Tumor Biol. DOI 10.1007/s13277-014-1904-2

REVIEW

Running for time: circadian rhythms and melanoma Elitza P. Markova-Car & Davor Jurišić & Nataša Ilić & Sandra Kraljević Pavelić

Received: 14 January 2014 / Accepted: 27 March 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Circadian timing system includes an input pathway transmitting environmental signals to a core oscillator that generates circadian signals responsible for the peripheral physiological or behavioural events. Circadian 24-h rhythms regulate diverse physiologic processes. Deregulation of these rhythms is associated with a number of pathogenic conditions including depression, diabetes, metabolic syndrome and cancer. Melanoma is a less common type of skin cancer yet more aggressive often with a lethal ending. However, little is known about circadian control in melanoma and exact functional associations between core clock genes and development of melanoma skin cancer. This paper, therefore, comprehensively analyses current literature data on the involvement of circadian clock components in melanoma development. In particular, the role of circadian rhythm deregulation is discussed in the context of DNA repair mechanisms and influence of UV radiation and artificial light exposure on cancer development. The role of arylalkylamine N-acetyltransferase (AANAT) enzyme and impact of melatonin, as a major output factor of circadian rhythm, and its protective role in melanoma Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-1904-2) contains supplementary material, which is available to authorized users. E. P. Markova-Car (*) : N. Ilić : S. Kraljević Pavelić Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, 51000 Rijeka, Croatia e-mail: [email protected] N. Ilić e-mail: [email protected] S. Kraljević Pavelić e-mail: [email protected] D. Jurišić Department for Plastic and Reconstructive Surgery, Clinic for Surgery, University Hospital Centre Rijeka, Krešimirova 42a, 51000 Rijeka, Croatia e-mail: [email protected]

are discussed in details. We hypothesise that further understanding of clock genes’ involvement and circadian regulation might foster discoveries in the field of melanoma diagnostics and treatment. Keywords Clock genes . Circadian rhythm . Melatonin . Cancer . Melanoma

Introduction It has been recently accepted that the deregulation of circadian rhythm in mammals is tightly linked to various pathological states. Indeed, circadian rhythms are among the most important regulators of our physiology and were endogenously set during the evolution due to day-night switches in nature with a period length of 24-h [1]. In mammals, the master clock is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus [2, 3], which receives environmental light-dark information, and the daily changes in light intensities are thought to be the major environmental signal involved in circadian entrainment. However, multiple circadian clocks that are functional in most body cells [4, 5] are located in the peripheral organs such as the liver, kidney, heart or skin. Those peripheral self-sustained and cell-autonomous oscillators are mutually synchronized through the neural and humoral signals from the SCN [6, 7]. Circadian rhythms include the sleep-wake cycle and hormone production rhythms. Any alteration of this fine-tuned mechanism leads to clock lesions that might result in arrhythmic behaviour, irregular sleep patterns [1] or even severe pathological states such as cancer-related phenotypes [8] (Table 1). Clock genes’ expression was extensively reviewed by Lengyel et al. in human cancer tissue biopsies in comparison with patient matched non-tumorous tissue biopsies where a clear correlation of deregulated circadian clock genes has been described [35].

Tumor Biol. Table 1 Associations of clock genes in cancer and other pathogenic conditions Disease

Clock gene

Genotype

Reference

Melanoma Pancreatic ductal adenocarcinoma (PDA) Head and neck squamous cell carcinoma (HNSCC) Colorectal cancer

Per1,2; Clock; Cry1 Per1,2,3; Bmal1; Clock; Cry1,2; Tim Per1,2,3; Cry1,2; Bmal1 Tim Per1,2,3; Cry2

Down-regulation Down-regulation Down-regulation Up-regulation Down-regulation

[9] [10] [11]

Lung cancer

Tim Clock/Bmal1 Per1

[12] [13–15] [16]

Tim Per1,2; Cry2 Per1,2,3; Npas2/Clock Per1 Per1,2 Bmal1 Per1,2,3; Cry1,2; Bmal1 Per2

