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.

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

Fig. 1

Integration of scRNA seq and proteomics following Ref-1 downregulation under hypoxia. TSNE plot of the scRNA-seq data colored by experimental conditions (A) and inferred cell clusters from unsupervised cell clustering (B). C Heatmap of the top 250 genes with largest dispersion in the scRNA-seq data. High and low expression are colored by yellow and purple, respectively. The column color code represents the experimental condition of each cell annotated in (A). D The top pathways in scRNA-seq data enriched by the down- (blue) and up- (red) regulated genes in Ref-1 KD vs Scr control under hypoxia. The x-axis is -log(p.value) assessed by hypergeometric test. E Heatmap of the proteomics data with the same color code as in (C). F The top pathways observed in the proteomics data enriched by the down- (blue) and up- (red) regulated proteins in siRef-1 vs Scr control under hypoxia. G Gene expression profile of selected genes with significant differential expression through the cell clusters. The color code is the same as the cell clusters annotated in (B)

Fig. 2

Blockade of Ref-1 under hypoxia causes downregulation of metabolic pathways. Differentially expressed central metabolic genes in siRef-1 vs Scr Control under hypoxia (A) and normoxia (B) conditions. The up- and down- regulated genes were colored by light (0.001 < p < 0.05) or dark (p < 0.001) red and green, respectively. Differentially expressed mitochondrial complex and ATP synthase genes in siRef-1 vs Scr control under hypoxia (C) and normoxia (D). E Gene expression profile of key genes involved in the central metabolism through different cell groups. The color code is the same as Fig. 1G

Fig. 3

Ref-1 inhibition downregulates mitochondrial complex genes as well as Ref-1 PD marker genes. A-C Validation of selected mitochondrial complex genes from the scRNA-seq data using qRT-PCR in Pa03C (n = 3), Pa02C (n = 3), and Panc10.05 (n = 2) cells (Scr/siRef-1 – 30 nM, 1% hypoxia for 24 h, p < 0.05–0.0001). D Western Blots representing downregulation of mitochondrial metabolic proteins with Ref-1 knockdown 72 h post transfection. E–G Expression of mitochondrial complex genes after treatment with Ref-1 redox inhibitor (APX2009-10 µM for Pa03C, 15 µM for Pa02C and 20 µM for Panc10.05 cells for 28 h) under normoxia and hypoxia (1%O₂ for 24 h) (n = 2, p < 0.05–0.0001). H Mitochondrial complex gene expression following Ref-1 redox inhibition (APX2009-10 µM 28 h) under normoxia and hypoxia (1%O₂ for 24 h) in CAFs (n = 3). I Mitochondrial complex gene panel after treatment with Ref-1 redox inhibitor (APX2009, 5 µM) compared to vehicle control (DMSO) in Pa03C 3D spheroids (n = 3, p < 0.05–0.0001) and J Images representing the spheroids of Pa03C cells. (Scale Bar – 100 µm, 10X mag). Relative fold change refers to the gene expression changes when compared to Scr or vehicle-treated cells under normoxia

Fig. 4

Ref-1 genetic or pharmacological inhibition reduces TCA cycle substrates. Mitochondrial functional assays in Pa03C cells transfected with Scr vs 10 nM siRef-1 (An = 3, *p < 0.05, ^##0.0001) and a representative image of the plate. Avg rate of reaction refers to slope of absorbance at 590 vs time. Western blot image of the Pa03C cells after transfection with Ref-1 or Scr siRNA (B). Vinculin is used as the loading control. Average rate of reaction in Pa03C cells treated with Ref-1 redox inhibitor (APX2009, n = 3, *p < 0.05, ^##0.0001) or inactive Ref-1 redox inhibitor analog (RN7-58, n = 2) (C) and their representative plate images (D) for 24 h. E Average rate of reaction in CAF02 cells treated with 5 µM APX2009 for 24 h (n = 3). F Fold change of the ratio of NADPH/NADP + in Pa03C cells treated with APX2009 (20 µM) or RN7-58 (20 µM) (n > 2, ^##p < 0.0001). G Boxplots show estimated flux of NADP + consuming reactions in one metabolic module using scRNA seq data from Scr vs siRef-1. The two metabolite names on top of each plot are the input and output of each metabolic module. H Measurement of ATP levels by CellTiter-Glo Luminescent Cell Viability Assay in OXPHOS proficient cells (143B WT) or treated with 300 μM phenformin and OXPHOS deficient cells (143B CytB) after treatment with Ref-1 inhibitor APX2009 at the indicated concentrations for 24 h (**p < 0.01, ^##0.0001). All data represent Mean ± SE

