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European Journal of Cell Biology 93 (2014) 36–41

Contents lists available at ScienceDirect

European Journal of Cell Biology

jo ur nal home p age: www.elsev ier .com/ locate /e jcb

eview

xidative stress in melanocyte senescence and melanomaransformation

venja Meierjohanna,b,∗

University of Wurzburg, Department of Physiological Chemistry I, Biocenter, Am Hubland, 97074, Wurzburg, GermanyComprehensive Cancer Center Mainfranken, University Clinic Würzburg, 97078, Würzburg, Germany

r t i c l e i n f o

rticle history:eceived 14 September 2013eceived in revised form5 November 2013ccepted 15 November 2013

a b s t r a c t

Melanoma is a severe type of skin cancer with a high metastasis potential and poor survival rates oncemetastasized. The causes of melanoma formation are multifactorial and not fully understood. Severalsignaling cascades such as the RAS/RAF/ERK1/2 pathway, the PI3K/AKT pathway, RAC1 and NF-�B areinvolved in melanoma initiation and progression. Reactive oxygen species (ROS) are induced by these sig-nal transduction cascades, and they play a fundamental role in melanomagenic processes. Cells derivedfrom the melanocytic lineage are particularly sensitive to an increase in ROS, and thus, melanoma cells

eywords:elanoma

eactive oxygen speciesranssulfuration pathwayarcinogenesis

rely on efficient antioxidant measures. This review summarizes the causes and consequences of ROS gen-eration in melanocytes and melanoma and discusses the potential of pro-oxidant therapy in melanomatreatment.

© 2013 Elsevier GmbH. All rights reserved.

enescenceelanocytes

Reactive oxygen species can be considered as second messen-er molecules. When they are present in low amounts, they firsteact with easily accessible cysteine residues which are e.g. foundn protein tyrosine phosphatases including PTP1B, SHP1 and -2 andTEN (Boivin et al., 2008; Salsman et al., 2005). This abolishes thehosphatase‘s activity and enhances intracellular receptor tyrosineinase signaling and proliferation (Monteiro et al., 2008). Further-ore, ROS are activators of NF-�B signaling (Gloire et al., 2006;

revisi et al., 2010), and result in the generation of oxidized lipids,hich can also aid proliferation and prevent apoptosis (Trevisi et al.,

010). However, at higher concentrations, ROS damage cell mem-ranes and organelle function and impact cellular vitality. As aonsequence, cells undergo senescence or apoptosis (Wang and Yi,008).

Melanoma belongs to the most aggressive forms of skin can-er and its incidence is continually rising worldwide (Godar,011). Melanomas arise from melanocytes of cutaneous or extra-utaneous origin. In approximately one third of cases, they developrom nevi (or “moles”), which are therefore considered as a benignrecursor form of melanoma. Multiple oncogenes contribute toelanoma development, among them the serine/threonine kinase

RAF, the GTPases NRAS, RAC1, GNAQ, and GNA11 and the recep-or tyrosine kinases c-KIT and ERBB4 (Mehnert and Kluger, 2012).

elanocytes are characterized by their capacity to produce melanin

∗ Tel.: +49 9313181348; fax: +49 931 888 4150.E-mail address: svenja.meierjohann@biozentrum.uni-wuerzburg.de

171-9335/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.ejcb.2013.11.005

as means to protect the skin from damaging UV irradiation.Although this constitutes an important protection device, it alsoemerges as factor which renders cells of melanocytic origin par-ticularly sensitive towards reactive oxygen species (ROS) such ashydrogen peroxide (H2O2) or the superoxide radical (O2

•−).

