Hereditary cancer syndromes

Considerable insight into the genetic basis of sporadic skin cancers has come from the elucidation of specific genes or genetic loci that define hereditary skin tumor syndromes (Table II) (^{Halpern and Altman, 1999}). Perhaps the best defined and most broadly relevant are the DNA repair genes that comprise the complementation groups of skin cancer prone xeroderma pigmentosum families (^{van Steeg and Kraemer, 1999}). At least six independent genes, on distinct chromosomal loci, define proteins involved in nucleotide excision repair. Among these are proteins that recognize and bind to sites of DNA damage (XPA, XPC), helicases (XPB, XPD), and endonuclease components (XPG, XPF), defects in any of which give a skin cancer prone phenotype. Potential polymorphisms with functional consequences in these and other DNA repair genes may contribute to susceptibility states in the general population as well (^{Wei et al, 1994}). Chromosomal mapping studies in the basal cell nevus syndrome, coupled with genetic and functional studies of Drosophila development, revealed the Sonic hedgehog pathway, and specifically mutations in the PTCH1 gene, as the basis for hereditary and many sporadic basal cell cancers (^{Bale and Yu, 2001}). Likewise the mapping of the inheritance pattern of the dysplastic nevus syndrome focused attention on the INK4a locus and specifically mutations in the p16^INK4a gene in the etiology of heredity-prone and sporadic melanoma (^{Hussussian et al, 1994}). Subsequently, defects in p16^INK4a or other components of the cyclin-CDK signaling pathway have been associated with both melanoma and nonmelanoma skin cancer (^{Soufir et al, 1999}). Detection of specific mutations in Cowden's syndrome (PTEN), Muir–Torre syndrome (MSH2, MLH1), pilomatricoma (CTNNB [-catenin]), and trichoepithelioma (PTCH1, p16^INK4aA) has illuminated the underlying pathways associated with adnexal tumors. The delineation of the specific genes mutated in other syndromes where locus mapping is confirmed should reveal even more insight into the broader spectrum of skin neoplasms.

Cutaneous malignant melanoma

Etiology and genetics

Cutaneous malignant melanoma (CMM) typically presents as a pigmented lesion that evolves either from a pre-existing nevus or arises de novo in normal-appearing skin. Compared with benign nevi, CMM frequently exhibit one or more of the following features (the ABCD of melanoma): asymmetry, border irregularity, color variegation, and diameter greater than 6 mm. CMM is notorious for its highly aggressive nature and its resistance to currently existing radiation and chemotherapeutic modalities. In fact, no standard therapy exists for patients with disseminated melanoma, who have a dismal prognosis. Recently, this potentially fatal disease has exhibited an alarming increase in incidence (^{Rigel et al, 1996}), evolving into a considerable health crisis. Epidemiologic evidence now points to a causal role for exposure to the UV spectrum of sunlight in the etiology of CMM (^{IARC, 1992;Armstrong and Kricker, 1995}), although the relative contributions of UVA (320–400 nm) and UVB (280–320 nm) are still disputed. Whereas UVA is thought to incite oxidative DNA damage, UVB can suppress cell-mediated immunity and induce the formation of characteristic pyrimidine dimers (^{De Gruijl, 2000}). Notably, unlike the more common squamous cell carcinoma (SCC), which is associated with cumulative lifetime UV exposure, melanoma appears to be induced by intense, intermittent exposure to UV, particularly during childhood (^{Holman et al, 1983;Whiteman et al, 2001}). In addition to sun exposure, other strong melanoma risk factors include the total number of nevi and dysplastic nevi, skin and hair color, and germline mutations in specific tumor suppressor genes (^{Goldstein and Tucker, 1993;Marks, 2000}).

