Increased Dietary Vitamin D Suppresses MAPK Signaling ... · otec), and F4/80 (MCA497, Serotec). Signals were detected using biotinylated goat anti-rat (Jackson ImmunoResearch) followed

Prevention and Epidemiology

Increased Dietary Vitamin D Suppresses MAPK Signaling,Colitis, and Colon Cancer

Stacey Meeker1, Audrey Seamons1, Jisun Paik1, Piper M. Treuting1, Thea Brabb1, William M. Grady2,3, andLillian Maggio-Price1

AbstractEpidemiologic studies associate low serum vitamin D levels with an increased risk of colon cancer and

inflammatory diseases such as inflammatory bowel disease (IBD). 129-Smad3tm1Par/J (Smad3�/�) mice are amodel of bacteria-driven colitis and colon cancer when infected with Helicobacter bilis (H. bilis). Thus, we usedthis mouse model to determine whether increased dietary vitamin D would reduce inflammation and coloncancer. Smad3�/� mice were fed purified diet with either maintenance (1 IU vitamin D/g diet; maintenance) orincreased concentrations of vitamin D (5 IU vitamin D/g diet; high vitamin D). One week after diet initiation,mice were inoculated with broth or H. bilis and were necropsied at several time points postinoculation to assessinflammation, dysplasia, and neoplasia incidence. At 16 weeks postinfection, 11% of mice fed high vitamin Ddiet had cancer compared with 41% of mice fed maintenance diet (P ¼ 0.0121). Evaluation at an early timepoint (1 week postinfection) showed that animals fed high vitamin D had decreased MAPK (p-P38 and p-JNK)activation in lamina propria leukocytes as well as decreased NFkB activation in colonic epithelial cells.Reduction in MAPK and NFkB activation correlated with decreased IBD scores (2.7 vs. 15.5; P < 0.0001) as wellas decreased inflammatory cell infiltrates and reduced expression of proinflammatory cytokines in cecal tissue.These findings suggest that increased dietary vitamin D is beneficial in preventing inflammation-associatedcolon cancer through suppression of inflammatory responses during initiation of neoplasia or early-stagecarcinogenesis. Cancer Res; 74(16); 4398–408. �2014 AACR.

IntroductionColorectal cancer is the third most commonly diagnosed

cancer in both men and women (1). Studies indicate that dietand lifestyle choices play a significant role in the developmentof and prognosis of colon cancer (2). Vitamin D status of anindividual is also influenced by both diet and lifestyle (3). Thelink between vitaminD and colon cancer was first suggested byGarland and colleagues who observed that populations resid-ing in the northeastern United States had an increased inci-dence of colon cancer–related mortality compared with thoseliving in the southern United States (4). Since then, adequateserum vitamin D levels have been associated with decreasedincidence of colon cancer and decreased mortality in patientsdiagnosed with colon cancer (5, 6). Adequate serum vitamin Dlevels also correlate with reduced risk for developing inflam-

matory diseases, such as Crohn's disease, a risk factor for coloncancer (3, 7).

The risk of colon cancer is increased in patients diagnosedwith IBD (Crohn's disease or ulcerative colitis) compared withthe general population, supporting the notion that colonicinflammation affects cancer development (8). The chronicinflammation seen in IBD, likely due in part to dysregulatedmucosal immune responses to enteric antigens (8), is believedto progress to cancer through the promotion of angiogenesis,tumor-promoting cytokine production, tumor cell–invasivebehavior and cellular proliferation (9).

Vitamin D has a protective effect against colon cancer invarious mouse models (10, 11). However, these cancer models,such as ApcMin/þ mice or mice given a chemical mutagen suchas azoxymethane (AOM), are not driven by inflammation (12–15). Currently, the protective effect of vitamin D on inflam-mation-associated colon cancer is not known.

To investigate the potential chemopreventive effects of ele-vated dietary vitamin D on inflammation-associated coloncancer, we utilized Smad3�/� (Smad3tm1Par/J) mice, which havedefective TGFb signaling due to the absence of the transcriptionfactor Smad3 (16). In humans, the TGFb signaling pathway iscommonly mutated in colon cancer, including colitis-associat-ed colorectal cancer (17). After being infected with an entericmicroorganism, Helicobacter bilis (16), Smad3�/� mice developtransient colitis followed months later by colon cancer makingthem a useful model for studying inflammation-associatedcolon cancer. Using this model, we demonstrate that elevated

1Department of Comparative Medicine, University of Washington, Seattle,Washington. 2Clinical ResearchDivision, FredHutchinsonCancerResearchCenter, Seattle, Washington. 3Division of Gastroenterology, University ofWashington Medical School, Seattle, Washington.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Lillian Maggio-Price, Department of ComparativeMedicine, University of Washington, 1959 NE Pacific Street, Box 357190Seattle, WA 98195. Phone: 206-685-3257; Fax: 206-685-3006; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-2820

�2014 American Association for Cancer Research.

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dietary vitamin D increases serum vitamin D and protectsH. bilis (Hb)-infected Smad3�/� mice from developing coloncancer. These protective effects are mediated through theinteractions between vitaminD and proinflammatory signalingpathways during the early stages of disease development.

