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

ADAR1-mediated RNA editing is a novel oncogenic process in thyroid cancer and regulates miR-200 activity

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. A-to-I editing of RNA is a widespread posttranscriptional process that has recently emerged as an important mechanism in cancer biology. A-to-I editing levels are high in several human cancers, including thyroid cancer, but ADAR1 editase-dependent mechanisms governing thyroid cancer progression are unexplored. To address the importance of RNA A-to-I editing in thyroid cancer, we examined the role of ADAR1. Loss-of-function analysis showed that ADAR1 suppression profoundly repressed proliferation, invasion, and migration in thyroid tumor cell models. These observations were validated in an in vivo xenograft model, which showed that ADAR1-silenced cells had a diminished ability to form tumors. RNA editing of miRNAs has the potential to markedly alter target recognition. According to TCGA data, the tumor suppressor miR-200b is overedited in thyroid tumors, and its levels of editing correlate with a worse progression-free survival and disease stage. We confirmed miR-200b overediting in thyroid tumors and we showed that edited miR-200b has weakened activity against its target gene ZEB1 in thyroid cancer cells, likely explaining the reduced aggressiveness of ADAR1-silenced cells. We also found that RAS, but not BRAF, modulates ADAR1 levels, an effect mediated predominantly by PI3K and in part by MAPK. Lastly, pharmacological inhibition of ADAR1 activity with the editing inhibitor 8-azaadenosine reduced cancer cell aggressiveness. Overall, our data implicate ADAR1-mediated A-to-I editing as an important pathway in thyroid cancer progression, and highlight RNA editing as a potential therapeutic target in thyroid cancerThis work was supported by grants SAF2016-75531-R from Ministerio de Ciencia e Innovación (MICIN), Spain, Fondo Europeo de Desarrollo Regional FEDER, B2017/BMD-3724 from Comunidad de Madrid, and GCB14142311CRES from Fundación Española contra el Cáncer (AECC) (To PS), and by the Ludwig Center at Harvard (To FJS). JR-M hold a FPU fellowship from MECD (Spai

Role-playing games para la adquisición de competencias en el Grado en Ingeniería Química

Se propone la realización de un juego de rol con el objetivo de que los alumnos comprendan una asignatura a priori tediosa, Gestión Integrada en la Industria Química, de una forma práctica y amena. Este tipo de prueba facilita la evaluación de varias competencias a la vez, englobando tanto habilidades técnicas, como la comunicación oral y la puesta en escena. En este caso concreto los alumnos se han introducido en la situación de una auditoría de los distintos sistemas de gestión de la Universidad de Alicante, incluyendo calidad, medio ambiente y prevención de riesgos laborales. Los profesores han valorado las competencias adquiridas y los alumnos han realizado una encuesta para comprobar si ellos tienen la percepción de haberlas adquirido. Además se estudia su utilidad en un entorno laboral, en prácticas en empresa, ya que varios alumnos realizan las asignaturas de Prácticas Externas después de haber cursado la asignatura de Gestión Integrada en la Industria Química. El trabajo se completa con el estudio del posible interés de los juegos de rol en otras asignaturas de la titulación