Up-regulation Mutation/up-regulation Down-regulation, promoter hypermethylation, histone deacetylation Up-regulation Down-regulation, promoter methylation Down-regulation/up-regulation Down-regulation Down-regulation Down-regulation, promoter hypermethylation Down-regulation; Per2,3 promoter hypermethylation Down-regulation

Per1,2 Per1 Per2 Cry1 Cry2 Bmal1 Bmal1

Down-regulation Down-regulation Deficient Up-regulation Down-regulation Deficient Up-regulation

[28] [29] [30] [31] [32] [33] [34]

Breast cancer Endometrial carcinoma Prostate cancer Hematologic malignancies Chronic myeloid leukaemia (CML) Acute myeloid leukaemia (AML) Glioma Buccal squamous cell carcinoma (BSCC) Lymphoma, mouse model Alzheimer’s disease Bipolar disorder Metabolic syndrome, mouse model Early stage of cartilage degeneration in the DMM mouse model of OA

The generally accepted hypothesis is that major circadian clock genes exert a tumour suppressor role in cells and are directly involved in the proliferation of malignant cells; deregulation of the circadian clock is then thought to lead to the deregulation of the cell cycle control. This is important as, among all other diseases, cancer remains one of the major clinical problems of modern times. Indeed, improvements in diagnostics and therapy still fail to cure many patients. According the World Health Organization predictions, the deaths from cancer worldwide are projected to continue rising, with estimated 13.1 million deaths in 2030 [36]. This is due to a highly intricate pathogenesis of cancer that includes a number of different molecular mechanisms and factors [37]. In particular, deregulation of cellular mechanisms including genome instability, proliferation, evasion of cell death and inflammation seem to be the major driving forces during early stages of tumour cell development [38], and it is worth mentioning that the majority of these processes in cancer are strictly regulated by circadian rhythms [24, 39]. Moreover, the circadian variation of mammal immunity appears to be hormonally modulated via the hypothalamic-pituitary-adrenal axis [40] or to coincide with changes of immune mediators [41]. The role of the immune system has already been recognized in cancer

[12–14]

[17] [18, 19] [18, 20–22] [23] [24] [25] [26] [27]

development, and for example, chronic inflammation characterized by Th2 dominance is typical for advanced melanoma stages [40]. Published in vivo data strongly support the role of circadian genes in tumour development. In vivo models have been especially useful in proving the tumour suppressor role of several circadian components, including the Per2 gene in murine sarcoma cell lines, in C57BL/6 mice transplanted with Lewis lung carcinoma [42, 43] and Bmal1, Tim and Per1 that are involved in the cellular response to genotoxic stresses [44, 45]. All these genes bear a central role within the mammalian circadian system regulated by positive and negative transcriptional-translational feedback loops [46] (Fig. 1). The positive regulators within this system are bHLH-PAS (basic helix-loop-helix-PAS) transcriptional factors, i.e. BMAL1 (brain and muscle ARNT-like protein 1) and CLOCK (circadian locomotor output cycles kaput) or NPAS2 (neuronal PAS domain protein) in neuronal tissue. BMAL1 and CLOCK/NPAS2 heterodimerise and positively stimulate daily accumulation of Period genes (Per1, Per2 and Per3) as well as expression of Cryptochrome (Cry1 and Cry2) genes through binding to an E-box (CACGTG), an enhancer found in the upstream regulatory region of Per and Cry genes [47–49]. In contrary, PER and CRY proteins appear to act as

Tumor Biol.

Fig. 1 Schematic representation of molecular clock in mammals. The transcriptional activators BMAL1 and CLOCK/NPAS2 heterodimerise, form the positive limb of the regulatory loop, and up-regulate the expression of Per and Cry by binding to the E-box regulatory region of these genes. PER and CRY proteins form the negative limb of the regulatory loop. After reaching a critical concentration, PER and CRY heterodimerise and enter the nucleus where they bind to BMAL1CLOCK/NPAS2 complex, repressing its transcriptional activity. In the

