Ferritin contributes to melanoma progression by modulating cell growth and sensitivity to oxidative stress.

PURPOSE: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease. EXPERIMENTAL DESIGN: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry. RESULTS: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients. CONCLUSIONS: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.     

A lymph node metastatic mouse model reveals alterations of metastasis-related gene expression in metastatic human oral carcinoma sublines selected from a poorly metastatic parental cell line

BACKGROUND: Greater than 40% of patients with squamous cell carcinoma (SCC) of the oral cavity have lymph node metastasis at the time of diagnosis and a 5-year survival rate of less than 50%. Changes in gene expression that regulate metastasis of SCC to lymph nodes have not been identified. METHODS: To study metastasis of oral SCC, highly metastatic oral SCC cell lines from a poorly metastatic oral SCC cell line were established by in vivo selection using a lymph node metastatic mouse model. The metastatic potential of the cells was studied using Matrigel invasion and cell surface protein adhesion assays. mRNA and protein encoded from metastasis-related genes in the metastatic derivatives and in their parental cells were examined using Northern blot analysis, immunoblotting, rapid analysis of gene expression, and a cDNA microarray technique. RESULTS: The in vivo selected metastatic cells showed much higher Matrigel invasion capability than the parental cells. They also showed alterations in their adhesion properties to three cell surface proteins. Comparison of metastatic and nonmetastatic cells revealed several significant alterations in the expression of metastasis-related genes, including up-regulation of the urokinase-type plasminogen activator receptor, integrin beta1, membrane type 1-matrix metalloproteinase, and down-regulation of protease-activated receptor-1. CONCLUSIONS: To the authors' knowledge, the current study is the first to report on gene expression analysis using a lymph node metastatic mouse model of human oral SCC. The data suggest that certain alterations of metastasis-related gene expression favor invasion of oral SCC and that cell surface proteins may play major roles in the metastasis of oral SCC to the lymph nodes.     

Expression of the apoptosis inhibitor protease inhibitor 9 predicts clinical outcome in vaccinated patients with stage III and IV melanoma.

PURPOSE: There have been reports of successful treatment of metastatic melanoma patients with active specific immunotherapy (ASI) using irradiated autologous tumor cell vaccination. It is still unknown why some patients respond and others do not. Tumor cells can evade the immune system, for example through interference with antigen presentation by down-regulation of MHC molecules or expressing proteins interfering with cytotoxic lymphocyte-induced apoptosis like the granzyme B antagonist protease inhibitor 9 (PI-9). EXPERIMENTAL DESIGN: PI-9 expression was detected in melanoma cell lines. To investigated if PI-9 is important in the response to ASI, paraffin-embedded tissues from stage III or IV melanoma patients were stained. RESULTS: PI-9 is expressed in melanoma cells and expression in metastasized melanoma cells is, in this group of patients, an adverse prognostic marker with regard to overall and disease-free survival. Moreover, loss of MHC-1 expression frequently occurs during tumor progression but is not associated with poor clinical outcome. Interestingly, melanoma patients with a favorable clinical outcome after ASI therapy usually have high percentages of activated (granzyme B-positive) tumor-infiltrating lymphocytes at time of first diagnosis and low percentages of activated lymphocytes at time of recurrent tumor. CONCLUSIONS: Expression of PI-9 in metastatic melanoma cells is associated with unfavorable clinical outcome whereas MHC-1 down-regulation is not. Although it cannot be proven that PI-9 expression is directly responsible for failure of immunotherapy, these data suggest that expression of PI-9 could be an important immune escape mechanism and that modulation of this inhibitor may enhance the efficacy of immunotherapy.     

