Toward a postbiotic era of microbiome science: Opportunities to advance immunotherapies for hepatocellular carcinoma.

The microbiome has revolutionized the field of cancer immunology and checkpoint therapeutics for gastrointestinal malignancies. Combating hepatocellular carcinoma (HCC) by immune checkpoint blockade (ICB) is a unique challenge due in part to chronic complications that arise from local and systemic metabolic dysfunctions. Gut microbial metabolites modulate key immunological processes that influence liver cancer susceptibility and resistance to ICB. This review discusses recent progresses in linking microbiota functions to HCC tumor immunity and highlights their therapeutic potential.

Lactobacillus gallinarum modulates the gut microbiota and produces anti-cancer metabolites to protect against colorectal tumourigenesis.

OBJECTIVE: Using faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in colorectal tumourigenesis. DESIGN: The tumor-suppressive effect of L. gallinarum was assessed in murine models of CRC. CRC cell lines and organoids derived from patients with CRC were cultured with L. gallinarum or Escherichia coli MG1655 culture-supernatant to evaluate cell proliferation, apoptosis and cell cycle distribution. Gut microbiota was assessed by 16S ribosomal DNA sequencing. Antitumour molecule produced from L. gallinarum was identified by liquid chromatography mass spectrometry (LC-MS/MS) and targeted mass spectrometry. RESULTS: L. gallinarum significantly reduced intestinal tumour number and size compared with E. coli MG1655 and phosphate-buffered saline in both male and female murine intestinal tumourigenesis models. Faecal microbial profiling revealed enrichment of probiotics and depletion of pathogenic bacteria in L. gallinarum-treated mice. Culturing CRC cells with L. gallinarum culture-supernatant (5%, 10% and 20%) concentration-dependently suppressed cell proliferation and colony formation. L. gallinarum culture-supernatant significantly promoted apoptosis in CRC cells and patient-derived CRC organoids, but not in normal colon epithelial cells. Only L. gallinarum culture-supernatant with fraction size <3 kDa suppressed proliferation in CRC cells. Using LC-MS/MS, enrichments of indole-3-lactic acid (ILA) was identified in both L. gallinarum culture-supernatant and the gut of L. gallinarum-treated mice. ILA displayed anti-CRC growth in vitro and inhibited intestinal tumourigenesis in vivo. CONCLUSION: L. gallinarum protects against intestinal tumourigenesis by producing protective metabolites that can promote apoptosis of CRC cells.

Aspirin Modulation of the Colorectal Cancer-Associated Microbe Fusobacterium nucleatum.

Aspirin is a chemopreventive agent for colorectal adenoma and cancer (CRC) that, like many drugs inclusive of chemotherapeutics, has been investigated for its effects on bacterial growth and virulence gene expression. Given the evolving recognition of the roles for bacteria in CRC, in this work, we investigate the effects of aspirin with a focus on one oncomicrobe-Fusobacterium nucleatum We show that aspirin and its primary metabolite salicylic acid alter F. nucleatum strain Fn7-1 growth in culture and that aspirin can effectively kill both actively growing and stationary Fn7-1. We also demonstrate that, at levels that do not inhibit growth, aspirin influences Fn7-1 gene expression. To assess whether aspirin modulation of F. nucleatum may be relevant in vivo, we use the ApcMin/+ mouse intestinal tumor model in which Fn7-1 is orally inoculated daily to reveal that aspirin-supplemented chow is sufficient to inhibit F. nucleatum-potentiated colonic tumorigenesis. We expand our characterization of aspirin sensitivity across other F. nucleatum strains, including those isolated from human CRC tissues, as well as other CRC-associated microbes, enterotoxigenic Bacteroides fragilis, and colibactin-producing Escherichia coli Finally, we determine that individuals who use aspirin daily have lower fusobacterial abundance in colon adenoma tissues, as determined by quantitative PCR performed on adenoma DNA. Together, our data support that aspirin has direct antibiotic activity against F. nucleatum strains and suggest that consideration of the potential effects of aspirin on the microbiome holds promise in optimizing risk-benefit assessments for use of aspirin in CRC prevention and management.IMPORTANCE There is an increasing understanding of the clinical correlations and potential mechanistic roles of specific members of the gut and tumoral microbiota in colorectal cancer (CRC) initiation, progression, and survival. However, we have yet to parlay this knowledge into better CRC outcomes through microbially informed diagnostic, preventive, or therapeutic approaches. Here, we demonstrate that aspirin, an established CRC chemopreventive, exhibits specific effects on the CRC-associated Fusobacterium nucleatum in culture, an animal model of intestinal tumorigenesis, and in human colonic adenoma tissues. Our work proposes a potential role for aspirin in influencing CRC-associated bacteria to prevent colorectal adenomas and cancer, beyond aspirin's canonical anti-inflammatory role targeting host tissues. Future research, such as studies investigating the effects of aspirin on fusobacterial load in patients, will help further elucidate the prospect of using aspirin to modulate F. nucleatumin vivo for improving CRC outcomes.

