Melanoma
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《新英格兰医药杂志》
Although melanoma accounts for only 4 percent of all dermatologic cancers, it is responsible for 80 percent of deaths from skin cancer; only 14 percent of patients with metastatic melanoma survive for five years.1 The intractability of advanced melanoma shows how much we have to learn about the changes that facilitate the vertical growth and deep invasion of melanoma and about the mechanisms that block the effectiveness of chemotherapy.
The Clark model of the progression of melanoma emphasizes the stepwise transformation of melanocytes to melanoma (Figure 1). The model depicts the proliferation of melanocytes in the process of forming nevi and the subsequent development of dysplasia, hyperplasia, invasion, and metastasis.2 Numerous molecular events, many of them revealed by genomic3 and proteomic4 methods, have been associated with the development of melanoma. But rather than catalogue all the molecular lesions in this tumor, we will focus on connections between molecular pathways and risk factors for melanoma, the different steps of neoplastic transformation, and the patterns of molecular changes in melanoma (Figure 2).
Figure 1. The Clark Model (Hematoxylin and Eosin).
Melanocytes progress through a series of steps toward malignant transformation. The frequency of both the progression of nevi toward becoming malignant and the regression of nevi is unknown. The model emphasizes the histopathological changes that occur in the progression of melanoma. Normal melanocytes progressively develop a malignant phenotype through the acquisition of various phenotypic features. The particular histologic features characterizing each step of progression are the visible manifestations of underlying genetic changes.
Figure 2. Biologic Events and Molecular Changes in the Progression of Melanoma.
At the stage of the benign nevus, BRAF mutation and activation of the mitogen-activated protein kinase (MAPK) pathway occur. The cytologic atypia in dysplastic nevi reflect lesions within the cyclin-dependent kinase inhibitor 2A (CDKN2A) and phosphatase and tensin homologue (PTEN) pathways. Further progression of melanoma is associated with decreased differentiation and the decreased expression of melanoma markers regulated by microphthalmia-associated transcription factor (MITF). The vertical-growth phase and metastatic melanoma are notable for striking changes in the control of cell adhesion. Changes in the expression of the melanocyte-specific gene melastatin 1 (TRPM1) correlate with metastatic propensity, but the function of this gene remains unknown. Other changes include the loss of E-cadherin and increased expression of N-cadherin, V3 integrin, and matrix metalloproteinase 2 (MMP-2).
Environmental and Genetic Interactions
Risk Factors
The strongest risk factors for melanoma are a family history of melanoma, multiple benign or atypical nevi, and a previous melanoma. Immunosuppression, sun sensitivity, and exposure to ultraviolet radiation are additional risk factors. Each of these risk factors corresponds to a genetic predisposition or an environmental stressor that contributes to the genesis of melanoma. Each factor is understood to various degrees at a molecular level. For example, 25 to 40 percent of the members of melanoma-prone families have mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A)5 (Table 1 lists all genes mentioned in this article), and a few rare kindreds have mutations in cyclin-dependent kinase 4 (CDK4). There is a rational basis for a link between susceptibility to melanoma and a mutation in CDKN2A or CDK4 since both are tumor-suppressor genes. They will be discussed later in the context of disease progression. In addition, sensitivity to ultraviolet light is associated with a polymorphic genetic determinant that affects susceptibility to melanoma, thereby highlighting an important genetic–environmental interaction.
Table 1. Important Genes in Melanoma.
Photosensitivity, Tanning, and Melanoma
The effect of exposure to ultraviolet light is governed by variations in particular genes (polymorphisms) that affect both the defensive response of the skin to ultraviolet light and the risk of melanoma. Ultraviolet radiation causes genetic changes in the skin, impairs cutaneous immune function, increases the local production of growth factors, and induces the formation of DNA-damaging reactive oxygen species that affect keratinocytes and melanocytes.6,7 The tanning response is a defensive measure in which melanocytes synthesize melanin and transfer it to keratinocytes, where it absorbs and dissipates ultraviolet energy.7 Clinically, variations in pigmentation and the tanning response to ultraviolet light are associated with variations in susceptibility to melanoma.8,9
At the molecular level, exposure to ultraviolet light increases skin pigmentation, in part through the action of -melanocyte–stimulating hormone (-MSH) on its receptor, the melanocortin receptor 1 (MC1R) (Figure 3). Binding of the hormone to the receptor stimulates intracellular signaling in melanocytes, and this signaling increases the expression of enzymes involved in the production of melanin. Light-skinned and redheaded people often carry germ-line polymorphisms in the MC1R gene10,11 that reduce the activity of the receptor.12 Such polymorphisms increase the risk of melanoma considerably.13 In light-skinned people, therefore, the basis of increased susceptibility to melanoma is a genetic impairment in the production of melanin, the main defense of melanocytes against ultraviolet radiation.
Figure 3. Microphthalmia-Associated Transcription Factor (MITF) and -Catenin Pathways.
In the MITF pathway, MITF is regulated at both transcriptional and post-translational levels. The post-translational activation can occur through the ERK component of the MAPK pathway. The chief transcriptional pathways that are activated by extracellular signals are the melanocortin and WNT pathways. The melanocortin pathway regulates pigmentation through the MC1R. MC1R activates the cyclic AMP (cAMP) response-element binding protein (CREB). Increased expression of MITF and its activation by phosphorylation (P) stimulate the transcription of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome tautomerase (DCT), which produce melanin; melan-A, silver homologue, and melastatin 1 (TRPM1) are melanoma markers; inhibitor of kinase 4A (INK4A) leads to cell-cycle arrest, and BCL-2 suppresses apoptosis. In the -catenin pathway, -catenin plays a central role in cell adhesion and cell signaling. Signals from WNT ligands block the breakdown of -catenin. When WNT proteins bind the G-protein–coupled receptor (called frizzled), they inactivate the kinase GSK3, an enzyme that phosphorylates -catenin and targets it for destruction in the proteosome. Then -catenin accumulates in the cytoplasm and translocates to the nucleus, where it binds to LEF–TCF transcription factors and increases the expression of several genes, including MITF, the cell-cycle mediator cyclin D1 (CCND1), and matrix metalloproteinase 7 (MMP-7).
Although the tanning response to ultraviolet radiation appears dose-dependent, the nature of the exposure is also a factor. Melanoma occurs most frequently after intermittent exposure to the sun and in people with frequent sunburns. Epidemiologic observations suggest that chronic or low-grade exposures to ultraviolet light induce protection against DNA damage, whereas intense, intermittent exposures cause genetic damage.7
A Molecular Model of Melanoma Progression
The Clark model (Figure 1) describes the histologic changes that accompany the progression from normal melanocytes to malignant melanoma.2 We will relate these histologic changes to particular gene mutations (Table 1) in melanoma and discuss how these mutations affect molecular signaling to contribute to the progression from normal melanocytes to melanoma (Figure 2).
