APhaseIbStudyoftheEffectsofBlackRaspberriesonRectal Polyps ...€¦ · Polyps in Patients with Familial Adenomatous Polyposis Li-Shu Wang1, Carol A. Burke3, Henrietta Hasson3, Chieh-Ti

Research Article

APhase IbStudyof theEffectsofBlackRaspberriesonRectalPolyps in Patients with Familial Adenomatous Polyposis

Li-Shu Wang1, Carol A. Burke3, Henrietta Hasson3, Chieh-Ti Kuo1, Christine L. Sardo Molmenti6,Claire Seguin4, Pengyuan Liu2, Tim H.-M. Huang7, Wendy L. Frankel5, and Gary D. Stoner1

AbstractFamilial adenomatous polyposis (FAP) is characterized by the early onset of colonic polyposis and a

high risk for colorectal cancer. FAP is treated by colectomy followed by lifelong removal of rectal polyps.

This study determined whether black raspberries (BRBs) might regress rectal polyps in patients with FAP.

Fourteen patients with FAP were treated with BRBs daily for 9 months. Seven patients received BRB powder

orally plus two BRB suppositories inserted into the rectum at bedtime. The other 7 received an oral placebo

plus the suppositories. Rectal polyp counts and polyp sizes were obtained at time zero and after 9 months

of BRB treatment. Polyps and adjacent normal tissue were collected at both time points. The burden (P ¼0.036) but not number (P¼ 0.069) of rectal polyps was significantly decreased. No benefit was noted with

the addition of oral BRBs. Three patients were nonresponders. BRBs significantly decreased cellular

proliferation, DNA methylation methyl transferase 1 protein expression, and p16 promoter methylation,

but not promoter methylation of the Wnt pathway antagonists, SFRP2 and WIF1, in rectal polyps

(adenomas) from responders but not from nonresponders. The MBD-seq assay revealed more demethy-

lated transcription start sites (TSS), including those for miRNAs, in BRB-treated adenomas from the

responders. In conclusion, BRB suppositories seem sufficient for regressing rectal polyps in patients with

FAP. Cancer Prev Res; 7(7); 666–74. �2014 AACR.

IntroductionFamilial adenomatous polyposis (FAP) is characterized

by colonic polyposis and a lifetime risk of subsequentcolorectal cancer of nearly 100% due to inherited germlinemutations in the adenomatous polyposis coli (APC) gene.Total abdominal colectomy with ileorectal anastomosis ortotal proctocolectomy with ileal pouch anal anastomosisare the traditional management strategies for colonic poly-posis. Lifelong endoscopic surveillance of the rectum isrequired for the management of recurrent polyposis and

does not obviate the development of uncontrolled rectalpolyposis or rectal cancer thatmay require proctectomy (1).

Nonsteroidal anti-inflammatory drugs (NSAIDs) werefirst reported to cause regression of colonic polyps in sub-jects with FAP over two decades ago. Case series and manyrandomized controlled trials have confirmed this observa-tion for nonselective NSAIDs such as sulindac (2–6). How-ever, the gastrointestinal toxicity of nonselectiveNSAIDs ledto thedevelopment of selectiveCOX-2 inhibitors. Celecoxiband rofecoxib have been shown in randomized controlledtrials to induce regression of colonic adenomas in patientswith FAP (7, 8). Celecoxib is the FDA approved for theregression of adenomatous colorectal polyps in patientswith FAP, as an adjunct to standard endoscopic manage-ment. Unfortunately, the increased risk of cardiovascular,thromboembolic, and cerebrovascular events led to thewithdrawal of rofecoxib from the market and remains aconcern for celecoxib (9). Therefore, an effective chemo-preventive agent with no or minimal systemic toxicitywould be a substantial advance for patients with FAP.

In one small study, the combined use of the dietarysupplements curcumin and quercetin led to a reduction inthe number and size of rectal polyps in patients with FAPwith minimal side effects or toxicity (10). Black raspberries(BRBs) are a source of multiple nutritive and nonnutritivecompounds, including vitamins A, C, E, and folic acid;calcium and selenium; b-sitosterol, ellagic acid, ferulic acid,quercetin, and several anthocyanins (11). Many of thesecompounds have demonstrated chemopreventive activity,

Authors' Affiliations: 1Division of Hematology andOncology, Departmentof Medicine; 2Department of Physiology, Medical College of Wisconsin,Milwaukee, Wisconsin; 3Department of Gastroenterology and Hepatology,ClevelandClinic, Cleveland; 4ComprehensiveCancer Center; 5Departmentof Pathology, Ohio State University, Columbus, Ohio; 6Department ofEpidemiology, Columbia University, New York, New York; and 7Depart-ment of Molecular Medicine, Cancer Therapy and Research Center, Uni-versity of Texas Health Science Center, San Antonio, Texas

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

L.-S. Wang and C.A. Burke share first authorship.