Identificación de mecanismos de regulación del RNA en cáncer de tiroides

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 19-02-2021RNA networks are fundamental in the control of gene regulation, and recent years have witnessed a strong interest in non-coding RNAs, such as microRNAs (miRNAs). These products of the noncoding genome have key regulatory roles in cellular biology, and their aberrant expression/function is associated with numerous diseases, including cancer. In the present thesis, we identified novel RNA regulatory mechanisms important for thyroid tumorigenesis, the most common endocrine cancer. We show that miRNA dysregulation is a common event in this cancer, and involves not only individual changes in the expression of miRNAs, but also disruption of the biogenesis machinery and/or modifications to miRNA sequences by RNA editing. We explored the role and mechanisms of action of the most upregulated miRNA in thyroid cancer, miR-146b, and describe three novel targets that help explain miR-146b-induced cell aggressiveness: PTEN, E-cadherin and DICER1. We also demonstrate that all three proteins have tumor-suppressive activity in thyroid cancer. Specifically, miR-146b induces hyperactivation of the oncogenic PI3K pathway by inhibiting PTEN, promotes cell motility by suppressing E-cadherin, and triggers the global reduction of the miRNA network by downregulating DICER1. Importantly, these events are frequent in thyroid tumors. We also investigated the involvement of the high adenosine-toinosine (A-to-I) RNA editing levels in thyroid tumors and how this process adds complexity to the post-transcriptional regulation of gene expression. We demonstrate that ADAR1, the enzyme responsible for A-to-I RNA editing in human thyroid cells, induces tumorigenesis both in vitro and in vivo. This process is dependent, at least in part, on the overediting of the seed sequence of the tumor-suppressor miR-200b, impairing its ability to inhibit its target ZEB1. Our results highlight potential therapeutic approaches based on miRNA inhibition, restoring global miRNA levels via the DICER1 pathway, and suppressing RNA editing, overall providing a basis for new thyroid cancer treatment

MiRNA-directed regulation of the main signaling pathways in thyroid cancer

© 2019 Ramírez-Moya and Santisteban.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.We acknowledge the support of grants SAF2016-75531-R (MINECO/FEDER, UE); Asociación Española Contra el Cancer (AECC; GCB141423113); CIBERONC from the Instituto de Salud Carlos III (ISCIII) and B2017/BMD-3724 Tironet2 (Comunidad de Madrid). JR-M, holds a FPU fellowship from Spanish Ministry of Education

A positive feedback loop between DICER1 and differentiation transcription factors is important for thyroid tumorigenesis

© Julia Ramírez-Moya and Pilar Santisteban, 2020.[Background]: DICER1 plays a central role in microRNA biogenesis and functions as a tumor suppressor in thyroid cancer, which is the most frequent endocrine malignancy with a rapidly increasing incidence. Thyroid cancer progression is associated with loss of cell differentiation and reduced expression of thyroid differentiation genes and response to thyrotropin (TSH). Here we investigated whether a molecular link exists between DICER1 and thyroid differentiation pathways. [Methods]: We used bioinformatic tools to search for transcription factor binding sites in the DICER1 promoter. DICER1, NKX2-1, PAX8, and CREB expression levels were evaluated by gene and protein expression in vitro and by interrogation of The Cancer Genome Atlas (TCGA) thyroid cancer data. Transcription factor binding and activity were assayed by chromatin immunoprecipitation, band-shift analysis, and promoter–reporter gene activity. Gene-silencing and overexpression approaches were used to elucidate the functional link between DICER1 and differentiation. [Results]: We identified binding sites for NKX2-1 and CREB within the DICER1 promoter and found that both transcription factors are functional in thyroid cells. TSH induced DICER1 expression in differentiated thyroid cells, at least in part, through the cAMP/PKA/CREB pathway. TCGA analysis revealed a significant positive correlation between CREB and DICER1 expression in human thyroid tumors. NKX2-1 overexpression increased DICER1 promoter activity and expression in vitro, and this was significantly greater in the presence of CREB and/or PAX8. Gain- and loss-of-function assays revealed that DICER1 regulates NKX2-1 expression in thyroid tumor cells and vice versa, thus establishing a positive feedback loop between both proteins. We also found a positive correlation between NKX2-1 and DICER1 expression in human thyroid tumors. DICER1 silencing decreased PAX8 expression and, importantly, the expression and activity of the sodium iodide symporter, which is essential for the diagnostic and therapeutic use of radioiodine in thyroid cancer. [Conclusions]: The differentiation transcription factors NKX2.1, PAX8, and CREB act in a positive feedback loop with DICER1. As the expression of these transcription factors is markedly diminished in thyroid cancer, our findings suggest that DICER1 downregulation in this cancer is mediated, at least partly, through impairment of its transcription.This work was supported by grants SAF2016-75531-R, Fondo Europeo de Desarrollo Regional, and PID2019-1053 03RB-I00/AEI/10.13039/501100011033 from Ministerio de Ciencia e Innovacio´n (MICIN), Spain, B2017/BMD-3724 from Comunidad de Madrid, and GCB14142311CRES from Fundacio´n Espan˜ola Contra el Ca´ncer (AECC). J.R.-M. holds an FPU fellowship from MECD (Spain)

