The metabolic microenvironment of melanomas: Prognostic value of MCT1 and MCT4

BRAF mutations are known drivers of melanoma development and, recently, were also described as players in the Warburg effect, while this reprogramming of energy metabolism has been identified as a possible strategy for treating melanoma patients. Therefore, the aim of this work was to evaluate the expression and prognostic value of a panel of glycolytic metabolism-related proteins in a series of melanomas. The immunohistochemical expression of MCT1, MCT4, GLUT1, and CAIX was evaluated in 356 patients presenting melanoma and 20 patients presenting benign nevi. Samples included 20 benign nevi, 282 primary melanomas, 117 lymph node and 54 distant metastases samples. BRAF mutation was observed in 29/92 (31.5%) melanoma patients and 17/20 (85%) benign nevi samples. NRAS mutation was observed in 4/36 (11.1%) melanoma patients and 1/19 (5.3%) benign nevi samples. MCT4 and GLUT1 expression was significantly increased in metastatic samples, and MCT1, MCT4 and GLUT1 were significantly associated with poor prognostic variables. Importantly, MCT1 and MCT4 were associated with shorter overall survival. In conclusion, the present study brings new insights on metabolic aspects of melanoma, paving the way for the development of new-targeted therapies.     

Abdominal wall and labial edema presenting in a girl with Henoch-Schönlein purpura: a case report

Introduction

We report a rare case of gastrointestinal perforation following dacarbazine infusion for metastatic melanoma. The condition is attributed to a responding malignant melanoma in the gastrointestinal tract.

Case presentation

A 52-year-old Caucasian man presented with abdominal pain and distension, malaise, night sweats, dysphagia and early satiety. A computed tomography scan showed massive ascites, lymphadenopathy and liver lesions suspect for metastases. An upper gastrointestinal endoscopy was performed and revealed multiple dark lesions of 5 mm to 10 mm in his stomach and duodenum. When his skin was re-examined, an irregular pigmented lesion over the left clavicle measuring 15 mm × 8 mm with partial depigmentation was found. Histological examination of a duodenal lesion was consistent with a diagnosis of metastatic melanoma. The patient deteriorated and his level of lactate dehydrogenase rapidly increased. The patient was started on systemic treatment with dacarbazine 800 mg/m² every three weeks and he was discharged one day after the first dose. On the sixth day he was readmitted with severe abdominal pain. A chest X-ray showed the presence of free intraperitoneal air that was consistent with gastrointestinal perforation. His lactate dehydrogenase level had fallen from 6969U/L to 1827U/L, supporting the conclusion that the response of gastrointestinal metastases to dacarbazine had resulted in the perforation of the patient's bowel wall. A laparotomy was discussed with the patient and his family but he decided to go home with symptomatic treatment. He died 11 days later.

Conclusion

Melanoma can originate in, as well as metastasize to, the gastrointestinal tract. Gastrointestinal perforations due to responding tumors are a well-known complication of systemic treatment of gastrointestinal lymphomas. However, as the response rate of metastatic melanoma to dacarbazine is only 10% to 20%, and responses are usually only partial, perforation due to treatment response in metastatic melanoma is rare. Medical oncologists should be aware of the risk of bowel perforation after starting cytotoxic chemotherapy on patients with gastrointestinal metastases.     

Chromosomal imbalances in primary and metastatic melanomas revealed by comparative genomic hybridization

Characteristic genetic changes underlying the metastatic progression of malignant melanoma is incompletely understood. The goal of our study was to explore specific chromosomal alterations associated with the aggressive behavior of this neoplasm. Comparative genomic hybridization was performed to screen and compare genomic imbalances present in primary and metastatic melanomas. Sixteen primary and 12 metastatic specimens were analyzed. We found that the pattern of chromosomal aberrations is similar in the two subgroups; however, alterations present only in primary and/or metastatic tumors were also discovered. The mean number of genetic changes was 6.3 (range 1-14) in primary and 7.8 (range 1-16) in metastatic lesions. Frequent losses involved 9p and 10q, whereas gains most often occurred at 1q, 6p, 7q, and 8q. Distinct, high-level amplifications were mapped to 1p12-p21 and 1p22-p31 in both tumor types. Amplification of 4q12-q13.1, 7q21.3-qter and 8q23-qter were detected only in primary tumors. The 20q13-qter amplicon was present in a metastatic tumor. The number of genetic alterations were significantly higher in primary tumors which developed metastases within one year after the surgery compared to tumors without metastasis during this time period. Fluorescence in situ hybridization with centromeric and locus-specific probes was applied to validate CGH results on a subset of tumors. Comparison of FISH and CGH data gave good correlation. The aggressive behavior of melanoma is associated with accumulation of multiple genetic alterations. Chromosome regions, which differ in the primary and metastatic lesions, may represent potential targets to identify metastases-related chromosomal alterations.     

