The Dynamic Roles of Cell-Surface Receptors in Melanoma Development

169

The Dynamic Roles of Cell-Surface Receptors in Melanoma Development

Dong Fang and Meenhard Herlyn

C

ONTENTS

I

NTRODUCTION

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ADHERIN

M

OLECULES IN

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ELL

–C

ELL

A

DHESION AND

S

IGNAL

T

RANSDUCTION

R

OLES OF

F

RIZZLED

R

ECEPTORS

C

ADHERIN

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HIFT BY

M

ELANOCYTES AND

T

HEIR

P

RECURSORS

D

URING

M

URINE

D

EVELOPMENT

A

SSOCIATION OF

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ADHERIN

W

ITH

T

YROSINE

K

INASE

R

ECEPTORS

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EFERENCES

9

Summary

Cell-surface receptors on melanocytic cells change dramatically during transformation. These mol- ecules facilitate adhesive functions and modulate growth, survival, and invasion. In this review, we focus on cadherins for cell–cell adhesion and frizzled (Fzd) receptors for growth control. Both receptors acti- vate the E-catenin pathway, which is critical for melanocyte differentiation and melanoma progression.

Cadherins are physically associated with receptor tyrosine kinases that mediate growth signals. The switch from epithelial cadherin in melanocytes to neural cadherin in melanoma cells appears to pro- foundly change cell-signaling partners for each cell type.

Key Words: Melanoma; transformation; cadherin; Wnt-signaling pathway; tyrosine kinase receptor.

INTRODUCTION

Pigment-producing melanocytes are localized in the basal layer of the skin. Each melanocyte is coordinately associated with 5 to 10 basal keratinocytes, forming the

“epidermal-melanin unit” of up to 35 cells (1). Within this unit, each melanocyte trans- ports melanin-containing melanosomes into neighboring keratinocytes. Pigmentation deposited in the skin protects us from the deleterious effects of ultraviolet radiation. The functions and biological properties of melanocytes are finely controlled by their symbi- otic keratinocytes. The regulation of melanocytes by keratinocytes is facilitated through cell surface receptors that either establish adhesive contacts or mediate ligands from the other cells. Disturbances in normal melanocyte–keratinocyte contacts may lead to a transformed phenotype of melanocytes. For example, epithelial (E)-cadherin, which is

From: From Melanocytes to Melanoma: The Progression to Malignancy Edited by: V. J. Hearing and S. P. L. Leong © Humana Press Inc., Totowa, NJ

170 From Melanocytes to Melanoma

the major adhesion molecule mediating melanocyte–keratinocyte adhesion, is downregulated in melanoma cells before their invasion into the adjacent dermal com- partment and is accompanied by an increase in neural (N)-cadherin expression (2). This chapter will focus on the alterations in cell surface receptors of melanocytic cells and their cellular signaling pathways during transformation of melanocytes. We will pre- dominantly discuss cadherin and Wnt-signaling pathways.

CADHERIN MOLECULES IN CELL–CELL ADHESION AND SIGNAL TRANSDUCTION

Adhesion molecules have important roles in cell–cell communication. Cadherins are a family of functionally related glycoproteins involved in calcium-dependent cell–cell adhesion and signaling. They are transmembrane proteins comprised of an extracellular domain, a membrane-spanning domain, and a highly conserved cytoplasmic domain.

Cadherins contribute to epithelial cell–cell adhesion, cell shape, differentiation, epithe- lial to mesenchymal transition, and invasion. Cadherins are expressed in a cell- and tissue-specific manner. The expression patterns of major cadherins in skin cells are summarized in Table 1. E-cadherin serves as the major adhesion molecule in the adher- ence junction of keratinocytes in the skin, whereas N-cadherin is expressed by both endothelial cells and fibroblasts. E-cadherins bind to unphosphorylated E-catenin, J- catenin (plakoglobin), and p120-catenin, forming an E-cadherin–catenin unit at the cell membrane to functionally maintain cell–cell contact. The link to the actin cytoskeleton is established through D-catenin ( Fig. 1). E-cadherin mediates cellular signaling by dissociating E-catenin from the complex, which then accumulates in the cytoplasm, enters the nucleus to bind the transcription factor lymphoid enhancer factor/T-cell factor (LEF/TCF), and then transactivates downstream target genes.

