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Targeted Therapies in Melanoma: Successes and Pitfalls

Written By

Giuseppe Palmieri, Maria Colombino, Maria Cristina Sini, Paolo Antonio Ascierto, Amelia Lissia and Antonio Cossu

Submitted: 25 April 2012 Published: 30 January 2013

DOI: 10.5772/53624

From the Edited Volume

Melanoma - From Early Detection to Treatment

Edited by Guy Huynh Thien Duc

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1. Introduction

Incidence of melanoma is steadily rising worldwide [1]. Lifetime risk of developing melanoma in Caucasians is estimated as 1 in 50 individuals [2-3]. The incidence of melanoma varies according to the geographical origins of the population and the extent of sun exposure. In Australia and United States, an incidence of melanoma higher than observed in the European countries (with the notable exception of Sweden) has been reported [4-5]. There is a gradient of melanoma incidence from north to south in Europe, with highest frequencies in the northern counties. This suggests that initiation and development of melanoma is due to a combination of the damaging effects of UV and a predisposing genetic background [5].

Melanoma arises from melanocytes, neural crest-derived cells that are located in the basal layer of the epidermis and skin appendages in humans. Melanocytes, by synthesizing melanin pigments and exporting them to adjacent keratonocytes play a key role in protecting the skin from the damaging effects of ultraviolet (UV) and other solar radiation [6]. Melanocytes can proliferate to form nevi (common moles), initially in the basal epidermis (junctional nevus) and later by limited local dermal infiltration (compound nevus). Nevi develop during embryonic life (congenital nevus) and in children and adults, (acquired nevus) partly as a result of solar exposure in the latter two populations. Further progression of melanocytic tumors relates to factors that include intermittent exposure to UV radiation (though a direct relationship between risk of melanoma and UV exposure remains somehow unclear), a history of sunburn and endogenous factors such as skin type and elevated numbers of nevi (especially dysplastic nevi, also known as atypical moles) [7-8].

Considering the growth patterns, four histological types of melanoma have been historically recognized: superficial spreading melanoma (SSM), lentigo maligna melanoma (LMM), nodular melanoma (NM), and acral lentiginous melanoma (ALM) [9]. Comparative genomic hybridization revealed that several genomic regions (mostly, 11q13, 22q11-13, and 5p15) were abnormally amplified in ALM [10]; such regions were different from those found altered in superficial SSM or NM (mainly, 9p21 and 1p22) [11]. Recently, a new classification of melanoma including the site of primary tumour and the degree of chronic sun-induced damage of the surrounding skin has been introduced [12]. Based on these criteria, melanomas are classified into four groups; melanoma on skin with chronic sun-damage (CSD melanoma), melanoma on skin without chronic sun-damage (non-CSD melanoma), melanoma on palms, soles and nail bed (acral melanoma), and melanoma on mucous membrane (mucosal melanoma) [12]. Non-CSD melanomas are characterized by high frequency of BRAF or NRAS mutations (which are mutually exclusive), while CSD, acral, and mucosal melanomas show a low frequency of BRAF/NRAS mutations but a high incidence of alterations in additional genes, such as mutations of receptor tyrosine kinase KIT gene, amplifications of cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) genes [7, 12-13]. All genes affected into the different types of melanoma are involved in regulating cell-cycle progression and cell survival [12-13]. On the other hand, such a difference of genetic alterations indicates distinct genetic pathways in the pathogenesis of melanoma depending on the anatomical site of the primary lesion. Trying to merge the two classifications, it could be affirmed that non-CSD melanoma roughly corresponds to SSM, CSD melanoma to LMM, and acral melanoma to ALM. Since NM may arise at any anatomical site, this histological type can not be included in any of the subgroups of the latter classification (indeed, no distinct genetic pathway has been so far correlated with NM).

During recent past years, melanocytic transformation is being demonstrated to occur as a sequential accumulation of genetic and molecular alterations [13-14]. In this sense, it is becoming an unquestionable certainty that molecular classification of melanoma patients could be achieved through the assessment of the molecular profile of primary tumors and/or the correspondent metastases, by unveiling which gene or pathway is truly affected. Although pathogenetic mechanisms underlying melanoma development are still largely unknown, several genes and metabolic pathways have been shown to carry molecular alterations in melanoma.

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2. Main genes and related pathways

2.1. BRAF and MAPK pathway

The mitogen-activated protein kinase (MAPK) signal transduction pathway regulates cell growth, survival, and invasion. MAPK signaling is initiated at the cell membrane, either by receptor tyrosine kinases (RTKs) binding ligand or integrin adhesion to extracellular matrix, which transmits activation signals via RAS on the cell membrane inner surface (Figure 1).

Figure 1.

Main pathways involved in melanomagenesis. Arrows, activation signals. Interrupted lines, inhibition signals.

RAS proteins are small GTPases thatare activated by extracellular stimuli and regulate signal transduction of the BRAF-MEK1/2-ERK1/2 and AKT/PI3K pathways, controlling crucial cellular processes such as proliferation, differentiation, cell adhesion, apoptosis, and cell migration [14-16]. RAS gene is mutated in an estimated 20-30% of all cancers [17]. RAS proteins are constituted by three main isoforms - NRAS, KRAS, and HRAS, which present a similar function but a different specificity for tissue distribution [18]. On this regard, KRAS alterations mostly occur in gastrointestinal cancer [17-18], HRAS alterations are frequently observed in bladder cancer, and NRAS is mutated in 15-25% of melanomas [17-18]. Despite the huge amount of knowledge implicating RAS in tumour initiation and promotion, RAS itself has not become a successful target of therapy.

The RAF kinase family consists of three members - ARAF, BRAF, and CRAF, all of which can activate a series of protein kinases; such a signaling cascade culminate in the phosphorylation and activation of the extracellular signal-regulated kinase (ERK) downstream protein [19]. In melanoma, the most commonly mutated component of this pathway is the BRAF gene; the prevalent BRAF mutation (in nearly, 90% of cases) being a substitution of valine with glutamic acid at position 600 (V600E) [20]. Mutated BRAF induces constitutive ERK activation; activated ERK then translocates to the nucleus and initiates the transcription of a variety of growth-related genes, stimulating cell proliferation and survival [20]. Indeed, the increased activity of ERK seems to be implicated in rapid melanoma cell growth, enhanced cell survival and resistance to apoptosis [21]. High levels of activated ERK may further induce the metastatic potential of melanoma by increasing the expression of invasion-promoting integrins [22-23]. Presence of BRAF mutations in benign and dysplastic nevi supports the hypothesis that activation of the RAF/MEK/ERK pathway is an early event in melanoma progression [24-25]. In other words, BRAF activation is necessary but not sufficient for the development of melanoma and additional co-operating genetic events are required to achieve full malignancy.

In a study aimed to better define the role of BRAF in melanomagenesis, a transgenic zebra fish expressing V600EBRAF showed dramatic development of patches of ectopic melanocytes (designated as fish-nevi) [26]. Remarkably, activated BRAF in p53-deficient zebra fish induced the formation of melanocytic lesions that rapidly developed into invasive melanomas that resembled human melanomas in terms of their histology and biological behaviors [26]. These data provide direct evidence that the p53 and BRAF pathways interact functionally during melanomagenesis.

The BRAF gene also cooperates with the cyclin-dependent kinase inhibitor p16CDKN2A (see below). Activating BRAF mutations have been reported to constitutively induce up-regulation of p16CDKN2A and cell cycle arrest (this phenomenon appears to be a protective response to an inappropriate mitogenic signal). In particular, mutant BRAF protein induces cell senescence by increasing the expression levels of the p16CDKN2A protein, which, in turn, may limit hyperplastic growth caused by BRAF mutations [25]. Therefore, inactivation of p16CDKN2A gene may promote the melanocytic proliferation depending on oncogenic BRAF. In this sense, several factors seem to be able to induce the arrest of the cell cycle and cell senescence caused by BRAF activation [27-28].

Finally, it has been showed that primary melanomas arising from chronically sun-damages skin and from mucosal sites, which typically do not harbour BRAF and NRAS mutations, have increased copy number of the CCND1/Cyclin D1 gene [12]. In contrast to primary melanomas, a subset (>15%) of metastatic melanoma samples with BRAF mutations also exhibit amplification of CCND1/Cyclin D1. These melanomas are resistant to BRAF inhibitors highlighting the need for combination therapy [29-30].

