Does Mutated K-RAS Oncogene Attenuate the Effect of ......Research Article Does Mutated K-RAS Oncogene Attenuate the Effect of Sulindac in Colon Cancer Chemoprevention? Photini F.S

Research Article

Does Mutated K-RAS Oncogene Attenuate theEffect of Sulindac in Colon CancerChemoprevention?Photini F.S. Rice1, Kevin G. Ehrichs1, Mykella S. Jones1, Hwudarw Chen2, Chiu-Hsieh Hsu3,Edward R. Abril2, Raymond B. Nagle4, David G. Besselsen5, Jennifer K. Barton1,2, andNatalia A. Ignatenko2,6

Abstract

The NSAID sulindac has been successfully used alone or incombination with other agents to suppress colon tumorigen-esis in patients with genetic predisposition and also showed itsefficacy in prevention of sporadic colon adenomas. At thesame time, some experimental and clinical reports suggestthat a mutant K-RAS oncogene may negate sulindac antitumorefficacy. To directly assess sulindac activity at suppressingpremalignant lesions carrying K-RAS mutation, we utilized anovel mouse model with an inducible colon-specific expres-sion of the mutant K-ras oncogene (K-rasG12D). Tumor devel-opment and treatment effects were monitored by minimallyinvasive endoscopic Optical coherence tomography. Expres-sion of the mutant K-ras allele accelerated azoxymethane(AOM)-induced colon carcinogenesis in C57BL/6 mice, astrain otherwise resistant to this carcinogen. Sulindac

completely prevented AOM-induced tumor formation in K-raswild-type (K-ras wt) animals. In K-rasG12D–mutant mice, a 38%reduction in tumor number, an 83% reduction in tumorvolume (P � 0.01) and an increase in the number of adeno-ma-free mice (P ¼ 0.04) were observed. The partial response ofK-RasG12D animals to sulindac treatment was evident by thedecrease in mucosal thickness (P < 0.01) and delay in pro-gression of the precancerous aberrant crypt foci to adenomas.Molecular analyses showed significant induction in cyclooxy-genase 2 (COX-2), cleaved caspase-3 (CC3), and Ki-67 expres-sion by AOM, but not sulindac treatment, in all genotypes.Our data underscore the importance of screening for K-RASmutations in individuals with colon polyps to provide morepersonalized interventions targeting mutant K-RAS signalingpathways. Cancer Prev Res; 1–11. �2017 AACR.

IntroductionColorectal cancer is the third most common cancer in the

United States and is a leading cause of cancer-related deathworldwide (1). If detected early, colorectal cancer is a treatablecancer with a 5-year survival rate of 90% (1). However, the 5-yearsurvival rate drops to as low as 11% if the disease is found inadvanced stages. Key factors for early detection and prevention ofcolon cancer include defining risk factors and understanding thestages of this disease.

Colon cancer develops gradually in normal colon epitheliumthrough precancerous aberrant crypt foci (ACF), which can prog-ress to early and intermediate adenomas and then malignant

tumors via an accumulation of chromosomal abnormalities,genetic mutations, and epigenetic changes (2–4). Inactivation oftumor suppressor genes and DNA mismatch repair genes, andactivation of oncogenes are important genetic mutations in colonneoplastic progression (5).K-RASmutations are frequently foundin colorectal cancer along with mutations of the adenomatouspolyposis coli (APC) and p53 genes (5, 6). K-RAS is mutated inabout one quarter of advanced colon adenomas (7) and in nearlyone half of colon cancers (5, 8). Constitutive activation of K-RASin colon cancer generally occurs by substitution of amino acidresidues in codons 12 and 13 and, less frequently, in codon 61(6, 9, 10). K-RAS mutations can be detected early in carcino-genesis, between the stages of early and intermediate adenoma,and maintain a constant incidence in late adenoma and incarcinoma (11, 12). K-RAS is a member of three highly homol-ogous oncogenes and encodes a monomeric protein of 21 kDa(p21ras), which is able to bind and hydrolyze GTP. This mem-brane-associated guanine nucleotide binding protein acts as amolecular switch for signal cascades that modulate manyaspects of cell behavior, including proliferation, differentiation,motility, and death (13). Activated RAS triggers uncontrolledproliferation and morphologic alteration, contributing to themalignant phenotype of transformed cells. Oncogenic RASinfluences different cellular processes in a cell type- anddisease-specific manner via a variety of signaling pathways,particularly those that include the serine/threonine proteinkinase Raf-1, Rho small GTPases and the AKT proto-oncogene(14, 15).

1Department of Biomedical Engineering, University of Arizona, Tucson, Arizona.2University of Arizona Cancer Center, University of Arizona, Tucson, Arizona.3Mel andEnidZuckermanCollegeof Public Health, University ofArizona, Tucson,Arizona. 4Department of Pathology, University of Arizona, Tucson, Arizona.5University Animal Care, University of Arizona, Tucson, Arizona. 6Department ofCellular and Molecular Medicine, University of Arizona, Tucson, Arizona.

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

Corresponding Author: Natalia A. Ignatenko, Department of Cellular andMolecular Medicine, University of Arizona, 1515 North Campbell Ave., Tucson,AZ 85724. Phone: 520-626-8044; Fax: 520-626-4979; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-17-0230

�2017 American Association for Cancer Research.

CancerPreventionResearch

www.aacrjournals.org OF1

Association for Cancer Research. on January 24, 2020. © 2017 Americancancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst November 8, 2017; DOI: 10.1158/1940-6207.CAPR-17-0230

http://crossmark.crossref.org/dialog/?doi=10.1158/1940-6207.CAPR-17-0230&domain=pdf&date_stamp=2017-12-13

http://cancerpreventionresearch.aacrjournals.org/

NSAIDs, including sulindac, inhibit the cyclooxygenase iso-zymes COX-1 and COX-2, which catalyze the rate-limiting stepin the metabolic conversion of arachidonic acid to prostaglandins.Sulindac, a nonselective COX-2 inhibitor, is a prodrug that ismetabolized by the liver and intestinal flora to the sulfide andsulfone forms (16). Sulindac sulfide is the active anti-inflammatorymetabolite, primarily responsible for blockage of prostaglandinsynthesis, whereas sulindac sulfone has no COX-inhibitoryactivity (16, 17). Sulindac is known to induce apoptosis incolon cancer cells (18, 19) and to cause regression of colonpolyps in patients with familial adenomatous polyposis (FAP)and with sporadic adenomas (20). At the same time, long-termtreatment with sulindac in FAP patients does not inhibit theprogression of polyps toward malignancy (21). Analysis ofmolecular changes in adenomas of FAP patients that failed torespond to sulindac therapy suggested that K-RAS mutationsmay be associated with this resistance (22). Mutant K-RAS–associated resistance to the sulindac metabolite sulindac sulfidealso has been reported in vitro, in the rat intestinal cell line IEC-18 transformed with K-RAS oncogene (23). In this study, ourgoal was to directly assess the effect of sulindac treatment onthe formation of colon adenomas that express the mutant K-RAS oncogene. We conducted this study using a novel mousemodel of colon cancer with the colon-specific and inducibleexpression of mutant K-RasG12D allele. We applied minimallyinvasive optical coherence tomography (OCT) to monitor thedevelopment of colon adenomas in this mouse model throughtime serial imaging, which enabled quantification of the che-mopreventive effect of sulindac during the course of wild-type(wt) and mutant K-RAS--driven colon tumorigenesis.

