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FRole of nodal signaling and the microenvironmentunderlying melanoma plasticity

Lynne-Marie Postovit1,2,*, Naira V. Margaryan1,

Elisabeth A. Seftor1 and Mary J. C. Hendrix1

1Children’s Memorial Research Center, Cancer Biology and

Epigenomics Program, Robert H. Lurie Comprehensive Cancer

Center, Northwestern University’s Feinberg School of Medicine,

Chicago, IL, USA2Department of Anatomy and Cell Biology, Schulich School of

Medicine and Dentistry, University of Western Ontario, London,

ON, Canada

*Address correspondence to Lynne-Marie Postovit,

e-mail: lynne.postovit@schulich.uwo.ca1

Abstract

The incidence of melanoma has increased dramati-

cally over the last 50 yr, and although melanoma

accounts for only 10% of all skin cancers, it is respon-

sible for over 80% of skin cancer deaths. Recent stud-

ies have uncovered critical molecular events

underlying melanocytic transformation and mel-

anomagenesis. Among these noteworthy observa-

tions are the acquisition of stem cell-associated

proteins, such as the Notch receptors and Nodal,

which have also been implicated in melanoma pro-

gression. For example, we have demonstrated that

Nodal expression is limited to invasive vertical

growth phase and metastatic melanoma lesions, and

that inhibition of Nodal signaling promotes the rever-

sion of metastatic melanoma cells toward a more

differentiated, less invasive non-tumorigenic pheno-

type. In addition, molecular cross-talk exists between

the Notch and Nodal signaling pathways. Interest-

ingly, the acquisition of stem cell-associated plastic-

ity is often acquired via epigenetic mechanisms, and

is therefore receptive to reprogramming in response

to embryonic microenvironments. Here, we review

the concept of melanoma plasticity, with an empha-

sis on the emerging role of Nodal as a regulator of

melanoma tumorigenesis and progression, and pres-

ent findings related to epigenetic reprogramming.

Key words XXXX ⁄ XXXX ⁄ XXXX2

Received 24 January 2008, revised and accepted for

publication 19 March 2008

doi: 10.1111/j.1755-148X.2008.00463.x

Melanoma defined

Melanoma arises from the transformation of neural

crest derived melanocytes that reside in the basal layer

of the epidermis. Normally, melanocytes are evenly dis-

persed into epidermal-melanin units, consisting of

approximately 36 keratinocytes per melanocyte. Each

melanocyte transfers pigment-containing melanosomes

to the keratinocytes in its unit via dendritic processes.

The melanin contained in these melanosomes absorbs

and scatters ultraviolet radiation, thereby shielding the

nucleic acids in the skin from damage. Interestingly,

in vitro skin models have demonstrated that the kerati-

nocytes dynamically regulate this process by controlling

dendrite growth, melanocyte proliferation and melanin

production (Chin, 2003; Hsu et al., 2002).

During melanoma progression there is a general dys-

function of this complex epidermal-melanin unit. Initially,

the melanocyte:keratinocyte ratio increases, resulting in

the formation of common nevi (moles) which may lead

to dysplastic nevi with structural atypia. Dysplastic nevi

can subsequently progress into a radial growth phase

(RGP) melanoma, characterized by lateral growth that is

largely confined to the epidermis. Radial growth phase

tumors may then acquire the ability to invade into the

dermis and subcutaneous tissue, to form a vertical

growth phase (VGP) melanoma. Histologically, VGP mel-

anomas are best characterized as expansive nodules of

malignant cells that have penetrated the epidermal

basement membrane. Unlike RGP melanomas, which

remain dependent on keratinocyte-derived growth

factors and cannot undergo anchorage-indepen-

dent growth, VGP melanomas have escaped keratino-

cyte control, can undergo anchorage-independent

growth and have acquired metastatic competency (Ben-

nett, 2008; Chin, 2003; Hsu et al., 2002). Metastatic

melanoma is characterized by a high mortality rate of

over 80% and a median survival of only 7.5 months. In

the last 40 yr, the incidence of melanoma in the USA

has increased by 15-fold and cutaneous melanoma has

become the most common cancer afflicting young

adults. When diagnosed prior to the onset of VGP

disease, melanoma is generally curable with surgery

(Chin et al., 2006; Chudnovsky et al., 2005b). However,

patients with metastatic melanoma have few clinical

options, because of a high resistance to therapy,

exacerbated by a very rapid disease progression. It is

P C R 4 6 3 B Dispatch: 11.4.08 Journal: PCR CE: Ulagammal

Journal Name Manuscript No. Author Received: No. of pages: 10 PE: Aswini

ª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard 1

Pigment Cell Melanoma Res. INVITED REVIEW

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therefore imperative that the molecular events that

characterize the melanocytic neoplasia be determined,

so that targets for early detection and intervention can

be developed.

Multipotent melanoma cells

There is a notable body of literature detailing the molec-

ular signature of aggressive melanoma cells and embry-

onic stem cells (ESCs), which reveals an intriguing

similarity in the pluripotent gene expression patterns

that characterize these cell types (Hendrix et al., 2007).

In the field of melanoma research, studies have utilized

comparative global gene analyses to decipher some of

the major gene expression patterns that arise as a con-

sequence of genomic and epigenetic transforming

events and that characterize the transition of melano-

cytes to poorly and then highly metastatic melanoma

cells (Bittner et al., 2000; Hendrix et al., 2003; Hoek,

2007; Smith et al., 2005). With this approach, we and

others have shown that aggressive melanoma cells

manifest a functional plasticity characterized by the

simultaneous expression of genes from a variety of cell

types, including stem cells, concomitant with a reduc-

tion in the expression of genes specific to their parental

cell lineage (Table 1). For example, aggressive mela-

noma cells aberrantly express genes (and proteins),

such as Vascular Endothelial Cadherin (VE-Cadherin),

which are normally associated with endothelial cells,

and also express Keratins, which are intermediate fila-

ments characteristically associated with epithelial cells

(Hendrix et al., 2003). Furthermore, the expression of

melanocyte-specific markers is dramatically reduced,

and sometimes absent, in aggressive melanoma cells:

Melan A is reduced by more than fivefold and Tyrosi-

nase, which catalyses the conversion of tyrosine to the

pigment melanin, is reduced by more than 35-fold in

aggressive melanomas relative to their poorly aggres-

sive counterparts (Hendrix et al., 2003). In addition,

reduced Tyrosinase levels are associated with immune

evasion (Takeuchi et al., 2003). Collectively, this gene

expression pattern confers a functional plasticity upon

aggressive melanoma cells that enables them to thrive

and metastasize. For example, VE-Cadherin expression

by melanoma cells is essential for the formation of

tumor-derived vascular networks, thought to provide the

tumor with a paravascular perfusion pathway, while the

expression of Keratins is associated with enhanced inva-

sion and metastasis (Hendrix et al., 1992, 2001).