2.2. CDKN2A and senescence/apoptosis pathways

The cyclin-dependent kinase inhibitor 2 (CDKN2A; at chromosome 9p21) gene encodes two proteins, p16CDKN2A and p14CDKN2A (a product of an alternative splicing), that are known to function as tumor suppressors [31-33]. The cyclin proteins are regulatory effectors able to bind and activate the cyclin-dependent kinases (CDKs) that bear catalytic kinase activity. Several distinct cyclin/CDK complexes have been identified and functionally assigned to specific phases of the cell cycle: Cyclin D/CDK4 complex leads the passage from the pre-replicative (G1) to the DNA duplication (S) phase; the Cyclin E/CDK2 complex promotes the progression through the S phase and the Cyclin B/CDK1 complex induces cells to enter mitosis [31-32]. In such a functional network, proteins like p16CDKN2A and p14CDKN2A act as inhibitors of the cell cycle, negatively interfering with the activity of the cyclin/CDK complexes and, in this way, ensuring the control of the cell replication [33]. In particular, p16CDKN2A is part of the G1–S cell cycle checkpoint mechanism that involves the retinoblastoma-susceptibility tumor suppressor protein (pRb). The p16CDKN2A inhibits the Cyclin D/CDK4 complex, which, in turn, phosphorylates pRb and allows progression through the G1–S checkpoint (Figure 1) [33]. The Cyclin D (CCND1) and CDK4 genes are found altered in less than 5% of total melanomas [12], though with an heterogeneous prevalence according to the distinct types of melanoma (see above). Somatic CDK4 amplification is relatively common in acral and mucosal melanomas [12], whereas germline CDK4 mutations are observed in a limited fraction of melanoma-prone families [34]. The CCND1 gene amplifications is primarily found in ALM lesions (more than one third of cases) and to a lesser degree in other types (11% for LMM and 6% for SSM) [35]. Regarding the alternative CDKN2A gene product, p14CDKN2A is an antagonist of the murine double minute 2 (MDM2) protein, which targets p53 to degradation by ubiquitination and proteasome processing, thus abrogating p53 control of cell growth (Figure 1) [32-33]. In particular, the p14CDKN2A protein exerts a tumor suppressor effect by inhibiting the oncogenic actions of the downstream MDM2 protein, whose direct interaction with p53 blocks any p53-mediated activity and targets the p53 protein for rapid degradation [32-33]. The p53 is a transcription factor that functions as a major negative regulator of cell proliferation and survival, being activated by different adverse signals (i.e. growth factors withdrawal, DNA damage, oncogenic aberrations, hypoxia, etc.) and driving cells to either interrupt progression into the cell-cycle or enter apoptosis program, in order to avoid reproduction of altered cells [33, 36]. In normal conditions, expression levels of p53 within cells are low. In response to DNA damage, p53 accumulates and prevents cell division. Therefore, inactivation of the TP53 gene results in an intracellular accumulation of genetic damage which promotes tumor formation [36]. In melanoma, such an inactivation is mostly due to functional gene silencing since the frequency of TP53 mutations is low (less than 10% of cases) [37]. Impairment of the p14CDKN2A-MDM2-p53 cascade, whose final effectors are the Bax/Bcl-2 proteins, has been implicated in defective apoptotic responses to genotoxic damage and, thus, to anticancer agents (in most cases, high expression levels of Bcl-2 protein have been demonstrated to reduce apoptosis and sensitivity of melanoma cells to proapoptotic stimuli, contributing to further increase tumor aggressiveness and refractoriness to therapy) [33].

More in general, genetic loss or rearrangement in the CDKN2A locus may result in impairing or silencing p16CDKN2A, p14CDKN2A or both genes, with the consequence of losing the mechanisms controlling cell proliferation and/or survival. In melanoma, the CDKN2A gene is somatically inactivated by genomic deletions (approximately 50% of cases) or point mutations (about 10% of cases); in addition, this gene is often transcriptionally silenced by promoter hypermethylation [38]. A reduced expression of the p16CDKN2A protein seems to be strictly associated with malignant tumor invasion, varying from 5% to about 15% in benign melanocytic lesions, from 10% to about 50% in primary melanomas, and from 50% to about 60% in melanoma metastases [39]. The CDKN2A gene is frequently mutated at germline level in patients with a strong familial history of melanoma (three or more affected family members), indicating that it represents a key susceptibility gene for familial melanoma [40]. In melanoma, CDKN2A mutations typically occur in the p16CDKN2A gene, either alone or in combination with p14CDKN2A gene (some families harbor however mutations only in this latter gene) [33, 40].

A recent meta-analysis of studies conducted in independent populations indicated that multiple variants of the melanocortin-1 receptor (MC1R) gene increase the melanoma risk in CDKN2A mutation carriers [41]. The MC1R gene encodes a G-protein coupled receptor. In the skin, two types of melanin pigment, dark-protective eumelanin and red-photo reactive pheomelanin, are present [42]. MC1R plays an important role in determining the ratio of eumelanin and pheomelanin production. After stimulation by UV, keratinocytes produce alpha melanocyte stimulating hormone (MSH) that binds to the MC1R on melanocytes and shifts the balance of these two pigments in the direction of eumelanin [42]. In particular, stimulation of MC1R by MSH mediates activation of adenylate cyclase, subsequent elevation of cAMP levels, and activation of the microphthalmia transcription factor (MITF; see below). Activated MITF binds to a conserved region found in the promoters of the tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and DOPAchrome tautomerase (DCT) genes, stimulating the transcriptional up-regulation of these proteins and inducing maturation of the melanosomes [43]. This ultimately results in increased eumelanin production and darkening of the skin or hair.

New findings have shed light on the mechanisms by which MC1R contributes to melanoma risk. In vitro studies showed that acute UV irradiation of melanocytes with impaired MC1R results in an increased production of free radicals [44]. Melanomas that arise on body sites only intermittently exposed to sun, and which therefore lack marked signs of chronic solar damage, were found to have a high frequency of BRAF mutations [12]. One could speculate that induction of BRAF mutations may occur only when solar exposure is not sufficiently prolonged to induce the striking tissue changes that generate the hallmark signs of solar damage. Several MC1R variants, that impairing relevant protein function, have been associated with BRAF mutation in melanoma arising in Caucasian populations from United States and Europe [45-48]. On the basis of such indications, it is possible that increased production of free radicals following UV exposure is combination with impairment of MC1R may induce mutations in the BRAF gene.

Additional mechanisms promoting susceptibility to pathogenetic mutations of the BRAF gene may however exist since there is no demonstrable association between germ line MC1R status and the prevalence of somatic BRAF mutations in melanomas from Australian population, even after classifying the melanomas by their location relative to intermittent and chronic sun-exposure [49].

2.3. PTEN and mTORC pathways

Phosphatase and tensin homolog deleted in chromosome ten (PTEN) has a key role in cellular signal transduction by decreasing intracellular phosphatidylinositol [3,4-bisphosphate (PIP2) and 3,4,5-triphosphate (PIP3)] that are produced by the activation of phoshoinosite 3-kinase (PI3K) [50]. In the absence of extracellular growth stimuli mediated by cell surface receptors and G-proteins, PTEN dephosphorylates PIP3 generating the PIP2 phospholipid, unable to stimulate phosphorylation of the PI3K protein; this in turn maintains suppression of cell cycle progression and cell growth. In other words, there is a balance between PIP2 and PIP3 which is maintained by the opposite activities of PTEN and PI3K, which instead converts PIP2 into PIP3 [50]. Upon growth stimulation, mainly obtained by triggering the RAS kinase, PI3K is constitutively activated (Figure 1), resulting in an increase of intracellular levels of PIP3 and a consequent activation of AKT by phosphorylation [50-51]. Activated AKT in turn phosphorylates its substrate, the serine/threonine kinase mTOR, leading to increased synthesis of target proteins that promote cell division and survival as well as apoptotic escape [51]. The mechanisms associated with the ability of AKT to suppress apoptosis include the phosphorylation and inactivation of many pro-apoptotic proteins, such as BAD (Bcl-2 antagonist of cell death) and MDM2, as well as the activation of NF-kB [52] (Figure 1).

Three AKT genes have been described in humans: AKT1, which is involved in apoptosis and protein synthesis; AKT2, which is involved in controlling the glucose metabolism; and AKT3, whose increased activity (often associated with the amplification of the AKT3 locus at chromosome 1q43-44) is mainly involved in stimulating cell growth and has been implicated in many cancers including melanoma [51-52]. More than two thirds of primary and metastatic melanomas exhibit higher levels of phosphorylated AKT [52], suggesting that such an alteration might be considered as an early event in melanoma pathogenesis.

Overall, PI3K expression is higher in malignant melanomas, as compared to nevi, and seems to correlate with a worse prognosis [53]. In primary melanomas, since activating mutations of PI3K are quite rare (about 1%), and comparative genomic hybridization did not reveal amplification at this gene locus [12, 37], activation of the PI3K pathway is mostly due to functional silencing of the tumour suppressor gene PTEN. Inactivation of PTEN gene is mainly due to hypermethylation-based epigenetic mechanisms, with a low incidence (less than 10%) of somatic mutations and/or allelic deletions; loss of (or reduced) PTEN protein is observed by immunohistochemistry in 20-40% of melanoma tissues [54-55]. Consistent with its role in the PI3K-AKT pathway, vast majority (more than 80%) of melanoma samples with loss of PTEN protein presents a significant increase in expression of phosphorylated AKT [37].

PTEN inactivation has been mostly observed as a late event in melanoma, although a dose-dependent down-regulation of PTEN expression has been implicated in early stages of tumorigenesis, often occurring in conjunction with mutations in BRAF gene (which have been demonstrated to indeed play a role in induction of the melanocytic proliferation and early steps of melanoma development) [50]. PTEN downregulation In addition, alterations of the BRAF-MAPK pathway are frequently associated with PTEN-AKT impairment [7, 56]. In summary, the combined effects of the inactivation of PTEN gene and activation of the PI3K-AKT effectors may result in aberrant cell growth, apoptosis escape, and abnormal cell spreading and migration [33, 50].

2.4. MITF and melanocytic differentiation

The microphthalmia-associated transcription factor (MITF) is a transcription factor that is involved in differentiation and maintenance of melanocytes, playing a role in melanoma development and pathogenesis [43, 57]. MITF is activated by the MAPK pathway as well as by the cAMP pathway (Figure 1), and leads to transcription of genes involved in pigmentation (TYR, TYRP1, and DCT; see above) as well as cell cycle progression and survival [43]. The MITF gene is amplified in melanoma (about 20% of cases); MITF amplification correlated with increased resistance to chemotherapy and decreased overall survival [57].

The connection between MITF and melanoma development is complex because it plays a double role of inducer/repressor of cellular proliferation. High levels of MITF expression lead to G1 cell-cycle arrest and differentiation, through induction of the cell cycle inhibitors p16CDKN2A and p21 [58-59] (Figure 1). Very low or null MITF expression levels predispose to apoptosis whereas inter-mediate MITF expression levels promote cell proliferation [57-59]. Therefore, it is thought that melanoma cells have developed strategies to maintain MITF levels in the range compatible with tumorigenesis. It has been shown that constitutive ERK activity, stimulated by V600EBRAF in melanoma cells, is associated with MITF ubiquitin-dependent degradation [60]. Nevertheless, continued expression of MITF is necessary for proliferation and survival of melanoma cells, because it also regulates CDK2 and Bcl-2 genes [61-62]. It has been recently shown that oncogenic BRAF may control intracellular levels of the MITF protein through a fine balance of two opposite mechanisms: a direct reduction of MITF levels, by inducing protein degradation, and an indirect increase of MITF levels, by stimulating transcription factors which increase protein expression levels [63]. Oncogenic BRAF mutations are associated with MITF amplification in a low fraction (10-15%) of melanomas [63], suggesting that other mechanisms are likely to be involved in ERK-dependent degradation of MITF.

