Transglutaminase-2 (TGase-2) is a GTP-binding protein/protein crosslinking enzyme implicated in various pathological conditions including coeliac disease, neurodegenerative disorders, and aggressive cancers. The mechanistic basis by which TGase-2 contributes to many of these disorders is poorly understood. Recently, we obtained exciting new clues regarding the roles of TGase-2 in human cancers by identifying it as a unique EGF receptor (EGFR)-signaling partner. Specifically, we found TGase-2 enhances EGFR expression by protecting EGFRs against the actions of c-Cbl, as well as functions as a novel scaffold to mediate PI-3K activation. TGase-2 expression is inversely correlated with brain cancer survival, and it is specifically expressed together with an oncogenic variant of the EGFR, the EGFRvIII, in a highly aggressive subset of glioma initiator (stem) cells (GSCs), called mesenchymal (MES) GSCs. Moreover, TGase-2 and EGFRvIII are among the cargo of microvesicles (MVs) shed by MES GSCs, offering a potential mechanism by which MES GSCs transfer these important signaling proteins to surrounding cells to re-program their microenvironment. In this proposal, we will build on these discoveries to establish the mechanisms by which TGase-2 contributes to disease states. We will focus on its role in glioblastoma (GBM) through the combined efforts of two laboratories with complimentary expertise in EGFR-signaling (Cerione) and translational studies of GSCs (Nakano), as follows: 1) Test the hypothesis that TGase-2 plays novel roles in the actions of the oncogenic EGFRvIII and other receptors implicated in GBM. Based on our recent findings, we will determine whether TGase-2 adversely affects patient survival by protecting the EGFRvIII, as well as other receptors linked to GBM, against c-Cbl-catalyzed degradation and/or by enhancing their signaling to PI-3K/Akt/mTORC1. 2) Test the hypothesis that TGase-2 and the EGFRvIII play an essential part in the intercellular signaling mediated by MVs from MES GSCs. We will determine whether this extremely aggressive sub-set of GSCs use MVs to ‘communicate’ with less aggressive GSCs or with differentiated cancer cells to re-program them for tumor formation. 3) Test the idea that targeting TGase-2 through different strategies blocks the actions of MES GSCs in mouse models for GBM. The expectation is these studies will be broadly relevant toward understanding the biological roles of TGase-2 in disease, as well as identify novel therapeutic targets for EGFR/EGFRvIII-driven cancers such as GBM.