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

• Published in: Cancer research (Chicago, Ill.) Ill.), 2013-04, Vol.73 (8_Supplement), p.4388-4388
• Main Authors: Siebring-van Olst, Ellen, de Menezes, Renee X., van der Meulen, Ida H., Smit, Egbert F., van Beusechem, Victor W.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2013-4388

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 th