Background HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ) a HER2-directed antibody used as a first line treatment for this disease. and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. In vivo studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions. Results The gefitinib and RAD001 combination inhibited cell growth in vitro in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated in vitro with cell line dependent increases in cytotoxicity and cytostasis while treatment in vivo promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (in vitro and in vivo) and EGFR signaling in vivo relative to the single drugs. Conclusions The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status. Background HER2 overexpression is present in 13-30% of all breast cancers [1 2 and it correlates with poor disease outcome high rates of metastasis and resistance to conventional treatment modalities [1-5]. Trastuzumab (TZ; Herceptin?) a Ammonium Glycyrrhizinate monoclonal antibody that targets the HER2 receptor and interferes with its function is effective in treating some HER2-positive breast cancers [6-8]. However many patients with HER2-positive disease are insensitive to TZ both as first line treatment or following a relapse after conventional chemotherapy [6-9]. Furthermore the majority of patients with metastatic disease that initially respond to TZ ultimately develop clinically relevant resistance to this agent [8 9 As TZ treatment has recently been expanded into the adjuvant setting [10] intrinsic and acquired resistance represents an important clinical problem that urgently awaits a discovery of novel drugs and development of innovative drug combinations to improve outcome for patients with advanced HER2-positive and TZ refractory disease. Numerous studies have demonstrated that HER2 is often co-expressed in breast cancers with epidermal growth factor receptor (EGFR) [1 5 8 11 It has been established that dimerization of HER2 and EGFR generates a potent signaling response mediated primarily through activation of the phosphatidylinositol 3-kinase (PI3K)/AKT and the RAS-Raf-mitogen-activated protein kinase (MAPK) pathways that sustain cancer cell growth proliferation and survival [5 8 Co-expression of EGFR and HER2 in breast cancer cell lines has been shown to induce drug resistance including resistance to TZ [17 18 and has been correlated with a negative prognosis for breast cancer patients [1 11 These data suggested that EGFR Ammonium Glycyrrhizinate constitutes an important therapeutic target in breast cancers and have prompted investigators to consider gefitinib (ZD1839 Iressa?) a reversible small molecule inhibitor of the EGFR tyrosine kinase for treatment of HER2 overexpressing and EGFR co-expressing breast malignancies [19]. The preclinical data have demonstrated that gefitinib exerts positive therapeutic effects in models of HER2 Ammonium Glycyrrhizinate overexpressing breast cancer which have been attributed to blocking activity of the Rabbit Polyclonal to CKLFSF1. PI3K/AKT and the MAPK pathways increased apoptosis induction of cytostasis through G1/G0 cell cycle arrest and downregulation of cyclin D1 as well as inhibiting angiogenesis [12-14 20 21 However our previous study conducted in animals bearing HER2 overexpressing MCF7-HER2 and MDA-MB-435/LCC6-HER2 breast cancer xenografts showed that gefitinib monotherapy results in only modest reduction of tumor volume [12]. The same study also showed that when gefitinib was used in combination with TZ the in vivo efficacy has been improved as judged by inhibition of tumor Ammonium Glycyrrhizinate growth but the data obtained by measuring multiple endpoints of therapeutic activity revealed that the combination was not beneficial [12]. These results have been recapitulated in a clinical trial demonstrating that the TZ and gefitinib combination should not be used for treatment in patients with HER2-positive breast cancer [19]. More recently it has been shown.