Materials and MethodsMice and dietsStudymicewere colony-bred 129-Smad3tm1Par/J (Smad3�/�)

mice (age 6–14 weeks) housed in a specific pathogen-freefacility. Mice were screened for rodent pathogens as previouslydescribed (18) except that sentinels were collected three timesyearly rather than quarterly. In addition to referenced patho-gens, annual screens were performed for minute virus of mice,lymphocytic choriomeningitis virus, and ectromelia virus.Mice were maintained in a Helicobacter and Mouse Noro-virus-free colony as previously described (19). The mice weregroup housed in ventilated cages and fed a purified, irradiateddiet with 1 IU vitamin D (5SRH, maintenance), 5 IU vitamin D(5AAA, high vitamin D), or 0 IU vitamin D per gram (5AV4,AIN93Null). All diets were manufactured by PMI NutritionInternational (LabDiet/TestDiet) based on AIN93M diet that isformulated for the maintenance of rodents' health. All animalprocedures were approved by the University of WashingtonInstitutional Animal Care and Use Committee.

Experimental designWhile no age-associated alterations in endpoint disease

have been noted in our experience with Smad3�/� mice, carewas taken to evenly distribute mice across treatment groupswith regard to age and sex. Independent studies were per-formed to evaluate the effects of high and lowdietary vitaminDlevels on cancer. For high vitamin D studies, mice were startedon eithermaintenance or high vitamin D diets one week beforeinfection. For vitamin D–deficient studies, maintenance orAIN93Null diets were initiated two weeks before infection(Supplementary Fig. S1). For each diet, mice were infectedwith either approximately 2� 107 CFU Hb in Brucella broth orBrucella broth alone (controls) by oral gavage as previouslydescribed (Supplementary Fig. S1; ref. 16). Helicobacter infec-tion status was monitored by fecal PCR using previouslypublished primer sequences (18). Fecal samples were collectedat 3, 6, and 14 days postinfection for subjective fecal scoringand at 7 days postinfection for fecal cytokine analysis. Micewere weighed weekly and monitored at least three timesweekly for dehydration, diarrhea, lethargy, or weight loss.Animals were euthanized by CO2 asphyxiation at the desig-nated end points.

Serum vitamin D and calcium determination and tissuecollectionFollowing euthanasia, blood was obtained via cardiac punc-

ture. Serum samples were submitted to Heartland Assays forquantification of 25-hydroxyvitamin D (radioimmunoassay)and calcium levels. Mesenteric lymph nodes, cecum, colon,and rectum were fixed in 10% phosphate-buffered formalinand processed for routine histologic examination. For study of

the early inflammatory phase, cross-sections of proximal colon(5 mm) were prepared for immunohistochemistry (IHC) andhistology, and cross-sections of proximal colon (5 mm) andcecum (3 mm) were stored in RNA later (Qiagen) for cytokineanalysis by quantitative real-time PCR (qRT)-PCR. For evalu-ation of Hb colonization, 3 cm sections of mid-jejunum,proximal colon, distal colon, and whole cecumwere harvested,rinsed gently in sterile PBS to remove fecalmaterial, and storedat �20�C until DNA extraction. For fecal cytokine analysis,individual fecal samples were collected, homogenized in 250mL RNAlater stabilization solution (Qiagen), and storedat �80�C until RNA extraction. At the time of collection, asubjective fecal score was assigned to each animal, rankingpresence of diarrhea and blood in the stool as a clinicalmeasure of IBD (20).

Histopathology and immunohistochemistryWhole colon and cecumwere evaluated by a board-certified

veterinary pathologist (P.M. Treuting) blinded to experimentalgroups to assess the severity of colitis and incidence of neo-plasia. An overall IBD score was determined as described (21)with the exceptions that scores were summed from cecum,proximal, mid, and distal colons and none were weighted.Analysis of the rectum was included with the distal colon. IBDscores incorporate the severity of mucosal epithelial changes,degree of inflammation, and extent of lesions. A dysplasia scorewas also generated by determining the degree of dysplasiapresent in each of four segments as described (22): cecum,proximal colon, mid colon, and distal colon. For each segment,a score ranging from 0–4 was assigned: 0, none; 1, indefinite; 2,low grade; 3, high grade; and 4, high grade with frank invasionbeyond tunica muscularis and distinguished from mucosalherniation. Cancers were classified as adenocarcinomas andmucinous adenocarcinomas (16, 22). The four individual seg-mental scores were summed to generate the overall dysplasiascore for each animal.

For study of the early inflammatory phase, colonic expres-sion of CD3, F4/80, cleaved caspase-3, and MHCII were eval-uated on transverse cross-sections of proximal colon in ani-mals euthanized 1 week postinfection. IHC staining within themucosa, excluding any gastrointestinal lymphoid tissue, wasscored by a pathologist blinded to groups (P.M. Treuting),using a range of 0–4: 0, no positive cells; 1, few single positivecells; 2, few (3 or less) scattered small clusters of positive cells; 3,many (>3) small clusters of positive cells or larger clusters ofpositive cells; and 4, large or coalescing clusters of positivecells. For early time points, in addition to IHC staining, cecumand serial transverse cross-sections of proximal colon wereevaluated histologically for evidence of IBD.

