Overt Increase of Oxidative Stress and DNA Damage in Murine and Human Colitis and Colitis-Associated Neoplasia

Author Manuscript Published OnlineFirst on January 29, 2018; DOI: 10.1158/1541-7786.MCR-17-0451 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Overt Increase of Oxidative Stress and DNA Damage in Murine and Human Colitis and Colitis-associated Neoplasia

Adrian Frick1, †, Vineeta Khare1, †, Gregor Paul1, Michaela Lang1, Franziska Ferk2, Siegfried Knasmüller2, Andrea Beer3, Georg Oberhuber4 and Christoph Gasche1,*

1 Department of Internal Medicine III, Division of Gastroenterology and Hepatology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria

2 Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria

3 Clinical Institute of Pathology, Medical University of Vienna, Vienna Austria

4 Pathologie Soleiman, A.ö. Landeskrankenhaus-Universitäts-Kliniken Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria

† These authors contributed equally to this work.

* Correspondence: Christoph Gasche, M.D., Div. of Gastroenterology and Hepatology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria

Phone: +43 40400 47640 Fax: +43 40400 47350 Email: [email protected]

Running Title: Oxidative stress and colon carcinogenesis

Keywords: IL-10, Ulcerative Colitis, Colitis-associated cancer, Oxidative stress, DNA repair, Double-strand breaks

Financial Support: This study was supported by the Federal Ministry of Economy,

Family and Youth and the National Foundation for Research, Technology and

Development and in part by the Austrian Science Fund (P27681 to CG)

Disclosures: none

Word count: abstract 225, manuscript 4939, references 58; figures 4

Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 29, 2018; DOI: 10.1158/1541-7786.MCR-17-0451 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Frick, A. et al. Oxidative stress and colon carcinogenesis

Abstract

Patients with inflammatory bowel disease (IBD) have a higher risk of developing colitis-associated-cancer (CAC), however, the underlying processes of disease progression are not completely understood. Here, the molecular processes of inflammation-driven colon carcinogenesis were investigated using IL-10 deficient mice (IL-10 KO). IL-10 KO mice were euthanized after development of colitis and dysplasia. Immunohistochemistry (IHC) was performed for markers of colitis-induced

DNA damage (CIDD): oxidative-DNA-lesions (8-oxoG), double-strand breaks (DSB;

γH2AX) and DSB-repair. MSI, LOH (Trp53, Apc) and global methylation (CIMP) were assessed on microdissected tissue. Comet-assay for DNA damage, immunofluorescence, and immunoblotting were performed on intestinal organoids from WT and IL-10 KO mice. Sequential biopsies and surgical specimens from IBD and CAC patients were used for IHC analysis. Severity of inflammation correlated with number of dysplasia. 8-oxoG and γH2AX positive cells were significantly increased in inflamed and dysplastic areas along with activation of DSB repair. The amount of positively stained cells strongly correlated with degree of inflammation (8- oxoG: R=0.923; γH2AX: R=0.858). Neither CIMP, MSI nor LOH was observed.

Enhanced DSBs in IL-10 KO organoids were confirmed by comet-assay and increased expression of γH2AX. Human clinical specimens exhibited significantly higher γH2AX and 8-oxoG in IBD, dysplasia and CAC compared to normal mucosa.

These data indicate that inflammation-driven colon carcinogenesis in IL-10 KO mice and IBD patients is associated with oxidative DNA damage and overt presence of

DSB.

Frick, A. et al. Oxidative stress and colon carcinogenesis

Introduction

Inflammatory bowel disease (IBD), a chronic inflammation of the gut, is linked to a high risk of developing colitis-associated cancer (CAC). The molecular mechanisms involved in colitis-associated carcinogenesis are incompletely understood.

Development of CAC shows mechanistic differences to sporadic colorectal cancer

(CRC) despite shared mechanisms such as chromosomal instability, microsatellite instability and CpG-island hypermethylation (CIN, MSI and CIMP, respectively).(1) In contrast to the adenoma-carcinoma sequence of sporadic CRC, CAC lesions are often multifocal throughout the colonic mucosa and progression is associated with certain mutations, chromosomal aberrations (such as change in number) and clonal expansion of those.(2, 3)

Chronic inflammation is considered to drive carcinogenesis by oxidative stress.(4-6)

The upregulated production of reactive oxygen and nitrogen species (ROS and RON, respectively) cause cellular damage to lipids, proteins and nucleic acids due to an outrun of scavenging potential. As a consequence, nucleotides can be oxidized (such as guanine to 8-oxoG) resulting in base transversion (GC-TA), abasic sites and DNA single- and double-strand breaks.(7-12) For cellular integrity, DNA double-strand breaks (DSB) can be fatal and their repair is initiated by the cellular DNA damage response (DDR) consisting of a multi-protein signalling cascade.(13, 14) An early event in DSB response is phosphorylation of the H2A subtype histone H2AX at ser-

