Pathobiology of actinic keratosis: Ultraviolet-dependent keratinocyte proliferation

Pathobiology of actinic keratosis: Ultraviolet-dependent keratinocyte proliferation Brian Berman, MD, PhD,a and Clay J. Cockerell, MDb,c Miami, Florida, and Dallas, Texas Actinic keratoses are proliferations of transformed neoplastic keratinocytes in the epidermis that are the result of cumulative ultraviolet (UV) radiation from sun exposure. They are commonly found on sites of sun-exposed skin such as the face, balding scalp, and back of the hand. Although UV exposure does exert certain beneficial effects on the skin, excessive exposure to UV radiation induces multiple cascades of molecular signaling events at the cellular level that produce inflammation, immunosuppression, failure of apoptosis, and aberrant differentiation. Cumulatively, these actions result in mutagenesis and, ultimately, carcinogenesis. This article provides a brief overview of the key mediators that are implicated in the pathobiology of actinic keratosis. Three evolutionary possibilities exist for these keratoses in the absence of treatment: (1) spontaneous remission, which can be common; (2) remaining stable, without further progression; or (3) transformation to invasive squamous cell carcinoma, which may metastasize. Because the effects of UV radiation on the skin are complex, it is not yet fully clear how all of the mediators of actinic keratosis progression are interrelated. Nonetheless, some represent potential therapeutic targets, because it is clear that directing therapy to the effects of UV radiation at a number of different levels could interrupt and possibly reverse the mechanisms leading to malignant transformation. ( J Am Acad Dermatol 2013;68:S10-9.) Key words: actinic keratosis; apoptosis; immunosuppression; keratinocyte carcinogenesis; p53.

A

ctinic keratosis (AK) is a common skin lesion of disordered keratinocyte proliferation in the disease continuum of photodamaged skin that may lead to invasive squamous cell carcinoma (SCC). The most important risk factor for AK is cumulative ultraviolet (UV) radiation exposure, and therefore AKs are typically found on sun-exposed skin, such as the face, balding scalp, and back of the hand, especially in fair-skinned individuals of advanced age.1 UV radiation is a complete carcinogen in that it both induces the initial genetic mutations in keratinocytes and promotes tumor cell expansion. UVexposure induces protective responses in the cell, but when the burden of exposure and damage becomes excessive, the cell may undergo apoptosis to eliminate mutant cells from the epidermis. When the apoptotic systems fail, the collective genomic, inflammatory, and immunosuppressive disruptions

From the Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicinea; Cockerell and Associates/Dermpath Diagnostics Dermatopathology Laboratories, Dallasb; and Cockerell Dermatopathology Consultants PA, Dallas.c Publication of this article was supported by a grant from LEO Pharma Inc., Parsippany, NJ. Disclosure: Dr Berman is on the advisory boards of Graceway Pharmaceuticals, LEO Pharma Inc, Medicis, and PharmaDerm; a

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Abbreviations used: AK: BRG1: BRM: COX: EGFR: Foxp3: HPV: IL: MIF: MMP: PAF: PKC: ROS: SCC: TLR: Tregs: TSG: UCA: UV:

actinic keratosis brahma-related gene 1 brahma cyclo-oxygenase epidermal growth factor receptor forkhead box p3 human papillomavirus interleukin macrophage migration inhibitory factor matrix metalloproteinase platelet-activating factor protein kinase C reactive oxygen species squamous cell carcinoma toll-like receptor regulatory T cells tumor suppressor gene urocanic acid ultraviolet

consultant for Graceway Pharmaceuticals, Medicis, and LEO Pharma Inc; and a speaker for LEO Pharma Inc, Medicis, and PharmaDerm. Dr Cockerell is a consultant for LEO Pharma Inc. Reprint requests: Panagiotis Zografos, LEO Pharma Inc., 1 Sylvan Way, Parsippany, NJ 07054. E-mail: [email protected]. 0190-9622/$36.00 Ó 2012 by the American Academy of Dermatology, Inc. http://dx.doi.org/10.1016/j.jaad.2012.09.053

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result in aberrant proliferation, leading to the appearance of AKs, with the associated risk of progression to invasive SCC. AKs are genetically and biochemically diverse, demonstrating that this aberrant proliferative lesion can arise by disruption of a variety of pathways. We will describe several cellular pathways that are affected by UV radiation and how they may contribute to keratinocyte transformation.

is the site of dividing cells and is therefore more likely to give rise to a cancer cell after mutagenesis than are the nondividing cells of the upper epidermis.

