Purpose: NRAS and BRAF mutations are common in cutaneous melanomas, although rarely detected mutually in the same tumor. Distinct clinical correlates of these mutations have not been described, despite in vitro data suggesting enhanced oncogenic effects. This study was designed to test the hypothesis that primary human cutaneous melanomas harboring mutations in NRAS or BRAF display a more aggressive clinical phenotype than tumors wild type at both loci.Experimental Design: Microdissection of 223 primary melanomas was carried out, followed by determination of the NRAS and BRAF mutational status. Genotypic findings were correlated with features known to influence tumor behavior including age, gender, Breslow depth, Clark level, mitotic rate, the presence of ulceration, and American Joint Committee on Cancer (AJCC) staging.Results: Breslow depth and Clark level varied significantly among the genotypes, with NRAS mutants showing the deepest levels and wild-type tumors the least depth. Ulceration also differed significantly among the genotypes, with BRAF mutants demonstrating the highest rate. In addition, tumors with mutated NRAS were more likely to be located on the extremities. Patients whose tumors carried either mutation presented with more advanced AJCC stages compared with patients with wild-type tumors, and specifically, were more likely to have stage III disease at diagnosis. Overall survival did not differ among the 3 groups.Conclusions: Distinct clinical phenotypes exist for melanomas bearing NRAS and BRAF mutations, whether considered together or separately, and are associated with features known to predict aggressive tumor behavior. The impact of these mutations is most evident at earlier stages of disease progression.

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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. A feature of malignant tumors that is considered therapeutically exploitable is the loss of cellular differentiation. This has lead to a search for tissue-specific differentiation factors that might be reintroduced into tumors in order to modify their growth and ability to metastasize. In the case of melanoma, several unique genes have been isolated by Jiang et al. 1 from a melanoma cell line induced to differentiate by interferon-␤ and mezerein. One of the most interesting and promising of these genes is the melanoma differentiation associated gene-7 (mda-7). Since its initial isolation from melanoma cells and subsequent molecular characterization, the mda-7 gene and MDA-7 protein have been studied in numerous other tumor types, some of which have included carcinoma of the lung, breast, prostate and cervix. 2 Experiments using an adenovirus/mda-7 vector construct (Ad-mda-7) to infect normal and malignant cells have consistently demonstrated growth-inhibitory effects on various tumor types, but no inhibitory effects on normal cells. 3,4 These findings have prompted the development of the Ad-mda-7 as a potential therapeutic agent, and plans for a phase I clinical trial are under way. Although the initial description of mda-7 occurred in melanoma, most of the clinically relevant research has focused on the more prevalent malignancies. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cellsOur laboratory has an ongoing interest in the development of prognostic tools and therapeutic interventions for advanced melanoma. We have therefore undertaken a study to better define the significance of MDA-7 in this disease. We describe in this paper our data from human tumors and cell lines demonstrating that MDA-7 protein can be found in normal melanocytes, early stage melanomas and smooth muscle cells. However, in keeping with a role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanom...

Prostate cancer is one of the malignant tumors which exhibit resistance to anticancer drugs, at least in part due to enhanced antiapoptotic mechanisms. Therefore, the understanding of such mechanisms should improve the design of chemotherapy against prostate cancer. Galectin-3 (Gal-3), a multifunctional oncogenic protein involved in the regulation of tumor proliferation, angiogenesis, and apoptosis has shown antiapoptotic effects in certain cell types. Here, we show that the expression of exogenous Gal-3 in human prostate cancer LNCaP cells, which do not express Gal-3 constitutively, inhibits anticancer drug-induced apoptosis by stabilizing the mitochondria. Thus, Gal-3-negative cells showed 66.31% apoptosis after treatment with 50 Mmol/L cis-diammine-dichloroplatinum for 48 hours, whereas two clones of Gal-3-expressing cells show only 2.92% and 1.42% apoptotic cells. Similarly, Gal-3-negative cells showed 43.8% apoptosis after treatment with 300 Mmol/L etoposide for 48 hours, whereas only 15.38% and 14.51% of Gal-3-expressing LNCaP cells were apoptotic. The expression of Gal-3 stimulated the phosphorylation of Ser 112 of Bcl-2-associated death (Bad) protein and down-regulated Bad expression after treatment with cis-diammine-dichloroplatinum. Gal-3 also inhibited mitochondrial depolarization and damage after translocation from the nuclei to the cytoplasm, resulting in inhibition of cytochrome c release and caspase-3 activation. These findings indicate that Gal-3 inhibits anticancer drug-induced apoptosis through regulation of Bad protein and suppression of the mitochondrial apoptosis pathway. Therefore, targeting Gal-3 could improve the efficacy of anticancer drug chemotherapy in prostate cancer. (Cancer Res 2006; 66(6): 3114-9)

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Inducible nitric oxide synthase (iNOS) produces nitric oxide, which has growth promoting activity in melanoma. A preliminary study of tumors from patients with Stage III melanoma who had received neo-adjuvant therapy revealed an association of tumor iNOS expression with shortened survival. The objective of the present study was to determine whether iNOS expression in tumors of newly diagnosed, untreated Stage III patients is predictive of survival. iNOS expression was examined by immunohistochemistry in tumors from 132 patients. The staining was evaluated for percentage of positive cells (Number score) and the intensity of staining (Intensity score). The association of iNOS expression with overall and disease-specific survival was tested in univariate and multivariate Cox proportional hazards regression models that included other known prognostic factors. Results of the univariate analysis demonstrated that the presence of iNOS in a patient's tumor, whether graded on the basis of Number or Intensity score, was associated with a significant increase in the hazard ratio of death from melanoma. These findings were corroborated by median survival data estimated from Kaplan Meier analysis. In the multivariate model including iNOS number or intensity, gender, age, number of lymph nodes, macroscopic disease and in-transit disease, only iNOS expression predicted survival. We conclude that a significant association exists between tumor iNOS expression and shortened survival in untreated Stage III melanoma patients. The ability of iNOS to predict outcomes for these patients may be independent of other known prognostic factors, providing a new molecular marker with significant potential for clinical utility. ' 2006 Wiley-Liss, Inc.

Downregulation of MDA-7 expression in primary melanomas facilitates progression to invasive and metastatic stages. These data support the development of Ad-mda7 as gene therapy for advanced melanoma.

Human melanoma tumors cells are known to express the enzyme, inducible nitric oxide synthase (iNOS), which is responsible for cytokine induced nitric oxide (NO) production during immune responses. This constitutive expression of iNOS in many patients' tumor cells, as well as its strong association with poor patient survival, have led to the consideration of iNOS as a molecular marker of poor prognosis, as well as a possible target for therapy. The expression of iNOS in patient tumors was found to associate with nitrotyrosine, COX2, pSTAT3, and arginase. Using human melanoma patients' samples as well as cell lines, we have further evidence supporting intracellular NO production by detection of nitrotyrosine and also by use of DAF-2 DA staining. Experiments were performed to scavange the endogenous NO (with c-PTIO) resulting in melanoma cell growth inhibition; this was restored with SIN-1 (NO and O2-donor) providing data to support a functional role of this gas. Our goal is to understand the aberrant biology leading to this curious phenomenon, and to regulate it in favor of patient treatments.