Long-term persistence of gastric dysbiosis after eradication of Helicobacter pylori in patients who underwent endoscopic submucosal dissection for early gastric cancer.

BACKGROUND: Gastric microbiome, other than Helicobacter pylori, plays a role in the tumorigenesis of gastric cancer (GC). Patients who undergo endoscopic submucosal dissection for early GC have a high risk of developing metachronous GC even after successful eradication of H. pylori. Thus, we investigated the microbial profiles and associated changes in such patients after the eradication of H. pylori. METHODS: A total of 19 H. pylori-infected patients with early GC who were or to be treated by endoscopic resection, with paired biopsy samples at pre- and post-eradication therapy, were retrospectively enrolled. Ten H. pylori-negative patients were enrolled as controls. Biopsy samples were analyzed using 16S rRNA sequencing. RESULTS: H. pylori-positive patients exhibited low richness and evenness of bacteria with the deletion of several genera, including Blautia, Ralstonia, Faecalibacterium, Methylobacterium, and Megamonas. H. pylori eradication partially restored microbial diversity, as assessed during a median follow-up at 13 months after eradication therapy. However, post-eradication patients had less diversity than that in the controls and possessed a lower abundance of the five genera mentioned above. The eradication of H. pylori also altered the bacterial composition, but not to the same extent as that in controls. The microbial communities could be clustered into three separate groups: H. pylori-negative, pre-eradication, and post-eradication. CONCLUSION: Changes in dysbiosis may persist long after the eradication of H. pylori in patients with a history of GC. Dysbiosis may be involved in the development of both primary and metachronous GC after the eradication of H. pylori in such patients.

Bacteria pathogens drive host colonic epithelial cell promoter hypermethylation of tumor suppressor genes in colorectal cancer.

BACKGROUND: Altered microbiome composition and aberrant promoter hypermethylation of tumor suppressor genes (TSGs) are two important hallmarks of colorectal cancer (CRC). Here we performed concurrent 16S rRNA gene sequencing and methyl-CpG binding domain-based capture sequencing in 33 tissue biopsies (5 normal colonic mucosa tissues, 4 pairs of adenoma and adenoma-adjacent tissues, and 10 pairs of CRC and CRC-adjacent tissues) to identify significant associations between TSG promoter hypermethylation and CRC-associated bacteria, followed by functional validation of the methylation-associated bacteria. RESULTS: Fusobacterium nucleatum and Hungatella hathewayi were identified as the top two methylation-regulating bacteria. Targeted analysis on bona fide TSGs revealed that H. hathewayi and Streptococcus spp. significantly correlated with CDX2 and MLH1 promoter hypermethylation, respectively. Mechanistic validation with cell-line and animal models revealed that F. nucleatum and H. hathewayi upregulated DNA methyltransferase. H. hathewayi inoculation also promoted colonic epithelial cell proliferation in germ-free and conventional mice. CONCLUSION: Our integrative analysis revealed previously unknown epigenetic regulation of TSGs in host cells through inducing DNA methyltransferase by F. nucleatum and H. hathewayi, and established the latter as CRC-promoting bacteria. Video abstract.

Aspirin Reduces Colorectal Tumor Development in Mice and Gut Microbes Reduce its Bioavailability and Chemopreventive Effects.