Hyperplasia and Nevus Formation
In the Clark model, the first phenotypic change in melanocytes is the development of benign nevi, which are composed of neval melanocytes (Figure 1). The control of growth in these cells is disrupted, yet the growth of a nevus is limited — a nevus rarely progresses to cancer.2 The absence of progression is probably due to oncogene-induced cell senescence, in which growth that is stimulated by the activation of oncogenic pathways is limited.14 At a molecular level, abnormal activation of the mitogen-activated protein kinase (MAPK) signaling pathway (also called extracellular-related kinase ) stimulates growth in melanoma cells (Figure 4A).15,16,17 Activation of this pathway is the result of somatic mutations of N-RAS, which are associated with about 15 percent of melanomas, or BRAF, which are associated with about 50 percent of melanomas. These mutations, which occur exclusively of each other, cause constitutive activation of the serine–threonine kinases in the ERK–MAPK pathway.18,19,20
Figure 4. MAPK and PTEN Pathways and the CDKN2A Tumor-Suppressor Locus.
Panel A shows the pathway associated with N-RAS, BRAF, and mitogen-activated protein kinase (MAPK). MAPKs are involved in signaling from numerous growth factors and cell-surface receptors. There are many variations in the components of particular cascades from various cell-surface receptors. Typically, adapter proteins (not shown) link the growth-factor receptor to RAS proteins, including N-RAS. When activated, RAS proteins phosphorylate (P) the mitogen-activated protein kinase (MEK) kinases, which then act on extracellular-related kinase (ERK) kinases. ERK kinases phosphorylate many targets in the cytoplasm and interact with other pathways, including phosphatidylinositol 3 kinase (PI3K) and MITF. ERK kinases translocate to the nucleus, where they activate transcription factors that promote cell-cycle progression and proliferation by increasing the transcription of many genes, including CD1. In survival signaling associated with phosphatase and tensin homologue (PTEN) and AKT, also known as protein kinase B, PTEN inhibits growth-factor signaling by inactivating phosphatidylinositol triphosphate (PIP3) generated by PI3K. A variety of growth factors (PDGF, NGF, and IGF-1) bind to their respective receptor tyrosine kinases and activate PI3K. The activated molecule converts the plasma membrane lipid phosphatidylinositol 4,5-bisphosphonate to PIP3. PIP3 acts as a second messenger, leading to the phosphorylation and activation of AKT. AKT is itself a kinase that phosphorylates protein substrates that affect the cell cycle, growth, and survival. Often, these AKT targets are inactivated by phosphorylation. PTEN attenuates this pathway through dephosphorylation and inactivation of PIP3, suppressing signaling from growth factors by blocking the activation of AKT. In Panel B, CDKN2A encodes two distinct tumor-suppressor genes; separate first exons that are spliced into alternate reading frames (ARF) of the second and third exons permit the expression of two different proteins from the same genetic locus. The gene has 4 exons. Transcription of messenger RNA (mRNA) can be initiated at either E1B or E1A, and the initiation site determines which gene the locus will express. RNA that is transcribed from either exon is spliced with the remaining two exons, E2 and E3, to produce mRNA for either INK4A or ARF. However, ARF uses a different reading frame of the exon 2 and 3 codons. In the cell-cycle progression involving INK4A, ARF, and retinoblastoma protein (Rb), a family of cyclins and cyclin-dependent kinases (CDKs) regulate progression through the cell cycle, and a family of CDK inhibitors opposes this action. In particular, the two phases of the G1–S checkpoint are governed primarily by cyclin D associated with cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) at its early phase and cyclin A or E associated with CDK2 at the later restriction phase. INK4A encodes a cyclin-dependent kinase inhibitor that inhibits CDK4 and CDK6. Ordinarily, these two kinases associate with D-type cyclins and drive the cell cycle by phosphorylating Rb, releasing it from its inhibitory interaction with the E2F transcription factor, thereby allowing the expression of E2F-related genes and progression from G1 to S. The absence of INK4A leads to unopposed CDK4 or 6 activity and increased cell-cycle activity. In response to DNA damage, mouse double minute 2 (MDM2) protein binds to the transcriptional activation domain of protein 53 (p53), blocking p53-mediated gene regulation while simultaneously leading to p53 ubiquination, nuclear export, and proteosomal degradation. ARF opposes this action by sequestering MDM2. This disruption of the MDM2–p53 interaction stabilizes p53 and increases p53 activity. Depending on other events, p53 either activates DNA repair and cell-cycle arrest or causes apoptosis and the formation of BCL-2–associated X protein (BAX). In the absence of ARF, p53 levels are decreased and the response to DNA damage is blunted.
BRAF mutations occur at a similar frequency in benign nevi and in primary and metastatic melanomas.21 Since most nevi cease proliferation and remain static for decades, these similar frequencies suggest that nevi must acquire additional molecular lesions to free themselves of growth restraints and become malignant. Experiments in model systems support this hypothesis. In zebrafish, melanocyte-specific expression of a mutant BRAF protein causes an ectopic proliferation of melanocytes, analogous to human nevi.22 In these fish, the combination of a BRAF mutation and inactivation of the tumor-suppressor gene p53 causes melanocytes to become malignant.22 In human melanocytes, mutant BRAF protein induces cell senescence by increasing the expression of the cell-cycle inhibitor of kinase 4A (INK4A).23 INK4A limits hyperplastic growth caused by a BRAF mutation. The arrest of the cell cycle caused by INK4A can, however, be overcome by mutations in INK4A itself, as well as other cell-cycle factors.
In vitro, depletion of BRAF and N-RAS from melanoma cells suppresses their growth.24,25,26 Small molecules that inhibit BRAF are being tested clinically (BAY 43-9006) but have had only limited success as single agents.27 In mice, the growth of melanomas with BRAF mutations can be suppressed by the inhibition of the downstream MEK enzymes, providing a possible target for treatment.28
Cytologic Atypia and Tumor-Suppressor Genes
The Clark model suggests that the next step toward melanoma is the development of cytologic atypia in dysplastic nevi, which may arise from preexisting benign nevi or as new lesions. The molecular abnormalities at this stage of progression affect cell growth, DNA repair, and the susceptibility to cell death. In 25 to 40 percent of cases of familial melanoma,6 a genetic defect inactivates CDKN2A, a single gene that encodes two tumor-suppressor proteins, p16INK4A and p19ARF29,30; in 25 to 50 percent of nonfamilial melanoma,31,32 a different tumor-suppressor gene, phosphatase and tensin homologue (PTEN) (Figure 3), is inactivated by mutation.33,34 In murine models of melanoma, mutation of either CDKN2A or PTEN alone fails to cause melanoma, but when combined with each other or with mutations in other genes,35 melanomas do arise. Mutation of CDKN2A or PTEN is only one molecular step on the path to the development of melanoma, but it is unclear precisely when such mutations occur. The increased susceptibility to melanoma that is associated with loss of the germ-line CDKN2A gene suggests that this genetic lesion increases the probability that dysplastic nevi will become malignant or increases the rate of the development of new melanoma without a precursor.
