Global expression profiling reveals gain-of-function oncogenic activity of a mutated thyroid hormone receptor in thyroid carcinogenesis.
ABSTRACT: 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) (Thrb(PV/PV) mice) was used in the present study. The Thrb(PV/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 Thrb(PV/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 Thrb(PV/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 Thrb(PV/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.
Project description:<h4>Background</h4>Over the last decade, annual incidence rates for thyroid cancer have been among the highest of all cancers in the Western world. However, the genomic mechanisms impacting thyroid carcinogenesis remain elusive.<h4>Methods</h4>We employed an established mouse model of follicular thyroid cancer (FTC) with a homozygous proline to valine mutation (Thrb(PV/PV)) in the thyroid receptor ?1 (TR?1) and applied quantitative three-dimensional (3D) telomere analysis to determine 3D telomeric profiles in Thrb(PV)(/PV), Thrb(PV/)(+), and Thrb(+/+) mouse thyrocytes before and after histological presentation of FTC.<h4>Results</h4>Using quantitative fluorescent in situ hybridization (Q-FISH) and TeloView™ image analysis, we found altered telomeric signatures specifically in mutant mouse thyrocytes. As early as 1 month of age, Thrb(PV/PV) mouse thyrocytes showed more telomeres than normal and heterozygous age-matched counterparts. Importantly, at the very early age of 1 month, 3D telomeric profiles of Thrb(PV/PV) thyrocyte nuclei reveal genetic heterogeneity with several nuclei populations exhibiting different telomere numbers, suggestive of various degrees of aneuploidy within the same animal. This was detected exclusively in Thrb(PV/PV) mice well before the presentation of histological signs of thyroid carcinoma.<h4>Conclusions</h4>We identified quantitative 3D telomere analysis as a novel tool for early detection and monitoring of thyrocyte chromosomal (in)stability. This technique has the potential to identify human patients at risk for developing thyroid carcinoma.
Project description:That a knock-in mouse harboring a dominant-negative thyroid hormone receptor (TR)-? (Thrb) mutation develops metastatic thyroid cancer strongly suggests the involvement of TR? in carcinogenesis. Epigenetic silencing of the THRB gene is common in human cancers. The aim of the present study was to determine how DNA methylation affected the expression of the THRB gene in differentiated thyroid cancer (DTC) and how reexpression of the THRB gene attenuated the cancer phenotypes. We used methylation-specific PCR to examine the expression and promoter methylation of the THRB gene in DTC tissues. Thyroid cancer cells with hypermethylated THRB were treated with the demethylating agents 5'-aza-2'-deoxycytidine (5'-aza-CdR) and zebularine to evaluate their impact on the cancer cell phenotypes. THRB mRNA expression in DTC was 90% lower than in normal controls, and this decrease was associated with a higher tumor/lymph node staging. The promoter methylation level of the THRB gene had a significant negative correlation with the expression level of the THRB gene. Treatment of FTC-236 cells with 5'-aza-CdR or zebularine induced reexpression of the THRB gene and inhibited cell proliferation and migration. FTC-236 cells stably expressing TR? exhibited lower cell proliferation and migration through inhibition of ?-catenin signaling pathways compared with FTC-236 without TR?. 5'-Aza-CdR also led to suppression of tumor growth in an in vivo xenograft model using FTC-236 cells consistent with the cell-based studies. These finding indicate that TR? is a tumor suppressor and could be tested as a potential therapeutic target.
Project description: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 (Thrb(PV/PV) mice). Our recent studies show that the highly elevated level of thyroid stimulating hormone (TSH) in Thrb(PV/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 Thrb(PV/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 Thrb(PV/PV) and WT-PTU mice (87 genes had higher expression and 63 genes had lower expression in Thrb(PV/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.
