Association between gut microbiota and CpG island methylator phenotype in colorectal cancer.

The intestinal microbiota is an important environmental factor implicated in CRC development. Intriguingly, modulation of DNA methylation by gut microbiota has been reported in preclinical models, although the relationship between tumor-infiltrating bacteria and CIMP status is currently unexplored. In this study, we investigated tumor-associated bacteria in 203 CRC tumor cases and validated the findings using The Cancer Genome Atlas datasets. We assessed the abundance of Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae through qPCR analysis and observed enrichment of all four bacterial species in CRC samples. Notably, except for E. coli, all exhibited significant enrichment in cases of CIMP. This enrichment was primarily driven by a subset of cases distinguished by high levels of these bacteria, which we labeled as "Superhigh". The bacterial Superhigh status showed a significant association with CIMP (odds ratio 3.1, p-value = 0.013) and with MLH1 methylation (odds ratio 4.2, p-value = 0.0025). In TCGA CRC cases (393 tumor and 45 adj. normal), bacterial taxa information was extracted from non-human whole exome sequencing reads, and the bacterial Superhigh status was similarly associated with CIMP (odds ratio 2.9, p < 0.001) and MLH1 methylation (odds ratio 3.5, p < 0.001). Finally, 16S ribosomal RNA gene sequencing revealed high enrichment of Bergeyella spp. C. concisus, and F. canifelinum in CIMP-Positive tumor cases. Our findings highlight that specific bacterial taxa may influence DNA methylation, particularly in CpG islands, and contribute to the development and progression of CIMP in colorectal cancer.

Metabolic immaturity of newborns and breast milk bile acid metabolites are the central determinants of heightened neonatal vulnerability to norovirus diarrhea.

Noroviruses are the leading global cause of acute gastroenteritis, responsible for 685 million annual cases. While all age groups are susceptible to noroviruses, children are vulnerable to more severe infections than adults, underscored by 200 million pediatric cases and up to 200,000 deaths in children annually. Understanding the basis for the increased vulnerability of young hosts is critical to developing effective treatments. The pathogenic outcome of any enteric virus infection is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors. A central mediator in these complex relationships are host- and microbiota-derived metabolites. Noroviruses bind a specific class of metabolites, bile acids, which are produced by the host and then modified by commensal bacterial enzymes. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Considering these opposing effects, the microbiota-regulated balance of the bile acid pool may be a key determinant of the pathogenic outcome of a norovirus infection. The bile acid pool in newborns is unique due to immaturity of host metabolic pathways and developing gut microbiota, which could underlie the vulnerability of these hosts to severe norovirus infections. Supporting this concept, we demonstrate herein that microbiota and their bile acid metabolites protect from severe norovirus diarrhea whereas host-derived bile acids promote disease. Remarkably, we also report that maternal bile acid metabolism determines neonatal susceptibility to norovirus diarrhea during breastfeeding by delivering proviral bile acids to the newborn. Finally, directed targeting of maternal and neonatal bile acid metabolism can protect the neonatal host from norovirus disease. Altogether, these data support the conclusion that metabolic immaturity in newborns and ingestion of proviral maternal metabolites in breast milk are the central determinants of heightened neonatal vulnerability to norovirus disease.

Antibiotic-Induced Gut Microbiota Dysbiosis Modulates Host Transcriptome and m6A Epitranscriptome via Bile Acid Metabolism.

Gut microbiota can influence host gene expression and physiology through metabolites. Besides, the presence or absence of gut microbiome can reprogram host transcriptome and epitranscriptome as represented by N6-methyladenosine (m6A), the most abundant mammalian mRNA modification. However, which and how gut microbiota-derived metabolites reprogram host transcriptome and m6A epitranscriptome remain poorly understood. Here, investigation is conducted into how gut microbiota-derived metabolites impact host transcriptome and m6A epitranscriptome using multiple mouse models and multi-omics approaches. Various antibiotics-induced dysbiotic mice are established, followed by fecal microbiota transplantation (FMT) into germ-free mice, and the results show that bile acid metabolism is significantly altered along with the abundance change in bile acid-producing microbiota. Unbalanced gut microbiota and bile acids drastically change the host transcriptome and the m6A epitranscriptome in multiple tissues. Mechanistically, the expression of m6A writer proteins is regulated in animals treated with antibiotics and in cultured cells treated with bile acids, indicating a direct link between bile acid metabolism and m6A biology. Collectively, these results demonstrate that antibiotic-induced gut dysbiosis regulates the landscape of host transcriptome and m6A epitranscriptome via bile acid metabolism pathway. This work provides novel insights into the interplay between microbial metabolites and host gene expression.

