Wnt4 inhibits cell motility induced by oncogenic Ras.

Aberrant motility and invasive ability are relevant hallmarks of malignant tumor cells. Pathways regulating the movement of cancer cells from the site of primary tumor toward adjacent and/or distant tissues are not entirely defined. By using a model of malignant transformation induced by Ras, we identified Wnt4 as an early target of Ras oncogenic signaling. Here we show that Wnt4 is repressed by Ras and that forced Wnt4 expression inhibits Ras-induced cell motility. Accordingly, we found that Wnt4 is downregulated in human anaplastic thyroid carcinomas, the most malignant and metastatic thyroid cancer histotype. Wnt4 interferes with Ras-induced actin cytoskeleton reorganization through non-canonical pathways, by altering the balance between the activation of different Rho-family small guanosine triphosphatases (GTPases). Finally, we demonstrate that Wnt4 is post-transcriptionally repressed by miR-24, a Ras-induced micro RNA (miRNA) targeting the 3'-untranslated region (UTR) of Wnt4. Taken together our data highlight a novel Ras-regulated miRNA-dependent circuitry regulating the motile phenotype of cancer cells.

Upregulation of miR-21 by Ras in vivo and its role in tumor growth.

miR-21 is a microRNA (miRNA) frequently overexpressed in human cancers. Here we show that miR-21 is upregulated both in vitro and in vivo by oncogenic Ras, thus linking this miRNA to one of the most frequently activated oncogenes in human cancers. Ras regulation of miR-21 occurs with a delayed kinetic and requires at least two Ras downstream pathways. A screen of human thyroid cancers and non-small-cell lung cancers for the expression of miR-21 reveals that it is overexpressed mainly in anaplastic thyroid carcinomas, the most aggressive form of thyroid cancer, whereas in lung its overexpression appears to be inversely correlated with tumor progression. We also show that a LNA directed against miR-21 slows down tumor growth in mice. Consistently, a search for mRNAs downregulated by miR-21 shows an enrichment for mRNAs encoding cell cycle checkpoints regulators, suggesting an important role for miR-21 in oncogenic Ras-induced cell proliferation.

An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation.

The transcription factor AP-1 plays key roles in tumorigenesis, by regulating a variety of protein-coding genes, implicated in multiple hallmarks of cancer. Among non-coding genes, no AP-1 target has been described yet in tumorigenesis. MicroRNAs (miRNAs) are negative post-transcriptional regulators of protein-coding genes. miRNA expression signatures are highly relevant in cancer and several tumor-associated miRNAs (oncomirs) play critical roles in oncogenesis. Here, we show that the miRNA miR-21, which represents the most frequently upregulated oncomir in solid tumors, is induced by AP-1 in response to RAS. By analyzing validated miR-21 targets, we have found that the tumor suppressors PTEN and PDCD4 are downregulated by RAS in an AP-1- and miR-21-dependent fashion. We further show that, given the role of PDCD4 as negative regulator of AP-1, the miR-21-mediated downregulation of PDCD4 is essential for the maximal induction of AP-1 activity in response to RAS. Our data reveal a novel mechanism of positive autoregulation of the AP-1 complex in RAS transformation and disclose the function of oncomirs as critical targets and regulators of AP-1 in tumorigenesis.

The CRE-like element inside the 5'-upstream region of the rat sodium/iodide symporter gene interacts with diverse classes of b-Zip molecules that regulate transcriptional activities through strong synergy with Pax-8.