Project description:Undifferentiated thyroid carcinoma is one of the most aggressive human cancers. Although genetic changes underlying this aggressive cancer remain to be elucidated, RAS mutations have been frequently identified in it. Mice harboring a mutant thyroid hormone receptor Thrb(PV) (Thrb(PV/PV) ) spontaneously develop differentiated follicular thyroid carcinoma similar to human thyroid cancer. We recently demonstrated that targeting a RAS mutation (Kras(G12D) ) to the thyroid of Thrb(PV/PV) mice (Thrb(PV/PV) Kras(G12D) mice) promotes initiation and progression of undifferentiated thyroid cancer. To uncover genes destined to drive the aggressive cancer phenotype, we used cDNA microarrays to compare the gene expression profiles of thyroid cells of Kras(G12D) mice and thyroid tumor lesions of Thrb(PV/PV) and Thrb(PV/PV) Kras(G12D) mice. Analyses of microarray data identified 14 upstream regulators that were significantly altered in thyroid tumors of Thrb(PV/PV) and Thrb(PV/PV) Kras(G12D) mice. Most of these genes with altered expression function as key regulators in growth factor-induced signaling. Further analysis identified gene expression profiles of markedly elevated integrin levels, acting as upstream activators to stimulate ERBB2-mediated downstream signaling in thyroid tumors of Thrb(PV/PV) Kras(G12D) mice. The present studies uncovered integrin-activated ERBB2 signaling as one of the mechanisms in synergy between TR?PV and KRASG12D signaling to promote aggressive tumor growth in undifferentiated thyroid cancer.
Project description:Thyroid hormone (T(3)) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance, but the signaling pathways mediating these effects are poorly understood. Thrb(PV/PV) mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T(3) receptor (TR?(PV)) that cannot bind T(3) and interferes with the actions of wild-type TR. Thrb(PV/PV) mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and Thrb(PV/PV) mice. Activation of the canonical Wnt signaling in Thrb(PV/PV) mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during postnatal growth. By contrast, T(3) treatment inhibited Wnt signaling in osteoblastic cells, suggesting that T(3) inhibits the Wnt pathway by facilitating proteasomal degradation of ?-catenin and preventing its accumulation in the nucleus. Activation of the Wnt pathway in Thrb(PV/PV) mice, however, results from a gain of function for TR?(PV) that stabilizes ?-catenin despite the presence of increased thyroid hormone levels. These studies demonstrate novel interactions between T(3) and Wnt signaling pathways in the regulation of skeletal development and bone formation.
Project description:Thyroid cancers are the most common malignancy of the endocrine system in humans. To understand the molecular genetic events underlying thyroid carcinogenesis, we have generated a mouse model that spontaneously develops follicular thyroid carcinoma similar to human thyroid cancer (Thrb(PV/PV) mouse). This mutant mouse harbors a dominant-negative mutated thyroid hormone receptor ? (denoted PV). The PV mutation was identified in a patient with resistance to thyroid hormone (TH). Thrb(PV/PV) mice exhibit highly elevated serum thyroid-stimulating hormone levels and increased TH. We have previously shown that thyroid-stimulating hormone is required, but not sufficient to induce metastatic follicular thyroid cancer in Thrb(PV/PV) mice. However, whether the elevated TH also contributes to the thyroid carcinogenesis of Thrb(PV/PV) mice was not elucidated. To understand the role of TH in thyroid carcinogenesis, we blocked the production of TH by treating Thrb(PV/PV) mice with propylthiouracil (Thrb(PV/PV)-PTU mice) and compared the development of thyroid cancer in Thrb(PV/PV)-PTU and untreated Thrb(PV/PV) mice. We found that thyroid tumor growth was reduced by ?42% in Thrb(PV/PV)-PTU mice as compared with Thrb(PV/PV) mice. Analysis by bromodeoxyuridine-nuclear labeling showed decreased incorporation of bromodeoxyuridine in thyroid tumor cells of Thrb(PV/PV)-PTU mice, indicative of decreased tumor cell proliferation. However, cleaved-caspase 3 staining showed no apparent changes in apoptosis of tumor cells in Thrb(PV/PV)-PTU mice. Molecular studies identified a marked attenuation of the PI3K-AKT-?-catenin signaling pathway that led to decreased protein levels of cyclin D2, thereby decreasing tumor cell proliferation in Thrb(PV/PV)-PTU mice. Furthermore, matrix metalloproteinase-2, a downstream target of ?-catenin and a key regulator during tumor invasion and metastasis, was also decreased. Thus, the present study uncovers a critical role of TH in promoting the thyroid carcinogenesis of Thrb(PV/PV) mice via membrane signaling events. Importantly, these findings suggest that anti-thyroid drugs could be considered as possible therapeutic agents of thyroid cancer.
