TWO TYPES OF MELANOMAS IN A NEW EXPERIMENTAL SYSTEM OF PLATYFISHSWORDTAIL HYBRIDS

A new experimental system for investigating platyfish-swordtail hybrid melanomas was developed using ornamental platyfish ( Xiphophorus maculatus ) and swordtails ( Xiphophorus helleri ). The incidence of melanomas in the hybrids was studied in relation to individual age factors. Based on the age of the onset of the melanomas, two types of the melanomas were discerned and were designated as the fry and the adult melanomas, respectively. The fry melanomas were characterized by early onset, wide-ranged lesions, an invasive nature owing to densely pigmented cells, and a tendency toward regression. The adult melanomas were characterized by occurrence in the later stage of life, a small number of primary sites, and rapid, expanding, and invasive growth due to sparsely pigmented cells.     

Multi-step genetic regulation of oncogene expression in fish hereditary melanoma

Melanoma occurring spontaneously in Xiphophorus fish hybrids is a model system in which involvement of cellular oncogenes and multi-step regulation of their expression have been identified by classical genetics. The macromelanophore gene in platyfish (Xiphophorus maculatus) is a sex-linked codominant gene which determines the black spot patterns of macromelanophores in the skin. The macromelanophore locus includes a cellular oncogene which potentially induces neoplasms of the pigment cells. Expression of the oncogene is regulated by a multi-step genetic process and brings about a characteristic phenotype associated with pigment cell differentiation at each step. The multi-step genetic regulation of oncogene expression can be recognized by interspecific hybridization of the platyfish with swordtails (Xiphophorus helleri) which have not developed the macromelanophore gene. When platyfish are hybridized with swordtails, the F1 offspring carrying this gene develop a preneoplastic state. When the F1 offspring are back-crossed to swordtails, the backcross offspring develop a heritable form of melanoma with a characteristic inheritance pattern. This heritable form of melanoma occurs at an early age and has a well differentiated character. Thus, the first and second steps of oncogene expression bring about a preneoplastic state in the F1 offspring and a heritable form of melanoma in the backcross offspring, respectively. These steps may be due to progressive substitution of platyfish chromosomes with swordtail chromosomes in germ line cells, resulting in a progressive reduction of the dosage of regulatory genes in the platyfish genome. The third step of oncogene expression brings about a sporadic form of melanoma in the hybrid offspring bearing the preneoplastic state and heritable form of melanoma spontaneously or through induction by carcinogens. This form of melanoma has a poorly differentiated character. The incidence of this form is considerably enhanced by aging in adult life, thus exhibiting age-specific incidence. It is likely that this step is due to mutational events in regulatory genes, which occur in somatic cells following chromosome substitution in germ line cells by hybridization. The albino gene enhances the malignancy of the two forms of melanoma and the incidence of the sporadic form of melanoma, possibly by suppressing the differentiation of transformed pigment cells. These facts and speculations are summarized in Fig. 6. The molecular identification of oncogenes in this melanoma system and their transfer into the swordtail eggs may provide a useful means for studying oncogene expression during development, growth, and aging of animals.     

Cellular Heterogeneity in the Late-onset Form of Hereditary Melanomas in the Xiphophorus Fish Hybrids

The late-onset form of melanomas occurring in the Xiphophorus , fish hybrids carrying a macro-melanophore gene Sp was investigated for its cellular heterogeneity. The melanoma tissues were dissociated enzymatically and cultured for a short term. The cultured melanoma cells were characterized according to cell size, cell shape, pigmentation, and response to epinephrine. The melanoma cells were considerably heterogeneous in these phenotypic traits. Various combinations of these heterogeneous cells gave a great heterogeneity to individual melanomas. The stability of the phenotypic traits was followed during the course of tumor growth. Cell size and cell shape were stable, but pigmentation and response to epinephrine varied. The results are discussed in relation to cell differentiation and tumor progression.     