second loop, BMAL1-CLOCK/NPAS2 heterodimers bind the E-boxes in the promoters of Rev-erbα and Rorα genes and promote their transcription. RORα and REV-ERBα compete for the ROR element (RORE) in the Bmal1 promoter. Consecutively, RORα promotes and REV-ERBα suppresses Bmal1 expression. In addition, PER is phosphorylated by casein kinase 1 epsilon (CK1ε) and tagged for proteasome-mediated degradation

negative feedback loop components. PER and CRY proteins interact in the cytoplasm with each other, form heterotypic complexes, and consequently enter the nucleus. Once translocated to the nucleus, CRY and PER specifically inhibit the CLOCK-BMAL1-mediated transcription by directly binding to these transcription factors [50]. There is a second loop which regulates the expression of the Bmal1 gene. In the nucleus, BMAL1/CLOCK or BMAL1/NPAS2 heterodimers bind to E-boxes in the promoters of retinoic acid-related orphan nuclear receptors Rev-erbα and Rorα genes, which compete for the ROR element (RORE) in the Bmal1 promoter. In turn, RORα up-regulates and REV-ERBα down-regulates the expression of Bmal1, and oscillations of Bmal1 and Rorα/ Rev-erbα are out of phase [51, 52]. At last, a number of kinases and phosphatases are an integral part of the clock function as they posttranslationally modify a number of clock proteins [53]. This mechanism, along with chromatin

remodelling, appears to be crucial for stable circadian rhythmicity [54].

Transcriptional deregulation of circadian genes in disease It is intriguing that the transcription of about 10 % of the genes, the so-called clock-controlled genes (CCGs), in a given cell is rhythmic and is under control of the circadian clock [55]. Many of these CCGs are tissue specific and differ among organs examined so far [56–59]. For example, Young et al. have investigated the diurnal variation in the intrinsic properties of the heart, at the levels of cardiac function, metabolism and gene expression and found that the intrinsic cardiac power, oxygen consumption and carbohydrate oxidation were greatest in the middle of the night [60]. Similarly, it has been proven that the disruption of the clock components CLOCK

Tumor Biol.

and BMAL1 may lead to metabolic imbalance and some associated pathological states including hypoinsulinaemia and diabetes [61]. Consequently, it is not surprising that the deregulation of circadian rhythms is associated with other pathogenic conditions as well (for the review of published data, please see Table 1). Analysis of gene expression patterns might be thus useful to study such circadian alterations, even though transcriptional regulation of the clock mechanism is extremely complex. For instance, human Tim gene, which is a mammalian homolog of circadian clock gene Timeless (Tim) in Drosophila but with somewhat uncertain role in mammalian circadian system [62], was found to be involved in human malignancies [17]. Microarray expression analysis of lung cancer cell lines revealed an elevated expression of Tim compared to normal lung controls, and in addition, high TIM protein expression correlated with poor survival of patient [17]. Gossan et al., in an attempt to characterize the circadian clock in murine cartilage tissue, performed time-series microarray analyses of mouse tissue and identified the first circadian transcriptome in cartilage which revealed that around 3.9 % of the genes were expressed in a circadian manner. Moreover, some clock genes showed disruption in the early stage of cartilage degeneration in the destabilization of the medial meniscus (DMM) model of osteoarthritis (OA) in mouse [34]. A whole-genome expression microarray analysis was performed in vitro, and the results suggest that Cry2 silencing in the breast cancer MCF-7 cell line significantly affected processes relevant for the immune response, cancer development in general as well as genes relevant for hematologic malignancies [63]. Lower levels of Clock expression were documented in healthy tissue relative to normal or tumour tissue from patients with breast cancer, suggesting that an aberrant overexpression of Clock may be an early event in cancer development. In addition, a whole-genome expression microarray analysis after Clock silencing revealed that Clock may play a particularly prominent role in regulating breast cancer-related biological pathways [20]. Importantly, as Yu and Weaver pointed out, circadian feedback loop may be disrupted in different ways which may cause different effects on gene expression. According to the authors, one has to be cautious in interpreting phenotypes that result from circadian gene disruption as this may be due to gene expression, loss of rhythmicity or disruption of specific effects [8]. Recent investigations were focused on the understanding of relationships between circadian clocks and gene expression and showed that direct interactions between master clock transcription factors and genomes are highly complex and surprisingly widespread. This indicates that core clock transcription factors can act in a ubiquitous, yet still flexible manner, to generate tissue-specific rhythmic patterns of global gene expression [64]. In addition, epigenetic factors should also be considered in a transcriptional regulation of clock genes. Indeed, Taniguchi et al. showed that Bmal1 gene was transcriptionally

