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Title Page

Downregulation of Hepatoma-derived Growth Factor Contributes to Retarded Lung

Metastasis via inhibition of Epithelial-Mesenchymal Transition by Systemic POMC

Gene Delivery in Melanoma

Han-En Tsai1, Guei-Sheung Liu2, Mei-Lang Kung3, Li-Feng Liu4, Jian-Ching Wu5,

Chia-Hua Tang6, Ching-Hui Huang1, San-Cher Chen7, Hing-Chung Lam8,

Chieh-Shan Wu9, Ming-Hong Tai1,2,6,7

1Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804,

Taiwan

2Centre for Eye Research Australia, Department of Ophthalmology, University of

Melbourne, Victoria 3002, Australia.

3Department of Chemistry and Center for Nanoscience and Nanotechnology, National

Sun Yat-Sen University, Kaohsiung 804, Taiwan

4Department of Biological Science and Technology, I-Shou University, Kaohsiung

824, Taiwan.

on January 25, 2021. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 6, 2013; DOI: 10.1158/1535-7163.MCT-12-0832

http://mct.aacrjournals.org/

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5Doctoral degree Program in Marine Biotechnology, National Sun Yat-Sen

University, Kaohsiung 804, Taiwan

6Department of Medical Education & Research, Kaohsiung Veterans General

Hospital, Kaohsiung 813, Taiwan

7Center for Neuroscience, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.

8Division of Endocrinology and Metabolism, Kaohsiung Veterans General Hospital,

Kaohsiung 813, Taiwan.

9Division of Dermatology, Kaohsiung Veterans General Hospital, Kaohsiung 813,

Taiwan

Correspondence to:

Ming-Hong Tai, Ph. D.

Institute of Biomedical Sciences, National Sun Yat-Sen University

70 Lien-Hai Rd., Kaohsiung 804, Taiwan

Tel: 886-7-5252000 Ext. 5816, FAX: 886-7-5250197

E-mail: [email protected]

Running Title: POMC Gene Therapy for Metastatic Melanoma




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Keywords: Pro-opiomelanocortin (POMC), Melanoma, Metastasis, Hepatoma-derived

Growth Factor (HDGF), Epithelial-Mesenchymal Transition




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Abstract

The prognosis of malignant melanoma is poor due to high incidence of metastasis, underscoring the

demand for development of novel therapeutic strategies. Stress hormone pro-opiomelanocortin

(POMC) is the precursor for several anti-inflammatory peptides that hold promise for management

of cancer-related diseases. The present study evaluated the anti-metastatic potential and mechanism

of POMC therapy for metastatic melanoma. Adenovirus-mediated POMC gene delivery potently

inhibited the invasiveness of human and mouse melanoma cells. Moreover, after induction of lung

metastasis, systemic POMC expression significantly reduced the foci formation and

neovascularization in lungs. Mechanistic studies revealed POMC therapy inhibited the

epithelial-mesenchymal transition (EMT) of melanoma cells by upregulation of E-cadherin and

downregulation of vimentin and α-smooth muscle actin (α-SMA). In addition, microarray analysis

unveiled POMC gene transfer reduced the mRNA level of multiple pro-metastatic factors including

hepatoma-derived growth factor (HDGF). Cell culture and immunohistochemical studies further

confirmed that POMC gene delivery significantly decreased the HDGF expression in melanoma cells

and tissues. Despite of stimulating the invasion and EMT, exogenous HDGF supply only partially

attenuated the POMC-mediated invasion inhibition and EMT change in melanoma cells. Finally, we

delineated the contribution of melanocortins to POMC-induced inhibition of invasion, HDGF

downregulation and E-cadherin upregulation. Together, these results indicate that HDGF

downregulation participates in POMC-induced suppression of metastasis and EMT in melanoma.




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Introduction

Cutaneous melanoma, which begins with benign nevi and progresses to radial and vertical

growth, is one of the fastest rising malignancies worldwide (1). Metastasis or the spread of cancer

cells accounts for the major cause of cancer mortality including melanoma (2, 3). Melanoma is

notorious for resistance to conventional chemotherapy and radiotherapy (4). Moreover, melanoma

metastases are extremely aggressive and the average survival for patients with metastatic melanoma

is only between six to nine months (5). Therefore, novel modalities are demanded for control of

metastatic melanoma.

Hepatoma-derived growth factor (HDGF) is an acidic heparin-binding protein originally

isolated from the conditional medium of human hepatoma cell line, HuH-7 (6, 7) and could stimulate

with the proliferation of fibroblast (8), vascular smooth muscle cells (9), endothelial cells (8), and

several tumor cells, including hepatoma and melanoma (10-12). HDGF has also been recognized as

an important prognostic marker that HDGF overexpression was correlated with advanced stages and

poor survival outcome in several types of cancers including liver cancer (13) and esophageal

carcinoma (14). Furthermore, we have recently demonstrated that HDGF overexpression promoted

epithelial-to-mesenchymal transition (EMT) by E-cadherin down-regulation and vimentin

up-regulation, thereby promoting the invasion and metastasis in breast cancer cell (15). However, the

upstream molecules or signalling mechanisms that modulate HDGF expression and therefore affect

cancer progression remain largely unknown.