Aggressive melanomas also express stem cell-associ-

ated proteins (including the Notch receptors, CD133,

Wnt-5a, and Nodal) which have been shown to play a

role in the maintenance of pluripotency (Balint et al.,

2005; Frank et al., 2005; Hendrix et al., 2003; Hoek

et al., 2004; Weeraratna et al., 2002). These intriguing

findings support the premise that aggressive melanoma

cells acquire a multipotent, plastic phenotype, a concept

that challenges our current thinking of how to target

tumor cells with stem cell-like properties. Indeed, while

previous therapeutic strategies have focused on elimi-

nating a homogeneous tumor expressing traditional bio-

markers, new treatment modalities should attempt to

target a heterogeneous population of cancer cells

whose stem cell-like phenotype facilitates adaptation

and consequently survival (Hendrix et al., 2007). There-

fore, pluripotency-promoting pathways, which maintain

tumor cell plasticity, would be ideal targets for early

diagnosis and therapeutic intervention.

Nodal as a melanoma plasticity biomarker

The recent studies in our laboratory have revealed a

new regulator of melanoma plasticity and tumorigenic-

ity, called Nodal (Topczewska et al., 2006). Nodal is a

member of the Transforming Growth Factor Beta (TGF-

b) superfamily and is a pivotal inhibitor of hESC differen-

tiation (James et al., 2005; Mesnard et al., 2006; Vallier

et al., 2005). Indeed, Nodal has been shown to maintain

the pluripotency of ESCs and is one of the first genes

to be down-regulated as totipotent hESCs differentiate

during embryoid body formation. Moreover, inhibition of

the Nodal signaling pathway, through pharmacological

inhibition of its receptor, results in hESC differentiation

(Vallier et al., 2004). We recently discovered that Nodal

expression is positively associated with melanoma

tumor progression: As indicated by Western blot analy-

sis, tumorigenic melanoma cells lines (C8161, WM793,

and 1205Lu) express high levels of Nodal, whereas

Nodal is absent in normal melanocytes and in non-

tumorigenic melanoma cells (C81-61) (Figure 1A). Nodal

expression is also positively correlated with melanoma

progression clinically (Figure 1B–E). Indeed, immunohis-

Table 1 Molecular profile of aggressive melanoma cells

expressing multiple cellular phenotypesa

Gene Function Ratiob

ESM-1 Endothelial surface molecule 44

VE-Cadherin Endothelial adhesion molecule 10.7

TIE-1 Endothelial protein receptor >100

EphA2 (Eck) Epithelial cell kinase 77

Keratins 7,8,18 Epithelial intermediate filaments 20–80

Mart-1 ⁄ Melan A Melanocyte antigen 5.1flLSP1 Lymphocyte specific protein 4.8

HCLS1 Hematopoietic lineage protein 10

KIT Stem cell factor receptor 2.5

Nodal Embryonic stem cell marker

and morphogen

20

Notch Stem cell marker and receptor 5.3

aAltered gene expression in human melanoma cells was identified

by cDNA microarray analysis and confirmed by Western blot.bSelected genes are reported as a ratio for highly aggressive C8161

human cutaneous melanoma cells compared to poorly aggressive,

isogenically matched C81-61 cells.

Postovit et al.

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Ftochemical analysis has shown that Nodal protein is

absent in normal skin and rare in poorly invasive RGP

melanomas. This is in contrast to invasive VGP melano-

mas and melanoma metastases where Nodal expres-

sion is detectable in up to 60% of cases. We have also

demonstrated that Nodal plays an instrumental role in

the maintenance of melanoma cell plasticity and tumori-

genicity (Topczewska et al., 2006). Metastatic C8161

melanoma cells re-expressed Tyrosinase, a melanocyte

marker, and down-regulated VE-Cadherin and Keratin

8 ⁄ 18, markers of endothelial and epithelial lineages

respectively, in response to Nodal inhibition with a

Nodal specific Morpholino (MONodal) (Table 2). As a

complement to these findings, we utilized an orthotopic

mouse model to examine the effect of Nodal inhibition

on melanoma tumor formation (Figure 2). Palpable

subcutaneous tumors arose within 7 days following the

injection of only 250 000 control C8161 cells. In

contrast, knocking down Nodal expression resulted in a

significant reduction in C8161 tumorigenicity when the

same number of cells was injected (Figure 2A). Indeed,

a 30% diminution of tumor incidence in addition to a

decrease in tumor growth occurred when Nodal

expression was inhibited (Topczewska et al., 2006). Pre-

A

B C

D E

Figure 1.11 Nodal expression correlates

with melanoma progression. (A) Western

blot analysis of Nodal in: C8161, human

metastatic cutaneous melanoma cells;

normal human melanocytes; C81-61, non-

tumorigenic melanoma cells (isogenically

matched to C8161 cells); WM793 human

vertical growth phase cutaneous

melanoma cells; 1205Lu, melanoma cells

derived from an experimental metastasis

of WM793; and A375P, a tumorigenic

human cutaneous melanoma cell line.

Actin is used as a loading control. Nodal

expression is exclusive to the tumorigenic

melanoma cells lines. (B–E)

Immunohistochemical analysis of Nodal

staining (red color) in (B,C) primary

cutaneous melanomas and (D,E)

cutaneous melanoma metastases.

Tumor-associated mast cells are also

immunoreactive for Nodal protein. Isotype

controls are pictured in the insets. Brown

areas are melanotic cells, and bars equal

50 lm.

Table 2 Summary of biomarker

expression in metastatic melanoma cells

treated with a morpholino designed to

inhibit nodal expression (MONodal)

or exposed to a hESC-derived

microenvironment (hESC CMTX) as

compared to control cellsa

Nodal (stem cell)

Tyrosinase

(Melanocyte)

Melan A

(Melanocyte)

VE-Cadherin

(Endothelial)

Keratin 8 ⁄ 18

(Epithelial)

Control +++ – – +++ +++

MONodal – ››› flflfl flflflhESC CMTX flflfl ››› flflfl

aRelative expression of biomarkers indicative of melanoma plasticity (Nodal, VE-Cadherin and

Keratin 8 ⁄ 18) or a differentiated phenotype (Tyrosinase and Melan A) were assessed using

RT-PCR analysis and confirmed by Western blot.