Materials and MethodsStudy design

The mouse model with inducible colon-specific expression ofthemutantK-ras allele (Vil-Cre-ERT2K-ras G12D)was generated andcharacterized as described in Supplementary Materials. Animalswere housed at the University of Arizona's Animal Care Facility ingroups of one to five in microisolator cages under fluorescentlighting on a 12-hour cycle in accordance with the Universityof Arizona Institutional Animal Care and Use Committeeguidelines. Mice of three genotypes were used in this study:Vil-Cre-ERT2

–positive K-ras–mutant mice (K-rasG12D), Vil-Cre-ERT2

–positive K-ras wild-type mice (K-ras wt), and Vil-Cre–negative K-ras mutant mice (Vil-Cre–negative K-rasG12D).

Experimental protocols were approved by the InstitutionalAnimal Care andUse Committee. The study timeline is illustratedin Fig. 1A. Mice in each of three genotypes were divided into fourexperimental groups: control no sulindac, control sulindac, AOM-induced no sulindac, AOM-induced sulindac. The groups andnumber of mice per group are presented in Fig. 1B.

Mice were fed the defined pelleted synthetic diet AIN93G orAIN93G formulated with 100 ppm (0.1 g/kg) sulindac-TD.08740(Harlan Teklad). Mice in sulindac-treated groups were providedsulindac-containing diet beginning at 11 weeks of age, which was1weekpost-AOMtreatment. The approximate daily consumptionof sulindac was 25 mg/kg based on average mouse weight of 20 gand food consumption of 5 g per day. The dietary application ofsulindac has been successfully used in the murine cancer modelsin many prior chemoprevention studies, and amounts are basedon the recommended human doses (24–26). Treatment with the

colon-specific carcinogen azoxymethane (AOM, Sigma) was per-formed to induce adenomas. AOMwas injected intraperitoneallyat concentration of 10 mg/kg in saline once a week for 4 weeksbeginning at 7weeks of age. Controlmice received 0.2mL normalsaline using the same regime of injections. Irradiated reverseosmosis water was available ad libitum for the duration of theexperiment.

OCT procedure and analysisThe endoscopic ultrahigh resolution OCT system employed in

this study has been previously described in detail (27). The lightsource was a superluminescent diode (Superlum Broadlighter,Moscow, Russia) centered at 890 nmwith a 150-nm full-width-at-half-maximum (FWHM) bandwidth. The endoscope optics con-sisted of a fiber, gradient index lens, and right angle prism thatdirected the light out the side of a 2-mm diameter endoscope,which could be inserted approximately 30 mm into the colon.Linear and rotational motion of the optics within a protectiveglass envelope enabled sampling of the distal colon. Cross-sectional images 2 mm depth by 30 mm length (1,024 �5,000 pixels) were obtained at 45-degree increments, resultingin 8 images around the circumference of the colon lumen. Thegenerated images have an axial resolution of 3.5 mm in air and alateral resolution of 5 mm. Depth of imaging was limited to1.4 mm; consequently, the deep boundary of larger tumors wasnot completely visible.

Imaging protocol.. Eighteen- to 24 hours prior to imaging with theOCT system, foodwas withheld from themice in an effort to clearthe colon. Themice were removed from the standard individuallyventilated cages (IVC) and placed into empty microisolator cageswith wire bottom inserts to keep them dry and to preventcoprophagia. Mice had free access to nonflavored pediatric elec-trolyte replacement supplement solution. The mice were thentransferred to standard IVC cage and bedding and transported forcolonic OCT imaging. Mice were anesthetized prior to imagingwith a mixture of 100 mg/kg ketamine, 10 mg/kg xylazineadministered intraperitoneally (IP), or 2.5%Avertin IP. The distalcolonwas lavagedwith 0.9% sodium chloride irrigation solution,USP, to clear residual fecal material. Following colon lavage, micewere placed on a warming pad and imaged with the OCT system.They were kept onwarming pads until recovered from anesthesia.Mice were imaged starting at 13 weeks of age through 34 weeks ofage at 4-week intervals.

Tumor analysis from OCT imagesEach set of 8OCT imageswas analyzed for allmice in this study,

at each time point, to determine tumor count and tumor burden.The criteria for adenoma identification was previously describedin detail by Hariri and colleagues (28) and includes mucosalthickness twice the local average, attenuation of signal, and faintboundaries between themucosa and submucosa tissue boundary.Adenomaswere identified, counted, and themaximumwidthwasmeasured. The volume of each tumor was calculated using themaximumwidth as the tumor diameter and assuming a sphericalshape.

Mucosal thickness measurementMucosal thickness for each control group genotype, with and

without sulindac treatment, was determined by analyzing theventral-most OCT image from each image set at each time point.

Rice et al.

Cancer Prev Res; 2017 Cancer Prevention ResearchOF2




The average thickness of the proximal 10mmof themucosal layerof the colon was measured using custom MATLAB software. Ablinded observer identified the luminal and deep boundaries ofthe mucosa, and the software calculated the average thickness.

Tissue collection and histologyAfterfinal imaging at 34weeks of age, themicewere euthanized

using CO2 asphyxiation. A 2-mm diameter lubricated glass rodwas inserted 30-mm into the colon. After the abdomen wasopened and the colon exposed, tissue marking ink was used tomark dorsally, to orientwithOCTpositions. After excision, colonswere placed on filter paper, cut longitudinally along the markedline, and flattened. The colons were examined visually and photo-graphed. Another piece of filter paper was placed over the colonto keep it flat for histology processing. The colons were fixed in10% buffered formalin or Histochoice (Ameresco), paraffin-embedded, and cut longitudinally into 6-mm sections every250 mm through the entire colon. The slides were stained withhematoxylin and eosin (H&E). Histologic features of colontumors from the entire colon were evaluated by a certified veter-inary pathologist (one animal per each genotype and treatment).The grade of dysplasia for each adenoma examined histologicallywas determined on the basis of criteria outlined in a review on thepathology of mouse models of intestinal cancer (29).

The H&E-stained sections of the colons of all animals wereanalyzed by an independent observer for tumor number andburden. Tumors were counted and volume was determined bymeasuring the maximum diameter of each discrete tumor usingthe Olympus Microsuite FIVE (2007) software, and assuming aspherical tumor, as described previously (30). A comparison of

final time point OCT- and histology-determined tumor numberand burden was made.

