Tissue specificity of oncogenic BRAF targeted to lung and thyroid through a shared lineage factor.

Cells of origin in cancer determine tumor phenotypes, but whether lineage-defining transcription factors might influence tissue specificity of tumorigenesis among organs with similar developmental traits are unknown. We demonstrate here that tumor development and progression markedly differ in lung and thyroid targeted by Braf mutation in Nkx2.1CreERT2 mice heterozygous for Nkx2-1. In absence of tamoxifen, non-induced Nkx2.1CreERT2;BrafCA/+ mutants developed multiple full-blown lung adenocarcinomas with a latency of 1-3 months whereas thyroid tumors were rare and constrained, although minute BrafCA activation documented by variant allele sequencing was similar in both tissues. Induced oncogene activation accelerated neoplastic growth only in the lungs. By contrast, NKX2-1+ progenitor cells were equally responsive to constitutive expression of mutant Braf during lung and thyroid development. Both lung and thyroid cells transiently downregulated NKX2-1 in early tumor stages. These results indicate that BRAFV600E-induced tumorigenesis obey organ-specific traits that might be differentially modified by a shared lineage factor.

Tissue architecture delineates field cancerization in BRAFV600E-induced tumor development.

Cancer cells hijack developmental growth mechanisms but whether tissue morphogenesis and architecture modify tumorigenesis is unknown. Here, we characterized a new mouse model of sporadic thyroid carcinogenesis based on inducible expression of BRAF carrying a Val600 Glu (V600E) point mutation (BRAFV600E) from the thyroglobulin promoter (TgCreERT2). Spontaneous activation of this Braf-mutant allele due to leaky activity of the Cre recombinase revealed that intrinsic properties of thyroid follicles determined BRAF-mutant cell fate. Papillary thyroid carcinomas developed multicentrically within a normal microenvironment. Each tumor originated from a single follicle that provided a confined space for growth of a distinct tumor phenotype. Lineage tracing revealed oligoclonal tumor development in infancy and early selection of BRAFV600E kinase inhibitor-resistant clones. Somatic mutations were few, non-recurrent and limited to advanced tumors. Female mice developed larger tumors than males, reproducing the gender difference of human thyroid cancer. These data indicate that BRAFV600E-induced tumorigenesis is spatiotemporally regulated depending on the maturity and heterogeneity of follicles. Moreover, thyroid tissue organization seems to determine whether a BRAF-mutant lineage becomes a cancerized lineage. The TgCreERT2;BrafCA/+ sporadic thyroid cancer mouse model provides a new tool to evaluate drug therapy at different stages of tumor evolution.

Asynchrony of Apical Polarization, Luminogenesis, and Functional Differentiation in the Developing Thyroid Gland.

Follicular thyroid tissue originates from progenitors derived from a midline endodermal primordium. Current understanding infers that folliculogenesis in the embryonic thyroid designates the latest morphogenetic event taking place after the final anatomical shape and position of the gland is established. However, this concept does not consider the fact that the thyroid isthmus develops chronologically before the lobes and also contains all progenitors required for lobulation. To elucidate whether cells committed to a thyroid fate might be triggered to differentiate asynchronously related to maturation and developmental stage, mouse embryonic thyroid tissues from E12.5-17.5 were subjected to immunofluorescent labeling of biomarkers (progenitors: NKX2-1; differentiation: thyroglobulin/TG); folliculogenesis: E-cadherin/CDH1; luminogenesis: mucin 1/MUC1; apical polarity: pericentrin/PCNT; basement membrane: laminin; growth: Ki67), quantitative RT-PCR analysis (Nkx2.1, Tg, Muc1) and transmission electron microscopy. Tg expression was detectable as early as E12.5 and gradually increased >1000-fold until E17.5. Muc1 and Nkx2.1 transcript levels increased in the same time interval. Prior to lobulation (E12.5-13.5), MUC1 and TG distinguished pre-follicular from progenitor cells in the developing isthmus characterized by intense cell proliferation. Luminogenesis comprised redistribution of MUC1+ vesicles or vacuoles, transiently associated with PCNT, to the apical cytoplasm and the subsequent formation of MUC1+ nascent lumens. Apical polarization of pre-follicular cells and lumen initiation involved submembraneous vesicular traffic, reorganization of adherens junctions and ciliogenesis. MUC1 did not co-localize with TG until a lumen with a MUC1+ apical membrane was established. MUC1 delineated the lumen of all newly formed follicles encountered in the developing lobes at E15.5-17.5. Folliculogenesis started before establishment of a complete follicular basal lamina. These observations indicate that embryonic thyroid differentiation is an asynchronous process consistent with the idea that progenitors attaining a stationary position in the connecting isthmus portion undergo apical polarization and generate follicles already at a primordial stage of thyroid development, i.e. foregoing growth of the lobes. Although the thyroid isthmus eventually comprises minute amounts of the total thyroid volume and contributes little to the overall hormone production, it is of principal interest that local cues related to the residence status of cells - independently of a prevailing high multiplication rate - govern the thyroid differentiation program.

