DANGER is involved in high glucose-induced radioresistance through inhibiting DAPK-mediated anoikis in non-small cell lung cancer

18F-labeled fluorodeoxyglucose (FDG) uptake during FDG positron emission tomography seems to reflect increased radioresistance. However, the exact molecular mechanism underlying high glucose (HG)-induced radioresistance is unclear. In the current study, we showed that ionizing radiation-induced activation of the MEK-ERK-DAPK-p53 signaling axis is required for anoikis (anchorage-dependent apoptosis) of non-small cell lung cancer (NSCLC) cells in normal glucose media. Phosphorylation of DAPK at Ser734 by ERK was essential for p53 transcriptional activity and radiosensitization. In HG media, overexpressed DANGER directly bound to the death domain of DAPK, thus inhibiting the catalytic activity of DAPK. In addition, inhibition of the DAPK-p53 signaling axis by DANGER promoted anoikis-resistance and epithelial-mesenchymal transition (EMT), resulting in radioresistance of HG-treated NSCLC cells. Notably, knockdown of DANGER enhanced anoikis, EMT inhibition, and radiosensitization in a mouse xenograft model of lung cancer. Taken together, our findings offered evidence that overexpression of DANGER and the subsequent inhibitory effect on DAPK kinase activity are critical responses that account for HG-induced radioresistance of NSCLC.


Real-time quantitative RT-PCR (qRT-PCR)
ITPRIP gene expression was measured using real-time qRT-PCR as previously described [1]. Aliquots of a master mix containing all of the reaction components with the primers were dispensed into a real-time PCR plate (Applied Biosystems, Foster City, CA). All PCR reagents were from a SYBR Green core reagent kit (Applied Biosystems). Sequences of the ITPRIP-specific primers were as follows: 5′-AGAACCTGCTGTGTGCCACAGA-3′ (forward), 5′-CAGGTCGAACTCGTACTTGTGG-3′ (reverse). ITPRIP gene expression was measured in triplicate in the reaction plate. qRT-PCR was performed using an Applied Biosystems-7900 HT qRT-PCR instrument. PCR was performed for 40 cycles of 95°C for 15 s and 60°C for 1 min followed by thermal denaturation. The expression of each gene relative to that of GAPDH was determined using the 2 − ΔCT method [2]. To simplify data presentation, relative expression values were multiplied by 10 2 .

Western blot analysis, immunoprecipitation (IP), transient transfection, and kinase assay
For Western blot analysis, whole cell lysates (WCL) were prepared as previously described [3]. WCL were extracted using radioimmunoprecipitation assay lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl, 1% Triton X-100, 25 mM NaF, 1 mM DTT, 20 mM EGTA, 1 mM Na 3 VO 4 , 0.3 mM PMSF, and 5 U/mL Aprotinin). Protein concentration of the lysates was verified using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). To prepare the cytoplasmic extract (CE), cells were suspended in buffer A (10 mM HEPES, pH 7.9; 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, 1 μg/mL Aprotinin, 5 μg/mL Leupeptin, and 1 μg/mL Pepstatin A) and then incubated for 20 min on ice. An equal volume of buffer B (buffer A + 0.1% NP-40) was then added and the cells were incubated for 20 min on ice. Next, the samples were centrifuged at 5,000 g for 2 min to remove cellular debris and the CE was collected. Proteins in the samples were then separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked with 5% skim milk in TBS with Tween 20 (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20) for 30 min at room temperature. The membranes were subsequently probed using specific primary antibodies and peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Antibody binding was visualized using an enhanced chemiluminescence detection system (Roche Applied Science, Penzberg, Germany).
IP studies were performed as previously described [4]. Briefly, protein samples were immunoprecipitated overnight with specific primary antibody and protein A/Gagarose beads (Santa Cruz Biotechnology). After washing three times with lysis buffer, the immunoprecipitates were boiled in 2 × SDS sample buffer for 10 min followed by centrifugation. The samples were then analyzed by Western blotting.
For transient transfection, cells were plated at a density of 5 × 10 5 cells in 6-well dishes and incubated for 4 h. Next, the cells were transiently transfected with the indicated plasmid using Lipofectin (Invitrogen, Carlsbad, CA) or siRNA oligonucleotides (10 nM) using DharmaFECT 1 (Dharmacon, Lafayette, CO) according to the manufacturers' instructions.
For the kinase assay, cells were transfected or cotransfected with the indicated gene construct for 24 h. After the desired treatment, the lysates were immunoprecipitated overnight with specific primary antibody. Western blotting was then conducted using phosphor-specific antibody.

Adhesion assay
96-well plates were coated with 10 ng/mL fibrinogen at 4°C overnight, washed with PBS, and blocked with 0.1% BSA for 1 h at 37°C. Cells were suspended in HEPES-Tyrode's buffer containing 0.1% BSA and 1 mmol/L MgCl 2 , transferred to the plate at a density of 1 × 10 5 in 200 μL/well, and allowed to attach for up to 60 min at 37°C in 5% CO 2 . The plates were washed to remove floating cells. The remaining attached cells were incubated with para-nitrophenol phosphate (5 mg/mL in 50 mmol/L sodium acetate and 1% Triton X-100, pH 5.2) for 30 min and quantified at 405 nm after adding 0.3 mol/L sodium hydroxide.

