Human Melanoma Progression Research Articles

Expression of glucose‐regulated stress protein GRP78 is related to progression of melanoma

Glucose-regulated protein 78 (GRP78) is a protein translated in response to endoplasmic reticulum (ER) stress that has been implicated in the pathogenesis and resistance to therapy of a variety of cancers. The aim of this study was to investigate its expression and role in the development and progression of human melanoma. The immunohistochemical expression of GRP78 in naevi, primary melanoma and melanoma metastases from 171 patients was correlated with clinicopathological factors and patient survival. The GRP78 immunoreactivity score (IRS) was 0.2 in compound naevi, 0.65 in dysplastic naevi, 4.65 in naevi adjacent to primary melanoma, 2.4 in melanoma in situ, 11.2 in thin (</=1.0 mm) and 18.1 in thick (>1.0 mm) primary melanoma. It was 18 and 17.3 in subcutaneous and lymph node metastases, respectively (P < 0.0001). GRP78 expression was positively correlated with increasing tumour thickness (P = 0.001) and with increasing dermal tumour mitotic index (P = 0.0004). Disease-free survival (chi(2) = 8.0703, P = 0.0045) and overall survival (chi(2) = 6.2633, P = 0.0123) in melanoma patients with IRS >25 were significantly lower than in melanoma patients with IRS <25. GRP78 expression appears to correlate with known correlates of melanoma progression and survival and requires further evaluation as a prognostic biomarker in melanoma.

Nitric oxide initiates progression of human melanoma via a feedback loop mediated by apurinic/apyrimidinic endonuclease-1/redox factor-1, which is inhibited by resveratrol

It is well recognized that nitric oxide (NO) is involved in tumor progression, including melanoma. Measurement of proliferative and metastatic capacity by MTS and Matrigel invasion assays, respectively, was done and showed that NO-treated melanoma cells exhibited a higher capacity compared with control, especially metastatic Lu1205 cells. Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) is a multifunctional protein and its role in tumor biology has attracted considerable attention. To determine whether APE/Ref-1 plays a role in mediating NO stimulation of melanoma progression, we investigated the effect of DETA/NO on levels of APE/Ref-1 and related downstream targets [activator protein-1 (AP-1)/JunD, matrix metalloproteinase-1 (MMP-1), Bcl-2, and inducible nitric oxide synthase (iNOS)] by Western blot and reverse transcription-PCR analysis. Following DETA/NO treatment, APE/Ref-1 and other downstream molecules were induced. Knockdown of APE/Ref-1 or AP-1/JunD by specific small interfering RNA markedly reversed the induction by NO stress of target proteins. These results present evidence for the existence of a functional feedback loop contributing to progression and metastasis of melanoma cells. Resveratrol has been shown to be an APE/Ref-1 inhibitor and significant decreases in AP-1/JunD, MMP-1, Bcl-2, and iNOS protein levels occurred after exposure to resveratrol. This phenolic antioxidant may be an appropriate choice for combining with other compounds that develop resistance by up-regulation of these molecules.

Abstract A137: NO initiates progression of human melanoma via a feedback loop mediated by APE/Ref-1, new opportunities for chemoprevention.

Abstract A137 It is well recognized that nitric oxide (NO) is involved in tumor progression, including melanoma. Measurement of proliferative and metastatic capacity by MTS and Matrigel-invasion assays respectively were done. NO-treated melanoma cells(especially metastatic Lu1205) exhibited a higher capacity compared with control, especially metastatic Lu 1205 cells. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE/Ref-1) is a multifunctional protein, and its role in tumor biology has attracted considerable attention. To determine whether APE/Ref-1 plays a role in mediating NO-stimulation of melanoma progression, we investigated the effect of DETA/NO on levels of APE/Ref-1 and related downstream targets (Activator Protein-1 (AP-1)/Jun D, Matrix Metalloproteinase-1 (MMP-1), Bcl-2, and inducible nitric oxide synthase (iNOS)) by Western Blot and RT-PCR analysis. Following DETA/NO treatment, APE/Ref-1 and other downstream molecules were induced. Knockdown of APE/Ref-1 or AP-1/Jun D by specific siRNA markedly reversed the induction by NO stress of target proteins. These results present evidence for the existence of a functional feedback loop contributing to progression and metastatic capacity of melanoma cells. Resveratrol has been demonstrated to be an APE/Ref-1 inhibitor, and significant decreases in AP-1/JunD, MMP-1, Bcl-2, and iNOS protein levels occurred after exposure to resveratrol. This phenolic antioxidant as well as other inhibitors of Ref-1 may be an appropriate choice for combining with other compounds against melanoma cells that develop resistance by up-regulations of these molecules. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A137.