Cytogenetic, linkage, and molecular analyses have provided compelling evidence for a strong underlying genetic basis for the genesis and progression of CMM (^{Chin et al, 1998}) (Figure 1). With few exceptions, however, these same studies have failed to identify consistent melanoma-associated genetic alterations. One significant exception is the INK4a locus at 9p21, which encodes in alternative reading frames two distinct tumor suppressor genes, p16^INK4a and p19^ARF (^{Quelle et al, 1995}). Germline mutations in the p16^INK4a gene, a negative regulator of the cell cycle, and subsequent loss of heterozygosity (LOH) in arising melanoma, are frequently observed in CMM-prone kindreds (^{Hussussian et al, 1994;Kamb et al, 1994;FitzGerald et al, 1996}). In sporadic CMM, INK4a mutations are less frequent, but functional loss of p16^INK4a in tumors can also occur through epigenetic mechanisms, such as promoter hypermethylation (^{Gonzalgo et al, 1997;Funk et al, 1998}). The significance of the pRB pathway, of which p16^INK4a is a major regulator in melanomagenesis, can be gleaned from the identification of germline mutations in two additional melanoma-prone kindreds of cyclin-dependent kinase 4 (CDK4), a promoter of cell cycle progression and a target of p16^INK4a inhibition (^{Zuo et al, 1996;Russo et al, 1998;Soufir et al, 1998}). Interestingly, p53, situated at the regulatory nexus of cellular growth and apoptosis, and the most common mutational target in human cancer (^{Cariello et al, 1994}), is infrequently mutated in primary melanoma (^{Zerp et al, 1999;Bardeesy et al, 2001};and references within). It has been postulated that this rarity can be at least partially accounted for by the positioning of p19^ARF, often inactivated in melanoma as a component of the INK4a locus, upstream of p53 (^{Chin et al, 1998}). Such a configuration would render inactivating mutations in both genes redundant with respect to cell cycle regulation.

Figure 1.

Genetic changes associated with melanoma progression. The multistage evolution of metastatic melanoma is depicted schematically with frequently associated stage-specific genetic changes detailed below. Trisomy of 7q, detected in invasive vertical growth phase melanoma, would amplify the listed tyrosine kinase receptors (EGFR, c-MET) the ligand HGF, and BRAF, but a causal relation to progression is not proven. The loss of APAF-1 may occur through gene deletion or gene silencing.

Full figure and legend (6K)

A second exception where specific genetic alterations have been clearly associated with CMM is in the MAPK signaling pathway. Although activating mutations in members of the RAS proto-oncogene family are among the most common in human cancer, demonstrating a causal role in CMM has proven elusive; however, more recent molecular analysis of primary and metastatic melanoma has implicated N-RAS and H-RAS mutations in CMM progression (^{Chin et al, 1997}). More significantly, a recent report has identified activating mutations in BRAF, which acts downstream of Ras, in almost 70% of human CMM (^{Davies et al, 2002}).

Point mutations with characteristic UV-associated signatures have been found in genes situated within those molecular pathways described above, including p16^INK4a (^{Kamb et al, 1994;Pollock et al, 1995}), N-RAS (^{van't Veer et al, 1989;van Elsas et al, 1997;Jiveskog et al, 1998}), and p53 (^{Zerp et al, 1999}), but not ARF (^{Peris et al, 1999}). These findings suggest that such critical genes can be the target of both hereditary and environmental insults. Recent epidemiologic data indicate that INK4a-associated melanoma-prone kindreds in geographic areas with the greatest exposure to sunlight are also at highest risk for melanoma, linking UV irradiation to the INK4a locus (^{Bishop et al, 2002}). Therefore, assessing the consequences of exposure to combinations of such risk factors represents an area of great experimental opportunity; however, at this time functional relationships between candidate genes and environmental factors in melanomagenesis are largely unknown.

Other classes of molecules have been implicated in melano-magenesis as well. Receptor tyrosine kinases (RTK), critical modulators of virtually all fundamental cellular behavior, including growth, differentiation, motility, and survival, play significant roles in normal melanocyte development and function (^{Bennett, 1993;Halaban, 1996}). These include c-Kit (^{Witte, 1990}), c-Met (^{Halaban et al, 1992;Takayama et al, 1996;Kos et al, 1999}), and the platelet-derived growth factor receptor (^{Stephenson et al, 1991;Soriano, 1997}). Multiple reports have either supported or refuted the role of specific RTK in primary and metastatic melanoma cells and their cultured derivatives (^{Albino, 1992;Shih and Herlyn, 1994;Halaban, 1996}). Of central importance to the aspiring melanoma cell is the acquisition of autonomous growth control through the creation of autocrine RTK signaling loops, i.e., a cell is able to manufacture both an RTK and its associated ligand. Examples of such loops include basic fibroblast growth factor (bFGF)-FGF receptor, considered a hallmark of CMM development, and transforming growth factor (TGF)-epidermal growth factor (EGF) receptor.