ALTERED SIGNAL TRANSDUCTION

Excessive skin exposure to UV radiation induces a wide range of adverse consequences that include DNA damage, inflammation, and immunosuppression, CAPSULE SUMMARY leading to premature photoMECHANISMS OF AK aging and carcinogenesis.4 Excessive exposure to ultraviolet PATHOGENESIS UV exposure induces phosradiation results in DNA damage and UV radiation causes major phorylation of membrane tyalterations in immune responses that alterations in the pathways rosine kinases, alterations to lead to premature photoaging and that regulate cell growth epidermal growth factor recarcinogenesis of the skin. and differentiation, inflamceptors (EGFRs),43 activation Absorption of both ultraviolet A and B mation, and immunosupof Ras and Raf,5 and dissociradiation produces disruptions in pression, which are involved ation and activation of nuintracellular signaling, cytokine in the development of AK clear factor kB from the regulation, and protective apoptotic (Fig 1). Table I provides a inhibitor B complex.6,7,44 mechanisms, which together result in brief summary of each casThese events precipitate cyactinic keratosis. cade, key targets, and actokine production, including tions.2-41 production of interleukin Understanding the mediators of aberrant (IL)-1, tumor necrosis factor, keratinocyte proliferation will help to and IL-6. These signal casEFFECT OF UV identify opportunities to treat actinic cades also lead to the activaRADIATION ON DNA keratosis. tion of the arachidonic acid Despite the beneficial cascade, which stimulates effects of UV exposure on oxidative and phosphorylative reactions that induce stimulating cutaneous synthesis of vitamin D, UV signal transduction and initiate the release of histaradiation is probably the most frequently encounmine secondary to mast cell degranulation and tered carcinogen that human beings experience. UV release from keratinocytes.8,9 These pathways cause radiation consists of 3 spectral regions based translocation of transcription factors into the nuon wavelength and biologic effects. The majority cleus, where they lead to changes in gene (94%-97%) of UV exposure is UVA of 320- to 400-nm expression.45 wavelength. The ozone layer filters much of UVB light (290-320 nm), such that it makes up only 3% to INFLAMMATORY EFFECTS 6% of exposure. Very little UVC radiation (200-290 Absorbed light induces photochemical changes in nm) reaches the earth’s surface, as a result of atmochromophores and is the source of the initial UV spheric absorption.42 The higher-energy UVB has insult, resulting in erythema and inflammation. long been recognized as a potent carcinogen Because different chromophores absorb different because of its direct mutagenic effects on DNA, wavelengths, the inflammation may be variable. including the signature cyclobutane pyrimidine Shorter wavelengths of light induce greater effects dimers and 6e4 photoproducts that result in the on nucleic acids, amino acids, urocanic acid (UCA), characteristic transitions C/T and CC/TT.2 and melanin, whereas longer wavelengths of light The more abundant UVA penetrates the skin more exert their effects through melanin.10 UV light podeeply, produces reactive oxygen species (ROS), and tentiates the production of metabolites of arachicauses oxidative damage in nucleic acids, membrane donic acid, proinflammatory cytokines, adhesion lipids, and proteins.42 Oxidative damage disrupts molecules, and mast cellederived mediators. The normal signal transduction pathways and cellular ROS that result from UV exposure induce lipid interactions, leading to abnormal proliferation. The peroxidation and cellular destruction.4,10 mutagenic effects of UVA are generally regarded to originate from oxidative damage to DNA and formaCyclo-oxygenase tion of 8-hydroxyguanine adducts, to yield UVA The significance of the inflammatory response in signature T/G mutations, distinct from UVBthe progression of AK is based on the observation that, induced mutations.3 The basal layer of the epidermis d

