The photobiology of melanocytes modulates the impact of UVA on sunlight-induced melanoma

Abstract

Ultraviolet radiation is responsible for melanoma. In this review, we address the role of the different UV spectra in melanoma. The data suggest that only UVB is capable of initiating melanoma, and that both UVA and UVB are involved in the progression of the disease. The etiology of sunlight-induced melanoma may be different for chronically-exposed tumors and for those located on body surfaces with considerably less exposure. Solar signature mutations are most likely associated with the progression of chronically-exposed tumors. The unique relationship between UVA and melanocytes, and the role of melanin in photocarcinogenesis is discussed. The current state of knowledge strongly indicates that UVA, regardless of its source, is involved in melanoma and should be avoided to deter progression of incipient tumors.

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UVA and the melanoma epidemic

Cutaneous malignant melanoma (CMM) continues to increase in the Caucasian population at a rate greater than any other cancer. About 160 000 new cases of CMM are diagnosed and about 48 000 melanoma-related deaths occur annually worldwide.¹ Epidemiological data indicate that the annual increase in CMM has been in the order of 3–7% per year for fair-skinned populations living in temperate latitudes, and is projected to double every 10–20 years.^2–4 The increase in CMM has been particularly acute in Australia and New Zealand, which show the highest melanoma rates in the world, with the northern latitude countries Sweden, Denmark, the Netherlands, the United States, Canada and Iceland showing lower, but still alarming increases as well. Although the rate of incidence has slowed over the past few years in many countries, in some, particularly in the emerging economies of Eastern Europe and elsewhere, it remains a present and growing concern.

The causes underlying the dramatic increase in CMM are not fully understood, but it is very likely that increased exposure of fair-skinned peoples to solar and artificial ultraviolet radiation (UVR) is an important contributing factor, along with increased public awareness and subsequent positive screening for early stage CMM. Brief intense exposure to solar UV (i.e., sunburn), particularly in children and young adults, is a risk factor for CMM. Paradoxically, it appears that the use of sunscreen, rather than reducing exposure, actually increases the time spent in the sun before erythema occurs.⁵ Because sunscreens do not protect against UVA (315–400 nm) as well as UVB (280–315 nm) and because SPF values are based on UVB-induced erythema, the absorbed UVA dose can vary and be extremely high after long periods of exposure. High cumulative exposures are also associated with the use of artificial solaria that rely primarily on UVA to stimulate tanning.⁶

A dramatic illustration of the apparent effects of sunbed usage on CMM can be seen in the “Iceland melanoma epidemic” that occurred between 1995 and 2005.⁷ A conspicuous spike in CMM that lagged behind increased sunbed usage in Iceland during the mid-1980s was most obvious in young women and involved primarily truncal melanomas associated with acute high exposures. Indeed, during this period, the rate of CMM on the trunk of women actually exceeded that observed in the lower extremities, opposite to trends observed prior to 1995. It is believed that the intensive use of artificial tanning devices beginning in the mid-1980s contributed to this phenomenon and that education efforts by the government in response to this public health crisis in turn contributed to the decline in CMM in Icelandic women after 2005. Albeit a very intriguing study, epidemiologists caution its conclusion based on the complexity of potential ecological factors contributing to CMM.⁸

Meta-analyses of human CMM strongly suggest that UVA is an important component in the etiology of sunlight-induced melanoma, and in 2009 the International Agency for Research on Cancer (IARC) determined that UVR from sunlight and artificial tanning devices, including UVA, is a potent (Group 1) human carcinogen, potentially equivalent to tobacco in its effects on human health (for a review, see Autier et al.⁹). In July 2009, the IARC released a report that categorized tanning beds as “carcinogenic to humans.” Indeed, it has been proposed that the combination of increased UVA exposure and decreased UVB-mediated vitamin D metabolism may be responsible for the increase in CMM.¹⁰ In this review, we will examine the similarities and differences in the photochemistry and photobiology of UVA and UVB radiation with particular regard to melanocytes, melanin and melanoma. In the process, we will highlight and discuss recent publications that bear on this theme, and offer a perspective on current dogma and future directions.

