Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. ...In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3′ splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5′ ss increases ETR-3 binding at the 3′ ss and subsequent E9 skipping, whereas strengthening of the 5′ ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5′ ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.
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•Alternative splicing is coupled to transcriptional elongation•Slow elongation either up- or downregulates exon inclusion depending on the exon•In CFTR exon 9, slow elongation favors recruiting the negative splicing factor ETR3•Delaying transcription of exon 9 5′ ss favors ETR-3 binding and exon 9 skipping
Splicing takes place cotranscriptionally and is coupled to transcription. Many alternative splicing decisions depend on the rate RNA Pol II elongation. Although the prevailing paradigm indicates that slow elongation promotes exon inclusion, Dujardin et al. describe a mechanism explaining why, in some exons, slow elongation has the opposite effect.
Gastric cancer (GC) is the third cause of cancer-related mortality worldwide and is often diagnosed at advanced stages of the disease. This makes the development of more comprehensive models and ...efficient treatments crucial. One option is based on repurposing already marketed drugs as adjuvants to chemotherapy. Accordingly, we have previously developed the combination of docetaxel and the cholesterol-lowering drug, lovastatin, as a powerful trigger of HGT-1 human GC cells' apoptosis using 2D cultures. Because 3D models, known as spheroids, are getting recognized as possibly better suited than 2Ds in toxicological research, we aimed to investigate the efficacy of this drug combination with such a model. We established monocellular spheroids from two human (GC) cell lines, HGT-1 and AGS, and bicellular spheroids from these cells mixed with cancer-associated fibroblasts. With these, we surveyed drug-induced cytotoxicity with MTT assays. In addition, we used the Incucyte live imaging and analysis system to follow spheroid growth and apoptosis. Taken together, our results showed that the lovastatin + docetaxel combination was an efficient strategy to eliminate GC cells grown in 2D or 3D cultures, lending further support in favor of repurposing lovastatin as an adjuvant to taxane-based anticancer treatment.
Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various ...genotoxic stress inducers, including camptothecin (CPT), inhibit the interaction between Ewing's sarcoma proto-oncoprotein (EWS), an RNA polymerase II-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene, which encodes the main p53 ubiquitin ligase. This reversible exon skipping participates in the regulation of MDM2 expression that may contribute to the accumulation of p53 during stress exposure and its rapid shut-off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to CPT and EWS-YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, which results in widespread exon skipping and plays a central role in the genotoxic stress response.
Gastric cancer (GC), which includes cancer of the esophagus, the oesophagogastric junction, and the stomach fundus, is highly deadly with strong regional influence, Asia being the most affected. GC ...is often detected at late stages, with 30% of metastatic cases at diagnosis. Many authors have devised models to both unravel the mechanisms of GC development and to evaluate candidate therapeutics. Among these models, 2D-cell cultures are progressively replaced by 3D-cell cultures that recapitulate, much more comprehensively, tumor cellular and genetic heterogeneity, as well as responsiveness to environmental changes, such as exposure to drugs or irradiation. With respect to the specifics of GC, there are high hopes from such model systems, especially with the aim of identifying prognostic markers and novel drug targets.
Abstract Virtually any cell type in a mammalian organism uses Acetyl CoA to yield mevalonate, through the activity of the 3-hydroxy-3-methyl-glutaryl-CoA reductase enzyme and, ultimately, ...cholesterol. Statins have long and quite successfully been used as cholesterol lowering drugs. They reversibly inhibit the 3-hydroxy-3-methyl-glutaryl-CoA reductase activity, which is rate limiting in the early steps of the cholesterol synthesis pathway. In addition to these effects, it has also been amply shown that statins may efficiently trigger cancer cell apoptosis, making them a plausible therapeutic option for the treatment of cancer. Whether statins may prevent cancer occurrence is a matter of debate and an unanswered question; undoubtedly experimental models have clearly demonstrated the potential of statins as direct cytotoxic agents, which can reduce tumour development or metastasis spread, even more so when combined with cytotoxic drugs. Until now, however, only few data in humans support the idea that statins could rightfully belong to the group of anticancer drugs. Nevertheless, as cancer cell metabolism is being thoroughly revisited, the mevalonate pathway has recently been reported as truly oncogenic, presenting the attractive possibility that mevalonate pathway inhibitors, such as statins, may join the ranks of anticancer drugs.
Abstract Background Colorectal cancer (CRC) mainly develops from colorectal adenomas (CRAs). MicroRNAs (miRs) are short non-coding transcripts that regulate gene expression by binding to target ...mRNAs, preventing their expression. It was suggested that miRs were involved in cancer as tumour suppressors or oncogenes, thereby being also potential cancer biomarkers. We conducted an expression analysis of miRNAs and several of their target mRNAs, by using microarrays and quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) (RT-qPCR), in CRA and CRC, as compared to normal mucosa (NOR), in order to identify candidate miRNAs involved in CRC progression. Results Microarray, together with confirmatory RT-qPCR analyses, showed 17 significantly deregulated miRNAs in colorectal lesions. While, as expected, some miRNAs have been previously reported to be associated with CRC, including miR-21 and miR-145, others were new (miR-125a-5p and miR-320 family). Some miRNAs were specific for the CRC versus NOR comparison (miR-320b), or for the CRA versus NOR comparison (miR-15b or miR-16), but several of them (miR-21, miR-24, miR-145, mir-150, miR-378) were deregulated in both CRAs and CRCs, as compared to NOR. The impact of these changes in miR expression on target genes is suggested by the associated deregulation of these genes in CRA and CRC. Conclusions We confirmed that several miRNAs were abnormally expressed in colorectal lesions, identified new deregulated miRs, and showed that several miRNAs could mark the transition from NOR to CRA, thereby marking progression from the early steps of cancer.
