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, AgilentMouse 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/normallog2 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 establishedfrom a spontaneous gastric adenocarcinoma of a Villin-Cre;Smad4F/F;Trp53F/F;Cdh1F/+mouse (9). All cells were grown in RPMI1640with 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 vectorsfor 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 shRNAwerecloned into a modifiedpCDH-CMV-MCS-EF1-Puro-copGFP vector from pCDH-CMV-MCS-EF1-copGFP (System Biosciences, Mountain View, CA)by fusingpuromycin resistance gene to copGFP for puromycin selection. We applied human U6 promoter andTTCAAGAGA loop (3) for our shRNA system. Sequence for human negative control (targetingmouse 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). Full coding sequence of mouse Adrm1 was cloned into the pCDH-CMV-MCS-EF1-Puro-copGFP vector. All cloned constructs were confirmedfor the DNA sequence by Sanger sequencing. Transduction oflentiviral 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 Countessautomated 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. The optical 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. For proliferation assays for SNU-216 cells RNA, cells were seeded on 60 mmdishes. After 24 hours, cells were transfected either with 20 nM of the negative control siRNAorthe siRNA targeting ADRM1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). ADRM1-targeting siRNA (SI04336241) sequence of QIAGEN (Valencia, CA) was usedfor 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 usingan 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 106empty 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 Ain PBS) at RT for 30 minutes. Cell cycles were then analyzed using FACSCalibur (BD Biosciences, San Jose, CA). 20,000 cells were counted in sextuplicate for SNU-601 cells and in quintuplicates for ImSt cells. Raw data were analyzed by ModFit LT software(Verity Software House, Topsham, ME). Differences in the S phase fraction were analyzed using Student’st-test.
Quantitative real-time PCR (QPCR) for copy number with genomic DNA
Genomic DNA was isolated from cell linesusing a DNeasy blood tissue kit (Qiagen, Valencia, CA)according to the manufacturer’s instructions. PCR reactions were performedon 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 valueusing 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 were5'-FAM-AACTTCTGCTGGCTCTGTCTGCG-BHQ-1-3'for ADRM1and5'-FAM-CTGAACAACCTGCTCCTGAATGACTACTG-BHQ-1-3'for LINE1. The 2-ΔΔCT method was used to calculate relative changes (7).
Fluorescence in situ hybridization (FISH)
FISH was performedwith formalin-fixed, paraffin-embeddedslides for ADRM1-amplified gastric cancers,as previously described (8). Briefly, after slide preparation including deparaffinizationin 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,washingwith 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 hybridizationof 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-diamidino-2-phenylindole (DAPI) were performed. Cy3-labeled probes for ADRM1 and FITC-labeled probes for 20p11.21 were used. Fluorescence signals were observed by Leica DMRXA2 microscope (Leica Microsystems, Wetzlar, Germany).
Immunohistochemistry (IHC)
A tissuemicroarray (TMA) slide (CQ2; SuperBioChips Laboratories, Seoul, Korea) containing 54 human gastric cancer tissues was purchased. Clinical information of the 54 tumors, such as lymph node status, wereprovided by the manufacturer. These 54 samples and 3 ADRM1-amplified human gastric cancer samples were fixed in 10% phosphate buffered formalin, processed in a routine mannerand embedded in paraffin. The 5μm FFPE sections were dewaxed, rehydrated and subjected to antigen retrieval for immunostaining by heating at 100°C for 20 minutes in 0.01M 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; sc-166754). 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. Negative controls were performed by omitting the primary antibody. The stainsunequivocally darker than the background wereregardedas positive. For grading, semiquantitative assessmentwas conducted for nuclear ADRM1 protein innormal 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%.
REFERENCES
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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 / RMACancer / Normal / Ratio / P
HRH3 / 220447_at / 4.4 / 3.8 / 1.5 / 0.0004
OSBPL2 / 209221_s_at / 7.7 / 7.6 / 1.1 / 0.5439
ADRM1 / 201281_at / 9.2 / 7.8 / 2.7 / <0.0001
LAMA5 / 210150_s_at / 7.7 / 6.6 / 2.2 / 0.0008
RPS21 / 200834_s_at / 13.2 / 12.8 / 1.4 / 0.0021
CABLES2
C20orf151
GATA5
C20orf200
C20orf166
SLCO4A1 / 219911_s_at / 7.4 / 7.1 / 1.2 / 0.3606
NTSR1 / 207360_s_at / 5.4 / 4.8 / 1.5 / 0.0004
C20orf20 / 218586_at / 6.3 / 4.8 / 2.8 / <0.0001
OGFR / 202841_x_at / 7.6 / 6.6 / 2.0 / 0.0001
COL9A3 / 204724_s_at / 5.6 / 5.0 / 1.6 / 0.0725
TCFL5 / 204849_at / 7.4 / 6.3 / 2.2 / 0.0001
DIDO1 / 213213_at / 7.6 / 7.1 / 1.4 / 0.0466
C20orf11 / 218448_at / 8.9 / 8.0 / 1.8 / <0.0001
C20orf59
BHLHB4
YTHDF1 / 221741_s_at / 9.1 / 8.8 / 1.3 / 0.0047
BIRC7 / 220451_s_at / 5.4 / 4.6 / 1.8 / 0.0011
NKAIN4
ARFGAP1 / 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 datafor three ADRM1-amplified patient tumors. Red and green arrows indicate ADRM1 and 20p11.21 loci, respectively. More than 3 copies were frequently observedforADRM1(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 7days after the initialADRM1 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 NCC-S1M as compared with its parental cell line NCC-S1that was established from a spontaneous gastric adenocarcinoma of a Villin-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mouse. PARP was used as a loading control.
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