Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease.

OBJECTIVE: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. DESIGN: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. RESULTS: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. CONCLUSION: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.

Regulation of the positive transcriptional effect of PLZF through a non-canonical EZH2 activity.

The transcription factor PLZF (promyelocytic leukemia zinc finger protein) acts as an epigenetic regulator balancing self-renewal and differentiation of hematopoietic cells through binding to various chromatin-modifying factors. First described as a transcriptional repressor, PLZF is also associated with active transcription, although the molecular bases underlying the differences are unknown. Here, we reveal that in a hematopoietic cell line, PLZF is predominantly associated with transcribed genes. Additionally, we identify a new association between PLZF and the histone methyltransferase, EZH2 at the genomic level. We find that co-occupancy of PLZF and EZH2 on chromatin at PLZF target genes is not associated with SUZ12 or trimethylated lysine 27 of histone H3 (H3K27me3) but with the active histone mark H3K4me3 and active transcription. Removal of EZH2 leads to an increase of PLZF binding and increased gene expression. Our results suggest a new role of EZH2 in restricting PLZF positive transcriptional activity independently of its canonical PRC2 activity.

A metagenomics investigation of carbohydrate-active enzymes along the goat and camel intestinal tract.

Studies of the digestive microbiota of ruminant animals most often focus on the bacterial diversity in the rumen or the feces of the animals, but little is known about the diversity and functions of their distal intestine. Here, the bacterial microbiota of the distal intestinal tract of two goats and two camels was investigated by metagenomics techniques. The bacterial taxonomic diversity and carbohydrate-active enzyme profile were estimated for samples taken from the small intestine, the large intestine, and the rectum of each animal. The bacterial diversity and abundance in the small intestine were lower than in the rectal and large intestinal samples. Analysis of the carbohydrate-active enzyme profiles at each site revealed a comparatively low abundance of enzymes targeting xylan and cellulose in all animals examined, similar to what has been reported earlier for sheep and therefore suggesting that plant cell wall digestion probably takes place elsewhere, such as in the rumen.

[Gut microbiota and digestion of polysaccharides]. / Le microbiote intestinal et la digestion des polysaccharides.

The distal gut microbiota corresponds to all the microorganisms, essentially bacteria, that reside commonly in the colon. The microbial population is characterized by a large taxonomical diversity, counting approximately a thousand distinct bacterial species for a single individual. The pace of investigations of this microbial system has greatly accelerated these last few years, fuelled by the advent of metagenomics techniques, which do not rely on bacterial cultivation, but utilize high throughput DNA sequencing. In just a few years studies of the intestinal microbiota have become fashionable, albeit with often contradictory results when attempting to correlate changes in microbial composition to diverse pathologies. The article focuses on one of the essential functions of the distal gut microbiota: the digestion of the immense variety of polysaccharides from our diet that enzymes of the host cannot breakdown.

Cytidine Deaminase Resolves Replicative Stress and Protects Pancreatic Cancer from DNA-Targeting Drugs.

Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. SIGNIFICANCE: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

Pancreatic ductal adenocarcinoma ubiquitination profiling reveals specific prognostic and theranostic markers.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been widely studied at multiomics level. However, little is known about its specific ubiquitination, a major post-translational modification (PTM). As PTMs regulate the final function of any gene, we decided to establish the ubiquitination profiles of 60 PDAC. METHODS: We used specific proteomic tools to establish the ubiquitin dependent proteome (ubiquitinome) of frozen PDXs (Patients' derived xenographs). Then, we performed bioinformatics analysis to identify the possible associations of these ubiquitination profiles with tumour phenotype, patient survival and resistance to chemotherapies. Finally, we used proximity ligation assays (PLA) to detect and quantify the ubiquitination level of one identified marker. FINDINGS: We identified 38 ubiquitination site profiles correlating with the transcriptomic phenotype of tumours and four had notable prognostic capabilities. Seventeen ubiquitination profiles displayed potential theranostic marker for gemcitabine, seven for 5-FU, six for oxaliplatin and thirteen for irinotecan. Using PLA, we confirmed the use of one ubiquitination profile as a drug-response marker, directly on paraffin embedded tissues, supporting the possible application of these biomarkers in the clinical setting. INTERPRETATION: These findings bring new and important insights on the relationship between ubiquitination levels of proteins and different molecular and clinical features of PDAC patients. Markers identified in this study could have a potential application in clinical settings to help to predict response to chemotherapies thereby allowing the personalization of treatments. FUNDING: Fondation ARC (PJA 20181208270 and PGA 12021010002840_3562); INCa; Canceropôle PACA; DGOS; Amidex Foundation; Fondation de France; and INSERM.