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Fvious results indicated that down-regulation of Nodal

expression using MONodal lasted for approximately

14 days – during which time there was no significant

tumor formation. By 17 days, Nodal was re-expressed

in the melanoma cells, and tumorigenicity resumed

(Topczewska et al., 2006). To establish a mechanism for

the reduction in tumorigenicity, we have examined the

effects of this treatment on in vivo tumor cell prolifera-

tion and apoptosis (Postovit et al., 2008). Using immu-

nohistochemical staining for Ki67 as a measure of

proliferation, and terminal deoxynucleotidyl transferase

biotin-dUTP nick-end labeling (TUNEL) as a measure of

apoptosis, we determined that inhibition of Nodal

expression with MONodal decreases proliferation and

increases apoptosis in orthotopic melanoma tumors

(Figure 2B). These in vivo data support a role for Nodal

in the maintenance of melanoma tumorigenicity and

implicate the potential involvement of apoptotic path-

ways. Furthermore, our data suggest that Nodal is a bio-

marker of melanoma progression – from a treatable

RGP disease to a more aggressive VGP disease, to the

presence of metastases.

Given the emerging role of Nodal in melanoma pro-

gression, we must understand how this gene is regu-

lated, so that its aberrant expression may be prevented

and ⁄ or reversed. The Nodal signaling pathway is tightly

regulated by a complex array of transcriptional regula-

tors, post-translational modifications and extracellular

factors (see Figure 3 for summary). The human Nodal

gene, containing three exons, is located on Chromo-

some 10q22.1. In mice, Nodal expression is enhanced

by at least three separate transcriptional regulatory

regions, the Node Specific Enhancer (NDE), approxi-

mately 10 kb upstream of the gene locus, the Left Side

specific Enhancer (LSE), approximately 4 kb upstream

of the translational start site; and the ASymmetric

Enhancer (ASE), located in the first intron. (Norris and

Robertson, 1999; Saijoh et al., 2005; Vincent et al.,

2004). Studies have determined that the LSE and the

ASE are regulated by Nodal via a positive-feedback loop

that culminates in the activation of FoxH1. In contrast,

the NDE has been shown to induce Nodal expression in

response to Notch signaling (Krebs et al., 2003; Raya

et al., 2003). Gene alignments indicate that the human

Nodal locus contains similar enhancer elements, so it is

likely that human Nodal expression is regulated in a sim-

ilar manner. Indeed, a positive-feedback loop, similar to

that described for the LSE and ASE in mice, has been

documented to sustain Nodal expression in human

ESCs and, most recently, melanoma cell types (Besser,

2004; Hendrix et al., 2007; Topczewska et al., 2006).

Moreover, our preliminary studies indicate that like

mouse Nodal, human Nodal is up-regulated by Notch

signaling in melanoma cells (Postovit et al., 2007b).

There are four known mammalian Notch receptors

(Notch1-4) and five ligands (Jagged1, Jagged2, Delta1,

Delta3, and Delta4) (Bray, 2006; Pinnix and Herlyn,

2007). The Notch receptors are activated by binding

ligands expressed on adjacent cells. Upon activation,

the Notch ectodomain is cleaved by a metalloproteinase

and the Notch IntraCellular Domain (NICD) is subse-

quently released as a consequence of c-secretase-medi-

ated cleavage. The NICD translocates to the nucleus

where it interacts with CSL, a protein that binds to the

DNA consensus sequence CGTGGGAA and normally

inhibits transcription by associating with co-repressor

proteins. The NICD generated upon ligand binding

competes with these repressor proteins to form a

NICD-CSL complex which is recognized by Master-

mind ⁄ Lag (MAML). This complex initiates transcriptional

A

B

Figure 2. Nodal inhibition diminishes tumorigenicity in an

orthotopic model. (A) In vivo tumor formation in a nude mouse

injected with C8161 cells treated with either MOControl or MONodal.

Previous work has shown that Nodal expression is down-regulated

for approximately 14 days using MONodal. Values represent the

median tumor volume (mm3) ±interquartile range, and the

MOControl and MONodal tumor volumes were significantly different

at the time points indicated by an asterisk (*) (n = 5, P < 0.05). (B)

Immunohistochemical analysis of Ki67 expression (red ⁄ brown) and

TUNEL (red fluorescence) staining in orthotopic melanoma (C8161)

tumors. Prior to injection into a mouse, cells were treated with

MONodal or left untreated (control). Proliferation is indicated by

Ki67 staining and apoptotic nuclei were detected with confocal

microscopy as red fluorescence staining localized to the nuclei of

apoptotic C8161 cells. For the TUNEL analyses, cell nuclei are

counterstained blue with DAPI. Bar equals 25 lm.

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Factivation of target genes such as c-myc (Bray, 2006;

Krebs et al., 2003; Raya et al., 2003). Of note, the NDE

of the Nodal gene contains two CSL binding sites, and

this region has been shown to respond to Notch signal-

ing (Krebs et al., 2003; Raya et al., 2003). We have

recently determined that inhibiting Notch in metastatic

melanoma cells with a c-secretase inhibitor (DAPT)

results in decreased Nodal expression. Moreover, using

specific siRNAs, we have found that Notch-4 may pref-

erentially regulate Nodal expression in these cells

(Postovit et al., 2007b).

As a complement to canonical regulators of transcrip-

tion, Nodal expression is also governed by gene methyl-

ation and miRNA-directed degradation. For example, we

have determined that there is a sizable CpG island

(>1300 base pairs) near the transcription start site (TSS)

of the Nodal gene, and that this site may regulate Nodal

expression (Postovit et al., 2007a). Moreover, a novel

miRNA (miR-430) has been shown to block the transla-

tion of a Nodal homolog, squint, in zebrafish (Choi et al.,

2007). MiR-430 target sites are also present in the

mammalian Nodal gene; and so it is likely that Nodal

expression is similarly affected by miRNA-mediated

degradation in humans.

Nodal is also regulated post-translationally by subtili-

sin-like pro-protein convertases, including PACE-4 and

Furin (Beck et al., 2002), and by glycosylation. In a

manner akin to most TGF-b family members, Nodal is

synthesized as a pro-protein that is activated following

proteolytic processing by covertases at R-X-(K ⁄ R)-R and

R-X-X-R consensus sequences (Schier, 2003). Removal

of the pro-domain potentiates autocrine signaling but

reduces Nodal stability and signaling range, thereby

promoting autocrine signaling (Le Good et al., 2005).

Conversely, glycosylation of mature Nodal affords the

protein with increased stability so that it can potentiate

paracrine signaling events (Le Good et al., 2005). Hence,

post-translational modifications of Nodal are important

mediators of Nodal signaling outcomes.

Nodal propagates its signal by binding to hetero-

dimeric complexes between type I (ALK 4 ⁄ 7) and type II

(ActRIIB) activin-like kinase receptors. Assembly of this

complex results in the phosphorylation and activation of

ALK 4 ⁄ 7 by ActRIIB, followed by the ALK 4 ⁄ 7-mediated

Figure 3.11 Regulation of the nodal signaling pathway. Nodal is secreted from the cell where it can act as an autocrine or paracrine factor. The

Nodal precursor is cleaved and activated by the pro-protein convertases (SPC), Pace-4, and Furin. Nodal propagates its signal by binding to

heterodimeric complexes between type I (ALK 4 ⁄ 7) and type II (ActRIIB) activin-like kinase receptors, resulting in the phosphorylation and

activation of ALK 4 ⁄ 7 by ActRIIB, followed by the ALK 4 ⁄ 7-mediated phosphorylation of Smad-2 and possibly Smad-3. The epidermal

growth factor-coreceptor (EGF-CFC), Cripto-1 (Cripto), is often a component of this receptor complex and can enhance Nodal signaling.

Phosphorylated Smad-2 ⁄ 3 associates with Smad-4 and translocates to the nucleus where it regulates gene expression through the

association with transcription factors such as FoxH1. Nodal up-regulates its own expression by stimulating transcriptional activation at the

Left Side specific and Asymmetric Enhancers (LSE and ASE). Extracellular Nodal inhibitors, most notably Lefty A and B (Lefty), spatially and

temporally restrict Nodal signaling levels through antagonism of Nodal and ⁄ or Cripto-1. Notch receptors become activated following binding to

ligands, such as Delta and Jagged, expressed on adjacent cells. Once activated, the intracellular component of the Notch receptor (NICD) is

released following a c-secretase-dependent cleavage. The NICD translocates to the nucleus where it interacts with CSL, a protein that binds

to the DNA and inhibits transcription by associating with co-repressor proteins. The NICD competes with these repressor proteins to form

a NICD-CSL complex enabling the transcriptional activation of target genes, including Nodal, which has CSL binding domains in its Node

Specific Enhancer. Nodal levels may also be regulated by miRNA and possibly DNA methylation of a CpG island.

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phosphorylation of Smad-2 and possibly Smad-3

(outlined in Figure 3). Phosphorylated Smad 2 ⁄ 3subsequently associates with Smad-4 and then

translocates to the nucleus where it regulates gene

expression through an association with transcription

factors such as FoxH1 and Mixer (Schier, 2003). Genetic

studies in zebrafish and mice have defined an

essential role for Cripto-1, an Epidermal Growth Factor–

Cripto-1 ⁄ FRL1 ⁄ cryptic (EGF–CFC) family member, in

Nodal function. Indeed, embryological studies have

determined that Cripto-1 directly associates with ALK 4

(with its CFC domain) and Nodal (with its EGF domain)

and that these associations may be required for Nodal

to propagate its signal (Bianco et al., 2002; Yeo and

Whitman, 2001). This prerequisite is perhaps best

exemplified in Cripto-1 null mice which die at day 7.5 as

a result of the inability to gastrulate (a Nodal-dependent

phenomenon) (Ding et al., 1998; Liguori et al., 1996).

Studies have determined that Nodal may also signal in a

Cripto-1-independent fashion. For example, Reissmann

et al. (2001) revealed that Nodal can bind to activate

ALK 7 in the absence of Cripto-1, but that Cripto-1 mark-

edly enhances this process. Another study determined

that the Nodal precursor can bind to ALK 4 in the extra-

embryonic ectoderm of the developing mouse embryo

in a Cripto-1-independent manner, and that this binding

results in the expression of Nodal-responsive genes

(Ben-Haim et al., 2006). Finally, using murine knock out

models, Liguori et al. (2007) recently demonstrated that

Nodal can signal extensively and control axis specifica-

tion in the absence of Cripto, if its inhibitor Cerberus is

also knocked out.

Nodal up-regulates its own transcription via a positive-

feedback loop. Hence, to control the levels of this

potent morphogen, hESCs also secrete Nodal inhibitors

such as Lefty A, Lefty B, Cerberus and Tomoregulin-1

(Schier, 2003; Tabibzadeh and Hemmati-Brivanlou,

2006). Of these factors, the Lefty molecules, highly

divergent members of the TGFb superfamily, are

expressed to the greatest extent. In fact, studies have

demonstrated that in conjunction with Nodal and Oct

3 ⁄ 4, Lefty A and B are among the most enriched genes

expressed in hESCs (Sato et al., 2003; Tabibzadeh and

Hemmati-Brivanlou, 2006). Extracellular Nodal inhibitors

control Nodal signaling by spatially and temporally

restricting the Nodal-mediated activation of ALK 4 ⁄ 7.

For example, Lefty A and B specifically antagonize the

Nodal signaling pathway by binding to and interacting

with Nodal and ⁄ or with Cripto-1 in a manner that blocks

ALK activation (Schier, 2003; Shen, 2007). This restric-

tion of Nodal signaling can occur in the extracellular

microenvironment, where Nodal and sometimes Cripto-

1 are present, as well as at the cell surface. Of note,

the Lefty proteins have not been found to bind ALK4 or

ActRIIB; hence these Lefty proteins are not competitive

inhibitors of the ALK receptor complex. Furthermore, in

embryological systems, the Lefty genes are often

downstream targets of Nodal signaling, thereby provid-

ing a powerful negative-feedback loop for this pathway

(Schier, 2003; Shen, 2007). In contrast, we have deter-

mined that Nodal-expressing melanoma cells do not

express Lefty, thereby allowing Nodal signaling to go

unchecked in this tumor-associated system (Postovit

et al., 2007a, 2008).

The myriad of regulatory mechanisms characterizing

the Nodal signaling pathway likely underlies its propen-

sity for aberrant expression in melanoma. However, this

complexity also affords a number of putative strategies

for the inhibition of Nodal signaling and the circumven-

tion of melanoma progression. One such approach

involves the epigenetic silencing of Nodal expression.

Epigenetic reprogramming of multipotentmelanoma cells

Although the role of genetic mutations in oncogenic

transformation is indisputable, a great deal of evidence

suggests that the tumorigenic potential of a transformed

cell is also attributable to epigenetic modifications.

Unlike genetic changes, epigenetic adjustments do not

affect the primary DNA sequence. Rather, they involve

interactions among cells and cell products, which lead

to alterations in reversible phenomena such as cell sig-

naling and DNA modifications (Postovit et al., 2007a;

Rothhammer and Bosserhoff, 2007). Exemplifying the

importance of epigenetics in melanomagenesis is a

recent study by Jaenisch et al. in which nuclear trans-

plantation of a melanoma nucleus into an oocyte gave

rise to ESCs with the capacity to differentiate into non-

tumorigenic cell types such as melanocytes and fibro-

blasts (Hochedlinger et al., 2004). Nuclear transplanta-

tion into an oocyte induces a dramatic hypomethylation

of the donor DNA, exposing promoters and enabling

transcription (Lotem and Sachs, 2006). These phenom-

ena confer the broad developmental spectrum observed

when normal or neoplastic somatic nuclei are trans-

planted. As differentiation and cell fate specification

ensue, there is a marked increase in DNA methylation

leading to the down-regulation of most genes and a

consequential specialization in gene expression (Lotem

and Sachs, 2006). In Jaenisch’s study, the tumorigenic

potential of the melanoma nuclei was temporarily

reversed in response to nuclear transplantation, even

though genetic mutations persisted (Hochedlinger et al.,

2004). Hence, the mutations which characterized the

melanoma genome worked in concert with epigenetic

factors to sustain transformation in the donor melanoma

cells.

Epigenetic modifications, initiated via microenviron-

mental factors, have also emerged as major players

in melanocyte transformation and transdifferentiation

(Bedogni et al., 2005; Carreira et al., 2006)3 . For

example, Goding et al. recently determined that the

microphthalmia-associated transcription factor, Mitf, epi-

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genetically regulates diaphanous-related formin (Dia1)

expression. Dia1, which promotes actin polymerization

and coordinates the actin cytoskeleton and microtubule

networks at the cell periphery, inhibits invasion and

induces cell-cycle arrest. Hence, by regulating Dai1 tran-

scription, alterations in Mitf, which occur in response to

microenvironmental factors, influence melanoma cell

proliferation and invasion (Carreira et al., 2006). In

another study, Bedogni et al. (2005) demonstrated that

hypoxia, which characterizes the microenvironment of

many solid tumors and has been shown to promote

melanoma cell invasion and metastasis, also contributes

to melanocyte transformation (Bedogni et al., 2005).

This study revealed that constitutively active Akt, which

is observed in a high percentage of melanomas, can

transform melanocytes exclusively when oxygen levels

are low (Bedogni et al., 2005). Furthermore, the skin’s

distance from superficial blood vessels renders it is

mildly hypoxic with oxygen levels between 1 and 5%.

This microenvironmental milieu permits melanocytes to

stabilize the transcriptional co-factor hypoxia inducible

factor 1 alpha (HIF-1a), which promotes hypoxia-associ-

ated gene expression. It was discovered that this

up-regulation of HIF-1a enhances melanocyte transfor-

mation by synergizing with constitutively active Akt to

promote anchorage-independent growth in vitro, and

tumor formation in vivo (Bedogni et al., 2005). This

finding exemplifies how the microenvironment can

complement aberrant genetic changes to promote

melanomagenesis. Moreover, these findings highlight

the importance of epigenetic phenomena (and resultant

gene expression patterns) in melanocyte transformation

and melanoma progression.

Epigenetic phenomena are theoretically reversible.

Hence, the plastic, stem cell-like phenotype of aggres-

sive tumor cells should be receptive to reprogramming

(i.e. redifferentiation) (Gerschenson et al., 1986). In sup-

port of this concept, embryonic microenvironments

have been shown to inhibit the tumorigenicity of a vari-

ety of cancer cell lines (Gerschenson et al., 1986; Pierce

et al., 1982; Podesta et al., 1984). For example, B16

murine melanoma cells were unable to form tumors

and appeared to differentiate toward a neuronal pheno-

type following exposure to microenvironmental factors

derived from the embryonic skin of a developing mouse

(Gerschenson et al., 1986). In another set of experi-

ments, Bissell and colleagues documented that Rous

sarcoma virus, which causes a rapidly growing tumor

when injected into hatched chicks, is non-tumorigenic

when injected into 4-day-old chick embryos, despite viral

replication and v-src oncogene activation (Dolberg and

Bissell, 1984).

More recently, we employed an in vitro 3D model

to examine whether the microenvironment of human

embryonic stem cells (hESCs) could similarly reprogram

the metastatic melanoma cell phenotype (Postovit et al.,

2006;4 Postovit et al., 2008). In this model, hESCs were

allowed to ‘condition’ a 3D matrix (CMTX), which would

subsequently receive multipotent metastatic melanoma

cells. Because the hESCs were removed prior to the

addition of melanoma cells, the melanoma cells were

exposed only to the extracellular microenvironment of

the hESCs, thereby removing the complexity of cell–cell

interactions from the vast array of mechanisms that

may be working to epigenetically modulate cell behav-

ior. Utilizing this approach we determined that, similar

to Nodal inhibition, exposure of melanoma cells to a

hESC microenvironment results in the re-expression of

Melan-A, a melanocyte specific marker, as well as a

reduction in the expression of VE-Cadherin (Table 2)

(Abbott et al., 2008; Hendrix et al., 2007; Topczewska

et al., 2006). Aggressive melanoma cells exposed to

hESC microenvironments also experienced an 87%

reduction in Nodal expression concomitant to a signifi-

cant decrease in tumorigenicity (Postovit et al., 2008).

Indeed, exposure to the hESC microenvironment signifi-

cantly diminished the ability of human metastatic mela-

noma cells to undergo anchorage-independent growth,

a phenomenon that was rescued by the inclusion of

rNodal (100 ng ⁄ ml). Moreover, exposure of these cells

to hESC-derived CMTX resulted in an inhibition of tumor

growth in an orthotopic mouse model (Hendrix et al.,

2007; Postovit et al., 2008). In a manner similar to Nodal

inhibition, we found that exposure to hESC CMTX

decreased proliferation and increased apoptosis in the

orthotopic tumors (Postovit et al., 2008), implicating the

potential involvement of apoptotic pathways in the

tumor suppressive effects of the hESC microenviron-

ment. Collectively, these findings illuminate the remark-

able ability of hESC-derived factors to inhibit melanoma

tumorigenicity and suggest that this tumor-suppressive

phenomenon is mediated via an inhibition of Nodal

expression and signaling.

The ability of hESCs to reprogram aggressive mela-

noma cells is reversible over time (Abbott et al., 2008;

Postovit et al., 2007a, 2008). As such, this phenomenon

is likely because of epigenetic alterations, such as DNA

methylation. Given the location of a sizable CpG island

near the TSS of the Nodal gene, together with the

marked down-regulation in Nodal expression observed

in melanoma cells exposed to hESC microenvironments,

we hypothesized that the Nodal CpG island is differen-

tially methylated in cells exposed to a hESC micro-

environment. Using bisufite-sequencing technology, we

determined that exposure of aggressive melanoma cells

to matrices conditioned by hESCs resulted in a marked

increase in site-specific methylation in the Nodal CpG

island. Although hESC microenvironments did not drasti-

cally affect global methylation, we observed specific

areas, in the first half of the CpG island, where a 32%

increase in DNA methylation occurred (Postovit et al.,

2007a). Sequence analyses determined that these areas

contain putative consensus sequences for transcription

factors including Sp1, Egr-1, and GATA-4. It is therefore

Nodal and melanoma plasticity

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plausible that hESC-derived microenvironments can alter

Nodal expression in melanoma cells by epigenetically

methylating transcription factor binding sites. These

modifications may canonically decrease the accessibility

of the Nodal promoter for transcriptional activators,

thereby decreasing Nodal expression commensurate

with differentiating melanoma cells and abrogating their

tumorigenicity. Our ongoing studies will further explore

this possibility to decipher some of the epigenetic

mechanisms by which embryonic microenvironments

reprogram aggressive cancer cells.

Conclusions and future perspectives

There is consensus in the melanoma research commu-

nity and affiliated patient advocacy groups that new

therapeutic strategies are needed to treat advanced

stages of this disease. However, one of the greatest

obstacles to achieving success has been a lack of

understanding of the basic biology underlying the onco-

genic transformation of melanocytes, the aggressive

plasticity of aggressive melanoma, and the possible epi-

genetic regulators of the metastatic phenotype. What

we have come to appreciate is that aggressive mela-

noma cells share many characteristics with embryonic

progenitor cells. In fact, our work has illuminated a new

pathway in melanoma which is directly related to tumor

cell plasticity, and represents the convergence of

embryonic and tumorigenic signaling pathways – via

Nodal, a member of the TGF-b superfamily responsible

for the pluripotency of hESCs.

Protein and immunohistochemical analyses of Nodal

demonstrate that this embryonic morphogen is aber-

rantly expressed in melanoma cells with tumorigenic

potential and in VGP and metastatic lesions. These obser-

vations suggest that Nodal expression may be associated

with the acquisition of an aggressive phenotype in mela-

noma. Additional studies are needed to determine the

prognostic value of Nodal as a new biomarker for disease

progression. Compelling evidence supporting a direct

relationship between Nodal expression, tumorigenicity

and plasticity is provided in Morpholino experiments

showing that when Nodal was down-regulated in aggres-

sive melanoma cells, tumor formation was significantly

diminished and apoptosis was induced. However, tumori-

genicity resumed when Nodal was re-expressed in these

same melanoma cells. Equally noteworthy is the finding

linking down-regulation of Nodal expression in melanoma

to loss of plasticity markers, such as VE-Cadherin and

Keratin 8 ⁄ 19, and the re-expression of melanocyte differ-

entiation pathway specific genes such as Tyrosinase and

Melan-A. Future studies will focus on new strategies to

down-regulate Nodal expression for a longer duration. In

addition, the molecular cross-talk revealed between

Notch-4 and Nodal may provide novel approaches to tar-

geting a broader signaling pathway underlying melanoma

aggressiveness and plasticity.

Based on the plastic phenotype of aggressive mela-

noma cells and their similar characteristics to stem

cells, we tested the possibility that the microenviron-

ment of hESCs could reprogram the multipotent pheno-

type. This unique experimental approach generated

important clues related to the epigenetic reprogram-

ming of plastic melanoma cells, including an 87%

reduction in Nodal expression and a 32% increase in

DNA methylation near the transcriptional start site of

the Nodal gene. These exciting results have stimulated

additional studies related to the identification of factor(s)

in the hESC microenvironment that might contribute to

this important reprogramming. As a consequence of

exposure to the normal hESC microenvironment, the

melanoma cells lost plasticity markers, regained mela-

noma differentiation markers, and underwent apoptosis;

similar to that described for Nodal down-regulation. Col-

lectively, these observations begin to elucidate a new

pathway in melanoma progression that deserves addi-

tional scientific scrutiny. Targeting Nodal and associated

pathways with molecular cross-talk may provide valu-

able new insights into managing the plastic melanoma

phenotype.

Acknowledgements

This work was supported by grants from the Illinois Regenerative

Medicine Institute, U.S. National Institutes of Health (CA50702 and

CA121205) and Charlotte Geyer Foundation to M.J.C.H., and a

Canadian Institutes of Health Research Post-doctoral Fellowship to

L.M.P. The authors wish to thank Drs Brian Nickoloff and Bento

Soares for helpful scientific discussions. We apologize to those col-

leagues whose studies were not cited in this review because of

space limitations.

References

Abbott, D.E., Bailey, C.M., Postovit, L.M., Seftor, E.A., Margaryan,

N.V., and Hendrix, M.J. (2008). The epigenetic influence of

tumor and embryonic microenvironments: how different are

they? Cancer Microenvironment. In press.5

Balint, K., Xiao, M., Pinnix, C.C., Soma, A., Veres, I., Juhasz, I.,

Brown, E.J., Capobianco, A.J., Herlyn, M., and Liu, Z.J. (2005).

Activation of Notch1 signaling is required for beta-catenin-medi-

ated human primary melanoma progression. J. Clin. Invest. 115,

3166–3176.

Beck, S., Le Good, J.A., Guzman, M., Ben, H.N., Roy, K., Beermann,

F., and Constam, D.B. (2002). Extraembryonic proteases regulate

Nodal signalling during gastrulation. Nat. Cell Biol. 4, 981–985.

Bedogni, B., Welford, S.M., Cassarino, D.S., Nickoloff, B.J., Giac-

cia, A.J., and Powell, M.B. (2005). The hypoxic microenvironment

of the skin contributes to Akt-mediated melanocyte transforma-

tion. Cancer Cell 8, 443–454.

Ben-Haim, N., Lu, C., Guzman-Ayala, M., Pescatore, L., Mesnard,

D., Bischofberger, M., Naef, F., Robertson, E.J., and Constam,

D.B. (2006). The nodal precursor acting via activin receptors

induces mesoderm by maintaining a source of its convertases

and BMP4. Dev. Cell 11, 313–323.

Bennett, D.C. (2008). How to make a melanoma: what do we

know of the primary clonal envents? Pigment Cell Melanoma

Res. 21, 27–38.

Postovit et al.

8 ª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

UN

CO

RR

EC

TED

PR

OO

F

Besser, D. (2004). Expression of nodal, lefty-a, and lefty-B in undif-

ferentiated human embryonic stem cells requires activation of

Smad2 ⁄ 3. J. Biol. Chem. 279, 45076–45084.

Bianco, C. et al. (2002). Cripto-1 activates nodal- and ALK4-depen-

dent and -independent signaling pathways in mammary epithelial

Cells. Mol. Cell. Biol. 22, 2586–2597.6

Bittner, M. et al. (2000). Molecular classification of cutaneous

malignant melanoma by gene expression profiling. Nature 406,

536–540.

Bray, S.J. (2006). Notch signalling: a simple pathway becomes

complex. Nat. Rev. Mol. Cell Biol. 7, 678–689.

