PROJECT SUMMARY (unchanged from Parent R37) Metastasis causes >90% of cancer death. The persistence and lethality of metastasis is driven by cells capable of self-renewal, slow cell-cycling, tumor re-initiation, and therapy resistance, termed metastasis stem cells (MetSCs). Development of effective strategies for eliminating metastasis requires a better understanding of the mechanisms that MetSCs exploit for survival. We recently demonstrated that (1) disseminating colorectal cancers (CRC) undergo a dynamic phenotypic switch from an LGR5+ tumor-initiating cancer stem cell (CSC) state to a distinct LGR5lowL1CAM+ state required for metastasis. (2) L1CAM+ MetSCs are functionally distinct from intestinal tumor-initiating LGR5+ CSCs: L1CAM is required for organoid formation, the regeneration of intestinal epithelium after colitis, and tumor formation after metastatic dissemination. But unlike LGR5, it is dispensable for epithelial homeostasis or intestinal tumor initiation. In contrast to tumor initiation, where homeostatic stem cells undergo oncogene-driven hyper proliferation in intact tissues, metastasis subverts a regenerative mechanism to survive and regrow outside an intact epithelial niche. (3) We have shown that the principal driver of L1CAM expression is loss of epithelial integrity itself, acting via loss of E-cadherin intercellular adherens junctions to transiently displace the transcriptional silencer REST/NSRF from chromatin in quiescent MetSCs, in turn derepressing expression of L1CAM and other genes required for tissue regeneration1. Proliferation, restoration of epithelial structures, and macrometastatic outgrowth, on the other hand, require high REST levels. Our evidence suggests that MetSCs cells are regenerative stem cells that emerge directly in response to loss of epithelial integrity to drive repair, a phenotype of physiological wound healing that is redeployed in MetSCs. Molecular mechanism by which REST chromatin binding is dynamically regulated in MetSCs, and how this in turn enables cell fate plasticity from stemness to proliferation, is unknown. In neurons, ZFP36L1/2, members of the tristetraproline family of CCCH RNA binding proteins, have been shown to bind to AU-rich elements in the REST 3’UTR and target REST mRNA for degradation2. ZFP36L1/2 are required for self- renewal of early burst forming unit erythroid progenitors3, and for maintenance of quiescence in lymphocytes and muscle progenitors4. Project hypothesis: The ZFP36L1/2-REST axis is a master regulator of cell fate plasticity in intestinal epithelial progenitors, with loss of ZFP36L1/2 function impairing progenitor self-renewal and metastatic seeding, but in turn promoting proliferation. Aim 1: Define the function of the ZFP36L1/2-REST axis in normal and neoplastic intestinal stem cell self renewal, differentiation, and proliferation. Aim 2: Dissect the molecular mechanism of ZFP36L1/2-mediated cell fate plasticity. Aim 3: Determine the functional consequences of ...