of thyroid tumor cells, but requires the collaboration of the oncogenic action of TRβPV to empower the tumor cells to undergo distant metastasis. To uncover genes destined to drive the metastatic process, we used cDNA microarrays to compare the genomic expression profile of laser capture microdissected thyroid tumor lesions of ThrbPV/PV mice with that of hyperplastic thyroid cells of wild-type mice having elevated TSH induced by treatment with the anti-thyroid drug propylthiouracil (WT-PTU mice). Analyses of microarray data indicated that the expressions of 150 genes were significantly altered between ThrbPV/PV and WT-PTU mice (87 genes had higher expression and 63 genes had lower expression in ThrbPV/PV mice than in WT-PTU mice). Thirty-six percent of genes with altered expression function as key regulators in metastasis. The remaining genes were involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/extracellular matrix signaling. The present studies have uncovered novel genes responsible for the metastatic spread of FTC and, furthermore, have shown that the metastatic process of thyroid cancer requires effective collaboration among genes with diverse cellular functions. Importantly, the present studies indicate that the tumor cells in the primary lesions are endowed with the genes destined to promote metastasis. Thus, our study has provided new insights into the understanding of the metastatic spread of human thyroid cancer.

Study of molecular actions of thyroid hormone receptor β (TRβ) mutants in vivo has been facilitated by creation of a mouse model (TRβPV mouse) that harbors a knockin mutant of TRβ (denoted PV). PV, which was identified in a patient with resistance to thyroid hormone, has lost T3 binding activity and transcription capacity. The striking phenotype of thyroid cancer exhibited by TRβPV/PV mice has allowed the elucidation of novel oncogenic activity of a TRβ mutant (PV) [PAS1]beyond nucleus-initiated transcription. PV was found to physically interact with the regulatory p85α subunit of phosphatidylinositol 3-kinase (PI3K) in both the nuclear and cytoplasmic compartments. This protein-protein interaction activates the PI3K signaling by increasing phosphorylation of AKT, mammalian target of rapamycin (mTOR), and p70S6K. PV, via interaction with p85α, also activates the PI3K-integrin-linked kinase-matrix metalloproteinase-2 signaling pathway in the extra-nuclear compartment. The PV-mediated PI3K activation results in increased cell proliferation, motility, migration, and metastasis.

In addition to affecting these membrane-initiated signaling events, PV affects [PAS2]the stability of the pituitary tumor-transforming gene (PTTG) product. PTTG (also known as securin), a critical mitotic checkpoint protein, is physically associated with TRβ or PV in vivo. Concomitant with T3-induced degradation of TRβ, PTTG is degraded by the proteasome machinery, but no such degradation occurs when PTTG is associated with PV. The degradation of PTTG/TRβ is activated by the direct interaction of the T3-bound TRβ with the steroid receptor coactivator-3 (SRC-3) that recruits a proteasome activator (PA28γ). PV that does not bind T3 cannot interact directly with SRC-3/PA28γ to activate proteasome degradation, and the absence of degradation results in an aberrant accumulation of PTTG. The PV-induced failure of timely degradation of PTTG results in mitotic abnormalities. PV, via novel protein-protein interaction and transcription regulation, acts to antagonize the functions of wild-type TRs and contributes to the oncogenic functions of this mutation.

Peroxisome proliferator-activated receptors (PPARs) and thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily. They are ligand-dependent transcription factors that interact with their cognate hormone response elements in the promoters to regulate respective target gene expression to modulate cellular functions. While the transcription activity of each is regulated by their respective ligands, recent studies indicate that via multiple mechanisms PPARs and TRs crosstalk to affect diverse biological functions. Here, we review recent advances in the understanding of the molecular mechanisms and biological impact of crosstalk between these two important nuclear receptors, focusing on their roles in adipogenesis and carcinogenesis.