139 (also known as γH2AX) by the phosphatidylinositol 3-like kinases ATM, ATR and

DNA-PK.(15, 16) Phosphorylation of H2AX spreads from the site of a DSB into both directions, resulting in initiation and maintenance of DDR. Hence, γH2AX is a

Frick, A. et al. Oxidative stress and colon carcinogenesis

common marker for DSB formation.(17, 18) Through indirect as well as direct interactions with proteins like BRCA1 and MRE11 with its BRCT domain, γH2AX recruits several DDR proteins at DSBs and activates DNA repair either by homologous recombination (HR) or non-homologous end joining (NHEJ) (19). NHEJ is a repair process in which the two DNA ends are re-ligated without the need for a homologue, a pathway that puts DNA integrity above repair fidelity potentially leading to significant DNA alterations such as mismatches, deletions and even CIN.(20, 21)

In contrast, HR utilizes the sister-chromatid as a template strand to reinstate a correct

DNA sequence.

IL-10 KO mice have been extensively used as a model for IBD and also CAC. These mice develop spontaneous enterocolitis and concomitant colitis-associated neoplasia.(22) Interestingly, the mechanism underlying the inflammation-driven carcinogenesis remains unclear. In a previous study neither MSI or CIN nor k-RAS, p-53 or APC gene mutations were identified in tumours from IL-10 KO mice.(23) Our aim was to challenge the molecular pathway of colitis-associated carcinogenesis in

IL-10 KO mice. Here we identified colitis-induced DNA damage (CIDD) and induction of error-prone NHEJ as drivers of dysplasia in IL-10 KO mice, independent of MSI and CIMP. Human samples exhibited a similar CIDD with disease progression from

IBD to LGD, HGD and CAC.

Material and Methods

Animal Experiments

C57BL/6 IL-10 KO mice were a kind gift from Dr. Terrence Barrett (University of

Kentucky, Lexington, USA) and bred under specific pathogen free (SPF) conditions.

For experiments, 98 mice were conventionally housed at the Biomedical Research

Frick, A. et al. Oxidative stress and colon carcinogenesis

Facility at the Medical University of Vienna. Animals were kept for 27 weeks, a group of 10 animals was euthanized one week after transfer to non-SPF and used as inflammation-free control samples. After one week of conventional housing, 43 mice received piroxicam (Sigma-Aldrich) in their chow (C1000, Altromin, Germany) at a concentration of 200 ppm for 7 days. Body weight and health status were checked at least once a week. Animals were euthanized in case of significant weight loss (>20% within one week) or symptoms of severe inflammation like bloody diarrhoea and rectal prolapse. Animal studies were performed under the Austrian Animal

Experiments Regulation and approved by the animal experiment committee in accordance with the institutional good scientific practice guidelines (BMWF-

66.009/0099_II/3b/2011).

Histological Analysis and Inflammation Score

Intestines were dissected, flushed with PBS and coiled up as Swiss rolls followed by

10% formalin treatment as previously described.(24) Haematoxylin and eosin (H&E) staining was performed on paraffin-embedded sections from mouse intestinal tissue.(25) Independent histological analysis was carried out by two scientists, one of which was a pathologist, scoring the number of tumours, the number of high grade dysplasias (HGD) and the grade of inflammation.(26) Human samples were selected from endoscopic biopsies or surgical specimens of patients with IBD and CAC.

Written informed consent was obtained from patients. The study was approved by the local ethics committee (EC number: 1354/2012) and complying with international and institutional guidelines for good scientific practice.

Immunohistochemistry

Frick, A. et al. Oxidative stress and colon carcinogenesis

The human samples were obtained from the Clinical Institute of Pathology at Medical

University of Vienna. Immunohistochemistry of formalin fixed paraffin-embedded slides was performed as described previously(27) with primary antibodies against 8- oxoG, γH2AX, MRE11, KU70, BRCA1, Nrf2 and p53 (Supplementary Table 1, supplementary methods). Briefly, slides were deparaffinised with 2 cycles of xylol and concomitant rehydration with descendent alcohol series. Heat-induced antigen retrieval was carried out either with citrate buffer (pH 6) or with Tris EDTA (pH 9) for nuclear proteins. Normal goat serum buffer was used as blocking solution, followed by optional M.O.M. (Vector Labs) blocking and subsequent primary antibody incubation (4°C overnight). Staining was performed using biotinylated antibody,

Avidin-Biotin-HRP-Complex (Vectastain, PK-6100) and diaminobenzidine. Slides were counterstained with haematoxylin (Merck 5174).

