A list of all oligonucleotides used here is reported in Suppl. Table S3. A list of all antibodies is reported in Suppl. Table S4.
NSCLC cells (A549, H1437 and H226, H1299 and H1650) were grown in RPMI 1640 supplemented with 5% fetal bovine serum (FBS). All cell lines were fingerprinted using the GenePrint fluorescent STR system (Promega, Madison, WI). Prior to injection in mice, A549 and H1437 were transduced with a lentivirus expressing luciferase (ViraPowerTM T-Rex Lentiviral expression system, Grand Island, NY; see below for cloning details). Some experiments were performed in cell lines re-derived from mice’s lungs after necropsies obtained through mechanical dissociation of tumor nodules. After 5 passages, cell lines contained 100% human cells as assessed by Q-PCR using species-specific primers for GAPDH.
γ-secretase inhibitor MRK-003, the humanized monoclonal IgG1 antibody against IGF-1R MK-0646, and the pan-Akt inhibitor MK-2206 were a generous gift of Merck & Co. (Whitehouse Station, NJ). Erlotinib (N-3-ethylnylphenyl)-6,7-bis(2-methoxyethoxy)-4 quinazolinamine) was donated by patients of the Loyola University Chicago Medical Center who had become unresponsive to it. IGF-1 was from Insight Genomics (Fall Church, VA). Cisplatin and NH4Cl were from Sigma-Aldrich (St. Louis, MO). MG-132 was from Calbiochem (San Diego, CA). The working concentrations and the duration of exposure used in each experiment are specified in the text and in figure legends. For TUNEL assays on 8-µm-thick slides obtained from frozen lungs, we first stained by immunofluorescence regions overexpressing glucose transporter 1 (GLUT-1) in green, and then we proceeded with TUNEL assays using the In Situ Cell Death Detection Kit, TMR red (Roche, Indianapolis, IN) following the manufacturer’s protocol.
Five-week-old female NOD.CB17-Prkdcscid/J mice (Jackson Laboratories, Bar Harbor, ME) were injected in the tail vein with 2.5 × 106 cells in 100 µl of sterile saline solution. Mice were housed in a pathogen-free animal facility at Loyola University Medical Center. All procedures were performed in accordance with the Institutional Animal Care and Use Committee of the Loyola University Chicago Medical Center. Animals were treated as indicated in the text and were monitored daily until they reached one of the end points (observed: dyspnea or weight loss). At euthanasia, human cells comprised 93% ± 0.8% of the total lungs of mice. Tumor burden quantification was performed using bioluminescence imaging (Xenogen VivoVision IVIS 100 In Vivo Imaging System, Caliper Life Science, Hopkinton, MA) and by Q-PCR measuring human versus mouse GAPDH after euthanasia. Mice were monitored weekly for bioluminescence. All data analysis was performed with the Living Image 3.2 Software (Caliper) after i.p. injection of 0.25 mg of D-Luciferin (Gold Biotechnology, St Louis, MO) with animals under isoflurane anesthesia. Q-PCR was performed using human and mouse specific GAPDH primers designed after sequence alignments of human and GAPDH genomic regions (Clustalw software) to discriminate the 2 genes (Suppl. Table S3). Primers were carefully validated, and calibration curves to match Q-PCR results to cell number were developed. Lungs, livers, and brains were dissected and flash frozen for molecular analyses.
Plasma glucose concentration was measured from 100 µl of blood drawn from the lateral tail vein of mice prior euthanasia using the Glucose Assay Kit (Eton Bioscience Inc., Cambridge, MA) following the manufacturer’s instructions.
Plasmids and lentiviral systems
Constitutively active Akt1 (NH2-terminal myristoylatable Akt1, Myr-Akt1) and dominant negative Akt1, K179M mutant Akt1) cloned into the pUSEamp(+) expression plasmid (pUSE empty vector was used as the negative control) were purchased from Upstate (Millipore, Temecula, MA). Transient transfections were done using Fugene HD (Promega, Madison, WI) according to the manufacturer’s instructions. Efficiency of transfection was greater than 95%.
