Microarray analysis of phosphatase gene expression in human melanoma.

BACKGROUND: Tyrosine phosphate is abnormally elevated in malignant melanoma, and this has been interpreted to reflect the activity of oncogenic protein tyrosine kinases. However, elevation may also arise due to decreased protein tyrosine phosphatase (PTP) expression. OBJECTIVES: To survey phosphatase gene expression in melanoma cell lines, a benign naevus and normal melanocytes: we searched for downregulation of phosphatase gene expression in malignant cells that may indicate a role as melanoma suppressor genes. METHODS: Microarray analysis was used to compare gene expression for 133 phosphatase genes, comprising 39 PTPs, 16 dual-specificity phosphatases (DSPs), 47 serine/threonine phosphatases and 31 acid/alkaline and lipid-based phosphatases. Northern blotting analysis was used to study gene expression in human melanoma biopsies. RESULTS: There was decreased expression of four DSP genes (including PTEN); eight receptor PTP genes were downregulated in melanoma, among which were PTP-KAPPA and PTP-PI (consistent with our previous data). In addition, PTP-RF/LAR was downregulated in 13 of 22 metastatic melanomas. CONCLUSIONS: The expression of multiple PTP receptors is decreased in melanoma; this may be a mechanism which stimulates autonomous growth in advanced melanoma.

Tyrosine phosphate in melanoma progression.

BACKGROUND: Cellular tyrosine phosphorylation is regulated by two large families of enzymes. Protein tyrosine kinases (PTK) mediate addition, and protein tyrosine phosphatases (PTP), removal of phosphate from protein substrates. PTKs are oncogenes and PTPs have been hypothesized to function as tumour suppressor genes. OBJECTIVES: To determine changes in tyrosine phosphate and PTP activity that occur during melanoma progression. METHODS: Immunohistochemistry was used to study phosphotyrosine in melanocytic lesions. In addition, PTP activity of normal melanocytes and melanoma cell lines was measured using an enzyme-linked immunosorbent assay-based system. RESULTS: Melanocytes in normal skin and most (67%) benign naevi were not immunostained. Neither were early malignant lesions (80% of malignant melanoma in situ and radial growth phase melanomas) stained. However, most advanced melanomas (100% of vertical growth phase, and 90% of metastatic melanomas) were immunoreactive. When total PTP enzyme activity was assayed in normal melanocytes and malignant melanoma cell lines, there was a significant increase in activity associated with melanoma progression. CONCLUSIONS: Taken together, the data suggest increased phosphotyrosine signalling occurs during melanoma progression at the stage when cells first become competent for metastasis.

Protein tyrosine phosphatase genes downregulated in melanoma.

Phospho-tyrosine levels are increased in melanoma, apparently consistent with reports of elevated protein tyrosine kinase activity. Some protein tyrosine kinases are encoded by oncogenes and have been implicated in melanoma genesis. Decreased protein tyrosine phosphatase activity may also increase phospho-tyrosine. Protein tyrosine phosphatase genes are candidate tumor suppressors and loss of expression may contribute to melanoma genesis. Here we survey protein tyrosine phosphatase expression in pigment cells. Protein tyrosine phosphatase genes were cloned by reverse transcriptase polymerase chain reaction using degenerate primers based upon conserved sequences within the phosphatase catalytic domain. Reaction products were cloned and sequenced: 118 and 113 partial protein tyrosine phosphatase products were isolated from normal melanocytes and melanoma cells, respectively. Northern blotting analysis was used to study expression of 15 protein tyrosine phosphatase genes. Expression of PTP-kappa and PTP-pi was absent or downregulated in more than 20% of melanoma cell lines and in some unmanipulated melanoma biopsies. These closely related enzymes are members of the 2B receptor protein tyrosine phosphatase family previously implicated in contact inhibition. Loss of protein tyrosine phosphatase expression may contribute to the abnormal tyrosine phosphorylation seen in melanoma; these genes are candidate tumor suppressors.

Protein tyrosine kinases in malignant melanoma.