Laser Capture Microdissection

Tissue sections mounted on PEN (polyethylene naphthalate) slides (Leica

Microsystems®, Wetzlar, Germany) were dissected using the LMD6000 laser microdissection microscope (Leica Microsystems®) to obtain samples from non- inflamed, inflamed and dysplastic intestinal epithelium. DNA was extracted from the microdissected samples using the QIAamp DNA FFPE tissue kit (Qiagen, Venlo,

Netherlands) according to the manufacturer’s instructions.

Loss of Heterozygosity (LOH) and MSI-Analysis

LOH was examined for Trp53 (chromosome 11; 42,8cM) and Apc (chromosome 18;

18,5cM) with 3 loci-aligning dinucleotide PCR amplifications, which were selected from the ABI prism mouse mapping primer list (supplementary methods). For Trp53

Frick, A. et al. Oxidative stress and colon carcinogenesis

the primer pairs were specific for segments D11mit86 (25.1cM), D11mit4 (36.1cM) and D11mit285 (49.2cM). Apc markers were specific for segments D18mit12 (9.8cM),

D18mit177 (14.2cM) and D18mit91 (19.7cM). For amplification the Multiplex PCR Kit

(Qiagen®, Venlo, Netherlands) was used. All primers were labelled with fluorescent dye (FAM, NED, VIC, supplementary table 2). The PCR reaction consisted of 7.5 µL master mix, 1.5 µL Solution Q, 0.5 µL per primer (6 primers per reaction) and a DNA amount of 5-10 ng. For MSI analysis reagents were combined prior to analysis:

8.6 µL formamide, 0.4 µL GeneScan 500 LIZ size standard (Applied Biosystems) and

0.4 µL PCR product followed by a denaturation step at 95°C and immediately cooled on ice. Carried out under following PCR conditions: 15 min at 95°C, 35 cycles at

94°C for 30 s; 60°C for 30 s and 72°C for 30 s; followed by 72°C for 10 min. Analysis was done by comparing non-inflamed, inflamed and dysplastic tissue using

GeneMapper Software 4.0. To additionally analyse the microsatellite status 4 dinucleotide and 2 polyA repeat markers were used (supplementary table 3) and handled and analysed as previously described.(28) All experiments were carried out on a Genetic Analyzer 3500 (Applied Biosystems).

Methylation Analysis

To determine the global DNA methylation status, DNA from microdissected tissue was converted using EpiMark® Bisulfite Conversion Kit (New England Biolabs) according to the manufacturer’s protocol (supplementary methods).

TUNEL Staining

To assess the number of apoptotic cells in mouse tissue samples TUNEL staining

(TdT-mediated dUTP-biotin nick end labelling) was performed according to

Frick, A. et al. Oxidative stress and colon carcinogenesis

manufacturer’s protocol, using the In Situ Cell Death Detection kit (Roche®). See supplementary methods.

Isolation of Small Intestinal Organoids and Western Blotting

Small intestinal and large bowel organoids (SIOs, LBOs, respectively) were prepared from IL-10 KO and wild type (WT) mice.(29) Organoids were cultured in matrigel with a passage every 3-5 days in a 1:2 ratio. Treatment of organoids was carried out with

TNFα (10 ng/mL, eBioscience BMS301), IL-6 (10 ng/mL, eBioscience BMS341), recombinant murine IL-10 (rmIL-10, 10 ng/mL, Preprotech, 210-10), rmIL-22

(10ng/mL, eBioscience 14-8221-63) and sulforaphane (10µM, Sigma Aldrich S4441) for 24 h or with H2O2 (100 µM) for 1 h. For Western blotting organoids were washed and re-suspended in 1 mL PBS and transferred into a precooled Eppendorf tube followed by centrifugation at 400 g for 5 min at 4 °C. Supernatant was discarded and the washing step repeated. The cell pellet was lysed in RIPA buffer (on ice for 10 min) followed by a short sonication and subsequent cooling on ice for 30 min. Using

Bradford assay, protein concentration was measured and 20 µg of protein was used for SDS-PAGE. Immunoblotting was performed on a PVDF membrane. The protein bands were visualized with IRDye coupled anti-rabbit or anti-mouse antibodies (either or both mouse/rabbit; LI-COR) and scanned on Odyssey imager (LI-COR

Biotechnology).

Immunofluorescence Microscopy

For immunofluorescence (IF) SIOs and LBOs were fixed in 4% paraformaldehyde at room temperature for 30 min and blocked in horse serum with 0.1% Triton X-100 for

1h. Samples were incubated with primary antibody in 1% BSA/PBS overnight at 4°C, then washed and incubated with secondary antibody (Alexa Fluor® 488) for 1 h at

Frick, A. et al. Oxidative stress and colon carcinogenesis

room temperature. Then samples were washed, dried and mounted with DAPI containing medium (Vector Laboratories), and imaged on an Olympus BX51 microscope. Evaluation of γH2AX staining was carried out using ImageJ

(supplementary methods)

Alkaline Single Cell Gel Electrophoresis Assay (Comet-Assay)

The experiments were carried out according to the international guidelines.(30) To detect formation of oxidative stress, additional experiments with a lesion-specific enzyme (formamidopyrimidine DNA glycosylase; FPG) were conducted as described

(supplementary methods).(31)

Statistics

Means of normal distributed data were compared using unpaired Student’s t-test; non-parametric Mann-Whitney U test was used not normally distributed data. Tumour incidence and inflammation analysis were tested with Pearson’s Chi Square test.

