Gastric cancer multicellular spheroid analysis by two-photon microscopy.

Gastric cancer (GC) is highly deadly. Three-dimensional (3D) cancer cell cultures, known as spheroids, better mimic tumor microenvironment (TME) than standard 2D cultures. Cancer-associated fibroblasts (CAF), a major cellular component of TME, promote or restrain cancer cell proliferation, invasion and resistance to drugs. We established spheroids from two human GC cell lines mixed with human primary CAF. Spheroid organization, analyzed by two-photon microscopy, showed CAF in AGS/CAF spheroids clustered in the center, but dispersed throughout in HGT-1/CAF spheroids. Such differences may reflect clonal specificities of GC cell lines and point to the fact that GC should be considered as a highly personalized disease.

Identification of 8-Hydroxyquinoline Derivatives That Decrease Cystathionine Beta Synthase (CBS) Activity.

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 cell death analyzed by live cell imaging of spheroids.

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.

[Spheroids to organoids: Solid cancer models for anticancer drug discovery]. / Des sphéroïdes aux organoïdes : modèles de cancers solides pour la découverte de molécules anticancéreuses.

Cell culture is an important and necessary technology in oncology research. Currently, two-dimensional (2D) cell culture models are the most widely used, but they cannot reproduce the complexity and pathophysiology of tumors in vivo. This may be a major cause of the high rate of attrition of anticancer drugs entering clinical trials, the rate of new anticancer drugs entering the market being less than 5 %. One way to improve the success of new cancer drugs in the clinic is based on the use of three-dimensional (3D) cell culture models, more able to represent the complex environment and architecture of tumors. These 3D culture systems are also a powerful research tool for modeling the evolution of cancer from early stages to metastasis. Spheroids and organoids, the most adaptable models among 3D culture systems, are beginning to be used in pharmaceutical research and personalized medicine. In this article, we review the use of spheroids and organoids by highlighting their differences, discussing their impact on drug development, and looking at future challenges.

Prognostic impact of cancer stem cell markers ABCB1, NEO1 and HIST1H2AE in colorectal cancer.

Colon cancer develops according to a defined temporal sequence of genetic and epigenetic molecular events that may primarily affect cancer stem cells. In an attempt to identify new markers of such cells that would help predict patient outcome, we performed a comparative transcriptome analysis of colon cancer stem cells and normal colon stem cells. We identified 162 mRNAs, either over- or under-expressed. According to Cox multivariate regression with our set of 83 colorectal cancers, low expression of ABCB1, NEO1, tumor size and the presence of distant metastases were predictive factors for overall survival. Combined expression of ABCC1 and NEO1 was a significant predictor for overall survival in our cohort, which was confirmed by external validation in 221 colorectal cancers from the Cancer Genome Atlas (TCGA) portal. Tumor size, lymph node involvement and HIST1H2AE expression were also independently correlated with disease-free survival. Taken together, our results suggest that molecular markers of colorectal cancers ABCB1, NEO1 and HIST1H2AE are prognostic factors in colorectal cancer patients. It can be proposed that surveying expression of these marker genes should help better characterizing CRC prognosis, and help selecting the best therapeutic options.

Three-Dimensional Culture Systems in Gastric Cancer Research.

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.

TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1.

We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.

Consequences of blunting the mevalonate pathway in cancer identified by a pluri-omics approach.

We have previously shown that the combination of statins and taxanes was a powerful trigger of HGT-1 human gastric cancer cells' apoptosis1. Importantly, several genes involved in the "Central carbon metabolism pathway in cancer", as reported in the Kyoto Encyclopedia of Genes and Genomes, were either up- (ACLY, ERBB2, GCK, MYC, PGM, PKFB2, SLC1A5, SLC7A5, SLC16A3,) or down- (IDH, MDH1, OGDH, P53, PDK) regulated in response to the drug association. In the present study, we conducted non-targeted metabolomics and lipidomics analyses by complementary methods and cross-platform initiatives, namely mass spectrometry (GC-MS, LC-MS) and nuclear magnetic resonance (NMR), to analyze the changes resulting from these treatments. We identified several altered biochemical pathways involved in the anabolism and disposition of amino acids, sugars, and lipids. Using the Cytoscape environment with, as an input, the identified biochemical marker changes, we distinguished the functional links between pathways. Finally, looking at the overlap between metabolomics/lipidomics and transcriptome changes, we identified correlations between gene expression modifications and changes in metabolites/lipids. Among the metabolites commonly detected by all types of platforms, glutamine was the most induced (6-7-fold), pointing to an important metabolic adaptation of cancer cells. Taken together, our results demonstrated that combining robust biochemical and molecular approaches was efficient to identify both altered metabolic pathways and overlapping gene expression alterations in human gastric cancer cells engaging into apoptosis following blunting the cholesterol synthesis pathway.

A transcriptome-based protein network that identifies new therapeutic targets in colorectal cancer.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Acquisition of anticancer drug resistance is partially associated with cancer stemness in human colon cancer cells.

