Supplementary Information
Supplementary Methods
Mouse
Villin-Cre mice (B6.Cg-Tg(Vil-Cre)20Sy), Pdx-1-Cre mice (B6.FVB-Tg(Ipf1-cre)1Tuv), and FVB.129Trp53tm1Brn mice (p53F/F) were obtained from the Mouse Models of Human Cancers Consortium
(MMHCC) repository (NCI Frederick Cancer Research Center (Frederick, MD)). B6.129-Cdh1tm2Kem/J
(Cdh1F/F) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Conditional Smad4
knockout mice (Smad4F/F) of the Black Swiss and B6 and 129 background were generous gifts from
Dr. Chuxia Deng. Mouse studies were conducted with the approval of the Animal Care and Use
Committees of National Cancer Center of Korea and the National Cancer Institute, Bethesda, MD.
Compound conditional knockouts of Smad4, p53, and E-cadherin were bred with Villin-Cre mice and
Pdx-1-Cre mice to perform targeted deletion for these genes in gastric epithelium, respectively.
Offspring mice were genotyped using polymerase chain reaction (PCR) assays for tail DNA. Mice
positive for Villin-Cre and Pdx-1-Cre genes were monitored until they became moribund or showed
signs of distress, at which time necropsies were performed.
Cell culture
Human gastric cancer cells AGS, mouse conditionally immortalized stomach epithelial cells (ImSt) [1],
and mouse fibroblast cells NIH3T3 were used as control cells to characterize the primarily cultured
mouse gastric cancer cells. ASG and ImSt cells were cultured in RPMI-1640 medium (Gibco, Grand
Island, NY, USA) containing 10 % FBS (Gibco), 1% penicillin and streptomycin (Invitrogen
Biotechnology, Grand Island, NY, USA). NIH3T3 cells were cultured in DEME (Gibco, Grand Island,
NY, USA) containing 10% FBS, 1% penicillin and streptomycin.
Genotyping PCR
Mouse tail genomic DNA was isolated using Genomic DNA Mini Kit (Geneaid, New Taipei, Taiwan).
PCR genotyping primers for Cdh1 were F: 5'-CTTATACCGCTCGAGAGCCGGA-3' and R: 5'GTGTCCCTCCAAATCCGATA-3'. Amplicons of 900 and 980 bp were expected for wild-type and
floxed alleles, respectively. PCR genotyping primers for Trp53 were F: 5'TGGAGATATGGCTTGGAGTAG-3' and R: 5'-CAACTTACTTCGAGGCTTGTC-3'. PCR products of
420 and 500 bp were expected for wild-type and floxed alleles, respectively. PCR genotyping primers
for Smad4 were F: 5'-GGGCAGCGTAGCATATAAGA-3' and R: 5'-GACCCAAACGTCACCTTCAC-3'.
PCR products of 390 and 480 bp were expected for wild-type and floxed alleles, respectively. Primers
for Villin-Cre were F: 5'-TCCTCTAGGCTCGTCCCG-3' and R: 5'-CAGATTACGTATATCCTGGCAG-3'.
Primers for Pdx1-Cre were F: 5'-CTGGACTACATCTTGAGTTGC-3' and R: 5'-
CAGATTACGTATATCCTGGCAG-3’.
Necropsy protocols for gastrointestinal tumors
For necropsy of Villin-Cre-positive and Pdx-1-Cre-positive mice, the entire gastrointestinal tract was
immediately removed, and the stomach was incised along the greater curvature. The stomach was
spread onto a piece of filter paper and fixed in neutral buffered 10% formalin. 24 hours after fixation,
the stomach was cut into six strips and processed by standard methods and embedded in paraffin.
Then, 5 µm paraffin-embedded sections were stained with hematoxylin and eosin (H&E).
Immunohistochemistry
The excised gastric tumor was fixed in neutral buffered 10% formalin, processed by standard
methods. The ABC method (Vectastain Elite ABC kit and Vectastain M.O.M. kit, Vector Laboratories)
was used for immunohistochemical detection of c-myc, p53, Smad4, E-cadherin, mucin 6, and TFF2.
The following antibodies were used in this study; rabbit polyclonal anti E-cadherin antibody (1:200;
Cell Signaling, #3195), rabbit polyclonal anti c-Myc antibody (1:50; Abcam, ab32072), rabbit
polyclonal anti p53 antibody (1:100; Santa Cruz, sc-6243), mouse monoclonal anti Smad4 (1:50; sc7966), mouse monoclonal anti mucin 6 antibody (1:100; Novus Biologicals, NB120-11335), and
mouse monoclonal anti trefoil factor 2 (TFF2) antibody (1:50; Abcam, ab49536). Briefly, the cross
section slides were dewaxed, rehydrated, and then antigen retrieval was performed by heating at
100°C for 20 minutes in 0.01 M citrate buffer (pH 6.0). The slides were immersed 3% hydrogen
peroxide for 10 minutes in order to block endogenouse peroxidase activity. To reduce non-specific
binding, the slides were incubated with blocking reagents with the kit. The slides were then incubated
for 20 minutes at room temperature with the diluted primary antibodies. The sections were then
incubated with biotinylated secondary antibody for 30 minutes at room temperature, followed by
incubation with ABC reagent for 30 minutes at room temperature. Subsequently, the slides were
subjected to colorimetri detection with ImmPact DAB substrate (Vector Laboratories, SK-4105). The
slides were counterstained with Mayer’s hematoxylin for 10 seconds. Negative controls were
performed by omitting the primary antibody and substitution with diluent. The stain that was
unequivocally deeper than the background was identified to be positively stained for each marker.
