Summary of low-penetrance candidate melanoma susceptibility genes
Melanoma genetics has been for a long time a great challenge to cancer biologists, in part due to a complete lack of a single candidate gene to melanoma development. Different from breast and colorectal cancers, where BRCA-1/2 and
However, alterations in those genes, either by mutations or by epigenetic alterations do not account for all melanoma cases. Moreover, the mutations found in the classical melanoma genes are not typical UV signature mutations (such as C to T transitions). This observation poses an interesting problem in melanoma biology. Extensive epidemiological data indicates that intermittent exposure to UV radiation, mainly UVB is a major etiologic factor for melanoma development. On the other hand, genes commonly mutated in melanomas lack UV signature mutations. Thus, evidence so far for the presence of UVB-generated signature mutations in melanoma that could be defined as driver mutations has been less than compelling. Two critical questions need therefore to be answered; (1) If the classical melanoma genes do not account for the majority of cases, what other genes are involved in melanomagenesis? And, (2) what is the real relationship between the mutagenic potential of UV radiation and melanoma genetics?
In the following pages, we will discuss the new findings about the biology of this neoplasia, besides discussing the known genes involved in melanomagenesis. A systematic review of to date GWAS data, deep-sequencing data and functional genomics will serve as the background for this discussion. As examples, GWAS studies have identified genetic variations in genes related to pigmentation that confer susceptibility to melanomas. The importance of these studies resides in the identification of new variants that can represent low penetrance susceptibility genes. Other classes of genes that have emerged as critical genes to melanoma are DNA repair genes, especially NER genes (Nucleotide Excision Repair – a pathway that repair typical UV DNA damages). New studies have identified polymorphisms in those genes that confer higher risk to melanoma development. This susceptibility, in an interesting manner, seems to be influenced by the UV index of a certain region. On the other hand, microarray studies have suggested that DNA repair genes may be critical to metastasis sucess of melanomas, through stabilization of a “metastatic genome”. Deep-sequencing studies of melanoma cells have also identified genes and patterns of mutational status that correlate with UV signatures, bringing new clues to melanoma genetics. Are these driver or passenger mutations? The importance of other genes and pathways is also highlighted. One good example of a gene involved with melanoma progression is the
Thus, in this chapter we discuss both the “old” and the “new” genetics of melanoma susceptibility and progression. A discussion that will allow for the readers a systematic overview of what is known about the classical melanoma genetics, at the same time that may provide the basis to explore the new concepts that are emerging in this field.
2. UV exposure, deep-sequencing and melanomas – Understanding the melanoma development in depth
Skin constitutes the first defense barrier in protection of internal environment and it is therefore subjected to several aggressions by pathogenic microorganisms or by chemical or physical damaging agents. Among these several agents, sunlight ultraviolet radiation (UV) is considered the most potent carcinogenic factor for skin cancers, although the precise relationship between dose, time and nature of sunlight exposure to skin cancer development remains controversial . Ultraviolet radiation can be classified according to its wavelength in UVA (320-400 nm), UVB (280-320 nm) and UVC (200-280 nm). Despite the fact that UVA is more abundant in sunlight (90 %), UVB is about 1000-fold more efficient to cause sunburns and DNA damage than UVA . Skin exposure to UV light affects epidermal and dermal cell survival and proliferation, besides other cutaneous functions . Acute effects of UV exposure are usually the most harmful, including DNA damage, apoptosis, erythema, immunosuppression, all factors contributing to aging and skin cancer .
One of the main effects of UV exposure on cancer development is direct damages to DNA. Photoreactions due to absorption of UV (mainly UVB) by DNA lead to the establishment of covalent linkages of adjacent pyrimidine bases (cytosine or thymine) thus forming cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts. CPDs are constituted by the ligation of C-4 and C-5 carbons in both pyrimidines, whereas 6-4 photoproducts are produced between C-6 and C-4 carbons of two adjacent pyrimidines, more frequently between TC and CC residues. CPDs are also considered more carcinogenic than 6-4 photoproducts, the frequency of CPD formation is three times higher and are less efficiently repaired . If not repaired, both photoproducts lead to genetic mutations such as C→T and CC→TT transitions, besides single and double strand breaks on DNA . Other genotoxic agents associated with excessive exposure to UV are reactive oxygen species (ROS), characterizing an indirect effect of radiation . ROS can promote deanimation and adduct formation, leading to errors in base pairing and, thus, mutations and chromosomal reorganization, contributing to carcinogenic process.
Epidemiologic studies have indicated that a pattern of intense and intermittent exposure to sunlight is a major risk factor for developing melanoma . History of skin sunburns have been frequently used as measure to intermittent exposure and can be a marker for high risk of melanoma development. One or more severe sunburns on younger ages increase the risk of melanoma [9-11]. Besides the clear risk attributed to sunlight and thus UV exposure in melanomagenesis, the lack of a typical signature of UV mutation in genes classically related to melanoma development and progression had intrigued researchers for years. First,
In a seminal study, a comparison of four distinct sets of melanomas at the genomic level gave important clues about the role of UV in melanomagenesis . The authors compared the number of copies of DNA and the mutational status of two critical genes to melanoma development,
The unanswered question about the real impact of UV light on melanoma genetics began to be solved with the development of new technologies in DNA sequencing, the so called “deep-sequencing method”. With this technology, the researchers could perform large-scale sequencing, covering the whole genome. In one of the first studies using deep-sequencing methods, the authors reported more than 1000 mutations using 210 diverse human cancers, including melanomas . This study covered 274 megabases (Mb) and was restricted to 518 protein kinase genes. The results showed that melanomas (in that case, melanoma cell lines), had a high prevalence of mutations showing a mean number of 18.54 mutations per Mb of DNA. The main result from this first study was that of 144 mutations in melanomas, more than 90% was C to T mutations, the typical transition of UV-related mutations. Most somatic mutations found were classified as “passengers” mutations, i.e. those which do not contribute directly to carcinogenesis. “Driver” mutations, those mutations that contribute to carcinogenesis, were found in approximately 120 genes.