Human autologous tumor-specific T cells in malignant melanoma

T cell lines and clones with autologous tumor-specific activity have been developed in malignant melanoma by stimulating peripheral blood lymphocytes (PBL), lymph node lymphocytes or tumor-infiltrating lymphocytes (TIL) with autologous melanoma cells in the presence of recombinant interleukin 2 (rIL2). T-cell lines and clones have been developed with specific cytotoxicity and/or proliferative responses for autologous melanoma targets but not for allogeneic melanoma tumor cells, autologous normal cells or natural killer (NK)-sensitive targets. The concentration of rIL2 is critical for the generation of autologous tumor-specific T-cell lines, with low rIL2 concentrations (up to 800 IU/ml) facilitating the growth of T-cell lines with tumor-specific activity. The T-cell receptor (TCR) and the CD3 antigen are involved in specific cytotoxicity and/or proliferative responses of these T-cell lines and clones. An oligoclonal pattern of -chain TCR gene rearrangements was observed on T-cell lines and clones with autologous tumor-specific cytotoxicity, suggesting that they are comprised of T cells that have undergone a clonal expansion in response to particular antigen. Autologous tumor-specific cytotoxic T cells are HLA-restricted and recognize on the melanoma tumor cells HLA Class I or possibly Class II antigens plus a tumor-specific determinant. TIL from patients with metastatic melanoma have unique characteristics in comparison with PBL and lymph node lymphocytes and they appear to contain substantial proportions of T cells that have been locally sensitized to autologous tumor cells. Single stimulation of TIL with autologous tumor cells in the presence of rIL2 is sufficient for the generation of T cell lines with autologous tumor-specific activity, whereas, multiple stimulation of PBL and lymph node lymphocytes was required to achieve the same purpose. TIL-derived T cell lines have been expanded in rIL2 in vitro by at least 1,500-fold without losing their activity. Approximately, 40% of the patients exhibited complete or partial responses to adoptive immunotherapy with melanoma TIL and rIL2.     

Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal-regulated kinase signaling in melanoma cell lines

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.     

Human cytotoxic T cells specific for autologous melanoma cells: successful generation from lymph node cells in seven consecutive cases

Human T-cell populations specifically cytotoxic for autologous melanoma cells have been successfully generated from lymph node cells obtained from seven consecutive patients. The lymph node cells were stimulated in vitro with autologous irradiated melanoma cells; stimulation was repeated every 10-15 days at a tumor cell-to-lymphocyte ratio of approximately 1:20. Cytotoxic activity was assessed by a 4-hour 51Cr release assay. Mean lysis of autologous tumor cells was 47% at an effector-to-target cell ratio of 20:1, while mean lyses of the human myeloid leukemia cell line K562, allogeneic melanoma cells, and an osteosarcoma cell were 20%, 13%, and 11%, respectively. There was no lysis of autologous fibroblasts, fresh lymphocytes, or phytohemagglutinin-stimulated blasts. Three grades of specificity developed sequentially. In grade I, lysis of autologous tumor cells exceeded lysis of allogeneic tumor cells but did not exceed lysis of K562 cells. In grade II, lysis of autologous tumor cells exceeded lysis of K562 cells and all allogeneic tumor cells tested. In grade III, potent lysis of autologous tumor cells (greater than 40%) exceeded lysis of K562 cells and of all allogeneic tumor cells tested. All seven lymphocyte populations reached or exceeded grade I. Six reached or exceeded grade II. Two progressed to grade III. The generated cells were T cells, as determined by phenotypic analysis with flow cytometry. CD4+ cells and CD8+ cells accounted for 83%-100% of the cells. CD8+ T cells were separated from CD4+ T cells by panning with OKT8 and OKT4 antibodies. The resulting CD8-enriched and CD4-enriched populations were compared as effectors in cytotoxicity assays. The results suggest that the cell responsible for lysis of autologous tumor cells is CD8+. The methods used in this study have repeatedly resulted in the successful generation of cytotoxic T lymphocytes specifically cytotoxic for autologous melanoma cells; it is suggested that these cells have potential application for adoptive immunotherapy of melanoma.     