Western blot anaylsis
To isolate total proteins from mouse cell lines and tissues, cell pellets and tissues were lysed with TPER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Hudson, NH, U.S.A) supplemented
with protease inhibitor (0.8 μM aprotinin, 20 μM leupeptin, 10 μM pepstatin A, 40 μM bestatin, 1 mM
phenylmethylsulfonyl fluoride (PMSF)) and phosphatase inhibitor (1 mM sodium fluoride, 1 mM
sodium pyrophosphate dehydrate, 1 mM sodium orthovanadate). To isolate nuclear proteins from
mouse gastric cancer cell lines, we used Qproteome nuclear protein extraction kit (Qiagen, Valencia,
CA) according to the manufacturer’s instructions. Protein concentration was quantitated by using a
BCA reagent kit (Thermo Fisher Scientific, Hudson, NH, U.S.A) according to manufacturer’s
instruction. Protein sample was prepared by making a 3 in 4 dilution with 4x Laemmli sample buffer
(250 mM Tris-HCl (pH 6.8), 4% SDS, 40% glycerol, 0.05% bromphenol blue, 4% 2-mercaptoethanol)
and boiling for 5 minute. Equal amounts of protein were separated on SDS-polyacrylamide gel and
transferred onto nitrocellulose membrane by electrophoresis and blotting apparatus (Bio-Rad,
Hercules, CA, U.S.A). The proteins were probed with the relevant primary antibodies and horseradish
peroxidase (HRP)-conjugated secondary antibodies at the recommended dilutions. The mouse
monoclonal anti pan-cytokeratin (1:1000; Santa Cruz, sc-8018), mouse monoclonal anti PCNA
antibody (1:1000, Santa Cruz, sc-56), rabbit polyclonal anti E-cadherin antibody (1:1000; Cell
Signaling, #3195), rabbit polyclonal anti c-Myc antibody (1:1000; Abcam, ab32072), rabbit polyclonal
anti EGFR antibody (1:1000; Santa Cruz, sc-03), rabbit polyclonal anti p-Akt1/2/3 (Thr 308) antibody
(1:1000, Santa Cruz, sc-16646-R), mouse monoclonal anti p44/42 MAPK (pErk1/2) antibody (1:1000,
Cell Signaling, #9106), rabbit monoclonal anti Cyclin D1 antibody (1:1000, Cell Signaling, #2978S),
mouse monoclonal anti smad4 antibody (1:1000; Santa Cruz, sc-7966), rabbit polyclonal anti p53
antibody (1:1000; Santa Cruz, sc-6243), rabbit monoclonal anti vimentin antibody (1:1000; Cell
signaling, #5741), mouse monoclonal anti β-catenin antibody (1:1000; BD, 610154), mouse
monoclonal anti p84 antibody (1:1000; GeneTex, GTX70220) and mouse monoclonal anti GAPDH
antibody (1:1000, Santa Cruz, sc-32233) were applied. Immunodetection were performed by using an
enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific, Hudson, NH, U.S.A).
Band densities were measured using ImageJ software (http://imagej.nih.gov/ij/) and normalized to
GAPDH.
Quantitative real-time RT-PCR (QRT-PCR)
Total RNA was isolated from mouse gastric cancer cells using AllPrep DNA/RNA/Protein Mini Kit
(Qiagen, Valencia, CA) according to the manufacturer’s instructions. 0.3 μg of total RNA was reverse
transcribed using random hexamers and amfiRivertII Reverse Transcriptase (GenDEPOT, Barker, TX)
according to the manufacturer’s standard protocols. PCR reactions were performed on a Roche
LC480 (Roche Diagnostics, Penzberg, Germany) using 5 μl of 2× QuantiTect SYBR Green PCR
Master Mix (Qiagen), 400 nM of each primer, and 2 μl of cDNA sample which was diluted 1:5 in water
in a total volume of 10 μl. Cycling conditions were as follows: 15 min at 95ºC, followed by 55 cycles
each consisting of 20 s at 94ºC, 20 s at 57ºC and 20 s at 72ºC. Data were analyzed using the LC480
software (Roche Diagnostics). QRT-PCR primers were F: 5'-GCTGCAGGTCTCCTCATG-3' and R: 5'CATCCTTCAAATCTCACTCTGC-3' for Cdh1, F: 5'-CACACGCTGCCTTGTGTCT-3' and R: 5'GGTCAGCAAAAGCACGGTT-3' for Snai1, F: 5'-CCTTGGGGCGTGTAAGTCC-3' and R: 5'TTCTCAGCTTCGATGGCATGG-3' for Snai2, F: 5'-TGATGAAAACGGAACACCAGATG-3' and R: 5'GTTGTCCTCGTTCTTCTCATGG-3' for Zeb1, F: 5'-AGCGACACGGCCATTATTTAC-3' and R: 5'GTTGGGCAAAAGCATCTGGAG-3' for Zeb2, F: 5'-GGACAAGCTGAGCAAGATTCA-3' and R: 5'CGGAGAAGGCGTAGCTGAG-3' for Twist1, , F: 5'-ACGAGCGTCTCAGCTACGCC-3' and R: 5'AGGTGGGTCCTGGCTTGCGG-3' for Twist2, F: 5'-GCAGTTGGAGAACATGGAGAC-3' and R: 5'AATAGGTTGGTACCAGTGACATCC-3' for Mmp3, F: 5'- CTTCAAGGAGCGATGGTTCTG-3' and R:
5'- TTGCCATCCTTCCTCTCGTAG-3' for Mmp14, F: 5'-GCAGGCCGTAGGACAGTATA-3' and R: 5'CCGCGCTATCATACTTCTCC-3' for Wnt5a, F: 5'-CTCAAGCGCGGTTTCCGTGA-3' and R: 5'-
CTAAGCCGGTCTTGCTCACC-3' for Wnt10b, F: 5'-ACTCGCAGTACTTCCACCTG-3' and R: 5'GGTTGTCAAGGCTCTGGTTG-3' for Fzd8 and F: 5'-GGTCGGTGTGAACGGATTTG-3' and R: 5'GTGAGTGGAGTCATACTGGAAC-3' for Gapdh. The 2-ΔΔCT method was used to calculate relative
changes.