d

d

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Fig 1. Mechanisms by which ultraviolet (UV) radiation causes actinic keratosis. UV radiation is absorbed by DNA, membrane phospholipids, and trans-urocanic acid (UCA). Generation of DNA photoproducts leads to mutations in p53 and additional tumor suppressor genes (TSGs), production of membrane lipid-derived mediators such as arachidonic acid and plateletactivating factor (PAF ), and cis-UCA; these effectors alter intracellular signaling. Dysregulation of cytokine milieu leads to inflammation, changes in T-cell homeostasis, and immunosuppression. Loss of p53-induced protective mechanisms results in accumulation of additional mutations and chromosome instability, culminating in abnormal keratinocyte proliferation. ROS, Reactive oxygen species.

before AK progresses to SCC, there is a period during which the lesions may become tender and inflamed.46 Cyclo-oxygenases (COXs) are rate-limiting enzymes in arachidonic acid metabolism and prostaglandin production. COX-2 is the primary UV-responsive COX isoform in human skin, and it plays a key role in UV-induced skin inflammation, immunosuppression, and apoptosis. As a result, pharmacologic inhibition of this pathway is one mechanism that has been used in the treatment of AK.47 Fas (CD95) A stepwise differentiation appears to signal the progression from asymptomatic AK, to inflamed AK, to SCC. The intense inflammatory response includes a significant increase in T lymphocytes and Langerhans cells. The number of infiltrating cells has been reported to diminish with advancement of AK to SCC, and the decrease accompanies an increase in immunoreactive p53 and the apoptosis inhibitor bcl-2. During this process, a decrease has been observed for Fas (CD95) and Fas ligand (members of the tumor necrosis family of proteins), which are critical in the role of initiating apoptosis.11 Fas may be one of the most important apoptotic defense mechanisms in the protection against UV-induced carcinogenesis, as it eliminates DNA-damaged cells that have the potential to undergo transformation. Keratinocytes in normal-appearing skin expressed CD95 in cytoplasmic membranes and intercellular bridges in the basal layer. In chronically

sun-exposed keratinocytes, CD95 expression is upregulated and observed throughout the entire thickness of the epidermis. However, in AK, a complete absence of Fas has been observed in the majority of cases.12 Although Fas is up-regulated early in chronically sun-exposed keratinocytes, further UVexposure results in a rapid down-regulation of this important mechanism, and the degree of attenuation appears to be proportional to the degree of dysplasia.12-14 Macrophage migration inhibitory factor Growing evidence suggests that the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is an important link between chronic inflammation and the development of nonmelanoma skin cancer. Mice lacking macrophage migration inhibiting factor (MIF / ), as compared with wild-type mice, have a diminished acute inflammatory response to UVB exposure, increased p53 activity, and a significantly reduced tumor burden in response to chronic exposure.15 In contrast, transgenic mice that overexpress MIF display reduced expression of the apoptosis regulatory genes p53 and p21, fewer apoptotic epidermal cells in response to acute UVB exposure, and both an earlier onset and higher numbers of skin tumors compared with wild-type mice.16 Immunohistochemical analyses of MIF in human skin samples demonstrated a progressive increase in expression of MIF from normal-appearing skin, to AK, to SCC. A similar progressive increase in CD74, an

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Table I. Mechanisms and mediators in actinic keratosis Mechanism