Protein tyrosyl phosphorylation is an essential component in intracellular signalling, with diverse and crucial functions including mediation of cell proliferation, survival, death, differentiation, migration and attachment. It is regulated by the balance between the activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. A number of PTKs are encoded by proto-oncogenes or viral oncogenes, and are thus strongly implicated in cancer. While a role for PTKs in human melanoma is less firmly established, human melanomas or melanoma cells have been reported to contain more tyrosine phosphate than normal melanocytes, and some receptor PTKs (EPH-A2/ ECK and EPH-B3) are overexpressed in over 90% of melanoma cell lines. Other specific PTKs are also frequently overexpressed, including KDR and fibroblast growth factor receptor-4 (FGF-R4), while, interestingly, yet others, such as KIT and FES, are consistently downregulated in melanoma cell lines. All of these differentially expressed PTKs are candidates for gene products important in melanoma development. In addition, PTKs expressed in significant amounts in both benign and malignant melanocytes, such as insulin-like growth factor-1 receptor (IGF1-R), FGF-R1, HER2/NEU and FAK, are likely to play a role in melanoma genesis and progression.

Requirement for focal adhesion kinase in tumor cell adhesion.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase and a major phosphotyrosine-containing protein. FAK is found in cell-matrix attachment sites (focal adhesions), and is activated on integrin-ligand binding and by other signaling pathways. Several roles have been proposed for FAK; here we report a novel function. We observed abundant FAK protein in all human melanoma cell lines tested except COLO839, a line that grows predominantly in suspension and was derived from peripheral blood. Five adherent lines, isolated from solid metastases in the same patient as COLO839, did express FAK. We derived four adherent sublines from COLO839. These did express FAK, even when plated on bacteriological plastic, to which they did not adhere. Thus, substrate attachment was not required for FAK expression. Three of the adherent sublines were then grown in the presence of antisense oligonucleotides to the initial FAK coding sequence. All showed substantially reduced FAK expression and, interestingly, the cells largely detached from the substrate while continuing to grow. Similar results were obtained with an independent melanoma line, DX3. Thus, FAK expression appears to be required by melanoma cells for substrate adhesion.

Cloning and characterisation of the chicken gene encoding the telomeric protein TRF2.

The telomere-associated protein TRF2 binds as a homodimer to double-stranded (TTAGGG)n arrays in vitro and localises to chromosome ends in vivo. Inhibition of TRF2 in human cell lines and recent electron microscopy analyses suggest that TRF2 plays a crucial role in the maintenance of telomere integrity. To study the role of TRF2 in vertebrate telomere biology using an alternative model system, we report the isolation and characterisation of the chicken TRF2 locus. The TRF2 protein is highly conserved between mammals and birds, particularly within the dimerisation and myb-type DNA binding domains. However, the chicken ORF predicts an additional protein domain consisting of 15 copies of a degenerate 13 amino acid repeat. Indirect immunofluorescence reveals the localisation of a FLAG-tagged version of the chicken TRF2 protein at chromosome ends in both chicken and human cells suggesting that the protein is functionally conserved.

Up-regulation of ephrin-A1 during melanoma progression.

Ephrin-A1, formerly called B61, is a new melanoma growth factor; it is angiogenic and chemoattractant for endothelial cells. EPH-A2, or ECK (a receptor for ephrin-A1), is ectopically expressed in most melanoma cell lines; the pathology where this expression is first manifested and the possible role of the receptor in tumor progression are unknown. To determine these, we studied the expression of this ligand and receptor in biopsies of benign and malignant melanocytic lesions. EPH-A2 was not detected in normal melanocytes, benign compound nevi or advanced melanomas, though it was found in 2 of 9 biopsies of malignant melanoma in situ. Ephrin-A1 was present in occasional early lesions and in advanced primary melanomas (43%) and metastatic melanomas (67%). Expression of ephrin-A1 was induced in melanoma cells by pro-inflammatory cytokines. Our findings are consistent with 2 possible roles for ephrin-A1 in melanoma development: it may promote melanocytic cell growth or survival and induce vascularization in advanced melanomas. Both effects may be potentiated by inflammatory responses. Our data are consistent with earlier observations that an inflammatory infiltrate is associated with poor prognosis in thin primary melanomas.

Impaired growth and differentiation of diploid but not immortal melanoblasts from endothelin receptor B mutant (piebald) mice.

