Abstract
Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a new therapeutic vulnerability to drive differentiation to the normal cell phenotype.
Highlights
Malignant melanoma is a potentially deadly type of skin cancer that occurs as a result of melanocyte transformation [1]
To better understand processes underlying the phenotype-switching of melanoma cells, we adopted a 3D-culture system, as numerous studies have indicated that formation of melanoma 3D-spheroids enriches for stem cell-like growth [19,27,28,35,36,37]
Five human metastatic melanoma cell lines, 1205Lu, 451Lu, SKMEL-28, C8161 and MNT1, were surveyed for their ability to form adherent 3D-spheroids when cultured in human embryonic stem cell medium (Fig. 1A)
Summary
Malignant melanoma is a potentially deadly type of skin cancer that occurs as a result of melanocyte transformation [1]. Melanoma is relatively rare, it has become a major concern due to an increased incidence over the past two decades. Melanoma is generally curable with surgical intervention, yet once metastasized to organ sites, the prognosis becomes very poor. As melanoma is highly resistant to many conventional therapies, there is an urgent need for new diagnostic, prognostic, and treatment approaches. The genetic lesions in melanoma, including NRAS and BRAF mutations, are well characterized, and activated BRAF kinase has been demonstrated to be an effective drug target for melanoma therapy [2]. An immunomodulatory approach for melanoma treatment has recently shown exciting promise [4]
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