Supplementary Information
Supplementary Methods
Patients and mRNA expression array analyses
Tissue samples were collected from metastatic gastric cancer patients by endoscopy before chemotherapy
(cisplatin in combination with either fluorouracil or capecitabine) and frozen in liquid nitrogen from 2001 to
2006, under a protocol approved by the Institutional Review Board (IRB) of the National Cancer Center
Hospital in Goyang, Korea.
All patients signed IRB-approved informed consent forms.
Tissue samples were
collected and processed for total RNA and genomic DNA extraction as previously described (1).
Also,
grossly-normal gastric mucosa tissue samples were collected from antrum of 21 healthy volunteers.
1 µg of
total RNA was subject to mRNA expression array analysis using Affymetrix (Santa Clara, CA) HG-U133A
gene expression microarrays. Microarray data were normalized using RMA, and Student t-test was performed
to identify differential gene expression.
Array CGH
Agilent 4x44k HD-CGH Microarrays containing 44,000 features (Agilent Technologies, Santa Clara, CA) were
used. 0.5-1 µg of tumor genomic DNA samples and the same amount of human genomic DNA from multiple
anonymous female donors (Promega, Madison, WI) were digested with AluI (50 units) and RsaI (50 units) for 2
h at 37°C.
5 µl of Random Primer was mixed with the digested DNA template.
The reference and sample
DNA were labeled using Agilent’s Labeling Kit PLUS, which includes 5x buffer, 10x dNTP, Cy-3/5 dUTP (1.0
mM), and Exo-Klenow Fragment.
The probe mixture of Cy3 labeled sample DNA, Cy5 labeled reference
DNA (39 µl), 5 µl of human Cot-1 DNA (Life Technologies), 11 µl of Agilent 10× blocking agent and 50 µl of
Agilent 2× hybridization buffer was denatured at 95 °C for 3 min and incubated at 37 °C for 30 min. The
probe was applied to the array using an Agilent microarray hybridization chamber, and hybridized for 21 h at
65°C in a rotating oven at 20 rpm.
Arrays were washed according to the manufacturer's recommendations,
dipped in Agilent’s Stabilizing and Drying Solution, and scanned using an Agilent 2565AA DNA microarray
scanner.
For array CGH analyses on mouse gastric cancer cell lines NCC-S1 and NCC-S1M, Agilent Mouse
Genome CGH 4x44K arrays were used.
The Agilent’s Scan Program Control Program 7.0 and Agilent’s Feature Extraction Software Program 9.5.1
were used for data processing.
Array CGH data were analyzed using Agilent's CGH Analytics software
(version 3.5.14). The ADM-2 algorithm was used (Threshold of ADM-2: 6.0; Centralization: ON (Threshold:
6.0, Bin Size: 10); Fuzzy Zero: ON; Aberration Filters: ON (minProbes = 5, minAvgAbsLogRatio = 0.5,
maxAberrations = 10,000, and percentPenetrance = 0).
Average tumor/normal log2 ratio > 0.5 for 5 or more
consecutive probes was identified as the copy number gain.
Cell lines and culture
Human gastric cancer cells (SNU-601, SNU-216, SNU-638, and SNU-484) were purchased from Korean Cell
Line Bank (Seoul, Korea).
ImSt immortalized mouse gastric epithelial cells were kindly gifted from Dr.
Jonathan C. A. Skipper in Ludwig Institute for Cancer Research LTD.
NCC-S1 and its metastatic derivative
NCC-S1M cells were established from a spontaneous gastric adenocarcinoma of a VillinCre;Smad4F/F;Trp53F/F;Cdh1F/+ mouse (9).
All cells were grown in RPMI 1640 with 10% fetal bovine serum
(PAA laboratories, Cölbe, Germany) and 0.5% Zell Shield (Minerva biolabs, Berlin, Germany) at 37ºC in a 5%
CO2 humidified incubator.
Lentiviral vectors for knockdown and overexpression of ADRM1
A PCR-based method was used for cloning of short-interfering RNA (shRNA) vector, as described (2).
The
ADRM1-targeting shRNA and control shRNA were cloned into a modified pCDH-CMV-MCS-EF1-PurocopGFP vector from pCDH-CMV-MCS-EF1-copGFP (System Biosciences, Mountain View, CA) by fusing
puromycin resistance gene to copGFP for puromycin selection.
TTCAAGAGA loop (3) for our shRNA system.
