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Identification of Substrates of the Her2/neu Tyrosine Kinase in Breast Cancer Cell Lines using Chemical Rescue of a Her2/neu Catalytic Loop Point Mutation
Her2/neu is an EGF receptor family member that is overexpressed in 20-30% of human breast cancers and has both prognostic as well as therapeutic importance in the clinical management of breast cancer. Her2/neu tyrosine kinase activity is largely ligand independent and this constitutive activation has created difficulty in characterizing in vivo substrates. While much is known about downstream signaling proteins activated by other EGF receptor family members, the signaling pathways of Her2/neu in epithelial cells have not been fully defined. Inhibition of baseline Her2/neu activity in a manner that can be rescued by small molecule activators would offer a powerful means to study the Her2/neu signaling pathway in cell culture. The mentor’s laboratory has established that a point mutation of the catalytic loop arginine in the tyrosine kinase, Csk, reduced the enzymatic activity in a manner that was reversible by addition of small, cell-permeable molecules such as imidazole and has utilized this mutation in transfected cell lines to study the biology of Csk. As Csk and Her2/neu have identical catalytic loop protein sequences, this chemical rescue approach is also predicted to be applicable to Her2/neu. Construction and characterization of such a mutant Her2/neu protein will be performed. Using transfected breast cancer cell lines, candidate Her2/neu substrates will be assessed by immunoprecipitation experiments and novel substrates will be identified and sequenced by mass spectroscopy. The further elucidation of Her2/neu substrates and downstream signaling proteins in an experimental system relevant to breast cancer would be of considerable importance to both basic science and clinical oncology. Potential benefits from this study include the identification of novel drug targets or biomarkers in the Her2/neu pathway. These biomarkers may define patients who have Her2/neu activation in the absence of conventional Her2/neu overexpression and enhance our understanding of pharmacologic inhibition of the pathway, thereby expanding the role and clinical benefit obtained from Her2/neu directed antibodies and small molecule inhibitors.
Elevated levels of the protein, Her2/neu, is found in 20-30% of breast cancers and negatively affects the prognosis of these affected women. Her2/neu functions in the cell as a protein kinase and drives cancer cell proliferation by phosphorylating key cellular proteins. An antibody directed against Her2/neu, called Herceptin or Trastuzumab, is in clinical use and yields significant tumor shrinkage in 35% of treated women. While these key cellular proteins which mediate the action of Her2/neu have been studied for many years, this research has been hampered by the lack of ability to rapidly and directly control the activity of the Her2/neu kinase. Development of an experimental system in which Her2/neu kinase activity could be switched on and off by a simple chemical substance would greatly facilitate further study of this protein. The sponsor’s laboratory has developed a method to chemically control the activity of other protein kinases in cell culture. In this project, we propose to apply this method to Her2/neu and further study the proteins it phosphorylates. We will first make the necessary mutation in the Her2/neu protein by standard molecular biology techniques and then obtain purified Her2/neu protein for testing. Next, we will introduce this mutation into breast cancer cell lines that are maintained in tissue culture and identify the proteins phosphorylated by Her2/neu through the use of antibodies and mass spectroscopy, a powerful modern technique that can characterize minute quantities of proteins. Through these experiments, we hope to identify new proteins which are vital for Her2/neu’s function. Once identified and characterized, these proteins may themselves be targets for new chemotherapy drugs or may be markers of the effectiveness of Herceptin therapy. Through further studies on Her2/neu, we hope to change the prognosis of this type of breast cancer.