Correlation between grade of inflammation and expression of certain markers was assessed by Spearman’s rank correlation. Two-sided analysis of variance (ANOVA) with Dunnet post-hoc analysis was used to compare all subgroups together. If p- values were equal or less 0.05 they were considered as statistically significant

(*p≤0.05; **p<0.01; ***p<0.001). GraphPad Prism 6.01 software (GraphPad Software,

La Jolla California, USA) was used to analyse all data.

Results

Inflammation and Tumour Incidence in IL-10 KO Mice

IL-10 KO mice were used as model to investigate the mechanism underlying inflammation-driven carcinogenesis. To induce a high degree of inflammation and

Frick, A. et al. Oxidative stress and colon carcinogenesis

synchronize inflammation onset (and secondary tumorigenesis), half of the mice were treated with piroxicam for 1 week.(26, 32) Distal inflammation and dysplasia were assessed by colonoscopy after 5 month, at which point piroxicam fed animals exhibited more signs of inflammation and higher number of dysplastic or hyperplastic lesions compared to untreated IL-10 KO mice (Figure 1A). Animals were euthanized after 27 weeks and intestines were prepared for further analysis. Histological inflammation scoring revealed an average inflammation grade of 2.86±0.63

(95%CI=2.66-3.06) with a prevalence of 100% for the piroxicam fed mice (n=43), and

1.8±0.81 (95%CI=1.55-2.04) with a prevalence of 84.1% for the group without piroxicam (n=45, p<0.001). Tumour counting showed a total number of 13 high-grade dysplastic lesions (HGD) and 4 invasive carcinomas were found in untreated IL-10

KO. Piroxicam fed IL-10 KO mice had 25 HGD (p<0.05) and 6 invasive carcinomas

(Figure 1B, Supplementary figure 1A, Supplementary Table 4). The severity of inflammation correlated to the number of dysplastic lesions (R=0.728, p<0.01; Figure

1C) in piroxicam-treated mice. Due to the low number of affected mice in the untreated group, all following experiments were carried out with samples from piroxicam-treated mice only.

Absence of MSI, LOH and CIMP in IL-10 KO Mice

To examine the molecular pathways leading to colorectal carcinogenesis in IL-KO mice, we analysed MSI, LOH and CIMP in DNA from microdissected samples (non- inflamed, inflamed and dysplastic). Fragment analysis was performed for MSI and

LOH analysis using certain markers. Three markers, each aligning the Trp53 or Apc gene, were investigated for LOH, whereas 6 markers were additionally utilized for

MSI analysis.(25) Of all analysed dysplastic samples, LOH could not be detected at any marker when comparing dysplastic or inflamed tissue to non-inflamed mucosa

Frick, A. et al. Oxidative stress and colon carcinogenesis

samples. No peak loss was observed and calculated peak-ratios revealed no change.

Fragment analysis also revealed no change at any of the six microsatellites in any of the dysplastic or inflamed samples compared to non-inflamed tissue

(Supplementary figure 2A, B). These data indicate that neither LOH at Trp53 or

Apc nor MSI is involved in colorectal tumour development in this CAC model.

To assess the CIMP, the murine B1 element was analysed using primers specific for the methylated or unmethylated sequence. Tumour-DNA from dysplasia bearing mice was bisulfite converted and compared to converted non-inflamed tissue samples as control. Quantification of the target sequences revealed no significant change in the levels of methylated or unmethylated CIMPs(Supplementary figure 2C), indicating that global methylation was not affected as well.

CIDD Increases with Mucosal Inflammation and Neoplastic Transformation

Resulting in DSBs and Associated DDR

As the common molecular pathways of sporadic colorectal carcinogenesis were not affected we further tested for the presence of oxidative stress and DNA damage response. The formation of DNA lesions, such as 8-oxoG, due to generation of ROS and NOS, is considered as a marker of oxidative stress. We examined dysplastic, inflamed and non-inflamed samples for 8-oxoG lesions and assessed the ratio of positive stained cells/per crypt at 400x magnification (Figure 2, left panel). A significant correlation was detected between the inflammation grade and the presence of 8-oxoG positive cells (R=0.923; p<0.01), indicative for a mucosal increase in oxidative DNA damage with the degree of intestinal inflammation. This effect was also observed with disease progression as there was a significant increase in 8-oxoG positive cells in dysplastic tissue (Figure 2, right panel).

