Abstract
Despite recent advances in melanoma treatment, metastasis and resistance to therapy remain serious clinical challenges. NME1 is a metastasis suppressor, a class of proteins which inhibits metastatic spread of cancer cells without impact on growth of the primary tumor. We have identified a rare subpopulation of cells with markedly reduced expression of NME1 (NME1LOW) in human melanoma cell lines. To enable isolation of viable NME1LOW cells for phenotypic analysis by fluorescence-activated cell sorting (FACS), a CRISPR-Cas9-mediated approach was used to attach an EGFP coding module to the C-terminus of the endogenous NME1 gene in melanoma cell lines. NME1LOW cells displayed enhanced collective invasion in vitro when implanted as 3D aggregates in Matrigel. NME1LOW cells were also highly metastatic to lung and liver when xenografted subcutaneously in immune-deficient NSG mice. RNA-seq analysis revealed that NME1LOW cells express elevated levels of genes associated with tumor aggressiveness, as well as with morphogenesis of tissues of neural crest-like origin (melanocytes and neurons, bone and heart tissues; GO: 0009653). The highly malignant NME1LOW variant of melanoma cells has potential to provide novel therapeutic targets and molecular markers for improved clinical management of patients with advanced melanoma.
Highlights
Despite recent advances in melanoma treatment, metastasis and resistance to therapy remain serious clinical challenges
Having recently observed that spheroids derived from melanoma cell lines exhibit cellular heterogeneity in expression of the metastasis suppressor NME117, we investigated the expression pattern of NME1 under monolayer culture conditions
Through analysis of the protein expression pattern for the metastasis suppressor protein, NME1, we have discovered a previously unidentified subpopulation present within melanoma cell lines that is highly metastatic and may well represent a barrier to successful therapy in melanoma patients
Summary
Despite recent advances in melanoma treatment, metastasis and resistance to therapy remain serious clinical challenges. While multiple studies have reported an association between reduced NME1 expression and more aggressive forms of melanoma in human patients[14], others observed little or no correlation[15,16] As these measurements of NME1 expression were conducted in whole tumor specimens, they did not address the possible existence of rare subpopulations of NME1-deficient cells which could well possess enhanced metastatic properties and represent a stronger index of melanoma progression. To this end, we recently demonstrated the presence of a rare subpopulation with nearly undetectable levels of NME1 expression in spheroid cultures derived from different melanoma cell lines[17]. Our study suggests the possible existence of NME1LOW cells in melanoma tumors that possess enhanced potential for tumor progression and metastatic activity
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