All animal experiments adhered to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the Mount Sinai School of Medicine and were performed according to the Office of Laboratory Animal Welfare (OLAW, National Institutes of Health) and Animal Welfare Act (AWA, United States Department of Agriculture) guidelines.
All peptides were purchased from Genscript (Piscataway, NJ). The high performance liquid chromatography reports indicated at least 92% purity and the peptide masses were confirmed by mass spectrometry. Antibodies for phospho-Akt (S473), Akt, phospho-Erk, Erk, phospho-JNK, JNK, phospho-p38, p38 and EGFR were purchased from Cell Signaling Technology (Danvers, MA). The phospho-EGFR Y1173 antibody was purchased from Millipore. The human mitochondria antibody was purchased from Abcam (Cambridge, MA). The EGFR-specific tyrosine kinase inhibitor (TKI; Cyclopropanecarboxylic acid-(3-(6-(3-trifluoromethyl-phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide) was purchased from Calbiochem (USA).
The MDA-MB-231, SK-BR-3, MDA-MB-435, MDA-MB-468, BT-474, DLD-1, A-549, MIA-PaCa-2 and SK-N-MC cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to ATCC guidelines. The Hep-G2 and HCT-116 cell lines were generously provided by Dr. Arthur Cederbaum and Dr. Stuart Aaronson, respectively, of the Mount Sinai School of Medicine, NY, were originally from the ATCC and cultured according to ATCC guidelines. The NR6 cells were generously provided by Dr. Alan Wells of the University of Pittsburgh, PA and cultured in MEM-α supplemented with non-essential amino acids, 7.5% FBS and antibiotics (penicillin/streptomycin) 
. The human mammary epithelial cell (HMEC) lines were established and generously provided by Dr. Martha Stampfer of Lawrence Berkley National Laboratory, CA 
. As described previously 
, HMEC lines were cultured in 50% mammalian epithelial growth medium (MEGM, Lonza, Allendale, NJ) and 50% DMEM/F12 medium with various supplements at 37°C and 5% CO2
. MEGM was supplemented with bullet kit (50%, Lonza, Allendale, NJ) containing transferring, isoproterenol and glutamine. DMEM/F12 media was supplemented with insulin, tri-iodothyronine, β-estradiol, hydrocortisone, fetal calf serum, EGF, glutamine and cholera toxin.
Cell Viability Assay
Cells were plated into a 96-well plate (5,000 to 10,000 cells per well) in full growth media. The following day, media is exchanged for serum starved media (0.1% BSA in growth media without FBS) and incubated overnight. Cells were treated for 24 hours with peptides at varying concentrations range (0.78 to 200 µM). The cell viability is read using luminescent cell viability dye (CellTiter-Glo, Promega Corporation, USA) by adding 20 µL of dye to each well containing 100 µL of treated media. The cell viability is calculated by dividing each luminescent reading by the average of the luminescent readings for control, untreated cells. Assays are run in triplicate. Dose-response curves were generated and fitted in Prism 5.0 (GraphPad Software, Inc., USA). The EC50
values were generated using the log inhibitor-normalized response variable slope function (Y
values are shown with standard deviation values (S.D.) from at least three independent experiments. For comparison of MDA-MB-231 with HMEC, the cells were not serum starved and plated and treated in HMEC media.
Fluorescent Confocal Microscopy
MDA-MB-231 cells (1×105 cells per plate) were plated on 35 mm glass-bottom plates (MatTek Corporation, Ashland, MA), allowed to adhere overnight, then serum starved overnight and analyzed the following day in HBSS (Hank’s balanced salt solution). For real-time uptake of the FAM-labeled peptides, after recording background images, the FAM-labeled peptides were added and cells were imaged every two minutes over approximately 30 minutes. After 30 minutes, images were taken less frequently. Images from representative timepoints are shown. For overnight treatment, the cells were treated in serum-starved media, exchanged into HBSS the following day and imaged.
Colony Formation in Soft Agar
Cells (1×105 to 2×105 per plate) were suspended in soft agar containing 5% serum and dosed with vehicle, Tat peptide or the TE-64562 peptide and allowed to grow for 2 to 3 weeks with periodic dosing to keep the dosing media fresh and the agar hydrated. Viable colonies were stained with iodonitrotetrazolium chloride at 0.5 mg/mL overnight. Colonies larger than 0.3 mm in each field were manually scored using a light microscope.
MDA-MB-231 cells were plated in 10 cm dishes, grown to 80% confluence and serum starved overnight. The TE-64562 peptide was added to at the indicated concentrations and incubated at 37°C for 18 hours (overnight). Cells were collected by trypsination, washed and suspended in binding buffer and stained according the manufacturer’s protocol (BD Biosciences, Annexin-V-FITC Apoptosis detection kit). Cells were analyzed on a FACscan instrument using BD Biosciences (USA) CellQuest software. For caspase-3 cleavage, treated cells were lysed using RIPA and analyzed by SDS-PAGE followed by Western blotting using an antibody specific for cleaved caspase-3 (Cell Signaling Technology, Danvers, MA).
MDA-MB-231 Xenografts in Nude Mice
NCR-nude female athymic mice were purchased from Taconic Farms, Inc. Mice were injected in the flank region with 1.5×106
MDA-MB-231 cells, while anesthetized with ketamine and xylazine. Prior to treatment, tumors were measured in three dimensions using a caliper and tumor volume was calculated by multiplying the three measured dimensions by 0.5 (V
0.5*l*w*h). Once the tumors reached a minimum size of 100 mm3
, mice were injected intraperitoneally, twice a week, with 200 µL of the TE-64562 peptide (40 mg/kg; 7 µmol/kg), Tat peptide in PBS (20 mg/kg; 7 µmol/kg) or PBS. Endpoints were defined as the tumor size reaching 2000 mm3
or 20 mm in any dimension, significant weight loss occurring or if the mouse appearing unhealthy according to body conditioning scoring standards. For histological analysis, organs were collected post-sacrifice and fixed in 4% formaldehyde in PBS, followed by paraffin embedding. Sections (6 µm thick) were mounted onto positively charged slides and H&E stained. Images were captured at 20× resolution on a Zeiss Axioplan 2 microscope.
