The supportive data for this hypothesis includes the in vitro observations that these are genes induced when an EGFR Dorsomorphin BMP dependent cell line is freed from growth inhibition via EGFR inhibitors and the in vivo associations between the high expression of these signa tures and genes including HRAS, KRAS and EGFR itself. Regardless of the classical markers of activation of the http://www.selleckchem.com/Wnt.html EGFR RAS MEK pathway, the strong associations between these expression profiles and patient outcomes in two dif ferent data sets suggest that they are important profiles. Currently, we have chosen only to validate our profiles using additional microarray U0126 order data sets, as opposed to using western blots or quantitative PCR of the training set, since each of these signatures represents a large number of genesproteins. Conclusion The EGFR pathway is a complex signaling network and differences in gene expression levels of its various compo nents can be observed across the breast cancer subtypes. EGFR associated gene expression profiles derived in vitro were prognostic in two independent breast tumor data sets. Using these EGFR associated gene expression pro files, and gene expression levels of known genes within the EGFR pathway, we have identified key differences in this pathway across the subtypes. A better understanding of each subtypes EGFR signaling pathway will have an impact on identifying and determining treatment as the gene expression signature may more readily be associated with activation of the pathway than EGFR status alone. Methods Cell culture SUM102 and SUM149 cells were a gift from Steve Ethier of Wayne State University and represent cell lines derived from ER and HER2 basal like breast tumors.
The SUM cell lines were maintained in an Epithelial Growth Medium developed by the Tissue Culture Facility at the University of North Carolina at Chapel Hill, and the SUM149 line was further supplemented with 5% FBS. The MCF 7, ZR 75 1, HME CC and ME16C cell lines were obtained and maintained as previously described. Cytotoxicity assay Cell line sensitivities to drugs were assessed using a mito chondrial dye conversion assay as described previously with the following modifications. Cells were seeded into trip licate 96 well plates and allowed to adhere over night. Cells were treated for 72 h with a range of doses of individual drugs. Carboplatin, doxorubicin, 5 fluorour acil, paclitaxel, and LY294002 were purchased from Sigma. Gefitinib was a gift from Astra Zeneca and cetuximab was purchased from the UNC Hos pitals Pharmacy Storeroom. U0126 was purchased from Cell Signaling. The inhib itory concentration that caused a 50% reduction in MTT dye conversion dose was determined as previously described. Drug combination interactions were analyzed using methods developed by Chou and Talalay. Using cell lines plated as described above, seven treatment combina tions consisting of constant ratios of IC50 doses were applied to cells and growth compared to untreated controls using the MTT assay. Four treatment schedules were tested 72 h concurrent, 72 h inhibitor followed by 72 h chemotherapeutic, 72 h chemotherapeutic followed by 72 h inhibitor, and a 144 h concurrent dose with a media change at 72 h.