BACKGROUND & AIMS: Alterations in the intestinal microbiota affect development of colorectal cancer and drug metabolism. We studied whether the intestinal microbiota affect the ability of aspirin to reduce colon tumor development in mice. METHODS: We performed studies with APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium to induce colorectal carcinogenesis. Some mice were given antibiotics to deplete intestinal microbes, with or without aspirin, throughout the entire experiment. Germ-free mice were studied in validation experiments. Colon tissues were collected and analyzed by histopathology, quantitative reverse-transcription polymerase chain reaction, and immunoblots. Blood samples and gut luminal contents were analyzed by liquid chromatography/mass spectrometry and an arylesterase activity assay. Fecal samples were analyzed by 16S ribosomal RNA gene and shotgun metagenome sequencing. RESULTS: Administration of aspirin to mice reduced colorectal tumor number and load in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium that had been given antibiotics (depleted gut microbiota), but not in mice with intact microbiota. Germ-free mice given aspirin developed fewer colorectal tumors than conventionalized germ-free mice given aspirin. Plasma levels of aspirin were higher in mice given antibiotics than in mice with intact gut microbiota. Analyses of luminal contents revealed that aerobic gut microbes, including Lysinibacillus sphaericus, degrade aspirin. Germ-free mice fed L sphaericus had lower plasma levels of aspirin than germ-free mice that were not fed this bacterium. There was an inverse correlation between aspirin dose and colorectal tumor development in conventional mice, but this correlation was lost with increased abundance of L sphaericus. Fecal samples from mice fed aspirin were enriched in Bifidobacterium and Lactobacillus genera, which are considered beneficial, and had reductions in Alistipes finegoldii and Bacteroides fragili, which are considered pathogenic. CONCLUSIONS: Aspirin reduces development of colorectal tumors in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium, depending on the presence of intestinal microbes. L sphaericus in the gut degrades aspirin and reduced its chemopreventive effects in mice. Fecal samples from mice fed aspirin were enriched in beneficial bacteria, with reductions in pathogenic bacteria.

A novel faecal Lachnoclostridium marker for the non-invasive diagnosis of colorectal adenoma and cancer.

OBJECTIVE: There is a need for early detection of colorectal cancer (CRC) at precancerous-stage adenoma. Here, we identified novel faecal bacterial markers for diagnosing adenoma. DESIGN: This study included 1012 subjects (274 CRC, 353 adenoma and 385 controls) from two independent Asian groups. Candidate markers were identified by metagenomics and validated by targeted quantitative PCR. RESULTS: Metagenomic analysis identified 'm3' from a Lachnoclostridium sp., Fusobacterium nucleatum (Fn) and Clostridium hathewayi (Ch) to be significantly enriched in adenoma. Faecal m3 and Fn were significantly increased from normal to adenoma to CRC (p<0.0001, linear trend by one-way ANOVA) in group I (n=698), which was further confirmed in group II (n=313; p<0.0001). Faecal m3 may perform better than Fn in distinguishing adenoma from controls (areas under the receiver operating characteristic curve (AUROCs) m3=0.675 vs Fn=0.620, p=0.09), while Fn performed better in diagnosing CRC (AUROCs Fn=0.862 vs m3=0.741, p<0.0001). At 78.5% specificity, m3 and Fn showed sensitivities of 48.3% and 33.8% for adenoma, and 62.1% and 77.8% for CRC, respectively. In a subgroup tested with faecal immunochemical test (FIT; n=642), m3 performed better than FIT in detecting adenoma (sensitivities for non-advanced and advanced adenomas of 44.2% and 50.8% by m3 (specificity=79.6%) vs 0% and 16.1% by FIT (specificity=98.5%)). Combining with FIT improved sensitivity of m3 for advanced adenoma to 56.8%. The combination of m3 with Fn, Ch, Bacteroides clarus and FIT performed best for diagnosing CRC (specificity=81.2% and sensitivity=93.8%). CONCLUSION: This study identifies a novel bacterial marker m3 for the non-invasive diagnosis of colorectal adenoma.

High-fat diet-mediated dysbiosis exacerbates NSAID-induced small intestinal damage through the induction of interleukin-17A.

Non-steroidal anti-inflammatory drugs (NSAIDs) cause damage in the small intestine in a bacteria-dependent manner. As high-fat diet (HFD) is a potent inducer of gut dysbiosis, we investigated the effects of HFD on bacterial flora in the small intestine and NSAID-induced enteropathy. 16S rRNA gene analysis revealed that the population of Bifidobacterium spp. significantly decreased by fold change of individual operational taxonomic units in the small intestine of mice fed HFD for 8 weeks. HFD increased intestinal permeability, as indicated by fluorescein isothiocyanate-dextran absorption and serum lipopolysaccharide levels, accompanied by a decrease in the protein expressions of ZO-1 and occludin and elevated mRNA expression of interleukin (IL)-17A in the small intestine. HFD-fed mice exhibited increased susceptibility to indomethacin-induced damage in the small intestine; this phenotype was observed in normal diet-fed mice that received small intestinal microbiota from HFD-fed mice. Administration of neutralizing antibodies against IL-17A to HFD-fed mice reduced intestinal permeability and prevented exacerbation of indomethacin-induced damage. Thus, HFD-induced microbial dysbiosis in small intestine caused microinflammation through the induction of IL-17A and increase in intestinal permeability, resulting in the aggravation of NSAID-induced small intestinal damage.

VSTM2A suppresses colorectal cancer and antagonizes Wnt signaling receptor LRP6.