We applied human U6 promoter and
Sequence for human negative control (targeting mouse
Ccne1) was 5’-GATGTTGGCTGCTTAGAAT-3’.
ADRM1-targeting siRNA (SI04336241) sequence of
QIAGEN (Valencia, CA) was used for subcloning.
shEGFP (SHC005) purchased from Sigma Inc. (St. Louis, MO).
cloned into the pCDH-CMV-MCS-EF1-Puro-copGFP vector.
DNA sequence by Sanger sequencing.
Full coding sequence of mouse Adrm1 was
All cloned constructs were confirmed for the
Transduction of lentiviral vectors was performed as described (4).
Briefly, lentiviral vectors were produced by cotransfection of 293FT cells (Invitrogen, Carlsbad, CA) with the
each cloned construct, pMD2.G, and psPAX2 vectors (Addgene, Cambridge, MA) by using Lipofectamine 2000
(Invitrogen, Carlsbad, CA).
The media containing lentiviral vectors were harvested 48 hours after transfection
and filtered through a 0.45 μm syringe filter.
After ultracentrifugation and titration, 0.1 multiplicity of
infection (MOI) of lentiviral vectors were transduced to SNU-601 and ImSt cells.
Transduced cells were
selected under 2 μg/mL of puromycin.
Cell proliferation assays
3 x 105 shEGFP-transduced and shADRM1-transduced SNU-601 cells were grown for 4 days on 150 mm dishes
and the cells were counted each day by Countess automated cell counter (Invitrogen, Carlsbad, CA) as
recommended by manufacturer’s instructions.
1 x 104 empty vector-transduced and Adrm1-overexpressing,
immortalized mouse gastric epithelial ImSt cells were grown for 4 days on 12 well plates.
MTT [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assays were performed
daily (5).
For MTT assays, 100 μL of MTT (Sigma, St. Louis, MO) solution (5 mg/mL in PBS) was added to
500 μL of growth medium to 12 well plate, and incubated at 37ºC with 5% CO2 for 15 minutes.
density was measured at 570 nm with a microplate reader.
The normalized absorbance was calculated by
subtracting the absorbance of DMSO blank from the absorbance of each well.
SNU-216 cells RNA, cells were seeded on 60 mm dishes.
The optical
For proliferation assays for
After 24 hours, cells were transfected either with 20
nM of the negative control siRNA or the siRNA targeting ADRM1 using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA).
ADRM1-targeting siRNA (SI04336241) sequence of QIAGEN (Valencia, CA) was used for
the ADRM1 knockdown.
The cells were then additionally transfected with the same siRNA 3 days later.
To compare proliferation rates, cell numbers or MTT optical densities of the same days were compared using
Student’s t-test.
Repeated measure ANOVA test was performed to compare entire growth curves, using proc
GLM provided by SAS software (version 9.1, Cary, NC).
Western blot analysis
Cells (at 70-80% confluency in 2-4 days) were collected and lysed with T-PER Tissue Protein Extraction
Reagent (Thermo Fisher Scientific, Hudson, NH) supplemented with protease inhibitors (0.8 μM aprotinin, 20
μM leupeptin, 10 μM pepstatin A, 40 μM bestatin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)) and
phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium pyrophosphate dehydrate, and 1 mM sodium
orthovanadate).
After removal of cellular debris protein concentration was quantitated by using a BCA reagent
kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions.
Protein samples were
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 by boiling for 5 minutes.
Fifteen
micrograms of total protein was separated on SDS-polyacrylamide gel and the resolved proteins were
transferred to nitrocellulose membranes by electrophoresis and blotting apparatuses (Bio-Rad, Hercules, CA).
After blocking with 2% skim milk and 1% BSA-containing phosphate-buffered saline with Tween-20 (PBS-T),
the proteins were probed with anti-ADRM1 (sc-166754, Santa Cruz Biotechnology, Santa Cruz, CA), anti-p21
(sc-393), anti-UCH37 (sc-271002), anti-RPN2/S1 (ab2941, Abcam, Cambridge, UK), anti-UBE2C (ab12290),
anti-PARP (sc-7150), and anti-β-Actin (sc-47778) primary antibodies at 4ºC overnight.
Blots were then
hybridized with horseradish peroxidase (HRP)-conjugated secondary antibodies (GenDEPOT, Barker, TX) at
RT for 2 hours at the recommended dilutions in 5% BSA-containing PBS-T.
Immunodetection was performed
on X-ray film by using an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific,
Waltham, MA).
