Abstract
(Englisch)
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Liver selected B16-LS9 melanoma cells show a dramatic overexpression of the proto-oncogene c-met, the cellular receptor for hepatocyte growth factor/scatter factor. As a consequence, c-met becomes constitutively active, and the cells become more responsive to hepatocyte growth factor stimulation. We have investigated the molecular mechanisms regulating c-met expression in both the parental line B16-F1, which has low expression levels, and the liver-specific B16-LS9, overexpressing c-met.
C-met overexpression is observed at the protein and mRNA levels, however without further evidence of gene amplification or rearrangement. C-met promoter activity was higher in B16-LS9 than -F1 cells, and also a nuclear run-off showed higher transcription levels in B16-LS9 cells. AP1, but not Sp1 transcription factor appeared to be involved in this regulation. Moreover, we found that c-met mRNA had a longer half-life in B16-LS9 cells, thus indicating also the involvement of post-transcriptional regulation mechanisms. Finally, we found evidence that autonomous activation of the melanocortin receptor-1 (MCR-1) is at least partially responsible for c-met upregulation in B16-LS9 cells, since treatment of the cells with a potent MSH antagonist (the agouti peptide) has strong down-regulatory effects. Based on this observation, it would be expected that introduction of a transgene coding for the agouti should downregulate the expression of c-met in B16 cells. We have cloned the agouti cDNA from newborn mouse skin by RT-PCR, however we had not enough time to develop a permanent transfected cell line to evaluate its phenotypic effects.
Ocular melanoma arises from uveal melanocytes. It is estimated to account for 12% of all melanomas, and between 30 and 80% of noncutaneous melanomas. Intriguingly, ocular melanomas metastasize primarily, and initially exclusively, to the liver. The hepatic metastases may not appear until many years after enucleation, and delays of 10 to 15 years are not uncommon . Patients with metastatic uveal melanoma have a poor prognosis: more than half of all the patients die within 5 months of diagnosis of the metastasis. Even with therapy, survival is from 1 to 59 months only, with a median of 5 to 8 months.
In a separate collaboration with Dr. Hans Grossniklaus from the Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia (USA), we have shown that the B16-LS9 model system is the only murine syngeneic model able to spontaneously produce liver metastasis from a primary tumour grown in the posterior eye chamber. This is a very interesting, albeit intriguing finding, since B16-LS9 cells are unable to give rise to liver metastasis once grown as a subcutaneous tumour. It is tempting to speculate that some specific interactions within the eye microenvironment could support the intrinsic liver metastatic capability of these cells. Therefore, since we have correlated c-met overexpression with liver metastatic ability, we have started to investigate whether growth of B16-LS9 cells in presence of different eye components (Vitreous or aqueous humour, corneal or conjunctival epithelial cells, retinal pigmented epithelial cells) could influence their c-met expression levels. Results obtained so far suggest that this is not the case. Alternatively, hepatocyte growth factor, which is present in different eye compartments in moderate amounts (much less than in the lungs), could favour local invasion from B16-LS9 cells growing as primary tumour. This could allow an early escape of malignant cells, and settlement of liver metastases before the liver metastatic phenotype is lost during growth of tumour cells in an environment different from the liver, where they have been originally selected. The relative experiments are now under way.
(This part of the work has been conducted by Dr. Dario Rusciano at the research facilities of the ophthalmic company Sifi, SpA, in Catania, Italy.)
Publications acknowledging EC (BBW) grant support:
Rusciano, D. (2000) Differentiation and metastasis in melanoma. Crit. Rev. Oncogenesis, 11:147-163.
Elia, G., Ren, Y., Lorenzoni, P., Zarnegar, R. and Rusciano, D. Mechanisms regulating c-met overexpression in liver-metastatic B16-LS9 melanoma cells. J. Cell. Biochem. (in press).
Rusciano, D., Welch, D. and Burger, M.M. (2000) Cancer metastasis: experimental approaches. Laboratory Techniques in Biochemistry and Molecular Biology. Elsevier, Amsterdam, New York, Oxford.
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