Recently, progress has been made in identifying new candidate therapeutic targets associated with melanoma cell survival. Soengas et al (2001) demonstrated that melanoma cells avoid apoptotic destruction through inactivation of apoptotic protease activating factor-1 (APAF-1), a requisite caspase-9 activator functioning downstream of p53. Common APAF-1 loss of function, which can occur by allelic loss and methylation silencing, also helps account for the relative rarity of p53 mutations in CMM. Significantly, restoration of APAF-1 pro-apoptotic function by the methylation inhibitor 5aza2dC rendered melanoma cells chemosensitive (^{Soengas et al, 2001}). In a related development,^{Griffith et al (2000)} showed that adenovirus-mediated expression of TNF-related apoptosis-inducing ligand (TRAIL), which induces caspase-8-associated apoptosis, can serve as gene therapy in the destruction of melanoma cells.

Experimental models of CMM

A major impediment to the study of melanoma has been the lack of relevant, tractable experimental animal models. Historically, the animal models studied in most detail have been the Xiphophorus hybrid fish and the South American opossum, as well as a number of rodent models (^{Kusewitt and Ley, 1996}) (Table III). The problem with all such models, however, is that arising melanocytic tumors do not resemble human CMM at the histopathologic level, and extensive genetic manipulation is not possible. The mouse represents an especially attractive system because of the availability of an extensive genetic base upon which to build. Unfortunately, murine melanocytes, unlike those in human skin, are typically confined to hair follicles and are exceedingly resistant to both spontaneous and UV-induced melanoma. Moreover, melanomas that arise do so with low penetrance, long latencies, and poor metastatic capacity.

Genetically engineered mouse models have proven to be amenable to the genetic dissection of molecular pathways in tumorigenesis, and a number of melanoma-prone, transgenic mice have recently been described (^{Satyamoorthy et al, 1999;Tietze and Chin, 2000}). Among these, melanoma induction in transgenic mice has been achieved through melanocytic expression of the oncogenes SV40 T-antigen, c-RET and H-RAS (^{Bradl et al, 1991;Klein-Szanto et al, 1994;Kato et al, 1998;Bardeesy et al, 2001}). The success of these models can be credited, in part, to earlier seminal work identifying candidate oncogenic pathways in human CMM: T-antigen disrupts pRB and p53 function in a fashion analogous to loss of both p16^INK4a and p19^ARF at the INK4a locus; c-RET, although not normally expressed in melanocytes, is a potent RTK whose presence would unbalance multiple kinase signaling pathways; and the potential role of activated RAS genes has already been noted. In an elegant set of genetic experiments, the activated H-RAS transgene was placed on a background deficient in Ink4a resulting in the efficient development of spontaneous nonmetastatic melanoma (^{Chin et al, 1997}). Moreover, when the H-RAS transgene was configured with a tetracycline-regulatable promoter, it could be demonstrated that activated H-RAS was required for melanoma maintenance in this model (^{Chin et al, 1999}). Recently, mice bearing p16^Ink4a-specific inactivating mutations, or the Cdk4 R24C mutation described in patients with familial CMM, were reported to be susceptible to DMBA-induced melanoma (^{Krimpenfort et al, 2001;Sharpless et al, 2001;Sotillo et al, 2001;Rane et al, 2002}), strongly supporting a role for the p16^INK4a/Cdk4/Rb pathway in melanomagenesis. As in other animal models, however, murine melanocytic neoplasms arising in these various transgenic mice do not closely resemble human lesions, in that they arise within the dermis and lack the epidermal component characteristic of conventional human CMM.

In melanoma-prone transgenic mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF), the c-Met ligand, metastasis occurs in approximately 15% of tumor-bearing animals (^{Takayama et al, 1996;1997;Otsuka et al, 1998}). Unlike other genetically engineered animals, melanocytes in HGF/SF transgenic mice demonstrate extra-follicular survival, residing within the epidermis, dermis, and epidermal–dermal junction. The resulting "humanized" skin has the potential to yield melanomas with a histopathologic profile reminiscent of human CMM (^{Noonan et al, 2001}). As an alternative to making mouse skin morphologically resemble human, human/mouse chimeras have been created in which human skin is grafted onto immunodeficient mice, and then subjected to experimental analysis (^{Satyamoorthy et al, 1999}). Such chimeras provide an orthotopic milieu in which to study spontaneous and UV-induced melanomagenesis in vivo. In fact, UVB exposure was capable of inducing melanocytic lesions, including a nodular melanoma, in xenografted human skin in concert with either DMBA treatment or adenovirus-mediated bFGF overexpression (^{Atillasoy et al, 1998;Berking et al, 2001}).

