👤 Laurent Schaeffer

GATA family transcription factors are somatically variable (SV) in esophageal adenocarcinomas (EAC) and inducible by simulated reflux. Our study examines the mechanisms whereby GATA family members (GATA4, GATA6, and the atypical TRPS-1) influence oncogenesis during the Barrett's esophagus (BE) metaplasia-dysplasia transition preceding EAC. RNAseq analyses of esophageal cell lines and lesion-derived adult stem cells (ASCs) in conjunction shRNA- or CRISPR-facilitated gene silencing, together with reanalysis of The Cancer Genome Atlas data, spatial transcriptomics, and organ-on-a-chip studies were used. Although a gastroesophageal reflux disease history positively correlated with GATA4/6 somatically variable and a columnar-associated gene signature (ANPEP/GATA4) in The Cancer Genome Atlas EAC cases, it negatively associated with a squamous lineage-linked signature (TP63/SOX15) containing TRPS1. In experimental data, opposing effects on regulators of squamous and columnar lineage identity were uncovered between TRPS1 and classical GATA factors (GATA4/6). Interrogation of this GATA "fulcrum" defined further genes (CGN, IL6R, and GPRC5B) targeted for TRPS1-mediated suppression or GATA4/6 activation. A novel spatial transcriptomic signature of BE-associated high-grade dysplasia (HGD) captured GATA fulcrum action, through GPRC5B expression. Functionally, GPRC5B was found to be low-pH-responsive, to increase proliferative and colony formation rates, and when overexpressed facilitate a hyperproliferative HGD-like transformation of BE-ASCs. Using an organ-on-a-chip platform, cellular overgrowth, reduced luminal villus structures, lower goblet cell numbers, and loss of intestine-associated marker gene expression (TFF3/MUC2) were observed following GPRC5B overexpression in BE-ASCs, mirroring HGD. This study identifies critical GATA factor-mediated processes underlying cellular phenotype in the BE-HGD-EAC transition and identifies GATA-inducible GPRC5B as a functional marker and possible driver of progression through HGD to EAC. Show less

Recurrent somatic mutations of the RPS6KA3 gene encoding for the serine/threonine kinase RSK2 were identified in hepatocellular carcinomas (HCCs), suggesting its tumour-suppressive function. Our goal was to demonstrate the tumour suppressor role of RSK2 in the liver and investigate the functional consequences of its inactivation. We analysed a series of 1,151 human HCCs for RSK2 mutations and 20 other driver genetic alterations. We then modelled RSK2 inactivation in mice in various mutational contexts recapitulating or not those naturally found in human HCC, using transgenic mice and liver-specific carcinogens. These models were monitored for liver tumour appearance and subjected to phenotypic and transcriptomic analyses. Functional consequences of RSK2 rescue were also investigated in a human RSK2-deficient HCC cell line. RSK2-inactivating mutations are specific to human HCC and frequently co-occur with AXIN1-inactivating or β-catenin-activating mutations. Modelling of these co-occurrences in mice showed a cooperative effect in promoting liver tumours with transcriptomic profiles recapitulating those of human HCCs. By contrast, there was no cooperation in liver tumour induction between RSK2 loss and BRAF-activating mutations chemically induced by diethylnitrosamine. In human liver cancer cells, we also showed that RSK2 inactivation confers some dependency to the activation of RAS/MAPK signalling that can be targeted by MEK inhibitors. Our study demonstrates the tumour suppressor role of RSK2 and its specific synergistic effect in hepatocarcinogenesis when its loss of function is specifically combined with AXIN1 inactivation or β-catenin activation. Furthermore, we identified the RAS/MAPK pathway as a potential therapeutic target for RSK2-inactivated liver tumours. This study demonstrated the tumour suppressor role of RSK2 in the liver and showed that its inactivation specifically synergises with AXIN1 inactivation or β-catenin activation to promote the development of HCC with similar transcriptomic profiles as found in humans. Furthermore, this study highlights that activation of the RAS/MAPK pathway is one of the key signalling pathways mediating the oncogenic effect of RSK2 inactivation that can be targeted with already available anti-MEK therapies. Show less

