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• W2955542857 abstract "Malignant melanoma is an extremely aggressive form of cancer, resulting in the majority of all skin-cancer related deaths. Despite developments in melanoma treatment, tumor recurrence and metastasis, and resistance to therapy remain serious challenges to successful management of the disease. Melanoma tumors contain subpopulations of cells that display genomic and phenotypic variation. NME1 is an established metastasis suppressor protein, which inhibits the metastatic spread of melanoma cells. In the current study, we sought to determine whether melanoma cell lines contain a subpopulation of cells that express reduced expression of NME1 and, thus, potentially exhibit aggressive tumor and metastatic activities. Through immunostaining and flow cytometry, we discovered a rare subpopulation of cells with markedly reduced NME1 expression (NME1 Low ) in two melanoma cell lines (WM9 and WM278). To enable the isolation and further characterization of NME1-based subpopulations, we employed CRISPR-Cas9 technology to introduce the EGFP coding sequence at the C-terminus of the endogenous NME1 gene in both WM9 and WM278 cell lines. The resulting NME1-EGFP fusion protein is thus expressed under the regulatory control of the endogenous NME1 promoter. Fluorescence-activated Cell Sorting (FACS) was used to isolate cells that expressed the NME1-EGFP fusion protein at different levels of expression, from which multiple clones were isolated and expanded for analysis of their tumor growth and metastatic properties. Clones were sequenced and genotyped to ensure accurate EGFP incorporation at the NME1 locus. Clones derived from cells expressing low levels of NME1-EGFP (NME1 Low ) retained their low expression phenotype over 10 passages in monolayer culture conditions. NME1 Low clones derived from both melanoma cell lines were much more invasive than clones expressing normal levels of NME1-EGFP (NME1 Normal ) when embedded in Matrigel. In addition, RNA-seq analysis demonstrated that NME1 Low and NME1 Normal clones differed greatly in their RNA expression profiles (>200 differentially expressed genes), with NME1 Low clones expressing elevated levels of genes associated with morphogenesis (GO: 0009653). In particular, genes were identified relating to development of neural, bone and heart tissues, suggesting relevance to neural crest cell differentiation. Studies are ongoing to assess the growth and metastatic properties of these distinct cell subpopulations in vivo using a xenograft approach in immunocompromised mice. Overall, these studies strongly suggest that a subpopulation of cells that expresses low levels of NME1 represents a primitive and malignant component of melanoma lesions. These cells have potential for providing new therapeutic targets and molecular markers in advanced melanoma. Citation Format: Devin E. Snyder, Ying Wang, David M. Kaetzel. A rare subpopulation of melanoma cells with low expression of the metastasis suppressor gene NME1 is highly invasive [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1127." @default.
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• W2955542857 date "2019-07-01" @default.
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• W2955542857 title "Abstract 1127: A rare subpopulation of melanoma cells with low expression of the metastasis suppressor geneNME1is highly invasive" @default.
• W2955542857 doi "https://doi.org/10.1158/1538-7445.sabcs18-1127" @default.
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