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Redox effector factor-1 (Ref-1) CRISPR-Engineered Cys93 and Cys99 Mutants Reveal Cooperative and Secondary Roles of Redox Signaling and PDAC Progression

ABSTRACT: APE1/Ref-1 is a multifunctional protein implicated in pancreatic ductal adenocarcinoma (PDAC) progression through its redox regulation of oncogenic transcription factors such as HIF-1α, NF-κB, and others. While Cys65 is recognized as the primary redox-active residue, the functions of other cysteines remain only partially understood. Here, we describe the first CRISPR-engineered homozygous PDAC cell lines with single (C93A, C99A) and double (C93A+C99A) point mutations in Ref-1. These models enabled detailed analysis of the individual and combined roles of Cys93 and Cys99 in Ref-1 redox signaling and their impact on various cancer phenotypes. A single mutation at Cys93 led to decreased transcription factor activation and reduced tumor cell survival in vitro and in vivo. These effects were similar but not as profound in cells expressing C99A. The C93A+C99A double mutant further impaired redox signaling and significantly decreased tumor growth and metastasis in orthotopic PDAC mouse models. All of these perturbations specifically disrupted Ref-1’s redox activity without impacting its DNA repair function. Due to reduced levels of Ref-1 protein in the C93A+C99A tumor cells, add-back experiments were performed with wt-Ref-1 and C93A+C99A-Ref-1 to examine the impact on cell survival and activation of transcription factors downstream of Ref-1. Transcriptomic analysis revealed distinct changes in redox and metabolic pathways, though these were less pronounced than those observed with the previously studied C65A mutant.

ORGANISM(S): Homo sapiens

PROVIDER: GSE347008 | GEO | 2026/09/15

REPOSITORIES: GEO

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Project description: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 and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its 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.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.

Project description:The presence of oxidized DNA lesions, such as 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic sites (AP sites), has been described as epigenetic signals that are involved in gene expression control. In mammals, Apurinic-apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is the main AP endonuclease of the base excision repair (BER) pathway and is involved in active demethylation processes. In addition, APE1/Ref-1, through its redox function, regulates several transcriptional factors. However, the transcriptional control targets of each APE1 function are not completely known. In this study, a transcriptomic approach was used to investigate the effects of chemical inhibition of APE1/Ref-1 redox or DNA repair functions by E3330 or methoxyamine (MX) in an inflammatory cellular model. Under lipopolysaccharide (LPS) stimulation, both E3330 and MX reduced the expression of some cytokines and chemokines. Interestingly, E3330 treatment reduced cell viability after 48 h of the treatment. Genes related to inflammatory response and mitochondrial processes were downregulated in both treatments. In the E3330 treatment, RNA processing and ribosome biogenesis genes were downregulated, while they were upregulated in the MX treatment. Furthermore, in the E3330 treatment, the cellular stress response was the main upregulated process, while the cellular macromolecule metabolic process was observed in MX-upregulated genes. Nuclear respiratory factor 1 (NRF1) was predicted to be a master regulator of the downregulated genes in both treatments, while the ETS transcription factor ELK1 (ELK1) was predicted to be a master regulator only for E3330 treatment. Decreased expression of ELK1 and its target genes and a reduced 28S/18S ratio were observed, suggesting impaired rRNA processing. In addition, both redox and repair functions can affect the expression of NRF1 and GABPA target genes. The master regulators predicted for upregulated genes were YY1 and FLI1 for the E3330 and MX treatments, respectively. In summary, the chemical inhibition of APE1/Ref-1 affects gene expression regulated mainly by transcriptional factors of the ETS family, showing partial overlap of APE1 redox and DNA repair functions, suggesting that these activities are not entirely independent. This work provides a new perspective on the interaction between APE1 redox and DNA repair activity in inflammatory response modulation and transcription.

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Redox effector factor-1 (Ref-1) CRISPR-Engineered Cys93 and Cys99 Mutants Reveal Cooperative and Secondary Roles of Redox Signaling and PDAC Progression

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