Ref-1 redox activity alters cancer cell metabolism in pancreatic cancer: exploiting this novel finding as a potential target

• Published in: Journal of experimental & clinical cancer research 2021-08, Vol.40 (1), p.251-251, Article 251
• Main Authors: Gampala, Silpa, Shah, Fenil, Lu, Xiaoyu, Moon, Hye-Ran, Babb, Olivia, Umesh Ganesh, Nikkitha, Sandusky, George, Hulsey, Emily, Armstrong, Lee, Mosely, Amber L, Han, Bumsoo, Ivan, Mircea, Yeh, Jing-Ruey Joanna, Kelley, Mark R, Zhang, Chi, Fishel, Melissa L
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Cancer; Cancer associated fibroblasts (CAFs); Cancer therapies; Dehydrogenases; DNA binding proteins; DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism; Fibroblasts; Gene expression; Genes; Genetic transcription; Genomes; Genomics; Health aspects; Humans; Hypoxia; Medical prognosis; Metabolism; Mice; Mitochondrial DNA; OXPHOS; Pancreatic cancer; Pancreatic Neoplasms - genetics; Pancreatic Neoplasms - pathology; Peptides; Physiological aspects; Prevention; Protein kinases; Proteins; Proteomics; Redox function; Ref-1; RNA; RNA sequencing; scRNA-seq; Transfection
• ISSN: 1756-9966; 0392-9078; 1756-9966
• DOI: 10.1186/s13046-021-02046-x

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 conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading 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. 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 identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. 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. 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 Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. 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.