We previously demonstrated that transcription of the rat sodium/iodide symporter (NIS) gene is regulated by NUE, an upstream enhancer located between nucleotides -2264 and -2495 of the 5'-flanking region. To elucidate the mechanism of TSH/cAMP-mediated regulation of NIS gene expression, we have characterized the putative cAMP response element (CRE)/activator protein (AP)-1 site (termed NUC) that is closely located between the two Pax-8 (paired box domain transcription factor-8) binding sites within NUE. In two different approaches using either gel supershift analyses or dominant-negative inhibitors of b-Zip molecules, we have shown that NUC can be recognized by several members of the AP-1 and CREB family transcription factors that modulate the transcriptional activity of NUE. Using tethered dimers of b-Zip molecules, we have also demonstrated that specific homo- or heterodimers of AP-1 can synergistically stimulate NUE activity in concert with Pax-8. To demonstrate further that NUC is a bona fide CRE, we made an artificial promoter with the five-time tandem repeat of this sequence (5xNUC). In comparison to the canonical CRE (5xCRE), 5xNUC manifested greater transcriptional activity and broader response to cAMP signaling. Hence, we postulate that the significance of this evolutionally conserved CRE-like site may lie in its broader cell type specificity.

Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland.

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSHTSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pit(dw)pit(dw)) or of a functional TSHR (tshr(hyt)tshr(hyt) and TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.

Cititf1 and endoderm differentiation in Ciona intestinalis.

Studies on the initial formation of the endoderm have lead to the identification, mostly in Xenopus, of numerous genes relevant for the formation of this tissue during early embryogenesis (reviewed by Date, Curr. Biol. 9 (1999) R812-R815 and by Yasuo and Lemaire, Curr. Biol. 9 (1999) 869-879). In ascidians, the most primitive chordates, endoderm differentiation is an autonomous process which is almost complete at 64 cells stage. Cititf1, a gene homologous to mammalian Titf1, is the first specific endodermal marker isolated from the ascidian Ciona intestinalis (Ristatore et al., Development 126 (1999) 5149-5159). Here we study the in vivo role of Cititf1 by using two different approaches: interference and ectopic expression. The results presented here show that interference with Cititf1 function seems to affect gastrulation movements, while ectopic expression of Cititf1 mRNA into the notochord alters differentiation of this tissue probably by recruiting notochord blastomeres to an endodermal fate. These data together with studies, still in progress in our laboratory, on the transcriptional regulation of Cititf1, strongly indicate that Cititf1 plays an important role in the process of endoderm formation in Ciona embryogenesis.

Hormonal control of the transcription factor Pax8 and its role in the regulation of thyroglobulin gene expression in thyroid cells.

The transcription factor Pax8 plays an important role in the expression of the differentiated phenotype of thyroid follicular cells. It has recently been shown that Pax8 is necessary for thyroglobulin (Tg) gene expression in the fully differentiated rat thyroid cell line PC. We have used the PC model system to investigate the role of Pax8 as a mediator of TSH regulation of Tg gene expression. We have demonstrated that Pax8 expression, as well as Tg expression, is severely reduced in cells grown in the absence of hormones and serum. The re-addition of TSH or forskolin to the culture medium is able to restore to wild-type levels the expression of both Pax8 and Tg. We have determined that the action of TSH/forskolin on Pax8 is at the transcriptional level. However, the re-expression of Pax8 can be observed several hours before that of Tg, suggesting that either another factor is needed or that Pax8 itself must be post-translationally modified by a newly synthesized protein to become active. To distinguish between these two possibilities we have stably transfected into PC cells an exogenous Pax8 that is expressed independently of TSH. Our results indicate that in these cells the Tg promoter is still dependent on TSH despite the constitutive presence of Pax8. Furthermore, we also show that in this condition Tg gene transcription requires de novo protein synthesis. In conclusion, TSH regulates the expression of Pax8 at a transcriptional level and also regulates the activity of Pax8 by controlling the expression of one or more as yet unknown factors.

Characterization of the upstream enhancer of the rat sodium/iodide symporter gene.

