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Molecular Cancer Therapeutics
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Preclinical Development

A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer

Wendy L. Allen, Leanne Stevenson, Vicky M. Coyle, Puthen V. Jithesh, Irina Proutski, Gail Carson, Michael A. Gordon, Heinz-Josef D. Lenz, Sandra Van Schaeybroeck, Daniel B. Longley and Patrick G. Johnston
Wendy L. Allen
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Leanne Stevenson
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Vicky M. Coyle
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Puthen V. Jithesh
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Irina Proutski
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Gail Carson
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Michael A. Gordon
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Heinz-Josef D. Lenz
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Sandra Van Schaeybroeck
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Daniel B. Longley
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Patrick G. Johnston
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DOI: 10.1158/1535-7163.MCT-11-0510 Published January 2012
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  • Figure 1.
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    Figure 1.

    Results from unsupervised classification analysis using PCA for clinical data displaying PC1 against PC3 (A), in vitro 5-FU basal data displaying PC1 against PC2 (B), in vitro 5-FU inducible data displaying PC1 against PC2 (C), in vitro SN38 basal data displaying PC1 against PC2 (D), and in vitro SN38 inducible data displaying PC1 against PC2 (E). A solid black line denotes the point of separation in the respective PC. Each sample group is displayed in either red or blue.

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    Figure 2.

    Results from the initial siRNA screen in the HCT116 cells. The graphs show the relative toxicity, as assessed from MTT and toxiLight assays, for all positive genes from the clinical analysis (A), the 5-FU in vitro analysis (B), and the SN38 in vitro analysis (C). Displayed are those genes that show an interaction between gene silencing and either 5-FU or SN38 treatment as measured by 2-way ANOVA. Synergistic interactions are highlighted as *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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    Figure 3.

    Results from the cell line panel siRNA screen. The graphs show the relative toxicity, as assessed from MTT and toxiLight assays, for SART1, PTRF, and RAPGEF2 for the LoVo (A), RKO (B) LS174T (C), HT29 (D), and SW620 (E) cell lines. Displayed is the effect of gene silencing alone and in combination with either 5-FU or SN38 treatment. Statistical significance between gene silencing alone (siRNA) and gene silencing in combination with chemotherapy was assessed by an unpaired 2-way t test. Statistical significance are highlighted as *, P < 0.05; **, P < 0.01; ***, P < 0.001. Two siRNA sequences were included for each gene, indicated by (1) and (2). ns, not significant.

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    Figure 4.

    A, Western blot analysis of SART1 expression and PARP cleavage in HCT116 cells transfected with control (−) or SART1 (+) siRNAs and cotreated with 5-FU (IC30(48h)) or SN38 (IC30(48h)) for 24 and 48 hours. B–D, flow cytometric analyses of apoptosis following SART1 silencing alone or in combination with either 5-FU (IC30(48h)) or SN38 (IC30(48h)) in HCT116 (B), RKO (C), and LS147T (D). All cells were reversed transfected with SART1 siRNA for 24 hours before a 48 hours treatment with chemotherapy.

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    Figure 5.

    Cell viability assays were conducted in HCT116 (A and B), LS174T (C and D), and RKO cell lines (E and F) in response to SART1 siRNA (0.5, 1 and 5 nmol/L) and either SN38 (1, 5, and 10 nmol/L) or 5-FU (1, 5 and 10 μmol/L) for 72 hours. To evaluate the interaction between chemotherapy and SART1 silencing, we used the method of Chou and Talalay (26). CI values <1, = 1, and >1 indicate synergism, additivity, and antagonism, respectively. For synergistic interactions, CI values between 0.8 and 0.9 indicate slight synergy, 0.6 and 0.8 indicate moderate synergy, 0.4 and 0.6 indicate synergy and those less than 0.4 indicate strong synergy.

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    Figure 6.

    A, flow cytometric analysis of apoptosis in HCT116 cells following treatment with control (SC) or SART1 siRNA (1 nmol/L) alone or in combination with chemotherapy [IC30 (48h)] for 72 hours in the presence and absence of the pan-caspase inhibitor, ZVAD (10 μmol/L). B, flow cytometric analysis of apoptosis in HCT116 cells following transfection with SC or SART1 siRNA (5 nmol/L) alone or in combination caspase-8 siRNA (10 nmol/L). C, Caspase-Glo assay measuring-caspase-8 activity or caspase-3/7 activity in HCT116 cells following transfection with SC or SART1 siRNA (5 nmol/L) alone or in combination caspase-8 siRNA (10 nmol/L). D, Western blot analysis of SART1, procaspase-8, and FLIPL expression following transfection with SC or SART1 siRNA (5 nmol/L) alone or in combination caspase-8 siRNA (10 nmol/L). All experiments are representative of 3 independent experiments. E, flow cytometric analysis of apoptosis in parental and c-FLIPL overexpressing cells (FL17 and FL24) following transfection with SC or SART1 siRNA (5 nmol/L) for 24 hours. Statistical significance was assessed by using an unpaired 2-tailed t test with ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Tables

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  • Additional Files
  • Table 1.

