Main integrins found altered in metastatic melanomas
Metastasis is one of the most important parameters that affect cancer patient’s prognosis. It is associated with resistance to treatments, high recurrence rates and poor cancer patient survival rates . Although melanoma is an aggressive type of skin cancer that carries significant morbidity and mortality, the prognosis for patients with localized tumor is quite good, the aggravation being the metastatic capacity of the tumor. When the process of metastasis takes place, the prognosis is generally very poor, with the metastatic melanoma being rarely curable [2-3].
The establishment of metastatic lesions is a complex process and requires a series of sequential steps, each of which is rate limiting, and although substantial progress has been made in understanding the molecular mechanisms of metastasis, new data suggest this process is perhaps even more complicated than originally suspected . Another issue in defining the factors involved in the melanoma metastasis is that melanoma is highly heterogeneous and for this its classification and staging is until today a challenge for specialists . Because of that, there is still a lot unclear understanding about the progression of the disease, making it difficult to ensure which molecular pathways are involved in each step of the disease development. Therefore, for the development of new prognostic and therapeutic targets of melanoma metastasis, it is important to elucidate which pathways are involved specifically in the progression of the localized tumor to its metastasis . For that, a deep review of the literature, analysing carefully which pathologic classification each study considered, may clarify the molecular alterations involved specifically in the most aggressive step in melanoma progression.
Signaling pathways, such as the canonical Wnt pathway, the c-kit receptor and the MITF transcription fator are among pathways found altered during melanoma . The reactivation of melanocyte-specific programs seems to contribute, in combination with other oncogenic changes, with melanoma aggressiveness. Others pathways that are classically altered in melanoma metastasis are the MAPK and PI3K pathways, with gain-of-function mutations leading to melanoma metastasis progression .
Besides these pathways that are consistently involved in the metastasis of melanoma, there are some other potentially altered pathways in this process. For example, it is known that the tumor microenvironment has an important role in determining melanoma progression to metastasis , and in this scope the importance of metalloproteinases is being highlighted. In addition, numerous studies report modulation of the metastatic properties of melanoma cells by microRNAs, suggesting this as a common occurrence. MicroRNAs regulate tumor suppressor genes and oncogenes, each miRNA being able to regulate a great number of genes. Besides, due to its stable and lineage-specific expression nature, microRNAs are attractive candidate as biomarkers and therapeutic targets .
Although there are a great number of studies trying to elucidate the molecular mechanisms of melanoma progression, the process of melanoma metastasis is still unclear. Finding core mediators of different processes in metastasis is a formidable challenge, and may provide opportunities for developing new prevention and treatment strategies. This chapter will discuss basic concepts of malignant melanoma metastasis, focusing on the pathways that the literature indicate as consistently altered in this process and will describe how the host's environment influences the biological behavior of metastatic cells.
2. Melanoma metastasis
Cutaneous melanoma is the most aggressive type of skin cancer, and the high mortality rates worldwide caused by this disease is due to its great ability to form metastases and resist to current therapies [8,9,10]. Genetic and epigenetic alterations contribute to the development of cells able to invade and metastasize . These changes ensure the features that allow cells to modulate the microenvironment, and change their interactions with the extracellular matrix and other cells . Although our knowledgement about the molecular pathogenesis of melanoma has increased, the molecular changes occuring during the malignant transformation of melanocytes are not very well described.
3. Adhesion molecules
One of the main characteristics of metastatic melanoma is cell heterogeneity . This characteristic provides the cell ability to invade and colonize different tissues . Local invasion and metastasis processes are responsible for the morbidity and mortality associated with melanoma. The development of invasive cells occurs in the vertical growth phase (VGP) when melanoma cells are able to penetrate the basement membrane, grow in the dermis and metastasize . Moreover, these cells have many cytogenetic abnormalities, suggesting a significant genomic instability . The development of metastatic melanoma from primary VGP melanomas occurs when these cells dissociate from the primary tumor, migrate through the adjacent stroma and invade the lymphatic and/or blood vessels to form tumors at distant sites . The invasion and migration of melanoma are related to changes in cell adhesion. Typically, cell adhesion controls cell migration, organization, organogenesis and tissue architecture . Disturbances in adhesion contribute to tumor invasion, tumor-stroma interactions and signaling between tumor cells and normal cells . There are numerous cell adhesion molecules, which are sub-grouped based on their structural similarity and are categorized as integrins, cadherins, immunoglobulin superfamily or selectins. The expression of these molecules is influenced by the environment, microenvironment and genetic/epigenetic factors . Therefore, determining changes in the expression of these molecules during metastasis may help to define future therapeutic targets. Several adhesion molecules have been reported to play a role in melanoma progression.