Evidence and impact of neutrophil extracellular traps in malignant melanoma

Ulceration of melanoma is associated with neutrophil infiltrates and lower survival rates opposite to non‐ulcerated melanoma. Neutrophils release neutrophil extracellular traps (NETs) that are chromatin structures loaded with antimicrobial proteins. Since NETs have been correlated with tumor progression, we investigated whether NETs appear in melanoma and affect melanoma cells. Indeed, human primary melanoma biopsies revealed neutrophils releasing NETs in all of 27 ulcerated melanomas, whereas NETs were absent in all of 7 non‐ulcerated melanomas. However, the quantity of intratumoral NETs did not correlate with tumor progression of melanoma. Interestingly, in vitro assays showed that melanoma cells attach to NETs via integrin‐mediated adhesion and that NETs inhibit tumor cell migration. Moreover, co‐culturing of NETs and melanoma cells had a cytotoxic effect on melanoma cells resulting in necrosis. Hence, we discovered in vitro an antineoplastic role of NETs in melanoma.     

Galectin-1-mediated biochemical controls of melanoma and glioma aggressive behavior

Gliomas and melanomas are associated with dismal prognosis because of their marked intrinsic resistance to proapoptotic stimuli,such as conventional chemotherapy and radiotherapy,as well as their ability to escape immune cell attacks.In addition,gliomas and melanomas display pronounced neoangiogenesis.Galectin-1 is a hypoxia-sensitive protein,which is abundantly secreted by glioma and melanoma cells,which displays marked proangiogenic effects.It also provides immune tolerogenic environments to melanoma and glioma cells through the killing of activated T cells that attack these tumor cells.Galectin-1 protects glioma and melanoma cells against cytotoxic insults(including chemotherapy and radiotherapy) through a direct role in the unfolded protein response.Altogether,these facts clearly point to galectin-1 as an important target to be combated in gliomas and melanomas in order to:(1) weaken the defenses of these two types of cancers against radiotherapy,chemotherapy and immunotherapy/vaccine therapy;and(2) reinforce antiangiogenic therapies.In the present article,we review the biochemical and molecular biology-related pathways controlled by galectin-1,which are actually beneficial for melanoma and glioma cells,and therefore detrimental for melanoma and glioma patients.     

Tumor Gene Expression and Interphase Chromatin Appearance in Xiphophorus

SYNOPSIS. Xiphophorus maculatus (platyfish) exhibits spots whichconsist of neoplastically transformed pigment cells (T-cells).The spots actually represent extreme benign melanomas. Transformationto T-cells is mediated by a "tumor gene" (Tu). Platyfish whichdevelop from X-irradiated embryos reveal an increase of Tu-expressionresulting in an overproduction of T-cells to benign melanomaswhich can be compared to that observed in certain hybrids betweenthe platyfish and Xiphophorus helleri (swordtail). Both theX irradiation-induced and the crossing conditioned increaseof Tu-expression represent a heritable alteration which mightbe related to a conversion from dispersed to condensed chromatinin the interphase nuclei.     

Structural organization, mapping, characterization and evolutionary relationships of CDKN2 gene family members in Xiphophorus fishes

Xiphophorus fishes and their hybrids are used as models for the study of melanoma and other diseases. The cyclin-dependent kinase inhibitor gene family in humans is comprised of four members, including CDKN2A (P16), and dysregulation of this gene is implicated in numerous neoplasms including melanomas. We have investigated the status of the gene family in the southern platyfish X. maculatus. Xiphophorus harbors at least two such loci, which we now term CDKN2A/B and CDKN2D. Both loci map to Xiphophorus linkage group 5, a genomic area that has long been known to harbor the DIFF tumor suppressor locus. Within this report, we report on the complete cloning, genomic exon/intron boundary delineation, linkage mapping and expressional characteristics of Xiphophorus CDKN2D. We also compare and contrast this expression to that of the previously isolated CDKN2AB locus in normal and neoplastic tissues derived from non-hybrid and hybrid fishes. The hypothetical evolutionary relationships of gene family members and their involvement in melanoma is evaluated. In comparison to CDKN2A/B, the RNA expression of Xiphophorus CDKN2D differs in normal tissues and is not associated with melanotic/pathologic tissues, confirming functional divergence between obvious homologues.     

