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Non-melanoma skin cancers
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Vol. 5, No. 1 2008
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Skin diseases MECHANISMS
Non-melanoma skin cancers Ashraf Hashim Ahmed1,a, H. Peter Soyer1, Nicholas Saunders2, Petra Boukamp3,*, Michael S. Roberts1 1
University of Queensland, School of Medicine, Southern Clinical Division, Princess Alexandra Hospital, QLD, Australia University of Queensland, Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, QLD, Australia 3 Division of Genetics of Skin Carcinogenesis, German Cancer Research Center, Heidelberg, Germany 2
This review examines the nature, incidence and molecular pathogenesis associated with non-melanoma skin cancers (NMSC). These comprise the basal cell carci-
Section Editor: Michael Roberts – School of Medicine, University of Queensland, Australia
nomas (BCCs), the most frequent though rarely metastasizing skin tumours, the more aggressive squamous cell carcinomas (SCCs), which are a problem especially in immunosuppressed organ transplant recipients. UV radiation is a key risk factor for both BSC and SCC. Whereas BCCs are associated with a limited number of sunburns (may be a one or more severe sunburns), SCCs usually occur after with recurrent UV exposure – with sun damage induced actinic keratoses as a well-established precursor. Each carcinoma may be associated with several chromosomal mutations and prevention is preferred to treatment, of which surgery is the first choice.
Background Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), also known as non-melanoma skin cancer, are the most frequent tumours in the world today and their incidence is increasing. Figure 1 shows some examples of these tumours, together with actinic keratosis, a common precursor for SCCs. BCC and SCC tumours are associated with a low rate of actual death but a high rate of disfigurement when skin lesions are located on the head and neck [1]. *Corresponding author: P. Boukamp ([email protected]) a On leave from Department of Pharmacology, Mosul Medical College, Mosul, Iraq. 1740-6765/$ ß 2008 Published by Elsevier Ltd.
DOI: 10.1016/j.ddmec.2008.05.007
SCC occurs less frequently than basal cell carcinoma (in a ratio 1:4) but is generally more aggressive [2]. SCC, a malignant epidermal keratinocyte neoplasm, often manifests as a rough keratotic papule, a wart-like growth, or a fast growing tender protuberance, frequently with a central keratinous core. It is suggested that about 10% of the SCCs arise from actinic keratosis (actinic signifying sun, and keratosis denoting a scaly spot) which, in turn, may be owing to atypic Malpighian cells spreading through the dermal basement membrane into the dermis [3]. Furthermore, it was recently proposed that also keratoacanthomas (KAs) have to be regarded as precursor lesions [4]. KAs are characterized by a rapid growth phase for the first 4–8 weeks giving rise to a tumour with a histology strongly resembling that of a well differentiated SCCs and a possible spontaneous self-induced regression after 3–6 months. KAs and SCCs generally arise at sites of extreme UV exposure, commonly on the face, head, neck, back, hand and forearm. Cutaneous SCC has a typical metastasis rate of 1%, but higher values have been reported for certain locations such as the lip [5]. By contrast, BCC is a malignant neoplasm that develops from the basal germinative cells, has no visible pre-malignant phase and is characterized by slow growth, and nodular, superficial, morpheaform or ulcerating subtypes may be differentiated [3,6]. The typical presentation of a nodular BCC, which usually appears on sun-exposed skin, is often characterized by a smooth and insensitive telangiectasis, often with an e55
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Figure 1. Pathology of non-melanoma skin cancer: (a) a fibrosing basal cell carcinoma (BCC); (b) a nodular basal cell carcinoma (BCC) in the corner of the eye; (c) multiple actinic keratoses and squamous cell carcinomas (SCC) on the forehead; (d) an 85-year-old man with a reddish, centrally eroded nodule on his right cheek. The clinical differential diagnoses include nodular basal-cell carcinoma, anaplastic squamous cell carcinoma, amelanotic melanoma, cutaneous metastasis and large-cell anaplastic lymphoma. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
inner ulceration [7]. BCCs generally develop on the face, nevertheless, about one third is on normally sun-protected locations such as the inner canthus or retroauricular region. BCCs have a metastasis rate of <0.1%, however, they are highly invasive and locally destructive. Sometimes it is difficult to define the type of skin cancer clinically and several differential diagnoses should be considered (Fig. 1d).
Epidemiology In Anglo-Saxon populations, melanoma and non-melanoma skin cancer are now the most common types of cancer and incidence rates are considered to be of epidemic proportions [8]. With annual cases in the United States surpassing 600,000, non-melanoma skin cancer constitutes more than one-third of all cancers [9]. Of these 600,000 cases, approximately 500,000 are for BCC and 100,000 are for SCC. Miller and Weinstock estimated a later incidence that was almost double the figure suggested by Silverberg [10]. In 1998, in Townsville, Australia, the incidence of BCCs was 2055 in males and 1195 in females, whilst that for SCCs was 1332 males and 755 females [11]. In Wales, UK, Holme et al. recorded 128 male and 105 female incidences of BCCs and 25 male and 9 female incidences of SCCs [12]. Trakatelli et al. emphasized the fact that in most countries cancers are not e56
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reported at all, and therefore the exact figures of BCC and SCC are unknown [1]. The authors claimed that the average annual increase of non-melanoma skin cancer in European, United States, Canadian and Australian Anglo populations raised since the 1960s from 3% to 8%. They also claim that non-melanoma skin cancer rates are on the increase in every country with a record system, although rates are lower in countries further away from the equator. Accordingly, Lear et al. claim that BCCs comprise roughly 70 to 80% of all skin cancers in North America [13]. They allege SCC to be the second most frequent cutaneous malignancy. Lear and others reported that in 2005, one million new BCC were detected in the United States, and more than 78,000 cases of BCC and SCC were diagnosed in Canada.
