Aspirin and Low Dose Nitric Oxide-Donating Aspirin Suppress. Tumorigenesis and Increase Life Span in a Lynch Syndrome Mouse Model

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1 Aspirin and Low Dose Nitric Oxide-Donating Aspirin Suppress Tumorigenesis and Increase Life Span in a Lynch Syndrome ouse odel ichael A. cilhatton, Jessica Tyler, Laura A. Kerepesi, Tina Bocker-Edmonston, elanie H. Kucherlapati, Winfried Edelmann, Raju Kucherlapati, Levy Kopelovich and Richard Fishel Supplementary Data Supplementary ethods Supplementary Figures & Tables Figure S1 Figure S2 Figure S3 Figure S4 Supplementary Tables Table S1 Table S2 Table S3

2 Supplementary ethods Glutathione and thioredoxin reductase assays Groups of sh2 flox/flox VpC +/+ and VpC +/+ mice (n=6) were treated with either 400 mg/kg ASA or 720 mg/kg NO-ASA for 120 days. ice had been weaned and were ~25 days old when treatment was started. Untreated control groups were also included for each strain. ice were then euthanized, their livers were removed, placed on ice and apportioned equally for separate determination of glutathione and thioredoxin reductase levels. These were assayed with kits obtained from Cayman Scientific (Glutathione Assay Kit, Catalog No ; Thioredoxin Reductase Assay Kit Catalog No ). The protocols accompanying the kits were followed exactly. Final concentrations of glutathione and thioredoxin reductase were calculated according to methods for data analysis outlined in each protocol. 1

3 wild-type allele sh2 locus targeting vector Neo F 184R 130F 165R homologous recombination targeted allele 11 Neo flipase recombination 130F 165R Neo deleted sh2 flox/flox F 184R Cre/loxP recombination exon 12 deleted sh2 floxδ F 165R loxp Frt Supplementary Figure S1. Targeting strategy used to generate sh2 flox/flox mice. The above figure outlines the procedures that were adopted to generate conditional sh2 knockout mice that had exon 12 flanked by two loxp sites. 184F, 184R, 130F and 165 indicate the approximate location of primers that were used in subsequent genotyping reactions to confirm that the correct targeting events had occurred. sh2 flox/flox mice were then crossed with mice carrying a Cre transgene, B6.SJL-Tg(Vilcre)997Gum/J. Expression of Cre recombinase in the intestine from the villin gene (Vil1) promoter, (VpC=Villin promoter Cre recombinase), resulted in tissue-specific ablation of sh2 in this cellular compartment, thus creating the sh2 flox/flox VpC +/+ strain of mice. 2

4 728 bp (genomic sequence) sh2 +/+ allele primers 130F/165R 984 bp (exon 12 + loxp intact) sh2 flox/flox allele primers 130F/165R 341 bp (exon 12 deleted) sh2 floxδ12 allele primers 184F/165R Supplementary Figure S2. Cre-mediated deletion of sh2 exon 12 occurs only in the intestinal tissues of sh2 flox/flox VpC +/+ mice. DNA was isolated from various tissues of two sh2 flox/flox VpC +/+ mice, Cr016 and Cr014, and analyzed at the molecular level by PCR with primers 130F, 165R and 184F. A 984 bp product, corresponding to the intact floxed sh2 allele was amplified from all tissues examined. The 341 bp product, corresponding to the sh2 floxδ12 allele, was only amplified from intestinal tissues, including the colon. Samples were not micro-dissected to obtain more homogenous populations of epithelial cells, which is why both PCR products were amplified from intestinal tissues. The 728 bp product corresponds to the wild-type, unfloxed genomic sequence around sh2 exon 12. It was amplified from DNA from a wild-type control C57Bl/6J mouse. 3