silenced by promoter CpG island hypermethylation in hematologic malignancies, where it might prevent the development of the physiologic circadian rhythm [25]. Promoters of hPer3 and 40 % of hPer2 genes were similarly hypermethylated in patients with chronic myeloid leukaemia (CML) where association between the methylation frequency of hPer3 and the clinical phases of CML has been assessed [26]. Similarly, Gery et al. proved the association between circadian epigenetic regulation and cancer development, providing evidence that DNA hypermethylation and histone H3 acetylation were the potential mechanisms for silencing Per1 expression in nonsmall cell lung cancer [16]. Epigenetic regulation of gene expression is central both to physiological regulation of gene expression and cancer development. For example, hallmarks of cancer include evasion of apoptosis, autocrine growth regulation, resistance to antigrowth signals, sustained angiogenesis, limitless replicative potential, invasiveness and ability of forming metastases [65]. For instance, it has been documented that the Cry gene is important in the regulation of p53-independent apoptosis driven by inflammatory stimuli through an activation of NF-κB signalisation [66]. Moreover, clock genes were found to be correlated with disease outcome in breast cancer patients where they account for aggressiveness and therapeutic sensitivity of breast cancer [67]. Similarly, Zhao et al. suggested that the aberrant expression of PER1 is correlated with the growth, proliferation and metastasis of buccal squamous cell carcinoma, and it might act as an anti-oncogene [29]. These findings are supported by other results on negative limbs of the molecular loop, namely, PER2 and CRY1 role in a periodical inhibition of hypoxia-induced vascular endothelial growth factor (VEGF), a key molecule in tumour-induced angiogenesis subject to circadian fluctuation [68]. Indeed, anti-angiogenic agents showed an enhanced anti-tumour efficacy if administered at the time of increased VEGF production, which illustrates the rational for the chronopharmacotherapy approach [68, 69]. More recent findings in developing zebrafish embryonic model corroborate this assumption as circadian clock played a major role in the regulation of vascular development in zebrafish embryos, where BMAL1 and PER2 probably had opposing functions in the modulation of angiogenesis [70]. Several lines of evidence suggest the existence of molecular links between components of the circadian machinery and molecules involved in the control of cell cycle progression and present evidence for a putative connection between dysfunction of circadian clock, cell cycle and cancer progression [45, 71]. In fact, microarray analyses revealed that key cell cycle elements including cyclin D1, cyclin B1, cyclin E, cyclin A, P53, Wee1, c-myc, Mdm2 and Gadd45 exhibit circadiandependent expression [71]. For example, RORα and REVERBα influence the G 1 /S progression via p21 and CDK2/cyclin E-dependent inhibition/activation. PER1 and

Tumor Biol.

