Inhibition of mitochondrial glutaminase activity reverses acquired erlotinib resistance in non-small cell lung cancer - PubMed (original) (raw)

Inhibition of mitochondrial glutaminase activity reverses acquired erlotinib resistance in non-small cell lung cancer

Caifeng Xie et al. Oncotarget. 2016.

Abstract

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib has been approved based on the clinical benefit in non-small cell lung cancer (NSCLC) patients over the past decade. Unfortunately, cancer cells become resistant to this agent via various mechanisms, and this limits the improvement in patient outcomes. Thus, it is urgent to develop novel agents to overcome erlotinib resistance. Here, we propose a novel strategy to overcome acquired erlotinib resistance in NSCLC by inhibiting glutaminase activity. Compound 968, an inhibitor of the glutaminase C (GAC), when combined with erlotinib potently inhibited the cell proliferation of erlotinib-resistant NSCLC cells HCC827ER and NCI-H1975. The combination of compound 968 and erlotinib not only decreased GAC and EGFR protein expression but also inhibited GAC activity in HCC827ER cells. The growth of erlotinib-resistant cells was glutamine-dependent as proved by GAC gene knocked down and rescue experiment. More importantly, compound 968 combined with erlotinib down-regulated the glutamine and glycolysis metabolism in erlotinib-resistant cells. Taken together, our study provides a valuable approach to overcome acquired erlotinib resistance by blocking glutamine metabolism and suggests that combination of EGFR-TKI and GAC inhibitor maybe a potential treatment strategy for acquired erlotinib-resistant NSCLC.

Keywords: epidermal growth factor receptor; erlotinib; glutaminase inhibitor-968; non-small cell lung cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1

Figure 1. HCC827ER cells are resistant to erlotinib

(A) Cell growth assay. HCC827 and HCC827ER cells were cultured in RPMI 1640 supplemented with 10% FBS, and were either treated with increasing concentrations of erlotinib for 48 h, or untreated. Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001. (B) Soft agar assays. HCC827 and HCC827ER cells were mixed with RPMI 1640 supplemented with 0.3% agrose and 10% FBS and plated on top of RPMI 1640 supplemented with 0.5% agrose and 10% FBS. Cells were treated with erlotinib (1 μM), or untreated. Colonies were scored after 14 days of growth. 100% represents 500 cells counted. (C) Statistical analysis of colony formation in soft agar assays described above. The data represent the average of three independent experiments (mean ± SD). **P < 0.01.

Figure 2

Figure 2. The growth of HCC827 and HCC827ER cells depends on glutamine

(A) HCC827 (1 × 105 cells per well) and HCC827ER cells (1 × 105 cells per well) were cultured in RPMI 1640 medium with glutamine or without for 6 days. The cell numbers were counted every day. The number of cells cultured in medium with glutamine (300 mg/L L-Glutamine) was calculated as control. Data are shown as means ± S.D. of three experiments. ***P < 0.001. (B) HCC827 cells were transfected with control siRNA or siRNA1 and siRNA2 targeting GAC. Cells were grown for the indicated number days and counted. Untreated cells were used as control. Data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01. (C) HCC827ER cells were transfected with control siRNA or siRNA1 and siRNA2 targeting GAC. Cells were grown for the indicated number days and counted. Untreated cells were used as control. Data represent the average of three independent experiments (mean ± SD). **P < 0.01, ***P < 0.001. (D) The efficiencies of the siRNA knocking down of GAC in both HCC827 and HCC827ER cells were determined by Western blot (top panel). The rescue efficiencies of GAC in HCC827 and HCC827ER cells were determined after the transfection of pCDNA 3.1-V5-GAC (bottom panel). (E) HCC827 cells were transfected with control siRNA or siRNA1 and siRNA2 targeting GAC. After 24 hours, V5-GAC plasmid was transfected into HCC827 cells. The cell numbers were counted on the indicated days. Data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01. (F) HCC827ER cells were transfected with control siRNA or siRNA1 and siRNA2 targeting GAC. After 24 hours, V5-GAC plasmid was transfected into HCC827ER cells. The cell numbers were counted on the indicated days. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001.

