Supporting Information Fujishita et al. 10.1073/pnas.0800041105 SI Text Polyp Scoring. Intestinal polyps were counted as described (1). Briefly, the small and large intestines were excised, washed with ice-cold PBS, and opened longitudinally. After fixation with 10% formalin-pbs, the number and size of polyps were counted under a dissection microscope. Animal Survival Assay. Drug-treated mice were kept until they became moribund, when the animals were euthanized. We stopped this experiment at 1.7 years, and, healthy mice that had survived were euthanized and subjected to polyp scoring. All mice were euthanized with carbon dioxide. Histological Analysis, Immunohistochemistry, Immunofluorescence, and Immunocytochemistry. Tissues were fixed in 10% formalin- PBS for mtor or 4% paraformaldehyde (PFA)-PBS for p-s6, VEGF, and von Willebrand factor (vwf). Samples embedded in paraffin were sectioned at 4- m thickness and stained with H&E or immunohistochemistry. Sections were stained with anti-p-s6 and anti-mtor (Cell Signaling Technology) antibodies with prior antigen retrieval by microwave in sodium citrate buffer (ph 6.0). For staining with anti-vwf (Dako) and anti-vegf (R&D Systems), sections were pretreated with trypsin (Sigma) for antigen retrieval. After incubation with primary antibodies, sections were incubated with biotinylated secondary antibodies (Vector Laboratories). The peroxidase was developed with a Vectorstain Elite kit (Vector Laboratories) with a DAB Substrate kit (Vector Laboratories). For immunofluorescence of tissues, they were embedded in OCT compound, frozen, and then sectioned at 6- m thickness. Anti-CD31 (BD Pharmingen) and anti-p-s6 antibodies were simultaneously used as primary antibodies. We used Alexa Fluor 488 donkey anti-rabbit IgG or Alexa Fluor 594 donkey anti-rat IgG (Molecular Probes) as a secondary antibody. For immunofluorescence of cells, the cells grown on coverslips were fixed with 4% PFA-PBS. Anti-mTOR and anti-active- -catenin (Upstate) antibodies were used as primary antibodies. We used Alexa Fluor 488 donkey anti-rabbit IgG or Alexa Fluor 594 donkey anti-mouse IgG (Molecular Probes) as a secondary antibody. Nuclei were stained by DAPI (Molecular Probes). Western Blot Analysis. The tissue and cells were homogenized and sonicated in lysis buffer [100 mm NaCl/10 mm Tris Cl (ph 7.6)/1 mm EDTA (ph 8.0)/1 mm sodium pyrophosphate/10 mm sodium fluoride/1 mm sodium orthovanadate/complete Mini (Roche Diagnostic)]. After centrifugation at 15,000 g, supernatants containing 20 50 g of protein were separated by 4/20% or 10/20% gradient gel (Daiichi Pure Chemicals) and transferred to Immobilon P membranes (Millipore). The membranes were probed with antibodies for p-s6 (Ser-235/236), S6, p-s6 kinase (Thr-389), S6 kinase, p-mtor (Ser-2448), mtor, p-akt (Ser- 473 or Thr-308), Akt, p-erk1/2 (Thr-202/Tyr-204), Erk1/2, p- AMPK (Thr-172), p-eif4g (Ser-1108) p-foxo1 (Ser-256), Foxo1 (Cell Signaling), -actin, -catenin (Sigma), COX-2 (Cayman Chemical), COX-1, cyclin A, and cyclin E (Santa Cruz Biotechnology) and detected by enhanced chemiluminescence using ECL Western Blotting Detection Reagents (GE Healthcare Bio-Sciences). The signal intensity was quantified by using the National Institutes of Health s Image program. TUNEL Analysis. Tissues were fixed in 10% formalin-pbs for TUNEL analysis. Samples embedded in paraffin were sectioned at 4- m thickness. Apoptotic cells were stained by using an Apoptosis in situ Detection Kit (Wako Pure Chemical Industries) and counted in high-magnification fields ( 400). BrdU Staining. Tissues were fixed in 70% ethanol for 2hafteri.p. injection of BrdU. Samples were embedded in paraffin, and then sectioned at 4- m thickness. BrdU staining was performed with a BrdU Labeling and Detection Kit II (Roche Diagnostics), and the stained cells were counted in high-magnification fields ( 400). Microvessel Density (MVD) and Antibody Array. MVD was determined by counting the number of capillaries composed of vwf-positive cells in high-magnification fields. Antibody array analyses were performed according to the manufacturer s instructions of Mouse Angiogenic Antibody Array 1.1 (RayBiotech). Cell Cultures and Transfection of RNA Oligonucleotides. We obtained human colon cancer cell line SW480 from M. Tsujii. SW480 was cultured in DMEM supplemented with 10% FBS (Biowest). Transfection of sirna was performed by using Lipofectamine 2000 (Invitrogen). Two sequences were designed to target human -catenin (CTNNB1 sirna-1: 5 -CCACAAGAUUA- CAAGAAACGGCUUU-3 and CTNNB1 sirna-2: we referred to ref. 2). Scramble RNA was used as a control (Hokkaido System Science). To investigate the effects of sirna on -catenin, we determined Wnt signaling dependent transcription activity by using -catenin-tcf reporter plasmids (Upstate) as described (3). Retroviral shrna Cloning, Production, and Infection. Desalted oligonucleotides were cloned into psuppressor Retro (IMGENEX) with the XhoI/XbaI sites at the 3 end of the human U6 promoter. We referred to ref. 4 for the sequences of the oligonucleotides. Plasmid transfection and retrovirus infection were guided by the manufacturer s instruction. The infected cells