PROJECT SUMMARY/ABSTRACT Due to the significant gaps in the understanding of mechanisms controlling tumor-promoting signaling pathways, mammary cancer remains the 2nd most common cancer diagnosed and the 2nd leading cause of death in women. Over the past few decades, hyperactive Wnt/?-catenin signaling has been linked to the development of multiple cancers, including mammary cancer, making identification of molecules regulating Wnt/?-catenin signaling crucial to the development of mammary cancer treatment strategies. Betaglycan is a transmembrane proteoglycan co-receptor that can exist with or without heparan and/or chondroitin sulfate glycosaminoglycan (GAG) modifications whose significance is underscored by embryonic lethality in mice and roles in regulating cancer cell biology, independent of its TGF-? coreceptor functions. The goal of this research proposal is to define the specific role of the differentially modified forms of Betaglycan on Wnt3a signaling in epithelial cancer models. To accomplish our goal, three aims will be pursued. Aim 1 will delineate the role of differentially modified Betaglycan in regulating Wnt3a signaling through the use of CRISPR/Cas replacements of full length Betaglycan with modified versions of Betaglycan lacking either its heparan sulfate (HS), chondroitin sulfate (CS) or both GAG chains. In this aim, we will test the hypothesis that HS Betaglycan suppresses Wnt signaling while CS Betaglycan enhances Wnt signaling. Aim 2 will focus on delineating changes in Wnt3a/Frizzled/Betaglycan interactions regulated by differentially modified Betaglycan by testing the hypothesis that HS Betaglycan sequesters Wnt3a through high affinity interactions resulting in less Frizzled/Betaglycan complexes while CS Betaglycan weakly interacts with Wnt3a promoting Frizzled/Betaglycan complex formation to enhance Wnt signaling. Aim 3, will determine the effects of differentially modified Betaglycan on mammary tumorigenesis in vitro and in vivo by testing the hypothesis that HS Betaglycan is responsible for suppressing epithelial tumor growth and metastasis while CS Betaglycan enhances epithelial tumor growth and metastasis, in part, through changes in Wnt signaling. Several combinations of biochemical techniques in vitro and syngeneic orthotopic studies in vivo will be used to address the aims. At the completion of these studies, we fully expect to define the specific roles of HS and CS modified BG in modulating Wnt activity and epithelial tumorigenesis. Our research will thus be relevant to public health as we will broaden our current understanding of mechanisms controlling tumorigenesis.