Melanocytes also express E-cadherin, allowing them to bind to keratinocytes (3).

Expression of E-cadherin by melanocytes is regulated by growth factors produced by either keratinocytes or fibroblasts. Keratinocyte-derived endothelin-1 (ET-1) down- regulates E-cadherin on melanocytes (4). Fibroblast-derived hepatocyte growth factor/

scatter factor can also downregulate the expression of E-cadherin (5). Similarly, platelet- derived growth factor (PDGF) can downregulate E-cadherin expressed by melanocytes (6). This downregulation will allow the melanocytes to decouple from keratinocytes before cell division or migration over the basement membrane. Keratinocytes and mel- anocytes also express desmoglein 1 (5), likely also desmocollin. Desmoglein 1 is regu- lated by growth factors similar to E-cadherin. The distribution pattern for P-cadherin in the epidermis is less clear, in large part because of difficulties with antibodies.

Keratinocytes express P-cadherin and likely also melanocytes. VE-cadherin is specific for endothelial cells but is expressed by melanoma cells (7).

Cadherins contribute to embryonic development of various tissues (Table 2). Gene

knockout experiments in mice show that, for example, E-cadherin is involved in the

development of trophectodermal epithelium (8); N-cadherin is required for embryonic

morphogenesis of the neural tube, heart, and somites (9); and P-cadherin expression is

associated with mammary gland development (10). Changes in expression of E-cadherin

correlate with malignant transformation in a variety of tumor systems (11). In the skin,

E-cadherin is negatively associated with melanoma development and progression and it

is absent in nevus or melanoma cells (12,13). Loss of E-cadherin on melanocytes appears

171

Table 1 Regulation and Function of Cadherins on Normal and Malignant Skin Cells Keratinocyte Squamous BasalSuperLangerhansEndothelialMelanomacarcinoma CadherinChromosome layer layerMelanocytescellsFibroblasts cells cells cells Type I: E-cadherin (cdh1)16q22.1+++––– N-cadherin (cdh2)18q11.2–––++++ P-cadherin (cdh3)16q22.1++–r Type II: VE-cadherin (cdh5)16q22.1–––++– Others: Desmoglein 1, 2, 3, 4 (DSG1, DSG2, DSG3, DSG4)18q12.1-q12.2+++ Desmocollin 1, 2, 3 (DSC1, DSC2,18q12.1++ DSC3) +, positive expression; r, variable expression; – not expressed; empty space, no information available.

172 From Melanocytes to Melanoma

Fig. 1. Signaling pathway mediated by cadherin. Epithelial (E)-cadherin molecules interact with neighboring cells in a zipper-like fashion. This homophilic interaction is mediated by the most amino-terminal cadherin domain on each E-cadherin molecule containing the histidine–alanine–

valine motif. D-catenin, E-catenin, J-catenin (plakoglobin), and p120-catenin form a cytoplasmic complex that links E-cadherin homodimers to the actin cytoskeleton. The cadherin–catenin complex is regulated by various pathways. In this case, E-catenin molecules are dissociated from the complex and released into the cytoplasm. Enhanced cytoplasmic E-catenin translocates into the nuclei to transactivate its target genes.

to be one of the first critical steps during melanoma progression (2). Restoring E-cadherin expression in melanoma cells inhibits invasion of melanoma cells into dermis through downregulation of melanoma cell adhesion molecule-CAM (Mel-CAM) and E3 integrin (13), suggesting that loss of adhesive capacity allows the melanocytes to escape from control by neighboring keratinocytes.

