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Aysegul Tura, Julia Lueke, Mihaela Reinsberg, Matthias Luecke, Salvatore Grisanti; Elevated NG2 expression and Rho-kinase activity in the uveal melanoma cells with monosomy-3. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5078.
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© ARVO (1962-2015); The Authors (2016-present)
Presence of monosomy-3 in the uveal melanoma (UM) cells is associated with a higher risk of mortality due to metastasis particularly into the liver. Currently, there are no effective treatment options to restrain the UM metastasis. The major liver-borne trophic factors like hepatocyte growth factor and insulin-like growth factor-1 (IGF-1) can activate the Rho/Rho-kinase pathway in diverse cell types. In this study, we analysed the outcomes of the conditioned medium from hepatocytes (HCM) on the metastatic potential of the UM cells together with the Rho-kinase activity and monosomy-3 status.
Circulating uveal melanoma cells (CMC) were immunomagnetically isolated from the freshly collected blood of n=21 UM patients. Cultures of UM cells were established from the primary tumor of a UM-patient operated in our clinic. Immuno-FISH was performed to detect chromosome-3 in the cells specifically expressing NG2, Ki67, or pMYPT1, the major downstream target of Rho-kinase. Cultured UM cells were incubated with the Rho-kinase inhibitor H-1152 for 1 to 3 days. Proliferation was analysed by Ki67 immunostaining and EdU incorporation. Cell motility was evaluated by the scratch assay. Levels of vimentin, beta-catenin, cleaved caspase-3, and IGF-1 were determined by immunocytochemistry and blotting.
The melanoma associated chondroitin sulphate proteoglycan (NG2), an upstream activator of RhoA/RhoC, was expressed at significantly higher levels in both the CMC and the cultured UM cells having monosomy-3. HCM led to a significant increase in the expression of vimentin and beta-catenin and the motility of the UM cells possibly due to the elevated levels of IGF-1. HCM also promoted the proliferation of UM cells having monosomy-3, together with an increase in Rho-kinase activity. Inhibition of Rho-kinase with H-1152 suppressed the proliferation of UM-cells with monosomy-3 (p<0.05), reduced the expression of beta-catenin and vimentin, and promoted apoptosis.
UM cells with monosomy-3 exhibited a higher expression level of NG2 and an associated increase in Rho-kinase activity, which may be involved in the elevated metastatic potential of these cells in response to hepatocyte-derived growth factors. Pharmacological inhibition of Rho-kinase therefore emerges as a noteworthy strategy to suppress UM metastasis into the liver.
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