Infiltration of primary and metastatic melanomas with macrophages of the 25F9-positive phenotype

Summary In order to gain insight into the role of macrophages in human melanoma, we studied fresh-frozen material from 15 dysplastic nevi, 199 primary melanomas, 107 melanoma metastases, and paraffin sections from 98 primary melanomas with the monoclonal antibody 25F9 which recognizes an 86×10³ dalton protein present on a subset of mature human macrophages. Considerable infiltration of tumors with 25F9-positive macrophages was observed in 2 dysplastic nevi (13%), 87 primary melanomas (44%), and 45 metastases (42%). The degree of intratumoral macrophage infiltration correlated with expression of class II HLA-DR antigens on tumor cells, in primary melanoma with a tumor thickness above 0.75 mm, and with the occurence of metastases within 2 years. In paraffin sections, intratumoral 25F9-positive macrophages also correlated with metastatic spread of primary tumors after longer follow-up. Metastases revealed a higher degree of macrophage infiltration following systemic or local immunotherapy, compared with untreated metastases, or metastases removed during chemotherapy. Of 38 patients who died within an observation period of 1 year, 19 (50%) had considerable infiltration of metastases with 25F9-positive macrophages, whereas this was found in only 4 of 12 patients (33%), who survived for longer than 2 years following metastases removal. A higher degree of 25F9-positive macrophages correlated with a shift towards the T8-positive subsets within the T cell compartment of the infiltrate. Our results suggest that accumulation of 25F9-positive macrophages in melanomas indicates more aggressive tumor properties.     

A Protein Deep Sequencing Evaluation of Metastatic Melanoma Tissues

Malignant melanoma has the highest increase of incidence of malignancies in the western world. In early stages, front line therapy is surgical excision of the primary tumor. Metastatic disease has very limited possibilities for cure. Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem. The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high. The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers. Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation). Fractionation produced higher numbers of protein identifications (4284). Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping). Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma “genomic subtypes”, (“pigmentation” and “high immune”) revealed several proteins showing differential levels of expression. In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications. The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725.     

A matter of the heart: myocardial metastases in neuroendocrine tumors

The aim of the present study was to evaluate frequency, clinical spectrum, and treatment of myocardial metastases in patients with histologically proven neuroendocrine tumors by analysis of our database and literature review. The literature on cardiac metastases in patients with neuroendocrine tumors published from 1973 to the present was reviewed for age, sex, primary tumor localization, metastases, symptoms, complications, treatment, diagnostic methods, and histology. Patient records from our institution were analyzed retrospectively for cardiac metastases detected by any diagnostic means and detailed patient histories are given. 4 patients with myocardial metastases could be identified in our database (n=550) while literature review identified 41 published cases. Mean age at initial diagnosis was 57.5 years (females=13, males=28), primary tumor localizations were foregut (n=7), midgut (n=28), hindgut (n=1), or unknown (n=3). Carcinoid syndrome was reported for 28 patients. Cardiac involvement was right-ventricular only (n=10), left-ventricular only (n=11), or biventricular (n=10). Diagnosis was obtained by echocardiography (n=21), CT/MRI (n=12) and other methods (n=9), or by autopsy (n=9). We describe visualization of cardiac metastases by (68)Ga-DOTATOC-PET/CT for the first time. Clinical presentation ranged from asymptomatic patients to cardiac arrest. Follow-up times ranged from <1 month up to 12 years. Clinicians treating patients with neuroendocrine tumors should be aware of the heart as a possible site of metastatic disease. Echocardiography and MRI are the methods of choice for follow-up, while PET/CT might contribute to earlier and more frequent detection. Management of cardiac metastases requires close cooperation between specialists of internal medicine, nuclear medicine, and cardiac surgery.     

Overlapping Cardiac Programs in Heart Development and Regeneration

Gaining cellular and molecular insights into heart development and regeneration will likely provide new therapeutic targets and opportunities for cardiac regenerative medicine,one of the most urgent clinical needs for heart failure.Here we present a review on zebrafish heart development and regeneration,with a particular focus on early cardiac progenitor development and their contribution to building embryonic heart,as well as cellular and molecular programs in adult zebrafish heart regeneration.We attempt to emphasize that the signaling pathways shaping cardiac progenitors in heart development may also be redeployed during the progress of adult heart regeneration.A brief perspective highlights several important and promising research areas in this exciting field.     

