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. 2016;24(1):55-64.
doi: 10.3727/096504016X14586627440192.

2-Deoxy-d-glucose Suppresses the In Vivo Antitumor Efficacy of Erlotinib in Head and Neck Squamous Cell Carcinoma Cells

Arya Sobhakumari  1 , Kevin P Orcutt, Laurie Love-Homan, Christopher E Kowalski, Arlene D Parsons, C Michael Knudson, Andrean L Simons
Affiliations

2-Deoxy-d-glucose Suppresses the In Vivo Antitumor Efficacy of Erlotinib in Head and Neck Squamous Cell Carcinoma Cells

Arya Sobhakumari et al. Oncol Res. 2016.

Abstract

Poor tumor response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a significant challenge for effective treatment of head and neck squamous cell carcinoma (HNSCC). Therefore, strategies that may increase tumor response to EGFR TKIs are warranted in order to improve HNSCC patient treatment and overall survival. HNSCC tumors are highly glycolytic, and increased EGFR signaling has been found to promote glucose metabolism through various mechanisms. We have previously shown that inhibition of glycolysis with 2-deoxy-d-glucose (2DG) significantly enhanced the antitumor effects of cisplatin and radiation, which are commonly used to treat HNSCC. The goal of the current studies is to determine if 2DG will enhance the antitumor activity of the EGFR TKI erlotinib in HNSCC. Erlotinib transiently suppressed glucose consumption accompanied by alterations in pyruvate kinase M2 (PKM2) expression. 2DG enhanced the cytotoxic effect of erlotinib in vitro but reversed the antitumor effect of erlotinib in vivo. 2DG altered the N-glycosylation status of EGFR and induced the endoplasmic reticulum (ER) stress markers CHOP and BiP in vitro. Additionally, the effects of 2DG + erlotinib on cytotoxicity and ER stress in vitro were reversed by mannose but not glucose or antioxidant enzymes. Lastly, the protective effect of 2DG on erlotinib-induced cytotoxicity in vivo was reversed by chloroquine. Altogether, 2DG suppressed the antitumor efficacy of erlotinib in a HNSCC xenograft mouse model, which may be due to increased cytoprotective autophagy mediated by ER stress activation.

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Conflict of interest statement

The authors have no conflict of interest.

Figures

Figure 1
Figure 1
Erlotinib suppresses glucose consumption in HNSCC cells. (A) Glucose concentrations in the cell culture media of FaDu, Cal-27, SCC-25, and SQ20B HNSCC cells were measured before and after 24 h DMSO (CON) or erlotinib (ERL) treatment. (B, C) SCC-25 cells were treated with DMSO or ERL for 24 and 48 h then analyzed for PKM2 mRNA levels by RT-PCR (B) and protein levels by Western blot (C). (D, E) FaDu cells were treated with DMSO or ERL, and glucose concentrations (D) and clonogenic survival (E) were measured 24, 48, and 72 h after treatment. Glucose concentrations were normalized to cell number. NSF, normalized surviving fraction. Error bars represent ± standard error of the mean (SEM) of at least N = 3 experiments. *p < 0.05 versus DMSO.
Figure 2
Figure 2
2-Deoxy-d-glucose (2DG) enhances the antitumor efficacy of erlotinib in vitro but not in vivo. (A) FaDu, Cal-27, SCC25, and SQ20B cells were treated with 2DG with or without erlotinib (ERL) and analyzed for cell survival by clonogenic assay. Error bars represent ± standard error of the mean (SEM) of at least N = 3 experiments. *p < 0.05 versus DMSO, ¥p < 0.05 versus 2DG/ERL. (B) FaDu cells were injected into the right flank of athymic nu/nu mice (n = 6) and treated with 2DG alone or in combination with ERL for 2 weeks. Saline was administered as a control (CON). Tumor growth was measured using Vernier calipers. (C) Kaplan–Meier plot of survival data. *p < 0.05 ERL versus CON, ≠p < 0.05 ERL versus 2DG, ¥p < 0.05 ERL versus 2DG/ERL.
Figure 3
Figure 3
2-Deoxy-d-glucose (2DG) induces markers or ER stress. (A) FaDu, Cal-27, and SQ20B cells were pretreated with mannose (MAN) for 2 h before treatment with 2DG and/or ERL then analyzed for CHOP (A) and GRP78/BIP (B) expression. Cells were treated with saline as a negative control (CON) and tunicamycin (TUN) as a positive control. β-Actin was used as a loading control.
Figure 4
Figure 4
Suppression of ER stress reverses drug-induced cell killing. (A) FaDu, Cal-27, and SQ20B cells were pretreated with mannose (MAN) for 2 h before treatment with 2DG + ERL then analyzed for cell viability. (B) FaDu cells were pretreated with MAN or salubrinal (SAL) for 2 h before treatment with 2DG + ERL then analyzed for clonogenic survival. Cells were treated with saline as a control (CON). Error bars represent ± standard error of the mean (SEM) of at least N = 3 experiments. *p < 0.05 versus CON, ¥p < 0.05 versus 2DG+ERL.
Figure 5
Figure 5
2-Deoxy-d-glucose (2DG) alters EGFR expression. (A) FaDu cells were treated with 2DG with or without erlotinib (ERL) and analyzed for phosphorylated EGFR (pEGFR) and Akt (pAkt) expression and total EGFR (tEGFR) and Akt (tAkt) expression by Western blot. (B) FaDu, Cal-27, and SQ20B cells were treated with 2DG with or without ERL and analyzed for total EGFR (EGFR) expression. (C) FaDu, Cal-27, and SQ20B cells were pretreated with mannose (MAN) for 2 h before treatment with 2DG + ERL then analyzed for EGFR expression. Cells were treated with saline as a negative control (CON) and tunicamycin (TUN) as a positive control. β-Actin was used as a loading control.
Figure 6
Figure 6
2-Deoxy-d-glucose (2DG) and erlotinib (ERL) induce autophagy. (A) FaDu, Cal-27, and SQ20B cells were pretreated with mannose (MAN) for 2 h before treatment with 2DG and/or ERL then analyzed for LC3BI/II expression. Cells were treated with saline as a negative control (CON) and tunicamycin (TUN) as a positive control. β-Actin was used as a loading control. (B) FaDu cells were injected into the right flank of athymic nu/nu mice (n = 6) and treated with 2DG in combination with ERL (2DG/ERL) with or without chloroquine (CLQ) for 9 days. Saline was administered as a control (CON). Tumor growth was measured using Vernier calipers. Error bars represent ± standard error of the mean (SEM) of N = 6 mice. *p < 0.05 versus CON, ≠p < 0.05 versus CLQ, ¥p < 0.05 versus 2DG/ERL.
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