All IHC staining was performed by Experimental Histopa-thology Services at Fred Hutchinson Cancer Research Center(Seattle, WA). Rat monoclonal antibodies were used to detectMHC class II (I-A/I-E, BD Pharmingen), CD3 (MCA1477, Ser-otec), and F4/80 (MCA497, Serotec). Signals were detectedusing biotinylated goat anti-rat (Jackson ImmunoResearch)followed by streptavidin HRP (Jackson ImmunoResearch).Cleaved caspase-3 antibody (Biocare Medical CP229B) wasfollowed by Mach 2 anti-rabbit HRP-labeled polymer (Biocare

Vitamin D Decreases MAPK Signaling, Colitis, and Cancer

www.aacrjournals.org Cancer Res; 74(16) August 15, 2014 4399




Medical RHRP520L). Staining was visualized with 3,30-diami-nobenzidine (Dako) and counterstained with hematoxylin(Dako). Concentration-matched isotype control slides wererun for each tissue sample (Jackson ImmunoResearch).

Cytokine analysis and Helicobacter quantification byquantitative real-time PCR analysis

For cytokine analysis, RNA was extracted from cecum, colon,and feces using the RNeasy Kit (Qiagen). Fecal RNA sampleswere further concentrated using the Qiagenmini-elution/clean-up kit. RNA was converted to cDNA using SuperScript FirstStrand Synthesis System (Invitrogen) followed by qRT-PCRusing Power Sybr Green Master Mix (Applied Biosystems) anda StratageneMx3005P analyzer (Agilent Technologies). Sampleswere run in duplicate. Cytokine levels were normalized tohypoxanthine-guanine phosphoribosyltransferase (HPRT) andexpressed relative to a control sample (RNA from cecum orproximal colon) that was run on every plate as a calibrator. ForHelicobacter quantification, DNA was extracted from mid-jeju-nal, cecal, proximal colon, and distal colon samples using apreviously published protocol (23). Primer sequences used areoutlined in Supplementary Table S1. Data were analyzed usingStratagene's MxPro v4.10 software (Agilent Technologies).

Epithelial and lamina propria cell preparationsProximal colon (4 cm sections) samples were rinsed in PBS

to remove fecal material, and epithelial and lamina proprialeukocyte (LPL) populations were isolated using the LaminaPropria Dissociation Kit and gentleMACS Dissociator accord-ing tomanufacturer's instructions (Miltenyi Biotec). Cells werepelleted, snap-frozen in liquid nitrogen, and stored at �80�C.

Western blottingProtein was extracted by homogenizing cell pellets in RIPA

buffer (Thermo Fisher Scientific) containing protease andphosphatase inhibitors (Thermo Fisher Scientific). Westernblot analysis was performed according to a previously pub-lished protocol (19) with the following modifications. Proteins(40 mg/lane) were run on gradient (4%–15%) Tris-HCl poly-acrylamide gels (Bio-Rad) and transferred to polyvinylidenefluoride membranes using 100 mmol/L N-cyclohexyl-3-amino-propanesulfonic acid buffer with 10% methanol. Primary anti-bodies used were vitamin D receptor (VDR, SC-1008; SantaCruz Biotechnology), NFkB signaling (Phospho-NF-kB p65(Ser536), and IkBa, L35A5; Cell Signaling Technology), MAPKsignaling (Phospho-MAPK Family, 9910, and MAPK Family,9926, Antibody sampler kits; Cell Signaling Technology), Bcl-xl(54H6; Cell Signaling Technology), and proliferating cell nucle-ar antigen (PCNA, PC10; Cell Signaling Technology). b-Actin(A5441; Sigma-Aldrich) was used as a loading control.

Statistical analysisBefore statistical analysis, distribution of data was assessed

for normality. If data were not normally distributed, transfor-mationwas attempted; if transformation did not normalize thedistribution, nonparametric tests were performed. Serum vita-min D, serum calcium, histologic scoring, fecal scoring, anddensitometries were analyzed using either an unpaired or

Mann–Whitney t test. qRT-PCR data were analyzed using theKruskal–Wallis nonparametric test followed by the Dunn posthoc test to adjust for multiple comparisons. Cancer anddysplasia incidence significance was determined by the Fisherexact test. All data are presented as mean � SEM. Differenceswith a P value of 0.05 or less were considered significant. Allstatistical analyses were performed using GraphPad Prismsoftware (Version 5.04, GraphPad Software Inc).

ResultsIncreased dietary vitamin D significantly increasesserum 25-hydroxyvitamin D without altering serumcalcium levels

Todeterminewhether highdietaryvitaminD increases serumvitaminD status without causing toxicity in Smad3�/�mice, wemeasured serum 25-hydroxyvitamin D and serum calcium inmice fed high vitamin D diet or maintenance diet (control) forone week. The high vitamin D diet significantly increased serum25-hydroxyvitamin D levels without altering serum calcium (25-hydroxyvitamin Dmean: 37.6 vs. 17.6 ng/mL, P¼ 0.016; calciummean: 9.6 vs. 10.6mg/dL,P¼ 0.1), demonstrating that thedietaryregimen rapidly elevated serum vitamin D levels without caus-ing hypercalcemia. Similarly, after 16 weeks on diet, Smad3�/�

mice fed high vitamin D diet had serum vitamin D levels thatwere roughly double that of mice fed maintenance diet (mean:38.4 vs. 14.4 ng/mL, P < 0.0001; Fig. 1A) while serum calciumlevels remained unchanged (Fig. 1B).