Colorectal cancer (CRC) is one of the most aggressive cancers worldwide. Several anticancer agents are available to treat CRC, but eventually cancer relapse occurs. One major cause of chemotherapy failure is the emergence of drug-resistant tumor cells, suspected to originate from the stem cell compartment. The aim of this study was to ask whether drug resistance was associated with the acquisition of stem cell-like properties. We isolated drug-resistant derivatives of two human CRC cell lines, HT29 and HCT116, using two anticancer drugs with distinct modes of action, oxaliplatin and docetaxel. HT29 cells resistant to oxaliplatin and both HT29 and HCT116 cells resistant to docetaxel were characterized for their expression of genes potentially involved in drug resistance, cell growth and cell division, and by surveying stem cell-like phenotypic traits, including marker genes, the ability to repair cell-wound and to form colonospheres. Among the genes involved in platinum or taxane resistance (MDR1, ABCG2, MRP2 or ATP7B), MDR1 was uniquely overexpressed in all the resistant cells. An increase in the cyclin-dependent kinase inhibitor p21, in cyclin D1 and in CD26, CD166 cancer stem cell markers, was noted in the resistant cells, together with a higher ability to form larger and more abundant colonospheres. However, many phenotypic traits were selectively altered in either HT29- or in HCT116-resistant cells. Expression of EPHB2, ITGß-1 or Myc was specifically increased in the HT29-resistant cells, whereas only HCT116-resistant cells efficiently repaired cell- wounds. Taken together, our results show that human CRC cells selected for their resistance to anticancer drugs displayed a few stem cell characteristics, a small fraction of which was shared between cell lines. The occurrence of marked phenotypic differences between HT29- and HCT116-drug resistant cells indicates that the acquired resistance depends mostly on the parental cell characteristics, rather than on the drug type used.

[Pre-mRNA splicing: when the spliceosome loses ground]. / L'épissage des ARN pré-messagers : quand le splicéosome perd pied.

Pre-mRNA splicing is an obligatory step required to assemble the vast majority of mRNAs in eukaryotes. In humans, each gene gives rise to at least two transcripts, with an average 6-8 spliced transcripts per gene. Pre-mRNA splicing is not unequivocal. Variations may occur, such that splicing can become alternative, thereby participating in increasing protein variability and restricting the gap that exists between the relatively low number of genes - between 20,000 and 25,000 in humans - and the much higher number of distinct proteins - at least 100,000. In addition, although alternative pre-mRNA splicing often fulfils cell-specific needs, many aberrant splicing events can happen and lead to either hereditary or acquired diseases such as neurodegenerative diseases or cancers. In those cases, alternative splicing events may serve as disease-associated markers, or even as targets for corrective approaches. In this review, we will summarize the main aspects of regulated alternative splicing. We will present the spliceosome, a large ribonucleoprotein complex that orchestrates the splicing reactions and that was recently identified as a preferential target for mutations in several pathologies. We shall discuss some spliceosome-associated defects linked to either cis (i.e on the DNA) or trans (e.g. in proteins) alterations of splicing machinery, like those that have been reported in genetic or acquired diseases.

The arachidonic acid-LTB4-BLT2 pathway enhances human B-CLL aggressiveness.

Deregulation of the oxidative cascade of poly-unsaturated fatty acids (PUFAs) has been associated with several cancers, including chronic lymphocytic leukemia (B-CLL). Leukotriene B4 (LTB4), a metabolite of arachidonic acid (AA), is produced by B-CLL and contributes to their survival. The aim of the present study was to analyze the activity of the oxidative cascade of PUFAs in B-CLL. Purified B cells from patients and normal B CD5 positive cells were subjected to flow cytometry, Western-blot and RT-qPCR analyses. LTB4 plasma and intracellular concentrations were determined by ELISA. Our results showed that aggressive B-CLL tumor cells, i.e. cells with an annual proliferation index above 2, over-expressed calcium-dependent and calcium-independent phospholipases A2 (cPLA2-alpha and iPLA2-beta, respectively), 5-lipoxygenase (5LOX) and leukotriene A4 hydroxylase (LTA4H). Intracellular LTB4 levels were lower in the most aggressive cells than in cells with a smaller proliferation index, despite equivalent plasma levels, and lower expression of cytochrome P450 4F3A (CYP4F3A), one major enzyme involved in LTB4 inactivation. Since BLT2, a LTB4 membrane receptor was also more often expressed on aggressive tumor cells, and since a BLT2 inhibitor significantly impaired B-CLL viability in vitro, we propose that LTB4 was efficiently trapped onto BLT2 present on aggressive tumors, thereby eliciting an autocrine response. Taken together our results demonstrate a major deregulation of the pathway leading to LTB4 synthesis and degradation in B-CLL cells, and provide a framework for understanding how these modifications promote cell survival and proliferation, especially in the most aggressive BCLL.

How slow RNA polymerase II elongation favors alternative exon skipping.