ROS in melanocytes

UV exposure leads to enhanced melanocytic pigment produc-tion which generally protects the skin from the deleterious DNA-damaging effects of UV-A and -B. However, when melanocytesare compared to non-pigmented cells, they display raised ROSlevels which are attributed to the presence of melanin (Jenkinsand Grossman, 2013). Melanins are complex pigments which arederived from tyrosine by repeated oxidation steps. They can occurin different polymerization forms and can either consist of theblack eumelanin or the reddish/brownish pheomelanin (Meyskenset al., 2001). In contrast to eumelanin, pheomelanin productionrequires the incorporation of cysteine. Although melanin has beenreported to aid in ROS neutralization (Bustamante et al., 1993),it can also mediate oxidative stress on the cellular level (Hillet al., 1997). The presence of melanin in cultured primary humanmelanocytes is associated with a higher degree of ROS accu-mulation and simultaneous reduction of the cellular antioxidant

glutathione (GSH) (Smit et al., 2008). Consequently, the inhibi-tion of melanin synthesis in melanocytes causes a reduction ofROS to levels comparable to fibroblasts (Jenkins and Grossman,2013). The irradiation of eu- or pheomelanin with UV light can

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lso enhance ROS production (Meyskens et al., 2001). In par-icular, UVA causes higher levels of oxidative DNA damage in

elanocytes compared to fibroblasts (Wang et al., 2010). The tumoruppressor protein p16INK4A plays a major role in the cell cycleontrol of melanocytes, which is demonstrated by the fact thateople with familial p16INK4A deficiency (also known as Leiden syn-rome) have a characteristic accumulation of nevi and an increasedelanoma risk (Hussussian et al., 1994). p16INK4A itself is involved

n the prevention of ROS accumulation in a manner which can bencoupled from its cell cycle regulatory function (Jenkins et al.,011) (Jenkins et al., 2013). Although p16INK4A deficiency was foundo raise ROS levels in several cell types, the effect was particularlytrong in melanocytes in a melanin-dependent manner (Jenkinsnd Grossman, 2013).

As a consequence of their increased sensitivity towards ROS,elanocytes have developed mechanisms to counteract aberrant

xidative damage. As an example, the transcription factor MITF,hich is both a master regulator of the melanocyte lineage and aelanoma oncogene, is not only involved in inducing pigmenta-

ion, but it also controls the cellular response to ROS by enhancinghe oxidative DNA damage repair enzyme apurinic apyrimidinicndonuclease 1 (APE-1/Ref-1) (Liu et al., 2009). Similar obser-ations were made for the melanocyte stimulating hormone�-MSH)-dependent signaling cascade. Pigment production is gen-rally induced by the binding of �-MSH to its receptor MC1R, whichs expressed on the melanocyte plasma membrane. The groupround Z. Abdel-Malek demonstrated recently that MC1R signal-ng also affects ROS-dependent damage independent of melaninroduction by increasing the levels of base excision repair enzymes-oxoguanine DNA glycosylase (OGG1) and APE-1/Ref-1 (Kadekarot al., 2012).

he role of ROS in premature melanocyte senescence

Many human nevi carry common melanoma oncogenes suchs BRAFV600E or NRASQ61K/R (Bauer et al., 2007; Poynter et al.,006). It is therefore assumed that oncogene-induced senescence

s at least partly involved in the limitation of nevus growth. Manyesearchers have described oncogene-induced melanocyte senes-ence in vitro and in vivo. We have found that strong receptoryrosine kinase (RTK)- or NRASQ61K signaling leads to the accu-

ulation of high ROS levels (Leikam et al., 2008). The ensuingenescence is mediated by the DNA damage response and cane entirely prevented by the antioxidant N-acetylcysteine (NAC).

nterestingly, the magnitude of oncogene signaling determineshether ROS levels are strong enough to cause senescence (Leikam

t al., 2008). Oncogene-induced ROS can be generated by severalechanisms. On the one hand, the superoxide-generating NADPH

xidases are induced by oncogenic RTKs or RAS, and their phar-acological or siRNA-mediated inhibition prevents the induction

f senescence ((Kodama et al., 2013) and own unpublished obser-ations). On the other hand, ROS can be caused by metabolictress as in case of aberrant mitochondrial engagement. In lineith this, BRAFV600E-induced senescence was recently described to

o along with activation of the mitochondrial gatekeeper enzymeyruvate dehydrogenase. This results in the predominant usage ofyruvate in the citric acid cycle and causes increased mitochon-rial respiration coupled with oxidative stress and a reduction ineduced/oxidized glutathione ratio (Kaplon et al., 2013).

