Abstract C139: Selective activation of mutant p53 through acetylation promotes cell death in lung SCC cells
Authors: Kong Li Ren , Cheok Chit Fang , Goh Boon Cher 
Published: May 2016

Abstract
Conventional chemotherapy has been applied indiscriminately and mostly produced unsatisfactory outcome. The development of small molecule inhibitors that target on specific oncogenic signaling has since revolutionized the treatment of cancer. From the perspective of lung cancer, it can be further characterized at the molecular level, such as EGFR, ALK, RET and ROS1, to guide treatment options. However, efforts at defining the molecular underpinnings of squamous cell carcinoma (SCC) of the lung have confirmed a paucity of somatic alterations among the common driver oncogenes, with a high frequency of TP53 mutation, particularly the R158 residue. In this study, we showed that combination of cisplatin and belinostat drastically enhanced cell death in a SCC cell line with loss-of-function p53R158G mutation, and intriguingly, this event is strong associated with p53 acetylation. Despite the emerging evidence demonstrating the gains of oncogenic functions in mutant p53, our data suggest for the first time a restoration of p53-dependent apoptosis through induction of acetylation in SCC cells. To examine this disparity, we generated stable clones expressing p53R158G mutant from Calu-1 cells. Compared with the parental p53-null cells, Calu-1-p53R158G cells were more sensitive to the belinostat/cisplatin combination as indicated by the increase in PARP cleavage and caspase 3 activation. Chip-seq and AmpliSeq were performed to determine the binding regions of p53R158G and its distance from nearest downstream genes, as well as the influence of this mutation and treatment on the transcriptional profiles. Overall, differences were seen in the binding behavior of the mutant protein as compared to the wild-type p53. In some cases, genes found to have differences in binding position, such as CDKN1A but not MDM2, also had significant differences (&gt;1.5 log2 fold change) in gene expression, suggesting a partial and selective activation of p53 pathway. Consistently, substitutions of lysine residues in p53R158G from site-directed mutagenesis effectively rescued apoptosis induced by belinostat/cisplatin combination, further supporting the significance of p53 acetylation in the observed cytotoxicity. Mechanistically, this combination was shown to inhibit the phosphorylation of both HDAC3 and sirtuin-1; whereas silencing of sirtuin-1, but not HDAC3, facilitated the acetylation of p53. Selisistat, a sirtuin-1 specific inhibitor, demonstrated strong synergistic combination with cisplatin in enhancing cytotoxicity and p53 acetylation. Overall, this study provides compelling evidences that inhibition of sirtuin-1 activity promotes cell death through acetylation of p53R158G. Given the prevalence of p53 mutation in lung SCC, treatment strategy subjecting mutant p53 to therapeutic intervention could be an appealing approach.
Citation Format: Li Ren Kong, Chit Fang Cheok, Boon Cher Goh. Selective activation of mutant p53 through acetylation promotes cell death in lung SCC cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C139.