A second study, in this time a comprehensive catalogue of the whole genome of a melanoma cell line and a lymphoblastoid cell line from the same person, provided the first catalogue of somatic mutations from an individual cancer . The numbers generated by the deep-sequencing are impressive. The study identified 33345 somatic mutations, where 32325 were single base mutations and 510 were double-base mutations. A total of 292 somatic base substitutions were in protein-coding sequences and of these, 187 were non-synonymous mutations leading to amino acid changes, including 172 missense mutations and 15 nonsense. Several individual substitutions highlighted novel candidate cancer genes such as mutations in
Of the total number of mutations found (33345), almost 25000 were C to T mutations, and of the 510 dinucleotide substitutions, 360 were CC to TT changes . The mutational spectrum observed is consistent with UV-associated mutations, fact that denotes the influence of UV on melanoma development. C to T and CC to TT changes were significantly more frequent in CpG dinucleotides than the expected by chance. The mutational pattern also indicated a strong relation of UV mutations with the nucleotide excision DNA repair pathway (NER) due to the high frequency of mutations in non-transcribed strands when compared to mutational frequency in transcribed strands. The transcription-coupled repair (a sub-pathway of NER system), which operates in transcribed strands, is credited to be more efficiently in repair UV lesions when compared to NER system that operates in non-transcribed strands. Finally, besides the majority of C to T mutations, the second commonest mutation frequency was substitution of G to T. High production of ROS can lead to oxidized guanines and in turn causes G to T changes. As UV exposure can also lead to ROS production, is tempting to suggest that besides the direct DNA damage caused by UV, contributing to C to T changes, indirect effects such as ROS production also may contribute to melanoma carcinogenesis. Thus, this first entire catalogue of mutations in melanoma by whole genome sequencing supports the notion that UV exposure plays a critical role in melanoma development.
A third whole genome sequencing study also confirmed the elevated mutational rate in melanomas, that in mean was about 30 mutations per Mb, and the C to T mutations were the most frequent, once again reinforcing the role of UV irradiation in melanomas . However, the great advantage of the study was using metastatic melanoma samples and also including melanomas from different body areas. Thus, the authors could present an interesting panel of mutational rate across the different melanoma subtypes. As example, acral melanomas showed mutational rates comparable to other solid tumor types (3 mutations per Mb), whereas melanomas from the trunk showed higher mutational rates. The sequencing of a melanoma from an individual with history of chronic sun exposure exhibited the higher mutational rate across the samples analyzed (111 somatic mutations per Mb). Moreover, that melanoma with the higher mutational rate showed 93% of C to T substitutions, while acral melanomas showed only 36% of such mutations. These data strongly support the contribution of sun exposure in melanoma etiology. From the most significantly genes, the authors identified frequent mutations in
Additional studies from exome sequencing have identified new genes related to melanoma development and also chemoresistance. From exome sequencing study performed in seven melanomas the authors found a total of 4933 somatic mutations, 3611 of which were located in protein-coding regions in 2586 genes . Confirming previous results, C to T transitions were the most representative mutations (ranging from 73 to 87% of all mutations). In order to get a more comprehensive view of melanoma genome, the authors looked to genes involved with MAPK pathway, which includes NRAS and BRAF. Two of seven melanomas analyzed showed a somatic G to A transition at homologous site in the
In an independent study , other melanoma exome sequencing also identified mutations in genes participating of MAPK pathway, more precisely
All of these genome sequence studies identified a great number of mutations, however most mutations are passenger mutations. In order to differentiate passenger from driver mutations, Linda Chin coordinated an effort to sequence exons and introns of melanoma samples, comparing their frequency in order to identify positively selected genes, based on enrichment of mutations in exons . The authors identified positive selection in melanoma genes including well-know genes such as
Innovative strategies exploiting deep sequencing will contribute to the understanding of the diversity of pathways involved with melanoma. We anticipate that studies of melanomas arising in different ethnic groups, and mainly from individuals who migrated from low-UV index regions to high-UV index regions in the globe will help us understanding more about the genes involved in melanomagenesis.
3. Melanoma genetics: Susceptibility genes
When we talk about susceptibility genes to diseases, especially to cancer, we are talking about inheritable genetic alterations. Such alterations in critical genes related to tumor suppression contribute to modulate the susceptibility to certain tumors. Inheritable alterations can be classified as mutations or polymorphisms (also known as single nucleotide polymorphism –SNP). Both genetic alterations have different features such as: (i) related to population allelic frequency (mutations < 1% and polymorphisms > 1%); (ii) related to its impact to gene functionality, where mutations cause deleterious alterations to the function while polymorphisms may modify the function, however not in a deleterious manner; (iii) related to penetrance, where mutations exerts its deleterious function in a high penetrance to development of the disease. Conversely, polymorphisms exert its function in a low penetrance to disease and may be more susceptible to environmental influence; (iv) age of tumor onset, where high penetrance mutated genes contribute to disease development in younger ages while polymorphisms are related to older ages to cancer development. Temporally, high susceptibility genes to melanoma were well established through the years, however low susceptibility genes have been identified recently. Appreciation of high penetrance genes came from multiple studies of melanoma-predisposed families studies; in which linkage analysis, cytogenetic and candidate gene studies helped to identify those genes. However, the high-penetrance genes account for 5 to 10% melanoma cases, indicating that other genes, including low penetrance genes may modulate the susceptibility. The development of new technologies has contributed to identify new susceptible genes and understand their roles to melanoma. In this section we discuss the “old” and the “new” genetics for melanoma susceptibility.
3.1. High penetrance genes: “The old genetics” for melanoma
3.1.1. CDKN2A – The classical susceptibility gene
The best-established gene for melanoma susceptibility is the
The role of p14/Arf in tumor suppression is related to regulation of p53 pathway. Its function is related to binding to HDM-2 protein and inhibition of its activity. The MDM-2 protein is a key regulator of p53 protein due to its ability to ubiquitinate p53, leading to p53 degradation. Thus, by p14/Arf function, MDM-2 is depleted and p53 is stabilized. Inactivation of p14/Arf functions is associated with MDM-2 accumulation, which in turn leads to p53 degradation and consequently loss of its tumor suppressor function. In summary, loss-of-function alterations in
Some studies have suggested that the penetrance of the
3.1.2. CDK4 – The second line in melanoma susceptibility
Another well known gene associated with melanoma susceptibility is the
3.1.3. Evidence of new susceptibility locus and other critical genes that confer risk to melanoma
Different from other familial cancer, such as breast and HNPCC colon cancers, a unique candidate gene seems not responsible to all familial cases of melanoma. As cited above, up to 40% of familial melanomas could be attributed to
Other germline mutations in critical genes responsible for cancer susceptibility, which melanoma is not a clinically feature, also increase the risk for melanoma, where some melanoma cases have been reported. Individuals harboring germline mutations in
3.2. Low penetrance genes: The “new genetics” of melanoma
The great development in low penetrance genes search for melanoma risk came with the development of genome-wide association studies (GWAS). With GWAS, several hundreds of thousands DNA variants can be detected and larger samples sizes can be used, thus increasing the power of analysis. A great advantage of using GWAS is the possibility to identify variants that are not located in protein coding regions. Coupled with the development of GWAS, the use of meta-analysis has also contributed to identify new low penetrance genes. Meta-analysis is a widely accepted method that summarizes the results from multiple published studies, then producing results with larger sample size and increasing statistical power. We discuss below the main findings regarding low-penetrance genes and melanoma of GWAS and meta-analysis studies in melanoma.