Treatment with increased dietary vitaminD significantlyreduces cancer incidence in Hb-infected Smad3�/� mice

Hb-infected mice fed increased dietary vitamin D had asignificantly reduced incidence of invasive colon cancer com-paredwithHb-infectedmice fedmaintenance diet (11% vs. 41%,P ¼ 0.0121; Fig. 1C). No neoplastic lesions developed in unin-fectedmice on either diet (Fig. 1C).Mucinous adenocarcinomaslocated in the proximal colon were the primary neoplasmdiagnosed, as previously noted in this model (Fig. 1D; ref. 16).Well-differentiated mucinous adenocarcinomas were charac-terized by expansile mucin-filled, epithelial-lined cysts thatdisrupt the muscular tunics, serosa and expand into mesentery(Fig. 1D). Consistent with the decreased incidence of invasiveadenocarcinoma, Hb-infected mice fed high vitamin D diethad an average 4-fold decrease in dysplasia scores comparedwith mice fed maintenance diet (mean: 0.71 vs. 2.85, P < 0.001;score range 0–16; Fig. 1E) with a significantly higher percentageof animals with no evidence of dysplasia (83% vs. 40%, P ¼0.0005). Dysplasia was primarily observed in the cecumand proximal colon. Minimal dysplasia was observed in themid colon of mice fedmaintenance diet but not in high vitaminD fed mice. No dysplasia was observed in the distal colonregardless of diet.

Clinical disease and colonic inflammatory cell infiltratesare reduced during the inflammatory phase in Hb-infected Smad3�/� mice fed increased dietary vitamin D

Smad3�/� develop acute inflammation approximately 3 to 7days after Hb infection, which is characterized by diarrhea,frank blood in the stool, dehydration, lethargy, and loss of body

Meeker et al.

Cancer Res; 74(16) August 15, 2014 Cancer Research4400




condition. Clinical signs typically resolve within 7 to 14 daysuntil the time that cancers develop (unpublished observations;ref. 16). To determine the effects of elevated dietary vitamin Don early disease stages, we assessed clinical disease parametersand alterations in inflammatory infiltrates in the colons of Hb-infected mice fed high vitamin D diet compared with Hb-infected mice fed maintenance diet. During the initial inflam-matory period, mice were assigned a subjective fecal score toassess clinical evidence of IBD. Animals fed high vitamin D diethad significantly decreased fecal scores at both 3 and 6 dayspostinfection (mean 0.1 vs. 0.8, P ¼ 0.0015 and 0.2 vs. 1.0 P ¼0.0003, respectively; Supplementary Fig. S2A and S2B) com-pared with animals fed maintenance diet. Broth-treated ani-mals showed no evidence of diarrhea, as expected, and by 14days postinfection all animals had minimal evidence of diar-rhea regardless of diet (Supplementary Fig. S2C). There was nosignificant difference in body weight change associated withdiet following Hb infection (data not shown).To determine whether improved clinical signs associated

with increased dietary vitamin D correlate with decreased

colonic inflammation during early disease, proximal colon, andcecum of mice fed high vitamin D or maintenance diet wereanalyzed for inflammation one week after Hb infection. Hb-infected animals fed high vitamin D diet had significantlyreduced colitis compared with infected animals fed mainte-nance diet (mean 2.7 vs. 15.5, P < 0.0001; Fig. 2 and Supplemen-tary Fig. S3A). Immunohistochemical studies further demon-strated that infectedmice fed high vitaminDdiet had decreasedinflammatory infiltrates compared with those fed maintenancediet [F4/80þ cells (mean � SEM: 2.7 � 0.19 vs. 3.7 � 0.11, P ¼0.0003), CD3þ T cells (mean� SEM: 2.1� 0.15 vs. 2.6� 0.17, P¼0.04), and MHC IIþ cells (mean � SEM: 2.05 � 0.20 vs. 2.85 �0.13, P¼ 0.001); Fig. 2 and Supplementary Fig. S3B–S3D]. Broth-treated controls had minimal inflammation regardless of diet.

Because inflammation is often associatedwith increased cellproliferation, we evaluated if increased dietary vitamin D wasassociated with changes in cell proliferation in either laminapropria or epithelial cell populationswithin the colon oneweekafter Hb treatment. PCNA was used as a marker of cellularproliferation by Western blot analysis. Analysis of PCNA

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Figure 1. Increased dietary vitamin D increases serum vitamin D and decreases dysplasia and cancer inHb-infected Smad3�/�mice. Serum 25-OH Vitamin D(A) and serum calcium (B) were measured 16 weeks after Hb infection in Hb-infected/maintenance (n ¼ 13) vs. high vitamin D (n ¼ 13) diet. ��, P < 0.0001;Mann–Whitney t test. C–E, colon and cecum were analyzed at 16 weeks after Hb infection for histopathologic evidence of invasive adenocarcinoma anddysplasia. C, cancer incidence is reduced in mice fed high vitamin D diet (�, P ¼ 0.0121; Fisher exact test). D, Hb-infected mice typically developgrossly visible tumors in the cecum or proximal colon as represented by the pale, multilobulated mass in the proximal colon (�). Note the mucin lakesand neoplastic epithelial cells penetrating the colonic wall and proliferating within the muscularis and serosa. H&E staining; original magnification,�20. Inset, subgross of the whole colon section; blue box, magnified region. E, Hb-infected animals fed high vitamin D diet had decreased mean dysplasiascores and an increased number of animals with no evidence of dysplasia. �, P < 0.01; ��, P < 0.001.






showed no significant differences associated with diet in eithercell population (LPLmean 0.73 vs. 0.67, epithelial cellmean 0.99vs. 0.85, high vitamin D diet vs. maintenance diet respectively,data not shown).

