Rol de la enzima ADAR en la proliferación y replicación del DNA durante la progresión del adenocarcinoma mamario

Grado de doctor en ciencias biomédicasLa enzima ADAR1 (Adenosina Deaminasa específica de RNA 1) presenta una expresión aumentada en diversos tumores, incluyendo el cáncer de mama. Múltiples análisis bioinformáticos y experimentales han revelado que la edición del mRNA mediada por esta proteína afecta preferentemente regiones intrónicas o regiones no traducibles del mRNA (UTRs), modificando la expresión o estabilidad de los transcritos editados por esta proteína. Adicionalmente, un grupo significativo de los genes editados en las regiones UTRs por ADAR1 están asociados a la respuesta al daño del DNA y puntos de control del ciclo celular, los cuales han sido relacionados a la progresión del cáncer de mama. A la fecha, no se ha determinado el efecto de la sobreexpresión de ADAR1 y el impacto de la edición de estos transcritos sobre la proliferación y la respuesta al daño del DNA de este tipo de cáncer, donde existe una alta expresión de esta enzima. Así, este trabajo propuso la siguiente Hipótesis: “La expresión de la enzima ADAR1 regula la estabilidad de los mRNAs de genes que participan en la respuesta al daño del DNA y puntos de control del ciclo celular, promoviendo el estrés replicativo y la proliferación celular en adenocarcinoma mamario” proponiendo como objetivo general determinar el efecto de la enzima ADAR1 sobre la expresión de genes asociados a los procesos de respuesta al daño del DNA y control del ciclo celular; y caracterizar su efecto sobre la replicación y proliferación celular, en el adenocarcinoma mamario. Para comprobar esta hipótesis se llevaron a cabo los siguientes objetivos específicos: 1. Analizar el efecto de la expresión de ADAR1, en la expresión y estabilidad de los mRNAs de genes asociados a los procesos de respuesta al daño del DNA y control del ciclo celular en las líneas celulares MCF-7 y ZR-751; 2. Determinar el efecto de la expresión de ADAR1 sobre el estrés replicativo en las líneas celulares MCF-7 y ZR-751; 3. Evidenciar el efecto de la expresión de ADAR1 sobre la proliferación en las líneas celulares MCF-7 y ZR-751; y 4. Analizar la expresión de ADAR1 y la edición de mRNAs de genes que participan en los procesos de respuesta al daño del DNA y control del ciclo celular, en muestras de pacientes con adenocarcinoma mamario. Los resultados muestran que la disminución de la expresión de ADAR1 en células ZR-751 produce cambios significativos en la estabilidad de los mRNAs editados por ADAR1 involucrados en los procesos de respuesta al daño del DNA, replicación y proliferación celular. Estos cambios se relacionaron con una disminución de la proliferación y un incremento en la apoptosis. Adicionalmente, células MCF7 y ZR-751 que presentan una disminución en la expresión de ADAR1, presentan una disminución significativa de la actividad de la cascada transduccional de respuesta al daño en el DNA y una acumulación en la fase S del ciclo celular. Por otro lado, el análisis de una cohorte de pacientes con cáncer de mama (The Cancer Genome Atlas, TCGA), mostró que los tumores presentan un mayor número de variantes, generadas por ADAR, en los UTRs analizados, en comparación a tejido mamario no tumoral. Finalmente, pacientes cuyos tumores presentan una mayor expresión de ADAR1 o número de variantes en sus UTRs, tienen una sobrevida significativamente menor que pacientes con baja expresión ADAR1. Así, este trabajo demuestra que ADAR1 desempeña un papel importante en la progresión de BRCA a través de la regulación de la estabilidad y expresión de genes que están involucrados en la replicación y la proliferación, afectando a la proliferación celular, viabilidad y respuesta al daño del DNA.The double stranded RNA-specific adenosine deaminase (ADAR1) enzyme has an increased expression in various tumors, including breast cancer (BRCA). Multiple bioinformatic and experimental analyzes have revealed that mRNA editing mediated by ADAR1 preferentially affects intronic or untranslated regions (UTRs) of the mRNA, modifying the expression or stability of the edited transcripts. In addition, a significant group of genes edited in UTRs regions are associated with DNA damage response and cell cycle control points, which have been linked to breast cancer progression. However, the effect of the overexpression of ADAR1 and the impact of the editing of these transcripts on the proliferation and response to DNA damage of this type of cancer, where there is a high expression of this enzyme, has not been determined.This work aims to determine ADAR1 expression role on genes associated in DNA damage response and cell cycle, to further characterize its effect on replicative stress and proliferation in breast cancer. Based on that, the following hypothesis was formulated: "ADAR1 expression regulates the mRNAs stability of genes involved in the DNA damage response and cell cycle check points, promoting replicative stress and cell proliferation in breast cancer". With the following specific aims: 1. To Analyze the ADAR1 expression role on mRNA expression and stability of genes associated with DNA damage response and cell cycle processes in MCF-7 cell lines and ZR-751. 2. To determine the ADAR1 expression effect on replicative stress in MCF-7 and ZR-751 cell lines. 3. To demonstrate the ADAR1 expression effect on proliferation in cell lines MCF-7 and ZR-751 and 4. To analyze the ADAR1 expression pattern and RNA editing on genes involved in the DNA damage response and cell cycle control from patients with breast adenocarcinoma. Our results demonstrate that decreased ADAR1 expression in ZR-751 cells produces significant changes on mRNAs stability of edited transcripts by ADAR1 involved in DNA damage response and DNA replication. Furthermore, ADAR1 knock down cells shown a decreased proliferation, viability and an increased apoptosis, in comparison to control cells. Moreover, MCF7 and ZR-751 knock down cells exhibit a significant decrease of their DNA damage response activation with a significant accumulation on S phase, suggesting an impair cell cycle progression of these cells. Finally, breast cancer patients from The Cancer Genome Atlas shown an increased number of A to G variants, addressable to ADAR1 in their UTRs, compared to control patients from this cohort. Finally, patients with an increased ADAR1 expression or variants counts in their UTRs have shown a decreased overall survival, suggesting that ADAR1 function has an implicit clinical outcome. Taken together, this work shows that ADAR1 plays an important role in BRCA progression through the regulation of mRNA stability and expression from genes that are involved in the replication stress and proliferation, affecting the cell proliferation, DNA damage response and viability allowing the development of malignant phenotypes in breast cancer cells.22/11/201

