Oxidative Damage-Induced KRAS Transcription: Investigating the Role of Base Excision Repair in Pancreatic Cancer

• Arya Gupta, Biochemistry, University of Delaware

• Amy Whitaker, Nuclear Dynamics & Cancer, Fox Chase Cancer Center – Temple University

Guanine-rich regions of DNA can form secondary structures known as G-quadruplexes, or G4s. Because guanine has a low oxidation potential, G4s are particularly susceptible to oxidative DNA damage. Although oxidative DNA damage is usually repaired through the Base Excision Repair (BER) pathway, it may also act as a signal that changes gene expression. This damage-induced regulation may be especially important for KRAS, an oncogene mutated in more than 90% of Pancreatic Ductal Adenocarcinoma (PDAC) cases. Due to resistance to treatment, PDAC has a 5-year survival rate of approximately 9%, highlighting the importance of understanding how KRAS expression is controlled. Although the KRAS promoter is regulated by a G4, the mechanism by which oxidative damage at this site activates KRAS transcription remains unclear. We hypothesize that BER proteins are required for oxidative damage-induced KRAS transcription. To test this, we are using the PDAC cell line, PANC1, in which genes encoding BER proteins, including APE1 and PARP1, have been knocked out using CRISPR-Cas9. Cells are treated with varying concentrations of hydrogen peroxide to mimic an increased oxidative environment and thereby elicit an increase of KRAS expression levels, which is measured by RT-qPCR. If a knocked-out protein is required for KRAS transcriptional activation, we expect KRAS expression levels to be lower in that cell line, even following oxidative damage. Identifying proteins involved in KRAS transcriptional activation may bring to light potential therapeutic targets for reducing KRAS expression in pancreatic cancer.