PROJECT SUMMARY Melanoma is the most lethal of skin cancers, with progression to local invasion and metastasis leading to poor patient outcomes, highlighting the need for better understanding of melanoma progression. During melanoma progression, tumorigenic cells must overcome growth restraints imposed by the microenvironmental keratinocytes. Although much is known of keratinocyte regulatory controls on normal melanocytes, less is known about their interactions in melanoma. Our preliminary data suggests that melanoma induces an epithelial-to- mesenchymal transition (EMT) program in adjacent keratinocytes In Aim 1, I will investigate the role of keratinocyte EMT on melanoma initiation. For this study, I will use the zebrafish as an animal model to study in vivo interactions between keratinocytes and melanoma cells. I will induce spontaneous melanoma formation in transgenic zebrafish lines with GFP labeled keratinocytes and use imaging to confirm morphological changes indicative of EMT in tumor-associated keratinocytes (TAKs). Then, I will assess them for EMT transcription factor and adhesion protein changes. We hypothesize that melanoma-induction of keratinocyte EMT will result in loss of keratinocyte regulation on melanoma proliferation. We will test this hypothesis by knocking out EMT transcription factors in keratinocytes and assess effects on melanoma initiation and proliferation in the zebrafish model. In addition, our preliminary data has also highlighted an upregulation of paracrine signals from TAKs involved in melanoma migration and invasion. In Aim 2, I will determine how keratinocyte-derived secreted factors affect migration and invasion of nascent melanoma. To study migration of melanoma in vivo, I will first optimize existing imaging pipelines in our lab to quantitatively to track cell migration by imaging of the zebrafish skin. I can then assess the effect of knocking out keratinocyte-derived factors such as endothelin, Wnt5A and BDNF using cell-type specific CRISPR-editing to determine their effects on migration. We hypothesize that given the migratory role of these paracrine factors from in vitro data, we will see reduced melanoma migration and invasion into adjacent tissues on migration tracking and histology by knocking-out these factors in keratinocytes. By characterizing the role of tumor-associated keratinocytes in the melanoma microenvironment, this proposal seeks to understand how melanoma modifies its microenvironment to overcome its natural growth restraints and identify new targets to limit melanoma progression.