Tumoricidal activity of syngeneic macrophages from melanoma-bearing mice: Changes with progressive tumor growth

Summary Changes in the cytostatic and cytotoxic activity of macrophages from tumor-bearing (TBM) and control mice were studied in a murine model of malignant melanoma. Syngeneic macrophages from TBM were initially noncytotoxic, but became cytotoxic and achieved their maximum destructive ability after 14 days of tumor growth. With continued tumor growth these macrophages either lost or had reduced cytotoxic activity. In contrast, macrophages from the same melanoma-bearing animals were significantly cytostatic at an earlier stage of tumor growth, but with continued melanoma growth these macrophages were no more cytostatic than controls. Moreover, melanomas grew slowly during the time when macrophages were observed to be cytostatic but grew rapidly at those stages when macrophages had a reduced ability to inhibit melanoma DNA synthesis. When these effector cells became cytotoxic melanomas were growing rapidly and changes in cytotoxicity had little effect on tumor mass. Thus, macrophages do not completely suppress melanoma proliferation and, although exhibiting cytotoxicity they were relatively ineffective in controlling a large mass of tumor cells.     

Constitutive aberrant endogenous interleukin-1 facilitates inflammation and growth in human melanoma

Interleukin (IL)-1-mediated inflammation is proposed to contribute to the development and progression of some cancers. IL-1 family member proteins are known to be expressed constitutively in many melanoma tumor cells, and we hypothesize that these support molecular pathways of inflammation and facilitate tumor growth. To investigate the expression of IL-1α and IL-1β in melanoma patients, and their association with disease progression, immunohistochemical staining was carried out on tissues from 170 patients including benign nevi, primary melanomas, and metastatic melanomas. IL-1β levels were low (or zero) in benign nevi and higher in primary and metastatic melanomas (P < 0.0001). IL-1α was expressed in about 73% of nevi and 55% of metastatic melanomas, with levels significantly higher in primary tumors (P < 0.0001); most (98%) primary melanoma samples were positive for IL-1α. In vitro studies with seven human melanoma cell lines showed that five cell lines expressed IL-1α and IL-1β proteins and mRNA. We identified for the first time several important downstream signaling pathways affected by endogenous IL-1, including reactive oxygen and nitrogen species, COX-2, and phosphorylated NF-κB inhibitor (IκB) and stress-activated protein kinase/c-jun-NH(2)-kinase; all of which were decreased by siRNA to IL-1s. Downregulation of IL-1α, IL-1β, or MyD88 substantially increased p21 and p53 levels. Treatment with IL-1 receptor type I neutralizing antibody or IL-1 pathway-specific siRNAs led to growth arrest in IL-1-positive melanoma cells. Furthermore, blocking the IL-1 pathway increased autophagy in IL-1-positive melanoma cells. These results indicate that the endogenous IL-1 system is functional in most human melanoma and interrupting its signaling inhibits the growth of IL-1-positive melanoma cells.     

Dual inactivation of RB and p53 pathways in RAS-induced melanomas

The frequent loss of both INK4a and ARF in melanoma raises the question of which INK4a-ARF gene product functions to suppress melanoma genesis in vivo. Moreover, the high incidence of INK4a-ARF inactivation in transformed melanocytes, along with the lack of p53 mutation, implies a cell type-specific role for INK4a-ARF that may not be complemented by other lesions of the RB and p53 pathways. A mouse model of cutaneous melanoma has been generated previously through the combined effects of INK4a(Delta2/3) deficiency (null for INK4a and ARF) and melanocyte-specific expression of activated RAS (tyrosinase-driven H-RAS(V12G), Tyr-RAS). In this study, we made use of this Tyr-RAS allele to determine whether activated RAS can cooperate with p53 loss in melanoma genesis, whether such melanomas are biologically comparable to those arising in INK4a(Delta2/3-/-) mice, and whether tumor-associated mutations emerge in the p16(INK4a)-RB pathway in such melanomas. Here, we report that p53 inactivation can cooperate with activated RAS to promote the development of cutaneous melanomas that are clinically indistinguishable from those arisen on the INK4a(Delta2/3) null background. Genomewide analysis of RAS-induced p53 mutant melanomas by comparative genomic hybridization and candidate gene surveys revealed alterations of key components governing RB-regulated G(1)/S transition, including c-Myc, cyclin D1, cdc25a, and p21(CIP1). Consistent with the profile of c-Myc dysregulation, the reintroduction of p16(INK4a) profoundly reduced the growth of Tyr-RAS INK4a(Delta2/3-/-) tumor cells but had no effect on tumor cells derived from Tyr-RAS p53(-/-) melanomas. Together, these data validate a role for p53 inactivation in melanomagenesis and suggest that both the RB and p53 pathways function to suppress melanocyte transformation in vivo in the mouse.     