‘Risk factors’ for skin cancer Sunlight, viral infection, diet, immunosuppression in organ transplant recipients and induction of genetic mutations has been given as causes for non-melanoma cancer (Fig. 2). We consider each in turn.
Sunlight Ultra violet radiation from the sun is the main cause of nonmelanoma skin cancer. According to Molho-Pessach and
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Figure 2. Diagram of the pathogenesis of non-melanoma skin cancer (for review see [71].
Lotem, solar UV radiation acts as a complete carcinogen or as a promoter of carcinogenesis [14]. Johnson et al. propose that SCC will develop from 10% of sun-provoked actinic keratoses [15]. Ultra violet radiation penetrates the epidermal cell layers where the precursor cells of non-melanoma skin cancer
are situated. UVB light interacts directly with DNA via induction of DNA damage although it can also generate reactive oxygen species, with UVA light acting mainly through induction of reactive oxygen species [16] (Fig. 2). DNA absorbs UV radiation and undergoes change in some genes within these www.drugdiscoverytoday.com
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cells. Major targets of UV radiation are the p53 gene for SCC and BCC, and the patched gene (PTCH) for BCC. Mutationdependent inactivation of these genes leads to cell propagation [17]. Daya-Grosjean and Sarasin observed typical UVinduced mutations of the patched gene in both xeroderma pigementosum and sporadic basal cell carcinoma though with a significantly higher level of mutations in xeroderma pigmentosum basal cell carcinomas [18]. Furthermore, Giglia-Mari and Sarasin analysed p53 mutations and they found that the majority of mutations corresponded to C–T transitions mainly at dipyrimidine sites known to be hotspots of DNA lesions after UVB exposure [19]. The role of UVA is less defined. However, evidence is increasing that UVA – as the major component of the UV radiation reaching the earth as well as the major source of UV radiation used in tanning devices – has a high damaging potential. Whereas the photon energy may be insufficient for direct DNA damage, UVA can penetrate into the epidermis down to the proliferative basal cells and even further to the dermal compartment where it contributes to photoaging (for review see [20]). In addition, there is now compelling support that UVA may not only be relevant for skin cancer development in albino hairless mice (for review see [21]) but that it induces DNA double strand breaks also in normal human skin keratinocytes and causes chromosomal aberrations that may lead to tumourigenic conversion. All this argues for an important role for UVA in the multistep process of cutaneous skin SCCs [22]. Constituent, genetic and acquired pigmentary features such as fair skin, red or blond hair, freckles and lack of tanning ability have been recognized as risk factors for both melanoma and non-melanoma skin cancers when combined with the environmental risk factor of high ultraviolet light exposure [23]. Kricker et al. stated that the incidence of skin cancer is higher among those with lighter skinned ethnic group that it is in darker skin [24].
Viral infection Based on the effect of UV irradiation on HPV 1, 2, 3, 5, 7, 20, 23, 27, 38, 41 and 77, de Villiers et al. concluded that human papilloma viral (HPV) infections may be involved in the etiology of non-melanoma skin cancer. Epidemiological data comparing non-melanoma cancers from normal skin (with potential HPV infection) and skin in patients treated with the HPV vaccine may confirm these conclusions [25]. Accordingly, Karagas et al. reported on a population-based casecontrol study with 252 SCC case patients, 525 BCC case patients, and 461 control subjects [26]. Using multiplex HPV serology, they found no difference in HPV seropositivity between BCC case patients and control subjects but a role for HPV beta types in the pathogenesis of SCCs. Patel et al. suggested a link between HPV and SCC when they found that a EVER2 gene polymorphism is associated with the e58
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presence of HPV antibodies and an increased risk of SCC [27]. Another indicator to the role of this virus in SCC is the conclusion of Nindl et al. [28]. They found that triggering of cutaneous HPV by UV-induced DNA damage and other factors like immunosuppression lead to increase HPV replication. This will cause persistent viral infections and accumulation of additional DNA mutations. A study conducted by Hall et al. showed that the combined effects of beta papilloma virus and presence of a susceptible phenotype like fair skin or prolong sun exposure resulted in a greater risk of SCC than either risk factor alone [29]. Furthermore, Tomassino and colleagues demonstrated that HPV 38 is able to transform human skin keratinocytes thus providing evidence for the aggressiveness of specific HPV types in skin carcinogenesis [30,31]. A possible explanation might be that by, for example blocking the action of the p53 tumour suppressor gene, the virus induced inhibition of DNA repair and apoptosis after UV-induced DNA damage [32].
Diet High intake of meat and fat increases the risk of developing SCC [33]. McNaughton et al., in a critical review on the role of diet in BCC and SCC development, concluded a possible positive relationship between BCC and SCC occurrence and fat intake [34]. Also higher intake of unmodified dairy, like whole milk and cheese may increase the risk of SCC in susceptible individuals [35].
Immunosuppression Skin cancer prevalence, annual incidence, and clinical risk factors in a white renal transplant population of 182 have been reported [36]. Non-melanoma skin cancer developed in 16.5% of patients post transplantation, with a SCC to BCC ratio of 3.8:1 and lentigo maligna melanoma was found in three patients (1.6% incidence). The potential for an increased skin cancer risk is also emphasized by the finding of increased numbers of precursor lesions for non-melanoma cancer, with 15.4% of actinic keratoses and 53% of viral warts being reported as resulting in non-melanoma cancer. The annual incidence rate of non-melanoma skin cancer in one UK transplant population was between 7.1% and 10.6% according to a study done by Harden et al. [37], and it is increased to 9.3% to 18.1% in patients under medical immunosuppression for 10 years or more. In Queensland, Australia, a study conducted by Ramsay et al. [38] showed that 187 of 361 transplant recipients developed non-melanoma skin cancer with inversed ratio of SCC/BCC from 1:3.7 before transplantation to 2:1 afterwards.
