The limited experimental evidence available shows that, in cancer cells, a cross regulation between FASN and HER2 exists, and also that pharmacological blockade of FASN with C75 can overcome acquired resistance to trastuzu mab. We have recently described a novel family of anti FASN compounds that exhibit in vitro anticancer activ ity, which do not exhibit customer reviews cross activation of b oxidation, and do not induce weight loss in animals. In the current study, we have characterised molecularly the in vivo anticancer activity of G28UCM in a model of FASN HER2 breast carcinoma. In addition, we have evaluated the pharmacological interaction Inhibitors,Modulators,Libraries of G28UCM with anti HER drugs, such as trastuzumab, lapatinib, erlotinib, gefitinib or cetuximab, at the cellular and molecular levels.

Finally, we report the effect of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib. Our data support the study of G28UCM as a potential therapeutic agent, either alone or in combi nation, against in vivo HER2 tumours that have pro gressed on trastuzumab and lapatinib. Materials and methods Chemicals, reagents and antibodies Erlotinib, gefitinib Inhibitors,Modulators,Libraries and lapatinib were provided by Roche, AstraZeneca and Glax oSmithKline, respec tively, and were restored in dimethyl sulfoxide, diluted in culture medium at 1,10,000 and stored at 20 C. Trastuzumab and cetuximab, provided by the Division of Pharmacy of the Catalan Institute of Oncol ogy, were directly diluted in cell culture medium at 1,1,000 or 1,10,000 and were stored at 4 C. EGCG, EDTA, dithiotreitol, Inhibitors,Modulators,Libraries acetyl CoA, malonyl CoA, NADPH and 3,4,5 dimethylthiazol 2 yl Inhibitors,Modulators,Libraries 2,5 diphenylte trazolium bromide were purchased from Sigma.

The primary antibody for FASN immunoblotting was a mouse IgG1 FASN monoclonal antibody from BD Biosciences Pharmingen. Monoclonal anti b actin mouse antibody was from Sigma. Rabbit monoclonal anti bodies against mTOR and phospo mTORSer2448 Inhibitors,Modulators,Libraries were monoclonal p185HER 2 neu were from Cell Signaling Technology. Peroxidase conjugated secondary antibody was from Calbiochem. 1,3 bis oxy naphthalene was synthesized as previously described. Cell culture and cell lines BT474 and AU565 breast carcinoma cells were obtained from the American Type Culture Collection. BT474 cells were cultured in DMEM F12 supplemented with 10% heat inactivated fetal bovine serum, 1% L gluta mine, 1% sodium pyruvate, 50 U mL penicillin, and 50 ug mL streptomycin.

AU565 cells were routi nely grown in Dulbeccos Modified Eagles Cisplatin FDA Medium supplemented as above. Trastuzumab resistant cells were developed by exposing AU565 cells continuously to trastuzumab for six months. Cells per plate were then pooled together and sensitivity to trastuzumab was determined by treating AU565 par ental and resistant cells with 2 uM trastuzumab and performing trypan blue exclusion assay periodically during 10 days.