Pseudogenes, traditionally considered non-functional gene copies resulting from evolutionary mutations, have garnered attention due to recent transcriptomics and proteomics revealing their unexpected expressions and consequential cellular functions. Ubiquitin, transcribed from UBA52 and RPS27A genes, fused to ribosomal proteins eL40 and eS31, and polyubiquitin precursors encoded by UBB and UBC genes, has additional pseudogenes labeled as non-functional. However, recent evidence challenges this notion, demonstrating that these pseudogenes produce ubiquitin variants with minimal differences from the canonical sequence, suggesting a new regulatory dimension in ubiquitin-mediated cellular processes. To systematically catalogue possible Ubiquitin (Ub) and Ubiquitin-like (Ubl) variants from pseudogenes, expression data was compiled, identifying potential functional variants. Among these pseudogenes, RPS27AP5 expresses both Ubiquitin variant (UbP5) and ribosomal protein variant (S27aP5), with precursor proteins maturing through cleavage and exhibiting behavior similar to their counterparts post-translation. Notably, S27aP5 integrates into translating ribosomes, increasing the 80S monosomal ribosomal fraction and indirectly influencing p16INK4A transcriptional activation. The discovery of a functional S27a pseudogene supports the concept that a subset of ribosomes may incorporate diverse subunits for specific translational functions.

DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/ GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.

Abstract The relationship between colon stem cells (SCs), its surrounding niche microenvironment like the extracellular matrix (ECM) and the mesenchymal niche cells is complex and dynamic. ECM proteins are not only a scaffolding system for SCs, but their biophysical properties also contribute to localizing signals and creating gradients leading to the SCs homeostasis. Foxl1+-Telocytes (TCFoxL1+) are mesenchymal niche cells implicated in cell-cell communication and the production of BMP ligands. Upon chronic inflammation, mice lacking BMP signaling in TCFoxL1+ (BmpR1aΔFoxL1+), developed colonic tumors with microenvironment exhibiting abnormal TCFoxL1+ structure, increased BMP expression and myofibroblastic-like cancer-associated fibroblasts (myCAF) subpopulation. Yet, how the TCFoxL1+ influences SCs niche ECM proteins during tumorigenesis is less understood. We aimed to investigate how the loss of BMP signaling in TCFoxL1+ affects ECM network impacting the niche microenvironment promoting the development of CAC. DSS-based chronic inflammatory challenge in mutant and control mice, quantitative-MS-based strategy was performed to determine the proteome of the transformed regions solely in epithelial-mesenchymal tissue following colon deconstruction. Expression of the proteins of interest were validated by Western Blots or immunostainings. Quantitative analysis of collagen network was assessed using phyton pipeline U-net in Sirius red-stained colon analyzed under polarized light. Differential significant expression of 798 proteins, with 342 upregulated and 456 downregulated, were found in the tumors of mutant mice compared to controls. Mfap5 involved in CAF activation was the highest upregulated protein. We found upregulated, laminin subunits α4 and β2, decorin, lumican as well as transgelin (myCAF marker), which are proteins known for contributing to the stiffening of the ECM during colorectal cancer. Adamdec1 which prevents aberrant ECM accumulation and thrombospodin-1, an ECM glycoprotein whose low levels have been related to tumor growth, were both found to be downregulated. Data extracted from Python's U-Net pipeline demonstrated that tumors, in mutant mice, exhibited higher frequencies of straighter collagen fibers compared to wavier fibers in controls. Collagen fiber distribution was shown to present an even angle distribution in controls whereas BmpR1aΔFoxL1+ mice presented a preferential orientation. These results suggest that defective TCFoxL1+ commits the surrounding niche microenvironment, especially its ECM, toward the development and progression of CAC. Citation Format: Vilcy Reyes Nicolas, Alain B. Alfonso, Jennifer Raisch, François M. Boisvert, Marc-Antoine Lauzon, Nathalie Perreault. BMP signaling impaired telocytes-Foxl1+disrupt the extracellular matrix network enabling the development of colitis-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4281.