Antimelanoma Antibodies in Swine With Spontaneously Regressing Melanoma

Sinclair swine provide a unique model for studying mechanisms of tumor regression because they are born with melanomas that spontaneously regress approximately 10 weeks after birth. To examine whether an antitumor immune response is present in these animals, and, if so, to study its relation to tumor regression, 38 sera specimens collected at different times from 13 swine born with melanomas were tested for melanoma antibodies by immunoprecipitation and SDS-PAGE analysis of ¹²⁵I labelled swine melanoma macromolecules. Antibodies to melanoma were present in 13 (100%) of the swine versus 1 of 3 control swine. The antibodies were directed to antigens of approximately 45, 68–75, or 100 kDa. These antigens were also expressed on human melanomas and normal melanocytes but on only one of five unrelated tumors. The incidence and level of these antibodies increased with time. Antibodies to the 45, 68–75, and 100 kDa antigens were present in 36%, 55%, and 9%, respectively, of sera collected prior to 7 weeks of age, but in 80%, 100%, and 37% of sera collected between 7 and 20 weeks (P<0.05). The rise in melanoma antibodies usually preceded or appeared together with tumor regression and loss of pigmentation. These findings indicate that Sinclair swine with melanomas have antibodies to antigens preferentially expressed on pigment cells, and support the hypothesis that the regression phenomenon and the vitiligo-like skin depigmentation result from immune responses to common antigens shared by normal and malignant swine pigment cells.     

Suppression of the maturation and activation of the dendritic cell line DC2.4 by melanoma-derived factors

Dendritic cells play critical roles in both innate and adaptive immunity, and their numerous functions are tightly linked to their maturation and activation status. Here, we characterize the murine dendritic cell line DC2.4 as a model for studying dendritic cell maturation and activation, and we evaluate the influence of melanoma tumor cells on these processes. Exposure of DC2.4 cells to the Toll-like receptor ligand lipopolysaccharide induces both maturation and activation of these cells, characterized by upregulation of costimulatory molecule expression and proinflammatory cytokine/chemokine production. This maturation and activation is suppressed by soluble factors derived from both the highly tumorigenic B16-F1 and the poorly tumorigenic D5.1G4 murine melanoma cell lines. Interestingly, the extent of DC2.4 immunosuppression by these melanomas correlates with their tumorigenicity, suggesting a potentially vital role for dendritic cell/tumor cell interactions in the regulation of anti-tumor immunity and tumor outgrowth.     

MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth

A microRNA expression screen was performed analyzing 157 different microRNAs in laser-microdissected tissues from benign melanocytic nevi (n = 10) and primary malignant melanomas (n = 10), using quantitative real-time PCR. Differential expression was found for 72 microRNAs. Members of the let-7 family of microRNAs were significantly downregulated in primary melanomas as compared with benign nevi, suggestive for a possible role of these molecules as tumor suppressors in malignant melanoma. Interestingly, similar findings had been described for lung and colon cancer. Overexpression of let-7b in melanoma cells in vitro downregulated the expression of cyclins D1, D3, and A, and cyclin-dependent kinase (Cdk) 4, all of which had been described to play a role in melanoma development. The effect of let-7b on protein expression was due to targeting of 3'-untranslated regions (3'UTRs) of individual mRNAs, as exemplified by reporter gene analyses for cyclin D1. In line with its downmodulating effects on cell cycle regulators, let-7b inhibited cell cycle progression and anchorage-independent growth of melanoma cells. Taken together, these findings not only point to new regulatory mechanisms of early melanoma development, but also may open avenues for future targeted therapies of this tumor.     

