Regulación transcripcional de NUAK1 por SALL2 frente a estrés metabólico.
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Date
2023
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad de Concepción.
Abstract
El estrés metabólico en cáncer es un paso crítico para la migración, invasión y metástasis. NUAK1, un miembro de la familia de quinasas relacionadas a AMPK (ARKs), se encuentra desregulado en algunos tipos de cáncer. Su sobreexpresión promueve migración, metástasis y supervivencia celular frente a distintos contex-tos, incluyendo estrés metabólico. Datos de nuestro laboratorio muestran que NUAK1 es inducido transcripcionalmente frente a la carencia de glucosa; sin embar-go, se desconocen los mecanismos que participan en esta respuesta. Se ha pro-puesto que SALL2, un factor de transcripción con roles duales en el cáncer podría estar involucrado. Aunque SALL2 generalmente actúa como un supresor tumoral, en condiciones de estrés metabólico su expresión aumenta y contribuye a la supervi-vencia en células MEF; sin embargo, el mecanismo mediante el cual SALL2 ejerce este rol no ha sido reportado. Para investigar la relación entre NUAK1 y SALL2, se realizaron análisis bioinformáticos utilizando bases de datos como miPanda y R2, los cuales mostraron una correlación positiva entre los niveles de SALL2 y NUAK1 en varios tejidos. De acuerdo con esos análisis, encontramos una dependencia de SALL2 para el incremento en la expresión de NUAK1 frente a estrés metabólico por carencia de glucosa. Con el fin de determinar si NUAK1 es un blanco directo de SALL2, se analizó el promotor de NUAK1 humano y Nuak1 de ratón, en los cuales se encontraron sitios consenso de unión a SALL2 conservados entre ambas especies. Ensayos de gen reportero de luciferasa demostraron que la ganancia de función de SALL2 incrementa la actividad del promotor Nuak1, mientras que estudios de inmu-noprecipitación de cromatina (ChIP) demostraron que SALL2 se une al promotor NUAK1. Al comprobar el rol pro-supervivencia de SALL2 ante estrés metabólico en diferentes modelos, se encontraron tendencias controversiales. Solo se observó el incremento en la supervivencia celular dependiente de SALL2 en modelos de pérdida de función (silenciamiento), y la tendencia fue similar a los datos previos usando células MEF silvestres y deficientes de Sall2. Más aún, nuestros estudios sugieren que existe una dependencia de NUAK1 para el rol pro-supervivencia de SALL2. De forma interesante, la inhibición de NUAK1 en condiciones de alta glucosa, induce la formación de vacuolas que se corresponden con el fenotipo de un tipo de muerte ce-lular conocida como methuosis, las cuales son más abundantes en células shCtrl respecto a las silenciadas (shSall2). Estos datos se condicen con observaciones pre-vias del laboratorio en células de cáncer de colon, asociándose SALL2 con la gene-ración de methuosis. En conjunto, nuestros datos sugieren a NUAK1 como un blanco transcripcional de SALL2 asociado a la supervivencia celular frente a estrés metabólico por carencia de glucosa. Dilucidar los mecanismos normales de regulación transcripcional de NUAK1 es esencial para comprender su desregulación en cáncer.
