The n e w e ng l a n d j o u r na l of m e dic i n e

review article

mechanisms of disease

Melanoma
Arlo J. Miller, M.D., Ph.D., and Martin C. Mihm, Jr., M.D.

A
lthough melanoma accounts for only 4 percent of all derma- From the Dermatopathology Unit, Massa-
tologic cancers, it is responsible for 80 percent of deaths from skin cancer; chusetts General Hospital, and Harvard
Medical School — both in Boston. Ad-
only 14 percent of patients with metastatic melanoma survive for five years.1 dress reprint requests to Dr. Mihm at the
The intractability of advanced melanoma shows how much we have to learn about Department of Dermatopathology, Massa-
the changes that facilitate the vertical growth and deep invasion of melanoma and chusetts General Hospital, 55 Fruit St.,
Warren 827, Boston, MA 02114.
about the mechanisms that block the effectiveness of chemotherapy.
The Clark model of the progression of melanoma emphasizes the stepwise trans- N Engl J Med 2006;355:51-65.
formation of melanocytes to melanoma (Fig. 1). The model depicts the proliferation Copyright © 2006 Massachusetts Medical Society.

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 (Fig. 2).

En v ironmen ta l a nd Gene t ic In t er ac t ions

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 sensi-
tivity, 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.

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 ultra-
violet 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,

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 The n e w e ng l a n d j o u r na l
where it absorbs and dissipates ultraviolet energy.7
Clinically, variations in pigmentation and the tan-
ning 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 α-melanocyte–stimulating hormone
(α-MSH) on its receptor, the melanocortin recep-
tor 1 (MC1R) (Fig. 3). Binding of the hormone to
the receptor stimulates intracellular signaling in
melanocytes, and this signaling increases the ex-
pression 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 mel-
anoma considerably.13 In light-skinned people,
therefore, the basis of increased susceptibility to
melanoma is a genetic impairment in the produc-
tion of melanin, the main defense of melanocytes
against ultraviolet radiation.
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. Epi-
demiologic 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 Mol e cul a r Model
of Mel a nom a Pro gr e s sion
The Clark model (Fig. 1) describes the histologic
changes that accompany the progression from
normal melanocytes to malignant melanoma.2
We will relate these histologic changes to particu-
lar gene mutations (Table 1) in melanoma and dis-
cuss how these mutations affect molecular sig-
naling to contribute to the progression from
normal melanocytes to melanoma (Fig. 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 (Fig. 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 pro-
gression is probably due to oncogene-induced
cell senescence, in which growth that is stimulat-
52 n engl j med 355;1
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of m e dic i n e
Figure 1 (facing page). 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 empha-
sizes the histopathological changes that occur in the
progression of melanoma. Normal melanocytes pro-
gressively develop a malignant phenotype through the
acquisition of various phenotypic features. The partic-
ular histologic features characterizing each step of pro-
gression are the visible manifestations of underlying
genetic changes.
ed by the activation of oncogenic pathways is lim-
ited.14 At a molecular level, abnormal activation
of the mitogen-activated protein kinase (MAPK)
signaling pathway (also called extracellular-related
kinase [ERK]) stimulates growth in melanoma
cells (Fig. 4A).15-17 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 per-
cent of melanomas. These mutations, which occur
exclusively of each other, cause constitutive acti-
vation of the serine–threonine kinases in the
ERK–MAPK pathway.18-20
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 fre-
quencies suggest that nevi must acquire addition-
al molecular lesions to free themselves of growth
restraints and become malignant. Experiments in
model systems support this hypothesis. In zebra-
fish, 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 induc-
es 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.24-26
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
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 mechanisms of disease