were selected for neomycin resistance by G418 (Sigma). RT-PCR Analysis. Total RNA from cultured cells and mouse tissue was extracted by using TRI Reagent (Sigma), according to the manufacturer s protocol. Each RNA sample (4 g) was reversetranscribed and subjected to PCR under the following conditions: denaturation at 94 C for 30 s, annealing at 55 C for 1 min, and extension at 72 C for 1 min, for 30 cycles. The primers for mtor were 5 -CTCAACATCGAGCATCGCATCATG-3 (forward primer) and 5 -ACCAGAAAGGGCACCAGC- CAATATAG-3 (reverse primer). The control RT-PCR for GAPDH was performed to normalize the sample amounts. Quantitative RT-PCR Analyses. Total RNA from cultured cells was extracted by using ISOGEN, and then reverse-transcribed. Thereafter, cdna was amplified and measured by using Taq- Man Gene Expression Assays (Assay ID: Hs00234522 m1) (Applied Biosystems) and an ABI-7700 DNA Sequence Detector (Applied Biosystems). TaqMan Ribosomal RNA Control Reagents were used for normalization. Statistical Analysis. Statistical analyses were carried out by unpaired Student s t test or Dunnett s multiple comparison test, and P 0.05 was considered significant. 1of6
1. Oshima M, et al. (1995) Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc Natl Acad Sci USA 92:4482 4486. 2. Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB (2003) Small interfering RNAs directed against -catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin Cancer Res 9:1291 1300. 3. Aoki K, Tamai Y, Horiike S, Oshima M, Taketo MM (2003) Colonic polyposis caused by mtor-mediated chromosomal instability in. Apc / 716 Cdx2 / compound mutant mice. Nat Genet 35:323 330. 4. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mtor complex. Science 307:1098 1101. 2of6
Fig. S1. RAD001 regresses large-size polyps in the Apc 716 mouse. (A) Schematic diagram of the RAD001 treatment schedule. Mice were treated with RAD001 once a day for 8 weeks. (B) Number of large size polyps ( 1.5 mm) per mouse scored in Apc 716 mice treated with placebo or RAD001. (C) Gross appearance of the small intestinal polyps. Duo, duodenum; Jej, jejunum; Ile, ileum. 3of6
Fig. S2. The mtor pathway activation in the Apc 716 intestinal polyps is independent of the PI3K-Akt, Erk-Mek, and AMPK pathways. (A) Western blot analysis of p-akt at Ser-473 or Thr-308, total Akt, and p-s6 in the normal ileal mucosa (N) and polyps (P) of the Apc 716 mouse that was treated with placebo, wortmannin (5 mg/kg), or RAD001 (10 mg/kg) for 3 days. -Actin is shown as loading control. (B) Western blot analysis of Erk1/2 phosphorylated at Thr-202/Tyr-204 (p-erk1/2) and total Erk in the normal ileal mucosa (N) and polyps (P) of the Apc 716 mouse that was treated with placebo or RAD001 (10 mg/kg) for 3 days. -Actin is shown as loading control. (C) Western blot analysis of AMPK phosphorylated at Thr-172 (p-ampk) and total AMPK in the normal ileal mucosa (N) and polyps (P) of the Apc 716 mouse that was treated with placebo or RAD001 (10 mg/kg) for 3 days or 8 weeks. (D) Effect of the starvation on S6 phosphorylation at Ser-235/236 in WT mice and Apc 716 mice. Placebo or RAD001 was administered for 2 weeks in WT (C57BL/6) mice. Apc 716 mice were administered with placebo for 2 weeks. Starvation of WT and Apc 716 mice were performed by food depletion for 16 h before euthanasia. Other mice were fed ad libitum. 4of6
Fig. S3. The activation of Wnt signaling increases expression level of mtor protein and mrna. (A D) Immunostaining of mtor protein in normal ileum (A and C) and polyp (B and D)oftheApc 716 mouse. Adenoma epithelium and crypt cytoplasm were strongly stained. C and D are higher magnifications of A and B, respectively. (Scale bars: A and B, 200 m; C and D,50 m.) (E and F) Quantitative PCR (E) and RT-PCR (F) analyses of mtor mrna expression. Two sirnas for -catenin with different sequences, CTNNB1 sirna-1 (40 nm) and CTNNB1 sirna-2 (40 nm), were used for transfection of SW480. Samples were prepared 72 h after transfection. G3PDH is shown as loading control in F.(G) RT-PCR analysis of mtor mrna expression in the normal ileal mucosa and polyps of the Apc 716 mouse. G3PDH is shown as loading control. (H) Effect of mtor knockdown on mtorc1 pathway activation in SW480. mtor was reduced by a retroviral shrna. S6 kinase, phosphorylated at Thr-389 (p-s6k), and its total protein (S6K) was detected by Western blot analysis. -Actin is shown as a loading control. 5of6
Fig. S4. mtor signaling in intestinal polyps of Apc 716 mice. (A) In the normal epithelium of intestine, -catenin is degraded by destruction complex containing APC, Axin, and GSK3. -Catenin is finally destructed through ubiquitin-proteasome pathway. Activity of mtorc1 signaling is regulated by nutrients, etc. (B) (Left) In the adenoma epithelium of intestinal polyps, loss of heterozygosity (LOH) of Apc gene results in -catenin stabilization and activation of the Wnt signaling and triggers off polyp initiation. mtor protein is increased in adenoma epithelium with Wnt signaling activation. (Right) In the angiogenic endothelial cells of polyps, mtorc1 signaling is activated by growth factors. Treatment with RAD001 inhibits both adenoma cell growth and angiogenesis. 6of6