Frick, A. et al. Oxidative stress and colon carcinogenesis

Oxidation of DNA bases can lead to single- and DSBs due to creation of abasic or complex DNA damage sites by several 8-oxoGs in close proximity.(7, 33) To assess the level of DSBs in colitis and dysplasia we stained for phosphorylated histone

H2AX, yH2AX, an early event in DSB response. Dysplastic, inflamed and non- inflamed samples were evaluated according to the ratio of positive nuclei per crypt at

400x magnification. The amount of positively stained nuclei was significantly increased in inflamed (p<0.001) and dysplastic tissue (p<0.001) compared to non- inflamed tissue. The increased staining was predominant in basal crypt region and in the intermediate zone (Figure 2, left panel). Additionally, the increase in DSBs positively correlated to the degree of inflammation (R=0.858, p<0.001). (Figure 2, right panel)

If not repaired, DSBs are hazardous for cell survival. In fact, phosphorylation of H2AX initiates DNA damage response and DSB repair. To assess the exact DDR mechanism, samples were stained for MRE11, representing the MRN-complex

(MRE11-RAD50-NBS1) that induces the DSB response. Furthermore, it interacts with

DSB repair proteins such as KU70 (which together with KU80 binds to DSB ends and is required for NHEJ and BRCA1 (involved in HR), which were further analysed.

Dysplastic samples were stained and compared to inflamed and non-inflamed samples. An increase of positive stained nuclei in inflamed (p<0.01) and dysplastic tissue compared to non-inflamed for each MRE11, BRCA1 and KU70 (p<0.001) was observed indicating an upregulation of DDR for both NHEJ and HR (Figure 2, left panel). Also, a positive correlation existed between grade of inflammation and rate of positive nuclei (MRE11: R=0.785, p<0.01; BRCA1: R=0.921, p<0.01, KU70:

R=0.851, p<0.01; Figure 2, right panel). This data support the notion that in contrast to MSI or CIN, DSBs are the major oxidative DNA lesion within the mucosal

Frick, A. et al. Oxidative stress and colon carcinogenesis

proliferative zone with induction of both HR and NHEJ which likely affects DNA fidelity in intestinal stem cells.

As a consequence of DSBs, damaged cells can either survive (after DNA repair) or undergo apoptosis. To test whether the lesions are associated with apoptosis,

TUNEL-staining was performed. We did not detect an increase in apoptotic cells in inflamed and dysplastic crypts (Supplementary figure 3) indicating that the DSBs did not trigger apoptosis.

Intestinal IL-10 KO Organoids Exhibit Increased DSBs

To validate the in vivo findings observed in IL-10 KO mice, an ex vivo model of intestinal organoids from WT and IL-10 KO mice was used. To examine whether

DNA damage could be induced in organoids they were treated with either pro- inflammatory cytokines TNF-α and IL-6 (for 24h) or with oxidative stress inducer hydrogen peroxide (H2O2, for 1h). γH2AX was increased upon treatment with TNF-α,

IL-6 or H2O2. When compared to WT, phosphorylated H2AX was higher in untreated

IL-10 KO organoids corroborating the observations in IL-10 KO mice (Figure 3A). An increase in spontaneous DSBs upon IL-10 deficiency was further confirmed by comet assay and modified FPG- comet assay (which detects oxidative DNA damage by strand break induction at oxidized DNA bases) of these SIOs (Figure 3B).

Immunofluorescence confirmed higher basal levels of nuclear γH2AX in IL-10 KO organoids (Figure 3C) again implying ongoing DSBs in IL-10 KO organoids compared to WT. Treatment with H2O2 further increased DSBs, while IL-10 treatment reverted the amount of DSBs in IL-10 KO organoids compared to untreated, presumably due to a reduction in cellular levels of ROS. IL-22 treatment showed no effect on γH2AX (Figure 3C, D). One of the main cellular sensors of oxidative stress is Nrf2, which coordinates the expression of antioxidant proteins.(34, 35) In fact, 13

Frick, A. et al. Oxidative stress and colon carcinogenesis

aberrant cytoplasmic accumulation of Nrf2 was observed in IL-10 KO organoids compared to WT (Supplementary figure 4A). Activating Nrf2 using sulforaphane

(SF) did not change the amount of γH2AX found in LBOs (supplementary figure

4B). We further investigated the level of STAT3 phosphorylation after treatment with

SF, IL-10 or IL-22. Activation of Nrf2 with SF showed no profound effect on STAT3 phosphorylation between WT and IL-10 KO LBOs. IL-22 treatment resulted in the strongest STAT3 phosphorylation in both WT and IL-10 KO organoids

(supplementary figure 4C). These data suggest increased basal levels of DSBs in

IL-10 KO organoids, likely due to an excessive presence of ROS and reduced scavenging potential indicated by cytoplasmic accumulation of Nrf2, a condition which can be attenuated by IL-10 treatment.