Fig. 5

Ref-1 inhibition with APX2009 results in diminished tumor growth. Tumor growth of Pa03C (A) or Panc10.05 + CAF19 (D) subcutaneous xenografts treated with PKT Vehicle or 35 mg/Kg APX2009 or 50 mg/Kg Devimistat (^##p < 0.0001, compared to vehicle control) twice a day, 8 h apart, continuously for 15 (for Pa03C xenografts) or 20 (for Panc10.05 + CAF19 xenografts) days. And corresponding tumor weights (B, E *p < 0.05, compared to vehicle control). C&F Graphs representing body weights over time for Pa03C or Panc10.05 + CAF19 xenografts. Representative images for H&E and IHC staining for vimentin positivity for Pa03C tumors and Panc10.05 + CAF19 (G). All data are represented as Mean ± SE

Fig. 6

Ref-1 inhibition in combination with Devimistat attenuates growth in two co-culture models of pancreatic cancer: 3D spheroids and i-TMOC. Representative pictures of two low passage patient-derived low passage cell lines, Pa03C (A) and Panc10.05 (D) plated as 3D co-cultures with CAF19 cells at a ratio of 1:4. These co-cultures were treated with increasing concentrations of Devimistat (0–50 µM) and in combination with APX2009 following intensity measurements on Days 4, 7, 10, and 14. For combination treatment in Pa03C cells, Devimistat was held constant at 25 µM and APX2009 at 5 µM, and in Panc10.05, Devimistat was held constant at 50 µM and APX2009 at 10 µM (*p < 0.05, ^##0.0001). Intensity of the tumor cells (red (B,E)) as well as the CAFs (green (C,F)) are represented as fluorescence intensity data normalized to Day 14 media control. Graphs are means with standard error of n = 3–4 and arrows correspond to treatment times. G Schematic of functional structure of PDAC iT-MOC and experimental timeline in (H). I Fluorescent microscopic observation of Panc10.05 (red) and CAF19 (green) in PDAC iT-MOC on Day 2 and Day 9. J Quantitation of cell survival in the iT-MOC system with single agent (APX2009 – 30 µM / Devimistat – 25 µM) and combination treatment (n ≥ 3, Mean ± S.E. *p < 0.05)

Fig. 7

Ref-1 inhibition in combination with Devimistat downregulates gene expression of the mitochondrial complex genes as well as mitochondrial function. Expression of mitochondrial genes (A) via qPCR in Pa03C 3D spheroids treated with DMSO or a combination of APX2009 (5 µM) and Devimistat (50 µM) (n = 3, p < 0.05–0.0001). The data with APX2009 is also in Fig. 3I and provided here for comparison to combination. B Mitochondrial functional assay in Pa03C with DMSO or a combination of APX2009 (5 & 10 µM, 24 h) and Deviminstat (50 µM, 24 h) (n = 3, p < 0.01, 0.0001) and the representative image of the plate following the reaction (C). Data represent Mean ± SE; Uppercase letters denote statistical significance of combination treatment to APX2009 (5 µM) and lowercase letters denote statistical significance of combination treatment to Devimistat (50 µM). D H&E staining of spheroids from Pa03C alone or cultures with CAF19 cells treated with APX2009 (5 µM) or Devimistat (50 µM) or a combination in comparison to DMSO control