Differentiation and the tumorigenic and metastatic phenotype of murine melanoma cells

Using B16 F10 murine melanoma cells and sublines generated from the JB/MS melanoma which exhibit various degrees of melanogenesis, the relationships among differentiation, tumorigenicity, and metastatic potential were examined. The effect of melanocyte-stimulating hormone (MSH), which specifically stimulates differentiation of melanocytes, was also studied. All melanoma lines tested were capable of growing as experimental pulmonary metastases but, surprisingly, the undifferentiated and amelanotic JB/MS-w cells failed to grow as primary subcutaneous tumors. JB/MS-w cells, which had few surface MSH receptors, did not respond to MSH with an increase in melanin production, unlike the other cell lines. Although in vitro treatment with MSH did not change the rates of growth of primary tumors by these cell lines, such treatment decreased the number of pulmonary metastases from B16 F10, JB/MS cells, JB/MS-b1 cells and JB/MS-w cells. Conversely, MSH treatment significantly increased the rates of pulmonary metastases from JB/MS-p cells. The expression of surface melanoma antigens, urokinase-type plasminogen activity and susceptibility to natural killer cells were examined. MSH did not significantly alter surface melanoma antigen expression, but increased the natural killer cell susceptibility of B16 F10, JB/MS and JB/MS-b1 cells, cells which possess abundant surface MSH receptors. There was an inverse correlation between differentiation (pigmentation) and proliferation in vitro, and the more pigmented melanoma cells (B16 F10, JB/MS and JB/MS-b1) expressed relatively lower levels of class-I MHC, relatively higher levels of class-II MHC and the highest metastatic capacity. These results demonstrate that MSH possesses the capacity to regulate not only melanogenesis, but also other factors critical to the metastatic growth of the cells.     

Effects of folate deficiency on the metastatic potential of murine melanoma cells

Experiments were designed to measure the effect of folic acid deficiency on a major determinant of cancer lethality, the propensity to form metastases. Murine B16 melanoma cells (F10 strain) were grown in folate-deficient and -supplemented media. After 3 days, cells in the deficient medium had restricted proliferative capacity, low folate levels by bioassay, increased cell volume, abnormal deoxyuridine suppression tests, accumulation of cells in S phase by flow cytometry, and increased numbers of DNA strand breaks. These folate-deficient cells consistently initiated more pulmonary metastases than control cells when injected into host mice. Cell size did not appear to be a major factor in pulmonary metastasis formation. In vitro growth rates and cloning efficiencies were comparable for cells in both types of medium as was subcutaneous growth of tumors. We conclude that folate deficiency increases the metastatic potential of cultured melanoma cells.     

IGFBP‐3 inhibits Wnt signaling in metastatic melanoma cells

In previous works, we have shown that insulin‐like growth factor‐binding protein‐3 (IGFBP‐3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP‐3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti‐tumoral effects of IGFBP‐3. We show that the anti‐tumoral effect of IGFBP‐3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP‐3 binds the Wnt signalosome interacting specifically with its component GSK‐3β. As a consequence, the β‐catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK‐3β is activated through dephosphorylation, becoming free to target cytoplasmic β‐catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP‐3 is a novel and effective inhibitor of Wnt signaling. As IGFBP‐3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.     

Adhesion-induce protein tyrosine phosphory-lation is associated with invasive and metastatic potentials in B16-BL6 melanoma cells

Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cell-ECM interaction and its role. Methods: To interrupt the cell-ECM interaction by suppression of adhesion-induced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16-B16 mouse melanoma cells. Results: When B16-BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30 μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein in response to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesion-induced protein tyrosine phosphorylation the genistein-treated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genistein-treated cells. Conclusion: It was suggested that protein tyrosine phosphorylation in cell-ECM interaction might be associated with invasive and metastatic potentials in cancer cells.     