Molecular pathogenesis Whereas it is believed that SCCs develop as a multistep process, and that actinic keratoses (AK) are precursor lesions, BCCs are rather thought to develop de novo. This is also
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supported by the fact that a cumulative UV damage is suggested only for SCCs whilst for BCCs one or more heavy UV exposures early in life may be causal events for tumour development.
P53 and skin cancer UV exposure induces an important aberration – mutation in the p53 tumour suppressor gene, the guardian of the genome. When the gene thereby gets inactivated, early genomic instability follows. These p53 gene mutations are the most prominent and best-studied aberrations in all skin cancers. It is now well established that more than 50% of all SCCs and BCCs show mutations and this frequency rises to 90% in skin cancers of patients with xeroderma pigmentosum (reviewed in [19]). Many of the p53 mutations show a very specific aberration, C to T transitions with even a high frequency of CC to TT double base changes, the so called UVB signature. Furthermore, and different from, for instance, colon cancer where the second p53 allele is often lost during tumour progression, also the second allele is frequently mutated in skin carcinomas and most importantly at different sites. This strongly supports the mutational power of UVB radiation [39,40]. The mutations do not appear at random but show a pattern of hot spots different from that of internal malignancies [41]. Furthermore, it is proposed that the mutation hot spot in codon 177 is specific for BCCs only, whereas mutations in codon 278, also found occasionally in certain internal cancers, are specific for SCCs [19]. Such specificity strongly argues for a selective advantage of these mutations, not only causing inactivation of the gene but also providing gain of function as suggested elsewhere [42]. Since these mutations can precede any visible tumour in skin, it is further suggested that p53 mutations are early if not initial events and it is tempting to suggest that this allows for a first pulse of genomic instability required for additional genetic events to occur.
The multistep model of skin cancer SCC
In agreement with the cumulative life time exposure of UV radiation, the genetic changes occurring during the multistep process of skin carcinogenesis can be explained in a sequencespecific manner. As discussed above, p53 mutations can already be detected in otherwise unsuspicious epidermis (summarized in [43]) and increase in frequency with age [44]. Accordingly, UV-type mutations are frequently found in the precancerous lesions of actinic keratosis (AK). Bowen’s disease or also known as carcinoma in situ (CIS) represent a preinvasive stage of the invasive skin SCCs. AK and CIS can occur at multiple sites, including in the vicinity of SCCs, supporting the hypothesis of field cancerization already suggested from the immuno-positive p53 distribution [45] and further substantiated by molecular- and cytogenetic studies
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[46–48]. Interestingly, a Japanese study which investigated micro-satellite instability and loss of heterogeneity (LOH) in normal skin, AKs, and SCCs suggested that at least in Japan AK was not likely to proceed SCCs [49]. Another potential early event is perturbation in glutathione peroxidase activity (GPX) leading to an elevated peroxide burden and thus risk for increased damage [50]. Indeed, Walshe et al. observed that reduced GPX in vivo can be reversed by agents that reduce the burden of oxidatives/ peroxidatives and concluded that a direct contributor to UV-induced SCC is disruption of peroxide and reactive oxygen species metabolism [50]. As a potential advanced step in the sequence of skin carcinogenesis keratoacanthomas (KA) already exhibit an increased aberrant genotype. Whereas previous LOH studies did not support the hypothesis that KAs are SCCs that regress as a result of external (host) influences [51] more recent data argue in favour of such an interpretation. Burnworth et al. demonstrated that many KAs were characterized by similar though less complex cytogenetic changes as SCCs [4]. Interestingly, in both tumour types the cell cycle regulator cyclin D1 was upregulated [52,53]. Whereas it suggested a role for a forced proliferation, the authors showed that overexpression of cyclin D1 caused abnormal tissue homeostasis and an altered interaction with the stromal environment. However, whereas in KAs this still correlated with increased expression of the cell cycle inhibitor p16 – likely still controlling the abnormal growth – p16 expression was lost in SCCs through further genetic or epigenetic silencing [4]. In addition, the SCCs had acquired an invasive phenotype most likely by loss of the angiogenic inhibitor thrombospondin 1 (TSP-1) which was still strongly expressed by KAs but absent or minimal in SCCs [4]. Since the latter correlated with the expression of the matrix degradation enzyme matrix metalloprotease 13 (MMP13) either expressed by the tumour cells or the surrounding stroma although largely absent in KAs (Burnworth and Boukamp, unpublished), these data suggest that inhibition of TSP-1 and upregulation of MMPs is a late event in tumour progression and is responsible for malignant progression. Using single nucleotide polymorphism (SNP) microarray analysis Purdie et al. [54] finally identified the protein tyrosine phosphatase receptor type D (PTPRD) locus, an emerging frequent target of homozygous deletion in lung cancer and neuroblastoma, as a candidate tumour suppressor gene in skin SCCs with a possible association with metastasis. Functional proof, however, is still awaited. Similarly inconclusive is the role of b-catenin in non-melanoma skin cancer. For instance, whilst Doglioni et al. found mutated b-catenin genes and suggested that also the TCF-1MITF-mediated pathway may be activated [55], Brasanac et al. [56] in noting the reported decrease in b-catenin level from actinic keratosis (AK) to SCC comments that correlation with SCC differentiation has not been established [56]. www.drugdiscoverytoday.com
