Asociación de SALL2 y vía WNT/β-CATENINA en cáncer de colon.
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Date
2023
Authors
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Publisher
Universidad de Concepción.
Abstract
SALL2 es un factor de transcripción implicado en el desarrollo embrionario, y
funcionalmente se relaciona con la regulación de la proliferación, migración y supervivencia celular. Análisis de datos masivos de cáncer versus tejidos normales indican que el ARNm de SALL2 se encuentra significativamente disminuido en el cáncer colorrectal (CCR), un tipo de cáncer caracterizado por la hiperactivación de la vía Wnt/β-catenina. De manera interesante, análisis previos de Inmunoprecipitación de cromatina-secuenciación (ChIPseq) del laboratorio sugirien que SALL2 regula genes asociados con la vía Wnt. Sin embargo, a la fecha no se ha caracterizado la expresión proteica de SALL2 en tejidos de colon normal o CCR. Por otra parte, se desconoce el rol de SALL2 en CCR y su relación con la vía Wnt. Para caracterizar la expresión de SALL2 en tejidos de colon, se construyó un tissue microarray usando 130 biopsias de archivo del Hospital Guillermo Gantt Benavente
incluyendo colon normal, adenoma y CCR, y se evaluó la expresión y localización de SALL2 mediante inmunohistoquímica. Encontramos que SALL2 se expresa en el núcleo y el citoplasma del epitelio, y en el estroma del colon normal se expresa en fibroblastos. Sin embargo, su expresión está disminuida en el adenoma y ausente en el CCR, coincidiendo con los resultados bioinformáticos previos. En biopsias de CCR observamos una
correlación negativa entre la expresión de SALL2 y la expresión de β-catenina nuclear en el frente migratorio, un marcador pronóstico. Esta correlacion se confirmó en células CCR con ganancia y pérdida de función de SALL2 a través de inmunofluorescncia y fraccionamiento subcelular. Además, evaluamos la dependencia de SALL2 en la expresión de blancos de la vía Wnt por Western blot y qRT-PCR, encontrando una correlación positiva entre SALL2 y AXIN2, un importante regulador negativo de la vía Wnt. El análisis del promotor de AXIN2 identificó varios sitios de unión putativos de SALL2. A nivel mecanístico demostramos que la inducción de SALL2 en modelos celulares doxiciclina inducible, aumenta la actividad del promotor de AXIN2. A través de ChIP-PCR demostramos que SALL2 se une a la porción proximal del promotor de AXIN2, lo que confirma una regulación transcripcional dependiente de SALL2. Finalmente, mostramos que el eje SALL2-AXIN2 es necesario para la respuesta de supervivencia a XAV939, un inhibidor de la vía Wnt. En conclusión, nuestro estudio confirma que SALL2 regula positivamente la transcripción de AXIN2. Se propone que la pérdida de la expresión de SALL2 durante la progresión del CCR contribuye a la disminución de los niveles de AXIN2, lo que contribuye a la hiperactivación de la vía Wnt.
SALL2 is a developmental transcription factor involved in the regulation of cell Proliferation, migration, and survival. Massive data analyses of cancer versus normal tissues indicated that SALL2 mRNA significantly decreases in colorectal cancer (CRC), a cancer type characterized by Wnt pathway hyperactivation. Interestingly, our unpublished ChIP-seq data analyses suggested that SALL2 regulates genes associated with the Wnt pathway, involved in the development and progression of solid tumors, including CRC. However, the role of SALL2 in CRC and its relationship with the Wnt pathway are yet unknown. We constructed a tissue microarray of 130 samples from Guillermo Gantt Benavente’s Hospital comprising normal colon, adenoma, and CRC to evaluate the expression and cellular location of SALL2 in normal tissues and during CRC progression by immunohistochemistry. We found that SALL2 expresses in the nucleus and cytoplasm of the normal colon epithelium and the stroma. However, its expression decreases in adenoma and is absent in CRC. We noted a negative correlation between SALL2 expression and the expression of nuclear β-catenin, a marker of progression and activation of the Wnt pathway, at the migratory front. Subcellular fractionation and immunofluorescence experiments in CRC cells with gain and loss of SALL2 function support the negative correlation between SALL2 expression and nuclear β-catenin. Additionally, we evaluated the expression of Wnt targets by Western blot and qRT-PCR, finding a positive correlation between SALL2 and AXIN2, a negative regulator of the Wnt pathway.Analysis of AXIN2 promoter identified several putative SALL2 binding sites, and the induction of SALL2 increases AXIN2 promoter activity in SALL2 knockout models. In addition, using ChIP-PCR we found that SALL2 binds to the proximal region in AXIN2 promoter, confirming a SALL2- dependent transcriptional regulation. Finally, we show that the SALL2-AXIN2 axis is required for the antiproliferative, and apoptotic response to XAV939, an inhibitor of the Wnt pathway. In conclusion our study confirms that SALL2 positively regulates the transcription of AXIN2. We proposed that the loss of SALL2 expression during CRC progression contributes to the decrease of AXIN2 levels, and the hyperactivation of the Wnt pathway.
SALL2 is a developmental transcription factor involved in the regulation of cell Proliferation, migration, and survival. Massive data analyses of cancer versus normal tissues indicated that SALL2 mRNA significantly decreases in colorectal cancer (CRC), a cancer type characterized by Wnt pathway hyperactivation. Interestingly, our unpublished ChIP-seq data analyses suggested that SALL2 regulates genes associated with the Wnt pathway, involved in the development and progression of solid tumors, including CRC. However, the role of SALL2 in CRC and its relationship with the Wnt pathway are yet unknown. We constructed a tissue microarray of 130 samples from Guillermo Gantt Benavente’s Hospital comprising normal colon, adenoma, and CRC to evaluate the expression and cellular location of SALL2 in normal tissues and during CRC progression by immunohistochemistry. We found that SALL2 expresses in the nucleus and cytoplasm of the normal colon epithelium and the stroma. However, its expression decreases in adenoma and is absent in CRC. We noted a negative correlation between SALL2 expression and the expression of nuclear β-catenin, a marker of progression and activation of the Wnt pathway, at the migratory front. Subcellular fractionation and immunofluorescence experiments in CRC cells with gain and loss of SALL2 function support the negative correlation between SALL2 expression and nuclear β-catenin. Additionally, we evaluated the expression of Wnt targets by Western blot and qRT-PCR, finding a positive correlation between SALL2 and AXIN2, a negative regulator of the Wnt pathway.Analysis of AXIN2 promoter identified several putative SALL2 binding sites, and the induction of SALL2 increases AXIN2 promoter activity in SALL2 knockout models. In addition, using ChIP-PCR we found that SALL2 binds to the proximal region in AXIN2 promoter, confirming a SALL2- dependent transcriptional regulation. Finally, we show that the SALL2-AXIN2 axis is required for the antiproliferative, and apoptotic response to XAV939, an inhibitor of the Wnt pathway. In conclusion our study confirms that SALL2 positively regulates the transcription of AXIN2. We proposed that the loss of SALL2 expression during CRC progression contributes to the decrease of AXIN2 levels, and the hyperactivation of the Wnt pathway.
Description
Tesis para optar al grado de Doctor en Ciencias Biológicas Área Biología Celular y Molecular.
Keywords
Genética bioquímica, Genética humana, Biopsia