Further, we investigated the antitumor activity of AZD8931 alone or in combination with paclitaxel in EGFR-overexpressed and HER2 non-amplified IBC models. Methods and materials Reagents and cell culture AZD8931 was synthesized and generously provided

by AstraZeneca [16]. SUM149 were obtained from Dr. Stephen Ethier (Kramanos Institute, MI, USA) and #CB-839 solubility dmso randurls[1|1|,|CHEM1|]# are commercially available (Asterand, Detroit, MI). SUM149 cells were cultured in Ham’s F-12 media supplemented with 10% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 5 μg/ml insulin and antibiotic-antimycotic. The FC-IBC-02 tumor cells were derived from primary human breast cancer cells isolated from pleural effusion of an IBC patient [14, 15]. Human samples used in this study were acquired with approval of the Fox Chase PF-562271 chemical structure Cancer Center’s Institutional Review Board. Importantly, written

informed consent was obtained from each participant. FC-IBC-02 cells were cultured in DMEM/F12 media with 10% FBS and 1% L-glutamine and antibiotic-antimycotic. Antibodies and immunoblot Following treatment with AZD8931 at the indicated concentration and time points, immunoblotting was performed as previously described [15]. In brief, cells were lysed in 1× lysis buffer (Cell signaling), and then the supernatant was collected by centrifuging at 10,000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA protein assay reagent kit (Pierce, Rockford, IL). Equal amounts of protein from cell lysates were resolved by SDS-PAGE electrophoresis. The membranes were incubated at 4°C overnight with the following antibodies: mouse anti-EGFR (1:1000; Cell Signaling), rabbit anti-AKT and rabbit anti-phospho-AKT (1:1000; Cell Signaling), mouse anti-β-actin (1:5,000; Santa Cruz). After incubation with anti-mouse IgG horseradish peroxidase conjugated secondary antibody (1:5,000; Amersham Pharmacia Biotech), immunoreactive proteins were visualized by the enhanced chemiluminescence reagents. Cell proliferation and apoptotic assay SUM149 and FC-IBC-02

cells (2 × 103) were seeded in triplicate in a 96-well plate and cultured overnight. Cells were treated TCL with AZD8931 at indicated concentration for 72 hrs. Cell proliferation was monitored at the indicated times, absorbance at 490 nm was measured using a microplate reader using the MTS assay (CellTiter 96 AQueous One Solution cell proliferation assay, Promega) according to the manufacturer’s instruction. Apoptotic cells were measured by Annexin V staining. Cells (1 × 105) were treated with 1 μM AZD8931 for 48 and 72 hrs. Cells were harvested and labeled with Annexin V-PE and 7-amino-actinomycin D (7-AAD) (Guava Technologies Inc, Burlingame, CA) according to the manufacturer’s instructions. The samples were then analyzed by Guava system on a GuavaPC personal flow cytometer (Guava Technologies).