As previously stated, the extracellular matrix (ECM) surrounding the cell provides a physical support for the cell adhesion. The anchorage of cell in the ECM is not just structural, considering that this binding stimulates signal transduction cascades that mediate signaling required for the proliferation, migration, differentiation and cell survival . The type of apoptosis triggered by the loss of anchorage is called "
Immunoglobulin superfamily adhesion molecules are cell surface glycoproteins that express a variable number of loops in its extracellular domain. Most of these molecules has a transmembrane domain but is linked to the cell surface only by a glycophosphatidilinositol anchor . Heterophilic interactions with members of the immunoglobulin superfamily, integrins, cadherins and extracellular matrix components may occur, as well as homophilic interactions, which are essential in Ca2+-dependent cell adhesion [36-37]. The family of immunoglobulins has an important relevance in the pathophysiology of melanoma, among which we can highlight the CD54 (ICAM), CD66 (CEACAM), CD146 (MCAM or Mel-CAM) and CD166 (ALCAM). These molecules appear to be important in the melanoma progression, although many of their functions are still uncertain. Through literature review, it is possible to note their participation in advanced cases of melanoma and metastatic disease [14, 38]. Particularly, CD54 (ICAM) has been associated with melanoma progression and risk of metastasis . Its expression is evident in melanoma when compared with nevi [26, 40-42]. The CD66 (CEACAM) is a glycoprotein surface molecule involved in intercellular adhesion and associated with diverse cellular functions that regulate growth and differentiation and play an important role in insulin homeostasis, vasculogenesis and immunomodulation. Furthermore, it implies in many intracellular signaling mediated processes involved in the growth and differentiation of tumor cells, performing thus a key role in the modulation of many types of cancer. A strong correlation between the expression of this molecule in primary tumors and subsequent development of metastatic disease was observed. An apparently gradual increase in CD66 expression in cutaneous melanocytic lesions in more advanced stages of neoplastic progression was observed, indicating that CD66 may play an important role in the development and progression of melanoma. Furthermore, this molecule interacts with integrins (especially with beta-3 subunit) and this interaction appears to be important in cell migration and metastasis . The CD146 molecule was first identified as a cell adhesion molecule specific for melanoma and capable of providing homologous and heterologous interactions between neoplastic melanocytes and endothelial cells in a calcium-independent manner . Also known as MELCAM, MCAM and MUC18 , CD146 plays a pro-migratory key role in the vascular system, normal development and tumor progression, displaying overexpression in many tumors, including melanoma, prostate cancer and cancer breast [45-47]. The expression of CD146 in melanocytes, nevi and melanoma cells from radial growth phase is environmentally regulated through direct contact cell-cell with keratinocytes, but the mechanisms of this regulation are not well-established . Moreover, in melanoma progression, the expression of this molecule increases gradually and reaches its peak in metastatic disease [14, 20, 49]. It is reported that CD146 displays strictly correlation with cadherins and experimental situations where an increased expression of cadherins is stimulated, CD146 levels return to normal levels . The CD166, also named ALCAM, was first identified on activated leukocytes, hematopoietic stem cells and myeloid progenitors. Furthermore, it is possible to observe its expression in neuronal cells, mesenchymal stem cells, stromal cells from bone marrow, but also in cultured metastatic melanoma cells . Like other adhesion molecules, CD166 shows homophilic and heterophilic interactions and has represented a marker of metastatic development similarly to beta-3 integrin, since its expression reaches peak levels in metastatic melanoma cells .
