Actin-Binding Protein, Espin: a Novel Metastatic Regulator For

Author Manuscript Published OnlineFirst on December 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0468-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Actin-binding Protein, Espin: a Novel Metastatic Regulator for

Melanoma

Takeshi Yanagishita1,3, Ichiro Yajima1,5, Mayuko Kumasaka1,5, Yoshiyuki Kawamoto2,

Toyonori, Tsuzuki4, Yoshinari Matsumoto3, Daisuke Watanabe3 and Masashi Kato1,5,6

1Units of Environmental Health Sciences and 2Immunology, Department of Biomedical

Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501,

Japan. 3Department of Dermatology, Aichi Medical University School of Medicine,

Nagakute, Aichi 480-1195, Japan. 4Department of Pathology, Nagoya Daini Red Cross

Hospital, Nagoya 466-8650, Japan. 5Department of Occupational and Environmental

Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku,

Nagoya, Aichi 466-8550, Japan.

6Correspondence: Masashi Kato, MD, PhD,

Department of Occupational and Environmental Health, Nagoya University Graduate

School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.

Tel & Fax: +81-52-744-2122, E-mail: [email protected]

Running title: Role of Espin in melanoma

Key words: Espin, melanoma, invasion, metastasis, actin

Word Count: 2242 words

Total number of figures and tables: 4 figures

Conflict of Interest: None

Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0468-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Espin is a multifunctional actin-bundling protein with multiple isoforms and has special

connections to hair cell stereocilia and microvillar specializations of sensory cells in the

inner ear. However, there have been no reports showing the expression and function of

Espin in cancers, including melanoma. Here it is demonstrated that Espin expression is

significantly increased in melanomas that spontaneously developed in RET-transgenic mice

(RET-mice). Importantly, the invasion capacity of Espin-depleted Mel-ret melanoma cells

derived from a tumor of the RET-mouse was dramatically less than that of control

melanoma cells with reductions of lamellipodia, focal adhesion kinase (FAK) and

GTP-Rac1 activities. Correspondingly, the ratio of metastatic foci in Espin-depleted

Mel-ret melanoma cells was significantly less than that of control melanoma cells in an in

vivo of melanoma metastasis model. Moreover, Espin could be a novel biomarker of

melanoma in humans, since our immunohistochemical analysis data reveal that percentages

of Espin-positive cells in human primary and metastatic melanomas were significantly

higher than the percentage of cells in melanocytic nevi. Combined, these results indicate

that Espin is not only a metastatic regulator for melanoma but also a potential biomarker of

disease progression.

Implications:

Actin-binding protein Espin is expressed in melanoma, affects metastasis, and is potential

target for melanoma therapy.

Introduction

The incidence of melanoma has been increased by 3.1% per year and its incidence

rate has doubled over a 10-year period (1). Melanoma is the most serious skin cancer and is

highly invasive and resistant to conventional therapy (2). Identification of a molecule

associated with melanoma growth, progression and metastasis will provide new insights

into design of a biomarker and a novel therapeutic strategy for melanoma. Preventing or

eliminating metastasis is one of the most important challenges for therapeutic intervention.

Several regulatory molecules for cancer invasion and metastasis have recently been

reported as candidates for clinical applications (3). However, an effective therapy for

melanoma has not yet been established (2).

The ESPIN gene has various isoforms and encodes an actin filament-binding protein

(4, 5) (Supplementary Fig. S1). All isoforms of ESPIN encode the same region of a

116-amino-acid actin-bundling module (ABM), which is necessary for potent

actin-bundling and micovillar PAB elongating activities (4, 5). Mutation in Wiskott-Aldrich

syndrome protein homology 2 (WH2) and ABM domains of ESPIN gene causes hereditary

deafness and vestibular dysfunction accompanied by stereociliary shortening in jerker mice

and humans (5, 6). Espins are also capable of affecting the actin cytoskeleton, resulting in a

special connection to microvillar specializations of sensory cells such as taste receptor cells,

solitary chemoreceptor cells and Merkel cells (7). On the other hand, dynamic remodeling

of the actin cytoskeleton is required for cancer invasion. To activate cancer invasion,

remodeling of the actin cytoskeleton results in the formation of lamellipodia, sheets of

F-actin, regulated by Rho family GTPases including Rac1, whose activity is regulated by

FAK (8). These previous findings indicate the possibility that ESPIN is functionally

correlated with invasion of cancer. To our knowledge, however, there is no information

about expression levels of Espin in cancer cells or its role in cancers including melanoma.