Skin layer-specific Melan-A expression during progression of human cutaneous melanoma: implications for diagnostic applications of the marker

Melan-A is widely used in the diagnostics of human melanoma. The immunogenicity of this glycoprotein makes it a potential target in immunotherapy and several authors have suggested its potential as a prognostic factor. Up to now there has been no clear direct evidence of changes of Melan-A expression during the progression of melanoma. We have performed objective immunohistochemical assessment of the expression of Melan-A in benign naevi and melanomas at different stages of progression. Our results show a complex pattern of changes in the expression of Melan-A in melanomas depending on the location of melanoma cells within individual skin layers. The expression of the antigen during tumour progression significantly decreases for melanoma cells located in the granular/spinous layer (r=-0.94, P=0.02) and increases for the papillary layer (r=0.99, P=0.002) and reticular layer (r=0.89, P=0.04). It should also be emphasized that from the Clark II level of progression the melanomas can be detected with high sensitivity and specificity using a simple cut-off test based on the determination of Melan-A expression in tumour cells located within the papillary layer.

The Impact of Genomics in Understanding Human Melanoma Progression and Metastasis

Recent technological advances in the analysis of the human genome have opened the door to improving our primitive understanding of the gene expression patterns in cancer. For the first time, we have an overview of the complexities of tumorigenesis and metastatic progression of cancer. The examination of the phenotypic and (epi)genetic changes in cutaneous melanoma has identified several genes deemed central to the development and progression of melanoma. A review of the recent literature was performed to determine the role of array-based high-throughput gene expression analysis in understanding the specific genes involved as well as the pathways and the comparative gene expression patterns of primary and metastatic melanoma. Most studies utilizing gene microarray analysis and other whole genome approaches reveal a wide array of genes and expression patterns in human melanoma. Furthermore, several of the same genes have been found in comparative studies, with some studies attempting correlation with clinical outcome. Several genes have been identified as potential prognostic markers of tumor progression and overall clinical outcome. High-throughput gene expression analysis has had a major impact in melanoma research. Several gene expression platforms have provided insight into the gene expression patterns in melanoma. Such data will provide the foundations for the future development of prognostic markers and improved targeted therapies for patients with melanoma.

Transforming Growth Factor-β Activation Promotes Genetic Context–Dependent Invasion of Immortalized Melanocytes

Accumulation of distinct sets of genetic/epigenetic alterations is thought to contribute to stepwise progression of human cutaneous melanomas. We found evidence of frequent tumor cell autonomous transforming growth factor-beta (TGF-beta) signal activation in both premalignant and malignant stages of human cutaneous melanoma histogenesis and investigated its potential causative roles using human organotypic skin cultures. PTEN deficiency and Braf activation, two common coincident genetic alterations found in primary cutaneous melanomas, were first introduced into human melanocytes previously immortalized by the SV40 large T antigen and telomerase. These changes individually supported anchorage-independent growth and conferred benign, hyperplastic growth in a skin-like environment. In addition, PTEN deficiency combined with Braf activation together induced a melanoma in situ-like phenotype without dermal invasion. Further addition of cell autonomous TGF-beta activation in the context of PTEN deficiency and Braf activation promoted dermal invasion in skin cultures without significantly promoting proliferation in vitro and in vivo. This proinvasive phenotype of cell autonomous TGF-beta activation is genetic context-dependent, as hyperactivating the TGF-beta type I receptor without PTEN deficiency and Braf activation failed to induce an invasive behavior. Evidence of genetic interactions among PTEN deficiency, Braf activation, and cell autonomous TGF-beta activation shows that distinct stages of human melanoma are genetically tractable in the proper tissue architecture.