Another major family of adhesion molecules found to be altered in metastatic melanoma is the cadherin family. Cadherins are calcium-dependent cell adhesion molecules with important functions in the formation and maintenance of normal tissue architecture . Cadherins are a superfamily of at least 30 different molecules, whose expression is temporally and spatially controlled. Classic cadherins are divided into three subtypes: N (neural), E (epithelial) and P (placental) . The E-cadherin molecule, which is expressed by epithelial cells, is the one most frequently altered in tumors. Different studies have shown that E-cadherin is frequently inactivated in the development of human carcinomas, including carcinomas of breast, colon, prostate, stomach, liver, esophagus, skin, kidney and lung and is associated with invasion and metastases formation in lymph nodes [53-54]. The inhibition of the E-cadherin function may occur by several mechanisms, including mutation or deletion of
4. Matrix metalloproteinases
Proteolytic enzymes, by their ability to degrade ECM proteins, become important components in the process of tumor progression. With the sequencing of the human genome, more than 500 genes were identified as encoding proteases or protease-like proteins, with a large number being associated with the tumor process . Among these, the matrix metalloproteinases (MMPs)-a group of 24 enzymes that degrade ECM components and the basal membrane -have been the focus of much research on cancer [63-64]. This family of glycoproteins is secreted as a latent pro-enzyme, activated by proteolysis of a conserved region present in the N-terminal domain and is divided into six groups depending on the type of substrate that degrades: collagenases, gelatinases, stromelysins and extracellular matrix metalloproteinases . MMPs are regulated both at transcriptional and post-transcriptional level. These regulation mechanisms operate to ensure their coordinated expression . MMPs activity occurs only where proteolysis is required. Cytokines and peptide factors, such as interleukin (IL)-4 and IL-10, growth factors (transforming growth factor (TGF-α), basic fibroblast growth factor (bFGF), and TGF-beta1, induce the expression of different members of MMPs family . However, malignant tumors have strategies that avoid these regulatory mechanisms and provide proteolytic activity that accompanies cancer development and metastasis . Increased MMP activity is associated with the stages of tumor invasion and metastasis and is frequently overexpressed in different cancers. Upon activation, MMP activity is modulated by common endopeptidase inhibitors, such as α2-macroglobulins (present in plasma and tissue fluids) and, more specifically, by the inhibitors of matrix metalloproteases (TIMPs) [63-65]. MMPs mediate the extracellular matrix and basement membrane degradation during early stages of tumorigenesis process, contributing to the formation of a microenvironment that promotes tumor growth. MMPs also participate in later stages of cancer development, promoting the sustained growth of both primary tumors and metastases by activating growth factors, inactivating protein binding to growth factors or releasing mitogenic molecules residing on the extracellular matrix. One of the first steps in the carcinoma invasion is the disruption of the basement membrane and subsequent migration through extracellular matrix . The basement membrane is composed of molecules such as laminin, type IV collagen and proteoglycans containing heparan sulfate . More recently, it was shown that laminin-5 (molecule present in basement membranes of epithelium) is associated with the control of melanoma cells migratory phenotype . The cleavage of laminin-5 by MMP-2 or MMP-14 shows a cryptic site of laminin that triggers cell motility . This laminin-5 cleaved form is found in experimental tumors. In human cancers, MMP-14 co-localizes with laminin-5, which suggests that it is associated with microinvasive cancers . E-cadherin is cleaved by MMP-3 or 7, and the release of E-cadherin promotes the invasion of tumor cells in a paracrine manner
5. Tissue inhibitors of metalloproteinases
Inhibitors of matrix metalloproteinases have been identified in various species, such as
6. Pathways classically involved in melanoma and their involvement in melanoma metastasis
The MAPK pathway is an important intracellular signal transduction pathway, which regulates cellular proliferation, differentiation, gene expression, cell survival and apoptosis . This pathway is activated by different factors through different receptors, as tyrosine kinases and G-protein-coupled receptors. The activation of those membrane receptors promotes RAS activation, which activates several effector proteins, as the ones in RAF family. RAF then activate the kinase cascade (MEK1/2 and ERK1/2), which can phosphorylate nuclear and cytoplasmic substrates involved in several cellular processes .
The most mutated gene in this pathway is the
Since the first report by Davies and colleagues in 2002, several studies have confirmed that activating BRAF mutations are present in approximately 60% of melanoma, and over 90% of them are BRAFV600E .
It is predicted that the constitutive activation of the MAPK pathway can lead to oncogenic transformation of cells by promoting cell proliferation, invasion, metastasis, migration, survival and angiogenesis . Specifically, MAPK is predicted to mediate melanoma metastasis by inducing proteolytic enzymes, as MMPs, which leads to degradation of basement membrane, and by regulating genes involved in cell migration, cell survival, and growth . However, this has been poorly studied.
