in-vivo-inhibition-of-microrna-to-decrease-tumor-growth-in-mice

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Instituto de Investigaciones Biomédicas, CSIC-UAM

Instituto de Salud Carlos III

Well-differentiated thyroid cancer has in general terms a very good outcome. It has a very slow growth rate and, although it metastasizes at a relatively high frequency, it has very high survival rates. Whereas the prevalence of nodular thyroid disease worldwide is high, malignant conversion from benign thyroid nodules is rare. Treatment of thyroid cancer is usually successful, but we still do not have effective therapies for patients with invasive or metastatic thyroid cancer if the disease does not concentrate radioiodine and it is not surgically resectable. On the other hand, from the same thyroid cell, one of the most aggressive human tumours can arise--undifferentiated or anaplastic thyroid carcinoma--leading to death in a few months. What features of this malignancy account for such paradoxical behaviour? The most common type of thyroid cancer--papillary thyroid carcinoma--stands out among solid tumours because many of the tumour-initiating events have been identified. All of them function in a single pathway--the RTK/RAS/RAF/MAPK pathway--and obey an 'exclusivity principle': one and only one component of the pathway is mutated in a single tumour. This highlights the requirement of this signal transduction pathway for the transformation to thyroid cancer and paves the way to targeted therapies against a tumour with a mutation in a known gene or any gene upstream of the target. However, it is also interesting to underscore the differences among the tumours arising from the different mutations. Studies in vitro and in vivo, including genomic profiling and genetically engineered mouse models, have clearly shown that each oncoprotein exerts its own oncogenic drive, conferring a distinct biological behaviour on thyroid tumours. In this review, we attempt to summarise the most recent advances in thyroid follicular cell-derived cancers research and their potential clinical impact that may change the management of thyroid cancer in the near future.

New insights in thyroid follicular cell biology and its impact in thyroid cancer therapy.

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FoxE1 is a thyroid-specific forkhead transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in adults. FoxE1 recognizes and binds to a short DNA sequence present in thyroglobulin (Tg) and thyroperoxidase (Tpo) promoters, but FoxE1 binding to regulatory regions other than Tg and Tpo promoters remains almost unexplored. Improving knowledge of the regulatory functions of FoxE1 is necessary to clarify its role in endocrine syndromes and cancer susceptibility.

New insights into FoxE1 functions: identification of direct FoxE1 targets in thyroid cells.

The sodium iodide symporter (NIS) mediates active transport of iodide into the thyroid and the lactating mammary glands and is highly expressed in thyroid and breast carcinomas. NIS is clinically very relevant because it allows the treatment with radioiodine of thyroid cancer patients.

NIS mediates iodide uptake in the female reproductive tract and is a poor prognostic factor in ovarian cancer.

The presence of differentiated thyroid cells in thyroid cancer is critical for the antitumor response to radioactive iodide treatment, and loss of the differentiated phenotype is a key hallmark of iodide-refractory metastatic disease. The role of microRNAs (miRNA) in fine-tuning gene expression has become a major regulatory mechanism by which developmental and pathologic processes occur. In this study, we performed next-generation sequencing and expression analysis of eight papillary thyroid carcinomas (PTC) to comprehensively characterize miRNAs involved in loss of differentiation. We found that only a small set of abundant miRNAs is differentially expressed between PTC tissue and normal tissue from the same patient. In addition, we integrated computational prediction of potential targets and mRNA sequencing and identified a master miRNA regulatory network involved in essential biologic processes such as thyroid differentiation. Both mature products of mir-146b (miR-146b-5p and -3p) were among the most abundantly expressed miRNAs in tumors. Specifically, we found that miR-146b-3p binds to the 3'-untranslated region of PAX8 and sodium/iodide symporter (NIS), leading to impaired protein translation and a subsequent reduction in iodide uptake. Furthermore, our findings show that miR-146b and PAX8 regulate each other and share common target genes, thus highlighting a novel regulatory circuit that governs the differentiated phenotype of PTC. In conclusion, our study has uncovered the existence of a miR-146b-3p/PAX8/NIS regulatory circuit that may be exploited therapeutically to modulate thyroid cell differentiation and iodide uptake for improved treatment of advanced thyroid cancer.

The miR-146b-3p/PAX8/NIS Regulatory Circuit Modulates the Differentiation Phenotype and Function of Thyroid Cells during Carcinogenesis.