PER2 repress indirectly the G1/S progression by inhibiting cmyc transcription and subsequently cyclin D1 inactivation. BMAL1/CLOCK or BMAL1/NPAS2 heterodimers and CRYs recognize three E-boxes represented in Wee1 tyrosine kinase promoter and, in that way, activate or repress Wee1 transcription, which consequently phosphorylates CDK1/cyclin B complex and, as a result, inhibits the G2/M transition. In addition, it was found that PER1 and TIM participate in DNA damage response. These two core clock proteins might act as co-factors that activate ataxia telangiectasia-mutated and ataxia telangiectasia and Rad3related protein kinases involved in the phosphorylation of checkpoint kinase 1 and checkpoint kinase 2, respectively, which in turn are responsible for cell cycle arrest [71]. Existing literature data provide evidence for a link between circadian regulation and immunological status in general, which might have an impact on cancer regulation. Interestingly, it was found that transcription factor CLOCK via its histone acetyltransferase (HAT) activity acetylates and represses the transcriptional activity of glucocorticoid receptor (GR), which functions as a hormone-activated transcription factor and mediates most of the known glucocorticoid actions [72, 73]. This acetylation-mediated epigenetic regulation of the GR might be essential for the maintenance of proper timeintegrated glucocorticoid action. This significantly influences human health conditions and longevity, since stress exposure promotes glucocorticoid secretion leading to alterations in physiology, behaviour, metabolism and modulation of immune functions [72]. For instance, dysregulation plus uncoupling of the circadian rhythm of cortisol driven by the central clock, and acetylation-mediated circadian changes in local tissue sensitivity to glucocorticoids driven by the peripheral clock, might lead to functional hypercortisolism in target tissues and development of pathologic conditions [72]. Interestingly, recent work by Valles et al. presents evidence that stress-related hormones like adrenocorticotropic hormone, corticosterone and noradrenaline promote growth of B16F10 melanoma metastases via interleukin 6 (IL-6) and glutathione-dependent mechanism, implying possibility that glucocorticoids and/or catecholamines might influence IL-6 production by growing metastatic cells [74]. In addition, treatment with GR blocker inhibited metastatic growth [74]. As CLOCK controls GR transcriptional activity, glucocorticoid therapy widely used in cancer treatment should be administered to patients at a specific circadian time to achieve a maximum effect along with minimum side effects.

Skin, melanoma and circadian rhythm The skin is the largest organ in the human organism and our major defence against external environment. The skin is affected by a direct influence of environmental stimuli, e.g.

light, which makes the skin essential in the regulation of circadian rhythms and autonomous circadian clock components. Zanello et al. presented molecular and immunologic evidence for the constitutive expression of circadian clockrelated genes in the skin. In particular, they demonstrated the expression of Clock and Per1 genes in cultured human keratinocytes, dermal fibroblasts and melanoma cells, although the regulation of their expression and activity has not been elucidated [75]. Further, circadian oscillation of the clock genes expression has been shown in the human skin and oral mucosa, the latter showing high correlation of the clock genes expression with specific cell cycle phase activity [76]. A more recent microarray study revealed circadian oscillations during a 1-day period of clock along with tumour suppressor genes in the human oral mucosa [77] where 33 differentially regulated clock and clock-controlled genes were identified, including tumour suppressor and oncogenes. Oscillators are present in epidermal keratinocytes and melanocytes as well, where they seem to act in coordination and drive rhythmic functions in the skin [78]. Sandu et al. proved the existence of a complex circadian organization in the skin where each cell type hosts a functional and distinct circadian machinery. These clockworks display specific periods and phase relationships between clock genes, and these autonomous oscillators might act locally and interact with signals from the central pacemaker for driving rhythmic skin functions [78]. The presence of molecular oscillators has also been discovered in mouse skin, and the findings support the idea of a functional canonical clock mechanism within the skin as well as its dependence on synchronizing signalling from the SCN [35, 79]. However, the functional roles of clock genes within the skin still remain to be revealed. Skin cancer is one of the most common forms of cancer in the USA and Europe, and melanoma, in particular, is the most rapidly increasing malignancy in the white population with high mortality rate [80–82]. Melanoma occurs among all generations and accounts for less than 5 % of skin cancer cases but causes a large majority of skin cancer deaths (for a comprehensive review on melanoma staging, outcome and treatment, please see Online Resource 1). Due to a highly malignant phenotype, novel prognostic biomarkers and new strategies toward treatments of melanoma are needed. Even though it is evident that melanocytes’ functional clock might be disrupted during melanoma development, little is known about these mechanisms in the malignant melanoma pathogenesis. Only recently, Lengyel et al. presented first clinical evidence on a possible link between circadian clock genes and human skin tumorigenesis [9]. The authors analysed the expression of circadian clock genes Per1, Per2, Clock and Cry1 in human melanoma biopsies and their possible associations with the histopathological characteristics of melanoma. The messenger RNA (mRNA) of studied genes, and corresponding protein levels in the nucleus were reduced by 30–60 % in