5 sh2 +/+ duodenum Lu sh2 +/+ jejunum Lu l l sh2 flox/flox VpC +/+ duodenum Lu sh2 flox/flox VpC +/+ jejunum Lu l l Supplementary Figure S3. sh2 protein is not expressed in the intestinal epithelial cells of sh2 flox/flox VpC +/+ mice. Tissues were prepared from wild-type (sh2 +/+ )VpC +/+ and sh2 flox/flox VpC +/+ mice, washed in PBS and fixed in formalin. They were embedded in paraffin and subjected to immunohistochemistry with an affinity purified rabbit antibody against sh2 (Bethyl Laboratories, catalogue number IHC-00082) at a 1:250 dilution. sh2 protein was visualized with horseradish peroxidase, indicated by a brown nuclear stain, and counter-stained with hematoxylin (blue). sh2 is clearly visible in the crypts, and to a lesser extent the villi, of the duodenum and jejunum of sh2 +/+ mice (upper panels). It is not detectable in the corresponding regions of sh2 flox/flox VpC +/+ mice due to localized Cre-mediated ablation of sh2 expression (deletion of exon 12) in these tissues. (l = muscularis; Lu = lumen) 4

6 A Total GSH (µ) untreated ASA NO-ASA untreated ASA NO-ASA B Thioredoxin Reductase Activity (µmol/min/ml/g protein) untreated ASA NO-ASA untreated ASA NO-ASA Supplementary Figure S4. Continuous treatment with NO-ASA does not significantly depress the glutathione or thioredoxin systems. A, Total glutathione levels in both sh2 flox/flox VpC +/+ and VpC +/+ mice livers were not significantly lowered by prolonged dietary exposure to NO-ASA. B, Similarly, levels of thioredoxin reductase were not significantly perturbed by long-term treatment with either NO- ASA or ASA. (Black bars = VpC +/+ ; grey bars = sh2 flox/flox VpC +/+ ; n=6 per group). Error bars show standard deviation around the mean. Differences between groups were compared by a t-test; none were statistically significant. 5

7 Supplementary Table S1. Histopathologic classification of tumors from sh2 flox/flox VpC +/+ mice Table S1 A. Untreated mice, plain food Block Location Diagnosis Cr049 invasive, poorly differentiated adenocarcinoma Cr065 (A) invasive, poorly differentiated adenocarcinoma arising in high grade dysplastic adenoma Cr065 (B) invasive, poorly differentiated adenocarcinoma Cr118 invasive, poorly differentiated adenocarcinoma Cr121 high grade dysplastic adenoma Cr143 (A) invasive, poorly differentiated adenocarcinoma Cr143 (B) high grade dysplastic adenoma Cr144 invasive, poorly differentiated adenocarcinoma Cr145 invasive, poorly differentiated adenocarcinoma Cr176 aberrant crypt foci Cr177 invasive, very poorly differentiated adenocarcinoma Cr194 invasive, very poorly differentiated adenocarcinoma Cr202 invasive, poorly differentiated adenocarcinoma Table S1 B. ice treated with 400 mg/kg ASA Block Location Diagnosis Cr139 invasive moderately differentiated adenocarcinoma and adenoma Cr157 invasive, poorly differentiated adenocarcinoma Cr158 invasive, poorly differentiated adenocarcinoma Cr162 (A) invasive moderately differentiated adenocarcinoma Cr162 (B) invasive, poorly differentiated adenocarcinoma Cr165 benign mucosa Cr178 invasive, poorly differentiated adenocarcinoma Cr180 high grade dysplastic adenoma with focal superficial invasion; peritumoral lymphoid infiltrate Cr186 moderately dysplastic adenoma Cr190 invasive, poorly differentiated adenocarcinoma Cr193 invasive, poorly differentiated adenocarcinoma Cr198 invasive, very poorly differentiated adenocarcinoma 6