The clinical trial of the study agent was performed under IND #: 73,366to the Cleveland Clinic Foundation.

Corresponding Author: Gary D. Stoner, Medical College of Wisconsin,8701 Watertown Plank Road, TBRC Room C4815, Milwaukee, WI 53226.Phone: 414-955-3618; Fax: 414-955-6059; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-14-0052

�2014 American Association for Cancer Research.

CancerPreventionResearch

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both in vitro and in animalmodels; for example, ellagic acidand the anthocyanins (11). Two mouse models of colorec-tal cancer were used to evaluate the effects of BRBs oncolorectal tumor development and to investigate the under-lying mechanisms (12, 13). A 12-week feeding of BRBssignificantly inhibited intestinal tumor formation in bothmodels; reducing tumor incidence by 45% and tumormultiplicity by 60% in Apc1638þ/� mice and tumor inci-dence and multiplicity by 50% in Muc�/� mice (12).Mechanistic studies showed that BRBs inhibit tumor devel-opment in Apc1638þ/� mice by suppressing b-cateninsignaling and in Muc�/� mice by reducing chronic inflam-mation (12). Intestinal cell proliferation was reduced inboth mouse models by BRBs; however, mucus differentia-tion was not affected in either model (12). Feeding of BRBsat 5% and 10% of the diet to F-344 rats for 32 weeks aftertreatment of the rats with the carcinogen, azoxymethane,resulted in a reduction of adenocarcinoma multiplicity of35% and 80%, respectively (13). In addition, dietaryadministration of BRBs has been shown to inhibit esoph-ageal, oral cavity, and mammary tumorigenesis in animalmodels (11). In humans, BRBs have been shown to be welltolerated and exert protective effects in multiple phase Ihuman clinical trials in patients with colorectal cancer (14),Barrett’s esophagus (15), and oral dysplasia (16, 17).We recently reported that dietary BRBs hypomethylate

p16 andWnt signaling pathway inhibitor genes in colorectaladenocarcinomas (14). This effect was associated withreduced activity of the enzyme, DNA methylation methyltransferase 1 (DNMT1) in colorectal tumors (14). DNMT1is chiefly responsible for DNA methylation homeostasis,whereas two other methyl transferases; i.e., DNMT3a andDNMT3b, primarily catalyze de novo hypermethylation(18). All three enzymes, DNMT1, DNMT3a, and DMNT3b,have been shown to be overexpressed in several tumortypes, including colorectal cancer (14, 19). Interestingly,both sporadic and FAP-related colon adenocarcinomashave common, for example, p16 and MGMT, as well asdifferent, for example, PAX3, tumor-suppressor genes thataremethylated, andhypermethylation seems tobe a generalfeature of both sporadic and FAP-related carcinomas (20,21). In this study, we determined whether BRBs adminis-tered in the form of a rectal suppository and/or in combi-nation with oral administration cause suppression of polypdevelopment in subjects with FAP, and whether BRBs affectDNA methylation in tissues collected from these patients.

Materials and MethodsFreeze-dried BRB powder, placebo powder, and BRBsuppositoriesThe procedures for the preparation of BRB powder for use

in clinical trials have been described in detail (11) and aregiven in Supplementary Materials and Methods. A 100-gsample of the berry powder was analyzed for content ofnutritive and nonnutritive constituents, most with knownchemopreventive potential, by Covance Laboratories andthese constituents are listed in Supplementary Table S1.BRBs contain four major polyphenolic anthocyanins that

are responsible for the color of the berries. Their content inthe powder was: cyanidin-3-glucoside (278.5 mg/100 g dryweight [DW]), cyanidin-3-sambubioside (56.0 mg/100 gDW), cyanidin-3-rutinoside (1,790 mg/100 g DW), andcyanidin-3-xylosylrutinoside (853.5 mg/100 g DW), result-ing in a total anthocyanin content of 2,978 mg/100 g DW.Wehave shownpreviously that the anthocyanins inBRBs arechemopreventive in a rat model of esophageal cancer (22).

The placebo agent was a purple colored powder thatcontained maltodextrin (75%) and dextrose (25%). Malto-dextrin is a nonsweet saccharide mixture that is freelysoluble in water. Dextrose is a water-soluble monosaccha-ride that is not as sweet as sugar. BRB suppositories wereprepared by the Central Ohio Compounding Pharmacyusing the BRB powder described above. Each BRB suppos-itory contained 720-mg BRB powder mixed in wax.