Circulating melanoma cells and survival in metastatic melanoma

A validated assay for the enumeration of circulating melanoma cells (CMCs) may facilitate the development of more effective therapies for metastatic melanoma patients. In this study CD146+ cells were immunomagnetically enriched from 7.5 ml of blood. Isolated cells were fluorescently stained with DAPI, anti-molecular weight melanoma-associated antigen (HMW-MAA), anti-CD45 and CD34 and Ki67. CMCs were identified as CD146+, HMW-MAA+, CD45-, CD34-, Ki67-/+ cells. Eighty-eight percent of spiked SK-MEL28 cells in 7.5 ml blood were recovered. In all 55 healthy donors ≤1 CMCs were detected in 7.5 ml of blood. A retrospective analysis was conducted comparing CMC counts and overall survival in 79 blood samples from 44 melanoma patients. CMCs ranged from 0 to 8,042 per 7.5 ml. Two or more CMCs were detected in 18 (23%) of the patients and 30-100% (mean 84%) of the CMCs expressed the proliferation marker Ki67. Patients with ≥2 CMCs per 7.5 ml of whole blood, as compared with the group with <2 CMCs, had a shorter overall survival (2.0 months vs. 12.1 months, P=0.001).     

Modulation of metastatic potential by cell surface urokinase of murine melanoma cells

We have carried out enzymatic, immunofluorescence, and surface iodination studies which show that B16 melanoma cells express the single chain form of the urokinase type plasminogen activator (uPA) on their cell surface, and that these cells are capable of plasminogen-dependent fibronectin degradation. The significance of the expression of surface single-chain uPA and uPA activity to the metastatic process was examined by preincubating melanoma cells with uPA modulating agents followed by i.v. injection of the cells into mice and enumeration of pulmonary nodules 17 days later. B16 cells that had been pretreated with anti-uPA immunoglobulins that were inhibitory to uPA activity invariably showed significantly decreased numbers of metastases compared to controls. On the contrary, pretreatment with plasmin, which is not only the product of the uPA catalyzed reaction but is also able to convert single-chain uPA to uPA, significantly increased the numbers of metastases. Control treatments, which included normal rabbit and mouse immunoglobulins, monovalent noninhibitory anti-uPA Fab fragments, and various monoclonal and polyclonal antibodies directed against other B16 cell surface antigens, did not affect the metastatic potential of the cells. Divalent inhibitory anti-uPA F(ab)2 fragments, on the contrary, inhibited metastasis as efficiently as intact IgG. The results support the hypothesis that proteolysis of extracellular matrix components by cell surface-localized uPA may be a critical step during the process of tumor cell invasion and metastasis.     

A new technique for implantation of tissue culture melanoma cells in a murine model of metastatic ocular melanoma

The aim of this study was to compare the transcorneal and transconjunctival techniques for the implantation of intraocular melanoma cells and development of metastasis in a murine model. Groups of C57BL/6 mice were given either transconjunctival or transcorneal inoculations of 2.5 x 10(5)/2.5 microl tissue culture B16-LS9 melanoma cells into the intraocular posterior compartment (PC). The eyes were enucleated at 4-11 days post-inoculation and histologically examined. The mice were sacrificed 14 days after enucleation and necropsies were performed with histological evaluation for visceral metastases. Intraocular and extraocular tumour growth was present in all of the eyes inoculated via the transconjunctival route. Pulmonary metastases were found in this group if the eye was enucleated 7 or more days post-inoculation. The melanoma remained confined to the inside of the eye in the transcorneal group until day 7. Haematogenous metastases to the lung and liver developed from the intraocular melanoma in this group. Transcorneal inoculation of tissue culture melanoma cells into the murine PC provides a useful animal model for visceral metastasis of ocular melanoma.     

