Fig 2: LAT2 is upregulated in pancreatic cancer. a The expression level of LAT2 in 71 pancreatic cancer tissue samples was higher than that in the matched paracancerous tissues (*, P < 0.05). b The Wilcoxon signed rank test was applied to compare the difference in LAT2 expression between the cancer and paracancerous tissues. c The expression of LAT2 in pancreatic cancer tissues was evaluated by immunohistochemistry. d The expression of LAT2 in pancreatic paracancerous tissues was evaluated by immunohistochemistry. e Kaplan-Meier survival analysis indicated that the overall survival time of the high-LAT2-level group is shorter than that of the low-LAT2-level group of patients with pancreatic cancer (P = 0.0154)

Fig 3: LAT2 binds to p-mTORSer2448 to upregulate glutamine synthetase and increase the intracellular level of glutamine in pancreatic cancer cells. a The Western blotting results showed that RAD001 could downregulate glutamine synthetase in MIA PaCa-2 and PANC-1 cells. b LAT2 knockout could inactivate mTOR and decrease the interaction between p-mTORSer2448 and glutamine synthetase, resulting in a reduced level of glutamine synthetase expression. c LAT2 knockout downregulated p-mTORSer2448 and glutamine synthetase in PANC-1 cells; both p-mTORSer2448 and glutamine synthetase were localized to the cytoplasm

Fig 4: LAT2 activates glycolysis in pancreatic cancer cells and alters glutamine metabolism to activate mTOR in vitro and in vivo. a, b LAT2 knockdown significantly decreased ECAR and OCR in MIA PaCa-2 and PANC-1 cells and promoted the transformation of cell metabolic phenotype from an energetic to a quiescent phenotype; LAT2 overexpression significantly increased ECAR and OCR in MIA PaCa-2 and PANC-1 cells and promoted the transformation of cell metabolic phenotype from a quiescent to an energetic phenotype (P < 0.05). c LAT2 knockdown significantly decreased glycoPER in MIA PaCa-2 and PANC-1 cells; LAT2 overexpression significantly increased glycoPER in MIA PaCa-2 and PANC-1 cells (P < 0.05). d LAT2 overexpression significantly increased the intracellular level of glutamine in PANC-1 cells. e The expression level of mTOR pathway molecules and key enzymes involved in glycolysis and glutamine metabolism was assessed, and the results revealed that LAT2 upregulated LDHB, PKM2, glutamine synthetase and p-mTOR and downregulated glutaminase. Otherwise, LAT2 did not regulate the expression of LDHA. The data are presented as the mean ± SD. (Student’s t-test; **, P < 0.01)

Fig 5: LAT2 activates mTOR to regulate apoptosis, glycolysis and gemcitabine sensitivity in pancreatic cancer cells. a, b LAT2 suppressed apoptosis in MIA PaCa-2 cells with LAT2 overexpression, which could be reversed by RAD001; gemcitabine induced apoptosis in MIA PaCa-2 cells, and RAD001 reversed the inhibition of apoptosis induced by gemcitabine in MIA PaCa-2 cells with LAT2 overexpression. c RAD001 increased gemcitabine sensitivity in MIA PaCa-2 cells with LAT2 overexpression. d-g Both ECAR and glycoPER were increased in MIA PaCa-2 and PANC-1 cells with LAT2 overexpression compared with the control cells, and this increase could be reversed by RAD001. h RAD001 downregulated the expression of LDHB and glutamine synthetase in MIA PaCa-2 and PANC-1 cells with LAT2 overexpression; RAD001 downregulated LAT2 expression in PANC-1 cells with LAT2 overexpression. i LAT2 knockout upregulated glutaminase expression, inhibited the mTOR pathway (thereby downregulating p-4EBP1) and downregulated AMPKa1/2 expression; and gemcitabine suppressed the mTOR pathway in PANC-1 cells dependent on LAT2. The data are presented as the mean ± SD. (Student’s t-test; *, P < 0.05)