Thyroid cancer is the most common endocrine malignancy giving rise to one of the most indolent solid cancers, but also one of the most lethal. In recent years, systematic studies of the cancer genome, most importantly those derived from The Cancer Genome Altas (TCGA), have catalogued aberrations in the DNA, chromatin, and RNA of the genomes of thousands of tumors relative to matched normal cellular genomes and have analyzed their epigenetic and protein consequences. Cancer genomics is therefore providing new information on cancer development and behavior, as well as new insights into genetic alterations and molecular pathways. From this genomic perspective, we will review the main advances concerning some essential aspects of the molecular pathogenesis of thyroid cancer such as mutational mechanisms, new cancer genes implicated in tumor initiation and progression, the role of non-coding RNA, and the advent of new susceptibility genes in thyroid cancer predisposition. This look across these genomic and cellular alterations results in the reshaping of the multistep development of thyroid tumors and offers new tools and opportunities for further research and clinical development of novel treatment strategies. 

ENDOCRINE TUMOURS: Advances in the molecular pathogenesis of thyroid cancer: lessons from the cancer genome.

Organization of epithelial cells during follicular lumen formation is crucial for thyroid morphogenesis and function of the thyroid gland; however, the molecular mechanisms underlying this are poorly understood. To investigate this process, we established three-dimensional (3D) epithelial culture model systems using Fischer rat thyroid (FRT) cells or murine primary thyrocytes that developed polarized spherical structures with a central lumen, mimicking thyroid follicles. Using microarray-based differential expression analysis of FRT cells grown under 2D or 3D conditions, followed by RNA-mediated interference (RNAi) and morphogenetic analysis, we identified a key role for the thyroid transcription factor Pax8 and its target cadherin-16 (Cdh16) in the generation of polarized follicle-like structures. Silencing Pax8 expression inhibited the acquisition of apical-basal membrane polarity and impaired lumen formation. Both laminin and β1-integrin (Itgb1) expression was reduced, and cell cytoskeleton polarized distribution was altered. Silencing Cdh16 expression also led to the formation of defective structures characterized by very low laminin expression at the follicle-matrix interface, downregulation of Itgb1, and unpolarized distribution of cell cytoskeleton. Our results demonstrate that Pax8 controls apical-basal follicular polarization and follicle formation through Cdh16.

Pax8 controls thyroid follicular polarity through cadherin-16.

Whole genome sequencing approaches have provided unprecedented insights into the genetic lesions responsible for the onset, progression and dedifferentiation of various types of thyroid carcinomas. Through these efforts, the MAPK and PI3K signaling cascades have emerged as the main activation pathways implicated in thyroid tumorigenesis. The nature of these essential pathways is highly complex, with hundreds of components, multiple points of crosstalk, different subcellular localizations and with the ability to potentially regulate many cellular processes. Small-molecule inhibitors targeting key kinases of these pathways hold great promise as novel therapeutics and several have reached clinical trials. However, while some remarkable responses have been reported, the development of resistance remains a matter of concern and limits the benefit for patients. In this review, we discuss the latest findings on the major components of the MAPK and PI3K pathways, including their mechanisms of activation in physiological and pathological contexts, their genetic alterations with respect to the different types of thyroid carcinomas and the more relevant drugs designed to block their activity.

Key signaling pathways in thyroid cancer.

Recent studies have shown that miR-146b is the most upregulated microRNA in thyroid cancer and has a central role in cancer progression through mechanisms that remain largely unidentified. As phosphoinositide 3-kinase/protein kinase-B (PI3K/AKT) signaling is a fundamental oncogenic driver in many thyroid cancers, we explored a potential role for miR-146b and its target genes in PI3K/AKT activation. Among the predicted target genes of miR-146b, we found the tumor-suppressor phosphatase and tensin homolog (PTEN). Constitutive overexpression of miR-146b in thyroid epithelial cell lines significantly decreased PTEN mRNA and protein levels by direct binding to its 3'-UTR. This was accompanied by PI3K/AKT hyperactivation, leading to the exclusion of FOXO1 and p27 from the nucleus and a corresponding increase in cellular proliferation. Moreover, miR-146b overexpression led to protection from apoptosis and an increased migration and invasion potential, regulating genes involved in epithelial-mesenchymal transition. Notably, with the single exception of E-cadherin expression, all of these outcomes could be reversed by PTEN coexpression. Further analysis showed that miR-146b directly inhibits E-cadherin expression through binding to its 3'-UTR. Interestingly, miR-146b inhibition in human thyroid tumor xenografts, using a synthetic and clinically amenable molecule, blocked tumor growth when delivered intratumorally. Importantly, this inhibition increased PTEN protein levels. In conclusion, our data define a novel mechanism of PI3K/AKT hyperactivation and outline a regulatory role for miR-146b in suppressing PTEN expression, a frequent observation in thyroid cancer. Both events are related to a more aggressive tumoral phenotype. Targeting miR-146b therefore represents a promising therapeutic strategy for the treatment of this disease.

