Glycosylation as a Regulator of Tropism of Melanoma Metastasis - Resubmission - 1

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Glycosylation as a regulator of tropism of melanoma metastasis Malignant melanoma is a type of cancer arising from melanocytes, the pigmented cells of the skin. Metastases of tumors to secondary sites are the cause of 90% of cancer mortality. A salient feature of metastasis is the ability of a primary tumor to colonize secondary organs. This has prompted a quest to identify the factors and mechanisms that support melanoma metastasis to specific organs as secondary sites (e.g. brain, lung, liver). One of the most devastating complications of melanoma is that around 50% of patients with metastatic melanoma develop brain metastasis, after which most patients survive less than 6 months. Patients with brain metastasis don't benefit from new therapies and have extremely poor prognosis. Understanding the basis to brain and other organs adaptation may reveal new therapies. Carbohydrates, which are altered in tumors, are involved in immune evasion, homing of cells to tissues, survival, and anchorage. Our recent clinically relevant study identified distinct glycosylation patterns of primary and metastatic melanoma. Moreover, our preliminary studies of patient samples suggest that specific glycosylation patterns are a site- specific feature of metastasis. I hypothesize that adaptation of tumor cells to different secondary sites requires specific changes in cell surface glycosylation. My proposed work will use innovative approaches to identify therapeutically relevant glycan structures and glycosylation enzymes as targets for anti-metastatic therapies. I will identify candidate glycans and glycogenes to regulate site-specific metastasis through glycan profiling of relevant in vivo models and high-throughput screens with a barcoded pooled shRNA library of glycogenes. Candidate glycans and glycogenes will be validated by lectin fluorescence and IHC analysis of melanoma patient FFPE samples. We will further validate candidate glycogenes using in vivo xenograft models and dissect their mechanism of action through various in vitro assays. Further, we will use a glycoproteomic strategy, coupling lectin-affinity purification with LC/MS shotgun protein identification, to identify glycosylated proteins with a specific glycan motif. Finally, site-specific metastasis related glycoproteins would be investigated for their mechanism of action. The identification of glycans and glycosylation enzymes actively participating in melanoma tropism as well as a precise understanding of their mechanism of action has the potential to provide a trove of glycan epitopes and enzymes, that are unexplored as anti-tumor targets for the treatment of site-specific metastases.