3.2.1. MC1R gene – Coloring the knowledge of melanoma susceptibility
Epidemiological studies have indicated that red hair and fair skin are host characteristics predisposing to melanoma . This phenotype is known to be more sensitive to harmful effects of UV exposure, mainly because the low capacity of tanning in red hair and fair skin individuals. As certain
In a recent meta-analysis, of 9
As it is well established, melanin is one of the major protective factors against ultraviolet radiation DNA damage that results in melanoma development. The formation of this pigment is triggered by melanocyte-stimulating hormone, a peptide hormone coded by the the proopiomelancortin gene (
There is a positive feedback loop in melanocytes caused by UV radiation damage, which increases melanin production and blocks cell cycle progression via MITF until DNA damage is no longer detected . Given its protective nature, melanoma researchers have spent significant effort testing skin coloration genes derived from animal studies or genetic association studies identified as targets of
3.2.3. Other pigmentation genes
Genetic epidemiological studies have recently identified a subset of other pigmentation genes that are associated with risk for melanoma and other cutaneous malignancies as well as photosensitivity for MITF-regulated solute carrier family 45 member 2 gene -
Another pigmentation gene extensively studied in melanoma is
Genome-wide association studies (GWAS) have unveiled single nucleotide polymorphisms (SNPs) or genetic variants in other genes involved with pigmentation pathways that can contribute to melanoma susceptibility. Examples follow; two pore segment channel 2
3.2.4. DNA repair genes – Polymorphisms contributing to a mutator phenotype?
Epidemiological and experimental data suggest that UV radiation is the main carcinogenic agent responsible for melanoma development. While UV-B radiation (290–320 nm) induces critical damage to DNA in the form of cyclobutane pyrimidine dimers (CPD) and pyrimidine photoproducts, UV-A radiation (320–400 nm) induces single strand breaks and generates free radicals that cause oxidative damage . While UV-induced DNA damage often activates distinct DNA repair pathways that maintain genome integrity, the main processes involve the Base Excision Repair (BER), which operates mainly to repair damage caused by oxidative stress and single strand breaks and Nucleotide Excision Repair (NER) that acts to neutralize photoproducts such as CPD and 6–4 dimers .
The differences in DNA repair capacity among individuals are genetically determined in function of mutations and polymorphisms in many genes implicated in these pathways and it has been examined in relation to cutaneous malignant melanoma. According with a recent review , some studies found significant association between variations in DNA repair genes and melanoma. The gene
In other repair pathways significant association has been described, for example variants in exon 7 of
A summary of the main founds regarding low penetrance genes and melanoma risk can be found in Table 1.
|1.45 (P = 1.2 x 10-9)|
|910873/ 1885120||1.72 (1.53, 2.01)|||
PAT+; 6A,Gln allele
4. Melanoma genetics: Progression genes
4.1. “Old genetics” of melanoma progression
The here called “old-genetics” of melanoma progression consist of known genes which its functions are well described and are also related to several other cancer types, mainly due to its function in controlling survival and proliferation pathways. An overview of such “old-genetics” of melanoma is shown in Figure 1.
4.1.1. Mitogen-Activated Protein Kinases (MAPK) pathway – MAPing the first melanoma progression pathway
Several molecular pathways are activated/deactivated during tumor formation and some of them are responsible for the development of specific phases of tumor progression. Among them, is the Mitogen-activated protein kinases (MAPK) pathway. The pathway consists in a chain-like activation cascade of serine/threonine-specific protein kinases, where one protein must be phosphorylated to activate another. The proteins involved in this pathway are the RAS oncogene, discovered in the early 80s, with three known isoforms (H-Ras, K-RAS and N-RAS); RAF kinase, with also three isoforms (A-RAF, B-RAF and C-RAF or RAF-1); MEK kinase and ERK kinase, which have cytoplasmic targets or can phosphorylate transcription factors in the nucleus. The MAPK pathway is one of the most well-known pathways involved not only in melanoma formation, but probably in most types of tumors. The pathway is responsible to conduct an extracellular signal, like growth signal, from receptors in cell surface towards cell nucleus. After activation of RAS, the first protein of the cascade, a multitude of cellular responses, like protein synthesis, regulation of cell survival, differentiation and proliferation can be observed, showing the importance of this pathway for melanoma progression. Mutations in MAPK pathway are necessary for the development of early stages melanomas, as the transfection of constitutively activated MEK into immortalized melanocytes is sufficient to induce tumorigenesis in nude mice, activation of the angiogenic switch, and increased production of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs) .
The most common mutations found in MAPK proteins in melanomas are in RAS protein, more specific in N-RAS and in RAF proteins, in B-RAF. The RAS proteins are members of a large superfamily of low molecular-weight GTP-binding proteins. The activation state of RAS proteins depends on whether they are bound to GTP (in which case, they are active and are able to engage downstream target enzyme) or GDP (in which case, they are inactive and fail to interact with these effectors). In normal cells, the activity of RAS proteins is controlled by the ratio of bound GTP to GDP . N-RAS mutations can be found in over 15% of all melanoma cases and are most commonly the result of the substitution from leucine to glutamine at position 61 . It is correlated to the vertical growth phase of melanoma progression. Although initially thought to occur mainly at the plasma membrane, there is increasing evidence that isoform-specific RAS signaling can take place at different cellular compartments and within different regions of the plasma membrane. Such compartmentalization and trafficking of endogenous RAS oncogenes is likely to play an important role in regulating downstream signaling processes involved in tumorigenesis . For its activation and function as a signal transducer, N-RAS needs to be modified by a farnesylation near its C-terminal domain. Several farnesylation inhibitors were tested in the clinics and all results were disappointing . In part, the failure of the clinical trials can be explained due to the fact that the farnesylation inhibitors may work in Rho (a subfamily of RAS superfamily) rather than RAS, or the fact that the inhibitors works on normal and mutated RAS.
Other important component of MAPK pathway that is mutated in melanomas is the RAF kinase B-RAF, the primer mediator of RAS protein. Some reports have shown that over 60% of all melanoma cases have mutation in B-RAF . RAF mutations occur in the kinase domains and the most common mutation found in melanomas, approximately 80%, is the substitution of valine at position 600 with glutamic acid also called B-RAFV600E mutation. This mutation creates a constitutively active status for B-RAF, independently of a previous activation by RAS oncogene and extracellular stimulus and it is more frequently found in skin of individuals with intermittent sun exposure than unexposed or chronically sun-damaged skin. Interestingly, B-RAF mutation frequency in benign melanocytic nevi seems to be equal or even higher than in that for melanomas. The frequency also varies, like melanomas, from 0% in Spitz nevi up to 90% in intradermal nevi. These differences, between B-RAF mutation in nevi and melanomas make the assessment of the impact of these mutations on prognosis difficult to determine . B-RAF mutation in nevi might be a critical step in melanoma development, suggesting its importance in early stages of the disease.