To understand the roles of thyroid hormone receptors (TRs) in adipogenesis, we adopted a loss-of-function approach. We generated 3T3-L1 cells stably expressing either TRα1 mutant (TRα1PV) or TRβ1 mutant (TRβ1PV). TRα1PV and TRβ1PV are dominant negative mutations with a frameshift in the C-terminal amino acids. In control cells, the thyroid hormone, tri-iodothyronine (T3), induced a 2·5-fold increase in adipogenesis in 3T3-L1 cells, as demonstrated by increased lipid droplets. This increase was mediated by T3-induced expression of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), which are master regulators of adipogenesis at both the mRNA and protein levels. In 3T3-L1 cells stably expressing TRα1PV (L1-α1PV cells) or TRβ1PV (L1-β1PV cells), adipogenesis was reduced 94 or 54% respectively, indicative of differential inhibitory activity of mutant TR isoforms. Concordantly, the expression of PPARγ and C/EBPα at the mRNA and protein levels was more repressed in L1-α1PV cells than in L1-β1PV cells. In addition, the expression of PPARγ downstream target genes involved in fatty acid synthesis – the lipoprotein lipase (Lpl) and aP2 involved in adipogenesis – was more inhibited by TRα1PV than by TRβ1PV. Chromatin immunoprecipitation assays showed that TRα1PV was more avidly recruited than TRβ1PV to the promoter to preferentially block the expression of the C/ebpα gene. Taken together, these data indicate that impaired adipogenesis by mutant TR is isoform dependent. The finding that induction of adipogenesis is differentially regulated by TR isoforms suggests that TR isoform-specific ligands could be designed for therapeutic intervention for lipid abnormalities.

We previously showed that two thyroid hormone receptor (TR) isoforms – TRα1 and TRβ1 – differentially regulate thyroid hormone (triiodothyroxine, T3)-stimulated adipogenesis in vivo. This study aims to understand the role of the nuclear receptor corepressor, NCoR1, in TR isoform-dependent adipogenesis. We found that T3-stimulated adipogenesis of 3T3-L1 cells was accompanied by progressive loss of NCoR1 protein levels. In 3T3-L1 cells stably expressing a mutated TRα1, PV (L1-α1PV cells), the T3-stimulated adipogenesis was more strongly inhibited than that in 3T3-L1 cells stably expressing an identical mutation in TRβ1 (L1-β1PV cells). The stronger inhibition of adipogenesis in L1-α1PV cells was associated with a higher NCoR1 protein level. These results indicate that the degree of loss of NCoR1 correlates with the extent of adipogenesis. siRNA knockdown of NCoR1 promoted adipogenesis of control 3T3-L1 cells and reversed the inhibited adipogenesis of L1-α1PV and L1-β1PV cells, indicating that NCoR1 plays an essential role in TR isoform-dependent adipogenesis. An ubiquitin ligase, mSiah2, that targets NCoR1 for proteasome degradation was upregulated on day 1 before the onset of progressive loss of NCoR1. NCoR1 was found to associate with mSiah2 and with TR, TRα1PV, or TRβ1PV, but a stronger interaction of NCoR1 with TRα1PV than with TRβ1PV was detected. Furthermore, TRα1PV–NCoR1 complex was more avidly recruited than TRβ1PV–NCoR1 to the promoter of the C/ebpα gene, leading to more inhibition in its expression. These results indicate that differential interaction of NCoR1 with TR isoforms accounted for the TR isoform-dependent regulation of adipogenesis and that aberrant interaction of NCoR1 with TR could underlie the pathogenesis of lipid disorders in hypothyroidism.

For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid β-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid β-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism.

Akt activation is common in progressive thyroid cancer. In breast cancer, Akt1 induced primary cancer growth, but is reported to inhibit metastasis in vivo in several model systems. In contrast, clinical and in vitro studies suggest a metastasis-promoting role for Akt1 in thyroid cancer. The goal of this study was to determine the functional role of Akt1 in thyroid cancer growth and metastatic progression in vivo using thyroid hormone receptor βPV/PV knock-in (PV) mice which develop metastatic thyroid cancer. We crossed Akt1-/- and PV mice and compared tumor development, local progression, metastasis, and histology in TRβPV/PV/Akt1+/+ (PVPV-Akt1WT) and TRβPV/PV/Akt1-/- (PVPV-Akt1KO) mice. Mice were sacrificed at 3, 6, 9, 12, and 15 months; necropsy was performed and serum TSH was measured. Thyroid hyperplasia occurred in both groups beginning at three months; the thyroid size was greater in the PVPV-Akt1WT mice (p<0.001). In comparison with PVPV-Akt1WT mice, thyroid cancer development was delayed in the PVPV-Akt1KO mice (P=0.003) and the degree of tumor invasion was reduced. The PVPV-Akt1WT mice displayed pulmonary metastases at 12 and 15 months of age, by contrast PVPV-Akt1KO mice did not develop distant metastases at 15 months of age. Despite continued expression of Akt2 or Akt3, pAkt levels were decreased, and there was evidence of reduced Akt effect on p27 in the PVPV-Akt1KO thyroids. TSH levels were similarly elevated in PV mice regardless of Akt1 expression. In conclusion, thyroid cancer development and progression in TRβPV/PV mice are Akt1-dependent, consistent with a tumor progression-promoting role in this murine thyroid cancer model.