The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms

The incidence of cutaneous melanoma is steadily increasing. Although several molecular abnormalities have been associated with melanoma progression, the mechanisms underlying the differential gene expression are still largely unknown and targeted therapies are not yet available. Noncoding small RNAs, termed microRNAs (miR), have been recently reported to play important roles in major cellular processes, including those involved in cancer development and progression. We have identified the promyelocytic leukemia zinc finger (PLZF) transcription factor as a repressor of miR-221 and miR-222 by direct binding to their putative regulatory region. Specifically, PLZF silencing in melanomas unblocks miR-221 and miR-222, which in turn controls the progression of the neoplasia through down-modulation of p27Kip1/CDKN1B and c-KIT receptor, leading to enhanced proliferation and differentiation blockade of the melanoma cells, respectively. In vitro and in vivo functional studies, including the use of antisense "antagomir" oligonucleotides, confirmed the key role of miR-221/-222 in regulating the progression of human melanoma; this suggests that targeted therapies suppressing miR-221/-222 may prove beneficial in advanced melanoma.

The release of fibroblast growth factor-1 from melanoma cells requires copper ions and is mediated by phosphatidylinositol 3-kinase/Akt intracellular signaling pathway

Melanoma is a highly invasive tumor with elevated mortality rates. Progression and aggressiveness appear related to the achievement of an angiogenic phenotype. Melanoma cells express several angiogenic factors, including fibroblast growth factor (FGF)-1 and FGF-2. The autocrine production and release of FGFs and the subsequent activation of FGF receptors, have a central role in melanoma tumor progression. We demonstrated that FGF-1 is secreted from a human melanoma cell line, A375, under conditions of serum deprivation. The release of FGF-1 is inhibited by the copper chelator ammonium tetrathiomolybdate, suggesting a role of copper in the secretory pathway, and is triggered by activation of phosphatidylinositol 3-kinase (PI3K)/Akt intracellular signaling. Interestingly, overexpression or activation of Akt has been correlated with poor prognosis in melanoma patients. Our data indicate a novel role for Akt in supporting the progression of human melanomas and advocate the need for new treatments targeting PI3K/Akt signaling pathway, to control tumor development and progression.

Loss of Hugl-1 Expression Associates With Lymph Node Metastasis in Endometrial Cancer

Mutation of neoplastic tumor suppressor genes, scribble, discs large, and lethal giant larvae (lgl), causes disruption of cell polarity and overproliferation of Drosophila epithelial cells and neuroblasts. Reduced expression of human homologue of lgl, Hugl-1, has been reported to be involved in development and progression of human colon cancer and malignant melanoma. To explore the association between Hugl-1 expression and clinical character in endometrial cancer, we examined the expression of Hugl-1 in primary endometrial cancer tissues. The expression of Hugl-1 mRNA in 86 primary endometrial cancer tissues was examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). All samples were categorized into two groups: Hugl-1 positive and Hugl-1 negative. Clinical data of each group were analyzed by Fisher's exact probability test and survival rates of each group were compared by Kaplan-Meier method and Log-rank test. Loss of Hugl-1 expression had correlation with the higher incidence of lymph node metastasis, but not to the patient's age at onset, distant metastasis, clinical stage, lymph or venous vessel invasion, or histopathological grade of differentiation. The Hugl-1-positive group had poorer prognosis compared with the Hugl-1-negative group. These results indicate that loss of Hugl-1 expression in endometrial cancer may contribute to lymph node metastasis and it can be a factor of poor prognosis.

Prognostic Significance of Nuclear ING3 Expression in Human Cutaneous Melanoma

The novel tumor-suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. Our previous study showed that ING3 promotes UV-induced apoptosis via the Fas/caspase-8-dependent pathway in melanoma cells. To investigate the putative role of ING3 in the development of melanoma, we examined the expression of ING3 in melanocytic lesions at different stages and analyzed the correlation between ING3 expression and clinicopathologic variables and patient survival. Using tissue microarray and immunohistochemistry, we evaluated nuclear and cytoplasmic ING3 staining in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas. Nuclear ING3 expression was remarkably reduced in malignant melanomas compared with dysplastic nevi (P<0.001), which was significantly correlated with the increased ING3 level in cytoplasm (P<0.05). Furthermore, the reduced nuclear ING3 expression was significantly correlated with a poorer disease-specific 5-year survival of patients with primary melanoma, especially for the high-risk melanomas (thickness >or=2.0 mm) with the survival rate reducing from 93% for patients with strong nuclear ING3 staining in their tumor biopsies to 44% for those with negative-to-moderate nuclear ING3 staining (P=0.004). Strikingly, our multivariate Cox regression analysis revealed that reduced nuclear ING3 expression is an independent prognostic factor to predict patient outcome in primary melanomas (P=0.038). Our data indicate that ING3 may be an important marker for human melanoma progression and prognosis as well as a potential therapeutic target.