We previously established RET-transgenic mice of line 304/B6 (RET-mice)

carrying oncogenic RET (RFP/RET) under the control of the metallothionein-I promoter (9).

Hyperpigmented skin, benign melanocytic tumors and melanomas with metastases develop

stepwise in RET-mice (9). Since histopathological characteristics of melanoma in

RET-mice are similar to those of melanoma in humans, RET-mice have been used as a

standard model of cutaneous melanoma in recent studies (9, 10). In this study, we examined

the effect of Espin on the pathogenesis of melanoma and found for the first time a

correlation between Espin and cancer.

Materials and Methods

Cells.

A murine Mel-ret melanoma cell line derived from a tumor in the RET-mouse (11)

and human melanoma cell lines (G361, SK-Mel28, Colo679, HMVII, A375P and A375M)

(10) were cultured in RPMI1640 supplemented with 10% fetal bovine serum. G361,

SK-Mel28, Colo679, HMVII were provided by the Riken Bioresource Center Cell Bank,

and A375P and A375M were kindly provided by Dr. Dorothy C Bennett (St. George's,

University of London, UK). Primary culture of normal human epithelial melanocytes

(NHEM) was performed according to the manufacturer’s protocol (Cell Applications).

Immunobloting, immunohistochemistry and immunofluoresense analysis.

An anti-Espin-ru rabbit polyclonal antibody was developed using peptides

(SSSSTGSTKSFN) in this study (Supplementary Fig. S1). Rabbit polyclonal antibodies

against phosphorylated tyrosine 397 in FAK (Invitrogen) and phosphorylated tyrosine 181

in Paxillin (EPITOMICS) and mouse monoclonal antibodies against Gapdh (Cell Signaling

Technology, Inc.), FAK (BD Transducton Laboratories), Vinculin (SIGMA) and Paxillin

(BD) were used as primary antibodies. A pull-down assay was performed using RhoA and

Rac1 Activation Assay Kits (CELL BIOLABS) according to the manufacturer’s protocol.

Immunohistochemical estimation of ESPIN (positive/negative) was performed using the

software program WinROOF (MITANI Corporation) according to the method previously

reported (10). Methods for immunoblotting, immunofluoresense analysis and

immunohistochemistry using human tissue array slides (ME103; US Biomax, Inc.) are

described in Supplementary Materials and Methods.

Wound healing assay and in vitro invasion assay.

Wound healing assays using shRNA stably expressed and control Mel-ret cells

seeded on type I collagen (10 µg/ml)-coated 6-well plastic dishes at a density of 2 x 105

cells per well were performed according to the method previously described (13). In vitro

invasion assays using 2 x 105 cells with stably expressed shRNA and 2 x 105 control

Mel-ret cells were performed according to the method previously described (14).

Melanoma xenograft studies.

Mel-ret melanoma cells (1x106 in PBS) stably transfected with Espin shRNA or

control shRNA were injected into the lateral veins of 6-week-old female mice (n=5 in each

group), and the mice were sacrificed at 42 days after inoculation. GFP-positive metastatic

melanomas in lungs were analyzed by MZ16F light microscopy (Leica).

Ethics statement.

This study was performed in Chubu University, Japan. The study was approved by

the Animal Care and Use Committee (approval no. 2410030), Recombination DNA

Advisory Committee (approval no. 10-08) and Ethical Committee (approval no. 20008-10)

in Chubu University.

Results

Expression and localization of Espin in murine melanomas.

We first showed that Espin transcript expression levels in melanomas were more

than 200-fold higher than those in benign melanocytic tumors in RET-mice by qRT-PCR

analysis using primers (primer set Total Espin shown in Supplementary Fig. S1), which

recognize all isoforms of Espin transcript (Supplementary Fig. S2A). These results

indicated that Espin transcript is highly expressed in melanomas in RET-mice. We then

examined the isoform of Espin transcript that is highly expressed in melanoma using

various primers (primer sets 1, 2A-A’, 2B-B’, 1-3 and 4) shown in Supplementary Fig. S1.

