The role of UV induced lesions in skin carcinogenesis: an overview of oncogene and tumor suppressor gene modifications in xeroderma pigmentosum skin tumors

Mutation Research 571 (2005) 43–56

Review

The role of UV induced lesions in skin carcinogenesis: an overview of oncogene and tumor suppressor gene modifications in xeroderma pigmentosum skin tumors Leela Daya-Grosjean∗ , Alain Sarasin Laboratory of Genetic Instability and Cancer, UPR2169 CNRS, IFR 54, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France Received 29 July 2004; received in revised form 27 October 2004; accepted 3 November 2004 Available online 25 January 2005

Abstract Xeroderma pigmentosum (XP), a rare hereditary syndrome, is characterized by a hypersensitivity to solar irradiation due to a defect in nucleotide excision repair resulting in a predisposition to squamous and basal cell carcinomas as well as malignant melanomas appearing at a very early age. The mutator phenotype of XP cells is evident by the higher levels of UV specific modifications found in key regulatory genes in XP skin tumors compared to those in the same tumor types from the normal population. Thus, XP provides a unique model for the study of unrepaired DNA lesions, mutations and skin carcinogenesis. The high level of ras oncogene activation, Ink4a-Arf and p53 tumor suppressor gene modifications as well as alterations of the different partners of the mitogenic sonic hedgehog signaling pathway (patched, smoothened and sonic hedgehog), characterized in XP skin tumors have clearly demonstrated the major role of the UV component of sunlight in the development of skin tumors. The majority of the mutations are C to T or tandem CC to TT UV signature transitions, occurring at bipyrimidine sequences, the specific targets of UV induced lesions. These characteristics are also found in the same genes modified in sporadic skin cancers but with lower frequencies confirming the validity of studying the XP model. The knowledge gained by studying XP tumors has given us a greater perception of the contribution of genetic predisposition to cancer as well as the consequences of the many alterations which modulate the activities of different genes affecting crucial pathways vital for maintaining cell homeostasis. © 2005 Elsevier B.V. All rights reserved. Keywords: Xeroderma pigmentosum; Skin tumor; Mutation; Oncogene; Tumor suppressor gene; DNA repair; UV

∗

Corresponding author. Tel.: +33 42 11 63 35/33 42 11 51 18; fax: +33 42 11 50 08. E-mail address: [email protected] (L. Daya-Grosjean).

0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.11.013

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Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.

Xeroderma pigmentosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3.

Skin cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4.

Multi-step model of UV light induced skin cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5.

Oncogene activation in XP skin tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6.

Modified tumor suppressor genes in XP skin cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. p53 gene mutations in XP tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. p53 mutation spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. INK4a-ARF mutations in XP skin cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7.

Alterations of the SHH signaling pathway in XP skin tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Patched gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Smoothened gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. SHH gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50 50 51 51

8.

High frequency of UV signature CC → TT tandem mutations in XP tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9.

DNA strand specificity of mutations in XP tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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10.

Multiple gene modifications in XP skin cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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11.

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction It is widely accepted that the cardinal feature of cancer is the acquisition of inherent genomic instability caused by or leading to the accumulation of numerous mutations. Many roads can lead to genetic instability including epigenetic alterations such as DNA methylation or remodeling of chromatin via histone protein modifications, the disruption of the different highly conserved repair pathways including recombination, base excision repair (BER), mismatch repair (MMR) and nucleotide excision repair (NER) [1]. It is clear that mutations in genes governing these pathways can drive carcinogenesis as attested by the existence of human cancer syndromes like Xeroderma pigmentosum

(XP) in which germline mutations of genes involved in NER predispose individuals to skin cancers [2,3]. NER is the most versatile of the repair systems and deals with a wide class of helix distorting lesions most of which arise from exogenous sources including the photoproducts caused by sunlight, a major carcinogen in our natural environment. Thus, hypersensitivity to sunlight and a dramatic >2000-fold incidence of skin tumors are the principal hallmarks of the cancer-prone, nucleotide excision repair deficient XP patients [4]. Normal human skin is a natural barrier against a variety of different types of external stress and is well adapted to UV irradiation but acute effects of high dose UV can cause erythema, tanning and local or systemic immuno-suppression. The ultimate effects