Clinical trialPatients with FAPwith at least 5,�2-mm rectal polyps on

baseline endoscopy and who met the other inclusion cri-teria were eligible to participate. The subjects were random-ly assigned to one of two treatment arms. The investigatorsand patients were blinded to the oral treatment arm assign-ment (Supplementary Fig. S1). This study was approved bythe Institutional Review Boards of the Cleveland Clinic andTheOhio StateUniversityMedicalCenter (Columbus,OH).

Treatment Schema: Arm 1 (N ¼ 7), 20 g of placebopowder administered as an oral placebo (maltodextrin/dextrose) slurry three times per day (60 g/d total), plus twoBRB rectal suppositories (each containing 720-mg BRBpowder) administered at bedtime. Arm 2 (N ¼ 7): 20 g ofBRB powder administered orally three times per day (60 g/dtotal), plus two BRB rectal suppositories administered atbedtime. Photographs of a coffee cup containing BRBpowder in water and of the rectal suppositories are shownin Supplementary Fig. S2.

Inclusion criteria. Patients accrued to the trial met thefollowing criteria: (i) A willingness and ability to giveinformed consent; (ii) �18 years of age; (iii) nonpregnantand nonlactating female subjects either surgically sterile,postmenopausal, or using adequate birth control; lactatingfemale subjects who chose to stop breastfeeding, or, malesubjects; (iv) negative urine pregnancy test (if applicable);(v) diagnosis of FAPwith at least 5,�2-mm rectal polyps onbaseline endoscopy; (vi) have an endoscopically accessiblerectal segment; (vii) have not taken chronic NSAIDs orselective COX-2 inhibitors for 2 months before participa-tion in the study andwilling to remain off NSAIDs for studyduration. Discontinuation of NSAIDs or selective COX-2inhibitors required approval by the physician treating thepotential study participant; (viii) willingness to travel tostudy center for initial study procedures and for the 36-weekevaluation; and (ix) have adequate home freezer storage fora 9-week supply of the BRB formulations.

Exclusion criteria. Patients excluded from the trial hadone of the following: (i) known allergies or hypersensitivityto berries, including BRBs; (ii) subjects taking NSAIDs orCOX-2 inhibitorswho could not be takenoff themedication

Berries Regress Rectal Polyps in Patients with FAP

www.aacrjournals.org Cancer Prev Res; 7(7) July 2014 667




due to their clinical condition; (iii) pregnant or lactatingsubjects; (iv) active peptic ulcer disease; (v) any gastrointes-tinal problem that in the opinion of the investigator wouldaffect absorption (including chronic use of antacids) orability to consume the oral preparations of BRBs; (vi) clin-ically significant hepatic or renal dysfunction; (vii) diabetesmellitus; (viii) on baseline evaluations: AST > 1.5 � upperlimit of normal, ALT > 1.5� upper limit of normal, alkalinephosphatase > 1.5� upper limit of normal, total bilirubin >2� upper limit of normal, serum creatinine or BUN > 1.5�upper limit of normal.

Distribution of powders and suppositories and monitoringfor toxicity and compliance. BRBs and placebo powders fororal consumption were packaged in hermetically sealedaluminum bags each containing 20 g of powder by theCentral Ohio Compounding Pharmacy. Two-month sup-plies of either berry or placebo powder and of the BRBsuppositories were sent by the Compounding Pharmacy tosubjects in the trial. The subjects were instructed to store thebags and the suppositories in a freezer (�20�C) uponreceipt, and to transfer the number of bags and supposito-ries required for one week’s use from their freezer to arefrigerator. Research personnel from the Central OhioCompounding Pharmacy telephoned each subject at 7 to10 days after randomization to the trial to evaluate com-pliance and adverse event monitoring. Subsequent evalua-tions were done at monthly intervals by phone. Subjectswere given instructions onhow to complete adiary to recordthe date and time the berry or placebo powder was con-sumed and also to record the date and time the twosuppositories were inserted.

