Colorectal cancer (CRC) has a U.S. incidence rate of almost 150,000 cases per year, encompassing over 10% of new cancer diagnoses. In 2016, within the Veterans Administration Healthcare System alone, 3,400 new patients were diagnosed with CRC, including 614 with advanced disease and very limited treatment options. Understanding the basic biology of the underlying malignant transformation is the key to identifying new therapeutic targets and intervention strategies. Epithelial junctional dysfunction is common in CRC. In early iterations of this research plan, we discovered that BVES, a tight junction-associated protein, was underexpressed via extensive promoter hypermethylation in colonic tumors, even at the earliest adenoma stage. We and others have further demonstrated that restoring BVES expression attenuated pro-tumorigenic phenotypes in a variety of cancer cell lines. In the prior funding period, we determined that BVES-deficient mice had increased tumor burden and more severe dysplasia in both genetic and chemical tumor models. Collectively, these data suggested that BVES functions as a tumor suppressor in epithelial malignancy; thus, BVES may represent a new diagnostic marker and/or therapeutic target in cancer. Mechanistically, we are beginning to understand that BVES functions as a scaffolding protein, orchestrating protein complex formation to facilitate outside ® in signal transduction to coordinately regulate intracellular signaling pathways to alter cellular phenotypes in response to microenvironmental cues. For example, we discovered that BVES interacts with PR61a:PP2A to reduce cellular c-Myc levels and attenuate WNT signaling by restricting LRP6 levels. Recently, BVES was discovered to modulate cAMP signaling in the heart. We determined that BVES interacts with AKAP11, a cAMP/PKA nanodomain partitioning protein, and that BVES loss was associated with reduced PKA activity as evidenced by reduced phosphorylated CREB. Thus, BVES likely regulates another signaling pathway highly relevant to tumor biology. Lastly, we have established a bank of Stage II and III human CRC 3D cultures and developed techniques to genetically modify and orthotopically xenograft them. This will serve as a platform for testing whether BVES can modify the growth of established CRC and determining the relative contributions of BVES-regulated signaling. In this proposal, we structure three Specific Aims designed to understand the role of BVES in tumor biology. First, we will further our understanding of the BVES:AKAP11:PKA axis. In the second aim, we will determine the functional impact of restoring BVES in early tumor biology (adenomas) ex vivo. In the last aim, we will test the hypothesis that restoring BVES can attenuate the growth of human CRC and test whether this restoring BVES sensitizes CRC to chemotherapeutic and targeted therapies in 3D culture and murine models. Through these studies, we will gain fundamental insights into how BVES loss contributes to ...