Christina E. DeStefano Shields

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis.

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, N1,N4-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.

Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria

Biofilms provide refuge for cancerous bacteria Familial adenomatous polyposis (FAP) causes benign polyps along the colon. If left untreated, FAP leads to a high incidence of colon cancer. To understand how polyps influence tumor formation, Dejea et al. examined the colonic mucosa of FAP patients. They discovered biofilms containing the carcinogenic versions of the bacterial species Escherichia coli and Bacteroides fragilis. Colon tissue from FAP patients exhibited greater expression of two bacterial genes that produce secreted oncotoxins. Studies in mice showed that specific bacteria could work together to induce colon inflammation and tumor formation. Science, this issue p. 592 Bacterial biofilms are linked to colon cancer. 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.

Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention.

Polyamines, including spermine, spermidine, and the precursor diamine, putrescine, are naturally occurring polycationic alkylamines that are required for eukaryotic cell growth, differentiation, and survival. This absolute requirement for polyamines and the need to maintain intracellular levels within specific ranges require a highly regulated metabolic pathway primed for rapid changes in response to cellular growth signals, environmental changes, and stress. Although the polyamine metabolic pathway is strictly regulated in normal cells, dysregulation of polyamine metabolism is a frequent event in cancer. Recent studies suggest that the polyamine catabolic pathway may be involved in the etiology of some epithelial cancers. The catabolism of spermine to spermidine utilizes either the one-step enzymatic reaction of spermine oxidase (SMO) or the two-step process of spermidine/spermine N1-acetyltransferase (SSAT) coupled with the peroxisomal enzyme N1-acetylpolyamine oxidase. Both catabolic pathways produce hydrogen peroxide and a reactive aldehyde that are capable of damaging DNA and other critical cellular components. The catabolic pathway also depletes the intracellular concentrations of spermidine and spermine, which are free radical scavengers. Consequently, the polyamine catabolic pathway in general and specifically SMO and SSAT provide exciting new targets for chemoprevention and/or chemotherapy.

Reduction of Murine Colon Tumorigenesis Driven by Enterotoxigenic Bacteroides fragilis Using Cefoxitin Treatment

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.

Mismatch repair proteins initiate epigenetic alterations during inflammation-driven tumorigenesis

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.

Distinct Immunomodulatory Effects of Spermine Oxidase in Colitis Induced by Epithelial Injury or Infection

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.

Abstract LB-098: Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC

Combination epigenetic treatment as a therapeutic approach holds much promise. However, to unlock the true potential of these combinatorial paradigms, further optimization is required. We have elucidated in our current study, a treatment schedule combining Azacitidine with Histone Deacetylase Inhibition (HDACi) which considers both schedule and isoform specificity. We have deployed a sequential schedule with HDACi administered post Azacitidine in a chronic low dose manner. This schedule takes advantage of two key parameters altered by Azacitidine: dsRNA induced immune signature and MYC depletion. In our system, HDACis amplify an existing immune signature and the MYC depleted environment acts as a potent sensitizer. HDACi induced augmentation of immune gene transcription seems driven predominantly through the inhibition of HDAC1/2 and HDAC6 isoforms, with broad spectrum HDACi inhibitors demonstrating the greatest potency. While the combination treatment induced perturbation of proliferation is driven by inhibition of HDAC1/2/3 isoforms. Applying this concept to a genetically engineered mouse model of NSCLC we observe a potent anti-tumor response as evidenced by a reduction in tumor burden, progression and proliferation. These phenotypic parameters are correlated with modification of both immune and proliferative gene signatures as well as alteration of key immune populations in the tumor microenvironment. We propose that this combinatorial alteration of both immune and proliferative parameters holds the promise to be a more robust treatment for NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Michael Topper, Michelle Vaz, Katherine Chiappinelli, Christina DeStefano Shields, Alyssa Wenzel, Cynthia Zahnow, Pam Strissel, Reiner Strick, Stephen Baylin. Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-098. doi:10.1158/1538-7445.AM2017-LB-098

Abstract A56: Induction of spermine oxidase by enterotoxigenic Bacteroides fragilis contributes to tumorigenesis in a model of colitis-associated colorectal cancer

It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, though the molecular events linking inflammation and the necessary carcinogenic mutations remain to be clarified. In the context of gastrointestinal disease, the bacterial pathogens Helicobacter pylori and enterotoxigenic Bacteroides fragilis (ETBF) are significant sources of chronic inflammation and have been implicated as risk factors for the development of gastric and colorectal cancer, respectively. We have previously demonstrated that H. pylori and the cytokine tumor necrosis factor-α (TNF-α) rapidly induce spermine oxidase (SMO), resulting in increased cellular reactive oxygen species (ROS) and DNA damage, in gastric and lung epithelial cells. In addition, tissue microarray studies revealed that increased SMO expression was associated with prostatic intraepithelial neoplasia and prostate cancer lesions and that benign prostate epithelial tissue from men with these diseases exhibits higher SMO expression than normal controls. We now demonstrate that purified B. fragilis toxin (BFT) upregulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in wild-type or APC+/− Min C57Bl/6 mice is associated with increased SMO expression. Inhibition of SMO activity by MDL 72,527 in vivo (20 mg/kg i.p. 3 days per week) significantly reduces ETBF-induced chronic inflammation, proliferation, and induction of Th17-associated cytokines. Most importantly, in Min mice, treatment with the SMO inhibitor reduces ETBF-induced colon tumorigenesis by 69% (p Citation Information: Cancer Prev Res 2010;3(12 Suppl):A56.