SKBr3 cells were obtained from ATCC and maintained at 37°C and 5%CO2 in DMEM:Ham's F12 1:1 media supplemented with 10% heat-inactivated Fetal Bovine Serum, 100 units/ml penicillin, 100 ug/ml streptomycin, and 4mM L-glutamine. LY29004, wortmannin, and 4-OH-tamoxifen were from EMD/Calbiochem. All compounds were reconstituted in DMSO, and 4-hydroxy-tamoxifen was reconstituted in ethanol. BEZ235 was obtained from Novartis. Lapatinib and erlotinib were purchased as tablets and the active ingredient purified by organic extraction as described in the supplementary materials section. In all TKI treatment experiments extending longer than 24 hours, the media was replaced with fresh media containing freshly mixed TKI every 24 hours.
Cell growth was assayed by seeding 20,000 cells per well in a 24-well cluster plate. After 24 hours, cells were treated with 200nM lapatinib or vehicle only. Media with fresh drugs was changed every 24 hours, and daily cell counts from triplicate wells were obtained for 7 days with a hematocytometer. Cell growth is reported as a percentage of the cell count obtained at time point “0”.
Total cellular lysates were obtained by harvesting cells in modified radioimmunoprecipitation assay (RIPA) buffer supplemented with leupeptin, aprotinin, phenylmethylsulphonyl fluoride, sodium vanadate and phosphatase inhibitor cocktail (Roche). Western blotting was performed by separating 50 ug of lysates on an SDS-PAGE, transferring to polyvinylidene fluoride membrane, and immunoblotting with the indicated antibodies followed by enhanced chemoluminescence visualization. Immunoprecipitation of HER2 was performed with antibody to HER2 (SC-284) (Santa Cruz Biotechnology). Antibodies used for western blot analysis were against p-Y1248-HER2, pS473-AKT, pT202/Y204-MAPK, MAPK, AKT, p-S235/236-S6, S6 ribosomal protein (Cell Signaling), β-actin, HER2 SC-284, HER3 5A12, p-Tyr PY99 (Santa Cruz Biotechnology) and p-T246-PRAS40 (Biomol). Polyclonal antibodies against pY1289-HER3 were generated in rabbits with a phosphopeptide spanning Y1289, and antisera were affinity purified over a phosphopeptide column and counter-purified over a non-phosphopeptide column. The phosphospecificity of the antibody was verified in cells treated with phosphorylated or non-phosphorylated HER3 and treated with heregulin or lapatinib, or with overexpression of HER3.
Phospho-RTK profiling was done with the R&D systems Proteome Profiler Phospho-RTK Antibody Array according to manufacturer's instructions. Briefly, the array membranes were blocked in blocking buffer and incubated with diluted cell lysates overnight at 4C. After washing, the array membranes were incubated with the detection antibody, washed again, and developed with standard chemiluminescent reagents.
Apoptosis was assayed by fluorescence-activated cell sorting (FACS) analysis of nuclear degradation as described (39
). In brief, cell nuclei were prepared and stained with ethidium bromide, and DNA content analyzed on FACS CaliburII with Modfit software. Apoptotic cells were identified by their Sub-G1 DNA content as analyzed by Modfit, and data averaged over triplicate experiments.
Total cellular RNA was isolated using the RNeasy miniprep kit with on-column DNAse treatment as per the manufacturer's protocols (Qiagen). Reverse transcription and real-time PCR amplification was perfomed as described using the IQ SybrGreen Supermix on a MyIQ I-cycler (BioRad) (40
). Normalization was performed against GAPDH or β-microglobulin and relative expression was obtained using Pfaffl ratios. Data represents average from triplicates. The primers used were 5’CCCTGCCATGAGAACTGCAC and 5’TCACTGTCAAAGCCATTGTCAGAT for HER3, 5’AACTGCACCCACTCCTGTGT and 5’TGATGAGGATCCCAAAGACC for HER2, 5’GGTCTCCTCTGACTTCAACA and 5’AGCCAAATTCGTTGTCATAC for GAPDH, 5’TGCTGTCTCCATGTTTGATGTATCT and 5’TCTCTGCTCCCCACCTCTAAGT.
SkBr3 cells were engineered to express an inducible form of Akt. This construct was generated previously by fusing a myristylated Akt to a mutated ligand binding domain of the estrogen receptor (myrAktΔER) (41
). Myristylated Akt is constitutively active, however the myrAktΔER fusion construct is auto-inhibited by the ER fragment. Treatment with the ER ligand 4-hydroxy tamoxifen (4HT) relieves the auto-inhibition leading to activation of myrAkt. Negative control is provided by the identical construct containing an inactivating mutation within the myristylation sequence (myr*AktΔER). SkBr3 cells were infected with retroviral particles generated with the pWZLneo-myrAkt-ΔER or the pWZLneo-myr*Akt-ΔER vectors and selected in neomycin.
Where indicated, statistical analysis was performed with t-tests based on two-tailed distribution and unequal variance; the calculated p values are stated in the figure legends.
Mouse experiments were done under an institutional IACUC approved protocol. Briefly 2×106 HCC1569 cells were orthotopically implanted into the mammary fat pad of 7-9 week old female nu/nu mice and allowed to grow into tumors. When tumors reached approximately 100mm3, mice were randomized and treated according to the experimental arms. Lapatinib was administered as a suspension in 0.5% hydroxypropylmethylcellulose, 0.2% tween-80 by oral gavage in two daily doses. Tumor sizes were measured once or twice weekly with calipers.
For biochemical and plasma analysis, some mice were sacrificed 4 hours after the morning dose of the 5th
day, the tumors rapidly dissected and flash frozen, and the plasma collected and frozen. Tumor lysates were prepared in RIPA buffer and western blotting performed as described. Plasma lapatinib concentrations were analyzed using a previously described method (43
) with a sensitivity of 1 ng/ml and a precision and accuracy within 15%.