The ability to evaluate the consequences of UV irradiation on melanoma development is a paramount feature in any animal model, as exposure to sunlight is thought to be a causal agent in up to 80% of CMM. With respect to genetically tractable transgenic mouse models of melanoma, some demonstrate UV sensitivity, although responses have been relatively inefficient (^{Kelsall and Mintz, 1998;Broome et al, 1999}). The extra-follicular melanocytes in the HGF/SF transgenic skin, however, appear to be highly susceptible to neonatal UV irradiation (^{Noonan et al, 2000; 2001}), linking both the power of genetic manipulation and the relevance of environmental challenge within the same model system. Taken together, these advances brighten future prospects of creating relevant mouse models to rigorously assess genetic and environmental melanoma risk factors, facilitate development of efficacious sun protection strategies, and establish effectual antimelanoma therapeutics.

Basal cell carcinoma (BCC)

Etiology and genetics

BCC is a very common, slow-growing, locally invasive tumor that typically presents as a pink or pearly papule with superficial telangiectasia and occasional ulceration. There are three clinical variants: nodular (the most common), superficial, and sclerosing. In contrast to cutaneous SCC, BCC precursor lesions have not been identified, there is no evidence of neoplastic progression, and metastases are exceedingly rare (^{Miller, 1995}). BCC are probably derived from hair follicles, and analogous to hair follicle epithelium, BCC growth is dependent on proper signaling between neoplastic keratinocytes and surrounding mesenchymal cells. This may account, in part, for the low incidence of BCC metastases, as well as the difficulty in establishing and maintaining BCC as xenografts in vivo or as immortalized cell lines in vitro.

Chromosomal losses involving 9q have been found in both sporadic and inherited BCC. The latter tumors occur in patients with the autosomal dominant disorder nevoid basal cell carcinoma syndrome (NBCCS), which is characterized by a predisposition to BCC (frequently multiple and appearing at an early age), an increased incidence of several other tumors, and a variety of developmental anomalies (^{Gorlin, 1987}). These clinical features suggested that the gene involved in BCC formation is also important during embryogenesis, and this notion was confirmed with the discovery in NBCCS patients of germline mutations of PTCH1 (^{Hahn et al, 1996;Johnson et al, 1996}), a homolog of the Drosophila ptc gene involved in embryonic development. BCC from NBCCS patients had lost the remaining normal PTCH1 allele, which was also found to be deficient in spontaneous BCC, suggesting that PTCH1 is a tumor suppressor. A second PTCH gene has subsequently been identified (PTCH2) (^{Zaphiropoulos et al, 1999}), but its role in BCC development is not yet known.

Ptch1 is a 12-pass transmembrane molecule that functions as a receptor for Sonic hedgehog (Shh), a secreted ligand that regulates proliferation and patterning of multiple tissues and organs during embryogenesis (^{Chuang and Kornberg, 2000}). According to the current model (Figure 2), Ptch1 normally antagonizes signaling activity by repressing Smoothened (Smo), a cell-surface molecule with homology to G protein-coupled receptors. Shh initiates signaling in responsive cell types by inhibiting Ptch, resulting in derepression of Smo and, ultimately, the activation of Shh target genes. Two "universal" Shh target genes are Gli1 (which encodes a transcription factor) and Ptch1, and the expression level of these transcripts has proven to be a reliable indicator of physiologic and pathologic Shh signaling. Under normal conditions, activation of this pathway is dependent on Shh, whose expression is tightly regulated both in space and in time. In human BCC, however, loss of PTCH1 results in constitutive signaling that is irreversible and independent of SHH. In addition to loss-of-function PTCH1 mutations, gain-of-function (oncogenic) SMO mutations have been found in some BCC where PTCH1 appears to be normal (^{Xie et al, 1998;Lam et al, 1999}). Together, PTCH1 or SMO mutations have been identified in less than 75% of BCC whereas hedgehog target genes are upregulated in essentially all tumors examined (^{Dahmane et al, 1997}), indicating that additional mechanisms must exist for uncontrolled activation of this pathway. The current data suggest that constitutive Shh signaling, regardless of how this is brought about, plays a central role in the genesis of BCC. PTCH1 gene alterations have also been found in trichoepitheliomas (^{Vorechovsky et al, 1997}) and nevus sebaceous of Jadassohn (^{Xin et al, 1999}), suggesting that deregulation of Shh signaling plays a role in the genesis of other follicle-derived tumors. Although mutations in p53 have been found in up to 50% of BCC, their involvement in the development or maintenance of these tumors is not known, particularly as most BCC fail to exhibit the genomic instability associated with other cancers where p53 function is compromised.

Figure 2.

