MicroRNAs diferencialmente expresados en cáncer de tiroides: funciones de miR-146b y miR-30a en la diferenciación y progresión tumoral tiroidea

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid. Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 01 de abril de 2016Para caracterizar integralmente el transcriptoma y específicamente el miRNoma en el carcinoma tiroideo, realizamos un análisisis de expresión génica, por secuenciación masiva, de 8 carcinomas papilares tiroideos (CPT) y de su tejido tiroideo adyacente sano. Basándonos en predicciones bioinformáticas de potenciales dianas, hemos descrito una red de regulación que relaciona los genes más infraexpresados con los miRNAs más sobreexpresados en CPT. También hemos identificado un conjunto de miRNAs sobreexpresados que tienen elementos de reconocimiento en los 3'UTR de genes esenciales para la diferenciación tiroidea como NIS, TPO, TSHR, PAX8 and NKX2-1. Entre estos miRNAs observamos que el miR- 146b-3p -un miR cuyas dos hebras estaban entre las más sobreexpresadas en CPT- tenía como potenciales dianas a varios de estos genes. Mediante ensayos funcionales demostramos que el miR-146b-3p reduce los niveles proteicos NIS, PAX8 y FOXE1. Además, demostramos que tanto la inhibición sobre NIS, como PAX8, es ejercida a través de la unión a su 3‘UTR. Estos datos indican que la sobreexpresión tumoral de miR-146b-3p contribuye a la desdiferenciación tiroidea y en especial a la pérdida de la función de NIS mediante al menos 2 mecanismos complementarios: i) una regulación postranscripcional directa y ii) mediante el silenciamiento de PAX8 y FOXE1. El resultado global, fue una reducción significativa en la captación de yodo. Por otra parte, hemos identificado al supresor tumoral PTEN como una nueva diana de miR- 146b-5 en el carcinoma tiroideo. La sobreexpresión del miR-146b redujo los niveles proteicos de PTEN mediante la unión a su 3'UTR promoviendo la hiperactivación oncogénica de la vía PI3K. Además, hemos demostrado que la sobreexpresión del miR-146b redujo los niveles nucleares de FOXO1 y p27 e incrementó los niveles de BCL-2. Inversamente, nuestros resultados mostraron que, tanto el oncogén RAS como el efector de la PI3K IGF-1, incrementaron la expresión del miR-146b. Así, en este trabajo proveemos de un nuevo mecanismo explicativo para: i) la actividad prooncogénica del miR-146b y ii) la reducción de PTEN observada en los tumores tiroideos. Por último, en este trabajo hemos descrito un eje regulatorio entre el miR-30a, uno de los más infraexpresados en CPT y la proteína oncofetal de unión a RNA, LIN28B. Nuestro resultados indican que ambos miembros se reprimen mutuamente y que comparten dianas comunes a las que regulan inversamente. También, confirmamos que la inhibición ejercida por el miR-30a sobre LIN28B está mediada por su 3'UTR. Por otra parte, determinamos los niveles de proteína de LIN28B en un panel de líneas celulares de carcinomas tiroideos y observamos que su expresión estaba relacionada con la existencia de mutaciones activadoras de la vía PI3K en líneas derivadas de carcinoma anaplásico. Además, validamos como dianas del eje a los inductores de la transición epitelio mesénquima HMGA2, SIX1 y EYA1, y a los efectores de la vía PI3K RAS, PI3Kß, BCL-2, CDK6 y p27(Kip). El efecto a nivel celular de la expresión del miR-30a fue una reducción de la invasión y la proliferación en las líneas celulares tiroideas. Inversamente, la sobreexpresión de LIN28B. Nuestros resultados indican la existencia de un eje LIN28B/miR-30a, de retroalimentación negativa doble, cuya desregulación oncogénica conlleva al silenciamiento del miR-30a y a la expresión aberrante de LIN28B contribuyendo a la progresión tumoral tiroidea.To comprehensively characterize microRNA (miR) and mRNA expression in thyroid cancer, we performed next-generation sequencing expression analysis of 8 papillary thyroid carcinomas (PTC) with paired samples of normal thyroid tissue. In this work, we identified, based on computational predictions of potential targets, a network relating the most abundant overexpressed miRs with the most downregulated targets in PTC. Also, we identified a set of upregulated microRNAs that target genes essential for thyroid differentiation (or thyroid iodide-metabolizing genes), such as NIS, TPO, TSHR, PAX8 and NKX2-1. Among these microRNAs, we found miR-146b-3p -a miR whose both strands were among the most highly overexpressed miRs in thyroid tumors - to putatively inhibit several of them. Performing functional studies we demonstrated that miR-146b-3p reduces NIS, PAX8, and FOXE1 protein expressión. Furthermore, we unveiled that both NIS and PAX8 inhibitions are specifically exerted through a direct binding to their 3‘UTRs. These data suggest that tumor overexpression of miR-146b-3p widely contributes to thyroid dedifferentiation and specially to NIS loss of function through at least two complementary mechanisms: (i) direct postranscriptional level, and (ii) through PAX8 and FOXE1 silencing. The general outcome was a significant decrease in iodide uptake in miR-146b-3p overexpressing cells. Furthermore, we identified the tumor suppressor PTEN as a novel target of miR-146b-5 in thyroid cancer. MiR-146b decreased PTEN mRNA and protein levels by direct binding to its 3‘UTR promoting tumoral hyperactivation of PI3K/AKT pathway. Additionally, mir-146b overexpressing cells showed decreased FOXO1 and p27 nuclear levels, and an increase of total BCL-2, mediating higher proliferation and survival rates. Conversely, our results showed that both constitutive overexpression of RAS and treatment with PI3K pathway effector IGF-1 resulted in increased expression of mir-146b in thyroid follicular cells. Thus, we provide a new mechanism to explain how miR-146b leads to a more aggressive tumoral behavior and an explanation on why PTEN levels are reduced in thyroid carcinomas. Next, we described a novel regulatory axis composed by PTC downregulated miR-30a and the oncofetal miRNA-binding protein LIN28B. Thus, we demonstrated that both members repress each other's expression and have a common pool of targets, which are inversely regulated by each of the axis members. Mir-30a-mediated LIN28B repression was found to be exerted by direct binding to its 3'UTR. We determined the expression levels of LIN28B in a panel of thyroid carcinoma cell lines and we showed it was linked to PI3K hyperactivating mutations in ATC derived cells. Moreover, we validated EMT inducers HMGA2, SIX1 and EYA1 and PI3K effectors RAS, PI3Kß, BCL-2, CDK6 and p27(Kip) as targets regulated by the axis. The general outcome was a significant decrease in invasion and proliferation in mir-30a overexpressing cells and, conversely, an increase in these parameters in LIN28B overexpressing cells. These data suggest the existence of a LIN28B/30a axis, with a double negative feedback regulation, whose tumoral shift, leading to overexpression of LIN28B and silencing of mir-30a, widely contributes to thyroid cancer progression