Melanomas usually do not have B-RafV600E mutation at the same time they have mutations in any RAS isotype. However, some small proportions of cases carry mutation in both B-RAF and any RAS isoform, but in these cases, B-RAF mutation almost never is in V600E locus . Recently, a link between B-RAF and the cell cycle controller E2F has been shown. B-RAF is able to phosphorylate the retinoblastoma (Rb) protein and release E2F transcription factor family to work . E2F family is a classic cell cycle controller, but can also induce DNA repair, regulates autophagy and MMPs expression. The link between MAPK pathway and E2F transcription factor family may provide new strategies for melanoma treatment. New drugs using the B-RafV600E mutation as a target is currently being used in the clinics. Vemurafenib (PLX-4032) is a novel treatment for metastatic disease for melanomas with the V600E mutation. Vemurafenib treatment has demonstrated improved progression-free and prolonging overall survival in three months, compared with chemotherapy in a randomized trial, and represents a new standard of care in patients with advanced melanoma harboring a BRAF-V600 mutation . However, Vemurafenib treatment induces several resistance pathways in B-RafV600E cells and is expected to failure after a few months, but it is the best treatment for melanoma disease so far. Among the resistance pathway induced by the drug are MEK activation by MAP3K8 , up regulation of N-RAS  and activation of fibroblast growth factor receptor 3 (FGFR3) .
4.1.2. PI3K pathway – Supporting MAPK pathway to melanoma progression
RAS can also activate other effectors pathways rather than RAF. RAS can interact directly with phosphatidylinositol 3-kinases (PI3Ks), activating other molecular pathways. One of the pathways activated by PI3Ks is the AKT/PKB pathway, which has a strong anti-apoptotic function by phosphorylating various targets and seems to be an important part of the survival signal that is generated by RAS activation.
MAPK activation is necessary for early stages melanomas, but is not sufficient for the development of advanced disease. Other molecular mechanisms are necessary for melanoma invade other tissues and survive in different microenvironments. AKT/PKB seems to be important for the development of radial growth melanomas, from cell lines which are characterized as radial growth melanomas. In this model, AKT overexpression induced VEGF expression and switched to a more glycolytic metabolism . The AKT family consists of three members, AKT1–3 and 43–50% of melanomas have a selective constitutively active AKT3. AKT3 overexpression may occur as a result of copy number increases in the long arm of chromosome 1. Another mechanism for PI3K/AKT pathway activation in melanoma is through the acquisition of activating E17K mutations in AKT3. AKT has a critical role in cancer development through its ability to block apoptosis through the direct phosphorylation of BAD as well as through its effects in many other pathways, including the inhibition of forkhead signaling and the inhibition of glycogen synthase kinase-3. One of the most critical regulators of AKT is the phosphatase and tensin homolog (PTEN), which degrades the products of PI3K, preventing AKT activation. The mechanism by which the PI3K/AKT pathway is activated in melanoma may involve the loss of expression or functional inactivation of PTEN . However, PI3K pathway mutations, though more heterogeneous, were present in 41% of the melanoma, with PTEN being the highest mutated gene of the PI3K pathway in melanomas (22%) .
4.1.3. WNT5A – Progression to the edges, leading to melanoma metastasis
The metastatic disease does not have fixed histopathological subclasses. That is why there is a need to look for genetic profiles that could predict a behavior in advanced stages. WNT5A, a protein of Wnt family, was identified as the gene that best defined the new subclasses of tumors. The Wnt family of proteins has over 19 members, all of which are secreted, that are very closely structurally related. The activation of Wnt signaling can have very different results depending on which members of the family are involved. Wnt proteins work through three different pathways: the
4.2. “New genetics” of melanoma progression
Melanoma is a complex genetic disease. Recent studies have begun to characterize the mechanisms underlying melanoma plasticity, relating to intratumoral switching between varying malignant capacities, such as proliferation, invasion, or tumorigenesis. The rate at which somatic and germline genetic alterations have been cataloged in melanoma has accelerated greatly in recent years. The ability to modulate genes and proteins of interest, even when pharmacologic agents are not available, has provided preclinical evidence that many putative oncogenes represent potential therapeutic targets . At the same time, the notorious resistance of melanoma to treatments with its strong potential to metastasize represents the major clinical obstacle in the treatment of these tumors. These observations allow the scientists to improve staging and subtype classification and lead them to design better therapeutic agents and approaches. New insights about genetics of melanoma, including high-throughput strategies such as gene expression microarrays, comparative genomic hybridization, mutation analysis by deep sequencing and microRNAs gene regulation have helped researchers to elucidate the crucial cell-signaling pathways or validate the already postulated pathways as modified in melanomas. The genes and pathways discussed below for the “new genetics” of melanoma progression are represented in Figure 2.
4.2.1. Activating Transcription Factor 2 (ATF2) – Helping melanoma progression activation
The ATF2 (Activating Transcription Factor-2 or cAMP response element [CRE]) it was first identified as an inducible enhancer of genes that can be transcribed in response to increased cAMP levels and mediates various transcriptional regulatory effects, for example, ATF2/Jun complex is implicated in multiple cellular processes [94,95]. The ATF2 transcriptional targets genes is divided into (a) regulation of transcription factors and proteins engaged in stress and DNA damage response (b) regulation of genes associated with growth and tumorigenesis (c) regulation of genes important for maintenance and physiological homeostasis . In addition to its function as a transcription factor, ATF2 was found to play an important role in DNA damage response. After damage occurs, ATF2 is phosphorylated by ATM and its results in rapid localization of ATF2 to ionizing radiation (IR) induced foci (IRIF), which contain DNA repair proteins and chromatin-modifying enzymes. Furthermore, ATF2 phosphorylated is required for an intact intra-S-phase checkpoint response necessary to stop DNA replication . In recent years, the study of ATF2 activity in melanoma cells has revealed a probably oncogenic function. In the early '90s, Ronai and Weinstein  elucidated the cellular response to UV irradiation. The authors characterized a UV-responsive element (URE;TGACAACA) and soon after, its binding proteins, AP1 and ATF family members . Interesting, the URE was found within the promoter sequences of stress-responsive genes, including c-jun, DNA polymerase B, and cyclin A, as well as on regulatory regions of viruses that respond to UV irradiation . Differences in transcriptional activities of URE-bound proteins were found after UV-irradiation of keratinocytes, melanocytes and melanoma, and also in repair deficient cells of patients with
An approach to selectively inhibit ATF2 activity in human melanoma was designed, based on peptides derived from ATF2 trans-activating domain which affect ATF2 transcriptional activity. In an attempt to sensitize melanoma cells to UV irradiation, Ronai
Even with these encouraging results, one question remains unanswered: how ATF2 inhibition induces apoptosis in melanoma cells? It was demonstrated that ATF250–100 induced apoptosis by sequestering ATF2 to the cytoplasm, thereby inhibiting its transcriptional activities . In addition, mutations within the c-Jun N-terminal kinases (JNK) binding region of ATF250–100 or expression of TAM67, a dominant negative of the Jun family of transcription factors, or JunD-RNA interference attenuate inhibition of melanoma tumorigenicity by ATF250–100. The JNKs are kinases responsive to stress stimuli, such as ultraviolet irradiation used in this study. These results were crucial to show that inhibition of ATF2 in concert with increased JNK/Jun activities is central for the sensitization of melanoma cells to apoptosis and inhibition of their tumorigenicity. Furthermore, ATF250–100 increases ATF2 localization within the cytoplasm. Indeed, one study evaluating the ATF2 as a prognostic marker among patients with melanomas validated this result. A study to determine the prognostic value of ATF2 evaluating the pattern and level of its expression in a tissue microarray was conducted . Cytoplasmic ATF2 expression was associated with primary tumor rather than metastases and with better patient survival whereas nuclear ATF2 expression was associated with metastatic tumor and with poor survival. Nuclear ATF2 seems to be transcriptionally active while cytoplasmic ATF2 probably represents an inactive form. These findings support one preclinical finding in which transcriptionally active ATF2 is involved in tumor progression-proliferation in melanoma, suggesting that ATF2 might be a useful prognostic marker in early-stage melanoma. Although the use peptide ATF250–100 have shown good results to sensitize melanoma cells to treatments, Ronai group´s continued investigating peptides with smaller size but producing the same effect. In 2004, Bhoumik
