Drugs and Cell Lines
Lapatinib was provided by GlaxoSmithKline (Philadelphia, PA) and trastuzumab was provided by Genentech (San Francisco, CA) through a Materials Transfer Agreement.
The human MDA-MB-231-BR “brain-seeking” breast cancer cell line (hereafter referred to as 231-BR cells; kindly provided by Dr Toshyaki Yoneda, University of Texas at San Antonio, San Antonio, TX) was previously described (31
). The 231-BR cells were transduced to express enhanced green fluorescent protein (EGFP) and transfected to overexpress HER2 as described in Palmieri et al. (21
). Briefly, the retroviral vector pLEGFP-C1 (BD Biosciences, San Jose, CA) was transfected into the murine fibroblast PT67 packaging cell line. After 24 hours, EGFP-expressing cells were selected in the presence of 1 mg/mL G418 (Invitrogen, Carlsbad, CA) and colonies were expanded. EGFP virus was harvested from the PT67 cells and used to infect 231-BR cells. The following day, 231-BR cells were selected in the presence of 0.8 mg/mL G418. EGFP-expressing cells were then co-transfected with pCMV4.HER2 full-length human cDNA (kindly provided by Dr Dihua Yu, M. D. Anderson Cancer Center, Houston, TX) and pSVzeo to confer antibiotic resistance. The sequence of the HER2
insert in pCMV4.HER2 was confirmed by sequencing. Stable colonies were selected in the presence of 0.750 mg/mL zeocin. A vector control cell line was simultaneously established by transfecting both pCMV4 that lacked inserted cDNA and pSVzeo into the 231-BR-EGFP cells and selecting stable colonies in the presence of 0.750 mg/mL zeocin. The 231-BR cells that were transfected with vectors that contained or lacked the HER2
cDNA were maintained in Dulbecco's modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin–streptomycin solution (Invitrogen). The human breast cancer SKBr3 cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM with 10% FBS.
Anchorage-Independent Cell Proliferation
231-BR-HER2 and SKBr3 cells (10
000 per well) were plated in 1 mL of culture medium containing 0.3% (wt/vol) top agar in 24-well plates as described previously (21
). After 14 days in culture, colonies (>50 cells) were counted. Results are representative of three independent experiments, each performed in triplicate.
EGFR Gene Silencing by Transfection With Small Interfering RNA
231-BR-vector and 231-BR-HER2 cells (3 × 105) were seeded in 10-cm plates and incubated overnight. The cells were transiently transfected with a small interfering RNA (siRNA) targeted against the EGFR gene or a control siRNA containing a sequence not homologous to any human, mouse, or rat gene (siGENOME SMARTpool Human ERBB1 and siCONTROL Non-Targeting siRNA Pool, respectively; Dharmacon, Chicago, IL). siRNAs were transfected into cells using HiPerFect reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were seeded into 6-well plates (for immunoblot analysis) or 96-well plates (for cell proliferation assays).
Immunoblot Analysis of HER Family Members and Downstream Signaling Proteins
231-BR-vector, 231-BR-HER2, and SKBr3 cells (3 × 106) were seeded in 10-cm plates and serum starved overnight. The cells were then treated with 0.5 or 1.0 μM lapatinib (in dimethyl sulfoxide [DMSO]) or an equal volume of diluent (DMSO) for 24 hours, followed by stimulation with with 100 ng/mL epidermal growth factor (EGF; Peprotech Inc., Rocky Hill, NJ) for 10 minutes before lysing. The cells were lysed in RIPA buffer (20 mM Tris–HCl [pH 8], 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) containing Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche Molecular Biochemicals, Bassel, Switzerland) and Phosphatase Inhibitor Cocktails 1 and 2 (Sigma, St Louis, MO). Total lysates (50 μg per lane) were resolved by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunoblot analysis was performed using the following rabbit polyclonal antibodies (Cell Signaling Technology, Danvers, MA), all at a 1:1000 dilution: anti-HER2, anti-phospho HER2 (Tyr877), anti-phospho HER2 (Tyr1221/1222), anti-EGFR, anti-phospho EGFR (Tyr845, 992, 1045, and 1068), anti-p42/44 mitogen activated pathway kinase (MAPK), anti-phospho p42/44 MAPK (Thr202/Tyr204), anti-AKT, anti-phospho AKT (Ser473), anti-p38 MAPK, anti-PLCγ1, and anti-phospho PLCγ1 (Tyr771). In addition, the following rabbit monoclonal antibodies (Cell Signaling Technology) were used at a 1:1000 dilution: anti-HER3, anti-phospho HER3 (Tyr1289), and anti-phospho p38 MAPK (Thr180/Tyr182). Mouse monoclonal antibodies against p21 and tubulin were purchased from Calbiochem (Gibbstown, NJ) and used at a 1:1000 and 1:2000 dilution, respectively.
Horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used at dilutions of 1:5000. Antibody binding was detected using enhanced chemiluminescence (Cell Signaling Technology) and autoradiography.
Cell Proliferation Assay
231-BR-vector and 231-BR-HER2 cells were plated at a density of 5 × 103 cells per well in 96-well plates in DMEM plus 10% FBS and incubated overnight to allow cells to adhere to the substratum. The cells were treated with various concentrations (3–10 μM) of lapatinib or with DMSO (ie, the diluent for lapatinib) as a control. We determined the number of viable cells at 72, 96, and 120 hours after lapatinib addition by adding 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma) at a final concentration of 0.5 mg/mL to each well. After a 2-hour incubation at 37°C, DMSO was added to the wells to dissolve the cells and solubilize the MTT, and absorbance was measured at 570 nm. Data are shown as a percentage of the vehicle-treated control cells at each time point tested. Three separate experiments were performed, with six replicate wells for each data point.
Cell Migration Assay
Cell migration was examined with the use of 48-well Boyden chemotaxis chambers, as previously described (21
). Briefly, the top and bottom compartments of the chambers were separated by polycarbonate (polyvinylprrolidone-free) nucleopore filters (8-μm pore size, Neuro Probe, Gaithersburg, MD) coated with 0.01% collagen (BD Biosciences). FBS (1%) in DMEM was used as the chemoattractant in the bottom chamber. 231-BR-vector and 231-BR-HER2 cells were pretreated for 24 hours with lapatinib (1 or 3 μM) or diluent (DMSO). The pretreated cells (3 × 105
cells/mL) were added to the top chamber in DMEM supplemented with 1 or 3 μM lapatinib or diluent (DMSO). The chambers were incubated for 4 hours in a 37°C incubator with 5% CO2
. The chambers were disassembled and the filters were fixed and stained with the use of a Diff-Quik kit (Fischer Scientific, Pittsburgh, PA). Cells that had migrated to the undersurface of the membrane were counted with the use of a light microscope. Three separate experiments were performed with four replicate wells for each data point in experiment 1 and three replicate wells in experiments 2 and 3.
Mice and Imaging
Animal experiments were conducted under a National Cancer Institute–approved Animal Use Agreement. In two experiments, a total of 140 female BALB/c nude mice (5–7 weeks old; Charles River Laboratories, Frederick, MD) were anesthetized with isoflurane/O2 and injected in the left cardiac ventricle with 231-BR-vector or 231-BR-HER2 cells (1.75 × 105 cells in 0.1 mL serum-free medium; n = 35 mice per cell line per experiment). Lapatinib treatment began 5 days after cell injection. Mice were randomly assigned to receive vehicle (0.5% hydroxypropylmethylcellulose with 0.1% Tween 80 in water) or lapatinib (30 or 100 mg/kg body weight) twice daily by oral gavage for 24 days (n = 22–26 mice per treatment group). Mice were euthanized by CO2 asphyxiation at the end of treatment or when they showed signs of neurological impairment. The whole brain was removed from the skull and subjected to fluorescence imaging to detect the presence of the injected 231-BR cells. EGFP fluorescence was detected in whole brains with the use of a Maestro 420 In Vivo Spectral Imaging System (Cambridge Research and Instrumentation, Woburn, MA) and data acquisition and processing software that could distinguish or unmix images of fluorescence from multiple sources (Nuance Technology, Burlington, MA).
After fluorescence imaging, each brain was bisected along the sagittal plane and the left hemisphere was immediately frozen in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA); these samples were used for histology. The right hemisphere was fixed in 4% paraformaldehyde for 24 hours at 4°C, transferred to 20% sucrose and incubated overnight at 4°C, and then frozen; these samples were used for immunohistochemistry (see below). Brain sections (10 μm thick) were serially cut from the left hemisphere and stained with hematoxylin and eosin (H & E) according to standard procedures.
