High-throughput RNAi screening identifies sensitizing targets to improve doxorubicin chemotherapy

Treatment of cancer often involves the use of chemotherapeutic agents that preferentially target tumor cells. Doxorubicin is an anthracycline antibiotic that induces an anticancer effect through intercalation into DNA, and inhibition of topoisomerase II. However, not all patients benefit from doxorubicin therapy and heterogeneity in the response to this agent has limited its clinical efficacy. In order to improve the efficiency of doxorubicin in a greater number of patients, we used RNA interference (RNAi) knockdown experiments to determine the extent to which a change in gene expression can increase cellular sensitivity to the anticancer effects of doxorubicin. RNAi is a naturally occurring mechanism that can be experimentally exploited by introducing double-stranded RNA molecules of ~21 base pairs termed short interfering RNAs (siRNAs) into cells to achieve sequence specific silence of gene expression. We have applied high-throughput RNAi analysis using a library of 83 siRNAs against 40 cancer-associated genes to assess the effects of gene silencing on HeLa cell survival with and without doxorubicin treatment. Eighteen hours after siRNA transfection, cells were treated with either a low (ineffective) dose of doxorubicin or vehicle control and cell viability was measured 72 hours after siRNA transfection. Control siRNA did not significantly affect cell survival either alone, or in combination with an ineffective dose of doxorubicin. Analysis of effects on cell survival revealed that the silencing of three of the 40 genes significantly increased sensitivity to doxorubicin. The three effective siRNA lead to decreased cell viability by 14, 6 and 25% alone, but when a low dose of doxorubicin was added a synergistic increase was observed that resulted in a decrease in cell viability by 73, 81 and 93%. These results indicate that these genes could serve as putative therapeutic targets for sensitization of cancer cells to doxorubicin. Furthermore, these findings demonstrate the effectiveness of using high-throughput RNAi as a tool for functional chemogenomics to identify sensitizing targets to chemotherapeutic agents.