Based on these findings, ATF2 present oncogenic action, but could it act as one tumor suppressor molecule? Although genetic changes in ATF2 have not been identified in human tumors, many data sustain the notion that ATF2 is not only oncogenic, whereas its altered expression and sub cellular localization is associated with tumor stage and prognosis in melanomas, but it also acts as a tumor suppressor molecule, under specific conditions. This hypothesis arose from independent studies with skin and mammary tumors. Studies from a mouse mammary tumor model revealed that loss of ATF2
Present knowledge positions ATF2 as important transcription factor and DNA damage response protein, which is also implicated in the regulation of cellular growth control. Along the growing complexity of understanding ATF2 regulation and function are the observations that point to its ability to elicit oncogenes or tumor suppressor functions, depending on the tissue type. Based on these findings, it was proposed one model for ATF2 oncogenic
4.2.2. Microphthalmia-associated Transcription Factor (MITF) the conductor of melanoma players
Recent observations of reversible phenotypic heterogeneity in melanoma have proposed a novel “phenotypic plasticity model” of cancer, whereas MITF seems to be one of the central players in melanoma phenotypic plasticity. The “dynamic epigenetic model” or rheostat model proposes that variations in the tumor microenvironment result in epigenetic lesions, leading to alterations observed in melanomas [for review 120]. In this model, high expression levels of MITF regulate genes involved with differentiation and cell cycle arrest. When MITF is expressed at average levels, melanoma cells proceed through cell cycle, while reduction of MITF to low levels switches off the cell proliferation program, inducing cell cycle arrest, and promotes invasion and metastasis. For example, prolonged MITF depletion leads melanomas to either quiescence or senescence . So, MITF regulates distinct functions in melanocytic cells at different levels of expression. While MITF lower levels are commonly observed in melanoma cells rather than in melanocytes, high levels of MITF activate the expression of differentiation-associated genes implicated on melanosome function and promote a differentiation-associated cell cycle arrest via up regulation of the p16 (
One interesting example from different levels of MITF action came from an elegant translational study . The authors demonstrated that the transcription factor ATF2 negatively regulates
Another possible mechanism that could explain the different levels of expression of
Recent studies have shown the role of germline mutations associated with MITF function. Evidence for germline mutations in melanomas comes from studies with relatives of patients with melanoma with increased risk of melanoma development, indicating the presence of mutations in genes with high penetrance [for review 133]. A study conducted by Bertolotto et al. involving patients with melanoma and renal cell carcinoma (RCC) supports the hypothesis of genetic predisposition for both cancers . MITF stimulates the transcription of HIF1A, the pathway of which is targeted by kidney cancer susceptibility genes, indicating that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. A germline missense substitution in
4.2.3. MITF as therapeutic strategy?
The understanding of the tumor stage, microenvironment, and mechanisms employed in phenotype switching have significant implications for clinical strategies in melanoma management. The description of
One therapeutic strategy is target one or more of the post-translational processes that determine MITF activity, stability, or degradation. Another approach is targeting the melanocyte-specific mechanisms controlling MITF expression. Nonspecific histone deacetylases seem to function in such a manner . Furthermore, MITF and its target genes have been used as diagnostic markers for melanoma . As cited above, MITF-M isoform is involved in the
4.2.4 DNA repair genes – Dual effect of DNA repair genes in melanoma progression
Exposure to UV radiation from sunlight induces DNA damage, which can lead to melanocyte carcinogenesis when not efficiently corrected. UV radiation may induce direct alterations through formation of pyrimidine dimmers, indirect alterations through formation of reactive oxygen species that may oxidize DNA bases and also induce DNA breaks. In a scenario where such alterations may facilitate the carcinogenic process, DNA repair systems are critical to suppress malignant transformation. There are different DNA repair systems inside the cells, which may repair a variety of DNA lesions, since mismatch base pairing formation during replication process, oxidized DNA bases, bulky addictions, intra and interstrand damages and single and double strand breaks. The main DNA repair systems are: Base excision repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Repair (HR) and Non-Homologous End-Joinning Repair (NHEJ) .
The critical role of DNA repair systems in cancer suppression is observed in a diversity of cancer predisposition syndromes which the main cause is due to mutations in DNA repair genes. Mutations in genes of nucleotide excision repair (NER), which preferentially corrects UV damages, caused the so-called
As discussed above, genetic variants that may alter the functionality of DNA repair genes, mainly genes from NER repair systems, may also modulate the susceptibility for melanoma. DNA repair systems were pointed as a functional network that could contribute to melanocyte carcinogenesis process by complete inactivating (such as in XP patients) or by differential functionality due to genetic variants associated with environmental factors such as UV exposure. However, this intuitive thought regarding the role of DNA repair systems restrict to the initials steps of melanoma development has changed in the last years. A study published in 2008  has suggested a new role of DNA repair systems in melanoma progression and metastasis. Aiming at the better understanding of primary melanoma to metastasis progression, the authors used a collection of frozen primary melanomas to study their gene expression by microarray. Those patients that had primary melanomas included in the study had a follow up for four years. After that, 26 of 60 patients showed metastasis while the other 34 patients did not. Gene expression of primary melanomas that originated metastasis (called M+ by the authors) was compared with the gene expression of primary melanomas that did not originate metastasis (M-). The results indicated a high and robust significant expression of genes involved with DNA replication (
The study indentified a total of 48 genes with higher expression in M+, which are related to DNA repair genes and genes related to maintenance of genetic stability in replication process . Among those genes, genes from the BER repair systems (a repair system strongly related to repair oxidized bases and single strand breaks) such as
As example, a gene overexpressed in M+ melanomas was
Following studies, confirmed the high expression of FANC DNA repair genes in melanoma samples when compared to normal skin and non-melanoma skin cancers . Moreover, there is a positive correlation regarding FANC genes and melanoma thickness by Breslow index. Conversely, NER genes were significantly decreased in melanomas, albeit its expression was not correlated with melanoma thickness. Immunohistochemistry of independent melanoma and non-melanoma skin cancers, confirmed the results previous discovered in gene expression regarding FANC genes and melanomas. Interestingly, down regulation of NER genes may have contributed to initial steps of melanomagenesis, however, the high expression of gene products of DNA repair pathways, mainly those regarding to solve double strand breaks, may be related to melanoma progression.