In 2012, Colombino and colleagues analyzed primary and metastatic melanoma samples and found that overall 43% of primary melanomas have mutated BRAF with no significant frequency increase in metastatic lesions . However, Shinozaki and collaborators in 2004 analyzed the BRAF mutation in tumor specimens and showed that this mutation is more frequent in melanoma metastasis (57%) than in primary melanomas (31%), which suggests that it can be acquired during metastasis. They also analyzed 13 pairs of primary melanomas with their respective metastases. Four pairs were not mutated in the primary or the metastatic tumor, other four pairs were mutated in both stages and five pairs (38%) present the wild type gene in the primary tumors and the mutated one in the metastatic tumors. This suggests that BRAF is not a key factor for the development of metastasis, although it can be acquired during this process. However, the frequency of mutation found in the study was lower than the report in the literature .
Another report that analyzed metastatic tumors in an Australian cohort demonstrated that 48% of the patients had a BRAF mutation, with 74% being the BRAFV600E. Interestingly, the presence of the BRAF mutation led to a poorer survival unless patients were treated with BRAF inhibitor .
Another study shows that inhibition of BRAFV600E expression causes a significant decrease in the metastatic ability of the metastatic cell lines Lu1205 and UACC 903M. This impaired ability to metastasize was due the reduction of cell extravasation through the endothelium, process mediated by IL-8 and ICAM . Mutated BRAF can also participate of metastasis development by the generation of new blood vessels by promoting macrophage inhibitory cytokine-1 (MIC-1) and vascular endothelial growth factors (VEGF) secretion . MIC-1 was shown to be upregulated in metastatic melanoma cell lines and patient biopsies in comparison with melanocytes. A trend was also observed in comparison with primary melanomas . A RNA interference targeting BRAFV600E blocks melanoma cell invasion
Mutation in BRAF can also activate ERK1/2, which was seen activated by Jorgensen and collaborators in 54% of primary melanomas and 33% of metastatic tumors . However, Mirmohammadsadegh and collaborators analyzed the expression of phosphorylated ERK1/2 (pERK) in human cells and specimens and saw that it levels was low in melanocytes, upregulated in melanoma cell lines and abundant in melanoma metastasis. Yet, this study did not distinguish between non-metastatic and metastatic cell lines and did not analyze samples of primary tissue .
The successful treatment of metastatic melanomas with BRAFV600E inhibitors  and the events here forehead mentioned suggests that this mutation is involved somehow in the process of metastasis, but the exact mechanisms involved are still unclear .
Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and regulates survival, growth, differentiation and pigmentation of these cells . There are several isoforms of MITF described, but in melanocytes and melanoma there is a predominance of the isoform MITF-M .
The levels of MITF expression are very important to cell fate. High levels of MITF induce cell cycle arrest and differentiation, while low levels promote cell cycle arrest, apoptosis and even invasion and metastasis. If MITF levels are depleted for long time, melanoma can enter quiescence or senescence. Thus, only an intermediate level of MITF favors cell proliferation [1, 90]. Wherefore, levels of MITF have to be tightly regulated during melanocytic development and even during melanoma progression.
Several reports indicate that most human melanomas express MITF , and that MITF is expressed in high levels in benign and primary tumors . MITF is genomically amplified in 10% of primary melanomas and 21% of metastatic melanoma, and the amplification in the metastatic samples correlates with a significant decrease in the 5-year survival rate of patients . However, expression of MITF in metastatic lesion is variable. Beyond the specimens that present MITF amplification and therefore high levels of expression, other metastatic lesions present predominantly downregulation of MITF . A report demonstrated that depletion of MITF expression in mouse and human cells is sufficient to induce experimental lung metastasis . Consistently, high levels of MITF in melanoma patients are associated with low invasiveness and fewer metastases . Because some metastatic lesions present low expression of MITF while others present MITF amplification, it is proposed that MITF can have paradoxical effects in different sub-groups of melanomas. For example, the decrease in MITF expression in metastatic melanomas can be beneficial for tumor growth because it reduces pigmentation, and therefore the cellular energy utilized for pigment production . However, in specimens with high expression of MITF, it can regulate c-Met expression, which upregulation appears to have a functional role in metastatization of melanoma .
Because MITF is part of a complex pathway, alterations in factors upstream or downstream to MITF can affect melanoma development . However, this has not been properly investigated in metastatic melanoma. Until now, the expression and functional role of MITF in metastatic melanoma is not determined.