We previously demonstrated the presence of an enhancer that is located between nucleotides - 2264 and - 2495 in the 5' flanking region of the rat sodium/iodide symporter (NIS) gene (Ohno et al., 1999). When attached to NIS or heterologous promoters, this 232 bp fragment, which we call NUE, is able to stimulate transcription in a thyroid-specific and cAMP-dependent manner. A paired-domain transcription factor Pax8 binds to this enhancer and can stimulate the transcription in non-thyroid cells that do not normally support the NUE activities. Cotransfection of PKA, a downstream effector of cAMP, further potentiates the Pax8-mediated transactivation. However, this transcriptional machinery containing pax8 seems to require contributions from the neighboring cis-acting element that is similar to CRE/AP-1 consensus sequences. Modification of this putative CRE/AP-1 site not only represses the NUE transcriptional activities by 90% in FRTL-5 cells, but also nullifies the synergistic effect of PKA on pax8-mediated transactivation in HeLa cells. In this report, we have further characterized the putative CRE/AP-1 site within the NIS upstream enhancer using gel mobility shift assay. An oligonucleotide probe with NIS CRE/AP-1 sequence produced complex binding patterns in both FRTL-5 and HeLa cell, reflecting the presence of diverse classes of binding factors. When compared with CRE or AP-1 elements in other genes, the mobility shift pattern of NIS CRE/AP-1 was similar to those of collagenase TRE, c-Jun TRE, and somatostatin CRE, but the relative intensities of the binding complexes were quite different. This observation raises a possibility that the NIS CRE/AP-site is regulated by a novel mechanism.

The DNA glycosylase T:G mismatch-specific thymine DNA glycosylase represses thyroid transcription factor-1-activated transcription.

The transcription factor thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein that belongs to the NK2 family of genes involved in organogenesis. TTF-1 is required for normal development of the forebrain, lung, and thyroid. In a search for factors that regulate TTF-1 transcriptional activity, we isolated three genes (T:G mismatch-specific thymine DNA glycosylase (TDG), homeodomain-interacting protein kinase 2 (HIPK2), and Ajuba), whose products can interact with TTF-1 in yeast and in mammalian cells. TDG is an enzyme involved in base excision repair. In the present paper, we show that TDG acts as a strong repressor of TTF-1 transcriptional activity in a dose-dependent manner, while HIPK2 and Ajuba display no effect on TTF-1 activity, at least under the tested conditions. TDG-mediated inhibition occurs specifically on TTF-1-responsive promoters in thyroid and non thyroid cells. TDG associates with TTF-1 in mammalian cells through the TTF-1 carboxyl-terminal activation domain and is independent of the homeodomain. These findings reveal a previously unsuspected role for the repair enzyme TDG as a transcriptional repressor and open new routes toward the understanding of the regulation of TTF-1 transcriptional activity.

Immediate early genes induced by H-Ras in thyroid cells.

Expression of oncogenic v-H-Ras in the thyroid cell line FRTL-5 (FRTL-5(Ras)) results in uncontrolled proliferation, loss of thyroid-specific gene expression and tumorigenicity. Concomitant expression of constitutively activated MEK and Rac, two major H-Ras downstream effectors, in FRTL-5 (FRTL-5(MEK/Rac)) recapitulates H-Ras effects on proliferation and morphology. In contrast to FRTL-5(Ras), however, FRTL-5(MEK/Rac) cells remain differentiated and are not tumorigenic. To find H-Ras induced genes potentially responsible for tumorigenicity and loss of differentiation, we have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, between de-differentiated and tumorigenic FRTL-5(Ras) cells and differentiated and non-tumorigenic FRTL-5(MEK/Rac) cells. We examined 800 of the cDNA clones obtained after subtraction and verified their levels of expression in the two cell lines by reverse northern, identifying 337 H-Ras induced genes. By sequence analysis, we clustered 57 different genes. Among these, 39 were known genes (involved in diverse signal transduction processes regulating mitogenic activity, cell survival, cytoskeletal reorganization, stress response and invasion) while the remaining 18 clones were novel genes. Among the 57 H-Ras specific clones, we identified those genes whose expression is induced early by H-Ras. We suggest that these immediate-early genes may play a crucial role in H-Ras-mediated transformation in thyroid epithelial cells.

A unique combination of transcription factors controls differentiation of thyroid cells.

