The increased ability of a range of melanoma cell lines to form spheres in hESCM-KSR as compared to hESCM-FBS medium indicates that bovine serum may contain inhibitory factors preventing the cells from reacting to stem cell like conditions that may be enhanced during metastatic transformation (Mani et al., 2008
). FBS is known to contain growth factors promoting cell attachment as well as differentiation both of which may counteract sphere formation. Another recent study has described the melanoma sphere forming ability of a single melanoma cell line WM-266-4, in which there was reported to be a small endogenous floating population of cells able to assume this state upon serum depletion and growth in neural stem cell-enriched media (Na et al., 2009a
). We have also tested this same cell line and found it to form spheres in both types of media using our conditions (Supplementary Table 1
). Notably, sphere formation of the WM-266-4 cell line could be promoted by treatment with a specific developmentally staged embryo extract from Zebrafish, possibly through TGF-β like signalling molecules (Na et al., 2009b
). Thus, melanoma sphere formation is directly influenced by the microenvironment the cells are subjected to, with embryonic conditions stimulating the adoption of this state of growth and plasticity (Fang et al., 2005
). Switching from radial to vertical cell growth states is a major determinant of melanoma metastasis which results in altered cell adhesive, invasive and migratory properties (Alonso et al., 2007
; Smit et al., 2007
). This is possibly linked to the changes in regulatory proteins accompanying the switch between melanoma cells grown in an adhesive or melanoma sphere states.
A general characteristic of growth as melanoma spheres compared to their adherent growth pattern was the reduction in the levels of the transcription factors assayed including BRN2, MITF, NOTCH1 and the JAG2 and DLL1 ligands. The NOTCH pathway participates in a variety of cellular processes including cell fate specification, proliferation, adhesion and migration, with NOTCH signalling associated with maintaining the undifferentiated melanoblast stem cell phenotype (Hoek and Goding, 2010
). More recently NOTCH1 has been implicated in conferring a transformed phenotype when expressed in normal human melanocytes (Pinnix et al., 2009
) and progression of melanoma to metastasis (Pinnix and Herlyn, 2007
). A link between the BRN2-MITF controlled gene network and regulation of the NOTCH pathway as seen with the reciprocal expression level changes upon repression of each molecule () has not previously been reported, but may have been anticipated. BRN2 has been reported to interact with the proneural basic-helix-loop helix transcription factor Mash1 (Castro et al., 2006
). This synergistic transcriptional activation occurs via an evolutionarily conserved octamer-E-box motif (termed the Mash1/Brn motif) first identified within the mouse Mash1-specific enhancer of the Delta1 gene, which codes for one of several ligands for the Notch receptor. Using a bioinformatics approach, the co-ordinate Mash1/Brn motif could be identified in the promoters of 21 other genes, and which also included several other members of the Notch signalling pathway (Castro et al., 2006
). Thus BRN2 has been shown to interact with these elements and the presence of the E-Box that is a core element for the MITF target binding site (Cheli et al., 2010
) means there are potentially two levels of interaction that may regulate the NOTCH pathway in melanocytic cells, though not all Mash1 sites will represent MITF target sequences. The first is some direct BRN2-MITF joint interaction on the regulatory region of some of these genes to result in reciprocal expression patterns upon target sequence binding. The other is a simple indirect mechanism whereby BRN2 levels are repressed with increasing MITF levels, thus reducing NOTCH pathway signalling through the absence of BRN2.
In a genome-wide scan for BRN2 target genes modulating melanoma cell phenotype, a ChIP-chip analysis was performed using the 501Mel human melanoma cell line to directly tag and capture promoter regions bound by the BRN2 transcription factor. This experimental approach combined with bioinformatic analysis indentified 2108 in vivo
BRN2 binding sites located within 1700 potential target genes (Kobi et al., 2010
). Although this did not include any NOTCH pathway members, this study suggested that elevated levels of BRN2 may induce autocrine and/or paracrine KIT ligand signalling, cell cycle regulated and Wnt-β cantenin pathways, and as such modulate the proliferative and invasive properties of melanoma tumour cells. The direct binding of BRN2 to any NOTCH component pathway member regulatory region in melanoma cells remains to be demonstrated.