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 The n e w e ng l a n d j o u r na l
growth of melanomas with BRAF mutations can
be suppressed by the inhibition of the down-
stream 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 preex-
isting benign nevi or as new lesions. The molecu-
lar abnormalities at this stage of progression
affect cell growth, DNA repair, and the suscepti-
bility 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) (Fig. 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 mela-
nomas do arise. Mutation of CDKN2A or PTEN is
only one molecular step on the path to the devel-
opment of melanoma, but it is unclear precisely
when such mutations occur. The increased sus-
ceptibility 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 increas-
es 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 (Fig. 4B).
In some familial and sporadic cases of melano-
ma,36,37 the CDKN2A locus is lost by homozygous
deletion of a portion of chromosome 9.36-38 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-depen-
dent kinases. INK4A (an inhibitor of CDK4) sup-
presses 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
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of m e dic i n e
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 down-
stream of INK4A, and they are also mutated in
some melanomas. These targets of INK4A func-
tion together as part of a complex that promotes
the progression of the cell cycle by phosphorylat-
ing retinoblastoma (Rb) protein, a cell-cycle reg-
ulator. 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 hu-
man 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 anti-
sense CCND1) causes apoptosis of human mela-
noma xenografts implanted in immunodeficient
mice, without an apparent effect on normal mela-
nocytes.
Alternative splicing of various exons within
CDKN2A yields two distinct tumor-suppressor pro-
teins, INK4A and alternate reading frame (ARF)
(Fig. 3).39 The ARF gene (also called p14ARF) de-
rives its name from the use of an alternative
reading frame of the exons it shares with INK4A.
ARF functions as a tumor suppressor by arrest-
ing 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, allow-
ing for repair of damaged DNA or the induction
of apoptosis.45,46 In cells, ARF deficiency abro-
gates oncogene-induced senescence and increases
susceptibility to transformation.47 In vitro, im-
mortalization 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;
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 mechanisms of disease

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 cy-
tologic 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 expres-
sion of melanoma markers regulated by microphthalmia-associated transcription factor (MITF). The vertical-growth phase and meta-
static 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, αVβ3 integrin, and matrix metalloproteinase 2 (MMP-2).

when both gene products of CDKN2A (INK4A and some 10.33,34,50 PTEN encodes a phosphatase that
attenuates signaling by a variety of growth factors
ARF) are deficient, the latent period is even short-
that use phosphatidylinositol phosphate (PIP3) as
er.49 These data suggest how ARF facilitates the
progression of melanoma and indicate that the an intracellular signal. In the presence of such
low frequency of p53 mutations in melanoma is growth factors, intracellular levels of PIP3 rapidly
increase. This increase triggers the activation of
partly related to loss of ARF, which renders the
p53 pathway inactive.39 protein kinase B (PKB, also called AKT) by phos-
phorylation (Fig. 3). Activated AKT phosphory-
PTEN, AKT, and Cell Death lates and inactivates proteins that suppress the
A second chromosomal region that is frequently cell cycle or stimulate apoptosis, thereby facilitat-
affected by homozygous deletion in melanoma ing the proliferation and survival of cells. PTEN
and other cancers is the PTEN locus on chromo- normally keeps PIP3 levels low; in its absence,

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 56
Table 1. Important Genes in Melanoma.

Pathway Gene or Protein* Function Changes in Melanoma

RAS and MAPK
N-RAS Oncogene Sporadic activating mutation at G13R
BRAF Oncogene Sporadic activating mutation at codon V600E in
nevi and melanoma
Mitogen-activated protein kinase–extracellular- Signal transduction Up-regulated in radial-growth and vertical-growth
related kinase (MEK) phases
Extracellular-related kinase 1 or 2 (ERK1 or ERK2) Signal transduction Increased activity
or mitogen-activated protein kinase (MAPK)
INK4A, CDK, and Rb
The

Cyclin-dependent kinase inhibitor 2A or inhibitor
of kinase 4A (CDKN2A or INK4A)
Cyclin-dependent kinase 4 (CDK4)
Cyclin D1 (CCND1)
Tumor suppressor–negative regulator of cell prolif-
eration
Promoter of cell proliferation
Promoter of cell proliferation
Germ-line mutations in some familial melanomas;
sporadic deletions, promoter inactivation, loss
of heterozygosity in many melanomas
Protein insensitive to inhibition by INK4A due to
rare familial germ-line mutations at R24C
Sporadic amplification in acral melanoma

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Retinoblastoma (Rb) Tumor suppressor–negative regulator of cell prolif- Phosphorylation leads to progression from
eration G1 to S
ARF and p53
Alternate reading frame (ARF) Tumor suppressor, degrades MDM2 Germ-line mutations in some familial melanomas;

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n e w e ng l a n d j o u r na l

sporadic deletions, promoter inactivation, in

The New England Journal of Medicine

of

many melanomas
Tumor protein 53 (p53) Tumor suppressor that induces apoptosis and sup- Expression usually present in melanoma
pressed proliferation after DNA damage

july 6, 2006
Mouse double minute 2 (MDM2) Targeter of p53 for ubiquination and destruction Up-regulated in presence of ARF mutation

Copyright © 2006 Massachusetts Medical Society. All rights reserved.