FRIZZLED7 Is Required for Tumor Inititation and Metastatic Growth of Melanoma Cells

Metastases are thought to arise from cancer stem cells and their tumor initiating abilities are required for the establishment of metastases. Nevertheless, in metastatic melanoma, the nature of cancer stem cells is under debate and their contribution to metastasis formation remains unknown. Using an experimental metastasis model, we discovered that high levels of the WNT receptor, FZD7, correlated with enhanced metastatic potentials of melanoma cell lines. Knocking down of FZD7 in a panel of four melanoma cell lines led to a significant reduction in lung metastases in animal models, arguing that FZD7 plays a causal role during metastasis formation. Notably, limiting dilution analyses revealed that FZD7 is essential for the tumor initiation of melanoma cells and FZD7 knockdown impeded the early expansion of metastatic melanoma cells shortly after seeding, in accordance with the view that tumor initiating ability of cancer cells is required for metastasis formation. FZD7 activated JNK in melanoma cell lines in vitro and the expression of a dominant negative JNK suppressed metastasis formation in vivo, suggesting that FZD7 may promote metastatic growth of melanoma cells via activation of JNK. Taken together, our findings uncovered a signaling pathway that regulates the tumor initiation of melanoma cells and contributes to metastasis formation in melanoma.     

Further characterization of a clinically relevant model of melanoma metastasis and an effective vaccine

A major problem in evaluating the effectiveness of tumor cell vaccination and other biological therapies is the variability of experimental models. In this study we have further developed and characterized a model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination: stage I-growth of the primary (local) tumor, stage II-dissemination to regional lymph nodes, and stage III-metastasis to distant organs (lungs). C57BL/6 mice were challenged subcutaneously with B16 F10 murine melanoma cells in the midtail, and within 3 weeks 100% of the mice had local tumors growing in their tails. By 5–7 weeks after challenge, most of the mice had developed metastases to the inguinal lymph nodes and subsequently had metastatic colonies in the lungs and in the bone marrow. Preimmunization of mice with a formalinized extracellular antigen vaccine, derived from B16F10 melanoma cells, provided partial inhibition of the growth of the primary melanoma tumors, as well as reducing the number of metastases to the regional (inguinal) lymph nodes and lungs along with concomitantly increasing survival time. This model for melanoma metastasis provides a reasonable and reproducible test system for the study of anti-melanoma immunity and the different cellular and humoral mechanisms involved.This work was supported in part by National Institutes of Health grants R37 CA45148 and R30 CA13943     

The role of surgery in the treatment of malignant melanoma

Surgical interventions have important role in the treatment of all stages of malignant melanoma. Surgery is the primary treatment of localized cutaneous melanoma. Excision of the primary tumor makes it possible to set up the histological diagnosis and to determine pathological prognostic factors. Appropriate surgical margin is important for local disease control. Sentinel lymph node biopsy with detailed histological examination has gained prominent importance for correct histological staging and for determining adjuvant oncological treatment. Surgery is the primary treatment of isolated regional metastases. Surgical methods also have a role in the palliative management of distant metastatic melanoma. In the present review the most important issues of the surgical treatment of malignant melanoma have been discussed in detail.     

Mda-9/syntenin is expressed in uveal melanoma and correlates with metastatic progression

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.     

Combination therapy with dacarbazine and statins improved the survival rate in mice with metastatic melanoma

Malignant melanoma is a highly aggressive skin cancer, and the overall median survival in patients with metastatic melanoma is only 6–9 months. Although molecular targeted therapies have recently been developed and have improved the overall survival, melanoma patients may show no response and acquisition of resistance to these drugs. Thus, other molecular approaches are essential for the treatment of metastatic melanoma. In the present study, we investigated the effect of cotreatment with dacarbazine and statins on tumor growth, metastasis, and survival rate in mice with metastatic melanomas. We found that cotreatment with dacarbazine and statins significantly inhibited tumor growth and metastasis via suppression of the RhoA/RhoC/LIM domain kinase/serum response factor/c-Fos pathway and enhanced p53, p21, p27, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase 1 expression in vivo. Moreover, the cotreatment significantly improved the survival rate in metastasis-bearing mice. Importantly, treatment with dacarbazine plus 100 mg/kg simvastatin or fluvastatin prevented metastasis-associated death in 4/20 mice that received dacarbazine + simvastatin and in 8/20 mice that received dacarbazine + fluvastatin (survival rates, 20% and 40%, respectively). These results suggested that cotreatment with dacarbazine and statins may thus serve as a new therapeutic approach to control tumor growth and metastasis in melanoma patients.     

RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line

The formation of melanoma metastases from primary tumor cells is a complex phenomenon that involves the regulation of multiple genes. We have previously shown that the receptor for advanced glycation end products (RAGE) was up-regulated in late metastatic stages of melanoma patient samples and we hypothesized that up-regulation of RAGE in cells forming a primary melanoma tumor could contribute to the metastatic switch of these cells. To test our hypothesis, we overexpressed RAGE in the WM115 human melanoma cell line that was established from a primary melanoma tumor of a patient. We show here that overexpression of RAGE in these cells is associated with mesenchymal-like morphologies of the cells. These cells demonstrate higher migration abilities and reduced proliferation properties, suggesting that the cells have switched to a metastatic phenotype. At the molecular level, we show that RAGE overexpression is associated with the up-regulation of the RAGE ligand S100B and the down-regulation of p53, ERK1/2, cyclin E and NF-kB. Our study supports a role of RAGE in the metastatic switch of melanoma cells.     

Heparanases and tumor metastasis

The successful penetration of endothelial basement membranes is an important process in the formation of hematogenous tumor metastases. Heparan sulfate (HS) proteoglycan is a major constituent of endothelial basement membranes, and we have found that HS-degradative activities of metastatic B16 melanoma sublines correlate with their lung-colonizing potentials. The melanoma HS-degrading enzyme is a unique endo-beta-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites and is detectable in a variety of cultured human malignant melanomas. The treatment of B16 melanoma cells with heparanase inhibitors that have few other biological activities, such as N-acetylated N-desulfated heparin, results in significant reductions in the numbers of experimental lung metastases in syngeneic mice, indicating that heparanase plays an important role in melanoma metastasis. HS-degrading endoglycosidases are not tumor-specific and have been found in several normal tissues and cells. There are at least three types of endo-beta-D-glucuronidases based on their substrate specificities. Melanoma heparanase, an Mr approximately 96,000 enzyme with specificity for beta-D-glucuronosyl-N-acetylglucosaminyl linkages in HS, is different from platelet and mastocytoma endoglucuronidases. Elevated levels of heparanase have been detected in sera from metastatic tumor-bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and extent of metastases. The results suggest that heparanase is potentially a useful marker for tumor metastasis.     

Metastases of human tumor cells in immunosuppressed newborn rats

The metastatic ability of human tumour cells can easily be evaluated by using as an experimental model the production of metastases in newborn rats immunosuppressed by an optimal dose of anti-thymocyte serum. Thus, following sub-cutaneous injection of 10(6) cells, a human melanoma cell line, tumorigenic but non metastatic in nude mice, produces within 3 weeks tumours in all inoculated rats and lymph node and pulmonary metastases in 50% of the animals. The cloning of this cell line in semi-solid agar shows its heterogeneity and demonstrates that it contains poorly tumorigenic but highly metastatic cells.     

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.     

Riluzole is a radio‐sensitizing agent in an in vivo model of brain metastasis derived from GRM1 expressing human melanoma cells

Approximately 50% of patients having metastatic melanoma develop brain metastases during the course of their illness. Evidence exists that melanoma cells have increased aptitude for the repair of sublethal DNA damage caused by ionizing radiation therapy. To address the radio‐resistance of melanoma, many groups adopted radiotherapy schedules that deliver larger daily fractions of radiation, but due to the risk of neurotoxicity, these large fractions cannot be delivered to the whole brain for patients with brain metastases. Here, we used orthotopic implanted GRM1 expressing human melanoma cell xenografts in mice, to demonstrate that animals receiving concurrent glutamate signaling blockade (riluzole) and radiation led to a decrease in intracranial tumor growth compared to either modality alone. These preclinical results suggest riluzole may cause radio‐sensitization that offers enhanced efficacy for a subset of human melanoma patients undergoing radiotherapy for brain metastasis.     

Microimaging characterization of a B16-F10 melanoma metastasis mouse model

Metastatic mouse models of melanoma have been characterized by gross necropsy examination, histopathology, and optical imaging. To determine if the time progression, extent, and metabolism of melanoma metastases could be monitored noninvasively, serial micro-CT and small-animal PET imaging studies were performed by using a mouse model of melanoma. Juvenile female C57BL/6 mice were injected intravenously with syngenic B16-F10 melanoma cells. Serial micro-CT imaging studies were performed on anesthetized mice. Mice were necropsied at the development of adverse clinical signs or at postinjection Day 30, and tissues were collected for histopathology. In a separate study of four mice, tumor viability was assessed with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) and studied by using small-animal PET imaging. A total of 59% of the mice developed metastatic tumors. Micro-CT image analysis was able to identify and follow up to 36% of metastatic lesions. Examples of metastatic lesions identified and followed up by micro-CT imaging included a lung metastasis, mandibular metastasis, subcutaneous metastasis, and tibial/femoral metastasis. Micro-CT and small-animal PET fusion imaging successfully correlated anatomic localization of glucose metabolism of the metastatic tumors. Micro-CT and small-animal PET imaging were found to be highly effective in detection and characterization of lesions produced by this metastatic melanoma model.     

RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf^V600E-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.     

RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf^V600E-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.