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- Exploring the effect of the pro-inflammatory microenvironment on the expression of the tumor-related RAC1B splice variant in colorectal cancer cellsPublication . Bessa, Cláudia; Pereira, Joana Filipa de SousaAn inflammatory tumor cell microenvironment has been identified as a critical tumor-promoting condition providing survival signals to which cancer cells respond with changes in their gene expression. Interestingly, alternative splicing (AS) is one key gene regulatory mechanism that responds to extracellular signals directly affecting cancer progression. For example RACB1, a RAC1 AS variant, previously identified by our group in a subset of BRAF-mutated colorectal tumors [3], was found increased in colon mucosa under inflammatory conditions, both in samples from inflammatory bowel disease patients or following experimentally-induced acute colitis in a mouse model. Based on these findings, the main goal of this work was to determine which pro-inflammatory signals from stromal cells lead to an increase in RAC1B expression levels in colorectal cancer (CRC) cells. For that, we use a more physiologically relevant culture model consisting of a 2D polarized monolayer of Caco-2 CRC cells grown on porous membranes, and then co-cultured with stromal cells, including fibroblasts, monocytes and macrophages. RAC1B expression was analyzed in Caco-2 cells by RT-qPCR, Western blot and confocal fluorescence microscopy. In fact, co-culture experiments revealed that the combined presence of fibroblasts and/or M1 macrophages induced a transient increase in RAC1B mRNA and protein levels in Caco-2 cells, accompanied by a loss of epithelial organization. Besides RAC1B, a panel of 6 additional pairs of tumor-related AS variants was analyzed, being observed an alteration of the expression ratios in 3 out of the 6 variants tested. Moreover, using a human inflammation array, we were able to identify from the conditioned co-culture media three cytokines that associated with increased RAC1B: IL-1, IL-6 and IL-8. Remarkably, the incubation of polarized Caco-2 cells with described purified cytokines was sufficient per se to trigger an increase in RAC1B expression in a dose-dependent manner. As a whole, our data indicate that pro-inflammatory signals can modulate AS in colon epithelial cells, particularly leading to an increase in RAC1B levels. Future identification of the interleukin-regulated signaling pathways involved in altered AS should provide details on how these talk to the splicing machinery, and may represent a promising approach to discovery novel therapeutic targets for suppressing oncogenic splice variants.
- Dissecting how cancer cells regulate the alterntative splicing of RAC1BPublication . Jordan, PeterRAC1B is an alternative splicing variant of the small GTPase RAC1 and overexpressed in several tumor types. In colon cancer, RAC1B overexpression occurs in distinct genetic subtypes, in particular those carrying an oncogenic mutation in the BRAF gene [1]. RAC1B encodes a protein with additional 19 amino acids resulting from in-frame inclusion of alternative exon 3b, and adopts predominantly the GTP-bound active conformation in vivo. Its signaling activity favors cell cycle progression and cell survival through activation of the transcription factor NF-κB [2]. The splicing factor SRSF1 was identified as a key promoter of RAC1B splicing in colorectal cells and binds an exonic splice enhancer element in the alternative exon [3]. We found that RAC1B levels depended on two protein kinases, SRPK1 and GSK3β, which both act via the phosphorylation status of SRSF1 that determines its nuclear translocation and concomitant inclusion of exon 3b [4]. Recent evidence suggests that the described increase in RAC1B expression can be triggered in colorectal cells by pro-inflammatory signals from the tumor cell microenvironment. Together, our results demonstrate how signal transduction pathways can deregulate alternative splicing in tumor cells and that pharmacological intervention with the protein kinases involved may be of therapeutic value.
- Ibuprofen disrupts a WNK1/GSK3β/SRPK1 protein complex required for expression of tumor-related splice variant RAC1B in colorectal cellsPublication . Gonçalves, VâniaA major risk factor promoting tumor development is chronic inflammation and the use of nonsteroidal anti-inflammatory drugs (NSAID), including ibuprofen, can decrease the risk of developing various types of cancer, including colorectal cancer (CRC). Although the molecular mechanism behind the antitumor properties of NSAIDs has been largely attributed to inhibition of cyclooxygenases (COXs), several studies have shown that the chemopreventive properties of ibuprofen also involve multiple COX-independent effects. One example is its ability to inhibit the alternative splicing event generating RAC1B, which is overexpressed in a specific subset of BRAF-mutated colorectal tumors and sustains cell survival. Here we describe the mechanism by which ibuprofen prevents RAC1B alternative splicing in a BRAF mutant CRC cell line: it leads to decreased translocation of SRPK1 and SRSF1 to the nucleus and is regulated by a WNK1/GSK3β/SRPK1 protein kinase complex. Surprisingly, we demonstrate that ibuprofen does not inhibit the activity of any of the involved kinases but rather promotes disassembly of this regulatory complex, exposing GSK3β serine 9 to inhibitory phosphorylation, namely by AKT, which results in nuclear exclusion of SRPK1 and SRSF1 hypophosphorylation. The data shed new light on the biochemical mechanisms behind ibuprofen’s action on alternative spliced RAC1B and may support its use in personalized approaches to CRC therapy or chemoprevention regimens.