The luciferase gene was excised from pGL2 basic vector (Promega) by restriction digestion at the KpnI and BamHI sites and ligated into the KpnI and EcoRI sites of pENTR4 (Invitrogen, Grand Island, NY; the incompatibility between the BamHI and EcoRI sites was circumvented using a linker oligonucleotide in the ligation mixture). The luciferase sequence was transferred via Gateway recombination into the lentiviral backbone plasmid pLenti4/TO/V5-DEST (ViraPower T-Rex Lentiviral Expression System, Invitrogen). To generate luciferase stable cell lines, cells were infected with packaged lentivirus. Transduction and selection of cells were done as recommended (Invitrogen). Luciferase expression was verified by Dual-Luciferase Reporter Assay System (Promega) as recommended by the manufacturer, and relative light units (RLUs) were measured using a Femtomaster FB15 luminometer (Zylux Corporation, Maryville, TN).
Human Phospho-RTK (Human Phospho-RTK Array Kit, R&D Systems Inc., Minneapolis, MN) arrays were used according to the manufacturer’s instructions. Briefly, tumor tissue was washed with cold PBS and homogenated in NP-40 lysis buffer (1% NP-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin), and 250 µg of cell homogenate was incubated with preblocked membranes overnight. Membranes were subsequently washed and incubated with freshly diluted detection antibody (anti-phospho-tyrosine-HRP) for 2 hours at room temperature (R.T.) on a rocking platform shaker. Spots were detected using enhanced chemiluminescence (ECL) and exposed to x-ray films.
Protein phosphorylation expression and analysis
Western blot analyses were performed as previously described.7
After ECL development, as an additional control for equal loading, nitrocellulose membranes were stained with Ponceau S solution (Sigma-Aldrich). The working dilutions for each primary antibody were those recommended by the various manufacturers. For immunoprecipitation, lysates were incubated overnight at 4°C with a specific primary antibody followed by 1 hour of incubation with Protein G-agarose beads (Santa Cruz Biotech, CA). The immunoprecipitates were washed 3 times with lysis buffer and resolved by SDS-PAGE.
Gene expression analysis
Quantitative reverse transcriptase real time PCR (Q-RT-PCR) using SYBR green incorporation was performed as previously described.7
Total RNA from cultured cells was extracted with the RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was synthesized using iScript Reverse Transcription Supermix RT-qPCR (Bio-Rad Laboratories, Hercules, CA). Quantitative real-time PCR was done with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in an ABI 7300 thermal cycler (Applied Biosystems). For each sample, serial dilutions of cDNA templates were measured in triplicate. Non–reverse transcription reactions served as controls. All measurements were normalized for human ribosomal protein RPL13A mRNA.
Cell surface biotinylation
For biotinylation of cell membrane proteins, cells were washed with ice-cold PBS and then incubated for 1 hour at 4°C with Sulfosuccinimidobiotin (EZ-Link Sulfo-NHS-Biotin; Pierce Biotechnology, Rockford, IL) freshly dissolved in H2O (2 mM final concentration). The reaction was blocked by rinsing the cells 3 times with 100 mM glycine in PBS. Cell lysates were incubated for 1 hour at 4°C with streptavidin-agarose (Sigma-Aldrich) to pull down biotinylated membrane proteins. The supernatant was stored for further manipulation (first fraction). Streptavidin-biotin complexes (surface protein enriched fraction) were incubated overnight at 4°C with a primary antibody specific for EGF-R. The suspension was then incubated for 1 hour with protein A conjugated to magnetic beads (Millipore), and immunocomplexes were captured using a dedicated magnet. To estimate the amount of EGF-R not biotinylated (therefore not present on the plasma membrane), the first fraction was immunoprecipitated using an antibody specific for EGF-R.
Cell viability and proliferation
NSCLC cells were plated in 100 µl of RPMI with 5% FBS into 96-well cell culture plates (5,000 cells/well). Cell viability was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell MTT assay kit (Roche) according to the manufacturer’s instructions. The spectrophotometric absorbance was measured at 575 nm using a fluorescent multiplate reader (Polar Star Omega, Ortemberg, Germany). The results were expressed as mean ± SD of 3 independent experiments. In parallel, dye exclusions and cell count assays were performed.
Proliferation was analyzed by bromodeoxyuridine (BrdU) incorporation (FITC BrdU Flow kit, BD Pharmingen, San Diego, CA)/7-aminoactinomycin D (7-AAD, Sigma-Aldrich) staining followed by FACS analysis using a BD FACS Canto II instrument (Becton Dickinson, San Jose, CA) measuring 30,000 events for each sample.
We performed statistical analysis by Student t test. Values were considered statistically significant at P < 0.05 in 2-tailed tests. Survival curves were analyzed using the Mantel-Cox and Gehan-Breslow-Wilcoxon tests (Prism 5 software).