Polyp size, number, and tissue collection. Subjects under-went flexible sigmoidoscopy before study treatment (base-line) and at end of study (EOS) at 36weeks (þ10 days) afterthe initiation of study treatment. One endoscopist per-formed all endoscopic examinations (C.A. Burke). The sizeand number of rectal polyps between the anal verge andileorectal anastomosiswere counted at each visit andphoto-documented. The size of each polyp wasmeasured by usingan open or closed biopsy forceps. The polyp burden wascalculated as the sum of diameters of all adenomas�2mm.Indigo carmine dye spray was used after polyp counting todistinguish normal mucosa from polyps. All polyps over 1cm were removed at baseline and sent for local histologicassessment. Up to two polyps were removed for biomarkerstudies at baseline. Biopsies were obtained of normal rectalmucosa (�8) at baseline and end of study. All rectal polypswere harvested at the endof the study. Tissueswere placed in10% buffered formalin for histologic analysis. Any polypsremoved at baseline were accounted for in the analysis. Thedifference in the number of �2-mm rectal polyps betweenbaseline and end of study was compared in those subjectsthat completed 36 weeks of therapy (after adjustment forbaseline polyp removal). All tissue specimens were classi-fied histologically by a pathologist (W.L. Frankel) withinterest in gastrointestinal diseases. All rectal polyps wereclassified as tubular adenoma (hereafter referred to asadenoma). Histologically confirmed paraffin-embedded

rectal polyps were used for immunohistochemical analysesand for subsequent molecular analysis.

Laboratory analysesImmunohistochemical staining and computer-assisted

image analysis. Baseline and EOS adjacent normal tissueand adenomas were cut into 4-mm sections and placed onslides. Methods for staining and quantification of Ki-67,cMyc, p16, DNMT1, DNMT3b, or TUNEL are detailed inSupplementary Materials and Methods. The commercialsources of the antibodies are given in SupplementaryTable S2.

Stained tissue was viewed and photographed at �200magnificationwith a bright-fieldmicroscopemountedwitha high-resolution spot camera. The camera was interfacedwith a computer containing a matrix frame grabber boardand image analysis software (Simple PCI Imaging Systems;Compix Inc.) as described before (14).

Analysis of DNA methylation. DNA extraction and bisul-fite conversion. Paraffin-embedded tissueswere cut into 10-mm sections and DNA was extracted using a PicoPure DNAKit (MDS Analytical Technologies). Extracted DNA waspurified using the QIAquick PCR purification Kit (Qiagen).Of note, 500 ng of extracted DNA was bisulfite-convertedusing the EZDNAMethylation Kit (ZymoResearch) accord-ing to the manufacturer’s instructions.

MassARRAY. Bisulfite-converted DNA was amplifiedwith primers (primer sequences are listed in SupplementaryTable S3), the PCR products spotted on a 384-pad Spectro-CHIP (Sequenom), and spectrally acquisited on a MassAR-RAY analyzer. Methylation data of individual units (1–4CpG sites/unit) were generated by EpiTyper software(Sequenom).

Pyrosequencing. Bisulfite-converted DNA was amplifiedand sequenced using the PyroMark LINE-1 Kit (Qiagen),which contains PCR primers and a sequencing primerprovided by the company. PCR-cycling conditions were95�C (30 s), 50�C (30 s), and 72�C (30 s) for 35 cycles.The PCR product was purified and methylation quantifiedusing the PSQ HS 96 Pyrosequencing System (Pyrosequen-cing Inc.).

MBDCap-seq, mapping, and normalization for genome-widemethylation analysis. As will be noted below, adenomasfrom a total of 2 subjects treated with orally administeredBRBs plus BRB suppositories and 1 subject treated withBRB suppositories only did not respond to berry treat-ment. These subjects had more rectal adenomas at the endof the trial than at baseline, and the adenomas were usedfor the MBDCap-seq assay to determine whether differ-ences in the response of adenomas from nonrespondersand responders might be due to differences in effects ofthe BRBs on genome-wide DNA methylation (23). Unfor-tunately, due to other uses, adjacent normal tissues fromtwo of the nonresponders were not available for theMBDCap-seq assay. Therefore, adjacent normal tissuesfrom 3 responders and 1 nonresponder as well as ade-nomas from 3 responders and 3 nonresponders were usedin the assay.

Wang et al.

Cancer Prev Res; 7(7) July 2014 Cancer Prevention Research668




MBDCap-seq,mapping, andnormalizationwere detailedin Supplementary Materials and Methods. The methylatedregions could be any length, but 8 kb was used because themajority of CpG islands are within 2 kb up- or downstreamof the transcription start site (TSS), and CpG island shoresare up to 2-kb distance relative to each gene’s CpG islands(24).

Statistical analysisAdenoma number and burden (the sum of diameters of

all adenomas�2mm) as well as data fromMassARRAY andimmunohistochemical staining were compared by the Wil-coxon signed-rank test. All analyses were two-sided, and aP value of less than 0.05 was considered significant.

