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

Journal of Experimental & Clinical Cancer Research

Jing-Ruey Joanna Yeh¹  Nikkitha Umesh Ganesh¹  Xiaoyu Lu²  Chi Zhang³  Emily Hulsey⁴  George Sandusky⁵  Olivia Babb⁶  Lee Armstrong⁶  Silpa Gampala⁶  Fenil Shah⁶  Mark R. Kelley⁷  Melissa L. Fishel⁸  Amber L. Mosely⁹  Mircea Ivan^1,10  Hye-ran Moon^1,11  Bumsoo Han^1,12 

[1] Cardiovascular Research Center, Massachusetts General Hospital, 02115, Charlestown, MA, USA;Department of Medicine, Harvard Medical School, 02115, Boston, MA, USA;Department of Medical and Molecular Genetics, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Department of Biohealth Informatics, IUPUI, 46202, Indianapolis, IN, USA;Department of Medical and Molecular Genetics, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Department of Biohealth Informatics, IUPUI, 46202, Indianapolis, IN, USA;Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, 1044 W Walnut St. R4-321, 46202, Indianapolis, IN, USA;Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, 1044 W Walnut St. R4-321, 46202, Indianapolis, IN, USA;Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, 1044 W Walnut St. R4-321, 46202, Indianapolis, IN, USA;Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Department of Pharmacology and Toxicology, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, 1044 W Walnut St. R4-321, 46202, Indianapolis, IN, USA;Department of Pharmacology and Toxicology, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, 1044 W Walnut St. R4-321, 46202, Indianapolis, IN, USA;Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;Indiana University Simon Comprehensive Cancer Center, Indiana University School of Medicine, 1044 W Walnut St. R4-321, 46202, Indianapolis, IN, USA;Department of Microbiology and Immunology, Indiana University School of Medicine, 46202, Indianapolis, IN, USA;School of Mechanical Engineering, Purdue University, 47906, West Lafayette, IN, USA;School of Mechanical Engineering, Purdue University, 47906, West Lafayette, IN, USA;Purdue Center for Cancer Research, Purdue University, 47906, West Lafayette, IN, USA;

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

DOI  :  10.1186/s13046-021-02046-x

来源: Springer

【 摘 要 】

BackgroundPancreatic 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.MethodsscRNA-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.ResultsDistinct 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.ConclusionRef-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.

【 授权许可】

CC BY