BCL-2–associated X protein (BAX) Inducer of cell death Variable but usually down-regulated
m e dic i n e

PTEN and AKT

Phosphatase and tensin homologue (PTEN)
Phosphatidylinositol 3 kinase (PI3K)
Protein kinase B (AKT or PKB)
BCL-2 antagonist of cell death (BAD)
Forkhead receptor (FKHR)
Tumor suppressor, represses PI3K
Signaling molecule for many growth factors
Oncogene that is activated by PI3K, leading to
increased cell survival
Inducer of cell death
Growth supression
Sporadic deletion of chromosomal region
Active in presence of PTEN mutation
Amplified in some melanomas
Variable but often down-regulated
Activated in response to PI3 pathway

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 MSH and MITF
Pro-opiomelanocortin or α-melanocyte–stimulating Signaling molecule important in pigmentation Increased melanoma vertical-growth phase
hormone (POMC or α-MSH)
Melanocortin receptor 1 (MC1R) Receptor for α-MSH Polymorphic gene affecting hair and skin color
and response to ultraviolet radiation
Adenylate cyclase (AC) Producer of cyclic AMP Up-regulated
cAMP response element–binding protein (CREB) Transcription factor Up-regulated; affects MITF and melanocyte
differentiation
Microphthalmia-associated transcription factor Transcription factor Sporadic amplification of chromosomal region
(MITF)
Tyrosinase (TYR) Pigment synthesis Decreased expression
Tyrosinase-related protein 1 (TYRP1) Pigment synthesis Decreased expression
Dopachrome tautomerase (DCT) Pigment synthesis Decreased expression
Melan-A (MLANA) Antigen recognized by melan-A and melanoma anti- Decreased expression

n engl j med 355;1

gen recognized by T-cells 1 (MART1) antibodies
Silver homologue (SILV) Antigen recognized by HMB-45 antibody Decreased expression

Melastatin 1 (TRPM1) Unknown Decreased expression in metastatic melanoma

BCL-2 Cell survival Variable up-regulation in various phases of

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melanoma
mechanisms of disease

Cell adhesion
Wingless-type mammary tumor virus integration- Protooncogene, secreted growth factor that inacti- Pathway up-regulated
site family (WNT) vates

The New England Journal of Medicine

july 6, 2006
GSK3-B
Glycogen synthase kinase 3β (GSK3β) Serine–threonine kinase that targets β-catenin for Variable; affected by WNT pathway
degradation
β-Catenin Adherens junction protein, transcriptional co- Sporadic stabilizing mutations
activator

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T-cell factor–lymphoid-enhancing factor (TCF–LEF) Transcription factor Up-regulated
E-cadherin Cell-adhesion molecule Decreased expression in vertical-growth phase
N-cadherin Cell-adhesion molecule Aberrant expression in vertical-growth phase
αVβ 3integrin Dimer that forms cell-adhesion molecule Aberrant expression in vertical-growth phase

* Abbreviated forms of the gene are given in parentheses.

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57
 The n e w e ng l a n d j o u r na l of m e dic i n e

Figure 3. Microphthalmia-Associated Transcription Factor (MITF) and β-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 β-catenin pathway, β-catenin plays a central role in cell adhesion and cell signaling. Signals from
WNT ligands block the breakdown of β-catenin. When WNT proteins bind the G-protein–coupled receptor (called frizzled), they inacti-
vate the kinase GSK3β, an enzyme that phosphorylates β-catenin and targets it for destruction in the proteosome. Then β-catenin accu-
mulates 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).

levels of PIP3 and active (phosphorylated) AKT in-
crease. Increased AKT activity prolongs cell sur-
vival through the inactivation of BCL-2 antago-
nist of cell death (BAD) protein and increases
cell proliferation by increasing CCND1 expres-
sion, 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 pro-
tein. Restoration of PTEN in cultured mouse me-
lanocytes 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 mela-
nomas implanted in immunodeficient nude mice.53