Hyperactivation of Wnt/ß-catenin signaling pathway is a critical step in colorectal tumorigenesis. In this study, we identified that V-set and transmembrane domain containing 2A (VSTM2A) was a top-downregulated secreted protein that negatively regulated Wnt singling pathways in colorectal cancer (CRC). We investigated the functional mechanisms and clinical implication of VSTM2A in CRC. Methods: Function of VSTM2A was investigated in vitro and in vivo. VSTM2A binding partner was identified by mass spectrometry, immunoprecipitation and Western blot. The clinical impact of VSTM2A was assessed in 355 CRC patients and TCGA cohort. Results: VSTM2A protein was prominently silenced in CRC tumor tissues and cell lines mediated by its promoter hypermethylation. VSTM2A DNA promoter hypermethylation and VSTM2A protein downregulation was associated with poor survival of CRC patients. Ectopic expression of VSTM2A inhibited colon cancer cell lines and organoid growth, induced CRC cells apoptosis, inhibited cell migration and invasion, and suppressed growth of xenograft tumors in nude mice. VSTM2A was released from CRC cells through a canonical secretion pathway. Secreted VSTM2A significantly suppressed Wnt signaling pathway in colon cancer cells. Wnt signaling co-receptor LDL receptor related protein 6 (LRP6) was identified as a cell membrane binding partner of VSTM2A. Using deletion/mutation and immunoprecipitation, we demonstrated that VSTM2A bound to LRP6 E1-4 domain with its IgV domain. VSTM2A suppressed LRP6 phosphorylation in a time and dose dependent manner, and induced LRP6 endocytosis and lysosome-mediated degradation, which collectively contributing to the inactivation of Wnt signaling. Conclusions: VSTM2A is a novel antagonist of canonical Wnt signaling by directly binding to LRP6 and induces LRP6 endocytosis and degradation. VSTM2A is a potential prognostic biomarker for the outcome of CRC patients.

Enteric fungal microbiota dysbiosis and ecological alterations in colorectal cancer.

OBJECTIVES: Bacteriome and virome alterations are associated with colorectal cancer (CRC). Nevertheless, the gut fungal microbiota in CRC remains largely unexplored. We aimed to characterise enteric mycobiome in CRC. DESIGN: Faecal shotgun metagenomic sequences of 184 patients with CRC, 197 patients with adenoma and 204 control subjects from Hong Kong were analysed (discovery cohort: 73 patients with CRC and 92 control subjects; validation cohort: 111 patients with CRC, 197 patients with adenoma and 112 controls from Hong Kong). CRC-associated fungal markers and ecological changes were also validated in additional independent cohorts of 90 patients with CRC, 42 patients with adenoma and 66 control subjects of published repository sequences from Germany and France. Assignment of taxonomies was performed by exact k-mer alignment against an integrated microbial reference genome database. RESULTS: Principal component analysis revealed separate clusters for CRC and control (p<0.0001), with distinct mycobiomes in early-stage and late-stage CRC (p=0.0048). Basidiomycota:Ascomycota ratio was higher in CRC (p=0.0042), with increase in Malasseziomycetes (p<0.0001) and decrease in Saccharomycetes (p<0.0001) and Pneumocystidomycetes (p=0.0017). Abundances of 14 fungal biomarkers distinguished CRC from controls with an area under the receiver-operating characteristic curve (AUC) of 0.93 and validated AUCs of 0.82 and 0.74 in independent Chinese cohort V1 and European cohort V2, respectively. Further ecological analysis revealed higher numbers of co-occurring fungal intrakingdom and co-exclusive bacterial-fungal correlations in CRC (p<0.0001). Moreover, co-occurrence interactions between fungi and bacteria, mostly contributed by fungal Ascomycota and bacterial Proteobacteria in control, were reverted to co-exclusive interplay in CRC (p=0.00045). CONCLUSIONS: This study revealed CRC-associated mycobiome dysbiosis characterised by altered fungal composition and ecology, signifying that the gut mycobiome might play a role in CRC.

CAB39L elicited an anti-Warburg effect via a LKB1-AMPK-PGC1α axis to inhibit gastric tumorigenesis.