8 Supplementary Table S1. (cont.) Table S1 C. ice treated with 720 mg/kg NO-ASA Block Location Diagnosis Cr076 Cr086 Cr088 Cr089 (A) Cr089 (B) Cr093 no neoplasm invasive, poorly differentiated adenocarcinoma; superficial high grade adenoma adenoma with high grade dysplasia/intramucosal carcinoma; superficial high grade adenoma, transition to benign mucosa invasive adenocarcinoma (x2); superficial adenomas with high grade dysplasia (x2) multiple foci of superficial adenoma with moderate to high grade dysplasia multiple foci of superficial adenoma with moderate to high grade dysplasia Cr096 (A) invasive, poorly differentiated adenocarcinoma Cr096 (B) invasive, poorly differentiated adenocarcinoma Cr096 (C) adenoma with high grade dysplasia/intramucosal carcinoma Cr097 (A) Cr097 (B) Cr097 (C) intramucosal carcinoma high grade, possibly invasive; several foci of superficial high grade adenoma several foci of adenoma with moderate to high grade dysplasia; invasive, poorly differentiated adenocarcinoma several foci of superficial high grade adenoma; possible intramucosal carcinoma Table S1 D. ice treated with 1500 mg/kg NO-ASA Block Location Diagnosis Cr216 (A) invasive, poorly differentiated adenocarcinoma Cr216 (B) adenoma with moderate dysplasia Cr217 high grade dysplastic adenoma Cr224 invasive, poorly differentiated adenocarcinoma Cr228 invasive, poorly differentiated adenocarcinoma Cr229 invasive, poorly differentiated adenocarcinoma Cr232 high grade dysplastic adenoma Cr234 high grade dysplastic adenoma Cr237 high grade dysplastic adenoma, focally invasive Cr241 invasive, poorly differentiated adenocarcinoma Cr245 high grade dysplastic adenoma Cr246 high grade dysplastic adenoma Cr252 superficially invasive, poorly differentiated adenocarcinoma 7

9 Supplementary Table S2. SI analysis of ear, normal (N) and tumor (T) intestinal tissues S2 A. VpC +/+ mice, 720 mg/kg NO-ASA S2 B. sh2 flox/flox VpC +/+ mice, plain food Tissue N= DNA S 01 S 02 S 03 S 04 S 05 il Co Co Co N= T= A Comparative SI is for (N v. T) intestinal tissues only. Du=duodenum; Je=jejunum; il=ileum; Co=colon - not applicable A Co m. SI SS Tissue N= DNA S 01 S 02 S 03 S 04 S 05 A Co m. SI N1=Je T1=Je SS N=Je SI- -2, - -1, - T=Je 0 0 0, -2 H 4 2 N1=Je T1=Je SS Kidne y N2=Je SI- T2=Je L Splee n N3=Je T3=Je SS Du SI- Je L N1=Je SI- -2, - T1=Je H 4 N1=Du SI- -1, - T1=Du L 2 Liver N2=Je T2=Je SS N=Je , - 2 SI- T=Je , - L 2 N1=Du 0, SI- T1=Du H N2=Je SI- T2=Je L N=Je SI- -1, - T=Je L 2 N=Je , -1 T=Je , -1 SS N1=Du 0 0 0, SI- T1=Du , -1 H N=Je SI- -2, - -4, - T=Je H 4 6 N1=Je SI- 8

10 202 T1=Je , - H N1=Je , -1 SI- T1=Je L Supplementary Table S2. (cont.) S2 C. sh2 flox/flox VpC +/+ mice, 400 mg/kg ASA Tissue N= DNA S 01 S 02 S 03 S 04 S 05 A Co m. SI N1=Je T1=Je SS N2=il 0, SI- T2=il L Liver Du , -1 SI- Co L N=Du T=Du SS N=Je , -1 T=Je , -1 SS N=Je SI- T=Je 0 0 0, H N=Je T=Je SS Du 0 0 0, SI- Co L N=Je SI- T=Je H N=Du SI- -1, - T=Du L 2 N=Je SI- T=Je L N1=Du SI- T1=Du H N1=Du SI- T1=Du L N2=Je 0, SI- -2, - T2=Je 0, H 4 N=Je , -1 SI- T=Je L N=Je SI- T=Je L N=Du 0 0 0, SI- 200 T=Du L N=Je SI- T=Je L S2 D. sh2 flox/flox VpC +/+ mice, 72 mg/kg NO-ASA Tissue N= DNA S 01 S 02 S 03 S 04 S 05 A Co m. SI N=Je 0, SI- T=Je H N=Je SI- T=Je H N1=Je SI- T1=Je H N2=Du SI- T2=Du L N= SI- T=Du L N=Je SI- T=Je L N=Je SI- T=Je H Ear , -2 0 N=Je SI- T=Je L N=Du SI- T=Du L N=Je SI- T=Je H N=Du T=Du SS N=Je SI- T=Je H N=Du T=Du SS A Comparative SI is for (N v. T) intestinal tissues only. Du=duodenum; Je=jejunum; il=ileum; Co=colon 9