Cyclooxygenase-2 (COX-2): first immunohistochemical marker distinguishing early cutaneous melanomas from benign melanocytic skin tumours

We have reported recently that changes in expression level of COX-2 are correlated with development and progression of human melanoma. In this study, we investigated whether the COX-2 expression level might be a useful immunohistochemical marker for distinguishing cutaneous melanomas from benign melanocytic lesions. Up to now, immunohistochemical markers have not ensured satisfactory sensitivity and specificity of differential pathologic diagnosis of melanoma. The expression of COX-2 was determined immunohistochemically in formalin-fixed, paraffin-embedded specimens of 33 early Clark I/II melanomas and 58 naevi. Mean COX-2 expression in melanomas was significantly stronger than in naevi (P approximately 10(-13)). A simple diagnostic algorithm using threshold values of the COX-2 expression level allows for differentiation between early melanomas and naevi with high sensitivity (Se) and specificity (Sp) (for Se between 91 and 100%, Sp values change between 96.5 and 51.7%). Areas under the receiver operating characteristic curves were, respectively, 0.97+/-0.02 and 0.86+/-0.04 for the COX-2 expression in central and border regions of the lesions. For all the melanomas (not only the early ones),the respective areas under the ROC curve values were 0.98+/-0.01 and 0.97+/-0.02. In conclusion, COX-2 is the first immunohistochemical marker that allows the distinguishing of early melanomas from benign melanocytic lesions with both high sensitivity and specificity.

Expression of lumican, a small leucine-rich proteoglycan with antitumour activity, in human malignant melanoma

The family of small leucine-rich proteoglycans (SLRPs), which includes decorin, lumican, biglycan and fibromodulin, constitutes an abundant component of the skin extracellular matrix. We previously demonstrated that human lumican inhibits melanoma growth and progression in a mouse experimental model, by regulating cell migration, proliferation and apoptosis. The aim of this study was to investigate the expression of lumican and decorin in human malignant melanoma and adjacent peritumoral tissue, to understand better their role in the control of growth and invasion of human melanoma. Expression of both proteoglycans was studied by immunohistochemistry using specific antibodies in 34 malignant melanomas, 12 Hutchinson's melanotic freckles and 4 cutaneous metastatic melanomas. We showed that lumican and decorin are located in the dermis and in the peritumoral stroma of malignant melanoma, but are not found in melanoma cells or dense tumour tissue. In the healthy dermis, distant from the tumour, the increasing ratio of lumican to decorin was inversely correlated with the proliferation of the tumour cells (P = 0.035). The comparison of the level of expression of lumican protein in superficial vs. nodular subtypes of malignant melanomas showed a decrease of lumican but not decorin in the peritumoral stroma of nodular subtypes. In the peritumoral stroma, the level of expression of lumican but not decorin decreased significantly (P = 0.016) with increasing Clark levels. In addition, immunocytochemical and reverse transcription PCR analyses of malignant melanoma cell lines (A-375, HT-144) and of MRC-5 and dermal fibroblasts from healthy donors in vitro confirmed that dermal fibroblasts are responsible for lumican and decorin synthesis in skin. CONCLUSIONS. Lumican may regulate vertical progression of human malignant melanoma, but further study is necessary to clarify the antitumour mechanism and the downstream signal transduction pathways involved.

Quantitative Analysis of Melanocytic Tissue Array Reveals Inverse Correlation between Activator Protein-2α and Protease-Activated Receptor-1 Expression during Melanoma Progression

The identification of molecular markers of melanoma progression is needed to more accurately stage and identify treatments for patients with malignant melanoma. Previously, we demonstrated that loss of the activator protein-2alpha (AP-2alpha) expression results in overexpression of the protease-activated receptor-1 (PAR-1) in human melanoma cell lines. Here, we used a tissue microarray platform that consisted of 64 melanocytic lesions, including dysplastic nevi (N=21), primary melanoma (N=20), and metastatic melanoma (N=23). We analyzed the expression of AP-2 and PAR-1 simultaneously by immunofluorescent microscopy with an automated quantification laser scanning cytometer. AP-2 was highly expressed in normal cutaneous melanocytes and dysplastic nevi but not in melanoma metastases. We observed a significantly higher number of AP-2-positive cells in the dysplastic nevi (P=0.0013) and primary melanoma (P=0.0023) compared to the metastatic melanoma. In contrast, we observed a significantly higher percentage of PAR-1-positive cells in the metastatic melanoma compared to dysplastic nevi (P=0.0072) and primary melanoma (P=0.0138). Increased expression of PAR-1 in metastatic melanomas contributes to tumor progression by modulating expression of genes, such as IL-8, matrix metalloproteinase-2, vascular endothelial growth factor, platelet-derived growth factor, and integrins. These findings support our hypothesis that loss of AP-2 is a crucial event in the progression of human melanoma and contributes to the acquisition of the metastatic phenotype via upregulation of PAR-1.

