FBW7 increases the chemosensitivity of pancreatic cancer cells to gemcitabine through upregulation of ENT1 - PubMed (original) (raw)

. 2017 Oct;38(4):2069-2077.

doi: 10.3892/or.2017.5856. Epub 2017 Jul 28.

Yi Qin 1, Bo Zhang 1, Chen Liang 1, Shunrong Ji 1, Si Shi 1, Wenyan Xu 1, Jinfeng Xiang 1, Dingkong Liang 1, Quanxing Ni 1, Xianjun Yu 1, Jin Xu 1

Affiliations

FBW7 increases the chemosensitivity of pancreatic cancer cells to gemcitabine through upregulation of ENT1

Qiangsheng Hu et al. Oncol Rep. 2017 Oct.

Abstract

F-box and WD repeat domain-containing 7 (FBW7) has been characterized as a tumor suppressor, and its mutation or decreased expression has been observed in many types of human cancers. Our recent studies have uncovered that in pancreatic cancer, the KRAS mutation decreased FBW7 expression through phosphorylation and subsequent ubiquitination. Moreover, FBW7 inhibited aerobic glycolysis in pancreatic cancer via induction of thioredoxin-interacting protein (TXNIP), a mitochondrial localized tumor suppressor. The roles of FBW7 in anti-apoptosis and drug resistance via proteosomal degradation of myeloid cell leukemia-1 (MCL-1), which is an anti-apoptotic factor have been reported. However, the role of FBW7 in the chemotherapeutic resistance of pancreatic cancer to gemcitabine has seldom been reported. In the present study, we demonstrated that overexpression of FBW7 in pancreatic cancer cells rendered increased sensitivity to gemcitabine. Mechanistically, FBW7 promoted gemcitabine sensitivity via upregulation of equilibrative nucleoside transporter 1 (ENT1) at the protein level rather than the transcriptional level. In depth analysis demonstrated that the ENT1 protein level could be increased by lysosome inhibition. Taken together, our results demonstrated that FBW7 could be a target for improving the therapeutic efficacy of gemcitabine by induction of ENT1.

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Figures

Figure 1.

Figure 1.

FBW7 increases the sensitivity of pancreatic cancer cells to gemcitabine. (A) FBW7 was introduced into PANC-1 and Mia PaCa-2 cells by lentiviral-mediated transfection, and the overexpression efficacy was assessed by detection with FLAG antibodies. (B) PANC-1 and Mia PaCa-2 cells were treated with increasing concentrations of gemcitabine (GEM) (0.01–100 µM) for 72 h. The cell viability ratios were measured by CCK-8 assays. In PANC-1 cells, FBW7 decreased the IC50 value from 10 to 2 µM, and in Mia PaCa-2 cells, the IC50 value changed from 2 to 0.5 µM. (C and D) Gemcitabine increased the cell apoptosis in the FBW7-overexpressing PANC-1 and Mia PaCa-2 cells as reflected by analysis of apoptosis. (E) The protein level of cleaved PARP1 was significantly increased in the gemcitabine-treated, FBW7-overexpressing PANC-1 and Mia PaCa-2 cells, indicating that FBW7 renders pancreatic cancer cells sensitive to gemcitabine. GEM, gemcitabine.

Figure 2.

Figure 2.

FBW7 regulates the ENT1 protein level. (A) Overexpression of the FBW7 substrate, anti-apoptotic factor MCL-1, is commonly regarded as a factor that regulates drug resistance. However, in the FBW7-overexpressing PANC-1 and Mia PaCa-2 cells, we detected no significant change in MCL-1 level, indicating that other molecular mechanisms underlie gemcitabine resistance. (B) Schematic representation of gemcitabine activation. ENT1 is responsible for gemcitabine transport, dCK catalyzes the phosphorylation of gemcitabine for subsequent activation, and RRM1 is inhibited by activated gemcitabine. ENT1, dCK and RRM1 are indicators of gemcitabine efficacy in pancreatic cancer. (C) FBW7 did not participate in the regulation of ENT1, dCK and RRM1 at the transcriptional level. (D) FBW7 increased the protein levels of ENT1 in PANC-1 and Mia PaCa-2 cells, while it had slight influence on the RRM1 protein level. dCK was not detected in the present study. GEM, gemcitabine.

Figure 3.

Figure 3.

FBW7 regulates ENT1 membrane distribution. (A) Membrane-bound ENT1 is responsible for efficient gemcitabine uptake. In FBW7-overexpressing PANC-1 and Mia PaCa-2 cells, we detected an increase in the membrane level of ENT1, indicating a positive role of FBW7 in the ENT1 protein level and membrane distribution regulation. (B) Immunofluorescence studies demonstrated that in FBW7-overexpressing PANC-1 and Mia PaCa-2 cells, the membrane localized ENT1 was higher than that in the control empty vector (EV)-transfected cells. (C) IHC staining in pancreatic cancer patient tissues demonstrated that patients with higher FBW7 displayed higher ENT1 expression, further validating the positive correlation between FBW7 and ENT1. (D) Proteosomal inhibitor MG132 did not increase the protein level of ENT1. (E) Pancreatic cancer cells PANC-1 and Mia PaCa-2 were treated with chloroquinone (CQ), a lysosome inhibitor. In addition, upon CQ treatment, the protein level of ENT1 was increased significantly. (F) CQ treatment increased gemcitabine sensitivity in PANC-1 and Mia PaCa-2 cells. (G) CQ treatment increased the level of cleaved PARP1, which also supported the hypothesis that gemcitabine resistance may involve the lysosome degradation of ENT1. GEM, gemcitabine.

Figure 4.

Figure 4.

FBW7 upstream regulator ERK regulates ENT1 abundance. (A) PANC-1 and Mia PaCa-2 cells were treated with UO126, which regulated FBW7 protein stability in pancreatic cancer. In addition, upon UO126 treatment, the protein level of ENT1 was increased significantly. (B) Immunofluorescence studies demonstrated that UO126 treatment not only increased total ENT1 level, but also increased cell membrane distribution of ENT1 in PANC-1 and Mia PaCa-2 cells. (C) Membrane purification and western blotting further indicated that UO126 treatment could increase membrane levels of ENT1 in PANC-1 and Mia PaCa-2 cells.

Figure 5.

Figure 5.

Illustration of the working model. FBW7 overexpression or induction by ERK inhibition increased gemcitabine sensitivity by induction of gemcitabine transporter ENT1. Further studies demonstrated that the protein level of ENT1 was regulated by lysosomes, and inhibition of lysosomes could increase the ENT1 level. Therefore, the FBW7/ENT1 axis may function as a novel therapeutic target in the treatment of pancreatic cancer. GEM, gemcitabine.

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