Molecular mechanisms controlling human melanoma progression and metastasis

Progression from normal melanocyte to metastatic melanoma is a multistep, multigenic process. While relatively easy to conceptualize, the stages of melanoma progression are not necessarily discrete, nor are they unambiguously defined at the morphologic or molecular levels. The ability to stage the lesions more precisely would afford clinicians a greater confidence when designing therapeutic strategies. Unfortunately, a molecular definition of cells at each stage does not yet exist. Toward that end, the purpose of this review is twofold: (1) to highlight recent advances in our understanding of the molecular genetics of human cutaneous melanoma predisposition, and (2) to construct a molecular model of melanoma progression toward metastasis.     

Molecular biology of human melanoma development and progression

In the United States, Australia, Northern Europe, and Canada, malignant melanoma is increasing at a faster rate than any other cancer, with the exception of lung cancer in women. Major advances have been made in the molecular biology and immunology of melanoma. These advances in basic science have led to more rational approaches to specifically targeting melanoma cells, with promising results in the clinic. An increased understanding of how melanoma spreads has led to more selective, less invasive surgical procedures that do not compromise patient health. Combinations of chemotherapy and immunotherapy are now available for patients with advanced melanoma that affect both the length and quality of the patients' lives. This review of the molecular biology of melanoma development and progression discusses the disease's etiology, molecular genetics, cell-surface antigens, experimental models, biological markers, and new forms of treatment. As we continue to learn more about malignant melanoma, we will be able to devise more specific and effective treatments that will give patients with this potentially deadly disease longer and more productive lives.     

Identification of molecules associated with the development of metastasis in human malignant melanoma

Differential antibody reactivity has been used to identify molecules which change in expression or which are modified during tumor progression in human malignant melanoma. Such molecules may play a role in the development of the metastatic capacity of this tumor. Two of these molecules (ICAM-1 and MUC18) have been identified as cell adhesion molecules which are potentially involved in tumor-leukocyte-endothelial interactions.     

Endothelial P-selectin expression is reduced in advanced primary melanoma and melanoma metastasis

To evaluate the possible antigoitrogenic effect of somatostatin, the influence of long-acting somatostatin analog--octreotide--on experimental goiter developed in rats treated with propylthiouracil was examined. Goiter formation was assessed by measurement of the main histological compartments of the thyroid as well as by morphometric analysis of the vascularization and blood supply of the gland. Although treatment with octreotide did not prevent the goiter formation, it clearly reduced blood supply and vascularization of the thyroid and counteracted propylthiouracil-induced increase in the relative volume of follicular epithelium. To conclude, the somatostatin analog--octreotide--is effective in reduction of goiter vascularisation. This finding provides a rationale for the clinical trials of the treatment of hypervascular goiter by somatostatin analogs.     

Molecular pathology of melanoma

There has long been a clinical need for improved molecular pathology in melanoma, particularly in the histopathology laboratory where the differentiation of melanoma from its benign counterparts is commonly difficult. The need for improved molecular pathology has recently increased as immunotherapeutic options for the treatment of the tumour evolve. It seems likely that in the relatively near future tumour typing before and during immunotherapy will be needed. The identification of the tumour suppressor gene coding for the protein p16 as an important gene in the pathogenesis of melanoma is of great interest but the identification of oncogenes having a significant role in melanoma carcinogenesis has been slow.     

Cell shape changes and cytoskeleton reorganization during transendothelial migration of human melanoma cells

An in vitro system has been established to study the migration of human melanoma cells through a monolayer of endothelial cells. Endothelial cells were cultured to confluence on Matrigel before the seeding of melanoma cells. Laser scanning confocal microscopy showed that, prior to migration, melanoma cells appeared round and showed cortical F-actin staining. The initial stage of transmigration was characterized by numerous membrane blebs protruding from basolateral surfaces of the melanoma cells, and contact regions showed an abundance of filaments arising in the underlying endothelial cells. Later, pseudopods from the melanoma cells inserted into contact regions between endothelial cells. Eventually, the melanoma cells intercalated with the endothelial cells. At this stage, many endothelial filament bundles terminated at contacts between the endothelial cells and the transmigrating melanoma cell, suggesting active interactions between the two cell types. Upon contact with the Matrigel, melanoma cells began to spread beneath the endothelium, displaying a fibroblastic morphology with prominent stress fibers. To reestablish the monolayer, adjacent endothelial cells extended processes over the melanoma cell. Tumor necrosis factor alpha did not affect the transmigration of melanoma cells from cell lines isolated from several stages of metastasis. However, tumor necrosis factor did promote the transmigration of melanoma cells derived from a non-metastatic lesion. These results thus define cell attachment and cell penetration of the monolayer as two distinct steps in transmigration and suggest that tumor necrosis factor may enhance the metastatic potential of tumor cells.     

