Effect of an inflammatory microenvironment on alternative splicing-mediated gene expression plasticity in colorectal cells

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Effect of the microenvironment on alternative splicing in colorectal cells

Publication . Pereira, Joana FS; Jordan, Peter

The mucosa of our gastrointestinal tract forms a barrier that separates the gut lumen from the surrounding tissues. Perturbation of the barrier function characterizes inflammatory bowel diseases, which constitute a risk factor for colon cancer development, because an inflammatory microenvironment is a tumor-promoting condition that provides survival signals to which cancer cells respond with changes in their gene expression. One key regulatory mechanism is alternative splicing. One example is RAC1B, a RAC1 alternative splicing variant, that the host laboratory previously identified in a subset of BRAF-mutated colorectal tumors, and was found increased in samples from inflammatory bowel disease or following experimentally-induced acute colitis in a mouse model. The main goal of this work was to generate co-culture models to understand whether colorectal cancer cells respond to a pro-inflammatory microenvironment with changes in alternative splicing of RAC1B. For this, culture conditions for polarized 2D and 3D models were established as physiologically more relevant colon cell models. Caco-2 colorectal cancer cells were grown as polarized cells on porous membranes and then co-cultured with stromal cells: fibroblasts, monocytes and macrophages. RAC1B expression was analyzed in Caco-2 by qRT-PCR, Western blot and confocal fluorescence microscopy. Co-culture experiments revealed that the presence of fibroblasts and/or M1 macrophages induced a transient increase in RAC1B protein levels in the colorectal cells, accompanied by a loss of epithelial organization. Through the use of a human inflammation array, we were able to identify three cytokines in co-culture media that associated with increased RAC1B: IL-1β, IL-6 and IL-8. In addition, the analysis of cytokine-stimulated signaling pathways allowed to identify the phosphorylation of ERK 1/2 and STAT3 as a major response to the external signals. Overall, our data present evidence that a pro-inflammatory environment triggers an increase in RAC1B expression in colon epithelial cells. Since RAC1B was shown to sustain tumor cell survival and promote escape form oncogene-induced senescence, the data further strengthen the causal connection between inflammatory conditions and the development of colorectal cancer, and may indicate RAC1B as a potential biomarker.

2021-06Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

Número da atribuição

SFRH/BD/109162/2015