ACF and small intestinal and colon tumor scoringAn additional set of K-ras wt and K-rasG12D mice described in

Supplementary Table S3A was sacrificed at the age of 18 weeks byCO2 inhalation, and the whole small intestine and colon seg-ments were removed, flushed with buffered saline, opened lon-gitudinally and laid flat, mucosal surface up. Tissues were fixed in10% neutral-buffered formalin for 4 hours. After 4 hours, smallintestines and colons were washed with cold PBS and stored in70% ethanol at 4�C. Fixed tissues were briefly stained with 0.5%methylene blue, and the colons were evaluated for the presence ofthe ACF and tumors. Morphologic evaluation of ACF and tumorswas performed using a brightfield Nikon microscope at �20magnification. Tumors in the proximal, middle, and distal por-tions of the small intestines were also counted where applicable.Tumors were counted starting at a diameter of 0.5 mm.

IHC and scoringIHC was performed using the automated Ventana platform

from Ventana Medical Systems (VMS) using a streptavidin DABDetection Kit from VMSi bed as described previously (30). Thetissue was stained for proliferationmarker Ki-67, apoptosis mark-er cleaved caspase-3, and inflammation marker COX-2. Stainingconditions and antibody dilution are described in SupplementaryMaterials. The appropriate positive control tissue was used foreach antibody assay. Analysis of the Ki-67 and CC3 staining wasdone bymanually counting the number of positively stained cellsin the crypts directly adjacent to the muscularis in 50 colonic

Figure 1.

A, Experimental protocol withtreatment and OCT imaging timepoints. B, Number of animals bygenotype and treatment groups usedin the study.

Sulindac Chemoprevention of K-RAS–Mutant Colon Adenomas

www.aacrjournals.org Cancer Prev Res; 2017 OF3




crypts per slide. The slides stained for COX-2 were read by anexperienced pathologist (R.B. Nagle) with 30 years of experience,who was blinded to treatment categories. Results are presented asa long score based on the sum of intensity of staining multipliedby the percent of stained tissue area. The following scoring criteriawere used: 0, no staining; 1þ, week diffuse staining (may containstronger intensity in <10% of the cells); 2þ, moderate staining in10% to 90% of the cells, and 3þ, more than 90% of the cellsstained with strong intensity. Staining for all endpoints wasanalyzed in three fields per slide.

Statistical analysisComparison between genotypes and groups (e.g., sulindac) on

tumor latency, tumor number, and tumor burdenwas statisticallyanalyzed using the following techniques. Cox regression wasperformed to determine the latency (timing of first adenomadetection). Wilcoxon rank sum /Kruskal–Wallis tests were per-formed to compare the tumor number derived fromOCT analysisat 34 weeks between groups/genotypes. Square root transforma-tionwas applied to the tumor number, and then, linear regressionwas performed to determine whether the genotypes modulatedthe group effects on the tumor number. On the basis of thesubstantial number of mice without tumors, Fisher exact testswere also performed to compare the percentage of mice with atleast one tumor between groups/genotypes, and logistic regres-sion was performed to determine whether the genotypes modu-lated the group effects. Wilcoxon rank sum /Kruskal–Wallis testswere also performed to compare tumor burden derived fromOCTanalysis at 34 weeks between groups/genotypes. Linear regressionwas performed to determine whether the genotypes modulatedthe group effects on tumor burden. Similar techniques were usedfor comparisons of tumor number and tumor burden fromhistology. For tumor grade analysis, the Dunnett test and Bon-ferroni multiple comparison test were used. For IHC stainingand analysis of mucosa thickness, linear mixed effects modelswith random intercept were fitted to account for within-mousecorrelation and compare between groups (genotype, sulindac,and AOM).

ResultsEffect ofK-RASmutationon tumorigenesismeasured fromOCTimages

Through the analysis of common downstream targets ofmutant K-RAS oncogene, we confirmed that our model success-fully recapitulates the main features of activated K-RAS signalingreported in humans (Supplementary Material; SupplementaryFig. S2).

Examples of OCT images obtained from the AOM-inducedexperimental groups of K-rasG12D and K-ras wt genotypes at thefinal imaging time point are presented in Fig. 2 along withcorresponding histology. Control animals of Vil-Cre–negativeK-rasG12D and K-ras wt genotypes did not develop any colontumors over the course of the study, but a low number of tumorswas detected in K-rasG12D mice (Table 1A). Treatment with AOMresulted in a significant increase in the number of colon adenomasand tumor burden in K-rasG12D animals (on average 6.91 colontumors and 50.43mm3 tumor burden permouse) comparedwiththe K-ras wtmice (average 0.83 tumor per mouse and total tumorburden of 10.89 mm3; Tables 1A and 1B, P < 0.0001).

In AOM-induced K-rasG12D mice, colon adenomas weredetected by OCT imaging beginning at 18 weeks of age (8 weeks

after the last AOM injection, second imaging time point). Incontrast, in AOM-induced K-ras wt, and Vil-Cre–negative K-rasG12D

mice, the first adenomas were detected at 26 and 30 weeks of age,respectively. This time difference was significant in K-rasG12D

compared with K-ras wt mice (P < 0.0001) and K-rasG12D com-pared with Vil-Cre–negative K-rasG12D mice (P < 0.0001). Accord-ing to OCT image measurements (Table 1A), sulindac treatmentdecreased the number of adenomas for both AOM-induced K-raswt (P ¼ 0.02) and K-rasG12D (P < 0.01) mice (Tables 1A and 1C,P < 0.001) and the decrease depended on the genotype of AOM-inducedmice (P < 0.001; Table 1C). However, sulindac treatmentdid not alter the time of the first adenoma appearance in AOM-induced K-rasG12D.

No significant differences in the rate of the tumorigenesis werenoted between the AOM-induced control genotypes (K-ras wt andVil-Cre–negative K-rasG12D) and sulindac completely preventedadenoma formation in these animals. In contrast, 61.54% ofsulindac-treated K-rasG12D mice developed colon adenomas(Table 1C). However, sulindac treatment did significantlydecrease the percentage of mice with at least one adenoma(P ¼ 0.04) and tumor burden in AOM-induced K-rasG12D miceby 83% (P < 0.01) (Table 1A; Fig. 3B), according to OCT imagemeasurements. Similarly to the tumor number, the decrease fortumor burden depended on the genotype of AOM-induced mice(Table 1C, P < 0.0001).

Histologic assessment of tumor number and burden in AOM-induced K-rasG12D mice in the no sulindac group at the time ofsacrifice identified an average of 8 tumors per mouse and a tumorburden of 62.88mm3permouse (Supplementary Table S1A). Themeasurements of tumornumber andburdenobtainedby imagingwere slightly lower than those obtained from histologic evalua-tion. The percentage of mice with at least one adenoma identifiedduring histologic evaluation on histology was nearly identical tothat obtained by OCT imaging (Supplementary Table S1).