CDKN2A
The G1–S checkpoint that governs the commitment of a cell to DNA replication during the S phase (synthesis of DNA) is a site where many pathways that control cell division converge36,37 (Figure 4B). In some familial and sporadic cases of melanoma,36,37 the CDKN2A locus is lost by homozygous deletion of a portion of chromosome 9.36,37,38 One of the genes in this locus encodes INK4A, (p16INK4A), a protein that blocks the cell cycle at the G1–S checkpoint by inhibiting cyclin-dependent kinases. INK4A (an inhibitor of CDK4) suppresses the proliferation of cells with damaged DNA or activated oncogenes and also acts when cells are old or crowded.39 Mice lacking INK4A appear normal but are abnormally sensitive to carcinogens and prone to the development of tumors.40 The development of melanoma in such mice requires mutations in other genes, such as an activating mutation in H-RAS, an upstream component in MAPK signaling, which triggers MEK signaling.41 Genes that encode CDK4 and cyclin D1 (CCND1) encode proteins that act downstream of INK4A, and they are also mutated in some melanomas. These targets of INK4A function together as part of a complex that promotes the progression of the cell cycle by phosphorylating retinoblastoma (Rb) protein, a cell-cycle regulator. Rare melanoma kindreds carry germ-line mutations in CDK4 that disrupt cell-cycle control by preventing the molecular interaction that allows INK4A to repress CDK4.42 Mice that carry the human CDK4 mutation are prone to melanoma when exposed to various carcinogens.43
The D-type cyclin CD1 may have an oncogenic role in acral melanoma, in which amplification of the CCND1 gene and overexpression of cyclin CD1 protein occur more frequently than in melanoma at other sites.44 Inhibition of CCND1 (with antisense CCND1) causes apoptosis of human melanoma xenografts implanted in immunodeficient mice, without an apparent effect on normal melanocytes.
Alternative splicing of various exons within CDKN2A yields two distinct tumor-suppressor proteins, INK4A and alternate reading frame (ARF) (Figure 3).39 The ARF gene (also called p14ARF) derives its name from the use of an alternative reading frame of the exons it shares with INK4A. ARF functions as a tumor suppressor by arresting the cell cycle or promoting cell death after DNA damage or when various oncogenes or loss of Rb stimulate aberrant cell proliferation. ARF participates in the core regulatory process that controls levels of the p53 protein. It acts through the mouse double minute 2 (MDM2) protein, which triggers the ubiquination of p53, thereby instigating its destruction in the proteosome. ARF binds to MDM2, sequestering it from p53 and in this way causes p53 to accumulate; p53 then arrests the cell cycle at the G2–M site, allowing for repair of damaged DNA or the induction of apoptosis.45,46 In cells, ARF deficiency abrogates oncogene-induced senescence and increases susceptibility to transformation.47 In vitro, immortalization of cells often occurs with the loss of either ARF or p53.48 In animals, ARF deficiency shortens the time required for the development of melanoma after exposure to ultraviolet light; when both gene products of CDKN2A (INK4A and ARF) are deficient, the latent period is even shorter.49 These data suggest how ARF facilitates the progression of melanoma and indicate that the low frequency of p53 mutations in melanoma is partly related to loss of ARF, which renders the p53 pathway inactive.39
PTEN, AKT, and Cell Death
A second chromosomal region that is frequently affected by homozygous deletion in melanoma and other cancers is the PTEN locus on chromosome 10.33,34,50 PTEN encodes a phosphatase that attenuates signaling by a variety of growth factors that use phosphatidylinositol phosphate (PIP3) as an intracellular signal. In the presence of such growth factors, intracellular levels of PIP3 rapidly increase. This increase triggers the activation of protein kinase B (PKB, also called AKT) by phosphorylation (Figure 3). Activated AKT phosphorylates and inactivates proteins that suppress the cell cycle or stimulate apoptosis, thereby facilitating the proliferation and survival of cells. PTEN normally keeps PIP3 levels low; in its absence, levels of PIP3 and active (phosphorylated) AKT increase. Increased AKT activity prolongs cell survival through the inactivation of BCL-2 antagonist of cell death (BAD) protein and increases cell proliferation by increasing CCND1 expression, and affects many other cell-survival and cell-cycle genes through the activation of the forkhead (FKHR) transcription factor.32,51 AKT activity can also be increased in cells by mutations that cause the amplification and overexpression of the protein. Restoration of PTEN in cultured mouse melanocytes decreases the ability of the cells to form tumors.52 In model systems, suppression of AKT3, a member of the AKT family, reduces the survival of melanoma cells and the growth of human melanomas implanted in immunodeficient nude mice.53 As compared with normal melanocytes, increased levels of the active form of AKT were found in the radial-growth phase.53
MITF and Melanocyte Differentiation
Clark proposed that many nevi regress through differentiation and that the failure of differentiation is necessary for dysplasia.2 The normal process of melanocyte differentiation requires exit from the cell cycle and the expression of genes that encode proteins necessary for the production of pigment — two processes that are deregulated in melanoma. The microphthalmia-associated transcription factor (MITF) regulates the development and differentiation of melanocytes54 and maintains melanocyte progenitor cells in adults.55,56
MITF in Development
Mice lacking functional MITF are albino because they lack melanocytes, whereas those with partial MITF function have premature graying owing to the death of melanocytes. These experiments show that MITF is important in the differentiation and maintenance of melanocytes.57,58 MITF appears to contribute to melanocyte survival by increasing the expression of the BCL-2 gene, a key antiapoptotic factor.59 In mice, deficiencies of both MITF and BCL-2 cause gray hair due to a loss of differentiated melanocytes. The loss of melanocytes is due to the apoptosis of melanocyte progenitor cells in the hair follicle.55 In melanoma cell lines, a reduction in BCL-2 protein also causes cell death, suggesting that the survival of malignant melanocytes depends on BCL-2.60
MITF in Differentiation
MITF functions in a key pathway leading to melanocyte pigmentation (Figure 3). Intracellular signaling induced by -MSH acting on MC1R increases MITF expression, which in turn increases the transcription of genes underlying melanin synthesis: tyrosinase, tyrosinase-related-protein 1, and dopachrome tautomerase.61 MITF also regulates the transcription of the melanocyte-specific genes silver homologue (SILV)62,63 and melan-A (MLANA),62 whose immunohistochemical detection points to the diagnosis of melanoma. In addition, MITF causes cell-cycle arrest by the induction of INK4A.64
MITF in Melanoma
Decreased or absent pigmentation and decreased or absent expression of SILV and MLANA accompany the progression from nevus to melanoma. Tumors that are deficient in these proteins have a poor prognosis.65,66,67,68 Expression of the melastatin 1 (TRPM1) gene, whose function is unknown, is also controlled by MITF.69 Melanomas that are deficient in melastatin have a poor prognosis.70 The mechanism of decreased expression of these genes is a puzzle because MITF is present in nearly all melanomas.71,72,73
Although MITF causes differentiation and cell-cycle arrest in normal melanocytes, melanoma cells do not have these characteristics. Recently, a large-scale search for genomic changes in melanoma with the use of high-density single-nucleotide polymorphisms (SNPs) found an increased copy number (4 to 119 copies per cell) of a region of chromosome 3 that includes the MITF locus.74 This increase was accompanied by the increased expression of MITF protein. The overexpression of both MITF and BRAF could transform primary cultures of human melanocytes, implicating MITF as an oncogene. Notably, MITF amplification occurs most frequently in tumors that have a poor prognosis and is associated with resistance to chemotherapy.74 Interference with MITF function increased the chemosensitivity of a melanoma cell line, making MITF a potential target for treatment.