Endothelin 3 (Edn3) and its preferred receptor, endothelin receptor B (Ednrb), are implicated in development, especially that of two neural-crest-derived cell lineages: melanocytes and enteric ganglion cells. Mice and humans with a null mutation at either locus can show major deficiencies in both cell types: congenital white spotting and aganglionic megacolon (Hirschsprung disease in human). Numbers of early (migrating) embryonic melanoblasts are low in Ednrb(ls) mutant mice, while added Edn3 appears to promote the growth of melanocyte precursors in neural crest cultures. However, it is hard to assess cell differentiation in these mixed cultures, and it is not known whether Ednrb has any role in the postnatal melanocytic lineage. We have therefore studied primary cultures of neonatal melanoblasts homozygous for the piebald (Ednrb(s)) mutation. These mutant melanoblasts showed severe impairment of both net cell growth and differentiation compared to wild-type melanoblasts. They were also unresponsive to stimulation of growth by cholera toxin. We have established three immortal lines of melanoblasts and one of melanocytes homozygous for Ednrb(s). These immortal lines, however, had no detectable deficiency of growth or differentiation as judged by cell counts, induced pigmentation and immunocytochemistry for melanocytic markers. Consistent with this, neither Ednrb nor Edn3 mRNA was detected in 3/3 tested immortal lines of mouse melanoblasts and 5/5 lines of melanocytes, of various genotypes. We also report for the first time a method to grow immortal melanoblasts in pure culture, without feeder cells.

Brachyury-related transcription factor Tbx2 and repression of the melanocyte-specific TRP-1 promoter.

Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position -237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.

recessive spotting: a linked locus that interacts with W/Kit but is not allelic.

BACKGROUND: The murine coat-colour mutation recessive spotting (rs) maps very closely to the W/Kit locus, encoding the proto-oncoprotein Kit, the protein tyrosine kinase receptor for stem cell factor. Kit is important in the development of melanocytes, germ cells, interstitial cells of Cajal (ICC) and haemopoietic lineages, including mast cells. rs has never been genetically separated from Kit, and interacts with Kit mutations, suggesting that it is a recessive allele of Kit. Here we have tested this possibility. We have shown previously that diploid rs/rs melanocytes proliferated more slowly than did +/+ melanocytes, as did an immortal line of rs/rs melanocytes, melan-rs. RESULTS: The Kit mRNA level in rs/rs melanocytes was indistinguishable from that of other melanocyte lines. The Kit cDNA sequence from rs/rs melanocytes and the kinase activity of Kit in rs/rs mast cells appeared to be normal. No deficiency of mast cells or ICC was observed in rs/rs mice. Moreover, following the overexpression of a normal Kit cDNA, proliferation of rs/rs melanocytes was retarded further, but that of +/+ melanocytes was increased, indicating an intracellular interaction between rs and Kit. Of other closely linked tyrosine kinase genes, melanocytes and melanoblasts did not express mRNA for Pdgfra, Flk-1 or Txk, but both expressed Tec, encoding a nonreceptor kinase that interacts with Kit. CONCLUSIONS: rs is not a mutation in Kit, although we have confirmed that rs interacts with Kit. It seems unlikely that rs affects Pdgfra, Flk-1 or Txk, but Tec remains a candidate for rs.

Melanosomal defects in melanocytes from mice lacking expression of the pink-eyed dilution gene: correction by culture in the presence of excess tyrosine.

Mutations in the murine pink-eyed dilution (p) gene, or its human homologue P, result in oculocutaneous albinism. Melanocytes cultured from mice lacking p gene expression exhibit defective melanogenesis, but following culture in the presence of high concentrations of L-tyrosine, increased melanin deposition is observed. Electron microscopy and image analysis demonstrated that untreated p mutant melanocytes exhibited small melanosomes, largely of stages I-II. Following tyrosine treatment, increased proportions of stage III-IV melanosomes, almost normal in size, were observed. Levels of tyrosinase protein and to a lesser extent of tyrosinase-related protein-1 (TRP-1) were subnormal but rose dramatically following stimulation by tyrosine. Levels of TRP-2 and Pmel17/silver gene product were not altered, nor were the levels of mRNA for tyrosinase, TRP-1, TRP-2, or the Pmel17/silver gene product. As expected, the 110-kDa product of the p gene was absent from both stimulated and unstimulated p mutant cells. In a melanoblast line derived from the same mice, excess tyrosine failed to stimulate visible melanogenesis or increase the low levels of tyrosinase. The melanosomes in these cells were smaller still than those in the mutant melanocytes even when cultured in the presence of excess tyrosine. Thus, absence of the p gene product affects melanosomal structure and protein composition at the posttranscriptional level. These defects are correctable at least in part by supplementation with L-tyrosine.