Apoptosis assay
Apoptosis was assessed by measuring Caspase 3/7 activities using a Caspase-Glo 3/7 assay kit (Promega, Madison, WI) according to the manufacturer's protocol. Briefly, treated cells (10 4 cells/mL) in 100 μL of culture medium were transferred to a 96-well microplate. Subsequently, 100 μL of Caspase-Glo 3/7 reagent containing Caspase 3/7 substrate was added to each well. After contents of the wells were gently mixed at 300-500 rpm for 30 s, the plate was incubated at room temperature for 1 h. Luminescence of each sample was measured using a Glomax multi-detection system (Promega).
Apoptosis induction was also assessed by analyzing cytoplasmic histone-associated DNA fragmentation. In brief, cells were plated in 96-well plates and allowed to attach overnight. The cells were then subjected to transfection, irradiation, and/or drug treatment. Cytoplasmic histone-associated DNA fragmentation was monitored using a cell death detection kit (Roche Applied Science) according to the manufacturer's instructions.

Transwell cell migration assay
To measure the migration capacity of NSCLC cells, a Transwell cell migration assay was conducted as previously described [5]. The assay was performed using a 24-well Transwell chamber (Corning, Inc., Corning, NY). Cells (1 × 10 4 in serum-free RPMI-1640) cultured in the presence or absence of various conditions (transfection, IR, and/ or drugs) for 72 h were seeded in the upper chamber with a 5-μm pore size insert. The lower chamber was filled with 600 μL of RPMI-1640 containing 2% FBS. Six h later, the upper membrane surface was wiped with a cotton swab to remove cells that had not migrated into the lower chamber. Cells that had migrated and attached to the lower membrane surface were fixed with 4% paraformaldehyde and stained with hematoxylin for counting. The migration index was calculated relative to the number of untreated cells that had migrated. The results are expressed as fold-increase of migration compared to the control group and based on the relative number of cells in a randomly selected field for three representative experiments.

Immunofluorescence (IF)
For IF, DAPK-and DANGER-overexpressed cells were grown on glass slides. Following the experimental treatments, the cells were fixed and permeabilized in cold acetone and then washed with cold PBS. After blocking with 1% bovine serum albumin/PBS, the cells were incubated overnight with anti-DAPK and anti-DANGER antibody at 4°C. Next, the cells were washed three times with cold PBS and incubated with DyLight 488-conjugated secondary antibodies and DyLight 649-conjugated ones (Thermo Scientific). After washing and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma), the glass slides were mounted with VECTASHIELD Hard-Set Mounting Medium (Vector Laboratories, Burlingame, CA) and visualized with an Olympus IX71 fluorescence microscope (Olympus Optical Co. Ltd., Tokyo, Japan).

Animal protocol
Six-wk-old male BALB/c athymic nude mice (Central Lab Animals Inc., Seoul, Republic of Korea) were used for the in vivo experiments. The protocols used were approved by the Institutional Animal Care and Use Committee of Pusan National University (Busan, Republic of Korea), and performed in accordance with the provisions of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The mice were housed individually or in groups of up to five in sterile cages. They were maintained in animal care facilities in a temperatureregulated room (23 ± 1°C) with a 12-h light/dark cycle and quarantined for 1 wk prior to the study. The animals were fed water and a standard mouse chow diet ad libitum.

Statistical analysis
All numeric data are presented as the mean ± SEM for at least three independent experiments. The results were analyzed using a one-way ANOVA for ranked data followed by a Tukey's honestly significant difference test. Prism 5 software (GraphPad Software, San Diego, CA) was used to perform all statistical analyses. A pvalue < 0.05 was considered statistically significant. Figure S5: The radiosensitivity of NSCLC cells is reduced by HG treatment or DANGER overexpression. A. Caspase 3/7 activation by IR in NSCLC cells were measured with a Caspase 3/7 activity assay. HG-or overexpressed DANGER-induced increase of Caspase 3/7 activity was reversed by DANGER knockdown. The Western blot results suggested that each transfection was efficient. Data are represented as mean ± SEM (n = 3); * p < 0.05 compared to non-irradiated cells, ** p < 0.05 compared to cells treated with irradiation-alone, *** p < 0.05 compared to HG-treated and irradiated cells. B. Functional involvement of DANGER knockdown in HG-and IR-induced DNA damage responses was measured with a DNA fragmentation assay. Data are represented as mean ± SEM (n = 3); * p < 0.05 compared to non-irradiated cells, ** p < 0.05 compared to cells treated with irradiation alone, *** p < 0.05 compared to HG-treated and irradiated cells.