The reduction of regulatory protein levels within melanoma spheres may reflect two independent processes. Adherent cells have unrestrained access to nutrients in the media and are uninhibited by contact with other cells that might set a limit to their growth expansion in two dimensions, at least until confluency is reached, when they then must overgrow the sheet of cells that have formed. In contrast, the cells within melanoma spheres quickly reach a barrier to active growth by the limits set in the rate of diffusion of nutrients and the immediate block to expansion being constrained by the spatial surroundings within the sphere. For this reason there may be a general suppression of the transcriptional and translational machinery in the majority of cells within the melanoma sphere, apart from those in the outer shell, resulting in a reduced requirement for general and specific transcription factors. An alternate explanation, but by no means mutually exclusive, is the induction of different cell states through a variety of cell-cell contacts and interactions, resulting in heterogeneity of protein expression patterns within individual melanoma sphere cells. This is indeed what we have found upon sectioning through individual melanoma spheres and assaying expression via immunofluoresence detection methods, as others have also recently reported (Perego et al., 2010
There have been few reports directly examining the degree, nature and consequences of melanoma tumour heterogeneity and the variation of cell morphology and immunohistochemical patterns within single melanocytic lesions of patients (Ohsie et al., 2008
), this is surprising given a direct relationship of melanogenesis and differentiation status of melanoma cells (Houghton et al., 1987
). For reasons of simplicity the melanoma field has pursued the relative homogenous nature of stable cell clones in adherent culture. Melanoma cell lines do possess an innate ability to respond to extracellular cues provided by the extracellular matrix and associated signalling molecules, with phenotypic switching apparent when introduced into an embryonic microenvironment illustrating the plasticity of these cells (Hendrix et al., 2007
). A recent model of melanoma progression has been put forward by Hoek and colleagues (2008)
in which there is epigenetic switching of the gene expression pattern of cells from a proliferative to an invasive signature (Hoek et al., 2008
), moreover these are proposed to represent distinct yet interchangeable states which are regulated by signals from the microenvironment (Hoek and Goding, 2010
). The existence of two separate states within melanoma tumour xenografts studied in this work (Hoek et al., 2008
) was supported by histochemical staining for MITF and Ki67 antigens, which appeared higher in tumours derived from proliferative cell lines.
This association of MITF with the proliferative gene expression pattern has recently been supported by the finding of two distinct heterogeneous subpopulations within melanoma tumour biopsies as being largely MITF positive or negative. The more invasive fraction being less proliferative and expressing low levels of MITF and high levels of p27Kip1
(Carreira et al., 2006
). The ability of the BRN2 regulatory molecule (Cook and Sturm, 2008
) to act as a direct repressor of the MITF gene promoter (Goodall et al., 2008
; Kobi et al., 2010
), combined with the discovery that MITF and BRN2 can be expressed in different subpopulations of cells within melanoma tumours, has lead to the hypothesis that individual cells may switch between BRN2+/MITF− and BRN2−/MITF+ states depending upon the extracellular cues. Alternatively this could be based on a stochastic mechanism in the absence of any signal (Dodd et al., 2007
), and that neither of these populations will reach clonal dominance within a tumour. Finally, remarkable images have recently been obtained using intravital microscopy to demonstrate that migrating B16-GFP tagged melanoma cells within tumours contain little or no pigment and high levels of BRN2 promoter activity (Pinner et al., 2009
), showing that it is a subset of undifferentiated melanoma cells that exit the primary tumour and give rise to the bulk of the resultant MITF positive differentiating cells during metastasis.
We have shown that melanoma cells grown in the adherent state or as short term spheroid-aggregates express high levels of both BRN2 and MITF unlike melanoma in situ. Induction of melanoma sphere formation and growth as tumour xenografts restores in part the BRN2+/MITF− or BRN2−/MITF+ reciprocal cellular expression patterns as seen in human melanoma tumours () (Goodall et al., 2008
). Our results demonstrate the importance of a BRN2-MITF axis of gene expression in efficient melanoma sphere formation as ablation of BRN2 results in poor sphere formation, and as we previously demonstrated a lack of ability to establish tumours in mouse xenografts (Thomson et al., 1995
). Thus the study of melanoma sphere formation should provide a useful approach to gain insight into the processes that induce metastasis of melanoma. As a model, it is much more representative of melanoma in situ, and so potentially will provide more successful outcomes for novel drug therapies than present models where results in vitro cannot be reproduced in the patient (Smalley et al., 2006