High dietary vitamin D decreases cecal, proximal colon,and fecal proinflammatory cytokines 1 week after Hbinfection

Because we previously observed elevated inflammatorycytokines in this model associated with Hb infection (16),proinflammatory cytokine expression in cecal and proximalcolon tissues one week after Hb infection were evaluated todetermine whether increased vitamin D would dampen theinflammatory response induced by infection. Increased expres-sion of Il1b, macrophage chemotaxis factor 1a (Mip1a), Il6,Tnfa, and Ifng were noted in cecal tissue from Hb-infectedanimals fed maintenance diet compared with broth-treatedcontrols (Fig. 3) as seen previously with this model (16).However, there was a significant reduction in expression ofthose same cytokines inHb-infectedmice fed increased dietaryvitamin D compared with those fedmaintenance diet (Fig. 3A–E). Interestingly, there were no changes in expression of theanti-inflammatory cytokine, Il10 in response to diet or Hbinfection (Fig. 3F). Expression patterns of proinflammatorycytokines in the proximal colon were similar to those observedin cecal tissue (Supplementary Fig. S4A–S4F).

Fecal cytokines have been used in the Hb-infectedSmad3�/� mouse model to characterize the inflammatory

response and predict development of cancers (24). To deter-mine whether fecal cytokine expression correlated withtissue cytokine expression, expression of Il1b and Mip1awas evaluated in fecal pellets collected from animals 1, 2,and 3 weeks after Hb infection. Similar to the expressionpattern of Il1b and Mip1a observed in cecal and proximalcolon tissues at 1 week after infection (Fig. 3A and B andSupplementary Fig. S4A and S4B), expression of Il1b andMip1a in feces were significantly increased in Hb-infectedanimals fed maintenance diet compared with broth-treatedanimals (Supplementary Fig. S4G and S4H). Accordingly, Hb-infected mice fed high vitamin D diet had an average 3-folddecrease in fecal Il1b and 1.5-fold decrease in fecal Mip1aexpression compared with mice fed the maintenance diet.However, these fecal cytokine changes were transient asthere were no significant differences in Il1b and Mip1aexpression between any treatment group at 2 or 3 weeksafter infection (data not shown).

Increaseddietary vitaminDdecreasesp-P38MAPK in thecolon

Helicobacter species have been shown to elicit proinflam-matory cytokine production through TLR4-dependent activa-tion of the MAPK and NFkB pathways (25). Thus, we deter-mined whether decreased colonic inflammation in Smad3�/�

mice in response to increased dietary vitaminDwas associatedwith altered MAPK and NFkB signaling pathways during earlydisease (1 week after Hb infection).

Figure 2. Increased dietary vitamin D decreases proinflammatory infiltrates 1 week after Hb infection. Serial sections of paraffin-embedded proximalcolon were stained with H&E for orientation and immunohistochemically for F4/80, CD3, and MHCII antigen. Images from representative samples werecaptured at the same low (�4; scale bars, 500 mm) and high (�10; scale bars, 200 mm; all bottom rows have the same magnification) magnifications. Broth-treated controls fed maintenance diet are shown. No significant differences were noted between maintenance and high vitamin D diet groups treated withbroth.

Meeker et al.





For changes in the MAPK pathway, relative levels of proteinexpression of activated forms of P38 (p-P38), JNK (p-JNK), andERK1/2 (p-ERK) in LPL and colonic epithelial cell populationswere determined. LPLs of Hb-infected animals fed increaseddietary vitamin D had a 7-fold decrease in p-P38 comparedwith maintenance diet-fed animals (P ¼ 0.012, Fig. 4A andSupplementary Fig. S5). Interestingly, similar changes were notdetected in colonic epithelial cells (Fig. 4A and SupplementaryFig. S5). There was a trend toward decreased p-JNK expressionin colonic tissue from animals fed increased dietary vitamin D;however, these differences were not statistically significant

(LPL: 4.5-fold decrease, P ¼ 0.14 and epithelial cells: 2-folddecrease, P¼ 0.052; Fig. 4B and Supplementary Fig. S5). Therewere no notable differences in p-ERK1/2, total P38, or total JNKin either cell population (data not shown).

For alterations in the NFkB pathway, phosphorylated P65(p-P65) was evaluated by Western blot analysis in LPL andepithelial cell populations. Mice fed high vitamin D diet had a4.5-fold decrease in p-P65 in colonic epithelial cells comparedwith mice fed maintenance diet (P ¼ 0.028, Fig. 4C andSupplementary Fig. S5). We did not detect p-P65 in LPLregardless of diet while IkBa, an inhibitor of NFkB activation,

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Figure 3. High dietary vitamin Ddecreases cecal tissue expressionof proinflammatory andchemotactic cytokines1weekafterHb infection. Expression of Il1b (A),Mip1a (B), Il6 (C), Tnfa (D), Ifng (E),and Il10 (F) in cecal tissue fromSmad3�/� mice fed high vitamin Dormaintenance diet with or withoutHb infection was determined byqRT-PCR. Hb-infected/maintenance diet (n ¼ 20),Hb-infected/high vitamin D diet(n ¼ 20), broth-treated/maintenance diet (n ¼ 10), andbroth-treated/high vitamin D diet(n¼10). Notedifferences in scale inD and E. Kruskal–Wallisnonparametric test and Dunn posthoc test �, P < 0.05; ��, P < 0.001.






was present in both LPL and colonic epithelial cells. (Supple-mentary Fig. S5).