Fluorescence activated cell sorting (FACS) cell cycle analysis
FACS was performed 4 days after 2 x 105 shControl- and shADRM1-transduced SNU-601 cells were grown on
100 mm dishes for 4 days.
FACS was also performed 1 day after 1.8 x 106 empty vector-transfected and
Adrm1-transfected ImSt cells were grown on 150 mm dishes for 24 hours.
Cells were harvested, washed with
PBS, fixed in 70% ethanol at 4ºC overnight, washed with cold PBS, and stained with propidium iodide (PI)
staining solution (50 μg/mL PI and 0.1 mg/mL RNase A in PBS) at RT for 30 minutes.
analyzed using FACSCalibur (BD Biosciences, San Jose, CA).
SNU-601 cells and in quintuplicates for ImSt cells.
Software House, Topsham, ME).
Cell cycles were then
20,000 cells were counted in sextuplicate for
Raw data were analyzed by ModFit LT software (Verity
Differences in the S phase fraction were analyzed using Student’s t-test.
Quantitative real-time PCR (QPCR) for copy number with genomic DNA
Genomic DNA was isolated from cell lines using a DNeasy blood & tissue kit (Qiagen, Valencia, CA) according
to the manufacturer’s instructions.
PCR reactions were performed on Roche LC480 (Roche Diagnostics,
Penzberg, Germany) in a total volume of 10 μL containing 5 μL of 2x QuantiTect Probe PCR Master Mix
(Qiagen, Valencia, CA), 400 nM of primers, 200 nM of TaqMan probe, and 60 ng of genomic DNA.
Cycling
conditions were as follows: 15 min at 95ºC, followed by 99 cycles each consisting of 20 s at 95ºC, 20 s at 57ºC
and 20 s at 72ºC.
Data were normalized to LINE1 repetitive sequence that is abundant in human genome (6),
and analyzed for CT value using the LC480 software.
Primer sequences were 5'-GCTCATGCAGCTCATCGG-
3' and 5'-CAGCTTGCGGACACACAG-3' for ADRM1, and 5'-AAAGCCGCTCAACTACATGG-3' and 5'-
TGCTTTGAATGCGTCCCAGAG-3' for LINE1.
TaqMan probe sequences were 5'-FAM-
AACTTCTGCTGGCTCTGTCTGCG-BHQ-1-3' for ADRM1 and 5'-FAMCTGAACAACCTGCTCCTGAATGACTACTG-BHQ-1-3' for LINE1.
The 2-ΔΔCT method was used to
calculate relative changes (7).
Fluorescence in situ hybridization (FISH)
FISH was performed with formalin-fixed, paraffin-embedded slides for ADRM1-amplified gastric cancers, as
previously described (8).
Briefly, after slide preparation including deparaffinization in xylene, rehydration in
100%, 95%, and 70% ethanol, pretreatment with 0.01 N HCl at RT for 20 minutes and 8% sodium thiocyanate
at 80ºC for 1 hour, washing with 2 x SSC, protease (0.05% pepsin) digestion with 0.01 N HCl, incubation with
1% formaldehyde at RT for 10 minutes, and dehydration in 70%, 95%, and 100% ethanol denaturation of probes
at 75ºC for 5 minutes and hybridization of probes at 37ºC for 16 hours in a probe/hybridization mixture using
MacProbeTM kit (Macrogen, Seoul, Korea) were carried out.
After the hybridization washing with 0.5x
SSC/0.1% SDS solution, dehydration in 70%, 95%, and 100% ethanol, and counterstaining with 4',6-diamidino2-phenylindole (DAPI) were performed.
20p11.21 were used.
Cy3-labeled probes for ADRM1 and FITC-labeled probes for
Fluorescence signals were observed by Leica DMRXA2 microscope (Leica
Microsystems, Wetzlar, Germany).
Immunohistochemistry (IHC)
A tissue microarray (TMA) slide (CQ2; SuperBioChips Laboratories, Seoul, Korea) containing 54 human
gastric cancer tissues was purchased.
provided by the manufacturer.
Clinical information of the 54 tumors, such as lymph node status, were
These 54 samples and 3 ADRM1-amplified human gastric cancer samples were
fixed in 10% phosphate buffered formalin, processed in a routine manner and embedded in paraffin.
The 5 μm
FFPE sections were dewaxed, rehydrated and subjected to antigen retrieval for immunostaining by heating at
100°C for 20 minutes in 0.01 M citrate buffer (pH 6.0).