CBS encodes a pyridoxal 5′-phosphate-dependent enzyme that catalyses the condensation of homocysteine and serine to form cystathionine. Due to its implication in some cancers and in the cognitive ...pathophysiology of Down syndrome, the identification of pharmacological inhibitors of this enzyme is urgently required. However, thus far, attempts to identify such molecules have only led to the identification of compounds with low potency and limited selectivity. We consequently developed an original, yeast-based screening method that identified three FDA-approved drugs of the 8-hydroxyquinoline family: clioquinol, chloroxine and nitroxoline. These molecules reduce CBS enzymatic activity in different cellular models, proving that the molecular mechanisms involved in yeast phenotypic rescue are conserved in mammalian cells. A combination of genetic and chemical biology approaches also revealed the importance of copper and zinc intracellular levels in the regulation of CBS enzymatic activity—copper promoting CBS activity and zinc inhibiting its activity. Taken together, these results indicate that our effective screening approach identified three new potent CBS inhibitors and provides new findings for the regulation of CBS activity, which is crucial to develop new therapies for CBS-related human disorders.
Gastric cancer is one of the most common cancers in the world. The "economically developed countries" life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may ...lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate.
A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy.
Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.
Colon cancer occurrence is increasing worldwide, making it the third most frequent cancer. Although many therapeutic options are available and quite efficient at the early stages, survival is ...strongly decreased when the disease has spread to other organs. The identification of molecular markers of colon cancer is likely to help understanding its course and, eventually, to uncover novel genes to be targeted by drugs. In this study, we compared gene expression in a set of 95 human colon cancer samples to that in 19 normal colon mucosae, focusing on 401 genes from 5 selected pathways (Apoptosis, Cancer, Cholesterol metabolism and lipoprotein signaling, Drug metabolism, Wnt/beta-catenin). Deregulation of mRNA levels largely matched that of proteins, leading us to build in silico protein networks, starting from mRNA levels, to identify key proteins central to network activity.
Among the analyzed genes, 10.5% (42) had no reported link with colon cancer, including the SFRP1, IGF1 and ADH1B (down), and MYC and IL8 (up), whose encoded proteins were most interacting with other proteins from the same or even distinct networks. Analyzing all pathways globally led us to uncover novel functional links between a priori unrelated or rather remotely connected pathways, such as the Drug metabolism and the Cancer pathways or, even more strikingly, between the Cholesterol metabolism and lipoprotein signaling and the Cancer pathways. In addition, we analyzed the responsiveness of some of the deregulated genes essential to network activities, to chemotherapeutic agents used alone or in presence of Lovastatin, a lipid-lowering drug. Some of these treatments could oppose the deregulations occurring in cancer samples, including those of the CHECK2, CYP51A1, HMGCS1, ITGA2, NME1 or VEGFA genes.
Our network-based approach allowed discovering genes not previously known to play regulatory roles in colon cancer. Our results also showed that selected drug treatments might revert the cancer-specific deregulation of genes playing prominent roles within the networks operating to maintain colon homeostasis. Among those genes, some could constitute novel testable targets to eliminate colon cancer cells, either directly or, potentially, through the use of lipid-lowering drugs such as statins, in association with selected anticancer drugs.
Cancers that belong to the microsatellite instability (MSI) class can account for up to 15% of all cancers of the digestive tract. These cancers are characterized by inactivation, through the ...mutation or epigenetic silencing of one or several genes from the DNA MisMatch Repair (MMR) machinery, including
,
,
,
,
,
,
and
. The unrepaired DNA replication errors turn into mutations at several thousand sites that contain repetitive sequences, mainly mono- or dinucleotides, and some of them are related to Lynch syndrome, a predisposition condition linked to a germline mutation in one of these genes. In addition, some mutations shortening the microsatellite (MS) stretch could occur in the 3'-intronic regions, i.e., in the
(ATM serine/threonine kinase),
(MRE11 homolog) or the
(Heat shock protein family H) genes. In these three cases, aberrant pre-mRNA splicing was observed, and it was characterized by the occurrence of selective exon skipping in mature mRNAs. Because both the
and
genes, which as act as players in the MNR (
/
(Nibrin)/RAD50 (RAD50 double strand break repair protein) DNA damage repair system, participate in double strand breaks (DSB) repair, their frequent splicing alterations in MSI cancers lead to impaired activity. This reveals the existence of a functional link between the MMR/DSB repair systems and the pre-mRNA splicing machinery, the diverted function of which is the consequence of mutations in the MS sequences.
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