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Show less

Prolonged fasting is characterized by consecutive phases, a short period of adaptation (phase 1), phase 2 (P2) characterized by fat oxidation, and phase 3 (P3) during which energy requirements are mostly derived from increased protein utilization. At this latter stage, food seeking behavior is induced. Very few circulating biomolecules have been identified that are involved in the response to prolonged fasting. To this end, rat plasma samples were compared by a proteomic approach, using 2-DE. The results revealed a selective variation of the levels of apolipoprotein A-IV, A-I, and E, haptoglobin, transthyretin, plasma retinol binding-protein, and vitamin D binding-protein in P2 and P3. The variations in protein levels were confirmed by ELISA. Changes in mRNA levels encoding these proteins did not systematically correlate well with protein concentrations, and tissue-specific regulation of mRNA expression was observed, underlining the complex metabolic regulation in response to food deprivation. In late fasting, the marked reduction of apolipoprotein A-IV levels could contribute to the alarm signal that triggers refeeding. The variations of the other differentially expressed proteins are more likely related to lipid metabolism and insulin signaling alterations. Show less

The present study gives further evidence for the recently found association between variants of the fatty acid desaturase 1 fatty acid desaturase 2 (FADS1 FADS2) gene cluster and PUFA in blood phospholipids and explores this association for cellular fatty acids in erythrocyte membranes. In a subgroup of adults participating in the Bavarian Nutrition Survey II, a cross-sectional population-based study conducted in Bavaria, Germany, allelic variation in three selected loci of the FADS1 FADS2 gene cluster was analysed and used for haplotype construction. Associations with plasma phospholipid PUFA (n 163) and PUFA in erythrocyte membranes (n 535) were investigated by regression analysis. All haplotypes of the original five-loci haplotypes of our previous study could be replicated. In addition, associations with serum phospholipid PUFA were confirmed in the present data set. Although less pronounced, associations between FADS1 FADS2 haplotypes and PUFA in erythrocyte membranes, particularly arachidonic and dihomo-gamma-linolenic acid, could be established. We provide the first replication of the association of the FADS1 FADS2 gene cluster with PUFA in blood phospholipids. For the first time, such associations were also shown for PUFA in cell membranes. Show less

The delta-5 and delta-6 desaturases, encoded by FADS1 and FADS2 genes, are key enzymes in polyunsaturated fatty acid (PUFA) metabolism that catalyze the conversion of linoleic acid (LA) into arachidonic acid (AA) and that of alpha-linolenic acid (ALA) into eicosapentaenoic acid (EPA). Single-nucleotide polymorphisms (SNPs) in FADS1 and FADS2 have been associated with different concentrations of AA and LA, and those associations have possible functional consequences for desaturase activity. We aimed to evaluate the possible association among FADS genotypes, desaturase activity, inflammation, and coronary artery disease (CAD). Thirteen FADS SNPs and the ratio of AA to LA (AA/LA) on red blood cell (RBC) membranes, a marker of desaturase activity, were evaluated in 876 subjects with (n = 610) or without (n = 266) angiographically documented CAD. Both AA/LA and the ratio of EPA to ALA (EPA/ALA) were higher in patients with CAD than in those without CAD, but, in a multiple logistic regression model, only a higher AA/LA resulted an independent risk factor for CAD (odds ratio: 2.55; 95% CI: 1.61, 4.05 for higher compared with lower ratio tertile; P for trend < 0.001). Furthermore, concentrations of high-sensitivity C-reactive protein increased progressively across tertiles of AA/LA. Graded increases in high-sensitivity C-reactive protein concentrations and CAD risk were related to the carriership of FADS haplotypes, including the alleles associated with a higher ratio. In populations following a Western diet, subjects carrying FADS haplotypes that are associated with higher desaturase activity may be prone to a proinflammatory response favoring atherosclerotic vascular damage. Show less

