Microenvironmental effects on alternative splicing in malignant progression of colorectal tumor cells

Funder

Organizational Unit

Publications

Pro-Inflammatory Cytokines Trigger the Overexpression of Tumour-Related Splice Variant RAC1B in Polarized Colorectal Cells

Publication . Pereira, Joana F. S.; Bessa, Cláudia; Matos, Paulo; Jordan, Peter

Simple Summary: Tumours are now known to develop more quickly when the tumour cell mass is located in a tissue that shows signs of chronic inflammation. Under such conditions, inflammatory cells from the surrounding tumour microenvironment provide survival signals to which cancer cells respond. We have previously found that some colorectal tumours overexpress the protein RAC1B that sustains tumour cell survival. Here we used a colon mucosa-like in vitro cell model and found that the presence of cancer-associated fibroblasts and pro-inflammatory macrophages stimulated colorectal cells to increase their RAC1B levels. Under these conditions, the secreted survival signals were analysed, and interleukin-6 identified as the main trigger for the increase in RAC1B levels. The results contribute to understand the tumour-promoting effect of inflammation at the molecular level.

2022-03-09Journal article

Open access

Targeting tumour-related alternative splice variant RAC1B with anti-sense oligonucleotides

Publication . Bessa, Cláudia; Gonçalves, Vânia; Jordan, Peter

Over 90% of human protein coding genes are able to generate more than one transcript due to alternative splicing. In cancer, certain alternative splicing variants are frequently overexpressed and contribute to tumour progression and aggressiveness. This is well illustrated by RAC1B, a variant of the small GTPase RAC1 that is overexpressed in tumours from colon, breast, lung, pancreas and thyroid. RAC1B is generated by inclusion of alternative exon 3b and the resulting protein contains 19 additional amino acids, which alter regulation and signalling properties. In colorectal cancer, RAC1B is overexpressed in the subgroup of BRAF mutated tumours. Using HT29 cells as a model, RAC1B was shown to exist predominantly in the active GTP-bound conformation in cells and signal via NF-κB to sustain tumour cell survival. To exploit the suitability of this highly gene-specific event for oligonucleotide-based therapeutic intervention, we tested different anti-sense oligonucleotides to interfere with RAC1B expression levels in cancer cells. We found that synthetic siRNAs that induced transcript degradation were able to efficiently suppress RAC1B transcript and protein levels in cells. Phosphorodiamidate morpholino-modifed oligonucleotides (PMO, morpholinos) present higher affinity and stability than conventional oligonucleotides. Thus, a morpholino was designed that targets the intron3–exon3b splice junction including the exonic splice enhancer sequence for SRSF1 binding that we previously identified. When validated in HT29 colorectal tumour cells, this morpholino performed poorly on levels of RAC1B transcript or protein and did not affect RAC1B-dependent cell viability. Indeed, morpholinos do not easily enter mammalian cells in culture but have shown efficacy in animal models in vivo and in human clinical trials. In order to validate, whether the morpholino’s target sequence was able to mediate RAC1B inhibition, we employed the nuclease resistant 2′-O-methyl-modified (2OM) oligonucleotides directed to this and three other exon 3b sequences. We found that all four 2OM oligos reduced RAC1B transcript and protein, comparable to the efficiency of the synthetic siRNA and sustained the downregulating effect for up to 72 h post-transfection.

2021-07-09Conference object

Restricted access

RAC1B alternative splicing regulation in colorectal cancer cells

Publication . Rosado, Telma; Gonçalves, Vânia; Jordan, Peter

Colorectal cancer (CRC) is a common malignancy with significant mortality, which presents different subtypes due to genetic heterogeneity. The focus of this work was on a subtype that is characterized by microsatellite instability and mutations in the BRAF gene, being associated, in most cases, with RAC1B overexpression. RAC1B is a splicing variant of the RAC1 gene and is characterized by the inclusion of an additional exon through alternative splicing, designated as exon 3b, presenting some regulation and signalling differences when compared to RAC1. RAC1B has been described as a promoter of tumour progression, therefore, it is considered as a potential therapeutic target, as it may allow the design of targeted therapies for patients with this subtype of CRC, but for this, understanding in more detail the mechanisms of RAC1B regulation is extremely important. Previous research projects in the host lab had already identified SRSF1 and SRSF3 as two splicing factors involved in the regulation of RAC1B splicing in CRC, so the first goal of this thesis work was to determine the effect of other selected proteins on the expression of RAC1B in colorectal cell lines. The proteins of interest are a nucleoporin, RANBP2, and four splicing factors, PTBP1, ESRP1, DIS3L2 and hnRNP U, which were previously described as upregulated in CRC or involved in the RAC1B splicing process in other cell lines. The effect of these proteins on RAC1B alternative splicing was assessed through their overexpression and depletion in two colorectal cell lines, NCM460 and HT29, and the analysis of RAC1 exon 3b inclusion was made at the transcript level by RT-PCR, and at the protein level through SDS-PAGE and Western blot. The obtained results provided strong evidence that, in fact, these proteins influence RAC1B expression, both at the transcript and protein level. Although the overexpression experiments performed in this work did not lead to many clear conclusions about the effect of the proteins of interest on the RAC1B expression, the siRNA assays were rather conclusive. Taking into account the results obtained through depletion experiments, RANBP2 seems to act as a RAC1B inhibitor in NCM460 cells, while PTBP1, ESRP1, DIS3L2 and hnRNP U are probably enhancers of that RAC1 isoform. In the HT29 cell line, RANBP2 and DIS3L2 were pointed as inhibitors of exon 3b inclusion, while PTBP1, ESRP1 and hnRNP U are most likely enhancers of that alternative splicing event. The second goal of this work was to identify PTBP1 and ESRP1 RNA-binding motifs in the RAC1 transcript sequence. Here, two predicted ESRP1 binding motifs in the RAC1 transcript were identified and mutated in a RAC1 minigene, in order to verify if any of these changes could avoid ESRP1 binding and consequently affect the splicing process. The presence of the mutations was confirmed through DNA sequencing, however, it was not possible in the remaining time to reclone the mutant fragments into the original minigene vector, to assure they did not have undesired mutations, allowing to proceed to the planned experiments. Future studies should be performed in order to confirm the obtained results, as not all of them resulted in statistically significant variations on RAC1B expression levels, and it is also imperative to clarify how the nucleoporin RANBP2 affects RAC1B expression and what binding sites are recognized by the studied splicing factors in the RAC1 pre-mRNA.

2021-02-26Master thesis

Restricted access

Microenvironment-induced changes in expression of tumor-promoting RAC1B in colorectal cells

Publication . Pereira, Joana; Gonçalves, Vânia; Matos, Paulo; Jordan, Peter

Introduction: 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.

2020-03-03Conference object

Restricted access