Valproic acid induces apoptosis, p16INK4A upregulation and sensitization to chemotherapy in human melanoma cells

It is known that melanoma develops as a consequence of multifactorial alterations. To date several studies indicate the effective implication of p16 as a tumor suppressor gene with a major role in either the development or progression of human melanoma. Deregulation of melanoma cell growth has been widely associated with mutations in the p16-cyclin D/cdk4-pRb pathway. Recently anticancer therapies are focused on restoration of p16 CDK inhibitory function and other proteins unregulated in melanoma cell cycle pathway (e.g., c-myc, p27). A combined strategy for restoration of normal homeostasis in the melanoma skin with targeted delivery of apoptosis-inducing agents does not seems to be far obtain. New class of antitumoral agents are emerging: histone deacetylase (HDAC) inhibitors have attracted much interest because of their ability to arrest cell growth, induce cell differentiation, and in some cases, induce apoptosis of cancer cells. Recently, attention has been focused on the ability of HDAC inhibitors to induce perturbation in cell cycle regulatory protein (e.g., p21CIP1) and down-regulation of survival signalling pathway. In the present study, we have examined the effect of valproic acid (VPA) on M14 human melanoma cell line. Here we observed that VPA induces cell cycle arrest and apoptosis sensitising melanoma cells to cis-platin and etoposide treatment. IC50 dose (2,99 mM) of VPA was able to induce G1 arrest (up to 75 %) in association with up-regulation of p16, p21 and cyclin-D1 related to Rb ipo-phosphorilation. In addition VPA activated apoptosis (50 %) in M14 cells, when given alone or in combination with antitumoral agents. The ability of valproic acid to re-established the G1 pathway in melanoma cells suggests a potential application of VPA in melanoma therapeutic protocols.

Molecular spectrum of pigmented skin lesions: from nevus to melanoma

Melanoma develops through a series of architectural and phenotypically distinct stages and becomes progressively aggressive, culminating in a metastatic disease. Considerable progress has been made in understanding the biological, pathological and immunological aspects of human melanoma progression. However, the diagnosis, prognosis and treatment of melanoma remain challenging tasks owing to the heterogeneous and complex nature of the molecular aberrations. By incorporating new molecular technology, key genes, involving signal pathways of malignant transformation and progression, continue to be uncovered. This revolutionizes current concepts of melanoma etiology and pathogenesis and introduces the most current diagnostic and prognostic techniques, as well as potential therapeutic approaches. A significant breakthrough is the discovery of the key role of the BRAF mutation in melanoma development and progression, including the most important signal pathway during melanoma development, the MAPK pathway. Addi...

Prognostic Significance of Nuclear Factor-κB p105/p50 in Human Melanoma and Its Role in Cell Migration

Transcriptional factor nuclear factor-kappaB (NF-kappaB) family has been shown to play an important role in tumor pathogenesis and serve as a potential target in cancer therapy. However, it is necessary to clarify the specific functions of NF-kappaB members, which would provide the basis for the selective blockade and reduction of therapeutic side effects resulting from unspecific inhibition of NF-kappaB members. In this study, we explored the role of NF-kappaB p105/p50 in melanoma pathogenesis in vitro and in vivo. We found that the expression of NF-kappaB p105/p50 significantly increased in dysplastic nevi, primary melanoma, and metastatic melanoma compared with normal nevi (P = 0.0004, chi(2) test). Furthermore, NF-kappaB p105/p50 nuclear staining increased with melanoma progression and strong NF-kappaB p105/p50 nuclear staining was inversely correlated with disease-specific 5-year survival of patients with tumor thickness >2.0 mm (P = 0.014, log-rank test). Multivariate Cox regression analysis revealed that nuclear expression of NF-kappaB p105/p50 is an independent prognostic factor in this subgroup. Moreover, we found that up-regulation of NF-kappaB p50 enhanced melanoma cell migration, whereas small interfering RNA knockdown inhibited cell migration. In addition, overexpression of NF-kappaB p50 induced RhoA activity and Rock-mediated formation of stress fiber in melanoma cells. Taken together, our data indicate that NF-kappaB p105/p50 may be an important marker for human melanoma progression and prognosis as well as a potentially selective therapeutic target.