Metabolic dysfunction is a hallmark of gastric cancer (GC). In this study, we reported the identification of Calcium Binding Protein 39-Like (CAB39L) as a novel regulator of tumor metabolism in GC. CAB39L mRNA was frequently silenced by promoter methylation in GC cell lines and tissues. Functional studies suggested that CAB39L functions as a tumor suppressor, as overexpression of CAB39L elicited suppression of multiple cancer phenotypes both in GC cells and an orthotopic mouse model; whilst its knockdown promoted tumorigenesis. Mechanistically, CAB39L interacted with LKB1-STRAD complex and induced LKB1, leading to the phosphorylation and activation of AMPKα/ß. LKB1-AMPK activation in GC cell lines was tumor suppressive, as metformin (an AMPK activator) inhibited GC cell growth in the CAB39L-silenced cells. Moreover, knockdown of LKB1 reversed growth inhibitory effect of CAB39L, indicating that tumor suppression by CAB39L depended on LKB1-AMPK. RNAseq and gene set enrichment analysis revealed that CAB39L was closely correlated with oxidative phosphorylation and mitochondrial biogenesis. Consistently, CAB39L-induced p-AMPK elicited PGC1α phosphorylation and increased the expression of genes involved in mitochondrial respiration complexes. Accordingly, CAB39L reversed the Warburg effect in GC, as evidenced by enhanced oxygen consumption rate and reduced extracellular acidification rate; inversely, CAB39L knockdown promoted a metabolic shift towards the Warburg phenotype. In GC patients, CAB39L promoter hypermethylation was correlated with poor prognosis. Our data demonstrate that CAB39L is a novel tumor suppressor which suppresses tumorigenesis by promoting LKB1-AMPK-PGC1α axis, thereby preventing a metabolic shift that drives carcinogenesis. CAB39L methylation is a potential prognostic biomarker for GC patients.

Association Between Bacteremia From Specific Microbes and Subsequent Diagnosis of Colorectal Cancer.

BACKGROUND & AIMS: Colorectal cancer (CRC) development has been associated with increased proportions of Bacteroides fragilis and certain Streptococcus, Fusobacterium, and Peptostreptococcus species in the intestinal microbiota. We investigated associations between bacteremia from specific intestinal microbes and occurrence of CRC. METHODS: We performed a retrospective study after collecting data on 13,096 adult patients (exposed group) in Hong Kong hospitalized with bacteremia (identified by blood culture test) without a previous diagnosis of cancer from January 1, 2006 through December 31, 2015. We collected data on intestinal microbes previously associated with CRC (genera Bacteroides, Clostridium, Filifactor, Fusobacterium, Gemella, Granulicatella, Parvimonas, Peptostreptococcus, Prevotella, Solobacterium, and Streptococcus). Clinical information, including patient demographics, comorbid medical conditions, date of bacteremia, and bacterial species identified, were collected. The incidence of biopsy-proved CRC was compared between the exposed and unexposed (patients without bacteremia matched for age, sex, and comorbidities) groups. RESULTS: The risk of CRC was increased in patients with bacteremia from B fragilis (hazard ratio [HR] = 3.85, 95% CI = 2.62-5.64, P = 5.5 × 10-12) or Streptococcus gallolyticus (HR = 5.73, 95% CI = 2.18-15.1, P = 4.1 × 10-4) compared with the unexposed group. In addition, the risk of CRC was increased in patients with bacteremia from Fusobacterium nucleatum (HR = 6.89, 95% CI = 1.70-27.9, P = .007), Peptostreptococcus species (HR = 3.06, 95% CI = 1.47-6.35, P = .003), Clostridium septicum (HR = 17.1, 95% CI = 1.82-160, P = .013), Clostridium perfringens (HR = 2.29, 95% CI = 1.16-4.52, P = .017), or Gemella morbillorum (HR = 15.2, 95% CI = 1.54-150, P = .020). We observed no increased risk in patients with bacteremia caused by microbes not previously associated with colorectal neoplasms. CONCLUSIONS: In a retrospective analysis of patients hospitalized for bacteremia, we associated later diagnosis of CRC with B fragilis and S gallolyticus and other intestinal microbes. These bacteria might have entered the bloodstream from intestinal dysbiosis and perturbed barrier function. These findings support a model in which specific members of the intestinal microbiota promote colorectal carcinogenesis. Clinicians should evaluate patients with bacteremia from these species for neoplastic lesions in the colorectum.

Zinc-finger protein 471 suppresses gastric cancer through transcriptionally repressing downstream oncogenic PLS3 and TFAP2A.