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 mechanisms of disease

As compared with normal melanocytes, increased MITF in Melanoma

levels of the active form of AKT were found in the Decreased or absent pigmentation and decreased
radial-growth phase.53 or absent expression of SILV and MLANA accom-
pany the progression from nevus to melanoma.
MI TF a nd Mel a no c y te Tumors that are deficient in these proteins have
Differ en t i at ion a poor prognosis.65-68 Expression of the mela-
statin 1 (TRPM1) gene, whose function is unknown,
Clark proposed that many nevi regress through is also controlled by MITF.69 Melanomas that are
differentiation and that the failure of differentia- deficient in melastatin have a poor prognosis.70
tion is necessary for dysplasia.2 The normal pro- The mechanism of decreased expression of these
cess of melanocyte differentiation requires exit genes is a puzzle because MITF is present in nearly
from the cell cycle and the expression of genes all melanomas.71-73
that encode proteins necessary for the produc- Although MITF causes differentiation and cell-
tion of pigment — two processes that are de- cycle arrest in normal melanocytes, melanoma
regulated in melanoma. The microphthalmia- cells do not have these characteristics. Recently,
associated transcription factor (MITF) regulates a large-scale search for genomic changes in mela-
the development and differentiation of melano- noma with the use of high-density single-nucleo-
cytes54 and maintains melanocyte progenitor cells tide polymorphisms (SNPs) found an increased
in adults.55,56 copy number (4 to 119 copies per cell) of a region
of chromosome 3 that includes the MITF locus.74
MITF in Development This increase was accompanied by the increased
Mice lacking functional MITF are albino because expression of MITF protein. The overexpression
they lack melanocytes, whereas those with par- of both MITF and BRAF could transform primary
tial MITF function have premature graying owing cultures of human melanocytes, implicating MITF
to the death of melanocytes. These experiments as an oncogene. Notably, MITF amplification oc-
show that MITF is important in the differentiation curs most frequently in tumors that have a poor
and maintenance of melanocytes.57,58 MITF ap- prognosis and is associated with resistance to
pears to contribute to melanocyte survival by in- chemotherapy.74 Interference with MITF function
creasing the expression of the BCL-2 gene, a key increased the chemosensitivity of a melanoma
antiapoptotic factor.59 In mice, deficiencies of both cell line, making MITF a potential target for
MITF and BCL-2 cause gray hair due to a loss of treatment.
differentiated melanocytes. The loss of melano-
cytes is due to the apoptosis of melanocyte pro- Cel l A dhe sion a nd In va sion
genitor cells in the hair follicle.55 In melanoma
cell lines, a reduction in BCL-2 protein also causes Local invasion and metastatic spread are respon-
cell death, suggesting that the survival of malig- sible for the morbidity and mortality in melano-
nant melanocytes depends on BCL-2.60 ma. In the Clark model, invasive characteristics
appear in the vertical-growth phase, when mela-
MITF in Differentiation noma cells not only penetrate the basement mem-
MITF functions in a key pathway leading to me- brane but also grow intradermally as an expand-
lanocyte pigmentation (Fig. 3). Intracellular sig- ing nodule (Fig. 2). Metastatic melanoma develops
naling induced by α-MSH acting on MC1R in- when tumor cells dissociate from the primary
creases MITF expression, which in turn increases lesion, migrate through the surrounding stroma,
the transcription of genes underlying melanin and invade blood vessels and lymphatics to form
synthesis: tyrosinase, tyrosinase-related-protein 1, a tumor at a distant site.75 Clinically, the absolute
and dopachrome tautomerase.61 MITF also regu- depth of local invasion, measured directly by histo-
lates the transcription of the melanocyte-specific pathologic analysis (the Breslow index), is the
genes silver homologue (SILV)62,63 and melan-A principal prognostic factor and primary criterion
(MLANA),62 whose immunohistochemical detec- in melanoma staging.76 Invasion and spread of
tion points to the diagnosis of melanoma. In addi- melanoma are related to alterations in cell adhe-
tion, MITF causes cell-cycle arrest by the induction sion. Normally, cell adhesion controls cell migra-
of INK4A.64 tion, tissue organization, and organogenesis,77