To evaluate LOH for the Cdh1 gene of NCC-S1M, genomic DNAs were isolated from mouse gastric
cancer cell lines and Cre-negative gastric mucosa using AllPrep DNA/RNA/Protein Mini Kit (Qiagen)
according to the manufacturer’s instructions. Genomic DNA real-time PCR was performed as
previously described [2]. LOH was defined as the average log2 ratio of three probes (for exons 6, 8,
and 10 of Cdh1 gene) of tumor to normal DNA < -1.5.
Bisulfite sequencing
Each 0.3 μg of genomic DNA from mouse gastric cell lines was bisulfite-converted by using EZ DNA
MethylationTM kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. Bisulfitemodified genomic DNA was PCR-amplified with Cdh1-BGS-F (5'-GTGGAATAGGAAGTTGGGAAGTT3') and Cdh1-BGS-R (5'-CAAAACCCTCCACATACCTACAAC-3'). After PCR products were purified
using gel extraction kit (Macrogen, Seoul, Korea), the purified PCR products were cloned into the
TOPO-TA vector (Invitrogen) and transformed of E-coli according to the manufacturer’s instructions.
After isolation of plasmid DNA from 8-10 clones per each sample using plasmid Mini-Prep kit
(Macrogen), each plasmid sample was sequenced with M13-F (-20) primer (5'GTAAAACGACGGCCAG-3').
In vitro 5-aza-2'-deoxycitidine (5-Aza) challenge
5-aza-2'-deoxycytidine (5-Aza) was purchased from Sigma-Aldrich (St. Louis, MO). 5-Aza was
dissolved in DMSO to a concentration of 10 mM. 12 hours before 5-Aza treatment, 5 x 104 S1M cells
were seeded in 6 well plates. The cells were collected after 48 hours exposure to 10 μM and 20 μM of
5-Aza. Total proteins extraction from the collected cells was performed using T-PER Tissue Protein
Extraction Reagent (Thermo Fisher Scientific).
In vivo response to the anti-4-1BB
1 x 106 NCC-S1M cells were injected into the subcutaneous flank tissue of syngeneic mice. When the
heterotopic allografts reached approximately 100 mm 3 in volume, the monoclonal antibody was
injected to 5 mice per group. 200 μg of the agonistic form of anti-4-1BB monoclonal antibody (anti-41BB; 3E1 clone) or rat IgG as a control were intraperitoneally injected at days 0 and 7. Anti-4-1BB
was a kind gift from Dr. Robert Mittler (Emory University, Atlanta, GA). Tumor responses were plotted
and statistically analyzed with nonlinear regression curve fit program (GraphPad Prism 4.0, San
Diego, CA).
Measurement of β-catenin activity
β-catenin activity was evaluated by using Cignal TCF/LEF reporter assay kit (CCS-018L, SA
Biosciences, Frederick, MD). 2 x 105 primary cultured cells were suspended in 1ml of Opti-MEM
medium (Life Technologies, Grand Island, NY) and the suspension cells were seeded in 12 well plates.
These cells were transiently transfected in suspension with the Tcf/Lef reporter plasmid using
Lipofectamine 2000 transfection reagent (Life Technologies). 24 hours after transfection, Opti-MEM
medium was changed to RPMI 1640 containing 0.5% FBS. 48 hours after transfection, luciferase
assays were carried out using the dual luciferase reporter assay system (Promega, Madison, WI)
according to the manufacturer’s protocol. Light emission was quantified with a Victor 3 1420
luminescence microplate reader (Perkin-Elmer, Waltham, MA). The signals were normalized for
transfection efficiency to the internal Renilla control.
Establishment of stable β-catenin knock down cells using lentiviral shRNA
The lentiviral Ctnnb1 shRNA constructs were purchased from Sigma-Aldrich (St. Louis, MO) with
pLKO.1-puro eGFP control vector (Sigma, SHC005). The target set was generated from accession
number NM_007614.2: (1)
CCGGGCGTTATCAAACCCTAGCCTTCTCGAGAAGGCTAGGGTTTGATAACGCTTTTT, (2)
CCGGCCCAAGCCTTAGTAAACATAACTCGAGTTATGTTTACTAAGGCTTGGGTTTTT. Lentiviruses
were produced by cotransfecting shRNA-expressing vector and pMD2.G and psPAX2 constructs
(Addgene) into 293T cells by using lipofectamine 2000 (Invitrogen). Viral supernatants were
harvested 48 hours after transfection, filtered though a 0.45 μm filter, titered, and used to infect NCCS1M cells with 10 μg/mL polybrene. Cells were treated by 2 μg/mL puromycin at 48 hours after viral
transduction and were selected for 3 days.