Effect of K-Ras mutation on mucosal thickness measured fromOCT images

An increase in the mucosal thickness of the colon in animalswith conditional activationof themutantK-Ras allelewasobviousfrom OCT imaging. K-rasG12D mice had a significantly largermucosal thickness than K-ras wt mice (P < 0.0001; Table 2).Sulindac treatment decreased the mucosal thickness significantly,when the values for all treated versus untreated mice were com-pared (P < 0.01). The effect of sulindac on thickness depended ongenotype (P ¼ 0.01): specifically, in mice of K-rasG12D genotypesulindac significantly decreased the thickness of mucosa (P ¼0.02),whereas inK-raswtmice, the effect of sulindacwasmarginal(P ¼ 0.07; Table 2B).

Measurements of mucosal thickness in AOM-induced K-ras wtandK-rasG12Dmice were not practical because of the difficulties inaccurate assessment of the mucosal layer thickness when tumorswere present.

Analysis of histopathologic endpointsAdenoma grade analysis.. Histolopathologic analysis of colontumors in a subset of AOM-induced experimental groups wasperformed. As the animals for this analysis were selected ran-domly from three genotypes and groups, the subset is considereda random sampling of all of the animals in the study. Analysisshowed that animals expressing mutant K-ras developed all low-grade adenomas (Supplementary Table S2). K-rasG12Dmice had a

Rice et al.





significantly higher number of low-grade adenomas (6.39� 0.42adenoma/mouse) compared with control genotypes (K-ras wt,0.38 � 0.11 adenoma/mouse P < 0.0001 and Vil-Cre–negative K-rasG12D, 0.14�0.08 adenoma/mouse,P<0.0001; SupplementaryTable S2). Sulindac treatment decreased the number of low-grade

adenomas in K-rasG12Dmice (0.5� 0.15 adenoma/mouse) to thelevel comparable with the number of adenomas found in nosulindac control genotypes (Supplementary Table S2). There wasno significant difference between K-ras wt and Vil-Cre–negativeK-rasG12D mice (P ¼ 0.16).

Figure 2.

OCT images for a single 34-week-oldAOM-induced mouse at a singlerotation. OCT images are 30 mm inlength and 1.4 mm in depth. A, AOM-induced Vil-Cre-ERT2K-ras wt mice ofno sulindac and sulindac-treatedgroups, and corresponding histology.B, AOM-induced Vil-Cre-ERT2K-rasG12D

mice of no sulindac and sulindacgroups, and corresponding histology.AN, anus; AD, adenoma.






Effect of sulindac on ACF formation in K-rasG12D mice. We per-formed evaluation of the ACF and tumors formed in the subset of18-week-old animals ofK-rasG12D andK-raswt genotypes from thecontrol and AOM-induced groups listed in Supplementary Table

S3A (8 weeks after the last AOM injection, when applicable).AOM-induced K-ras wt mice developed more ACF than controlmice, when fed a regular diet, and sulindac treatment lowered thenumber of ACF formed in this group (P � 0.02; Supplementary

Table 1. Statistical analysis of endpoints generated using OCT in 34 week old mice. Number of animals in different groups is listed in Figure 1BTable 1A. Summary for total number of adenomas, % of mice with �1 adenoma, and total tumor burden deriving from imaging

Total # of adenomas Total tumor burdenGroup Mean � SD Range % (>0) Mean � SD Range

Vil-Cre negative K-rasG12D

AOM-induced no sulindac 0.20 � 0.41a 0–1 20.00 1.39 � 2.88 0–7.15Control no sulindac 0 � 0 0 0.0 0 � 0 0AOM-induced sulindac 0 � 0 0 0.0 0 � 0 0Control sulindac 0 � 0 0 0.0 0 � 0 0

K-ras wtAOM-induced no sulindac 0.83 � 1.27 0–4 41.67 10.89 � 18.76 0–62.74Control no sulindac 0 � 0 0 0.0 0 � 0 0AOM-induced sulindac 0 � 0 0 0.0 0 � 0 0Control sulindac 0 � 0 0 0.0 0 � 0 0

K-rasG12D

AOM-induced no sulindac 6.91 � 4.93 1–17 100.0 50.43 � 27.70 5.34–94.34Control no sulindac 0 � 0 0 0.0 0 � 0 0AOM-induced sulindac 1.85 � 2.54 0–9 61.54 8.57 � 14.37 0–51.87Control sulindac 0.11 � 0.33 0–1 11.11 1.34 � 4.01 0–12.04

aMean � SD.

Table 1B. Summary of P values for the analysis of the total number of adenomas, total tumor burden, and for % of mice with �1 adenoma between genotypes

Group Number of adenomas, P valuea Tumor burden, P valuea % of mice with �1 adenoma, P valueb

AOM-inducedNo sulindac <0.0001 <0.0001 <0.0001Sulindac <0.0001 <0.0001 <0.0001

ControlNo sulindac 1.00 1.00 NASulindac 0.26 0.26 0.27

SulindacControl 0.26 0.26 0.27AOM-induced <0.0001 <0.0001 <0.0001

No sulindacControl 1.00 1.00 NAAOM-induced <0.0001 <0.0001 <0.0001

aFor the comparison between the 3 genotypes within each group derived from Kruskal–Wallis test.bFor the comparison between genotypes within each group derived from Fisher exact test.

Table 1C. Summary of P valuesa for the comparison between groups within each genotype

Group Vil-Cre negative K-rasG12D K-ras wt K-rasG12D Interaction effectb

AOM-induced No sulindac vs. sulindacTotal number of adenomas 0.10 0.02 <0.01 <0.001Total tumor burden 0.10 0.02 <0.01 <0.0001% of mice with �1 adenoma 0.22 0.01 0.04 NAc

Control No sulindac vs. sulindacTotal number of adenomas 1.00 1.00 0.33 0.17Total tumor burden 1.00 1.00 0.33 0.2% of mice with �1 adenoma NA NA 0.45 NA

Sulindac Control vs. AOM-inducedTotal number of adenomas 1.00 1.00 0.03 <0.001Total tumor burden 1.00 1.00 <0.05 0.08% of mice with �1 adenoma NA NA 0.03 NA

No sulindac Control vs. AOM-inducedTotal number of adenomas 0.13 <0.01 <0.001 <0.0001Total tumor burden 0.13 <0.01 <0.001 <0.0001% of mice with �1 adenoma 0.23 <0.01 <0.001 NA

aP value for the comparison between groups within each genotype derived from Wilcoxon rank sum test.bP value for the interaction effects between group and genotype derived from a linear regression model for the square root transformed total number of adenomas.cP value for the interaction effects between groups and genotypes derived from a logistic regression model.

Rice et al.