Chemotherapy-induced microbiota exacerbates the toxicity of chemotherapy through the suppression of interleukin-10 from macrophages.

The gut microbiota has been shown to influence the efficacy and toxicity of chemotherapy, thereby affecting treatment outcomes. Understanding the mechanism by which microbiota affects chemotherapeutic toxicity would have a profound impact on cancer management. In this study, we report that fecal microbiota transplantation from oxaliplatin-exposed mice promotes toxicity in recipient mice. Splenic RNA sequencing and macrophage depletion experiment showed that the microbiota-induced toxicity of oxaliplatin in mice was dependent on macrophages. Furthermore, oxaliplatin-mediated toxicity was exacerbated in Il10-/- mice, but not attenuated in Rag1-/- mice. Adoptive transfer of macrophage into Il10-/- mice confirmed the role of macrophage-derived IL-10 in the improvement of oxaliplatin-induced toxicity. Depletion of fecal Lactobacillus and Bifidobacterium was associated with the exacerbation of oxaliplatin-mediated toxicity, whereas supplementation with these probiotics alleviated chemotherapy-induced toxicity. Importantly, IL-10 administration and probiotics supplementation did not attenuate the antitumor efficacy of chemotherapy. Clinically, patients with colorectal cancer exposed to oxaliplatin exhibited downregulation of peripheral CD45+IL-10+ cells. Collectively, our findings indicate that microbiota-mediated IL-10 production influences tolerance to chemotherapy, and thus represents a potential clinical target.

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer.

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer (CAC) with experimental CH driven by Dnmt3a+/Δ, we found higher tumor penetrance and increased tumor burden compared with controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia, and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency and rebalanced hematopoiesis. This study provides conceptually novel insights into non-tumor-cell-autonomous effects of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies.

The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors.

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as "pks" encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

Microbiota Influences on Hematopoiesis and Blood Cancers: New Horizons?

Hematopoiesis governs the generation of immune cells through the differentiation of hematopoietic stem cells (HSC) into various progenitor cells, a process controlled by intrinsic and extrinsic factors. Among extrinsic factors influencing hematopoiesis is the microbiota, or the collection of microorganisms present in various body sites. The microbiota has a profound impact on host homeostasis by virtue of its ability to release various molecules and structural components, which promote normal organ function. In this review, we will discuss the role of microbiota in influencing hematopoiesis and how disrupting the microbiota/host network could lead to hematologic malignancies, as well as highlight important knowledge gaps to move this field of research forward. SIGNIFICANCE: Microbiota dysfunction is associated with many pathologic conditions, including hematologic malignancies. In this review, we discuss the role of microbiota in influencing hematopoiesis and how disrupting the microbiota/host network could lead to hematologic malignancies. Understanding how the microbiota influences hematologic malignancies could have an important therapeutic impact for patients.

Western diet influences on microbiome and carcinogenesis.

The intestinal microbiota composition and associated bioactivities are sensitive to various modifier cues such as stress, inflammation, age, life-style and nutrition, which in turn are associated with susceptibility to developing cancer. Among these modifiers, diet has been shown to influence both microbiota composition as well as being an important source of microbial-derived compounds impacting the immunological, neurological and hormonal systems. Thus, it is necessary to take a holistic view when considering effect of diet on health and diseases. In this review, we focus on the interplay between western diet, the microbiota and cancer development by dissecting key components of the diet and leveraging data from human interventions and pre-clinical studies to better understand this relationship. We highlight key progress as well as stressing limitations in this field of research.

