Our long-term objective is to elucidate how growth-regulatory signaling networks regulate the multiplication and survival of normal and neoplastic mammary epithelial cells. Our current studies focus on biological and biochemical functions of the oncoprotein encoded by the Tumor Susceptibility Gene 101 (Tsg101). Tsg101 is a key member of the endosomal sorting complex required for transport (ESCRT). This pathway is crucial for the transport, sorting, and lysosomal degradation of ubiquitinated cell surface receptors, in particular such as the EGFR and ErbB2 (Her2/neu) that are important receptor tyrosine kinases implicated in breast tumorigenesis and other human malignancies. A primary objective of our current research is to examine how Tsg101 engages with and modifies active ErbB-signaling complexes. In particular, we are interested in the function of Tsg101's intrinsic PTAP amino acid motif to regulate the activity of Tsg101 and its effect on the downregulation of oncogenic Her2/neu in normal and neoplastic mammary epithelial cells. Our central hypothesis is that altering the expression of Tsg101 or modifying its activity through inhibition of its self-regulatory PTAP domain will affect the onset and progression of Her2/neu-mediated mammary tumorigenesis. In the first specific aim of this proposal, we will determine how altering the expression level of wildtype Tsg101 modifies the growth properties of mouse and human mammary cancer cells in culture as well as tumor progression in vivo. We will also examine whether Tsg101 deficiency affects the survival of Trastuzumab-resistant human breast cancer cells. The second specific aim focuses on the mechanisms of the activation of Tsg101 that mediate an accelerated ubiquitination and degradation of the Tsg101 protein and its cargo (i.e. ErbB2). We will study the interaction of constitutively active Tsg101 with the ubiquitin ligase Tal as well as known members of the ESCRT complex. Additionally, we will use a proteomics approach to identify novel regulators for the activation of Tsg101. In the third specific aim, we will address whether activating Tsg101 will result in a more efficient downregulation of oncogenic ErbB2 in mammary cancer cells in vitro and in vivo. We propose the development of advanced genetically engineered models to predict the outcome of a possible targeted therapy against Her2/neu-mediated breast cancer by modifying the activity of Tsg101. Using these tools we might be able to herald the efficacy of this therapeutic strategy before pharmaceutical agents become available.
A significant subset of invasive breast cancers exhibit Her2/neu gene amplification with an unfavorable prognosis. The objective of this proposal is to elucidate how altering the expression of Tsg101 or modifying its activity will affect the onset and progression of Her2/neu-mediated breast cancer. The genetic and molecular studies outlined in this application will help to predict the potential value of Tsg101 as a therapeutic target before a drug will be developed and tested in preclinical trials.
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