Our group could recently demonstrate that ROS-inducedelanocyte senescence is counteracted by an activated transsul-

uration pathway, which causes a raise of glutathione levels andhereby increases the amount of ROS scavengers (Leikam et al.,013). The transsulfuration pathway serves the production ofysteine from methionine, and the expression of the CTH gene

Cell Biology 93 (2014) 36–41 37

encoding cystathionase – the last enzyme in the transsulfurationpathway, which is expressed in melanoma cells, but not inmelanocytes – was sufficient to prevent ROS-induced melanocytesenescence.

The role of ROS in melanocyte apoptosis

The massive induction of melanocyte apoptosis is a character-istic feature of vitiligo, a benign chronic disease which describesthe depigmentation of certain skin areas in affected patients(Glassman, 2011). The loss of functional melanocytes is the reasonfor the observed depigmentation. Although the underlying path-omechanisms are not completely understood, a role of ROS in thedevelopment of the disease is undisputed. The affected vitiligoskin has been shown to suffer from enhanced oxidative stress,mainly caused by H2O2 concentrations in the millimolar range(Schallreuter et al., 1999). At this concentration, H2O2 has dele-terious effects and leads e.g. to lipid peroxidation and apoptosis(Boissy et al., 1991a,b; Zhang et al., 2013). The fact that catalaselevels are very low in vitiligo skin contributes to the strong H2O2increase (Schallreuter et al., 1991). Various exogenous ROS sourcessuch as phenols/catechols and tetrahydrobiopterin were further-more found to be increased in vitiligo patients (Chavan et al., 2009;Morrone et al., 1992; Schallreuter et al., 1994). In particular, phenolsand catechols can serve as competitive targets of tyrosinase, whichon the one hand hampers the production of melanin and on theother hand leads to the production of reactive quinones (Westerhofand d’Ischia, 2007). Elevated H2O2 can even further raise reac-tive quinone generation. In addition to the cell-autonomous actionof oxidative species, ROS-dependent autoimmunity seems to beinvolved in the elimination of melanocytes (Laddha et al., 2013).Importantly, treatment of pediatric vitiligo patients with the pseu-docatalase PC-KUS, which releases the oxidative stress in the skin,leads to repigmentation in large areas of the body (Schallreuteret al., 2008).

Altogether, melanocyte senescence and melanocyte apoptosiscan be caused by an imbalance between ROS production and ROSdetoxication.

ROS in melanoma

The fact that cells from the melanocytic lineage are particularlysensitive to ROS represents a double-edged sword with respect tomelanoma development: While aberrant ROS levels easily lead tothe described anti-tumor programmes, sublethal amounts allowthe accumulation of potentially oncogenic mutations. During theprocess of melanoma development, melanoma cells have accu-mulated numerous of these oncogenic mutations which changescellular signal transduction and metabolism as well as the interac-tion with neighboring cells. This again alters the cellular responsetowards UV and ROS compared to melanocytes.

The relevance of ROS in melanoma development was previ-ously demonstrated in a UV-induced mouse melanoma model: Inpresence of N-acetylcysteine, UV-induced oxidative damage wasreduced and the mean onset of melanocytic tumors was stronglydelayed (Cotter et al., 2007). Oral administration of NAC was there-fore proposed as preventive measure against UV-induced oxidativestress (Goodson et al., 2009).

In the following, the function of ROS in melanoma developmentas well as progression of the disease will be discussed.

ROS in melanoma initiation and progression

One of the factors which enhances the risk of developingmelanoma during lifetime is the presence of the red hair/fair

38 S. Meierjohann / European Journal of Cell Biology 93 (2014) 36–41

Fig. 1. (A) Sources of ROS in pigment cells (melanocytes and melanoma), as explained in the text. B: Antioxidant measures in pigment cells, as explained in the text. Mutatedv t, delet , transp

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ersions of the respective genes are highlighted in red. RH/FS, red hair/fair skin; muhe phosphatase PTEN or amplification of AKT3. PDH, pyruvate dyhydrogenase. TSPathway is shown in darker green.