The transforming growth factor-β (TGF-β) is a cytokine implies in several cellular processes, as cell proliferation, differentiation and survival. TGF-β interacts with its receptor, leading to the receptor phosphorylation, which then phosphorylates the cytoplasmatic proteins SMAD. The SMADs proteins accumulate in the nucleus and acts as transcription factors. TGF-β can also signal through a non-canonical pathway. In this respect, TGF-β can activate phosphatidylinositol-3-kinase (PI3K) and several mitogen activated protein kinases (MAPKs), leading to cell signaling independent of SMAD .
There are three isoforms of TGF-β in mammalian, TGF-β1, TGF-β2, TGF-β3.
The overexpression of an inhibitory SMAD, SMAD 7, is effective in reducing secretion of MMPs and impairing bone metastasis development. The blockage of TGF-β receptor has the same effect. TGF-β also promotes the epithelial to mesenchymal transition (EMT), an event associated with increase in metastasis, increase in MMP2 and MMP9 expression, and angiogenesis by modulating IL-8, MMPs, VEGF, among other important factors [95, 97].
TGF-β also promotes metastasis through expression induction of the transcription factor GLI2. High expression of GLI2 correlated with increased cell invasiveness and bone metastasis emergence. In a murine model of induced metastasis, cells with elevated expression of GLI2 caused bone metastasis, while cells with low level of this factor have reduced and heterogenic capacity to form metastasis. In addition, the GLI2 knockdown in an aggressive melanoma cell line (1205Lu) dramatically diminishes their ability to form bone metastasis .
6.4. Wnt (β-catenin and WNTs)
β-catenin is an adherent junction protein and a transcriptional coativator. It mediates cell adhesion, proliferation, survival and migration . It participates in the WNT pathway and the interaction between WNT ligands and receptors leads to the accumulation of β-catenin in the nucleus, where it promotes transactivation of target genes .
The role of β-catenin in melanoma it is still unclear. Some show β-catenin is frequently constitutively activated in melanomas and that its accumulation promotes metastasis, while others show its high expression suppresses invasion and indicates a better prognosis for patients [1, 99].
A report shows that β-catenin is an important regulator of melanoma metastasis to lymph nodes and lungs. In this case, β-catenin cooperates with PTEN loss and BRAFV600E to promote primary tumor and metastasis, but the mechanism was not enlightened . In other study, activation of β-catenin signaling increased metastatic potential in NRAS-driven melanoma in mice, although it repressed cell migration, indicating that β-catenin inhibits the initial steps of metastasis, as cell migration, but can be involved in the latest steps of this process .
However, another study that analyzed β-catenin expression by immunohistochemistry in melanocytic samples identified that β-catenin was strongly stained in 96% of melanocytic nevi, in 94% of radial growth phase primary melanoma, in 65% of vertical growth phase primary melanoma and in only 38% of metastatic melanomas . The involvement of β-catenin decrease in melanoma metastasis was also seen indirectly by decrease in WLS, an important component to WNT ligands secretion. The reduction of WLS expression by shRNA in the melanoma cell line A375 caused spontaneous metastasis in the lung of mice. Of 36 animals injected with WLS shRNA cells, 10 developed lung metastasis while none of the 18 animals injected with control shRNA cell presented metastasis. This was due to the inhibition of the WNT/ β-catenin pathway .
Therefore, β-catenin effects on melanoma metastasis are still not fully understood and may depend on cellular context . Some state that WNT signaling via the canonical pathway, which involves β-catenin, is associated with a less metastatic phenotype, while the non-canonical pathway, involving Wnt5A, would be related with increased malignancy . The overexpression of Wnt5A appears to be consistently associated with a more aggressive disease and metastasis development  and this is probably due the upregulation of metastatic markers, as CD44 and Snail, and the promotion of EMT .
Wnt5 activity is regulated by heparan sulfate proteoglycans (HSPGs), glycoproteins that are categorized based on the structure of their glycosaminoglycan (GAG) chains and although has been poorly studied in melanomas, has been recently associated with melanoma metastasis. For example, HSPGs are important to cell signaling through the non-canonical Wnt pathway. The HSPGs syndecans 1 and 4 are necessary to the presentation of Wnt5A to its receptor ROR2 and its consequent internalization and signaling. Cleavage of the syndecans GAGs result in less Wnt5A at the cell surface and a consequent decrease in the cell metastatic potential .
The phosphoinositide-3 kinase (PI3K) pathway is another key signaling cascade that controls cell survival, proliferation and motility. PI3K is activated by receptor tyrosine kinases or RAS leading to the release of second messenger PIP3, which can activate several downstream effectors, as AKT. PTEN (Phosphatase and Tensin Homolog) is an important negative regulator of this pathway .
