Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123 low ) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123 high progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123 low cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123 low cells overexpress HIF1a, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123 high cells to the Rh123 low phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1a transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123 low cells. Moreover, the Rh123 low cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123 low melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123 low G0 cells promotes the exit of cycling cells from the stem cell compartment.

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Metastatic cancer relapses following the reactivation of dormant, disseminated tumour cells; however, the cells and factors involved in this reactivation are just beginning to be identified. Using an immunotherapy-based syngeneic model of melanoma dormancy and GFP-labelled dormant cell-derived cell lines, we determined that vaccination against melanoma prevented tumour growth but did not prevent tumour cell dissemination or eliminate all tumour cells. The persistent disseminated melanoma tumour cells were quiescent and asymptomatic for one year. The quiescence/activation of these cells in vitro and the dormancy of melanoma in vivo appeared to be regulated by glucocorticoid-induced leucine zipper (GILZ)-mediated immunosuppression. GILZ expression was low in dormant cell-derived cultures, and re-expression of GILZ inactivated FOXO3A and its downstream target, p21CIP1. The ability of dormancy-competent cells to re-enter the cell cycle increased after a second round of cellular dormancy in vivo in association with shortened tumour dormancy period and faster and more aggressive melanoma relapse. Our data indicate that future cancer treatments should be adjusted according to the stage of disease progression.