Abstract NOT PUBLISHED AT AUTHOR’S REQUEST Please acknowledge all funding agencies listed below Funding Agencies CIHRDoctoral Scholarship Fonds de recherche du Québec (FRQS)

FoxL1+telocytes (TCFoxL1+) are novel gastrointestinal subepithelial cells that form a communication axis between the mesenchyme and epithelium. TCFoxL1+ are strategically positioned to be key contributors to the microenvironment through production and secretion of growth factors and extracellular matrix (ECM) proteins. In recent years, the alteration of the bone morphogenetic protein (BMP) signaling in TCFoxL1+ was demonstrated to trigger a toxic microenvironment with ECM remodeling that leads to the development of pre-neoplastic gastric lesions. However, a comprehensive analysis of variations in the ECM composition and its associated proteins in gastric neoplasia linked to TCFoxL1+ dysregulation has never been performed. This study provides a better understanding of how TCFoxL1+ defective BMP signaling participates in the gastric pre-neoplastic microenvironment. Using a proteomic approach, we determined the changes in the complete matrisome of BmpR1a△FoxL1+ and control mice, both in total antrum as well as in isolated mesenchyme-enriched antrum fractions. Comparative proteomic analysis revealed that the deconstruction of the gastric antrum led to a more comprehensive analysis of the ECM fraction of gastric tissues microenvironment. These results show that TCFoxL1+ are key members of the mesenchymal cell population and actively participate in the establishment of the matrisomic fraction of the microenvironment, thus influencing epithelial cell behavior.

Cullin-RING finger ligases represent the largest family of ubiquitin ligases. They are responsible for the ubiquitination of ∼20% of cellular proteins degraded through the proteasome, by catalyzing the transfer of E2-loaded ubiquitin to a substrate. Seven cullins are described in vertebrates. Among them, cullin 4 (CUL4) associates with DNA damage-binding protein 1 (DDB1) to form the CUL4-DDB1 ubiquitin ligase complex, which is involved in protein ubiquitination and in the regulation of many cellular processes. Substrate recognition adaptors named DDB1/CUL4-associated factors (DCAFs) mediate the specificity of CUL4-DDB1 and have a short structural motif of approximately forty amino acids terminating in tryptophan (W)-aspartic acid (D) dipeptide, called the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested that at least sixty WD40-containing proteins could act as adaptors for the DDB1/CUL4 complex. To better define this association and classification, the interaction of each DCAFs with DDB1 was determined, and new partners and potential substrates were identified. Using BioID and affinity purification-mass spectrometry approaches, we demonstrated that seven WD40 proteins can be considered DCAFs with a high confidence level. Identifying protein interactions does not always lead to identifying protein substrates for E3-ubiquitin ligases, so we measured changes in protein stability or degradation by pulse-stable isotope labeling with amino acids in cell culture to identify changes in protein degradation, following the expression of each DCAF. In conclusion, these results provide new insights into the roles of DCAFs in regulating the activity of the DDB1-CUL4 complex, in protein targeting, and characterized the cellular processes involved.

Intestinal epithelial self-renewal is tightly regulated by signaling pathways controlling stem cell proliferation, determination and differentiation. In particular, Wnt/β-catenin signaling controls intestinal crypt cell division, survival and maintenance of the stem cell niche. Most colorectal cancers are initiated by mutations activating the Wnt/β-catenin pathway. Wnt signals are transduced through Frizzled receptors and LRP5/LRP6 coreceptors to downregulate GSK3β activity, resulting in increased nuclear β-catenin. Herein, we explored if LRP6 expression is required for maintenance of intestinal homeostasis, regeneration and oncogenesis. Mice with an intestinal epithelial cell-specific deletion of Lrp6 (Lrp6IEC-KO) were generated and their phenotype analyzed. No difference in intestinal architecture nor in proliferative and stem cell numbers was found in Lrp6IEC-KO mice in comparison to controls. Nevertheless, using ex vivo intestinal organoid cultures, we found that LRP6 expression was critical for crypt cell proliferation and stem cell maintenance. When exposed to dextran sodium sulfate, Lrp6IEC-KO mice developed more severe colitis than control mice. However, loss of LRP6 did not affect tumorigenesis in ApcMin/+ mice nor growth of human colorectal cancer cells. By contrast, Lrp6 silencing diminished anchorage-independent growth of BRafV600E-transformed intestinal epithelial cells (IEC). Thus, LRP6 controls intestinal stem cell functionality and is necessary for BRAF-induced IEC oncogenesis.

HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.

