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Nieuwpoort, A. F. van. (2011, March 16). Biochemical and molecular studies of atypical nevi. Retrieved from https://hdl.handle.net/1887/16632
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1.1 Melanoma and its risk factors
Melanoma is a skin tumour that develops from melanocytes, the pigment (melanin) producing cells of the skin. Worldwide, the incidence of melanoma skin cancer is still on the rise even after improved public awareness [1,2]. If identified at an early stage, melanoma has a good prognosis. However, life expectancy figures drop quickly with an increasing thickness of the tumour [1]. For example, patients with a melanoma thinner than 1 mm have an overall 5‐year survival of 97%, while patients with a melanoma with a thickness over 4 mm have a 5‐year survival of 55%
[3].
With respect to melanoma risk factors, intermittent exposure to sunlight at a young age is the best known environmental risk factor [4,5]. The phenotypic characteristics of fair skin (inability to tan), light hair and eye colour, extensive freckling, high number of nevi (moles) (particular atypical nevi), but also a family history of melanoma and a previous melanoma are all well recognized risk factors for melanoma [4‐6].
Of all the risk factors for melanoma, nevi have by far the greatest clinical implication. Nevi, Latin for “nests”, are aggregations of melanocytes. Nevi can be either present at birth (congenital nevi) or develop after birth (acquired nevi). A recent pooled analysis of 15 case‐control studies showed that the presence of 90 or more nevi (>3 mm) on the whole body surface is a risk factor for melanoma with a pooled OR of 6.9 (95% CI: 4.4, 11.2) [8]. The observation that an increased number of nevi, and in particular atypical nevi, is a substantial risk factors for melanoma is in line with the Clark development and progression model [7].
The Clark model describes the histopathological changes that accompany the progression from normal melanocytes to malignant melanoma (figure 1).
One of the first steps within this model is the progression of an acquired nevus into a dysplastic nevus, also known as a clinical atypical nevus [7].
An atypical nevus is larger than 5 mm, has a flat component and fulfils at least 2 of the 4 following criteria: (1) irregular outline, (2) variable pigmentation, (3) irregular shape; (4) erythema (reddish hue). In line with the Clark progression model, Chang et al also showed that atypical nevi are more common in melanoma patients than in controls (OR 4.0 (95% CI: 2.8, 5.8)) [8].
Recent studies started to unravel the (molecular) mechanisms underlying the different melanoma progression stages (for an extensive review see [9]). Most studies have focused on the late, less curable stages, whereas only few studies addressed the curable, early progression stages. From this review it becomes apparent that our understanding of the mechanisms involved in these early stages of melanoma development is still limited. Therefore, the studies presented in this thesis focus particularly on differences between normal melanocytes and the melanocytes in atypical nevi.
Figure 1: The Clark model describes the histopathological changes that accompany the progression from normal melanocytes to malignant melanoma (Clark et al., 1984). In the Clark model, the first event is a proliferation of morphologically normal melanocytes leading to the benign nevus (fig 1, Stage 1). The next step towards melanoma is the development of atypia in atypical (dysplastic) nevi, which may arise from preexisting benign nevi or as new lesions (fig 1, stage 2). During the next step of the Clark progression model, the radial‐grow phase, the melanocytes acquire the ability to proliferate throughout the epidermis and papillary dermis (fig 1, stage 3). Lesions that progress to the vertical‐growth phase acquire the ability to further invade in the dermis (fig 1, stage 4). The final step in this model is the metastatic melanoma, in which stage melanoma cells disseminate to other areas of the skin and/or other organs (fig 1, stage 5). Figure adapted from Miller and Mihm [9].
There is evidence to suggest a relationship between the development of an atypical nevus and the degree of pigmentation. Individuals with red hair or fair skin are more likely to develop atypical nevi than individuals with a dark skin [10,11]. In addition, individuals with a dark skin have a lower risk to develop melanoma (around 1 per 100,000) than individuals with a fair skin (around 21.6 per 100,000) [10]. Apart from the degree of pigmentation, also the type of pigment produced by the (atypical) melanocytes may be important.
Within the melanocyte two types of pigment can be distinguished: the red/yellow pheomelanin and the dark brown/black eumelanin. While individuals with a dark skin (skin type IV) show a preference for eumelanogenesis, in individuals with a light skin (skin types I and II) relatively more pheomelanin is produced. Especially during the production of pheomelanin products are formed that can generate reactive oxygen species (ROS) [14,15]. If not removed properly, increased levels of ROS not only affect physiological processes in the melanocytes, but could also induce DNA damage and subsequently DNA alterations, and in this way contribute to melanoma development. To test this hypothesis the studies included in this thesis focus on pheomelanin production in normal melanocytes and atypical nevi in relation to the generation of ROS and subsequent DNA damage.