RNA sequencing, Gene expression array, and array CGH analysis
1 μg of total RNAs, isolated from NCC-S1 and NCC-S1M cells, and normal mouse gastric epithelium,
were subjected to RNA sequencing using HiSeq2000 sequencer and TruSeq protocol, according to
the manufacturer’s recommendation (Illumina Hayward, CA). Quantile-normalized FPKM (fragments
per kilobase of exon per million fragments mapped) was used as the expression level of each gene.
BRB-ArrayTools (ver 4.1) was used for hierarchical clustering and gene set comparison analysis.
Average linkage hierarchical clustering was performed using centered correlation as a distance metric,
after gene centering. For gene set comparison analysis, names of differentially expressed genes were
mapped to probe set IDs on the HG-U133A array (www.NetAffx.com). The LS P value is the
proportion of random sets of N genes with smaller average summary statistics than the LS summaries
computed for the real data. An LS P value < 0.05 was considered significant.
1 μg of total RNAs, isolated from NCC-S3 cells and normal gastric epithelium, were subjected to
GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA) and summarized with robust
multichip average (RMA) using R (version 2.15.2). Student t-test was used to identify differentially
expressed genes. Functional category analysis was performed using DAVID functional annotation tool
(http://david.abcc.ncifcrf.gov/).
CGH array analysis were performed using Mouse GE 4x44K v2 Microarrays and 0.5 µg of genomic
DNAs from NCC-S1 and NCC-S1M cells and the same amount of tail genomic DNA of the mouse
from which NCC-S1 cell line were generated, according to the manufacturer’s recommendation
(Agilent Technologies, Santa Clara, CA). Using Agilent's CGH Analytics software, the data were
mapped to mm8 mouse genome and analyzed using the ADM-2 algorithm (Threshold of ADM-2: 6.0;
Centralization: ON; Fuzzy Zero: ON; Aberration Filters: ON (minProbes = 3 AND minAvgAbsLogRatio
= 0.25 AND maxAberrations = 10000000 AND percentPenetrance = 0)). An average
tumor/normal log2 ratio > 0.5 for 5 or more consecutive probes in a genomic locus was identified as
the amplification.
Table S1. DAVID pathway analysis on the genes up-regulated by > 3-fold in S1 and S3 cells
compared with normal gastric mucosa
Up-regulated genes in NCC-S1
KEGG_PATHWAY
P-Value
mmu04110:
Cell cycle
<0.001
mmu03030:
DNA replication
<0.001
mmu04510:
Focal adhesion
mmu04010:
MAPK signaling
pathway
mmu03440:
Homologous
recombination
mmu04512:
ECM-receptor
interaction
mmu00670:
One carbon pool by
folate
mmu04115:
p53 signaling
pathway
mmu05200:
Pathways in cancer
mmu05222:
Small cell lung
cancer
mmu04810:
Regulation of actin
<0.001
<0.001
Genes
E2F1, DBF4, TGFB3, TTK, CHEK1, CHEK2, PTTG1, TGFB1,
TGFB2, CDC45, MCM7, TFDP2, BUB1, CCNA2, MYC, CDC7,
CDK1, SKP2, ESPL1, CDC20, CDK6, MCM2, CDC25C, CDK4,
MCM3, MCM4, MCM5, CCNB1, MAD2L1, CCNB2, CCND2, PLK1,
BUB1B, GADD45A
POLE, POLA1, POLA2, MCM2, RNASEH2A, MCM3, MCM4,
MCM5, DNA2, RPA2, RFC4, MCM7, RFC1, POLD1, FEN1
CAV2, CAV1, PDGFA, PGF, TNC, ITGB3, ITGB1, IGF1R, LAMB2,