Establishment of cell-to-cell contact by adoptively transferred adherent lymphokine-activated killer cells with metastatic murine melanoma cells

A murine model of pulmonary B16 melanoma was used to study the infiltration into metastases of lymphokine-activated killer (LAK) cells and adherent lymphokine-activated killer (A-LAK) cells and, specifically, to study whether A-LAK cells are able to leave the tumor microcirculation and establish cell-to-cell contact with malignant cells. Fluorescence microscopy demonstrated that A-LAK cells accumulated in metastases twice as efficiently as LAK cells during interleukin-2 stimulation. Electron microscopy of pulmonary metastases 16 hours after administration of 2.5 x 10(7) A-LAK cells revealed A-LAK cells, identified by the presence of typical two-compartment granules, in direct contact with melanoma cells. This finding was confirmed by using A-LAK cells prelabeled with polycationized ferritin. In conclusion, our observations demonstrate unambiguously the ability of adoptively transferred A-LAK cells to establish contact with extravascular metastatic melanoma cells.     

Downregulation of microRNA-211 is involved in expression of preferentially expressed antigen of melanoma in melanoma cells

MicroRNAs (miRNAs) are small non-coding RNAs whose aberrations are involved in the initiation and progression of human cancers. To seek unique miRNAs contributing to melanoma tumorigenesis, we investigated the global miRNA expression profile of 7 melanoma cell lines and 3 primary cultures of neonatal human epidermal melanocytes (NHEMs) using the stem-loop real-time PCR method. We found 7 miRNAs that were commonly downregulated and 18 that were upregulated in all of the melanoma cell lines in comparison with the 3 primary cultures of NHEMs. We focused on one commonly downregulated miRNA (miR-211), and analyzed its relationship to the expression of preferentially expressed antigen of melanoma (PRAME) protein, which is a potential target of miR-211. We found that all melanoma cell lines exhibited marked down--regulation of miR-211 and upregulation of PRAME mRNA/protein expression in comparison with NHEMs (P<0.05). A significant inverse correlation between miR-211 and PRAME protein expression was found in melanoma cell lines and primary cultures of NHEMs (correlation coefficient of -0.733, P<0.05). We demonstrated that overexpression of miR-211 induced a reduction of PRAME protein levels, and confirmed the target specificity between miR-211 and PRAME by luciferase reporter assay. These results suggest that downregulation of miR-211 may be partly involved in aberrant expression of the PRAME protein in melanoma cells.     

Heparan sulfate endoglycosidase and metastatic potential in murine fibrosarcoma and melanoma

Several fibroblast and melanoma cell lines were studied with respect to their ability to degrade heparan sulfate (HS). The optimum pH for HS degradation by HS endoglycosidase (heparanase) for all cell lines is about 5.6, but the activity of the enzyme is still present at physiological pH. The gel permeation analysis of degradation products revealed that heparanase cuts HS in fragments about one-seventh of their original size. Since the optimum pH of HS endoglycosidase activity and the terminal molecular weight of degraded HS are the same in both cell lines, it is likely that fibrosarcoma and melanoma heparanases are identical enzymes. Cell extracts and intact cells of metastatic sublines degrade HS faster than do their nonmetastatic counterparts. The degradative activity of intact cells parallels those of cell extracts, but at a much lower level; moreover, conditioned media do not appreciably degrade HS, suggesting that heparanase is scarcely released into the medium; thus, considering the differences in degradative activity between cell extracts and intact cells or conditioned medium and the occurrence of cell lysis in a tumor in vivo, we suggest that the measure of degradative activity of intact cells in vitro is not indicative of a relationship to metastasis. The total cellular content of lytic enzymes could represent the real metastatic potential of proliferating cells, but it is also necessary to find an in vitro model better representing the behavior of neoplastic cells in vivo.     

