DGH - Apresentações orais em encontros internacionais
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Browsing DGH - Apresentações orais em encontros internacionais by Subject "Alternative Splicing"
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- 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.
- Ibuprofen Inhibits Overexpression of Tumor-Related RAC1B through SRSF1Publication . Gonçalves, Vânia; Matos, Paulo; Pereira, Joana; Jordan, PeterThe serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression [1]. A previous analysis of colorectal tumors revealed that overexpression of splice variant RAC1B occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival [2]. Patients with inflamed human colonic mucosa also have increased expression of RAC1B as well as mice with experimentally induced colitis [3]. The increase of RAC1B in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen [3]. The objective of our study is to understand the molecular regulation of RAC1B alternative splicing event and how it contributes to tumorigenesis. HT29 colorectal cell line was used as model to test several signaling pathways after 48h of treatment with ibuprofen. For this we analyzed the proteins of interest by Western Blot and the transcript levels by RT-PCR. Mechanistic studies in cultured HT29 colorectal tumor cells revealed that ibuprofen inhibited RAC1B expression in a cyclooxygenase inhibition–independent manner and targets directly the alternative splicing event [3]. Here, we provide evidence that ibuprofen leads to a protein kinase dependent decrease in expression of SRSF1, a splicing factor that we previously identified to promote RAC1B alternative splicing. Together, our results suggest that stromal cues, namely, inflammation, can trigger changes in RAC1B expression in the colon and identify ibuprofen as a highly specific and efficient inhibitor of RAC1B overexpression in colorectal tumors. Our data identify an additional cyclooxygenase–independent action of ibuprofen and suggest it may be beneficial in the treatment of patients with the subtype of BRAF-mutated serrated colorectal tumors. References [1] Velho S, Moutinho C, Cirnes L, Albuquerque C, Hamelin R, Schmitt F, Carneiro F, Oliveira C, and Seruca R (2008). BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: primary or secondary genetic events in colorectal carcinogenesis? BMC Cancer 8, 255. [2] Matos P, Oliveira C, Velho S, Gonçalves V, da Costa LT, Moyer MP, Seruca R, and Jordan P (2008). B-Raf V600E cooperates with alternative spliced Rac1b to sustain colorectal cancer cell survival. Gastroenterology 135, 899–906. [3] Matos P, Kotelevets L, Goncalves V, Henriques AF, Henriques A, Zerbib P, Moyer MP, Chastre E, Jordan P (2013). Ibuprofen inhibits colitis-induced overexpression of tumor-related Rac1b. Neoplasia 15(1):102-11. Acknowledgements Funding Support: grant UID/MULTI/04046/2019, SFRH/BD/109162/2015 and PTDC/BIA-MOL/28386/2017 from FCT
- Microenvironment-induced changes in expression of tumor-promoting RAC1B in colorectal cellsPublication . Pereira, Joana; Gonçalves, Vânia; Matos, Paulo; Jordan, PeterIntroduction: 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 gene regulatory mechanism that responds to extracellular signals is alternative splicing. For example RAC1B, a RAC1 alternative splicing variant, that we previously identified in a subset of BRAF-mutated colorectal tumors, was found increased in samples from inflammatory bowel disease patients or following experimentally-induced acute colitis in a mouse model.The main goal of this work is to determine the pro-inflammatory signals from stromal cells that lead to increased RAC1B expression in colorectal cells. Material and Methods: Caco-2 colorectal cells were either grown as polarized cell monolayer on porous filter membranes and then co-cultured with different stromal cell lines (fibroblasts, monocytes and macrophages) or grown as cysts in 3D matrices. RAC1B expression was analyzed by RT-PCR, Western blot and confocal fluorescence microscopy. Results and Discussions: Culture conditions for polarized 2D and 3D models were established as physiologically more relevant colon cell models. Co-culture experiments with polarized cells 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 progressive loss of epithelial organization. The cytokines secreted by stromal cells are currently being identified. Conclusion: Our data indicate that extracellular signals from stromal cells can affect gene expression in colorectal cancer cells. The observed increase in alternatively spliced RAC1B will help to understand the tumor-promoting effect of inflammation and identify novel therapeutic strategies.