UVA does not induce melanoma in animal models

Initially, the Setlow action spectrum for melanomagenesis in the Xiphophorus hybrid fish model supported the idea that UVA was indeed responsible for melanoma in the human population.¹¹ Subsequently, a more robust investigation of the phenomenon in this model showed that UVA does not induce melanoma.¹² Although data from carcinogenesis experiments in mice show that UVA alone is a complete carcinogen, capable of inducing squamous cell carcinomas (SCC) after chronic exposure,¹³ it does not induce melanomas in animal models susceptible to UVB-induced melanoma.^12,14,15 Despite the divergent evolutionary paths of the three primary UV-induced melanoma models (i.e., opossum, mice and fish), in vivo experimentation has revealed several key elements of melanoma susceptibility that are shared across these distantly-related organisms. First, studies of UVR irradiation in these models demonstrate that the initiation of melanomas is not related to chronic UV exposures, and the concomitant accumulation and persistence of DNA lesions and mutations. Second, the action spectrum for melanoma formation is conserved. UVB but not UVA induces CMM in all the models tested.^12,15–18 Furthermore, the importance of early life acute exposures to UVB is a general phenomenon. Lastly, as with human melanoma formation,¹⁹ RTKs, Ras-Raf-Mapk and PTEN-PI3-kinase-Akt signaling pathways are quintessential to melanomagenesis in in vivo animal models.^20,21 The continued development and use of diverse animal models will be essential to further elucidate the elusive mechanisms underlying melanomagenesis.

DNA damage and mutation spectra are similar for UVA and UVB

Although the literature is not consistent, work over the past 10 years or so has shown that the DNA damage spectra induced by UVA and UVB radiation in the absence of melanin are surprisingly similar. UVB damage is dominated by the direct absorption of photons by DNA and the formation of covalent photoproducts such as cyclobutane pyrimidine dimers (CPDs), pyrimidine-pyrimidione (6–4) photoproducts [(6–4)PP] and various minor lesions (e.g., pyrimidine photohydrates). Over the past decade, it has been shown that CPDs are induced by UVAin vitro²² and in vivo^23,24 at frequencies comparable to or exceeding that of oxidative damage. Researchers suggest that they form via either a photosensitizer-mediated triplet energy transfer²² or a direct photochemical reaction.²⁵UVA and UVB generate predominantly two species of free radicals, including singlet oxygen, which specifically reacts with guanine residues²⁶ and hydroxyl radicals,^27,28 both of which react primarily with purines.²⁹

Since the mechanisms of ROS formation by UVA and UVB are essentially the same and the action spectrum for 8-oxoGuo induction varies little between 300–400 nm,³⁰ it is doubtful that there are significant differences in the relative frequencies of ROS and the type of DNA damage induced by UVA and UVB. Because ROS-mediated base damage is low compared to direct damage and because this type of damage is very rapidly repaired,³¹ the importance of these lesions in the UV damage response, particularly carcinogenesis, is problematic. Depending on wavelength, the amount of UVA damage is small compared to UVB, ranging from ∼10% at 320 nm to ∼0.001% at 360 nm. Even when the overwhelming proportion of UVA in total incident solar UVR is considered, the damage induced is still minimal compared to UVB.³² Coupled with the observation that UVA does not induce melanoma, it is thus unlikely that free radical damage to DNA is involved in initiating CMM. However, it should be kept in mind that UVA can reduce the rate of DNA synthesis and induce a strong G2/S block in melanocytes and melanoma cells, suggesting that the DNA damage induced by UVA can have potent effects.³³

Mutation spectra of irradiated cells and non-melanoma skin cancers (NMSC) mirror the DNA damage spectra.³⁴ For many years, the C→T transition mutation was considered the “signature mutation” of UVB since it dominates mutation spectra in UVB-irradiated human primary fibroblasts³⁵ and NMSC,^36,37 and correlates well with T–C dipyrimidine sites in tumor suppressor genes. Despite the observation that UVA induces primarily thymine-thymine CPDs, which have a low mutagenic capacity, it generates mutations similar to those produced by UVB,^34,35 with the C→T transition being the most common mutation found in vivo after UVA irradiation³⁸ (for a review, see Rünger³⁹). Indeed, the C→T transition is now considered a “solar signature mutation” rather than solely a UVB signature mutation; its preferential formation at ^meCpG sites, particularly after UVA irradiation, increases its biological potential (see below).

