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See related article on pg 1915
Could BRAF Mutations in Melanocytic Lesions Arise from DNA Damage Induced by Ultraviolet Radiation? Nancy E. Thomas1,2, Marianne Berwick3 and Marila Cordeiro-Stone1,4 Although BRAF V600 mutations in melanocytic tumors are not UVsignature mutations, it is plausible that they could still arise from errorprone replication of UV-damaged DNA. We propose a mechanism for their origin, taking into consideration melanocytic-specific BRAF tandem mutations, nearby potential pyrimidine dimer sites, the properties of specialized DNA polymerases, and biological selection. Journal of Investigative Dermatology (2006) 126, 1693–1696. doi:10.1038/sj.jid.5700458
Shortly after BRAF mutations were discovered and found to be present in about 60% of melanomas (Davies et al., 2002), their presence was reported in 20%–80% of melanocytic nevi in a variety of studies (Kumar et al., 2004; Pollock et al., 2003; Yazdi et al., 2003). Now, Lassacher et al. (2006, this issue) have extended our knowledge in this area by reporting their finding that the t1796a mutation at codon 599 (V599) of BRAF is also common in biopsies of lentigines from patients who have received psoralen plus UVA (PUVA) treatment for psoriasis. The authors concluded that PUVA lentigines might be precursors of cutaneous malignant melanomas and that the BRAF mutations could be related to the molecular and cellular effects of PUVA. Previous studies in TP53 of basal-cell carcinomas (Seidl et al., 2001), INK4A/ARF of squamous-cell carcinomas (KreimerErlacher et al., 2003), and TP53 and HRAS of keratoses (Wolf et al., 2004) from PUVA-treated psoriasis patients disclosed that the majority of the mutations in the analyzed genes were C>T or CC>TT transitions at dipyrimidine sites, which are recognized as UV-signature
mutations. The etiology of BRAF mutations in melanocytic lesions, however, is far from clear. The t1796a BRAF mutation is embedded in a sequence (5′GTG) where psoralen binding could possibly induce the T>A transversion, although mono-addition and cross-linking of psoralens occur preferentially at 5′ATA and 5′TA (Esposito et al., 1988). PUVA treatment may increase melanoma risk (Stern, 2001), but study of PUVA is confounded by the fact that patients treated with PUVA also typically have received multiple psoriasis treatments, including UVB, tar, and/or immunosuppressants, such as cyclosporine and methotrexate. In this Commentary, we would like to explore the possibility that BRAF mutations detected in melanocytic lesions from areas of the skin that are prone to sunburns could arise from UVB-induced DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs) and [6-4] pyrimidine-pyrimidone adducts ([6-4]PPs). (Note: The numbering of BRAF codon V599E (t1796a) reported by Lassacher et al. is the same as in the original article on BRAF mutations in melanomas by Davies et al. (2002). Later in this Commentary, we present
1
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA; 2Department of Dermatology, University of North Carolina, Chapel Hill, North Carolina, USA; Department of Medicine, Division of Epidemiology, University of New Mexico, Albuquerque, New Mexico, USA; and 4Department of Pathology, University of North Carolina, Chapel Hill, North Carolina, USA. 3
Correspondence: Dr. Nancy E. Thomas, Department of Dermatology, University of North Carolina, 3100 Thurston Bowles Building, CB#7287, Chapel Hill, North Carolina 27599, USA. E-mail:
[email protected]
the numbering according to the correction by which the National Cancer Institute gene bank renamed the V599E (t1796a) mutation as V600E (t1799a); accession number NM_004333.2.) Melanoma risk is complex, but considerable roles for intermittent sun exposure and sunburn history have been identified in epidemiologic studies (Gandini et al., 2005b). Number of common nevi is also an important risk factor for melanoma (Gandini et al., 2005a). The pathogenic effects of sun exposure could involve the genotoxic, mitogenic, or immunosuppressive responses to the damage induced in the skin by UV. Controversial is whether the UVB or the UVA component of solar radiation is more important in melanoma development (De Fabo et al., 2004; Wang et al., 2001). UVB represents only a small portion of the solar radiation reaching the earth’s surface (T transversions are the major mutations induced by UVA in mammalian cells (Besaratinia and Pfeifer, 2005). Several possible UV targets have been recognized, including the INK4A, TP53, and RAS genes, which can have mutations in melanomas; however, BRAF, which is often mutated in nevi and melanomas, may also deserve serious consideration as a UV target. Suggestive of a role for UV in the formation of BRAF mutations are findings that these mutations are more common in melanomas arising on intermittently than rarely sun-exposed anatomic sites (Edwards et al., 2004; Maldonado et al., 2003). Edwards et al. (2004) have discussed the possible contributions to www.jidonline.org 1693