In another study, expression of DNA repair genes was associated with prognosis, disease relapse, tumor thickness and response to chemotherapy in melanomas . In that study, high expression of genes
4.2.5. PAX3 – Back to stemness?
Together with SOX10, PAX3 regulate transcription of MITF  and c-RET  in melanocytes. PAX3 is a key transcription factor during the development of the neural crest and its derivatives in the embryo. The neural crest cells detach from the dorsal neural epithelium and give origin to a diverse set of cells, including melanocytes. PAX3 starts its expression in neural crest precursors that are further committed with melanocytic cells lineage, such as melanoblasts . PAX3 exerts its activity by expressing MITF and repressing Dct (Dopachrome-tautomerase), thus leading to an undifferentiated cell state . When MITF levels reach a threshold, a complex consisting of MITF and β-catenin binds to Dct promoter, abolishing PAX3 inhibition, which leads to Dct expression and melanocyte differentiation. It is thought that upon terminal differentiation, the expression of PAX3 is reduced as suggested by initial studies that reported no expression of PAX3 in normal skin melanocytes [148;149] PAX3 expression has been described in nevi, in most primary melanoma tumors, melanoma cell lines [150-152]. The first study described the expression of PAX3 in 8/8 melanoma cell lines . The study also showed that PAX3 was commonly expressed in primary melanoma samples (21/58) but significantly less frequently in benign pigmented lesions (9/75). However, the following studies found PAX3 expression in melanoblasts localized in hair follicles and also in mature melanocytes in hair follicles, in 100% of the nevi examined, 94% of primary melanomas and in 90% of metastatic melanomas examined [151;152].
A most complete study performed in melanocytic lesions , analyzed PAX3 expression in normal skin, nevi, primary melanoma and melanoma metastases by immunohistochemistry. PAX3 was expressed in all samples and in normal cells. PAX3 expression showed a pattern of distribution characteristic of melanocytes (at epidermal-dermal boundary and along the hair follicle). Moreover, PAX3-positive cells were fewer and had a weaker staining in normal skin, as compared to nevi and melanomas. Co-expression of PAX3 with MITF was also observed in all samples, however, in normal skin some cells expressed only MITF, highlighting the differences in melanocyte phenotype. PAX3-positive cells were also co-stained with markers of less or more melanocyte differentiation, such as HES1 and Melan-A respectively. The samples indicated PAX3-positive cells co-stained with either markers, showing then a variable differentiation status of epidermal and follicular melanocytes, however a higher proportion of PAX3 and Melan-A positive cells. Finally, to further describe the phenotype of PAX3-positive melanocytes and melanoma cells, antiapoptotic factor BCL2L1 and melanoma progression marker MCAM were also analyzed in those cells. Regarding BCL2L1, a high similar proportion of PAX3-positive cells were also BCL2L1 positive cells, in all samples, with exception of melanoma metastases. These results suggest a role for PAX3 in regulation of survival of melanocytes and melanomas. Regarding MCAM, all melanocytic lesions showed its expression. Co-staining of MCAM and PAX3 increased in proportion from nevi to primary melanoma to melanomas metastases. As suggested above, PAX3 also plays a role in regulating genes involved in protecting cancer cells from apoptosis, as indicated by studies where the down-regulation of PAX3 increased the levels of apoptosis [153;154]. One of the mechanisms by which PAX3 may be involved with resistance to apoptosis resides in the fact that PAX3 interacts with the enhancer element of Bcl-XL gene, triggering its activation . Another mechanism described for the anti-apoptotic role of PAX3 is via the regulation of tumor supressor PTEN . In melanoma cells, the down regulation of PAX3 showed a dose-dependent reduction of proliferation and induction of apoptosis when cells were treated with cisplatin . Indeed, PAX3 down-regulation lead to increase in p53 protein and also caspase3 (a critical protein involved with apoptosis).
Functional studies have clarified the PAX3 function on melanocytes/melanomas . PAX3, acting synergistically with SOX10, play a role in the regulation of MET expression. MET is a transmembrane receptor tyrosine kinase activated by Hepatocyte Growth Factor (HGF) and plays a role in normal development and in cell migration, growth, survival, differentiation, angiogenesis . The HGF-MET pathway is involved in melanocyte biology acting on survival and maintenance of specific genes. MET is commonly over-expressed in melanoma and is associated with a more aggressive phenotype in terms of invasion and metastasis [160;161]. A strong correlation of expression of MET with PAX3 and SOX10 in primary melanomas was observed . Thus, the expression of PAX3 may facilitate melanoma progression and metastasis through the expression of MET, a classical proto-oncogene involved in invasion, metastasis, resistance of apoptosis, and tumor cell expansion.
PAX3 activities as a transcription factor were also analyzed by comparing melanocytic and melanoma cell lines . Initially, PAX3 binding to promoter regions of specific genes was analyzed and a enrichment of binding in melanoma cells was observed in genes such as HES1, SOX9 and NES (genes related to maintenance of stemness phenotype), CCNA2 and TPD52 (genes related to proliferation), BCL2L1, PTEN and TGFB1 (genes related to survival) and MCAM, CSPG4 and CXCR4 (genes related to migration). Conversely, in melanocytic cell lines, enrichment of PAX3 binding was just observed in HES1, SOX9, MCAM, TGFB1 and CSPG4, however quantitative analysis indicated lower PAX3 binding activity in melanocyte promoters, as compared to melanomas. Finally, a correlation of PAX3 promoter binding levels in melanocyte/melanoma cell line with gene expression of those genes indicated up-regulation of SOX9, NES, CCNA2, TPD52, TGFB1, MCAM, CSPG4 and CXCR4 in melanoma. Regarding BCL2L1 and PTEN, lower levels were observed in melanoma. In general, the study described a correlation between PAX3 binding to the target gene and its expression level, identified possible PAX3-regulated genes and also suggested the differential activity of PAX3 in transcriptional activity in melanocytes and melanoma cells. The interpretation of the results indicates critical features of the PAX3 function. Those genes up-regulated are genes related to cancer progression (SOX9 and NES), genes involved with cell motility, spread and metastatic potential (MCAM, CSPG4 and CXCR4) and with proliferation (TPD52). Moreover, down regulation of PTEN also contributes to melanoma progression due to tumor suppression activity of PTEN. Decreased of CDK2, BCL2 and MelanA (a melanocyte differentiation marker) gene expression and inhibition of cell growth was observed with PAX3 knock-down in melanoma cell lines, although the results were strongly cell line dependent . Moreover, an induced cell cycle arrest in S and G2/M phases and increase in apoptosis was also observed in PAX3 knock-down melanoma cells, and in one cell line. Silencing of PAX3 induced terminal differentiation.