To determine whether the changes in p-P38 or p-P65 wereassociated with decreased Tlr4 expression, qRT-PCR wasperformed on cecal tissues collected from mice 1 week afterHb infection. Average Tlr4 expression in Hb-infected animalswas modestly increased (1.3-fold) compared with broth-trea-ted controls (P < 0.01; Fig. 4D). Although Tlr4 expression inHb-infected mice was lower when fed high vitamin D diet, thedifference was not statistically significant.

High dietary vitamin D does not alter Hb colonization inSmad3�/� mice

Changes in the gut microbiome or changes in bacterial loadcan influence disease severity in both human patients (26) andanimal models of IBD (26, 27). As Tlr4 expression levels tend tobe lower in high vitamin D-fed Smad3�/� mice following Hbinfection, we determined whether vitamin D alters Hb colo-nization in cecum and colon where they preferentially reside(16). qRT-PCR was used to compare the relative amount ofHelicobacter organisms in cecal, proximal, and distal colonictissues collected 8 weeks after Hb infection from mice fedeither increased or maintenance levels of vitamin D. While

cecal tissues had the highest concentration of Helicobacter aspreviously reported (16), no significant differences in Helico-bacter colonization were associated with diet (SupplementaryFig. S6).

High dietary vitamin D does not alter cecal expression ofvitamin D receptor or enzymes involved in vitamin Dmetabolism

To determine whether the protective effect of increaseddietary vitamin D was associated with changes in proteinsinvolved in vitamin D signaling and/or metabolism, qRT-PCRwas used to evaluate RNA expression of vitamin D receptor(Vdr) as well as two enzymes involved in conversion of vitaminD into its active and inactive forms, 25(OH)D3-1a-hydroxylase(Cyp27b1), and 1, 25(OH)2D3 24 hydroxylase (Cyp24a1) in cecaltissue at one week after Hb infection. A small yet significantincrease in VDR expression was detected in mice fed highvitamin D diet following Hb infection (Fig. 5A). However, therewere no changes in mRNA levels of Cyp27b1 and Cyp24a1associated with either diet or infection status (Fig. 5B and C).Although VdrmRNA was altered with diet, differences in VDRprotein expression were not detected using Western blotanalysis of proximal colon tissue (data not shown).

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Figure 4. Increased dietary vitaminD decreases MAPK and NFkBsignaling in Hb-infected Smad3�/�

mice. Whole-cell lysates wereisolated from proximal colon LPLand epithelial cells harvested 1weekafterHb infection. Expressionlevels of p-P38 (A), p-JNK (B), andp-P65 (C) were determined byWestern blot analysis anddensitometry. Densitometryresults of each protein level werenormalized to b-actin. An unpairedt test; �, P < 0.05; ��, P < 0.001.Hb-infected on maintenance diet(n ¼ 4) and Hb-infected on highvitamin D diet (n ¼ 4). D, colonicexpression of Tlr4 was determinedby qRT-PCR from mice fed highvitamin D or maintenance diet andinfected with and without Hb. Hb-infected/maintenance diet (n¼ 20),Hb-infected/high vitamin D diet(n ¼ 20), broth-treated/maintenance diet (n ¼ 10), andbroth-treated/high vitamin D diet(n ¼ 10). Kruskal–Wallisnonparametric test and Dunn posthoc test (�, P < 0.05;��, P < 0.001).

Meeker et al.





A vitamin D–deficient diet did not exacerbate colitis orcolitis-associated colon cancer in Hb-infected Smad3�/�

miceBecause decreased serum vitamin D is associated with an

increased risk for developing IBD as well as colon cancer inhumans (4–7), we hypothesized that a diet deficient in vitaminDwould exacerbate inflammation and potentially increase theincidence of colon tumors in Hb-infected Smad3�/� mice. Wetested this hypothesis by feedingmicemaintenance diet or dietdevoid of vitamin D (AIN93Null) and induced inflammation byHb infection. Serum 25-hydroxyvitamin D levels were signifi-cantly decreased in AIN93Null-fed mice compared with micefedmaintenance diet (mean: 5.7 vs. 12.2 ng/mL,P¼ 0.01) after 2weeks on the diet and were below the limit of detection at theend of the 16-week study (Fig. 6A and B). Despite decreasedserum vitamin D levels, mice maintained on AIN93Null dietshowed no differences in serum calcium compared with main-tenance diet–fed controls (Fig. 6C). Clinical disease during theearly inflammatory phase was assessed by monitoring subjec-tive fecal scores and body weight change following Hb infec-tion. Mice maintained on AIN93Null diet showed no differ-ences in fecal scores or body weight change compared withmaintenance diet–fed controls (data not shown). To determinewhether decreased dietary vitamin D exacerbated IBD at earlydisease stages, proximal colon and cecal tissue of mice fedmaintenance diet or AIN93Null were analyzed for inflamma-tion one-week after Hb infection. There were no differences inIBD associated with diet (Fig. 6D). To further evaluate anyeffect of AIN93Null diet, proinflammatory cytokine expressionin proximal colon tissues was measured to determine whetherdecreased dietary vitamin D would exacerbate the inflamma-tory response induced by Hb. In correlation with the lack ofeffect on IBD scores, no differenceswere noted in cytokine geneexpression associated with diet (Supplementary Fig. S7). Con-sistent with the lack of altered inflammation early in disease,there were no differences in cancer incidence or dysplasianoted between AIN93Null-fed mice compared with mainte-

nance diet–fed animals (Fig. 6E and F) when necropsied after16 weeks of Hb infection.