ITGB7, COL6A1, SHC1, THBS1, SHC2, AKT3, FN1, SPP1,
THBS4, FLT1, VAV3, FLT4, PIK3CD, ITGA2, ACTN1, ITGA3,
COL5A3, FLNC, FLNB, FLNA, PRKCB, VEGFC, LAMA4, ITGA5,
FYN, CCND2, LAMA5, VEGFA, MYLK, PARVB, DIAP1
MEF2C, FGF18, PDGFA, MRAS, TGFB3, DUSP10, NFKB2,
TGFB1, TGFB2, ATF2, MAP3K7, HSPA2, DUSP14, RASGRP4,
MAPT, B230120H23RIK, DUSP16, PRKACB, RAPGEF2, FGF1,
MYC, AKT3, HSPA8, CACNA2D1, RELB, NF1, NR4A1, MAPK11,
FLNC, FLNB, FLNA, DDIT3, PRKCB, RPS6KA5, MAP4K4,
DUSP3, MAPK12, RPS6KA2, CACNA1G, HSPB1, MAPK8IP3,
STMN1, DUSP9, CACNA1C, GADD45A, CD14, DUSP7, DUSP6
<0.001
RAD51C, RPA2, XRCC2, BLM, RAD51L1, POLD1, EME1,
MUS81, BRCA2, RAD52, RAD54L, RAD51
<0.001
TNC, NPNT, ITGA2, ITGA3, ITGB3, COL5A3, ITGB1, HMMR,
LAMA4, LAMB2, CD44, ITGA5, LAMA5, ITGB7, COL6A1, SV2A,
THBS1, SPP1, THBS4, FN1
<0.001
MTHFD1, MTHFD2, SHMT2, DHFR, ATIC, MTR, MTHFD1L,
GART
<0.001
0.001
0.004
0.004
CDK1, LRDD, CDK6, CHEK1, CHEK2, CDK4, GTSE1, SESN3,
CCNB1, CCNB2, CCND2, RRM2, TSC2, SERPINE1, THBS1,
IGFBP3, GADD45A
E2F1, CKS1B, FGF18, WNT16, PTGS2, PDGFA, PGF, STK36,
ARNT2, TGFB3, NFKB2, GLI3, ITGB1, TGFB1, SUFU, TGFB2,
IGF1R, LAMB2, PAX8, FGF1, MYC, CSF2RA, AKT3, FN1, BMP4,
DVL2, CTBP1, RXRB, PIK3CD, SKP2, ITGA2, BRCA2, ITGA3,
BIRC5, CDK6, FZD2, CDK4, FZD7, PRKCB, RAD51, SMO,
VEGFC, WNT7B, LAMA4, RASSF5, LAMA5, VEGFA
E2F1, CKS1B, PTGS2, RXRB, PIK3CD, SKP2, ITGA2, CDK6,
ITGA3, CDK4, ITGB1, LAMA4, LAMB2, LAMA5, MYC, AKT3, FN1
FGF18, FGD1, ENAH, PDGFA, MRAS, IQGAP3, ABI2, RDX,
ITGB3, ITGB1, TIAM2, ITGB7, MSN, FGF1, FGD3, FN1, VAV3,
cytoskeleton
ARHGEF1, LIMK1, PIK3CD, ITGA2, ACTN1, ITGA3, CHRM4,
ITGA5, CYFIP2, PIP4K2A, CD14, MYLK, DIAP1, PIP4K2B, F2R,
MYH10
mmu04114:
Oocyte meiosis 20
0.007
CDK1, SGOL1, CDC20, AURKA, ESPL1, PTTG1, CPEB1,
CDC25C, ITPR1, CCNB1, IGF1R, MAD2L1, CCNB2, SLK,
MAPK12, PLK1, RPS6KA2, BUB1, FBXO5, PRKACB
mmu04914:
Progesteronemediated oocyte
maturation
0.009
CDK1, PIK3CD, MAPK11, CPEB1, CDC25C, CCNB1, IGF1R,
MAD2L1, CCNB2, MAPK12, PLK1, RPS6KA2, BUB1, PRKACB,
CCNA2, AKT3
mmu05220:
Chronic myeloid
leukemia
mmu00260:
Glycine, serine and
threonine
metabolism
mmu00240:
Pyrimidine
metabolism
0.018
E2F1, CTBP1, PIK3CD, TGFB3, CDK6, CDK4, TGFB1, TGFB2,
PTPN11, GAB2, SHC1, MYC, SHC2, AKT3
0.022
CTH, SHMT2, MAOA, PHGDH, GCAT, PSPH, PSAT1, CBS
0.025
DCTD, POLR3H, CTPS, POLE, POLR1A, POLA1, CAD, POLA2,
POLR1B, UPRT, NME1, RRM2, POLD1, RRM1, DPYD, DUT
NRP1, LIMK1, ABLIM3, EFNB2, DPYSL2, SLIT1, ITGB1, EPHA5,
EPHA4, EPHA7, EPHB6, SEMA6C, RND1, SEMA6D, ROBO1,
FYN, UNC5C, SEMA3A, NFATC3, SRGAP2
mmu04360:
Axon guidance
0.026
mmu05212:
Pancreatic cancer
0.027
mmu04530:
Tight junction
0.034
PARD6A, MAGI3, PARD3, MPDZ, CLDN3, CLDN6, MRAS,
CSNK2B, ACTN1, CLDN20, CDK4, AMOTL1, CSDA, TJAP1,
PRKCB, B230120H23RIK, PPP2R2B, TJP2, AKT3, MYH10
0.036
CACNA2D1, SLC8A1, TGFB3, ITGA2, ITGA3, ITGB3, TTN,
ITGB1, TGFB1, TGFB2, TNNT2, ITGA5, ITGB7, PRKACB,
CACNA1C
0.037
CACNA2D1, SLC8A1, TGFB3, ITGA2, ITGA3, ITGB3, TTN,
ITGB1, TGFB1, TGFB2, TNNT2, ITGA5, ITGB7, CACNA1C
mmu03018:
RNA degradation
0.041
DIS3, EXOSC8, ENO2, EXOSC2, ENO3, CNOT1, EXOSC1,
LSM2, CNOT7, CNOT4, HSPA9
mmu03430:
Mismatch repair
0.043
EXO1, RPA2, RFC4, RFC1, POLD1, PMS2
0.048
VEGFC, PGF, ETS1, PDGFA, PIK3CD, ARNT2, VEGFA, TGFB3,
TGFB1, AKT3, TGFB2, PTPN11
mmu05414:
Dilated
cardiomyopathy
mmu05410:
Hypertrophic
cardiomyopathy
(HCM)
mmu05211:
Renal cell
carcinoma
E2F1, PGF, PIK3CD, TGFB3, BRCA2, CDK6, CDK4, TGFB1,
TGFB2, RAD51, VEGFC, VEGFA, AKT3
Up-regulated genes in NCC-S3
KEGG_PATHWAY
mmu03030:
DNA replication
mmu04110:
Cell cycle
P-Value
<0.001
<0.001
Genes
POLE, POLA1, POLA2, MCM3, MCM4, MCM5, POLD3, PRIM1,
RFC5, DNA2, RPA2, RFC3, RFC4, MCM7, POLE2, RFC1, RFC2,
POLD1, POLD2, FEN1
E2F1, E2F3, CDC14B, DBF4, TTK, CHEK1, CHEK2, CCNE1,
CDC45, CDKN2A, MCM7, BUB1, CCNA2, MYC, CDC7, CDK1,
RBL1, ANAPC4, SKP2, CDK6, ESPL1, CDC20, CDC25C, MCM3,
MCM4, SMC3, MCM5, CCNB1, CCND1, CCNB2, MAD2L1, PLK1,
BUB1B, GADD45B
mmu00240:
Pyrimidine
metabolism
<0.001
POLR2E, CTPS, NT5C1A, POLA1, DCK, CAD, POLA2, POLR2D,
TK1, PRIM1, TYMS, POLE2, UPRT, CDA, ENTPD3, POLR3G,
DCTD, POLR3F, POLR1E, POLE, POLR1B, NME6, POLD3,
NME5, RRM2, POLD1, RRM1, POLD2, DUT
mmu03440:
Homologous
recombination
<0.001
XRCC2, BLM, EME1, BRCA2, RAD54L, RAD50, RAD51, POLD3,
RPA2, RAD51L1, POLD1, POLD2, RAD54B
<0.001
POLD3, RFC5, EXO1, RPA2, RFC3, RFC4, RFC1, RFC2, POLD1,
POLD2, PMS2
<0.001
POLE, GTF2H3, GTF2H2, RFC5, POLD3, RPA2, RFC3, RFC4,
RFC1, POLE2, RFC2, POLD1, POLD2, ERCC1
mmu03430:
Mismatch repair
mmu03420:
Nucleotide excision
repair
mmu04115:
p53 signaling
pathway
<0.001
BID, CDK1, CDK6, CHEK1, CHEK2, GTSE1, CCNB1, CCNE1,
CCND1, CDKN2A, CCNB2, SIAH1B, RRM2, SERPINE1, THBS1,
GADD45B, IGFBP3
ADCY1, POLR2E, NT5C1A, POLA1, DCK, POLA2, POLR2D,
PRIM1, POLE2, ATIC, ENTPD3, POLR3G, POLR3F, POLR1E,
POLE, AK5, POLR1B, AMPD2, GART, NME6, POLD3, NME5,
RRM2, POLD1, POLD2, RRM1, PRPS1
mmu00230:
Purine metabolism
<0.001
mmu04114:
Oocyte meiosis
0.001
CDK1, ADCY1, ANAPC4, SGOL1, CDC20, AURKA, ESPL1,
CDC25C, SMC3, CCNB1, CCNE1, MAD2L1, CCNB2, MAPK12,
PLK1, RPS6KA2, BUB1, FBXO5
mmu00670:
One carbon pool by
folate
0.003
TYMS, SHMT2, DHFR, ATIC, MTHFD1L, GART
0.003
CDK1, ADCY1, ANAPC4, MAPK11, CDC25C, CCNB1, MAD2L1,
CCNB2, MAPK12, RPS6KA2, PLK1, BUB1, CCNA2, AKT3
0.003
E2F1, CKS1B, E2F3, PTGS2, SKP2, ITGA2, CDK6, CCNE1,
CCND1, LAMA3, LAMA5, MYC, AKT3, FN1
0.004
IL1R1, TNF, PDGFB, DUSP10, CACNB2, FGF13, NFKB2,
DUSP14, RAC3, B230120H23RIK, RRAS, MYC, IL1A, AKT3,
MAPK11, FLNC, STK3, FLNA, MAP4K4, DUSP3, MAPK12,
RPS6KA2, RRAS2, HSPB1, STMN1, GADD45B, DUSP9, CD14,
DUSP7, NGF
mmu04914:
Progesteronemediated oocyte
maturation
mmu05222:
Small cell lung
cancer
mmu04010:
MAPK signaling
pathway
mmu03410:
Base excision repair
mmu01040:
Biosynthesis of
unsaturated fatty
acids
mmu05200:
Pathways in cancer
0.004
POLD3, APEX2, POLE2, NEIL3, POLD1, POLD2, POLE, TDG,
FEN1
0.007
ACOT9, SCD1, ACOT7, SCD2, ELOVL5, FADS1, FADS2
0.010
BID, E2F1, CKS1B, E2F3, PDGFB, PTGS2, PGF, FGF13, NFKB2,
SUFU, CCNE1, CDKN2A, RAC3, MYC, AKT3, FN1, WNT10A,
TCF7, SKP2, ITGA2, BRCA2, BIRC5, CDK6, FZD2, FZD7, FZD6,
RAD51, VEGFC, CCND1, WNT7B, LAMA3, LAMA5, WNT7A
CLDN7, CLDN4, MPDZ, CLDN3, CLDN6, ACTN1, AMOTL1,
TJAP1, EPB4.1L2, B230120H23RIK, RRAS2, RRAS, PARD6G,