11 10

12 Supplementary Table S2. (cont.) S2 E. sh2 flox/flox VpC +/+ mice, 720 mg/kg NO-ASA Tissue N= DNA S 01 S 02 S 03 S 04 S 05 A Co m. SI N=Du 0, SI- T=Du H N=Je SI- T=Je H Je , SI- Co H N=Je , T=Je , SS N=Du SI- T=Du H N=Du , -1 SI- T=Du H N=Du SI- T=Du 0, H N=Je , -2 0 SI- T=Je , H N=Du SI- -2, - T=Du H 4 N=Du SI- T=Du L N1=Du SI- -2, - T1=Du H 4 Splee n N=Du SI- -2, - T=Du H 4 N=Je SI- T=Je L N=Du , -2-1 SI- T=Du H N1=Du 0, SI- T1=Du H Ear nd N=Du nd SI- T=Du nd L S2 F. sh2 flox/flox VpC +/+ mice, 1500 mg/kg NO-ASA Tissue N= DNA S S S S S A Com.SI (N v N=Du SI- T=Du , -2 L N=Je SI- T=Je 0 0-2, , -2 H N=Du SI- T=Du , -4-2 H N=Je SI- T=Je L Liver N=Je , -2 SI- T=Je L N=Je SI- T=Je , -2 L N1=Du SI- T1=Du H N2=Je SI- T2=Je , -2 L N=Je T=Je SS N=Je SI- T=Je L N=Je 0 0-2, SI- T=Je L N=Je SI- T=Je H N=Du 0 0-2, , -1 SI- T=Du H N=Je T=Je SS A Comparative SI is for (N v. T) intestinal tissues only. Du=duodenum; Je=jejunum; il=ileum; Co=colon 11

13 Supplementary Table S2. Five microsatellite loci (S01-S05) were evaluated for matched sets of ear (E), intestinal tumor (T) and adjacent normal (N) intestinal tissues from sh2 flox/flox VpC +/+ mice in each treatment group. Overall, the VpC +/+ control groups did not develop tumors, so usually tissue samples were taken from two different (normal) locations along the intestinal tract. The SS ear tissues provide constitutional microsatellite profiles. They represent the standard allele sizes of these markers, indicated by 0 in each column to signify 0 bp deviation from the established marker size. S01, 02, 03 and 04 are dinucleotide markers. Differences in the lengths of microsatellite markers between normal and tumor samples and their cognate ear samples were expressed in units of 2 (bp). S05 is a mononucleotide marker; any differences in allele lengths were expressed in units of 1 (bp). If two major alleles were observed, both were recorded. Comparative SI (N vs. T) was calculated by simply assessing any changes between only the normal and tumor samples at the different markers. The relative degree of SI in the tumors was subsequently scored as follows: SS, no changes in microsatellite status; low microsatellite instability (SI-L), SI in 1 of 5 microsatellite markers; high microsatellite instability (SI-H), SI in at least 2 of 5 markers. Note that normal and tumor samples may be SI-H if compared to the SS ear but in the context of comparative SI they may be scored as SS, SI-L or SI-H. All tissues examined for VpC +/+ mice treated with 720 mg/kg ortho NO-ASA were SS, including any of the rare intestinal tumors that did develop. The criteria for comparative SI were not applicable to these samples. 12

14 Supplementary Table S3. Primer sequences for microsatellite instability analysis primer sets amplicon size primer name primer sequence accession number S01 (TG 27 ) 137 bp S 01F S 01R GGA TCA CTC GAT GTA CGG CTA CTC CCA GGC AGG CAA AGC ATT TAT AC S02 (TA 27 ) 143 bp S 02F S 02R CAC CCC TTG CTA CCA CTA AGA AA CTC ATT GGA GTT TGA CCC ATC A AC S03 (GA 29 ) 154 bp S 03F S 03R CAG GAG GTC AAG GTC ATC CTA AG CCA CCA TGG TAG GAG CTT GCT A AC S04 (CT 25 /CA 27 ) 152 bp S 04F S 04R GGA GAT TCT GCT GTT TCA AAC AAG AC TTC CTA TAC ATG GGT GGA GTA GGA S05 (A 33 ) 140 bp S 05F S 05R TAC AGA GGA TTG TCC TCT TGG AG GCT GCT TCA CTT GGA CAT TGG CT AC