Proposed model depicting the molecular basis of BCC. In contrast to the multistep evolution of SCC and melanoma, deregulation of the SHH signaling pathway may be sufficient for BCC development. During physiologic activation in responsive cell types, SHH binds and inhibits PTCH1, which normally represses SMO. Derepression of SMO results in transient activation of SHH target genes, including PTCH1, GLI1, and cell type-specific genes, which are likely to play an important role in growth control. In BCC, mutations involving PTCH1 or SMO result in uncontrolled signaling and constitutive expression of SHH target genes.

Full figure and legend (7K)

What is the normal function of Shh signaling in skin? Analysis of genetically engineered mutant mice has revealed a crucial requirement for Shh during hair follicle growth and morphogenesis, but not terminal differentiation (^{St Jacques et al, 1998;Chiang et al, 1999}). In addition, both loss-of-function (^{Wang et al, 2000}) and gain-of-function (^{Sato et al, 1999}) studies implicate Shh signaling in the regulation of hair follicle growth during the anagen phase of the hair cycle. Thus, whereas physiologic Shh signaling in skin may govern growth of follicle keratinocytes at appropriate times, uncontrolled Shh signaling due to PTCH1 or SMO mutations may cause sustained proliferation, resulting in the development of follicle-derived tumors.

How does stimulation of Shh signaling alter cell function? Transcriptional responses in the highly homologous Drosophila hedgehog pathway are mediated by ci (reviewed in^{Aza-Blanc and Kornberg, 1999}), which belongs to the Gli family of zinc finger-containing transcription factors. Vertebrate ci homologs include Gli1, Gli2, and Gli3, all of which bind to the consensus DNA sequence GACCACCCA (^{Matize and Joyner, 1999}). In contrast to Gli3, which appears to function primarily as a transcriptional repressor, both Gli1 and Gli2 are transcriptional activators. Major emphasis has been placed on Gli1 as a potential effector of normal and constitutive Shh signaling for several reasons: it is consistently upregulated in cells where the Shh pathway is active, both during embryogenesis and in neoplasms; ectopic Gli1 expression can mimic responses to Shh in certain settings; and when overexpressed in frog skin, GLI1 can give rise to primitive skin tumors (^{Dahmane et al, 1997}). Despite these findings Gli1 knockout mice are phenotypically normal (^{Morris and Potten, 1999}), arguing against an essential role for this Gli protein in physiologic Shh signaling, whereas Gli2 knockout mice exhibit abnormalities in multiple organs whose development is dependent on Shh. In particular, loss of Gli2 function results in severe impairment of hair follicle growth (^{Grachtchouk et al, 2000a}).

Experimental models of BCC

Rats exposed to chemical carcinogens (MCA, DMBA) or ionizing radiation preferentially develop BCC rather than squamous tumors, and frequently at a high incidence (reviewed in^{Zackheim, 1985}; Table IV). Rat BCC appear to arise in the outer root sheath of hair follicles and have a slow growth rate, similar to human BCC. The molecular basis for experimentally induced BCC in rats is not yet known, but is expected to result in Shh pathway activation if the current model is accurate. In striking contrast to their strong predisposition to squamous tumor development, mice appear to be remarkably resistant to BCC induction. One notable exception is the chemical carcinogen dehydroretronecine, which is effective at inducing mouse BCC (and other tumors) following subcutaneous or topical administration (^{Johnson et al, 1978}).

The discovery of inactivating PTCH1 mutations in BCC fueled the development of a number of transgenic and knockout mouse models exploring the potential role of deregulated Shh signaling in BCC tumorigenesis. Overexpression of SHH using a K14 promoter resulted in upregulation of Shh target genes and development of basal cell-like proliferations in newborn mouse skin (^{Oro et al, 1997}), with similar results obtained using the K5 promoter to drive expression of a gain-of-function SMO mutant, M2SMO (^{Xie et al, 1998}). Overexpression of SHH in human keratinocytes followed by grafting onto SCID mice resulted in development of BCC-like changes as well (^{Fan et al, 1997}). These studies supported the hypothesis that constitutive activation of Shh signaling in keratinocytes is sufficient for BCC development, but analysis of tumor phenotypes in adult transgenic mice could not be performed due to impaired viability of these animals. When K14-SHH mouse skin was transplanted onto SCID mouse hosts, BCC-like proliferations were apparently replaced by well-differentiated hair-follicle-like structures (^{Oro et al, 1997}), supporting the proposed requirement for an appropriate tumor stroma to maintain BCC growth. Mouse models have also been developed in which Ptch gene function has been disrupted (^{Goodrich et al, 1997;Hahn et al, 1998}), and Ptch+/– mice have many features in common with NBCCS patients. Detailed analysis of skin from Ptch^lacZ/+ mice has revealed microscopic hair-follicle-derived proliferations, with the appearance of a variety of macroscopic skin tumors, including BCC, following exposure to ionizing or UV radiation (^{Aszterbaum et al, 1999}). Taken together, these findings strongly support the concept that deregulated Shh signaling plays a central role in BCC development.