γH2AX as Marker of CIDD in Human IBD

To evaluate the presence of CIDD in IBD and CAC we also examined γH2AX in colonic tissue sections from biopsies and surgical specimens of patients with normal mucosa, IBD, low-grade dysplasia (LGD), HGD, CAC, CAC-adjacent tissue, CRC and familial adenomatous polyposis samples (FAP) (Figure 4A). 8-oxoG staining was used to evaluate levels of oxidative stress in normal, IBD and CAC samples. Of all 52 analysed samples, sequential specimen from six patients were available

(Figure 4B, upper panel and coloured bars). They all exhibited a sequential increase in γH2AX from LGD or HGD to CAC. Biopsies identified as LGD already show distinct γH2AX positive areas, with a threshold above 20% positive cells. This is also true for IBD surveillance biopsies of patients, who later developed a LGD or

HGD. The CAC-adjacent tissue exhibited specific crypts, solely positive for γH2AX, potentially marking crypts indefinite for dysplasia. (Figure 4A). The highest level of

γH2AX was observed in LGD, HGD and CAC samples (p<0.001) as well as in CRC

Frick, A. et al. Oxidative stress and colon carcinogenesis

(p<0.01) (Figure 4C). Interestingly, the staining for p53 revealed a similar pattern as seen in HGD and CAC-γH2AX staining with nuclear accumulation of p53 (Figure

4B/D). Quantifying the marker for oxidative stress revealed already overt presence of

8-oxoG in IBD samples, with almost 70% positive nuclei (p<0.001) and also in CAC

(p<0.001) (Figure 4E). These findings show overt CIDD, marked by 8-oxoG and

γH2AX, and its involvement in the sequence from IBD to LGD to HGD to CAC.

Discussion

Patients with ulcerative colitis are at risk for developing colorectal cancer. The molecular events of inflammation-driven carcinogenesis are incompletely understood and differ from sporadic colorectal cancer as well as familial cancer syndromes.

Although oxidative DNA damage is considered the driving force of mutagenesis in this setting, the actual molecular DNA lesion and the associated DNA repair pathway have not been investigated. Here we studied these molecular events in IL-10- deficient mice, a well-established model of CAC. We confirm that MSI, LOH and

CIMP were not observed in these mice (23). Instead oxidative DNA lesions (i.e. 8- oxoG) were found in the crypts of IL-10 KO mice and were associated with upregulation of DSB response and repair proteins (like γH2AX, MRE11, BRCA1 and

KU70). Ex-vivo, IL-10 KO organoids displayed spontaneous DSBs. Human sections also exhibited an increase in 8-oxoG in IBD and CAC. γH2AX was induced in LGD,

HGD, tumour adjacent and CAC tissue. Therefore we believe that DSBs as the core molecular lesion in CIDD and inflammation-driven carcinogenesis. Activation of the

DSB-repair pathway promotes cell survival and potential accumulation of mutations leading to CAC.

Frick, A. et al. Oxidative stress and colon carcinogenesis

Both, RONs and ROS contribute to DNA damage.(6, 36, 37) In fact, 8-oxoG increased with the grade of inflammation and was present in dysplastic lesions of IL-

10 KO mice.(38, 39) Similar γH2AX and DSB repair proteins increased with the grade of inflammation and dysplasia without inducing apoptosis. Induction of DSBs were previously reported for UC, gastric cancer and in ATM-deficient mice upon dextran sodium sulphate.(40-43) Compared to HR, NHEJ is considered error prone because of the direct ligation of broken DNA ends without a homologous template. NHEJ is active in all cell cycle states and appears to be more effective than HR, yet results in more DNA deletions and translocations.(44) Our data suggests that besides HR also

NHEJ is active and a possible mutational driver in this setting. In the absence of alteration in other examined molecular mechanisms (MSI, LOH, CIMP), this seems to be a predominant pathway of carcinogenesis in IL-10 KO mice. Abundance of CIDD together with error prone repair might predispose to accumulation and expansion of pre-mutagenic lesion. Additionally, crypt cells under inflammatory stress have higher proliferation rates and are therefore under constant replication stress, which is another cause of increased DSBs.(8, 45)

In the ex-vivo model, IL-10-deficient organoids displayed spontaneous DSBs without the presence of inflammatory cells or cytokines, a condition which could be reverted by addition of recombinant IL-10. In parallel, Nrf2 was found mainly in its aberrant cytoplasmic location. As Nrf2 elicits its ROS scavenging function as a transcription factor, cytoplasmic accumulation indicates an impaired activity, thereby showing a diminished potential to reduce ROS in IL-10 KO organoids. Remarkably, Nrf2 was shown to facilitate HR and its activation protects from DNA. On the contrary, inhibition of Nrf2 activity led to increase in γH2AX and reduced DSB-repair.(46, 47)