Metabolic stress in cancer is critical for migration, invasion, and metastasis. NUAK1, a member of the AMPK-related kinases (ARKs) family, is deregulated in some types of cancer. NUAK1 overexpression promotes cell migration, metastasis, and survival in different contexts, including metabolic stress. Data from our labora-tory show that NUAK1 is transcriptionally induced by glucose deprivation; however, the mechanisms involved in this response are unknown. It has been proposed that SALL2, a transcription factor with dual roles in cancer, may be implicated. Although SALL2 generally acts as a tumor suppressor, under conditions of metabolic stress, its expression increases and contributes to cell survival in MEF cells; however, the mechanism by which SALL2 exerts this role has not been reported. Bioinformatic analyses were performed using databases such as miPanda and R2, which showed a positive correlation between SALL2 and NUAK1 levels in several tissues. According-ly, we found SALL2 dependence for the increase of NUAK1 expression against meta-bolic stress due to glucose deficiency. To determine whether NUAK1 is a direct target of SALL2, we analyzed the promoter of human NUAK1 and mouse Nuak1, in which we found consensus SALL2 binding sites conserved in both species. Through lucifer-ase reporter gene assays, we demonstrated that SALL2 gain-of-function increas-es Nuak1 promoter activity, while chromatin immunoprecipitation (ChIP) studies showed that SALL2 binds to the NUAK1 promoter. Controversial trends were found when testing the pro-survival role of SALL2 against metabolic stress in different mod-els. The SALL2-dependent increase in cell survival was only observed in loss-of-function models (silencing), similar to previous data using wild-type and SALL2-deficient MEF cells. Moreover, our results suggest a dependence on NUAK1 for the pro-survival role of SALL2. Interestingly, NUAK1 inhibition under high glucose conditions induced vacuole formation corresponding to the phenotype of a type of cell death known as methuosis, which is more abundant in shCtrl cells than silenced (shSall2) cells. These data are consistent with previous observations from the labora-tory in colon cancer cells, associating SALL2 with the generation of methuosis. Our data suggest NUAK1 as a transcriptional target of SALL2 associated with cell survival under metabolic stress by glucose deprivation. Elucidating the nor-mal mechanisms of transcriptional regulation of NUAK1 is essential to understanding its dysregulation in cancer.
Metabolic stress in cancer is critical for migration, invasion, and metastasis. NUAK1, a member of the AMPK-related kinases (ARKs) family, is deregulated in some types of cancer. NUAK1 overexpression promotes cell migration, metastasis, and survival in different contexts, including metabolic stress. Data from our labora-tory show that NUAK1 is transcriptionally induced by glucose deprivation; however, the mechanisms involved in this response are unknown. It has been proposed that SALL2, a transcription factor with dual roles in cancer, may be implicated. Although SALL2 generally acts as a tumor suppressor, under conditions of metabolic stress, its expression increases and contributes to cell survival in MEF cells; however, the mechanism by which SALL2 exerts this role has not been reported. Bioinformatic analyses were performed using databases such as miPanda and R2, which showed a positive correlation between SALL2 and NUAK1 levels in several tissues. According-ly, we found SALL2 dependence for the increase of NUAK1 expression against meta-bolic stress due to glucose deficiency. To determine whether NUAK1 is a direct target of SALL2, we analyzed the promoter of human NUAK1 and mouse Nuak1, in which we found consensus SALL2 binding sites conserved in both species. Through lucifer-ase reporter gene assays, we demonstrated that SALL2 gain-of-function increas-es Nuak1 promoter activity, while chromatin immunoprecipitation (ChIP) studies showed that SALL2 binds to the NUAK1 promoter. Controversial trends were found when testing the pro-survival role of SALL2 against metabolic stress in different mod-els. The SALL2-dependent increase in cell survival was only observed in loss-of-function models (silencing), similar to previous data using wild-type and SALL2-deficient MEF cells. Moreover, our results suggest a dependence on NUAK1 for the pro-survival role of SALL2. Interestingly, NUAK1 inhibition under high glucose conditions induced vacuole formation corresponding to the phenotype of a type of cell death known as methuosis, which is more abundant in shCtrl cells than silenced (shSall2) cells. These data are consistent with previous observations from the labora-tory in colon cancer cells, associating SALL2 with the generation of methuosis. Our data suggest NUAK1 as a transcriptional target of SALL2 associated with cell survival under metabolic stress by glucose deprivation. Elucidating the nor-mal mechanisms of transcriptional regulation of NUAK1 is essential to understanding its dysregulation in cancer.
Description
Tesis para optar al grado de doctor en Ciencias Biológicas, área Biología Celular y Molecular.