kin phenotype. People with this phenotype have mutations inhe MC1R which decrease the receptor’s activity and lead to theoss of eumelanin, whereas the pheomelanin levels are raised. It

as presumed for a long time that the enhanced UV sensitivity,aused by low eumelanin levels, is responsible for the elevatedancer risk. However, this hypothesis was recently challengedith the help of a mouse melanoma model based on the pigment

ell specific expression of BRAFV600E on MC1R-deficient geneticackground (Mitra et al., 2012). Comparable to the human sit-ation, MC1R-deficient mice have fair skin and a reddish coatolour. Melanocytic BRAFV600E expression in these mice leads toelanoma development without the need for additional geneticanipulation. The tumors displayed high levels of oxidative DNA

nd lipid damage. However, when BRAFV600E expression and MC1Reficiency were combined on an albino background which also pre-ented the synthesis of pheomelanin, mice were protected fromelanoma development. The data show that pheomelanin itself

an be a source of ROS and thereby enhances the risk of developingelanoma.ROS can also be generated by a large number of signaling

olecules which are causative for melanoma development. TheI3K/AKT pathway is activated in the majority of melanomas, e.g. byTEN mutation or AKT3 amplification (Mehnert and Kluger, 2012),nd determines the transition between benign nevi and primaryelanoma in mice, where activation of the pathway was shown to

vercome BRAFV600E-induced senescence (Vredeveld et al., 2012).n human melanoma, AKT activation is associated with transitionetween the skin-restricted radial growth phase (RGP) and theetastasis-associated vertical growth phase (VGP) (Govindarajan

t al., 2007). AKT is a potent inducer of ROS, as it induces acti-ation of NADPH oxidases (Edderkaoui et al., 2011) and has the

otential to prevent apoptosis of cells with mitochondrial DNAutations which result in mitochondrial ROS leakage. It was there-

ore proposed that the AKT-dependent transition to the VGP stages mediated by ROS (Govindarajan et al., 2007).

terious mutation, act, activating mutation. In case of AKT, this reflects e.g. defects insulfuration pathway. Transcription factors are marked in light green, the metabolic

In addition to the PI3K/AKT pathway, other classical melanomadeterminants cause the generation of ROS. NRAS and RAC1 arecommonly mutated oncogenes in melanoma. Both are involved inactivating NADPH oxidases and thereby account for elevated ROSlevels. RAC1 is an integral component of the NADPH complexes1 and 3 (Ueyama et al., 2006). NRAS is a upstream activator ofRAC1 (Li et al., 2012) and enhances the transcription of NOX1 andNOX2 isoforms in melanocytes (own unpublished observations),thereby regulating NADPH oxidases on several levels. Inhbition ofRAC1 blocks NRASQ61K-driven tumor growth, lymph node spreadand invasiveness (Li et al., 2012), but the ROS-specific contributionto these effects still remains to be determined. The sources of ROSin pigment cells are summarized in Fig. 1A.

The function of NADPH oxidases in melanoma was directlyaddressed by Meyskens and colleagues. They found that NOX1expression was elevated in melanoma cells compared tomelanocytes and that NOX1 overexpression enhanced melanomacell invasion and epithelial-to-mesenchymal (EMT)-like transitionprocesses (Liu et al., 2012a). NOX4 expression is also enhanced inmany melanoma cells, at least partly due to MITF activity, where itblocks melanogeneses and drives G2/M progression during the cellcycle (Liu et al., 2012b; Yamaura et al., 2009).

Although the pro-tumorigenic consequences of elevated ROSproduction have been described in melanoma and other cancertypes, there remains one crucial questions: What is the molecu-lar reason for the ensuing tumor transformation? Are these theelevated mutation rates, the altered signal transduction (e.g. bymodification of susceptible cysteine residues) or the productionof second messengers (e.g. oxidized lipids)? Most likely, all threemechanisms are implicated in the tumor generation processes.However, the transcription factor NF-�B belongs to one of the

most prominent examples of ROS-sensitive signaling components.The dimeric NF-�B transcription factor is kept under tight con-trol by its interaction partner inhibitor of kB (IkB), which masksthe nuclear translocation signal of NF-�B proteins and thereby

