Abstract
Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.