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. 2016 Jul;15(7):1757-67.
doi: 10.1158/1535-7163.MCT-15-0765. Epub 2016 Apr 12.

NQO1-Mediated Tumor-Selective Lethality and Radiosensitization for Head and Neck Cancer

Long-Shan Li  1 , Srilakshmi Reddy  1 , Zhen-Hua Lin  2 , Shuangping Liu  2 , Hyunsil Park  1 , Stephen G Chun  3 , William G Bornmann  4 , Joel Thibodeaux  5 , Jingsheng Yan  6 , Gaurab Chakrabarti  1 , Xian-Jin Xie  6 , Baran D Sumer  7 , David A Boothman  8 , John S Yordy  9
Affiliations

NQO1-Mediated Tumor-Selective Lethality and Radiosensitization for Head and Neck Cancer

Long-Shan Li et al. Mol Cancer Ther. 2016 Jul.

Abstract

Ionizing radiation (IR) is a key therapeutic regimen for many head and neck cancers (HNC). However, the 5-year overall survival rate for locally advanced HNCs is approximately 50% and better therapeutic efficacy is needed.

Nad(p)h: quinone oxidoreductase 1 (NQO1) is overexpressed in many cancers, and β-lapachone (β-lap), a unique NQO1 bioactivatable drug, exploits this enzyme to release massive reactive oxygen species (ROS) that synergize with IR to kill by programmed necrosis. β-Lap represents a novel therapeutic opportunity in HNC leading to tumor-selective lethality that will enhance the efficacy of IR. Immunohistochemical staining and Western blot assays were used to assess the expression levels of NQO1 in HNC cells and tumors. Forty-five percent of endogenous HNCs expressed elevated NQO1 levels. In addition, multiple HNC cell lines and tumors demonstrated elevated levels of NQO1 expression and activity and were tested for anticancer lethality and radiosensitization by β-lap using long-term survival assays. The combination of nontoxic β-lap doses and IR significantly enhanced NQO1-dependent tumor cell lethality, increased ROS, TUNEL-positive cells, DNA damage, NAD(+), and ATP consumption, and resulted in significant antitumor efficacy and prolonged survival in two xenograft murine HNC models, demonstrating β-lap radiosensitization of HNCs through a NQO1-dependent mechanism. This translational study offers a potential biomarker-driven strategy using NQO1 expression to select tumors susceptible to β-lap-induced radiosensitization. Mol Cancer Ther; 15(7); 1757-67. ©2016 AACR.

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Conflict of interest statement