2.5. iNOS and NF-kB pathways

Human melanoma cells are known to express the inducible nitric oxide synthase (iNOS) enzyme, which is responsible for synthesis of nitric oxide (NO), a free radical involved in several physiological processes such as neurotransmission, vasodilation, and regulation of immune responses [64]. The iNOS enzyme has been found to be frequently expressed in melanoma [65-66] and the subsequent increased concentrations of NO have been demonstrated to contribute to melanomagenesis through a sustained protection of the tumour from apoptosis [67]. However, the role of iNOS in melanoma progression remains controversial. Higher levels of iNOS have been found in subcutaneous and lymph node metastases of non-progressive melanoma as compared to metastases of progressive melanoma [68], however, iNOS was found to be expressed to a lesser extent in metastases as compared with nevi and primary melanomas [69]. Nevertheless, the expression of iNOS in patients with lymph nodes and in-transit metastases (stage III disease) has been proposed as an indicator of poor prognosis [70].

Recently, it has been reported that the constitutive iNOS expression in melanoma cells might be induced by activation of the MAPK pathway through stimulation of the activity of the Nuclear Factor-kB (NF-kB) [71-72]. NF-kB is a protein complex that acts as a transcriptional factor and regulates the transcription of several genes involved in many critical pathways [73]. In a quiescent status, proteins of the NF-kB complex are localized into the cytoplasm. NF-κB exists as cytoplasmic hetero- or homodimers associated with members of the inhibitor-of-kB (IkB) proteins (IkBa, IkBb and IkBe), which form complexes sequestering NF-kB into the cytoplasm [74]. Upon appropriate stimulation, the phosphorylation of IkB proteins is promoted, triggering their ubiquitination and degradation in the proteasome [74]. As a consequence, NF-kB may translocate to the nucleus where it binds to target DNA loci and induces transcription of several genes - including iNOS - associated with immune and inflammatory response, angiogenesis, cell proliferation, tumor promotion, and apoptosis [73-74].

Regarding the role of NF-kB in tumorigenesis, there are compelling evidence that activation of NF-kB controls multiple cellular processes in cancer due to its ability to promote cell proliferation, suppress apoptosis, promote cell migration, and suppress cell differentiation, opening the way for new therapeutic approaches against such a target [75-76]. In melanoma, NF-kB is constitutively activated since expression of the IkB proteins seems to be significantly reduced in comparison to nevi [77].

2.6. cKIT and tyrosinase kinase receptors

cKIT is a member of the transmenbrane receptor tyrosine kinase family that comprised five immunoglobin-like motifs, a single transmembrane region, an inhibitory cytoplasmic juxtamenbrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment [78]. Under physiological conditions, binding of the cKIT ligand stem-cell factor (SCF) to the extracellular domain of the receptor leads its dimerization, activation of the intracellular tyrosine kinase domain through autophosporylation of specific tyrosine residue [78]. The intracellular signaling through cKIT plays a critical role in the development of several mammalian cells, including growth, differentiation, migration, and proliferation of melanocytes [79]. It has been defined that cKIT recruits and activates a number of intracellular signaling pathways implicated in tumor progression, such as MAPK, PI3K/AKT, Src, activators of transcription (STAT), and phospholipase-C (PLC) [79-80].

Although the role of cKIT in melanomagenesis is still controversial, several studies have reported its downregulation during melanoma growth and invasion (in vertical growth phase of melanoma and metastatic lesions) [81-83]. Indeed, the majority of highly metastatic human melanomas do not express detectable levels of the cKIT receptor [83]. As a confirmation of this, over-expression of cKIT in metastatic melanoma cell lines led to important reduction in tumor growth, while cKIT activation through exposure to cKIT ligands induced apoptosis [83].

Specific mutations within the cKIT gene cause constitutive phosphorylation and activation of the kinase domain resulting in uncontrolled cell proliferation. [84]. Although such mutations seem to be more rare than BRAF and NRAS mutations, these may reflect the important role of cKIT tyrosine kinase in melanocyte development [84]. Sequencing of cKIT exons 11, 13, 17, and 18 revealed the most prevalent mutation to be K642, L576P, D816H-V, V559A [84]. The cKIT mutations are more common in mucosal and acral melanomas compared with cutaneous melanomas and are in most cases accompanied by an increase in gene copy number (40% mucosal or acral melanomas - frequently, associated with amplification of cyclin D1 - as well as 30% of melanomas on skin with chronic sun-induced damage) [84]. High expression levels of cKIT and CDK4 proteins have been identified in another subset of melanomas lacking BRAF mutations [85].

2.7. CTLA-4 and T-cell activation

The above-mentioned main intracellular molecular pathways are thus involved in tumor growth and survival, actively participating to the different phases of development and progression of melanoma. Additional extracellular factors, mainly represented by different components of the tumor microenvironment, have been implied to play a role in melanoma tissue invasion and metastatic dissemination. As an example, changes in the expression of adhesion molecules such as MCAM/MUC18, E-cadherin, and integrins occur in the transition from the radial growth phase (RGP) to the vertical growth phase (VGP) of melanoma; they are induced by both intracellular modifications [i.e., activation of the focal adhesion kinase (FAK) and integrin linked kinase (ILK) patways or high levels of activated ERK (phospoERK1-2)] and biological signals directly generated by the extracellular matrix (ECM), which is composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans in complex arrangements [22-23].

Among others, a block of the anti-tumor immune response induced by changes in pericellular microenvironment has been demonstrated to contribute to melanoma progression [86]. In recent past years, research has tried to better define the molecular mechanisms underlying the downregulation of the immune system by such pericellular components, in order to develop new therapeutic targets [87]. Actually, two immunomodulant antibodies, such as anti-CTLA4 and anti-PD1, have been demonstrated to be effective in inhibiting some down-regulators of the anti-tumor immune response [30, 88]. Moreover, drugs able to interfere with the differentiation of the myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg), which are both physiologically involved in controlling an abnormal immune response during the inflammatory processes and pathologically favoring tumor progression through suppression of T-cell activation, represent additional therapeutic strategies to be exploited [89].

For T-cell activation, melanoma antigens that are bound to the major histocompatibility complex (MHC) on antigen-presenting cells (APCs) require the costimulation of CD28 receptor on T-cells by CD80 or CD86 ligands on APCs [90]. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) can bind with greater affinity to CD80 and CD86, and thus disrupt the necessary costimulatory signal provided by APCs [88, 90]. This led to the hypothesis that blockade of CTLA-4 function may allow for optimal costimulation of CD28 receptors on T-cells by APC CD80/86, and enhanced T-cell activation [88, 90]. Ipilimumab (Yervoy Bristol-Myers Squibb, New York, NY) blocks the costimulatory signal required for T-cell activation [30, 88]. In particular, Ipilimumab is a recombinant human IgG1 monoclonal antibody that binds to CTLA-4 and blocks binding to CD80 or CD86 on APCs, thus increasing activation and proliferation of T cells [88]. Two randomized phase III trials have indicated a significant advantage in disease-free survival (DFS) and overall survival (OS) in either monotherapy or combination therapy [91-92]. The first trial compared monotherapy with Ipilimumab 3 mg/kg, combination of Ipilimumab with gp100 vaccine, and gp100 vaccine alone; the study demonstrated a significant advantage in OS for patients treated with Ipilimumab (regardless the addition off gp100) in comparison to those receiving the gp100 vaccine alone [median OS 10.0 vs 6.4 mesi; Hazard Ratio (HR) for death, 0.68; p<0,001) [91]. In the second study, Dacarbazine was administered as standard chemotherapeutic drug for melanoma patients, in association with Ipilimumab or placebo; the OS rate was significantly higher in the group of patients treated with Ipilimumab + Dacarbazine (11.2 vs. 9.1 months), with even more significant percentages of survival for such an association after one year (47.3% vs. 36.3%), two years (28.5% vs. 17.9%), or three years (20.8% vs. 12.2%) of follow-up [92]. The DFS and OS curves from the two studies have been indicated as largely overlapping, strongly demonstrating, for the first time in the history of medical treatments for the advanced disease, a clear survival benefit in metastatic (disease stage IV) melanoma. For these reasons, Ipilimumab has been recently approved by FDA and EMEA for the treatment of metastatic melanoma.

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3. Melanoma subtypes and targeted therapeutic options

The different molecular pathways involved into the pathogenesis of melanoma are functionally linked each other (Figure 1). There is thus a need to consider such biological cascades as part of a functional web, and the alterations detected in distinct components of the various pathways must be globally considered for the effects determining in such a functional web. This new vision helps in clarifying the reasons by which some alterations may coexist or not in specific melanoma subtypes. As an example, BRAF mutations may be observed in conjunction with PI3K alterations, but none of them may coexist with NRAS mutations; since BRAF and PI3K kinases act downstream NRAS protein, occurrence of NRAS mutations activating both MAPK and PI3K-AKT pathways makes unnecessary the further activation of BRAF and PI3K (upstream effectors of the MAPK and PI3K-AKT pathways, respectively). Analogously, oncogenic BRAF mutations are able to more intensively activate ERK protein, main last effector downstream the MAPK pathway, when inactivation of the mechanisms controlling senescence and apoptosis concomitantly occurs.

