Publication: The MC1R and MGRN1 genes : genoprotective and phenotype-determining role of melanoma cells
Authors
Cerdido Ochoa, Sonia
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Escuela Internacional de Doctorado
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García-Borrón Martínez, José Carlos ; Herraiz Serrano, Cecilia María
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
El gen MC1R es el principal determinante de la pigmentación de la piel y la sensibilidad a la RUV. Es un gen altamente polimórfico y sus variantes alélicas conocidas como RHC (Red Hair Color) están asociadas a un fenotipo caracterizado por tener la piel clara, pelo rojizo/rubio y mayor riesgo a desarrollar melanoma debido a mecanismos dependientes e independientes de la pigmentación. Este fenotipo se suele atribuir a un defecto en la activación de la vía del AMPc. Sin embargo, algunas variantes RHC muestran una actividad residual notable en esta vía, y además son capaces de activar eficientemente la vía de ERK1/2. Sin embargo, la señalización residual y otras acciones clave del MC1R silvestre y sus variantes nunca se había estudiado en condiciones estrictamente comparables, es decir, en células melanocíticas con el mismo fondo genético y con niveles de expresión similares y en el rango fisiológico. Por ello, diseñamos una estrategia basada en la anulación de la expresión endógena del MC1R mediante CRISPR/Cas9 en células de melanoma humano (CMH) con genotipo silvestre para los principales genes conductores mutados en melanoma (BRAF/NRAS/NF1), reconstituimos la expresión del MC1R y analizamos los clones que expresaban niveles comparables de MC1R silvestre y sus variantes RHC. Demostramos que las variantes presentaban una pérdida parcial de capacidad de señalización por la vía del AMPc, pero eran capaces de activar la ruta de ERK1/2 a niveles comparables con el MC1R silvestre. Además, observamos que las variantes RHC presentaban una protección parcial frente al daño oxidativo en el ADN, si bien dicha protección fue menor que la que confiere el MC1R silvestre. Estos datos indican que las variantes comunes del MC1R asociadas al melanoma muestran una señalización sesgada y una actividad genoprotectora significativa, aunque parcial frente al daño oxidativo. Mahogunin Ring Finger-1 (MGRN1) es una E3 ubiquitina ligasa involucrada en la regulación de la eumelanogénesis en el ratón. Interactúa físicamente con el MC1R alterando su señalización, pudiendo así afectar al desarrollo del melanoma. Estudios realizados en ratones demostraron que la deficiencia de MGRN1 se asociaba con un fenotipo celular menos agresivo. En los pacientes de melanoma, una mayor expresión de MGRN1 en el tumor se correlaciona con menor supervivencia de los enfermos. Sin embargo, la función de MGRN1 en CMH permanece poco estudiada. Gracias al análisis transcriptómico realizado por secuenciación de ARN, observamos que la anulación en la expresión de MGRN1 en CMH (MGRN1-KO) dio lugar a una inducción en genes involucrados en la adhesión célula-célula y célula-matriz, regulación de las GTPasas y a la represión de genes relacionados con la EMT. Asimismo, las células MGRN1-KO presentaron un fenotipo menos agresivo, con mayor adhesión y menor movilidad. El aumento en la adhesión intercelular se debió principalmente a un incremento en la expresión de E-cadherina, acompañado de mayor co-localización con B-catenina. Además, la regulación transcripcional de E-cadherina estuvo mediada probablemente por la disminución en los niveles de su represor ZEB1. Finalmente, ensayos de pulldown mostraron una actividad reducida de CDC42 tras la anulación de MGRN1, que se revirtió tras el silenciamiento de E-cadherina. Paralelamente, demostramos que las células MGRN1-KO presentaban mayor expresión de ITGA5 e ITGB1, siendo notable el incremento de la ITGB1 en la membrana. También observamos como el doble silenciamiento de ITGA5 e ITGB1 en redujo la adhesión celular y aumentó la migración, revirtiendo el fenotipo previamente observado. Finalmente, detectamos una mayor acumulación de vinculina en las adhesiones focales, siendo más grandes y elongadas. Además, también observamos una disminución en la activación de FAK. En conjunto, estos resultados establecen una nueva vía dependiente de MGRN1 que regula la forma, motilidad y potencial invasivo de las CMH.