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of chronic exposure to UV include photo-aging and photo-carcinogenesis which involve the accumulation of genetic alterations resulting in the development of skin tumors. Among the genetic alterations, the activation of proto-oncogenes and inactivation of tumor suppressor genes in target cells are the crucial molecular events in the multi-step cutaneous cancer progression where advantageous selective growth and clonal expansion occur [5,6]. It is important to note that the incidence of skin cancer, the most common cancer in man in fair skinned populations, is doubling every 15–20 years because of an aging population, changes in habit and behaviour towards sun-exposure and a possible increase in the UV-light fluence at the earth’s surface due to ozone depletion [7,8]. Cellular UV hyper-mutability of XP cells demonstrates a mutator phenotype with numerous mutations arising as a consequence of DNA replication through unrepaired lesions. Indeed, it has also been clearly shown that XP skin tumors present higher levels of UV specific mutations in key regulatory genes than the same tumors from DNA repair–proficient individuals. Thus, XP provides a unique human model for studying the relationship between unrepaired DNA lesions, mutations and skin carcinogenesis. The past two decades have seen great strides in our understanding of the genetic basis of skin cancer. This is mainly due to the contribution of molecular profiling of the germinal variants of the XP NER genes and the complex somatic genetic events identified in skin cancers and found accentuated in XP tumors, which have a profound effect on the development of an emerging cancer cell or cancerous clone in the skin. In this review, we will describe the alterations of key oncogenes and tumor suppressor genes involved in the molecular pathogenesis of cutaneous carcinomas in XP patients as well as in the general population. The knowledge gained from the analysis of XP tumors has provided a greater perception of the contribution of genetic predisposition to cancer as well as the consequences of the many alterations which modulate the activities of different genes affecting crucial pathways vital for maintaining cell homeostasis.

2. Xeroderma pigmentosum Xeroderma pigmentosum, first described in the late 19th century, is a rare inherited disease transmitted

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as an autosomal recessive trait whose occurrence is favoured by consanguinity and its frequency shows large geographical variations. The disease is associated with an extreme photophobia with bilateral cataracts and ocular lesions, an acute photosensitivity characterized by sunlight induced abnormal pigmentation, a dry parchment-like prematurely aged skin and precocious cutaneous lesions which result in multiple skin cancers arising at the median age of eight years. About 20% of severely affected XP patients present neurological disorders resulting from progressive neuronal death, which includes mental retardation (the most common) as well as spacticity and microcephaly. The frequency of internal malignancies is also higher in XP, including a 20-fold increase in brain tumors. Some XP patients have a partial deficiency in natural killer cell activity and reduced interferon production resulting in a lowered immunity which may play a part in enhancing tumor progression [9]. The heterogeneous XP phenotype can be a result of defects in any one of the seven classic NER XP genes A to G, some involved only in NER (XPA and XPC) whereas the others are also implicated in other processes including transcription and recombination [2,3,10]. About 20% of XP patients, who are less sensitive to UV, belong to the variant XPV group proficient in NER but defective in translesion synthesis due to a mutation in the low fidelity DNA polymerase, pol␩. XPV patients are also prone to skin cancer which, however, develop later at around the age of 20 years and often exhibit a less severe phenotype than classic XP patients [10]. It is important to note that various alterations of the XP NER genes or other repair genes can give rise to two other photosensitive disorders, with very different phenotypes, Cockayne’s syndrome and trichothiodystrophy which do not show any skin cancer predisposition [2,3].

3. Skin cancers XP patients are characterized by their predisposition to skin cancers on sun-exposed body sites, as early as 3–5 years of age, comprising mainly the nonmelanoma skin cancers (NMSC), the basal (BCC) and squamous cell carcinomas (SCC) [11,12]. This is in striking contrast with the general population where the mean age for NMSC is 50–60 years. NMSCs are de-

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rived from keratinocytes of the basal layer of the epidermis and BCCs are found to be slow growing and rarely metastasize whereas SCCs are fast growing, invasive tumors with the capacity to metastasize. The frequency of NMSC is 50% in young XP (under 10 years) and the SCCs in XP patients have a higher probability to metastasize (4%) compared to under 2% in the normal population. Malignant melanoma (MM) are derived from melanocytes and 15–20% occur at atypical and melanocytic nevi and intermittent UV exposure or painful sunburn of healthy skin is also implicated in their development. The role of UV in the induction of MM is not clear but XP patients develop MMs on sun-exposed sites, 65% located on the head and neck, 28% on the arms and legs and 7% on the rest of the body. In XP patients under 20 years of age, MM incidence is 2000-fold higher than in an age-matched population. The NMSCs account for 96% of sporadic skin cancers and MMs account for only 4% whereas in XP patients up to 22% of skin cancers are melanomas [13,14].

molecules that lead to modified gene expression, the so-called ‘UV response’. The immediate UV response includes genetic and epigenetic changes in transcription factors, signal transduction and cytoskeleton all contributing to modify the normal skin cell phenotype [15,16]. Molecular analysis of skin tumors and in particular XP skin cancers has allowed characterization of the genes targeted by UV for altered functions which contribute to skin cancer development. Among these are the gatekeeper genes which control cell growth and death and the caretaker genes which maintain genome integrity, both groups being widely implicated in cancer [17]. The following sections will outline ras oncogene activation, Ink4a-Arf and p53 tumor suppressor gene modifications and alterations of the different partners of the mitogenic sonic hedgehog (SHH) signaling pathway characterized in XP skin tumors.