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BCCs
Treatment
In addition to a high frequency in p53 mutations, BCCs are characterized by their aberrant sonic hedgehog-patchedsmoothened pathway. The finding that patients with nevoid basal cell carcinoma syndrome (NBCC) who develop multiple BCCs at an early age, showed frequent loss of chromosome 9q has allowed identification of the putative tumour suppressor gene on 9q22 to q32, the PTCH gene, the cell surface receptor of the secreted signalling molecule sonic hedgehog (SHH) (for review see [57]). In skin, sonic hedgehog (SHH) signalling has been implicated in hair follicle growth and morphogenesis. In the absence of SHH, patched1, the protein product of PTCH, inhibits smoothened (SMO), a G-protein-coupled-like receptor. SMO is released upon binding of SHH to patched1 and can initiate a signal transduction cascade that causes activation of the transcription factor Gli. Thus, deregulation of this pathway by either loss of PTCH or forced expression of SMO results in elevated levels of the transcription factor Gli1 and as a consequence induces hair follicle tumours. Accordingly, loss-of-function mutations of PTCH as well as activating mutations of SMO and Gli have been found in several sporadic human BCCs and even more so in xeroderma pigmentosum patients who suffer from a defect in nucleotide excision repair that is responsible for hyperphotosensitivity and a high incidence of skin cancer. Recently also SHH mutations were reported for these patients [58,59]. Interestingly, a recent study reported that most of the PTCH mutations showed the UV signature (C–T transitions or even CC–TT double base changes), arguing for UV as being important to abrogate the SHH pathway in sporadic BCCs [60]. Furthermore, there is indication that preventing Gli2 function may inhibit BCC formation. From these new studies, using a BCC mouse model, it is suggested that the function of Gli2 is to prevent apoptosis and to promote micro-vascularization [61]. From all of this it is now widely accepted that abrogation of the SHH pathway is the major cause of BCC development and because BCCs develop only in skin containing hair follicles and the SHH pathway is essential for hair follicle development it has been suggested that BCCs originate from hair follicle precursor cells. Besides involvement of the SHH pathway new data also suggest a role for the TGF-b Smad pathway in BCC pathogenesis. Gambichler et al. found significant mRNA overexpression of TGF-b1 and the Smad pathways constituents Smad 3 and Smad7 in the center of BCCs as compared to adjacent healthy skin sites [62]. The bcl-2 family members are other genes that may contribute to the pathogenesis of both SCC and BCC development. For instance, Delehedde et al. [63] asserted that in normal epidermis bcl-2 oncoprotein was present in keratinocytes of the basal layer and downregulated in suprabasal layers. However, the proapoptotic bax protein was minimally expressed in basal keratinocytes, upregulated in suprabasal layers and downregulated in the granular cell layer.
Prevention is preferred to cure and when the latter is needed, surgical removal is always of choice. There are five groups of used and potential preventative agents. Sunscreens and antioxidants minimize sun or UV exposure and are the preferred choice for prevention. Whereas a therapeutic vaccine against HPV infection has been advocated [64], the value of an effective therapeutic vaccine against HPV and other viral infections as a preventative non-melanoma skin cancer causing agent is untested. Isotretinoin, and possibly other vitamin A-derived retinoids, may potentially prevent or delay the onset of non-melanoma skin cancer [65]. As COX-2 expression is increased by UV light exposure and may have a role [66], early pharmacological intervention using COX-2 inhibitors has been advocated to reduce the risk of UVB-induced skin cancer in both experimental animals and humans [67]. Silibinin, an agent protecting against photocarcinogenesis, induces several potentially favourable effects, including: (a) inhibition of skin inflammation, DNA damage, epithelial cell proliferation and sunburn; (b) alteration of mitogenic, apoptotic and survival signalling; (c) activation of p53; (d) induction of cell cycle arrest; and (e) enhanced repair of DNA damage [68]. Alternative treatments for non-melanoma skin cancer when surgery is not possible include: ionizing radiation, cryotherapy, photodynamic therapy, topical immunotherapy and, in special cases, injection of interferons or cytostatic agents into the tumour. In certain metastatic cases systemic chemotherapy may be required. The interferon-upregulating substance imiquimod is often used in the treatment of multiple superficial lesions [69]. Imiquimod facilitates immunostimulation by binding to cell surface receptors (such as Toll receptor 7) and stimulating the discharge of a variety of cytokines. [70]. According to Chakrabarty and Geisse [65], fluorouracil (5-FU) is the most commonly studied topical chemotherapeutic agent to date. 5-FU, a structural analogue of thymidine, competes for thymidylate synthetase and interferes with DNA synthesis in dividing cells, resulting in cell death. Owing to inadequate penetration of 5-FU beyond the epidermis, the reported outcomes for 5-FU with SCCs in situ and superficial BCCs have been better than with invasive SCCs and BCCs. Topical chemotherapy with 5-FU is of most use in the management of actinic keratoses, the precursor lesions of SCC.
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Acknowledgements We thank Prof Adele Green (Queensland Institute of Medical Research) for her advice on the epidemiological aspects and Angelika Lampe for her help in preparing the manuscript. This work was in part supported by the Tumorzentrum Heidelberg-Mannheim and the Bmbf – UVA radiation damage (both to PB), the NHMRC (Australia) (to MR) and the Cancer Council of Queensland (to MR, HPS).
