Tumor necrosis factor receptor 1 functions as a tumor suppressor

Am J Physiol Gastrointest Liver Physiol 302: G195–G206, 2012. First published November 3, 2011; doi:10.1152/ajpgi.00209.2011.

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Physiology and GI Cancer

Tumor necrosis factor receptor 1 functions as a tumor suppressor Fengqi Chang,1 Michelle R. Lacey,2 Mostafa Bouljihad,3 Kerstin Höner zu Bentrup,4 and Ilana S. Fortgang1 1

Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Tulane University School of Medicine, New Orleans; 2Department of Mathematics, Tulane University, New Orleans; 3Tulane National Primate Research Center, Division of Comparative Pathology, Tulane University, Covington; and 4Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana Submitted 31 May 2011; accepted in final form 2 November 2011

Chang F, Lacey MR, Bouljihad M, zu Bentrup KH, Fortgang IS. Tumor necrosis factor receptor 1 functions as a tumor suppressor. Am J Physiol Gastrointest Liver Physiol 302: G195–G206, 2012. First published November 3, 2011; doi:10.1152/ajpgi.00209.2011.—Tumor necrosis factor (TNF) is a key player in inflammatory bowel disease and has been variably associated with carcinogenesis, but details of the cross talk between inflammatory and tumorigenic pathways remain incompletely understood. It has been shown that, in C57BL/6 mice, signaling via TNF receptor 1 (TNFR1) is protective from injury and inflammation in experimental colitis. Therefore, we hypothesized that loss of TNFR1 signaling would confer increased risk of developing colitis-associated carcinoma. Using three models of murine tumorigenesis based on repeated bouts of inflammation or systemic tumor initiator, we sought to determine the roles of TNF and TNFR1 with regard to neoplastic transformation in the colon in wild-type (WT), TNFR1 knockout (R1KO), and TNF knockout (TNFKO) mice. We found R1KO animals to have more severe disease, as defined by weight loss, hematochezia, and histology. TNFKO mice demonstrated less weight loss but were consistently smaller, and rates and duration of hematochezia were comparable to WT mice. Histological inflammation scores were higher and neoplastic lesions occurred more frequently and earlier in R1KO mice. Apoptosis is not affected in R1KO mice although epithelial proliferation following injury is more ardent even before tumorigenesis is apparent. Lastly, there is earlier and more intense expression of activated ␤-catenin in these mice, implying a connection between TNFR1 and Wnt signaling. Taken together, these findings show that in the context of colitis-associated carcinogenesis TNFR1 functions as a tumor suppressor, exerting this effect not via apoptosis but by modulating activation of ␤-catenin and controlling epithelial proliferation. inflammation; neoplastic transformation; cytokine signaling AT LEAST ONE-FIFTH OF HUMAN cancers occur in the context of chronic inflammation (6, 28). Most anti-inflammatory agents, such as COX-2 inhibitors, have been of little use in the treatment of colon and other cancers, but recent work suggests that diminution of the inflammatory response with mesalamine is protective (10). Lately, attention has focused on the role of inflammatory cytokines, specifically tumor necrosis factor (TNF), in neoplastic transformation (3, 5, 24). TNF is a key player in inflammatory bowel disease (IBD), a condition that

Address for reprint requests and other correspondence: I. S. Fortgang, Dept. of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Tulane Univ. School of Medicine, 1430 Tulane Ave., SL-37, New Orleans, LA 70112 (e-mail: [email protected]). http://www.ajpgi.org

predisposes to colitis-associated carcinoma (CAC), and antiTNF agents, such as infliximab, approved in 1998, have been useful in the treatment of IBD (53). The long-term effects of anti-TNF agents, however, particularly on children, and their overall influence on the progression to CAC are not yet known. Moreover, the deleterious effects of these biologics, including vulnerability to life-threatening infection and their association with the development of B and T cell lymphoma, give urgency to investigations into the role of the cytokine and its receptors in progression of and recovery from disease (2, 38). IBD flares are remarkable for a pathological influx of inflammatory cells, including macrophages, to the mucosal epithelium and, with this, an enduring surge of proinflammatory cytokines, among them TNF (46, 53, 61). TNF signals via two cell surface receptors, TNFR1 and TNFR2, resulting in several sometimes opposing cellular responses that vary also by context and cell nature (4, 17, 18, 31, 60). In the colonic mucosa, TNF is involved in both cell survival and cell death (13, 19, 20, 35). Signaling via TNFR1 has been shown most often to promote apoptosis and to modulate inflammation, whereas signaling via TNFR2 has been shown to be important in wound healing, cell migration, and proliferation (17, 18). As well, and despite its name, increased levels of TNF have been found in the setting of other cancers, including those of the pancreas, skin, and ovaries (36, 37, 43). With specific regard to colon carcinogenesis, TNF activity has been shown both to promote and to protect from neoplastic transformation (6, 9, 23, 25, 41, 47). Finally, there are case studies of development of cancer in other organ systems (lymphatic and skin) following the use of anti-TNF for IBD or rheumatological disease (3, 38). These conflicting data underscore the paradoxical nature of the cytokine and its signaling via its two receptors. Although the correlation between chronic inflammation and carcinogenesis has long been appreciated, the mechanisms responsible have not yet been fully identified (6, 57). Of particular interest is the connection between inflammatory pathways and the Wnt/␤-catenin signaling pathway, which plays a critical role in colon carcinogenesis and in control of proliferation and differentiation in the colonic stem cell compartment (15, 39, 51, 59). Work is underway to clarify the specific underlying regulatory cross talk between the cellular signaling pathways of inflammatory cytokines such as TNF and those pathways leading to tumor initiation, such as Wnt and Notch2 (48, 58).

