Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network.

IMPORTANCE: Severe COVID-19 and post-acute sequelae often afflict patients with underlying co-morbidities. There is a pressing need for highly effective treatment, particularly in light of the emergence of SARS-CoV-2 variants. In a previous study, we demonstrated that DCLK1, a protein associated with cancer stem cells, is highly expressed in the lungs of COVID-19 patients and enhances viral production and hyperinflammatory responses. In this study, we report the pivotal role of DCLK1-regulated mechanisms in driving SARS-CoV-2 replication-transcription processes and pathogenic signaling. Notably, pharmacological inhibition of DCLK1 kinase during SARS-CoV-2 effectively impedes these processes and counteracts virus-induced alternations in global cell signaling. These findings hold significant potential for immediate application in treating COVID-19.

Methylation Array Signals are Predictive of Chronological Age Without Bisulfite Conversion.

DNA methylation data has been used to make "epigenetic clocks" which attempt to measure chronological and biological aging. These models rely on data derived from bisulfite-based measurements, which exploit a semi-selective deamination and a genomic reference to determine methylation states. Here, we demonstrate how another hallmark of aging, genomic instability, influences methylation measurements in both bisulfite sequencing and methylation arrays. We found that non-methylation factors lead to "pseudomethylation" signals that are both confounding of epigenetic clocks and uniquely age predictive. Quantifying these covariates in aging studies will be critical to building better clocks and designing appropriate studies of epigenetic aging.

Targeting Doublecortin-Like Kinase 1 (DCLK1)-Regulated SARS-CoV-2 Pathogenesis in COVID-19.

Host factors play critical roles in SARS-CoV-2 infection-associated pathology and the severity of COVID-19. In this study, we systematically analyzed the roles of SARS-CoV-2-induced host factors, doublecortin-like kinase 1 (DCLK1), and S100A9 in viral pathogenesis. In autopsied subjects with COVID-19 and pre-existing chronic liver disease, we observed high levels of DCLK1 and S100A9 expression and immunosuppressive (DCLK1+S100A9+CD206+) M2-like macrophages and N2-like neutrophils in lungs and livers. DCLK1 and S100A9 expression were rarely observed in normal controls, COVID-19-negative subjects with chronic lung disease, or COVID-19 subjects without chronic liver disease. In hospitalized patients with COVID-19, we detected 2 to 3-fold increased levels of circulating DCLK1+S100A9+ mononuclear cells that correlated with disease severity. We validated the SARS-CoV-2-dependent generation of these double-positive immune cells in coculture. SARS-CoV-2-induced DCLK1 expression correlated with the activation of ß-catenin, a known regulator of the DCLK1 promoter. Gain and loss of function studies showed that DCLK1 kinase amplified live virus production and promoted cytokine, chemokine, and growth factor secretion by peripheral blood mononuclear cells. Inhibition of DCLK1 kinase blocked pro-inflammatory caspase-1/interleukin-1ß signaling in infected cells. Treatment of SARS-CoV-2-infected cells with inhibitors of DCLK1 kinase and S100A9 normalized cytokine/chemokine profiles and attenuated DCLK1 expression and ß-catenin activation. In conclusion, we report previously unidentified roles of DCLK1 in augmenting SARS-CoV-2 viremia, inflammatory cytokine expression, and dysregulation of immune cells involved in innate immunity. DCLK1 could be a potential therapeutic target for COVID-19, especially in patients with underlying comorbid diseases associated with DCLK1 expression. IMPORTANCE High mortality in COVID-19 is associated with underlying comorbidities such as chronic liver diseases. Successful treatment of severe/critical COVID-19 remains challenging. Herein, we report a targetable host factor, DCLK1, that amplifies SARS-CoV-2 production, cytokine secretion, and inflammatory pathways via activation of ß-catenin(p65)/DCLK1/S100A9/NF-κB signaling. Furthermore, we observed in the lung, liver, and blood an increased prevalence of immune cells coexpressing DCLK1 and S100A9, a myeloid-derived proinflammatory protein. These cells were associated with increased disease severity in COVID-19 patients. Finally, we used a novel small-molecule inhibitor of DCLK1 kinase (DCLK1-IN-1) and S100A9 inhibitor (tasquinimod) to decrease virus production in vitro and normalize hyperinflammatory responses known to contribute to disease severity in COVID-19.

Cellular Carcinogenesis: Role of Polarized Macrophages in Cancer Initiation.

Inflammation is an essential hallmark of cancer. Macrophages are key innate immune effector cells in chronic inflammation, parainflammation, and inflammaging. Parainflammation is a form of subclinical inflammation associated with a persistent DNA damage response. Inflammaging represents low-grade inflammation due to the dysregulation of innate and adaptive immune responses that occur with aging. Whether induced by infection, injury, or aging, immune dysregulation and chronic macrophage polarization contributes to cancer initiation through the production of proinflammatory chemokines/cytokines and genotoxins and by modulating immune surveillance. This review presents pre-clinical and clinical evidence for polarized macrophages as endogenous cellular carcinogens in the context of chronic inflammation, parainflammation, and inflammaging. Emerging strategies for cancer prevention, including small molecule inhibitors and probiotic approaches, that target macrophage function and phenotype are also discussed.

It takes a village: microbiota, parainflammation, paligenosis and bystander effects in colorectal cancer initiation.

Sporadic colorectal cancer (CRC) is a leading cause of worldwide cancer mortality. It arises from a complex milieu of host and environmental factors, including genetic and epigenetic changes in colon epithelial cells that undergo mutation, selection, clonal expansion, and transformation. The gut microbiota has recently gained increasing recognition as an additional important factor contributing to CRC. Several gut bacteria are known to initiate CRC in animal models and have been associated with human CRC. In this Review, we discuss the factors that contribute to CRC and the role of the gut microbiota, focusing on a recently described mechanism for cancer initiation, the so-called microbiota-induced bystander effect (MIBE). In this cancer mechanism, microbiota-driven parainflammation is believed to act as a source of endogenous mutation, epigenetic change and induced pluripotency, leading to the cancerous transformation of colon epithelial cells. This theory links the gut microbiota to key risk factors and common histologic features of sporadic CRC. MIBE is analogous to the well-characterized radiation-induced bystander effect. Both phenomena drive DNA damage, chromosomal instability, stress response signaling, altered gene expression, epigenetic modification and cellular proliferation in bystander cells. Myeloid-derived cells are important effectors in both phenomena. A better understanding of the interactions between the gut microbiota and mucosal immune effector cells that generate bystander effects can potentially identify triggers for parainflammation, and gain new insights into CRC prevention.

Doublecortin-like kinase 1 promotes hepatocyte clonogenicity and oncogenic programming via non-canonical ß-catenin-dependent mechanism.

Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active ß-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3ß at Ser9. This was associated with an induction of a 48-kDa active ß-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa ß-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa ß-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active ß-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active ß-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical ß-catenin-dependent mechanism.

Risks associated with enterococci as probiotics.

Probiotics are naturally occurring microorganisms that confer health benefits by altering host commensal microbiota, modulating immunity, enhancing intestinal barrier function, or altering pain perception. Enterococci are human and animal intestinal commensals that are used as probiotics and in food production. These microorganisms, however, express many virulence traits including cytolysin, proteases, aggregation substance, capsular polysaccharide, enterococcal surface protein, biofilm formation, extracellular superoxide, intestinal translocation, and resistance to innate immunity that can lead to serious hospital-acquired infections. In addition, enterococci are facile in acquiring antibiotic resistance genes to many clinically important antibiotics encoded on a wide variety of conjugative plasmids, transposons, and bacteriophages. The pathogenicity and disease burden caused by enterococci render them poor choices as probiotics. No large, randomized, placebo-controlled clinical trials have demonstrated the safety and efficacy of any enterococcal probiotic. As a result, no enterococcal probiotic has been approved by the United States Food and Drug Administration for the treatment, cure, or amelioration of human disease. In 2007, the European Food Safety Authority concluded that enterococci do not meet the standard for "Qualified Presumption of Safety". Enterococcal strains used or proposed for use as probiotics should be carefully screened for efficacy and safety.

Impact of antibiotic treatment and host innate immune pressure on enterococcal adaptation in the human bloodstream.

Multidrug-resistant enterococcal strains emerged in the early 1980s and are now among the leading causes of drug-resistant bacterial infection worldwide. We used functional genomics to study an early bacterial outbreak in patients in a Wisconsin hospital between 1984 and 1988 that was caused by multidrug-resistant Enterococcus faecalis The goal was to determine how a clonal lineage of E. faecalis became adapted to growth and survival in the human bloodstream. Genome sequence analysis revealed a progression of increasingly fixed mutations and repeated independent occurrences of mutations in a relatively small set of genes. Repeated independent mutations suggested selection within the host during the course of infection in response to pressures such as host immunity and antibiotic treatment. We observed repeated independent mutations in a small number of loci, including a little studied polysaccharide utilization pathway and the cydABDC locus. Functional studies showed that mutating these loci rendered E. faecalis better able to withstand antibiotic pressure and innate immune defenses in the human bloodstream. We also observed a shift in mutation pattern that corresponded to the introduction of carbapenem antibiotics in 1987. This work identifies pathways that allow enterococci to survive the transition from the human gut into the bloodstream, enabling them to cause severe bacteremia associated with high mortality.

Commensal-infected macrophages induce dedifferentiation and reprogramming of epithelial cells during colorectal carcinogenesis.

The colonic microbiome contributes to the initiation of colorectal cancer through poorly characterized mechanisms. We have shown that commensal-polarized macrophages induce gene mutation, chromosomal instability, and endogenous transformation through microbiome-induced bystander effects (MIBE). In this study we show that MIBE activates Wnt/ß-catenin signaling and pluripotent transcription factors associated with dedifferentiation, reprogramming, and the development of colorectal cancer stem cells (CSCs). Exposure of murine primary colon epithelial cells (YAMC) to Enterococcus faecalis-infected macrophages increased Wnt3α expression while suppressing Wnt inhibitor factor 1 (Wif1). Wnt/ß-catenin activation was confirmed by increased active ß-catenin and Tcf4. in vivo, active ß-catenin was evident in colon biopsies from E. faecalis-colonized Il10 knockout mice compared to sham-colonized mice. This effect was mediated, in part, by 4-hydroxy-2-nonenal and tumor necrosis factor α. MIBE also activated pluripotent transcription factors c-Myc, Klf4, Oct4, and Sox2 in YAMC cells and colons from E. faecalis-colonized Il10 knockout mice. These transcription factors are associated with cellular reprogramming, dedifferentiation, and induction of colorectal CSC progenitors. In support of this was an increase in the expression of Dclk1 and CD44, two colorectal CSC markers, in YAMC cells that were exposed to MIBE. Finally, compared to normal colon biopsies and hyperplastic polyps, DCLK1 expression increased in human tubular adenomas and invasive colorectal cancers. Blocking ß-catenin/TCF4 signaling using FH535 and CTNNB1-specific small interfering RNA decreased DCLK1 expression in HCT116 human colon cancer cells. These findings provide mechanism for microbiome-induced colorectal cancer and identify new potential targets for colorectal cancer prevention.

Microbiome-driven carcinogenesis in colorectal cancer: Models and mechanisms.

Colorectal cancer (CRC) is a leading cause of cancer death and archetype for cancer as a genetic disease. However, the mechanisms for genetic change and their interactions with environmental risk factors have been difficult to unravel. New hypotheses, models, and methods are being used to investigate a complex web of risk factors that includes the intestinal microbiome. Recent research has clarified how the microbiome can generate genomic change in CRC. Several phenotypes among a small group of selected commensals have helped us better understand how mutations and chromosomal instability (CIN) are induced in CRC (e.g., toxin production, metabolite formation, radical generation, and immune modulation leading to a bystander effect). This review discusses recent hypotheses, models, and mechanisms by which the intestinal microbiome contributes to the initiation and progression of sporadic and colitis-associated forms of CRC. Overall, it appears the microbiome can initiate and/or promote CRC at all stages of tumorigenesis by acting as an inducer of DNA damage and CIN, regulating cell growth and death, generating epigenetic changes, and modulating host immune responses. Understanding how the microbiome interacts with other risk factors to define colorectal carcinogenesis will ultimately lead to more accurate risk prediction. A deeper understanding of CRC etiology will also help identify new targets for prevention and treatment and help accelerate the decline in mortality for this common cancer.

Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury.

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of VillinCre;Dclk1f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

Defective Intestinal Mucin-Type O-Glycosylation Causes Spontaneous Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Core 1- and core 3-derived mucin-type O-linked oligosaccharides (O-glycans) are major components of the colonic mucus layer. Defective forms of colonic O-glycans, such as the Thomsen-nouveau (Tn) antigen, frequently are observed in patients with ulcerative colitis and colorectal cancer, but it is not clear if they contribute to their pathogenesis. We investigated whether and how impaired O-glycosylation contributes to the development of colitis-associated colorectal cancer using mice lacking intestinal core 1- and core 3-derived O-glycans. METHODS: We generated mice that lack core 1- and core 3-derived intestinal O-glycans (DKO mice) and analyzed them, along with mice that singly lack intestinal epithelial core 1 O-glycans (IEC C1galt1(-/-) mice) or core 3 O-glycans (C3Gnt(-/-) mice). Intestinal tissues were collected at different time points and analyzed for levels of mucin and Tn antigen, development of colitis, and tumor formation using imaging, quantitative polymerase chain reaction, immunoblot, and enzyme-linked immunosorbent assay techniques. We also used cellular and genetic approaches, as well as intestinal microbiota depletion, to identify inflammatory mediators and pathways that contribute to disease in DKO and wild-type littermates (controls). RESULTS: Intestinal tissues from DKO mice contained higher levels of Tn antigen and had more severe spontaneous chronic colitis than tissues from IEC C1galt1(-/-) mice, whereas spontaneous colitis was absent in C3GnT(-/-) and control mice. IEC C1galt1(-/-) mice and DKO mice developed spontaneous colorectal tumors, although the onset of tumors in the DKO mice occurred earlier (age, 8-9 months) than that in IEC C1galt1(-/-) mice (15 months old). Antibiotic depletion of the microbiota did not cause loss of Tn antigen but did reduce the development of colitis and cancer formation in DKO mice. Colon tissues from DKO mice, but not control mice, contained active forms of caspase 1 and increased caspase 11, which were reduced after antibiotic administration. Supernatants from colon tissues of DKO mice contained increased levels of interleukin-1ß and interleukin-18, compared with those from control mice. Disruption of the caspase 1 and caspase 11 genes in DKO mice (DKO/Casp1/11(-/-) mice) decreased the development of colitis and cancer, characterized by reduced colonic thickening, hyperplasia, inflammatory infiltrate, and tumors compared with DKO mice. CONCLUSIONS: Impaired expression of O-glycans causes colonic mucus barrier breach and subsequent microbiota-mediated activation of caspase 1-dependent inflammasomes in colonic epithelial cells of mice. These processes could contribute to colitis-associated colon cancer in humans.

Inflammatory and oncogenic roles of a tumor stem cell marker doublecortin-like kinase (DCLK1) in virus-induced chronic liver diseases.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. We previously showed that a tumor/cancer stem cell (CSC) marker, doublecortin-like kinase (DCLK1) positively regulates hepatitis C virus (HCV) replication, and promotes tumor growth in colon and pancreas. Here, we employed transcriptome analysis, RNA interference, tumor xenografts, patient's liver tissues and hepatospheroids to investigate DCLK1-regulated inflammation and tumorigenesis in the liver. Our studies unveiled novel DCLK1-controlled feed-forward signaling cascades involving calprotectin subunit S100A9 and NFκB activation as a driver of inflammation. Validation of transcriptome data suggests that DCLK1 co-expression with HCV induces BRM/SMARCA2 of SW1/SNF1 chromatin remodeling complexes. Frequently observed lymphoid aggregates including hepatic epithelial and stromal cells of internodular septa extensively express DCLK1 and S100A9. The DCLK1 overexpression also correlates with increased levels of S100A9, c-Myc, and BRM levels in HCV/HBV-positive patients with cirrhosis and HCC. DCLK1 silencing inhibits S100A9 expression and hepatoma cell migration. Normal human hepatocytes (NHH)-derived spheroids exhibit CSC properties. These results provide new insights into the molecular mechanism of the hepatitis B/C-virus induced liver inflammation and tumorigenesis via DCLK1-controlled networks. Thus, DCLK1 appears to be a novel therapeutic target for the treatment of inflammatory diseases and HCC.

Colorectal cancer: role of commensal bacteria and bystander effects.

For years the human microbiota has been implicated in the etiology of colorectal cancer (CRC). However, identifying the molecular mechanisms for how aneuploidy and chromosomal instability (CIN) arise in sporadic and colitis-associated CRC has been difficult. In this Addendum we review recent work from our laboratory that explore mechanisms by which intestinal commensals polarize colon macrophages to an M1 phenotype to generate a bystander effect (BSE) that leads to mutations, spindle malfunction, cell cycle arrest, tetraploidy, and aneuploidy in epithelial cells. BSE represents the application of a phenomenon initially described in the radiation biology field. The result of commensal-driven BSE on colon epithelial cells is aneuploidy, chromosomal instability (CIN), expression of stem cell and tumor stem cell markers and, ultimately, malignant transformation. Our findings provide a conceptual framework for integrating the microbiota with aging, cyclooxygenase (COX)-2, and inflammation as risk factors for CRC.

Commensal bacteria drive endogenous transformation and tumour stem cell marker expression through a bystander effect.

OBJECTIVE: Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect. DESIGN: Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)-an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis. RESULTS: Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice. CONCLUSIONS: These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect.

Fluvastatin interferes with hepatitis C virus replication via microtubule bundling and a doublecortin-like kinase-mediated mechanism.

Hepatitis C virus (HCV)-induced alterations in lipid metabolism and cellular protein expression contribute to viral pathogenesis. The mechanism of pleiotropic actions of cholesterol-lowering drugs, statins, against HCV and multiple cancers are not well understood. We investigated effects of fluvastatin (FLV) on microtubule-associated and cancer stem cell marker (CSC), doublecortin-like kinase 1 (DCLK1) during HCV-induced hepatocarcinogenesis. HCV replication models, cancer cell lines and normal human hepatocytes were used to investigate the antiviral and antitumor effects of statins. FLV treatment resulted in induction of microtubule bundling, cell-cycle arrest and alterations in cellular DCLK1 distribution in HCV-expressing hepatoma cells. These events adversely affected the survival of liver-derived tumor cells without affecting normal human hepatocytes. FLV downregulated HCV replication in cell culture where the ATP pool and cell viability were not compromised. Pravastatin did not exhibit these effects on HCV replication, microtubules and cancer cells. The levels of miR-122 that regulates liver homeostasis and provides HCV genomic stability remained at steady state whereas DCLK1 mRNA levels were considerably reduced during FLV treatment. We further demonstrated that HCV replication was increased with DCLK1 overexpression. In conclusion, unique effects of FLV on microtubules and their binding partner DCLK1 are likely to contribute to its anti-HCV and antitumor activities in addition to its known inhibitory effects on 3-hydroxy-3-methylglutary-CoA reductase (HMGCR).

Colon Macrophages Polarized by Commensal Bacteria Cause Colitis and Cancer through the Bystander Effect.

Intestinal commensal bacteria have recently been shown to trigger macrophages to produce diffusible clastogens (or chromosome-breaking factors) through a bystander effect (BSE) that mediates DNA damage and induces chromosomal instability in neighboring cells. Colon macrophages appear central to colon carcinogenesis and BSE through the expression of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). The former induces netrin-1, a regulator of intestinal epithelial cell apoptosis, and the latter generates trans-4-hydroxy-2-nonenal (4-HNE), an endogenous mutagen. To test whether colon macrophages are key effectors for BSE, we depleted these cells in interleukin-10 knockout mice colonized with Enterococcus faecalis using encapsulated liposomal clodronate (ELC), a bisphosphonate that causes macrophage apoptosis. We observed that E. faecalis polarizes colon macrophages to an M1 phenotype. In addition, depleting these cells suppressed COX-2 and TNF-α, blocked the formation of 4-HNE protein adducts, and inhibited up-regulation of netrin-1-all markers for BSE. Finally, treatment with ELC prevented colitis, ß-catenin activation, and cancer formation. These results show that selected human commensals can polarize colon macrophages to the M1 phenotype and, when activated, serve as the key effector for bacterial-induced BSE. Our findings suggest that depleting M1-polarized macro-phages is a mechanism for the chemopreventive activity of bisphosphonates and that it represents a new strategy for preventing colon cancer induced by intestinal commensals.

Incidence of potentially human papillomavirus-related neoplasms in the United States, 1978 to 2007.

BACKGROUND: Population-based studies comprehensively describing incidence patterns of human papillomavirus (HPV)-related preinvasive and invasive neoplasms prior to widespread HPV vaccination are sparse. METHODS: Age-adjusted incidence rates (IRs), IR ratios (IRRs), and annual percent changes (APCs) in IRs were calculated for potentially HPV-related tumors diagnosed in the Surveillance, Epidemiology and End Results (SEER) Program during 1978 through 2007. RESULTS: Overall IRs for preinvasive tumors were significantly higher than for invasive squamous cell tumors of cervix (IRR = 3.42), vulva (IRR = 1.87), and vagina (IRR = 1.19) and significantly lower for adenomatous cervical tumors (IRR = 0.43), and squamous cell tumors of penis (IRR = 0.64), anus (males, IRR = 0.53; females, IRR = 0.14), and head and neck (H&N) (males, IRR = 0.01; females, IRR = 0.02). Incidence of preinvasive squamous tumors of cervix, vagina, and penis rose rapidly over time and decreased for invasive neoplasms. The most rapid increases occurred for preinvasive (males, APC = 16.0; females, APC = 7.3) and invasive anal tumors (males, APC = 3.6; females, APC = 2.3). IR patterns were generally similar among evaluable racial/ethnic groups, with the exception of H&N invasive tumor IRs which increased exclusively among white males. CONCLUSIONS: Contrary to the opposing trends of preinvasive and invasive squamous tumors of cervix, vagina, and penis, preinvasive and invasive anal tumor IRs increased significantly over time by sex, age, and racial/ethnic groups. Successful HPV vaccination programs are needed to measurably reduce incidence of HPV-related neoplasms in the future, particularly for cancer sites with rising incidence rates for which effective screening modalities are limited. Cancer 2013;119:2291-2299. © 2013 American Cancer Society.

Cyclooxygenase-2 generates the endogenous mutagen trans-4-hydroxy-2-nonenal in Enterococcus faecalis-infected macrophages.

Infection of macrophages by the human intestinal commensal Enterococcus faecalis generates DNA damage and chromosomal instability in mammalian cells through bystander effects. These effects are characterized by clastogenesis and damage to mitotic spindles in target cells and are mediated, in part, by trans-4-hydroxy-2-nonenal (4-HNE). In this study, we investigated the role of COX and lipoxygenase (LOX) in producing this reactive aldehyde using E. faecalis-infected macrophages and interleukin (IL)-10-knockout mice colonized with this commensal. 4-HNE production by E. faecalis-infected macrophages was significantly reduced by COX and LOX inhibitors. The infection of macrophages led to decreased Cox1 and Alox5 expression whereas COX-2 and 4-HNE increased. Silencing Alox5 and Cox1 with gene-specific siRNAs had no effect on 4-HNE production. In contrast, silencing Cox2 significantly decreased 4-HNE production by E. faecalis-infected macrophages. Depleting intracellular glutathione increased 4-HNE production by these cells. Next, to confirm COX-2 as a source for 4-HNE, we assayed the products generated by recombinant human COX-2 and found 4-HNE in a concentration-dependent manner using arachidonic acid as a substrate. Finally, tissue macrophages in colon biopsies from IL-10-knockout mice colonized with E. faecalis were positive for COX-2 by immunohistochemical staining. This was associated with increased staining for 4-HNE protein adducts in surrounding stroma. These data show that E. faecalis, a human intestinal commensal, can trigger macrophages to produce 4-HNE through COX-2. Importantly, it reinforces the concept of COX-2 as a procarcinogenic enzyme capable of damaging DNA in target cells through bystander effects that contribute to colorectal carcinogenesis.

TNF-α mediates macrophage-induced bystander effects through Netrin-1.

Macrophage-induced bystander effects have been implicated as an important mediator of chromosomal instability and colon cancer triggered by Enterococcus faecalis, a human intestinal commensal bacteria. There is little understanding about how inflammatory cytokines mediate bystander effects, but questions in this area are important because of the pivotal contributions made by inflammatory processes to cancer initiation and progression. Here, we report that the central proinflammatory cytokine TNF-α acts as a diffusible mediator of the bystander effects induced by macrophages, an effect caused by a proliferation of macrophages that trigger epithelial cell production of Netrin-1, a neuronal guidance molecule. TNF-α-mediated bystander assays used a murine coculture system of primary colonic epithelial cells and E. faecalis-infected macrophages (in vitro), with an interleukin 10 (IL-10)-deficient mouse model of colon cancer that involves long-term colonization with E. faecalis (in vivo). In cell cocultures, we observed increased expression of the TNF-α receptor Tnfrsf1b and Netrin-1. These effects were blocked by anti-TNF-α antibody or by pretreatment with an inhibitor of NF-κB signaling. RNAi-mediated attenuation of Tnfrsf1b decreased TNF-α-induced netrin-1 production and augmented epithelial cell apoptosis in culture. Extending these observations, colon biopsies from E. faecalis-colonized IL-10(-/-) mice exhibited crypt hyperplasia and increased staining for macrophages, TNF-α, netrin-1, NF-κB, Tnfrsf1b, and the proliferation marker proliferating cell nuclear antigen while also displaying a reduction in epithelial cell apoptosis. Together, our results define a pathway for macrophage-induced bystander effects in which TNF-α triggers TNFRSF1b receptor signaling leading to increased production of Netrin-1, crypt hyperplasia, and decreased epithelial cell apoptosis. In elucidating an important commensal-associated proinflammatory mechanism in the intestinal microenvironment, our work highlights the role of Netrin-1 and a specific TNF-α receptor as candidate targets to prevent or treat colorectal cancer.