Glutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells

Abstract

BACKGROUND Cellular metabolism regulates stemness in health and disease. A reduced redox state is essential for self-renewal of normal and cancer stem cells (CSCs). However, while stem cells rely on glycolysis, different CSCs, including pancreatic CSCs, favor mitochondrial metabolism as their dominant energy-producing pathway. This suggests that powerful antioxidant networks must be in place to detoxify mitochondrial reactive oxygen species (ROS) and maintain stemness in oxidative CSCs. Since glutathione metabolism is critical for normal stem cell function and CSCs from breast, liver and gastric cancer show increased glutathione content, we hypothesized that pancreatic CSCs also rely on this pathway for ROS detoxification. AIM To investigate the role of glutathione metabolism in pancreatic CSCs. METHODS Primary pancreatic cancer cells of patient-derived xenografts (PDXs) were cultured in adherent or CSC-enriching sphere conditions to determine the role of glutathione metabolism in stemness. Real-time polymerase chain reaction (PCR) was used to validate RNAseq results involving glutathione metabolism genes in adherent vs spheres, as well as the expression of pluripotency-related genes following treatment. Public TCGA and GTEx RNAseq data from pancreatic cancer vs normal tissue samples were analyzed using the webserver GEPIA2. The glutathione-sensitive fluorescent probe monochlorobimane was used to determine glutathione content by fluorimetry or flow cytometry. Pharmacological inhibitors of glutathione synthesis and recycling [buthionine-sulfoximine (BSO) and 6-Aminonicotinamide (6-AN), respectively] were used to investigate the impact of glutathione depletion on CSC-enriched cultures. Staining with propidium iodide (cell cycle), Annexin-V (apoptosis) and CD133 (CSC content) were determined by flow cytometry. Self-renewal was assessed by sphere formation assay and response to gemcitabine treatment was used as a readout for chemoresistance. RESULTS Analysis of our previously published RNAseq dataset E-MTAB-3808 revealed up-regulation of genes involved in the KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Glutathione Metabolism in CSC-enriched cultures compared to their differentiated counterparts. Consistently, in pancreatic cancer patient samples the expression of most of these up-regulated genes positively correlated with a stemness signature defined by NANOG , KLF4 , SOX2 and OCT4 expression ( P < 10 -5 ). Moreover, 3 of the upregulated genes ( MGST1, GPX8, GCCT ) were associated with reduced disease-free survival in patients [Hazard ratio (HR) 2.2-2.5; P = 0.03-0.0054], suggesting a critical role for this pathway in pancreatic cancer p...

Inhibition of Mitochondrial Dynamics Preferentially Targets Pancreatic Cancer Cells with Enhanced Tumorigenic and Invasive Potential

“…Importantly, DRP1 genetic loss in PDAC impaired tumor growth and showed analogous features in terms of mitochondrial dysfunction [17], validating our experimental approach using the pharmacological inhibitor mDivi-1. As we have previously reported for the inhibition of mitochondrial activity by metformin [9,10], ROS accumulation and inhibition of respiration have different consequences for PDAC cells depending on their differentiation state: proliferation blockade in bulk tumor cells, and energy crisis and cell death in CSCs, as most PDAC CSCs are unable to switch to glycolysis in order to maintain their energy levels and their increased sensitivity to oxidative stress ( Figure S4) [9,11]. Importantly, other studies also described loss of mitochondrial respiration and cell death in brain and breast CSCs upon mDivi-1 treatment [28,36], suggesting that DRP1-mediated fission supports stemness in different tumor types.…”

“…In these conditions, cells enhance nutrient oxidation at maximal respiratory rate while mitochondrial fission favors uncoupled respiration (decreased ATP synthesis efficiency) in order to avoid ROS overproduction and prevent oxidative damage [33]. Since we have described that sustaining low mitochondrial ROS content is essential for maintaining self-renewal and full functionality of pancreatic CSCs [9,11], we hypothesize that a similar mechanism might be operative in oxidative pancreatic CSCs to prevent excessive ROS accumulation due to elevated proton leak as compared to their differentiated counterparts (unpublished data). Despite this apparent similarity between beta-cells and CSCs, metabolism of pancreatic beta-cells is clearly distinct and adapted to the regulation of insulin secretion: on the one hand, glucose sensing is controlled by supply-driven oxidative metabolism [34]; on the other hand, beta-cells downregulate antioxidant defenses to facilitate intracellular redox signaling, which is essential for insulin secretion [35].…”

Inhibition of mitochondrial dynamics preferentially targets pancreatic cancer cells with enhanced tumorigenic and invasive potential

“…Importantly, DRP1 genetic loss in PDAC impaired tumor growth and showed analogous features in terms of mitochondrial dysfunction [17], validating our experimental approach using the pharmacological inhibitor mDivi-1. As we have previously reported for the inhibition of mitochondrial activity by metformin [9,10], ROS accumulation and inhibition of respiration have different consequences for PDAC cells depending on their differentiation state: proliferation blockade in bulk tumor cells, and energy crisis and cell death in CSCs, as most PDAC CSCs are unable to switch to glycolysis in order to maintain their energy levels and their increased sensitivity to oxidative stress ( Figure S5) [9,11]. Importantly, other studies also described loss of mitochondrial respiration and cell death in brain and breast CSCs upon mDivi-1 treatment [28,35], suggesting that DRP1-mediated fission supports stemness in different tumor types.…”

“…In these conditions, cells enhance nutrient oxidation at maximal respiratory rate while mitochondrial fission favors uncoupled respiration (decreased ATP synthesis efficiency) in order to avoid ROS overproduction and prevent oxidative damage [34]. Since we have described that sustaining low mitochondrial ROS content, despite their elevated OXPHOS activity, is essential for self-renewal and full functionality of pancreatic CSCs [9,11], we hypothesized that a similar mechanism might be operative in oxidative pancreatic CSCs to prevent excessive ROS accumulation.…”

“…Essentially, pancreatic CSCs tightly control the expression balance between the glycolysis-promoting oncogene c-MYC and the mitochondrial biogenesis transcription factor PGC1-, favoring mitochondrial metabolism in order to maintain full stemness. In fact, we have demonstrated that perturbing mitochondrial function by either inhibition of the electron transport chain (ETC) with the antidiabetic agent metformin [9,10] or altering its redox state [9,11], significantly decreased pancreatic CSCs functionality and chemoresistance. Thus, our results identified mitochondrial activity as a key vulnerability for pancreatic CSCs.…”