Intestinal bacteria and colorectal cancer: etiology and treatment.

The etiology of colorectal cancer (CRC) is influenced by bacterial communities that colonize the gastrointestinal tract. These microorganisms derive essential nutrients from indigestible dietary or host-derived compounds and activate molecular signaling pathways necessary for normal tissue and immune function. Associative and mechanistic studies have identified bacterial species whose presence may increase CRC risk, including notable examples such as Fusobacterium nucleatum, Enterotoxigenic Bacteroides fragilis, and pks+ E. coli. In recent years this work has expanded in scope to include aspects of host mutational status, intra-tumoral microbial heterogeneity, transient infection, and the cumulative influence of multiple carcinogenic bacteria after sequential or co-colonization. In this review, we will provide an updated overview of how host-bacteria interactions influence CRC development, how this knowledge may be utilized to diagnose or prevent CRC, and how the gut microbiome influences CRC treatment efficacy.

Comparing Transgenic Production to Supplementation of ω-3 PUFA Reveals Distinct But Overlapping Mechanisms Underlying Protection Against Metabolic and Hepatic Disorders.

We compared endogenous ω-3 PUFA production to supplementation for improving obesity-related metabolic dysfunction. Fat-1 transgenic mice, who endogenously convert exogenous ω-6 to ω-3 PUFA, and wild-type littermates were fed a high-fat diet and a daily dose of either ω-3 or ω-6 PUFA-rich oil for 12 wk. The endogenous ω-3 PUFA production improved glucose intolerance and insulin resistance but not hepatic steatosis. Conversely, ω-3 PUFA supplementation fully prevented hepatic steatosis but failed to improve insulin resistance. Both models increased hepatic levels of ω-3 PUFA-containing 2-monoacylglycerol and N-acylethanolamine congeners, and reduced levels of ω-6 PUFA-derived endocannabinoids with ω-3 PUFA supplementation being more efficacious. Reduced hepatic lipid accumulation associated with the endocannabinoidome metabolites EPEA and DHEA, which was causally demonstrated by lower lipid accumulation in oleic acid-treated hepatic cells treated with these metabolites. While both models induced a significant fecal enrichment of the beneficial Allobaculum genus, mice supplemented with ω-3 PUFA displayed additional changes in the gut microbiota functions with a significant reduction of fecal levels of the proinflammatory molecules lipopolysaccharide and flagellin. Multiple-factor analysis identify that the metabolic improvements induced by ω-3 PUFAs were accompanied by a reduced production of the proinflammatory cytokine TNFα, and that ω-3 PUFA supplementation had a stronger effect on improving the hepatic fatty acid profile than endogenous ω-3 PUFA. While endogenous ω-3 PUFA production preferably improves glucose tolerance and insulin resistance, ω-3 PUFA intake appears to be required to elicit selective changes in hepatic endocannabinoidome signaling that are essential to alleviate high-fat diet-induced hepatic steatosis.

Prevalence of genotoxic bacteria in men undergoing biopsy for prostate cancer.

BACKGROUND: New evidence suggests that bacteria-produced DNA toxins may have a role in the development or progression of prostate cancer. To determine the prevalence of these genes in a noninfection (i.e., colonized) state, we screened urine specimens in men before undergoing a biopsy for prostate cancer detection. METHODS: We developed a multiplex polymerase chain reaction using three of the most described bacterial genotoxin gene primers: Colibactin (polyketone synthase [pks] gene island: clbN and clbB), cytotoxic necrotizing factor (cnf1) toxin, and cytolethal distending toxin B (cdtB) represented gene islands. After calibration on Escherichia coli samples of known genotypes, we used a training and validation cohort. We performed multiplex testing on a training cohort of previously collected urine from 45 men undergoing prostate biopsy. For the validation cohort, we utilized baseline urine samples from a previous randomized clinical trial (n = 263) with known prostate cancer outcomes. RESULTS: The prevalence of four common bacterial genotoxin genes detected in the urine before prostate biopsy for prostate cancer is 8% (25/311). The prevalence of pks island (clbN and clbB), cnf1, and cdt toxin genes are 6.1%, 2.4%, and 1.7%, respectively. We found no association between urinary genotoxins and prostate cancer (p = 0.83). We did identify a higher proportion of low-grade cancer (92% vs. 44%) in those men positive for urinary genotoxin and higher-grade cancer in those genotoxin negative (8% vs. 56%, p = 0.001). CONCLUSIONS: The prevalence of urinary genotoxins is low and does not correspond to a prostate cancer diagnosis. The urine was taken at one point in time and does not rule out the possibility of previous exposure.