The MC1R gene is the main determinant of skin pigmentation and sensitivity to UVR. It is a highly polymorphic gene and its allelic variants known as RHC (Red Hair Color), are associated with a phenotype characterized by fair skin, reddish or blond hair and an increased risk of developing melanoma, due to pigmentation-dependent and pigmentation-independent mechanisms. Mechanistically, this phenotype is usually attributed to a defect in the activation of the cAMP pathway. However, some RHC variants show remarkable residual activity in this pathway and are also capable of efficiently activating the MAPK kinase pathway ERK1/2. The comparative analysis of the residual activity of cAMP and other key actions of wild MC1R and its allelic variants had never been studied under strictly comparable conditions in melanocytic cells with the same genetic background and similar levels of expression of receptor isoforms in the physiological range. Therefore, we designed a strategy based on the abrogation of endogenous expression of MC1R using CRISPR/Cas9 technology in human melanoma cells with wild genotype for the main mutated driver genes in melanoma (BRAF, NRAS and NF1), we reconstituted the expression of MC1R labeled with the epitope FLAG and we analyzed clones that expressed comparable levels of MC1R wildtype and the RHC R151C or D294H variants. We showed that R151C and D294H variants had a partial loss of signaling in the cAMP pathway but were able to activate the ERK1/2 pathway at levels comparable to MC1R wildtype. In addition, we observed that the RHC variants presented partial protection against DNA oxidative damage, although this protection was lower than that conferred by MC1R wildtype. These data indicate that common melanoma-associated MC1R variants show biased signaling and significant, albeit partial, genoprotective activity against oxidative damage. Mahogunin Ring Finger-1 (MGRN1) is an E3 ubiquitin ligase involved in the regulation of eumelanogenesis in mice. It physically interacts with MC1R by altering its signaling, thus affecting the development of melanoma. Studies in mice showed that MGRN1 deficiency was associated with a less aggressive phenotype. In melanoma patients, higher expression of MGRN1 in the tumor correlates with lower patient survival. Based on these data, we aimed to study the influence of MGRN1 on the phenotype of human melanoma cells. Thanks to the transcriptomic analysis performed by RNA sequencing (bulk RNA-seq), we observed that the permanent abrogation of MGRN1 expression in human melanoma cells (MGRN1-KO) resulted in an induction of genes involved in cell-cell and cell-matrix adhesion, in the regulation of GTPases and the repression of genes related to the epithelial-mesenchymal transition. Likewise, MGRN1-KO cells showed a less aggressive phenotype, with higher dendricity and formation of larger cellular aggregates and less migration and invasion. The increase in the intercellular adhesion was mainly due to an enhance in E-cadherin expression, together with greater co-localization with B-catenin. In addition, the transcriptional regulation of E-cadherin was probably mediated by the decrease of its repressor ZEB1. Finally, pulldown assays showed reduced CDC42 activity after MGRN1 depletion, which was reverted after E-cadherin silencing. At the same time, we showed that MGRN1-KO cells had higher expression of ITGA5 and ITGB1, being especially notable the increase in ITGB1 at the plasma membrane. We also observed tnat the double silencing of ITGA5 and ITGB1 in the MGRN1-KO clone reduced cell adhesion and increased migration, reverting the phenotype previously observed. Finally, we detected an increase in total vinculin expression and a greater accumulation of vinculin in the focal adhesions, which were larger and more elongated. In addition, we also observed a decrease in FAK activation. Altogether, these results establish a novel MGRN1-dependent pathway that regulates the shape, motility, and invasive potential of melanoma cells.
The MC1R gene is the main determinant of skin pigmentation and sensitivity to UVR. It is a highly polymorphic gene and its allelic variants known as RHC (Red Hair Color), are associated with a phenotype characterized by fair skin, reddish or blond hair and an increased risk of developing melanoma, due to pigmentation-dependent and pigmentation-independent mechanisms. Mechanistically, this phenotype is usually attributed to a defect in the activation of the cAMP pathway. However, some RHC variants show remarkable residual activity in this pathway and are also capable of efficiently activating the MAPK kinase pathway ERK1/2. The comparative analysis of the residual activity of cAMP and other key actions of wild MC1R and its allelic variants had never been studied under strictly comparable conditions in melanocytic cells with the same genetic background and similar levels of expression of receptor isoforms in the physiological range. Therefore, we designed a strategy based on the abrogation of endogenous expression of MC1R using CRISPR/Cas9 technology in human melanoma cells with wild genotype for the main mutated driver genes in melanoma (BRAF, NRAS and NF1), we reconstituted the expression of MC1R labeled with the epitope FLAG and we analyzed clones that expressed comparable levels of MC1R wildtype and the RHC R151C or D294H variants. We showed that R151C and D294H variants had a partial loss of signaling in the cAMP pathway but were able to activate the ERK1/2 pathway at levels comparable to MC1R wildtype. In addition, we observed that the RHC variants presented partial protection against DNA oxidative damage, although this protection was lower than that conferred by MC1R wildtype. These data indicate that common melanoma-associated MC1R variants show biased signaling and significant, albeit partial, genoprotective activity against oxidative damage. Mahogunin Ring Finger-1 (MGRN1) is an E3 ubiquitin ligase involved in the regulation of eumelanogenesis in mice. It physically interacts with MC1R by altering its signaling, thus affecting the development of melanoma. Studies in mice showed that MGRN1 deficiency was associated with a less aggressive phenotype. In melanoma patients, higher expression of MGRN1 in the tumor correlates with lower patient survival. Based on these data, we aimed to study the influence of MGRN1 on the phenotype of human melanoma cells. Thanks to the transcriptomic analysis performed by RNA sequencing (bulk RNA-seq), we observed that the permanent abrogation of MGRN1 expression in human melanoma cells (MGRN1-KO) resulted in an induction of genes involved in cell-cell and cell-matrix adhesion, in the regulation of GTPases and the repression of genes related to the epithelial-mesenchymal transition. Likewise, MGRN1-KO cells showed a less aggressive phenotype, with higher dendricity and formation of larger cellular aggregates and less migration and invasion. The increase in the intercellular adhesion was mainly due to an enhance in E-cadherin expression, together with greater co-localization with B-catenin. In addition, the transcriptional regulation of E-cadherin was probably mediated by the decrease of its repressor ZEB1. Finally, pulldown assays showed reduced CDC42 activity after MGRN1 depletion, which was reverted after E-cadherin silencing. At the same time, we showed that MGRN1-KO cells had higher expression of ITGA5 and ITGB1, being especially notable the increase in ITGB1 at the plasma membrane. We also observed tnat the double silencing of ITGA5 and ITGB1 in the MGRN1-KO clone reduced cell adhesion and increased migration, reverting the phenotype previously observed. Finally, we detected an increase in total vinculin expression and a greater accumulation of vinculin in the focal adhesions, which were larger and more elongated. In addition, we also observed a decrease in FAK activation. Altogether, these results establish a novel MGRN1-dependent pathway that regulates the shape, motility, and invasive potential of melanoma cells.
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