In an attempt to simplify such complex processes underlying the different phases of development and progression of melanoma, the main pathogenetic molecular alterations may be grouped in the following way:

  • oncogenic BRAF mutations, genomic rearrangements (mainly represented by allelic deletions) at the 9p21 chromosome, and increased expression levels of the AKT3 protein are the main alterations involved into the phase of stimulation of the proliferation for normal melanocytes (initial preneoplastic phase);

  • impairments of the mechanisms controlling the cell senescence, apoptosis and cell survival (which particularly include alterations in the different components of CDKN2A pathways: functional deficit of p16CDKN2A gene, amplification of CDK4-Cyclin D1/CCND1 loci, inattivation of TP53 gene through a deregulation of the p14CDKN2A-MDM2-p53 functional cascade), oncogenic mutations in NRAS gene, activating mutations and, to a lesser extent, gene amplifications of cKIT are the main alterations involved into the phase of acquisition of the malignant phenotype which underlay the development of melanoma (intermediate neoplastic phase);

  • complete silencing of p16CDKN2A gene, functional loss of PTEN, activation of the PI3K-AKT pathway, and amplification of the MITF gene are the main alterations involved into the phase of acquisition of a more aggressive and invasive phenotype which underlay the progression and dissemination of melanoma (final metasticase phase).

All these findings clearly indicate the existence of a complex molecular machinery that provides checks and balances in normal melanocytes, allowing a physiologically controlled cell proliferation. Progression from normal melanocytes to malignant metastatic cell in melanoma patients is the result of a combination of down- or up-regulations of the various effectors acting into the different molecular pathways. According to this, it has been proposed a linear model of pathogenesis of melanoma based on the sequential accumulation of most of the previously-described molecular alterations (Figure 2A) [7, 13, 93]. In a limited fraction of cases, it has been recently hypothesized a second non-linear model of melanomagenesis based on accumulation of the same genetic alterations in tissue stem cells, with generation of malignant cells directly forming RGP or VGP or metastatic melanoma lesions (Figure 2B) [94]. This latter hypothesis has been derived by the evidence of some inconsistencies of the linear model in subgroups of melanomas (i.e., incidence of BRAF mutations higher in VGP lesions than that found in RGP lesions [94]).

Figure 2.

Models of development and progression for melanoma. A, sequential model. B, non-linear model.

Nevertheless, all these evidence represent a strong indication that the different molecular pathways associated with the melanomagenesis does correspond to different subsets of melanoma patients, with distinguished biological and clinical behavior of the disease. Identification of such different patients’ subsets should be introduced in clinical trials, in order to better assess the classification of all predictive and prognostic factors associated with the disease as well as more accurately address patients to the most effective therapeutic intervention according to their biological and molecular status.

On the basis of the presence of the specific molecular features, a discrimination of the main subtypes of melanoma, along with the more appropriate therapeutic option for each subtype, could be schematically reported.

  1. Subtype MAPK

Prevalence of BRAF mutations, with tendency to increased expression level of activated ERK (phospoERK1-2) in melanoma tissues. This subtype benefits by the treatment with inhibitors of BRAF.

After failure of BAY 43-9006 (which is not specific for mutated BRAF, but suppresses activity of several different kinases [95]), a second generation BRAF inhibitor (Vemurafenib, also known as PLX4032 or RO5185426) was highly specific for V600EBRAF mutation and appeared very promising from the clinical point of view [96]. Very recently, results from a phase III study comparing the vemurafenib to dacarbazine have been indeed reported, indicating a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine [97]. Preliminary data using an additional V600EBRAF inhibitor compound, Dabrafenib (previously known as GSK2118436), seemed to point out that this molecule is also active on V600KBRAF and V600DBRAF mutations [98]; actually, treatment with such a compound is under evaluation in a phase III study among BRAF mutation positive stage III-IV melanoma patients.

However, preliminary data seem to indicate that a large variety of induced alterations may drive resistance to BRAF inhibitors: upregulation of the receptor tyrosine kinase (RTK) effectors [99], mutation in NRAS gene and platelet-derived growth factor receptor β (PDGFRβ) [99], amplification of the CCND1/Cyclin D1 gene or lack of phosphatase-and-tensin homologue (PTEN) function [100], mutations in downstream MEK gene [101], activation of MAPK pathway agonists such as COT kinase [102], or enhancement of the IGF-1R/PI3K signaling [103]. These findings highlight the need for combination therapy.

The MEK inhibitors (AS703026, E6201, GSK1120212, GDC0973, MEK162) as single agents have activity against melanoma, in patients either carrying BRAF mutations and unexposed to prior BRAF inhibitor therapy or presenting NRAS mutations [30]. A new combination of MEK and BRAF inhibitors as first line therapy for BRAF mutated melanoma patients naïve to prior anti-BRAF treatment is showing great promise [30].

The BRAF mutations may coexist with additional molecular alterations, with subsequent constitution of further biological and molecular subgroups of melanoma patients:

  1. Impairment of the p16CDKN2A-CDK4/CCND1-RB or p14CDKN2A-MDM2-TP53 pathways, with reduced expression of the p16CDKN2A protein and tendency to amplification of the CDK4/CCND1 gene loci or inactivation of the TP53 gene (with consequent functional loss of the p53 protein), respectively. This subtype benefits by the treatment with inhibitors of the cyclin-dependent kinases.

Melanoma patients carrying genetic alterations affecting p16CDKN2A could potentially be treated with inhibitors of CDK4/CDK6. There are currently no validated therapeutic options for melanoma with mutated p14CDKN2A. Conversely, several CDK4 inhibitors (Alvocidib, AT-7519, P1446A-05, PD-0332991, Flavopiridol/alvocidib/HMR 1275, P276-00, R547, SNS-032/ BMS-387032, UCN-01, ZK 304709/MTGI) are currently under investigation for a variety of cancer types, including metastatic melanoma, and results are awaited. For p53, there are currently no drugs, approved or in trials, against such a target. Conversely, an anti-sense agent (Oblimersen) targeted at nuclear Bcl-2 has been evaluated in trials, failing to demonstrate a significant clinical benefit among patients with melanoma [104].

  1. Amplification of MITF ± associated with reduction of the protein expression levels.

No drug targeting MITF has been developed; however, expression of MITF has been demonstrated to be reduced by compounds inhibiting the multiple histone deacetylase (HDAC) complex [105]. Ongoing trials based on HDAC-inhibitors [LBH589 (Panobinostat) or Valproic acid (Vorinostat)] will elucidate whether a clinical benefit could be obtained by down-regulating intracellular level of MITF protein.

  1. Activation of NF-kB.

Proteasome inhibitors, such as Bortezomib (Velcade, previously known as PS-341), represent a new class of anticancer therapeutic agents which inhibit degradation of important cell cycle and/or regulatory proteins, including IkB [106-107]. Bortezomid has been demonstrated to contribute in maintaining integrity of the complexes sequestering NF-kB into the cytoplasm, thus reducing the NF-kB activity [106-107]. Phase 2 studies combinating Bortezomib with other chemotherapeutic agents, including paclitaxel, carboplatin, or temozolomide equally have been established [108-109]. A compound that more directly targets the NF-kB pathway is BMS-345541 (4(2'-aminoethyl) amino-1,8-dimethylimidazo(1,2-a)quinoxaline), identified as a selective inhibitor of the catalytic subunits of IKK that binds at an allosteric site of the enzyme [110].

Since mutational activation of BRAF in human melanomas has been demonstrated to contribute to constitutive induction of NF-kB activity through an increase of the IKK activity [111], inhibition of BRAF signaling using the above mentioned inhibitors may decrease the NF-kB transcriptional activity and sensitize melanoma cells to apoptosis.

  1. Subtype NRAS

Prevalence of NRAS mutation, with markedly increased expression level of activated ERK (phospoERK1-2) and eventual activation of AKT. This subtype benefits by the treatment with inhibitors of MEK or mTORC.

To date, two approaches have been considered in developing drugs against RAS. The first is based on the block of farnesylation. A small clinical trial using an inhibitor of the farnesyl transferase enzyme failed to be efficacious in a melanoma cohort; however, patients included into such a study were not selected on the basis of their NRAS mutation status [112-113]. In the light of recent successes of the target therapies based on anti-BRAF or anti-cKIT inhibitors, a more stringently selected cohort carrying alterations in NRAS gene would have increased responsiveness. On the other hand, a direct targeting of RAS has been demonstrated to be very difficult [114]; this is the reason why therapeutic strategies have focused on inhibiting downstream effectors into the pathways activated by RAS (i.e., MEK inhibitors for the MAPK pathway - see above - and mTORC inhibitors for the PI3K-AKT pathway - see below), which represent the second treatment approach against RAS.

  1. Subtype cKIT

Prevalence of cKIT mutations ± gene amplification and/or increased protein expression levels. This subtype benefits by the treatment with inhibitors of cKIT (in particular, patients carrying gene mutations, with some sequence variants - such as K642E e L576P - which are highly responsive).

Activating cKIT mutations have been implicated in a variety of cancers, mainly represented by gastrointestinal stromal tumors (GIST) and chronic myelogenous leukemia (CML). This is the reason why several drugs targeting cKIT have been developed and tested in clinical trials, including Imatinib (approved for Philadelphia chromosome–positive CML and cKIT-positive GIST) and Sunitib (approved for advanced kidney cancer and Imatinib-resistant GIST) as well as Nilotinib and Dasatinib (approved for CML and Philadelphia chromosome-positive acute lymphoblastic leukemia).