ResultsEffects of oral BRBs and BRB suppositories on thedevelopment of adenomasThemean age of patients was 48 years (30–67), including

7 men and 7 women. All patients were Caucasian (Supple-mentary Table S4). All adverse events of randomized sub-jects considered possibly, probably, or definitely related tostudy treatment are as follows: anal fissure (n¼ 2), bloating(n¼ 3), diarrhea (n¼ 5), flatulence (n¼ 2), nausea (n¼ 7),and rectal irritation (n ¼ 6). In addition, 8 randomizedsubjects (total 24 patients were randomized) experienceddifficulty retaining the rectal suppositories.When data from all 14 patients were combined, there

was a reduction in the number (�3.5, P ¼ 0.069) andburden (�8.5, P ¼ 0.036) of rectal adenomas (Supple-mentary Table S5). No additional benefit on adenomaswas noted in the group randomized to oral BRBs (Sup-plementary Table 5). Only 1 of 7 patients (#1–7) whoreceived oral placebo and BRB suppositories had moreadenomas. Of the 7 patients (#8–14) who received oralBRBs plus BRB suppositories, only 4 had reductions inadenoma number, 1 had no change in adenoma number,and 2 had more adenomas at the end of the study (Fig.1A). Figure 1B indicates the decrease in polyp burden thatparallels the regression in number of polyps (Fig. 1A).These data suggest that the BRB suppository seems suffi-cient to regress rectal polyps.

BRBs decreased cellular proliferation in adenomasfrom respondersWe then determined whether BRB treatments (oral plus

suppository and suppository only) led to different bio-marker alterations in responders versus nonresponders.As indicated above, 3 patients with FAP had more rectaladenomas at the end of the BRB treatment than in thebeginning (nonresponders). We determined whether thismight be related to differences in the effects of BRBs oncell proliferation and apoptosis in the adenomas fromnonresponders and responders. Ki-67 staining was sig-nificantly decreased in adenomas from responders butnot from nonresponders (Fig. 2A). There was no signif-icant change in TUNEL (Fig. 2B) and cMyc (Fig. 2C)

staining in adjacent normal or adenoma tissues fromresponders or nonresponders.

Effects of BRBs on DNA methylation in tissues fromresponders and nonresponders

As noted previously (14), colorectal adenocarcinomasfrom patients with cancer who had consumed BRBs (60 g/dtotal) orally for at least 4 weeks had reduced levels ofDNMT1 protein after berry treatment when compared withbaseline. This observation was correlated with reducedmethylation of p16 and multiple regulatory genes in theWnt signaling pathway. We asked, therefore, whether BRBsadministered as oral BRBs plus suppository and suppositoryonly differentially affect DNMTs and the promoter meth-ylation status of p16 and the Wnt pathway regulators, forexample, SFRP2 andWIF1, in both adjacent normal tissuesand adenomas from responders and nonresponders. Asshown in Fig. 3A, BRBs significantly decreased DNMT1protein expression in adenomas from responders. BRBs did

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Figure 1. Effects of oral placebo plus BRB suppository and oral BRBsplus BRB suppository on polyp number (A) and burden (B) in patients withFAP. Patients 1 to 7 received oral placebo plus BRB suppositories.Patients 8 to 14 received oral BRBs plus BRB suppositories. Note thatthere are a total of three nonresponders; 1 in oral placebo plus BRBsuppository group (patient 1) and two in oral BRBs plus BRB suppositorygroup (patients 8 and 9).






not change DNMT3b expression in adjacent normal tissuesor in adenomas from responders or nonresponders(Fig. 3B). p16 protein expressionwas significantly increasedin adjacent normal tissues from responders (Fig. 3C) andp16 promoter methylation was significantly decreased inadjacent normal tissues and in adenomas from responders

(Fig. 3D). However, BRBs did not affect promoter methyl-ation of SFRP2 (Fig. 3E) and WIF1 (Fig. 3F), Wnt antago-nists, in adjacent normal tissues or adenomas from respon-ders or nonresponders.

BRBs decreased levels of promoter methylation ofother genes identified by MBDCap-seq

We used MBDCap-seq to globally screen methylationchanges induced by BRBs in 3 nonresponders (1 receivedsuppository only, patient #1; and 2 received oral BRBs plusBRB suppository, patients #8 and #9) and in the 3 bestresponders (2 received suppository only, patients #2 and#3; and 1 received oral BRBs plus BRB suppository, patient#11). Adjacent normal tissues from 2 nonresponders wereexhausted and were not available for MBDCap-seq assay.Genome-wide methylation patterns for adjacent normaltissues from 3 responders and 1 nonresponder as well asadenomas from 3 responders and 3 nonresponders aredepicted in Fig. 4A. Methylation status increased immedi-ately before the TSS and dropped at the TSS. It thenincreased in the gene bodies and then dropped at thetranscription termination site (TTS). LINE1 global methyl-ation was not significantly altered by BRBs in adjacentnormal tissues or in adenomas from responders or non-responders (Fig. 4B). We then looked into the numbers ofregions being demethylated by BRBs. In general, moreregions were demethylated in adenomas than in adjacentnormal tissues in responders (Fig. 4C). Similarly,more TSSswere demethylated in adenomas than in adjacent normaltissues in responders (Fig. 4D).