The WNT/β-catenin signaling pathway controls stem and progenitor cell proliferation, survival and differentiation in epithelial tissues. Aberrant stimulation of this pathway is therefore frequently observed in cancers from epithelial origin. For instance, colorectal and hepatic cancers display activating mutations in the CTNNB1 gene encoding β-catenin, or inactivating APC and AXIN gene mutations. However, these mutations are uncommon in breast and pancreatic cancers despite nuclear β-catenin localization, indicative of pathway activation. Notably, the low-density lipoprotein receptor-related protein 6 (LRP6), an indispensable co-receptor for WNT, is frequently overexpressed in colorectal, liver, breast and pancreatic adenocarcinomas in association with increased WNT/β -catenin signaling. Moreover, LRP6 is hyperphosphorylated in KRAS-mutated cells and in patient-derived colorectal tumours. Polymorphisms in the LRP6 gene are also associated with different susceptibility to developing specific types of lung, bladder and colorectal cancers. Additionally, recent observations suggest that LRP6 dysfunction may be involved in carcinogenesis. Indeed, reducing LRP6 expression and/or activity inhibits cancer cell proliferation and delays tumour growth in vivo. This review summarizes current knowledge regarding the biological function and regulation of LRP6 in the development of epithelial cancers—especially colorectal, liver, breast and pancreatic cancers.

Background/Aims: Nerve growth factor (NGF), which is crucial for growth and survival of neurons, also can exert pro-angiogenic actions on some endothelial cells through its high affinity TrkA receptor.Melatonin, a pineal gland hormone that regulates sleep and circadian rhythm via its MT1 and MT2 receptors, is also produced locally in the gastric mucosa.The subcellular distribution of TrkA, MT1 and MT2 in gastric endothelial cells (GECs), and their regulation by NGF, have not been explored before.We tested the novel hypothesis that the actions of NGF and melatonin on GECs are mediated via mitochondrial signaling.Since mitochondria generate energy crucial for all GEC functions (including cell survival and angiogenesis), we focused on mitochondrial localization of TrkA, MT1 and MT2 receptors in GECs and mitochondrial structure and function reflected by mitochondrial membrane potential (MMP) that generate ATP.Methods: GECs were isolated from gastric mucosa of Fisher rats using anti-PECAM-1 selection and magnetic bead separation.Cultured GECs were treated with either PBS or NGF (10 -1,000 ng/ml) for 1 to 4 hr.Studies: 1) Cell viability using Calcein AM; 2) Mitochondrial structure and function -MMP using cellpermeant MitoTracker probe that stains mitochondria in live cells in a MMP-dependent manner; 3) Sub-cellular localization of TrkA, MT1 and MT2 using dual molecular imaging, with special focus on their expression in mitochondria and translocation upon NGF stimulation.In addition, we determined the effect of NGF on angiogenesis and identified the underlying signaling mechanisms using specific inhibitors of PI3 Kinase (LY294002), Akt (inhibitor IV), MAPK (PD98059), mTOR (rapamycin) and actin polymerization (latrunculin B).Results: TrkA, MT1& MT2 receptors were expressed not only on the cell membrane but also localized to the mitochondria of GECs.NGF treatment increased MMP by 2.1-fold (p<0.001) and induced nuclear translocation of TrkA (2.3-fold increase; p<0.001) vs. PBS treated GECs.NGF treatment increased the expression of MT1 and MT2 by 1.9-fold and 1.7-fold, respectively, and increased angiogenesis by 1.5-fold (all p<0.001) vs. PBS controls.Pre-treatment with LY294002, Akt inhibitor IV, PD98059, rapamycin and latrunculin B abolished the angiogenic effect of NGF.Conclusions: 1) This study showed that in GECs TrkA, MT1 & MT2 receptors localize not only to the cell membrane but also to the mitochondria; 2) NGF treatment increases MMP and expression of TrkA, MT1 and MT2 in GECs; 3) NGF mediates its angiogenic action though TrkA, PI3 Kinase/Akt, MAPK and mTOR signaling pathways, and involves actin polymerization.3) Expression of TrkA and melatonin receptors in mitochondria of GECs has important implications for regulation of these cells' functions via mitochondrial signaling by NGF and melatonin. Tu1275