The thyroid follicular cell type is devoted to the synthesis of thyroid hormones. Several genes, whose protein products are essential for efficient hormone biosynthesis, are uniquely expressed in this cell type. A set of transcriptional regulators, unique to the thyroid follicular cell type, has been identified as responsible for thyroid specific gene expression; it comprises three transcription factors, named TTF-1, TTF-2, and Pax8, each of which is expressed also in cell types different from the thyroid follicular cells. However, the combination of these factors is unique to the thyroid hormone producing cells, strongly suggesting that they play an important role in differentiation of these cells. An overview of the molecular and biological features of these transcription factors is presented here. Data demonstrating that all three play also an important role in early thyroid development, at stages preceding expression of the differentiated phenotype, are also reviewed. The wide temporal expression, from the beginning of thyroid organogenesis to the adult state, is suggestive of a recycling of the thyroid-specific transcription factors, that is, the control of different sets of target genes at diverse developmental stages. The identification of molecular mechanisms leading to specific gene expression in thyroid cells renders this cell type an interesting model in which to address several aspects of cell differentiation and organogenesis.

Identification and developmental expression of three Distal-less homeobox containing genes in the ascidian Ciona intestinalis.

Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C.

Pax8 has a key role in thyroid cell differentiation.

Transformation of rat thyroid cells with polyoma virus middle T antigen results in loss of the thyroid-differentiated phenotype, measured as the expression of the thyroglobulin (Tg), thyroperoxidase (TPO), and sodium/iodide symporter (NIS) genes. Among the transcription factors involved in the regulation of these genes, TTF-1 and TTF-2 were still detected at nearly wild-type levels, while a specific loss of the paired domain transcription factor Pax8 was observed. In this study, we used the PCPy cell line as a model system to study the role of Pax8 in thyroid differentiation. We demonstrate that the reintroduction of Pax8 in PCPy cells is sufficient to activate expression of the endogenous genes encoding thyroglobulin, thyroperoxidase, and sodium/iodide symporter. Thus, this cell system provides direct evidence for the ability of Pax8 to activate transcription of thyroid-specific genes at their chromosomal locus and strongly suggests a fundamental role of this transcription factor in the maintenance of functional differentiation in thyroid cells. Moreover, we show that Pax8 and TTF-1 cooperate in the activation of the thyroglobulin promoter and that additional thyroid-specific mechanism(s) are involved in such a cooperation. To identify the Pax8 domain able to mediate the specific activation of the thyroglobulin promoter, we transfected in PCPy cells three different Pax8 isoforms. The results of such experiments indicate that for the transcriptional activation of thyroid-specific genes, Pax8 uses an as yet unidentified functional domain.

The thyroid transcription factor 2 (TTF-2) is a promoter-specific DNA-binding independent transcriptional repressor.

The thyroid transcription factor TTF-2 is a forkhead-containing protein involved in thyroid-specific gene expression and necessary for thyroid morphogenesis. In this paper, we demonstrate that TTF-2 is able to inhibit the activity of the thyroid-specific transcription factors TTF-1 and Pax-8 only on certain promoters. We identified the minimal protein domain responsible for repressor activity, which behaves as an independent functional domain, and we show that repression by TTF-2 is DNA-binding independent. We suggest that TTF-2 is able to interfere with a specific cofactor required for TTF-1 and Pax-8 activity.

Multiple ras downstream pathways mediate functional repression of the homeobox gene product TTF-1.