In an attempt to determine whether BRB treatmentmight affect methylation differently in responders andnonresponders, we asked whether there are differences inthe genes demethylated by BRBs in adenomas fromresponders and nonresponders. As shown in Fig. 5A, theTSSs of a higher number of genes were demethylated inadenomas from responders than from nonresponders; i.e.,1,358 versus 327. In addition, more genes were commonlydemethylated by BRBs in adenomas from responders thannonresponders. For example, 27 genes were commonlydemethylated in all 3 responders and there was no genecommonly demethylated in all 3 nonresponders; 292genes were commonly demethylated in any 2 responders,whereas there were 30 genes commonly demethylated inany 2 nonresponders. Genes demethylated in all 3 and inany 2 responders (27þ 292¼ 319), and in all 3 and in any2 nonresponders (0 þ 30 ¼ 30) were used to make the piechart to illustrate the numbers of genes uniquely andcommonly demethylated in responders or nonresponders(Fig. 5A). There were 312 and 23 genes uniquely demethy-lated in adenomas from responders and nonresponders,respectively (Fig. 5A). Pathway analysis of the 312 and 23genes indicated that there are no signaling pathways thatwere significantly altered (data not shown). Interestingly,there were 37 miRNAs uniquely demethylated in adeno-mas from responders; however, there was only one miRNAuniquely demethylated in adenomas from nonresponders(Fig. 5A). Predicted targets for the 37 miRNAs were used

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Figure 2. BRBs decreased cellular proliferation in adenomas from allresponders. Immunohistochemical staining of Ki-67 (A), TUNEL (B), andcMyc (C) in adjacent normal tissues (N) and adenomas (T) from 11responders (R) and 3 nonresponders (NR) at baseline (Ba) and after 9months (EOS) of BRB treatment from both protocols; BRB suppositoryonly and oral BRBs plus BRB suppository. �, P < 0.05.

Wang et al.





for pathway analysis and pathways with P values <0.05 arepresented in Fig. 4B. Interestingly, the Wnt pathway wasthe most significantly altered signaling pathway followedby axon guidance, TGFb signaling, focal adhesion, MAPKsignaling, Notch signaling, tight junction, and insulinsignaling (Fig. 5B).We next asked the question whether there are differences

in the responses of adjacent normal tissues and adenomasfrom responders to BRB treatment. Interestingly, there wereno genes commonly demethylated by BRBs in adjacentnormal tissues from all 3 or any 2 responders; however,27 and 292 genes were commonly demethylated in adeno-mas from all 3 and any 2 responders, respectively (Fig. 6A).The numbers of genes demethylated in adenomas fromall 3and any 2 responders (27 þ 292 ¼ 319) and in adjacentnormal tissues from any responder (¼ 120) were used tomake the pie chart to illustrate the numbers of genesuniquely and commonly demethylated in adenomas andadjacent normal tissues from responders (Fig. 6A). Therewere 286 and 87 genes uniquely demethylated in adeno-mas and in adjacent normal tissues from responders,

respectively (Fig. 6A). Pathway analysis of the 286 and87 genes suggests that there were no pathways that weresignificantly altered (data not shown). Interestingly, therewere 33miRNAs uniquely demethylated in adenomas fromresponders; however, therewereonly threemiRNAsunique-ly demethylated in adjacent normal tissues from responders(Fig. 6A). Predicted targets for the 33miRNAs were used forpathway analysis and pathways with P values <0.05 arepresented in Fig. 6B. Interestingly, theWnt pathway is againthemost significantly altered signaling pathway followedbyaxon guidance, endocytosis, adherens junction, focal adhe-sion, TGFb signaling, ErbB signaling, etc. (Fig. 6B).