Cell Adhesion and Invasion
Local invasion and metastatic spread are responsible for the morbidity and mortality in melanoma. In the Clark model, invasive characteristics appear in the vertical-growth phase, when melanoma cells not only penetrate the basement membrane but also grow intradermally as an expanding nodule (Figure 2). Metastatic melanoma develops when tumor cells dissociate from the primary lesion, migrate through the surrounding stroma, and invade blood vessels and lymphatics to form a tumor at a distant site.75 Clinically, the absolute depth of local invasion, measured directly by histopathologic analysis (the Breslow index), is the principal prognostic factor and primary criterion in melanoma staging.76 Invasion and spread of melanoma are related to alterations in cell adhesion. Normally, cell adhesion controls cell migration, tissue organization, and organogenesis,77 but disturbances in cell adhesion contribute to tumor invasion, tumor–stroma interactions, and tumor-cell signaling.
Cadherins
Cadherins are multifunctional transmembrane proteins that sustain cell-to-cell contacts, form connections with the actin cytoskeleton, and influence intracellular signaling. The extracellular domain of cadherins binds to like cadherins on other cells in regions of cell contacts called adherens junctions. Cadherins are divided into three subtypes: E (epithelial), present in polarized epithelial cells in the epidermis, including melanocytes and keratinocytes; P (placental); and N (neural), found in mesenchymal cells in the dermis. The intracellular domain is associated with a large protein complex that includes -catenin and forms structural links with bundles of actin filaments.
Several signaling pathways cause -catenin to dissociate from the cell adhesion complex and transduce signals to the nucleus (Figure 3). One of these pathways is called the wingless-type mammary tumor virus integration-site family (WNT) pathway. WNTs are secreted proteins with important functions in development, especially in neural crest cells like melanocytes. When WNT proteins bind their receptors, they inactivate the kinase GSK3, an enzyme that phosphorylates -catenin and targets it for destruction in the proteosome.78,79 Tyrosine phosphorylation of -catenin disrupts the association between E-cadherin and -catenin,80 allowing -catenin to translocate to the nucleus, where it binds to lymphoid enhancer factor–T-cell factor (LEF–TCF). Mutations in the -catenin gene can stabilize the -catenin protein81 or increase its nuclear localization.82,83,84 Increased levels of nuclear -catenin increase the expression of MITF85 and CCND1,86 and these in turn increase the survival and proliferation of melanoma cells. Alterations in cadherin expression affect the interaction of melanoma cells with the environment and alter -catenin signaling. E-cadherin expression occurs in melanocytes and keratinocytes in the epidermis and causes melanocytes to associate with keratinocytes.87 In turn, contacts with undifferentiated keratinocytes from the basal-cell layer inhibit melanocyte proliferation, suppress the expression of melanoma markers, and cause melanocytes to become dendritic.88
Progression from the radial-growth phase to the vertical-growth phase of melanoma is marked by the loss of E-cadherin and the expression of N-cadherin89,90,91 (Figure 2). N-cadherin is a characteristic of invasive carcinomas and enables metastatic spread by permitting melanoma cells to interact with other N-cadherin–expressing cells, such as dermal fibroblasts and the vascular endothelium.87 Besides these changes in cell adhesion, decreased E-cadherin expression92 and aberrant N-cadherin expression increase the survival of melanoma cells by stimulating -catenin signaling.93,94
Integrins
The integrins mediate cell contacts with fibronectin, collagens, and laminin, components of the extracellular matrix.95 Transition from radial to vertical growth of melanoma is associated with the expression of V3 integrin.96 This integrin induces expression of matrix metalloproteinase 2, an enzyme that degrades the collagen in basement membrane.97,98,99 In addition, V3 integrin increases expression of the prosurvival gene BCL-2100 and stimulates the motility of melanoma cells through the reorganization of melanoma cytoskeleton.101 These observations form a rationale for the development of integrin antagonists to treat melanoma.102
Patterns of Genetic Alteration
The genetic changes in melanoma can be seen as particular combinations of molecular lesions that interrupt a precise set of pathways, each with a crucial role in the development of melanoma. The MEK pathway can be activated by a mutation in either NRAS or BRAF, and an NRAS mutation can activate both the MEK and PTEN pathways. Similarly, INK4A, CDK4, and CCND1 function in a unique pathway that affects the cell cycle; a mutation of INK4A has similar consequences as a mutation of CCND1 or CDK4.103,104,105
There are particular genetic changes in melanomas in different sites, consistent differences related to ultraviolet exposure on sites that are chronically exposed (head and neck) or intermittently exposed (chest and back) and in acral and mucosal skin. For example, CCND1 amplification occurs predominantly in acral regions,44 whereas activating mutations in BRAF occur most frequently in skin sites of intermittent sun exposure.106
Modeling Melanoma Progression
For many of the molecular lesions we have described, animal models have provided validation. A surprising new model is the zebrafish, in which premalignant and malignant lesions can be created by the expression of mutant BRAF with or without p53 mutation.22 This model is the only currently tractable system in which genetic screens can be performed for modifiers of melanoma.
Human melanomas that are grafted onto or injected into nude mice allow measures of the tumors' metastatic potential and have allowed for the testing of therapeutic interventions. Genetic manipulation of mice has validated the contribution of many genetic alterations in melanoma, but there are fundamental differences between mouse and human skin. Mouse melanocytes occur in hair follicles and the dermis, rather than in the epidermis, as in humans. To circumvent this problem, human melanocytes can be altered in cell culture and combined with keratinocytes to produce graft material. Using this system, the inactivation of p53 and the simultaneous introduction of activated N-RAS, CDK4, and telomerase led to darkly pigmented grafts that became grossly ulcerated and displayed histologic features of melanoma, including vertical invasion.107 This experimental system provides a novel model to test invasion and metastases of transformed human melanocytes in a host organism.
Supported by a grant (MCM202534) from the Cancer Research Institute of New York and a grant (T32-GM07753, to Dr. Miller) from the National Institute of General Medical Science. No other potential conflict of interest relevant to this article was reported.
We are indebted to Drs. David E. Fisher, Adriano Piris, Jennifer Y. Lin, and Jennifer C. Broder for their critical reading of the manuscript, and to Dr. Claudio Clemente for contributing images for Figure 1.
Source Information
From the Dermatopathology Unit, Massachusetts General Hospital, and Harvard Medical School — both in Boston.
