Poster presentation, P166

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

Subtilisin-kexin isoenzyme-1-a novel player in melanoma biology

BACKGROUND: Prohormone convertases (PCs) are Ca2+-dependent serine proteases which do not only process prohormones into biologically active peptides but also activate cancer-related proteins such as growth factors, growth factor receptors, adhesion molecules and matrix metalloproteases. Although it has previously been reported that some PCs are overexpressed in a number of solid tumors, their function in human pigment cells is poorly investigated. In order to clarify if PCs are involved in the pathogenesis of melanoma we focused on subtilisin-kexin isoenzyme-1 (SKI-1), one of the most recently identified PC members. METHODS: In vitro expression of SKI-1 in normal and transformed human melanocytes (NHM) as well as in 9 human melanoma cell lines was determined by quantitative real-time RT-PCR and Western immunoblotting. Regulation of SKI-1 mRNA was examined in NHM exposed to a panel of established natural and artificial growth factors, extracellular and intracellular stressors. Regulation of SKI-1 was further investigated by in silico promoter analysis. Finally, the biological function of SKI-1 was tested by treating normal and transformed melanocytes with decanoyl (dec)-RRLL-chloromethylketone (CMK), a cell-permeable pharmacological SKI-1 inhibitor. Metabolic activity, cell survival and apoptosis were assessed by XTT test, crystal violet test and various apoptosis read-outs. RESULTS: SKI-1 was found to be constitutively expressed at mRNA and protein level in both normal and transformed melanocytes. In silico promoter analysis revealed several transcription factor binding sites for transcription factors that are typically activated by melanocytes mitogens as well as by extracellular and intracellular stressors. However, among several growth factors and stimuli tested, only phorbol-12-myristate-13-acetate and tunicamycin affected SKI-1 expression in NHM. Interestingly, dec-RRLL-CMK led to a dose-dependent inhibition of the metabolic activity and proliferation of normal and transformed melanoma cells in vitro. In general, melanoma cells appeared to be more sensitive towards the SKI-1 inhibitor. Moreover, dec-RRLL-CMK dose-dependently led to apoptosis of melanoma cells as shown by cell death detection assay, Annexin-V staining and processing of poly-adenosine diphosphate-ribose polymerase 1/2. This effect was associated with suppression of caveolin-1 and glucose-regulated-protein 78, two prototypical SKI-1 target genes implicated in melanoma growth and progress.

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