PROJECT SUMMARY/ABSTRACT Phenotypic plasticity and its regulation by contextual signals is of central importance to tumor biology. TGFβ is a major regulator of cell phenotype during development, tissue homeostasis, regeneration, and cancer. Our long- term goal is to elucidate TGFβ signaling and the principles that govern its effects on normal and neoplastic cells. This proposal is based on our long-standing contributions to delineating the TGFβ signal transduction pathway, its context-dependent effects, and its aberrant activity in tumorigenesis and metastasis. The proposed work builds on recent progress towards understanding how TGFβ-activated SMAD transcription factors regulate differentiation in stem and progenitor cells (Aragón et al Genes Dev. 2019; Wang et al Cell Stem Cell 2017), the basis for TGFβ-mediated tumor suppression and the evasion of this effect (David et al Cell 2016; Huang et al Cancer Disc. 2019), and the development of experimental models of dormant metastasis to expose the role of TGFβ in this poorly understood, yet highly significant aspect of cancer (Malladi et al Cell 2016). Moreover, we recently elucidated how TGFβ triggers epithelial-mesenchymal transitions (EMTs) in pancreatic ductal adenocarcinoma (PDA), lung adenocarcinoma (LUAD), and embryonic stem (ES) cells, and how these phenotypic plasticity events are coupled either to fibrogenesis or to differentiation depending on the epigenetic context (Su et al Nature 2019). Based on these advances and unique experimental models and human tumor single-cell analytics that we have developed, we will address long-standing questions of growing importance: How does TGFβ signaling regulate epithelial cell plasticity in development and cancer? What is the role of TGFβ-induced intra-tumoral fibrosis during tumorigenesis? What is the relevance of this mechanism to TGFβ-induced organ fibrosis? How does TGFβ drive metastasis-initiating cells into EMT-linked growth arrest? Does this state render cancer cells immune-evasive during metastasis dormancy? To investigate these questions, we will dissect an obscure RAS effector, RREB1, which we recently identified as a key partner of TGFβ-activated SMAD transcription factors in the induction of fibrogenic and developmental EMTs. We will elucidate the role of EMT-linked intra-tumoral fibrosis in tumor growth and metastasis. Focusing on metastasis- initiating cells, we will follow recent evidence that TGFβ imposes a quiescent, immune evasive state that provides long-term survival to dormant metastasis cells and potentially resistance immunotherapy. Collectively, these studies will provide knowledge and experimental models to delineate the role of TGFβ in fibrosis, tumor invasion and metastasis, and will better define how and when to target TGFβ in cancer.