Molecular genetics of familial cutaneous melanoma

PURPOSE: A family history of melanoma is a significant risk factor for the disease, and recently several loci that determine susceptibility to the development of melanoma have been identified. The most important of these is p16/CDKN2A. We attempted to determine the degree to which the p16/CDKN2A gene has been implicated in the development of melanoma, and to identify other genetic factors that play a role as well. METHODS: We reviewed the literature published since the isolation of p16/CDKN2A and identified 13 studies that report the status of the gene in melanoma samples and 12 reports that examine p16/CDKN2A in melanoma kindreds. We also reviewed associated studies on CDK4 and RB1 involvement in melanoma, and examined the role of p16/CDKN2A in other inherited cancers. RESULTS: The evidence strongly implicates p16/CDKN2A in determining predisposition to malignant melanoma. Overall, approximately 20% of families that have been studied show mutations in the gene. However, because of clustering of sporadic cases in families, and potentially because of technical factors, this is likely an underestimate of the proportion of the genetic predisposition for melanoma that is due to p16/CDKN2A mutation. Rare families carry a mutated CDK4 gene that is also responsible for inherited melanoma. CONCLUSION: The gene p16/CDKN2A is an important determinant of melanoma risk. A commercial test is presently available to assess the status of this locus. However, because of uncertainties regarding the interpretation of the results of p16/CDKN2A genetic testing, we do not recommend routine clinical use of this test at this time.     

Fas and Fas ligand interactions suppress melanoma lung metastasis

Apoptosis induced by Fas (CD95) ligation is frequently lost during tumor progression; however, there is no direct evidence to support an association of Fas loss-of-function with metastatic tumor behavior. To determine whether Fas loss-of-function is critical for acquisition of the metastatic phenotype, we have compared the ability of Fas-sensitive K1735 murine melanomas to form spontaneous lung metastases in wild-type and Fas ligand-deficient mice. Fas-sensitive melanoma clones are highly tumorigenic but rarely metastatic in wild-type syngeneic mice. However, in Fas ligand-deficient mice, both the incidence and number of metastases are increased. These findings provide the first evidence that Fas-Fas ligand interactions can suppress metastasis and that tumor Fas loss-of-function may be causally linked to metastatic progression.     

N-ras oncogene expression changes the growth characteristics of human melanoma in two independent SCID-hu mouse models

Fifteen percent of all human melanomas carry mutations in ras genes, the majority of which are located in codon 61 of the N-ras gene. However, the biological significance of these mutations is as yet unknown. In this study, we investigated the influence of N-ras oncogene products mutated in codon 61 on the growth characteristics of human melanoma in vivo by establishing 2 SCID-hu mouse xenotransplantation models. Tumors grown in SCID mice injected with human melanoma carrying activated N-ras genes were significantly larger (p < 0.004) than tumors grown in animals injected with the appropriate control transfectants. Additionally, tumors with N-ras point mutations clearly showed a more pleomorphic phenotype than the control groups. Our results, obtained in 2 independent SCID-hu xenotransplantation models, suggest that mutated N-ras oncogene expression may be an important factor influencing growth characteristics of human melanoma without altering metastatic potential. These novel in vivo model systems provide a tool for further study of the biology of mutated ras in melanoma and should also prove useful for testing new and improved treatment strategies for human melanoma carrying mutated ras genes.     