PI3K pathway is commonly hyperactivated in melanomas. A report that analyzed 68 metastatic melanomas showed that 41% presented a mutation in this pathway . Besides 3% of metastatic melanomas presents activating mutation of PI3K and 5-20% of late-stage melanoma present mutations that leads to PTEN loss, which is the protein most commonly mutated in this pathway . Another study that analyzed PI3K protein expression demonstrated that PI3K expression was low in nevi and high in melanomas. Interestingly this expression was even higher in metastatic specimens .
Inactivation of PTEN by mutation leads to a constitutive activation of PI3K pathway. Because PTEN mutations are rarely found in primary melanomas, it is proposed that the activation of the PI3K pathway may be important to the late events of melanoma progression, as invasion and metastasis . An interesting study demonstrated that mice with PTEN loss and BRAFV600E develop malignant melanoma that is able to metastasize to lymph nodes and distant organs, while mice with only BRAFV600E produced benign melanocytic hyperplasia, showing that PTEN loss cooperate with BRAFV600E to induce metastasis .
AKT is also able to induce tumor cell invasion and metastasis to the lung in a mouse model by inhibiting the small GTPase RhoB . Moreover, a higher percentage of biopsies present strong phospho-AKT (the activated form of AKT) staining in metastatic melanoma than in primary melanomas and nevi and high levels of phospho-AKT are associated with poor patient survival rates (Dai 2005).
7. Other pathways
Telomerase is a ribonucleoprotein complex and its main function is to maintain the telomeric repeats that cap the ends of eukaryotic chromosomes and therefore preserve their integrity by preventing end-to-end fusions . In melanocytic lesions, is seen that nevi have low or even absent telomerase activity, primary melanomas have intermediate levels of activity and metastatic melanomas present increased telomerase activity. Telomerase promoter seems to be frequently mutated in melanoma and two mutational hotspots were found in 85% of melanoma metastases .
The inhibition of the RNA portion of the telomerase, called TER (telomerase RNA), results in severe decrease in metastatic tumor development. In an experiment, mice that received injection of melanoma cells with TER downregulated presented 70% fewer metastases in the lung then mice injected with control cells. The inhibition of TER also led to the downregulation of several genes, including genes involved in transcriptional regulation, cell proliferation and adhesion, chromatin assembly and others. Interestingly, it was also seen a regulation of genes of the glycolytic pathway, as aldolase, suggesting that telomerase can mediate its metastatic properties through activation of glycolysis in cancer .
The analysis of the expression of ACP5 (Tartrate-resistant acid phosphatase 5), a protein involved in bone development , in a melanoma tissue microarray comparing primary and metastatic specimens demonstrated that ACP5 is upregulated in the metastatic lesions. To confirm the role of ACP5 in metastasis, the authors overexpressed ACP5 in a human poorly metastatic melanoma cell line (1205Lu) and observed an increase in the metastatic ability of those cells
Chemokines are secreted chemotactic cytokines that were first identified as modulators of leukocytes trafficking to inflammatory sites. Chemokines realize its biological function through the binding in chemokine receptors, G-protein coupled receptors. It has been seen by several authors that melanoma cells have a high expression of chemokines and that its interaction with its receptors stimulates tumor growth, angiogenesis and metastasis .
The expression of CXCL8, a potent chemokine also known as IL-8, has been associated with tumor angiogenesis, progression and metastasis in some mouse models. In one case, the induction of CXCL8 expression by ultraviolet-B radiation potentiated tumor and metastasis development. In addition, metastatic melanoma cell lines express higher levels of CXCL8 than its non-metastatic variants . Moreover, in an analysis of tumor specimens, it was seen that radial growth tumors do not present CXCL8 expression, while half of the vertical growth tumors present it. A significant increase in CXCL8 expression was also seen in metastatic samples in comparison with thin melanomas . The CXCL8 receptor, CXCR2, was also seen to be involved in melanoma metastasis.
Chemokines receptors also appear to be important to specific organ metastasis. CXCR4 was identified as the most frequent expressed chemokine receptor in liver metastasis of paraffin-embedded tissue, with 89% of the samples expressing it . CCR9 was observed as expressed in a great majority of intestine metastasis of melanoma, but not in other organs .