Table S3B; Fig. 4A). The AOM-induced K-rasG12D mice developeda similar number of ACFs as control K-rasG12D group, when nottreated with sulindac. In addition, the small intestinal and colonadenomas were detected in AOM-induced K-rasG12D mice (Sup-plementary Table S3B). Sulindac-treated AOM-induced K-rasG12D

mice had a significantly higher number of ACFs formed, com-pared with AOM-induced no sulindac K-rasG12D mice. However,they had less colonic adenomas formed (on average one permouse) and no small intestinal tumors, in contrast to AOM-induced no sulindac K-rasG12D mice (Fig. 4A, P ¼ 0.01). No

Figure 3.

Time serial plots of average tumornumber and tumor burden for theAOM-induced groups, obtained fromOCT images. Data are for the averagenumber of tumors per mouse (A) andthe average tumor burden per mouse(B). The number of mice in each groupis listed in Table 1.

Table 2. Analysis of mucosal thickness (mm) in different treatment groups determined by OCT imaging at different time pointsTable 2A. Statistical analysis of effects of mutant K-RAS and Sulindac on mucosal thickness

Group Week 13 Week 18 Week 22 Week 26 Week 30 Week 34

Vil-Cre negative K-rasG12D

Control no sulindac 102 � 18a 103 � 14 109 � 14 106 � 18 109 � 13 120 � 26Control sulindac 107 � 17 111 � 32 110 � 17 118 � 18 120 � 27 108 � 17

K-ras wtControl no sulindac 125 � 20 127 � 24 131 � 35 128 � 31 124 � 27 124 � 24Control sulindac 108 � 2 119 � 33 107 � 24 111 � 25 105 � 23 126 � 11

K-rasG12D

Control no sulindac 186 � 37 191 � 32 182 � 40 178 � 47 178 � 29 169 � 35Control sulindac 137 � 37 148 � 50 150 � 37 151 � 49 151 � 37 145 � 34

aMean � SD.

Table 2B. Summary of P values for the analysis of mucosal thickness after adjusting for time

Control groups Vil-Cre negative K-rasG12D K-ras wt K-rasG12Db Interaction effectc

No sulindac vs. sulindaca 0.41 0.07 0.02 0.01aP < 0.01 for all genotypes.bP < 0.0001bK-ras wt vs. K-rasG12D no sulindac.cP value for the interaction effects between genotypes based on a linear mixed effects model with a random intercept.






Figure 4.

Effect of sulindac treatment on histologic and molecular endpoints during colon tumorigenesis. A, Analysis of ACF and tumor counts (when applicable) in18-week-old mice of K-ras wt and K-rasG12D genotypes from the control no sulindac and AOM-induced groups (no sulindac and sulindac), listed inSupplementary Table S3A. � , P � 0.02 (AOM-induced no sulindac vs. control no sulindac and AOM-induced sulindac), �� , P ¼ 0.01 (AOM-induced sulindac vs.AOM-induced, no sulindac). B, Images of IHC staining for COX-2, CC3, and Ki-67 in AOM-induced K-ras wt and K-rasG12D mice captured at �20 magnification.

Rice et al.





adenomas were detected in K-ras wt mice of the same category(these data were not analyzed due to the small sample size).

Analysis of molecular endpointsIHC analysis of colon tissues from different experimental

groups is presented in Supplementary Table S4. Sulindac treat-ment did not have a significant effect on COX-2 expression (P ¼0.10). AOM treatment caused a significant increase in COX-2expression (P < 0.01). Examples of COX-2 staining in thecolon of the AOM-induced Vil-Cre-ERT2K-ras wt and Vil-Cre-ERT2K-rasG12D mice are presented in Fig. 4B, COX-2.

Analysis of IHC scores of the apoptosis-related protein CC3showed that in control groups, sulindac-treated mice of all threegenotypes had a significantly higher CC3 expression than untreat-ed mice (P ¼ 0.02). Mice in all AOM-induced groups had asignificantly higher number of cells stained for CC3 than micewithout AOM (P ¼ 0.02). It is important to note that K-ras wtanimals treated with sulindac had a significantly higher numberof apoptotic cells, compared with sulindac-treated K-rasG12Dmice(more than 3-fold induction in CC3 staining based on cleavedcaspase IHC score in these groups, although these results were notanalyzed due to the small sample size). No significant differencewas found between sulindac-treated control genotypes (Vil-Cre–negative K-rasG12D and K-ras wt). Examples of CC3 staining in thecolon of K-ras wt and K-rasG12D mice are shown in Fig. 4B, CC3.

For proliferationmarker Ki-67, a significantly higher number ofcells positive for Ki-67 protein was observed in K-rasG12D micecompared with K-ras wt mice (P < 0.001) and Vil-Cre–negativeK-rasG1D (P < 0.01), respectively (Supplementary Table S4, Ki-67).There was no significant difference between K-ras wt and Vil-Cre–negative K-rasG12D mice. Mice of K-rasG1D genotype, both controland AOM-induced, had a significantly higher Ki-67 than K-ras wtmice of the same groups (P < 0.001) and Vil-Cre–negativeK-rasG12D mice (P < 0.01). All AOM-induced groups had thehigher Ki-67 IHC scores than control groups (P < 0.001). Exam-ples of Ki-67 staining in the colon of the AOM-induced K-ras wtand K-rasG12D mice are shown in Fig. 4B, Ki-67.

DiscussionMortality from hereditary and sporadic colorectal cancers has

been significantly reduced through colorectal surveillance withcolonoscopy. Colon cancer chemoprevention also has been val-idated as an important and efficient approach to suppress colontumorigenesis. Multiple studies have demonstrated that theNSAID sulindac significantly inhibits colorectal polyps in patientswith FAP (31, 32), although it was less effective in prevention ofduodenal adenomas in these patients (33). The selective COX-2inhibitor celecoxib showed some efficacy against duodenal andcolorectal polyps but is no longer approved for treatment of theseconditions due to possible side effects (34, 35). Combinationtreatments, such as of sulindac and the EGFR inhibitor erlotinib,showed the improved, albeit incomplete, efficacy in prevention ofduodenal polyps in the FAP patients after 6-month treatment(36).Oncogenicmutations in theK-RAS gene are found in 32% to40% of colorectal cancers and are located predominantly incodons 12 and 13 (85%–90%) and in codon 61 (6%; refs. 9,10). Earlier studies (21, 22) strongly suggest that the presence,even at low frequency, of K-RASmutations in patients with a highrisk for developing of colorectal cancer may diminish the effectof chemoprevention by NSAIDs. A randomized double-blind,

placebo-controlled phase III trial of the combination of sulindacwith a-difluoromethylornithine, an inhibitor of the downstreameffector of Wnt signaling pathway, showed highly significantefficacy in preventing recurrent colorectal polyps (25). However,a direct assessment of K-RAS mutations in colorectal polyps wasnot performed in this trial.