Anti-cell death activity promotes pulmonary metastasis of melanoma cells

Bcl-2 inhibits apoptosis from a variety of stimuli, and a Bcl-2-binding protein BAG-1 also functions in protection from apoptosis in concert with Bcl-2. Here, we provide evidence that prolonged cell survival introduced by overexpression of Bcl-2 or BAG-1 proteins strongly promotes experimental pulmonary metastasis of melanoma B16-BL6 cells. In murine melanoma cell line B16-BL6, gene transfer-mediated expression of the Bcl-2 or BAG-1 led to prolonged cell survival against serum-starved apoptosis in vitro. The Bcl-2-expressing B16 cells, B16-Bcl-2 and the BAG-1-expressing B16 cells, B16-BAG-1 strongly enhanced pulmonary metastasis in allogenic BALB/c nude mice and whole lung weights were increased by 2.4-fold and 1.4-fold, respectively, compared with control transfectants, suggesting that Bcl-2 is a stronger positive modulator of metastasis. When the viable B16-Bcl-2 and control transfectants were injected subcutaneously into BALB/c nude mice, the colony numbers of pulmonary metastasis of the B16-Bcl-2 transfectant increased by 5.6-fold compared with the control transfectants. These enhanced metastatic potentials in the B16-Bcl-2 and the B16-BAG-1 transfectants were well correlated with anti-cell death activity against serum-starvation and enhanced cell viability on limiting dilution. Analysis of the transfectants however revealed that their growth rates, invasive ability and cell motility were not significantly altered by overexpression of either Bcl-2 or BAG-1 proteins. Taken together, these studies demonstrate that prolonged cell survival is a crucial factor to promote metastasis of melanoma, thereby contributing to tumor progression.     

Molecular mimicry in chronic inflammatory demyelinating polyneuropathy and melanoma

Polyclonal immunoglobulin M antibodies to the monosialoganglioside GM2, sulfoglucuronyl glycolipids, and sulfatide were detected by thin-layer chromatography and enzyme-linked immunosorbent assay in the serum of a patient with melanoma and chronic inflammatory demyelinating polyneuropathy. Both the patient's serum and polyclonal antibodies against GM2 reacted strongly with a biopsy of melanomatous tissue from the patient, suggesting a process of molecular mimicry.     

Simultaneous enucleation of a pulmonary melanoma metastasis and myocardial revascularization

BACKGROUND: The incidence of late, recurrence of malignant melanoma, is a well known, but very rare clinical experience. CASE REPORT: We report a case of simultaneous myocardial revascularisation and resection of pulmonary melanoma metastasis. In 1963 an enucleation of the right eye was necessary due to an ocular melanoma. In 1993 the patient suffered acute left heart failure and a 3-vessel disease with severe reduced left ventricular function was diagnosed. Chest X-ray examination revealed a singular pulmonary lesion in the right lower lobe with a diameter of 5.5 cm. Myocardial revascularisation and resection of the pulmonary focus was performed simultaneously without complication. The histological examination documented a pulmonary late recurrence of malignant melanoma. Up to this time (3 years later) the patient is free of cardiac symptoms and there is no evidence of further late recurrence of malignant melanoma. CONCLUSION: The appreciable number of patients who, after a disease-free interval of 10 to 20 years, develop a late recurrence of a malignant melanoma, and in particular-as in the present case-a choroidal melanoma, is powerful evidence for a need to keep these patients under lifelong surveillance.     

Suppression of human melanoma metastasis following introduction of chromosome 6 is independent of NME1 (Nm23)

BACKGROUND: Nasal immunotherapy with single allergen extracts, following premedication with cromolyn, has been reported to be effective in treating seasonal and perennial allergic rhinitis. METHODS: We conducted a double-blind, placebo-controlled study to assess the efficacy, tolerability, and mechanism of action of nasal immunotherapy for allergic rhinitis caused by weed pollens from three unrelated families. Twenty-seven weed-allergic patients underwent baseline nasal provocation and titrated skin test with a mixed weed extract containing ragweed, sage, and Chenopod extracts. Patients were randomized to receive either mixed weed extract or placebo. Nasal immunotherapy was self-administered daily to alternate nostrils preceded by 5.2 mg intranasal cromolyn. Beginning with 1:2500 wt/vol the concentration was increased to 1:10 wt/vol over an average period of 36 days. The maintenance dose (1:10 wt/vol) was administered daily for 12 to 16 weeks through the weed pollen season. Patients recorded nasal and eye symptoms and the use of rescue medications throughout the study. A nasal lavage for cytokine levels and nasal scraping with Rhinoprobe for nasal cytology were performed at the peak of the weed season. Nasal provocation and titrated skin tests with mixed weed extract were repeated after the weed season. Nasal lavage and scraping were also performed before and 24 hours after the final nasal provocation. RESULTS: During the peak weeks of the weed season the group receiving mixed weed extract by nasal instillation, compared with those treated with placebo, had significantly lower total nasal symptom scores, total eye symptom scores, and symptom medication scores. There were no significant differences in the nasal cytology or cytokines levels between the two groups, except for elevated IL-10 in the nasal lavage in the treated group at the peak of the season. Nasal symptoms and medication use were higher preseasonally in the active treatment group. CONCLUSION: Nasal immunotherapy with aqueous mixed weed extract administered with cromolyn sodium pretreatment for 17 to 21 weeks was effective in reducing both nasal and ocular symptoms of weed pollen-induced allergic rhinitis. There were increased nasal symptoms in the treated group preseasonally.     

