RelA Is an Essential Target for Enhancing Cellular Responses to the DNA Repair/Ref-1 Redox Signaling Protein and Restoring Perturbated Cellular Redox Homeostasis in Mouse PDAC Cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a poor response to current treatment regimens. The multifunctional DNA repair-redox signaling protein Ref-1 has a redox signaling function that activates several transcriptional factors (TFs) including NF-κB (RelA), STAT3, AP-1. These have been implicated in signaling in PDAC and associated with cancer progression and therapy resistance. Numerous studies have shown a role for RelA in PDAC inflammatory responses and therapy resistance, little is known as to how these inflammatory responses are modulated through Ref-1 redox signaling pathways during pancreatic pathogenesis. RelA and STAT3 are two major targets of Ref-1 and are important in PDAC pathogenesis. To decipher the mechanistic role of RelA in response to Ref-1 inhibition, we used PDAC cells (KC3590) from a genetically engineered KrasG12D-driven mouse model that also is functionally deficient for RelA (Parent/Vector) or KC3590 cells with fully functional RelA added back (clone 13; C13). We demonstrated that RelA deficient cells are more resistant to Ref-1 redox inhibitors APX3330, APX2009, and APX2014, and their sensitivity is restored in the RelA proficient cells. Knockdown of STAT3 did not change cellular sensitivity to Ref-1 redox inhibitors in either cell type. Gene expression analysis demonstrated that Ref-1 inhibitors significantly decreased IL-8, FOSB, and c-Jun when functional RelA is present. We also demonstrated that PRDX1, a known Ref-1 redox modulator, contributes to Ref-1 inhibitor cellular response. Knockdown of PRDX1 when functional RelA is present resulted in dramatically increased PDAC killing in response to Ref-1 inhibitors. The enhanced cell killing was not due to increased intracellular ROS production. Although Ref-1 inhibition decreased the NADP/NADPH ratio in the cells, the addition of PRDX1 knockdown did not further this redox imbalance. This data suggests that the mechanism of cell killing following Ref-1 inhibition is at least partially mediated through RelA and not STAT3. Further imbalancing of the redox signaling through disruption of the PRDX1-Ref-1 interaction may have therapeutic implications. Our data further support a pivotal role of RelA in mediating Ref-1 redox signaling in PDAC cells with the KrasG12D genotype and provide novel therapeutic strategies to combat PDAC drug resistance.

APE1/Ref-1 Inhibits Adipogenic Transcription Factors during Adipocyte Differentiation in 3T3-L1 Cells

“…The redox function of APE1/Ref-1 has been studied as an important aspect in protecting cells from oxidative stress [ 11 , 35 ]. Specific inhibition of redox activity of APE1/Ref-1 by E3330, a redox inhibitor of APE1/Ref-1, enhanced ROS production [ 36 , 37 ]. In this study, we demonstrated that the anti-adipogenic effect of APE1/Ref-1 could be mediated by the inhibition of expression of adipogenic transcriptional factors such as C/EBP-α and PPAR-γ .…”

Identification of Novel Pathways Regulated by APE1/Ref-1 in Human Retinal Endothelial Cells

“…HRECs were lysed in RIPA extraction buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) [ 83 ]. Denatured samples (20 μg) were subjected to SDS-PAGE.…”

“…At least three independent experiments were performed. Relative quantity was determined using the 2 −ΔΔCt method as previously published [ 83 ].…”

RELA promotes the progression of oral squamous cell carcinoma via TFAP2A‐Wnt/β‐catenin signaling

“…As the inhibitor degrades, NF-kappa-B migrates to the nucleus, regulating the transcriptional activities. [6][7][8] It has been demonstrated that RELA is the transcription factor of liver X receptor alpha, which is upregulated in gastric cancer. RELA appears to have a dual role in gastric cancer cells, promoting differentiation while simultaneously suppressing proliferation and invasion.…”

“…In the cytoplasm, however, NF‐kappa‐B is inactive due to the role of inhibitors. As the inhibitor degrades, NF‐kappa‐B migrates to the nucleus, regulating the transcriptional activities 6–8 . It has been demonstrated that RELA is the transcription factor of liver X receptor alpha, which is upregulated in gastric cancer.…”