Ten H & E–stained serial sections every 300 μm through the left hemisphere of the brain were analyzed for the presence of metastatic lesions with the use of a Zeiss microscope outfitted with a 5× objective that contained an ocular grid with 0.8-mm2 squares. We counted micrometastases (ie, those ≤50 μm2) to a maximum of 300 per section and every large metastasis (ie, those >50 μm2) in each section. The >50-μm2 metric for large metastases represents the mouse equivalent of the proportion of a magnetic resonance imaging–detectable brain metastasis (5 mm) to the length of a human brain. All analyses were carried out by two investigators who were blinded to experimental group assignment. Two separate experiments were performed, and the data were pooled for statistical analysis.
Sections (5 μm thick) of frozen OCT-embedded mouse brains were fixed and permeabilized in ice-cold methanol. Sections from five mice per treatment group were then incubated overnight at 4°C with a primary antibody specific for a form of HER2 that is phosphorylated at tyrosine residues 1221 and 1222 (p-HER2, rabbit monoclonal, 1:25 dilution, Cell Signaling Technology) or a primary antibody specific for EGFR that is phosphorylated at tyrosine residue 1068 (p-EGFR, rabbit polyclonal, 1:25 dilution, Cell Signaling Technology). Binding of the primary antibodies was detected with the use of an EnVision+ HRP system (Dako, Carpinteria, CA) according to the manufacturer's instructions followed by hematoxylin counterstaining. The staining assay was confirmed by a pathologist (M.J. Merino), who compared the staining results in the mouse samples with those in human breast tumors that were known to overexpress HER2 or EGFR.
One stained section per mouse (n = 5 mice per treatment group) was evaluated at ×100 magnification. Each large metastasis and 25 randomly chosen micrometastases per section were scored. No more than three micrometastases were scored from any group of multiple micrometastatic lesions in a given region so that a representative sampling of the entire brain section could be obtained. Staining for p-HER2 and p-EGFR was scored on an intensity scale of 0–3+ (defined a priori): 0 corresponded to background staining intensity; 1+ lesions contained tumor cells with visible cytoplasmic and membrane staining above background but often contained some tumor cells that had background levels of staining (ie, unstained); 2+ staining appeared homogeneous and darker than 1+ staining; and 3+ staining was the darkest.
Generally, for both the in vitro and in vivo experiments, an analysis of variance (ANOVA) was performed on the data with experiment specified as the random effect. When appropriate, residuals were examined for normality with the Shapiro–Wilk test and were found to be normally distributed. For all ANOVAs, residuals were examined for homogeneity both graphically and numerically (the model with no variance partitioning was compared with models with partitioned variance with likelihood ratio test). Residuals were partitioned into homogeneous groups if heterogeneous variance was detected. P values for a priori comparisons to a control group were adjusted using Dunnett's method. Statistical significance was defined as P < .05. All statistical tests were two-sided. All analyses were performed with SAS statistical software (version 9.1; SAS Institute, Cary, NC).
For cell proliferation assays, a one-way ANOVA was performed, by time (72, 96, and 120 hours), with cell line (231-BR-vector, 231-BR-HER2) specified as the fixed effect. For analysis of cell proliferation in response to transfection with siRNA, a three-factor factorial ANOVA was performed, with lapatinib treatment (7, 8, 9, and 10 μM), cell line (231-BR-vector, 231-BR-HER2), and siRNA (EGFR, control) specified as the factors. Cell line by siRNA simple effect comparisons were specified a priori. For migration assay analysis, a one-way ANOVA was performed, by cell line (231-BR-vector, 231-BR-HER2), with lapatinib level (0, 1, and 3 μM) specified as the fixed effect. For the in vivo mouse experiments, data were pooled from two experiments and a two-factor factorial ANOVA was performed for each outcome, with cell line and lapatinib dose specified as the factors. A priori hypotheses tested were as follows: for each cell line 1) mean outcome was equal between lapatinib doses 0 and 30 mg/kg body weight and 2) mean outcome was equal between lapatinib doses 0 and 100 mg/kg body weight; and between the two cell lines 3) for each level of lapatinib, the mean outcome was equal between cell lines. For ANOVA of the immunohistochemisty data, we used a binomial distribution for the outcome variable and a logit link function. Higher-order effects (eg, three-way interactions) were dropped from the model if P was greater than .05.