RESUMEN
Colorectal cancer is multifaceted, with subtypes defined by genetic, histologic, and immunologic features that are potentially influenced by inflammation, mutagens, and/or microbiota. Colorectal cancers with activating mutations in BRAF are associated with distinct clinical characteristics, although the pathogenesis is not well understood. The Wnt-driven multiple intestinal neoplasia (MinApcΔ716/+) enterotoxigenic Bacteroides fragilis (ETBF) murine model is characterized by IL17-dependent, distal colon adenomas. Herein, we report that the addition of the BRAF V600E mutation to this model results in the emergence of a distinct locus of midcolon tumors. In ETBF-colonized BRAF V600E Lgr5 CreMin (BLM) mice, tumors have similarities to human BRAF V600E tumors, including histology, CpG island DNA hypermethylation, and immune signatures. In comparison to Min ETBF tumors, BLM ETBF tumors are infiltrated by CD8+ T cells, express IFNγ signatures, and are sensitive to anti-PD-L1 treatment. These results provide direct evidence for critical roles of host genetic and microbiota interactions in colorectal cancer pathogenesis and sensitivity to immunotherapy. SIGNIFICANCE: Colorectal cancers with BRAF mutations have distinct characteristics. We present evidence of specific colorectal cancer gene-microbial interactions in which colonization with toxigenic bacteria drives tumorigenesis in BRAF V600E Lgr5 CreMin mice, wherein tumors phenocopy aspects of human BRAF-mutated tumors and have a distinct IFNγ-dominant immune microenvironment uniquely responsive to immune checkpoint blockade.This article is highlighted in the In This Issue feature, p. 1601.
Asunto(s)
Bacteroides fragilis/fisiología , Neoplasias Colorrectales/microbiología , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Carcinogénesis , Transformación Celular Neoplásica , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/terapia , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , MutaciónRESUMEN
Polyamines have been implicated in numerous biological processes, including inflammation and carcinogenesis. Homeostatic regulation leads to interconversion of the polyamines putrescine and the downstream metabolites spermidine and spermine. The enzyme spermine oxidase (SMOX), which back-converts spermine to spermidine, contributes to regulation of polyamine levels, but can also have other effects. We have implicated SMOX in gastric inflammation and carcinogenesis due to infection by the pathogen Helicobacter pylori. In addition, we reported that SMOX can be upregulated in humans with inflammatory bowel disease. Herein, we utilized Smox-deficient mice to examine the role of SMOX in two murine colitis models, Citrobacter rodentium infection and dextran sulfate sodium (DSS)-induced epithelial injury. In C. rodentium-infected wild-type (WT) mice, there were marked increases in colon weight/length and histologic injury, with mucosal hyperplasia and inflammatory cell infiltration; these changes were ameliorated in Smox-/- mice. In contrast, with DSS, Smox-/- mice exhibited substantial mortality, and increased body weight loss, colon weight/length, and histologic damage. In C. rodentium-infected WT mice, there were increased colonic levels of the chemokines CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10, and the cytokines IL-6, TNF-α, CSF3, IFN-γ, and IL-17; each were downregulated in Smox-/- mice. In DSS colitis, increased levels of IL-6, CSF3, and IL-17 were further increased in Smox-/- mice. In both models, putrescine and spermidine were increased in WT mice; in Smox-/- mice, the main effect was decreased spermidine and spermidine/spermine ratio. With C. rodentium, polyamine levels correlated with histologic injury, while with DSS, spermidine was inversely correlated with injury. Our studies indicate that SMOX has immunomodulatory effects in experimental colitis via polyamine flux. Thus, SMOX contributes to the immunopathogenesis of C. rodentium infection, but is protective in DSS colitis, indicating the divergent effects of spermidine.
