Hexokinases II-mediated glycolysis governs susceptibility to crizotinib in ALK-positive non-small cell lung cancer

Abstract

Background: Activation of ALK leads to a high level of aerobic glycolysis related to crizotinib insensitivity in anaplastic lymphoma kinase-positive non-small cell lung cancer (ALK⁺ NSCLC). The strategy and mechanism of glycolysis inhibition in sensitizing ALK⁺ NSCLC cells to crizotinib requires further investigation.

Methods: The levels of glycolysis in H3122 and H2228 cells were evaluated through detection of glucose consumption and lactate production. MTT assay was used to explore the effects of glycolytic inhibitors on crizotinib sensitivity, and the potential mechanism of action were detected by colony formation, Ki67 incorporation assay, transwell assay, small interfering RNA technology and western blot analysis.

Results: ALK⁺ NSCLC cells exhibited significantly higher levels of glycolysis compared to ALK^- NSCLC cells. Long-term exposure to crizotinib could decrease the sensitivity of ALK⁺ NSCLC cells to crizotinib via increasing the levels of glycolysis related to hexokinases II (HK2). Crizotinib in combination with glycolysis inhibitor 2-deoxy-D-glucose (2DG) synergistically inhibited proliferation, glycolysis, colony formation and invasion ability of ALK⁺ NSCLC cells. 2DG sensitization crizotinib might be associated with the inhibition of HK2-mediated glycolysis and P-ALK/AKT/mTOR signaling pathway in H3122 and H2228 cells.

Conclusions: These results indicate that HK2-mediated glycolysis plays a crucial role in the increased tolerance of ALK⁺ NSCLC cells to crizotinib. 2DG may sensitize ALK⁺ NSCLC to crizotinib via suppression of HK2-mediated glycolysis and the AKT/mTOR signaling pathway.

Keywords: 2DG; aerobic glycolysis; anaplastic lymphoma kinase; crizotinib tolerance; hexokinases II.

FIGURE 1

Levels of glycolysis and expression of HK2 in ALK⁺ and ALK⁻ NSCLC cell lines. (a) and (b) Glucose consumption and lactate production were detected by glucose assay kit I and lactate assay kit I in both ALK⁻ NSCLC cell lines including H1299, H460, HCC827, PC‐9 and H1650, and ALK⁺ NSCLC cell lines including H3122 and H2228 (*, p < 0.05; &, p < 0.01, as compared to the value of glucose consumption and lactate production of H1299 cells). (c) Expression of P‐ALK/ALK and HK2 in a series of NSCLC cells were analyzed by western blot with indicated antibodies. (d) Relative expression of P‐ALK/ALK and HK2 was expressed as mean ± SD from three independent experiments (*,p < 0.05; &, p < 0.01, as compared to relative protein expression in H1299 cells)

FIGURE 2

Effects of long‐term exposure to crizotinib on drug sensitivity and levels of glycolysis in ALK⁺ NSCLC cells. H3122 and H2228 cells were treated with crizotinib (cri) for 1, 2, 3 and 4 month, followed by cell viability assessment using the MTT assay. (a) and (b) Cell survival curves and half‐maximal inhibitory concentrations (IC₅₀) of H3122 and H2228 cells to crizotinib. (c) and (d) Relative glucose consumption and lactate production of H3122 cells and H2228 cells. (e) Expression of P‐ALK /ALK and HK2 were detected in H2228 cells treated with increasing treatment duration of crizotinib. (f) Relative expression of P‐ALK/ALK and HK2 expressed as mean ± SD from three independent experiments (*, p < 0.05; &, p < 0.01, compared to the control group)

FIGURE 3

HK2 plays a crux role in the change of crizotinib‐sensitivity in ALK⁺ NSCLC cells. Expression of HK2 was interferenced by siRNA (si‐HK2) in H3122 and H2228 cells. (a) The relative expression of mRNA of HK2 in H3122 and H2228 cells (b) protein expression of HK2 and P‐ALK/ALK in H3122 and H2228 cells. (c) IC50 of H3122 and H2228 cells to crizotinib; (d) relative glucose consumption and lactate production of H3122 and H2228 cells (*, p < 0.05; &, p < 0.01, as compared with negative control (NC) group). H3122 and H2228 cells were transfected with HK2 overexpression (oe‐HK2) plasmids and vector plasmids. (e) Relative expression of mRNA of HK2, and protein expression of HK2 and P‐ALK/ALK in H3122 cells. (f) IC50 of H3122 cells to crizotinib. (g) Relative expression of mRNA of HK2, and protein expression of HK2 and P‐ALK/ALK in H2228 cells. (h) IC50 of H2228 cells to crizotinib. The data are presented as the mean ± SD from three independent experiments (*, p < 0.05; &, p < 0.01, compared to the vector group)

FIGURE 4

2DG synergistically enhanced crizotinib‐induced cytotoxicity in ALK⁺ NSCLC cell lines. (a) and (b) 2DG‐induced cytotoxicity in H3122 and H2228 cells. (c) Effects of 2DG on glucose uptake and production of lactate in H2228 cells. (d) and (e) Expression of P‐ALK /ALK and HK2 in H2228 cells treated with a series doses of 2DG. (f) and (g) Cell viabilities in H3122 and H2228 cells treated with indicated doses of crizotinib or/and 0.5 mM 2DG for 48 h. (h) and (i) Combination index (CI) values in H3122 and H2228 cells. (j) Glucose consumption and lactate production in H2228 cells treatment with combination of 2DG (0.5 mM) or/and cri (0.4 μM) (*, p < 0.05; &, p < 0.01, as compared to the control group; †, p < 0.01, compared to the crizotinib‐treated group)

FIGURE 5

2DG augmented crizotinib‐induced inhibitory effects on proliferation activity and invasion in ALK⁺ NSCLC cell lines. H2228 cells were treated with 0.5 mM 2DG, 0.4 μM crizotinib (cri), or combined treatment of both agents, (a) and (b) Ki67⁺ cell ratio and colony formation. (c) Cell invasive capability was measured after 48 h using transwell assays (*, p < 0.05; &, p < 0.01, compared to the control group; †, p < 0.01, compared to the crizotinib‐treated group)

FIGURE 6

Crizotinib in combination with 2DG notably inhibited the activity of the AKT/mTOR pathway in the ALK⁺ NSCLC cell lines. (a) and (b) H3122 cells were treated with 0.2 μM crizotinib(cri) with and without 0.5 mM 2DG for 48 h, and a western blot assay was used to detect the expression of P‐ALK/ALK, P‐AKT/AKT, HK2, P‐MTOR/MTOR, P‐S6/S6. (c) and (d) H2228 cells were treated with 0.4 μM crizotinib with and without 0.5 mM 2DG for 48 h. Whole cell protein lysates were immunoblotted with the indicated antibodies. Similar results were obtained in three independent experiments (*, p < 0.05; &, p < 0.01, compared to the untreated group)