Combining the antianginal drug perhexiline with chemotherapy induces complete pancreatic cancer regression in vivo.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the human cancers with the poorest prognosis. Interestingly, we found that mitochondrial respiration in primary human PDAC cells depends mainly on the fatty acid oxidation (FAO) to meet basic energy requirements. Therefore, we treated PDAC cells with perhexiline, a well-recognized FAO inhibitor used in cardiac diseases. Some PDAC cells respond efficiently to perhexiline, which acts synergistically with chemotherapy (gemcitabine) in vitro and in two xenografts in vivo. Importantly, perhexiline in combination with gemcitabine induces complete tumor regression in one PDAC xenograft. Mechanistically, this co-treatment causes energy and oxidative stress promoting apoptosis but does not exert inhibition of FAO. Yet, our molecular analysis indicates that the carnitine palmitoyltransferase 1C (CPT1C) isoform is a key player in the response to perhexiline and that patients with high CPT1C expression have better prognosis. Our study reveals that repurposing perhexiline in combination with chemotherapy is a promising approach to treat PDAC.

A glycosyltransferase gene signature to detect pancreatic ductal adenocarcinoma patients with poor prognosis.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an important heterogeneity, reflected by different clinical outcomes and chemoresistance. During carcinogenesis, tumor cells display aberrant glycosylated structures, synthetized by deregulated glycosyltransferases, supporting the tumor progression. In this study, we aimed to determine whether PDAC could be stratified through their glycosyltransferase expression profiles better than the current binary classification (basal-like and classical) in order to improve detection of patients with poor prognosis. METHODS: Bioinformatic analysis of 169 glycosyltransferase RNA sequencing data were performed for 74 patient-derived xenografts (PDX) of resected and unresectable tumors. The Australian cohort of International Cancer Genome Consortium and the microarray dataset from Puleo patient's cohort were used as independent validation datasets. FINDINGS: New PDAC stratification based on glycosyltransferase expression profile allowed to distinguish different groups of patients with distinct clinical outcome (p-value = 0.007). A combination of 19 glycosyltransferases differentially expressed in PDX defined a glyco-signature, whose prognostic value was validated on datasets including resected whole tumor tissues. The glyco-signature was able to discriminate three clusters of PDAC patients on the validation cohorts, two clusters displaying a short overall survival compared to one cluster having a better prognosis. Both poor prognostic clusters having different glyco-profiles in Puleo patient's cohort were correlated with stroma activated or desmoplastic subtypes corresponding to distinct microenvironment features (p-value < 0.0001). Besides, differential expression and enrichment analyses revealed deregulated functional pathways specific to different clusters. INTERPRETATION: This study identifies a glyco-signature relevant for a prognostic use, potentially applicable to resected and unresectable PDAC. Furthermore, it provides new potential therapeutic targets. FUNDING: This work was supported by INCa (Grants number 2018-078 and 2018-079), Fondation ARC (Grant number ARCPJA32020070002326), Cancéropôle PACA, DGOS (labelization SIRIC, Grant number 6038), Amidex Foundation and Ligue Nationale Contre le Cancer and by institutional fundings from INSERM and the Aix-Marseille Université.

Targeting Mitochondrial Complex I Overcomes Chemoresistance in High OXPHOS Pancreatic Cancer.

Mitochondrial respiration (oxidative phosphorylation, OXPHOS) is an emerging target in currently refractory cancers such as pancreatic ductal adenocarcinoma (PDAC). However, the variability of energetic metabolic adaptations between PDAC patients has not been assessed in functional investigations. In this work, we demonstrate that OXPHOS rates are highly heterogeneous between patient tumors, and that high OXPHOS tumors are enriched in mitochondrial respiratory complex I at protein and mRNA levels. Therefore, we treated PDAC cells with phenformin (complex I inhibitor) in combination with standard chemotherapy (gemcitabine), showing that this treatment is synergistic specifically in high OXPHOS cells. Furthermore, phenformin cooperates with gemcitabine in high OXPHOS tumors in two orthotopic mouse models (xenografts and syngeneic allografts). In conclusion, this work proposes a strategy to identify PDAC patients likely to respond to the targeting of mitochondrial energetic metabolism in combination with chemotherapy, and that phenformin should be clinically tested in appropriate PDAC patient subpopulations.

A Metagenomics Investigation of Carbohydrate-Active Enzymes along the Gastrointestinal Tract of Saudi Sheep.

