Xiao Hui Liao

A novel syndrome combining thyroid and neurological abnormalities is associated with mutations in a monocarboxylate transporter gene

Thyroid hormones are iodothyronines that control growth and development, as well as brain function and metabolism. Although thyroid hormone deficiency can be caused by defects of hormone synthesis and action, it has not been linked to a defect in cellular hormone transport. In fact, the physiological role of the several classes of membrane transporters remains unknown. We now report, for the first time, mutations in the monocarboxylate transporter 8 (MCT8) gene, located on the X chromosome, that encodes a 613-amino acid protein with 12 predicted transmembrane domains. The propositi of two unrelated families are males with abnormal relative concentrations of three circulating iodothyronines, as well as neurological abnormalities, including global developmental delay, central hypotonia, spastic quadriplegia, dystonic movements, rotary nystagmus, and impaired gaze and hearing. Heterozygous females had a milder thyroid phenotype and no neurological defects. These findings establish the physiological importance of MCT8 as a thyroid hormone transporter.

Targeted Expression of BRAFV600E in Thyroid Cells of Transgenic Mice Results in Papillary Thyroid Cancers that Undergo Dedifferentiation

The BRAFT1799A mutation is the most common genetic alteration in papillary thyroid carcinomas (PTC). It is also found in a subset of papillary microcarcinomas, consistent with a role in tumor initiation. PTCs with BRAFT1799A are often invasive and present at a more advanced stage. BRAFT1799A is found with high prevalence in tall-cell variant PTCs and in poorly differentiated and undifferentiated carcinomas arising from PTCs. To explore the role of BRAFV600E in thyroid cancer pathogenesis, we targeted its expression to thyroid cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Two Tg-BRAFV600E lines (Tg-BRAF2 and Tg-BRAF3) were propagated for detailed analysis. Tg-BRAF2 and Tg-BRAF3 mice had increased thyroid-stimulating hormone levels (>7- and approximately 2-fold, respectively). This likely resulted from decreased expression of thyroid peroxidase, sodium iodine symporter, and thyroglobulin. All lines seemed to successfully compensate for thyroid dysfunction, as serum thyroxine/triiodothyronine and somatic growth were normal. Thyroid glands of transgenic mice were markedly enlarged by 5 weeks of age. In Tg-BRAF2 mice, PTCs were present at 12 and 22 weeks in 14 of 15 and 13 of 14 animals, respectively, with 83% exhibiting tall-cell features, 83% areas of invasion, and 48% foci of poorly differentiated carcinoma. Tg-BRAF3 mice also developed PTCs, albeit with lower prevalence (3 of 12 and 4 of 9 at 12 and 22 weeks, respectively). Tg-BRAF2 mice had a 30% decrease in survival at 5 months. In summary, thyroid-specific expression of BRAFV600E induces goiter and invasive PTC, which transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAF-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis.

Mutations in SECISBP2 result in abnormal thyroid hormone metabolism

Incorporation of selenocysteine (Sec), through recoding of the UGA stop codon, creates a unique class of proteins. Mice lacking tRNASec die in utero, but the in vivo role of other components involved in selenoprotein synthesis is unknown, and Sec incorporation defects have not been described in humans. Deiodinases (DIOs) are selenoproteins involved in thyroid hormone metabolism. We identified three of seven siblings with clinical evidence of abnormal thyroid hormone metabolism. Their fibroblasts showed decreased DIO2 enzymatic activity not linked to the DIO2 locus. Systematic linkage analysis of genes involved in DIO2 synthesis and degradation led to the identification of an inherited Sec incorporation defect, caused by a homozygous missense mutation in SECISBP2 (also called SBP2). An unrelated child with a similar phenotype was compound heterozygous with respect to mutations in SECISBP2. Because SBP2 is epistatic to selenoprotein synthesis, these defects had a generalized effect on selenoproteins. Incomplete loss of SBP2 function probably causes the mild phenotype.

Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation

Advanced human thyroid cancers, particularly those that are refractory to treatment with radioiodine (RAI), have a high prevalence of BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations. However, the degree to which these cancers are dependent on BRAF expression is still unclear. To address this question, we generated mice expressing one of the most commonly detected BRAF mutations in human papillary thyroid carcinomas (BRAF(V600E)) in thyroid follicular cells in a doxycycline-inducible (dox-inducible) manner. Upon dox induction of BRAF(V600E), the mice developed highly penetrant and poorly differentiated thyroid tumors. Discontinuation of dox extinguished BRAF(V600E) expression and reestablished thyroid follicular architecture and normal thyroid histology. Switching on BRAF(V600E) rapidly induced hypothyroidism and virtually abolished thyroid-specific gene expression and RAI incorporation, all of which were restored to near basal levels upon discontinuation of dox. Treatment of mice with these cancers with small molecule inhibitors of either MEK or mutant BRAF reduced their proliferative index and partially restored thyroid-specific gene expression. Strikingly, treatment with the MAPK pathway inhibitors rendered the tumor cells susceptible to a therapeutic dose of RAI. Our data show that thyroid tumors carrying BRAF(V600E) mutations are exquisitely dependent on the oncoprotein for viability and that genetic or pharmacological inhibition of its expression or activity is associated with tumor regression and restoration of RAI uptake in vivo in mice. These findings have potentially significant clinical ramifications.