ATR is a MYB regulated gene and potential therapeutic target in adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a rare cancer that preferentially occurs in the head and neck, breast, as well as in other sites. It is an aggressive cancer with high rates of recurrence and distant metastasis. Patients with advanced disease are generally incurable due to the lack of effective systemic therapies. Activation of the master transcriptional regulator MYB is the genomic hallmark of ACC. MYB activation occurs through chromosomal translocation, copy number gain or enhancer hijacking, and is the key driving event in the pathogenesis of ACC. However, the functional consequences of alternative mechanisms of MYB activation are still uncertain. Here, we show that overexpression of MYB or MYB-NFIB fusions leads to transformation of human glandular epithelial cells in vitro and results in analogous cellular and molecular consequences. MYB and MYB-NFIB expression led to increased cell proliferation and upregulation of genes involved in cell cycle control, DNA replication, and DNA repair. Notably, we identified the DNA-damage sensor kinase ATR, as a MYB downstream therapeutic target that is overexpressed in primary ACCs and ACC patient-derived xenografts (PDXs). Treatment with the clinical ATR kinase inhibitor VX-970 induced apoptosis in MYB-positive ACC cells and growth inhibition in ACC PDXs. To our knowledge, ATR is the first example of an actionable target downstream of MYB that could be further exploited for therapeutic opportunities in ACC patients. Our findings may also have implications for other types of neoplasms with activation of the MYB oncogene.

Linking loss of sodium-iodide symporter expression to DNA damage.

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1nM) calicheamicin blocked NIS mRNA expression and transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress.

Revising the embryonic origin of thyroid C cells in mice and humans.

Current understanding infers a neural crest origin of thyroid C cells, the major source of calcitonin in mammals and ancestors to neuroendocrine thyroid tumors. The concept is primarily based on investigations in quail-chick chimeras involving fate mapping of neural crest cells to the ultimobranchial glands that regulate Ca(2+) homeostasis in birds, reptiles, amphibians and fishes, but whether mammalian C cell development involves a homologous ontogenetic trajectory has not been experimentally verified. With lineage tracing, we now provide direct evidence that Sox17+ anterior endoderm is the only source of differentiated C cells and their progenitors in mice. Like many gut endoderm derivatives, embryonic C cells were found to coexpress pioneer factors forkhead box (Fox) a1 and Foxa2 before neuroendocrine differentiation takes place. In the ultimobranchial body epithelium emerging from pharyngeal pouch endoderm in early organogenesis, differential Foxa1/Foxa2 expression distinguished two spatially separated pools of C cell precursors with different growth properties. A similar expression pattern was recapitulated in medullary thyroid carcinoma cells in vivo, consistent with a growth-promoting role of Foxa1. In contrast to embryonic precursor cells, C cell-derived tumor cells invading the stromal compartment downregulated Foxa2, foregoing epithelial-to-mesenchymal transition designated by loss of E-cadherin; both Foxa2 and E-cadherin were re-expressed at metastatic sites. These findings revise mammalian C cell ontogeny, expand the neuroendocrine repertoire of endoderm and redefine the boundaries of neural crest diversification. The data further underpin distinct functions of Foxa1 and Foxa2 in both embryonic and tumor development.

Role of EphA4 receptor signaling in thyroid development: regulation of folliculogenesis and propagation of the C-cell lineage.

Transcriptome analysis revealed that the tyrosine kinase receptor EphA4 is enriched in the thyroid bud in mouse embryos. We used heterozygous EphA4-EGFP knock-in mice in which enhanced green fluorescent protein (EGFP) replaced the intracellular receptor domain (EphA4(+/EGFP)) to localize EphA4 protein in thyroid primordial tissues. This showed that thyroid progenitors originating in the pharyngeal floor express EphA4 at all embryonic stages and when follicles are formed in late development. Also, the ultimobranchial bodies developed from the pharyngeal pouch endoderm express EphA4, but the ultimobranchial epithelium loses the EGFP signal before it merges with the median thyroid primordium. Embryonic C cells invading the thyroid are exclusively EphA4-negative. EphA4 expression continues in the adult thyroid. EphA4 knock-out mice and EphA4-EGFP homozygous mutants are euthyroid and have a normal thyroid anatomy but display subtle histological alterations regarding number, size, and shape of follicles. Of particular interest, the pattern of follicular abnormality differs between EphA4(-/-) and EphA4(EGFP/EGFP) thyroids. In addition, the number of C cells is reduced by >50% exclusively in animals lacking EphA4 forward signaling (EphA4(EGFP/EGFP)). Heterozygous EphA4 mutants have no apparent thyroid phenotype. We conclude that EphA4 is a novel regulator of thyroid morphogenesis that impacts on postnatal development of the two endocrine cell lineages of the differentiating gland. In this process both EphA4 forward signaling (in the follicular epithelium) and reverse signaling mediated by its cognate ligand(s) (A- and/or B-ephrins expressed in follicular cells and C cells, respectively) are probably functionally important.

Radiation-induced thyroid stunning: differential effects of (123)I, (131)I, (99m)Tc, and (211)At on iodide transport and NIS mRNA expression in cultured thyroid cells.

