PROVIDED. Over-expression of the receptor tyrosine kinase HER (ErbB2/Neu) plays an important role in breast carcinogenesis and response to therapy. The humanized monoclonal anti-HER2 antibody trastuzumab (Herceptin@), in combination with chemotherapy, extends survival of HER2+ breast cancer patients. Unfortunately, cancer often recurs following such regimens, and current treatment is unlikely to cure most patients. New combination approaches to trastuzmab therapy would be of substantial clinical beneiii. HER2+ tumors may resist trastuzumab therapy due to activation of other growth factor or cytokinesignaling pathways. The goal of this proposal is to develop a three-tiered "pipeline" approach for delineating new combinations of trastuzumab and other targeted signal transduetion inhibitors. In Aim 1, we use microarray and immunohistochemical techniques to delineate biomarkers for the early detection of optimal response to trastuzumab alone in a "brief exposure" setting. A Phase 1trial of trastuzumab plus the rapamyein analog CCI-779 will be evaluated in metastatic patients, and if the combination is found to be safe, moved to the brief exposure setting. Knowledge gained from the trastzumab exposure study will be used to assess the value of this and other combinations. In Aim 2, we will test combinations of trastuzumab and novel signal transduetion inhibitors in the drug development pipeline (Akt, Mek, PI3K, Jnk). We will also determine whether combination therapy can extend the therapeutic range of trastuzumab to breast tumor cells expressing low levels of HER2. In Aim 3 we will carry out a, high throughput siRNA screen kinase targets that enhance trastuzumab action, and a high throughput mutation screen for ErbB family members in HER2+ disease. We envision this pipeline will produce novel targets (Aim 3) that would progress to pre-clinical testing and prioritizationof promising drug candidates (Aim 2) that then move to rapid and efficient clinical testing (Aim 1). With these complementary approaches, the results of our research are likelyto have impact on the treatment of HER2+ breast cancer patients.