Abstract 4388: Genome-wide siRNA screens identify potent p53 inhibitors in NSCLC cells.

Abstract

Abstract Background: Non Small Cell Lung Cancer (NSCLC) remains the number one cause of cancer death worldwide. For a number of cancers, restoration of tumor suppressor p53 activity is being studied as a possible treatment strategy. Approximately 50% of NSCLCs harbor wild type p53. The p53 inhibitor RCHY1 was previously shown to be highly expressed in lung cancer and suggested to play a critical role in lung tumor progression by degrading p53 (Duan et al., J Natl Cancer Inst 96: 1718-1721). We hypothesized that more molecules could be inhibiting p53 in NSCLC. Here we set out to identify these inhibitors as potential therapeutic targets in NSCLC. Methods: Three siRNA screens were done with the Dharmacon siGENOME whole human genome siRNA library on a derivative of the A549 NSCLC cell line harboring the p53 dependent firefly luciferase reporter PG13Luc. Luciferase activity was measured three days after siRNA transfection using an in-house developed assay (Siebring-van Olst et al, J Biomol Screen, PMID: 23112084). Results: The screens showed excellent assay metrics (Z’ factors >0.5 and Spearman Rank Correlations >0.89). Hit selection threshold for stratification was set at a robust Z-score ≥ 3, which selected 54 genes. Silencing these candidates increased luciferase activity more than 5-fold in the primary screen, which exceeded commonly known p53 inhibitors such as MDM2 and MDM4 (2.5 and 3.2-fold, respectively). For 37 hits, the phenotype was confirmed with two or more individual siRNAs targeting that gene, as well as in a different A549-PG13Luc cell clone. In A549 cells carrying a shRNA construct suppressing p53, a reduction in p53 activity was seen for all 37 candidates, thus showing that the effects of gene silencing on luciferase activity were indeed p53 dependent. A second reporter system with a different p53 response element, the Cignal assay, confirmed p53 dependency for 14 hits. Silencing these 14 inhibitors in a second NSCLC cell line (NCI-H292), also resulted in p53 activation. Western blot analysis on siRNA-transfected A549 cells confirmed p53 activation by showing increased levels of p53 and/or increased levels of p21 (12 and 8 candidate genes, respectively). Interestingly, silencing 9 putative p53 inhibitors was accompanied with a subsequent loss of cell viability. Protein interaction analysis using STRING showed that six of these genes, i.e., CWC22, SF3A3, SF3B1, SF3B14, HNRPL and SNRPD3, cluster together in a network involved in RNA processing, while the other candidates appeared unrelated. Conclusion: We identified a number of potent inhibitors of p53 in NSCLC cells that have not previously been associated with p53 function. Some of these proteins could be potential therapeutic targets in cancers harboring wild type p53. Citation Format: Ellen Siebring-van Olst, Renee X. de Menezes, Ida H. van der Meulen, Egbert F. Smit, Victor W. van Beusechem. Genome-wide siRNA screens identify potent p53 inhibitors in NSCLC cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4388. doi:10.1158/1538-7445.AM2013-4388