Zinc-finger protein 471 (ZNF471) was preferentially methylated in gastric cancer using promoter methylation array. The role of ZNF471 in human cancer is unclear. Here we elucidated the functional significance, molecular mechanisms and clinical impact of ZNF471 in gastric cancer. ZNF471 mRNA was silenced in 15 out of 16 gastric cancer cell lines due to promoter hypermethylation. Significantly higher ZNF471 promoter methylation was also observed in primary gastric cancers compared to their adjacent normal tissues (P < 0.001). ZNF471 promoter CpG-site hypermethylation correlated with poor survival of gastric cancer patients (n = 120, P = 0.001). Ectopic expression of ZNF471 in gastric cancer cell lines (AGS, BGC823, and MKN74) significantly suppressed cell proliferation, migration, and invasion, while it induced apoptosis in vitro and inhibited xenograft tumorigenesis in nude mice. Transcription factor AP-2 Alpha (TFAP2A) and plastin3 (PLS3) were two crucial downstream targets of ZNF471 demonstrated by bioinformatics modeling and ChIP-PCR assays. ZNF471 directly bound to the promoter of TFAP2A and PLS3 and transcriptionally inhibited their expression. TFAP2A and PLS3 showed oncogenic functions in gastric cancer cell lines. Moreover, ZNF471 recruited KAP1 to the promoter of the target genes, thereby inducing H3K9me3 enrichment for transcriptional repression and inhibition of oncogenic TFAP2A and PLS3. In conclusion, ZNF471 acts as a tumor suppressor in gastric cancer by transcriptionally inhibiting downstream targets TFAP2A and PLS3. KAP1 is a co-repressor of ZNF471 at the promoter of the target genes. The promoter CpG-site methylation is an independent prognostic factor for overall survival of gastric cancer patients.

Multi-cohort analysis of colorectal cancer metagenome identified altered bacteria across populations and universal bacterial markers.

BACKGROUND: Alterations of gut microbiota are associated with colorectal cancer (CRC) in different populations and several bacterial species were found to contribute to the tumorigenesis. The potential use of gut microbes as markers for early diagnosis has also been reported. However, cohort specific noises may distort the structure of microbial dysbiosis in CRC and lead to inconsistent results among studies. In this regard, our study targeted at exploring changes in gut microbiota that are universal across populations at species level. RESULTS: Based on the combined analysis of 526 metagenomic samples from Chinese, Austrian, American, and German and French cohorts, seven CRC-enriched bacteria (Bacteroides fragilis, Fusobacterium nucleatum, Porphyromonas asaccharolytica, Parvimonas micra, Prevotella intermedia, Alistipes finegoldii, and Thermanaerovibrio acidaminovorans) have been identified across populations. The seven enriched bacterial markers classified cases from controls with an area under the receiver-operating characteristics curve (AUC) of 0.80 across the different populations. Abundance correlation analysis demonstrated that CRC-enriched and CRC-depleted bacteria respectively formed their own mutualistic networks, in which the latter was disjointed in CRC. The CRC-enriched bacteria have been found to be correlated with lipopolysaccharide and energy biosynthetic pathways. CONCLUSIONS: Our study identified potential diagnostic bacterial markers that are robust across populations, indicating their potential universal use for non-invasive CRC diagnosis. We also elucidated the ecological networks and functional capacities of CRC-associated microbiota.

Alterations in Enteric Virome Are Associated With Colorectal Cancer and Survival Outcomes.

BACKGROUND & AIMS: Patients with colorectal cancer (CRC) have a different gut microbiome signature than individuals without CRC. Little is known about the viral component of CRC-associated microbiome. We aimed to identify and validate viral taxonomic markers of CRC that might be used in detection of the disease or predicting outcome. METHODS: We performed shotgun metagenomic analyses of viromes of fecal samples from 74 patients with CRC (cases) and 92 individuals without CRC (controls) in Hong Kong (discovery cohort). Viral sequences were classified by taxonomic alignment against an integrated microbial reference genome database. Viral markers associated with CRC were validated using fecal samples from 3 separate cohorts: 111 patients with CRC and 112 controls in Hong Kong, 46 patients with CRC and 63 controls in Austria, and 91 patients with CRC and 66 controls in France and Germany. Using abundance profiles of CRC-associated virome genera, we constructed random survival forest models to identify those associated with patient survival times. RESULTS: The diversity of the gut bacteriophage community was significantly increased in patients with CRC compared with controls. Twenty-two viral taxa discriminated cases from controls with an area under the receiver operating characteristic curve of 0.802 in the discovery cohort. The viral markers were validated in 3 cohorts, with area under the receiver operating characteristic curves of 0.763, 0.736, and 0.715, respectively. Clinical subgroup analysis showed that dysbiosis of the gut virome was associated with early- and late-stage CRC. A combination of 4 taxonomic markers associated with reduced survival of patients with CRC (log-rank test, P = 8.1 × 10-6) independently of tumor stage, lymph node metastases, or clinical parameters. We found altered interactions between bacteriophages and oral bacterial commensals in fecal samples from patients with CRC compared with controls. CONCLUSIONS: In a metagenomic analysis of fecal samples from patients and controls, we identified virome signatures associated with CRC. These data might be used to develop tools to identify individuals with CRC or predict outcomes.