- Regulation of alternative splicing of tumor-related RAC1b by signal transduction pathwaysPublication . Jordan, PeterIn colon cancer, distinct genetic subtypes have been described and about 10% of cases carry an oncogenic mutation in the BRAF gene. These cases also show overexpression of alternative splicing variant RAC1b that encodes a protein with additional 19 amino acids resulting from in-frame inclusion of alternative exon 3b. RAC1b is a hyperactive variant that exists predominantly in the GTP-bound active conformation in vivo and promotes cell cycle progression and cell survival through activation of the transcription factor NF-κB. RAC1b overexpression functionally cooperates with the oncogenic mutation in BRAF-V600E to sustain colorectal tumor cell survival. The splicing factor SRSF1 was identified as an important key regulator of RAC1b alternative splicing in colorectal cells. It binds an exonic splice enhancer element in the alternative exon. We found that pro-inflammatory signals increase RAC1b expression in colorectal cell lines and studied how signaling pathways are involved in the deregulation of alternative splicing. The depletion of two protein kinases, SRPK1 and GSK3β, affected RAC1b levels and both do so via the phosphorylation status of splicing factor SRSF1. Reduced SRSF1 phosphorylation decreased its nuclear translocation and concomitantly the generation of RAC1b. Interestingly, GSK3β phosphorylation was identified to serve as target for the anti-inflammatory drug ibuprofen, which we previously found to inhibit RAC1b overexpression in a cyclooxygenase-independent manner. Together, our results demonstrate that oncogenic signal transduction pathways can deregulate alternative splicing in tumor cells and that pharmacological intervention with the protein kinases involved may be of therapeutic value.
- Regulation of the Alternative Splicing of Tumor-Related RAC1b by Signal Transduction PathwaysPublication . Gonçalves, Vânia; Matos, Paulo; Pereira, Joana; Henriques, Andreia; Jordan, PeterDistinct genetic subtypes have been described in colon cancer, one of which involves overexpression of RAC1b, a variant generated by alternative splicing. Aberrant splicing is known to occur in cancer and can be caused by mutation in a gene or splicing factor but also represents a dynamic response to oncogeneinduced cellular signaling and in this case it may be pharmacologically targeted. Here we explore how signaling pathways are involved in the deregulation of alternative RAC1b splicing in colorectal tumor cells.
- Signal Transduction Pathways Regulating the Alternative Splicing of Tumor Related RAC1bPublication . Gonçalves, Vânia; Matos, Paulo; Jordan, PeterDistinct genetic subtypes have been described in colon cancer, one of which involves overexpression of RAC1b, a variant generated by alternative splicing. Aberrant splicing is known to occur in cancer and can be caused by mutation in a gene or splicing factor but also represents a dynamic response to oncogene-induced cellular signaling and in this case it may be pharmacologically targeted. Here we explore how signaling pathways are involved in the deregulation of alternative RAC1b splicing in colorectal tumor cells. HT29 cells represent serrated colorectal tumors with BRAF gene mutation V600E in one allele and RAC1b overexpression. Cells were transfected with shRNA vectors directed against target candidate protein kinase transcripts and their effects on RAC1b levels analyzed 24h later by Western Blot and qRT-PCR. Treatment with kinase inhibitors or anti-inflammatory drugs was performed 24h and 48h prior to cell lysis. Two kinases, SRPK1 and GSK3β, were found required to sustain RAC1b levels and both were shown to act upon the phosphorylation of splicing factor SRSF1, which binds to and promotes the inclusion of the alternative exon in RAC1b. SRPK1 knockdown or pharmacological inhibition reduced SRSF1 phosphorylation decreasing its nuclear translocation and concomitantly RAC1b splicing. The same regulatory pathway was also found to be controlled by GSK3β. Interestingly, GSK3β phosphorylation was identified to serve as target for the anti-inflammatory drug ibuprofen, which inhibits RAC1b overexpression. Together, our results demonstrate that alternative splicing is deregulated by oncogenic signal transduction pathways and it may be drug revertable.