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with the presence of beta-catenin gene mutation. Am. J. Pathol. 163, 2277– 2287 Brasanac, D. et al. (2005) Cyclin A and beta-catenin expression in actinic keratosis, Bowen’s disease and invasive squamous cell carcinoma of the skin. Br. J. Dermatol. 153, 1166–1175 Toftgard, R. (2000) Hedgehog signalling in cancer. Cell Mol. Life Sci. 57, 1720–1731 Couve-Privat, S. et al. (2004) Functional analysis of novel sonic hedgehog gene mutations identified in basal cell carcinomas from xeroderma pigmentosum patients. Cancer Res. 64, 3559–3565 Reifenberger, J. et al. (2005) Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas. Br. J. Dermatol. 152, 43–51 Heitzer, E. et al. (2007) UV fingerprints predominate in the PTCH mutation spectra of basal cell carcinomas independent of clinical phenotype. J. Invest. Dermatol. 127, 2872–2881 Ji, J. et al. (2008) Gene silencing of transcription factor Gli2 inhibits basal cell carcinomalike tumor growth in vivo. Int. J. Cancer 122, 50–56 Gambichler, T. et al. (2007) Increased expression of TGF-beta/Smad proteins in basal cell carcinoma. Eur. J. Med. Res. 12, 509–514
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Delehedde, M. et al. (1999) Altered expression of bcl-2 family member proteins in nonmelanoma skin cancer. Cancer 85, 1514–1522 Lowy, D.R. and Schiller, J.T. (2006) Prophylactic human papillomavirus vaccines. J. Clin. Invest. 116, 1167–1173 Chakrabarty, A. and Geisse, J.K. (2004) Medical therapies for nonmelanoma skin cancer. Clin. Dermatol. 22, 183–188 Buckman, S.Y. et al. (1998) COX-2 expression is induced by UVB exposure in human skin: implications for the development of skin cancer. Carcinogenesis 19, 723–729 An, K.P. et al. (2002) Cyclooxygenase-2 expression in murine and human nonmelanoma skin cancers: implications for therapeutic approaches. Photochem. Photobiol. 76, 73–80 Singh, R.P. and Agarwal, R. (2005) Mechanisms and preclinical efficacy of silibinin in preventing skin cancer. Eur. J. Cancer 41, 1969–1979 Sterry, W. (2007) Skin diseases with high public health impact. Nonmelanoma skin cancer. Eur. J. Dermatol. 17, 562–563 Stanley, M.A. (2002) Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential. Clin. Exp. Dermatol. 27, 571–577 Boukamp, P. (2005) Non-melanoma skin cancer: what drives tumour development and progression? Carcinogenesis 26, 1657–1667
DRUG DISCOVERY
TODAY
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Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Skin diseases MECHANISMS
Non-melanoma skin cancers Ashraf Hashim Ahmed1,a, H. Peter Soyer1, Nicholas Saunders2, Petra Boukamp3,*, Michael S. Roberts1 1
University of Queensland, School of Medicine, Southern Clinical Division, Princess Alexandra Hospital, QLD, Australia University of Queensland, Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, QLD, Australia 3 Division of Genetics of Skin Carcinogenesis, German Cancer Research Center, Heidelberg, Germany 2
This review examines the nature, incidence and molecular pathogenesis associated with non-melanoma skin cancers (NMSC). These comprise the basal cell carci-
Section Editor: Michael Roberts – School of Medicine, University of Queensland, Australia
nomas (BCCs), the most frequent though rarely metastasizing skin tumours, the more aggressive squamous cell carcinomas (SCCs), which are a problem especially in immunosuppressed organ transplant recipients. UV radiation is a key risk factor for both BSC and SCC. Whereas BCCs are associated with a limited number of sunburns (may be a one or more severe sunburns), SCCs usually occur after with recurrent UV exposure – with sun damage induced actinic keratoses as a well-established precursor. Each carcinoma may be associated with several chromosomal mutations and prevention is preferred to treatment, of which surgery is the first choice.
Background Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), also known as non-melanoma skin cancer, are the most frequent tumours in the world today and their incidence is increasing. Figure 1 shows some examples of these tumours, together with actinic keratosis, a common precursor for SCCs. BCC and SCC tumours are associated with a low rate of actual death but a high rate of disfigurement when skin lesions are located on the head and neck [1]. *Corresponding author: P. Boukamp ([email protected]) a On leave from Department of Pharmacology, Mosul Medical College, Mosul, Iraq. 1740-6765/$ ß 2008 Published by Elsevier Ltd.
DOI: 10.1016/j.ddmec.2008.05.007
SCC occurs less frequently than basal cell carcinoma (in a ratio 1:4) but is generally more aggressive [2]. SCC, a malignant epidermal keratinocyte neoplasm, often manifests as a rough keratotic papule, a wart-like growth, or a fast growing tender protuberance, frequently with a central keratinous core. It is suggested that about 10% of the SCCs arise from actinic keratosis (actinic signifying sun, and keratosis denoting a scaly spot) which, in turn, may be owing to atypic Malpighian cells spreading through the dermal basement membrane into the dermis [3]. Furthermore, it was recently proposed that also keratoacanthomas (KAs) have to be regarded as precursor lesions [4]. KAs are characterized by a rapid growth phase for the first 4–8 weeks giving rise to a tumour with a histology strongly resembling that of a well differentiated SCCs and a possible spontaneous self-induced regression after 3–6 months. KAs and SCCs generally arise at sites of extreme UV exposure, commonly on the face, head, neck, back, hand and forearm. Cutaneous SCC has a typical metastasis rate of 1%, but higher values have been reported for certain locations such as the lip [5]. By contrast, BCC is a malignant neoplasm that develops from the basal germinative cells, has no visible pre-malignant phase and is characterized by slow growth, and nodular, superficial, morpheaform or ulcerating subtypes may be differentiated [3,6]. The typical presentation of a nodular BCC, which usually appears on sun-exposed skin, is often characterized by a smooth and insensitive telangiectasis, often with an e55
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Figure 1. Pathology of non-melanoma skin cancer: (a) a fibrosing basal cell carcinoma (BCC); (b) a nodular basal cell carcinoma (BCC) in the corner of the eye; (c) multiple actinic keratoses and squamous cell carcinomas (SCC) on the forehead; (d) an 85-year-old man with a reddish, centrally eroded nodule on his right cheek. The clinical differential diagnoses include nodular basal-cell carcinoma, anaplastic squamous cell carcinoma, amelanotic melanoma, cutaneous metastasis and large-cell anaplastic lymphoma. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
inner ulceration [7]. BCCs generally develop on the face, nevertheless, about one third is on normally sun-protected locations such as the inner canthus or retroauricular region. BCCs have a metastasis rate of <0.1%, however, they are highly invasive and locally destructive. Sometimes it is difficult to define the type of skin cancer clinically and several differential diagnoses should be considered (Fig. 1d).