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BRAF mutations of UV-induced DNA lesions other than CPDs and [6-4]PPs, including minor photoproducts and oxidative damage. Additional evidence that BRAF mutations might arise from UV exposure are findings that melanocytic lesions often include tandem BRAF mutations (Thomas et al., 2004), which are rarely found in other nonmelanocytic tumors harboring BRAF mutations (such as colon, thyroid, ovarian, and lung cancer) (Salvatore et al., 2004; Samowitz et al., 2005; Sieben et al., 2004). Only one V600 tandem mutation has been reported in a nonmelanocytic lesion (a cholangiocarcinoma) (Tannapfel et al., 2003), whereas approximately 25% of BRAF mutations
in primary invasive melanomas are tandem mutations (Maldonado et al., 2003; Thomas et al., 2004). Do these melanocytic-specific BRAF tandem mutations provide a mechanistic clue to genotoxic damage in melanomas? The most common BRAF mutation found in melanocytic nevi and melanomas, the t1799a substitution — that is, the V600E mutation — is not at a dipyrimidine site, and, thus, it is not generally viewed as resulting from error-prone replication of UVBdamaged DNA. However, this possibility cannot be ruled out when one takes into consideration the known properties of specialized DNA polymerases. These enzymes catalyze nucleotide
Figure 1. Single and tandem mutations in codon 600 of BRAF in melanocytic lesions. Nucleotide sequence surrounding V600 in wild-type BRAF is illustrated to show dipyrimidine sites where UVinduced DNA photoproducts could be formed (asterisks); these positions are color-coded to indicate the relative probability of cyclobutane pyrimidine dimer (CPD) formation as high (red), intermediate (green), or low (blue). Both CPDs and [6-4] pyrimidine-pyrimidone adducts ([6-4]PPs) can be formed at 5′TC sites at approximately the same frequency; because of the higher rate of removal of [6-4]PPs than CPDs by error-free nucleotide excision repair, it is more likely that a CPD at the 1800–1801 position might influence the fixation of the single or tandem BRAF mutations listed in this figure (see text for details).
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polymerization across template DNA lesions that stall the progression of replication forks. The BRAF sequence surrounding codon 600 includes several dipyrimidine sites where photoproducts could be formed by UVB absorption (Figure 1). As reviewed recently by Pfeifer et al. (2005), CPDs (rather than [6-4]PPs or other lesions) are associated with the great majority of UVB-induced mutations, and cytosine deamination could be responsible for most of the C>T substitutions. DNA polymerase η (pol η) represents an important safeguard against the mutagenic effects of CPDs; although these lesions are strong blocks to the replicative polymerases, the active site of pol η is flexible enough to accommodate the dimers, “read” their sequence composition, and replicate beyond the photoproduct with an error rate of 10–2 to 10–3 (Johnson et al., 2000). This means that pol η is an error-prone enzyme that makes a mistake for every 100 to 1,000 nucleotide incorporation events (including during replication of undamaged DNA) but allows human cells to complete replication in the presence of UV-induced DNA damage and survive with a low mutagenic burden. It is still not understood how pol η gains access to the stalled replication fork and, soon after completing synthesis across the CPD, hands the growing nascent DNA back to DNA polymerase δ (pol δ). Primer-extension studies with purified enzymes suggested that the processivity of pol η across the CPD and flanking bases is higher than on the undamaged sequence and influences the switching between pol η and pol δ before and after the dimer (McCulloch et al., 2004a; McCulloch et al., 2004b); this evidence implies that pol η could get access to the nascent DNA at the –1 position relative to the CPD. In the diagrams in Figure 2, we consider the possibility of accurate (Figure 2a) or inaccurate (Figure 2b) polymerization across the nucleotide just preceding a CPD at the 1800–1801 dipyrimidines. Also considered in Figure 2b is the evidence that pol η is not adept at extending a mismatched primer (Masutani et al., 2000). DNA polymerase ζ (pol ζ), however, is more efficient at extending from base mispairs created by another