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.

Statins: perspectives in cancer therapeutics.

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.

Human cytochrome P450 4F3: structure, functions, and prospects.

Cytochrome P450 4F3 (CYP4F3), originally identified as one of the leukotriene B4 ω-hydroxylases, belongs to a CYP gene family that comprises several members, which participate in the metabolism of various endobiotics, as well as some xenobiotics. The CYP4F gene family is clustered in a 0.5-Mb stretch of genomic DNA on the p13 region of chromosome 19. Apart from the ω-hydroxylation of leukotriene B4 and prostaglandins, CYP4F3 is the main catalyst in the oxidation of fatty acid epoxides. CYP4F3 expression results from the synthesis of two distinct enzymes, CYP4F3A and CYP4F3B, which originate from the alternative splicing of a single pre-mRNA precursor molecule. Remarkably, the selection of either isoform is part of a tissue-specific control through which CYP3F3A is mostly expressed in leukocytes and CYP4F3B mostly in the liver. Recently, CYP4F3 single nucleotide polymorphisms have been incriminated in the onset of pathologies, including celiac or Crohn's diseases. Although much has been discovered in the regulation and function of CYP4F2, the closest CYP4F subfamily member, analyses of CYP4F3 enzymes lag somewhat behind in the field of our knowledge. In this short review, emphasis will be placed on the regulation and the functional roles of human CYP4F3.

Statins increase cytochrome P450 4F3-mediated eicosanoids production in human liver cells: a PXR dependent mechanism.

In the present study, the ability of lovastatin, a competitive inhibitor of HMG-CoA reductase, to regulate the gene expression and function of Cytochrome P450 4F3B (CYP4F3B) was examined in the well differentiated HepaRG human hepatoma cell line. Statins induced CYP4F3B mRNA, protein and the production of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of arachidonic acid metabolism and a peroxisome proliferator activated receptor (PPAR) ligand. This response was not dependent on cholesterol shortage or on sterol regulatory element binding protein activation. By both a pharmacological and a siRNA approaches, we demonstrated that recruitment of the Pregnane X Receptor (PXR) was required to mediate CYP4F3 induction by lovastatin. Furthermore, the CYP4F3 gene promoter was transcriptionally activated by PXR, and responded to lovastatin. Finally, the expression of fatty acid-responsive genes was increased in response to the statin or 20-HETE in a CYP4F3-dependent way. We propose that metabolites produced by CYP4F3 could modulate lipid metabolism in response to lovastatin. These results suggest the existence of a novel pathway, operating in liver cells, through which statins could lower lipid levels.

Adaptation to statins restricts human tumour growth in Nude mice.

BACKGROUND: Statins have long been used as anti-hypercholesterolemia drugs, but numerous lines of evidence suggest that they may also bear anti-tumour potential. We have recently demonstrated that it was possible to isolate cancer cells adapted to growth in the continuous presence of lovastatin. These cells grew more slowly than the statin-sensitive cells of origin. In the present study, we compared the ability of both statin-sensitive and statin-resistant cells to give rise to tumours in Nude mice. METHODS: HGT-1 human gastric cancer cells and L50 statin-resistant derivatives were injected subcutaneously into Nude mice and tumour growth was recorded. At the end of the experiment, tumours were recovered and marker proteins were analyzed by western blotting, RT-PCR and immunohistochemistry. RESULTS: L50 tumours grew more slowly, showed a strong decrease in cyclin B1, over-expressed collagen IV, and had reduced laminin 332, VEGF and CD34 levels, which, collectively, may have restricted cell division, cell adhesion and neoangiogenesis. CONCLUSIONS: Taken together, these results showed that statin-resistant cells developed into smaller tumours than statin-sensitive cells. This may be reflective of the cancer restricting activity of statins in humans, as suggested from several retrospective studies with subjects undergoing statin therapy for several years.

Splicing defects in the CFTR gene: minigene analysis of two mutations, 1811+1G>C and 1898+3A>G.

BACKGROUND: Cystic fibrosis is caused by mutations of the Cystic Fibrosis Transmembrane conductance Regulator gene (CFTR). Among the 1795 reported mutations, 221 (12.31%) are believed to affect pre-mRNA splicing. Nevertheless, not all splicing mutations have been demonstrated, by functional assays, to affect splicing in living cells. METHODS: We have used a minigene-based approach, coupled to site-specific mutagenesis, to analyze the effects of presumptive pre-mRNA splicing mutations. RESULTS: We show here that the intron 11 1811+1G>C and the intron 12 1898+3A>G mutations strongly affected CFTR pre-mRNA splicing. The encoded proteins are predicted to be defective, which would thus participate in the disease phenotype of carrier individuals. CONCLUSIONS: These results further validate the minigene strategy for the study of presumptive splice mutations, and report unanticipated defects in splicing. Such assays should improve the analysis of genotype-phenotype correlations.

Cotranscriptional exon skipping in the genotoxic stress response.

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.

CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3.

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain.