HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells.

RNA-binding proteins play diverse roles in cancer, influencing various facets of the disease, including proliferation, apoptosis, angiogenesis, senescence, invasion, epithelial-mesenchymal transition (EMT), and metastasis. HuR, a known RBP, is recognized for stabilizing mRNAs containing AU-rich elements (AREs), although its complete repertoire of mRNA targets remains undefined. Through a bioinformatics analysis of the gene expression profile of the Hs578T basal-like triple-negative breast cancer cell line with silenced HuR, we have identified SOX9 as a potential HuR-regulated target. SOX9 is a transcription factor involved in promoting EMT, metastasis, survival, and the maintenance of cancer stem cells (CSCs) in triple-negative breast cancer. Ribonucleoprotein immunoprecipitation assays confirm a direct interaction between HuR and SOX9 mRNA. The half-life of SOX9 mRNA and the levels of SOX9 protein decreased in cells lacking HuR. Cells silenced for HuR exhibit reduced migration and invasion compared to control cells, a phenotype similar to that described for SOX9-silenced cells.

New Functions of Intracellular LOXL2: Modulation of RNA-Binding Proteins.

Lysyl oxidase-like 2 (LOXL2) was initially described as an extracellular enzyme involved in extracellular matrix remodeling. Nevertheless, numerous recent reports have implicated intracellular LOXL2 in a wide variety of processes that impact on gene transcription, development, differentiation, proliferation, migration, cell adhesion, and angiogenesis, suggesting multiple different functions for this protein. In addition, increasing knowledge about LOXL2 points to a role in several types of human cancer. Moreover, LOXL2 is able to induce the epithelial-to-mesenchymal transition (EMT) process-the first step in the metastatic cascade. To uncover the underlying mechanisms of the great variety of functions of intracellular LOXL2, we carried out an analysis of LOXL2's nuclear interactome. This study reveals the interaction of LOXL2 with numerous RNA-binding proteins (RBPs) involved in several aspects of RNA metabolism. Gene expression profile analysis of cells silenced for LOXL2, combined with in silico identification of RBPs' targets, points to six RBPs as candidates to be substrates of LOXL2's action, and that deserve a more mechanistic analysis in the future. The results presented here allow us to hypothesize novel LOXL2 functions that might help to comprehend its multifaceted role in the tumorigenic process.

FOXE1 regulates migration and invasion in thyroid cancer cells and targets ZEB1.

FOXE1 is a thyroid-specific transcription factor essential for thyroid gland development and maintenance of the differentiated state. Interestingly, a strong association has been recently described between FOXE1 expression and susceptibility to thyroid cancer, but little is known about the mechanisms underlying FOXE1-induced thyroid tumorigenesis. Here, we used a panel of human thyroid cancer-derived cell lines covering the spectrum of thyroid cancer phenotypes to examine FOXE1 expression and to test for correlations between FOXE1 expression, the allele frequency of two SNPs and a length polymorphism in or near the FOXE1 locus associated with cancer susceptibility, and the migration ability of thyroid cancer cell lines. Results showed that FOXE1 expression correlated with differentiation status according to histological sub-type, but not with SNP genotype or cell migration ability. However, loss-and-gain-of-function experiments revealed that FOXE1 modulates cell migration, suggesting a role in epithelial-to-mesenchymal transition (EMT). Our previous genome-wide expression analysis identified Zeb1, a major EMT inducer, as a putative Foxe1 target gene. Indeed, gene silencing of FOXE1 decreased ZEB1 expression, whereas its overexpression increased ZEB1 transcriptional activity. FOXE1 was found to directly interact with the ZEB1 promoter. Lastly, ZEB1 silencing decreased the ability of thyroid tumoral cells to migrate and invade, pointing to its importance in thyroid tumor mestastases. In conclusion, we have identified ZEB1 as a bona fide target of FOXE1 in thyroid cancer cells, which provides new insights into the role of FOXE1 in regulating cell migration and invasion in thyroid cancer.

Impaired Gene Expression Due to Iodine Excess in the Development and Differentiation of Endoderm and Thyroid Is Associated with Epigenetic Changes.

Background: Thyroid hormone (TH) synthesis is essential for the control of development, growth, and metabolism in vertebrates and depends on a sufficient dietary iodine intake. Importantly, both iodine deficiency and iodine excess (IE) impair TH synthesis, causing serious health problems especially during fetal/neonatal development. While it is known that IE disrupts thyroid function by inhibiting thyroid gene expression, its effects on thyroid development are less clear. Accordingly, this study sought to investigate the effects of IE during the embryonic development/differentiation of endoderm and the thyroid gland. Methods: We used the murine embryonic stem (ES) cell model of in vitro directed differentiation to assess the impact of IE on the generation of endoderm and thyroid cells. Additionally, we subjected endoderm and thyroid explants obtained during early gestation to IE and evaluated gene and protein expression of endodermal markers in both models. Results: ES cells were successfully differentiated into endoderm cells and, subsequently, into thyrocytes expressing the specific thyroid markers Tshr, Slc5a5, Tpo, and Tg. IE exposure decreased the messenger RNA (mRNA) levels of the main endoderm markers Afp, Crcx4, Foxa1, Foxa2, and Sox17 in both ES cell-derived endoderm cells and embryonic explants. Interestingly, IE also decreased the expression of the main thyroid markers in ES cell-derived thyrocytes and thyroid explants. Finally, we demonstrate that DNA methyltransferase expression was increased by exposure to IE, and this was accompanied by hypermethylation and hypoacetylation of histone H3, pointing to an association between the gene repression triggered by IE and the observed epigenetic changes. Conclusions: These data establish that IE treatment is deleterious for embryonic endoderm and thyroid gene expression.