Forkhead Box F2 Suppresses Gastric Cancer through a Novel FOXF2-IRF2BPL-ß-Catenin Signaling Axis.

DNA methylation has been identified as a hallmark of gastric cancer (GC). Identifying genes that are repressed by DNA promoter methylation is essential in providing insights into the molecular pathogenesis of gastric cancer. Using genome-wide methylation studies, we identified that transcription factor forkhead box F2 (FOXF2) was preferentially methylated in gastric cancer. We then investigated the functional significance and clinical implication of FOXF2 in gastric cancer. FOXF2 was silenced in gastric cancer cell lines and cancer tissues by promoter methylation, which was negatively associated with mRNA expression. Ectopic expression of FOXF2 inhibited proliferation, colony formation, G1-S cell-cycle transition, induced apoptosis of gastric cancer cell lines, and suppressed growth of xenograft tumors in nude mice; knockdown of FOXF2 elicited opposing effects. FOXF2 inhibited Wnt signaling by inducing ß-catenin protein ubiquitination and degradation independently of GSK-3ß. FOXF2 directly bound the promoter of E3 ligase interferon regulatory factor 2-binding protein-like (IRF2BPL) and induced its transcriptional expression. IRF2BPL in turn interacted with ß-catenin, increasing its ubiquitination and degradation. Multivariate Cox regression analysis identified FOXF2 hypermethylation as an independent prognostic factor of poor survival in early-stage gastric cancer patients. In conclusion, FOXF2 is a critical tumor suppressor in gastric carcinogenesis whose methylation status serves as an independent prognostic factor for gastric cancer patients.Significance: FOXF2-mediated upregulation of the E3 ligase IRF2BPL drives ubiquitylation and degradation of ß-catenin in gastric cancer, blunting Wnt signaling and suppressing carcinogenesis. Cancer Res; 78(7); 1643-56. ©2018 AACR.

Mucosal microbiome dysbiosis in gastric carcinogenesis.

OBJECTIVES: We aimed to characterise the microbial changes associated with histological stages of gastric tumourigenesis. DESIGN: We performed 16S rRNA gene analysis of gastric mucosal samples from 81 cases including superficial gastritis (SG), atrophic gastritis (AG), intestinal metaplasia (IM) and gastric cancer (GC) from Xi'an, China, to determine mucosal microbiome dysbiosis across stages of GC. We validated the results in mucosal samples of 126 cases from Inner Mongolia, China. RESULTS: We observed significant mucosa microbial dysbiosis in IM and GC subjects, with significant enrichment of 21 and depletion of 10 bacterial taxa in GC compared with SG (q<0.05). Microbial network analysis showed increasing correlation strengths among them with disease progression (p<0.001). Five GC-enriched bacterial taxa whose species identifications correspond to Peptostreptococcus stomatis, Streptococcus anginosus, Parvimonas micra, Slackia exigua and Dialister pneumosintes had significant centralities in the GC ecological network (p<0.05) and classified GC from SG with an area under the receiver-operating curve (AUC) of 0.82. Moreover, stronger interactions among gastric microbes were observed in Helicobacter pylori-negative samples compared with H. pylori-positive samples in SG and IM. The fold changes of selected bacteria, and strengths of their interactions were successfully validated in the Inner Mongolian cohort, in which the five bacterial markers distinguished GC from SG with an AUC of 0.81. CONCLUSIONS: In addition to microbial compositional changes, we identified differences in bacterial interactions across stages of gastric carcinogenesis. The significant enrichments and network centralities suggest potentially important roles of P. stomatis, D. pneumosintes, S. exigua, P. micra and S. anginosus in GC progression.

Gavage of Fecal Samples From Patients With Colorectal Cancer Promotes Intestinal Carcinogenesis in Germ-Free and Conventional Mice.