Using sulforaphane as an Nrf2 activator we could not detect a change in DSB levels

Frick, A. et al. Oxidative stress and colon carcinogenesis

in IL-10 KO organoids. Such an effect has been postulated for skin after UV radiation or in human lymphocytes after contact with DNA damaging substances. (48, 49) The abrogated Nrf2 signalling in IL-10 KO mice possibly enhances intracellular ROS,

DNA damage and NHEJ. The overt presence of ROS is likely further sustained as foci of γH2AX were shown to trigger and maintain pro-inflammatory cytokine secretion.(50) . Our experiments showed no overt phosphorylation of STAT3 or a difference in its phosphorylation levels between WT- and IL-10 KO organoids, after

Nrf2 activation or cytokine treatment. The effect of IL-10 itself might be attributable to its ability to regulate oxidative homeostasis. (51) Nevertheless, further research is needed to elucidate potential interaction and crosstalk between Nrf2 and the IL-10-

STAT3 signalling cascade. IL-10 itself is known to regulate oxidative homeostasis.(51)

Our mouse and ex-vivo findings were verified in human tissue section. The longitudinal analysis of IBD samples revealed a similar pattern of CIDD as observed in IL-10 KO mice. An increase in DSBs was seen in LGD, HGD and CAC samples compared to normal biopsies. Samples from IBD patients showed at least 20%

γH2AX positive nuclei (per crypt), if they developed LGD in later stages. We also examined p53 expression, considered as one of the first-hit targets in the sequela of

IBD to CAC and having a critical role in DDR.(52, 53) The areas with nuclei positive for p53 were mostly seen in HGD and CAC samples and were associated with

γH2AX positive areas. Although a normal p53 function cannot be ruled out, given the fact that these are already progressed neoplasias, it is likely that the observed nuclear accumulation of p53 indicates a non-functional mutated protein.(54, 55)

Hence, this creates a condition of cellular damage and survival resulting in carcinogenesis.

Frick, A. et al. Oxidative stress and colon carcinogenesis

Here, we propose γH2AX as a marker for CIDD and disease progression for surveillance endoscopy in IBD. Evaluating H2AX activation was already tested in various malignancies such as gastric, lung- and cervical cancer and it is discussed as a potential marker for cancer progression as well as for radiation- and chemotherapy response.(45, 56-58) In IBD, the risk of developing CAC is heterogeneous and relates to duration, extent and severity of disease. γH2AX-staining of biopsies from surveillance endoscopy may help separating those individuals at risk from those without. More studies are needed before we can use this biomarker for adjusting surveillance intervals or selecting patients who benefit most from chemoprevention or surgery.

Acknowledgement

This study was supported by the Federal Ministry of Economy, Family and Youth and the National Foundation for Research, Technology and Development and in part by the Austrian Science Fund (P27681 to CG)

IL-10 KO mice were gratefully provided by Dr. Terrence A. Barrett and Jeffrey B.

Brown, Northwestern University, Feinberg School of Medicine, Chicago, Illinois.

Author contributions: Study concept and design: VK, AF, GP, CG. Drafting of manuscript: AF, VK. Data acquisition and interpretation: AF, VK, GP, ML, FF, GO.

Critical revision of the manuscript: VK, CG. Statistical analysis: AF, ML. Obtained funding: CG. Technical or material support: SK, AB.

Frick, A. et al. Oxidative stress and colon carcinogenesis

FIGURE LEGENDS

Figure 1: Inflammation and disease progression in IL-10 KO mice.

A: Results of colonoscopy indicating percentage of dysplastic and inflamed lesions in investigated mice (IL-10 KO: n=20, IL-10 KO piroxicam n=9). Exemplary colonoscopy pictures showing non-inflamed, inflamed and dysplastic areas of the murine large bowel B: Percentage of mice with histologically determined high-grade dysplastic lesions and invasive carcinomas. C: Piroxicam fed mice exhibit higher grades of inflammation (t-test, two-tailed, ***p<0.001) compared to IL-10 KO mice fed with normal chow. The number of dysplastic lesions was positively correlated to the grade of inflammation (Spearman-Rho, R= 0.701 for control and R=0.728 for piroxicam- group, **p<0.01).