0193-1857/12 Copyright © 2012 the American Physiological Society

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In our earlier studies of acute inflammation, we noticed that C57BL/6 mice lacking TNFR1 developed dysplastic lesions when dosed with the detergent dextran sodium sulfate (DSS), dissolved in their drinking water. We did not see dysplastic changes in mice lacking TNFR2 or in animals deficient for the cytokine alone (I. S. Fortgang and D. B. Polk, unpublished observations). Given the overlapping associations between IBD and TNF, TNF and cancer, and cancer and IBD, the studies presented herein were designed to assess the role of TNF and TNFR1 in the progression of CAC. We hypothesized that absence of TNFR1 in these mice would result in a more avid inflammatory response and confer increased vulnerability to CAC. We based our experiments on the well-established model of azoxymethane (AOM)/DSS in which animals are given an intraperitoneal (ip) injection of the tumor initiator AOM followed by three treatments with DSS. This protocol has the advantage of widespread use and acceptance as a model for murine CAC, and it is known to produce tumors in at least 50% of treated C57BL/6 mice (52, 54). However, its reliance on AOM prompted us to employ two control protocols that have also been used as models for colon carcinogenesis in rodents: 1) phosphate-buffered saline (PBS)/DSS, consisting of an ip injection of PBS which is biologically inert, followed by repeated episodes of DSS-induced colitis; and 2) AOM-H2O, consisting of the ip injection of AOM followed by filtered, unadulterated drinking water (Fig. 1) (54). Here we report the significant differences in the responses of TNF- and TNFR1-deficient animals (TNFKO and R1KO, respectively) with regard to frequency of neoplastic transformation. We observed earlier and more invasive disease in R1KO animals, even in the absence of the tumor initiator AOM. However, although R1KO animals did have more inflammation following bouts of acute colitis, there was no direct, significant statistical correlation between this and the increase in tumor burden and grade. By contrast, TNFKO animals were relatively spared from developing tumors compared with R1KO animals. In addition, TNFKO mice were smaller and grew at slower rates and had extent and degrees of injury and inflammation comparable to wild-type (WT). Overall, TNFKO animals were not significantly protected from neoplastic trans-

Fig. 1. Time line depicting 3 treatment protocols and days of timed euthanasia. Day 0 animals were injected with either the tumor initiator azoxymethane (AOM) or, as a negative control, phosphate-buffered saline (PBS). Day 5 treatment with the detergent dextran sodium sulfate (DSS) in the drinking water was initiated in 1 of the AOM-treated groups and the PBS-treated group. As a second negative control, the second AOM-treated group was provided filtered, unadulterated water. Animals were euthanized at designated time points (TP-I, TP-II, TP-III).

Table 1. Distribution of mouse genotypes and treatment protocols Treatment

Genotype

Female/Male

Total (n ⫽ 218)

AOM/DSS

WT R1KO TNFKO WT R1KO TNFKO WT R1KO TNFKO

22/12 23/25 18/13 5/8 3/12 7/9 7/10 8/16 11/9

34 48 31 13 15 16 17 24 20

AOM/H2O PBS/DSS

AOM, azoxymethane; DSS, dextran sodium sulfate; WT, wild-type (C57B1/ 6); R1KO, TNF receptor 1 knockout; TNFKO, TNF knockout.

formation compared with WT. Finally, we found that the tumorigenesis was not preceded by a decrease in apoptosis in challenged R1KO animals; rather, we documented increased proliferative activity and activation of ␤-catenin before tumorigenesis, implying a link between TNFR1 and Wnt signaling. MATERIALS AND METHODS