Other transgenic mouse studies have focused on Gli proteins as potential mediators of constitutively activated Shh signaling in BCC. GLI1 and Gli2 have both been overexpressed in mouse skin using the same bovine K5 promoter with intriguingly different results. K5-GLI1 transgenic mice developed a variety of follicle-derived tumor types with relatively few BCC (^{Nilsson et al 2000}), whereas K5-Gli2 transgenic mice developed only BCC (^{Grachtchouk et al 2000b}). These findings, coupled with the results of knockout mouse studies described above, strongly implicate Gli2 both in physiologic (hair follicle growth) and pathologic (BCC development) Shh signaling in skin. Despite the compelling phenotypes produced in these mouse models, there is as yet no direct evidence that Gli transcription factors are required for tumorigenesis associated with PTCH1 or SMO mutations.

As it appears that deregulation of Shh signaling is sufficient for BCC development, these tumors may be uniquely responsive to mechanism-based therapeutic intervention. The steroidal alkaloid cyclopamine has been shown to inhibit Shh signaling by blocking SMO (^{Taipale et al, 2000}), but it is not yet known whether BCC could be successfully treated, or their appearance prevented, using this agent. In September 2001, one small-molecule inhibitor of Shh signaling had been granted FDA approval for phase I clinical trials in patients with BCC. Although this type of work is just getting under way, it may eventually lead to novel nonsurgical approaches to treating BCC and possibly other cancers associated with deregulated Shh signaling, such as medulloblastomas and a subset of rhabdomyosarcomas.

Cutaneous SCC

Etiology and genetics

Cutaneous SCC frequently presents as a firm, pink papule or nodule, with a conspicuous hyperkeratotic surface. Although they represent only about 20% of nonmelanoma skin cancers, SCC are generally more aggressive and occasionally lethal. SCC is more frequent with higher cumulative sunlight exposure, as cancers associated with occupational exposures, and in immunosuppressed patients. Hereditary syndromes uniquely associated with SCC risk have not been described although DNA repair defects in xeroderma pigmentosum substantially increase the risk for SCC development. Insight into the pathogenesis of cutaneous SCC has come from studies of the frequent precursor lesion, actinic keratosis (AK) (Figure 3). These benign hyperproliferative-hyperkeratotic lesions frequently have sunlight-induced clonal p53 mutations suggesting clonal expansion from a single cell carrying a specific p53 mutation. Most frequently, these mutations are in codon 278 or other codons of the DNA-binding domain of p53 that contain dipyrimidine sites (^{Hussain and Harris, 1998}). Of particular interest is the common finding of LOH at particular chromosomal sites such as 13q, 17p, 17q, 9p, and 9q (^{Rehman et al, 1994}). Frequently, multiple sites of allelic loss are detected in the same lesion. As actinic keratoses progress to SCC at an extremely low frequency, the challenge is to determine which of these genetic lesions, if any, contributes to premalignant progression. Analyses of SCC have also revealed frequent LOH and p53 mutations, but a modal allelic loss or specific p53 codon strongly associated with the acquisition of a malignant phenotype is yet to be identified (^{Quinn et al, 1994}). Other genetic changes have been associated with progression in SCC, and their possible involvement in pathogenesis has been explored in experimental studies. Mutations in the K-RAS and Ha-RAS gene are detected in both AK and SCC, and activation of the RAS pathway through mutation of the gene or growth factor stimulation may be extremely common in squamous tumors, particularly in sites with intense exposure to UV radiation (^{Pierceall et al, 1991;Kreimer-Erlacher et al, 2001}). Inactivating mutations or epigenetic silencing of p16^INK4a and activation of telomerase are other pathways associated with SCC development (^{Taylor et al, 1996;Soufir et al, 1999}). Constitutive activation of the EGF receptor (EGFR) by amplification or expression of ligands with the formation of an autocrine loop is a frequent finding in SCC (^{Moghal and Sternberg, 1999}). Although these correlations have provided clues to pathways involved in SCC pathogenesis, definitive causal associations in human SCC have not yet been confirmed. For this reason, model systems utilizing human and mouse keratinocytes in culture and animal models in vivo have been developed and utilized to test causal relationships at the molecular, cellular, and organism levels.

