Therapeutic Opportunities in Targeting the Pentose Phosphate Pathway in Colorectal Cancer

Abstract

Introduction: Colorectal cancer (CRC) is the third most common neoplasia and the second cause of cancer-related deaths worldwide. Unlike normal cells, tumor cells deregulate their metabolism and rely on aerobic glycolysis (Warburg effect). CRC cells upregulate the pentose phosphate pathway (PPP), and p53 is a crucial regulator. The PPP is a significant route for glucose catabolism and is required for DNA synthesis of rapidly-proliferating cells. Its oxidative phase, catalyzed by the rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD), provides the cell with nicotinamide adenine dinucleotide phosphate (NADPH) that has biosynthetic and detoxifying functions, particularly against the generation of reactive oxygen species (ROS). 5-Fluorouracil (5-FU) is the treatment of choice in CRC. However, 5-FU exhibits high toxicity and drug resistance. Therefore, we hypothesized that targeting the PPP might offer novel therapeutic opportunities in CRC and improve the response to 5-FU. We aimed to investigate and characterize the anti-tumor effect of G6PD inhibitors (6-aminonicotinamide (6-AN), dehydroepiandrosterone (DHEA), and polydatin) alone, or in combination with 5-FU on the PPP in CRC cells. Methods: Assess the effect of G6PD inhibitors and 5-FU alone or in combination using cell viability assays (MTT, SRB) on CRC cell lines of different p53 and 5-FU resistance status. Synergistic effects are estimated using Compusyn software. Determine the mechanism of action of the combination treatment on cell cycle and cell death using propidium iodide, and immunoblotting assays. PPP-related enzymes’ (G6PD, transketolase) product levels and/or activities are measured. ROS levels and glutathione peroxidase (GPx) activity are evaluated. Results: In silico analysis revealed elevated G6PD mRNA in CRC versus normal tissue. The tested G6PD inhibitors reduced CRC cell viability. 6-AN synergized with 5-FU treatment in HCT116 cells. Furthermore, 6-AN sensitized HCT116-5-FU resistant and HCT116 p53-/- cells to 5-FU treatment. The 6-AN/5-FU combination treatment induced cell cycle arrest in different phases and accumulation of treated HCT116 and HCT116 p53-/- cells in the sub-G1 phase. The 6-AN/5-FU combination reduced the activity of G6PD compared to single treatments while G6PD and transketolase levels remain unaltered. ROS levels and GPx activity increased upon treatment. CRISPR/Cas9 G6PD knockout HCT116 cells are being developed. Conclusion: These findings reveal that combining G6PD inhibitors with 5-FU decrease resistance and further sensitize CRC cells to 5-FU treatment independently of p53 and 5-FU drug resistance status. Exploiting the metabolic vulnerability of cancer offer a novel clinical approach in colorectal cancer management.