Tumor Biol.

melanoma and naevus biopsies in comparison with adjacent non-tumorous samples. Consecutively, they found upregulation of the Clock gene in non-tumorous cells of melanoma biopsies, but not in melanoma cells or naevus cells, suggesting a role in the impaired regulation of metabolism in malignant tumours. Observed transcriptional alteration of clock genes may, however, not be necessarily a feature of malignant cells themselves but may be a relevant mechanism occurring in the surrounding non-malignant cells [9, 35]. It may be therefore hypothesised that the altered expression of the clock genes in the human skin might be an early event during tumorigenesis. Additional evidence that altered circadian rhythms are involved in the development of melanoma skin cancer came from cytotoxic activity investigations in the peripheral blood of mononuclear cells derived from patients with malignant melanoma [83]. Significant alterations in circadian rhythms of NK cell cytotoxicity and phagocyte activity were found in patients with malignant melanoma in comparison with rhythms of healthy volunteers. The degree of the rhythm disruption depended on a disease stage, though the mechanisms involved in these alterations are difficult to explain [83]. Furthermore, some clock-controlled genes such as human Nocturnin (hccrn4l) and the oncogene like human Mothers against decapentaplegic homolog 5 (hsmad5) expressed in the human oral mucosa are also under circadian clock control [77], pointing out the importance of further investigations of asynchronous regulations of clock genes and CCGs in cutaneous malignant processes.

The role of melatonin and arylalkylamine N-acetyltransferase enzyme Melatonin is closely involved in vertebrates’ time keeping and circadian functions. It acts as an output factor that primarily translates the length of the photoperiod to the organism. It is synthesized in the pineal gland in a circadian and lightdependent manner under control of the endogenous oscillator with the highest levels at night [84]. The synthesis of melatonin begins with the N-acetylation of serotonin. Critical regulatory element of melatonin synthesis is the penultimate enzyme arylalkylamine N-acetyltransferase (AANAT). While serotonin levels are much lower at night, melatonin concentrations display a reverse rhythm with the highest concentration at night associated with elevated circulating levels of melatonin. The link between these two reciprocal rhythms is the rate-limiting enzyme for melatonin synthesis, serotonin Nacetyltransferase (AANAT) [84]. Melatonin synthesis occurs also in other tissues in the body, notably in the skin. Slominski et al. provided evidence that serotoninergic and melatoninergic systems were fully expressed in the human skin and in most skin cell populations.

They tested normal and pathologic human skin, including melanoma cell lines, and demonstrated expression of fully developed, local serotonin and melatonin biosynthetic pathways in the human skin [85]. Moreover, melatonin receptors are expressed in normal and malignant melanocytes as in other skin cell types, they mediate phenotypic action on cellular proliferation and differentiation, and their expression may be altered by numerous environmental stimuli, for instance, ultraviolet radiation (UVR) [86]. Beside its indispensable role in circadian regulation, melatonin acts as a multifunctional agent. For example, melatonin has been suggested to protect skin through several mechanisms. As an anti-oxidant, it is a strong radical and UV-induced reactive oxygen species (ROS) scavenger, and it is more potent than vitamin C or vitamin E [86–88]. In that way, melatonin prevents potential DNA damage that could promote cancer development. In addition, melatonin can trigger an entire endogenous enzymatic protective system against oxidative stress by up-regulating genes and activity of some anti-oxidative enzymes like Cu/Znsuperoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase and glutathione peroxidase (GPx) [87, 89]. Melatonin can also protect the skin as it efficiently prevents and reduces UVR damage [87, 90]. Melatonin retains anti-apoptotic and anti-tumour properties as well [86, 87] and exerts oncostatic effects on human melanoma cell lines [91]. The authors showed that melatonin at pharmacological concentrations (1×10−3–1×10−7 M) suppressed the proliferation of melanoma cell lines [91]. Furthermore, a number of studies suggested oncostatic and therapeutic potency of melatonin treatment in different tumours including metastatic melanoma, though the exact mechanisms of melatonin action are not clear [86, 87, 92]. These pleiotropic functions of melatonin and involvement in the circadian rhythm regulation point to its possible impact on the expression and function of the circadian clock genes and vice versa. Considering its protective role against harmful UV light, disruption in melatonin expression levels might contribute to the skin cancer development, particularly melanoma. The rate-limiting enzyme for melatonin synthesis, AANAT, produces N-acetyl serotonin (HIOMT), which finally synthesizes melatonin. Besides its expression in the pineal gland, it may be produced extrapineally, for example, in the skin. The AANAT gene expression and corresponding proteins were indeed identified in human skin and in cultured normal and malignant melanocytes [85]. Rhythmic AANAT expression is controlled by the cyclic adenosine monophosphate (cAMP) signal transduction pathway involving β-adrenergic receptors activation through cAMP-responsive elements (CREs) in the AANAT promoter region [84]. Rodent AANAT promoter possess a functional E-box element which can be activated by the BMAL1-CLOCK heterodimer, although results showed that E-box sequences are not required to drive circadian expression of the gene in the pineal gland, but