Epidemiology In Anglo-Saxon populations, melanoma and non-melanoma skin cancer are now the most common types of cancer and incidence rates are considered to be of epidemic proportions [8]. With annual cases in the United States surpassing 600,000, non-melanoma skin cancer constitutes more than one-third of all cancers [9]. Of these 600,000 cases, approximately 500,000 are for BCC and 100,000 are for SCC. Miller and Weinstock estimated a later incidence that was almost double the figure suggested by Silverberg [10]. In 1998, in Townsville, Australia, the incidence of BCCs was 2055 in males and 1195 in females, whilst that for SCCs was 1332 males and 755 females [11]. In Wales, UK, Holme et al. recorded 128 male and 105 female incidences of BCCs and 25 male and 9 female incidences of SCCs [12]. Trakatelli et al. emphasized the fact that in most countries cancers are not e56
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reported at all, and therefore the exact figures of BCC and SCC are unknown [1]. The authors claimed that the average annual increase of non-melanoma skin cancer in European, United States, Canadian and Australian Anglo populations raised since the 1960s from 3% to 8%. They also claim that non-melanoma skin cancer rates are on the increase in every country with a record system, although rates are lower in countries further away from the equator. Accordingly, Lear et al. claim that BCCs comprise roughly 70 to 80% of all skin cancers in North America [13]. They allege SCC to be the second most frequent cutaneous malignancy. Lear and others reported that in 2005, one million new BCC were detected in the United States, and more than 78,000 cases of BCC and SCC were diagnosed in Canada.
‘Risk factors’ for skin cancer Sunlight, viral infection, diet, immunosuppression in organ transplant recipients and induction of genetic mutations has been given as causes for non-melanoma cancer (Fig. 2). We consider each in turn.
Sunlight Ultra violet radiation from the sun is the main cause of nonmelanoma skin cancer. According to Molho-Pessach and
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Figure 2. Diagram of the pathogenesis of non-melanoma skin cancer (for review see [71].
Lotem, solar UV radiation acts as a complete carcinogen or as a promoter of carcinogenesis [14]. Johnson et al. propose that SCC will develop from 10% of sun-provoked actinic keratoses [15]. Ultra violet radiation penetrates the epidermal cell layers where the precursor cells of non-melanoma skin cancer
are situated. UVB light interacts directly with DNA via induction of DNA damage although it can also generate reactive oxygen species, with UVA light acting mainly through induction of reactive oxygen species [16] (Fig. 2). DNA absorbs UV radiation and undergoes change in some genes within these www.drugdiscoverytoday.com
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cells. Major targets of UV radiation are the p53 gene for SCC and BCC, and the patched gene (PTCH) for BCC. Mutationdependent inactivation of these genes leads to cell propagation [17]. Daya-Grosjean and Sarasin observed typical UVinduced mutations of the patched gene in both xeroderma pigementosum and sporadic basal cell carcinoma though with a significantly higher level of mutations in xeroderma pigmentosum basal cell carcinomas [18]. Furthermore, Giglia-Mari and Sarasin analysed p53 mutations and they found that the majority of mutations corresponded to C–T transitions mainly at dipyrimidine sites known to be hotspots of DNA lesions after UVB exposure [19]. The role of UVA is less defined. However, evidence is increasing that UVA – as the major component of the UV radiation reaching the earth as well as the major source of UV radiation used in tanning devices – has a high damaging potential. Whereas the photon energy may be insufficient for direct DNA damage, UVA can penetrate into the epidermis down to the proliferative basal cells and even further to the dermal compartment where it contributes to photoaging (for review see [20]). In addition, there is now compelling support that UVA may not only be relevant for skin cancer development in albino hairless mice (for review see [21]) but that it induces DNA double strand breaks also in normal human skin keratinocytes and causes chromosomal aberrations that may lead to tumourigenic conversion. All this argues for an important role for UVA in the multistep process of cutaneous skin SCCs [22]. Constituent, genetic and acquired pigmentary features such as fair skin, red or blond hair, freckles and lack of tanning ability have been recognized as risk factors for both melanoma and non-melanoma skin cancers when combined with the environmental risk factor of high ultraviolet light exposure [23]. Kricker et al. stated that the incidence of skin cancer is higher among those with lighter skinned ethnic group that it is in darker skin [24].
Viral infection Based on the effect of UV irradiation on HPV 1, 2, 3, 5, 7, 20, 23, 27, 38, 41 and 77, de Villiers et al. concluded that human papilloma viral (HPV) infections may be involved in the etiology of non-melanoma skin cancer. Epidemiological data comparing non-melanoma cancers from normal skin (with potential HPV infection) and skin in patients treated with the HPV vaccine may confirm these conclusions [25]. Accordingly, Karagas et al. reported on a population-based casecontrol study with 252 SCC case patients, 525 BCC case patients, and 461 control subjects [26]. Using multiplex HPV serology, they found no difference in HPV seropositivity between BCC case patients and control subjects but a role for HPV beta types in the pathogenesis of SCCs. Patel et al. suggested a link between HPV and SCC when they found that a EVER2 gene polymorphism is associated with the e58
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presence of HPV antibodies and an increased risk of SCC [27]. Another indicator to the role of this virus in SCC is the conclusion of Nindl et al. [28]. They found that triggering of cutaneous HPV by UV-induced DNA damage and other factors like immunosuppression lead to increase HPV replication. This will cause persistent viral infections and accumulation of additional DNA mutations. A study conducted by Hall et al. showed that the combined effects of beta papilloma virus and presence of a susceptible phenotype like fair skin or prolong sun exposure resulted in a greater risk of SCC than either risk factor alone [29]. Furthermore, Tomassino and colleagues demonstrated that HPV 38 is able to transform human skin keratinocytes thus providing evidence for the aggressiveness of specific HPV types in skin carcinogenesis [30,31]. A possible explanation might be that by, for example blocking the action of the p53 tumour suppressor gene, the virus induced inhibition of DNA repair and apoptosis after UV-induced DNA damage [32].
Diet High intake of meat and fat increases the risk of developing SCC [33]. McNaughton et al., in a critical review on the role of diet in BCC and SCC development, concluded a possible positive relationship between BCC and SCC occurrence and fat intake [34]. Also higher intake of unmodified dairy, like whole milk and cheese may increase the risk of SCC in susceptible individuals [35].