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

An ADAR1-dependent RNA editing event in the cyclin-dependent kinase CDK13 promotes thyroid cancer hallmarks

© The Author(s) 2021.[Background]: Adenosine deaminases acting on RNA (ADARs) modify many cellular RNAs by catalyzing the conversion of adenosine to inosine (A-to-I), and their deregulation is associated with several cancers. We recently showed that A-to-I editing is elevated in thyroid tumors and that ADAR1 is functionally important for thyroid cancer cell progression. The downstream effectors regulated or edited by ADAR1 and the significance of ADAR1 deregulation in thyroid cancer remain, however, poorly defined. [Methods]: We performed whole transcriptome sequencing to determine the consequences of ADAR1 deregulation for global gene expression, RNA splicing and editing. The effects of gene silencing or RNA editing were investigated by analyzing cell viability, proliferation, invasion and subnuclear localization, and by protein and gene expression analysis. [Results]: We report an oncogenic function for CDK13 in thyroid cancer and identify a new ADAR1-dependent RNA editing event that occurs in the coding region of its transcript. CDK13 was significantly over-edited (c.308A > G) in tumor samples and functional analysis revealed that this editing event promoted cancer cell hallmarks. Finally, we show that CDK13 editing increases the nucleolar abundance of the protein, and that this event might explain, at least partly, the global change in splicing produced by ADAR1 deregulation. [Conclusions]: Overall, our data support A-to-I editing as an important pathway in cancer progression and highlight novel mechanisms that might be used therapeutically in thyroid and other cancers.This work was supported by the following grants: SAF2016-75531-R, Fondo Europeo de Desarrollo Regional, and PID2019-105303RB-I00/AEI/10.13039/501100011033 from Ministerio de Ciencia e Innovación, Spain; B2017/BMD-3724 from Comunidad de Madrid, and GCB14142311CRES from Fundación Española Contra el Cáncer (AECC) (to PS); NIH (R35 CA232105) and the Ludwig Center at Harvard (to FJ-S); outstanding Investigator Award (R35CA232115) from the National Cancer Institute of the NIH (to RI-G). J.R-M holds an FPU fellowship from Ministerio de Educación, Cultura y Deporte (Spain)

Transcriptome profiling of ADAR1 targets in triple-negative breast cancer cells reveals mechanisms for regulating growth and invasion

ADARs catalyze adenosine-to-inosine (A-to-I) editing of double-stranded RNA and regulate global gene expression output through interactions with RNA and other proteins. ADARs play important roles in development and disease, and previous work has shown that ADAR1 is oncogenic in a growing list of cancer types. Here we show that ADAR1 is a critical gene for triple-negative breast cancer cells, as ADAR1 loss results in reduced growth (viability and cell cycle progression), invasion, and mammosphere formation. Whole transcriptome sequencing analyses demonstrate that ADAR1 regulates both coding and noncoding targets by altering gene expression level, A-to-I editing, and splicing. We determine that a recoding edit in filamin B (FLNB chr3:58156064) reduces the tumor suppressive activities of the protein to promote growth and invasion. We also show that several tumor suppressor miRNAs are upregulated upon ADAR1 loss and suppress cell-cycle progression and invasion. This work describes several novel mechanisms of ADAR1-mediated oncogenesis in triple-negative breast cancer, providing support to strategies targeting ADAR1 in this aggressive cancer type that has few treatment options.We acknowledge the NCI Outstanding Investigator Award (R35CA232105) and funding from the Ludwig Center at Harvard to F.J. Slack