Three novel human sporadic melanoma cell lines: signaling pathways controlled by MC1R, BRAF and β-catenins

We studied the behaviour of three novel human sporadic melanoma cell lines (hmel1, hmel9, hmel11) extracted from tumors with different degrees of malignancy, concerning the cell signalling pathways controlled by MC1R, BRAF, NRAS and β-catenins. The novel cell lines were compared to metastatic cell lines (HBL, LND1), wild type (wt) for MC1R and BRAF genes, that have been extensively characterised and were used as control. All the novel cell lines have silent or no MC1R mutations even though MC1R signalling is severely impaired. Conversely, they harbour BRAF mutations at the V600 residue. These mutations determine a constitutive ERK phosphorylation in all the three cell lines. Our new melanoma cell lines were BRAF mutated in hetero- and homozygosis, even with a wild type MC1R, and unresponsive to NDP-MSH treatment. Quantity and subcellular localization of β-catenin were analyzed in both novel and control cell lines. In HBL and LND1 there were high levels of beta-catenin distributed in the cytoplasm/nucleus, while in the novel melanoma cell lines β-catenins were less abundant and seemed to be located at the plasma membrane/cytoplasm and absent in the nucleus. We sequenced beta-catenin cDNA for all the melanoma cell lines, and found mutations in HBL, LND1 and hmel1, while hmel9 and hmel11 were wt. We found that beta-catenin levels were not influenced by the RAS/RAF/MAPK pathway because inhibition with PD98059 (a MEK inhibitor) did not produce any effect on beta-catenin stability and/or localization.

We studied the behaviour of three novel human sporadic melanoma cell lines (hmel1, hmel9, hmel11), having different degree of malignancy, for cell signalling pathways controlled by MC1R, BRAF,NRAS and bcatenins. The novel cell lines were compared to metastatic cell lines (HBL, LND1), wild type (wt) for MC1R and BRAF genes, extensively characterised and used as control. All the novel cell lines have silent or no MC1R mutations even though MC1R signalling is severely impaired. Conversely they harbour BRAF mutations at the V600 residue. These mutations determine a constitutive ERK phosphorylation in all three cell lines. Our new melanoma cell lines BRAF mutated in hetero- and homozygosis, even with a wild type MC1R, are unresponsive to NDP-MSH treatment. The quantity and subcellular localization of bcatenin were analyzed both in novel and control cell lines. In HBL and LND1 there are high levels of bcatenin distributed in the cytoplasm/nucleus, while in novel melanoma cell lines bcatenins are less abundant and seem to be located at plasma membrane/cytoplasm and absent in the nucleus. We sequenced bcatenin cDNA for all the melanoma cell lines, and we found it mutated in HBL, LND1 and hmel1, while hmel9 and hmel11 were wt. We found that bcatenin levels are not influenced by the RAS/RAF/MAPK pathway because its inhibition with PD98059 (a MEK inhibitor) does not produce any effect on bcatenin stability and/or localization.