Address reprint requests to Dr. Mihm at the Department of Dermatopathology, Massachusetts General Hospital, 55 Fruit St., Warren 827, Boston, MA 02114.
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The Clark model of the progression of melanoma emphasizes the stepwise transformation of melanocytes to melanoma (Figure 1). The model depicts the proliferation of melanocytes in the process of forming nevi and the subsequent development of dysplasia, hyperplasia, invasion, and metastasis.2 Numerous molecular events, many of them revealed by genomic3 and proteomic4 methods, have been associated with the development of melanoma. But rather than catalogue all the molecular lesions in this tumor, we will focus on connections between molecular pathways and risk factors for melanoma, the different steps of neoplastic transformation, and the patterns of molecular changes in melanoma (Figure 2).
Figure 1. The Clark Model (Hematoxylin and Eosin).
Melanocytes progress through a series of steps toward malignant transformation. The frequency of both the progression of nevi toward becoming malignant and the regression of nevi is unknown. The model emphasizes the histopathological changes that occur in the progression of melanoma. Normal melanocytes progressively develop a malignant phenotype through the acquisition of various phenotypic features. The particular histologic features characterizing each step of progression are the visible manifestations of underlying genetic changes.
Figure 2. Biologic Events and Molecular Changes in the Progression of Melanoma.
At the stage of the benign nevus, BRAF mutation and activation of the mitogen-activated protein kinase (MAPK) pathway occur. The cytologic atypia in dysplastic nevi reflect lesions within the cyclin-dependent kinase inhibitor 2A (CDKN2A) and phosphatase and tensin homologue (PTEN) pathways. Further progression of melanoma is associated with decreased differentiation and the decreased expression of melanoma markers regulated by microphthalmia-associated transcription factor (MITF). The vertical-growth phase and metastatic melanoma are notable for striking changes in the control of cell adhesion. Changes in the expression of the melanocyte-specific gene melastatin 1 (TRPM1) correlate with metastatic propensity, but the function of this gene remains unknown. Other changes include the loss of E-cadherin and increased expression of N-cadherin, V3 integrin, and matrix metalloproteinase 2 (MMP-2).
Environmental and Genetic Interactions
Risk Factors
The strongest risk factors for melanoma are a family history of melanoma, multiple benign or atypical nevi, and a previous melanoma. Immunosuppression, sun sensitivity, and exposure to ultraviolet radiation are additional risk factors. Each of these risk factors corresponds to a genetic predisposition or an environmental stressor that contributes to the genesis of melanoma. Each factor is understood to various degrees at a molecular level. For example, 25 to 40 percent of the members of melanoma-prone families have mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A)5 (Table 1 lists all genes mentioned in this article), and a few rare kindreds have mutations in cyclin-dependent kinase 4 (CDK4). There is a rational basis for a link between susceptibility to melanoma and a mutation in CDKN2A or CDK4 since both are tumor-suppressor genes. They will be discussed later in the context of disease progression. In addition, sensitivity to ultraviolet light is associated with a polymorphic genetic determinant that affects susceptibility to melanoma, thereby highlighting an important genetic–environmental interaction.
Table 1. Important Genes in Melanoma.
Photosensitivity, Tanning, and Melanoma
The effect of exposure to ultraviolet light is governed by variations in particular genes (polymorphisms) that affect both the defensive response of the skin to ultraviolet light and the risk of melanoma. Ultraviolet radiation causes genetic changes in the skin, impairs cutaneous immune function, increases the local production of growth factors, and induces the formation of DNA-damaging reactive oxygen species that affect keratinocytes and melanocytes.6,7 The tanning response is a defensive measure in which melanocytes synthesize melanin and transfer it to keratinocytes, where it absorbs and dissipates ultraviolet energy.7 Clinically, variations in pigmentation and the tanning response to ultraviolet light are associated with variations in susceptibility to melanoma.8,9
At the molecular level, exposure to ultraviolet light increases skin pigmentation, in part through the action of -melanocyte–stimulating hormone (-MSH) on its receptor, the melanocortin receptor 1 (MC1R) (Figure 3). Binding of the hormone to the receptor stimulates intracellular signaling in melanocytes, and this signaling increases the expression of enzymes involved in the production of melanin. Light-skinned and redheaded people often carry germ-line polymorphisms in the MC1R gene10,11 that reduce the activity of the receptor.12 Such polymorphisms increase the risk of melanoma considerably.13 In light-skinned people, therefore, the basis of increased susceptibility to melanoma is a genetic impairment in the production of melanin, the main defense of melanocytes against ultraviolet radiation.
Figure 3. Microphthalmia-Associated Transcription Factor (MITF) and -Catenin Pathways.
In the MITF pathway, MITF is regulated at both transcriptional and post-translational levels. The post-translational activation can occur through the ERK component of the MAPK pathway. The chief transcriptional pathways that are activated by extracellular signals are the melanocortin and WNT pathways. The melanocortin pathway regulates pigmentation through the MC1R. MC1R activates the cyclic AMP (cAMP) response-element binding protein (CREB). Increased expression of MITF and its activation by phosphorylation (P) stimulate the transcription of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome tautomerase (DCT), which produce melanin; melan-A, silver homologue, and melastatin 1 (TRPM1) are melanoma markers; inhibitor of kinase 4A (INK4A) leads to cell-cycle arrest, and BCL-2 suppresses apoptosis. In the -catenin pathway, -catenin plays a central role in cell adhesion and cell signaling. Signals from WNT ligands block the breakdown of -catenin. When WNT proteins bind the G-protein–coupled receptor (called frizzled), they inactivate the kinase GSK3, an enzyme that phosphorylates -catenin and targets it for destruction in the proteosome. Then -catenin accumulates in the cytoplasm and translocates to the nucleus, where it binds to LEF–TCF transcription factors and increases the expression of several genes, including MITF, the cell-cycle mediator cyclin D1 (CCND1), and matrix metalloproteinase 7 (MMP-7).
Although the tanning response to ultraviolet radiation appears dose-dependent, the nature of the exposure is also a factor. Melanoma occurs most frequently after intermittent exposure to the sun and in people with frequent sunburns. Epidemiologic observations suggest that chronic or low-grade exposures to ultraviolet light induce protection against DNA damage, whereas intense, intermittent exposures cause genetic damage.7
A Molecular Model of Melanoma Progression
The Clark model (Figure 1) describes the histologic changes that accompany the progression from normal melanocytes to malignant melanoma.2 We will relate these histologic changes to particular gene mutations (Table 1) in melanoma and discuss how these mutations affect molecular signaling to contribute to the progression from normal melanocytes to melanoma (Figure 2).