Project description:Correlative data suggest that thyroid hormone receptor-? (TR?) mutations could increase the risk of mammary tumor development, but unequivocal evidence is still lacking. To explore the role of TR? mutants in vivo in breast tumor development and progression, we took advantage of a knock-in mouse model harboring a mutation in the Thrb gene encoding TR? (Thrb(PV) mouse). Although in adult nulliparous females, a single ThrbPV allele did not contribute to mammary gland abnormalities, the presence of two ThrbPV alleles led to mammary hyperplasia in ?36% Thrb(PV/PV) mice. The ThrbPV mutation further markedly augmented the risk of mammary hyperplasia in a mouse model with high susceptibility to mammary tumors (Pten(+/-) mouse), as demonstrated by the occurrence of mammary hyperplasia in ?60% of Thrb(PV/+)Pten(+/-) and ?77% of Thrb(PV/PV)Pten(+/-) mice versus ?33% of Thrb(+/+)Pten(+/-) mice. The Thrb(PV) mutation increased the activity of signal transducer and activator of transcription (STAT5) to increase cell proliferation and the expression of the STAT5 target gene encoding ?-casein in the mammary gland. We next sought to understand the molecular mechanism underlying STAT5 overactivation by TR?PV. Cell-based studies with a breast cancer cell line (T47D cells) showed that thyroid hormone (T3) repressed STAT5 signaling in TR?-expressing cells through decreasing STAT5-mediated transcription activity and target gene expression, whereas sustained STAT5 signaling was observed in TR?PV-expressing cells. Collectively, these findings show for the first time that a TR? mutation promotes the development of mammary hyperplasia via aberrant activation of STAT5, thereby conferring a fertile genetic ground for tumorigenesis.
Project description:Studies using mice deficient in thyroid hormone receptors (TR) indicate that the two TR isoforms, TR?1 and TR?1, in addition to mediating overlapping biological activities of the thyroid hormone, T3, also mediate distinct functions. Mice harboring an identical dominant negative mutation (denoted PV) at the C terminus of TR?1 (Thra1(PV) mice) or ?1 (Thrb(PV) mice) also exhibit distinct phenotypes. These knockin mutant mice provide an opportunity to understand the molecular basis of isoform-dependent functions in vivo. Here we tested the hypothesis that the distinct functions of TR mutant isoforms are directed by a subset of nuclear regulatory proteins. Tandem-affinity chromatography of HeLa nuclear extracts showed that distinct 33 nuclear proteins including nuclear receptor corepressor (NCoR1) and six other proteins preferentially associated with TR?1PV or TR?1PV, respectively. These results indicate that recruitment of nuclear regulatory proteins by TR mutants is subtype dependent. The involvement of NCoR1 in mediating the distinct liver phenotype of Thra1(PV) and Thrb(PV) mice was further explored. NCoR1 preferentially interacted with TR?1PV rather than with TR?1PV. NCoR1 was recruited more avidly to the thyroid hormone response element-bound TR?1PV than to TR?1PV in the promoter of the CCAAT/enhancer-binding protein ? gene to repress its expression in the liver of Thra1(PV) mice, but not in Thrb(PV) mice. This preferential recruitment of NCoR1 by mutant isoforms could contribute, at least in part, to the distinct liver lipid phenotype of these mutant mice. The present study highlights a novel mechanism by which TR isoforms direct their selective functions via preferential recruitment of a subset of nuclear coregulatory proteins.