The digestive microbiota of humans and of a wide range of animals has recently become amenable to in-depth studies due to the emergence of DNA-based metagenomic techniques that do not require cultivation of gut microbes. These techniques are now commonly used to explore the feces of humans and animals under the assumption that such samples are faithful proxies for the intestinal microbiota. Sheep (Ovis aries) are ruminant animals particularly adapted to life in arid regions and in particular Najdi, Noaimi (Awassi), and Harrei (Harri) breeds that are raised in Saudi Arabia for milk and/or meat production. Here we report a metagenomics investigation of the distal digestive tract of one animal from each breed that (i) examines the microbiota at three intestinal subsites (small intestine, mid-colon, and rectum), (ii) performs an in-depth analysis of the carbohydrate-active enzymes genes encoded by the microbiota at the three subsites, and (iii) compares the microbiota and carbohydrate-active enzyme profile at the three subsites across the different breeds. For all animals we found that the small intestine is characterized by a lower taxonomic diversity than that of the large intestine and of the rectal samples. Mirroring this observation, we also find that the spectrum of encoded carbohydrate-active enzymes of the mid-colon and rectal sites is much richer than that of the small intestine. However, the number of encoded cellulases and xylanases in the various intestinal subsites was found to be surprisingly low, indicating that the bulk of the fiber digestion is performed upstream in the rumen, and that the carbon source for the intestinal flora is probably constituted of the rumen fungi and bacteria that pass in the intestines. In consequence we argue that ruminant feces, which are often analyzed for the search of microbial genes involved in plant cell wall degradation, are probably a poor proxy for the lignocellulolytic potential of the host.

Binding to RNA regulates Set1 function.

The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs (small nuclear RNAs). We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.

JAM-C Identifies Src Family Kinase-Activated Leukemia-Initiating Cells and Predicts Poor Prognosis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells that acquire somatic mutations, leading to disease and clonogenic evolution. AML is characterized by accumulation of immature myeloid cells in the bone marrow and phenotypic cellular heterogeneity reflective of normal hematopoietic differentiation. Here, we show that JAM-C expression defines a subset of leukemic cells endowed with leukemia-initiating cell activity (LIC). Stratification of de novo AML patients at diagnosis based on JAM-C-expressing cells frequencies in the blood served as an independent prognostic marker for disease outcome. Using publicly available leukemic stem cell (LSC) gene expression profiles and gene expression data generated from JAM-C-expressing leukemic cells, we defined a single cell core gene expression signature correlated to JAM-C expression that reveals LSC heterogeneity. Finally, we demonstrated that JAM-C controls Src family kinase (SFK) activation in LSC and that LIC with exacerbated SFK activation was uniquely found within the JAM-C-expressing LSC compartment. Cancer Res; 77(23); 6627-40. ©2017 AACR.

The abundance and variety of carbohydrate-active enzymes in the human gut microbiota.

Descriptions of the microbial communities that live on and in the human body have progressed at a spectacular rate over the past 5 years, fuelled primarily by highly parallel DNA-sequencing technologies and associated advances in bioinformatics, and by the expectation that understanding how to manipulate the structure and functions of our microbiota will allow us to affect health and prevent or treat diseases. Among the myriad of genes that have been identified in the human gut microbiome, those that encode carbohydrate-active enzymes (CAZymes) are of particular interest, as these enzymes are required to digest most of our complex repertoire of dietary polysaccharides. In this Analysis article, we examine the carbohydrate-digestive capacity of a simplified but representative mini-microbiome in order to highlight the abundance and variety of bacterial CAZymes that are represented in the human gut microbiota.

Development and validation of a microarray for the investigation of the CAZymes encoded by the human gut microbiome.

Distal gut bacteria play a pivotal role in the digestion of dietary polysaccharides by producing a large number of carbohydrate-active enzymes (CAZymes) that the host otherwise does not produce. We report here the design of a custom microarray that we used to spot non-redundant DNA probes for more than 6,500 genes encoding glycoside hydrolases and lyases selected from 174 reference genomes from distal gut bacteria. The custom microarray was tested and validated by the hybridization of bacterial DNA extracted from the stool samples of lean, obese and anorexic individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the finding that a gene encoding a GH6-family cellulase was present in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding CAZome core containing 46 families of glycoside hydrolases and polysaccharide lyases, which suggests the functional stability of the gut microbiota despite large taxonomical variations between individuals.

A metagenomics investigation of carbohydrate-active enzymes along the goat and camel intestinal tract

Studies of the digestive microbiota of ruminant animals most often focus on the bacterial diversity in the rumen or the feces of the animals, but little is known about the diversity and functions of their distal intestine. Here, the bacterial microbiota of the distal intestinal tract of two goats and two camels was investigated by metagenomics techniques. The bacterial taxonomic diversity and carbohydrate-active enzyme profile were estimated for samples taken from the small intestine, the large intestine, and the rectum of each animal. The bacterial diversity and abundance in the small intestine were lower than in the rectal and large intestinal samples. Analysis of the carbohydrate-active enzyme profiles at each site revealed a comparatively low abundance of enzymes targeting xylan and cellulose in all animals examined, similar to what has been reported earlier for sheep and therefore suggesting that plant cell wall digestion probably takes place elsewhere, such as in the rumen

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