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.

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.

Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells.

Cancer-associated fibroblasts (CAFs) comprise the majority of the tumor bulk of pancreatic ductal adenocarcinomas (PDACs). Current efforts to eradicate these tumors focus predominantly on targeting the proliferation of rapidly growing cancer epithelial cells. We know that this is largely ineffective with resistance arising in most tumors following exposure to chemotherapy. Despite the long-standing recognition of the prominence of CAFs in PDAC, the effect of chemotherapy on CAFs and how they may contribute to drug resistance in neighboring cancer cells is not well characterized. Here, we show that CAFs exposed to chemotherapy have an active role in regulating the survival and proliferation of cancer cells. We found that CAFs are intrinsically resistant to gemcitabine, the chemotherapeutic standard of care for PDAC. Further, CAFs exposed to gemcitabine significantly increase the release of extracellular vesicles called exosomes. These exosomes increased chemoresistance-inducing factor, Snail, in recipient epithelial cells and promote proliferation and drug resistance. Finally, treatment of gemcitabine-exposed CAFs with an inhibitor of exosome release, GW4869, significantly reduces survival in co-cultured epithelial cells, signifying an important role of CAF exosomes in chemotherapeutic drug resistance. Collectively, these findings show the potential for exosome inhibitors as treatment options alongside chemotherapy for overcoming PDAC chemoresistance.