Figure 3.

Genetic changes associated with human cutaneous SCC. The multistage evolution of invasive SCC is depicted schematically with frequently associated genetic changes detailed below. Single base mutations in early lesions frequently are characteristic of UV light-induced damage, whereas later changes are associated with genomic instability. Increased activity of telomerase (deletion of inhibitor) or EGFR tyrosine kinase (gene amplification) may also result from epigenetic changes.

Full figure and legend (15K)

Experimental models of cutaneous SCC

The induction of squamous cell cancers on mouse skin by chemical carcinogens or UV light has been an excellent model to study cancer pathogenesis in general and skin cancer development in particular. These models have shown remarkable phenotypic homology to human SCC development and have provided additional information on the contribution of specific genetic changes to particular stages of tumor progression. The addition of genetically modified mice to the mix of models available has further clarified the specific requirements for particular genes and their downstream pathways in benign and malignant tumor formation (Table V). Together with in vitro analysis of keratinocytes, the biochemistry of SCC development is being revealed.

Animal model studies indicate that heterozygous activating Ras gene mutations are sufficient to induce a benign squamous lesion, and this is coupled to constitutive activation of the EGFR (^{Yuspa, 1994}) (Figure 4). Activation of the keratinocyte EGFR through transgenic targeting of TGF to the epidermis can also produce a benign tumor phenotype in the absence of Ras mutations, but tumors regressed unless subjected to continuous exposure to tumor promoters (^{Vassar et al, 1992;Dominey et al, 1993;Jhappan et al, 1994}). This suggests that activation of the EGFR is not sufficient for autonomous tumor formation, and alterations in other pathways are required. Under conditions of high expression or homozygosity of a mutant Ras gene, progression to malignancy is enhanced (^{Quintanilla et al, 1986;Greenhalgh and Yuspa, 1988}), suggesting that the Ras pathway can recruit additional changes required for progression. The target cell for Ras activation in the epidermis may also determine the tumor phenotype as transgenic targeting of oncogenic Ras to keratinocytes committed to the differentiation program produces terminally benign tumors whereas targeting to less differentiated cells permits progression to SCC (^{Brown et al, 1998}). In contrast, suprabasal targeting of c-Myc produces the papilloma phenotype, possibly through reducing apoptosis, whereas basal cell targeting of c-Myc is not oncogenic (^{Pelengaris et al, 1999;Waikel et al, 2001}). Deletion of the cyclin/CDK inhibitor p21^waf1, a downstream effector of p53, increases the number of benign tumors but does not influence the rate of premalignant progression (^{Missero et al, 1996;Weinberg et al, 1999}). In contrast, p53 depletion enhances malignant progression but does not increase benign tumor formation (^{Kemp et al, 1993}). These results suggest that the p53 pathway involving cell cycle inhibition through p21^waf1 does not determine the risk for malignant conversion, but another p53 regulated pathway is critical for suppression of premalignant progression. TGF plays a dual role in experimental SCC development, suppressing premalignant progression while enhancing phenotypic progression from SCC to a spindle cell phenotype (^{Glick et al, 1994;Portella et al, 1998;Go et al, 1999}). Members of the AP-1 transcription factor family also play a dual role in experimental skin tumor development, where c-June is essential for papilloma and c-Fos is essential for SCC development (^{Saez et al, 1995;Young et al, 1999}). Other pathways now implicated in SCC development and progression to spindle cell tumors from experimental studies are cyclin D1, ornithine decarboxylase, p16^ink4A, p15^ink4A, and E-cadherin (^{Navarro et al, 1991;Clifford et al, 1995;Linardopoulos et al, 1995;Robles et al, 1998}). These pathways are frequently altered in human SCC, but their contribution to pathogenesis remains to be proven by studies that directly transform human keratinocytes in an in vivo setting.

Figure 4.

Genetic changes associated with chemically induced mouse cutaneous SCC. The multistage evolution of anaplastic or spindle cell tumors in this model is highly ordered both temporally and genetically. Ras mutations are characteristic of chemical mutagens used to initiate tumor formation. Early upregulation of cyclin D1 and later up-regulation of TGF1 occur through epigenetic mechanisms and appear to be important components of carcinogenesis.