Animals. R1KO, TNFKO, and WT mice on C57BL/6 genetic backgrounds were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were maintained in conventional conditions in the Tulane animal-care facility, and all experiments were approved by the Animal Care and Usage Committee of Tulane University. Study design. On the basis of previously established methods for promotion of murine colon tumorigenesis (22, 52, 54), 7-wk-old mice of each of the above-described genotypes were injected ip with AOM (12.5 mg/kg dissolved in sterile PBS, concentration of 1 ␮g/␮l) (Fig. 1 and Table 1). Five days later they were provided with filter-sterilized 2.5% (weight/volume) DSS solution, followed by 18 days of autoclaved drinking water. This cycle was repeated twice with slight variation: 5 days of 2.5% DSS solution followed by 11 days of autoclaved drinking water, followed by 5 days of 2% DSS. Mice were euthanized 10 days following the last administration of DSS. A second cohort of animals was given an ip injection of 12.5 ␮l/g sterile PBS and treated with the three courses of DSS. A third cohort received ip AOM as described above and was provided untreated, autoclaved water for the duration of the experiment. Animals chosen randomly from each cohort were euthanized at three set time points (TP). TP-I and TP-II were chosen to be 3 days following administration of DSS because our previous work had determined these to be times of intense inflammation and repair following acute injury. TP-III, 10 days following the last dose of DSS, was chosen according to previously described tumor-inducement protocols. Outcomes measured at these time points were weights and degree of hematochezia, determined by guaiac card analysis. Hematochezia scores were 0 for normal formed stool, 1 for diarrhea without occult blood, 2 for demonstration of occult blood, 3 for gross blood. Table 1 presents numbers of animals per treatment per time point. Tissue retrieval and preparation. Following euthanasia by CO2 asphyxiation, laparotomy with total colectomy, from anal verge to cecum, was performed. Cecums were removed and discarded. Colons were irrigated with cold PBS, flayed open, and bisected longitudinally. Mucosa was removed from one bisected half by scraping with a glass slide and placed in RNA extraction buffer provided in RNeasy Mini Kit (Qiagen). The remaining bisected half was prepared in a “Swiss roll” fashion, wrapped on itself so that the most distal region of the intestine became the innermost part of the roll. The tissue was fixed in 10% buffered-formalin for 24 h and placed in 70% ethanol before standard paraffin embedding.

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TNFR1 FUNCTIONS AS A TUMOR SUPPRESSOR

Microscopic evaluation. Hematoxylin and eosin (H&E)-stained colon sections (4 – 6 ␮m thick) were examined by light microscopy (Leica model DM 2500, Leica Microsystems CMS, Weltzar, Germany) and evaluated by a pathologist (M. Bouljihad) who was blinded to the experimental design, by a scoring method adapted from validated scoring systems in murine colitis models (16, 30, 32). Each colon section was scored for severity of inflammation with a range from 0 (none) to 4 (severe). Neoplasia scores were separated into three groups: 0 for no neoplasia; 1 for adenomatous or in situ disease; 2 for extensive mucosal or invasive disease. Animals not exposed to DSS had no histopathological evidence of any lesion, and, as a group, the average of their scores in each category was 0. Hence only scores of AOM/DSS- and PBS/DSS-treated animals were considered in our analysis of inflammation and neoplastic transformation. TUNEL assay. The apoptotic cells were counted by using DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s instructions. RNA extraction and quantitative real-time PCR. Total RNA was extracted from mucosal tissue with RNeasy Mini Kit (Qiagen) and treated with RNase-Free DNase I (Qiagen) according to the manufacturers’ protocols. One microgram total RNA was reverse-transcribed by using iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions. Quantitative real-time PCR with iTaq SYBR Green Supermix with ROX (Bio-Rad) was performed in triplicate by using the Stratagene Mx3000P (Agilent Technologies) and was normalized to endogenous ␤-actin mRNA levels for each

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reaction. PCR primer sequences for TNFR2, TNF, and ␤-actin are as follows: TNFR2 5=-CTTGGATGCCATGCTCACCGATT-3= and 5=-GTCCAGTATCTTGATTCCAGAGTGC-3=; TNF 5=GCACCACCATCAAGGACTCAA-3= and 5=-TCAGGGAAGAATCTGGAAAGGT-3=; ␤-actin: 5=- AGAGGGAAATCGTGCGTGAC-3= and 5=-CAATAGTGATGACCTGGCCG-3=. The thermal profile used was as follows: 95°C for 4 min, 45 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 20 s. Quantification was done by the standard ⌬⌬CT method. The scale bars represent the standard error of mean. Fluorescent immunohistochemistry analysis. Fluorescent immunostaining was performed on formalin-fixed and paraffin-embedded tissues. Five-micrometer paraffin sections were subjected to antigen retrieval (10 mM sodium citrate, pH 6.0) after deparaffinization and rehydration steps. Primary antibodies used were anti-phospho-histone H3 at 1:100 dilution, anti-cleaved caspase 3 at 1:100 dilution (Cell Signaling) and anti-active-␤-catenin at 1:100 dilution (Millipore). The signals were visualized with Tyramide Signal Amplification kit (Invitrogen) using donkey anti-rabbit or donkey anti-mouse secondary IgG antibodies (1:100, Jackson ImmunoResearch) performed as per manufacturer’s protocols. Ten randomly selected images of each section were acquired with a Zeiss Axioplan 2 microscope and analyzed for cell counts by use of Slidebook 5 software (Intelligent Imaging Innovations, Denver, CO). The imaging and data analysis were performed in a blinded manner. For each genotype, the whole