Hyperplasia and Nevus Formation
In the Clark model, the first phenotypic change in melanocytes is the development of benign nevi, which are composed of neval melanocytes (Figure 1). The control of growth in these cells is disrupted, yet the growth of a nevus is limited — a nevus rarely progresses to cancer.2 The absence of progression is probably due to oncogene-induced cell senescence, in which growth that is stimulated by the activation of oncogenic pathways is limited.14 At a molecular level, abnormal activation of the mitogen-activated protein kinase (MAPK) signaling pathway (also called extracellular-related kinase ) stimulates growth in melanoma cells (Figure 4A).15,16,17 Activation of this pathway is the result of somatic mutations of N-RAS, which are associated with about 15 percent of melanomas, or BRAF, which are associated with about 50 percent of melanomas. These mutations, which occur exclusively of each other, cause constitutive activation of the serine–threonine kinases in the ERK–MAPK pathway.18,19,20
Figure 4. MAPK and PTEN Pathways and the CDKN2A Tumor-Suppressor Locus.
Panel A shows the pathway associated with N-RAS, BRAF, and mitogen-activated protein kinase (MAPK). MAPKs are involved in signaling from numerous growth factors and cell-surface receptors. There are many variations in the components of particular cascades from various cell-surface receptors. Typically, adapter proteins (not shown) link the growth-factor receptor to RAS proteins, including N-RAS. When activated, RAS proteins phosphorylate (P) the mitogen-activated protein kinase (MEK) kinases, which then act on extracellular-related kinase (ERK) kinases. ERK kinases phosphorylate many targets in the cytoplasm and interact with other pathways, including phosphatidylinositol 3 kinase (PI3K) and MITF. ERK kinases translocate to the nucleus, where they activate transcription factors that promote cell-cycle progression and proliferation by increasing the transcription of many genes, including CD1. In survival signaling associated with phosphatase and tensin homologue (PTEN) and AKT, also known as protein kinase B, PTEN inhibits growth-factor signaling by inactivating phosphatidylinositol triphosphate (PIP3) generated by PI3K. A variety of growth factors (PDGF, NGF, and IGF-1) bind to their respective receptor tyrosine kinases and activate PI3K. The activated molecule converts the plasma membrane lipid phosphatidylinositol 4,5-bisphosphonate to PIP3. PIP3 acts as a second messenger, leading to the phosphorylation and activation of AKT. AKT is itself a kinase that phosphorylates protein substrates that affect the cell cycle, growth, and survival. Often, these AKT targets are inactivated by phosphorylation. PTEN attenuates this pathway through dephosphorylation and inactivation of PIP3, suppressing signaling from growth factors by blocking the activation of AKT. In Panel B, CDKN2A encodes two distinct tumor-suppressor genes; separate first exons that are spliced into alternate reading frames (ARF) of the second and third exons permit the expression of two different proteins from the same genetic locus. The gene has 4 exons. Transcription of messenger RNA (mRNA) can be initiated at either E1B or E1A, and the initiation site determines which gene the locus will express. RNA that is transcribed from either exon is spliced with the remaining two exons, E2 and E3, to produce mRNA for either INK4A or ARF. However, ARF uses a different reading frame of the exon 2 and 3 codons. In the cell-cycle progression involving INK4A, ARF, and retinoblastoma protein (Rb), a family of cyclins and cyclin-dependent kinases (CDKs) regulate progression through the cell cycle, and a family of CDK inhibitors opposes this action. In particular, the two phases of the G1–S checkpoint are governed primarily by cyclin D associated with cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) at its early phase and cyclin A or E associated with CDK2 at the later restriction phase. INK4A encodes a cyclin-dependent kinase inhibitor that inhibits CDK4 and CDK6. Ordinarily, these two kinases associate with D-type cyclins and drive the cell cycle by phosphorylating Rb, releasing it from its inhibitory interaction with the E2F transcription factor, thereby allowing the expression of E2F-related genes and progression from G1 to S. The absence of INK4A leads to unopposed CDK4 or 6 activity and increased cell-cycle activity. In response to DNA damage, mouse double minute 2 (MDM2) protein binds to the transcriptional activation domain of protein 53 (p53), blocking p53-mediated gene regulation while simultaneously leading to p53 ubiquination, nuclear export, and proteosomal degradation. ARF opposes this action by sequestering MDM2. This disruption of the MDM2–p53 interaction stabilizes p53 and increases p53 activity. Depending on other events, p53 either activates DNA repair and cell-cycle arrest or causes apoptosis and the formation of BCL-2–associated X protein (BAX). In the absence of ARF, p53 levels are decreased and the response to DNA damage is blunted.
BRAF mutations occur at a similar frequency in benign nevi and in primary and metastatic melanomas.21 Since most nevi cease proliferation and remain static for decades, these similar frequencies suggest that nevi must acquire additional molecular lesions to free themselves of growth restraints and become malignant. Experiments in model systems support this hypothesis. In zebrafish, melanocyte-specific expression of a mutant BRAF protein causes an ectopic proliferation of melanocytes, analogous to human nevi.22 In these fish, the combination of a BRAF mutation and inactivation of the tumor-suppressor gene p53 causes melanocytes to become malignant.22 In human melanocytes, mutant BRAF protein induces cell senescence by increasing the expression of the cell-cycle inhibitor of kinase 4A (INK4A).23 INK4A limits hyperplastic growth caused by a BRAF mutation. The arrest of the cell cycle caused by INK4A can, however, be overcome by mutations in INK4A itself, as well as other cell-cycle factors.
In vitro, depletion of BRAF and N-RAS from melanoma cells suppresses their growth.24,25,26 Small molecules that inhibit BRAF are being tested clinically (BAY 43-9006) but have had only limited success as single agents.27 In mice, the growth of melanomas with BRAF mutations can be suppressed by the inhibition of the downstream MEK enzymes, providing a possible target for treatment.28
Cytologic Atypia and Tumor-Suppressor Genes
The Clark model suggests that the next step toward melanoma is the development of cytologic atypia in dysplastic nevi, which may arise from preexisting benign nevi or as new lesions. The molecular abnormalities at this stage of progression affect cell growth, DNA repair, and the susceptibility to cell death. In 25 to 40 percent of cases of familial melanoma,6 a genetic defect inactivates CDKN2A, a single gene that encodes two tumor-suppressor proteins, p16INK4A and p19ARF29,30; in 25 to 50 percent of nonfamilial melanoma,31,32 a different tumor-suppressor gene, phosphatase and tensin homologue (PTEN) (Figure 3), is inactivated by mutation.33,34 In murine models of melanoma, mutation of either CDKN2A or PTEN alone fails to cause melanoma, but when combined with each other or with mutations in other genes,35 melanomas do arise. Mutation of CDKN2A or PTEN is only one molecular step on the path to the development of melanoma, but it is unclear precisely when such mutations occur. The increased susceptibility to melanoma that is associated with loss of the germ-line CDKN2A gene suggests that this genetic lesion increases the probability that dysplastic nevi will become malignant or increases the rate of the development of new melanoma without a precursor.