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 The
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 in-
fluence intracellular signaling. The extracellular
domain of cadherins binds to like cadherins on
other cells in regions of cell contacts called adhe-
rens junctions. Cadherins are divided into three
subtypes: E (epithelial), present in polarized epi-
thelial cells in the epidermis, including melano-
cytes and keratinocytes; P (placental); and N (neu-
ral), found in mesenchymal cells in the dermis.
The intracellular domain is associated with a large
protein complex that includes β-catenin and forms
structural links with bundles of actin filaments.
Several signaling pathways cause β-catenin to
dissociate from the cell adhesion complex and
transduce signals to the nucleus (Fig. 3). One of
these pathways is called the wingless-type mam-
mary tumor virus integration-site family (WNT)
pathway. WNTs are secreted proteins with impor-
tant functions in development, especially in neu-
ral crest cells like melanocytes. When WNT pro-
teins bind their receptors, they inactivate the
kinase GSK3β, an enzyme that phosphorylates
β-catenin and targets it for destruction in the pro-
teosome.78,79 Tyrosine phosphorylation of β-catenin
disrupts the association between E-cadherin and
β-catenin,80 allowing β-catenin to translocate to
the nucleus, where it binds to lymphoid enhancer
factor–T-cell factor (LEF–TCF). Mutations in the
β-catenin gene can stabilize the β-catenin pro-
tein81 or increase its nuclear localization.82-84 In-
creased levels of nuclear β-catenin increase the
expression of MITF85 and CCND1,86 and these in
turn increase the survival and proliferation of
melanoma cells. Alterations in cadherin expres-
sion affect the interaction of melanoma cells with
the environment and alter β-catenin signaling.
E-cadherin expression occurs in melanocytes and
keratinocytes in the epidermis and causes mela-
nocytes to associate with keratinocytes.87 In turn,
contacts with undifferentiated keratinocytes from
the basal-cell layer inhibit melanocyte prolif-
eration, suppress the expression of melanoma
60
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n e w e ng l a n d j o u r na l of m e dic i n e
Figure 4 (facing page). 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 vari-
ations 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 path-
ways, 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 transcrip-
tion of many genes, including CD1. In survival signaling associated with
phosphatase and tensin homologue (PTEN) and AKT, also known as pro-
tein kinase B, PTEN inhibits growth-factor signaling by inactivating phos-
phatidylinositol 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 af-
fect the cell cycle, growth, and survival. Often, these AKT targets are inacti-
vated by phosphorylation. PTEN attenuates this pathway through dephos-
phorylation 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 re-
maining 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 pro-
tein (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 gov-
erned 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 ki-
nase 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 progres-
sion 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 acti-
vation domain of protein 53 (p53), blocking p53-mediated gene regulation
while simultaneously leading to p53 ubiquination, nuclear export, and pro-
teosomal 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–asso-
ciated X protein (BAX). In the absence of ARF, p53 levels are decreased and
the response to DNA damage is blunted.
n engl j med 355;1 www.nejm.org july 6, 2006
 mechanisms of disease

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 The n e w e ng l a n d j o u r na l of m e dic i n e

markers, and cause melanocytes to become den-
dritic.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-cadherin89-91 (Fig. 2). N-cadherin is a character-
istic 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 β-catenin signaling.93,94
Integrins
The integrins mediate cell contacts with fibro-
nectin, collagens, and laminin, components of the
extracellular matrix.95 Transition from radial to
vertical growth of melanoma is associated with
the expression of αVβ3 integrin.96 This integrin
induces expression of matrix metalloproteinase 2,
an enzyme that degrades the collagen in basement
membrane.97-99 In addition, αVβ3 integrin increas-
es 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 de-
velopment of integrin antagonists to treat mela-
noma.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 mu-
tation of INK4A has similar consequences as a
mutation of CCND1 or CDK4.103-105
There are particular genetic changes in mela-
nomas in different sites, consistent differences
related to ultraviolet exposure on sites that are
chronically exposed (head and neck) or intermit-
tently 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 frequent-
ly in skin sites of intermittent sun exposure.106
Model ing Mel a nom a
Pro gr e s sion
For many of the molecular lesions we have de-
scribed, animal models have provided validation.
A surprising new model is the zebrafish, in which
premalignant and malignant lesions can be cre-
ated 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 mela-
noma.
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 contribu-
tion 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 intro-
duction of activated N-RAS, CDK4, and telomer-
ase 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 hu-
man 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, Jenni-
fer Y. Lin, and Jennifer C. Broder for their critical reading of the
manuscript, and to Dr. Claudio Clemente for contributing im-
ages for Figure 1.

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