Inflammation

Mediators d

d

d d d

Oxidative stress

d

Immunosuppression

d d d d d

Effects

Arachidonic acid4,10 - COX-2 - Prostaglandins

d d d

Adhesion molecules, proinflammatory cytokines4,10 Mast cells4,10 ROS4,10 MIF15-17 ROS DNA damage18,19 Foxp31 Tregs20,21 TLR22 HPV41 PAF and UCA23

d

Links between chronic inflammation and tumor progression via MIF15-17

d

mtDNA and genomic DNA damage, lipid peroxidation3,10,24,25 Local and systemic immune suppression18,19 Inhibition of effector genes of immune surveillance20 Potentiation of IL-10, TGF-b21 Inhibition of CD4 T-cell activation and proliferation21 Cutaneous immune defense22 Down-regulation of IL-8, leading to weakened response to UV radiation41 Early mediation of immune suppression23 Via suppression, elimination of activation of apoptotic mediators CD95 and TRAIL26 Via mutation of gene, elimination of proapoptotic tumor suppression28-30 Regulation of apoptotic activity of p5329 Inhibition of apoptosis39,40 Amplification/overexpression33 Disruptions implicated in codon encoding of TSGs27 Accumulation of additional genomic mutations, loss of transcriptional signaling2 Abnormal expression36 Via ROS, phosphorylation/inactivation, dysregulation of apoptosis via p38, ERK, AKT31 Suppressed interferon signaling32 Elevated expression in cancer37 Ras, Raf activation5,7 Dissociation of NF-kB-IB6 Cytokine production (IL-1, IL-6), TNF, IL, GM-CSF8,9 Activation of arachidonic acid pathways, inducing mast cell degranulation with release of histamine8 Degradation of basement membrane34 Tumor invasiveness34,35

d d

d d

d d

d

Apoptosis perturbations

d d d

26

p53 PKC-d28-30 HPV39,40

d

d

d d

Mutagenesis

d d d

Cellular growth and differentiation

d d d d

MYC proto-oncogene33 TSGs: p14ARF, p16INK14a, p15Ink4b27 p532 E-cadherin/catenin36 EGFR31 Interferon32 Osteopontin37

d d d

d d

d d

Tissue remodeling

d d

Cyclobutane pyrimidine dimers MMPs34,35

Lipid peroxidation4,10 Increase in T lymphocytes, Langerhans cells11 Increase in p53 and bcl-211 - Decrease in Fas (CD95), Fas-ligand11-14

d d d d

d d

AKT, A protein kinase; BCL, apoptosis regulatory protein; COX, cyclo-oxygenase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinases; Foxp3, forkhead box p3; GM-CSF, granulocyte-macrophage colony-stimulating factor; HPV, human papillomavirus; IL, interleukin; MIF, macrophage migration inhibitory factor; MMPs, matrix metalloproteinases; mtDNA, mitochondrial DNA; NF-kB-IB, nuclear factor kB-inhibitor IB; PAF, platelet-activating factor; PKC, protein kinase C; RAS and RAF, members of the mitogen-activated protein kinase pathway; ROS, reactive oxygen species; TGF, transforming growth factor; TLR, toll-like receptor; TNF, tumor necrosis factor; TRAIL, tumor necrosis factorerelated apoptosis-inducing ligand; Tregs, regulatory T cells; TSG, tumor suppressor gene; UCA, urocanic acid; UV, ultraviolet.

MIF receptor, was also observed. A low dose of UVB stimulated MIF production from normal and immortalized human keratinocytes in culture and from SCCderived cell lines.17 These data suggest a role for MIF, not only in a murine model of UV-induced SCC but also in human keratinocyte carcinogenesis.

