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 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.

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 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.

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 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.

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.

• Authors: Silpa Gampala, Fenil Shah, Xiaoyu Lu, Hye-Ran Moon, Olivia Babb, Nikkitha Umesh Ganesh, George Sandusky, Emily Hulsey, Lee Armstrong, Amber L Mosely, Bumsoo Han, Mircea Ivan, Jing-Ruey Joanna Yeh, Mark R Kelley, Chi Zhang, Melissa L Fishel
• Journal: Journal of experimental & clinical cancer research : CR (J Exp Clin Cancer Res) Vol. 40 Issue 1 Pg. 251 (Aug 10 2021) ISSN: 1756-9966 [Electronic] England
• PMID: 34376225 (Publication Type: Journal Article)
• Copyright: © 2021. The Author(s).

Chemical References

• APEX1 protein, human
• DNA-(Apurinic or Apyrimidinic Site) Lyase

Topics

• Animals
• DNA-(Apurinic or Apyrimidinic Site) Lyase (metabolism)
• Humans
• Mice
• Pancreatic Neoplasms (genetics, pathology)
• Transfection