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens

Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201⁺ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. Received: 31 October 1997 / Accepted: 4 August 1999     

Non-thermal Nanoelectroablation of UV-induced Murine Melanomas Stimulates an Immune Response

Non‐thermal nanoelectroablation therapy completely ablates UV‐induced murine melanomas. C57/BL6‐HGF/SF transgenic mice were exposed to UV radiation as pups and began to develop visible melanomas 5–6 months later. We have treated 27 of these melanomas in 14 mice with nanosecond pulsed electric field (nsPEF) therapy delivering 2000 electric pulses each 100 ns long and 30 kV/cm at a rate of 5–7 pulses per second. All nanoelectroablated melanoma tumors began to shrink within a day after treatment and gradually disappeared over a period of 12–29 days. Pyknosis of nuclei was evident within 1 h of nsPEF treatment, and DNA fragmentation as detected by TUNEL staining was evident by 6 h after nsPEF treatment. In a melanoma allograft system, nsPEF treatment was superior to tumor excision at accelerating secondary tumor rejection in immune‐competent mice, suggesting enhanced stimulation of a protective immune response by nsPEF‐treated melanomas. This is supported by the presence of CD4⁺‐T cells within treated tumors as well as within untreated tumors located in mice with other melanomas that had been treated with nanoelectroablation at least 19 days earlier.     

Evolutionary processes of melanomas from giant congenital melanocytic nevi

Melanoma can develop in a congenital melanocytic nevus (CMN). In fact, a large CMN is associated with a high risk of developing melanoma. Although melanomas arising from CMNs are thought to have a pathogenesis distinct from conventional melanomas, no studies have been conducted on the evolution or tumor heterogeneity of CMN melanomas. We applied multi‐region whole‐exome sequencing to investigate the clonal nature of driver events and evolutionary processes in CMNs and melanomas arising from CMNs. In two patients, we observed an independent subclonal evolution in cancerized fields of CMNs and chromosome 8q amplification in both melanomas arising from CMNs. The amplification of MYC, located in chromosome 8q, was correlated with the percentage of tumor cells expressing high levels of MYC protein detected in melanoma cells by immunohistochemistry. Our analysis suggests that each CMN cell may evolve sporadically and that amplification of MYC might be a key event for melanoma development in CMNs.     

Tyrosinase-Induced Free Radical Formation From VP-16,213: Relationship To Cytotoxicity

Tyrosinase-dependent activation of hydroxybenzenes forms reactive compounds, including catechols and o-quinones, and some of which show antitumor activity against pigmented melanomas. Since VP-16 is a phenoxy-containing antitumor drug, forms free radicals and reactive o-quinones during peroxidative activation, wc evaluated the cytotoxicity of VP-16 to both tyrosinase-containing and non-tyrosinasecontaining tumor cells. Our results show that VP-16 is significantly more cytotoxic to B-16/F-10 melanoma cells than human MCF-7 breast tumor cells. Phenylthiocarbamide, an inhibitor of tyrosinase activity, selectively decreased VP-16 toxicity only in melanoma cells. Furthermore, VP-16 was readily activated to its phenoxy free radical intermediate by purified tyrosinase, indicating tyrosinase may play a role in VP-16 toxicity in pigminted melanomas.     

Arsenite sensitizes human melanomas to apoptosis via tumor necrosis factor alpha-mediated pathway

Arsenic is a well established human carcinogen and is associated with a variety of cancers including those of the skin. Paradoxically, arsenic has also been used, amid at low doses, in the treatment of leukemia for over a century. Here we demonstrate that low to moderate concentrations of arsenite (2-10 microm) that has little or no effect on normal melanocytes may induce apoptosis of human melanomas including highly metastatic ones despite their low surface Fas levels. The two prerequisites that dictate apoptotic response of melanomas upon arsenite treatment are low nuclear NF-kappaB activity and an endogenous expression of tumor necrosis factor alpha. Under these conditions, melanoma cells acquired sensitivity to tumor necrosis factor alpha-mediated killing. On the other hand, signaling pathways including those of phosphatidylinositol 3-kinase-AKT, MEK-ERK, and JNK play a protective role against arsenite-induced oxidative stress and apoptosis in melanoma cells. Suppression of these pathways dramatically accelerates arsenite-induced apoptosis. Taken together, these data could provide potential approaches to sensitize melanomas to the cytotoxic effects of arsenite through modulating the signaling pathways.