Transient silencing of GLUT1, LDHA, and MCT4 affects glucose transport, glycolysis, and chemosensitivity in human pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC), often referred to as pancreatic cancer is one of the most aggressive and deadliest cancer types, with a 5-year survival rate of less than 10%. PDAC is characterized by vague, non-specific symptoms, late detection, early metastasis, a high degree of heterogeneity, significant resistance to existing treatments, and a poor prognosis. A reprogrammed glucose metabolism is one of the hallmarks of pancreatic cancer. Pancreatic cancer cells (PCCs) take up large amounts of glucose and convert it into lactate independent of oxygen availability, a process called the Warburg effect, which leads to higher glucose needs. Thus, several metabolic proteins such as GLUT1, LDHA, and MCT4, responsible for glucose transport, lactate production, and lactate transport, respectively, are often overexpressed in PDAC. Evidence suggests the possibility of using GLUT1, LDHA, and MCT4 as prognostic markers and therapeutic targets for PDAC. However, the contribution of these glycometabolic proteins to the chemosensitivity of PDAC remains unknown. The aim of this study is to investigate the impact of GLUT1, LDHA, and MCT4 expression on glycolysis and chemosensitivity in human PCCs. In this study, the expression of GLUT1, LDHA, or MCT4 was silenced for a short duration using siRNA-based transfections, and the impact of this on PCC glucose transport, glycolysis, viability, proliferation, and chemosensitivity was investigated using three different PCC lines (BxPC-3, Mia PaCa-2, Panc-1). Protein expression was determined by immunocytochemistry and western blot analysis. Phenotypic changes were determined by investigating MTT-based cell viability and proliferation by BrdU incorporation. Changes in glycolytic activity were determined using [3H]-2-deoxy-D-glucose to measure glucose transport and the Glycolysis Cell-Based Assay Kit to measure lactate secretion. Lastly, chemosensitivity for gemcitabine and 5-fluorouracil (5-FU), reflected by drug-induced reduction in cell viability, was determined by MTT assay. Expression analysis by immunocytochemistry and western blot analysis confirmed the successful silencing of GLUT1, LDHA, or MCT4, compared to the non-targeting control (NTC) in all three PCC lines. Discrete and mainly nonsignificant differences in viability and proliferation were observed when comparing the cells with suppressed GLUT1, LDHA, and MCT4 expression compared to NTC-transfected cells. Both BxPC-3 and Mia PaCa-2 showed significantly higher reductions in glucose transport following GLUT1 silencing compared to NTC. GLUT1 silencing resulted in a significant decrease in lactate secretion in all three PCC lines. Compared to the other PCCs, Panc-1 showed higher lactate secretion upon silencing of LDHA or MCT4. When comparing the PCCs with silenced glycometabolic proteins to the NTC controls, treatment with Gemcitabine resulted in a small increase in cytotoxic effect in Mia PaCa-2 and Panc-1 with silenced LDHA or MCT4 when grown in normal glucose (NG) conditions, whereas all three cell lines remained resistant to Gemcitabine treatment when grown in low glucose (LG) conditions. Treatment with 5-FU had little to no impact on the viability of Mia PaCa-2 and Panc-1, both in LG and NG conditions, suggesting low 5-FU efficacy in these cell lines. In contrast, in BxPC-3, treatment with 5-FU resulted in a significant decrease in viability, both in LG and NG conditions. Furthermore, an increase in 5-FU sensitivity was observed in the cells with suppressed expression of LDHA or MCT4 as compared to NTC transfected cells both in LG and NG conditions. In conclusion, a general trend of reduced glucose transport, lactate secretion, and unaffected or increased chemosensitivity was observed in PCCs with silenced GLUT1, LDHA, or MCT4 compared to PCCs transfected with NTC. However, it is important to note that the changes observed varied remarkably between the PCCs. Further investigation is needed to understand the underlying mechanisms that link chemoresistance with the changes induced by the silencing of GLUT1, LDHA, or MCT4