Fig. 2. Outcomes of DSS treatment vary by genotype. A–C: weight curves fitted from mixed-effects regression models present average growth for mice within each treatment group, stratified by genotype, and show that treatment with DSS impairs growth in wild-type (WT) and TNF receptor 1 knockout (R1KO) animals. Although weight gain in TNF knockout (TNFKO) mice was not significantly affected, R1KO and WT animals were comparable in their growth profiles, but TNFKO animals were consistently smaller than R1KO and WT animals (P ⫽ 0.03). A: WT animals treated with AOM/DSS (A/D) had significantly less weight gain (growth) compared with those treated with AOM/H2O (P ⬍ 0.05), but this was not significantly different compared with WT treated with PBS/DSS (P/D). B: R1KO mice treated with either AOM/DSS or PBS/DSS experienced significantly less weight gain than mice treated with AOM/H2O (P ⬍ 0.05). C: weight gain of TNFKO mice was not significantly affected by either the PBS/DSS or AOM/DSS treatment. D and E: proportion of mice experiencing hematochezia by day, stratified by genotype and treatment. x-Axis is progression of days; y-axis represents proportion of mice observed to have hematochezia with 1.0 equal to 100%. Apexes correspond to treatment cycle end points. D: significantly more R1KO animals have prolonged hematochezia following treatment with AOM/DSS, implying worse colitis in this group. E: all genotypes recover from DSS colitis almost completely if not treated with the tumor initiator AOM. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00209.2011 • www.ajpgi.org

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colon sections from three to six different animals were selected for analysis. shRNA and generation of stable clones in mouse colon cancer cells. Short hairpin RNA (shRNA) against TNFR1 was used to knock down TNFR1 in murine colon cancer cells (MC-38), obtained as a kind gift from Dr. W. T. Godbey (Tulane University, New Orleans, LA). Cells were maintained in DMEM (Invitrogen), and the cells were trans-

fected with SureSilencing shRNA plasmid for mouse Tnfsf1a and nonspecific control plasmid (SABiosciences) by using Lipofectamine transfection reagent and Plus reagent (Invitrogen) according to the manufacturer’s protocol. To generate stable clones, puromycin selection (12 ␮g/ml, Invitrogen) was started at 24 h after transfection and lasted for ⬃6 wk before the first clones were isolated. Knockdown of TNFR1 gene expression was validated by means of quantitative

Fig. 3. R1KO mice develop more tumors than do WT or TNFKO. Tumors in R1KO animals appear less differentiated and more invasive. A: representative macroscopic changes involving colonic mucosal surface of AOM/DSS-treated WT, R1KO and TNFKO animals at TP-III. Black arrows designate obvious tumors. R1KO animals develop multiple, multifocal-to-coalescing, raised, erythematous nodules whereas WT and TNFKO animals develop fewer, more circumscribed, paler lesions. B: representative microscopic changes involving colonic mucosa of AOM/DSS-treated animals at TP-I, TP-II, and TP-III [hematoxylin and eosin (H&E), ⫻2.5]. Following bouts of acute colitis (TP-I and -II), all animals show multifocal to segmental areas of surface epithelium erosion, loss of crypts, and locally extensive cellular infiltration with mononuclear cells and eosinophils. C: higher magnification (H&E, ⫻20) of areas highlighted in yellow in B. WT animals demonstrate numerous hyperplastic crypts with occasional mild to moderate crypt ectasia and adenomatous transformation. R1KO animals developed lesions such as this tubular adenocarcinoma. TNFKO animals were seen to have low-grade lesions, represented by this carcinoma in situ with high-grade dysplasia. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00209.2011 • www.ajpgi.org

TNFR1 FUNCTIONS AS A TUMOR SUPPRESSOR

real-time PCR with the primers 5=-CTTGCGATTCTGTATGCTGTGGTGG-3= and 5=-TGCATCTCCAGCCTCTCGATCTCG-3=, normalized to ␤-actin levels. Two stable knockdown clones (4E4 and 4F2) and one negative control (NC2) were identified and used to generate colonies for subsequent experiments (Western blot analysis and TOPflash assays). Western blot analysis. Cells were lysed in PhosphoSafe Extraction Reagent (EMD4Biosciences) containing 10 ␮l/ml protease inhibitor cocktail (Sigma-Aldrich); 50 ␮g of protein was separated in Novex 4 –20% Tris-glycine gel (Invitrogen), followed by electrotransfer to a nitrocellulose membrane (Thermo Scientific). Immunodetection was carried out with total ␤-catenin antibody (Cell Signaling) and ␤-actin antibody (Sigma-Aldrich), and the signals were visualized with IRDye-labeled secondary antibodies (LI-COR) and analyzed with Odyssey Infrared Imaging System (LI-COR) according to the manufacturer’s manual. TOPflash dual-luciferase reporter assays. TOPflash dual-luciferase reporter assays were performed as described elsewhere (11). Briefly, cells of negative control and knockdown clones were plated in six-well plates at a density of 2⫻105 cells/well. Plasmid mixtures containing 1.5 ␮g of TOPflash luciferase construct (Upstate Biotechnology) and 0.5 ␮g of Renilla luciferase driven by the SV40 promoter (Promega) were transfected into cells by use of Lipofectamine transfection reagent and Plus reagent (Invitrogen). After 24 h of transfection, cells were lysed, and luciferase activity was evaluated by use of the Dual-Luciferase assay kit (Promega). Values for TOPflash luciferase activity were normalized to those of Renilla activity. Statistical analysis. A mixed-effects regression model (45) was fit to the weight data to compare the effects of each treatment on each of the three mouse genotypes. Variables included as fixed effects were genotype, treatment type, time (in days), and treatment phase. Nonnumerical covariates were treated as unordered factors. Random effects terms were included to account for variation in initial weights and growth rates within each group. To model hematochezia, observations at each measured time point were converted to binary variables indicating the presence or absence of blood in each sample. A generalized mixed-effects model was then constructed to predict the response by logistic regression, again with genotype, treatment type, day, and treatment phase considered as predictors. As scores for histological variables were integer valued, differences among geno-

