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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

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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 Published OnlineFirst October 25, 2011; DOI: 10.1158/1535-7163.MCT-11-0510 Molecular Cancer Preclinical Development Therapeutics A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer Wendy L. Allen1, Leanne Stevenson1, Vicky M. Coyle1, Puthen V. Jithesh1, Irina Proutski1, Gail Carson1, Michael A. Gordon2, Heinz-Josef D. Lenz2, Sandra Van Schaeybroeck1, Daniel B. Longley1, and Patrick G. Johnston1 Abstract Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily because of drug resistance, so there is an urgent need to improve current treatment strategies. To identify novel determinants of resistance to the clinically relevant drugs 5-fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pretreatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemoresistance-determining genes, an unsupervised bioinformatics approach was used, and 50 genes were selected and then functionally assessed using custom-designed short interfering RNA (siRNA) screens. In the primary siRNA screen, silencing of 21 genes sensitized HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF, and SART1) were selected for further analysis in a panel of 5 colorectal cancer cell lines. Silencing SART1 sensitized all 5 cell lines to 5-FU treatment and 4/5 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 showed that its silencing induced apoptosis that was caspase-8 dependent. Furthermore, silencing of SART1 led to a downregulation of the caspase-8 inhibitor, c-FLIP, which we have previously shown is a key determinant of drug resistance in colorectal cancer. This study shows the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug- induced activation of caspase-8. Mol Cancer Ther; 11(1); 119–31. Ó2011 AACR. Introduction research to identify the key, clinically relevant molecular determinants of sensitivity to particular chemotherapy Resistance to chemotherapeutic drugs is a major prob- drugs, as these may constitute novel predictive biomar- lem in the treatment of many cancers. In colorectal cancer, kers of drug response and drug resistance. In addition, the long established antimetabolite drug 5-fluorouracil these key molecular determinants of drug sensitivity may (5-FU), even when used in combination with newer cyto- identify novel therapeutic strategies for enhancing the toxic drugs such as oxaliplatin and irinotecan (CPT-11), clinical effectiveness of chemotherapy. still produces a response in only 50% of patients with Recently, high-throughput technologies such as DNA advanced disease (1, 2). Hence, there is a pressing need for microarrays have been used to identify panels of markers that predict prognosis (3–8) or response to treatments (9–11) based on the expression profiles of those genes. In Authors' Affiliations: 1Centre for Cancer Research and Cell Biology, this article, we carried out unsupervised analyses of Queen's University Belfast, Belfast, Northern Ireland; and 2Division of Medical Oncology, University of Southern California/Norris Comprehen- microarray expression profiling data to identify gene lists sive Cancer Center, Keck School of Medicine, Los Angeles, California that segregate advanced colorectal cancer patients on the Note: Supplementary data for this article are available at Molecular Cancer basis of response to 5-FU/CPT-11 therapy. In parallel, we Therapeutics Online (http://mct.aacrjournals.org/). profiled and carried out unsupervised analyses of paired drug sensitive and resistant colorectal cancer cell lines to W.L. Allen, L. Stevenson, D.B. Longley, and P.G. Johnston contributed equally to this work. further identify genes associated with drug resistance. Furthermore, we have functionally tested whether any of Corresponding Author: Daniel B. Longley, Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 the genes contained within these lists are functionally 7BL, Northern Ireland. Phone: 44-28-90972764; Fax: 44-28-90972776; involved in chemotherapeutic resistance/sensitivity in E-mail: [email protected] colorectal cancer cell lines. For expression profiling in this doi: 10.1158/1535-7163.MCT-11-0510 study, we have used a colorectal cancer disease–specific Ó2011 American Association for Cancer Research. array, which contains 61,528 probesets and encodes 52,306 www.aacrjournals.org 119 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst October 25, 2011; DOI: 10.1158/1535-7163.MCT-11-0510 Allen et