UV-INDUCED IMMUNOSUPPRESSION A growing body of evidence suggests that oxidative stress is implicated in photocarcinogenesis. This is supported by the fact that ROS participate in a number of pathologic processes, including DNA damage and lipid oxidation, that are considered to

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be major factors in tumor progression in AK.10 UV-induced immunosuppression occurs as a result of DNA damage, altered cytokine release, and effects on antigen-presenting cells and immunosuppressive T cells, which together lead to diminished immune surveillance and the growth of tumor cells.18,19 Forkhead box p31 regulatory T cells Regulatory T cells (Tregs) expressing the marker forkhead box p3 (Foxp3) possess immunosuppressive properties that directly inhibit effector cells from acting against tumor cells; increased prevalence of Tregs is associated with progression of AK to SCC.20 In addition, Foxp31 Tregs induce immunosuppression by secretion of IL-10 and transforming growth factor-b, inhibition of CD4 T-cell activation and proliferation, and inhibition of dendritic-cell activation and cytokine production. Foxp31 Tregs also infiltrate SCC and other tumors, suppress immune responses, and enhance tumor tolerance, actions that result in tumor persistence with an accumulation of further proliferative modifications.20 Foxp31 Tregs can be suppressed by treatment with imiquimod, a toll-like receptor (TLR) 7/8 agonist.21,22 Toll-like receptors TLRs play a central role in the cutaneous immune defense system. A variety of TLRs have been described in major cell populations such as keratinocytes, fibroblasts, antigen-presenting cells, and melanocytes. The activation of TLRs leads to the production of inflammatory stimuli through different intracellular signaling pathways. This activation initiates chemosignals that transform skin into a functional state of defense. TLRs also play a role in tissue homeostasis and renewal.22 Activation of TLRs invokes the production of interferon-alfa and other cytokines, which also promotes antigen-specific T-helper type 1 cell-mediated immune response.22,48 Platelet-activating factor and UCA Two very early events in UV-induced immunosuppression are secretion of platelet-activating factor (PAF) and isomerization of the photoreceptor transUCA to the immune-suppressive cis-UCA. PAF, a phospholipid derived from arachidonic acid, is produced during UV-induced oxidative stress. Treatment of keratinocyte cultures with a metabolically stable form of PAF or UV radiation increased transcription of COX-2 and IL-10 messenger RNA, whereas pretreatment of cultures with a PAF antagonist blocked UV-induced increases in transcription.49 This indicated that UV-induced activation of cytokine transcripts was mediated by PAF through the PAF receptor. Treatment of UV-irradiated mice

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with PAF antagonists blocked immunosuppressive responses.49 cis-UCA induces immunosuppression by binding to the serotonin (5-hydroxytryptamine) 2A receptor (5-HT2A).50 Treatment of mice with PAF or 5-HT2A receptor antagonists blocked skin cancer induction and its progression.23 In addition to blocking immunosuppression, PAF or 5-HT2A receptor antagonists modulate DNA repair, accelerating nucleotide excision repair and reducing the formation of 8-oxo-deoxyguanosine, as shown both in vivo and in vitro.51 Given that both PAF and cis-UCA increase ROS, an action that can be blocked by their respective antagonists, ROS may link genetic damage, DNA repair, and immunosuppression driven by PAF and cis-UCA.51 Mitochondrial DNA Mitochondrial DNA is a sensitive indicator of UV exposure. Mitochondria are susceptible to ROS damage because of lack of both protective histones and extensive DNA repair mechanisms. The mitochondrial environment also is a source of ROS production from incomplete respiration.24 A comparison of mitochondrial chromosome aberrations in usually sun-exposed versus occasionally sun-exposed body sites found a greater frequency of both a common mitochondrial DNA deletion (P \.0001) and tandem duplications (P = .058) with greater sun exposure.52 A separate study demonstrated that the actual levels of a specific 3895ebase pair mitochondrial DNA deletion were significantly higher in dermis samples from skin that was usually exposed to the sun compared with samples from skin that was only occasionally exposed (P \.0009).25 This damage to the mitochondrial genome may impair the capacity for mitochondrial oxidative phosphorylation, which may increase ROS.25