Figure 2: Oxidative stress is upregulated with grade of inflammation and disease progression in IL-10 KO, resulting in increased DSBs and upregulated

DDR

Left panel with staining for γH2AX (showing DSBs), 8-oxoG (oxidative stress),

MRE11 (DDR), BRCA1 (representative for HR) and KU70 (representative for NHEJ) on non-inflamed, inflamed and dysplastic samples. Right panel with inflammation grading (grade 0-4) and disease progression. γH2AX positive nuclei are significantly increased in inflamed (***p<0.001) and dysplastic samples (*** p<0.001), indicating increase in DSBs with disease severity, predominantly in the basal crypt region and the intermediate zone. Disease progression shows increase of DSBs from non- inflamed to inflamed to dysplastic tissue. The number of DSBs significantly correlates with the grade of inflammation (R= 0.858, p<0.001). An increase in 8-oxoG positive cells is seen with increase in disease severity. Disease progression from non-

Frick, A. et al. Oxidative stress and colon carcinogenesis

inflamed to inflamed to dysplastic tissue shows a significant increase in oxidative

DNA damage (***p<0.001). A significant positive correlation is seen between grade of inflammation and 8-oxoG positive cells; (R=0.923, p<0.01). An increase of DDR and

DNA repair mechanism is detected in the crypts of inflamed and in dysplastic samples. IRS of DDR and DNA repair proteins indicate significant increase with disease severity (**p<0.01; ***p<0.001) and strong positive correlation with grade of inflammation (MRE11: R=0.785, p<0.01; BRCA1: R=0.921, p<0.01, KU70: R=0.851, p<0.01). Statistical analysis was done with ANOVA using Dunnet post hoc analysis and Spearman Rho for correlation analysis. DDR: DNA damage response, HR: homologous recombination, IHC: immunohistochemistry, IRS: immunoreactivity score, NHEJ: non-homologous end-joining

Figure 3: Increased DSBs in IL-10 KO intestinal organoids

A.Western blot analysis of proteins from small intestinal- and large bowel organoids

(WT, IL-10 KO) treated with pro-inflammatory cytokines TNF-α, IL-6 (24 h) and H2O2

(1 h) showed increase of γH2AX after pro-inflammatory treatment, profoundly in IL-10

KO organoids. Actin and tubulin were used as loading control. B: DSBs in WT and IL-

10 KO organoids were analysed using comet-assay, with and without FPG. FPG treatment induces further breaks at oxidized DNA bases, thereby detecting oxidative

DNA damage, (t-test, two-tailed, ***p<0.001). IL-10 KO organoids exhibited more

DSBs and oxidative DNA damage than WT organoids, in classic comet assay and

FPG-comet assay, respectively. C: IF with antibody against γH2AX (green) and DAPI to visualize nuclei in WT and IL-10 KO organoids. DSBs are increased in IL-10 KO organoids, while treatment with 10 ng/mL rmIL-10 reduced the amount of positive

γH2AX foci in IL-10 KO organoids compared to untreated IL-10 KO SIOs. D: Western blot of LBOs treated with either rmIL-10 or rmIL-22, showing a reduction in γH2AX

Frick, A. et al. Oxidative stress and colon carcinogenesis

after rmIL-10 treatment. FPG: Formamidopyrimidine glycosylase, IF:

Immunofluorescence, LBO: large bowel organoid, SIO: small intestinal organoid

Figure 4: γH2AX as a marker of CIDD in patients with IBD.

A: Exemplary pictures of γH2AX staining of human samples (normal, IBD, LGD,

HGD, tumour adjacent, CAC, CRC, FAP) indicating increase of DSBs with disease progression and severity. B: Analysis of patient samples stained for γH2AX, each bar represents a patient, coloured bars indicate the same patient with available sequential biopsy and surgical samples. Profound increase of γH2AX positive nuclei marking DSBs is seen in LGD, HGD and CAC samples. Below the x-axis corresponding p53 staining (D) is shown as either – (nuclei p53 negative) or + (nuclei p53 positive) C: Cumulative analysis of γH2AX staining showing significantly higher levels for CAC, HGD, LGD (***p<0.001) and CRC (**p<0.01) samples compared to normal mucosa. E: IHC for 8-oxoG in human colon samples from healthy subjects,

IBD- and CAC-patients, showing overt presence of oxidized DNA damage in IBD and

CAC samples. IRS of 8-oxoG levels are significantly increased in IBD (***p<0.001) and CAC (***p<0.001). Statistical analysis was done with two-sided ANOVA using

Dunnet post hoc analysis. CAC: colitis-associated cancer, HGD: high-grade dysplasia, IBD: inflammatory bowel disease, IHC: Immunohistochemistry, IRS:

Immunoreactivity score, LGD: low-grade dysplasia

Frick, A. et al. Oxidative stress and colon carcinogenesis

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Overt Increase of Oxidative Stress and DNA Damage in Murine and Human Colitis and Colitis-associated Neoplasia

Adrian Frick, Vineeta Khare, Gregor Paul, et al.

Mol Cancer Res Published OnlineFirst January 29, 2018.

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