Abstract PO025: Ref-1 redox function identified as mitochondrial metabolic regulator in pancreatic cancer cells but not in CAFs
Authors: Gampala Silpa , Shah Fenil , Lu Xiaoyu , Moon Hye-ran , Sandusky George E. , Hulsey Emily , Mosley Amber L. , Han Bamsoo , Zhang Chi , Kelley Mark R. , Fishel Melissa L. 
Published: March 2021

Abstract
Pancreatic cancer can survive under the harshest of conditions including nutrient deprivation and extreme hypoxia. Its complex microenvironment contributes to PDAC’s (pancreatic ductal adenocarcinoma) therapeutic resistance and aggressive metastasis. Previous research shows that PDAC cells highly rely on increased glycolysis for ATP thereby limiting the use of TCA (tricarboxylic acid) cycle and ETC (electron transport chain), especially under hypoxia. However, recent studies indicate a dynamic exchange of nutrients between PCCs (Pancreatic Cancer cells) and its TME (tumor microenvironment) thus allowing the tumor to rely on either aerobic glycolysis or traditional oxidative phosphorylation (OXPHOS) depending on the oxygen tension and glucose availability. With HIF-1a being a central player in PDAC’s hypoxic response, and Redox factor-1 (Ref-1) controlling HIF-1a along with several other transcription factors (TFs) like NFkB and STAT3, Ref-1 proffers to be an ideal target. Ref-1 reduces oxidized TFs, thus enhancing their DNA binding and transcription. Several studies show that perturbation of Ref-1 levels affect the function of mitochondria, warranting the investigation of its role in metabolism. We conducted single cell RNA-seq (scRNA-seq) with Ref-1 knockdown (KD) in PDAC patient-derived cells under hypoxia and identified TCA cycle to be the most downregulated pathway with glycolysis and OXPHOS also significantly down. These results were validated with proteomics analysis. From the most downregulated DEGs (differentially expressed genes), a subset of genes confined to mitochondrial ETC and a subset of metabolite intermediates from TCA cycle were chosen to study and validate the role of Ref-1 in PDAC mitochondrial metabolic signaling. PCCs as well as CAFs (cancer-associated fibroblasts) were subjected to Ref-1 KD or redox inhibition using a potent second-generation inhibitor, APX2009. CAFs were chosen with the intent of understanding the differences in metabolic pathways between tumor and TME cells. As observed from scRNA-seq data, qPCR results demonstrated significant downregulation of mitochondrial DEG subsets with Ref-1 KD and redox inhibition. Mitochondrial plate-based functional assays revealed that Ref-1 inhibition caused decreased uptake of TCA cycle substrates in PCCs, but no change in CAFs. These effects of mitochondrial inhibition on cell growth were confirmed with reduction in growth of PCCs, but not CAFs in 3D co-culture spheroid assays. CPI-613 (mitochondrial metabolic inhibitor) was used as a comparator compound and showed similar results. Likewise, in-vivo xenograft studies with co-implantation of PCCs and CAFs confirmed reduction of tumor growth similar to CPI-613. These results confirm Ref-1’s redox role in tumor cells’ ability to utilize TCA cycle substrates and that we can detect differences in the metabolic phenotype between PCCs and CAFs. To conclude, suppression of tumor growth with minimal impact on CAFs, suggests a significant therapeutic benefit of Ref-1 redox inhibition.
Citation Format: Silpa Gampala, Fenil Shah, Xiaoyu Lu, Hye-ran Moon, George E. Sandusky, Emily Hulsey, Amber L. Mosley, Bamsoo Han, Chi Zhang, Mark R. Kelley, Melissa L. Fishel. Ref-1 redox function identified as mitochondrial metabolic regulator in pancreatic cancer cells but not in CAFs [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO025.