5. Oncogene activation in XP skin tumors 4. Multi-step model of UV light induced skin cancers The major etiological factor in skin cancer is sunlight and in particular the UV component of solar emission of which the shortwave UVC and most of the UVB is filtered out by ozone resulting in only 5–10% of UVB and 90–95% of longwave UVA reaching the earth’s surface. Skin tumor formation proceeds through three steps involving UV light as a carcinogen, tumor initiation, promotion and progression. Thus, during the initiation step, unrepaired UV photoproducts formed throughout the genome can eventually give rise to mutations in coding regions of genes including oncogenes and tumor suppressor genes. Chronic UV exposure promotes benign tumor formation by allowing clonal expansion of epidermal cells carrying specific modified genes such as the ras proto-oncogenes or the p53 tumor suppressor gene. Continued UV irradiation allows tumor progression by selecting clones insensitive to UV induced apoptosis. Thus, further gene alterations including changes in gene copy number, additional gene mutations and rearrangements result in genetically unstable malignant skin cancers. Much of the effects of UV occur through alterations of signaling

Proto-oncogenes, which can code for growth factors, receptors, signal transduction protein kinases and nuclear transcription factors, play a crucial role in the regulation of normal cell growth, differentiation and apoptosis. Among these, the ras family of proto-oncogenes encoding GTP binding proteins, have played an important causal role in many cancer types through selective mutations inducing constitutive activation of ras signal transduction. Thus, among the proto-oncogenes that have been found altered in skin tumors are the ras gene family, N-ras, Ki-ras and Ha-ras, which are discussed here. The modification in skin tumors of the sonic hedgehog and smoothened proto-oncogenes involved in the sonic hedgehog signaling pathway will be discussed in a later section. The reported frequencies of ras gene modifications (point mutations, amplification, rearrangement) in skin cancers from the general population varies from study to study ranging from under 5% to up to 40% reflecting heterogeneity in samples and the type of analyses used. The few early studies of ras alterations in skin cancers, including one from our laboratory analysing XP NMSC, also reported varying but low frequencies of ras [18–20]. These differences could also arise from dif-

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ferent techniques used to identify the mutations. However, it should be underlined that samples were from XP patients of different ethnic origins, mainly North African or Japanese in these studies. Thus, the differences in preponderance of specific XP complementation groups in these populations (XPA in Japanese and XPC or XPD in the North African) may explain the varying gene mutation levels that reflect differences in repair capacities. When we carried out a comparative study of NMSC from the normal population and XP patients this allowed us to establish a more than two-fold higher mutation frequency (50%) of ras genes in XP tumors in contrast to those from the normal population (Fig. 1) [21]. The majority of mutations in both tumor groups were located at codon 12 and found in all three ras genes with a preponderance for N-ras alterations. All the mutations are located opposite bipyrimidine sequences, hot-spots for UV induced lesions, indicating the initiating role of unrepaired lesions in skin carcinogenesis in general. Interestingly, in XP tumors the high ras mutation frequency observed by us was accompanied by high levels of amplification and rearrangement of Ha-ras and c-myc, a mitogenic oncogene. In these tumors, amplification is probably a consequence of blocked DNA replication at UV lesions inducing the re-initiation of several abortive rounds of replication or an attempt to undergo genetic recombination. In sporadic melanomas, mutations of the ras regulated kinase BRAF (50%) and N-ras (10%) are frequently

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observed but no data is available for XP melanomas [22,23].

6. Modified tumor suppressor genes in XP skin cancers Tumor suppressor genes are negative growth regulators which are recessive at the cellular level and have to be mutated in both alleles to cause phenotypic changes in cells. The gatekeeper functions of tumor suppressor genes which suppress cancer development include triggering of cellular responses to damage, the most important being apoptosis or cell cycle arrest which can be transient or permanent when resulting in cellular senescence [24]. The best-known example of a tumor suppressor gene is the p53 gene which is found mutated in about half of all human cancers [25]. The p53 protein has many modulating functions in cells playing a role in gene transcription, cell cycle control, DNA repair and replication, senescence and apoptosis. In this section we will describe the analysis of p53 mutations in XP skin cancers which has proven to be a perfect target gene for correlating a mutation spectrum with the causative agent, UV light. Recent data have also identified mutations in skin tumors in a new locus, INK4a-ARF, mapping at chromosome 9p21 which codes for two tumor suppressor proteins involved in cell cycle regulation: (1) p16 INK4a which is an inhibitory protein of the cyclin dependent kinase (CDK)4, and (2) p14 ARF which stabilizes p53 by inhibiting MDM2-dependent p53 degradation, thereby specifically activating the p53 pathway [26,27]. 6.1. p53 gene mutations in XP tumors

Fig. 1. Comparison of gene mutations in XP and non-XP skin cancers. Higher levels of oncogene and tumor suppressor gene mutation frequencies are found in skin tumors from XP patients compared with those observed in the non-XP population.