BACKGROUND & AIMS: Altered gut microbiota is implicated in development of colorectal cancer (CRC). Some intestinal bacteria have been reported to potentiate intestinal carcinogenesis by producing genotoxins, altering the immune response and intestinal microenvironment, and activating oncogenic signaling pathways. We investigated whether stool from patients with CRC could directly induce colorectal carcinogenesis in mice. METHODS: We obtained stored stool samples from participants in a metagenome study performed in Hong Kong. Conventional (male C57BL/6) mice were given azoxymethane to induce colon neoplasia after receiving a course of antibiotics in drinking water. Mice were gavaged twice weekly with stool from 5 patients with CRC or 5 healthy individuals (controls) for 5 weeks. Germ-free C57BL/6 mice were gavaged once with stool from 5 patients with CRC or 5 controls. We collected intestinal tissues from mice and performed histology, immunohistochemistry, expression microarray, quantitative polymerase chain reaction, immunoblot, and flow cytometry analyses. We performed 16S ribosomal RNA gene sequencing analysis of feces from mice. RESULTS: Significantly higher proportions of conventional mice fed with stool from individuals with CRC than control stool developed high-grade dysplasia (P < .05) and macroscopic polyps (P < .01). We observed a higher proportion of proliferating (Ki-67-positive) cells in colons of germ-free mice fed with stool from patients with CRC vs those fed with stool from controls (P < .05). Feces from germ-free and conventional mice fed with stool from patients with CRC vs controls contained different microbial compositions, with lower richness in mice fed with stool from patients with CRC. Intestines collected from conventional and germ-free mice fed with stool from patients with CRC had increased expression of cytokines that modulate inflammation, including C-X-C motif chemokine receptor 1, C-X-C motif chemokine receptor 2, interleukin 17A (IL17A), IL22, and IL23A. Intestines from conventional and germ-free mice fed with stool from patients with CRC contained higher proportions of T-helper 1 (Th1) cells (2.25% vs 0.44%) and Th17 cells (2.08% vs 0.31%) (P < .05 for each) than mice fed with stool from controls. Real-time polymerase chain reaction arrays revealed up-regulation of genes involved in cell proliferation, stemness, apoptosis, angiogenesis, invasiveness, and metastasis in mice fed with stool from patients with CRC. CONCLUSIONS: We fed stool samples from patients with CRC and heathy individuals to germ-free mice and conventional mice with azoxymethane. We found stool from patients with CRC to increase the numbers of polyps, levels of intestinal dysplasia and proliferation, markers of inflammation, and proportions of Th1 and Th17 cells in colon, compared with stool from individuals without CRC. This study provides evidence that the fecal microbiota from patients with CRC can promote tumorigenesis in germ-free mice and mice given a carcinogen.

Peptostreptococcus anaerobius Induces Intracellular Cholesterol Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice.

BACKGROUND & AIMS: Stool samples from patients with colorectal cancer (CRC) have a higher abundance of Peptostreptococcus anaerobius than stool from individuals without CRC, based on metagenome sequencing. We investigated whether P anaerobius contributes to colon tumor formation in mice and its possible mechanisms of carcinogenesis. METHODS: We performed quantitative polymerase chain reaction analyses to measure P anaerobius in 112 stool samples and 255 colon biopsies from patients with CRC or advanced adenoma and from healthy individuals (controls) undergoing colonoscopy examination at hospitals in Hong Kong and Beijing. C57BL/6 mice were given broad-spectrum antibiotics, followed by a single dose of azoxymethane, to induce colon tumor formation. Three days later, mice were given P anaerobius or Esherichia coli MG1655 (control bacteria), via gavage, for 6 weeks. Some mice were also given the nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin. Intestine tissues were collected and analyzed histologically. The colon epithelial cell line NCM460 and colon cancer cell lines HT-29 and Caco-2 were exposed to P anaerobius or control bacteria; cells were analyzed by immunoblot, proliferation, and bacterial attachment analyses and compared in gene expression profiling studies. Gene expression was knocked down in these cell lines with small interfering RNAs. RESULTS: P anaerobius was significantly enriched in stool samples from patients with CRC and in biopsies from patients with colorectal adenoma or CRC compared with controls. Mice depleted of bacteria and exposed to azoxymethane and P anaerobius had a higher incidence of intestinal dysplasia (63%) compared with mice not given the bacteria (8.3%; P < .01). P anaerobius mainly colonized the colon compared with the rest of the intestine. Colon cells exposed to P anaerobius had significantly higher levels of proliferation than control cells. We found genes that regulate cholesterol biosynthesis, Toll-like receptor (TLR) signaling, and AMP-activated protein kinase signaling to be significantly up-regulated in cells exposed to P anaerobius. Total cholesterol levels were significantly increased in colon cell lines exposed to P anaerobius via activation of sterol regulatory element-binding protein 2. P anaerobius interacted with TLR2 and TLR4 to increase intracellular levels of reactive oxidative species, which promoted cholesterol synthesis and cell proliferation. Depletion of reactive oxidative species by knockdown of TLR2 or TLR4, or incubation of cells with an antioxidant, prevented P anaerobius from inducing cholesterol biosynthesis and proliferation. CONCLUSIONS: Levels of P anaerobius are increased in human colon tumor tissues and adenomas compared with non-tumor tissues; this bacteria increases colon dysplasia in a mouse model of CRC. P anaerobius interacts with TLR2 and TLR4 on colon cells to increase levels of reactive oxidative species, which promotes cholesterol synthesis and cell proliferation.