Non-pathogenic microbiota accelerate age-related CpG Island methylation in colonic mucosa.

DNA methylation is an epigenetic process altered in cancer and ageing. Age-related methylation drift can be used to estimate lifespan and can be influenced by extrinsic factors such as diet. Here, we report that non-pathogenic microbiota accelerate age-related methylation drift in the colon when compared with germ-free mice. DNA methylation analyses showed that microbiota and IL10KO were associated with changes in 5% and 4.1% of CpG sites, while mice with both factors had 18% alterations. Microbiota, IL10KO, and their combination altered 0.4%, 0.4%, and 4% of CpG island methylation, respectively. These are comparable to what is seen in colon cancer. Ageing changes were accelerated in the IL10KO mice with microbiota, and the affected genes were more likely to be altered in colon cancer. Thus, the microbiota affect DNA methylation of the colon in patterns reminiscent of what is observed in ageing and colorectal cancer.

Skin microbiome sampling in the preterm neonate.

Despite advances in our understanding of the human microbiome, there exist significant knowledge gaps in our understanding of the skin microbiome of the preterm neonate. Herein, we describe skin microbiome sampling of six preterm neonates at multiple timepoints, and compare the skin microbiome samples to environmental (crib/isolette swabs) and negative controls. Samples of the same type (skin, crib, control) were more similar than when compared by week or by patient.

Bacteriophage-mediated manipulations of microbiota in gastrointestinal diseases.

Although some gastrointestinal diseases could be managed using various antibiotics regimen, this therapeutic approach lacks precision and damages the microbiota. Emerging literature suggests that phages may play a key role in restoring the gut microbiome balance and controlling disease progression either with exogenous phage intervention or filtered fecal transplantation or even engineered phages. In this review, we will discuss the current phage applications aiming at controlling the bacterial population and preventing infection, inflammation, and cancer progression in the context of gastrointestinal diseases.

Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival.

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1-/-). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1-/- and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/- and hTS/Men1-/- mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

Intestinal microbiota modulates pancreatic carcinogenesis through intratumoral natural killer cells.

Preclinical data demonstrate that the gut microbiota can promote pancreatic ductal adenocarcinoma (PDAC), but mechanisms remain unclear. We hypothesized that intestinal microbiota alters anti-tumor innate immunity response to facilitate PDAC progression. Human PDAC L3.6pl cells were heterotopically implanted into Rag1-/- mice after microbiota depletion with antibiotics, while syngeneic murine PDAC Pan02 cells were implanted intrapancreatic into germ-free (GF) C57BL/6 J mice. Natural killer (NK) cells and their IFNγ expression were quantitated by flow cytometry. NK cells were depleted in vivo using anti-Asialo GM1 antibody to confirm the role of NK cells. Bacteria-free supernatant from SPF and GF mice feces was used to test its effect on NK-92MI cell anti-tumor response in vitro. SPF and ex-GF mice (reconstituted with SPF microbiota) developed larger PDAC tumors with decreased NK cell tumor infiltration and IFNγ expression versus GF-Rag1-/-. Microbiota-induced PDAC tumorigenesis was attenuated by antibiotic exposure, a process reversed following NK cell depletion in both Rag1-/- and C57BL/6 J mice. Compared to GF, SPF-Rag1-/- abiotic stool culture supernatant inhibited NK-92MI cytotoxicity, migration, and anti-cancer related gene expression. Gut microbiota promotes PDAC tumor progression through modulation of the intratumoral infiltration and activity of NK cells.

