4.7 Article

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

Journal

Publisher

BMC
DOI: 10.1186/s13046-021-02046-x

Keywords

scRNA-seq; Ref-1; Redox function; Metabolism; OXPHOS; Cancer associated fibroblasts (CAFs); Pancreatic cancer; Mitochondria

Categories

Funding

  1. IUSCC Cancer Center Support grant [P30 CA082709]
  2. Purdue Center for Cancer Research [P30 CA023168]
  3. National Cancer Institute [R01CA167291, R01CA167291-S1]
  4. NIH/NCI [R01CA205166, R01CA231267, R01HL140961, U01HL143403]
  5. CTSI grant [UL1 TR002529]
  6. NIGMS [1R01GM131399]
  7. Riley Children's Foundation
  8. NIH [R01 CA215410]
  9. National Institute of Health
  10. [R01CA211098]
  11. [NF180045]

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Inhibiting Ref-1 redox signaling alters cancer cell metabolism by causing TCA cycle dysfunction and reduces pancreatic tumor growth in vitro and in vivo.
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-1 alpha, STAT3 and NF kappa B 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.

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