    The top 50 genes identified from PCA of the clinical, 5-FU in vitro basal, 5-FU in vitro inducible, SN38 in vitro basal, and SN38 in vitro inducible transcriptional profiling experiments

    Probe IDGene symbolGene name
    ADXCRIH.1569.C1_s_atRPS27LHomo sapiens ribosomal protein S27-like (RPS27L) mRNA
    ADXCRAD_BI820604_s_atMAPK9Homo sapiens mitogen-activated protein kinase 9 (MAPK9) transcript
    ADXCRAG_AF536980_atPDE4DHomo sapiens cAMP-specific phosphodiesterase (PDE4D) mRNA 3 untranslated region p
    ADXCRIH.4.C1_atRPS9Homo sapiens ribosomal protein S9 (RPS9) mRNA
    ADXCRPDRC.2813.C1_s_atCYB5D2Cytochrome b5 domain containing 2 (CYB5D2) mRNA
    ADXCRAG_BX640769_x_atRAPGEF2Rap guanine nucleotide exchange factor (GEF) 2
    ADXCRAG_AL133646_s_atGMEB2Homo sapiens glucocorticoid modulatory element binding protein 2
    ADXCRAG_AL832400_x_atNLRP1NLR family pyrin domain containing 1
    ADXCRAG_NM_152345_s_atANKRD13BHomo sapiens ankyrin repeat domain 13B (ANKRD13B) mRNA
    ADXCRAD_CV571495_x_atTNFSF14Homo sapiens TNF (ligand) superfamily member 14
    ADXCRPD.6977.C1_atGARTHomo sapiens phosphoribosylglycinamide formyltransferase
    ADXCRIH.2762.C1_s_atMRPL21Homo sapiens mitochondrial ribosomal protein L21 (MRPL21) nuclear
    ADXCRAD_CN279751_s_atIRAK1Homo sapiens interleukin-1 receptor-associated kinase 1 (IRAK1)
    ADXCRAG_BC016330_s_atRAD51AP1Homo sapiens RAD51-associated protein 1 (RAD51AP1) mRNA
    ADXCRAD_BX415970_x_atAASDHPPTAminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase
    ADXCRAD_AL135445_atBTN3A2Homo sapiens butyrophilin subfamily 3 member A2 (BTN3A2) mRNA
    ADXCRPD.4326.C1_s_atOMA1Homo sapiens OMA1 homolog zinc metallopeptidase (S. cerevisiae)
    ADXCRAD_AL545542_x_atPTRFHomo sapiens polymerase I and transcript release factor (PTRF)
    ADXCRAG_NM_032242_s_atPLXNA1Homo sapiens plexin A1 (PLXNA1) mRNA
    ADXCRPD.7079.C1_x_atPRKCDBPHomo sapiens protein kinase C delta binding protein (PRKCDBP)
    ADXCRAD_BX100631_s_atSLC12A8Homo sapiens solute carrier family 12 (potassium/chloride
    ADXCRPD.888.C1_s_atASH2LHomo sapiens ash2 (absent small or homeotic)-like (Drosophila)
    ADXCRAD_AF155810_s_atSLC25A14Homo sapiens solute carrier family 25 (mitochondrial carrier)
    ADXCRPD.1085.C1_atTSFMHomo sapiens Ts translation elongation factor mitochondrial
    ADXCRIHRC.3317.C1_s_atPSMA2Homo sapiens proteasome (prosome macropain) subunit alpha type 2
    ADXCRPD.6687.C1_s_atCDK3Homo sapiens cyclin-dependent kinase 3 (CDK3) gene complete cds
    ADXCRAG_XM_031689_s_atMGAHomo sapiens MAX gene–associated (MGA) mRNA
    ADXCRAD_H10318_s_atBAT4Homo sapiens HLA-B–associated transcript 4 (BAT4) mRNA
    ADXCRPD.3851.C1_s_atFAM102AHomo sapiens family with sequence similarity 102 member A
    ADXCRPD.3229.C1_s_atTMC4Homo sapiens transmembrane channel-like 4 (TMC4) mRNA
    ADXCRPD.12486.C1_atAGPAT61-Acylglycerol-3-phosphate O-acyltransferase 6 (lysophosphatidic acid acyltransfase
    ADXCRAD_CB161421_s_atPIK3AP1Homo sapiens phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1)
    ADXCRPD.5659.C1_s_atPAPSS1Homo sapiens 3-phosphoadenosine 5-phosphosulfate synthase 1
    ADXCRPD.9008.C2_s_atCC2D1AHomo sapiens coiled-coil and C2 domain containing 1A (CC2D1A)
    ADXCRIH.3630.C1_s_atBTN3A2Homo sapiens butyrophilin subfamily 3 member A2 (BTN3A2) mRNA
    ADXCRAG_BX640630_s_atSLC30A7Homo sapiens solute carrier family 30 (zinc transporter) member 7
    ADXCRAD_CA776658_s_atWDR48Homo sapiens WD repeat domain 48 (WDR48) mRNA
    ADXCRAG_AL834333_s_atSUHW3Homo sapiens suppressor of hairy wing homolog 3 (Drosophila)
    ADXCRAG_BC016847_s_atE2F3Homo sapiens E2F transcription factor 3 (E2F3) mRNA
    ADXCRAD_AA311408_x_atLNPEPHomo sapiens mRNA for leucyl/cystinyl aminopeptidase variant protein.
    ADXCRPD.8916.C1_atCGREF1Homo sapiens cell growth regulator with EF-hand domain 1 (CGREF1)
    ADXCRAD_BE870333_atRPL28Homo sapiens ribosomal protein L28 (RPL28) mRNA
    ADXCRPD.8036.C1_atZNF669Homo sapiens zinc finger protein 669 (ZNF669) mRNA
    ADXCRSS.Hs#S634877_atRPL32Homo sapiens ribosomal protein L32 (RPL32) transcript variant 2
    ADXCRAD_BQ216862_atWDR68Homo sapiens WD repeat domain 68 (WDR68) mRNA
    ADXCRAD_BM542286_s_atRFC4Homo sapiens replication factor C (activator 1) 4 37kDa (RFC4)
    ADXCRAD_CN368539_s_atDOLPP1Homo sapiens dolichyl pyrophosphate phosphatase 1 (DOLPP1) mRNA
    ADXCRAD_BM793751_s_atBMS1Homo sapiens BMS1 homolog ribosome assembly protein (yeast)
    ADXCRAD_CN297084_s_atMAST2Homo sapiens microtubule-associated serine/threonine kinase 2
    ADXCRPD.5126.C1_s_atSART1Homo sapiens squamous cell carcinoma antigen recognized by T cells