CDKN2A
The G1–S checkpoint that governs the commitment of a cell to DNA replication during the S phase (synthesis of DNA) is a site where many pathways that control cell division converge36,37 (Figure 4B). In some familial and sporadic cases of melanoma,36,37 the CDKN2A locus is lost by homozygous deletion of a portion of chromosome 9.36,37,38 One of the genes in this locus encodes INK4A, (p16INK4A), a protein that blocks the cell cycle at the G1–S checkpoint by inhibiting cyclin-dependent kinases. INK4A (an inhibitor of CDK4) suppresses the proliferation of cells with damaged DNA or activated oncogenes and also acts when cells are old or crowded.39 Mice lacking INK4A appear normal but are abnormally sensitive to carcinogens and prone to the development of tumors.40 The development of melanoma in such mice requires mutations in other genes, such as an activating mutation in H-RAS, an upstream component in MAPK signaling, which triggers MEK signaling.41 Genes that encode CDK4 and cyclin D1 (CCND1) encode proteins that act downstream of INK4A, and they are also mutated in some melanomas. These targets of INK4A function together as part of a complex that promotes the progression of the cell cycle by phosphorylating retinoblastoma (Rb) protein, a cell-cycle regulator. Rare melanoma kindreds carry germ-line mutations in CDK4 that disrupt cell-cycle control by preventing the molecular interaction that allows INK4A to repress CDK4.42 Mice that carry the human CDK4 mutation are prone to melanoma when exposed to various carcinogens.43
The D-type cyclin CD1 may have an oncogenic role in acral melanoma, in which amplification of the CCND1 gene and overexpression of cyclin CD1 protein occur more frequently than in melanoma at other sites.44 Inhibition of CCND1 (with antisense CCND1) causes apoptosis of human melanoma xenografts implanted in immunodeficient mice, without an apparent effect on normal melanocytes.
Alternative splicing of various exons within CDKN2A yields two distinct tumor-suppressor proteins, INK4A and alternate reading frame (ARF) (Figure 3).39 The ARF gene (also called p14ARF) derives its name from the use of an alternative reading frame of the exons it shares with INK4A. ARF functions as a tumor suppressor by arresting the cell cycle or promoting cell death after DNA damage or when various oncogenes or loss of Rb stimulate aberrant cell proliferation. ARF participates in the core regulatory process that controls levels of the p53 protein. It acts through the mouse double minute 2 (MDM2) protein, which triggers the ubiquination of p53, thereby instigating its destruction in the proteosome. ARF binds to MDM2, sequestering it from p53 and in this way causes p53 to accumulate; p53 then arrests the cell cycle at the G2–M site, allowing for repair of damaged DNA or the induction of apoptosis.45,46 In cells, ARF deficiency abrogates oncogene-induced senescence and increases susceptibility to transformation.47 In vitro, immortalization of cells often occurs with the loss of either ARF or p53.48 In animals, ARF deficiency shortens the time required for the development of melanoma after exposure to ultraviolet light; when both gene products of CDKN2A (INK4A and ARF) are deficient, the latent period is even shorter.49 These data suggest how ARF facilitates the progression of melanoma and indicate that the low frequency of p53 mutations in melanoma is partly related to loss of ARF, which renders the p53 pathway inactive.39
PTEN, AKT, and Cell Death
A second chromosomal region that is frequently affected by homozygous deletion in melanoma and other cancers is the PTEN locus on chromosome 10.33,34,50 PTEN encodes a phosphatase that attenuates signaling by a variety of growth factors that use phosphatidylinositol phosphate (PIP3) as an intracellular signal. In the presence of such growth factors, intracellular levels of PIP3 rapidly increase. This increase triggers the activation of protein kinase B (PKB, also called AKT) by phosphorylation (Figure 3). Activated AKT phosphorylates and inactivates proteins that suppress the cell cycle or stimulate apoptosis, thereby facilitating the proliferation and survival of cells. PTEN normally keeps PIP3 levels low; in its absence, levels of PIP3 and active (phosphorylated) AKT increase. Increased AKT activity prolongs cell survival through the inactivation of BCL-2 antagonist of cell death (BAD) protein and increases cell proliferation by increasing CCND1 expression, and affects many other cell-survival and cell-cycle genes through the activation of the forkhead (FKHR) transcription factor.32,51 AKT activity can also be increased in cells by mutations that cause the amplification and overexpression of the protein. Restoration of PTEN in cultured mouse melanocytes decreases the ability of the cells to form tumors.52 In model systems, suppression of AKT3, a member of the AKT family, reduces the survival of melanoma cells and the growth of human melanomas implanted in immunodeficient nude mice.53 As compared with normal melanocytes, increased levels of the active form of AKT were found in the radial-growth phase.53
MITF and Melanocyte Differentiation
Clark proposed that many nevi regress through differentiation and that the failure of differentiation is necessary for dysplasia.2 The normal process of melanocyte differentiation requires exit from the cell cycle and the expression of genes that encode proteins necessary for the production of pigment — two processes that are deregulated in melanoma. The microphthalmia-associated transcription factor (MITF) regulates the development and differentiation of melanocytes54 and maintains melanocyte progenitor cells in adults.55,56
MITF in Development
Mice lacking functional MITF are albino because they lack melanocytes, whereas those with partial MITF function have premature graying owing to the death of melanocytes. These experiments show that MITF is important in the differentiation and maintenance of melanocytes.57,58 MITF appears to contribute to melanocyte survival by increasing the expression of the BCL-2 gene, a key antiapoptotic factor.59 In mice, deficiencies of both MITF and BCL-2 cause gray hair due to a loss of differentiated melanocytes. The loss of melanocytes is due to the apoptosis of melanocyte progenitor cells in the hair follicle.55 In melanoma cell lines, a reduction in BCL-2 protein also causes cell death, suggesting that the survival of malignant melanocytes depends on BCL-2.60
MITF in Differentiation
MITF functions in a key pathway leading to melanocyte pigmentation (Figure 3). Intracellular signaling induced by -MSH acting on MC1R increases MITF expression, which in turn increases the transcription of genes underlying melanin synthesis: tyrosinase, tyrosinase-related-protein 1, and dopachrome tautomerase.61 MITF also regulates the transcription of the melanocyte-specific genes silver homologue (SILV)62,63 and melan-A (MLANA),62 whose immunohistochemical detection points to the diagnosis of melanoma. In addition, MITF causes cell-cycle arrest by the induction of INK4A.64
MITF in Melanoma
Decreased or absent pigmentation and decreased or absent expression of SILV and MLANA accompany the progression from nevus to melanoma. Tumors that are deficient in these proteins have a poor prognosis.65,66,67,68 Expression of the melastatin 1 (TRPM1) gene, whose function is unknown, is also controlled by MITF.69 Melanomas that are deficient in melastatin have a poor prognosis.70 The mechanism of decreased expression of these genes is a puzzle because MITF is present in nearly all melanomas.71,72,73
Although MITF causes differentiation and cell-cycle arrest in normal melanocytes, melanoma cells do not have these characteristics. Recently, a large-scale search for genomic changes in melanoma with the use of high-density single-nucleotide polymorphisms (SNPs) found an increased copy number (4 to 119 copies per cell) of a region of chromosome 3 that includes the MITF locus.74 This increase was accompanied by the increased expression of MITF protein. The overexpression of both MITF and BRAF could transform primary cultures of human melanocytes, implicating MITF as an oncogene. Notably, MITF amplification occurs most frequently in tumors that have a poor prognosis and is associated with resistance to chemotherapy.74 Interference with MITF function increased the chemosensitivity of a melanoma cell line, making MITF a potential target for treatment.
