Identification of Src's role in governing chemotherapy resistance in oesophageal adenocarcinoma through functional genomic and high-throughput drug screening approaches

Introduction: Five-year survival rates for oesophageal adenocarcinoma (OAC) remain poor at just 15%. The development of drug-resistance limits the effectiveness of current chemotherapies, 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 in non-responders to chemotherapy compared to responders. 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. An Enzo compound screen of FDA-approved drugs was carried out in OE33 and FLO-1 cell lines in combination with cisplatin, and cell viability was measured by CellTiter Glo. Src inhibition using dasatinib and saracatinib was assessed with MTT viability assays, western blotting and flow cytometry to investigate the mechanisms of action. CI values were generated to assess synergy.

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 or 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 in OAC cell lines 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 Src was identified as a novel target associated with reduced cell viability following siRNA-mediated knockdown. By the use of small molecule inhibitors, the results of the siRNA screen were reinforced, verifying the clinical potential and synergistic effects of targeting Src alongside traditional chemotherapy treatments.