DiscussionUsing Smad3�/� mice, we have shown that increased

dietary vitamin D affords protection against the developmentof colon cancer. In this model, we have demonstrated thatincreased dietary vitamin D (i) induces elevated serum 25-hydroxyvitamin D without causing hypercalcemia, (ii) signif-icantly decreases inflammation, dysplasia, and tumor inci-dence following infection with Hb, and (iii) is associated withdecreased p-P38 (MAPK) and p-P65 (NFkB) expression dur-ing the acute inflammatory stage of disease. These studiesprovide evidence that the protective effect(s) of elevateddietary vitamin D supplementation in a model of inflamma-tion-associated colon cancer are mediated through suppres-sion of the inflammatory responses triggered following infec-tion with colitogenic bacteria.

Animals fed increased concentrations of dietary vitamin Ddemonstrated significant protection against inflammation andtumor formation. It should be noted that though serumvitamin D levels are increased in mice fed high vitamin D dietcompared with those fed maintenance diet, levels remainwithin the comparable recommended range for humans with-out reaching superphysiologic levels (28). Many animal andhuman studies that have shown antitumor effects of vitamin Dadminister metabolically active 1,25(OH)2D3, which results inhypercalcemia and vitamin D toxicities (10, 12, 15). Our studiesshow that dietary vitamin D supplementation offers a way toimprove vitamin D status and provide protection from inflam-mation-associated colon cancer while avoiding vitamin Dtoxicity.

Vitamin D supplementation decreases cancer incidence inseveral rodent models of colon cancer (12–15). These modelsrely on either genetic predisposition for the development ofgastrointestinal neoplasias as is the case withAPCMin/þmice or

Vdr Cyp27b1 Cyp24a1A B C2.5

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Figure 5. Increased dietary vitamin D increases Vdr mRNA expression but does not alter enzymes involved in vitamin D metabolism (Cyp27b1 and Cyp24a1).Expression levels of Vdr (A), Cyp27b1 (B), and Cyp24a1 (C) were determined with qRT-PCR using RNA from cecum (3 mm piece) isolated from mice1 week after Hb infection. Hb-infected/maintenance diet (n ¼ 20), Hb-infected/high vitamin D diet (n ¼ 20), broth-treated/maintenance diet (n ¼ 10), and broth-treated/high vitamin D diet (n ¼ 10). Kruskal–Wallis nonparametric test and Dunn post hoc test; ��, P < 0.001.






treatment with a chemical mutagen such as AOM to simulatethe adenoma to carcinoma progression that occurs during thedevelopment of sporadic colon cancer (8, 29). However, themolecular changes, disease progression, and pathology ofinflammation-associated cancer are distinct from thosechanges observed in sporadic or familial colon cancers andtherefore may influence the efficacy of vitamin D as well asmechanisms through which it promotes protection fromtumor formation.

Chronic inflammation is believed to frequently play a keyrole in carcinogenesis (9). Links between inflammation andcancer have not only been observed in colon cancer in humanpatients with IBD (8) but also in liver, pancreatic, stomach,esophageal, and prostate cancers (9). Several studies utilizingmouse models of colitis have shown that vitamin D can bebeneficial in preventing or ameliorating inflammation andclinical disease (30–32); however, thesemodels do not typicallyprogress to neoplasia. Recently, the DSS/AOM model, anothermodel of inflammation-associated cancer that does progressto dysplasia and tumor formation, was used to demonstratethat increasing concentrations of dietary vitamin D are pro-tective against preneoplasic lesions in a dose-dependent man-ner (33). Consistent with these findings, we have shown that

increased dietary vitamin D is effective at not only preventinginflammation and dysplasia, but subsequent invasive tumorformation as well.

Chronic inflammation is associated with increased pro-duction of proinflammatory cytokines, including TNFa,IL1b, and IL6, which contribute to carcinogenesis throughinfluences on cell proliferation, apoptosis, differentiation,and angiogenesis (34). Dietary vitamin D supplementationsignificantly lowered inflammatory cytokines induced inresponse to Hb in Smad3�/� mice. In human patients withcolon cancer, these proinflammatory cytokines are positivelyassociated with increased cancer growth, higher neoplasticgrade, and increased risk of mortality (34). Proinflammatorycytokines are also upregulated in patients with IBD, evenbefore the onset and progression to dysplasia or neoplasia(35), and vitamin D supplementation has been linked todecreased circulating proinflammatory cytokines in patientswith colorectal adenomas (36). Epidemiologic evidence sug-gests that treatments that limit inflammation may be ben-eficial in reducing the incidence of inflammation-associatedcolon cancer in high risk populations (37). Together, thesedata suggest that dietary vitamin D may be effectiveat decreasing the proinflammatory milieu in patients with