Tumor Biol.

mainly in the retina. The authors proposed that the E-box element is in a position to contribute significantly to circadian regulation of the AANAT gene in retina photoreceptors by the primary clock mechanism [93]. Humphries et al. confirmed their results and anticipated that E-box acts in the specification of rat AANAT gene expression and found a role for these enhancer sequences in the control of tissue/cellular specificity in mammals [94]. Moreover, in Per1-deficient mice, the amplitude in AANAT expression and enzyme activity was significantly elevated in the pineal gland as compared to the wild type, indicating that molecular clock alters the AANAT gene expression. The authors concluded that the core clock protein PER1 plays an important role in the modulation of rhythmic melatonin synthesis in the pineal gland [95]. Similarly, AANAT mRNA in the chicken pineal gland undergoes circadian rhythm as well. It has been shown that the chicken AANAT promoter contains an E-box element as well that binds the BMAL1CLOCK heterodimer that consequently enhances transcription of avian AANAT, providing evidence for the direct link between the clock genes and the output mechanism [96]. Considering these investigations and the existence of the AANAT E-box element in the promoter region, it may be plausible to assume a central role within the first-order clock-controlled genes. Our hypothesis is therefore that AANAT gene in the human skin and/or pineal gland might be directly regulated by the BMAL1-CLOCK complex. Disruption in some of these core clock gene expression or regulation mechanisms might lead to the direct deregulation of AANAT, leading to disruption in melatonin synthesis. However, more additional experiments and investigations are required to elucidate the highly complex systems which ensure a proper spatial and temporal pattern of gene expression and regulation in general and, importantly, how their disruption may particularly influence the development of malignant melanoma.

Influence of UV radiation and artificial light exposure as a risk factor for melanoma The UVR is thought to be one of the main causes of skin cancer as it induces DNA damage. According to some experiments performed on animals, UVA and UVB subsets of solar UV may be equally responsible for the malignant transformation of melanocytes since melanoma can be induced by exposure to UVR [81, 97]. Even though the sunlight is considered as the main input factor of circadian rhythm, there is no much information on the effect of UVR on the circadian rhythm. It was shown that low-dose UVB could down-regulate some of circadian core clock genes and alter their expression in human keratinocyte cell culture, suggesting a role in the circadian rhythm [98]. In humans and mice, the nucleotide excision repair system is responsible for removing UV-induced damages, and xeroderma pigmentosum group A (XPA) protein is

