A distinct octamer-binding protein present in malignant melanoma cells.

The octamer-binding proteins present in HeLa cells, B-cells and malignant melanoma cells were compared by a gel-electrophoresis DNA-binding assay. Using an extract from the malignant melanoma cells a complex was formed using a variety of octamer containing probes that was distinct from those found using either a HeLa or B-cell extract. DNAase 1 footprints and methylation interference patterns of the melanoma-specific octamer-binding protein were indistinguishable from those obtained with the HeLa factor NF-A1, except for preferential binding of the melanoma-specific factor to DNA methylated at two G residues 16 base-pairs 3' to the octamer motif. Competition analyses using a variety of wild-type and mutant probes showed that mutations affecting binding of NF-A1 similarly affected binding of the melanoma octamer-binding factor. These data also revealed the extreme flexibility of the octamer-binding site, with one probe sharing only 4 bases with the 8 base consensus sequence binding efficiently.     

Identification of proteins bearing beta1-6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary beta1-6-N-acetylglucosamine (beta1-6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing beta1-6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35--from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing beta1-6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing beta1-6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

Identification of proteins bearing β1–6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary β1–6-N-acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35 — from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing β1–6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing β1–6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

NOVA1 acts as an oncogene in melanoma via regulating FOXO3a expression

Increasing studies have suggested that dysregulation of RNA‐binding proteins (RBPs) contributes to cancer progression. Neuro‐oncological ventral antigen 1 (NOVA1) is a novel RBP and plays an important role in tumour development. However, the expression and role of NOVA1 in melanoma remain unknown. In this study, we indicated that NOVA1 expression was up‐regulated in melanoma samples and cell lines. Moreover, we demonstrated that knockdown of NOVA1 suppressed melanoma cell proliferation, migration and invasion in both A375 and A875 cell lines. In addition, we showed that suppressed expression of NOVA1 enhanced forkhead box O3a (FOXO3a) expression while inhibited AKT expression in melanoma cell. Furthermore, we demonstrated that inhibited expression of FoxO3A rescued NOVA1‐mediated cell proliferation, migration and invasion in melanoma cell line A375. These results suggested that NOVA1 acted as an oncogene in the development of melanoma partly through regulating FoxO3A expression.     

Cysteine 50 of the POU H domain determines the range of targets recognized by POU proteins.

The best target of POU proteins (Oct-1, Oct-2) is an octamer sequence ATGCAAAT. POU proteins also recognize, with weaker affinity, the TAAT-like targets of another group of regulatory factors, the homeoproteins. Up to now, it has not been known why Cys50 of the POUHdomain is absolutely conserved in contrast to that in homeoproteins. To assess the importance of Cys50 in determining the binding specificity of POU proteins, all possible amino acids were substituted for Cys at position 50, and the resulting mutants were tested with probes containing octamer (ATGCAAATNN) or homeospecific binding sites. Only the wild-type POU was shown to adequately discriminate between the octamer and homeospecific sites, and the protein affinity was only slightly affected by the nucleotide sequence flanking the octamer at the 3'-end. Any amino acid substitution at position 50 resulted in the mutant protein binding efficiently both to the octamer and the TAAT-like sequences. Moreover, in this case the 3'-flanking sequences influenced the binding to a much greater extent.     

Glycosylation profile of integrin alpha 3 beta 1 changes with melanoma progression

Glycosylation of integrins has been implicated in the modulation of their function. Characterisation of carbohydrate moieties of alpha(3) and beta(1) subunits from non-metastatic (WM35) and metastatic (A375) human melanoma cell lines was carried out on peptide-N-glycosidase F-released glycans using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). beta(1) integrin subunit from both cell lines displayed tri- and tetraantennary oligosaccharides complex type glycans, but only in A375 cell line was the sialylated tetraantennary complex type glycan (Hex(7)HexNAc(6)FucSia(4)) present. In contrast, only alpha(3) subunit from metastatic cells possessed beta1-6 branched structures. Our data indicate that the beta(1) and alpha(3) subunits expressed by the metastatic A375 cell line carry beta1-6 branched structures, suggesting that these cancer-associated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of alpha(3)beta(1) integrin. In direct ligand binding assays, alpha(3)beta(1) integrin from both cell lines binds strongly to fibronectin and to much lesser degree to placental laminin. No binding to collagen IV was observed. Enzymatic removal of sialic acid residues from purified alpha(3)beta(1) integrin stimulates its adhesion to all examined ECM proteins. Our data suggest that the glycosylation profile of alpha(3)beta(1) integrin in human melanoma cells correlates with the acquisition of invasive capacity during melanoma progression.     

Selective induction of cell death in melanoma cell lines through targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies.     

Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.     

P53 Mutation and c-fos Overexpression Are Associated With Detection of the Antigen VLA-2 in Human Melanoma Cell Lines

In this study, we examined the expression of c-fos, c-myc, mutant c-Ha-ras and mutant p53 proteins in three normal human melanocyte cell lines and the following 12 melanoma cell lines: M5, Mewo, A375, Bro, Mel 2a, O-Mel II, IgR 39, SkMel-13, -19, -28 Mel-57 and NKI-4, using an immunohistochemical assay (APAAP). An effort was made to correlate oncogene expression with growth parameters, differentiation antigens (HMB-45, vla-2, k.1.2.58, HLA-DR, HLA-I), and pigmentation. All melanocyte cell lines were negative for the oncogenes examined, whereas six of the melanoma cell lines were found also positive (three for c-fos, two for c-myc, one for c-Ha-ras, and four for p53). Three melanoma cell lines expressed one oncogene and three the combination c-fos/p53. These three melanoma cell lines were positive for the “late” tumor progression marker A. 1. 43 (vla-2 adhesion molecule) and negative for the differentiation marker k. 1. 2. 58. Positivity for A. 1. 43 combined with negative staining for k. 1. 2. 58 was found in six out of the 12 cell lines. The observed oncogene expression correlated neither with growth parameters nor melanin content. The present findings revealed a coexpression of mutant p53 and c-fos proteins being associated with a highly malignant phenotype in melanoma cell lines. Further studies are necessary to clarify the significance of the above findings