The analysis of p53 mutations, 75% of which are missense mutations mainly located in evolutionary conserved sequences at exons 5–9, has highlighted its usefulness as a probe in molecular epidemiology. Indeed, the specificity of p53 mutation spectra has also made it possible to implicate environmental carcinogens and endogenous processes in the etiology of human cancer. Thus, UV specific p53 mutations are found in sun-exposed skin and benign precursor lesions indicating that these modifications are early events in

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skin tumor development [28–30]. A study of the sunexposed normal skin from an XP patient also showed a multitude of UV specific p53 gene mutations all likely to result in a selective growth advantage [31]. Analysis of non-melanoma skin tumors [6] has shown that the frequency of p53 mutations, ranging from 50 to 90%, is higher than that seen in internal malignancies (<50%). Moreover, the p53 mutation spectra in skin cancers are also significantly different being mainly C→T transitions located at dipyrimidine sites which are hot spots for UV induced lesions. In internal malignancies nearly 50% of mutations are also the C→T substitutions but are largely found at CpG sequences and are probably caused by the spontaneous deamination of 5MeC. The most remarkable alterations specific to skin cancers are the frequent tandem CC→TT transitions considered to be the veritable UV signature and rarely found in internal tumors. Analysis of the p53 gene in skin tumors from the NER deficient XP patients has demonstrated that the high level of p53 mutations are directly caused by unrepaired UV induced DNA lesions by the presence of the C→T and the tandem CC→TT transitions located at dipyrimidine sites (Fig. 1) [32–36]. Strikingly, there is a significantly higher level of the tandem CC→TT transitions (<60%) in XP skin tumors compared to those found in non-XP skin cancers (10%) in which C→T transitions predominate and their distribution is also different, the majority occurring on the nontranscribed strand in XP tumors indicating preferential repair. 6.2. p53 mutation spectra The p53 mutation spectra observed in internal cancers show mutation hot spots in the highly conserved domains at codons 175, 245, 248, 249, 273 and 282. In skin tumors, the mutation spectra hot spots are significantly different and depend also on the tumor type, basal cell carcinoma, squamous cell carcinoma or malignant melanoma (Fig. 2). Surprisingly, the distribution of the p53 mutations found in XP skin tumors is also statistically different from that observed in sporadic skin cancers [36]. Thus, in sporadic skin cancers there is one common hot spot at codon 248 found in the three skin tumor types, which is also a hot spot in internal cancers. It is important to remember that the Arg 248 mutation can disrupt p53 DNA binding and

Fig. 2. Specific differences in gene mutation levels are observed between basal cell carcinomas and squamous cell carcinomas from XP patients. The sonic hedgehog signaling pathway genes, patched, smoothened and sonic hedgehog are found specifically modified in BCCs and higher levels of the tumor suppressor gene, p53 are observed. In squamous cell carcinomas ras and p16 modifications are prevalent as is the association of modifications of p16 with p53 in the same tumor.

transactivation functions which could result in a selective advantage for cells during tumor development. In XP, the codon 248 hot spot is also observed in the SCCs and MMs but not in XP BCCs. However, it should be noted that fewer total numbers of XP tumors have been analyzed compared to sporadic skin cancers. Nevertheless, SCCs present five distinct p53 mutation hot spots at codons 179, 196, 248, 278 and 282. It should be noted that p53 mutations, (mainly C > T transitions), are rare in sporadic MMs (10%) whereas XP melanomas show a high (>50%) p53 mutation frequency with a preponderance of CC > TT substitutions [37,38]. Their distribution is also statistically different from that observed in non-XP MMs which present a codon 213 hot spot not found in XP melanomas but share a hot spot at codon 247– 248. The association of specific p53 codon 72 polymorphisms has been identified in bladder and lung cancers but its relationship with skin cancer is unclear. In a study by McGregor et al. [39], a significant association between p53-72R homozygosity and NMSC is seen in renal transplant recipients but not in immunocompetent patients with NMSC. We have determined the genotype of the p53 codon 72 in a large number of XP patients, in order to examine whether there existed a specific p53 codon 72 polymorphism associated with skin cancer in XP who present some