Impaired microRNA processing by DICER1 downregulation endows thyroid cancer with increased aggressiveness.

The global downregulation of microRNAs (miRNAs) is emerging as a common hallmark of cancer. However, the mechanisms underlying this phenomenon are not well known. We identified that the oncogenic miR-146b-5p attenuates miRNA biosynthesis by targeting DICER1 and reducing its expression. DICER1 overexpression inhibited all the miR-146binduced aggressive phenotypes in thyroid cells. Systemic injection of an anti-miR-146b in mice with orthotopic thyroid tumors suppressed tumor growth and recovered DICER1 levels. Notably, DICER1 downregulation promoted proliferation, migration, invasion, and epithelial-mesenchymal transition through miRNA downregulation. Our analysis of The Cancer Genome Atlas revealed a general decrease in DICER1 expression in thyroid cancer that was associated with a worse clinical outcome. Administration of the small-molecule enoxacin to promote DICER1 complex activity reduced tumor aggressiveness both in vitro and in vivo. Overall, our data confirm DICER1 as a tumor suppressor and show that oncogenic miR-146b contributes to its downregulation. Moreover, our results highlight a potential therapeutic application of RNA-based therapies including miRNA inhibitors and restoration of the biogenesis machinery, which may provide treatments for thyroid and other cancers.post-print3221 K

Genome-wide microrna expression analysis of thyroid cancer by next generation sequencing identifies micrornas involved in thyroid differentiation