The gut microbiota acts as a real organ. The symbiotic interactions between resident micro-organisms and the digestive tract highly contribute to maintain the gut homeostasis. However, alterations to the microbiome caused by environmental changes (e.g. , infection, diet and/or lifestyle) can disturb this symbiotic relationship and promote disease, such as inflammatory bowel diseases and cancer. Colorectal cancer is a complex association of tumoral cells, non-neoplastic cells and a large amount of micro-organisms, and the involvement of the microbiota in colorectal carcinogenesis is becoming increasingly clear. Indeed, many changes in the bacterial composition of the gut microbiota have been reported in colorectal cancer, suggesting a major role of dysbiosis in colorectal carcinogenesis. Some bacterial species have been identified and suspected to play a role in colorectal carcinogenesis, such as Streptococcus bovis , Helicobacter pylori , Bacteroides Submit a Manuscript: http://www.wjgnet.com/esps/ Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx DOI: 10.3748/wjg.v22.i2.501 501 January 14, 2016|Volume 22|Issue 2| WJG|www.wjgnet.com World J Gastroenterol 2016 January 14; 22(2): 501-518 ISSN 1007-9327 (print) ISSN 2219-2840 (online) © 2016 Baishideng Publishing Group Inc. All rights reserved.

The gut microbiota maintains a relationship with its host with strong mutual benefits. Changes in the composition of the intestinal microbiota have been detected in colorectal cancer patients to the extent that it is now considered as a real contributing factor in this pathology. In this review, we focus on three commensal bacterial species, namely Bacteroides fragilis, Fusobacterium nucleatum, and Escherichia coli, which seem to emerge as pathogens and to contribute to colorectal carcinogenesis through their inflammatory and oncogenic properties.

The gut microbiota acts as a real organ. The symbiotic interactions between resident micro-organisms and the digestive tract highly contribute to maintain the gut homeostasis. However, alterations to the microbiome caused by environmental changes (e.g., infection, diet and/or lifestyle) can disturb this symbiotic relationship and promote disease, such as inflammatory bowel diseases and cancer. Colorectal cancer is a complex association of tumoral cells, non-neoplastic cells and a large amount of micro-organisms, and the involvement of the microbiota in colorectal carcinogenesis is becoming increasingly clear. Indeed, many changes in the bacterial composition of the gut microbiota have been reported in colorectal cancer, suggesting a major role of dysbiosis in colorectal carcinogenesis. Some bacterial species have been identified and suspected to play a role in colorectal carcinogenesis, such as Streptococcus bovis, Helicobacter pylori, Bacteroides fragilis, Enterococcus faecalis, Clostridium septicum, Fusobacterium spp. and Escherichia coli. The potential pro-carcinogenic effects of these bacteria are now better understood. In this review, we discuss the possible links between the bacterial microbiota and colorectal carcinogenesis, focusing on dysbiosis and the potential pro-carcinogenic properties of bacteria, such as genotoxicity and other virulence factors, inflammation, host defenses modulation, bacterial-derived metabolism, oxidative stress and anti-oxidative defenses modulation. We lastly describe how bacterial microbiota modifications could represent novel prognosis markers and/or targets for innovative therapeutic strategies.

Le microbiote intestinal entretient une relation mutualiste forte avec l’hôte. Depuis la mise en évidence de modifications de sa composition chez les patients atteints de cancer colorectal, le microbiote intestinal est considéré comme un facteur contribuant à part entière à cette pathologie. Nous avons focalisé notre attention dans cette revue sur trois espèces bactériennes commensales, Bacteroides fragilis, Fusobacterium nucleatum et Escherichia coli, qui semblent émerger sous les traits de pathogènes et participer au développement du cancer colorectal au travers de leurs propriétés inflammatoires et oncogéniques.

was to characterize diet-related changes in macroscopic and microscopic mucosal blood vessels in the colon as assessed using confocal microscopy (CM) and magnetic resonance imaging (MRI).METHODS: C57Bl6 mice (n=10) were fed standard AIN76A diet (StdD, n=5 mice) or Western diet (WD, n=5 mice) containing 20% lipid and low in vitamin D and calcium.Serial weights and confocal laser endomicroscopy (CLE) and MRI assessments were performed.Colonoscopy was performed using a rigid endoscope for small animals.CLE was performed with high molecular weight FITC dextran and the Cellvizio laser scanning unit with the ultra high definition probe inserted per rectum.For MRI studies, high-resolution T 2 -weighted images were acquired using a 9.4 Tesla Buker small animal scanner.Images from a time-of-flight (TOF) sequence were acquired to capture blood vessel images in the same region as anatomical MR images.Immediately prior to sacrifice, mice were injected with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) which labels endothelial cells and vessels were then imaged with CM ex vivo.Tissues were also collected for VEGF-A analysis by Western blotting (WB) and for fatty acid quantification.RESULTS: There were no colonoscopic differences in mucosal appearance in Std versus WD fed animals.MRI image analysis utilizing fat suppression settings demonstrated that WD induced: 1) 4fold increase in subcutaneous fat; 2) 6-fold increase in visceral fat; and 3) 10-fold increase in intra-muscular fat (p<0.01).Furthermore, WD increased total blood volume >2-fold and caused a 5-fold increase in dilated peri-colonic blood vessels.With regard to microscopic blood vessel architecture, both CLE imaging in vivo and Dil staining by CM ex vivo demonstrated a uniform regular honeycomb pattern in the StdD fed mice, whereas there was large variation in blood vessel architecture in WD fed mice, including irregular networks or redundant arcades.WD also induced 1.4-fold increase in VEGF-A (p, NS).Analyses of fatty acid composition are in progress.CONCLUSIONS: We have demonstrated that chronic WD alters macroscopic and microscopic features of mucosal colonic blood vessels.Additional studies are needed to determine whether these metabolically induced changes in vascularity contribute to diet-related colon cancer risk.Figure 1.StdD fed mice exhibited mucosal blood vessels with regular honeycomb patterns in all mice as shown by CLE (A) and Dil (B).In contrast, in WD fed mice, blood vessels were frequently ectatic and irregular, with sprouting and redundancy as shown by CLE (C) and Dil (D).