Vector ImmPRESS Peroxidase Polymer kit (Vector
Laboratories, Burlingame, CA) was used according to the manufacturer’s protocol.
Slides were then incubated
at RT for 30 minutes with the diluted primary antibody; mouse monoclonal anti-ADRM1 antibody (1:100; sc166754).
After slides were incubated with the peroxidase polymer-linked secondary antibody for 30 minutes,
they were subjected to colorimetric detection with ImmPact DAB substrate (Vector Laboratories, SK-4105).
Slides were then counterstained with Meyer’s hematoxylin for 10 seconds.
by omitting the primary antibody.
positive.
Negative controls were performed
The stains unequivocally darker than the background were regarded as
For grading, semiquantitative assessment was conducted for nuclear ADRM1 protein in normal
gastric epithelial and tumor cells.
Nuclear ADRM1 expression was categorized into 4 groups according to the
percentage of positive staining: 0, less than 5%; 1, between 5% and 30%; 2, between 30% and 70%; 3, more
than 70%.
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knockdown constructs with convergent H1 and U6 promoters. Eur J Cell Biol 85(5):433-440
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4.Kutner RH, Zhang XY, Reiser J. (2009) Production, concentration and titration of pseudotyped HIV-1-based
lentiviral vectors. Nat Protoc 4(4):495-505
5.Wang P, Henning SM, Heber D. (2010) Limitations of MTT and MTS-based assays for measurement of
antiproliferative activity of green tea polyphenols. PLoS One 5(4):e10202
6.Salani R, Davidson B, Fiegl M, Marth C, Müller-Holzner E, Gastl G, Huang HY, Hsiao JC, Lin HS, Wang TL,
Lin BL, Shih IeM. (2007) Measurement of cyclin E genomic copy number and strand length in cell-free DNA
distinguish malignant versus benign effusions. Clin Cancer Res 13(19):5805-5809
7.Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR
and the 2(-Delta Delta C(T)). Methods 25(4):402-408
8.Bayani J, Squire JA. (2004) Fluorescence in situ Hybridization (FISH). Curr Protoc Cell Biol Chapter 22:Unit
22.4
9.Park JW, Jang SH, Choi BG, et al. Establishment and characterization of metastatic gastric cancer cell lines
from murine gastric adenocarcinoma lacking Smad4, p53, and E-cadherin (manuscript under review).
Supplementary Tables
Table S1. Genes in recurrent amplicons identified in study patients
Chr17q21.2
KRTAP4-4, KRTAP4-3, KRTAP4-2, KRTAP4-1, KRTAP9-2, KRTAP9-3, KRTAP9-9, KRTAP9-8, KRTAP9-4,
KRTAP17-1, KRT33A, KRT33B, KRT34, KRT31, KRT37, KRT38, KRT32, KRT35, KRT36, KRT13, KRT15,
KRT19, KRT9, KRT14, KRT16, KRT17, EIF1, GAST, HAP1, JUP, SC65, FKBP10, NT5C3L, KLHL10, KLHL11,
ACLY
Chr8q24.13 - q24.21
NSMCE2, TRIB1, FAM84B, MYC
Chr20q13.33
HRH3, OSBPL2, ADRM1, LAMA5, RPS21, CABLES2, C20orf151, GATA5, C20orf200, C20orf166, SLCO4A1,
NTSR1, C20orf20, OGFR, COL9A3, TCFL5, DIDO1, DIDO1, DIDO1, DIDO1, C20orf11, C20orf59, BHLHB4,
YTHDF1, BIRC7, NKAIN4, ARFGAP1
Chr20q13.11 - q13.2
SFRS6, L3MBTL, SGK2, SGK2, IFT52, MYBL2, GTSF1L, TOX2, TOX2, TOX2, JPH2, JPH2, C20orf111,
GDAP1L1, C20orf142, R3HDML, HNF4A, HNF4A, HNF4A, HNF4A, C20orf121, SERINC3, SERINC3, PKIG,
ADA, WISP2, KCNK15, RIMS4, YWHAB, PABPC1L, TOMM34, STK4, KCNS1, WFDC5, WFDC12, PI3,
