Abstract 5854: Therapeutic targeting of p73-activated c-FLIP switches cell fate from growth arrest and survival to apoptosis in p53-deficient cancer cells

Abstract

Abstract The tumor suppressor p73, a member of the p53 family, transcriptionally activates numerous p53-target genes in cell cycle regulation and apoptosis. Therapeutic activation of p73 can restore p53-signaling in mutant p53-expressing cancer cells effectively bypassing the p53 deficiency. In addition to pro-apoptotic signaling, outcomes of p73 activation contain survival signals and this may provide insights in cell fate outcomes between cell survival and apoptosis after cellular stress. We report that cellular FLICE inhibitory protein (c-FLIP), a master antiapoptotic factor, is a novel transcriptional target of p73. To test if p73 regulates c-FLIP expression in p53-deficient cancer cells, we overexpressed p73 in p53-deficient cancer cells using p73-adenovirus. Overexpression of p73 dramatically increased c-FLIP at the protein and RNA levels. The effect of p73 on c-FLIP upregulation was blocked by knock-down of p73. ChIP assay showed that p73 binds to the c-FLIP promoter in a p73-level dependent manner. Furthermore, a luciferase reporter assay showed that p73 increased the c-FLIP-L-promoter-mediated luciferase reporter expression in p53-deficient cancer cells. To test the effect of the outcomes of p73 signaling on cell survival or death, we performed cell viability assay and found that p73 reduces cell viability in a p73-level dependent manner. We found an increase of both p21 and c-FLIP at the protein levels in response to overexpression of p73 in p53-deficient cancer cells, but no cleaved-PARP was detected and flow cytometric analysis showed the cells were arrested in G1 phase. These results suggest that p73 arrests the cell cycle and prevents apoptosis at the tested levels of p73. We knocked down c-FLIP using siRNA in p53-deficient cancer cells, and found that knockdown of c-FLIP enhanced cleaved-PARP, cleaved caspase- 3 and -8 correlated to high level of apoptotic signaling with Noxa and puma upregulation in response to the overexpression of p73. Our results suggest that the depletion of c-FLIP primes cellular apoptosis switching from p73-induced cell cycle arrest (cell survival) and sensitizes cells to p73-induced apoptotic signals in p53-deficient cells. The cell fate decision following p73 activation is determined by the adjustment of the balance of outcomes of p73 activation between p73-induced pro-apoptotic signaling and c-FLIP expression in p53-deficent cancer cells. Our discovery of the new function of p73 to upregulate c-FLIP provides a rationale for depleting c-FLIP to improve antitumor efficacy of p73-targeting cancer therapy. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Therapeutic targeting of p73-activated c-FLIP switches cell fate from growth arrest and survival to apoptosis in p53-deficient cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5854.