To directly address the efficacy of Sulindac for prevention ofcolon adenomas expressing the mutant K-RAS oncogene weutilized a novel mouse model of colon carcinogenesis with theconditional and inducible expression of themutant K-ras allele inthe colonic epithelium (Vil-Cre-ERT2K-rasG12D). Our initial anal-ysis of expression of downstream molecular targets of mutantK-ras in K-rasG12D model confirmed the upregulation of ERK,c-MYC, RalB GTPase and CAV-1 proteins, which are importantsignaling molecules that promote the proliferative signals ofoncogenic K-ras in the colon. Similarly to the reported earliermousemodels with constitutive activation of themutant K-Ras inthe colon (37, 38) we did not observe any tumorigenic effects ofthe K-ras mutation after activation of mutant K-Ras allele bytreatment with 4-OHT. We then introduced the DNA-damagingagent azoxymethane (AOM), which is commonly used to induceneoplastic progression in colon tissues. The chemically-inducedmouse colon tumor model recapitulates many histopathologicalfeatures associated with the multistage progression of humansporadic colorectal cancers. AOM has been successfully used togenerate noninvasive in situ adenocarcinomas at the progressionstage of carcinogenesis (20weeks old, 10weeks after the last AOMinjection), specifically within the proximal colon in the mousewith constitutive expression of oncogenic K-ras (39). Our modelutilizes the inducibleVil-Cre-ERT2-driven Cre recombinase, whichis expressed in the distal and proximal positions of the colon(Supplementary Fig. S1A), therefore, the adenomas were foundthroughout the colon.

Minimally invasive OCT quantified the growth of colonic ade-nomas in this mouse model over time, and traditional histologicand molecular methods were used to analyze the sulindac che-mopreventive effect in the presence of K-rasmutation at the finaltimepoint (34weeks ofmice age).Wehave previously shown thatOCT imaging is capable of identifying diseased tissue and mon-itoring the development of lesions individually and accurately inboth chemical-induced (AOM-induced) and geneticallymodified(ApcMin/þ)mousemodels (28, 30,40).Wealsohavedemonstratedan advantage of this highly accurate and efficient nondestructiveimaging technology to evaluate the effect of chemopreventiveagents on colon tumorigenesis in different colon cancer models(30, 41). This study confirmed once more the validity of the OCTmethod for monitoring of colon tumorigenesis.

Analysis of the number of colon tumors usingOCT showed thatAOM-induced animals expressing mutant K-RAS protein devel-opedonaverage7 timeshighernumberof tumors andhada5-foldincrease in tumor burden than their K-ras wt littermates. Analysisof the OCT images generated at multiple time points showed thatsulindac was completely effective in preventing the emergence ofcolon adenomas after AOM induction in K-ras wt and Vil-Cre–negative K-rasG12Dmice (reduction from42% incidence in untreat-ed animals to 0% incidence in treated), but was partially effectivein treating animals expressingmutantK-ras (reduction from100%incidence in untreated animals to 62% in treated). At the sametime, statistical analyses of tumor number, tumor burden, and thepercentage of mice with at least one adenoma, demonstrated asignificant efficacy of sulindac treatment against the mutant






K-RAS–expressing colon tumors (Table 1C). Sulindac treatmentwas particularly effective in suppressing tumor burden driven byK-ras mutation (83% reduction, P < 0.01). This finding indicatesthat sulindac has a potential advantage for colon cancer chemo-prevention in patients with colon tissue positive for K-RASmuta-tion. Because of the signs of ongoing colorectal decease, theremaystill be additional positive effects.

Image-based analysis of the tumor number and tumor burden(as a function of time (tumor growth rate) was especially valuablein this study as it provided information on colon carcinogenesisduring the promotion/progression stages. In particular, it dem-onstrated that mutant K-ras shortens the promotion stage ofAOM-induced colon carcinogenesis (time between the last AOMinjection and thedetectionof the colon adenomas) from26weeksin K-ras wt mice to 18 weeks in K-rasG12D mice. Treatment ofK-rasG12D mice with sulindac did not increase tumor-free dura-tion, but did reduce the number of adenomas detected at the 18-week time point.

OCT imaging also facilitated the analysis of the effects ofmutant K-ras on morphologic parameters of the colon tissue, forexample, mucosal thickness. In control animals, expression ofmutant K-ras caused a significant increase in the thickness ofcolonic mucosa, and sulindac prevented this increase effectively.Mucosal thickness measurement obtained in vivo with OCT maybe more accurate than those measured from histology, becausethe shrinkage effects of fixation are eliminated, and because theamount of stretch to the colon was standardized by the insertionof the snug-fitting 2-mm diameter endoscope.

Unfortunately, there is currentlynomethod todifferentiate low-grade from advanced adenomas with OCT. Histologic evaluationshowed that all adenomas in all genotypes were low grade. Thisfindingmaybeexplainedby the fact thatourmousemodel isbasedon the monoallelic expression of the mutant K-ras, whereas high-grade colon adenomas from clinical studies have a complexmutational profile of multiple mutations (i.e., APC, SMAD, etc.)ashasbeenassessedby thewhole-exomesequencinganalysis (42).

Prior studies suggest that aberrant crypts in the colon ofexperimental animals are precursor lesions of the adenoma–carcinoma sequence (43, 44). The ACF analysis performed in18-week-old mice showed that sulindac treatment significantlyincreased the number of ACF in AOM-induced K-rasG12Dmice. Atthe same time point, sulindac-treated AOM-induced K-rasG12D

micedidnot develop any adenomas in the small intestine andhada 3-fold decrease in the colon tumor number (Fig. 4B). Thissuggests that sulindac inhibits the progression of ACFs to adeno-mas in colon mucosa, which expresses mutant K-RAS protein.

In addition, the IHC analyses of colonic tissues from differenttreatment groups were done to evaluate the expression of COX-2(inflammation marker involved in the mechanism of sulindacantitumor activity), CC3 (apoptosis marker), and Ki-67 (prolif-eration marker).

On the basis of IHC scoring, COX-2 expression (inflammationmarker involved in themechanismof sulindac antitumor activity)was elevated in all AOM-treated animals, compared with controlanimals of all groups (P < 0.01). IHC scoring was not significantlyaltered bymouse genotype or sulindac treatment. Themechanismresponsible for the antitumor activity of sulindac metabolites hasbeen studied extensively and a number of COX-dependent, andindependent targets have been identified (45). We reportedpreviously that sulindac sulfide metabolite suppressed prosta-glandin synthase E2 (PGE2) production in colon cancer cells

expressingmutantK-RASwhile it increasedCOX-2protein level inthis model, but sulindac sulfone metabolite inhibited both PGE2levels and COX-2 expression in treated cancer cells (46). Previousstudies have concluded that inhibition of COX-2 enzyme activityis required for antitumor activity of NSAIDs, although othermechanisms have been reported (45).

High rates of proliferation and apoptosis were noted in AOM-treated groups comparedwith control groups (P<0.001 for Ki-67,P¼ 0.02 for CC3). The Ki-67 scores were not significantly affectedby sulindac treatment in these groups.We found an increase in theapoptosis marker CC3 staining in sulindac-treated mice of allgenotypes in both control and AOM-treated groups (P ¼ 0.02).The lack of significant changes in Ki-67 and CC3 expression insulindac-treated mice in the current study may be due to therelatively low dose of sulindac used (100 ppm) or could suggest aresistance to apoptosis in the tested mouse model.