Resumen del trabajo presentado al 37th Annual Meeting of the European Thyroid Association, celebrado en Leiden (Holanda) del 7 al 11 de septiembre de 2013.To comprehensively characterize microRNA (miRNA) expression in thyroid cancer, we performed next-generation sequencing expression analysis of this disease. Profiling of microRNAs by deep sequencing measures absolute abundance and allows for the discovery of novel microRNAs or other small RNA species. We sequenced small RNA from 8 papillary thyroid carcinomas with paired samples of normal thyroid tissue. Our results indicate that tumor identity is achieved mainly by variation in the expression levels of a common set of miRNAs rather than by tissue-especific miRNA expression. We observed that mir-146b was by far the most highly overexpressed miRNA in thyroid tumors followed by mir-21, mir-221, mir-222 and mir-31 (in order of abundance). These miRNAs have been previously associated with thyroid cancer. Additionally, we observed at least four miRNAs consistently overexpressed in thyroid cancer and not previously described. Based on computational prediction of potential targets, we identify a set of miRNAs that target genes essential for thyroid differentiation such as NIS, TPO, TG, TSHR, PAX8 and TTFI. Among these miRNAs, we found mir-146b to have one of the best scores in the predictions. We validated mir-146b through functional studies using the differentiated rat thyroid follicular cells PCCl3 and MDCK cells stably transfected with human NIS. Mir-146b is strongly downregulated by TSH in PCCl3 and its expression is recovered by TGFbeta, a strong repressor of thyroid differentiation. We demonstrate that mir-146b binds to the 3`UTR of NIS, destabilizing the mRNA and leading to an impaired translation of the protein and subsequently decreasing the iodide uptake of the cells. In conclusion, our work expands the number of known microRNAs in thyroid cancer and highlights the importance of microRNAs in thyroid differentiation.Financed by grants: BFU2010-16025, S2011/BMD-2328 and RD12/00361-0030Peer reviewe

Identification of miR-146b-3p as a key player in thyroid cancer dedifferentiation

Resumen del trabajo presentado al 38th Annual Meeting of the European Thyroid Association celebrado en Santiago de Compostela (España) del 6 al 10 de septiembre de 2014.Next-generation sequencing expression analysis of papillary thyroid cancer performed in our laboratory, revealed the existence of a set of abundant miRNAs differentially expressed in this pathology. MiR-146b-3p was unveiled as one of the most upregulated microRNAs (miRs) in this neoplasm, surpassing the minimum threshold amount to repress target mRNAs. Furthermore, our previous results showed that this miR binds to the 3'UTR of NIS, inhibiting its expression and consequently its activity. As NIS is a gene regulated by thyroid transcription factors, the aim of this work was to study the effect of miR-146b-3p on the thyroid cancer dedifferentiation process, focusing on the most relevant event for the clinical outcome: the loss of NIS function that leads to radioiodide therapy refractority. By computational predictions, we identified PAX8 and FOXE1 as potential targets of miR-146b-3p according to miRanda algorithm. We validated these predictions through functional studies in PCCl3, NThy-ori and, hNISMDCK cells. Mir-146b expression vector was transfected and reporter constructs containing 3'UTRs, miR specific binding sites, and NIS promoter were generated. The levels of mRNA and protein were determined by RT-qPCR and Western Blot; direct mRNA destabilizing effect was measured in luciferase assays. Overexpression of miR-146b-3p resulted in NIS, PAX8, and FOXE1 mRNA and protein silencing. Furthermore, we observed that as NIS, the PAX8 inhibition is specifically exerted through a direct binding to their 3'UTRs. Also, preliminary data suggest that FoxE1 and PAX8 silencing induced by miR-146b-3p overexpression was sufficient to repress NIS promoter activation. The general outcome was a significant decrease in iodide uptake in miR-146b-3p overexpressing cells. These data suggest that tumor overexpression of miR-146b-3p widely contributes to thyroid dedifferentiation and especially to NIS loss of function through at least two complementary mechanisms: (i) direct postranscriptional level, and (ii) through PAX8 and FOXE1 silencing.Peer reviewe