Mutations in BRAF and NRAS in human and mouse melanomas do not show the signatures expected from ROS-mediated damage, such as G→T transversions, but, in fact, show T→A transversions comprising 92% of BRAF mutations. The proportion of oxidized pyrimidines in UV-irradiated DNA is much greater than oxidized purines.^30,40 Hence, a T→A transversion should be a fairly rare UV-induced mutation in BRAF, although a mechanism has been proposed in which the error-prone replication of bases adjacent to a CPD could lead to the BRAF^(V600E) mutation.⁴¹ In light of these and other data, it is probable that the BRAF mutations associated with sporadic melanoma occur as downstream products of progression rather than as a direct result of UVR.^42,43

Unique UVA photobiology of melanocytes may contribute to tumorigenesis

The ratio of UVA/UVB increases with depth in the skin,⁴⁴ and melanocytes, which are located at the basal layer, at the base of hair follicles or in the dermis, receive proportionately more UVA than suprabasal (squamous cell) keratinocytes. Hence, under UVR conditions in which UVB is reduced or blocked and UVA is substantial (e.g., sunbathing with or without sunscreen, artificial tanning), most biological effects will occur at a greater depth in the skin where melanocytes are located. Because of this, it is important to visit recent data regarding complex interactions between UVA and melanocytes, some of which may increase melanoma risk and others which may protect against melanomagenesis.

Wood and co-workers⁴⁵ investigated melanin photosensitization by different monochromatic wavelengths of UVR using the same Xiphophorus fish model that Setlow and colleagues used for their original UVA melanoma studies.¹² In this report, electron paramagnetic resonance was used to generate an action spectrum for reactive melanin radicals in pigmented fish skin and showed a peak of formation in the UVA, which correlates with the Setlow melanoma action spectrum.¹¹ Since the ROS action spectrum shows only a ∼2-fold difference in the frequency of ROS generated by 313 nm (UVB) and 365 nm (UVA), and given that subsequent studies showed that, like marsupials and mice, the fish are refractory to melanoma induction by 365 nm but not 313 nm radiation, the significance of these photoproducts in melanomagenesis is problematic. This is made even more complex by the observation that oxidative damage in DNA is poorly repaired in human melanocytes⁴⁶ and, hence, could accumulate in significant frequencies after chronic exposure. The relationship between melanin free radicals, the damage induced by these radicals, the repair of this damage and melanoma is unclear (for a review, see Rünger⁴⁷), but it is clear that they are not sufficient by themselves to induce melanoma in this fish model, which shows robust UVB melanomagenesis.¹² Identifying and characterizing the DNA photoproducts produced by melanin free radicals and how they relate to the mutation spectra in truncal and sun-exposed CMM would be of considerable interest.

The “love–hate” relationship between UVR and melanin is well known.^48,49 On the one hand, melanin is photoprotective, absorbing UV photons that could otherwise damage the genetic material; on the other hand, it provides a substrate for generating a unique class of photodamage that adds to the burden of endogenous oxidative damage, which is potentially deleterious to the cell. Although research on this complex group of tyrosine-based molecules is difficult, many different melanin species have been identified and grouped into two main classes, eumelanin and pheomelanin [note: the etymology of these terms is as convoluted as its biochemistry: the Greek root “pheo” means brown; pheomelanin is red/yellow in contrast to eumelanin (“good” melanin), which is brown/black]. Ironically, pheomelanin is considered the “bad” melanin and is thought to be causally related to melanoma, although differences in the eumelanin/pheomelanin content in human skin do not correlate with carcinogenesis.⁵⁰ Both pheomelanin and eumelanin are induced in human skin by UVB and, whereas UVA has no effect on the concentration of either species, solar-simulated radiation (SSR), containing both UVA and UVB wavelengths, shows significantly higher melanin induction than UVB alone, suggesting that UVA contributes to melanogenesis synergistically through UVB.⁵¹ A function of UVA separate from UVB is the immediate tanning response, whereby melanin precursors are “activated” via photooxidation reactions; processes that could be associated with melanin free radical generation. UVB, on the other hand, is responsible for delayed tanning, which involves up-regulating melanin synthesis and increasing pigmentation coverage. Again, simultaneous exposure to UVA and UVB using SSR shows greater stimulation than UVB alone.⁵² One could evoke a teleology whereby UVA throws up a temporary protective screen (melanin) immediately after sunlight exposure to protect the DNA from further damage by UVB. However, UVA alone has little effective photoprotection but does have the ability to induce free radicals and, perhaps, oxidative damage through its interactions with melanin precursors.