Invasive tumor incidence Dysplasia

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Figure 6. AIN93Null diet does not exacerbate Hb-induced disease in Smad3�/� mice. Serum 25-hydroxyvitamin D was measured after two weeks (A) or 18weeks (B) on diet. Serum calcium levels (C) were determined at 18 weeks after diet initiation. Mann–Whitney t test. �, P < 0.05; ��, P < 0.001. One week afterHb infection, cecum and proximal colon were histologically scored for inflammation (D). Hb-infected/maintenance diet (n ¼ 14), Hb-infected/AIN93Nulldiet (n¼15), broth-treated/maintenancediet (n¼7), andbroth-treated/AIN93Null diet (n¼7).Mann–Whitney t test; ��,P<0.001. Sixteenweeks after infection,colon and cecum were analyzed for cancer incidence (E) and dysplasia (F). Incidence of no dysplasia is indicated by percent near brackets (F). Hb-infected/maintenance diet (n ¼ 20), Hb-infected/AIN93Null diet (n ¼ 20), broth-treated/maintenance diet (n ¼ 10), and broth-treated/AIN93Null diet (n ¼ 10).

Meeker et al.





IBD and serve as a useful adjunct treatment in certainpopulations.The mechanism by which vitamin D suppresses colon

cancer in Smad3�/� mice is not completely clear, althoughour data suggest the anti-inflammatory effects of vitamin D areimportant. Helicobacter species are Gram negative, microaer-ophilic bacteria that can induce local production of proin-flammatory cytokines and chemokines through TLR4 signalingand subsequent activation of the MAPK and NFkB pathways(25). Both of these pathways have been shown to be upregu-lated in human patients with IBD (38) and are thought to beimportant links between inflammation and cancer (38–40). Invitro evidence suggests that vitamin D is able to suppressMAPK activity and subsequent proinflammatory cytokineproduction through the upregulation of MAPK phosphatase-1 (41) andNFkB signaling through the upregulation of IkBa, aninhibitor of NFkB activation (42), or through decreased expres-sion of the NFkB component RelB, which can lead to inhibitionof dendritic cell differentiation andmaturation (43). During theearly inflammatory disease phase in Hb-infected Smad3�/�

mice, vitamin D–high diet was associated with dramaticdecreases in p-P38 in the lamina propria cells, decreased NFkBactivation in epithelial cell populations, and suppressed proin-flammatory cytokine expression compared with that observedin infected mice on maintenance diet. On the basis of thesedata, we propose a model where vitamin D suppresses inflam-mation by decreasing P38 MAPK activation in lamina propriacells, resulting in decreased proinflammatory cytokine pro-duction by those cells, which in turn decreases NFkBactivationin colonic epithelial cells.We have shown that while increased dietary vitamin D

affords protection against the development of colon cancer,decreased dietary vitamin D was not sufficient to exacerbatedisease in Smad3�/� mice. Because epidemiologic studies(5, 7, 30) as well as studies utilizing mouse models of colitissuggest that vitamin D deficiency or lack of vitamin D signalingcan exacerbate IBD (44–46), we hypothesized that decreaseddietary vitamin D would exacerbate disease in the Smad3�/�

mouse model. Interestingly, although the AIN93Null diet sig-

nificantly depleted circulating serum 25-hydroxyvitamin Dlevels, we did not see exacerbation of Hb-induced inflamma-tion or subsequent inflammation-associated colon cancer.These findings are consistent with the idea that modulationof inflammation is likely responsible for the protectionafforded by increased dietary vitamin D.

In conclusion, increased dietary vitamin D suppresses acuteinflammation and consequently neoplastic development in amouse model of bacterial-driven colon cancer. While addi-tional studies are needed to elucidate the molecular mechan-isms through which vitamin D and TGFb interact to affordprotection in this model, these findings suggest that vitamin Dsupplementation may prove useful in the treatment of IBD orpotentially the prevention of inflammation-associated cancerby limiting inflammation early in disease development.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Meeker, A. Seamons, J. Paik, P.M. Treuting, T. Brabb,W.M. Grady, L. Maggio-PriceDevelopment ofmethodology: S.Meeker, A. Seamons, P.M. Treuting, T. Brabb,L. Maggio-PriceAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.Meeker, A. Seamons, J. Paik, P.M. Treuting, T. Brabb,L. Maggio-PriceAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Meeker, A. Seamons, J. Paik, P.M. Treuting,W.M. Grady, L. Maggio-PriceWriting, review, and/or revision of the manuscript: S. Meeker, A. Seamons,J. Paik, P.M. Treuting, T. Brabb, W.M. Grady, L. Maggio-PriceAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Maggio-PriceStudy supervision: S. Meeker, A. Seamons, J. Paik, W.M. Grady, L. Maggio-Price

Grant SupportThis work was supported by grants AICR 09A136-Rev (L. Maggio-Price),

NIH 5T32DK007742-17 (John Inadomi, PI), and NIH R21 CA149995-01A1(L. Maggio-Price).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 30, 2013; revised May 13, 2014; accepted June 2, 2014;published OnlineFirst June 17, 2014.

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Meeker et al.




2014;74:4398-4408. Published OnlineFirst June 17, 2014.Cancer Res Stacey Meeker, Audrey Seamons, Jisun Paik, et al. and Colon CancerIncreased Dietary Vitamin D Suppresses MAPK Signaling, Colitis,

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Increased Dietary Vitamin D Suppresses MAPK Signaling ... · otec), and F4/80 (MCA497, Serotec). Signals were detected using biotinylated goat anti-rat (Jackson ImmunoResearch) followed