P53 AND APOPTOSIS Expression and activation of p53 in the epidermis occurs in response to UV radiation. P53 acts to arrest cell cycling and activate DNA repair systems in damaged cells. In the event of irreparable damage, the p53 transcription factor activates apoptosis, a pathway of programmed cell death.26 The role of the p53 tumor suppressor gene (TSG) in UV-induced skin carcinogenesis has been well established by the identification of frequent p53 mutations in SCC, AK, and perilesional sun-exposed skin.53-55 UV exposure induces expression of genes required for cell cycle arrest. Disruption of the TSG cluster p14ARF, p15INK4b, and p16INK4a at chromosome 9p21 contributes to carcinogenesis.27 Reports suggest that an increase in immunohistochemical expression of p16 is a marker of progression to

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SCC.56,57 Moreover, a significant percentage of adjacent sun-exposed, morphologically normal tissues near AK and SCC lesions showed similarly altered expression of p14ARF, p15INK4b, p16INK4a, and p53 transcripts, which is consistent with the field cancerization effects caused by UV radiation.27 Apoptosis is designed to eliminate continued replication of premalignant mutant cells. In keratinocytes, both mitochondrial and death receptore mediated apoptotic pathways are activated by p53.26 The p53 gene is a key positive regulator for the development of ‘‘sunburn cells,’’ or apoptotic keratinocytes.2 P53 drives expression of the members of the Bcl-2 family of proteins that reside in the mitochondrial membrane, leading to loss of cytochrome C and activation of proteolytic caspases.58 The p53 gene product also activates the death-receptor proteins Fas (CD95) and tumor necrosis factorerelated apoptosis-inducing ligand, and the proapoptotic Bcl-2 proteins.26 p53 mutations Mutations in p53 are an early step in skin tumorigenesis. When p53 acquires a UV-induced mutation, it loses its ability to regulate transcription of the protective mechanisms, and mutant cells proliferate. In the absence of DNA repair, cells accumulate additional mutations in TSGs and oncogenes, which lead to altered expression of additional proteins that stimulate cell proliferation. Loss of p53 function also leads to chromosome translocations and deletions, yielding cells with complex karyotypes characteristic of SCC with the capacity for invasive growth.59 Mutations in p53 are the most common genetic abnormality found in human cancers. The prevalence is over 80% in some histologic subtypes.60 P53 mutations have been identified in greater than 50% of premalignant AK lesions.53,54 The majority of these p53 mutations are characteristic of UVB mutations in p53 hot spots frequently correlated with UV exposure. The fact that these mutations are not present in the mutated p53 gene associated with internal cancers suggests that UV-induced p53 mutations are a significant factor in the progression of precancerous AK lesions to SCC.2,53,54 A study in mice using continued and discontinued regimens of chronic UVB treatment resulted in skin tumor development with 100% incidence.61 The process was delayed, but not averted, with chronic, discontinuous UVB exposure, suggesting that further avoidance of UVB merely delays the kinetics of tumor development.61 This emphasizes the critical need for treatment of AK in addition to sun avoidance. Thus, it appears that the p53 TSG protects against SCC. This concept is supported by evidence demonstrating that tumors emanating from p53

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mutations are predominately SCC and associated with premalignant lesions resembling AK.62 These mutations can be detected months before the gross appearance of skin tumors, which suggests that p53 mutations can serve as a surrogate early end point in skin cancer prevention studies.26

