Mechanisms Of Low Levels Of Sox2 Inducing Melanoma Tumor-repopulating Cell Dormancy

Despite significant progress in cancer therapeutics over the past few decades,tumor relapse following long periods of remission after treatment remains a challenging problem.Tumorigenic cells,when facing a hostile environment,may enter a dormant state,leading to long-term tumor survival,relapse,and metastasis.To date,the molecular mechanism of tumor cell dormancy remains poorly understood.Recently,a 3-dimentional(3D)soft fibrin gel culture system was used to mechanically select and grow highly malignant and tumorigenic tumor-repopulating cells(TRCs)from a general population of tumor cell lines.Our previous studies have shown that Sox2 is essential for proliferation and self-renewal of melanoma TRCs.Sox2,a stemness molecule that governs the pluripotency of embryonic stem cells,is dramatically upregulated in TRCs that grow in soft matrices.TRCs gradually lose Sox2 expression and become differentiated when cultured on 2D rigid plastic dish.When tumor cells are cultured in 3D stiff fibrin matrices,Sox2 expression becomes greatly downregulated and TRC proliferation substantially decreases and enter dormancy.From these reports,we hypothesize that altering Sox2 expression,independent of the mechanics of 3D matrices,changes the cell cycle state of TRCs to modulate their dormancy propensity.In this study,Sox2 shRNAs were used to silence Sox2 in melanoma TRCs.With the knockdown manipulation,Sox2 expression was maintained at a low level in the soft fibrin matrix.On the other hand,CRISPR/Cas9 was applied to generate frameshift mutations in the Sox2 coding sequence.By this way,Sox2 was complete depletion in melanoma TRCs.Control melanoma TRCs,TRCs with Sox2 knockdown,TRCs with Sox2 knockout,and 2D control melanoma cells were cultured for in vitro and in vivo experiments.Molecular biology approaches and single-cell analyses were performed to examine TRC dormancy and exit from dormancy.Under a low-expression condition,we show that Sox2,a stemness molecule of normal stem cells that participates in dormancy regulation of highly tumorigenic cells that can repopulate a tumor.Low levels of Sox2 induce the entry of melanoma TRCs into dormancy,mediated by activating the expression of the IDO1/AhR metabolic pathway,leading to upregulation of p27 and p21,contributing to TRC growth arrest and dormancy.Intriguingly,complete depletion of Sox2 via knockout results in dormancy exit and growth resumption of melanoma TRCs in culture and elevation of melanoma TRC apoptosis.Mice that are injected subcutaneously with Sox2-depleted melanoma TRCs do not form tumors and survive much longer than those injected with melanoma TRCs.We found that complete depletion of Sox2 promotes nuclear translocation of phosphorylated STAT3,where it binds to the p53 gene promoter,thus activating the p53-caspase3 cascade.These findings provide a novel insight into the role of the Sox2 in tumor cell stemness,tumor dormancy,and apoptosis.