Identifying Phased Mutations and Complex Rearrangements in Human Prostate Cancer Cell Lines through Linked-Read Whole-Genome Sequencing.

A limited number of cell lines have fueled the majority of preclinical prostate cancer research, but their genomes remain incompletely characterized. Here, we utilized whole-genome linked-read sequencing for comprehensive characterization of phased mutations and rearrangements in the most commonly used cell lines in prostate cancer research including PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b, RWPE-1, and four derivative castrate-resistant (CR) cell lines LNCaP_Abl, LNCaP_C42b, VCaP-CR, and LAPC4-CR. Phasing of mutations allowed determination of "gene-level haplotype" to assess whether genes harbored heterozygous mutations in one or both alleles. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy. In addition, comparison of parental and derivative CR lines revealed previously known and novel genomic alterations associated with the CR phenotype. IMPLICATIONS: This study therefore comprehensively characterized phased genomic alterations in the commonly used prostate cancer cell lines, providing a useful resource for future prostate cancer research.

Design and Structure-Activity Relationships of Isothiocyanates as Potent and Selective N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibitors.

N-Acylethanolamines are signaling lipid molecules implicated in pathophysiological conditions associated with inflammation and pain. N-Acylethanolamine acid amidase (NAAA) favorably hydrolyzes lipid palmitoylethanolamide, which plays a key role in the regulation of inflammatory and pain processes. The synthesis and structure-activity relationship studies encompassing the isothiocyanate pharmacophore have produced potent low nanomolar inhibitors for hNAAA, while exhibiting high selectivity (>100-fold) against other serine hydrolases and cysteine peptidases. We have followed a target-based structure-activity relationship approach, supported by computational methods and known cocrystals of hNAAA. We have identified systemically active inhibitors with good plasma stability (t1/2 > 2 h) and microsomal stability (t1/2 ∼ 15-30 min) as pharmacological tools to investigate the role of NAAA in inflammation, pain, and drug addiction.

Bacterial-Driven Inflammation and Mutant BRAF Expression Combine to Promote Murine Colon Tumorigenesis That Is Sensitive to Immune Checkpoint Therapy.

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.

Cytokine signature of inflammation mediated by autoreactive Th-cells, in calf muscle of claudicating patients with Fontaine stage II peripheral artery disease.

Peripheral artery disease (PAD), a severe atherosclerotic condition primarily of the elderly, afflicts 200 million individuals, worldwide, and is associated with lower extremity myopathy. Circulating markers of inflammation have been linked to risk and severity of PAD but the contribution of local inflammation to myopathy remains unknown. We evaluated, by ELISA, calf muscle of PAD patients (N = 23) and control subjects (N = 18) for local expression of inflammatory cytokines including Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF), Interleukin 17A (IL-17A), Interferon ϒ (IFN-ϒ), tumor necrosis factor α (TNF-α), and Interleukin 6 (IL-6). One or more of these cytokines were expressed in nineteen patients and 2 controls and coordinated expression of GM-CSF, IL-17A, IFN-ϒ, and TNF-α, a signature of activated, MHC Class II dependent autoreactive Th-cells, was unique to 11 patients. GM-CSF is the central driver of tissue-damaging myeloid macrophages. Patients with this cytokine signature had a shorter (P= 0.017) Claudication Onset Distance (17 m) compared with patients lacking the signature (102 m). Transforming Growth Factor ß1 (TGFß1) and Chemokine Ligand 5 (CCL5) were expressed coordinately in all PAD and control muscles, independently of GM-CSF, IL-17A, IFN-ϒ, TNF-α, or IL-6. TGFß1 and CCL5 and their gene transcripts were increased in PAD muscle, consistent with increased age-associated inflammation in these patients. Serum cytokines were not informative of muscle cytokine expression. We have identified a cytokine profile of autoimmune inflammation in calf muscles of a significant proportion of claudicating PAD patients, in association with decreased limb function, and a second independent profile consistent with increased "inflammaging" in all PAD patients.

Ablation of polyamine catabolic enzymes provokes Purkinje cell damage, neuroinflammation, and severe ataxia.

BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.

Abnormal Microvascular Architecture, Fibrosis, and Pericyte Characteristics in the Calf Muscle of Peripheral Artery Disease Patients with Claudication and Critical Limb Ischemia.

Work from our laboratory documents pathological events, including myofiber oxidative damage and degeneration, myofibrosis, micro-vessel (diameter = 50-150 µm) remodeling, and collagenous investment of terminal micro-vessels (diameter ≤ 15 µm) in the calf muscle of patients with Peripheral Artery Disease (PAD). In this study, we evaluate the hypothesis that the vascular pathology associated with the legs of PAD patients encompasses pathologic changes to the smallest micro-vessels in calf muscle. Biopsies were collected from the calf muscle of control subjects and patients with Fontaine Stage II and Stage IV PAD. Slide specimens were evaluated by Quantitative Multi-Spectral and Fluorescence Microscopy. Inter-myofiber collagen, stained with Masson Trichrome (MT), was increased in Stage II patients, and more substantially in Stage IV patients in association with collagenous thickening of terminal micro-vessel walls. Evaluation of the Basement Membrane (BM) of these vessels reveals increased thickness in Stage II patients, and increased thickness, diameter, and Collagen I deposition in Stage IV patients. Coverage of these micro-vessels with pericytes, key contributors to fibrosis and BM remodeling, was increased in Stage II patients, and was greatest in Stage IV patients. Vascular pathology of the legs of PAD patients extends beyond atherosclerotic main inflow arteries and affects the entire vascular tree-including the smallest micro-vessels.

Biochemical and Proteomic Characterization of Recombinant Human α/ß Hydrolase Domain 6.

Human alpha/beta hydrolase domain 6 (hABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol (2-AG), a potent agonist at both cannabinoid CB1 and CB2 receptors. In vivo modulation of ABHD6 expression has been shown to have potential therapeutic applications, making the enzyme a promising drug target. However, the lack of structural information on hABHD6 limits the discovery and design of selective inhibitors. We have performed E. coli expression, purification and activity profiling screening of different hABHD6 constructs and identified a truncated variant without N-terminal transmembrane (TM) domain, hΔ29-3-ABHD6, as the most promising protein for further characterization. The elimination of the TM domain did not affect 2-AG or fluorogenic arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE) substrates hydrolysis, suggesting that the TM is not essential for enzyme catalytic activity. The hΔ29-3-ABHD6 variant was purified in a single step using Immobilized Metal Affinity Chromatography (IMAC), in-solution trypsin digested, and proteomically characterized by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The N-terminal peptide without methionine was identified indicating on a post-translational modification of the recombinant protein. The mechanism of inhibition of hABHD6 with AM6701 and WWL70 covalent probes was elucidated based on MS analysis of trypsin digested hABHD6 following the Ligand Assisted Protein Structure (LAPS) approach. We identified the carbamylated peptides containing catalytic serine (Ser148) suggesting a selective carbamylation of the enzyme by AM6701 or WWL70 and confirming an essential role of this residue in catalysis. The ability to produce substantial quantities of functional, pure hABHD6 will aid in the downstream structural characterization, and development of potent, selective inhibitors.

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.

Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria.

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.

Bacteroides fragilis Toxin Coordinates a Pro-carcinogenic Inflammatory Cascade via Targeting of Colonic Epithelial Cells.

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.

Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer.

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.

Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury.

Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX) increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI). Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H2O2 and aminopropanal, significantly diminished the severity of cisplatin AKI. In vitro studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and enhances the expression of binding immunoglobulin protein BiP/GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153). The increased expression of these endoplasmic reticulum stress response (ERSR) markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP) and apoptosis (e.g. reduced activated caspase-3). These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI.

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.

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.

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 N (1)-acetyltransferase (SSAT) coupled with the peroxisomal enzyme N (1)-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.

Effect of elastic taping on postural control deficits in subjects with healthy ankles, copers, and individuals with functional ankle instability.

BACKGROUND: Ankle sprains are the most common injury among physically active people, with common sequelae including repeated episodes of giving way, termed functional ankle instability. Copers are a cohort in ankle research comprised of those who have sprained their ankle but have not suffered any further dysfunction. The use of an elastic tape, Kinesio Tape, in sports medicine practice has recently gained popularity and may help improve postural control deficits related to functional ankle instability. The purpose of this study was to examine the immediate and prolonged effects of Kinesio Taping on postural control in healthy, coper, and unstable ankles as measured through single-limb balance on a force plate. METHODS: Sixty physically active, college-aged participants (72.5 ± 9.7 cm, 74.2 ± 16.2 kg, 21.5 ± 2.6 years) were stratified into healthy, coper, or unstable groups using the Cumberland Ankle Instability Tool (CAIT) combined with their history of ankle injury. Dependent variables included time-to-boundary (TTB) measures and traditional center of pressure (COP) measures in both the mediolateral (frontal) and anteroposterior (sagittal) planes. Testing was performed prior to tape application, immediately after application of the tape, 24 hours following tape application, and immediately after tape removal. RESULTS: Significant differences between groups were observed for COP standard deviation and range in the sagittal plane. Significant differences between tape conditions for TTB absolute minima and standard deviation were also noted. Post hoc testing revealed large to medium effect sizes for the group differences and very small effect sizes for the differences between conditions. CONCLUSIONS: Our study did not reveal decisively relevant changes following application of Kinesio Tape to the ankle. However, we did observe sagittal plane postural control deficits in subjects with ankle instability measured through summary COP variables over 20-second trials. CLINICAL RELEVANCE: Ankle instability is a concern for many clinicians. Kinesio Taping, although a popular form of clinical intervention, remains understudied. Evidence from this study does not support the use of Kinesio Taping for improving postural control deficits in those with ankle instability.

Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands.

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

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, 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.