JAM2, PPP2R2C, AKT3, MYH10
mmu04530:
Tight junction
0.014
mmu05212:
Pancreatic cancer
0.017
E2F1, VEGFC, CCND1, E2F3, CDKN2A, PGF, RAC3, BRCA2,
CDK6, AKT3, RAD51
0.017
E2F1, VEGFC, CCND1, E2F3, CDKN2A, PGF, THBS1, MYC
0.024
ENAH, PDGFB, LIMK1, SSH2, IQGAP3, ITGA2, ACTN1, ABI2,
RDX, FGF13, TIAM2, RAC3, PAK3, RRAS2, RRAS, MSN,
PIP4K2A, CD14, FGD3, F2R, FN1, MYH10, PIP4K2B
0.028
POLR3G, POLR3F, POLR2E, POLR1E, POLR1B, POLR2D
mmu05219:
Bladder cancer
mmu04810:
Regulation of actin
cytoskeleton
mmu03020:
RNA polymerase
mmu04510:
Focal adhesion
mmu04060:
Cytokine-cytokine
receptor interaction
0.032
0.044
CAV2, CAV1, PDGFB, PGF, FLT4, ITGA2, ACTN1, FLNC, FLNA,
VEGFC, CCND1, LAMA3, RAC3, FYN, PAK3, LAMA5, THBS1,
AKT3, PARVB, SPP1, FN1
CXCL1, IL18R1, IL1R1, IL18RAP, TNF, CXCL5, PDGFB,
TNFRSF12A, LEPR, FLT4, CSF1, CCL9, IL24, TNFSF9, LIF,
IFNAR2, INHBA, VEGFC, IL23A, PPBP, CLCF1, CXCL16, IL5RA,
IL1A
Table S2. Copy numbers of chromosome in NCC-S1 estimated by array CGH
Chr
chr2:105540774
-105541174
chr3:40736750
-159781068
chr3:56084581
-79999266
chr3:119732201
-123455031
chr3:132611925
-133119982
chr5:3285447
-151449604
chr8:3151637
-132003081
chr11:3144144
-121517858
chr15:3228882
-103359097
chr19:3259656
-61194972
Amp=Amplification
Del=Deletion
Cytoband
qE3
qB-qH4
qC-qE3
qG1
qG3
qA1-qG3
qA1.1-qE2
qA1-qE2
qA1-qF3
qA-qD3
#Probes
Amp
3 1.632055
Del
Annotations
P-value
0
8.96E-16
1819
0.264959
0
7.47E-307
222
0.517655
0
2.13E-35
80
0.600934
0
9.40E-24
15
0.780683
0
1.67E-11
2636
0.346884
0
0.00E00
2196
0.540138
0
0.00E00
3001
0.310604
0
0.00E00
1663
0.545865
0
0.00E00
1356
0.916769
0
0.00E00
Elp4
Intu, Slc25a31, Hspa4l, etc
Nbea, Tm4sf1, Tm4sf4, etc
Ptbp2, Rwdd3,
2510027J23Rik, etc
Scye1, A630047E20Rik,
Npnt, etc
Cdk6, 5830415L20Rik,
1700109H08Rik, etc
Insr, A430078G23Rik,
Arhgef18, etc
Eif4enif1, Drg1, Patz1, etc
Sepp1, Ghr, Fbxo4, Myc etc
Ighmbp2, Mrpl21, Cpt1a,
etc
Table S3. DAVID pathway analysis on the genes up-regulated by > 2-fold in S1M cells compared with
S1 cells
KEGG_PATHWAY
P-Value
Genes
mmu04360:
Axon guidance
<0.001
NGEF, PLXNA3, DPYSL5, L1CAM, FES, CXCL12, EPHA1, NTN1, SLIT2,
EPHB2, SLIT3, PAK6, SEMA5A, SEMA6A, RAC3, RGS3, PAK3, SRGAP3,
NFATC4, SEMA3A, SRGAP1, RASA1
mmu04610:
Complement and
coagulation cascades
<0.001
KNG2, MASP1, C3, CFB, C4B, SERPING1, BDKRB1, C1S, BDKRB2,
C1RA, GM5077, THBD, TFPI, SERPIND1, PLAU
mmu04142:
Lysosome
0.001
HYAL1, CLN3, CTSZ, AP1M2, GUSB, ATP6AP1, ACP5, CTSA, DNASE2A,
LAMP1, AP1S2, GLA, IGF2R, GAA, ARSA, CTSB, IDUA
mmu04670:
Leukocyte
transendothelial
migration
0.008
CLDN7, CLDN4, CLDN3, ITGB2, CLDN11, VAV2, MMP2, CXCL12, CYBA,
MAPK13, PLCG2, CLDN1, PIK3R5, RAPGEF4, RAPGEF3
mmu04510:
Focal adhesion
0.010
COL3A1, HGF, VAV2, COL5A1, SRC, PAK6, VEGFB, LAMA3, RAC3,
PAK3, ITGB8, ITGB7, PDGFRA, PDGFRB, PIK3R5, PDGFD, THBS1,
SHC2, THBS3, PARVB, SPP1
mmu04650:
Natural killer cell
mediated cytotoxicity
0.010
H2-K1, KLRA18, TNF, KLRA33, H2-D1, ITGB2, VAV2, RAC3, ULBP1,
PLCG2, KLRA4, PIK3R5, NFATC4, KLRA1, SHC2, KLRC1, SH3BP2
mmu05200:
Pathways in cancer
0.011
TRAF1, WNT5A, FGFR1, FGF10, MMP2, RAC3, PIK3R5, RARB, RUNX1,
