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 Ca2+ 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.

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 DNAdamage 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 MYBpositive 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.

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.

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. Developmental Dynamics 237:3820 -3829, 2008.

Abstract131 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 À 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

A possible role of sonic hedgehog (Shh) in recruitment of C cell precursors to the Ultimobranchial Body (UB) and embryonic thyroid was investigated in Shh -/-mice. Nkx2.1 and Foxa2 co-expression distinguished UB originating in the fourth pharyngeal pouch from other derivatives of pharyngeal endoderm. In mutants UB formed a single structure that failed to bud and instead of fusing with the midline thyroid primordium adhered to the thymic rudiments. Mature C cells appeared in the UB remnant and ectopically in the thymic parenchyma, foregut endoderm and trachea. Thyroid did not contain C cells except minute numbers close to the tracheal interface. Tracing progeny in Shh-CRE/Rosa26R mice showed the vast majority of both UB and thyroid progenitors derived from Shh negative endoderm, but Shh expressing cells appeared in both thyroid primordia before fusion of the two. The findings indicate that Shh determines the endoderm territory for C cell differentiation and guides the migration of C cell precursors into the thyroid, presumably by regulating the separation of glandular domains in the pharyngeal pouch endoderm. A cell-autonomous role of Shh in thyroid morphogenesis is suggested.

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 ϩ/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 EphA4Ϫ/Ϫ and EphA4 EGFP/EGFP thyroids. In addition, the number of C cells is reduced by Ͼ50% 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. (Endocrinology 152: 1154 -1164, 2011) E ph receptors (originally named for the expression in an Erythropoetin-Producing Hepatocellular carcinoma cell line) and their cognate ligands, the ephrins, are increasingly recognized as regulators of embryonic organogenesis and of homeostatic control in adult organs (1, 2). In mammalians this ligand-receptor system consists of eight ephrins and 14 Eph members; Ephs therefore constitute the largest receptor tyrosine kinase family. The common mode of action is typically bidirectional in that the activated Eph receptor mediates forward signaling through its tyrosine kinase domain and the receptorbound ephrin transduces reverse signaling by recruitment of signal effectors in the ligand-expressing cell (3). This provides novel means by which cells communicate in concert with classical adhesion molecules (e.g., cadherins and integrins) and external signals (viz., hormones, growth factors, and transmitter substances) to jointly regulate cell behavior. Ephs and ephrins are both classified as A-and B-types based on their binding preferences. Recently, EphA-ephrinA bidirectional signaling was found to modify insulin secretion in adult pancreatic ␤-cells (4), providing the first compelling evidence for a local regulatory circuit of endocrine function involving this new class o...

The aim of the study was to examine the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/acetylsalicylic acid (ASA) on human lens epithelial cells (HLECs) during oxidative stress. HLECs were exposed to H₂O₂ in the absence or presence of indomethacin, diclofenac, celecoxib (NSAIDs) or ASA for 24 h. HLECs were assayed for changes in superoxide and peroxide production and for variations in glutathione. Mitochondrial depolarization was measured using the membrane potential-sensitive dye JC-1. The results of the study include reduction in superoxide and peroxide production as well as reduction in glutathione depletion in oxidatively stressed HLECs incubated with low concentrations of NSAIDs/ASA. However, no protection against H₂O₂-induced mitochondrial depolarization by NSAIDs/ASA could be seen. In conclusion, NSAIDs/ASA display reactive oxygen species-scavenging properties in H₂O₂-exposed HLECs in culture.