Differences in lipid characteristics of autologous human melanoma cell lines with distinct biological properties

Significant differences in lipid composition were found when six established human melanoma cell lines were compared. A pair of cell lines was initiated from a superficial spreading melanoma and the lymph node of the same patient; four others were also autologous, three of which originated from the same nodular melanoma and the other from its metastasis. Cell lines varied in pigmentation level and ability to grow in nude mice. Cell lines contained similar amounts of total cholesterol, glycerides, and phospholipids but different amounts of free cholesterol and cholesterol esters. In particular, the molar ratio of free cholesterol to phospholipid was increased in highly tumorigenic cell lines. No changes in phospholipid profiles were noted among cell lines, except an increase in sphingomyelin with a concomitant decrease in phosphatidylcholine in one cell line compared to the profiles of its counterpart cell line. The saturated-to-unsaturated fatty acid ratios in phosphatidylcholine and phosphatidylethanolamine were similar in all cell lines, but the monounsaturated-to-polyunsaturated fatty acid ratio in phosphatidylcholine was increased in highly tumorigenic cell lines. A significant variation in the latter ratio in phosphatidylethanolamine was also observed in the pair of autologous cell lines. These changes were unrelated to a depletion in linoleic acid in culture medium. Results obtained by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene were consistent with the differences in lipid composition between two autologous cell lines. The present results indicate that two lipid characteristics were significantly changed in highly tumorigenic cell lines as compared to cell lines with low tumorigenicity, but no correlation was found between either pigmentation level or origin (primary or metastatic) and lipid composition.     

Orthotopic human melanoma xenograft model systems for studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern.

Adequate tumour models are a prerequisite in experimental cancer research. The purpose of the present work was to establish and assess the validity of four new orthotopic human melanoma xenograft model systems (A-07, D-12, R-18, U-25). Permanent cell lines were established in monolayer culture from subcutaneous metastases of four different melanoma patients by using an in vivo-in vitro procedure, and cells from these lines were inoculated intradermally in Balb/c nu/nu mice to form tumours. Individual xenografted tumours of the same line differed substantially in growth and pathophysiological parameters, probably as a consequence of differences between inoculation sites in host factors which influence tumour angiogenesis. Nevertheless, xenografted tumours of different lines showed distinctly different biological characteristics. Several biological characteristics of the donor patients'' tumours were retained in the xenografted tumours, including angiogenic potential; growth, histopathological and pathophysiological parameters; and sensitivity to radiation, heat and dacarbazine treatment. Moreover, the organ-specific metastatic pattern of the xenografted tumours reflected the pattern of distant metastases in the donor patients. The organs of preference for distant metastases were lungs (A-07, D-12), lymph nodes (R-18) and brain (U-25). R-18 lymph node metastases and U-25 brain metastases developed in the absence of lung involvement. The four orthotopic human melanoma xenograft model systems show great promise for future studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern.     

Identification of the human PHLDA1/TDAG51 gene: down-regulation in metastatic melanoma contributes to apoptosis resistance and growth deregulation

To identify molecules involved in the progression of human melanoma to metastatic disease, autologous primary and metastatic melanoma cells were compared by differential mRNA display. One cDNA, expressed in primary but not in autologous metastatic cells in three different patients, was cloned and characterized, and shown to be the human homologue of the inducible, immediate early TDAG51/PHLDA1 (pleckstrin-homology-like domain family A, member1) gene. Monoclonal antibodies produced against the PHLDA1 protein revealed homogeneous strong expression by benign melanocytic nevi, and progressively reduced expression in primary and metastatic melanomas in vivo. Analysis of stable cDNA transfectants in two different cell lines revealed that constitutive PHLDA1 expression is associated with reduced cell growth, cloning efficiency, and colony formation but not with alterations in cell cycle parameters. However, PHLDA1 expression was associated with increased basal apoptosis as assessed by live cell annexin V binding, terminal deoxynucleotidyltransferase-dependent nucleotide incorporation, and with increased cleavage of poly(ADP-ribose) polymerase and caspase-9. Constitutive PHLDA1 expression greatly enhances the sensitivity of human melanoma cells to the chemotherapeutic agents doxorubicin and camptothecin. These results suggest that PHLDA1 is constitutively expressed by melanocytic nevi where it may contribute to their benign phenotype. The progressive loss of PHLDA1 expression in melanomas may play a role in deregulated cell growth and apoptosis resistance in these tumors.     

