Effect of Nanog overexpression on the metastatic potential of a mouse melanoma cell line B16-BL6

Abstract

Nanog, a marker and regulator of the undifferentiated state in embryonic stem cells were anticipated to be an effective enhancer of cancer metastasis. We have developed a Nanog overexpressing mouse melanoma cell line B16-BL6 (BL6). BL6 was well recognized as a cell line with a high metastatic potential. In vitro tests revealed the enhancement of cell proliferation, wound healing activity, and matrix metalloproteinase 9 (MMP9) activity. Nanog-induced up- or down-regulated genes were comprehensively analyzed by transcriptome sequencing using Nanog+BL6 and wild-type BL6. Principally, up-regulated genes were involved in vesicle-aided glucose transport and oxidative phosphorylation, while down-regulated genes were associated with immunosuppression and apoptosis. A marked finding was that TGF-β1 was down-regulated, because TGF-β1 has been well discussed about its suppressive/progressive dual role in cancer. In vivo test showed that the number and volume of metastatic colonies of BL6 to lung were as high as 115 colonies/lung and 5.6 mm3/lung. Under this condition, Nanog overexpression caused a progressive effect (150 colonies/lung, p = 0.25; 9.2 mm3/lung, p = 0.13) rather than a suppressive effect on the metastasis. In this study, the effectiveness of Nanog overexpression in enhancing the metastatic potential of melanoma cell lines has been demonstrated for the first time.

Anti-Metastatic Function of Extracellular Vesicles Derived from Nanog-Overexpressing Melanoma

“…In a previous study, we showed that Nanog overexpression caused a decrease in the expression of TGF-β1 in BL6 cells [ 18 ]. Here, we likewise found that the level of TGF-β1 expression in Nanog + F10 cells decreased by 0.37 fold compared to F10 cells ( Figure 3 A).…”

“…Previously, we modified the melanoma cell line B16-BL6 (BL6) to overexpress Nanog , a major factor for maintaining the undifferentiated state of embryonic stem cells, in the expectation that this would increase the metastatic potential of this cell line. The modified BL6 line did indeed show an enhanced level of metastatic potential [ 18 ]. A transcriptome sequencing analysis indicated that transforming growth factor (TGF)-β1 was the only secretory cytokine among the top15 up-regulated and top 16 down-regulated genes after Nanog overexpression in BL6 cells.…”

Novel Roles of Nanog in Cancer Cells and Their Extracellular Vesicles

“…The transcriptome analysis of mouse melanoma cells was conducted to clarify the differential expression intensities between a cell line, B16-BL6, and its Nanog -overexpressing cell line Nanog + BL6. The up-regulated top-16 genes and down-regulated top15 genes were depicted [ 19 ]. The functional roles of up-regulated top-7 genes and down-regulated top-3 genes are illustrated in Figure 3 A,B, respectively.…”

“…Since the expression level of Tgf-β1 was a controversial matter, it was analyzed at mRNA and protein levels. As a result, its expression was down-regulated at both levels [ 19 ]. Another study conducted elsewhere [ 18 ] demonstrated a conflicting result.…”

“…The expression of matrix metalloproteinase ( MMP )s was studied since MMPs were believed to be relevant to invasion, although they were not included in the top 31 genes. They observed the increase in MMP9 , a secretion-type MMP [ 19 ].…”