Wednesday, May 8, 2024
5/8/2024
Project Summary Keratinocytes and melanocytes together form the epidermal melanin unit, which is essential for the protective tanning response after ultraviolet radiation (UV) exposure. Keratinocytes respond to UV by releasing paracrine factors that induce melanocytes to increase melanin production and dendricity. My lab showed that the keratinocyte-specific desmosomal cadherin, desmoglein 1 (Dsg1), which is temporarily lost in response to acute UV exposure, plays a role in this protective tanning response. Acute exposure of melanocytes to conditioned media from Dsg1-deficient keratinocytes increase melanin secretion and dendricity. However, melanocytes chronically exposed to Dsg1-deficient keratinocytes exhibited features of transformation, including pagetoid melanocytosis. We found recently that melanoma cells in conditioned media from Dsg1-deficient keratinocytes exhibited increased migration, due at least in part to CXCL1/CXCR2 signaling. When keratinocytes were cultured in melanoma conditioned media, Dsg1 was downregulated along with its transcriptional activator Grainyhead like 1 (Grhl1), while in sections from patient melanoma tissues, Dsg1 was lost in keratinocytes surrounding melanomas. Overall, these data lead me to hypothesize that melanoma cells actively target Dsg1 for downregulation in keratinocytes for their own benefit, leading to increased production of paracrine factors that in turn promote melanoma progression. Our RNA sequencing results suggest that melanoma cells cultured in Dsg1-deficient media upregulate a neural crest stem cell (NCSC)-like signature, exhibited by a melanoma cell population with enhanced migration, invasion and drug resistance. Here, I propose to 1) determine how loss of keratinocyte Dsg1 leads to the reprogramming of melanoma cells to adopt more pro-tumorigenic cell fates, and 2) determine how melanoma cells downregulate keratinocyte Dsg1 by elucidating the upstream regulators that modulate Dsg1 expression and the mechanisms by which melanoma cells target Dsg1 in keratinocytes. Toward this end, I will culture melanoma cells in Dsg1-deficient keratinocyte conditioned media and use RNA sequencing and single molecule RNA FISH to identify changes in transcriptional signatures between migratory/non-migratory and invasive/non-invasive melanoma cell populations. To determine if melanoma cells adjacent to keratinocytes that lack Dsg1 exhibit an increase in NCSC-like markers, I will use human tissue sections and stain for NCSC- like markers. To identify the melanoma secreted factors that can inhibit Dsg1 in keratinocytes, I will take a parallel candidate and unbiased approach, focusing on growth factors that can activate Erk5, which was identified as an upstream regulator of Dsg1 by RNA sequencing. Factors that alter Dsg1 expression in keratinocytes will be genetically manipulated in melanoma cells and resulting conditioned media collected. Keratinocytes will be cultured in melanoma conditioned media to determine if loss of identified targets inhibits Dsg1 loss. Overall, these studies will elucidate novel paracrine signaling between keratinocytes and melanoma cells and identify the molecular mechanisms by which melanoma cells can control keratinocyte secretion of pro-melanoma factors.
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