Ski Is required for tri-methylation of H3K9 in major satellite and for repression of pericentromeric genes: Mmp3, Mmp10 and Mmp13, in mouse fibroblasts

Several mechanisms directing a rapid transcriptional reactivation of genes immediately after mitosis have been described. However, little is known about the maintenance of repressive signals during mitosis. In this work, we address the role of Ski in the repression of gene expression during M/G(1) transition in mouse embryonic fibroblasts (MEFs). We found that Ski localises as a distinct pair of dots at the pericentromeric region of mitotic chromosomes, and the absence of the protein is related to high acetylation and low tri-methylation of H3K9 in pericentromeric major satellite. Moreover, differential expression assays in early G(1) cells showed that the presence of Ski is significantly associated with repression of genes localised nearby to pericentromeric DNA. In mitotic cells, chromatin immunoprecipitation assays confirmed the association of Ski to major satellite and the promoters of the most repressed genes: Mmp3, Mmp10 and Mmp13. These genes are at pericentromeric region of chromosome 9. In these promoters, the presence of Ski resulted in increased H3K9 tri-methylation levels. This Ski-dependent regulation is also observed during interphase. Consequently, Mmp activity is augmented in Ski-/- MEFs. Altogether, these data indicate that association of Ski with the pericentromeric region of chromosomes during mitosis is required to maintain the silencing bookmarks of underlying chromatin.Comisión Nacional de Investigación Cientifica y Tecnológica (CONICYT) FONDECYT 1151435 1120222 PIA ACT172101 FONDAP 15090007 2111055

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response

International audienceThe molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis