Melanoma

T h e n e w e ng l a nd j o u r na l o f m e dic i n e

n engl j med 355;1 www.nejm.org july 6, 2006 51

review article

mechanisms of disease

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

From the Dermatopathology Unit, Massa-chusetts General Hospital, and Harvard Medical School both in Boston. Ad-dress reprint requests to Dr. Mihm at the Department of Dermatopathology, Massa-chusetts General Hospital, 55 Fruit St., Warren 827, Boston, MA 02114.

N Engl J Med 2006;355:51-65.Copyright 2006 Massachusetts Medical Society.

A lthough melanoma accounts for only 4 percent of all derma-tologic 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 trans-formation of melanocytes to melanoma (Fig. 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 (Fig. 2).

En v ironmen ta l a nd Gene tic In ter ac tions

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 geneticenvironmental 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,

The New England Journal of Medicine Downloaded from nejm.org on March 17, 2015. For personal use only. No other uses without permission.

Copyright 2006 Massachusetts Medical Society. All rights reserved.


n engl j med 355;1 www.nejm.org july 6, 200652

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 -melanocytestimulating hormone (-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 Molecul a r Model of Mel a nom a Pro gr ession

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-

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 serinethreonine kinases in the ERKMAPK 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

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.





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.

CDKN2AThe G1S 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 G1S 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

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 G2M 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;





when both gene products of CDKN2A (INK4A and ARF) are deficient, the latent period is even short-er.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 chromo-

some 10.33,34,50 PTEN encodes a phosphatase that attenuates signaling by a variety of growth factors that use phosphatidylinositol phosphate (PIP

3) as

an intracellular signal. In the presence of such growth factors, intracellular levels of PIP

3 rapidly

increase. This increase triggers the activation of protein kinase B (PKB, also called AKT) by phos-phorylation (Fig. 3). Activated AKT phosphory-lates and inactivates proteins that suppress the cell cycle or stimulate apoptosis, thereby facilitat-ing the proliferation and survival of cells. PTEN normally keeps PIP

3 levels low; in its absence,

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, V3 integrin, and matrix metalloproteinase 2 (MMP-2).





Tabl

e 1.

Impo

rtan

t Gen

es in

Mel

anom

a.

Path

way

Gen

e or

Pro

tein

*Fu

nctio

nC

hang

es in

Mel

anom

a

RA

S an

d M

APK

N-R

AS

Onc

ogen

eSp

orad

ic a

ctiv

atin

g m

utat

ion

at G

13R

BR

AF

Onc

ogen

eSp

orad

ic a

ctiv

atin

g m

utat

ion

at c

odon

V60

0E in

ne

vi a

nd m

elan

oma

Mito

gen-

activ

ated

pro

tein

kin

ase

extr

acel

lula

r-re

late

d ki

nase

(M

EK)

Sign

al tr

ansd

uctio

nU

p-re

gula

ted

in r

adia

l-gro

wth

and

ver

tical

-gro

wth

ph

ases

Extr

acel

lula

r-re

late

d ki

nase

1 o

r 2

(ER

K1

or E

RK

2)

or m

itoge

n-ac

tivat

ed p

rote

in k

inas

e (M

APK

)Si

gnal

tran

sduc

tion

Incr

ease

d ac

tivity

INK

4A, C

DK

, and

Rb

Cyc

lin-d

epen

dent

kin

ase

inhi

bito

r 2A

or

inhi

bito

r of

kin

ase

4A (

CD

KN

2A o

r IN

K4A

)Tu

mor

sup

pres

sor

nega

tive

regu

lato

r of

cel

l pro

lif-

erat

ion

Ger

m-li

ne m

utat

ions

in s

ome

fam

ilial

mel

anom

as;

spor

adic

del

etio

ns, p

rom

oter

inac

tivat

ion,

loss

of

het

eroz

ygos

ity in

man

y m

elan

omas

Cyc

lin-d

epen

dent

kin

ase

4 (C

DK

4)Pr

omot

er o

f cel

l pro

lifer

atio

nPr

otei

n in

sens

itive

to in

hibi

tion

by IN

K4A

due

to

rare

fam

ilial

ger

m-li

ne m

utat

ions

at R

24C

Cyc

lin D

1 (C

CN

D1)

Prom

oter

of c

ell p

rolif

erat

ion

Spor

adic

am

plifi

catio

n in

acr

al m

elan

oma

Ret

inob

last

oma

(Rb)

Tum

or s

uppr

esso

rne

gativ

e re

gula

tor

of c

ell p

rolif

-er

atio

nPh

osph

oryl

atio

n le

ads

to p

rogr

essi

on fr

om

G1

to S

AR

F an

d p5

3

Alte

rnat

e re

adin

g fr

ame

(AR

F)Tu

mor

sup

pres

sor,

deg

rade

s M

DM

2G

erm

-line

mut

atio

ns in

som

e fa

mili

al m

elan

omas

; sp

orad

ic d

elet

ions

, pro

mot

er in

activ

atio

n, in

m

any

mel

anom

as

Tum

or p

rote

in 5

3 (p

53)

Tum

or s

uppr

esso

r th

at in

duce

s ap

opto

sis

and

sup-

pres

sed

prol

ifera

tion

afte

r D

NA

dam

age

Expr

essi

on u

sual

ly p

rese

nt in

mel

anom

a

Mou

se d

oubl

e m

inut

e 2

(MD

M2)

Targ

eter

of p

53 fo

r ub

iqui

natio

n an

d de

stru

ctio

nU

p-re

gula

ted

in p

rese

nce

of A

RF

mut

atio

n

BC

L-2

asso

ciat

ed X

pro

tein

(B

AX

)In

duce

r of

cel

l dea

thV

aria

ble

but u

sual

ly d

own-

regu

late

d

PTEN

and

AK

T

Phos

phat

ase

and

tens

in h

omol

ogue

(PT

EN)

Tum

or s

uppr

esso

r, r

epre

sses

PI3

KSp

orad

ic d

elet

ion

of c

hrom

osom

al r

egio

n

Phos

phat

idyl

inos

itol 3

kin

ase

(PI3

K)

Sign

alin

g m

olec

ule

for

man

y gr

owth

fact

ors

Act

ive

in p

rese

nce

of P

TEN

mut

atio

n

Prot

ein

kina

se B

(A

KT

or P

KB

)O

ncog

ene

that

is a

ctiv

ated

by

PI3K

, lea

ding

to

incr

ease

d ce

ll su

rviv

alA

mpl

ified

in s

ome

mel

anom

as

BC

L-2

anta

goni

st o

f cel

l dea

th (

BA

D)

Indu

cer

of c

ell d

eath

Var

iabl

e bu

t oft

en d

own-

regu

late

d

Fork

head

rec

epto

r (F

KH

R)

Gro

wth

sup

ress

ion

Act

ivat

ed in

res

pons

e to

PI3

pat

hway





MSH

and

MIT

F

Pro-

opio

mel

anoc

ortin

or

-m

elan

ocyt

est

imul

atin

g ho

rmon

e (P

OM

C o

r

-MSH

)Si

gnal

ing

mol

ecul

e im

port

ant i

n pi

gmen

tatio

nIn

crea

sed

mel

anom

a ve

rtic

al-g

row

th p

hase

Mel

anoc

ortin

rec

epto

r 1

(MC

1R)

Rec

epto

r fo

r

-MSH

Poly

mor

phic

gen

e af

fect

ing

hair

and

ski

n co

lor

and

resp

onse

to u

ltrav

iole

t rad

iatio

n

Ade

nyla

te c

ycla

se (

AC

)Pr

oduc

er o

f cyc

lic A

MP

Up-

regu

late

d

cAM

P re

spon

se e

lem

ent

bind

ing

prot

ein

(CR

EB)

Tran

scri

ptio

n fa

ctor

Up-

regu

late

d; a

ffect

s M

ITF

and

mel

anoc

yte

diffe

rent

iatio

n

Mic

roph

thal

mia

-ass

ocia

ted

tran

scri

ptio

n fa

ctor

(M

ITF)

Tran

scri

ptio

n fa

ctor

Spor

adic

am

plifi

catio

n of

chr

omos

omal

reg

ion

Tyro

sina

se (

TYR

)Pi

gmen

t syn

thes

isD

ecre

ased

exp

ress

ion

Tyro

sina

se-r

elat

ed p

rote

in 1

(TY

RP1

)Pi

gmen

t syn

thes

isD

ecre

ased

exp

ress

ion

Dop

achr

ome

taut

omer

ase

(DC

T)Pi

gmen

t syn

thes

isD

ecre

ased

exp

ress

ion

Mel

an-A

(M

LAN

A)

Ant

igen

rec

ogni

zed

by m

elan

-A a

nd m

elan

oma

anti-

gen

reco

gniz

ed b

y T-

cells

1 (

MA

RT1

) an

tibod

ies

Dec

reas

ed e

xpre

ssio

n

Silv

er h

omol

ogue

(SI

LV)

Ant

igen

rec

ogni

zed

by H

MB

-45

antib

ody

Dec

reas

ed e

xpre

ssio

n

Mel

asta

tin 1

(TR

PM1)

Unk

now

nD

ecre

ased

exp

ress

ion

in m

etas

tatic

mel

anom

a

BC

L-2

Cel

l sur

viva

lV

aria

ble

up-r

egul

atio

n in

var

ious

pha

ses

of

mel

anom

a

Cel

l adh

esio

n

Win

gles

s-ty

pe m

amm

ary

tum

or v

irus

inte

grat

ion-

site

fam

ily (

WN

T)Pr

otoo

ncog

ene,

sec

rete

d gr

owth

fact

or th

at in

acti-

vate

sG

SK3-

B

Path

way

up-

regu

late

d

Gly

coge

n sy

ntha

se k

inas

e 3

(G

SK3

)Se

rine

thr

eoni

ne k

inas

e th

at ta

rget

s

-cat

enin

for

degr

adat

ion

Var

iabl

e; a

ffect

ed b

y W

NT

path

way

-C

aten

inA

dher

ens

junc

tion

prot

ein,

tran

scri

ptio

nal c

o-ac

tivat

orSp

orad

ic s

tabi

lizin

g m

utat

ions

T-ce

ll fa

ctor

lym

phoi

d-en

hanc

ing

fact

or (

TCF

LEF)