Selective drugs targeting dysregulated oncogenic pathways are promising cancer therapies. Because the mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in human follicular thyroid cancer (FTC), we hypothesized that its inhibition could block cancer development and progression. We, therefore, analyzed the effect of a treatment with a specific mTORC1 inhibitor (RAD001) in a faithful mouse model of FTC with constitutive mTORC1 activation (TRβPV/PVPten+/− mice). The treatment did not prevent capsular and vascular invasion of the thyroid and the occurrence of lung metastasis. However, it substantially decelerated thyroid tumor growth, thereby prolonging TRβPV/PVPten+/− mouse life span. RAD001 efficiently inhibited mTORC1 activity, as shown by the reduced phosphorylation of its downstream targets involved in the activity of the translation machinery, such as ribosomal S6 kinase (p70S6K), eukaryotic translation initiation factor 4E binding protein (4E-BP1) and the eukaryotic translation initiation factors eIF-4B and eIF-4G. Whereas mTORC1 signaling inhibition did not alter cell apoptosis, it induced a significant decrease in cell proliferation that was associated with the reduced abundance and altered activity of key regulators of cell cycle progression. Altogether, our data indicate that mTORC1 signaling plays a major role in the integration of the mitogenic signal in FTC. Therefore, our preclinical study with a relevant mouse model of FTC demonstrates for the first time that RAD001 efficaciously stabilizes cancer growth although it does not prevent its fatal outcome. In conclusion, our work underscores that in the treatment of FTC patients, RAD001 can only be used in combination with drugs and therapies inducing tumor shrinkage and blocking metastasis.

The thyroid hormone receptors (TRs) are transcription factors that mediate the pleiotropic activities of the thyroid hormone, T3. Four T3-binding isoforms, TRα1, TRβ1, TRβ2, and TRβ3, are encoded by two genes, THRA and THRB. Mutations and altered expression of TRs have been reported in human cancers. A targeted germline mutation of the Thrβ gene in the mouse leads to spontaneous development of follicular thyroid carcinoma (TRβPV/PV mouse). The TRβPV mutant has lost T3 binding activity and displays potent dominant negative activity. The striking phenotype of thyroid cancer exhibited by TRβPV/PV mice has recently led to the discovery of novel non-genomic actions of TRβPV that contribute to thyroid carcinogenesis. These actions involve direct physical interaction of TRβPV with cellular proteins, namely the regulatory subunit of the phosphatidylinositol 3-kinase (p85α), the pituitary tumor-transforming gene (PTTG) and β-catenin, that are critically involved in cell proliferation, motility, migration, and metastasis. Thus, a TRβ mutant (TRβPV), via a novel mode of non-genomic action, acts as an oncogene in thyroid carcinogenesis.

The thyroid hormone, T3, plays important roles in metabolism, growth, and differentiation. Germline mutations in thyroid hormone receptor beta (TRβ) have been identified in many individuals with resistance to thyroid hormone, a syndrome of reduced sensitivity to T3. A close association of somatic mutations of TRβ with several human cancers has become increasingly apparent, but how TRβ mutants could be involved in the carcinogenesis in vivo has not been addressed. The creation of a mouse model (TRβPV/PV mouse) that harbors a knockin mutation of TRβ (denoted TRβPV) has facilitated the study of the molecular actions of TRβ mutants in vivo. The striking phenotype of thyroid cancer and the development of pituitary tumors exhibited by TRβPV/PV mice have uncovered novel functions of a TRβ mutant in tumorigenesis. It led to the important findings that the oncogenic action of TRβPV is mediated by both genomic and non-genomic actions to alter gene expression and signaling pathways activity.

We previously created a knock-in mutant mouse harboring a dominantly negative mutant thyroid hormone receptor β (TRβPV/PV mouse) that spontaneously develops a follicular thyroid carcinoma similar to human thyroid cancer. We found that β-catenin, which plays a critical role in oncogenesis, was highly elevated in thyroid tumors of TRβPV/PV mice. We sought to understand the molecular basis underlying aberrant accumulation of β-catenin by mutations of TRβ in vivo. Cell-based studies showed that thyroid hormone (T3) induced the degradation of β-catenin in cells expressing TRβ via proteasomal pathways. In contrast, no T3-induced degradation occurred in cells expressing the mutant receptor (TRβPV). In vitro binding studies and cell-based analyses revealed that β-catenin physically associated with unliganded TRβ or TRβPV. However, in the presence of T3, β-catenin was dissociated from TRβ-β-catenin complexes but not from TRβPV-β-catenin complexes. β-Catenin signaling was repressed by T3 in TRβ-expressing cells through decreasing β-catenin-mediated transcription activity and target gene expression, whereas sustained β-catenin signaling was observed in TRβPV-expressing cells. The stabilization of β-catenin, via association with a mutated TRβ, represents a novel activating mechanism of the oncogenic protein β-catenin that could contribute to thyroid carcinogenesis in TRβPV/PV mice.