al. transcripts confirmed as being expressed in colorectal 2003 and was grown in McCoy’s 5A medium. LoVo cells cancer and normal colorectal tissue. This array contains were provided for AstraZeneca in 2003. HCT116, HT29, transcripts that have not been available for previous LoVo, and RKO cell lines were validated by short tandem expression analysis studies (12). The generation and util- repeat profiling by LGC Standards (18) in May 2011. The 5- ity of other disease-specific arrays using a similar techni- FU- and SN38-resistant HCT116 sublines and were gen- cal approach have been previously reported (13–15). erated in our laboratory as previously described (19). The The power of this study is that we have used a systems FLIP overexpressing HCT116 cell lines were previously biology approach of transcriptional profiling with func- described (20). All medium was supplemented with 10% tional testing of the identified genes to highlight poten- dialysed fetal calf serum, 50 g/mL penicillin–streptomy- tial novel drug targets and/or biomarkers, which could cin, 2 mmol/L L-glutamine, and 1 mmol/L sodium pyru- be used to potentially improve response rates for vate (all medium and supplements from Invitrogen Life advanced colorectal cancer. Technologies Corp.). All cell lines were maintained at 37 C in a humidified atmosphere containing 5% CO2. Materials and Methods Microarray analysis Patient samples Total RNA was extracted from 20 pretreatment meta- These have been previously described (16). Briefly, 20 static tumor biopsies from patients with advanced colo- patients with metastatic colorectal cancer were included rectal cancer and also profiled on the colorectal cancer in this study. All patients provided written fully informed disease-specific array (Almac Diagnostics). In addition, in consent as per Institutional Review Board guidelines in vitro analyses were also carried out using the colorectal the University of Southern California and approval was cancer disease-specific array. Briefly, HCT116 parental granted from this body. These patients underwent biopsy cells were either untreated (0 h control) or treated with of colorectal liver metastases before commencing irinote- either 5 mol/L 5-FU or 5 nmol/L SN38 for 24 hours can/5-FU chemotherapy on the IFL schedule: CPT-11 (acutely altered genes). Also, untreated 5-FU–resistant 125 mg/m2 i.v. over 90 minutes, leucovorin 20 mg/m2 as and SN38-resistant cells were analyzed to identify those intravenous bolus injection immediately before 5-FU genes that are basally deregulated between parental and and5-FU500mg/m2 asintravenousbolusinjectionadmin- resistant cells. Total RNA was isolated from 3 indepen- istered weekly for 4 weeks and repeated every 6 weeks. dent experiments using the RNA STAT-60 Total RNA CT imaging for response evaluation using WHO criteria isolation reagent (Tel-Test, Inc.) according to the manu- was done every 6 weeks. Of these 20 patients, 1 had a facturer’s instructions. For both the clinical and in vitro complete response to treatment, 10 had a partial response studies, RNA was sent to Almac Diagnostics for cDNA to treatment, and 9 had progressive disease on treatment. synthesis, cRNA synthesis, fragmentation, and hybrid- For the purpose of this study, we have further defined ization onto the colorectal cancer disease-specific array. responders as those patients with either complete or Detailed experimental protocols and raw expression data partial response and nonresponders as those patients with are available within the ArrayExpress repository [ref. 21; progressive disease. We had specifically excluded Accession number E-MEXP-1692 (clinical analysis) and E- patients with stable disease on chemotherapy from the MEXP-1691 (in vitro analysis)]. study. Unsupervised classification analysis Materials Unsupervised classification analysis was carried out 5-FU and SN38 were purchased from Sigma Chemical using Principal Components Analysis (PCA). All PCA Co. and Abatra Technology Co., Ltd, respectively. was carried out using the Partek software (version 6.3, Partek Inc.). Briefly, each microarray experiment (5-FU Cell culture basal, 5-FU inducible, SN38 basal, SN38 inducible, and All colorectal cancer cells were grown as previously 5-FU/irinotecan clinical) was initially normalized and described (17). Following receipt, cells were grown up, then underwent minimal flag filtering before PCA anal- and as soon as surplus cells became available, they were ysis. Following PCA analysis of each experiment, the frozen
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