DICER1 downregulation and impaired miRNA processing in thyroid tumorigenesis

Resumen del póster presentado al 6th Symposium on Biomedical Research: Advances and Perspectives in Molecular Endocrinology "In Homage to Gabriella Morreale", celebrado en el Instituto de Investigaciones Biomédicas Alberto Sols (IIBM-CSIC) el 31 de mayo de 2019.Thyroid cancer is a common endocrine malignancy that has rapidly increased its global incidence in recent decades. Currently the important role of microRNAs (miRNAs) in cancer has been described, including thyroid neoplasms. It is known that miRNAs are important modulators of cancer progression and are globally downregulated in several human tumor types, through mechanisms that remain largely undefined. Functional maturation of most miRNAs requires processing of the primary transcript by DICER1, an RNAse III-type enzyme essential for both mammalian development and cell differentiation. Here we identified DICER1 and miRNA downregulation as sentinel events in thyroid cancer, providing a clear cut evidence of the molecular mechanisms underlying this effect. We demonstrated that this key miRNA-processing enzyme is a target of the most upregulated miRNAs in thyroid cancer. Specifically, miR-146b reduced DICER1 expression and DICER1 overexpression inhibited all the miR-146b-induced aggressive phenotypes, both in cells and in tumor models. Our analysis of The Cancer Genome Atlas revealed a general decrease in DICER1 expression in thyroid cancer and suggested a clinical association between DICER1 and patient prognosis. Notably, DICER1 downregulation promoted proliferation, migration, invasion and epithelial-mesenchymal transition in thyroid cancer cell lines, whereas it suppressed the expression of pro-differentiation transcription factors. Interestingly, these factors transcriptionally upregulated DICER1 expression, supporting the existence of a positive feedback loop. Finally, administration of the small molecule enoxacin to promote DICER1-complex activity reduced cell aggressiveness in vitro and tumor growth in vivo. Overall, our data establish DICER1 as a new tumor suppressor in thyroid cancer, and highlight a potential therapeutic approach of RNA-based therapies including antagomiRs and restoration of the biogenesis machinery may provide treatments for thyroid cancer and other cancers.Peer reviewe

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 ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets

Identification of an interactome network between lncRNAs and miRNAs in thyroid cancer reveals SPTY2D1-AS1 as a new tumor suppressor

Thyroid cancer is the most common primary endocrine malignancy in adults and its incidence is rapidly increasing. Long non-coding RNAs (lncRNAs), generally defined as RNA molecules longer than 200 nucleotides with no protein-encoding capacity, are highly tissue-specific molecules that serve important roles in gene regulation through a variety of different mechanisms, including acting as competing endogenous RNAs (ceRNAs) that ‘sponge’ microRNAs (miRNAs). In the present study, using an integrated approach through RNA-sequencing of paired thyroid tumor and non-tumor samples, we have identified an interactome network between lncRNAs and miRNAs and examined the functional consequences in vitro and in vivo of one of such interactions. We have identified a likely operative post-transcriptional regulatory network in which the downregulated lncRNA, SPTY2D1-AS1, is predicted to target the most abundant and upregulated miRNAs in thyroid cancer, particularly miR-221, a well-known oncomiRNA in cancer. Indeed, SPTY2D1-AS1 functions as a potent tumor suppressor in vitro and in vivo, it is downregulated in the most advanced stages of human thyroid cancer, and it seems to block the processing of the primary form of miR-221. Overall, our results link SPTY2D1-AS1 to thyroid cancer progression and highlight the potential use of this lncRNA as a therapeutic target of thyroid cancer.This work was supported by grants SAF2016-75531-R and PID2019-105303RB-I00/AEI/10.13039/501100011033 from Ministerio de Ciencia e Innovación (MICIN); Fondo Europeo de Desarrollo Regional, B2017/BMD-3724 from Comunidad de Madrid; GCB14142311CRES from Asociación Española contra el Cáncer (AECC); and PI14/01980 from Instituto de Salud Carlos III (Spain). JR-M holds a FPU fellowship from MECD (Spain

The miR-146b-3p/PAX8/NIS regulatory circuit modulates the differentiation phenotype and function of thyroid cells during carcinogenesis

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.This work was supported by Grants BFU2010-16025 and SAF2013-44709-R from the Dirección General de Proyectos de Investigación; RD12/0036/0030 from FIS, Instituto de Salud Carlos III (ISCIII); and S2011/BMD-2328 TIRONET project from the Comunidad de Madrid (Spain) to P. Santisteban; and FIS-ISCIII; PI14/01980 to G. Riesco-Eizaguirre. L. Wert-Lamas and A. Sastre-Perona hold a predoctoral FPI and FPU fellowship, respectively, from the Spanish Government and L.P. Fernández was a JAE.doc postdoctoral from CSIC.Peer reviewe