GFPT2 expression is induced by gemcitabine administration and enhances invasion by activating the hexosamine biosynthetic pathway in pancreatic cancer

Abstract

Our previous studies revealed a novel link between gemcitabine (GEM) chemotherapy and elevated glutamine-fructose-6-phosphate transaminase 2 (GFPT2) expression in pancreatic cancer (PaCa) cells. GFPT2 is a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP). HBP can enhance metastatic potential by regulating epithelial-mesenchymal transition (EMT). The aim of this study was to further evaluate the effect of chemotherapy-induced GFPT2 expression on metastatic potential. GFPT2 expression was evaluated in a mouse xenograft model following GEM exposure and in clinical specimens of patients after chemotherapy using immunohistochemical analysis. The roles of GFPT2 in HBP activation, downstream pathways, and cellular functions in PaCa cells with regulated GFPT2 expression were investigated. GEM exposure increased GFPT2 expression in tumors resected from a mouse xenograft model and in patients treated with neoadjuvant chemotherapy (NAC). GFPT2 expression was correlated with post-operative liver metastasis after NAC. Its expression activated the HBP, promoting migration and invasion. Treatment with HBP inhibitors reversed these effects. Additionally, GFPT2 upregulated ZEB1 and vimentin expression and downregulated E-cadherin expression. GEM induction upregulated GFPT2 expression. Elevated GFPT2 levels promoted invasion by activating the HBP, suggesting the potential role of this mechanism in promoting chemotherapy-induced metastasis.

Keywords: Cancer; Chemotherapy-induced metastasis; Glutamine-fructose-6-phosphate transaminase 2; Hexosamine biosynthesis pathway; Neoadjuvant chemotherapy.

Fig. 1

GFPT2 expression in PaCa cells. a GFPT2 expression in PANC-1, SUIT2, BxPC3, and PK1 cells was evaluated via RT-qPCR. The relative expression level was calculated after normalization to the level of β-actin. b and c GFPT2 was overexpressed in PANC-1 cells. b RT-qPCR and c western blotting. (d and e) GFPT2 expression was suppressed by shRNA transfection in SUIT2 cells. d RT-qPCR and e western blotting. An empty vector was used as the control. β-Actin was used as an internal control. RT‑qPCR and western blotting were performed in a biological triplicate. GFPT2 glutamine-fructose-6-phosphate transaminase 2; PaCa pancreatic cancer; RT-qPCR reverse transcription-quantitative polymerase chain reaction; sh, short hairpin

Fig. 2

O-GlcNAc protein expression in PaCa cells a O-GlcNAc protein expression in GFPT2-suppressed SUIT2 cells. b O-GlcNAc protein expression in GFPT2-overexpressing PANC1 cells. β-Actin was used as an internal control. The data are expressed as the mean ± SD. All experiments were performed in a biological triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001. cont control; GFPT2 glutamine-fructose-6-phosphate transaminase 2; O-Glc-Nac O-N-acetylglucosamine; sh, short hairpin

Fig. 3

Migration of and invasion by PaCa cells. a, b Migration was assessed using a scratch assay. a Migrated cancer cells and the relative migration rate of GFPT2-suppressed SUIT2 cells. b Migrated cancer cells and the relative migration rate of GFPT2-overexpressing PANC-1 cells. Scale bar, 20 μm. c–e Transwell assays were performed to examine the association between cancer-cell invasion and GFPT2 expression. Invading cells were stained with lysis buffer/dye solution and quantified using a fluorescence plate reader with a 480/520 nm filter. c GFPT2-suppressed SUIT2 cells. d GFPT2-OE PANC-1 cells. e GFPT2-OE cells were treated with the HBP inhibitor, DON. The data are expressed as the mean ± SD. All experiments were performed in a biological triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001. GFPT2 glutamine-fructose-6-phosphate transaminase 2; cont, control; DON 6-diazo-5-oxo-L-norleucine; GFPT2 glutamine-fructose-6-phosphate transaminase 2; HBP hexosamine biosynthetic pathway; OE overexpression; sh short hairpin

Fig. 4

Proliferation and chemosensitivity of pancreatic cancer cells a, b Pancreatic cancer cell proliferation. a Proliferation curve for GFPT2-suppressed SUIT2 cells. b Proliferation curve for GFPT2-overexpressing PANC-1 cells. c, d Sensitivity of pancreatic cancer cells to gemcitabine. c IC₅₀ did not differ among the cont, sh1, and sh2 groups for GFPT2-suppressed SUIT2 cells or d between the control and OE groups for GFPT2-overexpressing PANC-1 cells. All experiments were performed in biological triplicate. Cont control; GFPT2 glutamine-fructose-6-phosphate transaminase 2; OE overexpression; sh short hairpin

Fig. 5

Expression of EMT-related proteins in PANC-1-OE cells. ZEB1 and vimentin expression was significantly elevated, while E-cadherin expression was significantly reduced. β-Actin was used as an internal control. The data are expressed as the mean ± SD. All experiments were performed in biological triplicate. *P < 0.05, **P < 0.01 and ***P < 0.001. GFPT2 glutamine-fructose-6-phosphate transaminase 2; OE overexpression

Fig. 6

GFPT expression after gemcitabine administration in a mouse xenograft model. GFPT2 expression was significantly greater in the high-dose gemcitabine-treated group than in the control group. GFPT1 expression was not significantly altered after gemcitabine treatment. β-Actin was used as an internal control. Data are expressed as the mean ± SD. All experiments were performed in biological triplicate. *P < 0.05. GFPT1, glutamine-fructose-6-phosphate transaminase 1; GFPT2 glutamine-fructose-6-phosphate transaminase 2; GEM gemcitabine

Fig. 7

Comparison of GFPT2 expression between patients receiving NAC and upfront surgery. GFPT2 expression was significantly higher in patients undergoing NAC (P = 0.022). GFPT2 glutamine-fructose-6-phosphate transaminase 2; NAC neoadjuvant chemotherapy; IRS immunoreactive score

Fig. 8

Kaplan–Meier curves of liver recurrence-free survival. Patients with high (n = 30) or low GFPT2 (n = 32) expression are represented by dots and lines, respectively. Liver recurrence: cancer recurrence occurring first in the liver following pancreatic cancer resection