Tran

scri

ptio

n fa

ctor

Up-

regu

late

d

E-ca

dher

inC

ell-a

dhes

ion

mol

ecul

eD

ecre

ased

exp

ress

ion

in v

ertic

al-g

row

th p

hase

N-c

adhe

rin

Cel

l-adh

esio

n m

olec

ule

Abe

rran

t exp

ress

ion

in v

ertic

al-g

row

th p

hase

V

3

inte

grin

Dim

er th

at fo

rms

cell-

adhe

sion

mol

ecul

eA

berr

ant e

xpre

ssio

n in

ver

tical

-gro

wth

pha

se

* A

bbre

viat

ed fo

rms

of t

he g

ene

are

give

n in

par

enth

eses

.





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

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-proteincoupled receptor (called frizzled), they inacti-vate the kinase GSK3, an enzyme that phosphorylates -catenin and targets it for destruction in the proteosome. Then -catenin accu-mulates in the cytoplasm and translocates to the nucleus, where it binds to LEFTCF transcription factors and increases the expression of several genes, including MITF, the cell-cycle mediator cyclin D1 (CCND1), and matrix metalloproteinase 7 (MMP-7).





As compared with normal melanocytes, increased levels of the active form of AKT were found in the radial-growth phase.53

MI TF a nd Mel a no c y te Differ en ti ation

Clark proposed that many nevi regress through differentiation and that the failure of differentia-tion is necessary for dysplasia.2 The normal pro-cess of melanocyte differentiation requires exit from the cell cycle and the expression of genes that encode proteins necessary for the produc-tion of pigment two processes that are de-regulated in melanoma. The microphthalmia-associated transcription factor (MITF) regulates the development and differentiation of melano-cytes54 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 par-tial 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 ap-pears to contribute to melanocyte survival by in-creasing 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 melano-cytes is due to the apoptosis of melanocyte pro-genitor 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 malig-nant melanocytes depends on BCL-2.60

MITF in Differentiation

MITF functions in a key pathway leading to me-lanocyte pigmentation (Fig. 3). Intracellular sig-naling induced by -MSH acting on MC1R in-creases MITF expression, which in turn increases the transcription of genes underlying melanin synthesis: tyrosinase, tyrosinase-related-protein 1, and dopachrome tautomerase.61 MITF also regu-lates the transcription of the melanocyte-specific genes silver homologue (SILV)62,63 and melan-A (MLANA),62 whose immunohistochemical detec-tion points to the diagnosis of melanoma. In addi-tion, 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 accom-pany the progression from nevus to melanoma. Tumors that are deficient in these proteins have a poor prognosis.65-68 Expression of the mela-statin 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.71-73

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 mela-noma with the use of high-density single-nucleo-tide 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 oc-curs 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.

Cel l A dhesion a nd In va sion

Local invasion and metastatic spread are respon-sible for the morbidity and mortality in melano-ma. In the Clark model, invasive characteristics appear in the vertical-growth phase, when mela-noma cells not only penetrate the basement mem-brane but also grow intradermally as an expand-ing nodule (Fig. 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 histo-pathologic 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 adhe-sion. Normally, cell adhesion controls cell migra-tion, tissue organization, and organogenesis,77





but disturbances in cell adhesion contribute to tumor invasion, tumorstroma 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 -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 (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 GSK3, an enzyme that phosphorylates -catenin and targets it for destruction in the pro-teosome.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 factorT-cell factor (LEFTCF). Mutations in the -catenin gene can stabilize the -catenin pro-tein81 or increase its nuclear localization.82-84 In-creased 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 expres-sion 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 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

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 G1S 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 MDM2p53 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-2asso-ciated X protein (BAX). In the absence of ARF, p53 levels are decreased and the response to DNA damage is blunted.





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-cadherinexpressing 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 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 V3 integrin.96 This integrin induces expression of matrix metalloproteinase 2, an enzyme that degrades the collagen in basement membrane.97-99 In addition, V3 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 ession

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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JOURNAL EDITORIAL FELLOWThe Journals editorial office invites applications for a one-year

research fellowship beginning in July 2007 from individuals at any stage of training. The editorial fellow will work on Journal projects

and will participate in the day-to-day editorial activities of the Journal but is expected in addition to have his or her own independent projects. Please send curriculum vitae and research interests to the Editor-in-Chief, 10 Shattuck St., Boston, MA 02115

(fax, 617-739-9864), by October 1, 2006.



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