CEACAM1 expression in cutaneous malignant melanoma predicts the development of metastatic disease.

PURPOSE: The cell adhesion molecule CEACAM1 is involved in intercellular adhesion and subsequent signal transduction events in a number of epithelia. CEACAM1 downregulation has been demonstrated in colorectal and prostate carcinomas. This study sought to analyze whether its expression in malignant melanoma is associated with metastasis. PATIENTS AND METHODS: CEACAM1 expression was immunohistochemically evaluated in 100 primary cutaneous malignant melanomas and correlated with metastasis in a 10-year follow-up. Furthermore, CEACAM1 expression was analyzed in metastatic lesions (11 distant metastases and six sentinel lymph node metastases). Univariate Kaplan-Meier analysis and multivariate Cox proportional hazard regression analysis adjusted for standard prognostic indicators were performed to assess the prognostic relevance of CEACAM1 expression. RESULTS: A total of 28 of 40 patients with CEACAM1-positive primary melanomas developed metastatic disease, compared with only six of 60 patients with CEACAM1-negative melanomas. Often, the strongest CEACAM1 expression was observed at the invading front. In addition, CEACAM1 expression was preserved in the metastatic lesions. Kaplan-Meier analysis revealed a highly significant association between CEACAM1 expression and metastasis (P <.0001); multivariate Cox regression analysis, including CEACAM1 expression status adjusted for tumor thickness, presence of ulceration, and mitotic rate, confirmed that CEACAM1 is an independent factor for the risk of metastasis and demonstrated that the predictive value of CEACAM1 expression is superior to that of tumor thickness. CONCLUSION: Expression of the cell adhesion molecule CEACAM1 in the primary tumors in melanoma patients is associated with the subsequent development of metastatic disease. This raises the possibility of a functional role for this cell adhesion molecule in the metastatic spread it indicates.     

Deletion in p16INK4a and loss of p16 expression in human skin primary and metastatic melanoma cells

p16INK4a gene mapped at chromosome 9p21 region encodes a tumor suppressor protein p16 which is frequently inactivated in human cancers, including skin melanoma. In order to clarify the importance of p16 alterations in melanoma, we examined the deletions of p16INK4a and expression of p16 protein in eight unselected primary and metastatic melanoma cell lines from human skin melanomas. Normal skin melanocytes were used as controls. Deletions of entire exons in the p16INK4a gene were detected by PCR technique and expression of the p16 protein was examined by Western blotting and immunocytochemistry. Results showed that the fragments from exons 2A, 2C and 3 in p16INK4a gene were totally deleted in the metastatic melanoma cell line, FM28.7 and the fragment from exon 3 was deleted in the metastatic melanoma cell line, FM55M2. P16 protein was strongly expressed in two of the primary melanomas cell lines (FM55P and RaH3). The p16 protein was weakly expressed in one of the metastatic melanoma cell lines (FM55M1) and negative in the other metastasis (FM55M2) as compared to their matched primary melanoma cells (FM55P). The p16 protein was strongly expressed in normal skin melanocytes. Immunocytochemistry showed that p16 protein was mainly localized in the nuclei of the melanoma cells and normal melanocytes, if it was expressed. Deletions of p16INK4a gene was uncommon and loss of p16 protein expression was common event in melanoma, especially in the later stages of melanoma.     

Metastatic potential of human melanoma cells in nude mice--characterisation of phenotype, cytokine secretion and tumour-associated antigens.