Expression of N-cadherin in melanoma cells is associated with increased invasive

potential during tumor progression (2). Altered N-glycans of N-cadherin were found in

metastatic but not in primary melanoma cells, suggesting different carbohydrate profiles

for N-cadherin in melanoma cells for different stages of progression (14). N-cadherin

enhances migration of melanoma cells over dermal fibroblasts (15). It also acts as a

survival factor for the malignant cells by upregulation of the antiapoptosis molecule, Bad

(15). N-cadherin mediates gap junctional communications between malignant cells,

between melanoma cells and fibroblasts, and between melanoma cells and endothelial

cells (16). Mel-CAM can act as a co-receptor for N-cadherin-mediated gap junctional

communication (17).

ROLES OF FRIZZLED RECEPTORS

Wnt signals are indispensable for embryonic development (Table 2). Wnt proteins may be differentially involved in the development of a variety of cells and tissues. For example, Wnt-1 is required for midbrain patterning, central nervous system develop- ment, and T-cell maturation (18–20); whereas Wnt3a is essential for somite and tailbud formation (21). Double knockouts of Wnt1 and Wnt3a cause deficiencies in neural crest derivatives, including lack of melanocytes (22). In avian and fish embryos, Wnt proteins act as the differentiation inducer of the neural crest (23,24), and Wnt3a induces melanocytic differentiation of neural crest cells in vitro (25,26). Wnt3a transactivates one of the melanocyte-associated transcription factors, the nacre/MITF gene (27,28).

Wnts are secreted glycoproteins that bind to frizzled (Fzd) seven-transmembrane span receptors and are thus involved in cell–cell signaling. Fzd receptors have been identified in both vertebrates and invertebrates. There are 10 known members in humans and mice, 4 in flies, and 3 in worms (29). Intracellular signaling of the Wnt pathway diversifies into at least three other pathways:

Table 2

Functional Consequences of Ablation of Genes in the Cadherin and Wnt Pathway

Gene Functional consequences of gene knockout Reference E-cadherin (cdh1)

Defects in embryonic development

8

of trophectodermal epithelium

N-cadherin (cdh2)

Defects in embryonic morphogenesis,

9

including the neural tube, early heart, somites, and so on.

P-cadherin (cdh3)

Defects in mammary gland development

10

E -catenin Defects in development of ectodermal

72,73

cell layer and anterior–posterior axis formation

Wnt-1

Defects in embryonic development

18–20

of midbrain patterning, T-cell maturation

Wnt-3

Defect in axis formation

74

Wnt-3a

Homozygous null mutants die at embryonic

21 and

day 10.5–12.5 with failed development http://www.inform- of caudal somites, notochord, and atics.jax.org structures rostral to hindlimbs

Wnt-1/Wnt-3a

Defects in expansion of neural crest

22

(compound and central nervous system progenitors, mutants) including melanocytes

Wnt-4

Defects in T-cell maturation

75

Wnt-1/Wnt-4^a

Defects in T-cell maturation and dysregu-

75

(compound lation of immature thymocyte precursors mutants)

aWnt-4 was shown to be primarily mediated by the calcium pathway instead of the E-catenin pathway.

174 From Melanocytes to Melanoma

1. The E-catenin pathway (canonical Wnt pathway; stimulated by Wnt1, Wnt2, Wnt3, Wnt3a, and Wnt8), which activates target genes in the nucleus.

2. The planar cell polarity pathway, which involves Jun N-terminal kinase (JNK) and cytoskeletal rearrangements.

3. The Wnt/calcium pathway (noncanonical Wnt pathway), which is stimulated by Wnt4, Wnt5a, Wnt5b, Wnt7, and Wnt11.

The relatively well-studied canonical and calcium pathways are outlined in Fig. 2. In humans, 19 distinct Wnt proteins have been identified (29). Distinct sets of Wnt and Fzd

