microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C

Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non-coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR-214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well-established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR-214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR-214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.     

Expression of CD40, CD44, bcl-2 antigens and rate of cell proliferation on fine needle aspirates from metastatic melanoma

The clinical behaviour of melanoma is often unpredictable using clinical and histological criteria. Tumour cell markers related to cell cycle regulation, apoptosis, cell-cell interactions and cell proliferation might improve the possibility of predicting the clinical course of melanoma. The aim of the present study was to refine prognostic criteria by an immunocytochemical investigation of CD44, CD40, bcl-2 antigens and cell proliferation in tumour cells aspirated from metastases of malignant melanoma. CD40 is a cell surface receptor shown to be expressed by lymphomas as well as carcinomas, and is thought to play a central role in the process of tumour progression. CD44 is a transmembrane glycoprotein, which is involved in growth signal transmission of importance in the binding of tumour cells to endothelium, cell migration and enhancement of cell motility, which makes it of interest to study in relation to the metastasizing capacity of tumours. The bcl-2 protein is active in the process of programmed cell death (apoptosis) as an antiapoptotic agent and its expression may reflect tumour progression. Mean/median percentages of tumour cell positivity were 8.5/3.0 for CD40, 76.1/86.3 for CD44 and 7.4/3.3 for bcl-2. A significant correlation was observed between expression of apoptosis-associated bcl-2 antigen and overall survival (r = 0.33). The CD44 positive cell fraction was higher in patients with short overall survival than those with long survival but this difference was not statistically significant. The expression of CD40 did not correlate with overall survival. The mean/median proliferation fraction assessed by MIB-1 monoclonal antibody was 25.8/23.9 and showed a significant correlation with survival after diagnosis of melanoma metastasis (r = 0.32). Lack of bcl-2 expression and a high proportion of tumour cells expressing Ki-67 antigen are predictors of poor prognosis that are independent of the traditionally accepted Breslow's thickness of the primary melanomas.     

The role of CAF derived exosomal microRNAs in the tumour microenvironment of melanoma

Exosomes play a crucial role in the crosstalk between cancer associated fibroblasts (CAFs) and cancer cells, contributing to carcinogenesis and the tumour microenvironment. Recent studies have revealed that CAFs, normal fibroblasts and cancer cells all secrete exosomes that contain miRNA, establishing a cell-cell communication network within the tumour microenvironment. For example, miRNA dysregulation in melanoma has been shown to promote CAF activation via induction of epithelial-mesenchymal transition (EMT), which in turn alters the secretory phenotype of CAFs in the stroma. This review assesses the roles of melanoma exosomal miRNAs in CAF formation and how CAF exosome-mediated feedback signalling to melanoma lead to tumour progression and metastasis. Moreover, efforts to exploit exosomal miRNA-mediated network communication between tumour cells and their microenvironment, and their potential as prognostic biomarkers or novel therapeutic targets in melanoma will also be considered.     

The multifaceted role of CD146/MCAM in the promotion of melanoma progression

Human malignant melanoma is a common primary malignant cutaneous tumour derived from transformed epidermal melanocytes. Patients with melanoma have a high rate of mortality due to resistance to chemotherapeutic drugs, a major obstacle to a successful treatment. Several reports have suggested that CD146 plays an important role as a signalling molecule in human melanoma. This role includes CD146 as a participant in inflammation, differentiation, adhesion, tumourigenicity, metastasis, invasion and angiogenesis among other processes, which suggests that this molecule promotes the progression of human melanoma as a multifaceted regulator. In this article, we explore the effects and corresponding mechanisms with respect to the role of CD146/MUC18 in the promotion of human melanoma progression. Collectively, the studies indicated that targeting CD146, because it is a suitable marker of poor patient outcome, might be useful in the design of future strategies for the prevention and treatment of human melanoma.     

Differential LEF1 and TCF4 expression is involved in melanoma cell phenotype switching

Recent observations suggest that melanoma cells drive disease progression by switching back and forth between phenotypic states of proliferation and invasion. Phenotype switching has been linked to changes in Wnt signalling, and we therefore looked for cell phenotype-specific differences in the levels and activity of β-catenin and its LEF/TCF co-factors. We found that while cytosolic β-catenin distribution is phenotype-specific (membrane-associated in proliferative cells and cytosolic in invasive cells), its nuclear distribution and activity is not. Instead, the expression patterns of two β-catenin co-factors, LEF1 and TCF4, are both phenotype-specific and inversely correlated. LEF1 is preferentially expressed by differentiated/proliferative phenotype cells and TCF4 by dedifferentiated/invasive phenotype cells. Knock-down experiments confirmed that these co-factors are important for the phenotype-specific expression of M-MITF, WNT5A and other genes and that LEF1 suppresses TCF4 expression independently of β-catenin. Our data show that melanoma cell phenotype switching behaviour is regulated by differential LEF1/TCF4 activity.     

Systematic classification of melanoma cells by phenotype-specific gene expression mapping

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.     