Asunto(s)
Colitis/etiología , Colitis/metabolismo , Inmunomodulación , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Animales , Citrobacter rodentium/fisiología , Colitis/patología , Citocinas/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Eliminación de Gen , Inmunidad Mucosa/genética , Inmunomodulación/efectos de los fármacos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Espermidina/metabolismo , Espermidina/farmacología , Espermina/metabolismo , Espermina/farmacología , Poliamino OxidasaRESUMEN
Pro-carcinogenic bacteria have the potential to initiate and/or promote colon cancer, in part via immune mechanisms that are incompletely understood. Using ApcMin mice colonized with the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) as a model of microbe-induced colon tumorigenesis, we show that the Bacteroides fragilis toxin (BFT) triggers a pro-carcinogenic, multi-step inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CECs). Although necessary, Stat3 activation in CECs is not sufficient to trigger ETBF colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1, that mediates the recruitment of CXCR2-expressing polymorphonuclear immature myeloid cells with parallel onset of ETBF-mediated distal colon tumorigenesis. Thus, BFT induces a pro-carcinogenic signaling relay from the CEC to a mucosal Th17 response that results in selective NF-κB activation in distal colon CECs, which collectively triggers myeloid-cell-dependent distal colon tumorigenesis.
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Toxinas Bacterianas/inmunología , Bacteroides fragilis/inmunología , Carcinogénesis/patología , Colon/inmunología , Neoplasias Colorrectales/etiología , Células Epiteliales/inmunología , Interleucina-17/inmunología , Metaloendopeptidasas/inmunología , Factor de Transcripción ReIA/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Toxinas Bacterianas/metabolismo , Bacteroides fragilis/patogenicidad , Línea Celular Tumoral , Colon/citología , Colon/microbiología , Neoplasias Colorrectales/microbiología , Neoplasias Colorrectales/patología , Activación Enzimática/inmunología , Femenino , Eliminación de Gen , Células HT29 , Humanos , Inflamación/inmunología , Inflamación/microbiología , Interleucina-17/genética , Masculino , Metaloendopeptidasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/inmunología , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/inmunología , Receptores de Interleucina-8B/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of Escherichia coli and Bacteroides fragilis Genes for colibactin (clbB) and Bacteroides fragilis toxin (bft), encoding secreted oncotoxins, were highly enriched in FAP patients' colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with E. coli (expressing colibactin), and enterotoxigenic B. fragilis showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria.
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Poliposis Adenomatosa del Colon/microbiología , Poliposis Adenomatosa del Colon/patología , Bacteroides fragilis/patogenicidad , Biopelículas , Carcinogénesis , Colon/microbiología , Neoplasias del Colon/microbiología , Escherichia coli/patogenicidad , Interleucina-17/análisis , Animales , Toxinas Bacterianas/genética , Bacteroides fragilis/genética , Bacteroides fragilis/aislamiento & purificación , Colon/patología , Neoplasias del Colon/patología , Daño del ADN , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Microbioma Gastrointestinal , Humanos , Mucosa Intestinal/química , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones , Péptidos/genética , Péptidos/metabolismo , Policétidos , Lesiones Precancerosas/microbiologíaRESUMEN
Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/ß-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/terapia , Quimioterapia Combinada , Neoplasias Pulmonares/terapia , Escape del Tumor/efectos de los fármacos , Animales , Presentación de Antígeno/efectos de los fármacos , Antineoplásicos/uso terapéutico , Azacitidina/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Inmunoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Ratones , Linfocitos T/inmunología , Transcriptoma , Microambiente TumoralRESUMEN
Aberrant silencing of genes by DNA methylation contributes to cancer, yet how this process is initiated remains unclear. Using a murine model of inflammation-induced tumorigenesis, we tested the hypothesis that inflammation promotes recruitment of epigenetic proteins to chromatin, initiating methylation and gene silencing in tumors. Compared with normal epithelium and noninflammation-induced tumors, inflammation-induced tumors gained DNA methylation at CpG islands, some of which are associated with putative tumor suppressor genes. Hypermethylated genes exhibited enrichment of repressive chromatin marks and reduced expression prior to tumorigenesis, at a time point coinciding with peak levels of inflammation-associated DNA damage. Loss of MutS homolog 2 (MSH2), a mismatch repair (MMR) protein, abrogated early inflammation-induced epigenetic alterations and DNA hypermethylation alterations observed in inflammation-induced tumors. These results indicate that early epigenetic alterations initiated by inflammation and MMR proteins lead to gene silencing during tumorigenesis, revealing a novel mechanism of epigenetic alterations in inflammation-driven cancer. Understanding such mechanisms will inform development of pharmacotherapies to reduce carcinogenesis. Cancer Res; 77(13); 3467-78. ©2017 AACR.