In general, there is convincing evidence that PAX3 is expressed in melanomas and in melanocytic lesions, such as nevi. Indeed, PAX3 expression in melanomas may play a role in progression regulating processes such as survival, proliferation, metastases and participating in the maintenance of stemness. However, PAX3 seems expressed in a subset of differentiated melanocytes. Further clarification of PAX3 function in these cells is necessary. Environmental stimuli may be related to PAX3 expression in melanocytic lesions, as reported by up-regulation of PAX3 under UV-induced loss of TFG-β signaling from keratinocytes . Thus, PAX3 may be a good target gene to understanding the melanomagenesis process and more studies regarding its function are required.
TP53gene and melanoma – What is its function?
However, the proportion of primary melanomas harboring
Some reports have indicated that high expression of p53 can be found in both melanoma samples and melanoma cell lines. In addition, others reports have also indicated that this high expression does not correlate with p53 functionality. Melanoma cell lines harboring wild-type p53 showed transcriptional inactivity , a feature of melanoma cell lines that corroborates with data showing different gene expression of p53 targets in melanomas compared with nevi, strongly suggesting a dysfunctional p53 . Moreover, melanoma cell lines with wild-type p53 shows an absent p53 DNA-binding activity . All these reports indicate that downstream mechanisms could be operating to down-regulate p53 pathway in melanomas. One of the challenges of melanoma genetics in the coming years is to identify and characterize those downstream mechanisms, which certainly will improve our knowledge about p53 dysfunction in melanoma biology as well as identifying possible windows for melanoma treatment. There are at this moment critical candidates genes to act as negative regulator of p53 activity. Proteins such as iASPP (Inhibitor of apoptosis-stimulating protein of p53) , delta Np73 , YB-1  and Parc protein  has been described as p53 inhibitors. Alternatively, posttranslational modifications may also be responsible to p53 transcriptional silencing, such as phosphorylation, acetylation, methylation, sumoylation and neddlyation. Some findings have suggested that accumulation and increase in wild type p53 expression during melanoma progression may be indicative of dysfunctional p53 activity, reflecting posttranslational p53 modifications. Cytoplasmatic functions of transcriptionally inactive p53 have also emerged as a good hypothesis to a new p53 activity in either limit or promote tumor growth .
Additional reports have also confirmed the p53 transcriptional inability in melanomas . The results from such study showed that p53 downstream genes involved in apoptosis have low expression in melanoma metastases and melanoma cell lines. Conversely, genes involved with cell cycle were over-expressed in melanoma cell lines. Curiously, little difference between melanomas with wild-type p53 and mutant p53 could be observed regarding expression of p53 target genes, which confirm the notion that possible negative regulators are involved in the suppression of the p53 pathway. Even with down-regulation of p53 by using short-harpin method, there was limited effect on p53 target genes in p53 wild-type melanomas, however to melanocytes, p53 inhibition leads to alteration of several p53-dependent transcripts. An interesting feature observed was related to the proliferative capacity in melanocytes and melanomas, down regulation of p53 in melanocytes resulted in a gene expression similar to melanomas and increased proliferation rates while in melanomas, down regulation of p53 contributed to decreased proliferation, corroborating the results described by an independent study  (discussed below).
Although melanomas may have an inability to exert p53 full transcriptional capability, the p53 accumulation observed in such melanomas may still have basal activity. A central question is to understand the role of this basal p53 transcriptional activity in progression of melanomas. Recent functional studies start to address this interesting question. Melanoma cells are described as largely adapted to certain stress such as endoplasmic reticulum (ER) stress , a situation where melanomas acquire resistance to ER stress-induced apoptosis as well as resistance to chemotherapy . This adaptative response may be attributed to expression of Mcl-1 protein, which acts antagonizing the pro-apoptotic proteins PUMA and NOXA. Under ER stress, melanoma cells accumulate p53, which in turn (even in basal activity) induces the transcription of the microRNA miR149* . The p53-dependent expression of miR149* decrease the activity of GSK3α, resulting in Mcl-1 increase and consequent resistance to apoptosis. Moreover, decrease of miR149* elevated the rate of cell death in these melanoma cells and inhibited melanoma growth in a xenograft model. Finally, elevated expression of miR149 was found in melanoma samples, associated with decrease of GSK3α and increase of Mcl-1.
Other elegant functional study indicated critical features of p53 role in melanocytes and melanoma cells . First, the study indicated that p53 may be dispensable for melanoma cells due to lack of increase in DNA damage and enhanced proliferative potential in p53 depleted cells. Conversely, depletion of p53 in melanocytes increased mitotic defects. This last result is consistent with animal models in which genetic depletion of p53 cooperates with cell transformation . Indeed, in melanoma cells p53 is kept in a basal state of functionality. This basal activity showed to be critical to melanoma growth, as: (i) basal p53 activity leads to HDM-2 expression, which in turn keeps the basal levels of p53; (ii) this basal level of p53 avoids the activation of a p53-dependent pro-senescence program; (iii) in a basal state, p53 does not induce expression of p21, which in turn does not inhibits E2F1. The following E2F1 activation contributes to melanoma cell proliferation; (iv) expression of HMD-2 leads to activation of E2F1 in a p53-independet manner, contributing to melanoma cell proliferation. Instead, the “so-called” HDM-2 addiction in melanoma cells seems not to be related to melanocytes due to maintenance of viability and absence of senescence when p53 is activated by MDM-2 depletion. In summary, this study  elucidates new functions of the p53-HDM-2 axis in melanomas. Besides, the p53-HDM-2 axis in melanomas is now suggested as a promising target for melanoma treatment, since the use of specific HDM-2 antagonist rescues the p53 activity, leading to melanoma growth suppression and melanoma cell death .
The identification of negative p53 regulators that keep p53 pathway dysfunctional seems critical for a better understanding of the involvement of p53-dependent pathways in melanomagenesis and progression. Further functional studies will elucidate the intriguing questions regarding the real function of p53 to melanoma biology: Why has TP53 low frequency of mutations? How is p53 basal state maintained? What are p53 functions in melanomas?