In this introductory chapter we will therefore first discuss pigment biosynthesis and pigment function, and the production and handling of ROS generated during pigment synthesis. Subsequently we will discuss potential effects of ROS in melanocytes and melanoma development. Finally, we will describe the aims of the studies included in this thesis.
1.2 Pigment biosynthesis and pigment function
The main function of pigmentation is darkening of the skin and thereby protection against ultraviolet (UV) radiation [11,12]. It is this very same exposure of the skin to UV radiation that initiates pigment production, which remarkably starts with hormone production in the keratinocyte and not in the melanocyte. Upon UV radiation keratinocytes start to produce proopiomelanocortin that is subsequently processed into α‐melanocyte‐stimulating hormone (α‐MSH) and
adrenocorticotropic hormone [13]. α‐MSH is the ligand for the melanocortin 1 receptor (MC1R), a seven‐pass transmembrane G‐protein–coupled receptor, expressed in melanocytes [14]. Stimulation of MC1R activates adenylate cyclase that results in elevated levels of cyclic adenosine monophosphate (cAMP) [15].
cAMP activates protein kinase A, which in its turn phosphorylates and activates cAMP responsive element binding protein (CREB) [16]. CREB increases the expression of the microphthalmia protein (MITF) that subsequently up‐regulates the expression of a specific enzyme tyrosinase. Tyrosinase is transported into membrane‐bound organelles termed melanosomes, where the actual pigment synthesis takes place (see figure 2).
Figure 2: MSH‐MC1R regulation of pigment genes. Upon binding of α‐MSH to MC1R the G proteins (Gα, Gβ, Gγ) stimulate the activation of the intracellular messenger adenylyl cyclase (AC). AC in its turn catalyses the activation of cyclic AMP (cAMP) leading to increased levels of cAMP in the cytoplasm of the melanocyte. cAMP in its turn activates PKA which migrates into the nucleus and phosphorylates the cAMP responsive element binding protein (CREB). CREB increases the expression of the microphthalmia protein (MITF) that subsequently up‐regulates the expression of a specific enzyme tyrosinase. Figure adapted from Chin [17].
In the melanosome, tyrosinase oxidises L‐tyrosine to the highly reactive intermediate dopaquinone [18] (figure 3). Dopaquinone is the starting point of the two chemically distinct types of melanin, brown‐black eumelanin and reddish‐
yellow pheomelanin [19]. In principle, dopaquinone undergoes a series of spontaneous reactions, leading to the production of eumelanin. However, in the presence of L‐cysteine the eumelanin synthesis is driven in the direction of pheomelanogenesis induced by production of 5‐S‐ and 2‐S‐cysteinyldopa that after oxidation gives rise to pheomelanin. Finally, protection against UV is realised by the transfer of the fully pigmented melanosomes to the surrounding keratinocytes, where the melanosomes form a supranuclear cap protecting the nucleus from UV radiation [20].
Thody et al. was the first to determine pheo/eumelanin ratios in melanocytes derived from suction blisters [21]. They and Hunt et al. showed that the ratio pheo/eumelanin in dark skin individuals in comparison with light skin individuals is lower, mainly due to the relative higher proportion of eumelanin present in the dark skin [25,26]. Hunt et al. also showed that the ratio
pheo/eumelanin sustained in cultured blister derived melanocytes thereby mimicking the in vivo situation [22]. Our group showed that ratios in
pheo/eumelanin can be influenced in melanocytes by adding L‐tyrosine to the culture medium [25]. A higher increase in the amount of pheomelanin and subsequently in the pheo/eumelanin ratio in skin type I compared to skin type VI was observed. This shows a tendency for pheomelanogenesis for the lighter skin type while skin type VI has a preference for eumelanogenesis, since the difference in pheo/eumelanin ratio was already present in the non‐stimulated melanocytes.
This in vitro melanogenesis model allows studies of the pigment biosynthesis and the effects thereof in more detail, not only in melanocytes obtained from different skin types, but also in various melanocytic lesions, such as nevi and melanoma.
