Role of Poly [ADP-ribose] Polymerase 1 in Activating the Kirsten ras (KRAS) Gene in Response to Oxidative Stress

Abstract

In pancreatic Panc-1 cancer cells, an increase of oxidative stress enhances the level of 7,8-dihydro-8-oxoguanine (8OG) more in the KRAS promoter region containing G4 motifs than in non-G4 motif G-rich genomic regions. We found that H2O2 stimulates the recruitment to the KRAS promoter of poly [ADP-ribose] polymerase 1 (PARP-1), which efficiently binds to local G4 structures. Upon binding to G4 DNA, PARP-1 undergoes auto PARylation and thus becomes negatively charged. In our view this should favor the recruitment to the KRAS promoter of MAZ and hnRNP A1, as these two nuclear factors, because of their isoelectric points >7, are cationic in nature under physiological conditions. This is indeed supported by pulldown assays which showed that PARP-1, MAZ, and hnRNP A1 form a multiprotein complex with an oligonucleotide mimicking the KRAS G4 structure. Our data suggest that an increase of oxidative stress in Panc-1 cells activates a ROS-G4-PARP-1 axis that stimulates the transcription of KRAS. This mechanism is confirmed by the finding that when PARP-1 is silenced by siRNA or auto PARylation is inhibited by Veliparib, the expression of KRAS is downregulated. When Panc-1 cells are treated with H2O2 instead, a strong up-regulation of KRAS transcription is observed.

Effect of DNA Glycosylases OGG1 and Neil1 on Oxidized G-Rich Motif in the KRAS Promoter

“…Preliminary data have shown that both conformers are recognized by the nuclear factors, albeit to different extents (in preparation). Evidence that 32R behaves as a regulatory element for KRAS transcription has been provided [ 35 , 37 , 39 , 40 ]. The mechanism by which the gene is regulated is rather complex and recent data suggest that guanine oxidation to 8OG might play a role, in particular under-enhanced oxidative stress, which is typical of cancer cells [ 26 , 39 ].…”

“…Evidence that 32R behaves as a regulatory element for KRAS transcription has been provided [ 35 , 37 , 39 , 40 ]. The mechanism by which the gene is regulated is rather complex and recent data suggest that guanine oxidation to 8OG might play a role, in particular under-enhanced oxidative stress, which is typical of cancer cells [ 26 , 39 ]. This is based on the following experimental observations: (i) guanines in 32R tend to oxidize more than isolated guanines; the guanine at the 5′ end of a G-run shows the lowest ionization potential, i.e., the highest tendency for oxidation [ 41 , 42 , 43 ]; (ii) the KRAS gene is responsive to ROS, as it is upregulated when the cellular oxidative stress is increased by H 2 O 2 or by photoactivated porphyrins [ 39 , 44 ]; (iii) pulldown and chromatin immunoprecipitation (ChIP) experiments show that 8OG and G4 co-localize in the same promoter region containing 32R [ 26 ].…”

“…Indeed, a ChIP qPCR analysis showed that 8OG is more abundant in the promoter region containing 32R than in other G-rich genomic sequences, which are unable to fold into a G4 [ 26 ]. We found that oxidized 32R stimulated the recruitment to the promoter of nuclear factors involved in KRAS transcription: PARP1, MAZ, Ku70, and hnRNP A1 [ 35 , 39 ]. Besides being components of the transcription initiation complex, MAZ and hnRNP A1 have the capacity to unfold the G4 [ 50 , 51 ].…”

“…Under oxidative stress, the guanines in G4 motifs are inclined to oxidize to 8OG due to their low redox potential [ 13 ]. Previous studies have shown that 8OG in 32R, the G4 motif in KRAS promoter critical for transcription, behaves as an epigenetic mark for the recruitment of transcription factors [ 26 , 39 ]. However, 8OG is mutagenic and is repaired by the cell.…”

A Comprehensive Analysis of the Role of hnRNP A1 Function and Dysfunction in the Pathogenesis of Neurodegenerative Disease

“…Transcription of oncogenes, like Kirsten Rat Sarcoma Viral Proto-Oncogene (KRAS) and Transformer 2 Beta Homolog (TRA2B), are promoted by hnRNP A1 binding to a G-quadruplex structure in the genes' promoters, utilizing hnRNP A1 DNA binding activity (Paramasivam et al, 2009;Nishikawa et al, 2019). Promotion of KRAS transcription has recently been shown to be regulated by the cell stress response regulator poly (ADP-ribose) polymerase (PARP-1), which itself PARylates hnRNP A1 (discussed below), although the impact of hnRNP A1 PARylation on KRAS transcription, and the function of KRAS expression in the context of oxidative stress are currently unclear (Table 1) (Duan et al, 2019;Cinque et al, 2020). Additionally, hnRNP A1 stabilizes SIRT1 mRNA (Wang et al, 2016); increased abundance of its protein product Sirtuin/SIRT1 prevents progression to cellular senescence, but also de-acetylates hnRNP A1 protein, which negatively regulates oncogenic cell proliferation by modulating alternative splicing of pro-survival mRNAs like PKM (Table 1) .…”