MicroRNA-146b promotes PI3K/AKT pathway hyperactivation and thyroid cancer progression by targeting PTEN.

Iodide (I) metabolism is crucial for the synthesis of thyroid hormones (THs) in the thyroid and the subsequent action of these hormones in the organism. I is principally transported by the sodium iodide symporter (NIS) and by the anion exchanger PENDRIN, and recent studies have demonstrated the direct participation of new transporters including anoctamin 1 (ANO1), cystic fibrosis transmembrane conductance regulator (CFTR) and sodium multivitamin transporter (SMVT). Several of these transporters have been found expressed in various tissues, implicating them in I recycling. New research supports the exciting idea that I participates as a protective antioxidant and can be oxidized to hypoiodite, a potent oxidant involved in the host defense against microorganisms. This was possibly the original role of I in biological systems, before the appearance of TH in evolution. I per se participates in its own regulation, and new evidence indicates that it may be antineoplastic, anti-proliferative and cytotoxic in human cancer. Alterations in the expression of I transporters are associated with tumor development in a cancer-type-dependent manner and, accordingly, NIS, CFTR and ANO1 have been proposed as tumor markers. Radioactive iodide has been the mainstay adjuvant treatment for thyroid cancer for the last seven decades by virtue of its active transport by NIS. The rapid advancement of techniques that detect radioisotopes, in particular I, has made NIS a preferred target-specific theranostic agent.

Role of iodide metabolism in physiology and cancer.

An oncogenic role for microRNA-146b in the thyroid.

N-methyladenosine (mA) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered mA homeostasis is linked to cancer. mA modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon. METTL3 can promote translation but the mechanism and relevance of this process remain unknown. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also mA-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.

mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis.

Current research on thyrotropin-activated proliferation in the thyrocyte needs to be aimed at a better understanding of crosstalk and negative-feedback mechanisms with other proliferative pathways, especially the insulin/IGF-1-induced phosphoinositol-3 kinase pathway and the serum-induced MAPK or Wnt pathways. Convergence of proliferative pathways in mTOR is a hotspot of current research, and combined treatment using double class inhibitors for thyroid cancer may bring some success. New thyroid-stimulating hormone receptor (TSHR)-interacting proteins, a better picture of cAMP targets, a deeper knowledge of the action of the protein kinase A regulatory subunits, especially their interactions with the replication machinery, and a further understanding of mechanisms that lead to cell cycle progression through G/S and G/M checkpoints are areas that need further elucidation. Finally, massive information coming from microarray data analysis will prompt our understanding of thyroid-stimulating hormone-promoted thyrocyte proliferation in health and disease.

Thyroid-stimulating hormone/cAMP-mediated proliferation in thyrocytes.

In the last two decades, great strides have been made in the study of microRNAs in development and in diseases such as cancer, as reflected in the exponential increase in the number of reviews on this topic including those on undifferentiated and well-differentiated thyroid cancer. Nevertheless, few reviews have focused on understanding the functional significance of the most up- or down-regulated miRNAs in thyroid cancer for the main signaling pathways hyperactivated in this tumor type. The aim of this review is to discuss the major miRNAs targeting proteins of the MAPK, PI3K, and TGFβ pathways, to define their mechanisms of action through the 3'UTR regions of their target genes, and to describe how they affect thyroid tumorigenesis through their actions on cell proliferation, migration, and invasion. Given the importance of miRNAs in cancer as diagnostic, prognostic and therapeutic candidates, a better understanding of this cross-talk might shed new light on the biomedical treatment of thyroid cancer.

Pilar Santisteban

Instituto de Investigaciones Biomédicas, CSIC-UAM

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice