Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer

Carcinogenesis, Feb 2018

Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factor-α (TNF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2’,7’–dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.

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Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer

Received: August Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer Nicolas Granofszky 2 Michaela Lang 2 Vineeta Khare 2 Gerald Schmid 2 Theresa Scharl 1 Franziska Ferk 0 Kristine Jimenez 2 Siegfried Knasmülle 0 Christoph Campregher 2 Christoph Gasche 2 0 Idnstitute of Cancer Research, Department of Medicine I, Medical University of Vienna , Vienna , Austria 1 aA,CIB GmbH, c/o Institute of Applied Statistics and Computing, University of Natural Resources and Life Sciences , Vienna , Austria a n 2 Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna , Vienna, Austri Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O 2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O 2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factαor(T-NF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNFα, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis. Introduction Ulcerative colitis (UC) is associated with an increased risk for the colorectal cancer (CAC)1(–5). Oxidative stress is a recognized development of colorectal cancer (CRC). Epithelial cells of the driver of mutations in CRC, colitis6)( and CAC ( 7 ). UC is considinflamed mucosa accumulate genetic alterations even before ered an ‘oxyradical overload’ disease in which degranulating any histological evidence of dysplasia or colitis-associated polymorphonuclear cells (PMNs) induce certain cellular damage Materials and methods Cell lines Various established EGFP-based reporter clones20( ,21 ) were created to compare mutations of mono-,di- and tetranucleotide repeats in human CRC cells [HCT116 + chr3 2( 2 ), which has one hmutL homologue 1 (MLH1) wild-type allele transferred through chromosome 3] and primary im-mor talized human colonic epithelial cells (HCEC)-1CT23)(. HCT116  + chr3 FPG single cell clones harboring G16, A10, [CA]13 and [CA]26 repeats were established previously 2(0,24). Insertion of a single plasmid was verified HCEC by Southern blot, and sequence analysis of the EGFP region containing H2O 2, the microsatellites within stable single cell clones was performed upon IL creation of the cells2(0,24). HCEC-1CT stably transfected with EGFP-based JAK plasmids (pIREShyg2-EGFP) harboring A10, [CA]13 and [CA]26 repeats were LPS newly established for this work as performed previously for HCEC-1CTMLH1 [AAAG]17 ( 19 ). Hygromycin B was used for selection of stable transfe-ct MMR ants. Single cell clones of HCEC-1CT cells did not prolifera1t9e).(Thus, MRs stable HCEC-1CT were mixed populations. Upon resuscitation of HCECMSI 1CT and HCT116  + chr3 clones for this study, cells were characterized morphologically and expression of MMR proteins (MSH2, MSH3 and MLH1) Na2EDTA was verified ( 19,25 ). HCEC-1CT were analyzed for E-cadherin expression by Western blot, villin and cytokeratin expression by immunofluorescence PMA upon receipt from Jerry W.  Shay laboratory in 200923() before plasmid PMNs transfection, to verify epithelial origin. In addition, HCEC-1CT reporter ROS cell lines were fluorescently stained for LGR5 to verify stem cell origin. SOD HCT116  + chr3 clones were cultured in IMDM (Gibco/life technologies) TNF-α containing 4 mM L-glutamine, 10% fetal bovine serum (Biochrom, Berlin, UC Germany), 150  µg/ml hygromycin B (Gibco) and 400  µg/ml G418 (Gibco). HCEC-1CT were grown in a 4:1 mixture of DMEM (Gibco) and Medium 199 including DNA mutations 8() and thereby contribute to mut-a (Gibco) supplemented with 2% calf serum (Hyclone), 50 µg/ml gentamicin genesis ( 1,9 ). It was hypothesized that reactive oxygen species (eSpiigdmeram),a1l  µggr/omwlthhyfadcrtoocror(tBiDsoBnieos(cSiiegnmcae)s,),250  nnMg/ msoldiruemco msebliennaitnet (hGuibmcoa)n, (ROS) or reactive nitrogen species may overwhelm intracellular 2 µg/ml transferrin (Gibco), 10 µg/ml insulin (Gibco) and 300 µg/ml hyg-ro DNA repair pathways leading to microsatellite instability (MSI) mycin B.  HCEC-1CT were cultured in 75  cm2 Primaria cell culture flasks ( 10 ), a characteristic of a subset of CRC and other cancers. In fact, (BD Falcon). Cells were incubated at 37°C, 95% humidity and 5% C2.OUpon MSI, due to deficiencies in the mismatch repair (MMR) system, reconstitution, cells were not kept longer than 3  months in culture. For is detected in 15% of all CRCs; 3% of them is caused by Lynch some experiments, HCT116 + chr3-[CA]26, -G16, -A10 or HCEC-1CT-[CA]26 syndrome (formerly named hereditary non-polyposis CRC), a were treated with 0.15 nM phorbol-12-myristate-13-acetate (PMA; Sigmacommon familial cancer syndrome1( 1 ). MSI-high tumors are Aldrich, Germany), 1–50 µM hydrogen peroxide (H2O 2; Sigma), 25 ng/ml IL-6 associated with immune cell infiltrates. More general, about 20% (BMS341, eBioscience, Vienna, Austria), 25 ng/ml IL-8 (14-8089-63, eBiosc-i of all cancers arise in association with chronic inflammation, ence), 25  ng/ml TNF-α (130-094-022, Miltenyi Biotec, Bergisch Gladbach, Germany), 5 mM 5-aminosalicylic acid (5-ASA; Shire, Dublin, Ireland) and/ and most solid tumors contain inflammatory infiltrates where or 2 µM tofacitinib, a Janus kinase (JAK) inhibitor (CP-690550, Invivogen, cytokines and chemokines may promote proliferation of tumor San Diego, CA) for 24 h, washed with PBS and measured by flow cytometry cells, perturb their differentiation and support their survival after 6 to 8 days. ( 12,13 ) and thus create a pro-tumorigenic microenvironment. Also, certain cytokines, such as interleukin (IL)-6, tumor nec-ro Isolation and activation of PMNs sis factor-α (TNF-α), can be produced by PMNs and are increa-s PMNs were freshly isolated from blood of healthy donors. Blood was mixed ingly recognized in promoting tumor initiation, progression and with ½ volume ice-cold dextran solution [2% Dextran T500 (Pharmacia), metastasis 1( 4–16 ). Only recently, it has been shown that IL-6 0.9% NaCl] followed by sedimentation for 30 min. The upper phase h-ar may induce elevated microsatellite alterations at selected t-etra boring the majority of PMNs was applied on a Ficoll-Paque gradient (GE nucleotide repeats (EMAST), which is also commonly found in Healthcare) followed by centrifugation for 20 min at g40a0nd 15°C. The inflamed mucosa ( 17 ). Although in the setting of infectious colitis pellet was resuspended in 15 ml chilled 0.2% NaCl for 45 s to lyse rem-ain the role of PMNs is considered primarily beneficia9l)(, the PMN ing erythrocytes. Physiological osmolarity was recovered by addition of 15 infiltration of colonic crypts resulting in crypt abscesses in UC ml chilled 1.6% NaCl. PMNs were centrifuged for 3 min at 4g00and 15°C, washed in Ca/Mg-free Hank’s balanced salt solution (HBSS) (Gibco) and counted using a hemocytometer. PMNs were activated with 0.5 nM PMA, if not stated otherwise, in cell culture medium. leads to mucosal injury, local inflammation and DNA damage. Here, we hypothesize that certain secreted products from degranulating PMNs enhance replication errors and frameshift mutations in colonic crypts. In this study, we established a direct Cell sorting, co-culture, flow cytometry, analysis of co-culture system with activated PMNs and colon epithelial cells mutations and calculation of mutation rates to test for the induction of frameshift mutation1s8,( 19 ). The reporter system involved various pIREShyg2-enhanced green fluorescent protein (EGFP) plasmids each harboring a mono-, disome experiments PMNs were treated with superoxide dismutase (SOD; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, S7571), apocynin (Santa Cruz, sc-203321) or catalase (Sigma, C3155) Sigma, M5655) was added to the medium and cells were incubated at at indicated concentrations. After 24 h, medium was renewed. 37°C for 3 h to allow formation of formazan crystals. Cells were subjected Target cells were detached using 160  µl accutase (PAA Laboratories to a 1:1 dimethyl sulfoxide:ethanol mixture for 10 min and solubilized GmbH, Linz, Austria) and analyzed on a CellLabs Quanta flow cytometer crystals were measured at 570 nm within 1 h on a plate reader (Anthos (Beckman Coulter, Brea, CA). Flow cytometric analysis and calculation of 2010). For measuring cell proliferation, the Cyquant NF cell proliferation mutation rates (MRs) were performed as described previousl1y8( ,20 ). Data assay (Invitrogen) was performed, according to protocol. Briefly, cells were are presented as fold changes to control cells (±95% confidence interval). washed with HBSS and incubated with 50 µl of ×1 dye binding solution Also for HCEC-1CT, although mixed clones, fold changes for MRs were c-al for 1 h at 37°C. Fluorescence intensity, as a measure of cellular DNA c-on culated, assuming one plasmid insertion in each clon1e8(). tent, was determined on a Chameleon V microplate reader (Hidex, Ex/Em = 485/535 nm). Measurements were carried out in duplicates. Analysis of PMN-released superoxide Comet assay Release of superoxide (O2−) was analyzed using a lucigenin-amplified chemiluminescence assay as described previously 2( 5 ). Briefly, 7.5 × 104 The comet assay, a single cell gel electrophoresis assay, with lesion--spe freshly isolated PMNs were activated with 0.1–5 nM PMA in 500 µl HBSS cific enzyme formamidopyrimidine glycosylase (FPG) for the detection and 20 µM lucigenin.·O2− release was measured between 10 min and of oxidized DNA bases, was performed. Inorganic salts, bovine serum a-l 3 h upon activation on a tube luminometer (Lumat LB 9507, Berthold bumin fraction V, dimethyl sulfoxide, ethidium bromide, NaOH, Trizma Technologies) and was expressed as relative light units. Single measu-re base, 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulphonic acid, Triton ments were performed. X-100, trypan blue, ethylenediaminetetraacetic acid disodium salt d-ihy drate (Na2EDTA) and FPG were purchased from Sigma-Aldrich (Steinheim, Analysis of PMN-released H2O2 Germany) and Dulbecco’s PBS (Ca-Mg-free, pH 7.4) from PAA Laboratories H2O 2 was fluorometrically detected using a2OH2 Assay (Abcam) according GmbH (Pasching, Austria). Low melting point agarose and normal melting to the manufacturer’s protocol. Briefly, 7×. 51 04 or 1.5 × 105 freshly isolated point agarose were obtained from Gibco (Paisley, UK). PMNs were activated with 0.5 or 10 nM PMA for 30 min. Fifty microliter HCT116 + chr3 cells were treated with 25 µM2HO2, 50–100 ng/ml IL-6 of the cell supernatant or2HO2 standard dilutions were mixed with 50 µl or 0.5 nM PMA for 24 h and subjected to comet assay analysis26(). 1.5 × horse radish peroxidase/OxiRed probe reaction mix. After 10 min, the red- 105 HCT116 + chr3 cells were transferred to tubes containing PBS, mixed fluorescent dye was measured using a Chameleon V microplate reader with 0.5% low melting point agarose and transferred to 1% normal m-elt (Ex/Em = 485/535 nm; Hidex, Turku, Finland) and absolute amounts of2HO 2 ing point agarose coated slides. Slides were immersed in lysis solution were calculated. Measurements were carried out in duplicates. Fo2rO H2 (pH 10, 0.1 M Na2EDTA, 2.5 M NaCl, 10 mM Trizma base, prior to use, 1% release upon apocynin treatment, 7.5×  104 freshly isolated PMNs were Triton X-100 and 10% dimethyl sulfoxide were added freshly) over night at activated with 10 nM PMA and treated with 0–200 µM apocynin for 30 min. 4ºC. After lysis, the slides were washed twice with enzyme reaction buffer Measurements were carried out in quadruplicates. {0.1 M KCl, 40 mM 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulphonic acid, 0.5 mM Na2EDTA, 0.2 mg/ml bovine serum albumin, pH 8} for 8 min. Multiplex immunoassay To optimize FPG concentrations, calibration experiments with different FPG dilutions (0, 1:1000, 1:3000, 1:5000, 1:7000) were performed. The nuclei A bead-based multiplex assay (Bio-Plex; BioRad, Hercules, CA) was used were exposed to either 50 µl of the FPG enzyme solution (1:3000 dilution) taoctdiveatetciotn.P2M×N1-0r6ePleMaNsesd/mclytwoekrieneasctuipvoantePdMwAitohr 0l.i5ponpMolPyMsaAcocrh1aroirde10(LµPgS/)- fSourbs3e0qmueinntalyt, 3D7N°CA woraswaitlhlotwheedetnozyumnwe irnedacfotrio3n0bmufinferinaelloencetraofptehrorleyssiiss. lmiqluLidPSnfitorro1g6enh..TShuepbeeranda-tbaanstesd wmeurletcipollelxecatsesdayawndasi mpemrfeodrimateedlyacfrcoozredninign rbiuefdfeoru(t0.f3oMr3N0amOHina(3n0d01mmAM,1.N0aV2E/cDmTAc,oprHre>sp1o3)n.dEilnegcttroop25hoVr)eastis4w°Ca.sSclaid-res to the manufacturer’s protocol and beads were measured on a Bio-plex were neutralized (0.4 M Trizma base, pH 7.5) twice for 8 min, rinsed in 200 instrument (Biorad). A total of 22 cytokines and growth factors (ma-cro distilled water and air-dried overnight. phage inflammatory protein-β1, IL-8, vascular endothelial growth factor, For each experimental condition, three slides were prepared and from intercellular adhesion molecule-1, IL-1 receptor antagonist, IL-17, MCP- each, 50 cells were examined under a fluorescence microscope (Nikon 1, interferon-γ, IL-6, IP-10, granulocyte-macrophage colony-stimulating EFD-3, Tokyo, Japan) using 25-fold magnification. The DNA was stained factor, TNF-α, IL-1β, IL-1α, IL-12, G-CSF, IL-13, IL-2, IL-7, IL-4, IL-10, IL-5) was with ethidium bromide (20  µg/ml) and the percentage of DNA in tail analyzed. Measurements were carried out in duplicates. was analyzed with a computer-aided system (Comet Assay IV, Perceptive Analysis of intracellular ROS production Instruments, UK). Cell viability was determined with trypan blue (0.4%). DNA damage was only analyzed in samples with ≥80% viability. To detect intracellular ROS, the ′,72′-dichlorofluorescein diacetate (DCFDA), Cellular ROS Detection Assay was used, according to man-u Immunocytochemistry facturer’s protocol (Abcam). The non-fluorescent, cell-permeable DCFDA diffuses into cells, where it is deacetylated by celluar esterases to 5 × 104 HCEC-1CT A10, CA13 and CA26 reporter cell lines were seeded into 2′,7′-dichlorodihydrofluorescin. In the presence of ROS, the dichlorodi-hy IBIDI 8-well 15 µm slides and grown for 24 h. Cells were fixed in 3.7% PFA drofluorescin is oxidized to the highly fluorescent 2’,7’-DCF and further for 15 min at 4°C, permeabilized with 0.1% Triton-X100 in PBS for 12 min at detected by fluorescence spectroscopy. 5× 103 HCEC-1CT cells/96-well 4°C and blocked with 3% bovine serum albumin, 0.03% NaN3in PBS (blockwere grown for 24 h to 70–80% confluence. Cells were treated with 25 ng/ ing buffer) for 1 h at room temperature (RT). Cells were incubated with ml TNF-α, IL-6 or IL-8 for 15 min, 30 min, 1 h, 2 h or 3 h. Subsequently, LGR5 primary antibody (1:100, Origene, TA503316) for 2 h at RT and rabbit cells were washed twice with PBS, 100 µl of 25 µM DCFDA in HBSS was anti-mouse IgG Alexa Fluor 594 (1:1000, ThermoFisher) secondary an-ti added and incubated for 30 min. Cells were washed with cold PBS, body for 1 h at RT. Nuclear counterstaining was performed using Hoechst, 100 µl HBSS was added and cells were incubated for 10 min at 37°C. and cells were embedded in citifluor AF1. HCEC-1CT cells were imaged on Subsequently, fluorescent cells were measured on a Chameleon V mic-ro a IX81 microscope (Olympus). plate reader (Hidex, Ex/Em = 485/535 nm). Measurements were carried out in triplicates. Statistics Total cell numbers, 2HO 2 and O2− release, cell proliferation, metabolic-ac Cell metabolic activity and proliferation assays tivity and DNA oxidation are presented as mean ± SD and significant 1 × 103 HCT116-chr3-A10 or -[CA]26 cells/well were seeded into differences were calculated using unpaired samplest-test. Statistical 96-well plates and were grown for 24 h. Cells were treated with analyses were performed using Graphpad Prism 6.02 (Graphpad Software, 0–1000 U/ml catalase, 0–1000 U/ml SOD or 0–800 µM apocynin in San Diego, CA). MRs were calculated as shown previously20() using the triplicates for 48 h. For the metabolic activity assay, 0.5 mg/ml maximum likelihood method and the statistical computing environment R Version 3.2.5. MRs are presented as fold changes compared with control To test whether PMA can also trigger degranulation of PMNs, (±95% confidence interval) by calculating fold change = [(MR treatment − superoxide production was measured using a lucigenin-amp-li mean MR control)/mean MR controlP].< 0.05 was considered statistically fied chemiluminescence assay (28,29). PMNs were incubated significant. with up to 5  nM PMA and 20  µM lucigenin for up to 3  h. The effect levels at 1  nM PMA F(igure  1B). The superoxide release Results lasted for at least 3 h with a peak after 30 min. Treatment with 0.5  nM PMA demonstrated robust superoxide release. Thus, in Establishment of a 24-well co-culture system the subsequent co-culture experiments, PMA was used at a nonTo simulate the interactions of PMNs with crypt epithelial cells MSI-inducing concentration of 0.5 nMF(igure 1A). in UC, we had previously utilized a co-culture system with PMA- The effector (PMNs) to target (reporter cell lines) ratio was activated PMNs and colon epithelial cells separated by a se-mi further optimized using HCT116  + chr3-derived reporter cell permeable membrane in 6-well plates25(). Here, we improved lines stably transfected with an EGFP-reporter plasmid -car this system by using 24-well plates without the use of a se-mi rying G16, A10, [CA]13 or [CA]26 repeats ( 20 ). Additionally, pr-i permeable membrane, thereby reducing the amount of freshly mary human colorectal epithelial reporter cell lines (HCEC-1CT isolated PMNs about 10-fold. A  direct cell-to-cell contact also( 19,23 )) were newly established to better understand early events improved the efficiency of such a system as certain ROS have a in colitis-associated tumorigenesis. HCEC-1CT-derived reporter very limited half-life, and the reduced distance between PMNs cells were stably transfected with EGFP-reporter plasmids- car and epithelial cells enhanceisn vitro potency of released ROS. rying A10, [CA]13 and [CA]26 repeats for this set of experiments PMA is known to be a strong activator for degranulation of similarly as described for the [AAAG]17 tetranucleotide reporter PMNs but has also several other effects in cells and tissue2s7)(. cells 1( 9 ). To verify crypt stem cell origin, HCEC-1CT-derived -re To establish a non-mutagenic PMA concentration,×5  103 EGFP- porter cells were stained for the stem cell marker LGR5+, which negative HCT116  + chr3-G16 cells were treated with 0.1–5  nM was expressed in the cytoplasm and on the membrane of all-re PMA for 24  h. A  dose-dependent decrease in cell proliferation porter cells tested, a finding which was reported previously for paralleled by an increase in MRs within the G16-repeat at c-on spheroid HT29 and HCT116 cells 3(0) and primary colonic ep-i centrations above 0.5  nM PMA were observed, after 6  days of thelial cells (HCECs)3( 1 ) (Supplementary Figure  1, available at culture F(igure 1A). Carcinogenesis Online). Induction of MSI by H O 2 2 Activated PMNs are known to release H2O 2 during degranulation. Indeed, in our system, PMNs released H2O 2 in a cell number- and PMA-dependent fashion (Figure  3A). 7.5  ×  104 PMNs released about 1 nmol/ml (=1 µM) H2O 2 upon activation with 0.5 nM PMA, which is equal to approximately ×8 109 H2O 2 molecules per cell. 5  ×  103 EGFP-negative cells were sorted into 24-well plates In contrast, non-activated PMNs did not secrete2OH2 above the and after 24  h co-culturing with PMA-activated PMNs at detection limit of the assay. effector:target ratios ranging from 25:1 to 75:1 for another 24 h, In order to verify H2O 2 as a potent driver of MSI, HCT116 + washed and kept in culture for further 6 days. A dose-depen-d chr3-A10 cells were treated with 2HO 2 for 24 h, medium was ent decrease in cell count was detected in all clones from exchanged and the MR was measured after 6 days by flow both HCT116  + chr3 and HCEC-1CT cellsF(igure  2A and B, re- cytometry. A dose-dependent decrease in cell proliferation spectively). HCT116  + chr3-G16, HCT116  + chr3-A10, HCT116  + and increase in MSI were found upon treatment with 1–50 chr3-[CA]13 and HCT116 + chr3-[CA]26 clones showed a sizable µM H2O 2 (Figure 3B). One micromolar H2O 2 was sufficient to increase in frameshift mutations between 1.6- and 2.2-fold at an induce frameshift mutations. To investigate whether PMNeffector:target ratio of 75:1Fi(gure 2A). HCEC-1CT reporter cells released H2O 2 is involved in induction of mutations within were more sensitive to co-culture-induced stress regarding cell microsatellites, catalase, an antioxidant enzyme catalyzing viability and revealed a similar increase in frameshift mutations the decomposition of H2O 2 to H O and O 2, was added to the 2 up to 1.9-fold (CA26) at an effector:target ratio of 50F:1ig(ure 2B). direct co-culture system3( 2 ). PMA-activated PMNs and target For subsequent experiments, A10 and [CA]26 reporter clones cells were co-incubated with 250 U/ml catalase, a conc-en were used. To investigate whether there is a time-dependent -in tration not affecting metabolic activity or cell proliferation crease in MRs, HCT116 + chr3 [CA]26 reporter cells were treated (Supplementary Figure 3A and D, available atCarcinogenesis with activated PMNs at a ratio of 75:1 for 5, 7 or 9 days. The MR Online) or MR if added to MSI-reporter cells alone (data not after 7 days increased by 1.5-foldP( = 0.07), after 9 days to 1.9- shown). Indeed, 250 U/ml catalase counteracted PMN-induced fold (P = 0.07) compared with the MR at day 5 (Supplementary frameshift mutagenesis in HCT116 + chr3-A10, HCT116 + chr3Figure  2, available atCarcinogenesis Online). Naturally, cell [CA]26 ( Figure 3C) and HCEC-1CT-A10 (Figure 3D) MSI-reporter number increased over time. cell lines by 24%, 31% and 23%, respectively. Increasing ca-ta lase concentration beyond 250 U/ml (up to 1000 U/ml) did not further decrease MR (data not shown), suggesting that besides H2O 2, other PMN-released factors may also act as mutagenic drivers. Although MSI was reduced, no rescue on cell growth was apparent F(igure 3C and D). In summary, these data ind-i cate that H2O 2 is released by activated PMNs and may induce MSI in the setting of colonic inflammation. SOD increases PMN-induced frameshift mutations system, PMNs were pre-incubated for 10–15  min with 75  µM SOD catalyzes the dismutation of the highly reactive superoxide apocynin, activated with 0.5 nM PMA and subsequently added anion radical (O2−), also released by PMNs 3( 3 ), into oxygen (O2) to HCT116 + chr3-A10 or HCT116 + chr3-[CA]26 cells. Apocynin and H2O 2. PMNs were activated with PMA and added to HCT16 did not affect cell proliferation and metabolic activFitigyu(re 4B, + chr3-A10 concomitantly with 100–500 U SOD, concentrations Supplementary Figure  3C and F, available atCarcinogenesis not affecting metabolic activity and cell growth, as evaluatedOnline) or MR if added to HCT116  + chr3 clones alone (data by MTT and Cyquant cell proliferation assay, respectively not shown) but reduced PMN-induced MSI by 47% and 31% in (Supplementary Figure 3B and E, available aCtarcinogenesis HCT116 + chr3-A10 and HCT116 + chr3-[CA]26 cells, respectively, Online). Unexpectedly, SOD fostered PMN-induced MR F(igure demonstrating only incomplete inhibition of PMN-induced MSI 4A ). As H 2O 2 is an end product of SOD activity and H2O 2 has a by blockage of the oxidative burst. Thus, other PMN-released strong pro-mutagenic ability itself, the increase in frameshift factors, such as cytokines, may add to the induction of MSI in mutations upon SOD might be a consequence of generated the setting of chronic inflammation. H2O 2. Inhibition of oxidative burst in PMNs is insufficient for complete prevention of MSI Immune cells secrete a wide range of cytokines. Therefore, we To investigate whether the blockage of the oxidative burst and quantitatively analyzed which soluble factors are released by the subsequent release of ROS/reactive nitrogen species from PMNs. 2 × 106 freshly isolated PMNs were activated with 0.5 nM PMNs is sufficient to reduce frameshift mutations, the NADPH- PMA or 1 or 10  µg LPS. Supernatants were analyzed using a oxidase inhibitor apocynin was added to the co-culture s-ys multiplex bead-based immunoassay. tem. Apocynin is a natural plant-derived compound, known Upon stimulation of PMNs with 0.5  nM PMA, the majo-r to block the translocation of the p47phox and p67phox s-ub ity of cytokines analyzed were secreted at pico- to nanomolar unit to the cell membrane, thereby inhibiting the assembly of concentrations F(igure  5A). The highest concentrations were NADPH oxidase ( 34) and subsequent O2− production, which can found for macrophage inflammatory proteinβ-/1CCL4, reachbe finally converted to 2HO 2 by SOD. Indeed, apocynin inhibited ing at 20  ng/ml, the upper detection limit of the assay. IL-8/ H2O 2 release by PMA-activated PMNs in a dose-dependent fash- CXCL8 was expressed at 6 ng/ml, followed by 2.2 ng/ml vascular ion (Supplementary Figure 4, available aCtarcinogenesis Online) endothelial growth factor, 1.3 ng/ml intercellular adhesion-mol and an IC50 of 16 µM (± 48). To test apocynin in our co-culture ecule-1, 0.57  ng/ml IL-1 receptor antagonist, 0.13  ng/ml IL-17, Identification of cytokines released by activated PMNs 0.12 ng/ml monocyte chemotactic protein-1 MCP-1/CCL2, 64 pg/ chr3-G16 with IL-6 also induced mutations (data not shown). ml interferonγ-, 39 pg/ml IL-6, 35 pg/ml IP-10/CXCL-10, 34 pg/ This increase in frameshift mutations was accompanied ml granulocyte-macrophage colony-stimulating factor and 28 by ROS production in HCEC-1CT cells upon cytokine trea-t pg/ml TNF-α. LPS stimulation at 1 or 10  µg/ml was similar to ment (Figure  5D). Beyond frameshift mutations, IL-6 also 0.5 nM PMA (Supplementary Figure 5, available aCtarcinogenesis induced oxidative DNA damage as measured by comet assay Online). In summary, PMNs release a wide range of cytokines (Figure 5E), emphasizing the importance of JAK-STAT signa-l and growth factors in cell culture, with macrophage inf-lam ing in cancer progression. matory protein-β1, IL-8 and vascular endothelial growth factor being most abundant. JAK inhibitor attenuates IL-6-induced MSI The JAK inhibitor tofacitinib is in clinical development for a Single cytokines induce MSI in colon epithelial cells series of immune-mediated diseases including UC 3( 6 ), celiac IL-6, IL-8 and TNF-α are cytokines which are highly relevant in disease (37) and is FDA approved for rheumatoid arthritis38(). inflammatory bowel diseases. IL-6, IL-8 and TNFα- are increas- Here, we investigated whether tofacitinib affects IL-6-induced ingly expressed at sites of inflammation compared with un-in MSI. Indeed, tofacitinib abolished IL-6-induced frameshift mut-a flamed areas of the intestinal tissue. Data from mouse models tions in HCT116 + chr3-CA (26) (Figure 6A), indicating that inhi-b indicate that IL-6, IL-8 and TNFα- may contribute to CAC ition of the JAK-STAT pathway is sufficient to block frameshift (35).To test whether certain cytokines may directly induce mutagenesis. mutagenesis in dinucleotide repeats, we treated HCT116  + chr3-A10 and HCEC-1CT-[CA]26 clones with IL-8, IL-6 or TNF- 5-ASA counteracts the mutagenic effect of IL-6 α (Figure 5B and C). All three cytokines decreased cell count, Mesalamine, or 5-ASA, is a well-established drug for the tre-at with TNF-α being most effective and HCEC-1CT-[CA]26 being ment of mild-to-moderate UC with antineoplastic properties most sensitive. Treatment with IL-6, IL-8 and TNFα-resulted and known to decrease MSI bothin vitro ( 21,39 ) and in vivo (40). in a 0.6-fold, 0.2-fold and 0.7-fold increase of MSI in HCT116 + To evaluate whether 5-ASA specifically reduces MRs induced chr3-A10, respectively. In HCEC-1CT-[CA]26, TNF-α treatment by IL-6, HCT116  + chr3-[CA]26 were treated with 5  mM 5-ASA. increased frameshift mutations by 3.4-fold, IL-6 and IL-8 by Similar to tofacitinib, also 5-ASA counteracted IL-6-induced MSI 0.4- and 0.3-fold, respectively. Similar treatment of HCT116 + (Figure 6B). Tofacitinib, but not 5-ASA counteracted PMN-induced MSI To evaluate the effect of 5-ASA, tofacitinib or a combination thereof in our co-culture model, HCT116  + chr3-[CA]26 were treated with 5  mM 5-ASA and/or 2  µM tofacitinib before the addition of PMA-activated PMNs. Tofacitinib reduced micros-at ellite mutations, whereas 5-ASA failed to do soF.i(gure  6C) No synergistic or additive effects for the reduction of microsatellite mutations were found upon combinatorial treatment. Discussion Besides·O 2− and H2O 2, which are catalyzed by SOD and catalase, respectively, other factors released by activated PMNs such as MSI, specifically at dinucleotide repeats, is an early event of CAC chlorinating agents (e.g. HOCl) or reactive nitrogen species may development and can be detected in chronic inflamed mucosa lead to cell cycle arrest (without inducing MSI) and thus inhibit ( 1–5 ). Inhibition of MSI in the setting of UC may help preventing proliferation. CAC. To simulate the carcinogenic environment in the setting of PMNs do not only produce ROS but also secrete a panel of UC, we co-cultured PMNs (the effector cells) with MSI-reporter cytokines, chemokines and growth factors, independent of the colon epithelial cells (the targets). Here, we demonstrate thatoxidative burst. Certainly, the inflammatory profile of PMNs PMN-derived ROS from oxidative burst play an important role at the site of inflammation might differ fromin vitro activated in the induction of MSI in colorectal cells. In addition, PMN- PMNs, but cytokines such as IL-8 5( 2 ), IL-6 (53) and TNF-α (54) derived cytokines including IL-8, IL-6 and TNFα- also contribute were shown to be elevated in active UC and Crohn’s disease. to frameshift mutations within dinucleotide repeats. Such Treatment of HCT116 + chr3 (MLH1 proficient, MSH3 deficient) cytokines inducede novo ROS production within colonic epith-e and HCEC-1CT (MMR proficient) cells with these cytokines lial cells, and JAK inhibition reduces mutations. Therefore, both induced MSI. Shown previously, IL-6 treatment of lung and ROS scavenging and inhibition of cytokine signaling may pr-e cancer cells leads to a nucleocytoplasmic shuttling of the MMR vent cancer development in UC. protein MSH3 and low-dose, long-term IL-6 treatment causes During the oxidative burst of degranulating PMNs, a vast EMAST ( 17 ). In our assay, we demonstrate that a single hit of amount of various ROS is released. ROS can induce mutations 25  ng/ml IL-6, IL-8 or TNF-α induced frameshift mutations and genomic instability4( 1–45 ). Exposure of mammalian cells to in mono- and dinucleotide repeats. Internal ROS production hyperoxia can lead to gross rearrangements of DNA rather than upon cytokine treatment increased, as also reported by Tsengpoint mutations or small deletion4s3(). Subjecting plasmid DNA Rogenski et al., supporting the hypothesis that oxidative stress carrying monkey kidney cells to 2HO 2 induces base substitutions by a single cytokine can induce frameshift mutations17(). To and short deletions of 1–3 base pairs4( 4 ). PMN exposure to repl-i counteract ROS production, we co-treated cells with IL-6 and cating human cells carrying plasmid DNA induced deletions and the potent ROS scavenger 5-ASA (39,55,56), a standard drug base substitutions, but no 1–2 base pair deletions45(), pointing for the treatment of UC. 5-ASA reduced IL-6-induced MSI, su-p to different mutational pathways and repair kinetics induced by porting its antineoplastic activity. This was in agreement with PMNs versus H2O 2-induced lesions (44). In addition, other factors previous studies on 5-ASA and MSI in vitro ( 21,39,57 ) and in vivo secreted by PMNs might contribute to DNA damage. Previously, (40). Even more effective, the JAK inhibitor tofacitinib, succes-s we could show that exposure of HCT11M6LH1-/- to activated PMNs fully tested in three phase 3 placebo-controlled clinical trials induces a G2/M cell cycle arrest, indicative of DNA damage repair for the treatment of UC3( 6 ), abolished IL-6-induced mutations. (25). In addition, PMNs, separated by a semipermeable mem- The JAK-STAT pathway is highly affected in inflammation and brane from colon epithelial cells, increase frameshift mutations CAC, with a specific role for IL-61( 6 ). IL-6 can alter DNA methyl-a within microsatellites in such. Herein, we provide evidence that tion via SOCS3 promoter hypermethylation induced by DNMT1 direct co-culture of activated PMNs with MLH1-proficient colon and subsequent STAT3 overactivation in CAC patients5( 8,59 ). epithelial cells induces MSI in mono- and dinucleotide repeats. Constitutive STAT3 activation leads to a cytoplasmic transl-oca Mono- and dinucleotide repeats were similarly affected in tion of hMSH3, similar to treatment with IL-167(). Counteracting HCT116 + chr3MSH3−/− and HCEC-1CT, whereas tetranucleotide r-e IL-6-induced mutations with the JAK inhibitor highlights the peat harboring HCEC-1CTs were most sensitive to PMN-induced importance of the JAK-STAT signaling pathway in DNA damage stress paralleled by cell death at low effector:target ratios. Ininduction upon oxidative stress. Targeting of IL-6 or TNαF-by fact, EMAST is found in chronic inflamed tissue and correlates monoclonal antibodies might dampen mutagenesis, thereby with a heterogeneous or decreased MSH3 protein1( 7,46,47 ). reducing CAC. Clinical data on the chemopreventive properties Ulcerated tumors4(6) or tumors with CD+8T-cell infiltrates48() of any of the anti-TNF drugs are still lacking. Although 5-ASA did may present EMAST. Lynch syndrome tumors and some spo-r not prevent microsatellite mutations in our co-culture model adic MSI-H tumors show a high abundance of tumor infiltrating with activated PMNs in contrast to treatment with IL-6 only, a lymphocytes 4( 9,50 ). Hence, it is not surprising that PMNs -in combined approach of another ROS scavenger and a JAK-STAT duce mutations not only in mono- and dinucleotide repeats but inhibitor, such as tofacitinib, may further delay or prevent CRC also affect tetranucleotide repeats as a major driver of genomic development in the setting of colitis. instability. As mentioned above, H2O 2 is secreted by degranulating PMNs Supplementary material and is capable of inducing genomic instability. In our search for the major culprit factor responsible for MSI in the setting Supplementary data are available aCtarcinogenesis online. of chronic inflammation, we identified 2HO 2 to induce frameshift mutations in mononucleotide repeats in a concentration- Funding dependent manner. Blocking H2O 2 with catalase or limiting2OH2 The financial support by the Federal Ministry of Economy, production with apocynin reduced MR significantly. Similarly, Family and Youth and the National Foundation for Research, single strand breaks resulting from the oxidative burst could be Technology and Development is gratefully acknowledged. This counteracted with catalase51(). In contrast, SOD, catalyzing the study was supported in part by the Austrian Science Fund (FWF; dismutation of O2− to O2 and H2O 2, increased MR, emphasizing P 24121 to CG). the importance of H2O 2 as an inducer of MSI. Although catalase was able to reduce MSI in our co-culture system, cell growth was not rescuedFi(gure  3C). Previously, Acknowledgements we could show that combined treatment with catalase and We would like to thank Andreas Spittler and Günther Hofbauer SOD was not able to counteract PMN-induced 2G/M arrest, a-l from the Core Facility Flow Cytometry, Medical University though such treatment reversed 2HO 2-induced G2/M arrest 2( 5 ). of Vienna, for cell sorting, Margarete Focke-Tejkl (Division Allergy Research , Center of Pathophysiology, Infectiology and with homozygous hMLH1 mutation . Cancer Res. , 54 , 4308 - 4312 . Immunology , Medical University of Vienna) for providing the Bio- 23. Roig , A.I. et al. ( 2010 ) Immortalized epithelial cells derived from human Cell Biology , Division of Digestive and Liver Diseases , University Gastroenterology, 138 , 1012 - 21 . e1 . 24 . Gasche , C. et al. ( 2001 ) Oxidative stress increases frameshift mutations of Texas Southwestern Medical Center, Dallas, Texas, for prov-id in human colorectal cancer cells . 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Granofszky, Nicolas, Lang, Michaela, Khare, Vineeta, Schmid, Gerald, Scharl, Theresa, Ferk, Franziska, Jimenez, Kristine, Knasmüller, Siegfried, Campregher, Christoph, Gasche, Christoph. Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer, Carcinogenesis, 2018, 146-157, DOI: 10.1093/carcin/bgx118