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revents its action. The IkB kinase IKK phosphorylates IkB andhereby causes its dissociation from NF-�B and nuclear transloca-ion of the transcription factor. H2O2 causes phosphorylation of IkBy IKK or the tyrosine kinase Syk and similarly causes IkB dissocia-ion (Gloire et al., 2006). NF-�B is active in a large number of human

elanomas and is involved in proliferation, survival, resistanceo apoptosis and metastasis (Madonna et al., 2012). Importantly,ndogenously produced ROS results in constitutive NF-�B activa-ion in melanoma cell lines (Brar et al., 2001).

Our group has recently revealed the surprisingly strongro-angiogenic ability of ROS-induced NF-�B in melanomaSchaafhausen et al., 2013). We used the transparent medaka as

elanoma animal model. Here, pigment cell specific expression ofhe fish melanoma oncogene Xmrk, an EGFR homologue, leads to

elanomas as well as fish-specific yellowish pigment cell tumorsith high penetrance (Schartl et al., 2010, 2012). Xmrk induces ROS

ery efficiently (Leikam et al., 2008), and Xmrk-bearing tumorsre characterized by compensatory increase of antioxidant pro-eins like e.g. peroxiredoxins or glutathione-S transferases (Lokajt al., 2009). When we crossed Xmrk-transgenic medaka fishesith fli::egfp medaka fishes which express enhanced GFP under the

ndothelial-specific fli promoter, we generated a melanoma ani-al model with very high blood vessel and pigment cell resolutionhich is easy to image due to the transparency of the fishes. Using

his model, we could show that even single transformed pigmentells were capable of causing tumor angiogenesis. This effect wasediated by NF-�B, which was activated by ROS in response tomrk activation. We could also demonstrate that the potent pro-ngiogenic factor angiogenin was strongly secreted by melanomaells with activated NF-�B (Schaafhausen et al., 2013). Interestingly,OS are also implicated in vasculogenic mimikry, a process whichescribes the formation of vascular channels without endothe-

ial cell contribution by cancer cells (Folberg et al., 2000). Thesetructures are also involved in the microcirculation and are oftenbserved in melanoma cells in vitro and in vivo (Paulis et al., 2010).he formation of capillary-like structures in melanoma cells wasnhibited by antioxidants, along with the expression of VEGF andEGF receptors (Vartanian et al., 2007). The association of the tumorith the blood vessel system provides the gate to metastasis, theext step in tumor progression. Along these lines, melanoma cell

ntravasation can also be dose-dependently induced by H2O2, andverexpression of thioredoxin or NAC treatment reverses this effectCheng et al., 2004).

nti-oxidant measures in melanoma

In order to profit from the pro-tumorigenic effect of raisedOS levels and to keep their various ROS sources under control,elanoma cells have to take measures to balance their oxidative

tress load to an acceptable level.Although mostly addressed as cell cycle regulators, the cyclin

ependent kinases CDK4/6 phosphorylate a substantial number ofubstrates apart from Rb. The transcription factor FOXM1 belongso their targets and plays a particuar role in the suppression ofenescence in melanoma (Anders et al., 2011). CDK4- or CDK6-ediated phosphorylation of FOXM1 stabilizes and activates the

ranscription factor, which blocks a ROS-dependent senescencerogramme. This effect can be reverted by CDK4/6 inhibition.uperoxide dismutase (MnSOD) and peroxiredoxins are amonghe transcriptional targets of FOXM1 (Park et al., 2009), therebyroviding at least a partial explanation for the FOXM1-mediatedrotection from oxidative stress.

There are also genetic mechanisms by which redox-relevantranscription factors are induced in melanoma. Exome sequencingf acral lentiguous melanoma (ALM) has uncovered a frameshiftutation in the gene encoding KEAP1 (Kelch-like ECH-associated

Cell Biology 93 (2014) 36–41 39

protein 1). KEAP1 is a regulator of the transcription factor nuclearfactor (erythroid-derived 2)-like 2 (NRF2), a master regulatorof oxidative stress response. The binding to KEAP1 leads tocytoplasmic retention, facilitated ubiquitination and proteasomaldegradation of NRF2, but the described mutation prevents theinteraction (Miura et al., 2013). The authors showed that NRF2 isdirectly involved in counteracting ROS in melanoma and providingresistance towards ROS inducers such as platinum compounds anddacarbazine.