Full figure and legend (14K)

Mouse models promise to reveal another essential aspect of skin cancer pathogenesis, that of individual susceptibility. Inbred mouse strains differ in susceptibility to particular exposures by several orders of magnitude (^{DiGiovanni et al, 1992}). Carcinogenesis studies on cultured mouse keratinocytes derived from different background strains or on skin from specific strains grafted to nude mice indicate that sensitivity is determined by the target tissue rather than systemically (^{Yuspa and Morgan, 1981;Yuspa et al, 1982;Glick et al, 1999}). Tumor induction on F1 hybrid backcrosses between sensitive and resistant strains followed by genome scans using microsatellite markers has revealed that determinants for susceptibility or resistance are multigenic and distinct for benign tumor formation or premalignant progression and malignant conversion (^{Nagase et al, 1995;Mock et al, 1998}). Analysis of congenic strains and other approaches indicate that specific loci are epistatic, and the interactions are specific for benign or malignant tumor formation (^{Nagase et al, 2001}). Genetic loci also determine the survival potential for tumor-bearing animals (^{Nagase et al, 1999}). As the specific genes are identified for these determinants, syntenic sites in the human genome that determine skin cancer susceptibility may be revealed.

Conclusion

The rapidly accumulating genetic details of cutaneous cancer pathogenesis combined with the illumination of interacting signaling pathways downstream from the genes involved provides an opportunity to model the biochemistry of cutaneous tumors, a scheme that must ultimately be understood for rational therapy or medical intervention. Currently any biochemical model would almost certainly be incomplete. Alternatively, a general biologic scheme that may fit the multistage development of both melanoma and squamous cell skin cancers might be constructed with current information. The scheme has value as an organizational foundation to focus on emerging understanding of the biochemistry of the individual biologic processes and to test the biochemical principles in cutaneous cancer models. Such a scheme (Figure 5) assigns aberrations in control of proliferation, normal differentiation, apoptosis or senescence, or developmental processes (in the case of some hereditary syndromes) as early events. As a consequence of these changes or coupled to p53 mutations, resistance to terminal cell death produces a survival advantage for the incipient cancer cells. Under selective pressure (cytotoxicity from sunlight or environmental chemicals being the most obvious exogenous sources), clonal expansion produces a clinically apparent benign lesion (AK) or dysplastic nevus. Premalignant progression is associated with genomic instability that could be inherent in a lesion with enhanced survival and a high proliferation rate. Nevertheless, other important changes must occur in this growing lesion that could be modulated by the host response. An inflammatory reaction could deposit cytokines in the lesional environment as well as mutagenic reactive oxygen or nitrogen species (^{Smith et al, 1998;Hussain et al, 2000;Fitzpatrick, 2001}). Angiogenesis and immune surveillance may play opposing roles or systemic immunosuppression may act in concert with angiogenesis to enhance progression (^{Prehn and Prehn, 1996;Smith-McCune et al, 1997}). Many cell generations may pass as the positive and negative influences of the organismal response interact with the endogenous activities of the neoplastic lesion. Subsequent clones evolve and subclones from these as further survival advantage is acquired and selected. Alternatively, lethal genetic aberrations may arise and account for the spontaneous regression of some AK or melanotic lesions. The tumor environment evolves concomitant with clonal selection, altering the interactions of stromal, epithelial, and inflammatory components and the tumor matrix itself. It is this balance that finally allows for the invasive properties of tumor cells to be displayed. Thus, we see experimental evidence for stromal and matrix determinants of tumor cell behavior (^{Arias, 2001;Liotta and Kohn, 2001}). The advantage the host has over the tumor cell is time. The opportunity for intervention is long. Our goal, in addition to education and lifestyle change, should be reduction in mortality, morbidity, and deformity. The knowledge to achieve this goal is rapidly evolving. Cutaneous cancer intervention and therapy is ideally suited to showcase the opportunities for rational approaches to cancer control and cancer cure.

Figure 5.

Stage-specific biologic changes associated with multistage cutaneous carcinogenesis. This scheme is proposed to focus on processes that may be common for multistage tumor development where precise biochemical pathways are still under study. Early events reflect changes occuring in incipient tumor cells whereas progression incorporates processes at the tissue and organismal level that must be addressed to fully comprehend cutaneous cancer pathogenesis.

Full figure and legend (21K)

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Acknowledgements

Due to the breadth of this review, we apologize for the unavoidable exclusion of references to work done by many outstanding investigators working in these areas. We wish to acknowledge the contribution of Dr. Luowei Li to the construction of the figures and Ms. Bettie Sugar for outstanding editorial assistance.