In vitro and In vivo evidence demonstrating chronic absence of Ref-1 Cysteine 65 impacts Ref-1 folding configuration, redox signaling, proliferation and metastasis in pancreatic cancer
M. Mijit,
E. Kpenu,
N.N. Chowdhury,
S. Gampala,
R. Wireman,
S. Liu,
O. Babb,
M.M. Georgiadis,
J. Wan,
M.L. Fishel,
M.R. Kelley
Affiliations
M. Mijit
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
E. Kpenu
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
N.N. Chowdhury
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
S. Gampala
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
R. Wireman
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
S. Liu
Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
O. Babb
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
M.M. Georgiadis
Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, Indianapolis, IN, USA
J. Wan
Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
M.L. Fishel
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
M.R. Kelley
Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, Indianapolis, IN, USA; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA; Corresponding author. Department of Pediatrics, Herman B Wells Center for Pediatric Research, 1044 W. Walnut, R4-302C, Indianapolis, IN, 46202, USA.
Ref-1/APE1 (Redox Effector/Apurinic Endonuclease 1) is a multifunctional enzyme that serves as a redox factor for several transcription factors (TFs), e.g., NF-kB, HIF-1α, which in an oxidized state fail to bind DNA. Conversion of these TFs to a reduced state serves to regulate various biological responses such as cell growth, inflammation, and cellular metabolism. The redox activity involves a thiol exchange reaction for which Cys65 (C65) serves as the nucleophile. Using CRISPR editing in human pancreatic ductal adenocarcinoma (PDAC) cells, we changed C65 to Ala (C65A) in Ref-1 to evaluate alteration of Ref-1 redox dynamics as well as chronic loss of Ref-1 redox activity on cell signaling pathways, specifically those regulated by NF-kB and HIF-1α. The redox activity of Ref-1 requires partial unfolding to expose C65, which is buried in the folded structure. Labeling of Ref-1 with polyethylene glycol-maleimide (PEGm) provides a readout of reduced Cys residues in Ref-1 and thereby an assessment of partial unfolding in Ref-1. In comparing Ref-1WT vs Ref-1C65A cell lines, we found an altered distribution of oxidized versus reduced states of Ref-1. Accordingly, activation of NF-kB and HIF-1α in Ref-1C65A lines was significantly lower compared to Ref-1WT lines. The bioinformatic data revealed significant downregulation of metabolic pathways including OXPHOS in Ref-1C65A expressing clones compared to Ref-1WT line. Ref-1C65A also demonstrated reduced cell proliferation and use of tricarboxylic acid (TCA) substrates compared to Ref-1WT lines. A subcutaneous as well as PDAC orthotopic in vivo model demonstrated a significant reduction in tumor size, weight, and growth in the Ref-1C65A lines compared to the Ref-1WT lines. Moreover, mice implanted with Ref-1C65A redox deficient cells demonstrate significantly reduced metastatic burden to liver and lung compared to mice implanted with Ref-1 redox proficient cells. These results from the current study provide direct evidence that the chronic absence of Cys65 in Ref-1 results in redox inactivity of the protein in human PDAC cells, and subsequent biological results confirm a critical involvement of Ref-1 redox signaling and tumorigenic phenotype.