The inhibitors of cKIT that may have a therapeutic benefit in melanoma, by inducing cell cycle arrest and apoptosis as well as significantly inhibiting cell migration and invasion of tumor cells, are:

Imatinib mesylate, formerly known as STI571, is designated chemically as 4-benzamide methanesulfonate. The efficacy of imatinib varies with the site of cKIT mutation; moreover, this drug can inhibit both the wild-type receptor activated by ligand and mutated receptor in the absence of ligand. However, imatinib is less effective at inhibiting the receptor with mutations in the enzymatic site (exon 17 mutations) [79];

Nilotinib (AMN107), which has been rationally designed based on the imatinib mesylate scaffold to have a more selective action. On this regard, Nilotinib inhibits both wild-type and cKIT mutants in exon 11 (V560del and V560G), exon 13 (K642E), double mutants involving exons 11, 13, and 17 as well as imatinib-resistant cKIT mutant (V560del/V654A) cells [115];

Dasatinib, which is a piperazinyl ethanol exhibiting increased potency but reduced selectivity compared with imatinib mesylate, has been demonstrated to inhibit both wild type and mutant cKIT in a dose-dependent manner, causing inhibition of cell migration and invasion through reduction of the phosphorylation of either Src kinase and FAK pathway [116-117].

  1. Subtype mTORC

Prevalence of PTEN loss (± PI3K mutations, which are mostly infrequent) and phosphorylation of AKT, with absence of concurrent mutations in BRAF gene. This subtype mainly benefits by the treatment with inhibitors of mTOR.

In melanoma cells, three potential targets may be considered for therapeutic intervention against this pathway: AKT, PI3K and mTOR. Restoration of functional PTEN or interfering with AKT and PI3K activity would increase chemosensitivity to apoptotic agents and improve the efficacy of anti-tumor treatment. Several inhibitors of PI3K (BKM120, BEZ235, BGT226, GDC0941, PX-866, SF1126, and XL147) and AKT (GSK690693, MK2206, and VQD-002) have been developed; results of ongoing trials are thus awaited. To date, clinical trials using agents against the PI3K/AKT pathway have failed to demonstrate significant efficacy [118]. However, one therapeutic approach which seems to inhibit this pathway is based on the use of mTOR inhibitors [119]: rapamycin, Temsirolimus (CCI-779), Everolimus (RAD001), Sirolimus and AZD8055. While controversial data have been reported for rapamycin (suppressing disease progression in some patients with glioblastoma but ineffective in controlling the disease in others) [120], a limited advantage in response rates has been so far described for Temsirolimus [121]. It is to be underlined that none of the trials with mTOR inhibitors included patients specifically selected for alterations in the AKT/PI3K pathway. Several clinical trials are investigating specific combinations of mTOR inhibitors and chemotherapy drugs in the treatment of melanoma.

  1. Subtype GNAQ/GNA11

Prevalence of GNAQ/GNA11 mutations, with increased expression level of phosphoERK. This subtype benefits by the treatment with inhibitors of MEK.

The GNAQ and GNA11 genes encodes specific GTP binding proteins that mediate signal transduction from the inner cell surface to the MAPK pathway through activation of the protein kinase C (PKC) enzyme [122]. Somatic mutations in GNAQ gene have been observed in about 90% of blue naevi, 50% of malignant blue naevi and 50% of primary uveal melanoma; conversely, the GNA11 mutations have been found in less than 10% of blue nevi, about one third of primary uveal melanomas, and about 60% of metastatic uveal melanoma [122]. Since mutations in these two genes have non been detected among all the remaining types of melanoma (cutaneous, acral, mucosal), a clinical trial aimed at testing efficacy of a MEK inhibitor, Dabrafenib/GSK1120212, has been focused on patients with metastatic uveal melanoma carrying GNAQ and/or GNA11 mutations [123].

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4. Diagnostic panel of molecular alterations

The most prevalent molecular alterations within the heterogeneous patterns of biological features which characterize the distinct subtypes of melanoma are here summarized, according to the anatomical site of melanoma onset, the degree of exposure to the sun, and the histologic characteristics of the tumor lesions.

  • Acral melanoma

Mutation ± amplification of cKIT; amplification of CDK4 or CCND1 (associated with increased expression levels of the related proteins); amplification of the 11q13, 22q11-13, and/or 5p15 genomic loci.

  • Melanoma of the head and neck district and melanoma on skin with chronic sun-induced damage (CSD)

Amplification of CDK4 and/or CCND1; increased expression levels of p53 protein; mutation ± amplification of cKIT (in about 5% of cases).

  • Melanoma of the trunk and melanoma on skin without chronic sun-induced damage (non-CSD)

Mutation of BRAF or, alternatively, NRAS (with eventual coexistence of molecolar alterations which may ba associated with BRAF mutations; see above); tendency to reduced expression of the p53 protein; occurrence of specific polymorphisms in MC1R gene.

  • Mucosal melanoma

Mutation ± amplification of cKIT; amplification of CDK4 or CCND1 (associated with increased expression levels of the related proteins); mutation of BRAF (in less than 10% of cases).

  • Uveal melanoma

Mutation ± amplification of cKIT; mutation of GNAQ and/or GNA11.

Knowledge of the principal molecular alterations to be tested in patients with such distinct subtypes of melanoma will be of great clinical importance, because it is likely to result in separate targeted therapeutic approaches and prevention strategies. To date, it has been already developed a panel of molecular tests to be performed in patients with melanoma from different anatomical locations (Figure 3). This initial “flow-chart” will surely become more detailed and enriched on the basis of the progressive identification and validation of additional genetics and molecular alterations correlated with the disease.

Finally, recent meta-analyses tried to define the prognostic role of majority of molecular alterations previously described [124-125]:

  • negative prognostic factors

loss of p53; over-expression of iNOS, AP-2, MMP-2 and metallothioneine; increased proliferation index (high expression levels of Ki-67);

  • positive prognostic factors

reduced expression or loss of p16CDKN2A; over-expression of Bcl-2 and ATF-2 (± associated with simultaneous increases expression of beta-catenin, fibronectin and p21 proteins).

Figure 3.

Principal genetic and molecular tests on tumor tissues for the different types of melanoma. Amp, gene amplification detected by fluorescence in situ hybridization (FISH) analysis. Exp, protein expression level detected by immunohistochemistry. Mut, gene sequence variation detected by mutation analysis. In red, tests for less prevalent alterations.

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5. Conclusion

Taken together all the described molecular mechanisms involved in melanoma genesis and progression, data seem to emphasize the fact that in melanoma, but probably in all types of cancer, it is unlikely that targeting a single component in the signalling pathway will yield significant anti-tumour responses. For this purpose, molecular analyses could help clinicians to define the prognosis (prognostic value) as well as to make a prediction, identifying the subsets of patients who would be expected to be more or less likely to respond to specific therapeutic interventions (predictive value).

In other words, it is becoming evident that combination therapies targeting simultaneously several signaling pathways might be a winning therapeutic strategy to treat melanoma patients. Preclinical studies using combination of anti BRAF and AKT3 siRNA demonstrated a significantly higher reduction of tumor growth compared to single agent administration [126-127]. There is also evidence of synergism among MEK and PI3K inhibitors as well as promising results have been obtained by combinations of the mTOR inhibitors and sorafenib or MEK inhibitors [121, 128-129]. In contrast to single agent activity, these combinations of target drugs resulted in recovering of apoptosis by complete down-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Cooperation between BRAF and MEK inhibitors has also been demonstrated in preclinical studies with a consistent increase of apoptosis and abrogation of ERK activation compared to BRAF inhibitor alone [130]. Such cooperation was based on the observation that MEK activation was not abrogated in melanoma cells that develop resistance to BRAF inhibitors [131-134]. BRAF and MEK targeted synergic therapy is currently tested in a phase I clinical trial (NCT01072175) which combines the selective RAF inhibitor GSK2118436 with the MEK inhibitor GSK1120212 in patients with BRAF mutant tumors [135].

After decades without perspective, the history of medical treatment for the advanced melanoma is rapidly changing. Combined therapeutic approaches do represent the next challenge for treatment of patients with such a disease.

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Acknowledgments

Authors are always grateful to patients for their cooperation into the various studies. Work was funded by Regione Autonoma della Sardegna and Ricerca Finalizzata Ministero della Salute.

