Role of post-translational modification in KRAS expression: interplay between PARP1, Reactive Oxygen Species and G-quadruplex DNA

Over 90% pancreatic ductal adenocarcinomas (PDACs) carry mutations in exon 1 of the KRAS gene causing the malignant transformation of the cells. One main function of mutant KRAS is to reprogram the metabolism in PDAC to generate biomass and reducing power to fuel a high proliferation rate. Previous studies have demonstrated that the KRAS promoter contains a G-rich sequence located immediately upstream of the transcription start site (TSS). Our laboratory reported for the first time in 2006 that this sequence (called 32R or G4 near) can fold in a non-canonical DNA structure, called G-quadruplex (or G4) under physiological conditions. Two years later, we found that the KRAS G4 is recognized by several transcription factors (TFs) including MAZ, hnRNP A1, Ku70 and PARP1. PDAC cells produce more reactive oxygen species (ROS) than healthy cells, leading to oxidation of lipids, proteins and DNA. Among the four nucleobases, guanine is more prone to oxidation to 7,8-dihydro-8-oxoguanine (8OG). Previous data from our laboratory revealed that an increase of oxidative stress results in a higher level of 8OG in the promoter G4 region than in non-G4 guanine-rich regions. This guanine lesion is likely to behave as an epigenetic mark for the recruitment of MAZ and hnRNP A1 to the promoter. A crucial report in this thesis is the existence of a strict correlation between KRAS and oxidative stress, as an increase of ROS significantly stimulates the expression of the gene. We reasoned that PARP1 could control transcription because when ROS are enhanced in PDAC cells, PARP1 is recruited to the KRAS promoter in the region near to TSS. PARP1 (Poly-ADP-Ribose Polymerase 1) is a nuclear protein that catalyses the transfer of poly ADP-ribose units (PARylation) or mono ADP-ribose unit (MARylation) onto target proteins, including itself. PARP1 is known to take part to the base excision repair (BER) mechanism by which an oxidized base is recognized, excised and repaired. We found that PARP1 tightly binds to 32R. Mobility-shift assays (EMSA) revealed that PARP1 forms two DNA-protein complexes of stoichiometry 1:1 and 1:2. Binding curves fit to the Hill equation giving KD values of about 2.5 x 10 -7 M and n=3, pointing to a cooperative type of binding. By measuring Trp fluorescence quenching upon addition of G4 to PARP1, we confirmed the DNA-protein 1:2 stoichiometry. More, upon binding to the G4s, but not a linear DNA strand, PARP1 undergoes auto PARylation, acquiring a net negative charge. PARylated PARP1 may behave as a recruitment platform for cationic TFs under physiological conditions like MAZ (pI = 8.1) and hnRNP A1 (pI = 9.2). According to this model, G4 and PARP1 play together a key role in activating the transcription of KRAS. By pulldown experiments carried out with biotinylated G4s and a nuclear extract from PDAC cells, we found G4 baits pulled down all the proteins forming this pre-initiation complex. When cells are treated with an oligonucleotide mimicking the KRAS G4, the expression of KRAS is strongly inhibited because exogenous G4s sequester TFs essential for KRAS transcription, in support of the hypothesis that G4 works as an antenna for recruiting TFs. Finally, both siRNA-mediated silencing of PARP1 and inhibition of its catalytic activity by Veliparib dramatically reduce KRAS expression. Together, these results provide evidence that PARP1 inhibition is a validated strategy to arrest PDAC cell proliferation. Besides this work, we addressed also the issue about the recognition of PARP1 by the G4 formed by the 32R motif. A recent NMR study reported that 32R folds in two structurally different G4 conformers (namely G25T and G9T) that are in slow equilibrium one another. We found by EMSA that both conformers interact with PARP1, forming stable DNA-protein complexes. However, pulldown assays showed that G25T has a higher capacity than G9T to form a multiprotein complex. Further experiments are required to clarify the role, if any, of G9T.