Our current study is significant as it demonstrates for the firsttime using in vivo OCT imaging that mutant K-RAS expressionresults in faster development of the colonic adenomas, andsulindac chemoprevention does not inhibit the onset of adeno-mas carrying a K-RAS mutation.

Our findings underscore the importance of targeted and tai-lored therapy aimed to suppress mutant K-RAS activity in indi-viduals with high risk of developing cancer.

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

DisclosureThe content is solely the responsibility of the authors and does not neces-

sarily represent the official views of the NIH.

Authors' ContributionsConception and design: J.K. Barton, N.A. IgnatenkoDevelopment of methodology: R.B. Nagle, J.K. BartonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.F.S. Rice, K.G. Ehrichs, M.S. Jones, R.B. Nagle,D.G. Besselsen, J.K. Barton, H. Chen, E.R. AbrilAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.F.S. Rice, K.G. Ehrichs, N.A. Ignatenko, C.-H. Hsu,J.K. BartonWriting, review, and/or revision of themanuscript:P.F.S. Rice, C.-H.Hsu,D.G.Besselsen, J.K. Barton, N.A. IgnatenkoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P.F.S. RiceStudy supervision: P.F.S. Rice, J.K. Barton

AcknowledgmentsWewould like to thankDr. Sylvia Robin, Institute Curie-CNRS, Paris, France,

for providing us with the inducible Vil-Cre ERT2 strain.

Grant SupportThis work was supported by NIH/NCIR01CA157595 (to N.A. Ignatenko),

NIH/NCIR01CA109835 (to J.K. Barton), Research reported in this publicationwas supported by theNCI of the NIH under award numbers R01CA109835 andR01CA157595. Research in this article was directly supported by the Experi-mental Mouse and Tissue Acquisition andMolecular Analysis Shared Resourcesfunded by the NCI Award P30CA023074 (to A.E. Kraft, Cancer Center Supportgrant).

The costs of publication of this articlewere defrayed inpart 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 July 27, 2017; revised September 15, 2017; accepted October 23,2017; published OnlineFirst November 8, 2017.

Rice et al.





References1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin

2016;66:7–30.2. Leslie A, Pratt NR, Gillespie K, Sales M, Kernohan NM, Smith G, et al.

Mutations of APC, K-ras, andp53are associatedwith specific chromosomalaberrations in colorectal adenocarcinomas. Cancer Res 2003;63:4656–61.

3. Mizumoto K, Tanaka M. Genetic diagnosis of pancreatic cancer. J Hepa-tobiliary Pancreat Surg 2002;9:39–44.

4. Cowgill SM, Muscarella P. The genetics of pancreatic cancer. Am J Surg2003;186:279–86.

5. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell1990;61:759–67.

6. Fearon ER.Molecular genetic studies of the adenoma-carcinoma sequence.Adv Intern Med 1994;39:123–47.

7. Einspahr JG, Martinez ME, Jiang R, Hsu CH, Rashid A, Bhattacharrya AK,et al. Associations of Ki-ras proto-oncogene mutation and p53 gene over-expression in sporadic colorectal adenomas with demographic and clin-icopathologic characteristics. Cancer Epidemiol Biomarkers Prev 2006;15:1443–50.

8. Vincenzi B, Cremolini C, Sartore-Bianchi A, Russo A,Mannavola F, PerroneG, et al. Prognostic significance of K-Ras mutation rate in metastaticcolorectal cancer patients. Oncotarget 2015;6:31604–12.

9. Shaw P, Tardy S, Benito E, Obrador A, Costa J. Occurrence of Ki-ras and p53mutations in primary colorectal tumors. Oncogene 1991;6:2121–8.

10. Yanez L, Groffen J, Valenzuela DM. c-K-ras mutations in human carcino-mas occur preferentially in codon 12. Oncogene 1987;1:315–8.

11. Forrester K, Almoguera C,HanK,GrizzleWE, PeruchoM.Detection of highincidence of K-ras oncogenes during human colon tumorigenesis. Nature1987;327:298–303.

12. Pretlow TP, Brasitus TA, Fulton NC, Cheyer C, Kaplan EL. K-ras mutationsin putative preneoplastic lesions in human colon. J Natl Cancer Inst1993;85:2004–7.

13. Shields JM, Pruitt K, McFall A, Shaub A, Der CJ. Understanding Ras: `it ain'tover til it's over'. Trends Cell Biol 2000;10:147–54.

14. Pruitt K, Der CJ. Ras and Rho regulation of the cell cycle and oncogenesis.Cancer Lett 2001;171:1–10.

15. Mitsushita J, Lambeth JD, Kamata T. The superoxide-generating oxidaseNox1 is functionally required for Ras oncogene transformation. Cancer Res2004;64:3580–5.

16. DugganDE,Hooke KF, Risley EA, Shen TY, ArmanCG. Identification of thebiologically active form of sulindac. J Pharmacol Exp Ther 1977;201:8–13.

17. Marnett LJ. Aspirin and the potential role of prostaglandins in colon cancer.Cancer Res 1992;52:5575–89.

18. Lawson KR, IgnatenkoNA, PiazzaGA, CuiH,Gerner EW. Influence of K-rasactivation on the survival responses of Caco-2 cells to the chemopreventiveagents sulindac and difluoromethylornithine. Cancer Epidemiol Biomar-kers Prev 2000;9:1155–62.

19. Tai WP, Hu PJ, Wu J, Lin XC. The inhibition of Wnt/beta-catenin signalingpathway in human colon cancer cells by sulindac. Tumori 2014;100:97–101.

20. Meyskens FL, McLaren CE, Pelot D, Fujikawa-Brooks S, Carpenter PM,Hawk E, et al. Difluoromethylornithine plus sulindac for the prevention ofsporadic colorectal adenomas: a randomized placebo-controlled, double-blind trial. Cancer Prev Res 2008;1:32–8.

21. Tonelli F, Valanzano R, Messerini L, Ficari F. Long-term treatment withsulindac in familial adenomatous polyposis: is there an actual efficacy inprevention of rectal cancer? J Surg Oncol 2000;74:15–20.

22. Keller JJ, Offerhaus GJA, Drillenburg P, Caspers E, Musler A, Ristim€aki A,et al. Molecular analysis of sulindac-resistant adenomas in familial ade-nomatous polyposis. Clin Cancer Res 2001;7:4000–7.

23. ArberN,HanEK, SgambatoA, PiazzaGA,Delohery TM, BegemannM, et al.A K-ras oncogene increases resistance to sulindac-induced apoptosis in ratenterocytes. Gastroenterology 1997;113:1892–900.