Fig. 2.E-catenin activation through outside signals. E-catenin plays a central role in various signaling pathways mediated by Wnt, insulin-like growth factor (IGF)-1, and epithelial (E)- cadherin. Wnt binds to frizzled (Fzd) seven-transmembrane span receptors. The proteoglycan dally acts as a co-receptor for Wnts. Interactions of Wnt/Fzd lead to stabilization of cytosolic E-catenin. In the absence of Wnts, E-catenin is phosphorylated by casein kinase ID (CKID) and/or CKIH at Ser45. Initial phosphorylation at Ser45, in turn, causes glycogen synthase kinase (GSK)-3E to phosphorylate serine/threonine residues 41, 37, and 33. Phosphorylation of these residues prompts ubiquitylation of E-catenin and degradation in proteasomes. Phosphorylation ofE-catenin occurs in a multiprotein complex containing the scaffold protein, axin, which can form a homodimer or a heterodimer with the related protein, conductin/axin2, the tumor suppres- sor gene product APC. In the presence of Wnts, disheveled (Dsh) blocks E-catenin degradation.

StabilizedE-catenin enters the nucleus and associates with LEF/TCF transcription factors, which leads to the transcription of Wnt target genes. Binding of the growth factor IGF-1 to the IGF-1 receptor tyrosine kinase induces a conformational change in the IGF-1 receptor tyrosinase kinase and its trans-phosphorylation, which, in turn, leads to phosphorylation of insulin receptor sub- strate (IRS-1). This activates PI(3)K. Activation of PI(3)K results in the production of phospha- tidylinositol-3,4,5-trisphosphate, which provides a membrane-binding site for the serine/

threonine kinase Akt/PKB. Akt is phosphorylated after translocation to the membrane by the kinase Pdk-1. Once activated, Akt blocks E-catenin degradation through phosphorylation of GSK-3E. E-catenin also mediates the signaling pathway of cadherin, as described above.

ligand-receptor pairs can activate each of these pathways and result in unique cellular responses. Because of their diversity, it is unclear how the Fzd members differ in func- tion and ligand specificity.

The E-catenin pathway is critically involved in development (Table 2). Defects in knockouts are observed primarily in ectodermal cell layers and anterior–posterior axis formation. A large number of target genes of E-catenin have been identified (Fig. 3), including c-myc (30), cyclin D (31,32), TCF (33), LEF1 (34), PPARG (35), c-Jun (36), MMP-7 (37), BMP4 (38), survivin (39), VEGF (40), sFRP-2 (41), Frizzled 7 (42), Id2 (43), fibronectin (43), connexin 43 (44), connexin 30 (45), twist (46), and keratin (47).

Few genes are downregulated by E-catenin signaling, including osteocalcin (48) and E-cadherin (49).

The Wnt/E-catenin signaling plays a critical role in self-renewal of stem cells and acts as their growth factor (50,51). Wnt signaling is endogenously activated in undifferen- tiated stem cells and appears to be indispensable for self-renewal and proliferation of stem and progenitor cells. After differentiation, Wnt expression is downregulated (52).

Our own preliminary studies suggest that Wnt signaling, mediated by Wnt3a, plays an important role in melanocytic differentiation of human embryonic stem cells. This dif- ferentiation process depends on continuous presence of the ligand.

In addition to the critical roles for Wnt–Fzd signaling during development, the path-

way is also involved in tumorigenesis, including melanoma development (53). Gene

Fig. 3. Nuclear targets for E-catenin signaling. The following genes are upregulated by E-catenin signaling: c-myc, Cyclin D, TCF, LEF1, PPAR-G, c-jun, MMP-7, BMP4, Survivin, VEGF, sFRP- 2, Frizzled 7, Id2, fibronectin, connexin 43, connexin 30, nacre/MITF, Twist, Keratin. The following genes are downregulated by E-catenin signaling: osteocalcin, E-cadherin.