4.2.7. MicroRNAs and melanoma – Another level of gene expression in melanomas
MicroRNAs (miRNAs) are small non-coding RNAs (21–23 nucleotides) encoded in the genome of plants, invertebrates, and vertebrates. These small molecules bind imperfectly to the 3´ untranslated (3´UTR) regions of target messenger RNAs (mRNAs) thus, miRNAs are central regulators of gene expression and can act both in a positive and a negative way to control protein levels in the cell. More than a thousand miRNAs exist in the human genome and each one can potentially regulate hundreds of mRNAs. Target prediction algorithms can be helpful in identifying potential mRNA targets of the miRNA of interest and further they should be validated by functional studies . MicroRNAs play an important role in many cellular processes, such as differentiation, proliferation, apoptosis, and stress response. Additionally, they are key regulators in many diseases, including cancer . These molecules regulate pathways in cancer by targeting various oncogenes and tumor suppressors and there is an increasing body of evidence suggesting that genomic instability regions harbor miRNA genes . The first study to associate genomic instability regions, miRNAs and cancer was published in 2002 . The authors found frequent deletions at 13q14 involving miR-15 and miR-16 genes in B-cell from chronic lymphocytic leukaemia. Since then, hundreds miRNAs have been reported acting as oncogenes or tumour suppressor genes in a wide variety of cancers [for review 183]. The first miRNAs described as involved in cancer formation was miR-let-7  and further the family of miRs let 7a and let 7b were reported to play a role in melanomas [for review 187]. For example, miR-let 7-b acts as a negative regulator of melanoma cell proliferation via regulation of cyclin D1, whereas miR-let-7a was demonstrated to regulate the expression of integrin-β3 and the Ras . So, modulation of miRNA expression is increasingly thought to be an important mechanism by which tumour suppressor proteins and oncoproteins exert some of their effects. Studies assessing the role of miRNAs in melanomas are still very recent and many efforts have been made to identify the ‘melano-miRs’. Despite the increasing number of studies (NCBI searching in September 2012 retrieved 162 results) a small number of miRNAs were identified to regulate genes involved specifically in melanomagenesis and some of them will be discussed here.
The linking between expression of miR-137 and
Besides miRNAs “
Other studies have identified a cluster of miRNAs that are either involved in melanomagenesis or predictors of survival. A study has identified the miR-506–514 cluster as a transforming oncogene that regulates melanoma progression and melanocyte transformation . Moreover, the authors showed that ectopic expression of this cluster in melanocytes was sufficient to transform them, activating cell growth, cell proliferation and migration/invasion along with inhibiting apoptosis. Although this study did not identify any direct gene targets of the miRNAs, further investigation is necessary because this cluster may reveal pathways that contribute to both the initiation and the maintenance of melanoma. As presented above, studies showed the increased expression of the miR-221/222 cluster associated with melanoma progression [for review 205]. A cascade involving
Approaches investigating miRNAs expression are also based on gene silencing by CpG methylation. Since miRNAs precursor genes are usually within regions of coding genes (intron sequences, for example), dysfunction of these protein-coding genes by epigenetic mechanisms may also be expected to cause aberrant regulation of the miRNA target genes . For example, miRNA-34a is highly methylated in melanoma cell lines and primary tumors and additionally, it was described that
All of these studies investigated the biological functions of miRNAs and their contribution to melanomagenesis. Other studies have attempted to identify miRNAs signatures for diagnostic and prognostic, melanoma progression by comparing the expression profiles in different stages of transformation, and others focused on specific pathways. Some of these studies will be presented here. In 2007, assays were performed using the well established NCI-60 cancer cell line panel and normal tissue . The study was able to discriminate between the malignancies, including melanomas cell lines whereas miR-146, miR-204 and miR-211 miRNAs shown to be highly expressed in melanomas. Large cohorts of miRNAs associated with malignant transformation as well as with the progression and with metastatic colonization in melanocytes and subsets of melanoma cell lines also was identified . Subsequently, down regulation of miRNA-200c in melanocytes, melanoma cell lines, and patient samples could be reported, whereas miRNA-205 and miRNA-23b were markedly reduced among patient samples . In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Using deep sequencing approach of a diverse set of melanoma and pigment cell libraries it was identified 539 known mature sequences along with the prediction of 279 novel miRNAs candidates . Some of these novel candidate miRNAs may be specific to the melanocytic lineage and as such could be used as biomarkers in the early detection of distant metastases by measuring the circulating levels in blood. The expression of 611 miRNAs in 59 metastatic specimens was profiled and the authors were able to identify a “miRNA classifier” consisting of miR-150, miR-342-3p, miR-455-3p, miR-145, miR-155 and miR-497 that were considered predictors of post-recurrence survival . Similarly the analyses of the miRNA expression profiling from melanoma lymph node metastases reported a unique signature consisting of down regulation of miR-191, combined with up regulation of miR-193a, miR-193b, miR-365, miR-338 and let-7. Together, this miRNAs also serves as predictors of short-term survival in melanoma patients . These findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their dysfunction can be impacted by inherited gene variants, supporting the hypothesis that miRNA dysfunction reflects biological differences in melanoma. Recently, the use of microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis identified differentially expressed microRNAs . The miR-203 and miR-205 were differentially expressed between primary and metastatic melanomas and functional
As such, miRNAs represent a new class of molecules that might prove to be powerful cancer biomarkers useful in future staging systems and used as stratification criteria in clinical trials as well as treatment of patients with disseminated disease. It was demonstrated that miR-214 is over-expressed in metastatic melanoma cell lines as well as tumor specimens. MiR-214 regulates the expression of two transcription factors, AP-2c and AP-2a . These molecules have been previously shown to play major roles in melanoma metastasis via regulation of genes involved in invasion and angiogenesis. Histological examination of skin biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis. For purpose of clinical management, the microRNA profiling of clinical melanoma subtypes samples considering the somatic and inherited mutations associated with melanomas, including the presence of one variant in a miRNA binding site in the 3’UTR of the
In this chapter we proposed to discuss the melanoma genetics, starting from the genes that may confer susceptibility to the genes that may be involved with progression. Moreover, we addressed the already known genes (here called as “old genetics”) as well as new genes that have been discovered as involved in melanoma (here called as “new genetics). It is noteworthy that the new technologies such as GWAS and deep-sequencing have improved our knowledge about melanoma genetics. Nowadays we have critical information about the disease, such as the clear involvement of UV in carcinogenic process and the many pathways that contribute significantly to it. As could be observed, conversely to other cancer types where single genes has great impact on susceptibility and progression, such as BRCA in breast and ovarian, mismatch repair pathways and colorectal cancer or
It is tempting to assume that the more we know about melanoma biology, including melanoma genetics, much more efficacious melanoma prevention and treatment will be. Heterogeneity within the very same tumor will certainly hamper treatment. We will need to take it in account in the days of personalized medicine. To this, improvement of technologies, coordinated studies of gene-environment interactions, allied to functional studies and critical clinical trials, will be necessary for the adequate translation of this body of information into patient benefit.