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immunodeficiency due to a defect in NK cell activity [9]. Our data show that no clear correlation exists between a specific codon 72 polymophism and skin tumors in XP patients (unpublished data). To date, no clear-cut diagnostic or prognostic indication of tumor aggressiveness in NMSC can be drawn from the analysis p53 mutation type, frequency or spectra [40]. 6.3. INK4a-ARF mutations in XP skin cancers The cyclin dependent kinase inhibitor 2A (CDKN2A) locus which maps to chromosome 9p21 codes for two tumor suppressor genes, p16INK4a and p14ARF . Indeed, this locus is the second most commonly altered gene locus in human cancers after p53 [26,27]. Inactivation of CDKN2A can occur by homozygous deletion, point mutation or methylation in many tumor types including skin cancers. Alternative reading frames on the INK4a-ARF locus encodes the two cell cycle regulator proteins, p16INK4a and p14ARF , which share exons 2 and 3 but have different exons 1 and show no sequence homology at the amino acid level [41]. Thus, exon 1␣-containing transcripts code for p16INK4a , a cyclin dependent kinase inhibitor that regulates cell cycle progression through the retinoblastome (Rb)-CDK pathway. The p16INK4a protein by binding to CDK4 and CDK6 blocks phosphorylation of the Rb protein, thus maintaining Rb in its active state and specifically inhibiting progression through the G1 phase of the cell cycle. The p14ARF protein encoded by exon1␤containing transcripts, can also arrest proliferation by specifically activating the p53 pathway. Thus, activation of p14ARF in response to oncogenic stimuli allows it to bind to the MDM2 protein resulting in the sequestration of MDM2 in the nucleus. This prevents MDM2-dependent p53 ubiquitin-mediated degradation as well MDM2-dependent Rb inactivation which can cause arrest at both G1 and G2 phases of the cell cycle [42,43]. Squamous epithelial cells seem particularly sensitive to growth regulation pathways involving the INKARF genes whose deregulation results in a wide variety of carcinomas, in particular the head and neck SCC [44]. Moreover, targeted disruption of the INKARF locus renders mice susceptible to SCC [45,46]. Finally, the accumulation of p16INK4a observed in HeLa

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cells after UV irradiation results in cell cycle arrest implicating p16INK4a alteration is important in UV induced tumorigenesis. Nevertheless, there is no strong evidence that UV is a major factor in CDKN2A alterations involved in skin cancers where only about 20% of mutations are UV specific CC→TT transitions found mainly in the squamous cell carcinomas of the skin [47,48]. In fact, a recent study has clearly shown that promoter methylation is the predominant mechanism for inactivation of both p16INK4a and p14ARF [49] in skin SCCs. Inactivation of the INK-ARF locus in basal cell carcinomas is less clear and none or few (<2%) mutations have been detected up to now [50,48]. Interestingly, high (43%) levels of UV specific mutations are found in XP skin tumors both in BCCs and SCCs compared to 25% in sporadic skin cancers [51]. Among the 10 XP BCCs analyzed, 2 (20%) presented 3 mutations and 6 of the 18 XP SCCs (33%) presented a total of 10 mutations, most of them occurring at the known mutational hotspots. These results also indicate a statistically significant higher frequency (46%) of the p16INK4a and p14ARF mutations in XP skin cancers compared to that (12%) seen in the general population. Moreover, probably due to the genetic instability of the repair deficient patients, accumulation of un-repaired lesions has resulted in three XP tumors with multiple mutations of p16INK4a and two XP tumors with multiple p14ARF alterations. Multiple mutations of p16INK4a and p14ARF have not been observed in sporadic NMSC. In XP tumors the majority of the mutations originated at the UV target dipyrimidine sequences, 38% being C→T transitions and 54% the CC→TT tandem UV signature mutations significantly higher than that found (25%) in sporadic skin cancers. Of significant importance in the XP tumors is the positive association of p16INK4a , p14ARF mutations and p53 alterations in 60% of XP tumors (Fig. 1). The simultaneous inactivation of p53 with p16INK4a or p14ARF is rarely found in sporadic tumors. It is postulated that de-stabilisation of the cooperative effects of the INKARF/p53/Rb pathways must be particularly important in promoting tumorigenesis and could account for the accelerated skin tumor formation observed in XP patients. To date, epigenetic changes such as hypermethylation of p16INK4a or p14ARF predominantly found in sporadic SCCs have not been analysed for in XP skin tumors.

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It should be noted that CDKN2A genes are associated with an increased risk of malignant melanoma and germline CDKN2A mutations are found in 20% of familial melanomas [52]. There are no published reports of CDKN2A alterations in XP melanomas.

7. Alterations of the SHH signaling pathway in XP skin tumors The hedgehog signaling pathway, first described in Drosophila and highly conserved in vertebrates, has been found to play a crucial role in embryonic development and adult tissue homeostasis. The vertebrate sonic hedgehog signaling pathway is important in regulating patterning, differentiation, cell cycle, proliferation and cell survival both in the embryo and the adult. Activation of the SHH pathway is triggered by the binding of the secreted N-terminal signal peptide of SHH, a glycoprotein, to its receptor patched (PTCH). Patched, a 12-pass trans-membrane molecule, is a key inhibitory regulator of the constitutively active 7-pass trans-membrane protein, smoothened (SMO). The mechanisms by which PTCH suppresses SMO activity is not clear but recent studies implicate a catalytic regulation through small molecules which may antagonize SMO activity directly or indirectly. The inactivation of patched by binding of the SHH ligand relieves repression of SMO allowing the activation of transcription of downstream genes. Among the target genes activated are the Gli transcription factors, Gli 1, 2 and 3, multifunctional large proteins which can behave both as an activators or repressors [53–55]. Activation or dysregulation of the SHH pathway by inactivating mutations of different members has been implicated in tumor initiation and growth of human cancers predominantly in tissues of ecdodermal origin such as skin and brain. In fact, the SHH signaling pathway known to play a key role in skin and hair follicle development is a major determinant in skin tumorigenesis notably in the specific formation of basal cell carcinomas [56]. The BCC is the most frequent skin cancer in the white population and occurs sporadically in relation to sun-exposure. Its incidence is also increased in patients with rare genetic disorders such as XP and the Gorlin’s syndrome also known as the nevoid BCC (NBCC) syndrome. NBCC