Notch4+ cancer stem‐like cells promote the metastatic and invasive ability of melanoma

Sphere formation in conditioned serum‐free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor (tumorospheres) is considered useful for the enrichment of cancer stem‐like cells, also known as tumor‐initiating cells. We used a gene expression microarray to investigate the gene expression profile of melanoma cancer stem‐like cells (MCSLCs). The results showed that MCSLCs highly expressed the following Notch signaling pathway molecules: Notch3 (NM_008716), Notch4 (NM_010929), Dtx4 (NM_172442), and JAG2 (NM_010588). Immunofluorescence staining showed tumorosphere cells highly expressed Notch4. Notch4^high B16F10 cells were isolated by FACS, and Western blotting showed that high Notch4 expression is related to the expression of epithelial–mesenchymal transition (EMT)‐associated proteins. Reduced invasive and migratory properties concomitant with the downregulation of the EMT markers Twist1, vimentin, and VE‐cadherin and the overexpression of E‐cadherin was observed in human melanoma A375 and MUM‐2B cells. In these cells, Notch4 was also downregulated, both by Notch4 gene knockdown and by application of the γ‐secretase inhibitor, DAPT. Mechanistically, the re‐overexpression of Twist1 by the transfection of cells with a Twist1 expression plasmid led to an increase in VE‐cadherin expression and a decrease in E‐cadherin expression. Immunohistochemical analysis of 120 human melanoma tissues revealed a significant correlation between the high expression of Notch4 and the metastasis of melanoma. Taken together, our findings indicate that Notch4+ MCSLCs trigger EMT and promote the metastasis of melanoma cells.     

Screening cellular proteins involved in the anti-proliferative effect of the ADAM15 disintegrin domain in murine melanoma cells

A disintegrin and metalloprotease protein 15 (ADAM15), a membrane-anchored glycoprotein, is believed to function in cell-cell interactions via an integrin binding motif within its disintegrin domain. To screen its target proteins, the recombinant ADAM15 disintegrin domain (RADD) was expressed in Escherichia coli, biotinylated and used in a protein pull-down assay in vitro. A total of eight kinds of proteins were identified by 2DE/LC-MS-MS. One of them, p38 kinase, was selected for further investigation of its involvement in the anti-proliferative effect of RADD on melanoma cells. Phosphorylation of p38 kinase in melanoma cells was detected upon treatment with RADD. Furthermore, the suppression of p38 kinase activity resulted in a decrease in the RADD inhibitory effect on melanoma cell proliferation. These results provide evidence that RADD inhibits melanoma cell proliferation partly through p38 kinase activation.     

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti‐NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti‐NCAM‐treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM‐140 knocked‐down by siRNA in LP07 cells pretreated or not with anti‐NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti‐NCAM treatment could be mimicking homophilic trans‐interactions and NCAM‐140 knocked‐down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression. Mol. Carcinog. © 2010 Wiley‐Liss, Inc.     

Phase I trial of the murine monoclonal anti-GD2 antibody 14G2a in metastatic melanoma.

In a phase I trial, 12 patients with GD2 antigen-positive metastatic melanoma received the murine anti-GD2 monoclonal antibody 14G2a. The monoclonal antibody was administered in four doses over an 8-day period with total dose ranging from 10 to 120 mg. All patients receiving greater than 10 mg of 14G2a experienced transient abdominal/pelvic pain during the antibody infusion. Five patients had a delayed extremity pain syndrome following the third and fourth antibody infusion. Four of the five patients developed neurological toxicity, including two patients with significant although reversible motor neuropathy. Two of the patients developed hyponatremia secondary to a syndrome of inappropriate antidiuretic hormone. All 12 patients developed high levels of human anti-14G2a antibody. The plasma half-life of 14G2a was 42 +/- 6 (SD) h. One patient each had a partial response, mixed response, and stable disease, respectively. The very modest antitumor activity accompanied by dose-limiting neurological toxicity at total doses greater than 80 mg may restrict the clinical utility of murine 14G2a.     