Loss of expression of receptor tyrosine kinase family genes PTK7 and SEK in metastatic melanoma.

Protein tyrosine kinases (PTKs) have been implicated in the development of many common human tumours including melanoma. Previously we isolated PTK gene sequences expressed in normal melanocytes. Here we examined expression of 9 of these genes in cell lines derived from defined stages of melanoma progression, by Northern blotting and in some cases immunoblotting. We also tested cells from 2 animal models of particular stages in progression, as well as uncultured biopsies of metastatic melanoma. The expression of 2 receptor kinase family members found in melanocytes, PTK7/CCK-4 and SEK/TYRO1, was decreased or lost in advanced melanomas. PTK7 mRNA was found in only 54% of melanoma cell lines and 20% of melanoma biopsies. Similarly, expression was lost in 2 advanced cell lines selected from an early melanoma line that did express PTK7 mRNA. SEK/TYRO1 expression was observed in 75% and 17% of cell lines from primary and metastastic melanomas, respectively. Conversely, mRNA for the non-receptor kinase PTK6/BRK was not detected in normal melanocytes or primary melanoma lines, but was found in 9% of metastatic melanoma cell lines.

Cloning of the murine homolog of the ocular albinism type 1 (OA1) gene: sequence, genomic structure, and expression analysis in pigment cells.

We report the isolation of the mouse homolog of OA1, the gene responsible for ocular albinism type 1. The mouse Oa1 gene encodes a putative protein of 405 amino acids displaying a high level of homology (78% identity, 87% similarity) to the human gene. All disease-associated missense mutations reported in patients with ocular albinism involve conserved amino acid residues in the mouse protein. Moreover, the murine homolog shows six putative transmembrane domains, as observed for the human gene, indicating that the overall structure of the two proteins is conserved. The genomic organization is also conserved between the two species across the entire coding region with splice sites located in the same positions. Like its human counterpart, the expression pattern of Oa1, apart from the eye, is restricted to the epidermal melanocyte lineage. A transcript of approximately 1.8 kb was readily detected by this probe in 5 out of 5 murine melanocyte lines, 4 out of 4 murine melanoblast lines, 1 out of 2 murine melanoma lines, and 1 out of 2 human melanoma lines tested, but it was not detected in 2 out of 2 lines of a developmentally earlier normal cell type, melanoblast precursor cells, suggesting that the gene is transcriptionally activated in epidermal melanocytes at the same stage as most other tested melanosomal proteins. Together, these data suggest that the function of the OA1 gene is conserved between human and mouse and point to the mouse as a model to facilitate the understanding of ocular albinism pathogenesis.

Expression of NM23 in human melanoma progression and metastasis.

NM23 is a putative metastasis-suppressor gene for some human cancers. Here we have studied NM23 expression during melanoma progression using Northern blotting and immunocytochemistry. There was no significant difference in the average amounts of NM23 mRNA between cell lines derived from metastatic and primary melanomas. The level of NM23 mRNA was also determined for three pairs of poorly metastatic parental (P) and their highly metastatic variant (M) cell lines; the ratios for M/P were 1.2, 0.98 and 0.80. Next we used immunocytochemistry to study NM23 protein in normal skin, benign naevi and primary and metastatic melanomas. Melanocytes in all normal skin and benign samples were positive for NM23; however most primary melanomas (7/11) were not stained by the antibody. All metastatic melanoma samples (5/5) were positively stained. Findings were similar with an antiserum reactive with both forms of NM23 (H1 and H2), and with an antibody specific for NM23-H1. No relationship was apparent between NM23 immunoreactivity in primary tumours and their aggressiveness or prognosis. Hence, in contrast to the situation described for murine melanoma, the amount of NM23 mRNA or protein in human melanoma did not correlate inversely with metastasis.