Carreira, S., Goodall, J., Denat, L., Rodriguez, M., Nuciforo, P.,

Hoek, K.S., Testori, A., Larue, L., and Goding, C.R. (2006). Mitf

regulation of Dia1 controls melanoma proliferation and invasive-

ness. Genes Dev. 20, 3426–3439.

Chin, L. (2003). The genetics of malignant melanoma: lessons from

mouse and man. Nat. Rev. Cancer 3, 559–570.

Chin, L., Garraway, L.A., and Fisher, D.E. (2006). Malignant mela-

noma: genetics and therapeutics in the genomic era. Genes Dev.

20, 2149–2182.

Choi, W.Y., Giraldez, A.J., and Schier, A.F. (2007). Target protectors

reveal dampening and balancing of Nodal agonist and antagonist

by miR-430. Science 318, 271–274.

Chudnovsky, Y., Adams, A.E., Robbins, P.B., Lin, Q., and Khavari,

P.A. (2005a). Use of human tissue to assess the oncogenic activity

of melanoma-associated mutations. Nat. Genet. 37, 745–749.7

Chudnovsky, Y., Khavari, P.A., and Adams, A.E. (2005b). Melanoma

genetics and the development of rational therapeutics. J. Clin.

Invest. 115, 813–824.

Ding, J., Yang, L., Yan, Y.T., Chen, A., Desai, N., Wynshaw-Boris,

A., and Shen, M.M. (1998). Cripto is required for correct orienta-

tion of the anterior–posterior axis in the mouse embryo. Nature

395, 702–707.

Dolberg, D.S., and Bissell, M.J. (1984). Inability of Rous sarcoma

virus to cause sarcomas in the avian embryo. Nature 309, 552–

556.

Frank, N.Y., Margaryan, A., Huang, Y., Schatton, T., Waaga-Gasser,

A.M., Gasser, M., Sayegh, M.H., Sadee, W., and Frank, M.H.

(2005). ABCB5-mediated doxorubicin transport and chemoresis-

tance in human malignant melanoma. Cancer Res. 65, 4320–

4333.

Gerschenson, M., Graves, K., Carson, S.D., Wells, R.S., and Pierce,

G.B. (1986). Regulation of melanoma by the embryonic skin.

Proc. Natl Acad. Sci. USA 83, 7307–7310.

Hendrix, M.J. et al. (1992). Coexpression of vimentin and keratins

by human melanoma tumor cells: correlation with invasive and

metastatic potential. J. Natl Cancer Inst. 84, 165–174.

Hendrix, M.J., Seftor, E.A., Meltzer, P.S., Gardner, L.M., Hess,

A.R., Kirschmann, D.A., Schatteman, G.C., and Seftor, R.E.

(2001). Expression and functional significance of VE-cadherin in

aggressive human melanoma cells: role in vasculogenic mimicry.

Proc. Natl Acad. Sci. USA 98, 8018–8023.

Hendrix, M.J., Seftor, E.A., Hess, A.R., and Seftor, R.E. (2003).

Vasculogenic mimicry and tumour-cell plasticity: lessons from

melanoma. Nat. Rev. Cancer 3, 411–421.

Hendrix, M.J., Seftor, E.A., Seftor, R.E., Kasemeier-Kulesa, J.,

Kulesa, P.M., and Postovit, L.M. (2007). Reprogramming meta-

static tumour cells with embryonic microenvironments. Nat. Rev.

Cancer 7, 246–255.

Hochedlinger, K., Blelloch, R., Brennan, C., Yamada, Y., Kim, M.,

Chin, L., and Jaenisch, R. (2004). Reprogramming of a melanoma

genome by nuclear transplantation. Genes Dev. 18, 1875–1885.

Hoek, K.S. (2007). DNA microarray analyses of melanoma gene

expression: a decade in the mines. Pigment Cell Res. 20, 644–

484.8

Hoek, K. et al. (2004). Expression profiling reveals novel pathways

in the transformation of melanocytes to melanomas. Cancer Res.

64, 5270–5282.

Hsu, M.Y., Meier, F., and Herlyn, M. (2002). Melanoma develop-

ment and progression: a conspiracy between tumor and host.

Differentiation 70, 522–536.

James, D., Levine, A.J., Besser, D., and Hemmati-Brivanlou, A.

(2005). TGFbeta ⁄ activin ⁄ nodal signaling is necessary for the

maintenance of pluripotency in human embryonic stem cells.

Development 132, 1273–1282.

Krebs, L.T. et al. (2003). Notch signaling regulates left-right asym-

metry determination by inducing Nodal expression. Genes Dev.

17, 1207–1212.

Le Good, J.A., Joubin, K., Giraldez, A.J., Ben-Haim, N., Beck, S.,

Chen, Y., Schier, A.F., and Constam, D.B. (2005). Nodal stability

determines signaling range. Curr. Biol. 15, 31–36.

Liguori, G., Tucci, M., Montuori, N., Dono, R., Lago, C.T., Pacifico,

F., Armenante, F., and Persico, M.G. (1996). Characterization of

the mouse Tdgf1 gene and Tdgf pseudogenes. Mamm. Genome

7, 344–348.

Liguori, G.L., Borges, A.C., D’Andrea, D., Liguoro, A., Goncalves,

L., Salgueiro, A.M., Persico, M.G., and Belo, J.A. (2007). Cripto-

independent Nodal signaling promotes positioning of the A–P

axis in the early mouse embryo. Dev. Biol. Epub ahead of print.9

Lotem, J., and Sachs, L. (2006). Epigenetics and the plasticity of

differentiation in normal and cancer stem cells. Oncogene 14,

7663–72.

Mesnard, D., Guzman-Ayala, M., and Constam, D.B. (2006). Nodal

specifies embryonic visceral endoderm and sustains pluripotent

cells in the epiblast before overt axial patterning. Development

133, 2497–2505.

Norris, D.P., and Robertson, E.J. (1999). Asymmetric and node-

specific nodal expression patterns are controlled by two distinct

cis-acting regulatory elements. Genes Dev. 13, 1575–1588.

Pierce, G.B., Pantazis, C.G., Caldwell, J.E., and Wells, R.S. (1982).

Specificity of the control of tumor formation by the blastocyst.

Cancer Res. 42, 1082–1087.

Pinnix, C.C., and Herlyn, M. (2007). The many faces of Notch

signaling in skin-derived cells. Pigment Cell Res. 20, 458–465.

Podesta, A.H., Mullins, J., Pierce, G.B., and Wells, R.S. (1984). The

neurula stage mouse embryo in control of neuroblastoma. Proc.

Natl Acad. Sci. USA 81, 7608–7611.

Postovit, L.M., Seftor, E.A., Seftor, R.E., and Hendrix, M.J. (2006).