Additional Files

  • Figures
  • Tables
  • Supplementary Data

    Files in this Data Supplement:

    • Supplementary Figure 1 - PDF file - 32K, QPCR validations.
    • Supplementary Table 1 - PDF file - 41K, QPCR validation.
    • Supplementary Table 2 - PDF file - 62K, unsupervised top 10 positive and negative correlated genes - clinical.
    • Supplementary Table 3 - PDF file - 61K, unsupervised top 10 positive and negative correlated genes - 5-FU basal.
    • Supplementary Table 4 - PDF file - 62K, unsupervised top 10 positive and negative correlated genes - 5-FU inducible.
    • Supplementary Table 5 - PDF file - 63K, unsupervised top 10 positive and negative correlated genes - SN38 basal.
    • Supplementary Table 6 - PDF file - 64K, unsupervised top 10 positive and negative correlated genes - SN38 inducible.
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Molecular Cancer Therapeutics: 11 (1)
January 2012
Volume 11, Issue 1
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A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer
Wendy L. Allen, Leanne Stevenson, Vicky M. Coyle, Puthen V. Jithesh, Irina Proutski, Gail Carson, Michael A. Gordon, Heinz-Josef D. Lenz, Sandra Van Schaeybroeck, Daniel B. Longley and Patrick G. Johnston
Mol Cancer Ther January 1 2012 (11) (1) 119-131; DOI: 10.1158/1535-7163.MCT-11-0510

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A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer
Wendy L. Allen, Leanne Stevenson, Vicky M. Coyle, Puthen V. Jithesh, Irina Proutski, Gail Carson, Michael A. Gordon, Heinz-Josef D. Lenz, Sandra Van Schaeybroeck, Daniel B. Longley and Patrick G. Johnston
Mol Cancer Ther January 1 2012 (11) (1) 119-131; DOI: 10.1158/1535-7163.MCT-11-0510
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