EGFR-peptide Biotin-binding Assay
The EGFR constructs (ICD 645–998; ΔJMA 663–998; ΔJM 677–998) were obtained from the laboratory of Dr. Graham Carpenter of Vanderbilt University and described previously 
. SK-N-MC cells were plated in 10-cm dishes, grown to 90% confluence, transfected with the indicated EGFR construct using Lipofectamine 2000 (Invitrogen, USA) for 6 hours and serum starved overnight. The cells were treated with the biotinylated peptides for 2 hours, washed with phosphate buffer containing 500 mM NaCl pH 7.4, and then lysed by sonication in immunoprecipitation buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and protease inhibitor cocktail (Halt, Thermo Scientific, USA). The lysates were incubated with pre-washed, streptavidin-coated beads overnight at 4°C with rotation. For the reverse experiment, biotinylated peptides were incubated with streptavidin-coated beads for 2 hours at 4°C with rotation and washed. Transfected SK-N-MC cells were lysed, as above and incubated with the peptide-conjugated beads overnight at 4°C with rotation. The resulting bead-precipitates were washed and analyzed by Western blot with the indicated antibodies following standard procedures and visualized by chemiluminescence.
Cell Treatment and Lysate Collection for Western Blot Analysis
Cells were plated in 10-cm dishes, grown to 80% confluence, serum starved overnight, treated and analyzed. For dose response assays, the peptides, EGFR-specific tyrosine kinase inhibitor (TKI) or control was added to each plate and incubated at 37°C for 30 minutes, followed by EGF (10 ng/mL) for an additional 10 minutes at 37°C. For timecourses, TE-64562 and/or EGF were added and the cells were incubated at 37°C for the indicated amounts of time. For EGFR dimer detection, cells were cross-linked after treatment using BS3 crosslinker (bis[sulfosuccinimidyl] suberate, Thermo scientific, Inc) according to the manufacturer’s instructions. Immediately following treatment, cells were put on ice, washed twice with cold Tris-buffered saline (TBS, pH 7.4) and lysed with radio-immunoprecipitation buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA and protease inhibitor cocktail). After protein concentration determination, cell lysates were analyzed by Western blot analysis with the indicated antibodies following standard procedures and visualized by chemiluminescence. Images were quantified using ImageJ Version 1.43 u.
Immunofluorescent and Western Blot Analysis of Tumor Tissue
Nude mice bearing subcutaneous, MDA-MB-231 xenographic tumors were injected with the TE-64562 peptide (40 mg/kg; 7 µmol/kg), Tat-peptide (20 mg/kg; 7 µmol/kg) or vehicle (saline), intraperitoneally for four days, once per day. On the last day, the mice were injected 30 minutes prior to extracting the tumor. For immunostaining, resected tumors were snap-frozen in isopentane submerged in liquid nitrogen and sectioned onto positive slides. Unstained frozen sections were fixed for 15 minutes in ice-cold acetone, dried, rehydrated in PBS and blocked in TBS containing 1% BSA, 10% goat serum and goat anti-mouse FAb (Jackson Immunoresearch, West Grove, PA) for 1 hour, followed by overnight (4°C) incubation with primary antibodies for phospho-Akt or phospho-Erk. After washing, Alexafluor 568-Goat anti-rabbit secondary antibodies (Invitrogen, USA) were incubated with the tissue for 1 hour at RT, followed by DAPI (Hoescht 33342, Molecular Probes, USA) staining. Staining was visualized using an Olympus MVX10 Macroview microscope with a 2X Apochromat lens with 5× zoom. Images were constructed into a montage using fluorescent tiling in the Olympus MicroSuite Biological Suite software.
For Western blot analysis, a 2 to 3 mm cross-sectional slice of the tumor was lysed in RIPA buffer by sonication and the resulting lysates were analyzed by Western blot following standard methods. Since samples contained both mouse and human tissue and cells, connective tissue and blood samples were taken from the mouse for comparison. The mouse samples have a high amount of total Erk (p44) and a negligible amount of basal phospho-Erk. In order to compare the level of phospho-Erk to the human tissue, the phospho-signal was normalized to a human tissue marker (human mitochondria).
TCGA Data Analysis
We utilized protein expression level data provided through the TCGA for breast invasive carcinoma (BRCA) for total EGFR (antibody reference: EGFR-R-C) and phospho-EGFR (antibody reference: EGFR_pY1173-R-C) for 354 individuals. The values were normalized across the population such that the average is zero and the standard deviation is one for both the total and phosphor-EGFR expression. Two sets were obtained by separating individuals that had a normalized total EGFR level more than one standard deviation above the average but a normalized phosphor-EGFR level below one standard deviation above the average (20). Two individuals that had total EGFR levels more than 6.62 and 5.67 standard deviations away from the average level were excluded to give a remaining set of 320.
Plots and statistics were generated using Prism 5.0 (GraphPad Software, Inc.). Unless otherwise indicated, one-tailed, nonparametric Mann-Whitney tests (95% confidence interval) were used to determine if the mean values for each treatment condition were significantly different from control groups. P values are reported for each analysis in the figure legends, P values of 0.05 (or less) were considered significant.