Abstract 317: Role of KLLN in DNA damage-induced apoptosis is associated with the regulation of p53 phosphorylation and acetylation in breast cancer cells

Abstract

Abstract KLLN was initially discovered as a target of p53 involved in S-phase checkpoint regulation. Germline hypermethylation in the promoter of KLLN is known to increase the predisposition for breast and clear cell renal cell carcinomas in PTEN-wildtype Cowden syndrome (CS) patients. KLLN is a known tumor suppressor protein and required for p53-mediated apoptosis. In this study, we explore the underlying mechanism by which KLLN regulates DNA damage response (DDR), specifically apoptosis. Our DDR studies were conducted on one breast normal and four breast cancer cell lines using doxorubicin as the DNA damaging agent. We initially showed that doxorubicin-induced DNA damage increases KLLN expression at 16h and 24h post-damage which could be abrogated by RNAi-mediated knockdown of KLLN expression. Response to DNA damage as determined by the presence of γH2AX foci formation and expression of γH2AX protein were both decreased after KLLN knockdown. Using TUNEL assay and cell viability studies, we demonstrated that a lack of KLLN expression decreases apoptosis and increases cell viability at 16h and 24h post-damage. Definitive markers of apoptosis such as cleavage of caspase 3 and PARP were also decreased. Since KLLN is a known transcription factor and regulates the expression of DDR genes such as TP53 and CHK1, we assessed the regulation of these genes in response to DNA damage and found that lack of KLLN expression did not affect the expression of either TP53 or CHK1. Cell cycle analysis results using flow cytometry also suggested that KLLN does not play a role in S-phase cell cycle regulation in response to DNA damage as was previously thought, since KLLN knockdown did not change accumulation of cells in S-phase after doxorubicin-induced DNA damage. DDR in cells is immediate and therefore we assessed the abrogation of DDR after KLLN knockdown at early timepoints (1h and 2h) post-damage. We found that lack of KLLN expression decreased Ser15 phosphorylation of p53 and Thr68 phosphorylation of CHK2 at 1h and 2h post-damage. KLLN regulation of p53-mediated apoptosis could also be associated with its interaction with deleted in breast cancer (DBC1). DBC1 is a known inhibitor of SIRT1, a deacetylase that is known for its inhibitory deacetylation of p53. Phosphorylated DBC1 binds to SIRT1 and inhibits its deacetylation of p53. We found that lack of KLLN expression decreased the phosphorylation of DBC1 on Thr454 in response to DNA damage. Therefore, KLLN displays a 2-pronged regulation of the activation of p53 and the downstream cascade of proteins involved in apoptosis. SIGNIFICANCE: Our study demonstrates a clear role for KLLN in DNA damage-induced apoptosis and elucidates the underlying mechanism through the regulation of p53 phosphorylation and deacetylation in response to DNA damage. Our observations add to the basis of KLLN as a tumor suppressor and as a susceptibility gene for inherited cancers. Citation Format: Madhav Sankunny, Charis Eng. Role of KLLN in DNA damage-induced apoptosis is associated with the regulation of p53 phosphorylation and acetylation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 317.