ADDITIONAL GENE PRODUCTS IMPLICATED IN KERATINOCYTE TRANSFORMATION Many proteins have been evaluated for evidence of altered expression in the progression of keratinocyte transformation. Several proteins that are implicated in AK and its progression are discussed below. Protein kinase C Protein kinase C (PKC) is a serine and threonine kinase family that consists of multifunctional isoforms that are key components in signal transduction pathways regulating development, proliferation, and apoptosis. PKC-d has been implicated as a tumor suppressor for SCC formation, as its expression is significantly reduced or absent in tumor tissue, and ectopic expression inhibits tumor growth by inducing apoptosis.28,29 Since the identification that PKC-d is a substrate for caspase-3, the literature has linked PKC-d to proapoptotic signaling. However, although PKC-d functions as a proapoptotic protein during DNA damageeinduced apoptosis, it also acts as an antiapoptotic protein during receptor-initiated cell death in some cell types.30 Nevertheless, the proapoptotic activity of PKC-d has made it an attractive target for antiproliferative agents in the treatment of malignancy. Epidermal growth factor receptor EGFR is activated in response to UV. UV-induced ROS lead to oxidative inhibition of receptor-type protein phosphatase, a negative regulator of EGFR, which keeps EGFR in its unphosphorylated, inactive state. Activated EGFR mediates pathways that enhance keratinocyte proliferation, and it suppresses apoptosis by suppression of p38, extracellular signalregulated kinases, and the kinases JNK and AKT.4,31 In a study of skin biopsy specimens from patients with AK or SCC, copy number aberrations in the EGFR gene and altered protein expression were detected in 52% of AKs and 77.1% of SCCs, thus supporting the common origin of AK and SCC.63 Interferons Interferons are cytokines that regulate proliferation, differentiation, and immune function via modulation of transcription factors, including interferon-stimulated gene factor-3 proteins.

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Suppression of type I interferon signaling proteins may occur early in the UV-damage continuum and has been implicated in skin malignancy. Significant reduction in 1 or more interferon-stimulated gene factor-3 proteins has been reported in 76% of patients with AK and 67% of patients with SCC.32 MYC In a study of MYC genomic aberrations, 35% of the AK and 63% of the SCC samples demonstrated MYC gene numerical aberrations, suggesting that there is a common origin of these lesions. As these aberrations were more frequent in poorly differentiated SCCs compared with well-differentiated SCCs, this oncogene may have a role in conferring a more aggressive tumor phenotype.33 Granulocyte colony-stimulating factor Granulocyte colony-stimulating factor receptors have been observed on the surface of several species of cancer cells. In AK, the percentage of cells positive for granulocyte colony-stimulating factor receptors has been reported as 49%, compared with 30% for normal-appearing skin, 51% for Bowen disease, and 78% for SCC. A significant, positive correlation has been noted between increased expression of granulocyte colony-stimulating factor and its receptor in skin tumors, indicating that they may form an autocrine loop for the proliferation of skin tumors.64 Matrix metalloproteinases, cadherin, and catenin Tumor cells must penetrate the basement membrane and span the extracellular matrix to invade surrounding structures and metastasize. Matrix metalloproteinases (MMPs) comprise a family of enzymes that are involved in the degradation of extracellular matrix. MMP-2, in particular, demonstrates degradative activity against the basement membrane and has been hypothesized to play a role in the progression of SCC.34 The overexpression of MMP-1, MMP-2, and MMP-3 has been correlated with tumor invasiveness and metastasis in a variety of cancer types.34,35 Ultimately, MMP expression may precede the development of SCC. AKs that demonstrate elevated MMP-1 messenger RNA may be in a more advanced dysplastic state, suggesting a greater likelihood of progression to SCC.34,35 Complexes of transmembrane E-cadherin protein and cytoplasmic catenin proteins have a critical role in the control of epithelial differentiation as a mediator of calcium-dependent cell adhesion. The probability of abnormal expression of this complex increases with the progression from benign tissue to premalignant lesions and to malignant nonmelanocytic skin