The nuclear receptor corepressor (NCoR) regulates the activities of DNA-binding transcription factors. Recent observations of its distribution in the extranuclear compartment raised the possibility that it could have other cellular functions in addition to transcription repression. We previously showed that phosphatidylinositol 3-kinase (PI3K) signaling is aberrantly activated by a mutant thyroid hormone β receptor (TRβPV, hereafter referred to as PV) via physical interaction with p85α, thus contributing to thyroid carcinogenesis in a mouse model of follicular thyroid carcinoma (TRβPV/PV mouse). Since NCoR is known to modulate the actions of TRβ mutants in vivo and in vitro, we asked whether NCoR regulates PV-activated PI3K signaling. Remarkably, we found that NCoR physically interacted with and competed with PV for binding to the C-terminal SH2 (Src homology 2) domain of p85α, the regulatory subunit of PI3K. Confocal fluorescence microscopy showed that both NCoR and p85α were localized in the nuclear as well as in the cytoplasmic compartments. Overexpression of NCoR in thyroid tumor cells of TRβPV/PV mouse reduced PI3K signaling, as indicated by the decrease in the phosphorylation of its immediate downstream effector, p-AKT. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells led to overactivated p-AKT and increased cell proliferation and motility. Furthermore, NCoR protein levels were significantly lower in thyroid tumor cells than in wild-type thyrocytes, allowing more effective binding of PV to p85α to activate PI3K signaling and thus contributing to tumor progression. Taken together, these results indicate that NCoR, via protein-protein interaction, is a novel regulator of PI3K signaling and could serve to modulate thyroid tumor progression.

Thyroid hormone (T3) is critical for growth, differentiation, and maintenance of metabolic homeostasis. Mice with a knock-in mutation in the thyroid hormone receptor α gene (TRα1PV) were created previously to explore the roles of mutated TRα1 in vivo. TRα1PV is a dominant negative mutant with a frameshift mutation in the carboxyl-terminal 14 amino acids that results in the loss of T3 binding and transcription capacity. Homozygous knock-in TRα1PV/PV mice are embryonic lethal, and heterozygous TRα1PV/+ mice display the striking phenotype of dwarfism. These mutant mice provide a valuable tool for identifying the defects that contribute to dwarfism. Here we show that white adipose tissue (WAT) mass was markedly reduced in TRα1PV/+ mice. The expression of peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipogenesis, was repressed at both mRNA and protein levels in WAT of TRα1PV/+ mice. Moreover, TRα1PV acted to inhibit the transcription activity of PPARγ by competition with PPARγ for binding to PPARγ response elements and for heterodimerization with the retinoid X receptors. The expression of TRα1PV blocked the T3-dependent adipogenesis of 3T3-L1 cells and repressed the expression of PPARγ. Thus, mutations of TRα1 severely affect adipogenesis via cross talk with PPARγ signaling. The present study suggests that defects in adipogenesis could contribute to the phenotypic manifestation of reduced body weight in TRα1PV/+ mice.

Overexpression of pituitary tumor–transforming 1 (PTTG1) is associated with thyroid cancer. We found elevated PTTG1 levels in the thyroid tumors of a mouse model of follicular thyroid carcinoma (TRβPV/PV mice). Here we examined the molecular mechanisms underlying elevated PTTG1 levels and the contribution of increased PTTG1 to thyroid carcinogenesis. We showed that PTTG1 was physically associated with thyroid hormone β receptor (TRβ) as well as its mutant, designated PV. Concomitant with thyroid hormone–induced (T3-induced) degradation of TRβ, PTTG1 proteins were degraded by the proteasomal machinery, but no such degradation occurred when PTTG1 was associated with PV. The degradation of PTTG1/TRβ was activated by the direct interaction of the liganded TRβ with steroid receptor coactivator 3 (SRC-3), which recruits proteasome activator PA28γ. PV, which does not bind T3, could not interact directly with SRC-3/PA28γ to activate proteasome degradation, resulting in elevated PTTG1 levels. The accumulated PTTG1 impeded mitotic progression in cells expressing PV. Our results unveil what we believe to be a novel mechanism by which PTTG1, an oncogene, is regulated by the liganded TRβ. The loss of this regulatory function in PV led to an aberrant accumulation of PTTG1 disrupting mitotic progression that could contribute to thyroid carcinogenesis.