176 From Melanocytes to Melanoma

Fig. 4. Cadherin shift during embryonic development determines melanocyte locations. Melano- cyte progenitors migrate along the dorsolateral pathway to the dermis during embryonic devel- opment. Types and levels of cadherin expression correlate to the locations of the progenitor cells.

expression profiling found that Wnt5a expression correlates with increasing tumor pro- gression stage (54). Immunohistology revealed changes in Wnt and Fzd expression patterns with progression of melanoma cells (55). Reduced activity or absence of soluble Fzd receptor protein 1 permits the transduction of noncanonical Wnt signals, at least in colorectal tumorigenesis (56). Much less is known in melanoma, but Wnt–Fzd signals are most likely involved in development of this disease as in other tumors (53,57,58).

E-catenin plays a central role in transducing multiple signals. Besides being activated by Wnt and E-cadherin signaling, E-catenin also mediates insulin-like growth factor (IGF)-1 signals (Fig. 2). IGF-1 induces survival and growth of biological early mela- noma cells through E-catenin stabilization, as indicated by nuclear and cytoplasmic localization (59,60). Potentially, this could occur through activating mutations in the E-catenin gene (61), but mutations are relatively rare.

CADHERIN SHIFT BY MELANOCYTES AND THEIR PRECURSORS DURING MURINE DEVELOPMENT

Epidermal melanocytes are derived from neural crest cells, which are a transient population of cells arising from the dorsal neural tube. Melanocyte precursors migrate along the dorsolateral pathway, and cross the basement membrane into the epidermis, where they differentiate to melanocytes (62). During migration and differentiation of murine melanoblasts, there is a significant shift in expression of cadherins (Fig. 4).

Dorsolaterally migrating melanoblasts in the dermis lack E-cadherin at 10.5 d post

coitum. Within 2 d, they become weakly positive. At 13.5 d, melanoblast migration into

the epidermis is accompanied by an approx 200-fold increase in E-cadherin expression

Fig. 5. Association of epithelial (E)-cadherin and neutral (N)-cadherin with receptor tyrosine kinase. E-cadherin can initiate signal transduction pathways through the involvement of tyrosine kinase receptors for epidermal growth factor (EGFR) and/or c-met. N-cadherin-mediated signal- ing pathways can be associated with fibroblast growth factor receptor (FGFR) and neural cell adhesion molecule (NCAM).

(63). One or two days later, most melanocytes downregulate E-cadherin, leave the epi- dermis, and migrate into hair follicles, where they express P-cadherin. Few remaining epidermal melanocytes continue to express E-cadherin, whereas dermal melanocytes express N-cadherin. After birth, epidermal melanocytes maintain high levels of E-cadherin expression, whereas dermal melanocytes continue to express N-cadherin and hair follicular melanocytes express P-cadherin (63,64). Pigmented murine melano- cytes are first detected at E16.5 and increase dramatically after birth (65,66).

ASSOCIATION OF CADHERIN WITH TYROSINE KINASE RECEPTORS

In addition to homophilic and heterophilic binding, cadherins can interact with growth

factor receptors. Hepatocyte growth factor receptor, c-met, forms a complex with

E-cadherin in tumor cells, including melanoma cells (Fig. 5) (5,67,68). In immortalized

nontumorigenic keratinocytes, E-cadherin also stimulates the MAPK pathway through

ligand-independent activation of epidermal growth factor receptor (EGFR) (67,68).

178 From Melanocytes to Melanoma

E-cadherin could negatively regulate ligand-dependent activation of divergent classes of receptor tyrosine kinases by inhibiting their ligand-dependent activation in associa- tion with decreases in receptor mobility and in ligand-binding affinity. EGFR regulation by E-cadherin was associated with complex formation between EGFR and E-cadherin, which is dependent on the extracellular domain of E-cadherin but was independent of E-catenin binding or p120-catenin binding ( 69). These results suggest that dysregulation of E-cadherin signaling or downregulation of expression may have profound effects on receptor tyrosine kinase signaling. Similar to E-cadherin, N-cadherin also appears to closely interact with a tyrosine kinase. N-cadherin promotes neuronal cell survival by activating fibroblast growth factor receptor (70,71). However, little is known about the underlying mechanisms.

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