TRP53, FZD8, AR, WNT10B, CTBP1, FLT3, FZD1, SMAD2, HGF, MECOM,
FZD6, VEGFB, CBLC, LAMA3, RASSF1, PLCG2, PDGFRA, PDGFRB,
PIAS2, WNT9A, WNT7A
mmu04310:
Wnt signaling
pathway
0.012
WNT5A, TRP53, FZD8, WNT10B, CTBP1, FZD1, SMAD2, FZD6, DKK2,
SFRP1, RAC3, SFRP2, NFATC4, SOX17, WNT9A, FOSL1, WNT7A
mmu04060:
Cytokine-cytokine
receptor interaction
0.013
CXCL1, TNF, CXCL5, FLT3, CRLF2, CXCL2, CNTFR, HGF, CX3CL1, IL24,
CXCL12, VEGFB, TNFRSF9, TSLP, IL23A, PPBP, CXCL16, IL1RAP,
PDGFRA, PDGFRB, IL5RA, PDGFD, IL1A, GHR
mmu00561:
Glycerolipid
metabolism
0.017
ALDH7A1, DGAT1, GLA, DGAT2, LIPG, ALDH2, DGKH, PPAP2A
mmu04530:
Tight junction
0.024
CLDN7, CLDN4, CLDN3, CRB3, PRKCH, CLDN11, AMOTL1, SRC,
EPB4.1L3, EPB4.1L1, CLDN1, MYH14, PARD6G, YES1, PPP2R2C
mmu04514:
Cell adhesion
molecules (CAMs)
0.032
H2-K1, CLDN7, CLDN4, CLDN3, H2-D1, ITGB2, L1CAM, CLDN11, H2-Q6,
H2-Q7, SDC2, H2-Q9, ITGB8, ITGB7, CLDN1, VCAN, NEGR1, SPN
mmu05217:
Basal cell carcinoma
0.038
TRP53, WNT5A, FZD8, WNT10B, FZD1, WNT9A, WNT7A, FZD6
mmu04810:
Regulation of actin
cytoskeleton
0.045
FGD1, FGFR1, FGF10, BDKRB1, ITGB2, BDKRB2, VAV2, PAK6, RAC3,
PAK3, ITGB8, GSN, ITGB7, SCIN, PDGFRA, PDGFRB, TMSB4X, PIK3R5,
MYH14, PDGFD
Table S4. DAVID pathway analysis on the 903 genes overexpressed in advanced (stage III/IV)
tumors than in localized (stage I/II) tumors at p<0.05.
KEGG_PATHWAY
hsa04310:
Wnt signaling
pathway
P-Value
0.017
Genes
WNT5A, DVL3, PPP2R5A, SMAD4, PRKCG, CTNNB1, CSNK2A2,
CCND1, SFRP1, GSK3B, PPP2CA, CACYBP, PPP3CC, PRKACB,
CAMK2A, APC
WNT5A, BID, TRAF1, FGF5, XIAP, EGLN3, CTNNB1, MAX,
CSF3R, RARB, TRAF6, FIGF, APC, DVL3, EPAS1, MAP2K1,
SMAD4, ITGA3, PRKCG, APPL1, CCND1, GSK3B, PDGFRA,
PTCH1, ABL1
hsa05200:
Pathways
in cancer
0.082
hsa00260:
Glycine, serine
and threonine
metabolism
0.085
CTH, SHMT2, GATM, GAMT, PIPOX
0.085
DVL3, CCND1, MAP2K1, GSK3B, PDGFRA, SMAD4, APPL1,
CTNNB1, APC
0.095
CD3G, CD3D, CD44, CD8B, FCGR1A, CSF3R, MME, CD1A, ITGA3
hsa05210:
Colorectal cancer
hsa04640:
Hematopoietic
cell lineage
Supplementary Figure Legends
Fig S1. Immunohistochemistry for the tissue origin of the gastric adenocarcinomas arising in a VillinCre;Smad4F/F;Trp53F/F;Cdh1F/+ mouse and a Pdx-1-Cre;Trp53F/F;Cdh1F/F mouse. These tumors
showed immunoreactivities for Mucin 6 and Trefoil factor 2 (TFF2).
Fig S2. Western blot analysis for Smad4, p53, and E-cadherin in mouse gastric cancer cell lines we
have established. The numerical value for band density normalized to GAPDH was indicated under
the band in each of the 5 lanes.
Fig S3. Genome overview for copy number aberration estimated by array CGH in NCC-S1.
Fig S4. Real-time PCR analysis to validate the Wnt-related gene alteration listed in Table S3.
Fig S5. (A) Western blot analysis for nuclear β-catenin expression in mouse gastric cancer cell lines.
The numerical value for nuclear β-catenin expression normalized to p84 was indicated under the band
in each of the 4 lanes. (B) The decrease in TCF/LEF1 reporter activity in S1M cells after β-Catenin
knockdown.
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