Project Summary It is estimated that approximately 90% of cancer mortality is the result of cancer cells gaining the ability to leave the primary tumor, activate survival mechanisms, invade surrounding tissues, enter the circulation and then exit by migrating into new tissues where they may form metastatic tumors, often after long latencies. Therefore, defining the signaling mechanisms that contribute to altered growth, metabolism, motility and survival associated with metastasis, are of critical importance. Our goal is to uncover the molecular basis of these signaling events using biochemical, cell biological and genetic approaches. It is well established that the Ras-MAP kinase/ERK pathway plays a role in most aspects of cancer cell biology. However, it remains unclear how ERK signaling can generate different cell fates. We have revealed the importance of subtle differences in ERK signal strength, location and duration as critical determinants of cellular outcomes. Additionally, we have more recently demonstrated that different ERK isoforms promote different cell fates. For example, ERK2 but not ERK1, plays a major role in promoting the epithelial to mesenchymal transition (EMT). Furthermore, we have found that different ERK2 docking domains, the CD domain and the DEF binding pocket (DBP), also regulate different cellular outcomes. Whereas low level, sustained wild type ERK2 activity promotes EMT, we discovered that ERK2 with CD domain mutations, which have been identified in several cancers and which signal through its DBP, robustly promotes the development of the EMT phenotype. This is the result of gain-of-function positive EMT signaling via the DBP and loss-of-function of negative inputs into EMT via the CD domain. It is therefore of critical importance to determine how ERK2 signaling promotes EMT and metastatic behavior. Understanding these mechanisms are part of the long-term goal of our basic research efforts to discover new potential targets and identify new biomarkers, and to help resolve this currently unmet clinical need of targeting the metastatic process. Thus, this grant proposes to investigate three novel areas associated with EMT and metastatic behavior and to take advantage of discoveries made during the previous funding period. In aim 1, we will investigate a unique connection between ERK2 and the histone H3.3 chaperone HIRA, how this regulates chromatin remodeling, and how this contributes to EMT in cells and in in vivo models. In aim 2, we will define the molecular basis for new links between ERK2 and the TGFb pathway as collaborators in the EMT process. In aim 3, we will define how ERK2 alters the metabolic landscape associated with EMT and determine how critical enzymes involved in a previously unknown link between amino acid metabolism and EMT are regulated and contribute to the invasive phenotype. In conclusion, there is an essential need for greater understanding of the mechanisms associated with EMT and metastatic behavior. Our expectations are that successful completion of the proposed work will impact cancer therapies through the identification of new biomarkers and novel drug targets that will yield small molecules that target the migration and survival of aggressive cancers.