References

  1. 1. Curado MP, Edwards B, Shin HR, Storm H, Ferlay J, Heanue M, Boyle P (eds). Cancer Incidence in Five Continents, Vol. IX, International Agency for Research on Cancer (IARC) Scientific Publications, No. 160, Lyon, IARC, 2007.
  2. 2. Linos E, Swetter S, Cockburn MG, et al. Increasing burden of melanoma in the United States. J Invest Derm. 2009;129: 1666-74
  3. 3. Rigel DS. Epidemiology of melanoma. Semin. Cutan. Med. Surg. 2010; 29: 204-209
  4. 4. Welch HG, Woloshin S, Schwartz LM. Skin biopsy rates and incidence of melanoma: population based ecological study. BMJ 2005: 331: 481;
  5. 5. de Vries E, Coebergh JW. Melanoma incidence has risen in Europe. BMJ. 2005: 331: 698
  6. 6. Jhappan C, Noonan FP, Merlino G. Ultraviolet radiation and cutaneous malignant melanoma. Oncogene 2003: 22: 3099-3112
  7. 7. Thompson JF, Scolyer RA, Kefford RF. Cutaneous melanoma. The Lancet 2005: 365: 687-701
  8. 8. Cho E, Rosner BA, Feskanich D, Colditz GA. Risk factors and individual probabilities of melanoma for whites. J Clin Oncol. 2005: 23: 2669-2675
  9. 9. Balch CM, Buzaid AC, Soong SJ, Atkins MB, Cascinelli N, Coit DG, Fleming ID, Gershenwald JE, Houghton A Jr, Kirkwood JM, McMasters KM, Mihm MF, Morton DL, Reintgen DS, Ross MI, Sober A, Thompson JA, Thompson JF. Final version of the American Joint Committee on Cancer Staging System for Cutaneous Melanoma. J Clin Oncol 2001; 19: 3635-48
  10. 10. Bastian BC, Olshen AB, LeBoit PE, Pinkel D. Classifying melanocytic tumors based on DNA copy number changes. Am J Pathol 2003;163:1765-70
  11. 11. Haluska FG, Tsao H, Wu H, Haluska FS, Lazar A, Goel V. Genetic alterations in signaling pathways in melanoma. Clin Cancer Res. 2006; 12: 2301-7
  12. 12. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, Cho KH, Aiba S, Bröcker EB, LeBoit PE, Pinkel D, Bastian BC. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005; 353:2135-47
  13. 13. Palmieri G, Capone ME, Ascierto ML, Gentilcore G, Stroncek DF, Casula M, Sini MC, Palla M, Mozzillo N, Ascierto PA. Main roads to melanoma. J Transl Med. 2009; 7: 86
  14. 14. Miller AJ, Mihm MC. Melanoma. N Engl J Med 2006; 355, 51-65
  15. 15. Giehl K. Oncogenic Ras in tumor progression and metastasis. Biol Chem. 2005;386:193–205
  16. 16. Heidorn SJ, Milagre C, Whittaker S, et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010 Jan 22;140:209-21
  17. 17. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature. 2002;418:934
  18. 18. Quinlan MP, Settleman J. Isoform-specific ras functions in development and cancer. Future Oncol. 2009; 5:105-16
  19. 19. Heidorn SJ, Milagre C, Whittaker S, et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010; 140:209-21
  20. 20. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA. Mutations of the BRAF gene in human cancer. Nature. 2002; 417:949-54
  21. 21. Smalley KSM. A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int J Cancer 2003; 104:527-32
  22. 22. McGary EC, Chelouche Lev D, Bar-Eli M. Cellular adhesion pathways and metastatic potential of human melanoma. Cancer Biol Ther 2002; 5: 459-465
  23. 23. Kuphal S, Bauer R, Bosserhoff AK. Integrin signaling in malignant melanoma. Cancer Metastasis Reviews 24: 195–222, 2005
  24. 24. Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, Moses TY, Hostetter G, Wagner U, Kakareka J, Salem G, Pohida T, Heenan P, Duray P, Kallioniemi O, Hayward NK, Trent JM, Meltzer PS. High frequency of BRAF mutations in nevi. Nat Genet. 2003, 33:19-20
  25. 25. Michaloglou C, Vredeveld LC, Soengas MS, Denoyelle C, Kuilman T, van der Horst CM, Majoor DM, Shay JW, Mooi WJ, Peeper DS. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 2005; 436, 720–724
  26. 26. Patton EE, Widlund HR, Kutok JL, Kopani KR, Amatruda JF, Murphey RD, Berghmans S, Mayhall EA, Traver D, Fletcher CD, Aster JC, Granter SR, Look AT, Lee C, Fisher DE, Zon LI. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr Biol. 2005;15:249–54
  27. 27. Wajapeyee N, Serra RW, Zhu X, et al. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell. 2008;132:363–374
  28. 28. Dhomen N, Reis-Filho JS, da Rocha Dias S, et al. Oncogenic Braf induces melanocyte senescence and melanoma in mice. Cancer Cell. 2009;15:294–303
  29. 29. Smalley KS, Lioni M, Dalla Palma M, Xiao M, Desai B, Egyhazi S, Hansson J, Wu H, King AJ, Van Belle P, Elder DE, Flaherty KT, Herlyn M, Nathanson KL. Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas. Mol Cancer Ther. 2008; 7: 2876-83
  30. 30. Ascierto PA, Grimaldi AM, Curti B, Faries MB, Ferrone S, Flaherty K, Fox BA, Gajewski TF, Gershenwald JE, Gogas H, Grossmann K, Hauschild A, Hodi FS, Kefford R, Kirkwood JM, Leachmann S, Maio M, Marais R, Palmieri G, Morton DL, Ribas A, Stroncek DF, Stewart R, Wang E, Mozzillo N, Marincola FM. Future Perspectives in melanoma research. Meeting report from the "Melanoma Research: a bridge from Naples to the World. Napoli, December 5th-6th 2011". J. Transl Med. 10: 83, 2012
  31. 31. Pomerantz J, Schreiber-Agus N, Lie´ geois NJ. The Ink4a tumor suppressor gene product, 19Arf, interacts with MDM2 and neutralizes DM2’s inhibition of p53. Cell 1998; 92: 713-23
  32. 32. Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 1998: 92: 725–734
  33. 33. Palmieri G, Capone ME, Ascierto ML, Gentilcore G, Stroncek DF, Casula M, Sini MC, Palla M, Mozzillo N, Ascierto PA. Main roads to melanoma. J Transl Med. 7: 86, 2009
  34. 34. Soufir N, Avril MF, Chompret A, Demenais F, Bombled J, Spatz A, Stoppa-Lyonnet D, Bénard J, Bressac-de Paillerets B. Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. The French Familial Melanoma Study Group. Hum Mol Genet. 1998; 7: 209-16
  35. 35. Sauter ER, Yeo UC, von Stemm A, Zhu W, Litwin S, Tichansky DS, Pistritto G, Nesbit M, Pinkel D, Herlyn M, Bastian BC. Cyclin D1 is a candidate oncogene in cutaneous melanoma. Cancer Res. 2002; 62:3200-6
  36. 36. Box NF, Terzian T: The role of p53 in pigmentation, tanning and melanoma. Pigment Cell Melanoma Res 2008, 21:525-533
  37. 37. Hocker TL, Singh MK, Tsao H. Melanoma genetics and therapeutic approaches in the 21st century: moving from the benchside to the bedside. J Invest Dermatol. 2008; 128:2575-95
  38. 38. Bennett DC. How to make a melanoma: what do we know of the primary clonal events? Pigment Cell Melanoma Res. 2008; 21:27-38
  39. 39. Sini MC, Manca A, Cossu A, Budroni M, Botti G, Ascierto PA, Cremona F, Muggiano A, D'Atri S, Casula M, Baldinu P, Palomba G, Lissia A, Tanda F, Palmieri G. Molecular alterations at chromosome 9p21 in melanocytic nevi and melanoma. Br. J. Dermatology 158: 243-250, 2008
  40. 40. Meyle KD, Guldberg P. Genetic risk factors for melanoma. Hum Genet. 2009; 126: 499-510
  41. 41. Fargnoli MC, Gandini S, Peris K, Maisonneuve P, Raimondi S. MC1R variants increase melanoma risk in famiglie with CDKN2A mutations: A meta-analysis. Eur J Cancer 2010; 46: 1413-20
  42. 42. Scott MC, Wakamatsu K, Ito S, et al. Human melanocortin 1 receptor variants, receptor function and melanocyte response to UV radiation. J Cell Sci. 2002; 115: 2349-55
  43. 43. Levy C, Khaled M, Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol. Med. 2006;12, 406–414
  44. 44. Hauser JE, Kadekaro AL, Kavanagh RJ, et al. Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes. Pigment Cell Res. 2006; 19:303-14
  45. 45. Landi MT, Bauer J, Pfeiffer RM, et al. MC1R germline variants confer risk for BRAF-mutant melanoma. Science 2006; 313:521-2
  46. 46. Fargnoli MC, Pike K, Pfeiffer RM, et al. MC1R variants increase risk of melanomas harboring BRAF mutations. J Invest Dermatol. 2008; 128:2485-90
  47. 47. Thomas NE, Kanetsky PA, Edmiston SN, et al. Relationship between germline MC1R variants and BRAF-mutant melanoma in a North Carolina population-based study. J Invest Dermatol. 2010; 130:1463-5
  48. 48. Scherer D, Rachakonda PS, Angelini S, et al. Association between the germline MC1R variants and somatic BRAF/NRAS mutations in melanoma tumors. J Invest Dermatol. 2010; 130:2844-8
  49. 49. Hacker E, Hayward NK, Dumenil T, et al. The association between MC1R genotype and BRAF mutation status in cutaneous melanoma: findings from an Australian population.J Invest Dermatol. 2010; 130:241-8
  50. 50. Wu H, Goel V, Haluska FG. PTEN signaling pathways in melanoma. Oncogene. 2003; 22: 3113-22
  51. 51. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer. 2002;2:489-501
  52. 52. Plas DR, Thompson CB. Akt-dependent transformation: there is more to growth than just surviving. Oncogene. 2005;24:7435–7442
  53. 53. Aziz SA, Davies M, Pick E, Zito C, Jilaveanu L, Camp RL, Rimm DL, Kluger Y, Kluger HM. Phosphatidylinositol-3-kinase as a therapeutic target in melanoma. Clin Cancer Res. 2009; 15: 3029-36