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types were analyzed by Poisson regression (33) and all P values were adjusted for multiple comparisons. Neoplasia scores were grouped into three levels to represent no (lev0), low-grade (lev1), or highgrade neoplastic lesion, and a multinomial logit model (1) was fit to determine the probabilities associated with each level on the basis of genotype and treatment. TOPflash data were also analyzed by using a mixed-effects model with random intercepts to account for variations in baseline levels associated with repeat cycles, and data from the terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) and fluorescent immunohistochemistry assays were independently analyzed by two-way ANOVA to assess differences among genotypes and time points. All analyses were performed by using the R statistical computing environment, and models were fit by using the contributed “nlme” and “mlogit” packages. In all cases, nested models were compared by using likelihood-ratio tests to determine the significance of added covariates and interactions, and terms that did not significantly improve the fit of the model were discarded. All findings reported to be statistically significant had corresponding P values of 0.05 or less. RESULTS

DSS treatment causes weight loss in R1KO and WT mice. TNFKO mice do not lose weight following DSS treatment, but they are consistently small. Because failure to thrive is a common presentation of pediatric inflammatory diseases and cachexia and weight loss are consequences of increased levels of TNF, we sought to characterize the effects of these three treatments on weight gain and growth among the different genotypes. In every genotype, animals treated with DSS, whether or not they were injected with AOM, experienced less weight gain than unchallenged animals, although this was significant only for WT and R1KO genotypes. Animals treated with AOM and unadulterated water gained more weight than did animals treated with DSS; their weight gain was at rates comparable to untreated animals in the respective colonies (data not shown). WT animals treated with AOM/DSS had significantly less weight gain compared with those treated with

Fig. 4. R1KO animals have the most severe inflammation in the setting of acute colitis and develop early and aggressive neoplastic lesions. A: box plots depicting the distributions of inflammation scores. R1KO animals demonstrate significantly higher inflammation scores than WT at TP-I (P ⫽ 0.017) and at TP-III (P ⬍ 0.001) but not at TP-II. At TP-III R1KO and TNFKO mice have statistically comparable scores (P ⫽ 0.074) and both have significantly higher inflammation scores than WT (P ⫽ 0.001). B: distribution of neoplasia grade by genotype and treatment at all TP, representing no (lev0), low- (lev1), or high-grade (lev2) lesions with each box indicating the number of animals demonstrating a particular lesion. R1KO animals treated with AOM/DSS have more tumors at TP-II and more tumors and high-grade lesions at TP-III than the other genotypes and are significantly more likely to develop high-grade lesions overall than either the WT or TNFKO mice (P ⬍ 0.01). TNFKO animals are not significantly protected from neoplastic transformation compared with WT. Finally, R1KO animals did not require treatment with AOM to develop tumors. Two of 5 R1KO animals developed advanced (lev2) lesions from repeated DSS treatment alone (P ⫽ 0.04) (Fig. 4B).

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AOM/H2O (P ⬍ 0.05), but this was not significantly different compared with WT treated with PBS/DSS (Fig. 2A).R1KO mice treated with either AOM/DSS or PBS/DSS experienced significantly less weight gain than mice treated with AOM/ H2O (P ⬍ 0.05) (Fig. 2B). Weight gain of TNFKO mice was not significantly affected by either the PBS/DSS or AOM/DSS treatment (Fig. 2C). Among the genotypes, regardless of treat-

ment protocol, TNFKO mice were consistently smaller than WT or R1KO animals (Fig. 2, A–C, P ⫽ 0.03). However, TNFKO mice did not demonstrate statistically significant weight loss following treatment with DSS (Fig. 2C). R1KO and WT mice grew comparably and both groups had statistically significant weight loss following treatments with DSS with comparable recoveries (Fig. 2, A–C, P ⬍ 0.05).

Fig. 5. Apoptosis is not affected in R1KO animals. A: terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) results at TP-I, -II, and -III, original magnification ⫻20. B: cleaved caspase 3 (CCasp3), original magnification ⫻20. C: box plots demonstrating increased rates of apoptosis in R1KO compared with WT animals treated with AOM/DSS at all TP demonstrated by TUNEL (P ⫽ 0.061) and cleaved caspase 3 (P ⫽ 0.04). DAPI, 4,6-diamidino-2-phenylindole.