A 3-D model to study the epigenetic effects induced by the

microenvironment of human embryonic stem cells. Stem Cells

24, 501–505.

Postovit, L.M., Costa, F.F., Bischof, J.M., Seftor, E.A., Wen, B.,

Seftor, R.E., Feinberg, A.P., Soares, M.B., and Hendrix, M.J.

(2007a). The commonality of plasticity underlying multipotent

tumor cells and embryonic stem cells. J. Cell. Biochem. 101,

908–917.

Postovit, L.M., Seftor, E.A., Seftor, R.E., and Hendrix, M.J.

(2007b). Targeting Nodal in malignant melanoma cells. Expert.

Opin. Ther. Targets 11, 497–505.

Postovit, L.M., Margaryan, N.V., Seftor, E.A., Kirschmann, D.A.,

Lipavsky, A., Wheaton, W.W., Abbott, D.E., Seftor, R.E.B., and

Hendrix, M.J. (2008). Human embryonic stem cell microenviron-

ment suppresses the tumorigenic phenotype of aggressive can-

cer cells. Proc. Natl Acad. Sci. USA. In press.10

Raya, A. et al. (2003). Notch activity induces Nodal expression and

mediates the establishment of left-right asymmetry in vertebrate

embryos. Genes Dev. 17, 1213–1218.

Reissmann, E., Jornvall, H., Blokzijl, A., Andersson, O., Chang, C.,

Minchiotti, G., Persico, M.G., Ibanez, C.F., and Brivanlou, A.H.

(2001). The orphan receptor ALK7 and the Activin receptor ALK4

Nodal and melanoma plasticity

ª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard 9

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

UN

CO

RR

EC

TED

PR

OO

F

mediate signaling by Nodal proteins during vertebrate develop-

ment. Genes Dev. 15, 2010–2022.

Rothhammer, T., and Bosserhoff, A.K. (2007). Epigenetic events in

malignant melanoma. Pigment Cell Res. 20, 92–111.

Saijoh, Y., Oki, S., Tanaka, C., Nakamura, T., Adachi, H., Yan, Y.T.,

Shen, M.M., and Hamada, H. (2005). Two nodal-responsive enh-

ancers control left-right asymmetric expression of Nodal. Dev.

Dyn. 232, 1031–1036.

Sato, N., Sanjuan, I.M., Heke, M., Uchida, M., Naef, F., and Brivan-

lou, A.H. (2003). Molecular signature of human embryonic stem

cells and its comparison with the mouse. Dev. Biol. 260, 404–413.

Schier, A.F. (2003). Nodal signaling in vertebrate development.

Annu. Rev. Cell Dev. Biol. 19, 589–621.

Shen, M.M. (2007). Nodal signaling: developmental roles and regu-

lation. Development 134, 1023–1034.

Smith, A.P., Hoek, K., and Becker, D. (2005). Whole-genome expres-

sion profiling of the melanoma progression pathway reveals

marked molecular differences between nevi ⁄ melanoma in situ

and advanced-stage melanomas. Cancer Biol. Ther. 4, 1018–1029.

Tabibzadeh, S., and Hemmati-Brivanlou, A. (2006). Lefty at the

crossroads of ‘stemness’ and differentiative events. Stem Cells

24, 1998–2006.

Takeuchi, H., Kuo, C., Morton, D.L., Wang, H.J., and Hoon, D.S.

(2003). Expression of differentiation melanoma-associated

antigen genes is associated with favorable disease out-

come in advanced-stage melanomas. Cancer Res. 63, 441–448.

Topczewska, J.M., Postovit, L.M., Margaryan, N.V., Sam, A., Hess,

A.R., Wheaton, W.W., Nickoloff, B.J., Topczewski, J., and Hen-

drix, M.J. (2006). Embryonic and tumorigenic pathways converge

via Nodal signaling: role in melanoma aggressiveness. Nat. Med.

12, 925–932.

Vallier, L., Reynolds, D., and Pedersen, R.A. (2004). Nodal inhibits

differentiation of human embryonic stem cells along the neuroec-

todermal default pathway. Dev. Biol. 275, 403–421.

Vallier, L., Alexander, M., and Pedersen, R.A. (2005). Activin ⁄ Nodal

and FGF pathways cooperate to maintain pluripotency of human

embryonic stem cells. J. Cell Sci. 118, 4495–4509.

Vincent, S.D., Norris, D.P., Le Good, J.A., Constam, D.B., and Rob-

ertson, E.J. (2004). Asymmetric Nodal expression in the mouse

is governed by the combinatorial activities of two distinct regula-

tory elements. Mech. Dev. 121, 1403–1415.

Weeraratna, A.T., Jiang, Y., Hostetter, G., Rosenblatt, K., Duray, P.,

Bittner, M., and Trent, J.M. (2002). Wnt5a signaling directly

affects cell motility and invasion of metastatic melanoma. Cancer

Cell 1, 279–288.

Yeo, C., and Whitman, M. (2001). Nodal signals to Smads through

Cripto-dependent and Cripto-independent mechanisms. Mol. Cell

7, 949–957.

Postovit et al.

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Author Query Form

Journal: PCR

Article: 463

Dear Author,

During the copy-editing of your paper, the following queries arose. Please respond to these by marking

up your proofs with the necessary changes/additions. Please write your answers on the query sheet if

there is insufficient space on the page proofs. Please write clearly and follow the conventions shown on

the attached corrections sheet. If returning the proof by fax do not write too close to the paper’s edge.

Please remember that illegible mark-ups may delay publication.

Many thanks for your assistance.

Query reference Query Remarks

1 Au: The first author has been retained as corresponding author. Please

check.

2 Au: Please supply up to six keywords for indexing.

3 Au: Bedgoni et al., 2005 has been changed to Bedogni et al., 2005 so that

this citation matches the list.

4 Au: Postovit et al., 2005 has been changed to Postovit et al., 2006 so that

this citation matches the list.

5 Au: Please update volume number, page detail in reference Abbott et al.

(2008).

6 Au: The journal style is to list the names of first 3 authors followed by

et al. for 11 or more authors. Please provide author names according to

journal style for all references in the list.

7 Au: Chudnovsky et al. (2005a) not cited. Please cite reference in text or

delete from the list.

8 Au: Please check the page detail in Hoek (2007).

9 Au: Please update volume number, page detail in reference Liguori et al.

(2007).

10 Au: Please update volume number, page detail in reference Postovit et al.

(2008).

11 Au: This figure has been supplied as a low resolution file. Please supply

high quality, high resolution figure files to the Production Editor along

with all other proof corrections. Please go to the following link to access

the Blackwell Publishing guidelines on supplying electronic artwork:

http://www.blackwellpublishing.com/bauthor/illustration.asp