Abstract
RNAi screening holds the promise of systemizing the search for combination therapeutic strategies. Here we performed a pooled shRNA library screen to look for promising targets to inhibit in combination with inhibition of the mitotic regulator polo-like kinase (PLK1). The library contained ~4,500 shRNAs targeting various signaling and cancer-related genes and was screened in four lung cancer cell lines using both high (IC80) and low (IC20) amounts of the PLK1 inhibitor GSK461364. The relative abundance of cells containing individual shRNAs following drug treatment was determined by microarray analysis, using the mock treatment replicates as the normalizing reference. Overall, the inferred influences of individual shRNAs in both high and low drug treatment were remarkably similar in all four cell lines and involved a large percentage of the library. To investigate which functional categories of shRNAs were most prominent in influencing drug response, we used statistical analysis of microarrays (SAM) in combination with a filter for genes that had two or more concordant shRNAs. The most significant functional categories that came out of this analysis included receptor tyrosine kinases and nuclear hormone receptors. Through individual validation experiments, we determined that the two shRNAs from the library targeting the nuclear retinoic acid receptor gene RARA did indeed silence RARA expression and as predicted conferred resistance to GSK461364. This led us to test whether activation of RARA receptor with retinoids could sensitize cells to GSK461364. We found that retinoids did increase the drug sensitivity and enhanced the ability of PLK1 inhibition to induce mitotic arrest and apoptosis. These results suggest that retinoids could be used to enhance the effectiveness of GSK461364 and provide further evidence that RNAi screens can be effective tools to identify combination target strategies.
Highlights
Developing combinations of chemotherapeutic agents that increase tumor cell toxicity was a major milestone in cancer treatment [1]
In this study looked for polo-like kinase 1 (PLK1)-combination targets in non-small cell lung cancer cells (NSCLC), a clinically important tumor type that is driven to a significant degree by mutations in TP53 and KRAS and that as a whole are sensitive to PLK1 inhibition [7]
We focused on four NSCLC cell lines, two that harbor mutant KRAS but are wild-type for TP53 (A549 and NCI-H460) and two that harbor mutant TP53 but are wild-type for KRAS (NCI-H522 and NCI-H322)
Summary
Developing combinations of chemotherapeutic agents that increase tumor cell toxicity was a major milestone in cancer treatment [1]. RNAi screening has the potential to systematize the search for genes to target in combination with specific anti-cancer agents. We used RNAi screening to look for sensitizers to the candidate cancer drug GSK461364A, a potent inhibitor of polo-like kinase 1 (PLK1) [5]. Initial motivation for developing inhibitors of PLK1 as candidate cancer drugs was the potential to avoid the toxicities of traditional antimitotics that target tubulin structures in both cancer and nondividing cells [6, 7]. Perhaps a more compelling rationale is based on findings that PLK1 inhibition is selectively potent for cells harboring mutant TP53 or mutant KRAS [8,9,10], which is the reverse of the usual situation where altered TP53 and mutant KRAS confer drug resistance
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