Browsing by Author "Henriques, Andreia"
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- Alternative splicing of tumour-related Rac1b in colorectal cells is regulated by protein phosphorylation of splicing factor SRSF1Publication . Gonçalves, Vânia; Henriques, Andreia; Pereira, Joana; Neves-Costa, Ana; Moyer, Pat; Ferreira Moita, Luís; Gama-Carvalho, Margarida; Matos, Paulo; Jordan, PeterThe pre-messenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications such as protein phosphorylation, which are determined by signal transduction pathways. Here we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells we found that depletion of AKT2, AKT3, GSK3β and SRPK1 significantly decreased endogenous Rac1b levels. Whereas knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insights into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.
- B-Raf-induced senescence in colorectal cells is antagonized by expression of tumour-related Rac1bPublication . Henriques, Andreia; Barros, Patrícia; Moyer, Mary; Matos, Paulo; Jordan, PeterMutations in the BRAF oncogene have been identified as a tumour-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumour progression. A previous analysis revealed that around 80% of colorectal tumours carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and analysed the effect on expression the senescence marker β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21. We provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence marker β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21. Upon co-expression of splice variant Rac1b, the B-Raf-induced senescence was relieved and expression of the cell-cycle inhibitor proteins downregulated. Our data indicate the selection for increased Rac1b expression as one potential mechanism by which colorectal tumour cells can escape from B-Raf-induced OIS.
- Efeito do oncogene BRAF e da GTPase Rac1b sobre a expressão de genes envolvidos no estado de senescência de células do cólon humanoPublication . Henriques, Andreia; Gomes, Rui; Jordan, PeterO cancro colo-rectal é uma das principais causas de morte no mundo ocidental. Trata-se de uma doença heterogénea com diferentes vias de desenvolvimento, uma das quais é a Via Serreada caracterizada pela ocorrência de mutações no gene BRAF nomeadamente a mutação BRAF V600E. No entanto ensaios feitos com a mutação BRAF V600E mostraram que esta não é suficiente para induzir a transformação maligna e provoca em vários tipos celulares Oncogene Induced Senescence – OIS. O facto de ter sido encontrada sobre expressão de Rac1b em amostras de tumores e ter sido demonstrada uma cooperação entre BRaf-V600E e Rac1b para a sobrevivência celular em células HT29 sugeriu esta GTPase como interveniente na transformação maligna quando a mutação BRAF V600E está presente. Estudos preliminares sugeriram então a hipótese de haver comunicação celular por via de citocinas, nomeadamente, PDGF-BB e IL-8 entre a célula tumoral e células do estroma durante o processo de progressão tumoral envolvendo a expressão do receptor CXCR4. Foi então criado um novo modelo celular de células normais de cólon, NCM460, transformadas com o sistema indutível (ProteoTuner – Clontech) para estudar os efeitos da expressão de Rac1b e BRaf-V600E. Os níveis de expressão dos inibidores de proliferação p14ARF e p15INK4b aumentaram nas células NCM460 transfectadas com pPTuner BRaf-V600E indicando indução de OIS. Já a expressão de Rac1b apresenta efeito antagónico, inibindo a expressão de marcadores de senescência e ainda um aumento da expressão de IL-8. Sendo o IL-8 um estímulo para a produção de SDF1 pelo estroma o efeito da activação do receptor de SDF1, o CXCR4, na expressão de Rac1b foi mimetizado pela sua sobre-expressão nas NCM460. Os resultados sugerem que CXCR4 provoca um aumento da expressão de Rac1b para níveis semelhantes aos encontrados em células tumorais. Os resultados deste trabalho sustentam o modelo de OIS induzido pela mutação BRaf-V600E numa célula de cólon normal e que Rac1b tem um efeito contrário. Posteriormente, por um processo que ainda é necessário definir ocorre na célula aumento da expressão de Rac1b criando um ciclo de retro alimentação entre a célula tumoral e as células envolventes do estroma pelo eixo IL-8- SDF-1- CXCR4 que sustenta a expressão de Rac1b e conduz a progressão tumoral.
- Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cellsPublication . Henriques, Andreia; Barros, Patrícia; Matos, Paulo; Jordan, PeterMutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.
- Ibuprofen inhibits colitis-induced overexpression of tumor-related Rac1bPublication . Matos, Paulo; Kotelevets, Larissa; Gonçalves, Vânia; Henriques, Andreia; Zerbib, Philipe; Moyer, Mary Ann; Chastre, Eric; 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. The analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant B-Raf and both events were shown to cooperate in tumor cell survival. Here we provide evidence for increased expression of Rac1b in samples from inflammatory bowel disease patients as well as following experimentally induced colitis in mice. The increase of Rac1b in the mouse model was specifically prevented by the non-steroidal anti-inflammatory drug ibuprofen, which also inhibited Rac1b expression in cultured HT29 colorectal tumour cells through a cyclooxygenase inhibition-independent mechanism. Accordingly, the presence of ibuprofen led to a reduction of HT29 cell survival in vitro and inhibited Rac1b-dependent tumor growth of HT29 xenografts. 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 suggest that the use of ibuprofen may be beneficial in the treatment of patients with serrated colorectal tumors and in cancer prophylaxis following colon inflammation disorders.
- Ibuprofen Inhibits Overexpression of Tumor-Related Rac1b through SRSF1Publication . Gonçalves, Vânia; Matos, Paulo; Pereira, Joana; Henriques, Andreia; Jordan, PeterAbstract: The 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. 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. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen. Purpose: The objective of our study is to understand the molecular regulation of Rac1b alternative splicing event and how it contributes to tumorigenesis. Experimental description: 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. Results: 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. Here, we provide evidence that ibuprofen leads to a 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. Conclusions: 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.
- Ibuprofen prevents inflammation-induced increase in Rac1 expression and growth of Rac1b-associated colorectal tumoursPublication . Matos, Paulo; Kotelevets, Larissa; Gonçalves, Vânia; Henriques, Andreia; Zerbib, Philipe; Chastre, Eric; Jordan, Peter
- Lessons from Colon Cancer: How Signal Transduction Pathways Regulate Alternative Splicing of RAC1bPublication . Gonçalves, Vânia; Matos, Paulo; Henriques, Andreia; Pereira, Joana; Jordan, PeterIn colon cancer distinct genetic subtypes have been described, one of which involves overexpression of RAC1b, a variant generated by alternative splicing.
- New insights into how cancer cells regulate glucose uptake by protein phosphorylationPublication . Henriques, Andreia; Matos, Paulo; Jordan, PeterIntroduction: Cancer cells require increased amounts of glucose to sustain their proliferation and upregulate plasma membrane expression of glucose transporter GLUT1. In insulin responsive cells, glucose uptake requires previous phosphorylation of TBC1D4, a negative regulator of endosomal GLUT traffic. Previous work published by the host lab has discovered that protein kinase WNK1 can also phosphorylate TBC1D4 and promote the translocation of GLUT1 to the cell surface. In vitro, WNK1 also phosphorylates the homologue TBC1D1 for which a role in cancer cell glucose uptake is not known. The extent to which WNK1 and both TBC1D proteins contribute to glucose uptake in cancer cells is not understood but its characterization is required for a systems- -based understanding of glucose metabolism.
- New insights into how cancer cells regulate glucose uptake by protein phosphorylationPublication . Henriques, Andreia; Jordan, PeterCancer cells require increased glucose supply to sustain proliferation. One mechanism involves increased expression of glucose transporter (GLUT) genes. But insulin has revealed that protein phosphorylation is another key mechanism in glucose uptake regulation: insulin binding to responsive cells triggers a signalling cascade with phosphorylation of TBC1D4, a negative regulator of endosomal GLUT trafficking, so that more transporters are inserted into the plasma membrane.
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