patients are characterized by a range of developmental abnormalities and a predisposition to various cancers. The main manifestations include crano-facial alterations, palmar and plantar hyperkeratosis and skeletal abnormalities [57]. Among the many types of tumors are the highly aggressive central nervous system tumors, the medulloblastomas and meningiomas, jaw cysts and ovarian fibromas, but the most frequent tumor is the BCC occurring in more than 80% of NBCC patients. 7.1. Patched gene mutations Genetic studies have shown that the NBCC phenotype is due to germline mutations of PTCH, a tumor suppressor gene located on chromosome 9q22.3 and 89% of the BCCs in Gorlins’ patients show LOH at this locus. The majority of the germline PTCH mutations are rearrangements which mainly result in truncated proteins. Interestingly, sporadic BCCs were also found to present frequent PTCH gene mutations, but only 30% are rearrangements whereas nearly 70% are point mutations [58]. The majority of these alterations are also predicted to result in premature protein termination. The mutation spectrum of the PTCH gene in sporadic BCCs from repair proficient patients indicates a major role for solar irradiation in tumor development as 50% of mutations are UV specific C to T or CC to TT transitions located at bipyrimidine sites (Fig. 1) [59–63]. The analysis of BCC from NER deficient XP patients reveal between 80% and 90% of PTCH gene mutations of which nearly 80% are UV specific (Fig. 1) [64,37]. In the study from our laboratory analyzing 22 XP tumors, 65% were the UV hallmark, the tandem CC to TT transitions and 23% were C to T transitions all targeted at bipyrimidine sites [64]. However, in the study by D’Errico et al. of eight XP BCCs, only one CC to TT tandem was identified among the 75% UV specific alterations. This difference may be explained by differences in the complementation group of the XP patients from whom tumors were studied. In fact, the majority of the XP tumors in our study are from XPC patients, where a specificity for the CC to TT tandem mutations has been characterized [36]. In the study by D’Errico et al., among the eight BCC presenting PTCH mutations, three were from XP patients of unknown complementation group and five from XPV patients, where C to T

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transitions represent the prominent gene modifications as found for other genes modified in XPV skin cancers [37]. A high proportion (42%) of the XP patched gene alterations are predicted to produce a truncated protein caused by a premature stop codon. For XP and sporadic BCC, the localization of patched gene mutations on the different domains of the protein is not significantly different but germline mutations are seen more often in the N-terminal half of the protein. The majority of the point mutations are distributed at random in the non-trans-membrane domains in particular in the large extra-cellular loops which may be important for interaction with the SHH ligand and in the large intracellular loop and C-terminal tail for protein-protein interactions [65]. 7.2. Smoothened gene mutations In a study of 30 BCCs from XP patients, a significantly high level (30%) of the SMO gene alterations were characterized, consisting of one silent and seven missense mutations (Fig. 1) [66]. As with the patched gene, the majority of these XP BCC mutations (89%) are UV specific, 69% of which are tandem CC → TT transitions not found in SMO modifications of sporadic BCCs. Interestingly, the majority of the mutations are located in the extra-membrane domains of the smoothened protein, two being located in the large extra-cellular loop and two in the intra-cellular cytoplasmic tail. Significantly, one mutation, a G to T transversion at codon 535, appears to be a hot spot for SMO modification being also found in the three studies analyzing sporadic BCCs [67–69]. Indeed, activating mutations in two codons (535 and 562) of the SMO proto-oncogene have been found in only about 9% of nearly 100 sporadic BCCs from the Caucasian population analyzed up to now, 30% of these alterations being UV specific (Figs. 1 and 2) [67,68]. In a study of BCCs from an Asian population only analyzing for the two codons 535 and 562, the 20 SMO mutations in 97 BCCs (20.6%) were G to T transversions targeted at codon 535. This codon lies in the seventh trans-membrane domain of the SMO protein and the G → T transversion modifies the highly conserved tryptophan residue to leucine which may alter the latent state of the SMO receptor protein and result in constitutive SHH signaling. It should be noted that the G to T hot spot SMO mutation can arise from replication of DNA containing 8-