24. Rao CV, Rivenson A, Simi B, Zang E, Kelloff G, Steele V, et al. Chemopre-vention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflam-matory agent. Cancer Res 1995;55:1464–72.

25. Jacoby RF, Seibert K, Cole CE, Kelloff G, Lubet RA. The cyclooxygenase-2inhibitor celecoxib is a potent preventive and therapeutic agent in the minmouse model of adenomatous polyposis. Cancer Res 2000;60:5040–4.

26. Saukkonen K, Tomasetto C, Narko K, Rio MC, Ristimaki A. Cyclooxygen-ase-2 expression and effect of celecoxib in gastric adenomas of trefoil factor1-deficient mice. Cancer Res 2003;63:3032–6.

27. Winkler AM, Rice PF,Weichsel J,Watson JM, BackerMV, Backer JM, et al. Invivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-tar-geted NIR fluorescent probe in the AOM-treated mouse model. MolImaging Biol 2011;13:1173–82.

28. Hariri LP, Qiu Z, Tumlinson AR, Besselsen DG, Gerner EW, Ignatenko NA,et al. Serial endoscopy in azoxymethane treated mice using ultra-highresolution optical coherence tomography. Cancer Biol Ther 2007;6:1753–62.

29. Boivin GP, Washington K, Yang K, Ward JM, Pretlow TP, Russell R, et al.Pathology of mouse models of intestinal cancer: consensus report andrecommendations. Gastroenterology 2003;124:762–77.

30. LeGendre-McGhee S, Rice PS,Wall RA, Sprute KJ, Bommireddy R, LuttmanAM, et al. Time-serial assessment of drug combination interventions in amouse model of colorectal carcinogenesis using optical coherence tomog-raphy. Cancer Growth Metastasis 2015;8(Suppl 1):63–80.

31. Giardiello FM, Hamilton SR, Krush AJ, Piantadosi S, Hylind LM, Celano P,et al. Treatment of colonic and rectal adenomas with sulindac in familialadenomatous polyposis. N Engl J Med 1993;328:1313–6.

32. Giardiello FM, Yang VW, Hylind LM, Krush AJ, Petersen GM, Trimbath JD,et al. Primary chemoprevention of familial adenomatous polyposis withsulindac. N Engl J Med 2002;346:1054–9.

33. Debinski HS, Trojan J, Nugent KP, Spigelman AD, Phillips RK. Effect ofsulindac on small polyps in familial adenomatous polyposis. Lancet1995;345:855–6.

34. Phillips RK, Wallace MH, Lynch PM, Hawk E, Gordon GB, Saunders BP,et al. A randomised, double blind, placebo controlled study of celecoxib, aselective cyclooxygenase 2 inhibitor, on duodenal polyposis in familialadenomatous polyposis. Gut 2002;50:857–60.

35. Arber N, Eagle CJ, Spicak J, R�acz I, Dite P, Hajer J, et al. Celecoxib for theprevention of colorectal adenomatous polyps. N Engl J Med 2006;355:885–95.

36. SamadderNJ,NeklasonDW,BoucherKM,ByrneKR,KanthP, SamowitzW,et al. Effect of sulindac and erlotinib vs. placebo on duodenal neoplasia infamilial adenomatous polyposis: a randomized clinical trial. JAMA2016;315:1266–75.

37. Johnson L, Mercer K, GreenbaumD, Bronson RT, Crowley D, Tuveson DA,et al. Somatic activation of the K-ras oncogene causes early onset lungcancer in mice. Nature 2001;410:1111–6.

38. Tuveson DA, Shaw AT, Willis NA, Silver DP, Jackson EL, Chang S, et al.Endogenous oncogenic K-ras(G12D) stimulates proliferation and wide-spread neoplastic and developmental defects. Cancer Cell 2004;5:375–87.

39. Calcagno SR, Li S, Colon M, Kreinest PA, Thompson EA, Fields AP, et al.Oncogenic K-ras promotes early carcinogenesis in the mouse proximalcolon. Int J Cancer 2008;122:2462–70.

40. McNally JB, Kirkpatrick ND, Hariri LP, Tumlinson AR, Besselsen DG,Gerner EW, et al. Task-based imaging of colon cancer in the Apc(Min/þ)mouse model. Appl Opt 2006;45:3049–62.

41. Tumlinson AR, Povazay B, Hariri LP, McNally J, Unterhuber A, Her-mann B, et al. In vivo ultrahigh-resolution optical coherence tomog-raphy of mouse colon with an achromatized endoscope. J BiomedOptics 2006;11:064003.

42. Lee SH, Jung SH, Kim TM, Rhee JK, Park HC, Kim MS, et al. Whole-exomesequencing identified mutational profiles of high-grade colon adenomas.Oncotarget 2017;8:6579–88.

43. McLellan EA, Bird RP. Aberrant crypts: potential preneoplastic lesions inthe murine colon. Cancer Res 1988;48:6187–92.

44. Takayama T, Katsuki S, Takahashi Y, Ohi M, Nojiri S, Sakamaki S, et al.Aberrant crypt foci of the colon as precursors of adenoma and cancer.N Engl J Med 1998;339:1277–84.

45. Tsioulias GJ, Go MF, Rigas B. NSAIDs and colorectal cancer control:promise and challenges. Curr Pharmacol Rep 2015;1:295–301.

46. Taylor MT, Lawson KR, Ignatenko NA, Marek SE, Stringer DE, Skovan BA,et al. Sulindac sulfone inhibits K-ras-dependent cyclooxygenase-2 expres-sion in human colon cancer cells. Cancer Res 2000;60:6607–10.






Published OnlineFirst November 8, 2017.Cancer Prev Res Photini F.S. Rice, Kevin G. Ehrichs, Mykella S. Jones, et al. Sulindac in Colon Cancer Chemoprevention?

Oncogene Attenuate the Effect ofK-RASDoes Mutated

Updated version

10.1158/1940-6207.CAPR-17-0230doi:

Access the most recent version of this article at:

Material

Supplementary

-17-0230.DC1

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2017/11/08/1940-6207.CAPRAccess the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.17-0230http://cancerpreventionresearch.aacrjournals.org/content/early/2017/12/12/1940-6207.CAPR-To request permission to re-use all or part of this article, use this link



http://cancerpreventionresearch.aacrjournals.org/lookup/doi/10.1158/1940-6207.CAPR-17-0230

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2017/11/08/1940-6207.CAPR-17-0230.DC1

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2017/11/08/1940-6207.CAPR-17-0230.DC1

http://cancerpreventionresearch.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerpreventionresearch.aacrjournals.org/content/early/2017/12/12/1940-6207.CAPR-17-0230

http://cancerpreventionresearch.aacrjournals.org/content/early/2017/12/12/1940-6207.CAPR-17-0230


Documents

Does Mutated K-RAS Oncogene Attenuate the Effect of ......Research Article Does Mutated K-RAS Oncogene Attenuate the Effect of Sulindac in Colon Cancer Chemoprevention? Photini F.S