  54. 54. Mirmohammadsadegh A, Marini A, Nambiar S, et al. Epigenetic silencing of the PTEN gene in melanoma. Cancer Res. 2006; 66: 6546-52
  55. 55. Goel VK, Lazar AJ, Warneke CL, Redston MS, Haluska FG. Examination of mutations in BRAF, NRAS, and PTEN in primary cutaneous melanoma. J Invest Dermatol. 2006; 126:154-60
  56. 56. Tsao H, Goel V, Wu H, et al. Genetic interaction between NRAS and BRAF mutations and PTEN/MMAC1 inactivation in melanoma. J Invest Dermatol 2004; 122: 337–41
  57. 57. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, Beroukhim R, Milner DA, Granter SR, Du J, Lee C, Wagner SN, Li C, Golub TR, Rimm DL, Meyerson ML, Fisher DE, Sellers WR. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature. 2005; 436:117-22.
  58. 58. Carreira S, Goodall J, Aksan I, et al. Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression. Nature 2005;433: 764-769
  59. 59. Loercher AE, Tank EM, Delston RB, Harbour JW. MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A. J Cell Biol 2005;168: 35-40
  60. 60. Wellbrock C, Marais R: Elevated expression of MITF counteracts B-RAF stimulated melanocyte and melanoma cell proliferation. J Cell Biol 2005;170: 703-708
  61. 61. McGill GG, Horstmann M, Widlund HR, et al. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 2002;109:707-718.
  62. 62. Du J, Widlund HR, Horstmann MA, et al. Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF. Cancer Cell 2004;6: 565-576
  63. 63. Wellbrock C, Rana S, Paterson H, et al. Oncogenic BRAF regulates melanoma proliferation through the lineage specific factor MITF. PLoS ONE 3:2734,2008
  64. 64. Ekmekcioglu S, Tang CH, Grimm EA. NO news is not necessarily good news in cancer. Curr Cancer Drug Targets. 2005; 5:103-15
  65. 65. Massi D, De Nisi MC, Franchi A, Mourmouras V, Baroni G, Panelos J, Santucci M, Miracco C. Inducible nitric oxide synthase expression in melanoma: implications in lymphangiogenesis. Mod Pathol. 2009; 22:21-30
  66. 66. Grimm EA, Ellerhorst J, Tang CH, Ekmekcioglu S. Constitutive intracellular production of iNOS and NO in human melanoma: possible role in regulation of growth and resistance to apoptosis. Nitric Oxide. 2008; 19:133-7
  67. 67. Sikora AG, Gelbard A, Davies MA, Sano D, Ekmekcioglu S, Kwon J, Hailemichael Y, Jayaraman P, Myers JN, Grimm EA, Overwijk WW. Targeted inhibition of inducible nitric oxide synthase inhibits growth of human melanoma in vivo and synergizes with chemotherapy. Clin Cancer Res. 2010; 16:1834-44
  68. 68. Tschugguel W, Pustelnik T, Lass H, Mildner M, Weninger W, Schneeberger C, Jansen B, Tschachler E, Waldhör T, Huber JC, Pehamberger H: Inducible nitric oxide synthase (iNOS) expression may predict distant metastasis in human melanoma. Br J Cancer 1999, 79:1609-1612
  69. 69. Ahmed B, Van den Oord JJ: Expression of the inducible isoform of nitric oxide synthase in pigment cell lesions of the skin. Br J Dermatol 2000, 142:432-40
  70. 70. Ekmekcioglu S, Ellerhorst JA, Prieto VG, Johnson MM, Broemeling LD, Grimm EA. Tumor iNOS predicts poor survival for stage III melanoma patients. Int J Cancer. 2006; 119:861-6
  71. 71. Dhawan P, Richmond A. A novel NF-kappa B-inducing kinase-MAPK signaling pathway up-regulates NF-kappa B activity in melanoma cells. J Biol Chem 2002; 277:7920-8
  72. 72. Uffort DG, Grimm EA, Ellerhorst JA. NF-kappaB mediates mitogen-activated protein kinase pathway-dependent iNOS expression in human melanoma. J Invest Dermatol. 2009; 129:148-54
  73. 73. Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kB. J Clin Invest 2001;107:241–246
  74. 74. Madonna G, Dansky Ullman C, Gentilcore G, Palmieri G, Ascierto PA. NF-kappaB as potential target in the treatment of melanoma. J. Transl Med. 2012; 10: 53
  75. 75. Chaturvedi MM, Sung B, Yadav VR, Kannappan R, Aggarwal BB. NF-kB addiction and its role in cancer: 'one size does not fit all'. Oncogene. 2011; 30:1615-30.
  76. 76. Kim HJ, Hawke N, Baldwin AS. NF-kappaB and IKK as therapeutic targets in cancer. Cell Death Differ. 2006; 13:738-47
  77. 77. McNulty SE, del Rosario R, Cen D, et al. Comparative expression of NFkappaB proteins in melanocytes of normal skin vs. benign intradermal naevus and human metastatic melanoma biopsies. Pigment Cell Res. 2004; 17:173-80
  78. 78. Clifford D, Mol, Kheng B. Lim. Structure of a c-Kit product complex reveals the basis for kinase transactivation. J. Biol Chem 2003; 278: 31461-64
  79. 79. Smalley KS, Sondak VK, Weber JS. c-KIT signaling as the driving oncogenic event in sub-groups of melanomas. Histol Histopathol. 2009; 24:643-50.
  80. 80. Alexeev V, Yoon K. Distinctive role of the cKit receptor tyrosine kinase signaling in mammalian melanocytes. J Invest Dermatol. 2006; 126: 1102-1110
  81. 81. Shen SS, Zhang PS, Eton O, Prieto VG. Analysis of protein tyrosine kinase expression in melanocytic lesions by tissue array. J Cutan Pathol 2003; 30: 539-547
  82. 82. Stefanou D, Batistatou A, Zioga A. Immunohistochemical expression of vascular endothelial growth factor (VEGF) and CKIT in cutaneous melanocytic lesions. Int J Surg Pathol 2004; 12: 133-138.
  83. 83. Curtin JA, Busam K, Pinkel D, Bastian BC. Somatic activation of KIT in distinct subtypes of melanoma. J. Clin. Oncol. 2006; 24: 4340-43
  84. 84. Beadling C, Jacobson-Dunlop E, Hodi FS, Le C, Warrick A, Patterson J, Town A, Harlow A, Cruz F 3rd, Azar S, Rubin BP, Muller S, West R, Heinrich MC, Corless CL. Kit gene mutations and copy numbering melanoma subtypes .Clin Cancer Res 2008; 14: 6821-6828
  85. 85. Riviera RS, Nagatsuka H, Gunduz M, Cengiz B, Gunduz E, Siar CH, Tsujigiwa H, Tamamura R, Han K N, Nagai N. C-kit protein expression correlated with activating mutations in KIT gene in oral mucosal melanoma.Virchows Arch. 2008; 452: 27-32
  86. 86. Postow MA, Harding J, Wolchok JD. Targeting immune checkpoints: releasing the restraints on anti-tumor immunity for patients with melanoma. Cancer J. 2012; 18:153-9
  87. 87. Melero J, Hervas-Strubbs S, Glennie M et al. Immunostimulatory monoclonal antibodies for cancer therapy. Nat Rev Cancer 2007; 7:95-106
  88. 88. Ascierto PA, Marincola FM, Ribas A. Anti-CTLA4 monoclonal antibodies: the past and the future in clinical application. J Transl Med. 2011 Nov 13;9:196
  89. 89. Ozao-Choy J, Ma G, Kao J, Wang GX, Meseck M, Sung M, Schwartz M, Divino CM, Pan PY, Chen SH. The novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune-based cancer therapies. Cancer Res. 2009 Mar 15;69(6):2514-22
  90. 90. Lee KM, Chuang E, Griffin M, et al. Molecular basis of T cell inactivation by CTLA-4. Science. 1998; 282: 2263–2266
  91. 91. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den Eertwegh AJ, Lutzky J, Lorigan P, Vaubel JM, Linette GP, Hogg D, Ottensmeier CH, Lebbé C, Peschel C, Quirt I, Clark JI, Wolchok JD, Weber JS, Tian J, Yellin MJ, Nichol GM, Hoos A, Urba WJ. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010; 363:711-23
  92. 92. Robert C, Thomas L, Bondarenko I, O'Day S, M D JW, Garbe C, Lebbe C, Baurain JF, Testori A, Grob JJ, Davidson N, Richards J, Maio M, Hauschild A, Miller WH Jr, Gascon P, Lotem M, Harmankaya K, Ibrahim R, Francis S, Chen TT, Humphrey R, Hoos A, Wolchok JD. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011; 364:2517-26
  93. 93. Miller AJ, Mihm MC. Melanoma. N Engl J Med 2006; 355: 51-65
  94. 94. Zabierowski SE, Herlyn M. Melanoma stem cells: the dark seed of melanoma. J Clin Oncol. 2008; 26: 2890-4
  95. 95. Eisen T, Ahmad T, Flaherty KT, Gore M, Kaye S, Marais R, Gibbens I, Hackett S, James M, Schuchter LM, Nathanson KL, Xia C, Simantov R, Schwartz B, Poulin-Costello M, O'Dwyer PJ, Ratain MJ. Sorafenib in advanced melanoma: a phase II randomised discontinuation trial analysis. Br J Cancer 2006; 95: 581-86
  96. 96. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O'Dwyer PJ, Lee RJ, Grippo JF, Nolop K, Chapman PB. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010; 363, 809-19
  97. 97. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M, Hogg D, Lorigan P, Lebbe C, Jouary T, Schadendorf D, Ribas A, O'Day SJ, Sosman JA, Kirkwood JM, Eggermont AM, Dreno B, Nolop K, Li J, Nelson B, Hou J, Lee RJ, Flaherty KT, McArthur GA; BRIM-3 Study Group. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364:2507-16.
  98. 98. Falchook GS, Long GV, Kurzrock R, Kim KB, Arkenau TH, Brown MP, Hamid O, Infante JR, Millward M, Pavlick AC, O'Day SJ, Blackman SC, Curtis CM, Lebowitz P, Ma B, Ouellet D, Kefford RF. Dabrafenib in patients with melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial. Lancet. 2012; 379:1893-901
  99. 99. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, Chen Z, Lee MK, Attar N, Sazegar H, Chodon T, Nelson SF, McArthur G, Sosman JA, Ribas A, Lo RS. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 2010; 468: 973-77