NEDD9 (neural precursor expressed, developmentally downregulated 9) is an adaptor protein that belong to the Cas family of signal transduction molecules. NEDD9 is genomically amplified in melanomas and its high expression correlates with melanoma progression and metastasis. NEDD9 is frequently overexpressed in human metastatic tumor in comparison to primary ones and its expression enhances invasion
MicroRNAs (miRNAs) are small non-coding RNA Molecules - 17 to 22 nucleotides - that regulate gene expression post-transcriptionally. MiRNAs are negative regulators of gene expression. These regulatory RNAs act by binding to mRNA molecules of specific gene targets and inducing their cleavage or translation repression. Because just approximately seven nucleotides of the miRNA – the so-called seed region - interact with the target mRNA, each miRNA may have a large number of targets. Therefore, a unique miRNA can influence the expression of even hundreds of proteins and, for that, miRNAs participate of several processes in the cell as development, cell proliferation, differentiation and metabolism [116-117].
Because of the critical role miRNAs have in the cell biology, changes in their expression patterns have impact in different disorders, as cancer itself . After miRNAs being related with tumorigenesis, it did not take long until they were specifically related with metastasis. The first microRNA linked with metastasis was described in 2007 by Ma, Weinberg and colleagues. In this paper, the authors demonstrated that microRNA-10b is highly expressed in metastatic breast cancer cells and that its overexpression in non-metastatic breast tumors leads to metastasis [91, 118]. Two years later, in 2009, Hurst termed the miRNAs involved in the metastasis process, metastamiRs. The metastamiRs modulate key signaling pathways and can promote or suppress metastasis .
Recently, miRNAs have been linked with melanoma metastasis, but there is still limited information about it. Most time, there is only one study analyzing the involvement of certain miRNA with melanoma aggressiveness. Here, we will present some of miRNAs that are consistently associated with melanoma metastasis, but are not necessarily defined as metastamiRs.
A miRNA that appears to act as a metastamiR in melanoma is the miR-214. MiR-214 expression was evaluated in the poorly metastatic lineage A375 and in its highly metastatic variant cell lines, originated from several passages
Other metastamiRs which involvement with melanoma has been recently discovered are miR-30b and miR-30d. These clustered miRNAs (miRNAs that are transcribed together) are upregulated in melanoma metastasis samples related to primary melanomas and nevi. Overexpression and underexpression assays
The miR-1908, miR-199a-5p and miR-199a-3p also promote human melanoma metastasis. These miRNAs are overexpressed in highly metastatic cell lines in comparison with their poorly metastatic parental cells. Ectopic expression of these miRNAs in the parental cell lines (MeWo) caused increased metastatic potential and individual inhibition of these miRNAs in the metastatic cells suppressed metastatic colonization. Interestingly, the expression of these miRNAs are upregulated in primary melanomas that had metastasized in comparison with those that had not, indicating that the expression of them in primary tumors is an early event that can be predictive of melanoma metastasis. Another indicator that those are metastamiRs is that they do not affect tumor growth .
An important genomic region involved in melanoma development is the 7q31-34 region, because it is commonly amplified in melanomas. This region includes the BRAF and c-MET oncogenes, and the miR-182. It was observed a higher expression of this miRNA in metastatic melanoma tumors compared with primary melanomas and nevi, and this is caused, at least in part, by gene amplification. It was seen that this overexpression promotes metastatic properties as cell ability to extravasate or to seed at a distant site, through the downregulation of FOX3 and MITF-M .
Another miRNA that is upregulated in metastatic melanoma in comparison with primary melanoma and dysplastic nevi is the miR-21. MiR-21 expression also correlates positively with Breslow thickness, advanced clinical stage and metastatic behavior [123-124]. miR-532-5p also presents higher expression levels in metastatic melanoma tumors than in primary melanomas. An important target of miR-532-5p in metastatic tumor is the transcription factor RUNX3, which may act through β-catenin .
miR-221/222 are also upregulated in metastatic cells relative to primary melanomas or melanocytes. They are not expressed in compound nevus, but are progressively increased in the melanoma progression, with the higher level being in the metastases samples. Although they have increased expression in metastatic melanomas, they increase during tumorigenesis, which suggests that they are not metastamiRs
In comparison with miRNAs that are overexpressed in melanoma, little is known about miRNAs that are diminished during melanoma progression , so even less is known about miRNAs downregulated in melanoma metastasis. Fortunately, there is some information in this area and we will present it here.
