Ref-1 Redox Activity Alters Cancer Cell Metabolism in Pancreatic Cancer: Exploiting This Novel Finding as a Potential Target  

Abstract

Background: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB . The conversion of these TFs from an oxidized to reduced state lead to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The impact of knocking down Ref-1 under hypoxia was dramatic on the mitochondria and metabolic pathways. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo and compared to Devimistat, a drug that disrupts mitochondrial metabolism.Results: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat.Conclusion: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.

Clinical and Preclinical Outcomes of Combining Targeted Therapy With Radiotherapy

“…APE1 contributes to the repair of ionizing radiation through its ability to repair a 3'-phosphoglycolate end within a DNA strand break that is generated following ionizing radiation (IR) (167). A decrease in expression of APE1/Ref-1 in cancer cells results in apoptosis, cell cycle arrest, a decrease in proliferative capacity, a blockade of mitochondrial metabolism, and sensitization to various anti-cancer agents including RT (166,(168)(169)(170). Biochemical studies using oligonucleotides with clustered damage sites as would be encountered in a cell following RT demonstrate that APE1/Ref-1 can repair these types of DNA lesions (171).…”

“…As mentioned previously, the rationale for targeting tumor metabolism to sensitize cancer cells to RT is well-established. Mitochondrial metabolism is crucial to cancer cell survival and RT-induced mitochondrial DNA damage as well as excess ROS generation provides an attractive target to suppress cancer cell proliferation and induce apoptosis ( 146 , 147 , 206 ). Glutamine metabolism facilitates cancer cell survival, and breakdown of glutamine is mediated by glutaminases, making them the focus for development of small molecule inhibitors.…”