  100. 100. Smalley KS, Sondak VK. Melanoma--an unlikely poster child for personalized cancer therapy. N Engl J Med. 2010; 363:876-8
  101. 101. Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, Kehoe SM, Johannessen CM, Macconaill LE, Hahn WC, Meyerson M, Garraway LA. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol. 2011; 29: 3085-96.
  102. 102. Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA, Emery CM, Stransky N, Cogdill AP, Barretina J, Caponigro G, Hieronymus H, Murray RR, Salehi-Ashtiani K, Hill DE, Vidal M, Zhao JJ, Yang X, Alkan O, Kim S, Harris JL, Wilson CJ, Myer VE, Finan PM, Root DE, Roberts TM, Golub T, Flaherty KT, Dummer R, Weber BL, Sellers WR, Schlegel R, Wargo JA, Hahn WC, Garraway LA. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 2010; 468, 968-72
  103. 103. Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, Cipolla AK, Wubbenhorst B, Xu X, Gimotty PA, Kee D, Santiago-Walker AE, Letrero R, D'Andrea K, Pushparajan A, Hayden JE, Brown KD, Laquerre S, McArthur GA, Sosman JA, Nathanson KL, Herlyn M. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 2010; 18, 683-95
  104. 104. Bedikian AY, Millward M, Pehamberger H, Conry R, Gore M, Trefzer U, Pavlick AC, DeConti R, Hersh EM, Hersey P, Kirkwood JM, Haluska FG; Oblimersen Melanoma Study Group. Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. J Clin Oncol. 2006; 24:4738-45
  105. 105. Yokoyama S, Feige E, Poling LL, Levy C, Widlund HR, Khaled M, Kung AL, Fisher DE. Pharmacologic suppression of MITF expression via HDAC inhibitors in the melanocyte lineage. Pigment Cell Melanoma Res. 2008; 21:457-63
  106. 106. Adams J. The proteasome: a suitable antineoplastic target. Nat Rev Cancer 2004;4:349–60
  107. 107. Amiri KI, Horton LW, LaFleur BJ, Sosman JA, Richmond A. Augmenting chemosensitivity of malignant melanoma tumors via proteasome inhibition: Implication for bortezomib (VELCADE, PS-341) as a therapeutic agent for malignant melanoma. Cancer Res 2004;64:4912–4918
  108. 108. Su Y, Amiri KI, Horton LW, Yu Y, Ayers GD, Koehler E, Kelley MC, Puzanov I, Richmond A, Sosman JA. A phase I trial of bortezomib with temozolomide in patients with advanced melanoma: toxicities, antitumor effects, and modulation of therapeutic targets. Clin Cancer Res 2010;16:348-57
  109. 109. Croghan GA, Suman VJ, Maples WJ, Albertini M, Linette G, Flaherty L, Eckardt J, Ma C, Markovic SN, Erlichman C. A Study of Paclitaxel, Carboplatin, and Bortezomib in the Treatment of Metastatic Malignant Melanoma. Cancer 2010;116:3463-8
  110. 110. López-Guerra M, Roué G, Pérez-Galán P, Alonso R, Villamor N, Montserrat E, Campo E, Colomer D. p65 activity and ZAP-70 status predict the sensitivity of chronic lymphocytic leukemia cells to the selective IkappaB kinase inhibitor BMS-345541. Clin Cancer Res. 2009; 15:2767-76
  111. 111. Liu J, Suresh Kumar KG, Yu D, Molton SA, McMahon M, Herlyn M, Thomas-Tikhonenko A, Fuchs SY. Oncogenic BRAF regulates β-Trcp expression and NF-kB activity in human melanoma cells. Oncogene 2007; 26:1954–1958
  112. 112. Niessner H, Beck D, Sinnberg T, Lasithiotakis K, Maczey E, Gogel J, Venturelli S, Berger A, Mauthe M, Toulany M, Flaherty K, Schaller M, Schadendorf D, Proikas-Cezanne T, Schittek B, Garbe C, Kulms D, Meier F. The farnesyl transferase inhibitor lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells. J Invest Dermatol. 2011; 131:468-79
  113. 113. Hong DS, Sebti SM, Newman RA, Blaskovich MA, Ye L, Gagel RF, Moulder S, Wheler JJ, Naing A, Tannir NM, Ng CS, Sherman SI, El Naggar AK, Khan R, Trent J, Wright JJ, Kurzrock R. Phase I trial of a combination of the multikinase inhibitor sorafenib and the farnesyltransferase inhibitor tipifarnib in advanced malignancies. Clin Cancer Res. 2009; 15:7061-8
  114. 114. Konstantinopoulos PA, Karamouzis MV, Papavassiliou AG. Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat Rev Drug Discov. 2007; 6:541-55
  115. 115. Tran A, Tawbi HA. A potential role for nilotinib in KIT-mutated melanoma. Expert Opin Investig Drugs. 2012; 21:861-9
  116. 116. Serrels A, Macpherson IR, Evans TR, Lee FY, Clark EA, Sansom OJ, Ashton GH, Frame MC, Brunton VG. Identification of potential biomarkers for measuring inhibition of Src kinase activity in colon cancer cells following treatment with dasatinib. Mol Cancer Ther. 2006; 5: 3014–3022
  117. 117. Gnoni A, Marech I, Silvestris N, Vacca A, Lorusso V. Dasatinib: an anti-tumour agent via Src inhibition. Curr Drug Targets. 2011; 12:563-78
  118. 118. Courtney KD, Corcoran RB, Engelman JA. The PI3K pathway as drug target in human cancer.J Clin Oncol. 2010; 28:1075-83
  119. 119. Abraham RT, Eng CH. Mammalian target of rapamycin as a therapeutic target in oncology.Expert Opin Ther Targets. 2008; 12:209-22
  120. 120. Cloughesy TF, Yoshimoto K, Nghiemphu P, Brown K, Dang J, Zhu S, Hsueh T, Chen Y, Wang W, Youngkin D, Liau L, Martin N, Becker D, Bergsneider M, Lai A, Green R, Oglesby T, Koleto M, Trent J, Horvath S, Mischel PS, Mellinghoff IK, Sawyers CL. Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med. 2008; 5:e8
  121. 121. Margolin KA, Moon J, Flaherty LE, Lao CD, Akerley WL 3rd, Othus M, Sosman JA, Kirkwood JM, Sondak VK. Randomized phase II trial of sorafenib with temsirolimus or tipifarnib in untreated metastatic melanoma (S0438). Clin Cancer Res. 2012; 18:1129-37
  122. 122. Van Raamsdonk CD, Griewank KG, Crosby MB, Garrido MC, Vemula S, Wiesner T, Obenauf AC, Wackernagel W, Green G, Bouvier N, Sozen MM, Baimukanova G, Roy R, Heguy A, Dolgalev I, Khanin R, Busam K, Speicher MR, O'Brien J, Bastian BC. Mutations in GNA11 in uveal melanoma. N Engl J Med. 2010; 363:2191-9
  123. 123. Khalili JS, Yu X, Wang J, Hayes BC, Davies MA, Lizee G, Esmaeli B, Woodman SE. Combination Small Molecule MEK and PI3K Inhibition Enhances Uveal Melanoma Cell Death in a Mutant GNAQ- and GNA11-Dependent Manner. Clin Cancer Res. 2012 Jul 19
  124. 124. Gould Rothberg BE, Bracken MB, Rimm DL. Tissue biomarkers for prognosis in cutaneous melanoma: a systematic review and meta-analysis. J Natl Cancer Inst. 2009; 101:452-74
  125. 125. Gould Rothberg BE, Berger AJ, Molinaro AM, Subtil A, Krauthammer MO, Camp RL, Bradley WR, Ariyan S, Kluger HM, Rimm DL. Melanoma prognostic model using tissue microarrays and genetic algorithms. J Clin Oncol. 2009; 27:5772-80
  126. 126. Cheung M, Sharma A, Madhunapantula SV, Robertson GP. Akt3 and mutant V600E B-Raf cooperate to promote early melanoma development. Cancer Res. 2008; 68, 3429–3439
  127. 127. Tran MA, Gowda R, Sharma A, Park EJ, Adair J, Kester M, Smith NB, Robertson GP. Targeting V600EB-Raf and Akt3 using nanoliposomal small interfering RNA inhibits cutaneous melanocytic lesion development. Cancer Res. 2008; 68, 7638–7649
  128. 128. Lasithiotakis KG, Sinnberg TW, Schittek B, Flaherty KT, Kulms D, Maczey E, Garbe C, Meier FE. Combined inhibition of MAPK and mTOR signaling inhibits growth, induces cell death, and abrogates invasive growth of melanoma cells. J Invest Dermatol. 2008;128:2013-23
  129. 129. Davies MA, Fox PS, Papadopoulos NE, Bedikian AY, Hwu WJ, Lazar AJ, Prieto VG, Culotta KS, Madden TL, Xu Q, Huang S, Deng W, Ng CS, Gupta S, Liu W, Dancey JE, Wright JJ, Bassett RL, Hwu P, Kim KB. Phase I study of the combination of sorafenib and temsirolimus in patients with metastatic melanoma. Clin Cancer Res. 2012;18:1120-8
  130. 130. Paraiso KH, Fedorenko IV, Cantini LP, Munko AC, Hall M, Sondak VK, Messina JL, Flaherty KT, Smalley KS. Recovery of phospho-ERK activity allows melanoma cells to escape from BRAF inhibitor therapy. Br J Cancer. 2010; 102:1724-30
  131. 131. Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, Reis-Filho JS, Springer CJ, Pritchard C, Marais R. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 2010;140:209-21
  132. 132. Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature. 2010; 464:427-30
  133. 133. Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G, Shi H, Atefi M, Titz B, Gabay MT, Salton M, Dahlman KB, Tadi M, Wargo JA, Flaherty KT, Kelley MC, Misteli T, Chapman PB, Sosman JA, Graeber TG, Ribas A, Lo RS, Rosen N, Solit DB. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 2011; 480: 387-90
  134. 134. Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, Alvarado R, Ludlam MJ, Stokoe D, Gloor SL, Vigers G, Morales T, Aliagas I, Liu B, Sideris S, Hoeflich KP, Jaiswal BS, Seshagiri S, Koeppen H, Belvin M, Friedman LS, Malek S. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 2010;464:431-5
  135. 135. Arkenau H-T, Kefford R, Long GV. Targeting BRAF for patients with melanoma. Br J Cancer 2011; 104: 392-398

Written By

Giuseppe Palmieri, Maria Colombino, Maria Cristina Sini, Paolo Antonio Ascierto, Amelia Lissia and Antonio Cossu

Submitted: 25 April 2012 Published: 30 January 2013