PROJECT 2: PROJECT SUMMARY We have found that the epidermal growth factor receptor (EGFR) is required to sustain activation of the oncogene STAT3 in response to IL-6 or oncostatin M (OSM), which signal through the common gp130 receptor. In the normal inflammatory response, the initial activation of STAT3 in response to IL-6 or OSM is soon down regulated by the STAT3-induced inhibitor SOCS3, a potent negative regulator of gp130 and JAK2. However, in cancer cells, STAT3 phosphorylation is re-established and sustained in a second wave of activation. A novel complex is formed, involving IL-6, IL-6 receptor, gp130, STAT3 and EGFR, in which Y1068 of EGFR, the known STAT3 docking site, is phosphorylated, thus facilitating the re-activation of STAT3 by a SOCS3-independent mechanism. We now seek to understand in detail how this second wave gp130-EGFR complex is formed and how it drives not only STAT3 phosphorylation, but also additional downstream signals that emanate from EGFR in response to activation by gp130 ligands. We also wish to investigate the functional importance of the second wave in cancers that rely heavily on the ability of gp130 ligands such as IL-6 and OSM to activate STAT3. In glioblastoma (GBM), we wish to understand the importance of EGFR in sustaining STAT3 activation through the mechanism we have identified. In breast cancer, we can now identify the gp130- EGFR interaction as an important contributor to an epithelial-mesenchymal transition (EMT) that is driven by OSM, leading to the acquisition of cancer stem cell (CSC) properties. Elevated OSM in the microenvironment is associated with increased risk of recurrence, while increased levels of OSM receptor on cancer cells are associated with poor outcomes. We hypothesize that prolonged STAT3 activation, facilitated by the gp130- EGFR interaction, is a key contributor to poor outcome in patients harboring tumors with elevated OSM. In Aim 1, we will evaluate many details of the novel interaction between gp130 and EGFR. Which tyrosine residues of EGFR are phosphorylated in the second wave complex and which downstream pathways do they drive? Which proteins are present in the complex and what are the functions of each? Which genes are activated by prolonged phosphorylation of STAT3 in the second wave and how do the encoded proteins contribute to tumorigenesis and EMT? In Aim 2, the consequence of suppressing or activating the second wave on the ability of OSM to induce EMT, cancer stem cell expansion, and resistance to chemotherapy will be assessed. In Aim 3, we will use syngeneic, orthotopic mouse models of triple-negative breast cancer and glioblastoma to determine the importance of how the presence of immune cells within the tumor microenvironment impacts the second wave and affects tumorigenesis in general. Our studies will significantly advance current understanding of how OSM and IL-6, derived from the tumor microenvironment, drive aberrant STAT3 and EGF...