Immunosuppression Skin cancer prevalence, annual incidence, and clinical risk factors in a white renal transplant population of 182 have been reported [36]. Non-melanoma skin cancer developed in 16.5% of patients post transplantation, with a SCC to BCC ratio of 3.8:1 and lentigo maligna melanoma was found in three patients (1.6% incidence). The potential for an increased skin cancer risk is also emphasized by the finding of increased numbers of precursor lesions for non-melanoma cancer, with 15.4% of actinic keratoses and 53% of viral warts being reported as resulting in non-melanoma cancer. The annual incidence rate of non-melanoma skin cancer in one UK transplant population was between 7.1% and 10.6% according to a study done by Harden et al. [37], and it is increased to 9.3% to 18.1% in patients under medical immunosuppression for 10 years or more. In Queensland, Australia, a study conducted by Ramsay et al. [38] showed that 187 of 361 transplant recipients developed non-melanoma skin cancer with inversed ratio of SCC/BCC from 1:3.7 before transplantation to 2:1 afterwards.
Molecular pathogenesis Whereas it is believed that SCCs develop as a multistep process, and that actinic keratoses (AK) are precursor lesions, BCCs are rather thought to develop de novo. This is also
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supported by the fact that a cumulative UV damage is suggested only for SCCs whilst for BCCs one or more heavy UV exposures early in life may be causal events for tumour development.
P53 and skin cancer UV exposure induces an important aberration – mutation in the p53 tumour suppressor gene, the guardian of the genome. When the gene thereby gets inactivated, early genomic instability follows. These p53 gene mutations are the most prominent and best-studied aberrations in all skin cancers. It is now well established that more than 50% of all SCCs and BCCs show mutations and this frequency rises to 90% in skin cancers of patients with xeroderma pigmentosum (reviewed in [19]). Many of the p53 mutations show a very specific aberration, C to T transitions with even a high frequency of CC to TT double base changes, the so called UVB signature. Furthermore, and different from, for instance, colon cancer where the second p53 allele is often lost during tumour progression, also the second allele is frequently mutated in skin carcinomas and most importantly at different sites. This strongly supports the mutational power of UVB radiation [39,40]. The mutations do not appear at random but show a pattern of hot spots different from that of internal malignancies [41]. Furthermore, it is proposed that the mutation hot spot in codon 177 is specific for BCCs only, whereas mutations in codon 278, also found occasionally in certain internal cancers, are specific for SCCs [19]. Such specificity strongly argues for a selective advantage of these mutations, not only causing inactivation of the gene but also providing gain of function as suggested elsewhere [42]. Since these mutations can precede any visible tumour in skin, it is further suggested that p53 mutations are early if not initial events and it is tempting to suggest that this allows for a first pulse of genomic instability required for additional genetic events to occur.
The multistep model of skin cancer SCC
In agreement with the cumulative life time exposure of UV radiation, the genetic changes occurring during the multistep process of skin carcinogenesis can be explained in a sequencespecific manner. As discussed above, p53 mutations can already be detected in otherwise unsuspicious epidermis (summarized in [43]) and increase in frequency with age [44]. Accordingly, UV-type mutations are frequently found in the precancerous lesions of actinic keratosis (AK). Bowen’s disease or also known as carcinoma in situ (CIS) represent a preinvasive stage of the invasive skin SCCs. AK and CIS can occur at multiple sites, including in the vicinity of SCCs, supporting the hypothesis of field cancerization already suggested from the immuno-positive p53 distribution [45] and further substantiated by molecular- and cytogenetic studies
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[46–48]. Interestingly, a Japanese study which investigated micro-satellite instability and loss of heterogeneity (LOH) in normal skin, AKs, and SCCs suggested that at least in Japan AK was not likely to proceed SCCs [49]. Another potential early event is perturbation in glutathione peroxidase activity (GPX) leading to an elevated peroxide burden and thus risk for increased damage [50]. Indeed, Walshe et al. observed that reduced GPX in vivo can be reversed by agents that reduce the burden of oxidatives/ peroxidatives and concluded that a direct contributor to UV-induced SCC is disruption of peroxide and reactive oxygen species metabolism [50]. As a potential advanced step in the sequence of skin carcinogenesis keratoacanthomas (KA) already exhibit an increased aberrant genotype. Whereas previous LOH studies did not support the hypothesis that KAs are SCCs that regress as a result of external (host) influences [51] more recent data argue in favour of such an interpretation. Burnworth et al. demonstrated that many KAs were characterized by similar though less complex cytogenetic changes as SCCs [4]. Interestingly, in both tumour types the cell cycle regulator cyclin D1 was upregulated [52,53]. Whereas it suggested a role for a forced proliferation, the authors showed that overexpression of cyclin D1 caused abnormal tissue homeostasis and an altered interaction with the stromal environment. However, whereas in KAs this still correlated with increased expression of the cell cycle inhibitor p16 – likely still controlling the abnormal growth – p16 expression was lost in SCCs through further genetic or epigenetic silencing [4]. In addition, the SCCs had acquired an invasive phenotype most likely by loss of the angiogenic inhibitor thrombospondin 1 (TSP-1) which was still strongly expressed by KAs but absent or minimal in SCCs [4]. Since the latter correlated with the expression of the matrix degradation enzyme matrix metalloprotease 13 (MMP13) either expressed by the tumour cells or the surrounding stroma although largely absent in KAs (Burnworth and Boukamp, unpublished), these data suggest that inhibition of TSP-1 and upregulation of MMPs is a late event in tumour progression and is responsible for malignant progression. Using single nucleotide polymorphism (SNP) microarray analysis Purdie et al. [54] finally identified the protein tyrosine phosphatase receptor type D (PTPRD) locus, an emerging frequent target of homozygous deletion in lung cancer and neuroblastoma, as a candidate tumour suppressor gene in skin SCCs with a possible association with metastasis. Functional proof, however, is still awaited. Similarly inconclusive is the role of b-catenin in non-melanoma skin cancer. For instance, whilst Doglioni et al. found mutated b-catenin genes and suggested that also the TCF-1MITF-mediated pathway may be activated [55], Brasanac et al. [56] in noting the reported decrease in b-catenin level from actinic keratosis (AK) to SCC comments that correlation with SCC differentiation has not been established [56]. www.drugdiscoverytoday.com