Figure 3

Figure 3. Combination of compound 968 and erlotinib has synergized inhibitory effects on the growth of HCC827ER cells

(A) Saturation density assay. Cells were treated with or without increasing concentrations of compound 968 for 6 days, trypsinized and counted. The data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01. (B) HCC827 and HCC827ER cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with compound 968 (10 μM), or erlotinib (1 μM), or compound 968 (10 μM) combined with erlotinib (1 μM) for 48 hours or untreated. Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001. (C) HCC827ER cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with erlotinib (1 μM) combined with increasing concentrations of compound 968 (5, 10, 20, and 40 μM), or treated with compound 968 (10 μM) combined with increasing concentrations of erlotinib (0.5, 1, 2, and 4 μM) for 48 hours or untreated. Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01. (D) Time course experiment. HCC827ER cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with compound 968 (10 μM), or erlotinib (1 μM), or compound 968 (10 μM) combined with erlotinib (1 μM) for different time or untreated. Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). *P < 0.05, ***P < 0.001.

Figure 4

Figure 4. Combination of compound 968 and erlotinib also decreases the growth of other NSCLC cell lines

(A) NCI-H1650, NCI-H1975 and A549 cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with different concentrations of erlotinib (0.01, 0.05, 0.1, 0.5, 1, and 2 μM) for 48 h or untreated. Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). (B) NCI-H1650, NCI-H1975 and A549 cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with different concentrations of compound 968 (0, 5, 10, and 20 μM) for 48 h or untreated. Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001. (C) NCI-H1650, NCI-H1975 and A549 cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with erlotinib (1 μM) combined with increasing concentrations of compound 968 (5, 10, and 20 μM). Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). **P < 0.01, ***P < 0.001. (D) NCI-H1650, NCI-H1975 and A549 cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with compound 968 (10 μM) combined with increasing concentrations of erlotinib (0.01, 0.1, 1, and 2 μM) for 48 hours or untreated. Cell growth was determined by crystal violet staining. Data represent the average of three independent experiments (mean ± SD). **P < 0.01, ***P < 0.001.

Figure 5

Figure 5. Compound 968 reverses acquired erlotinib resistance by blocking GAC activity in HCC827ER cells

(A) HCC827 cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with compound 968 at 0, 1, 5, and 10 μM concentrations for 48 hours (left panel) or treated with 10 μM compound 968 for 0, 12, 24 and 48 hours (right panel). The EGFR and GAC expressions were determined by Western blot. (B) HCC827ER cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with compound 968 at 0, 1, 5, and 10 μM concentrations for 48 hours (left panel) or treated with 10 μM compound 968 for 0, 12, 24 and 48 hours (right panel). The EGFR and GAC expressions were deterimine by Western blot. (C) HCC827 (left panel) and HCC827ER (right panel) cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with compound 968 (10 μM), or erlotinib (1 μM), or compound 968 (10 μM) combined with erlotinib (1 μM) for 48 hours respectively. The EGFR and GAC expressions were determined by Western blot. (D) HCC827 and HCC827ER cells were cultured in RPMI 1640 supplemented with 10% FBS, and were treated with compound 968 (10 μM), or erlotinib (1 μM), or compound 968 (10 μM) combined with erlotinib (1 μM) for 48 hours respectively, then mitochondria was isolated from different cells, and GAC activity was determined by glutaminase activity assay. Data represent the average of three independent experiments (mean ± SD). **P < 0.01, ***P < 0.001.

Figure 6

Figure 6. Combination of compound 968 and erlotinib inhibits the glutamine and glycolysis metabolism

The concentrations of Glutamine (A), glutamate (B), glucose (C), lactate (D), and ATP (E) were detected in HCC827, HCC827ER cells or HCC827ER cells treated with compound 968 (10 μM) combined with erlotinib (1 μM). Data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001. (F) A summary map of metabolites from glycolysis and glutamine metabolism in this study.

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