Expression of monoacylglycerol lipase as a marker of tumour invasion and progression in malignant melanoma.

BACKGROUND: Accumulating evidence suggests that the lipid lytic enzyme monoacylglycerol lipase (MAGL) promotes tumour invasion and metastasis through up-regulation of pro-tumorigenic signalling lipids in several tumour cell lines. However, the expression status of MAGL in clinical melanoma tissues and its clinicopathological significance remain unclear. OBJECTIVE: To correlate the tumour expression status of MAGL with the clinicopathological information of patients with malignant melanoma. METHODS: Polymerase chain reaction (PCR) array screening was performed, and the results were validated using immunocytochemical analysis of tumour and non-tumour melanocytic cell lines. Immunohistochemical staining for MAGL was performed for 74 melanoma samples, including 48 primary and 26 metastatic tumours, in which the expression of MAGL was determined by evaluating the percentage of MAGL-positive tumour cells and the MAGL staining intensity. Finally, we analysed the association of MAGL expression status with tumour progression, tumour thickness and vascular invasion of the primary lesion. RESULTS: Immunocytochemical analysis revealed that MAGL was expressed in all 12 melanoma cell lines, but not in normal human epidermal melanocytes. In the immunohistochemical analysis, positive staining for MAGL was noted in 32 of 48 (64.5%) primary lesions, 14 of 17 (82.4%) lymph node metastatic lesions and 7 of 9 (77.8%) skin metastatic lesions. Metastatic tumours had a significantly higher staining intensity (P = 0.033 for lymph node, P = 0.010 for skin). In the analysis of primary lesions, higher MAGL expression correlated with greater tumour thickness (P = 0.015) and the presence of vascular invasion (P = 0.017). On further evaluation of MAGL-positive primary lesions, staining intensity of MAGL tended to be higher in deeper areas of the tumour mass. CONCLUSIONS: The expression of MAGL in tumour cells reflects the aggressiveness of melanoma cells and may serve as a marker of tumour progression.

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma.

Activating mutations of the genes for NRAS and BRAF, components of the p44/42 mitogen-activated protein kinase (MAPK) pathway, are common findings in melanoma. Recent evidence in several nonmelanoma cell systems supports the regulation of the inducible nitric oxide synthase (iNOS) gene by this pathway. On the basis of our data showing that melanoma iNOS expression predicts shortened patient survival, we formulated the hypothesis that activating mutations of NRAS or BRAF, which lead to constitutive activation of the p44/42 MAPK pathway, drive iNOS expression in human melanoma. In the present study, we have shown that inhibition of melanoma iNOS activity by S-methylisothiourea leads to decreased cell proliferation, confirming the importance of iNOS activity for melanoma cell growth. Regulation of melanoma iNOS expression by the p44/42 MAPK pathway was demonstrated by inhibition of the pathway by U0126, and by BRAF RNA interference. To explore this regulatory pathway in human tissue, 20 melanoma tumors were examined for NRAS and BRAF mutations, immunohistochemical evidence of ERK phosphorylation, and iNOS expression. A significant association was found among these three features. We conclude that in human melanoma, activating mutations of NRAS and BRAF drive constitutive iNOS expression and, implicitly, nitric oxide production, contributing to the poor survival of these patients.

Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.

The melanoma differentiation associated gene-7 (mda-7) has a potential inhibitory role in melanoma progression, although the mechanisms underlying this effect are still unknown. mda-7 mRNA has been found to be present at higher levels in cultured normal melanocytes compared with metastatic melanoma cell lines. Furthermore, levels of mda-7 message have shown an inverse correlation with melanoma progression in human tumor samples, suggesting that mda-7 may be a novel tumor suppressor gene. We have designed this study to investigate MDA-7 protein expression in different stages of melanoma progression and to examine its antiproliferative effects in vitro. Our data demonstrate that MDA-7 protein can be found in normal melanocytes and early stage melanomas. It is also observed in smooth muscle cells in the skin. However, in keeping with a possible role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated antitumor effects of overexpressed MDA-7 on human melanoma cells in vitro. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes.

Inducible nitric oxide synthase and nitrotyrosine in human metastatic melanoma tumors correlate with poor survival.

Despite recognition of the malignant potential of human melanomas, the mechanisms responsible for the pathobiological characteristics contributing to tumor growth, vascular invasiveness, and distant organ metastasis remain undefined. Recent studies have shown that various human tumors express an inducible form of nitric oxide synthase (iNOS) and nitrotyrosine (NT), which suggests a mechanistic role of tumor-associated nitric oxide (NO) in tumorigenesis. We investigated iNOS and NT expression by immunohistochemistry in 20 human metastatic melanoma tissue specimens specifically with respect to iNOS-expressing cell types in the tumor area, pathological and clinical response to systemic therapy, potential role as a prognostic indicator, and NT formation. Our results showed that melanoma cells from 12 of 20 tumors express iNOS, yet the expression of this molecule in the tumor did not correlate with pathological or clinical response to therapy. More importantly, iNOS and NT expression by the melanoma cells strongly correlated with poor survival in patients with stage 3 disease (P < 0.001 and P = 0.020, respectively), suggesting a pathway whereby iNOS might contribute to enhanced tumor progression. In conclusion, our findings strongly suggest that iNOS expression has potential to be considered as a prognostic factor and NO as a critical mediator of an aggressive tumor phenotype in human metastatic melanomas.