UVA, initiation and progression

Although the DNA photochemistry and mutagenicity of UVA is similar to UVB, experimental evidence strongly suggests that UVA by itself cannot initiate melanoma.⁵³ If melanocyte initiation does indeed require a high acute dose and the high levels of damage that go along with it, then the inability of UVA to initiate melanoma may simply be a matter of dosage. We used HPLC-MS/MS to measure dimer frequencies in pigmented epidermis from animals (fish) exposed to doses of UVA and UVB used for tumorigenesis studies.⁵⁴ We found that the total direct damage induced by UVB in pigmented cellsin vivo was 3.2 lesions kJ⁻¹ m⁻²; we detected no damage after UVA irradiation. Using a much higher dose of UVA to purified DNAin vitro, we found that UVA induced direct damage at ∼0.1% the rate of UVB. With 20-fold greater UVA in sunlight compared to UVB, we calculate that ∼2% of the total direct damage caused by sunlight would have been due to UVA. If DNA damage thresholds participate in melanoma initiation, whatever that process might be, it is possible that UVA does not have the ability to exceed the thresholds required to initiate truncal, acute CMM.

Once initiated, incipient melanomas from different body sites will progress either in the absence of further sunlight exposure (sporadic truncal melanomas) or in the presence of continued chronic solar UV (melanomas of the face and extremities). The progression of truncal melanoma is probably more dependent on the genetics of the individual (atypical nevi) than DNA damage. In contrast, the progression of initiated melanomas chronically exposed to sunlight would be aggravated by the accumulation of DNA damage and mutations. Although the role of mutagenesis in melanoma initiation is problematical, work by Pleasance and co-workers⁵⁵ suggests that C→T transition mutations may play an important role in progression. The genome sequence from a metastatic melanoma is dominated by signature mutations associated with solar UVR (UVA and UVB). If the primary tumor for this metastasis was truncal, it is very doubtful that the high number of C→T mutations could have been generated without additional chronic exposure over a long period of time. Hence, it is likely that the primary site was on a sun-exposed surface, and that the accumulation of transition mutations reflected that location and contributed to progression.

The observation that UVA induces C→T mutations at ^meCpG sites more frequently than UVB³⁸ and the fact that these sites of damage correlate with mutation hotspots in tumor suppressor genes⁵⁶ suggests that UVA may play an important role in progression. Methylation at CpG islands (^meCpG) significantly increases CPD formation at these sites^57,58 and, consequently, the formation of C→T mutations. Indeed, cytosine deamination within a T<>^meC CPD located at a CpG island is greatly enhanced by the 3′ G and explains the targeting of these mutations to hotspots in tumor suppressor genes such as p53.⁵⁹ The role of UVA in the oxidation-mediated formation of hydroxymethylcytosine has not been investigated but may have a significant bearing on gene expression, epigenetic regulation and tumorigenesis (including melanoma). UVA has the potential to affect progression in a variety of ways in addition to mutagenesis, including signaling,^60,61apoptosis,^62,63 immunosuppression^64–66 and metastasis.^67,68UVA may also influence melanocyte behavior indirectly through its micro-environment by inducing growth factors in fibroblasts and keratinocytes, the paracrine activation of melanocytes, and subsequent progression to melanoma.⁶⁹

Conclusion

Although DNA damage and mutation data suggest that UVA should elicit photobiological responses similar to UVB, albeit at significantly higher doses, the fact remains that UVA does not induce melanoma in animals that are susceptible to UVB-induced melanoma. It is important to note that the protocols used to induce melanoma in animal models imitate the acute early life exposures presumably associated with human melanomagenesis. Many sunlight-induced melanomas occur on body sites not exposed to repeated solar UV (i.e., truncal melanomas) and do not show the solar signature mutations characteristic of carcinomas or melanomas occurring on sun-exposed sites (e.g., head, neck, arms). Whether this observation is due to different etiologies, as proposed by the “diversity hypothesis”,⁷⁰ or the same etiology under different environmental conditions⁵² is a matter for future research in animal models. The photobiological response of melanocytes at different stages of development (including stem cells) and of melanin to solar UVR is of considerable interest. Furthermore, understanding the role of ROS in the progression of truncal melanomas (in the absence of sunlight) would offer considerable insight into the role of photo-oxidative damage in the progression of sunlight-exposed melanoma. Current research suggests that UVB dominates the etiology of melanoma and may be prerequisite for initiation. However, UVA has potent biological effects that may make a significant contribution to the progression (e.g., rate of onset and severity) of the disease. Careless and casual exposure to UVA from sunlight or artificial tanning sources significantly enhances the risk of melanoma by facilitating progression and metastasis, and may account for recent trends in melanoma incidence attributed to natural and artificial UVA exposures.

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Footnote

† Contribution to the themed issue on the biology of UVA.

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