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Fig. 6. Epithelial cell proliferation is more vigorous in R1KO animals prior to tumor formation, and increased active ␤-catenin expression is seen prior to neoplastic transformation. A and B: phospho-histone H3 (PH3) results demonstrating increased proliferative activity at all TP in R1KO mice (P ⫽ 0.0132), original amplification ⫻20. C: active ␤-catenin (Active ␤-Cat)-stained sections at all TP. Representative results from 4 independent experiments are shown.

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Fig. 7. Knockdown of TNFR1 results in increased expression and activity of ␤-catenin. A: quantitative real-time PCR verifying suppression of gene expression following shRNA knockdown of TNFR1 in MC-38 cells: colonies from 1 shRNA-nontarget control clone (NC2) and 2 TNFR1 knockdown stable clones (4E4 and 4F2) were established. B: Western blot analysis with ␤-actin as a loading control. C: densitometry demonstrating significantly more expression of ␤-catenin in knockdown stable clones. Results are representative of 3 independent experiments. D: TOPflash assay (6 separate repeats) demonstrating significantly more activation of ␤-catenin in lysates from knockdown colonies than from WT (P ⬍ 0.0001).

R1KO mice had more ongoing bleeding than WT following AOM/DSS treatment. Frankly bloody diarrhea is the hallmark sign of one form of IBD, ulcerative colitis, and stool occult blood can persist for weeks after the initiation of treatment, even when other symptoms have subsided. Furthermore, tumors bleed. A significantly higher proportion of R1KO mice were observed to have ongoing bleeding compared with WT mice following treatment with AOM/DSS (P ⬍ 0.001). TNFKO mice were at intermittent risk but not significantly more or less so than WT or R1KO, respectively (Fig. 2D). In the group treated with PBS/DSS, R1KO, WT, and TNFKO mice had statistically comparable rates and durations of hema-

Fig. 8. AOM/DSS-treated R1KO mice have increased TNF and TNFR2 transcript levels. A: TNF expression in treated animals. B: TNFR2 expression in treated animals. Increase of both TNF and the TNFR2 are significant starting at TP-II in R1KO animals. Data are expressed as the fold change (means ⫾ SE). P value represents results of Student’s t-test (n ⫽ 5), adjusted for multiple comparisons by the Benjamini-Hochberg correction.

tochezia (Fig. 2E). In all groups the recovery rate was significantly higher for PBS/DSS-treated mice than in AOM/DSStreated animals. AOM/H2O-treated animals did not develop hematochezia (data not shown). R1KO mice develop early and aggressive neoplastic lesions even in the absence of tumor initiator. TNFKO mice are not significantly protected from tumorigenesis compared with WT. R1KO animals treated with AOM/DSS have more tumors at TP-II and more tumors and high-grade lesions at TP-III than WT or TNFKO mice (P ⬍ 0.01) (Figs. 3 and 4B). Finally, R1KO animals did not require treatment with AOM to develop tumors. Two of five R1KO animals developed advanced (lev2) lesions from repeated DSS treatment alone (P ⫽ 0.04) (Fig. 4B). TNFKO animals are not significantly protected from tumorigenesis compared with WT (Figs. 3 and 4B). R1KO animals have the most severe inflammation in the setting of acute colitis and in the presence of tumorigenesis. There was no significant difference in inflammation scores among cohorts of the same genotype dosed with AOM/DSS or PBS/DSS. Therefore, the inflammation scores of animals of the same genotype dosed with DSS were considered together. At TP-I R1KO animals had significantly higher inflammation scores than did WT or TNFKO, reflecting more intense inflammation in response to acute colitis (Fig. 4A, light gray box plots). At TP-II, following a second dose of DSS, all genotypes had higher scores than at TP-I, as could be explained by chronic colitis, and the differences among the genotypes was not significant (Fig. 4A, dark gray box plots). At TP-III, 10 days following administration of DSS and induction of a third bout of colitis, R1KO and TNFKO animals had significantly higher scores than WT, reflecting either chronic colitis or inflammation associated with tumorigenesis (Fig. 4A, charcoal gray box plots). For WT and TNFKO animals the correlation between inflammation and neoplasia scores at TP-III was significantly positive (P ⫽ 0.01), but among R1KO animals at