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BCCs
Treatment
In addition to a high frequency in p53 mutations, BCCs are characterized by their aberrant sonic hedgehog-patchedsmoothened pathway. The finding that patients with nevoid basal cell carcinoma syndrome (NBCC) who develop multiple BCCs at an early age, showed frequent loss of chromosome 9q has allowed identification of the putative tumour suppressor gene on 9q22 to q32, the PTCH gene, the cell surface receptor of the secreted signalling molecule sonic hedgehog (SHH) (for review see [57]). In skin, sonic hedgehog (SHH) signalling has been implicated in hair follicle growth and morphogenesis. In the absence of SHH, patched1, the protein product of PTCH, inhibits smoothened (SMO), a G-protein-coupled-like receptor. SMO is released upon binding of SHH to patched1 and can initiate a signal transduction cascade that causes activation of the transcription factor Gli. Thus, deregulation of this pathway by either loss of PTCH or forced expression of SMO results in elevated levels of the transcription factor Gli1 and as a consequence induces hair follicle tumours. Accordingly, loss-of-function mutations of PTCH as well as activating mutations of SMO and Gli have been found in several sporadic human BCCs and even more so in xeroderma pigmentosum patients who suffer from a defect in nucleotide excision repair that is responsible for hyperphotosensitivity and a high incidence of skin cancer. Recently also SHH mutations were reported for these patients [58,59]. Interestingly, a recent study reported that most of the PTCH mutations showed the UV signature (C–T transitions or even CC–TT double base changes), arguing for UV as being important to abrogate the SHH pathway in sporadic BCCs [60]. Furthermore, there is indication that preventing Gli2 function may inhibit BCC formation. From these new studies, using a BCC mouse model, it is suggested that the function of Gli2 is to prevent apoptosis and to promote micro-vascularization [61]. From all of this it is now widely accepted that abrogation of the SHH pathway is the major cause of BCC development and because BCCs develop only in skin containing hair follicles and the SHH pathway is essential for hair follicle development it has been suggested that BCCs originate from hair follicle precursor cells. Besides involvement of the SHH pathway new data also suggest a role for the TGF-b Smad pathway in BCC pathogenesis. Gambichler et al. found significant mRNA overexpression of TGF-b1 and the Smad pathways constituents Smad 3 and Smad7 in the center of BCCs as compared to adjacent healthy skin sites [62]. The bcl-2 family members are other genes that may contribute to the pathogenesis of both SCC and BCC development. For instance, Delehedde et al. [63] asserted that in normal epidermis bcl-2 oncoprotein was present in keratinocytes of the basal layer and downregulated in suprabasal layers. However, the proapoptotic bax protein was minimally expressed in basal keratinocytes, upregulated in suprabasal layers and downregulated in the granular cell layer.
Prevention is preferred to cure and when the latter is needed, surgical removal is always of choice. There are five groups of used and potential preventative agents. Sunscreens and antioxidants minimize sun or UV exposure and are the preferred choice for prevention. Whereas a therapeutic vaccine against HPV infection has been advocated [64], the value of an effective therapeutic vaccine against HPV and other viral infections as a preventative non-melanoma skin cancer causing agent is untested. Isotretinoin, and possibly other vitamin A-derived retinoids, may potentially prevent or delay the onset of non-melanoma skin cancer [65]. As COX-2 expression is increased by UV light exposure and may have a role [66], early pharmacological intervention using COX-2 inhibitors has been advocated to reduce the risk of UVB-induced skin cancer in both experimental animals and humans [67]. Silibinin, an agent protecting against photocarcinogenesis, induces several potentially favourable effects, including: (a) inhibition of skin inflammation, DNA damage, epithelial cell proliferation and sunburn; (b) alteration of mitogenic, apoptotic and survival signalling; (c) activation of p53; (d) induction of cell cycle arrest; and (e) enhanced repair of DNA damage [68]. Alternative treatments for non-melanoma skin cancer when surgery is not possible include: ionizing radiation, cryotherapy, photodynamic therapy, topical immunotherapy and, in special cases, injection of interferons or cytostatic agents into the tumour. In certain metastatic cases systemic chemotherapy may be required. The interferon-upregulating substance imiquimod is often used in the treatment of multiple superficial lesions [69]. Imiquimod facilitates immunostimulation by binding to cell surface receptors (such as Toll receptor 7) and stimulating the discharge of a variety of cytokines. [70]. According to Chakrabarty and Geisse [65], fluorouracil (5-FU) is the most commonly studied topical chemotherapeutic agent to date. 5-FU, a structural analogue of thymidine, competes for thymidylate synthetase and interferes with DNA synthesis in dividing cells, resulting in cell death. Owing to inadequate penetration of 5-FU beyond the epidermis, the reported outcomes for 5-FU with SCCs in situ and superficial BCCs have been better than with invasive SCCs and BCCs. Topical chemotherapy with 5-FU is of most use in the management of actinic keratoses, the precursor lesions of SCC.
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Acknowledgements We thank Prof Adele Green (Queensland Institute of Medical Research) for her advice on the epidemiological aspects and Angelika Lampe for her help in preparing the manuscript. This work was in part supported by the Tumorzentrum Heidelberg-Mannheim and the Bmbf – UVA radiation damage (both to PB), the NHMRC (Australia) (to MR) and the Cancer Council of Queensland (to MR, HPS).
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