Differential increase of Fas ligand expression on metastatic and thin or thick primary melanoma cells compared with interleukin-10.

Fas ligand (FasL), a cell surface molecule belonging to the tumour necrosis factor family, binds to its receptor Fas and thus induces apoptosis of Fas-bearing cells such as activated lymphocytes. In this paper, we report the expression of FasL on melanoma cell lines and patient tumour specimens, and compare it with the expression of interleukin-10 (IL-10), a putative immunosuppressive factor. Apoptosis of Fas-bearing Jurkat cells was increased after interferon-alpha treatment of the FasL-positive melanoma cell line A375, suggesting a regulation of FasL function. We also tested whether FasL and IL-10 were ever co-expressed. Immunohistochemistry studies showed that IL-10 expression was highly positive in the same tumour samples which expressed FasL. In the melanoma patients with thin primaries, 10 of the 12 primaries and six of the seven metastatic lesions were positive for IL-10. In the melanoma patients with thick primaries (> 0.75 mm), four of the five primary lesions and nine of the 10 metastatic lesions were positive for IL-10. In contrast, FasL was generally negative in primary tumours and positive in metastatic tumours. In the thin primary melanoma patients, two of the 12 primaries and five of the seven metastatic tumours were positive for FasL. From the thick melanomas, one of the five primaries and five of the 10 metastatic lesions were positive for FasL. The function of melanoma-derived FasL was confirmed by four different cytotoxicity assays.

Suppression of the immune response to an adenovirus vector and enhancement of intratumoral transgene expression by low-dose etoposide.

Adenoviral vectors are commonly used in gene therapy trials because of their efficiency in gene transfer. However, their use is limited by cellular and humoral immune responses that result in temporary transgene expression and reduced efficacy of repeated vector administration. We hypothesized that certain oncolytic agents commonly used to treat cancer patients could suppress the immune response to adenoviral vectors, and enable repeated adenovirus-mediated cancer gene therapy. Etoposide and cyclophosphamide were tested for their ability to suppress the humoral and cellular immune responses to an adenoviral vector in immunocompetent C3H mice. Intratumoral transgene expression was monitored in adenovirus-immunized animals treated with etoposide or cyclophosphamide. Neutralizing antibodies to adenovirus and cytotoxic T lymphocyte (CTL) lysis of virally transduced cells were significantly suppressed in mice treated with etoposide at 2 or 10 mg/kg/day or cyclophosphamide at 10 mg/kg/day compared with untreated mice (P < 0.05). Significantly larger areas of gene transduction were observed in treated animals compared with untreated mice or the mice treated with cyclophosphamide at 2 mg/kg/day (P < 0.05). Our results suggest that repeated adenovirally mediated gene therapy is achievable in cancer patients who are concurrently undergoing treatment with chemotherapy.

Germline mutations of the p53 gene in children with malignant solid tumors.

Although somatic mutations of p53 are the most common genetic changes observed to date, the frequency of germline p53 mutations is found to be very low in sporadic malignant tumors. It has been postulated that de novo germline p53 mutations may occur in a substantial population of patients in pediatric age group, who die of their disease and do not propagate the mutation. To determine the frequency and type of p53 germline mutations in pediatric patients, we screened 65 children who were consecutively admitted with primary malignant solid tumors.

Immunologic status in children with brain tumors and the effect of therapy.

The cellular and humoral immunological parameters (leucocyte, granulocyte, lymphocyte, total T, T4, T8 lymphocyte counts, lymphoproliferative response to PHA [LP-PHA], natural killer cell activity [NKCA], IgG, IgM and IgA levels) of 20 pediatric brain tumor patients were investigated before and after chemo-(CT) and radiotherapy (RT) administered according to the UIOI-PBT-91 protocol. The T4 and T8 cell percentages and the LP-PHA values before therapy were found to be significantly diminished in comparison to values obtained from 12 healthy children (p < 0.05). In patients receiving postoperative CT, all cellular immunity parameters except T8 cell number and NKCA; IgG and IgA levels were significantly decreased after two courses of CT (p < 0.05). In 7 patients given postoperative RT, a depression in all cellular immunity parameters was observed (p < 0.05). In 6 patients treated with 2 courses of postoperative CT followed by RT administered concomitantly with low dose CDDP, there was a decrease in all cellular and humoral immunity parameters, which was not found to be significant. In 5/18 patients infectious episodes in mild to moderate severity were observed, none causing mortality. It was concluded that the UIOI-PBT-91 protocol caused cellular immunosuppression both after CT and after RT and some humoral immunosuppression after CT, but was found to be tolerable in regard to acute immunological side effects.