DiscussionAlthough there were 3 nonresponders in this study, our

data suggest that BRBs have the ability to regress rectalpolyps in patientswith FAP; in particular, BRB suppositoriesalone might be sufficient to suppress rectal polyp develop-ment (Fig. 1, Supplementary Table S5). In other clinicaltrials with oral administration of berries, BRBs have been

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Figure 3. Effects of BRBs on DNAmethylation in tissues from allresponders and 3 nonresponders.Immunohistochemical staining ofDNMT1 (A), DNMT3b (B), and p16(C), and promoter methylation ofp16 (D), SFRP2 (E), and WIF1 (F) inadjacent normal (N) and adenoma(T) from 11 responders (R) and 3nonresponders (NR) at baseline(Ba) and after 9 months (EOS)of BRB treatment from bothprotocols; BRB suppository onlyand oral BRBs plus BRBsuppository. �, P < 0.05.






shown to be well tolerated and exert protective effects inpatients with colorectal cancer (14) and Barrett’s esophagus(15), and strawberries regress mild dysplastic esophageallesions that are precursors to squamous cell carcinoma (25).Accordingly, we anticipated that oral BRBs plus BRB sup-positories could be more effective than suppositories alonein regressing polyps in patients with FAP. Surprisingly,however, our data suggest that BRB suppositories alone aresufficient to regress polyps as no additional benefit wasnoted in the group randomized to oral BRBs (Supplemen-tary Table S5). However, with only 7 subjects in each arm, itis difficult to arrive at definitive conclusions and a moreexpanded study is necessary to draw final conclusions.

It is well known that FAP is caused by mutations in theAPC gene. The severity of FAP is known to be influenced bythe site of the APC mutation (26, 27). We asked whethernonresponders in this study have different somatic SNPs inthe APC gene than responders. Forty SNPs in APC geneswere assayed in adjacent normal and adenomas collected at

baseline from all 14 patients. There was no significantdifference in the 40 SNPs in the APC gene in respondersversus nonresponders in adjacent normal tissues and ade-nomas (Supplementary Fig. S3).

We previously reported that an average of 4 weeks of BRBconsumption decreased promoter methylation of p16 andof Wnt pathway regulator tumor-suppressor genes in biop-sies collected from patients with human colorectal cancer(14). Therefore, we decided to investigate whether BRB-induced promoter DNA demethylation might be differentin responders and nonresponders. Interestingly, our datashowed that thereweremore TSSs commonly demethylatedin adenomas from responders than nonresponders (Fig.5A). It is possible that resistance of these adenomas to BRBtreatment is due to decreased sensitivity of their responses toBRB-induced DNA demethylation.

Interestingly, BRBs demethylated approximately 10 timesfewer regions and TSSs in adjacent normal tissues fromresponders than in adenomas (1,358 vs. 120, Fig. 6A). Cell

Genome-wide DNA methylation distribution

Ba

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TSS TTS

LINE1

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0

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of

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Nu

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er

of T

SS

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Figure 4. MBDCap-seq assayidentified regions and TSSsdemethylated by BRB treatment.A, genome-wide DNA methylationdistribution from MBDCap-seqassay at baseline (Ba) and after 9months (EOS) of BRB treatmentfrom both protocols; BRBsuppository only and oral BRBsplus BRB suppository, from 3 bestresponders and 3 nonresponders.B, LINE-1–repetitive elementbisulfite/pyrosequencing assay forglobal methylation in adjacentnormal tissue (N) and adenomas (T)from 11 responders (R) and 3nonresponders (NR) at baseline(Ba) and after 9 months (EOS) ofBRB treatment using bothprotocols. Numbers of regions(C) and TSSs (D) demethylated inadjacent normal tissue (N) andadenomas (T) after 9 months (EOS)of BRB treatment using bothprotocols and from the best 3responders and the 3nonresponders.

Wang et al.





replication in normal tissues is low and two rounds of cellreplication are required to achieve a heritable demethyla-tion affect; first round produces hemi-demethylated DNAand second round produces fully-demethylated DNA (28).The observation that the berries demethylatedmore regionsin adenomas than in adjacent normal tissues is likely due todifferences in rates of cell replication in the two tissues.A previous study in our laboratory found that the pro-

moter regions of p16 and Wnt regulator genes in colorectaltumors were demethylated by oral consumption of BRBs(14). In contrast, in the present study, p16 promoter meth-ylation was decreased in adjacent normal tissues and inadenomas from responders (Fig. 3D). However, BRBsexerted no significant effects on promoter methylation ofSFRP2 andWIF1, Wnt regulators, in either adjacent normaltissues or in adenomas from responders andnonresponders(Fig. 3E and F). Although promoter methylation of exam-inedWnt regulatorswas not changed byBRB treatment (Fig.3E and F), miRNAs regulating Wnt pathway were uniquelydemethylated by BRBs in adenomas from responders (Figs.5A and 6A). These results suggest that BRBs regulate theWntpathway through demethylation of miRNAs in adenomasfrom patients with FAP. These miRNAs also regulate other

pathways associated with the development of colorectalcancer, for example, TGFb, MAPK, Notch, Tight junction,insulin signaling, and so forth. Thus, BRBs may regulatemultiple signaling pathways associated with the develop-ment of colorectal cancer through their effects on miRNAs.Finally, our results suggest that berry suppositoriesmight bean alternative toCelecoxib for the treatment of patientswithFAP.