of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Expression of NQO1 and Catalase in HNC and adjacent normal tissues
(A) Representative images from tissue microarray IHC staining from HNC patients. (B) A statistically significant inverse expression ratio exists between the relative intensity of NQO1 and Catalase staining in the tumor and adjacent normal tissue (*p < 0.05). See Supplemental Table 1 for the scoring for each individual patient. A two-by-two contingency table comparing NQO1 and Catalase expression levels for all patients (0 and 1 expression levels were grouped as negative and compared to 2 and 3 expression levels which were grouped as positive [see materials and methods]) is shown for (C) NQO1 and (D) Catalase, demonstrating a statistically significant difference in the elevated expression levels of NQO1 in tumor compared to adjacent normal tissue and Catalase in adjacent normal tissue compared to tumor (***p < 0.001; **p < 0.01). n, number of patients.
Figure 2
Figure 2. Expression of NQO1 and Catalase in HNC cell lines
(A) Forty-two independent HNC cell lines were probed for NQO1, Catalase and GAPDH expression, demonstrating significant inverse expression in the protein abundance between NQO1 and Catalase, (B) LD50 of β-lap in human primary IMR90 fibroblasts (passage 12, closed square) and in 41 HNC cell lines varying in NQO1 enzyme activity (open squares). (C) LD50 of β-lap in human primary IMR90 fibroblasts (P12, passage 12) and in 41 HNC cell lines varying in NQO1/Catalase ratios (closed circle is LD50 of IMR90 cell line). NQO1:Catalase expression ratios were calculated from relative radiographic intensities on Western blots using NIH Image J. MDA-MB-231 NQO1+ (231NQO1+) and NQO1- (231NQO1-) cells served as positive and negative controls for NQO1 expression. GAPDH served as the loading control.
Figure 3
Figure 3. Functional implications of NQO1 expression in response to β-lap
(A) Shown are four representative cell lines, including FaDu, SqCC/Y1 and Detroit 562 that express NQO1 and the *2 polymorphic UM-SCC-10A that lacks expression (refer to Fig 2A). Several approaches were taken to demonstrate the role of β-lap-induced NQO1-dependent cell death, including adding the specific NQO1 inhibitor, dicoumarol (DIC, 50 μM) or exogenous Catalase (2000 U). β-Lap alone induced significant cell death in a concentration-dependent manner in the three cell lines that expressed NQO1 (β-lap dose indicated on the x-axis) and co-treatment with DIC completely abrogated cell killing by β-lap. No cell death was seen in UM-SCC-10A cells after β-lap exposure. Likewise, co-treatment with exogenous Catalase partially blocked β-lap-induced NQO1-dependent cell death and had no effect when NQO1 was not expressed (UM-SCC-10A, bottom row). Shown are means ±SE (***p < 0.001). (B), Western blot analyses for NQO1 and Catalase expression in IMR90 primary fibroblasts (left panel). SqCC/Y1 cells served as positive controls for NQO1 and Catalase expression. GAPDH levels served as a loading control. Relative survival assays in IMR90 primary fibroblasts (right panel) were performed after treatment with β-lap alone for 2 h, with or without DIC (50 μM) or Catalase (2000 U). β-Lap did not induce any significant cell death at concentrations that produced complete cell death in the cell lines that expressed NQO1 (panel A). Supra-high dose β-lap-induced cell death was abrogated by co-treatment with DIC or Catalase. Shown are means ±SE (**p < 0.01). All experiments were performed three times in triplicate.
Figure 4
Figure 4. Functional consequences of reduced NQO1 expression and mechanisms of β-lap-induced NQO1-dependent cell death
(A) Lentiviral shRNA against NQO1 was used to stably reduce the protein abundance of NQO1 and a representative western blot for several different independent NQO1 knockdown clones is presented. A non-targeting scrambled shRNA (shSCR) was used to control for the lentiviral vector and GAPDH served as the loading control. (B) β-Lap-induced lethality is rescued in SqCC/Y1 shNQO1 knockdown clones. shSCR and shRNA-NQO1 knockdown cells #7 and #11 were evaluated by relative survival assay after various doses of β-lap exposure (μM, 2 h) as depicted on the x-axis demonstrating a β-lap induced NQO1 dependent cell death (***p < 0.001). (C) β-Lap-induced oxidative stress (reactive oxygen species [ROS], predominately H2O2) was monitored by DCFDA staining and was analyzed using high-throughput imaging analysis technology in FaDu and SqCC/Y1 cells after β-lap treatment (5 μM), with or without DIC (50 μM) at the indicated times in minutes across the x-axis. Hydrogen peroxide (500 μM, 15 min) served as the positive control. (D) Time-dependent increase in DNA strand breaks was demonstrated by the alkaline comet assay in the presence of β-lap alone (5 μM, 2 h) or co-treatment with β-lap and DIC (50 μM) in SqCC/Y1 cells, with the graphed comet tail lengths measured using Komet 5.5 software. Shown are representative micrographs