Darren J. Smit

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

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.

Osteonectin downregulates E‐cadherin, induces Osteopontin and Focal adhesion kinase activity stimulating an invasive melanoma phenotype

Osteonectin is recognised as a marker of metastasis progression in melanoma and has been implicated in the transition from radial to vertical growth phase. A Tetracycline‐inducible system was used to regulate Osteonectin protein levels in melanoma cell lines to examine the morphological, biochemical and invasive changes that accompany its altered expression. Assay of protein and phosphorylation changes showed a downregulation of E‐cadherin, upregulation of Osteopontin and a corresponding increase in phosphorylation of Focal Adhesion Kinase on Tyr397 and Tyr576 concomitant with Osteonectin induction. Melanoma cells overexpressing Osteonectin displayed increased invasive potential, whereas ablation of Osteonectin gene transcription using siRNA suppressed the invasive potential of these cells and resulted in the upregulation of E‐cadherin. The recently described interaction of Osteonectin with Integrin Linked Kinase leading to modulation of its activity suggests a mechanism relevant to the loss of E‐cadherin and cell adhesion that occurs during melanoma progression. These results indicate a central role for Osteonectin in the regulation of gene expression changes driving the progression of melanoma toward metastasis.

Melanocortin-1 receptor-mediated signalling pathways activated by NDP-MSH and HBD3 ligands

Binding of melanocortin peptide agonists to the melanocortin‐1 receptor of melanocytes results in eumelanin production, whereas binding of the agouti signalling protein inverse agonist results in pheomelanin synthesis. Recently, a novel melanocortin‐1 receptor ligand was reported. A β‐defensin gene mutation was found to be responsible for black coat colour in domestic dogs. Notably, the human equivalent, β‐defensin 3, was found to bind with high affinity to the melanocortin‐1 receptor; however, the action of β‐defensin as an agonist or antagonist was unknown. Here, we use in vitro assays to show that β‐defensin 3 is able to act as a weak partial agonist for cAMP signalling in human embryonic kidney (HEK) cells expressing human melanocortin‐1 receptor. β‐defensin 3 is also able to activate MAPK signalling in HEK cells stably expressing either wild type or variant melanocortin‐1 receptors. We suggest that β‐defensin 3 may be a novel melanocortin‐1 receptor agonist involved in regulating melanocyte responses in humans.

PPARγ agonists attenuate proliferation and modulate Wnt/β-catenin signalling in melanoma cells

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcriptional regulators. Accumulating evidence suggests that PPARgamma agonists such as the thiazolidinediones (TZDs) may prove to be useful anti-cancer agents exhibiting anti-proliferative and/or pro-apoptotic affects in a range of cancer cell types including melanoma, however, the mechanisms underlying this effect remain unclear. We have demonstrated the anti-proliferative effects of full and partial PPARgamma modulators in human melanoma cell lines. Ablation of PPARgamma expression in the MM96L melanoma cell line by siRNA mediated mechanisms attenuates the anti-proliferative effect of these agents suggesting this effect is directly mediated by PPARgamma. The mechanisms underlying the anti-proliferative effects of PPARgamma in melanoma cells involve the regulation of expression of a number of critical cell cycle genes and beta-catenin. Moreover, our data indicate that PPARgamma modulates Wnt/beta-catenin mediated signalling in melanoma cells in an agonist dependent manner.

Molecular analysis of common polymorphisms within the human Tyrosinase locus and genetic association with pigmentation traits

We have compared the melanogenic activities of cultured melanocytes carrying two common TYR alleles as homozygous 192S‐402R wild‐type, heterozygous and homozygous variant. This includes assays of TYR protein, DOPAoxidase activity, glycosylation and temperature sensitivity of protein and DOPAoxidase levels. Homozygous wild‐type strains on average had higher levels of TYR protein and enzyme activity than other genotypes. Homozygous 402Q/Q melanocytes produced significantly less TYR protein, displayed altered trafficking and glycosylation, with reduced DOPAoxidase. However, near wild‐type TYR activity levels could be recovered at lower growth temperature. In a sample population from Southeast Queensland, these two polymorphisms were present on four TYR haplotypes, designated as WT 192S‐402R, 192Y‐402R and 192S‐402Q with a double‐variant 192Y‐402Q of low frequency at 1.9%. Based on cell culture findings and haplotype associations, we have used an additive model to assess the penetrance of the ten possible TYR genotypes derived from the combination of these haplotypes.