AIM:To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients.METHODS:Phylogroups and the presence of cyclomodulin-encoding genes of mucosa-associated E. coli from colon cancer and diverticulosis specimens were determined by PCR. Adhesion and invasion experiments were performed with I-407 intestinal epithelial cells using gentamicin protection assay. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression in T84 intestinal epithelial cells was measured by enzyme-linked immunosorbent assay and by Western Blot. Gut colonization, inflammation and pro-carcinogenic potential were assessed in a chronic infection model using CEABAC10 transgenic mice. Cell proliferation was analyzed by real-time mRNA quantification of PCNA and immunohistochemistry staining of Ki67.RESULTS:Analysis of mucosa-associated E. coli from colon cancer and diverticulosis specimens showed that whatever the origin of the E. coli strains, 86% of cyclomodulin-positive E. coli belonged to B2 phylogroup and most harbored polyketide synthase (pks) island, which encodes colibactin, and/or cytotoxic necrotizing factor (cnf) genes. In vitro assays using I-407 intestinal epithelial cells revealed that mucosa-associated B2 E. coli strains were poorly adherent and invasive. However, mucosa-associated B2 E. coli similarly to Crohn's disease-associated E. coli are able to induce CEACAM6 expression in T84 intestinal epithelial cells. In addition, in vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation.CONCLUSION:In conclusion, these data bring new insights into the ability of E. coli isolated from patients with colon cancer to establish persistent colonization, exacerbate inflammation and trigger carcinogenesis.

It is with profound sadness that we note the loss, at age 59, of our dear friend and mentor Dr Arlette Darfeuille-Michaud on June 28, 2014, following a 15-month battle against cancer. Arlette was a Professor at Auvergne University, and director of an INSERM unit that investigates microbial-host interactions in intestinal inflammation, located in Clermont-Ferrand, France. Arlette advanced our understanding of intestinal diseases, and humanity in general, via pioneering research in gut microbiology, visionary leadership, energetic teaching, and exceptionally supportive mentorship.⇓ Arlette lived her early years in the tiny French village of Beyssenac (in the beautiful Correze region) where her family owned and operated a fruit orchard. Arlette earned her PhD in 1987 at Auvergne University in the lab of Dr Bernard Joly, studying Klebsiella pneumoniae strains involved in nosocomial infections and Escherichia coli pathogenesis. Arlette then joined the faculty at Auvergne University as a lecturer in 1989, and then was promoted to professor in microbiology and molecular biology in 1994. Arlette then initiated a line of experimentation that would ultimately help contribute to our understanding of the pathogenesis of inflammatory bowel disease (IBD). Briefly, working with gastroenterologist Dr Jean-Frederic Colombel and Dr Christel Neut, Arlette made the discovery, reported in Gastroenterology in 1998, that many ileal samples of Crohn's patients were colonized by a previously unappreciated class of E. coli strains. As Dr Colombel recently recalled. “Arlette called us, very excited and enthusiastic, exclaiming that she had discovered …