The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) is important in melanoma development, however its function is controversial, with some showing that miR-200 is upregulated during melanoma progression and some showing that it is downregulated. Importantly for metastasis, miR-200c was shown to be downregulated in metastatic melanoma compared with primary melanoma and melanocytic nevi . In addition, miR-200c levels were different in melanoma samples located at primary versus distant metastatic sites . The role in suppressing metastasis was confirmed by a miR-200c overexpression assay, which showed inhibition of melanoma growth and metastasis
One of the first discovered miRNAs, let-7b, is also related with the aggressiveness of melanoma. This miRNA has been implicated as a tumor suppressor in several tumors and its levels are diminished in metastatic melanoma cell lines compared to primary melanoma cell lines . The ectopic expression of let-7b in the highly metastatic cells B16-F10 originated a great decrease in the number of tumor nodules formed in the lung. The authors also suggested that the low expression of miR-7b facilitates metastasis through the upregulation of its target Basigin (an extracellular matrix metalloproteinase inducer), that consequently increases MMPs production .
MiR-9 is also frequently lost in metastatic melanoma tissues related to primary melanoma tissues. In addition, the same expression was observed in metastatic melanoma cell lines compared with cells from radial growth phase melanomas and vertical growth phase melanomas. The low expression of miR-9 correlates with high cell proliferation, migration, and metastasis development
Others miRNAs that are downregulated in metastatic melanoma in comparison with primary melanoma are the miR-20b and miR-145, however to confirm their role in metastasis it would be important to do other experiments beyond its expression analysis. The mechanism of action of miR-145 is still unknown , however miR-20b seems to act through the regulation of the proteinase-activated receptor 1 (PAR-1), a thrombin receptor that it is involved in thrombosis and hemostasis and appears to have a key role in the progress of malignant melanoma .
Although there are still limited studies about the role of miRNAs in the process of melanoma metastasis, it is already a fact that the miRNA circuit is altered in this process and therefore the miRNA processing machinery may be deregulated. However, this has been poorly studied. A study shows that TARBP2 e SND1 (two RISC components) are overexpressed in cutaneous malignant melanoma metastases related to primary cutaneous malignant melanoma. This study also shows that Dicer, Drosha and other molecules of the miRNA machinery are not altered in the metastasis process . In another study with tissue samples, it was observed Dicer upregulation in metastatic melanoma in comparison with
Cutaneous melanoma is a melanocytic tumor whose incidence and mortality are on the rise worldwide. Prognosis of patients with melanoma depends on the stage of the tumor and is usually based on microstaging and radiological evaluation of metastases. Among the numerous prognostic parameters, tumor thickness is the most sensitive in predicting the risk of metastasis. However, it is still difficult to determine the prognosis for melanoma patients individually. More specific prognostic indicators for metastatic melanoma are needed.
The homeostasis of skin melanocytes is controlled by the keratinocytes of the epidermis and the balance between these cells is maintained by regulating the division of melanocytes. Mutations in genes regulating growth, changes in the production of paracrine growth factors and loss of adhesion molecules (integrins, cadherins, selectins and family of immunoglobulins) contribute to the disruption of cell signaling. Thus, the melanocytes may escape regulation, spread and proliferate and thereby form melanoma.
Several genes are involved in the pathogenesis of malignant melanoma. The best known in metastatic melanoma are the mutations in BRAF, MITF and changes in WNT and PI3K pathways. Due to the heterogeneity of metastatic lesions many changes have been described for metastatic melanoma, thus leading to greater difficulty of classifying major pathways altered in melanoma. Therefore, studies on the molecular biology of melanoma seeking to identify molecular markers for prognosis are interesting. Immunohistochemical (Mel-CAM), enzyme (Tyrosinase), protein (integrins, ICAM-1, cyclin D1), microRNAs and genetic (CDKN2A, p53) markers could be used for this purpose.
In this way, although there are several molecular alterations described in melanoma metastasis, there are some inconsistent data, probably due driver factors, as the heterogeneity of metastatic lesions, difficulty of correctly classifying the different stages of the tumor and difficulty in obtaining appropriate samples. Moreover, there is complicacy in the classification of cell lines and use of inappropriate technique. For example, today it is well established that metastatic characteristics can only be analyzed
This work was supported by FAPESP (2012/08776-5 to MGJ, 2013/04829-0 to ACM, and 2010/18715-8 to MT) and CNPq (470681/2012-8 to MGJ).