SEMG1, SEMG2, SLPI, MATN4, RBPJL, SDC4, SYS1, C20orf10, DBNDD2, DBNDD2, DBNDD2, PIGT,
WFDC2, WFDC6, SPINLW1, SPINLW1, WFDC8, WFDC8, WFDC9, WFDC10A, WFDC11, WFDC10B,
WFDC13, SPINT4, WFDC3, DNTTIP1, UBE2C, UBE2C, TNNC2, SNX21, ACOT8, ZSWIM3, ZSWIM1,
C20orf165, NEURL2, CTSA, PLTP, C20orf67, ZNF335, MMP9, SLC12A5, NCOA5, CD40, CDH22, SLC35C2,
ELMO2, ZNF334, SLC13A3, SLC13A3, TP53RK, SLC2A10, EYA2, EYA2, EYA2, EYA2, ZMYND8, NCOA3,
SULF2, PREX1, ARFGEF2, CSE1L, STAU1, DDX27, ZNFX1, KCNB1, PTGIS, B4GALT5, SLC9A8, SPATA2,
ZNF313, SNAI1, UBE2V1, UBE2V1, UBE2V1, TMEM189-UBE2V1, TMEM189-UBE2V1, TMEM189, CEBPB,
PTPN1, C20orf175, PARD6B, BCAS4, ADNP, DPM1, MOCS3, KCNG1, KCNG1, NFATC2, ATP9A, SALL4,
ZFP64, ZFP64, TSHZ2, ZNF217, BCAS1, CYP24A1, PFDN4, DOK5
Chr10q26.13
FGFR2
Table S2. mRNA expression levels of genes in the common chr20q13.33 amplicon in 3
cancer patients harboring the gene amplification
Gene name
Probe
RMA
Cancer
Normal
Ratio
P
HRH3
OSBPL2
ADRM1
LAMA5
RPS21
CABLES2
C20orf151
GATA5
C20orf200
C20orf166
SLCO4A1
NTSR1
C20orf20
OGFR
COL9A3
TCFL5
DIDO1
C20orf11
C20orf59
BHLHB4
YTHDF1
BIRC7
NKAIN4
ARFGAP1
220447_at
209221_s_at
201281_at
210150_s_at
200834_s_at
4.4
7.7
9.2
7.7
13.2
3.8
7.6
7.8
6.6
12.8
1.5
1.1
2.7
2.2
1.4
0.0004
0.5439
<0.0001
0.0008
0.0021
219911_s_at
207360_s_at
218586_at
202841_x_at
204724_s_at
204849_at
213213_at
218448_at
7.4
5.4
6.3
7.6
5.6
7.4
7.6
8.9
7.1
4.8
4.8
6.6
5.0
6.3
7.1
8.0
1.2
1.5
2.8
2.0
1.6
2.2
1.4
1.8
0.3606
0.0004
<0.0001
0.0001
0.0725
0.0001
0.0466
<0.0001
221741_s_at
220451_s_at
9.1
5.4
8.8
4.6
1.3
1.8
0.0047
0.0011
217888_s_at
7.7
7.0
1.6
0.0005
RMA, Robust multichip average: Ratio, Affymetrix U133A signal ratio of cancer patients with 20q13.33 amplification (n=3) to healthy
volunteers (n=21): Blank indicates the unavailability of microarray data because these genes are not represented in U133A chips.
Supplementary Figures
Fig S1. FISH analyses on three ADRM1-amplified gastric cancer tissue samples. (A)
Validation of probes specific for ADRM1 and a centromere site (20p11.21) as the reference on
metaphase spread of normal male. Cy3-labeled probe for ADRM1 (red) and FITC-labeled
probe for 20p11.21 (green) are seen. (B, C, and D) Representative FISH data for three
ADRM1-amplified patient tumors. Red and green arrows indicate ADRM1 and 20p11.21
loci, respectively. More than 3 copies were frequently observed for ADRM1 (red spots), but
not for the reference (green spots).
Fig S2. Proliferation of SNU-216 cells after ADRM1 siRNA transfection. ADRM1
knockdown significantly decreased the number of cells 7 days after the initial ADRM1 siRNA
transfection, as compared with control siRNA treatment (p=0.034).
Fig S3. Western blot analyses on ADRM1 and p21 in ImSt cells after Adrm1 knockdown.
Protein expression of p21 was increased in the clone stably expressing shAdrm1.
Fig S4. Western blot analyses on ADRM1 in metastatic mouse gastric cancer cell line NCCS1M as compared with its parental cell line NCC-S1 that was established from a spontaneous
gastric adenocarcinoma of a Villin-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mouse. PARP was used
as a loading control.
.