Incidence and mortality of human malignant melanoma has risen rapidly over recent decades. Although the notorious resistance to treatment is characteristic for metastatic malignant melanoma, only a few experimental models have been established to study the metastatic cascade or to test new alternative treatment modalities. Thus, new human models are wanted. Here, we describe the metastatic behaviour of seven human melanoma cell lines derived from two primary cutaneous melanomas (WM 98-1, WM 1341) and five metastases established from liver (UKRV-Mel-4), skin (M7, M13), pleural effusion (UKRV-Mel-2) and lymph node (MV3). All cell lines were analysed for their capacity to grow in nude mice after s.c. and i.v. administration. M13 cells developed liver metastases spontaneously after s.c. injection, and subsequent passages of M13 and M7 melanoma cells caused liver metastases after i.v. injection, whereas MV3 and WM98-1 gave rise to lung metastases, using the same inoculation route. In contrast, WM 1341, UKRV-Mel-2 and UKRV-Mel-4 grew only very slowly in nude mice after s.c. injection and did not cause any metastases after i.v. or s.c. administration. The pattern of metastases or growth kinetics did not correlate with the interleukin 8 or tumour necrosis factor secretion of cell lines. Adhesion molecules and growth factor receptor expression on the cell lines differed widely, as determined by flow cytometry, with the low metastatic cell lines (UKRV-Mel-2, UKRV-Mel-4 and WM 1341) demonstrating a marked reduction in VLA-1 and VLA-5 expression compared with the metastatic lines (M7, M13, MV3 and WM 98-1). Expression of pigment-related proteins such as tyrosinase, TRP-1, TRP-2, Melan-A/MART-1, gp100, MAGE1 or MAGE-3 was not associated with growth and metastatic characteristics of the melanoma cell lines analysed. In conclusion, the established human melanoma cell lines exhibited diverse growth behaviour in nude mice in congruence with some early established prognostic markers such as VLA-1 and VLA-5. The xenografts provide good models for further study of metastatic processes as well as for evaluation of alternative treatment modalities including new pharmaceutical drugs and gene therapeutic targeting using tissue-specific gene regulatory elements for gene targeting.     

Clinical significance of CXCR3 and CXCR4 expression in primary melanoma

Tumor cell migration involved in metastases is a tightly regulated, nonrandom process. Chemokines have been identified as critical molecules guiding cell migration. We performed a prospective study to analyze a possible association between the expression of chemokine receptors CXCR3 and CXCR4 by primary melanoma and clinical outcome. Forty primary melanomas were available for analysis; 57% of the tumors expressed CXCR3 and 35% expressed CXCR4 by melanoma cells. At initial diagnosis, 5 patients had subclinical lymph node involvement and after a median follow-up time of 32 months, 2 additional patients developed regional lymph node metastases and 5 patients developed distant metastases. The expression of CXCR4, but not CXCR3, by melanoma cells in primary lesions was significantly associated with the presence of ulceration, increased tumor thickness, a greater risk of developing regional and distant metastases and a higher mortality rate. Our study underscores the value of CXCR4 expression as a useful marker for predicting outcome in patients with localized melanoma. In addition, our findings support that, among chemokine receptors, CXCR4 might be an appropriate therapeutic target for adjuvant therapy in patients at risk for metastatic disease.     

The role of sentinel lymph node biopsy for melanoma

Regional lymph nodes are a common site of melanoma metastases, and the presence or absence of melanoma in regional lymph nodes is the single most important prognostic factor for predicting survival. Furthermore, identification of metastatic melanoma in lymph nodes and excision of these nodes may enhance survival in a subgroup of patients whose melanoma has metastasized only to their regional lymph nodes and not to distant sites. Sentinel lymph node (SLN) biopsy was developed as a low morbidity technique to stage the lymphatic basin without the potential morbidity of lymphedema and nerve injury. The presence or absence of metastatic melanoma in the SLN accurately predicts the presence or absence of metastatic melanoma in that lymph node basin. When performed by experienced centers, the false-negative rate of SLN biopsy is very low. As such, the nodal basin that contains a negative SLN will usually be free of microscopic disease. Since occult micrometastatic disease affects only 12% to 15% of patients with melanoma, selective SLN dissection allows up to 85% of patients with melanoma to be spared a formal lymph node dissection, thus avoiding the complications usually associated with that procedure. While standard pathologic evaluation of lymph nodes may miss metastatic melanoma cells, more sensitive techniques are developing which may identify micrometastases more accurately. The clinical significance of these micrometastases remains unknown and is the subject of active investigations.