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    Research Grants Awarded

    Synergy Between ErbB2 And The Breast Tumor Kinase Brk

    Study Section:
    Tumor Cell Biology V

    Scientific Abstract:
    Background : Constitutive activation of tyrosine kinase signaling is believed to play a crucial role in breast cancer. The receptor tyrosine kinase ErbB2/HER-2 is amplified in 20 to 30% of human primary breast cancers, and expression of ErbB2 in tumor tissues is correlated with poor clinical prognosis. Brk (Breast tumor kinase) is a nonreceptor tyrosine kinase that was identified in a study of kinase expression in human metastatic breast tumors. Recent data from our laboratory and from others indicate that ErbB2 and Brk are overexpressed simultaneously in breast cancer, and synergize to promote tumor formation. Hypothesis : We hypothesize that Brk and ErbB2 synergize in breast cancer cells by means of an interaction between autophosphorylation sites on ErbB2 and the SH2 domain of Brk. This interaction activates Brk towards specific downstream signaling proteins. Specific Aims : (1) We will investigate the synergy between Brk and ErbB2 using a mouse in vivo tumor transplantation model. (2) We will study the biochemical basis for the interaction between ErbB2 and Brk. (3) We will carry out proteomic experiments to identify proteins that interact with Brk in cells co-expressing Brk and ErbB2. Study Design : For Specific Aim 1, we will collaborate with another group to use the mammary fat pad transplantation assay. We will measure tumor formation after transplanting cells expressing ErbB2 or Brk alone, or cells expressing both tyrosine kinases. For Aim 2, we will test for activation of Brk in ErbB2/Brk-coexpressing cells. We will produce phosphopeptides based on ErbB2 autophosphorylation sites, and study their interaction with the SH2 domain of Brk. We will also carry out experiments with mutant forms of ErbB2 lacking specific phosphorylation sites. For Aim 3, lysates from cells expressing FLAG-tagged Brk and ErbB2 will be subjected to anti-FLAG immunoprecipitation, and proteins will be identified by mass spectrometry. We will also use proteomics and phage display experiments to identify proteins that specifically interact with the SH3 domain of Brk. Potential Outcomes and Benefits of the Research : The goal of this project is to understand the biochemical basis for the synergy between Brk and ErbB2, and to elucidate the signaling pathways that emanate from this complex. The information provided by these studies will be important in determining how Brk activity is deregulated in breast cancer, and may provide a framework for strategies to block the activity of Brk.

    Lay Abstract:
    Background : Normal cells respond to signals from their environment that cause them to grow and divide. These signals are sensed and processed by cellular proteins such as receptors, enzymes, and regulatory proteins. Tyrosine kinases are enzymes found in mammalian cells that are components of these signaling systems. In normal cells, tyrosine kinases are under tight control; they are activated transiently in response to specific stimuli. In many forms of cancer, however, tyrosine kinases are inappropriately active. This project investigates two tyrosine kinases that are overproduced in breast cancer cells: ErbB2 and Brk. ErbB2 is amplified in 20 to 30% of human primary breast cancers, and high production of ErbB2 in tumor tissues is correlated with poor clinical prognosis. Brk (Breast tumor kinase) is a tyrosine kinase that was identified in a study of kinases in human metastatic breast tumors. Recent data from our laboratory and from others indicate that ErbB2 and Brk are overproduced simultaneously in breast cancer, and act together to promote tumor formation. Hypothesis : We hypothesize that Brk and ErbB2 act synergistically in breast cancer cells by means of an interaction between the two kinases. This interaction increases the activity of Brk towards specific signaling proteins. Specific Aims : (1) We will investigate the synergy between Brk and ErbB2 by tumor transplantation experiments in mice. (2) We will study the biochemical basis for the interaction between ErbB2 and Brk. (3) We will carry out experiments to identify proteins that interact with Brk in cells co-expressing Brk and ErbB2. Study Design : For Specific Aim 1, we will collaborate with another group to use the mammary fat pad transplantation assay. We will measure tumor formation after transplanting cells expressing ErbB2 or Brk alone, or cells expressing both tyrosine kinases. For Aim 2, we will test for activation of Brk in ErbB2/Brk-coexpressing cells. We will also test for a direct interaction between the two tyrosine kinases. For Aim 3, we will use mass spectrometry to identify proteins that interact with Brk in cells that express both ErbB2 and Brk. Potential Outcomes and Benefits of the Research : The information provided by these studies will be important in determining how Brk activity is deregulated in breast cancer, and may provide a framework for strategies to block the activity of Brk.