Transcript expression levels of Espin-1, -2 and -3 and Espin-4 isoforms detected by primer

sets 1-3 and 4 (Supplementary Fig. S1) in melanomas were 286-fold and 28-fold higher

than those in benign tumors, respectively (Supplementary Fig. S2B). However, transcript

expression levels of Espin-1, Espin-2A and -2A’ and Espin-1, 2B and -2B’ isoforms

detected by primer sets 1, 2A-2A’ and 2B-B’ (Supplementary Fig. S1) in melanomas were

comparable to those in benign tumors (Supplementary Fig. S2C). These results showed

higher expression of Espin-3, lower expression of Espin-4, and no expression of Espin-1

and Espin-2 transcripts in melanomas. Since ESPIN-1 and -3, but not ESPIN-2 and 4, were

detected in humans in previous studies (5, 7), we newly developed a rabbit polyclonal

anti-Espin-ru antibody targeting the common sequence for Espin-3 protein in mice and

humans (Supplementary Fig. S1 and S3).

Corresponding to the results of qRT-PCR analysis, immunoblot analysis using

anti-Espin-ru-antibody revealed a more than 44-fold increased Espin protein expression

level in melanomas compared to that in benign tumors in RET-mice (Fig. 1A).

Immunofluorescence analysis showed that Espin protein was localized in the cytoplasm and

lamellipodia (Fig. 1B) and was co-localized with F-actin protein in lamellipodia (Fig. 1B,

arrowheads). Disruption of lamellipodia formation and rugged shape were observed in

stable clones of Espin-depleted Mel-ret cells (Clone E4 in Fig. 1C and D), while

lamellipodia were organized in the mobile edge of cells in the control clones (Clone C6 in

Fig. 1C and D). Cortical thick stress-fibers were also identified in the Espin-depleted clone

(arrows of Clone E4 in Fig. 1D). Statistical analysis further showed about 55% (Clones C3

and C6) and about 12% (Clones E2 and E4) of lamellipodia formed in control (Clones C3

and C6 in Fig. 1D) and Espin-depleted Mel-ret cells (Clones E2 and E4 in Fig. 1D),

respectively.

Effects of Espin on migration and invasion activities of melanoma cells.

We next performed wound-healing assays and in vitro invasion assays in stable

clones of control and Espin-depleted Mel-ret cells. Activities of cell migration in the

Espin-depleted clones (Clones E2 and E4 in Fig. 2A) were about 77% reduced compared

with those in the control cells (Clones C3 and C6 in Fig. 2A) in wound-healing assays.

Activities of cell invasion in Espin-depleted cells (Clones E2 and E4 in Fig. 2B) were also

about 80% reduced compared with those in control cells (Clones C3 and C6 in Fig. 2B) in

invasion assays. Levels of FAK, Paxillin and Rac1 activities and Vinculin expression in

Espin-depleted cells (Clone E4) were decreased compared to those in control cells (Clone

C6) (Fig. 2C). However, activity levels of RhoA were comparable in control cells and

Espin-depleted cells (Fig. 2C).

Effects of Espin on metastasis of melanoma cells in vivo.

Since many studies have showen that migration and invasion involve metastasis

(15), we then examined the effect of Espin on melanoma metastasis in vivo. Green

fluorescence protein (GFP) tagged-Espin-depleted Mel-ret cells (Clone E4; n=5) and

control cells (Clone C6; n=5) were injected into tail veins of nude mice (Fig. 3).

Macroscopic analysis for fluorescence intensity on the surface of the lung showed that the

number of metastatic foci in Espin-depleted cells (Clone E4) was smaller than that in

control cells (Clone C6) (Fig. 3A). Statistical analysis also showed that the number of

GFP-positive metastatic foci on the surface of the lung in Espin-depleted cells (Clone E4)

was about 25% of that in control cells (Clone C6) (Fig. 3B). In addition, lung metastasis in

Espin-depleted Mel-ret cells (Clone E4) and control cells (Clone C6) was microscopically

confirmed by HE staining (Fig. 3A).

ESPIN expression in human melanomas.

We finally examined ESPIN protein expression levels in melanoma cell lines and

tissues in humans. Immunoblot analysis showed that ESPIN was expressed all of the six

melanoma cell lines (G361, SK-Mel28, Colo679, HMVII, A375P and A375M) but not in

NHEM (Fig. 4A). Immunohistochemical analysis also showed that ESPIN protein was

expressed in more than 80% and 90% of primary melanomas (n = 77) and metastatic

melanomas for lymphnodes (n = 23), respectively, while no expression of ESPIN was

detected in more than 60% of melanocytic nevi (n =36) (Fig. 4B and C). Statistical analysis

showed that percentages of ESPIN-positive primary and metastatic melanomas were

significantly higher (p<0.01) than the percentage of ESPIN-positive melanocytic nevi (Fig.