Expression of oncogenic Ras in thyroid cells results in loss of expression of several thyroid-specific genes and inactivation of TTF-1, a homeodomain-containing transcription factor required for normal development of the thyroid gland. In an effort to understand how signal transduction pathways downstream of Ras may be involved in suppression of the differentiated phenotype, we have tested mutants of the Ras effector region for their ability to affect TTF-1 transcriptional activity in a transient-transfection assay. We find that V12S35 Ras, a mutant known to interact specifically with Raf but not with RalGDS or phosphatidylinositol 3-kinase (PI3 kinase) inhibits TTF-1 activity. Expression of an activated form of Raf (Raf-BXB) also inhibits TTF-1 function to a similar extent, while the MEK inhibitors U0126 and PD98059 partially relieve Ras-mediated inactivation of TTF-1, suggesting that the extracellular signal-regulated kinase (ERK) pathway is involved in this process. Indeed, ERK directly phosphorylates TTF-1 at three serine residues, and concomitant mutation of these serines to alanines completely abolishes ERK-mediated phosphorylation both in vitro and in vivo. Since activation of the Raf/MEK/ERK pathway accounts for only part of the activity elicited by oncogenic Ras on TTF-1, other downstream pathways are likely to be involved in this process. We find that activation of PI3 kinase, Rho, Rac, and RalGDS has no effect on TTF-1 transcriptional activity. However, a poorly characterized Ras mutant, V12N38 Ras, can partially repress TTF-1 transcriptional activity through an ERK-independent pathway. Importantly, concomitant expression of constitutive activated Raf and V12N38 Ras results in almost complete loss of TTF-1 activity. Our data indicate that the Raf/MEK/ERK cascade may act in concert with an as-yet-uncharacterized signaling pathway activated by V12N38 Ras to repress TTF-1 function and ultimately to inhibit thyroid cell differentiation.

Expression and functional analysis of Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.

In solitary ascidians the fate of endoderm is determined at a very early stage of development and depends on cytoplasmic factors whose nature has not been determined. We have isolated a member of the NK-2 gene family, Cititf1, from the ascidian Ciona intestinalis, showing high sequence homology to mammalian TITF1. The Cititf1 gene was expressed in all endodermal precursors at the pregastrula and gastrula stages, and is thus the first specific regulatory endodermal marker to be isolated from an ascidian. Cititf1 expression was downregulated at the end of gastrulation to reappear at middle tailbud and larval stages in the most anterior and ventral parts of head endoderm, regions which give rise, after metamorphosis, to the adult endostyle, where Cititf1 mRNA was still present. Microinjection of Cititf1 mRNA into fertilized eggs resulted in tadpole larvae with abnormalities in head-trunk development consequent to the formation of excess endoderm, perhaps due to recruitment of notochord precursors to an endodermal fate. These data suggest that Cititf1 plays an important role in normal endoderm differentiation during ascidian embryogenesis.

Ascidian homologs of mammalian thyroid peroxidase genes are expressed in the thyroid-equivalent region of the endostyle.

The endostyle is a pharyngeal organ for the internal filter feeding of urochordates, cephalochordates, and larval lamprey. This organ is also considered to be homologous to the follicular thyroid gland of higher vertebrates. Thyroglobulin (Tg) and thyroid peroxidase (TPO) are specifically expressed in the thyroid gland of higher vertebrates, and they play an important role in iodine metabolism for the synthesis of thyroid hormones. Previous histochemical observations showed that iodine-concentrating and peroxidase activities were detected in zones 7, 8, and 9 of the ascidian endostyle, suggesting that these zones contains cells that are equivalent to those in the vertebrate follicular thyroid. In order to investigate the molecular developmental mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the thyroid gland, in the present study, we isolated and characterized cDNA clones for TPO genes, CiTPO from Ciona intestinalis and HrTPO from Halocynthia roretzi. Northern blot and in situ hybridization analyses revealed that the expression of the ascidian TPO genes was restricted to zone 7, one of the elements equivalent to the thyroid. These results provide the first evidence at the gene expression level for shared function between a part of the ascidian endostyle and the vertebrate follicular thyroid gland. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:158-169, 1999.

Cloning, chromosomal localization and identification of polymorphisms in the human thyroid transcription factor 2 gene (TITF2).

The human gene encoding the thyroid transcription factor 2 (TTF-2) was cloned and mapped to human chromosome 9q22. Three polymorphisms were identified in the gene by SSCP and direct sequencing: two consist of a third base substitution in the triplet encoding Leu129 and Ser273, and the third is an alanine stretch that varies from 12 to 17 residues. TTF-2 plays a critical role during thyroid morphogenesis in mice, and in man the TITF2 gene is associated with congenital hypothyroidism and cleft palate with thyroid dysgenesis. The polymorphisms identified in this study can be used as markers to study the role of the TITF2 gene in other cases of thyroid dysgenesis, especially in familial cases.