Le cancer colorectal (CCR) est au 3eme rang des cancers les plus repandus dans le monde. Il est bien etabli que l'inflammation est un acteur cle de la carcinogenese colorectale. Parmi les cellules de l'infiltrat inflammatoire tumoral, les macrophages jouent un role moteur dans la carcinogenese en secretant un arsenal de cytokines/chimiokines et facteurs protumoraux tels que la prostaglandine E2 (PGE2), produit enzymatique de la cyclo-oxygenase 2 (COX-2). Des dysbioses du microbiote intestinal, en particulier une augmentation d’Escherichia coli (E. coli) appartenant au phylogroupe B2 et producteurs de cyclomodulines ont ete rapportees chez les patients atteints de CCR. Mon doctorat s'inscrit dans lacaracterisation du potentiel carcinogene de souches d'E. coli B2 isolees de patients atteints de CCR, et ceci notamment au travers de leur interaction avec les macrophages, principale population de l'infiltrat inflammatoire tumoral. La caracterisation de souches d'E. coli de phylogroupe B2 isolees de patients atteints de CCR a revele qu'elles possedent de faibles capacites d'adhesion et d'invasion aux cellulesepitheliales intestinales comparativement a la souche adherente et invasive d'E. coli LF82. En revanche, les E. coli associes au CCR sont capables d'induire l'expression du recepteur CEACAM6 par les cellules epitheliales coliques humaines T-84. Nous avons egalement montre que la souche d'E. coli 11G5 associee au CCR est capable, dans un modele murin d'infection chronique exprimant CEACAM6, de coloniser la muqueuse colique, d'induire une inflammation colique et des dommages epitheliaux, ainsi qu'une augmentation de la proliferation cellulaire, suggerant que les E. coli associes au CCR pourraient participer a l'etablissement d'un epithelium hyperproliferatif. Par ailleurs, nous avons montre que les E. coli isoles de patients atteints de CCR sont capables de survivre en macrophages humains THP-1, suggerant que les macrophages pourraient representer un niche de replication pour ces bacteries. Les souches d'E. coli isolees de CCR sont capables d'induire des niveaux de COX-2 significativement superieur a celui induit par une souche de la souche d'E. coli commensale ED1a. L'expression de COX-2 induite par les souches d'E. coli associees au CCR est dependante du nombre et de la viabilite des bacteries internalisees. Enfin, l'analyse des voies de signalisation a revele que la voie de signalisation p38 permet de controler le nombre de bacteries intracellulaires et l'expression de COX-2, suggerant que cette voie de signalisation pourrait etre impliquee dans la survie et la multiplication des E. coli associes au CCR. Ce travail de these a contribue a une meilleure caracterisation des souches d'E. coli associes au cancer du colon et a ouvert de nouvelles pistes sur la comprehension de leurcapacite a influencer la carcinogenese colorectale. Plus largement, ces donnees suggerent que les bacteries associees aux tumeurs pourraient jouer un role actif dans la progression tumorale, et ceci notamment au travers de leur interaction avec les macrophages. En plus d'etre une cible, le tropisme particulier des E. coli associes au CCR pour les macrophages en ferait de bons candidats pour le developpement de nouvelles strategies therapeutiques.

Intestinal dysbiosis has been reported in patients with colorectal cancer, and there is a high prevalence of Escherichia coli belonging to B2 phylogroup and producing a genotoxin, termed colibactin. Macrophages are one of the predominant tumor-infiltrating immune cells supporting key processes in tumor progression by producing protumoral factors such as cyclooxygenase-2 (COX-2). Here, we investigated whether B2 E. coli colonizing colon tumors could influence protumoral activities of macrophages. In contrast to commensal or nonpathogenic E. coli strains that were efficiently and rapidly degraded by macrophages at 24 h after infection, colon cancer-associated E. coli were able to resist killing by human THP-1 macrophages, to replicate intracellularly, and to persist inside host cells until at least 72 h after infection. Significant increases in COX-2 expression were observed in macrophages infected with colon cancer E. coli compared with macrophages infected with commensal and nonpathogenic E. coli strains or uninfected cells at 72 h after infection. Induction of COX-2 expression required live bacteria and was not due to colibactin production, as similar COX-2 levels were observed in macrophages infected with the wild-type colon cancer-associated E. coli 11G5 strain or a clbQ mutant unable to produce colibactin. Treatment of macrophages with ofloxacin, an antibiotic with intracellular tropism, efficiently decreased the number of intracellular bacteria and suppressed bacteria-induced COX-2 expression. This study provides new insights into the understanding of how tumor- infiltrating bacteria could influence cancer progression through their interaction with immune cells. Manipulation of microbes associated with tumors could have a deep influence on the secretion of protumoral molecules by infiltrating macrophages.