CXCR4 signaling regulates metastasis of chemoresistant melanoma cells by a lymphatic metastatic niche

Highly metastatic and chemotherapy-resistant properties of malignant melanomas stand as challenging barriers to successful treatment; yet, the mechanisms responsible for their aggressive characteristics are not fully defined. We show that a distinct population expressing CD133 (Prominin-1), which is highly enriched after administration of a chemotherapeutic drug, dacarbazine, has enhanced metastatic potential in vivo. CD133(+) tumor cells are located close to tumor-associated lymphatic vessels in metastatic organs such as the regional lymph nodes and lung. Lymphatic endothelial cells promote the migratory activity of a CD133(+) subset to target organs and regulation of lymphatic growth efficiently modulates the metastasis of CD133(+) tumor cells. We found that lymphatic vessels in metastatic tissues stimulate chemokine receptor 4 (CXCR4)(+)/CD133(+) cell metastasis to target organs by secretion of stromal cell-derived factor-1 (SDF-1). The CXCR4(+)/CD133(+) cells exhibited higher metastatic activity compared with CXCR4(-)/CD133(+) cells and, importantly, blockade of CXCR4 coupled with dacarbazine efficiently inhibited both tumor growth and metastasis; dacarbazine alone could not attenuate tumor metastasis. The current study demonstrates a previously unidentified role of the lymphatic microenvironment in facilitating metastasis of chemoresistant melanoma cells via a specific chemotactic axis, SDF-1/CXCR4. Our findings suggest that targeting the SDF-1/CXCR4 axis in addition to dacarbazine treatment could therapeutically block chemoresistant CD133(+) cell metastasis toward a lymphatic metastatic niche.     

Cell cycle control and adhesion signaling pathways in the development of metastatic melanoma

Metastatic melanoma is a fatal malignancy which is remarkably resistant to treatment. It is not entirely clear what determines transition from primary local to metastatic melanoma. Recent gene profiling studies shed light onto the complexity of pathogenesis of melanoma progression. An interaction between cell cycle signaling, adhesion pathways and epithelial-mesenchimal transition program appears to be critical in the development of metastatic disease. An isolated deregulation of either of those pathways may not be sufficient to initiate tumor evolution towards an aggressive phenotype. Here we review how they act in concert to make such a transition possible.     

Immunological detection of altered signaling molecules involved in melanoma development

To understand immune responses to human cancer and develop more effective immunotherapy, human tumor antigens has been isolated using various immunological methods with tumor reactive T cells or antibodies obtained from patients with melanoma. During the process of tumor antigen isolation, various molecules with genetic alterations or over-expression in tumor cells, which may be involved in proliferation, differentiation, or survival of various cancer cells, were identified. In melanoma, abnormal molecules with mutations including β -catenin, CDK4, and BRAF, and molecules with increased expression including Survivin, were immunologically detected. Therefore, immunological isolation of human tumor antigens contributes to the identification of important molecules including altered signaling molecules involved in melanoma formation.     

Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells

The chemokine receptors CC chemokine receptor (CCR) 7 and CXC chemokine receptor (CXCR) 4 have been implicated in cancer metastasis. To evaluate whether CXCR4 is sufficient to increase tumor metastasis in an organ-specific manner, we transduced murine B16 melanoma cells with CXCR4 (CXCR4-B16) and followed the metastatic fate of the transduced cells in both i.v. and s.c. inoculation models of metastasis. CXCR4-B16 cells demonstrated marked increases (>10-fold) in pulmonary metastasis compared with vector (pLNCX2)-B16 after i.v. and s.c. inoculation of tumor cells. The increase in metastasis could be completely inhibited by T22, a small peptide antagonist of CXCR4. As early as 24 and 48 h after i.v. injection, CXCR4-B16 cells were significantly increased in the lung compared with control B16 cells by 5- and 10-fold (P < 0.05), respectively. CXCR4-B16 cells adhered better to both dermal and pulmonary microvascular endothelial cells relative to control B16 cells. Moreover, CXCL12 promoted the growth of CXCR4-B16 cells in vitro. Whereas expression of CXCR4 in B16 cells dramatically enhanced pulmonary metastasis, metastasis to the lymph nodes, liver, and kidney was rare. Immunohistochemical staining of both primary human cutaneous melanoma and pulmonary metastases revealed CXCR4 expression. Thus, CXCR4 plays a potentially important role in promoting organ-selective metastasis, possibly by stimulating tumor adhesion to microvascular endothelial cells and by enhancing the growth of tumor cells under stress.