4 C).

Discussion

We first demonstrated that expression of Espin in skin melanomas is definitely

higher than that in benign melanocytic tumors in RET-mice. Since our previous study

showed that melanomas developed from benign melanocytic tumors in RET-mice (9), the

results suggest that increased Espin expression level in melanoma is associated with

malignant transformation. We also demonstrated that levels of ESPIN expression in

melanoma cell lines and primary and metastatic melanomas are higher than those in NHEM

and melanocytic nevi, respectively, in humans. These results suggest that ESPIN could be a

potential biomarker for primary and metastatic melanomas.

We then examined the biological significance of increased Espin expression in

melanoma cells. We showed that Espin was co-localized with F-actin in lamellipodia.

Lamellipodia are formed when cancer cells migrate on an ECM such as the type-I collagen

or fibronectin matrix (16, 17). The structure is induced in many biological processes

including cell migration and drives progression of cancer invasion and metastasis via

dynamic remodeling of the actin cytoskeleton (18). Since various actin-binding proteins

constructing lamellipodia were previously reported to play an important role in cancer

invasion (14), we hypothesized that Espin regulates the migration and invasion of

melanoma cells. We found localization of Espin at lamellipodia in migrating melanoma

cells on the type-I collagen matrix as an ECM in addition to decreased migration activity in

Espin-depleted melanoma cells with cortical thick stress-fibers, which inhibit cell motility

(18), in the wound healing assay. Together with the results showing decreased invasion

activity in Espin-depleted melanoma cells, these results suggest that Espin regulates the

dynamics of actin polymerization at lamellipodia and is one of the drivers of melanoma cell

motility.

Cells in the process of migration must acquire a spatial asymmetry enabling them to

turn intracellularly generated forces into net cell body translocation (19). One manifestation

of this asymmetry is a polarized morphology, which is clearly distinct between the cell

front and cell rear (19). Since central regulators of the cell front are Rho family small

GTP-binding proteins (Rho GTPases) including Rac and Rho, they are pivotal regulators of

actin and adhesion organization and control the formation of lamellipodia (20). Rac1 is a

direct regulator of the organization of lamellipodial structure (8, 16). A previous study

showed that Rac1 is downstream of signaling from the complex of Paxillin, FAK and

Vinculin in focal adhesions and cell-cell contacts (8). FAK is not only a central regulator in

focal adhesions but also a critical player in melanoma cell motility via interaction with the

ECM and subsequent activation of downstream signals towards a malignant phenotype (8).

Since our results showed decreased levels of FAK, Paxillin and Rac1 activities and

Vinculin expression in Espin-depleted cells with disruption of lamellipodia, Espin may be

associated with lamellipodia formation via regulation of FAK/Paxillin/Vinculin/Rac1

signaling in melanoma cells. Together with our results showing an approximately 75%

decrease of lung metastasis in Espin-depleted cells inoculated in nude mice, the results

indicated that Espin is a molecular target for therapy of metastatic melanoma.

In summary, we demonstrated for the first time that Espin/ESPIN is expressed in

melanoma and affects metastasis of melanoma by regulation of migration and invasion with

modulation of FAK/Paxillin/Vinculin/Rac1 signaling. Our results indicate that ESPIN is

potentially useful for therapy of metastatic melanoma as well as a biomarker for melanoma.

Acknowledgements

We would like to thank Ms Kazumi Shigeta and Ms Yoko Kato for technical

assistance.

Grant Support

This study was supported in part by Grants-in-Aid for Scientific Research (B)

(24390157, 24406002), and (C) (25340052), Research Fellow of Japan Society for the

Promotion of Science (25-40080), Grant-in-Aid for Challenging Exploratory Research

(23650241) and Grant-in-Aid for Scientific Research on Innovative Areas (24108001) from

the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Uehara

Memorial Foundation, the Naito Foundation and the Cosmetology Research Foundation.

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Figure legends

Figure 1: Morphological effects of Espin expression on melanoma cells.

A, Espin (Espin-3; 35 kDa) protein expression levels in the skin of WT- mice (WT) and

RET-mice (RET) and in benign melanocytic tumors and melanoma from RET-mice are

shown (left). Intensities of bands are compared between benign melanocytic tumors (mean

± SD; n=4) and melanoma (mean ± SD; n=3) (right). B, Confocal immunofluorescence

images of merged Espin (green) and F-actin (magenta) in Mel-ret cells are presented.