The POU domain transcription factor Brn-2: elevated expression in malignant melanoma and regulation of melanocyte-specific gene expression.

Previous work has shown that melanoma cell lines express a distinct octamer binding protein. Given the role of octamer-binding proteins in cell differentiation and development, the role this factor is a key issue in understanding melanocyte differentiation and transformation. Using a proteolytic clipping assay, we show that the melanoma-specific octamer factor is Brn-2/N-Oct3, a POU domain protein previously known to be expressed in adult brain and in the developing nervous system. N-Oct3 mRNA was detected in a range of human melanoma cell lines and was around 10-fold elevated compared to normal human melanocytes while mRNA for Brn-2 was also detected in a mouse melanoblast cell line. Expression of Brn-2/N-Oct3, in melanoma cells in cotransfection assays activated the expression of the MHC class II DR alpha promoter but repressed the activity of the melanocyte-specific tyrosinase promoter. Repression correlated with Brn-2/N-Oct3 binding in a mutually exclusive fashion with basic-helix-loop-helix-leucine-zipper (bHLH-LZ) transcription factor USF in vitro and with Brn-2 expression preventing activation of the tyrosinase promoter by the bHLH-LZ factor Microphthalmia in vivo. The potential role of Brn-2/N-Oct3 in melanocyte differentiation and gene expression is discussed.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

Abnormal protein tyrosine kinase gene expression during melanoma progression and metastasis.

Protein tyrosine kinases have been implicated in tumor initiation and progression. Here we used Northern blotting to study expression of their genes in cultured normal melanocytes and 19 melanoma cell lines from different stages of tumor progression. We detected transcripts for 2 cytoplasmic (ABL and FES) and 6 receptor (ECK, ERB-B2, FGF-R4, IGFI-R, KDR and TIE) kinases but not for receptors RET or TRK-A. Genes for ECK, FGF-R4 and TIE were expressed ectopically in melanomas (not in normal melanocytes). Similarly, ECK protein was detected by immunoblotting in metastatic melanomas but not in normal melanocytes. ECK mRNA levels tended to increase again during late melanoma progression. ECK and TIE mRNAs were also detected in highly metastatic variant cells but not in the corresponding poorly metastatic parental lines. Conversely, FES and KDR gene expression was lost in most advanced primary and metastatic melanomas. These findings suggest positive and negative roles for specific tyrosine kinases during progression.

Novel and known protein tyrosine kinases and their abnormal expression in human melanoma.

We have used the polymerase chain reaction and Northern blotting to identify protein tyrosine kinases that may play an important role in the process of melanoma initiation and progression. Degenerate primers from the conserved catalytic domain of tyrosine kinase genes were used to amplify and clone partial cDNA sequences from a human melanoma cell line (DX3-LT5.1) and normal human melanocytes. When the melanoma reaction products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named MEK (for melanocytic kinase). Of the remaining 12 known kinases, only two, ERB-B2 and IGF1-R, have previously been reported in pigment cells. Reaction products from melanocytes included only eight of these 13 sequences. To test for quantitative differences in tyrosine kinase expression between normal and malignant cells, a panel of eight melanoma lines and normal melanocytes was analyzed by Northern blotting. Two tyrosine kinases (JTK-14/TIE and TYRO-9) were detected in some melanomas but were not found in normal melanocytes, whereas others, including MEK, appeared to be overexpressed in some malignant lines. A minority of kinases showed either no change or a reduction in the level of mRNA. Expression of tyrosine kinases varied independently, and individual lines contained various combinations of these enzymes. Our findings are consistent with an increased overall expression of these putative growth factor receptors during melanoma development.

Characterization and mapping of the human SOX4 gene.

The SOX genes comprise a large family related by homology to the HMG-box region of the testis-determining gene SRY. We have cloned and sequenced the human SOX4 gene. The open reading frame encodes a 474 amino acid protein, which includes an HMG-box. The non-box sequence is particularly rich in serine residues and has several polyglycine and polyalanine stretches. With somatic cell hybrids, human SOX4 has been mapped to Chromosome (Chr) 6p distal to the MHC region. There is no evidence for clustering of other members of the SOX1, -2, and -3 or SOX4 gene families around the SOX4 locus.