Project description:<h4>Background</h4>The thyroid hormone triiodothyronine (T3) is critical for vertebrate development and affects the function of many adult tissues and organs. Its genomic effects are mediated by thyroid hormone nuclear receptors (TRs) present in all vertebrates. The discovery of patients with resistance to thyroid hormone (RTH?) >50 years ago and subsequent identification of genetic mutations in only the THRB gene in these patients suggest that mutations in the THRA gene may have different pathological manifestations in humans. Indeed, the recent discovery of a number of human patients carrying heterozygous mutations in the THRA gene (RTH?) revealed a distinct phenotype that was not observed in RTH patients with THRB gene mutations (RTH?). That is, RTH? patients have constipation, implicating intestinal defects caused by THRA gene mutations.<h4>Methods</h4>To determine how TR?1 mutations affect the intestine, this study analyzed a mutant mouse expressing a strong dominantly negative TR?1 mutant (denoted TR?1PV; Thra1<sup>PV</sup> mice). This mutant mouse faithfully reproduces RTH? phenotypes observed in patients.<h4>Results</h4>In adult Thra1<sup>PV/+</sup> mice, constipation was observed just like in patients with TR? mutations. Importantly, significant intestinal defects were discovered, including shorter villi and increased differentiated cells in the crypt, accompanied by reduced stem-cell proliferation in the intestine.<h4>Conclusions</h4>The findings suggest that further analysis of this mouse model should help to reveal the molecular and physiological defects in the intestine caused by TR? mutations and to determine the underlying mechanisms.
Project description:Thyroid cancer is the most common endocrine malignancy. Although an association between inflammation and thyroid cancer has long been recognized, a cause-effect relationship at the molecular level has yet to be elucidated. We explored how inflammation could contribute to thyroid carcinogenesis in <i>Thrb<sup>PV/PV</sup>Pten<sup>+/-</sup></i> mice. The <i>Thrb<sup>PV/PV</sup>Pten<sup>+/-</sup></i> mouse expresses a dominantly negative thyroid hormone receptor ? (denoted as PV) and a deletion of one single allele of the Pten gene. This mutant mouse exhibits aggressive follicular thyroid cancer similarly as in patients. We found significantly increased infiltration of inflammatory monocytes in thyroid tumors of <i>Thrb<sup>PV/PV</sup>Pten<sup>+/-</sup></i> mice, while no apparent changes in monocyte homeostasis in the bone marrow and blood of tumor-bearing mice. Using global gene expression profiling, we found altered expression of inflammation mediators in that the expression of Ptgs1, Sphk1, OPN, Chil1, Tnfrsf18, IL6, and Ccl12 genes was significantly increased and expression of Kit, Ly96, Ephx2, CD163, IL15, and Ccr2 was significantly decreased. Subsequent validation of the gene expression by mRNA analysis prompted us to further delineate the inflammatory role of osteopontin (OPN) in thyroid carcinogenesis because of its critical role in monocyte/macrophage functions and proinflammatory responses. We found that the protein abundance of OPN and its receptor, integrin ?1, was highly increased and, concurrently, the downstream effectors AKT and NF-?B were significantly elevated to drive thyroid tumor progression of <i>Thrb<sup>PV/PV</sup>Pten<sup>+/-</sup></i> mice. These results demonstrated that increased inflammation driven by elevated expression of immune-related genes and cytokines promoted thyroid cancer progression. Importantly, we uncovered OPN as a novel regulator in inflammatory response during thyroid carcinogenesis. These preclinical findings suggested that OPN can be a potential target for thyroid cancer therapy via modulation of inflammatory signaling.