Tissue availability of polyunsaturated fatty acids (PUFAs) is of major relevance for health, and it depends on both dietary intake and metabolic turnover. We found close associations between variants in the human genes of Delta5- and Delta6-desaturase, FADS1 and FADS2, and serum phospholipid contents of PUFAs and long-chain PUFAs (LCPUFAs). Polymorphisms and reconstructed haplotypes of FADS1 and the upstream region of FADS2 showed strong associations with levels of the n-6 LC-PUFA arachidonic acid (20:4n-6). Carriers of the less common polymorphisms and their respective haplotypes also had a lower prevalence of allergic rhinitis and atopic eczema. Our data demonstrate for the first time that the fatty acid composition of serum phospholipids is genetically controlled by the FADS1 FADS2 gene cluster. The investigated single nucleotide polymorphisms in this cluster explain 28% of the variance of serum phospholipid arachidonic acid and up to 12% of its precursor acids. Based on this genetic variation, individuals may require different amounts of dietary PUFAs or LC-PUFAs to achieve comparable biological effects. We strongly recommend including analyses of FADS1 and FADS2 polymorphism in future cohort and intervention studies addressing the biological effects of PUFAs and LC-PUFAs, which should enhance the sensitivity and precision of such studies. Show less

Polymorphisms of the human Delta-5 (FADS1) and Delta-6 (FADS2) desaturase genes have been recently described to be associated with the level of several long-chain n-3 and n-6 polyunsaturated fatty acids (PUFAs) in serum phospholipids. We have genotyped 13 single nucleotide polymorphisms (SNPs) located on the FADS1-FADS2-FADS3 gene cluster (chromosome 11q12-13.1) in 658 Italian adults (78% males; mean age 59.7 +/- 11.1 years) participating in the Verona Heart Project. Polymorphisms and statistically inferred haplotypes showed a strong association with arachidonic acid (C20:4n-6) levels in serum phospholipids and in erythrocyte cell membranes (rs174545 adjusted P value for multiple tests, P < 0.0001 and P < 0.0001, respectively). Other significant associations were observed for linoleic (C18:2n-6), alpha-linolenic (C18:3n-3) and eicosadienoic (C20:2n-6) acids. Minor allele homozygotes and heterozygotes were associated to higher levels of linoleic, alpha-linolenic, eicosadienoic and lower levels of arachidonic acid. No significant association was observed for stearidonic (C18:4n-3), eicosapentaenoic (C20:5n-3) and docosahexaenoic (C22:6n-3) acids levels. The observed strong association of FADS gene polymorphisms with the levels of arachidonic acid, which is a precursor of molecules involved in inflammation and immunity processes, suggests that SNPs of the FADS1 and FADS2 gene region are worth studying in diseases related to inflammatory conditions or alterations in the concentration of PUFAs. Show less

Fatty acid composition in membranes plays an important role in cellular processes and has shown to be associated with the aetiology of several complex diseases in humans. We report strong associations between variants in the human delta-5 and delta-6 desaturase genes FADS1 FADS2 and fatty acid composition in serum phospholipids. Eighteen polymorphisms located in this gene cluster were genotyped in 727 adults from Erfurt, a German centre of the European Community Respiratory Health Survey. The cluster is located at chromosome 11q12-11q13.1, a region repeatedly found to be linked with atopy and other complex diseases. Polymorphisms and statistically reconstructed haplotypes of FADS1 and the upstream region of FADS2 showed strongest associations with the level of the direct precursor of inflammatory eicosanoids, the n-6 fatty acid arachidonic acid (C20:4n-6), also strong associations with levels of the n-6 fatty acids C18:2n-6, C18:3n-6, C20:2n-6, C20:3n-6, C22:4n-6 and of the n-3 fatty acids C18:3n-3, C20:5n-3 and C22:5n-3 (P-values < 1.0 x 10(-13)). Carriers of the rare alleles of several SNPs and their respective haplotypes had a lower prevalence of allergic rhinitis and atopic eczema. No association was found for total and specific IgE levels. Show less