Disclosure of Potential Conflicts of InterestG. Stoner has ownership interest (including patents) in BerriProducts,

Inc., Corvallis, Oregon. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: C.A. Burke, T.H.-M. Huang, G.D. StonerDevelopment of methodology: C.A. BurkeAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): L.-S. Wang, C.A. Burke, H. Hasson, W.L. Frankel

Nonresponders

adenoma

(NR_T)

Responders

adenoma

(R_T)Number of TSSs

demethylated

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In all 3 patients

In any 2 patients

In any patient

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292

1,358

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37 miRNAs 312 7 23 1 miRNA

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ht ju

nction

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–2

–4

–6

–8

–10

Pathways for predicted targets for 37 miRNAs

Log P

valu

e

Figure 5. More TSSs were commonly demethylated in adenomas fromresponders than fromnonresponders after 9months ofBRB treatment. A,number of TSSsdemethylated in adenomas (T) from responders and fromnonresponders. B, pathways for predicted targets for miRNAs uniquelydemethylated in adenomas (T) from responders but not fromnonresponders.

Responders

adenoma

(R_T)No. of TSSs

demethylated

A

B

Responders

normal

(R_N)

In all 3 patients 27 0

In any 2 patients 292 0

In any patient 1,358 120

R_T R_N

33 miRNAs 286 33 87 3 miRNAs

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Wnt

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odiu

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–10

Pathways for predicted targets for 33 miRNAs

Log P

valu

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Figure 6. More TSSs were commonly demethylated in adenomas thanin adjacent normal tissues from responders after 9 months of BRBtreatment. A, number of TSSs demethylated in adenomas (T) andadjacent normal tissues (N) from responders. B, pathways for predictedtargets for miRNAs uniquely demethylated in adenomas (T) but not inadjacent normal tissues (N) from responders.






Analysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): L.-S. Wang, C.A. Burke, P. Liu,G.D. StonerWriting, review, and/or revision of the manuscript: L.-S. Wang,C.A. Burke, C.L.S. Molmenti, W.L. Frankel, G.D. StonerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.A. Burke, H. Hasson, C.-T. Kuo,C.L.S. Molmenti, C. Seguin, T.H.-M. HuangStudy supervision: L.-S. Wang, C.A. Burke, G.D. Stoner

AcknowledgmentsThe authors thank the patients who participated in this trial and took

either placebo or berry powder orally and rectal suppositories for a period of9 months. Their patience and high degree of compliance is appreciated. Theauthors thank the Stokes Raspberry Farm in Wilmington, Ohio, for thefreeze-dried BRBs, and Tony Buchta of the Central Ohio CompoundingPharmacy in Worthington, Ohio, for making the rectal suppositories andberry packets anddistributing them to thepatients. Finally, the authors thank

Dr. Dennis Pearl of the Ohio State University for initial statistical analysis ofthe data.

Grant SupportThis work was supported by NIH grants 5 R01 CA148818 04 and ACS,

RSG-13-138-01—CNE (to L.-S. Wang); NCI grants 7 R01 CA069065 15, 5U54 CA113001 09, 5 U54, CA113001 09, 5 P50 CA134254 03, 5 R01ES017594 06, 5 U01 ES019482 04 (to T.H-M. Huang); grant 5 R01CA103180 09 as well as USDA award nos. 2005-38903-02313 and 2008-38903-03660 to TheOhio StateUniversity (toG.D. Stoner); and SubcontractNo. 60010819 to the Cleveland Clinic Foundation (C.A. Burke).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 10, 2014; revised April 1, 2014; accepted April 15, 2014;published OnlineFirst April 25, 2014.

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Wang et al.




2014;7:666-674. Published OnlineFirst April 24, 2014.Cancer Prev Res Li-Shu Wang, Carol A. Burke, Henrietta Hasson, et al. Polyps in Patients with Familial Adenomatous PolyposisA Phase Ib Study of the Effects of Black Raspberries on Rectal

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APhaseIbStudyoftheEffectsofBlackRaspberriesonRectal Polyps ...€¦ · Polyps in Patients with Familial Adenomatous Polyposis Li-Shu Wang1, Carol A. Burke3, Henrietta Hasson3, Chieh-Ti