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TP-III correlation of degree of inflammation and neoplastic transformation was close to 0 and completely insignificant. Hence, in this model, with regard to loss TNFR1 signaling, inflammation is required for neoplastic transformation but not linearly related. AOM/DSS-treated R1KO mice had increased rates of apoptosis, proliferation, and increased expression of activated ␤-catenin. Because TNFR1 is known to promote apoptosis, we wanted to know whether the increased tumorigenesis in DSStreated R1KO mice could be attributed to a decreased rate of apoptosis. In fact, we saw more apoptosis in the colons of DSS-treated R1KO compared with WT at all time points, assayed by TUNEL staining (P ⫽ 0.061) and IHC for cleaved caspase 3 (P ⫽ 0.04) (Fig. 5). By contrast, increased rates of proliferation were seen at all three time points in colons from R1KO animals compared with WT, and this, too, was statistically significant (P ⫽ 0.0132) (Fig. 6, A and B). ␤-Catenin is a bifunctional protein related to cell adhesion and gene transcription that when activated by Wnt pathway accumulates in the cytosol and, subsequently, translocates into the nucleus (7). We saw a clear and obvious increase in activated ␤-catenin beginning at TP-I. This increased signal in R1KO animals became most obvious at TP-III in AOM/DSStreated animals even in tissue without obvious tumor formation as seen grossly and histologically (Fig. 6C). Enumeration and quantification of the signal generated by activated ␤-catenin proved to be challenging, enabling us to provide only qualitative statements about the respective signal. Therefore, we performed an in vitro assay to validate the link between loss of TNFR1 and activation of ␤-catenin. We used shRNA techniques to knock down the TNFR1 expression in MC-38 cells and obtained one stable negative control clone (NC2) and two stable knockdown clones (4E4 and 4F2) (Fig. 7A). Western blot analysis followed by quantitative densitometry demonstrated increased total ␤-catenin in cells with successful knockdown of TNFR1 (Fig. 7, B and C). TOPflash analysis showed significantly increased ␤-catenin activity in TNFR1 knockdown clones (P ⫽ 0.0001) (Fig. 7D). In the absence of TNFR1, transcription of TNF and TNFR2 is increased following injury. Quantitative real-time PCR analysis showed insignificantly increased levels of TNF and TNFR2 mRNA from AOM/DSS-treated R1KO mice compared with AOM/DSS-treated WT at TP-I. However, the increased levels of TNFR2 and TNF in R1KO mice were significant at

TP-II and TP-III (Fig. 8), suggesting that loss of TNFR1 permits increased binding of TNF to TNFR2, hence promoting increased proliferation. DISCUSSION

Herein we present work carried out on 218 animals on C57BL/6 backgrounds showing that loss of TNFR1 confers increased vulnerability to CAC. R1KO animals had worse colitis, higher inflammation scores, and increased tumor formation. Furthermore, we found that absence of TNF does not significantly alter the course of disease, favorably or unfavorably, compared with WT. Thus the role of TNFR1 in protection from CAC is crucial and independent of its ligand TNF. The risk of developing CAC has been related to extent and duration of inflammation (8, 49, 57). Recently, however, the linear nature of this relationship has become less certain (22, 40, 44). Although we found the correlation between inflammation and neoplasia scores at TP-III to be significantly positive (P ⫽ 0.01) for WT and TNFKO animals, this most likely reflects relatively mild colitis and benign tumorigenesis. R1KO mice as a group had significantly higher inflammation scores initially and developed significantly more tumors at all time points (Figs. 3 and 4), but because mice at TP-I and TP-II were euthanized prior to tumor formation, we were unable to use inflammation scores at TP-I and TP-II to predict which animals would develop tumors. However, for R1KO animals at TP-III the correlation of degree of inflammation and neoplastic transformation was close to 0 (data not shown). Taken together, these findings suggest that, in the absence of TNFR1, inflammation is required for neoplastic transformation, possibly as an initiating event, but extent, severity, and duration of inflammation are irrelevant to the process. Our results are consistent with those of Onizawa et al. (40), who found that antibodies to TNF diminished expression of NF-␬B and protected from tumorigenesis despite failing to modulate inflammation. Not only did the neoplastic lesions in R1KO mice occur earlier and appear more invasive than in WT, but in four of nine of R1KO mice, the systemic tumor initiator AOM was not required for tumor formation, further underscoring the link between mucosal injury, disruption of TNFR1 signaling and neoplastic transformation. These data suggest that inflammation giving rise to cancer (inappropriate immune response), such as seen in this study with the loss of TNFR1, may be

Fig. 9. Putative pathway by which TNFR1 functions as a tumor suppressor. A: intact TNFR1 signaling modulates Wnt signaling or otherwise maintains ␤-catenin inactivated in the cytosol. Normal levels of TNF bind to normal numbers of both receptors and maintain gut homeostasis. B: absent TNFR1 signaling, increased levels of TNF bind to increased numbers of TNFR2 and result in increased epithelial cell proliferation. As well, possibly because of unmitigated Wnt signaling, activated ␤-catenin translocates to the nucleus, driving proliferation and neoplastic transformation.