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oxoguanine, a base modification that is often produced by UVA induced reactive oxygen species and arsenic compounds. Interestingly, arsenic is a well-known food contaminant in Asia and this could explain the high frequency of the codon 535 G to T transversions observed in the study by Lam et al. [69]. 7.3. SHH gene mutations Our recent analysis of XP BCCs has firmly established a role for SHH in human skin tumorigenesis by the finding of six SHH gene mutations in 33 tumors analyzed [70]. In fact, mutations of the postulated proto-oncogene SHH are extremely rare in sporadic BCCs and only one potential gain of function mutation (H133Y) has been reported among 74 sporadic BCCs analyzed to date (Figs. 1 and 2) [71,68]. A later study, looking only for codon 133 alterations, found no modifications in 36 sporadic BCCs analyzed [72]. All of the six XP SHH mutations we have characterized are UV-specific, C → T transitions or CC → TT tandem substitutions. Four alterations are missense mutations (12%) in exons 1 and 2, in highly conserved codons, 57 and 64 in exon 1, and 147 and 155 in exon 2 at the N-terminal domain of the SHH protein. One silent mutation was found in exon 3 and a base substitution was detected in intron 1 near the junction to exon 1. The unique SHH alteration, H133Y, detected in 1 of 43 sporadic BCCs was also found in a medulloblastoma and a breast carcinoma by Oro et al. in the same study [71]. This unique exon 2 mutation (gcCa397gcTa) is also a C → T transition located at a bipyrimidine sequence but is probably not a hot spot for UV induced lesions in the SHH gene as it was not found in the XP BCCs and was identified in internal cancers not related with UV exposure. All the SHH mutations identified are located in the N-terminal domain of SHH known to retain the signaling activities of the protein, whereas the C-terminal domain is responsible for the intramolecular precursor processing [73,74]. We carried out structural modeling studies of the four XP SHH proteins altered at the surface residues, G57S, G64K, D147N and R155C, as well as the sporadic BCC alteration H133Y which showed that they do not significantly affect the protein conformation. Interestingly, they are all located on one face of the compact SHH protein suggesting they may have altered affinity for different partners which may be im-

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portant in modulating different functions. We carried out a functional analysis of the altered SHH proteins identified in XP BCCs and did not find mutations to be activating for its postulated proto-oncogene function, as they do not show transforming activity and induce differentiation or stimulate proliferation to the same level as the wild-type SHH protein. This is also true for the sporadic BCC mutant, SHH H133Y protein [70]. Germline mutations of the SHH gene are the cause of the Holoprosencephly (HPE) syndrome presenting forebrain malformation associated with mental retardation and craniofacial anomalies indicating the importance of SHH signaling in human development. However, none of the SHH mutations found in BCCs are identified in HPE where a variety of alterations are found including frameshift, nonsense and missense mutations. Finally, our analysis of both squamous cell and basal cell carcinomas from XP patients for patched, smoothened and sonic hedgehog gene alterations has clearly shown that the SHH signaling pathway gene modifications are specific for BCC development none having been detected in XP SCCs [64,66,70]. 8. High frequency of UV signature CC → TT tandem mutations in XP tumors All the gene modifications characterized in XP skin cancers have revealed significantly high levels of the UV hallmark mutations, the tandem CC → TT substitution caused by adjacent mis-pairing opposite two damaged pyrimidines during translesion synthesis. This mutation is rare in internal cancers (<1%), but relatively frequent in sporadic skin cancers (<14%) and significantly high levels are found in XP skin tumors, ranging from 36% to 90% depending on the skin tumor type and the gene analyzed [32,35,36,64,51,66,70]. Interestingly, sequence analysis of the p53 gene has revealed that this tandem substitution occurs more frequently at methylated CpG sites which may be preferential targets for UV induced CPD lesions [75,76]. We postulate that deamination of this lesion, spontaneous or UV induced, occurs more frequently in skin cells resulting in the signature UV, CC→TT transitions found in genes modified in skin cancers. This deamination is accentuated in the repair deficient XP cells where unrepaired lesions persist, resulting in the very high levels

of this tandem UV signature mutation seen in different genes modified in XP tumors [36].

9. DNA strand specificity of mutations in XP tumors Data from our laboratory studying p53 mutations in XP skin tumors, mainly XPC patients, allowed the first demonstration of the existence of preferential repair in human cells. It is evident that UV induced lesions are equally distributed on both DNA strands and the p53 mutations in the XPC tumors are only found on the non-transcribed strand. This is due to the fact that XPC cells can efficiently repair transcribed strands of active genes but are deficient in global genome repair. Thus, replication errors at un-repaired lesions on the non-transcribed strand yield the non-transcribed strand specific mutations. This also explains the difference in distribution of p53 mutations seen between non-XP patients as well as XPA, XPD, XPF and XPV patients where mutations are distributed more or less equally on both DNA strands. The strand specificity of mutations in XPC skin tumors was confirmed in our later studies analyzing for other gene modifications including p16/p14, patched, smoothened and sonic hedgehog.

