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

Oncogene volume 30, pages 3036–3048 (2011)Cite this article

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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 with 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 the 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 the growth as adherent melanoma cells.

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Acknowledgements

We thank Professor N Hayward (QIMR) for providing some of the cell lines that were tested for melanoma sphere formation as part of this manuscript. RAS is a Senior Research Fellow of the Australian NHMRC, and in conducting part of this work, was supported by an SSP award from the University of Queensland. This work was supported by grants from the Cancer Council Queensland to RAS and JHL and NIH grant CA-25874 to MH.

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Authors and Affiliations

  1. Institute for Molecular Bioscience, Melanogenix Group, The University of Queensland, Brisbane, Queensland, Australia

    A E Thurber, G Douglas, E C Sturm, D J Smit, S N Ramakrishnan & R A Sturm

  2. The Wistar Institute, Philadelphia, PA, USA

    S E Zabierowski, M Herlyn & R A Sturm

  3. Queensland Institute of Medical Research, Brisbane, Queensland, Australia

    E Hacker & J H Leonard

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  1. A E Thurber
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  2. G Douglas
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Correspondence to R A Sturm.

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Thurber, A., Douglas, G., Sturm, E. et al. Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway. Oncogene 30, 3036–3048 (2011). https://doi.org/10.1038/onc.2011.33

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