Project Summary/Abstract Melanoma arises from transformation of melanocytes (MCs) in the basal layer of the epidermis where they are surrounded by keratinocytes (KCs). While promote extrinsic much effort has been devoted to determining how MC driver mutations melanoma development, tumorigenesis also depends on non-genetic environmental factors. How factors cooperate with driver mutations to promote MCs to adopt a particular cell fate is poorly understood. We identified the keratinocyte (KC)-specific cadherin Desmoglein 1 (Dsg1) as a mediator of KC:MC communication. Best known as a keratinocyte-specific adhesion molecule of stratified mammalian epithelia, we demonstrated that its transient downregulation like that occurring in response to ultraviolet radiation (UV) initiates a protective tanning response in MCs. However, chronic Dsg1 loss stimulates pathogenic pro-inflammatory cytokine production. Our work supports the hypothesis that melanoma cells hijack UV response pathways to downregulate Dsg1 in neighboring KCs, creating a pro-inflammatory environment that drives transforming MCs and melanoma cells to adopt new cell fates and bypass check points to tumor progression. To test this, we will: 1) Determine the stage-wise mechanisms that promote MC transformation and melanoma development through KC Dsg1 loss. We propose that KC Dsg1 loss alters adjacent MCs through paracrine and contact- mediated signaling, acting as a non-genetic hit to drive melanomagenesis. Using novel transcriptional reporters, barcoding with scRNA-Seq to track cell fates, and label free phase imaging to track single cell behaviors, we will address how KC Dsg1 loss helps a) MCs with driver mutations in BRAF and the tumor suppressor CDKN2A and b) paired non-invasive and invasive melanoma cells adopt cell fates that promote melanoma progression. Human 3D epidermal co-cultures and a novel Dsg1-deficient cKit-driven oncogene mouse model will be used to determine how cell fates adopted in response to Dsg1 loss drive oncogenesis in the context of the epidermal environment. 2) Determine how melanoma cells downregulate KC Dsg1 to promote their own tumorigenic potential. We hypothesize that mutant MCs or melanoma cells chronically suppress Dsg1 in perilesional KCs to create a pro-tumorigenic KC:melanoma cell niche. Like UV, paracrine signaling from melanoma cells increases the activity of the transcriptional repressor SNAI2/Slug, decreasing nuclear Grhl1, a known transcriptional regulator of Dsg1. We will determine how melanoma cells mediate transcriptional reprogramming of KCs for their own benefit. The extent to which oxidative stress, present in both UV-exposed and melanoma tissues, cooperates with melanoma-derived factors to reduce Dsg1 will be determined. Transcriptional and proteomic analysis will be used to validate the spatiotemporal progression of changes in human tumors that downregulate Dsg1. This work promises to illuminate a new pathway by which extrinsic factors drive m...