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polymerase (reviewed by Prakash and Prakash, 2002), a characteristic that appears to underlie the finding that UV-induced mutagenesis is strongly dependent on pol ζ (Gibbs et al., 1998). Therefore, it is the encounter of a template CPD by the replication machinery that would set the stage for the switch of pol δ for pol η, followed by accurate translesion synthesis before handing back of the growing strand to pol δ one or two nucleotides beyond the dimer (Figure 2a). If the 3′C at the dimer were to undergo deamination, then accurate translesion synthesis by pol η would result in a silent C>T substitution at the 1800 position (Figure 2a). Figure 2b envisions the possibility of the error-prone pol η inserting an A at the 1799 position and this mispairing causing it to dissociate from the growing strand; then, elongation of the mispaired primer by pol ζ would lead to the t1799a transversion mutation, or the tg1799–1800aa tandem mutation when combined with deamination at the dimer. Induction of the V600D tandem mutation (Figure 1) is more difficult to explain; it would require pol ζ to extend the mispair introduced by pol η and make another one of its own at position 1800. Finally, acquisition of the tandem mutations leading to V600R or V600K could possibly be influenced by a CPD at the 1797–1798 dipyrimidines. Even though CPDs are formed at very low frequency at 5′CT, a C>T transition facilitated by misincorporation by pol η at the +1 position could explain these tandem mutations. In closing, it is important to recognize that the explanations offered above for how common BRAF mutations could be linked to the presence of nearby pyrimidine dimers require the convergence of several rare events. Hence, if they do occur as predicted, the common finding of these mutations in BRAF, but not in other gene targets, might reflect a powerful biological selection for mutations at codon 600; this mutation activates the BRAF oncogene, and expression of the active oncogene stimulates mitogenic signaling in melanocytes at least briefly (Michaloglou et al., 2005). Theoretically, repeated blasts of intense sun exposure, similar to that resulting in sunburn during vacation,
a
b
Figure 2. Modeling the acquisition of codon 600 BRAF somatic mutations on the basis of error-prone translesion synthesis of a UVB-induced CPD. Polymerase η (pol η) is a specialized DNA polymerase that contributes to the extension of nascent DNA strands when the replicative polymerases (for example, pol δ) are blocked at CPDs. (a) Pol η reduces the probability of UV-induced mutagenesis by catalyzing translesion synthesis accurately in more than 99% of CPDs on template DNA. Deamination of cytosines to uracil before replication could result in the C:G>T:A transition at the nucleotide position 1800; this substitution, however, would be a codon 600 silent mutation, as both GTG and GTA code for valine. (b) Mutation fixation across a UV-induced CPD may involve the participation of more than one polymerase and is strongly dependent on another specialized DNA polymerase, pol ζ. In the scenario depicted in this diagram, pol δ is blocked by the dimer and its exonucleolytic activity removes the last incorporated nucleotide before pol η gets access to the stalled replication fork (see text for details). With an overall fidelity of 10–2 to 10–3 (even on undamaged templates), pol η could introduce the wrong nucleotide before dissociating from the primer template, as this enzyme cannot extend a mismatched primer. Pol ζ, however, can carry out such extension quite well. This combination of circumstances could lead to the fixation of the t1799a mutation. Deamination at the 3′C of the dimer could lead to the tg1799–1800aa tandem mutation. NER, nucleotide excision repair.
might lead to the accumulation of multiple rare events. However, the current state of knowledge makes it impossible
to determine with certainty whether UV is involved in the induction of BRAF mutations and, if it is, whether www.jidonline.org 1695
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UVB or UVA is the culprit. Whereas UVB is a better mutagen, more UVA reaches the skin and penetrates deeper. UVA can induce CPDs at high doses (Pfeifer et al., 2005), although oxidative damage predominates. In addition, melanin might contribute to production of somatic mutations through incompletely understood photosensitization and/or pro-oxidant effects (Meyskens et al., 2004; Samokhvalov et al., 2005). Further epidemiologic and basicscience studies will be necessary to unravel the contribution that UVA or UVB might make in the production of BRAF mutations. CONFLICT OF INTEREST The author states no conflict of interest.
ACKNOWLEDGMENTS This work was supported in part by National Cancer Institute Awards CA102096, CA112243, and CA055065.
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