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different than inflammation arising in cancer (appropriate immune response) (6, 14, 57). It has been shown that, in C57BL/6 mice, signaling via TNFR1 is protective from injury and inflammation (17, 27, 34). Our results in this study reaffirm that and, furthermore, suggest that this anti-inflammatory function of TNFR1 signaling protects from CAC. However, in 2008 Popivanova et al. (47) reported that the ablation of TNFR1 in BALB/c mice was protective as was interference with ligand binding by administration of a decoy receptor, etanercept. A possible explanation for our disparate findings may be the differences in backgrounds of the experimental animals. BALB/c mice are more vulnerable to AOM/DSS-induced injury and tumorigenesis, and AOM has been shown to induce colonic adenomas in these animals without requiring treatment with DSS (21, 52). Secondly, of all the TNF antagonists, etanercept, which also binds lymphotoxin, is an inferior therapeutic agent in the treatment of IBD than are the monoclonal antibodies to TNF such as infliximab and adalimumab (12, 50, 56). It may be that the systemic effects of AOM in a more vulnerable mouse combined with the failure of etanercept to decrease colonic inflammation might have actually been protective from tumorigenesis. Reconciliation of the discrepancies of our two studies may rest on varying methodologies and could lead to better understanding of the functioning of TNFR1 in various contexts. In our experiments absence of TNFR1 correlated with significantly increased vulnerability to carcinogenesis, whereas absence of TNF did not. Furthermore, although TNFKO animals were less susceptible to all categories of disease than R1KO, they were not significantly more protected, and sometimes less, compared with WT, nor did they grow as well. Given the efficacy and widespread use of anti-TNF agents in a number of inflammatory diseases, these discrepancies between function of ligand and receptor were unexpected but reconcilable. Firstly, although it is certain that increased levels of TNF are pathological in IBD and other inflammatory diseases, normal levels of TNF are required for homeostasis (4, 13, 27, 34, 35). Although a genetic knockout is not equivalent to antibody interference, complete absence of the cytokine, in our model, correlates with growth inhibition and may raise concerns about administration of anti-TNF to children, heightening the urgency for a treatment more refined than current biologics. A second reason that absence of the cytokine is not equivalent to absence of the receptor is that there are two TNFR, which have different and sometimes opposing functions (4, 17, 18, 31, 60), and both of these receptors can be bound by TNF or by other ligands (29, 55). Given that we found that, in the absence of TNFR1, mice treated with AOM/DSS had increased levels of TNF and TNFR2, it may be that TNFR1, regardless of which ligand binds to it, serves as a brake on inflammatory and proliferative pathways, and its loss but not that of the cytokine confers susceptibility to neoplastic transformation (Fig. 8). TNFR1 has long been associated with control of apoptosis via death domain-dependent signaling, and impaired apoptosis is a hallmark of carcinogenesis (4, 26). However, TNFR1 has also been identified as an antiapoptotic influence, promoting Raf activation of MAPK and NF-␬B pathways (19, 20). In fact, we saw increased rates of apoptosis in R1KO mice compared with WT at all time points (Fig. 5). Although the TUNEL

assays were not statistically significant, assays for cleaved caspase 3 were. Importantly, these experiments demonstrate that loss of TNFR1 does not increase vulnerability to neoplastic transformation because of a nonfunctioning apoptotic pathway. Rather, primarily, absence of TNFR1 results in significantly increased rates of proliferation (Fig. 6, A and B). This may be attributed to increased transcription of TNF and TNFR2, which, together and unopposed, promote proliferation (Fig. 8). Dysregulation of the canonical Wnt signaling pathway has been well established as a proliferative carcinogenic precursor (42). Direct activation of the Wnt pathway from other inflammation-associated pathways would establish the basis for a causal molecular connection (39). We found increased activated ␤-catenin following injury compared with AOM/DSStreated WT mice (Fig. 6C), implying a link between intact TNFR1 signaling and modulation of Wnt/␤-catenin signaling pathways. To validate this observation, we assayed for ␤-catenin in murine colon cancer cells treated with shRNA against TNFR1 and found increased total ␤-catenin and increased ␤-catenin activity in knockdown cells (Fig. 7). However, the signaling cross talk between TNFR and Wnt remain to be defined. Whether this is modulated by TNFR1 or TNFR2 is not clear and bears further investigation. In summary, our results suggest that TNFR1 functions as a tumor suppressor, exerting this effect not via control over apoptosis but by signaling cross talk that modulates Wnt activity or other influences on activated ␤-catenin (Fig. 9). This suggests that TNFR1 signaling modulates the inflammatory response either by constitutive activation in the absence of ligand or via a second ligand. The clinical implications of these findings are that manipulation and augmentation of the receptor, or an alternative ligand, may be efficacious in the treatment of certain inflammatory states and neoplastic diseases. ACKNOWLEDGMENTS The authors thank Laura Levy and Samir El-Dahr for thoughtful and insightful discussions about the project. As well, acknowledgment is due Brent Polk and David Jones for early training, guidance, and inspiration. GRANTS This work was supported by National Institutes of Health Grant P20RR020152. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES 1. Agresti A. Categorical Data Analysis. New York: Wiley, 1990. 2. Askling J, Dixon W. The safety of anti-tumour necrosis factor therapy in rheumatoid arthritis. Curr Opin Rheumatol 20: 138 –144, 2008. 3. Askling J, Fahrbach K, Nordstrom B, Ross S, Schmid CH, Symmons D. Cancer risk with tumor necrosis factor alpha (TNF) inhibitors: metaanalysis of randomized controlled trials of adalimumab, etanercept, and infliximab using patient level data. Pharmacoepidemiol Drug Saf 20: 119 –130, 2011. 4. Baker SJ, Reddy EP. Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 12: 1–9, 1996. 5. Balkwill F. Tumor necrosis factor or tumor promoting factor? Cytokine Growth Factor Rev 13: 135–141, 2002. 6. Balkwill F. Tumour necrosis factor and cancer. Nat Rev Cancer 9: 361–371, 2009. 7. Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382: 638 –642, 1996.

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