There is a huge variation in skin type among individuals. Even within races variation in skin type exists. For instance, in Caucasians skin types can vary from skin type I till skin type IV. This constitutive make up of pigmentation determining skin colour is regulated by more than 120 genes (for an extensive review: [23]). One of the key genes is the above described melanocortin 1 receptor [27]. The gene encoding MC1R is located on chromosome 16q24 and is highly polymorphic. Till today more than 70 genetic variants have been described. Several studies conducted in populations of Northern European origin showed that risk of melanoma is higher among MC1R variant carriers than among non‐carriers, with the strongest risk effects observed for carriers of multiple variants [24‐27]. These studies thereby identified MC1R variants as an additional, this time genetic risk factor (for variants in two MC1R alleles) with odds ratios ranging between 1.42 and 2.45 for melanoma, irrespective of skin type.
Only a few genetic variants have been functionally characterized, which complicates the establishment of accurate correlations between the signalling properties of mutant alleles and pigmentation phenotypes. Three variants (R151C, R160W, and D294H) that are strongly associated with red hair colour, poor tanning ability, pale/fair skin colour, and extensive freckling (24,28) have shown impaired cAMP levels upon stimulation of the receptor [29‐31].
These R151C, R160W, and D294H variants also associate with an increased pheomelanin synthesis [30,32]. Melanocytes with non‐functional MC1R had the least DNA repair in comparison with melanocytes with a functional MC1R [37]. In addition, cells with a MC1R based impaired repair system displayed a higher level of (oxidative) DNA damage [38,39].
Figure 3: The production of melanin in the melanosomes starts with dopaquinone. Dopaquinone is synthesized either by the hydroxylation and oxidation of L‐tyrosine via tyrosinase or by the oxidation via oxy‐tyrosinase of L‐DOPA. Dopaquinone has three potential chemical fates that alternatively generate black‐brown (eumelanin), or reddish‐yellow (pheomelanin) pigment. The synthetic pathway favoured and thereby determining the type of pigment produced, depends on the relative influx of L‐tyrosine and L‐cysteine into the melanosome, the activities of the three melanogenic enzymes (tyrosinase, TRP‐1, and TRP‐2), and the pH of the melanosome. In the presence of L‐cysteine, a sulphur containing amino acid (concentration is 10−7 M), formation of 5‐S‐cysteinyldopa is favoured in one synthetic pathway resulting in pheomelanin. In the other synthetic pathway dopaquinone is oxidized into dopachrome which tautomerizes spontaneously at a moderate rate to form dihydroxyindole‐2‐carboxylic acid (DHICA) or partially decarboxylates to form dihydroxyindole (DHI). Polymerization of DHI generates a dark coloured, high molecular weight, insoluble pigment, termed DHI‐melanin. Polymerization of DHICA yields a lighter coloured, lower molecular weight pigment, called DHICA‐melanin, which is slightly more soluble than DHI‐melanin. DHI‐melanin and DHICA‐melanin are collectively termed eumelanin. Figure adapted from [33].
1.3 Generation and handling of ROS during pigment synthesis
ROS are generated in any cell as byproducts of reactions in which oxygen is involved. Well known is the utilization of oxygen by the mitochondria in energy production (ATP). In comparison to other cells melanocytes contain an additional ROS producing system, namely melanogenesis. During melanogenesis
intermediates are generated. Borovansky et al. showed that various intermediates can leak out of the melanosome into the cytoplasm of the melanocyte [40]. Others observed that especially intermediates of pheomelanogenesis via specific
interactions generate reactive oxygen species (ROS) [34‐36]. In addition, our group has shown that melanin precursors can also liberate iron from ferritin, the iron stores in the melanocyte, which can subsequently increase the level of ROS via the Fenton reaction [37].
In general, ROS comprise a family of radical (containing a free electron such as the hydroxyl radical (•OH)) and nonradical species (such as H2O2). ROS can immediately react with its nearest molecule resulting in, for example, loss of protein function or disturbance of membrane integrity or diffuse away from their sites of formation having an impact at a distance [38]. Although the interaction of ROS with cellular components might give the impression that ROS have a
predominantly damaging effect on the cell, ROS also have a physiological function.
Low amounts of ROS are involved in signalling pathways that induce gene transcription [39,40], cell proliferation [40] and differentiation [39]. However excessive amounts can result in oxidative DNA damage, such as 8‐hydroxydeoxy guanosine (8‐OHdG) [41]. Other effects of high levels of ROS are lipid and protein peroxidation [42].
Therefore, under normal conditions, homeostasis between ROS production and ROS reducing mechanisms (anti‐oxidant system) exists in the cell to maintain a physiological level of ROS (for an overview see [38]). An imbalance between the
formation of ROS and the anti‐oxidant system is named oxidative stress and results in a permanently increased ROS level in the cell [43].