Expression of cyclooxygenase-2 in benign naevi and during human cutaneous melanoma progression

Cyclooxygenase-2 (COX-2) is an enzyme that plays an important role in the production of prostaglandins. Numerous studies have demonstrated increased levels of COX-2 in human cancers of different types. It is thought that COX-2 may be involved in the development and progression of malignant tumours. However, data on the changes in COX-2 expression during the development and progression of human melanoma are relatively limited. Moreover, the results reported by different groups disagree to a large extent. The aim of this work was to evaluate whether COX-2 protein might be considered a potential molecular marker of melanoma progression. The expression of COX-2 was determined immunohistochemically in formalin-fixed, paraffin-embedded specimens of 64 human melanocytic skin tumours (17 naevi, 36 primary cutaneous melanomas and 11 lymph node melanoma metastases, with six pairs of primary and metastatic lesions obtained from the same patients). It was found that the expression level of COX-2 was dependent on both the stage and histopathological type of the melanoma. Collectively, our data indicate that changes in the expression level of COX-2 are correlated with the development and progression of human melanoma, and imply that the COX-2 protein may be considered a potential prognostic and predictive marker in malignant melanoma.

Apaf‐1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf-1, an important mediator of mitochondrial-dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf-1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf-1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf-1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf-1 mRNA by fluorescence in situ hybridization and of Apaf-1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf-1. A significant decrease in Apaf-1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf-1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf-1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf-1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf-1 may be an important contributory factor in the development of the disease.

Activation of Notch1 signaling is required for -catenin-mediated human primary melanoma progression

Notch is a highly conserved transmembrane receptor that determines cell fate. Notch signaling denotes cleavage of the Notch intracellular domain, its translocation to the nucleus, and subsequent activation of target gene transcription. Involvement of Notch signaling in several cancers is well known, but its role in melanoma remains poorly characterized. Here we show that the Notch1 pathway is activated in human melanoma. Blocking Notch signaling suppressed whereas constitutive activation of the Notch1 pathway enhanced primary melanoma cell growth both in vitro and in vivo yet had little effect on metastatic melanoma cells. Activation of Notch1 signaling enabled primary melanoma cells to gain metastatic capability. Furthermore, the oncogenic effect of Notch1 on primary melanoma cells was mediated by beta-catenin, which was upregulated following Notch1 activation. Inhibiting beta-catenin expression reversed Notch1-enhanced tumor growth and metastasis. Our data therefore suggest a beta-catenin-dependent, stage-specific role for Notch1 signaling in promoting the progression of primary melanoma.

Tumour-associated macrophage infiltration, neovascularization and aggressiveness in malignant melanoma: role of monocyte chemotactic protein-1 and vascular endothelial growth factor-A

The objective of this study was to evaluate the role of tumour-associated macrophages (TAMs) in malignant melanoma progression, invasion and angiogenesis. We examined the levels of macrophage infiltration and monocyte chemotactic protein-1 (MCP-1), neovascularization and vascular endothelial growth factor-A (VEGF-A) in different Clark's level melanomas with varying thicknesses and metastases. The level of TAM density was significantly higher in thick (>0.75 mm) than thin (<or=0.75 mm) melanomas, and positively correlated with melanoma invasiveness and metastasis. In contrast, MCP-1 expression was significantly lower in thick (>0.75 mm) than thin (<or=0.75 mm) melanomas and negatively correlated with melanoma aggressiveness and invasion. We did not observe any significant difference in the levels of neovascularization between thin and thick melanomas, and no correlation with VEGF-A expression, TAM density or melanoma aggressiveness and invasion. Interestingly, levels of VEGF-A were significantly higher in metastatic melanoma than in thick melanoma. In addition, we observed lower levels of MCP-1 messenger RNA (mRNA) expression in more aggressive melanomas and in cell lines with higher metastatic potential. In summary, our data suggest a distinct pattern of TAM infiltration, MCP-1 expression, neovascularization and VEGF-A expression during human melanoma progression, and a complex interaction between TAMs and melanoma cells in the regulation of melanoma progression, angiogenesis and metastasis.