Abstract 123: Reactive oxygen species (ROS) as mediators of the cancer cell’s transition into the polyaneuploid cancer cell (PACC) state
Authors: Kuczler Morgan D. , Pienta Kenneth J. , Amend Sarah R. 
Published: June 2022

Abstract
Metastatic cancer is lethal and incurable due to the emergence of therapeutic resistance, resulting in the deaths of 10 million people each year. Our data suggests that therapeutic resistance is mediated by stress-induced transition into a protective polyaneuploid cancer cell (PACC) state. The PACC state is characterized by large size, polyploidization of the original aneuploid genome, and pause in cell cycle. Once released from stress, some PACCs survive to repopulate a non-polyploid, resistant cancer cell population. Using Nanostring technology to profile cisplatin-induced PACCs in prostate cancer PC3 and breast cancer MDA-MB-231 cells, we observed significant increase in gene expression related to reactive oxygen species (ROS) and the DNA damage response (DDR). We hypothesize that ROS mediate a cancer cell’s ability to enter the PACC state to survive chemotherapy.ROS are produced as byproducts of cellular respiration and are present in all cells. ROS radicals are damaging to cellular macromolecules, particularly DNA. Cancer cells have evolved to withstand high ROS, but the increase in radicals contribute to genomic instability. ROS-induced DNA damage activates the DDR leading to four possible fates: continued cycling, DNA repair, apoptosis, or cell cycle arrest. Therapy significantly increases DNA damage while also increasing total ROS levels. At this level of DNA damage, the likely cell fates are death or cycle arrest. We induced the PACC state in PC3 cells with cisplatin and monitored ROS during PACC formation (24, 48, and 72 hours) using fluorescent microscopy and flow cytometry techniques. After 72 hours, the only surviving cells are in the PACC state. We found a step wise increase in the amount of ROS per cell across treatment time. By 72 hours, total ROS is 7-fold higher in PACCs compared to the parental control. PACCs are substantially larger than parental controls, so cell size must be considered. After adjusting for unit area, we found treated cells have a higher level of ROS compared to parental cells. The greatest increase in ROS per area was seen at 24hrs, suggesting there is an initial spike in ROS at the introduction of therapy. This suggests that the ability to enter the PACC state may be due to cell cycle arrest as a response to the extensive ROS caused by therapy. To assess cells that survive therapy in the PACC stateand produce resistant progeny, we induced PACC formation, then released therapy and monitored ROS for 10 days. Adjusted for area, ROS levels remained stable and elevated compared to parental control for 10 days following therapy release. The increase in ROS per unit area shows high ROS is not simply due to increased size. The stability over time suggests PACCs are able to manage ROS levels to prevent death by oxidative stress. Future work will evaluate PACCs induced by non-platinum chemotherapies, as well as, in additional cell lines.
Citation Format: Morgan D. Kuczler, Kenneth J. Pienta, Sarah R. Amend. Reactive oxygen species (ROS) as mediators of the cancer cell’s transition into the polyaneuploid cancer cell (PACC) state [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 123.