UNLABELLED: Recent clinical and experimental data demonstrate that thyroid stunning is caused by previous irradiation and may influence the efficacy of (131)I radiation therapy of thyroid cancer and possibly hyperthyroidism. To avoid stunning, many clinics have exchanged (131)I for (123)I for pretherapeutic diagnostic imaging and dose planning. Furthermore, recent in vitro studies indicate that (131)I irradiation reduces iodide uptake by downregulating the expression of the sodium iodide symporter (NIS). The rationale for this study was therefore to study effects on iodide transport and NIS messenger RNA (mRNA) expression in thyrocytes exposed to both (123)I and (131)I in addition to some other potentially interesting radionuclides. METHODS: Thyrotropin-stimulated thyroid cell monolayers were exposed to 0.5 Gy of (123)I, (131)I, (99m)Tc, or (211)At, all being radionuclides transported via NIS, in the culture medium for 6 h, or to various absorbed doses of (123)I or (131)I for 48 h. NIS mRNA expression was analyzed using quantitative reverse-transcriptase polymerase chain reaction. RESULTS: Iodide transport and NIS mRNA expression were reduced by all radionuclides. At the same absorbed dose, iodide transport was reduced the most by (211)At, followed by (123)I and (99m)Tc (equally potent), whereas (131)I was least effective. The onset of NIS downregulation was rapid (<1 d after irradiation) in cells exposed to (123)I or (211)At and was delayed in cells irradiated with (131)I or (99m)Tc. Iodide transport and NIS expression were recovered only for (211)At. (123)I reduced the iodine transport and the NIS mRNA expression more efficiently than did (131)I at an equivalent absorbed dose, with a relative biological effectiveness of about 5. CONCLUSION: The stunning effect per unit absorbed dose is more severe for (123)I than for (131)I. Despite the lower absorbed dose per unit activity for (123)I than for (131)I, stunning by (123)I cannot be excluded in patients. The degree to which iodide transport capacity and NIS mRNA expression are reduced seems to be related to the biological effectiveness of the type of radiation delivering the absorbed dose to the target, with (211)At (which has the highest relative biological effectiveness) causing the highest degree of stunning per unit absorbed dose in the present study.

Expression of Islet1 in thyroid development related to budding, migration, and fusion of primordia.

The LIM homeodomain transcription factor Isl1 was investigated in mouse thyroid organogenesis. All progenitor cells of the midline thyroid diverticulum and lateral primordia (ultimobranchial bodies) expressed Isl1. This pattern persisted until the growing anlagen fused at embryonic day (E) 13.5. In Isl1 null mutants thyroid progenitors expressing Nkx2.1 and Pax8 were readily specified in the anterior endoderm but the size of the thyroid rudiment was reduced. In late development, only immature C-cells expressed Isl1. In the adult gland the number of Isl1+ cells was small compared with cells expressing calcitonin. Analysis of microarray profiles indicated a higher level of Isl1 expression in medullary thyroid carcinomas than in tumors derived from follicular cells. Together, these findings suggest that Isl1 may be a novel regulator of thyroid development before terminal differentiation of the endocrine cell types. Isl1 is an embryonic C-cell precursor marker that may be relevant also in cancer developed from the mature C-cell.

Down-regulation of the sodium/iodide symporter explains 131I-induced thyroid stunning.

(131)I radiation therapy of differentiated thyroid cancer may be compromised by thyroid stunning (i.e., a paradoxical inhibition of radioiodine uptake caused by radiation from a pretherapeutic diagnostic examination). The stunning mechanism is yet uncharacterized at the molecular level. We therefore investigated whether the expression of the sodium/iodide symporter (NIS) gene is changed by irradiation using (131)I. Confluent porcine thyroid cells on filter were stimulated with thyroid-stimulating hormone (TSH; 1 milliunit/mL) or insulin-like growth factor-I (IGF-I; 10 ng/mL) and simultaneously exposed to (131)I in the culture medium for 48 h, porcine NIS mRNA was quantified by real-time reverse transcription-PCR using 18S as reference, and transepithelial iodide transport was monitored using (125)I(-) as tracer. TSH increased the NIS expression >100-fold after 48 h and 5- to 20-fold after prolonged stimulation. IGF-I enhanced the NIS transcription at most 15-fold but not until 5 to 7 days. (131)I irradiation (7.5 Gy) decreased both TSH-stimulated and IGF-I-stimulated NIS transcription by 60% to 90% at all investigated time points. TSH and IGF-I stimulated NIS synergistically 15- to 60-fold after 5 days. NIS expression was reduced by (131)I also in costimulated cells, but the transcription level remained higher than in nonirradiated cells stimulated with TSH alone. Changes in NIS mRNA always correlated with altered (125)I(-) transport in cultures with corresponding treatments. It is concluded that down-regulation of NIS is the likely explanation of (131)I-induced thyroid stunning. Enhanced NIS expression by synergistically acting agents (TSH and IGF-I) partly prevents the loss of iodide transport expected from a given absorbed dose, suggesting that thyroid stunning might be pharmacologically treatable.