A pathways-based approach identifying determinants of drug resistance in oesophageal adenocarcinoma
Introduction: Five-year survival rates for oesophageal adenocarcinoma (OAC) remain poor at approximately 15%. The development of drug-resistance limits the effectiveness of current chemotherapies, with less than 20% of patients experiencing complete pathological response, so the discovery of underlying resistance mechanisms and novel agents to target these pathways is crucial. Materials and Methods: 273 pre-treatment OAC biopsies were transcriptionally profiled using the Almac Diagnostics Xcel array™. The Broad Institute’s Gene Set Enrichment Analysis (GSEA) software was used to identify pathways enriched between non-responders to chemotherapy and responders, identifying genes differentially regulated between the two groups. A focused siRNA screen of 80 target genes identified from the GSEA results was then devised and completed, highlighting the role of Src in chemotherapy non-responders. A compound screen of FDA-approved drugs was carried out in the OE33 and FLO-1 cell lines in combination with cisplatin, and cell viability was measured by CellTiter Glo. This identified the additive effect of combining dasatinib with cisplatin in an OAC setting. Src inhibition using dasatinib and saracatinib was assessed with MTT viability assays, assessing the efficacy of combining Src-inhibition with traditional chemotherapies. CI values were generated to assess synergy. Western blotting, flow cytometry and high-content costaining was performed to investigate the mechanisms of action governing the synergistic effects identified. A model of cisplatin-resistance (OE33-CDDPR) was used to assess Src’s role in acquired resistance. Results: GSEA identified pathways associated with resistance to chemotherapy in OAC. Candidate genes were selected according to predefined criteria and 80 genes were taken forward in a siRNA screen to study the effects on cell viability of gene silencing alone or in combination with cisplatin/5-FU. Twelve genes were found to have an additive effect with chemotherapy to improve chemo-efficacy across an OAC cell line panel, including SRC. A high-throughput drug screen highlighted a number of novel compounds that displayed additive effects when combined with cisplatin, including dasatinib, a Src inhibitor. Src was then validated in an in vitro OAC setting, investigating the effects of Src inhibition using siRNA, dasatinib and saracatinib. In vitro combination experiments showed that combining Src inhibitors with traditional chemotherapies provides synergistic effects. Conclusions: Using a functional genomic approach we identified Src as a novel target associated with reduced cell viability following siRNA-mediated knockdown. By the use of small molecule inhibitors, we reinforced the results of the siRNA screen, verifying the clinical potential and synergistic effects of targeting Src alongside traditional chemotherapy treatments in a setting of OAC.
oesophageal, cancer, chemotherapy, resistance, src, adenocarcinoma, translational, oncology
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