the rate-limiting subunit of excision repair. Kang et al. demonstrated that XPA in the mice liver and brain is under control of the circadian clock mechanism [99, 100]. They found that the circadian oscillation of XPA is done by regulation of transcription through CRY and by regulation at the posttranslational level by the HERC2 ubiquitin ligase [99]. Recently, Gaddameedhi et al. reported that XPA and the excision repair rate exhibit circadian oscillations in the mouse skin, reaching maximum in the evening and minimum in the morning. The authors also found that mice exposed to UVB light at low excision repair activity developed skin cancer faster and at fivefold higher frequency than mice exposed to UVR in the evening hours when excision repair was at its peak. Based on these findings, it seems that the daily period of exposure to UVR is an important factor for carcinogenicity in mice and probably in humans as well [101]. For instance, Wang et al. provided evidence for UV-induced mutations in tumour suppressor genes, i.e. p53 and PTEN (phosphatase and tensin homologue) in XP melanoma patients. This finding supports the role of UV in the induction of melanoma [102]. In addition, recent investigation on the role of the clock within the mouse skin revealed that BMAL1-dependent circadian variation is modulated by UVB-induced DNA damage, pointing out the possible relevance of circadian mechanism in general mechanisms of the epidermal carcinogenesis. It was indeed observed that BMAL1 might suppress the number of cells in the S-phase by reducing oxidative phosphorylation during the day when the ROS levels are high. Similarly, in the mice epidermis, the highest susceptibility to UVB-induced DNA damage was observed late at night during the S-phase peak. If this mechanism is true for diurnal humans when the UV intensity is high and DNA damage susceptibility should be highest, which is opposed to the S-phase peak and mitosis in epidermis to that of nocturnal mice, human skin might be particularly vulnerable to UV-induced skin cancer [103]. However, circadian regulation in humans and mice might not be exactly alike, and more profound experiments on molecular and population level need to be done in order to uncover exact mechanisms underlying UV-induced carcinogenesis and its potential role in melanoma development. In the context of melanoma, it is also noteworthy that our circadian rhythm and particularly melatonin synthesis could be altered by the wide use of artificial light. Moreover, UVR exposure does not suffice to explain all types of skin cancers, particularly melanoma, where the connection with sunlight exposure is evident, but still not that implicit as with other types of skin cancers [97]. Increased incidences of melanoma were observed in recent years in indoor and office workers exposed to fluorescent light. Kvaskoff and Weinstein hypothesised that exposure to artificial light at night might be linked to an increased risk of melanoma as it promotes melatonin suppression. This so-called light at night (LAN) theory might explain at least partially the increasing incidence

Tumor Biol.

of breast cancer via inhibition of melatonin secretion as the use of artificial light at night, and industrialisation is in constant growth [92]. In conclusion, light pulse at night can shift the circadian rhythm and strongly suppress melatonin secretion thus promoting progression of melanoma or other malignancies. Effects on melatonin should not be considered as sole consequences of LAN: artificial light alters circadian rhythms in general and disrupt clock gene regulation, which might lead to cell cycle disruption and/or apoptosis with a plethora of biological effects.

Conclusion Even though deregulation of circadian rhythm in mammals is known to be tightly linked to various pathological states, the exact mechanisms have not been elucidated so far. In particular, these mechanisms in cancer development should be investigated in more details as they may provide novel possibilities for prevention or treatment. For example, literature data provide evidence that the AANAT gene, a penultimate enzyme of melatonin synthesis, might be directly regulated by the BMAL1-CLOCK complex and that any disruption of the circadian rhythm might lead to a direct deregulation of AANAT and disruption in melatonin production. A role of melatonin disruption might be particularly interesting for elucidation of melanoma pathogenesis. Considering the rapidly increasing melanoma malignancy rate in the white population with still very high mortality rate, analysis of this functional link between AANAT gene, melatonin and circadian rhythms might provide novel insights for medical doctors dealing with this problem. To date, little is known about molecular clock functions in melanoma, in particular related to the wide use of artificial light or prolonged exposure to UV light, both factors that directly alter melatonin expression levels particularly in the skin. Besides alteration of melatonin production, disruption of circadian rhythms has a plethora of biological effects that still have to be exactly established. For that purpose, more research emphasis should be put into detailed analyses of disease pathogenesis by the use of high-throughput genomics and proteomics methods [37, 104] that might reveal novel molecular mechanisms behind melanoma. Acknowledgments This work was supported by the Croatian Ministry of Science, Education and Sports grant (335-0000000-3532). Conflicts of interest None

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