10. Multiple gene modifications in XP skin cancers Cancer tends to involve multiple gene mutations and the hypermutability of XP cells by UV is correlated to their high predisposition to cutaneous cancers. Thus, to create a cancer cell, it requires that the brakes on cell proliferation, the tumor suppressor genes, are released and also that the accelerators of cell growth, the oncogenes, are activated. In XP cells, as in skin cells from the general population, solar UV irradiation plays an unambiguous central role in initiation and multi-step skin cancer progression involving the accumulation of mutations in the different oncogenes and tumor suppressor genes that have been described above (Fig. 3). Indeed, different studies on XP skin tumors have shown the association of the occurrence of several mutated genes in the same tumor. Thus, the p53 gene mutations, considered as early genetic changes, have been detected in sun-exposed skin, benign skin lesions

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Fig. 3. The role of UV modified genes in skin carcinogenesis. Different caretaker and gatekeeper genes have been found altered in sporadic and XP skin cancers development. As shown these genes, when modified, can act at different levels leading to genetic instability and cancer formation. Dotted arrows indicate this path is not specific for all the genes identified in the boxes.

as well as the malignant cancers. In XP skin cancers, p53 gene mutations have been found with mutations of one or another of the p16INK4a , p14ARF , PTCH-1, SMO and SHH genes. Interestingly, in XP BCCs, some which were mutated for p53, we found SHH gene mutations together with alterations of PTCH and SMO, other members of the SHH signaling pathway. This complex accumulation of lesions in the same tumor, which results in an inherent genomic instability, could be due to clonal expansion of cells harboring the different mutated genes and/or that cells require modification of several key genes for tumor development (Fig. 3).

11. Conclusions Our understanding of the dramatic consequences of genetic instability leading to skin cancer has been greatly enhanced by the analysis of the DNA repair deficient XP patients. Unequivocal evidence accumu-

lated over the last 20 years has shown that the inability of XP cells to repair UV induced DNA lesions results in enhanced mutation levels which when accumulated in key regulatory genes, leads to malignant cell transformation resulting in skin cancer. The molecular analysis of XP skin tumors with a defect in NER which results in enhanced mutation levels permits the identification of crucial regulatory genes involved in carcinogenesis. Significantly, these are the same as those altered at lower levels in skin cancers from the normal population. In fact, the analysis of XP tumors has allowed us to confirm the importance of SHH gene modifications in BCCs described above and further analysis of different members of the SHH pathway should provide new insights into BCC developement. The specificity of the SHH signaling pathway in BCCs infers that SCC formation is through the modulation of other pathways or that different stem cells in the skin are involved in the development of the two tumor types. Mouse models of XP have been particularly informative and it should

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be noted only SCCs are induced following UV treatment of the XP mice. However, the specificity of SHH signaling in skin cancer is revealed by heterozygous patched gene transgenic mice which develop BCC like lesions following chronic UV irradiation, characterized by high levels of UV specific p53 mutations [77]. In conclusion, it is clear that our understanding of skin carcinogenesis has gained much from the investigation of the XP syndrome and further insight into the molecular mechanisms involved in skin cancer formation should help in their prevention and also in the development of new strategies for targeted skin cancer therapies. Acknowledgements We would like to thank the Association de Recherche sur le Cancer (Villejuif), the Ligue Nationale Contre le Cancer (Creteil), the Groupement des Entreprises franc¸aises dans la Lutte contre cancer (Charenton) and l’Electricit´e de France (EDF, Paris) for their support in providing grants for the work presented here. References [1] J.H. Hoeijmakers, Genome maintenance mechanisms for preventing cancer, Nature 411 (2001) 366–374. [2] E.C. Friedberg, How nucleotide excision repair protects against cancer, Nat. Rev. Cancer 1 (2001) 22–33. [3] A.R. Lehmann, DNA repair-deficient diseases, xeroderma pigmentosum Cockayne syndrome and trichothiodystrophy, Biochimie 85 (2003) 1101–1111. [4] A. Stary, A. Sarasin, The genetics of the hereditary xeroderma pigmentosum syndrome, Biochimie 84 (2002) 49–60. [5] F.R. de Gruijl, Skin cancer and solar UV radiation, Eur. J. Cancer 35 (1999) 2003–2009. [6] Y. Matsumura, H.N. Ananthaswamy, Molecular mechanisms of photocarcinogenesis, Front. Biosci. 7 (2002) d765–d783. [7] J.C. van der Leun, F.R. de Gruijl, Climate change and skin cancer, Photochem. Photobiol. Sci. 1 (2002) 324–326. [8] F.R. de Gruijl, J. Longstreth, M. Norval, A.P. Cullen, H. Slaper, M.L. Kripke, Y. Takizawa, J.C. van der Leun, Health effects from stratospheric ozone depletion and interactions with climate change, Photochem. Photobiol. Sci. 2 (2003) 16–28. [9] A.A. Gaspari, T.A. Fleisher, K.H. Kraemer, Impaired interferon production and natural killer cell activation in patients with the skin cancer-prone disorder, xeroderma pigmentosum, J. Clin. Invest. 92 (1993) 1135–1142. [10] A. Stary, A. Sarasin, Molecular mechanisms of UV induced mutations as revealed by the study of DNA polymerase eta in human cells, Res. Microbiol. 153 (2002) 441–445.

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