Fusobacterium is enriched in oral cancer and promotes induction of programmed death-ligand 1 (PD-L1).

Recently, increased number of studies have demonstrated a relationship between the oral microbiome and development of head and neck cancer, however, there are few studies to investigate the role of oral bacteria in the context of the tumor microenvironment in a single head and neck subsite. Here, paired tumor and adjacent normal tissues from thirty-seven oral tongue squamous cell carcinoma (SCC) patients were subjected to 16S rRNA gene sequencing and whole exome sequencing (WES), in addition to RNA sequencing for tumor samples. We observed that Fusobacterium was significantly enriched in oral tongue cancer and that Rothia and Streptococcus were enriched in adjacent normal tissues. A decrease in alpha diversity was found in tumor when compared to adjacent normal tissues. While increased Fusobacterium in tumor samples was not associated with changes in immune cell infiltration, it was associated with increased PD-L1 mRNA expression. Therefore, we examined the effects of Fusobacterium on PD-L1 expression in head and neck SCC cell lines. We demonstrated that infection with Fusobacterium species can increase both PD-L1 mRNA and surface PD-L1 protein expression on head and neck cancer cell lines. The correlation between Fusobacterium and PD-L1 expression in oral tongue SCC, in conjunction with the ability of the bacterium to induce PD-L1 expression in vitro suggests a potential role for Fusobacterium on modulation of the tumor immune microenvironment in head and neck cancer.

Human Colon Cancer-Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient-derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. SIGNIFICANCE: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825.

Group 3 innate lymphoid cell pyroptosis represents a host defence mechanism against Salmonella infection.

Group 3 innate lymphoid cells (ILC3s) produce interleukin (IL)-22 and coordinate with other cells in the gut to mount productive host immunity against bacterial infection. However, the role of ILC3s in Salmonella enterica serovar Typhimurium (S. Typhimurium) infection, which causes foodborne enteritis in humans, remains elusive. Here we show that S. Typhimurium exploits ILC3-produced IL-22 to promote its infection in mice. Specifically, S. Typhimurium secretes flagellin through activation of the TLR5-MyD88-IL-23 signalling pathway in antigen presenting cells (APCs) to selectively enhance IL-22 production by ILC3s, but not T cells. Deletion of ILC3s but not T cells in mice leads to better control of S. Typhimurium infection. We also show that S. Typhimurium can directly invade ILC3s and cause caspase-1-mediated ILC3 pyroptosis independently of flagellin. Genetic ablation of Casp1 in mice leads to increased ILC3 survival and IL-22 production, and enhanced S. Typhimurium infection. Collectively, our data suggest a key host defence mechanism against S. Typhimurium infection via induction of ILC3 death to limit intracellular bacteria and reduce IL-22 production.

MarZIC: A Marginal Mediation Model for Zero-Inflated Compositional Mediators with Applications to Microbiome Data.

BACKGROUND: The human microbiome can contribute to pathogeneses of many complex diseases by mediating disease-leading causal pathways. However, standard mediation analysis methods are not adequate to analyze the microbiome as a mediator due to the excessive number of zero-valued sequencing reads in the data and that the relative abundances have to sum to one. The two main challenges raised by the zero-inflated data structure are: (a) disentangling the mediation effect induced by the point mass at zero; and (b) identifying the observed zero-valued data points that are not zero (i.e., false zeros). METHODS: We develop a novel marginal mediation analysis method under the potential-outcomes framework to address the issues. We also show that the marginal model can account for the compositional structure of microbiome data. RESULTS: The mediation effect can be decomposed into two components that are inherent to the two-part nature of zero-inflated distributions. With probabilistic models to account for observing zeros, we also address the challenge with false zeros. A comprehensive simulation study and the application in a real microbiome study showcase our approach in comparison with existing approaches. CONCLUSIONS: When analyzing the zero-inflated microbiome composition as the mediators, MarZIC approach has better performance than standard causal mediation analysis approaches and existing competing approach.