Arrowheads indicate lamellipodia. C, Espin expression levels in stable clones established

from Mel-ret cells transfected with control (Clones C3 and C6) and Espin-shRNA (Clones

E2 and E4) vectors are shown (top). Intensities of bands are presented as percentages (mean

± SD; n=3) relative to control Clone C3 (bottom). D, Morphological change of Mel-ret cells

by stable transfection of shRNA vectors. Cells were stained with Alexa568-phalloidin to

visualize the structures of actin filaments. Arrowheads indicate lamellipodia in Mel-ret cells.

Arrows indicate cortical thick stress fibers in Espin-depleted Mel-ret cells (Clone E4) (left).

Percentages of lamellipodia formation (n=100) are compared between control cells (Clones

C3 and C6) and Espin-deleted cells (Clones E2 and E4) (right). Significantly different (**,

p<0.01) from the control (Clone C3) by Student’s t-test. Scale bars: 20 µm.

Figure 2: Decreased cell migration and invasion of Espin-depleted melanoma cells.

A, Wound healing assay was performed with control (Clones C3 and C6) and Espin-deleted

(Clones E2 and E4) Mel-ret cells (left). The graph shows the wound filled area evaluated as

percentage (mean ± SD; n=3) at 16 hrs after scratching (right). B, Matrigel-invasion assay

was performed with control (Clones C3 and C6) and Espin-deleted (Clones E2 and E4)

Mel-ret cells. Photographs of cells invading the membrane stained with HE are shown (left).

After invading cells had been counted in five random microscopic fields in each

matrigel-invasion assay, the results of 3 independent assays were normalized and are

presented as an invasion index (right). C, Phosphorylation and expression levels of FAK

and Paxillin and expression levels of Vinculin and Gapdh in control (Clone C6) and

Espin-deleted (Clone E4) Mel-ret cells are shown. Amounts of GTP-bound forms and total

Rac1 and RhoA proteins in control (Clone C6) and Espin-deleted (Clone E4) Mel-ret cells

are also shown. Intensities of bands are presented as percentages (mean ± SD; n=3) relative

to control Clone C3. Significantly different (**, p<0.01; *, p<0.05) from the control (C6)

by Student’s t-test. Scale bar: 200 μm.

Figure 3: Decreased lung metastasis in Espin-depleted melanoma cells in vivo.

A, Results of morphological analysis of lung metastasis of control (Clone C6) and

Espin-deleted (Clone E4) Mel-ret cells injected into tail veins of nude mice (n=5) are

presented. Animals were dissected to observe lung metastases at 42 days after inoculation.

Lung metastases were macroscopically visualized by GFP fluorescence images. Metastatic

foci (arrow) derived from control (Clone C6) and Espin-deleted (Clone E4) cells were

microscopically confirmed by HE staining. B, Number of GFP-positive metastatic foci per

lung surface (mean ± SD; n=5) is presented. Significantly different (**, p<0.01) from the

control (C6) by Student’s t-test.

Figure 4: ESPIN expression in human melanoma cell lines and melanoma tissues.

A, Expression levels of ESPIN protein (ESPIN-3; 34 kDa) in normal human epithelial

melanocytes (NHEM) and six human melanoma cell lines are shown. Intensities of bands

are presented as ratio (mean ± SD; n=3) relative to NHEM. Significantly different (**,

p<0.01) from NHEM by Student’s t-test. B, HE-stained nevus (top), primary melanoma

(middle) and lymphnode metastasis of melanoma (bottom) in humans are presented (Scale

bar: 100 μm). Representative ESPIN protein expressions (purple) in melanocytic nevus

(top), primary melanoma (middle) and lymphnode metastasis of melanoma (bottom)

evaluated by immunohistochemical analysis counterstained by methyl green with

anti-Espin-ru antibody are presented in lower (Scale bar: 100 μm) and higher (Scale bar: 20

μm) magnifications. C, Percentages of ESPIN-positive (Black) and -negative (white) cells

in melanocytic nevus, primary melanomas and lymphnode metastases of melanomas are

presented. Number of ESPIN-positive cells was divided by number of total cells in five

fields with 200-fold magnification in each tissue. Significantly different (**, p<0.01) from

nevi by Fisher’s exact test.

Actin-binding Protein, Espin: a Novel Metastatic Regulator for Melanoma

Takeshi Yanagishita, Ichiro Yajima, Mayuko Kumasaka, et al.

Mol Cancer Res Published OnlineFirst December 13, 2013.

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