Both FOXM1 and NRF2 are also transcriptional targets of c-MYC,which is characteristically expressed in melanoma cells (Blanco-Bose et al., 2008; DeNicola et al., 2011). However, our group foundthat the induction of the c-MYC target gene CTH plays a dominantrole in preventing cellular stress. The presence of CTH, which blocksROS-induced senescence in melanocytes as described above, is alsorequired in a large fraction of melanoma cell lines in order to avoidthe accumulation of DNA damage (Leikam et al., 2013). Pharma-cological or siRNA-mediated inhibition of CTH strongly inhibitedmelanoma cell propagation by inducing senescence or apoptosis. Ascysteine source, CTH provides the major component for intracellu-lar antioxidants. Cysteine is not only active as cellular antioxidanton its own, but it represents the functional core for glutathioneand thioredoxin, which are co-factors for numerous antioxidantenzymes (e.g. GSH peroxidase, peroxiredoxins) and provide cellu-lar building blocks (e.g. by enabling the ribonucleotide reductasereaction). In addition, cysteine is involved in the formation of Fe/Sclusters and in binding metal ions at active sites of enzymes whichcatalyse redox reactions. A strong supply of cysteine is therefore theprerequisite for an intact redox balance and efficient ROS detoxi-fication and might thus constitute a good target for cancer cellswith high ROS production. The antioxidant measures of pigmentcells are summarized in Fig. 1B. Interestingly, the import of cys-tine, which is considered as regular source for cysteine, is lessimportant for melanoma cells. Inhibition of the cystine-glutamatetransporter SLC7A11 does not affect melanoma cell viability onits own, but only in combination with additional oxidative stress((Vene et al., 2011) and own unpublished observations). The rea-son for this might be the compensatory induction of CTH and theactivation of the transsulfuration pathway which goes along withinhibition of cystine transport (Leikam et al., 2013). The data indi-cate that the cysteine supply by de novo cysteine synthesis plays amore important role than cystine import, although the reasons forthis have yet to be uncovered.

Elevating ROS in cancer therapy

The increase of ROS levels by exogenous stress inducers isa strategy which is widely used in cancer treatment. Manychemotherapeutic agents such as cisplatin, doxorubicin and azi-dothymidine have the potential to raise ROS levels (Deavall et al.,2012). However, their therapeutic success in melanoma is ratherlimited, and the levels of cellular damage do not suffice to allowlongterm tumor regression. Researchers therefore keep looking formore efficient ROS inducers. The plant compound piperlonguminespecifically induces ROS in cancer cells including melanoma cellsand induces cell death (Raj et al., 2011), further indicating that highROS loads are a characteristic feature of cancer cells. However, thepresence of enhanced detoxification capacities, as e.g. mediated byPGC1� expression in melanoma cells, weakens the pro-apoptoticeffect of piperlongumine (Vazquez et al., 2013).

Conclusion and therapeutical implications

In summary, melanomas bear many sources of oxidative specieswhich are important mediators of tumor transformation and

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rogression of the disease. To profit from the pro-tumorigenicffects of ROS and prevent their toxic accumulation, the devel-pment and maintenance of antioxidant measures is necessary.hese antioxidant enzymes and their regulators provide valuableargets for tumor treatment. The application of CDK4/6 or CTHnhibitors might represent promising approaches to take thera-eutical advantage of the dependency of melanoma cells towards

ntracellular antioxidants. A combination of an inhibitor of cancer-pecific antioxidation pathways with a ROS inducer poses a gooduture strategy to specifically hit the tumor cells, particularly in casef melanoma.

cknowledgements

SM is funded by the German Cancer Aid (Melanoma Researchetwork), the German Research Foundation (TRR-17), the inter-isciplinary center for clinical research (IZKF) Wurzburg, and theiege Foundation against skin cancer.

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