In vivo activation and expansion of T cells by a bi-specific antibody abolishes metastasis formation of human melanoma cells in SCID mice

Bi-specific antibody fragments (bAB) are used in tumour therapy as a means to redirect and to strengthen effector cell function. It would be of great therapeutic advantage if, in addition, recruitment, expansion and the state of activity of effector cells are influenced by targeting through a bAB. This question was explored in the melanoma-bearing SCID mouse. The chemically coupled Fab' fragments of an anti-CD3 and an anti-p97 monoclonal antibody (MAB) were characterized in vitro for dual binding specificity and support of lymphokine-activated-killer-cell (LAKC) cytotoxicity towards a highly aggressive human melanoma line, which was significantly increased and exceeded levels of antibody-dependent cellular cytotoxicity observed in the presence of the anti-p97 MAB. The in vivo efficacy was tested in the SCID mouse: 5, 10 and 15 days after i.p. application of tumour cells, mice received LAKC (2 x 10(7)) together with bAB (150-100 microg). The application of bAB was repeated at days 20 and 25. Application of LAKC to melanoma-bearing SCID mice prolonged the mean survival time from 22 days of the untreated control group to 41 days. Anti-p97 did not exert any additive effect. In the presence of bAB, melanoma cells did not grow in 3 out of 8 mice. The mean survival time of the 5 mice developing tumours was 45 days. Importantly, none of the mice receiving bAB developed metastases, which were seen in 100% of animals receiving tumour cells or tumour cells plus LAKC or tumour cells plus LAKC plus anti-p97. As revealed by LAKC recovered from the SCID mice, the efficacy of the bAB was based on prolonged persistence of CD8-positive cells as well as on expansion and activation of CD4-positive cells, which was observed only in bAB-treated tumour-bearing mice. The efficiency in recruiting cytotoxic and, in particular, helper T cells suggests bAB as a valuable additive in immunotherapeutic treatment of melanoma patients.     

Adenosine receptor mediates motility in human melanoma cells

Cell motility is an essential component of tumor progression and metastasis. A number of factors, both autocrine and paracrine, have been found to influence cell motility. In the present study, adenosine and adenine nucleotides directly stimulated chemotaxis of A2058 melanoma cells in the absence of exogenous factors. Three adenosine receptor agonists stimulated motility in the melanoma cells and two adenosine receptor antagonists strongly inhibited the chemotactic response to both adenosine and AMP. The chemotactic stimulation by adenosine and AMP was pertussis toxin sensitive. Otherwise unresponsive Chinese hamster ovary cells which were transfected with the adenosine A1 receptor cDNA acquired the direct, pertussis toxin sensitive, chemotactic response to adenosine, and this response was inhibited by adenosine receptor antagonists. These findings demonstrate that adenosine and adenine nucleotides are capable of stimulating chemotaxis of tumor cells mediated through an adenosine receptor, probably of the A1 subtype. The possibility of antimetastatic therapies based on inhibition of adenosine receptor activity is raised.     

Phaeomelanic pigments from a human melanoma

The pigments in melanomas from 2 patients were studied with regard to solubility and chemical composition. Melanoma pigment from a patient with red-blonde hair was alkali-soluble and contained 9 or 10% sulfur and was thus of phaeomelanic type. Melanoma pigment from a patient with red-brown hair was insoluble in 0.2 N NaOH. Its sulfur content was 6%. This pigment was eumelanic with regard to solubility characteristics but the sulfur content was higher than previously observed for eumelanin.     

Interleukin 10 production by human melanoma

Interleukin 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these 35 samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.