Cell Adhesion and Invasion
Local invasion and metastatic spread are responsible for the morbidity and mortality in melanoma. In the Clark model, invasive characteristics appear in the vertical-growth phase, when melanoma cells not only penetrate the basement membrane but also grow intradermally as an expanding nodule (Figure 2). Metastatic melanoma develops when tumor cells dissociate from the primary lesion, migrate through the surrounding stroma, and invade blood vessels and lymphatics to form a tumor at a distant site.75 Clinically, the absolute depth of local invasion, measured directly by histopathologic analysis (the Breslow index), is the principal prognostic factor and primary criterion in melanoma staging.76 Invasion and spread of melanoma are related to alterations in cell adhesion. Normally, cell adhesion controls cell migration, tissue organization, and organogenesis,77 but disturbances in cell adhesion contribute to tumor invasion, tumor–stroma interactions, and tumor-cell signaling.
Cadherins
Cadherins are multifunctional transmembrane proteins that sustain cell-to-cell contacts, form connections with the actin cytoskeleton, and influence intracellular signaling. The extracellular domain of cadherins binds to like cadherins on other cells in regions of cell contacts called adherens junctions. Cadherins are divided into three subtypes: E (epithelial), present in polarized epithelial cells in the epidermis, including melanocytes and keratinocytes; P (placental); and N (neural), found in mesenchymal cells in the dermis. The intracellular domain is associated with a large protein complex that includes -catenin and forms structural links with bundles of actin filaments.
Several signaling pathways cause -catenin to dissociate from the cell adhesion complex and transduce signals to the nucleus (Figure 3). One of these pathways is called the wingless-type mammary tumor virus integration-site family (WNT) pathway. WNTs are secreted proteins with important functions in development, especially in neural crest cells like melanocytes. When WNT proteins bind their receptors, they inactivate the kinase GSK3, an enzyme that phosphorylates -catenin and targets it for destruction in the proteosome.78,79 Tyrosine phosphorylation of -catenin disrupts the association between E-cadherin and -catenin,80 allowing -catenin to translocate to the nucleus, where it binds to lymphoid enhancer factor–T-cell factor (LEF–TCF). Mutations in the -catenin gene can stabilize the -catenin protein81 or increase its nuclear localization.82,83,84 Increased levels of nuclear -catenin increase the expression of MITF85 and CCND1,86 and these in turn increase the survival and proliferation of melanoma cells. Alterations in cadherin expression affect the interaction of melanoma cells with the environment and alter -catenin signaling. E-cadherin expression occurs in melanocytes and keratinocytes in the epidermis and causes melanocytes to associate with keratinocytes.87 In turn, contacts with undifferentiated keratinocytes from the basal-cell layer inhibit melanocyte proliferation, suppress the expression of melanoma markers, and cause melanocytes to become dendritic.88
Progression from the radial-growth phase to the vertical-growth phase of melanoma is marked by the loss of E-cadherin and the expression of N-cadherin89,90,91 (Figure 2). N-cadherin is a characteristic of invasive carcinomas and enables metastatic spread by permitting melanoma cells to interact with other N-cadherin–expressing cells, such as dermal fibroblasts and the vascular endothelium.87 Besides these changes in cell adhesion, decreased E-cadherin expression92 and aberrant N-cadherin expression increase the survival of melanoma cells by stimulating -catenin signaling.93,94
Integrins
The integrins mediate cell contacts with fibronectin, collagens, and laminin, components of the extracellular matrix.95 Transition from radial to vertical growth of melanoma is associated with the expression of V3 integrin.96 This integrin induces expression of matrix metalloproteinase 2, an enzyme that degrades the collagen in basement membrane.97,98,99 In addition, V3 integrin increases expression of the prosurvival gene BCL-2100 and stimulates the motility of melanoma cells through the reorganization of melanoma cytoskeleton.101 These observations form a rationale for the development of integrin antagonists to treat melanoma.102
Patterns of Genetic Alteration
The genetic changes in melanoma can be seen as particular combinations of molecular lesions that interrupt a precise set of pathways, each with a crucial role in the development of melanoma. The MEK pathway can be activated by a mutation in either NRAS or BRAF, and an NRAS mutation can activate both the MEK and PTEN pathways. Similarly, INK4A, CDK4, and CCND1 function in a unique pathway that affects the cell cycle; a mutation of INK4A has similar consequences as a mutation of CCND1 or CDK4.103,104,105
There are particular genetic changes in melanomas in different sites, consistent differences related to ultraviolet exposure on sites that are chronically exposed (head and neck) or intermittently exposed (chest and back) and in acral and mucosal skin. For example, CCND1 amplification occurs predominantly in acral regions,44 whereas activating mutations in BRAF occur most frequently in skin sites of intermittent sun exposure.106
Modeling Melanoma Progression
For many of the molecular lesions we have described, animal models have provided validation. A surprising new model is the zebrafish, in which premalignant and malignant lesions can be created by the expression of mutant BRAF with or without p53 mutation.22 This model is the only currently tractable system in which genetic screens can be performed for modifiers of melanoma.
Human melanomas that are grafted onto or injected into nude mice allow measures of the tumors' metastatic potential and have allowed for the testing of therapeutic interventions. Genetic manipulation of mice has validated the contribution of many genetic alterations in melanoma, but there are fundamental differences between mouse and human skin. Mouse melanocytes occur in hair follicles and the dermis, rather than in the epidermis, as in humans. To circumvent this problem, human melanocytes can be altered in cell culture and combined with keratinocytes to produce graft material. Using this system, the inactivation of p53 and the simultaneous introduction of activated N-RAS, CDK4, and telomerase led to darkly pigmented grafts that became grossly ulcerated and displayed histologic features of melanoma, including vertical invasion.107 This experimental system provides a novel model to test invasion and metastases of transformed human melanocytes in a host organism.
Supported by a grant (MCM202534) from the Cancer Research Institute of New York and a grant (T32-GM07753, to Dr. Miller) from the National Institute of General Medical Science. No other potential conflict of interest relevant to this article was reported.
We are indebted to Drs. David E. Fisher, Adriano Piris, Jennifer Y. Lin, and Jennifer C. Broder for their critical reading of the manuscript, and to Dr. Claudio Clemente for contributing images for Figure 1.
Source Information
From the Dermatopathology Unit, Massachusetts General Hospital, and Harvard Medical School — both in Boston.
Address reprint requests to Dr. Mihm at the Department of Dermatopathology, Massachusetts General Hospital, 55 Fruit St., Warren 827, Boston, MA 02114.
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