To reduce damaging radical species, the anti‐oxidant system contains enzymes, such as superoxide dismutase, catalase and glutathione peroxidase, that e.g. inactivate •OH by reducing it into H2O2 and ultimately harmless H2O. The system also includes binding catalysts such as ferritin which binds free bivalent iron.
A third anti‐oxidant process is scavenging. The latter process is very well illustrated for melanocytes and plays a role in the reduction of melanin precursors [44]. As a scavenger, glutathione transferase catalyzes the addition of a glutathione molecule to the pheomelanin quinone intermediates thereby prohibiting subsequent reactivity of these quinone intermediates [45]. L‐cysteine is an amino acid that is used in the production of both glutathione (GSH) and pheomelanin. We
hypothesize that in the melanocyte less L‐cysteine becomes available for GSH production during increased pheomelanogenesis resulting in diminished levels of GSH and subsequently higher levels of ROS.
1.4 Effects of ROS in relation to melanocyte progression and melanoma development
Generalised oxidative stress and consequently DNA damage could lead to DNA mutations (46‐48). Especially mutations in critical genes could drive
subsequent stages of melanoma development. Remarkably, alterations in genes in early melanoma progression stages have been hardly reported. Just mutations in two genes, BRAF and NRAS, both members of the mitogen‐activated protein kinase (MAPK) pathway, have been observed in normal, atypical nevi and melanoma [49‐
51].
The MAPK pathway is one of the most well known signal transduction pathways involved in oncogenesis due to its role in a wide variety of cellular functions as cell proliferation, cell‐cycle arrest, terminal differentiation and apoptosis (for a review see [52]).
The most common mutation in the BRAF gene is a substitution of a valine to a glutamic acid at codon 600 (V600E, T/A). This mutation has been described in 80% of both acquired nevi and atypical nevi [49], 60% of primary melanomas [51]
and 68% of the melanoma cell lines [53]. The two most common mutations for NRAS involve a substitution at codon 61 of glutamine into a lysine (Q61K) or into a leucine (Q61L) that are present in 19% of the acquired nevi and 15% of melanoma [54]. It is notable that neither the NRAS Q61K (C/A) and NRAS Q61L (A/T) nor the BRAF V600E (T/A) mutations observed in nevi or melanoma demonstrate
characteristic UV induced signature changes of cytosine into thymine (C/T) or CC/TT [51,55,56].
In this light it is also of interest that the activating V600E BRAF mutation occurs more frequently in melanomas arising at body sites exposed to intermittent UV exposure (65%) in comparison to melanoma at body sites with frequent sun exposure (33%) [57].
Lack of UV related mutations and poor association with UV exposure favor the hypothesis that cellular biochemical processes, such as sustained oxidative stress rather than UV radiation could play a role in the acquisition of mutations in critical genes, e.g. NRAS and BRAF driving melanoma development. However nevi can be indolent for decades despite the presence of activating BRAF or NRAS mutations. This suggests that BRAF or NRAS activation on its own is insufficient for the development of melanoma [49,54,58].
1.5 Aim and outline of the thesis
The central theme of this thesis is that pigment biosynthesis, and
especially pheomelanin production, could play an important role in early melanoma progression by inducing ROS, which can result in DNA damage ultimately resulting in mutations driving melanoma progression. In the following chapters, this theme was mostly studied by comparing normal melanocytes and atypical melanocytes.
In chapter 2 we first focused on melanin production in normal skin. Using x‐ray microanalysis (XRMA) and HPLC the total amount and type of pigment (pheo/eumelanin ratio) as well as the location of pigment production was
investigated in cultured melanocytes obtained from individuals with light and dark skin types. After having established the location of melanin production and melanin composition in normal melanocytes, in chapters 3 and 4 melanin content and composition was measured in normal and atypical melanocytes and correlated with levels of ROS and oxidative stress induced DNA damage. Using XRMA the
composition of melanin was investigated in archival material of benign dermal nevi, atypical nevi and melanomas and compared with that of cultured normal
melanocytes. Using various redox‐sensitive probes levels of ROS were measured in cultured melanocytes from normal skin and atypical nevi from the same donors by FACS analysis, and correlated with pheomelanin levels and oxidative stress induced DNA damage, as determined by the comet assay.
To gain a better understanding of the genetic events involved in early melanoma development, in chapter 5 gene expression profiling was performed on cultured melanocytes from normal skin and from atypical nevi obtained from the same donors. In order to verify whether the observed gene expression differences are reflected at the protein level, in chapter 6 protein expression profiles were investigated by means of 2‐dimensional gel electrophoresis on the same normal and atypical melanocytes used in chapter 5. The results obtained in this thesis are summarised and discussed in Chapter 7.
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