Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway

Abstract

The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared to adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression respectively. This links BRN2 as an activator and conversely MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense ablated cell lines decreased melanoma sphere forming capability, cell adhesion during 3D-spheroid formation, and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than growth as adherent melanoma cells.

Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression

“…This heterogeneous downregulation of ZEB2 expression is reminiscent to the profile of MITF expression in human melanoma. 38 Interestingly we found that human melanoma metastases express high nuclear ZEB2 levels suggesting reversible loss of expression during melanoma dissemination (Supplementary Figure S3c). We extended this study by analyzing 178 primary melanoma samples on a tissue array.…”

Lunasin reduces the melanoma stem cell population in vitro and inhibits tumor proliferation in vivo.

“…In support of these findings, inhibition of MITF increased expression of stem cell markers Nanog and Oct4 resulting in enhanced tumor growth in C57/B6 mice [102]. Again, culture conditions played a major role in expression of differentiation and stem markers [100,103]. Hoek and Goding summarize: "If the combination favours the proliferative phenotype over the invasive, a tumour may grow rapidly, but for a given size, it will seed fewer metastases than a tumour whose cells are not subject to a proliferative phenotype bias."…”

A POU3F2-MITF-SHC4 Axis in Phenotype Switching of Melanoma Cells

“…Thus, the change from monolayer to 3 dimensional culture in stem cells medium strongly influences MITF expression. Thurber et al, (Thurber et al, 2011) also showed using siRNA silencing that POU3F2 was an activator and MITF a repressor of the Notch pathway. Loss of POU3F2-MITF signaling resulted in decreased capacity to form melanospheres and invasion through a collagen matrix.…”

“…Spontaneous switching can also take place to a certain extent in cell culture, however, the low frequency of this event makes it a difficult process to follow. More recently however, Thurber et al (Thurber et al, 2011), showed that growth of high MITF expressing cells as non-adherent 'melanospheres' dramatically enhanced the frequency of low MITF/high POU3F2 expressing cells. Similarly, when we grew 501Mel cells expressing high levels of MITF in human embryonic stem cell medium under non-adherent conditions they readily form melanospheres, where we observe the appearance of a significant fraction of low MITF expressing cells in the spheres (Fig.…”