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Carcinogénesis/genética , Carcinogénesis/patología , Reparación de la Incompatibilidad de ADN , Enzimas Reparadoras del ADN/genética , Inflamación/genética , Inflamación/patología , Animales , Metilación de ADN , Epigénesis Genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Chronic inflammation and composition of the colon microbiota have been associated with colorectal cancer in humans. The human commensal enterotoxigenic Bacteroides fragilis (ETBF) is linked to both inflammatory bowel disease and colorectal cancer and, in our murine model, causes interleukin 17A (IL-17A)-dependent colon tumors. In these studies, we hypothesized that persistent colonization by ETBF is required for tumorigenesis. METHODS: We established a method for clearing ETBF in mice, using the antibiotic cefoxitin. Multiple intestinal neoplasia mice were colonized with ETBF for the experiment duration or were cleared of infection after 5 or 14 days. Gross tumors and/or microadenomas were then evaluated. In parallel, IL-17A expression was evaluated in wild-type littermates. RESULTS: Cefoxitin treatment resulted in complete and durable clearance of ETBF colonization. We observed a stepwise increase in median colon tumor numbers as the duration of ETBF colonization increased before cefoxitin treatment. ETBF eradication also significantly decreased mucosal IL-17A expression. CONCLUSIONS: The timing of ETBF clearance profoundly influences colon adenoma formation, defining a period during which the colon is susceptible to IL-17A-dependent tumorigenesis in this murine model. This model system can be used to study the microbiota-dependent and molecular mechanisms contributing to IL-17A-dependent colon tumor initiation.
Asunto(s)
Carcinogénesis/efectos de los fármacos , Cefoxitina/efectos adversos , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias del Colon/complicaciones , Neoplasias del Colon/tratamiento farmacológico , Enterotoxinas/efectos adversos , Enterotoxinas/uso terapéutico , Animales , Bacteroides fragilis/química , Colon/microbiología , Neoplasias del Colon/microbiología , Humanos , RatonesRESUMEN
It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, although the direct molecular events linking inflammation and carcinogenesis are poorly defined. In the context of gastrointestinal disease, the bacterium enterotoxigenic Bacteroides fragilis (ETBF) is a significant source of chronic inflammation and has been implicated as a risk factor for colorectal cancer. Spermine oxidase (SMO) is a polyamine catabolic enzyme that is highly inducible by inflammatory stimuli resulting in increased reactive oxygen species (ROS) and DNA damage. We now demonstrate that purified B. fragilis toxin (BFT) up-regulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent generation of ROS and induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in C57BL/6 mice is associated with increased SMO expression and treatment of mice with an inhibitor of polyamine catabolism, N(1),N(4)-bis(2,3-butandienyl)-1,4-butanediamine (MDL 72527), significantly reduces ETBF-induced chronic inflammation and proliferation. Most importantly, in the multiple intestinal neoplasia (Min) mouse model, treatment with MDL 72527 reduces ETBF-induced colon tumorigenesis by 69% (P < 0.001). The results of these studies indicate that SMO is a source of bacteria-induced ROS directly associated with tumorigenesis and could serve as a unique target for chemoprevention.