Expression of hypothalamic regulatory peptides in thyroid C cells of different mammals.

Besides intervening in calcium homeostasis by means of calcitonin, C cells are also implicated in the synthesis of an increasing number of regulatory peptides that could exert a paracrine regulation on the neighbouring follicular cells. Among the latest peptides reported in C cells, there are several characteristic hypothalamic peptides, such as TRH, CART, and ghrelin, which are mainly involved in the regulation of the metabolism at hypothalamic-pituitary-thyroid axis. The main aim of the present work has been to study the synthesis of the referred hypothalamic peptides by normal and neoplastic C cells of different mammals as well as in C-cell lines of both rat (CA-77, 6-23) and human (TT) origins in order to elucidate whether this is a fact in this kind of vertebrates. With that objective, we have applied the immunoperoxidase technique to analyze the presence of TRH, CART, ghrelin, and somatostatin in thyroid tissues of different species, and immunofluorescence to study those same peptides in C-cell cultures. Furthermore, we have investigated their expression at mRNA level by RT-PCR analysis. Our results demonstrate immunocolocalization of CART, ghrelin, somatostatin and TRH with calcitonin in normal C cells of different mammals, as well as in rat and human neoplastic C cells. We also confirm the expression of those peptides in rat and human C-cell lines by RT-PCR. Consequently, we can conclude that the synthesis of those peptides by C cells is a general event characteristic of the thyroid gland in mammals.

Melatonin-synthesizing enzymes and melatonin receptor in rat thyroid cells.

Melatonin is an indoleamine with a wide spectrum of biological activities other than transmitting photoperiod information, including antioxidant, oncostatic, anti-aging and immunomodulatory properties. Although melatonin is synthesized mainly in the pineal gland, other tissues have the same capacity. In the present study, we examined whether two key enzymes in melatonin biosynthesis, arylalkylamine Nacetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT) and its receptor MT1 are expressed in the two endocrine thyroid cells of the rat, follicular cells and C cells. Reverse transcriptase polymerase chain reaction analyses demonstrated that both AANAT and HIOMT mRNAs are expressed in the rat thyroid C-cells, and MT1 expression has been detected in C cells and follicular cells. Immunofluorescence revealed that AANAT protein is localized in C-cell cytoplasm, and MT1 protein in both cell populations. These findings demonstrate that the rat thyroid expresses AANAT, HIOMT, and its receptor MT1, showing that C cells are the main melatonin-synthesizing sites in the thyroid. This local C-cell-secreted melatonin may protect follicular cells from the oxidative stress inherent to the thyroid gland, and could also have paracrine and autocrine functions.

Functional expression of the thyrotropin receptor in C cells: new insights into their involvement in the hypothalamic-pituitary-thyroid axis.

Thyroid C cells, or parafollicular cells, are mainly known for producing calcitonin, a hormone involved in calcium homeostasis with hypocalcemic and hypophosphatemic effects. Classically, the main endocrine activity of this cell population has been believed to be restricted to its roles in serum calcium and bone metabolism. Nonetheless, in the last few years evidence has been accumulating in the literature with regard to local regulatory peptides secreted by C cells, such as somatostatin, ghrelin, thyrotropin releasing hormone or the recently described cocaine- and amphetamine-related transcript, which could modify thyroid function. As thyrotropin is the main hormone controlling the hypothalamic-pituitary-thyroid axis and, accordingly, thyroid function, we have examined the functional expression of the thyrotropin receptor in C-cell lines and in thyroid tissues. We have found that rat and human C-cell lines express the thyrotropin receptor at both mRNA and protein levels. Furthermore, incubation of C cells with thyrotropin resulted in a 10-fold inhibition of thyrotropin-receptor expression, and a concomitant decrease of the steady-state mRNA levels for calcitonin and calcitonin gene-related peptide determined by quantitative real-time PCR was found. Finally, thyrotropin receptor expression by C cells was confirmed at protein level in both normal and pathological thyroid tissues by immunohistochemistry and immunofluorescence. These results confirm that C cells, under regulation by thyrotropin, are involved in the hypothalamic-pituitary-thyroid axis and suggest a putative role in local fine-tuning of follicular cell activity.