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tumors. Abnormal expression has been observed in SCC, Bowen disease, and intraepithelial dysplastic lesions such as AK, which suggests that this complex may be an early indicator of the neoplastic process.36 Osteopontin Osteopontin is a highly inducible adhesive matricellular glycoprotein secreted by tumor cells and stromal cells that may alter the cells’ microenvironment. Elevated expression has been observed in several human cancers and in the serum of patients with cancer. Immunohistochemical analysis demonstrated expression in 100% of SCC (n = 20 cases) and AK (n = 16 cases) samples, but it was absent or minimally expressed in solid basal cell carcinoma (n = 17).37 In addition, osteopontin functions as a proinflammatory cytokine through a diverse set of integrin receptors. Human papillomavirus Human papillomavirus (HPV) is regarded as a cocarcinogen in the development of AK. The E6 and E7 viral proteins from a broad spectrum of HPV types prevent apoptosis, independent of the status of p53.39,40 The apoptosis-resistant cells are vulnerable to the accumulation of additional UV-induced genetic damage, which contributes to unregulated cell proliferation. Further, in primary keratinocytes, the E6 protein of HPV5 and HPV8 can down-regulate the expression of IL-8, a prominently expressed cytokine induced upon UV exposure, which weakens the response to UV-induced DNA damage.41 Brahma and brahma-related gene 1 The preceding discussion has described some of the changes in gene expression that occur in the course of keratinocyte transformation. Loss of p53 function is generally recognized as the earliest and most commonly detected event. The relative importance of other genes, and the sequence of their activities in the carcinogenic process, is less clear. It is likely that carcinogenesis depends on the acquisition of changes in many genes over time. Two recent reports describing expression changes in Brahma (BRM) and BRM-related gene 1 (BRG1) have identified events that may occur late in the AK-to-SCC transformation process. BRM and BRG1 appear to be products of key TSGs that regulate the conversion of AK lesions to SCC. BRM and BRG1 are 2 alternative ATPase subunits of the SWI/SNF chromatin remodeling complex required for cellular differentiation and tumor suppression. The promoter of the oncogene c-myc is a direct target of the SWI/SNF (SWItch/Sucrose NonFermentable) complex.65,66 A genetic analysis

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of BRM identified a common point mutation in 1 of 10 SCCs and in 2 of 6 basal cell carcinoma lesions that was absent from AKs and normal-appearing skin samples, suggesting that BRM is involved in disease progression.67 A subsequent study reported that expression of BRM and BRG1 proteins was reduced approximately 10-fold in 100% of SCC (n = 11) and BCC (n = 5) specimens, but not in AK (n = 11) samples, which were similar to normal-appearing skin (n = 9).68 These data suggest that loss of BRM and BRG1 expression is a late and necessary event in the progression of nonmelanoma skin cancer.68

SUMMARY Environmental exposures to UVradiation initiate a multifactorial cascade of molecular, cellular, viral, immune, and genetic events that influence the progression of AK to malignancy. Although particular alterations in gene expression are strongly associated with invasive SCC, there is no mechanism to predict which AKs are at increased risk for malignant progression to invasive SCC. Certainly, it appears that discontinuation of sun exposure alone may be insufficient to avert the progression of AK to invasive SCC once these cascades are activated. Prompt and efficacious treatment of AK is essential to reduce disease progression. The application of molecular biologic techniques, in addition to studies in murine models of targeted gene ablation, has identified many genes that contribute to skin carcinogenesis. The resulting information suggests potential opportunities to test therapeutic approaches with novel mechanisms of action to inhibit and reverse the process of AK transformation. Editorial assistance was provided by Tanya MacNeil, PhD, of p-value communications. REFERENCES 1. Frost CA, Green AC. Epidemiology of solar keratoses. Br J Dermatol 1994;131:455-64. 2. Brash DE, Ziegler A, Jonason AS, Simon JA, Kunala S, Leffell DJ. Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion. J Investig Dermatol Symp Proc 1996;1:136-42. 3. Agar NS, Halliday GM, Barnetson RS, Ananthaswamy HN, Wheeler M, Jones AM. The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis. Proc Natl Acad Sci U S A 2004;101:4954-9. 4. Wang L, Eng W, Cockerell CJ. Effects of ultraviolet irradiation on inflammation in the skin. Adv Dermatol 2002;18:247-86. 5. Engelberg D, Klein C, Martinetto H, Struhl K, Karin M. The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals. Cell 1994;77:381-90. 6. Devary Y, Rosette C, DiDonato JA, Karin M. NF-kB activation by ultraviolet light not dependent on a nuclear signal. Science 1993;261:1442-5.

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