Role of SOCS-1 Gene on Melanoma Cell Growth and Tumor Development

Melanoma is the most aggressive form of skin cancer, and its incidence has increased dramatically over the years. The murine B16F10 melanoma in syngeneic C57Bl/6 mice has been used as a highly aggressive model to investigate tumor development. Presently, we demonstrate in the B16F10-Nex2 subclone that silencing of SOCS-1, a negative regulator of Jak/Stat pathway, leads to reversal of the tumorigenic phenotype and inhibition of melanoma cell metastasis. SOCS-1 silencing with short hairpin RNA affected tumor growth and cell cycle regulation with arrest at the S phase with large-sized nuclei, reduced cell motility, and decreased melanoma cell invasion through Matrigel. A clonogenic assay showed that SOCS-1 acted as a modulator of resistance to anoikis. In addition, downregulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptor. In vivo, silencing of SOCS-1 inhibited subcutaneous tumor growth and metastatic development in the lungs. Because SOCS-1 is expressed in most melanoma cell lines and bears a relation with tumor invasion, thickness, and stage of disease, the present results on the effects of SOCS-1 silencing in melanoma suggest that this regulating protein can be a target of cancer therapy.     

Expression of the alpha7beta1 laminin receptor suppresses melanoma growth and metastatic potential.

The alpha7beta1 integrin is a laminin-binding receptor that was originally identified in melanoma. Here, we show that, in clonally derived mouse K1735 melanoma variant cell lines with high (M-2) and low (C-23) metastatic potential, elevated expression of alpha7 correlates with reduced cell motility, metastasis, and tumor growth. Both cell lines showed similar beta1 integrin-dependent adhesion to laminin-1 and the E8 laminin fragment. However, the highly metastatic M-2 cells rapidly migrated on laminin, whereas the nonmetastatic C-23 cells were minimally motile. Laminin-binding integrin profiles showed that the M-2 cells expressed moderate amounts of alpha1 and abundant alpha6 but low or undetectable levels of alpha2 and alpha7. By contrast, C-23 cells expressed low or undetectable levels of alpha1, alpha2, and alpha6 but had up-regulated levels of alpha7. Consistent with the protein data, Northern blot analysis showed that levels of alpha7 mRNA were highest in the poorly metastatic variant cells, whereas alpha6 message was not detected; in contrast, alpha6 mRNA was elevated in the highly metastatic cells, whereas alpha7 message was not detected. Forced expression of alpha7 in the M-2 cells suppressed cell motility, tumor growth, and metastasis. Collectively, these results indicate that, during melanoma progression, acquisition of a highly tumorigenic and metastatic melanoma phenotype is associated with loss of the alpha7beta1 laminin receptor.     

Overexpression of collagenase 1 (MMP-1) is mediated by the ERK pathway in invasive melanoma cells: role of BRAF mutation and fibroblast growth factor signaling

Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.     

Heterotypic signaling between dermal fibroblasts and melanoma cells induces phenotypic plasticity and proteome rearrangement in malignant cells

The signaling events triggered by soluble mediators released from both transformed and stromal cells shape the phenotype of tumoral cells and have significant implications in cancer development and progression. In this study we performed an in vitro heterotypic signaling assays by evaluating the proteome diversity of human dermal fibroblasts after stimulation with the conditioned media obtained from malignant melanoma cells. In addition, we also evaluated the changes in the proteome of melanoma cells after stimulation with their own conditioned media as well as with the conditioned medium from melanoma-stimulated fibroblasts. Our results revealed a clear rearrangement in the proteome of stromal and malignant cells upon crosstalk of soluble mediators. The main proteome signature of fibroblasts stimulated with melanoma conditioned medium was related to protein synthesis, which indicates that this process might be an early response of stromal cells. In addition, the conditioned medium derived from ‘primed’ stromal cells (melanoma-stimulated fibroblasts) was more effective in altering the functional phenotype (cell migration) of malignant cells than the conditioned medium from non-stimulated fibroblasts. Collectively, self- and cross-stimulation may play a key role in shaping the tumor microenvironment and enable tumoral cells to succeed in the process of melanoma progression and metastasis. Although the proteome landscape of cells participating in such a heterotypic signaling represents a snapshot of a highly dynamic state, understanding the diversity of proteins and enriched biological pathways resulting from stimulated cell states may allow for targeting specific cell regulatory motifs involved in melanoma progression and metastasis.     

Tenascin-C Signaling in melanoma

Tenascin-C (TNC), a multifunctional matricellular glyco-protein, is highly expressed in the majority of melanoma cell lines and has been implicated in the progression of melanoma. A growing body of evidence has implicated the role of TNC in the process of invasion and metastasis for melanoma. However, the mechanism and individual signaling pathways by which TNC drives melanoma progression have not been illuminated. Herein we provide perspectives from the investigation of TNC in other settings that may hint at the mechanistic role of TNC in this disease.