Generation of functional thyroid from embryonic stem cells

The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of growth, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse embryonic stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion

The mechanism of thyroid hormone (TH) secretion from the thyroid gland into blood is unknown. Humans and mice deficient in monocarboxylate transporter 8 (MCT8) have low serum thyroxine (T4) levels that cannot be fully explained by increased deiodination. Here, we have shown that Mct8 is localized at the basolateral membrane of thyrocytes and that the serum TH concentration is reduced in Mct8-KO mice early after being taken off a treatment that almost completely depleted the thyroid gland of TH. Thyroid glands in Mct8-KO mice contained more non-thyroglobulin-associated T4 and triiodothyronine than did those in wild-type mice, independent of deiodination. In addition, depletion of thyroidal TH content was slower during iodine deficiency. After administration of 125I, the rate of both its secretion from the thyroid gland and its appearance in the serum as trichloroacetic acid-precipitable radioactivity was greatly reduced in Mct8-KO mice. Similarly, the secretion of T4 induced by injection of thyrotropin was reduced in Mct8-KO in which endogenous TSH and T4 were suppressed by administration of triiodothyronine. To our knowledge, this study is the first to demonstrate that Mct8 is involved in the secretion of TH from the thyroid gland and contributes, in part, to the low serum T4 level observed in MCT8-deficient patients.

Thyrotrophin receptor signaling dependence of Braf-induced thyroid tumor initiation in mice

Mutations of BRAF are found in ∼45% of papillary thyroid cancers and are enriched in tumors with more aggressive properties. We developed mice with a thyroid-specific knock-in of oncogenic Braf (LSL-BrafV600E/TPO-Cre) to explore the role of endogenous expression of this oncoprotein on tumor initiation and progression. In contrast to other Braf-induced mouse models of tumorigenesis (i.e., melanomas and lung), in which knock-in of BrafV600E induces mostly benign lesions, Braf-expressing thyrocytes become transformed and progress to invasive carcinomas with a very short latency, a process that is dampened by treatment with an allosteric MEK inhibitor. These mice also become profoundly hypothyroid due to deregulation of genes involved in thyroid hormone biosynthesis and consequently have high TSH levels. To determine whether TSH signaling cooperates with oncogenic Braf in this process, we first crossed LSL-BrafV600E/TPO-Cre with TshR knockout mice. Although oncogenic Braf was appropriately activated in thyroid follicular cells of these mice, they had a lower mitotic index and were not transformed. Thyroid-specific deletion of the Gsα gene in LSL-BrafV600E/TPO-Cre/Gnas-E1fl/fl mice also resulted in an attenuated cancer phenotype, indicating that the cooperation of TshR with oncogenic Braf is mediated in part by cAMP signaling. Once tumors were established in mice with wild-type TshR, suppression of TSH did not revert the phenotype. These data demonstrate the key role of TSH signaling in Braf-induced papillary thyroid cancer initiation and provide experimental support for recent observations in humans pointing to a strong association between TSH levels and thyroid cancer incidence.

Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo.

Thyroid hormone action is mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. DNA-binding is presumed to be essential for all nuclear actions of thyroid hormone. To test this hypothesis in vivo, the DNA-binding domain of TR-beta was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-beta DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homozygous mutant (TR-betaGS/GS) mice displayed abnormal T3 regulation of the hypothalamic-pituitary-thyroid axis and retina identical to abnormalities previously observed in TR-beta KO (TR-beta-/-) mice. However, TR-betaGS/GS mutant mice maintained normal hearing at certain frequencies and did not display significant outer hair cell loss, in contrast to TR-beta-/- mice. DNA-binding, therefore, is essential for many functions of the TR, including retinal development and negative feedback regulation by thyroid hormone of the hypothalamic-pituitary-thyroid axis. Inner ear development, although not completely normal, can occur in the absence of TR DNA-binding, suggesting that an alternative and perhaps novel thyroid hormone-signaling pathway may mediate these effects.

Oncogenic Kras Requires Simultaneous PI3K Signaling to Induce ERK Activation and Transform Thyroid Epithelial Cells In vivo

Thyroid tumors arising from the follicular cells often harbor mutations leading to the constitutive activation of the PI3K and Ras signaling cascades. However, it is still unclear what their respective contribution to the neoplastic process is, as well as to what extent they interact. We have used mice harboring a Kras oncogenic mutation and a Pten deletion targeted to the thyroid epithelium to address in vivo these questions. Here, we show that although each of these two pathways, alone, is unable to transform thyroid follicular cells, their simultaneous activation is highly oncogenic, leading to invasive and metastatic follicular carcinomas. In particular, phosphatidylinositol-3-kinase (PI3K) activation suppressed Kras-initiated feedback signals that uncouple mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and ERK activation, thus stunting MAPK activity; in addition, PI3K and Kras cooperated to drastically up-regulate cyclin D1 mRNA levels. Finally, combined pharmacologic inhibition of PI3K and MAPK completely inhibited the growth of double-mutant cancer cell lines, providing a compelling rationale for the dual targeting of these pathways in thyroid cancer.

Dominant Role of Thyrotropin-releasing Hormone in the Hypothalamic-Pituitary-Thyroid Axis

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the β isoforms of TH receptors (TRβ KO), or both (double KO). TRβ knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.