of studies performed at least three times and the graphed comet tail lengths are means ±SE from three experiments. Student's t tests were performed to assess significance (***p < 0.001). (E) Poly ADP-ribosylation (PAR) levels were determined by western blot for total PAR in the presence of 5 μM β-lap without or with co-treatment with DIC (50 μM) in FaDu and SqCC/Y1 cells. GAPDH served as loading control. (F) ATP levels are dramatically reduced by exposure to β-lap at the indicated doses and this response is blocked by co-treatment with DIC (50 μM). Student's t tests were performed to assess for significance (***p < 0.001). (G) The percent TUNEL positive FaDu and SqCC/Y1 cells is elevated with β-lap exposure at the indicated doses and abrogated with the co-treatment of DIC (50 μM). Results are means ±SE for studies performed three times. Student's t tests were performed to assess for significance (***p < 0.001).
Figure 5
Figure 5
Combination low dose radiation and sublethal doses of β-lap result in decreased cell survival, and increased ROS, TUNEL positivity, γH2AX foci formation and ATP consumption. (A) Relative cell survival assays were performed by treating with β-lap at the specified concentration immediately after IR at the indicated doses for cell lines, FaDu, SqCC/Y1, Detroit 562 and UM-SCC-10A. Data represent relative cell survival assays as described in ‘Materials and Methods’. Triple asterisks represent statistically significant differences between β-lap (1.5 μM and 2.5 μM) and DMSO (2.5 μM) treatments (***p < 0.001). (B) Oxidative stress (reactive oxygen species [ROS], predominately H2O2) was monitored by DCFDA staining and analyzed using high-throughput imaging analysis technology in SqCC/Y1 HNC cells after combined treatment of low dose IR (Gy) and β-lap (μM) at 20 min time points. β-Lap (5 μM) with or without DIC (50 μM) served as positive and negative controls, respectively. The asterisk represents statistically significant differences between β-lap (2.5 μM) + 2 Gy vs. β-lap (2.5 μM) or 2 Gy alone (*p < 0.05). (C) Percent TUNEL+ SqCC/Y1 cells were significantly increased with the combination of low dose β-lap (2.5 μM) + 2 Gy or 3 Gy compared to single agents alone. Co-treatment with DIC (50 μM) rescued the combined treatment induced cell death. Results are means ±SE for studies performed three times. Student's t tests were performed to assess for significance (*p < 0.05; **p < 0.01). (D) Representative images of γH2AX foci formation in SqCC/Y1 HNC cells in the presence of increasing doses of β-lap, without or with 2 Gy. Quantitation of means ±SE of these foci from 100 cells across treatment group combinations demonstrated an increased number of foci at 2 h in the presence of the indicated concentration of β-lap with 2 Gy versus controls (bar chart). Student's t-tests were performed to assess statistical significance between identical β-lap concentrations, without or with IR (***p < 0.001). A lethal dose of β-lap (5 μM), with or without DIC (50 μM) was used as controls. (E) IR enhanced NAD+ and ATP loss in SqCC/Y1 HNC cells in the presence of sublethal doses of β-lap in a dose-dependent manner for both β-lap with or without IR as indicated. Student's t-tests were performed comparing β-lap alone and β-lap + IR (***p < 0.001).
Figure 6
Figure 6. Cooperative antitumor efficacy using a combination of β-lap and IR to treat HNC xenograft models
Mice bearing 30 mm3 SqCC/Y1 HNC xenografts with high levels of NQO1 expression were treated with 2 Gy every other day for five treatments (10 Gy total dose). β-lap-HPβ-CD 10 mg/kg was intravenously administered by tail-vein injection immediately following 2 Gy treatment. Vehicle alone (HPβ-CD) served as a control cohort (Materials and Methods). Results (means ± SE) are representative of repeated similar experiments (n=10 for each group). Student's t-tests (*** p < 0.001) were performed comparing treated vs control groups. (A) Tumor volume measurements and (B) Kaplan-Meier overall survival over the indicated number of days is graphed for control (HPβ-CD), β-lap-HPβ-CD 10 mg/kg alone, 2 Gy alone and a combination of 2 Gy plus β-lap-HPβ-CD 10 mg/kg. Log-rank analyses were performed comparing survival curves using various IR + β-lap-HPβ-CD regimens (*** p < 0.001 for the combined treatment compared to each single treatment). Survival curves show equivalency between HPβ-CD and β-lap-HPβ-CD 10 mg/kg. (C-E) Short-term in vivo pharmacodynamic studies to assess γH2AX foci formation and ATP levels in SqCC/Y1 HNC cells 4 h after combined treatment with 2 Gy followed by administration of β-lap-HPβ-CD as indicated. (C) Representative micrographs of γH2AX foci formation demonstrating (D) a significant increase in foci formation in the combined treatment group compared to all other treatments. (E) ATP levels are significantly reduced in the combined treatment and not in the control or single treatments (** p < 0.01).
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