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.

Autor:	Mijit M; 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., Kpenu E; Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA., Chowdhury NN; 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., Gampala S; 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., Wireman R; Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA., Liu S; 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., Babb O; Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA., Georgiadis MM; Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, Indianapolis, IN, USA., Wan J; 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., Fishel ML; 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., Kelley MR; 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. Electronic address: mkelley@iu.edu.
Jazyk:	angličtina
Zdroj:	Redox biology [Redox Biol] 2024 Feb; Vol. 69, pp. 102977. Date of Electronic Publication: 2023 Dec 01.
DOI:	10.1016/j.redox.2023.102977
Abstrakt:	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-1 WT vs Ref-1 C65A 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-1 C65A lines was significantly lower compared to Ref-1 WT lines. The bioinformatic data revealed significant downregulation of metabolic pathways including OXPHOS in Ref-1 C65A expressing clones compared to Ref-1 WT line. Ref-1 C65A also demonstrated reduced cell proliferation and use of tricarboxylic acid (TCA) substrates compared to Ref-1 WT lines. A subcutaneous as well as PDAC orthotopic in vivo model demonstrated a significant reduction in tumor size, weight, and growth in the Ref-1 C65A lines compared to the Ref-1 WT lines. Moreover, mice implanted with Ref-1 C65A 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. Competing Interests: Declaration of competing interest MRK is CSO and consultant of Apexian Pharmaceuticals LLC and a medical consultant with Ocuphire Pharma. Both companies have or are using clinical compounds licensed from MRK through Indiana University Research and Technology Corporation. Apexian Pharmaceuticals, and Ocuphire Pharma had no control or oversight of the studies, interpretation, writing or presentation of the data in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze:	MEDLINE
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