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The initiation of metastasis is characterized by the increased motility and invasiveness of

cancer cells. To acquire such invasive abilities, tumor cells undergo physiological changes such as

EMT. a process in which cells lose generally immotile epithelial characteristics and gain motile

mesenchymal properties (16, 17). EMT is mediated through several transcription repressors, such as

Snail, Slug, Twist and ZEB1, mesenchymal markers Vimentin and N-cadherin, and these EMT

inducers typically suppress the transcription of the E-cadherin gene, an epithelial cell marker and a

potent suppressor of tumor cell invasion and metastasis (18, 19). Therefore, identifying the

mechanisms that block the loss of E-cadherin expression is critical for preventing malignant

progression via suppression of the EMT.

Stress hormone pro-opiomelanocortin (POMC) is the precursor of several anti-inflammatory

neuropeptides including adrenocorticotropin (ACTH), melanocortins (α-, β- and γ-MSH), and

β-endorphin (β-EP). Recent studies indicate that the anti-inflammatory POMC therapy effectively

suppresses the growth of murine primary tumors, including B16-F10 (20, 21) and Lewis lung

carcinoma (22). Furthermore, POMC-derived peptide, α-MSH, has been reported to trigger

melanoma/melanocyte differentiation (23, 24) and potently inhibits both the in vitro and in vivo

invasion of highly invasive B16-BL6 melanoma cells (25, 26). A recent study has indicated that

prophylactic α-MSH treatment decreased the metastatic potential of melanoma cells in vitro and in

vivo (27). These findings suggested peripheral POMC expression may inhibit invasive and

metastasis. However, the underlying mechanism by which POMC-derived peptides inhibit melanoma




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metastasis has not been fully understood. Therefore, it will be valuable to examine whether

POMC-derived peptides could be a novel regulator of EMT.

In this study, we first evaluated the therapeutic efficacy of POMC gene therapy for lung

metastasis after administration of melanoma cells into circulation by bio-imaging and histological

analysis. Subsequently, the therapeutic mechanism underlying POMC therapy was explored by

examining tumor angiogenesis and EMT profiles. Finally, microarray analysis was employed to

identify the involvement of HDGF downregulation in POMC-induced metastasis suppression.




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Materials and methods

Reagents- Generation of recombinant HDGF protein and anti-HDGF were previous described (10).

TGF-β was purchased from Sigma. Rhodamine B-dextran (10,000 Mw) was purchased from

Invitrogen. Anti-mouse E-cadherin, Vimentin antibodies were from Santa Cruz. Anti-mouse α-SMA

antibody was from Sigma. Anti-mouse β-actin was from Millipore. ACTH, α-MSH, β-MSH, and

γ-MSH were purchased from Bachem.

Cell culture - Mouse B16-F0, B16-F10 and human A2058 melanoma cells were purchased from

ATCC (Manassas, VA) and cultured in DMEM (Invitrogen; Carlsbad, CA) medium containing 10%

fetal calf serum (FCS), 2 mM glutamine, 100 mg/ml streptomycin and 100 U/ml penicillin at 37 oC

in 5% CO2 incubator. Cells were initially grown and multiple aliquots were cryopreserved. All the

cell lines were used within 6 months after resuscitation, and no further cell line authentication was

conducted. The GFP- and luciferase-expressing B16-F10 melanoma cells were generated as

previously described (20).

Preparation of adenovirus vectors - The recombinant adenovirus vectors containing green

fluorescent protein (Ad-GFP) and POMC (Ad-POMC) were generated as previously described (20,

28).

Metastatic melanoma model and gene delivery - Male C57BL/6JNarl mice (4-6 weeks old) were

purchased from the National Laboratory Animal Center (Taipei, Taiwan), and housed under specific




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pathogen-free conditions. All animal experiments were carried out under protocols approved by

Animal Care and Use Committee (IACUC) of National Sun Yat-Sen University (Kaohsiung, Taiwan;

approval ID, NSC 98-2320-B-110-004-MY3/97014).

In the metastasis model, 5 × 105 B16-F10 cells were re-suspended in 0.1 ml PBS and injected

into the tail vein of C57BL/6 mice at day 0 to induce the pulmonary metastasis. For liver-based gene

delivery, C57BL/6 mice are injected with adenovirus vectors: Ad-POMC (1 x 109 pfu) or Ad-GFP (1

x 109 pfu) via tail vein at day 1. Metastatic progression was monitored and quantified by using

non-invasive bioluminescence as previously described (29). The mice were sacrificed on day 14 and

the lungs were harvested for consequence analysis.

Immunohistochemistry - Immunohistochemistry was performed as described previously (20). The

primary antibodies are CD31 (1:100; Novocastra), α-SMA (1:200), E-cadherin (1:250) and Vimentin

(1:200).

Western blot analysis - Whole cell protein extracts were prepared as described previously (20).

Proteins were separated on 8% to 16% AmershamTM ECLTM gels and western blotted with

anti-α-SMA (1:1000), E-cadherin, Vimentin, (1:500) and β-actin (1:5000).

Flow cytometry - 1 x 106 infected-B16-F10 cells were harvested and washed twice with PBS and

incubated with blocking buffer (5% BSA in PBS) for 1 hour. For surface molecules content analysis,

cells were incubated with primary antibodies for 1 hour (E-cadherin, 1:500), and followed by

incubation with Alexa 488-conjugated secondary antibodies for 30 minutes (Molecular Probes). The




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data from Flow cytometry was analyzed by Cell Lab QuantaTM SC (Beckman Coulter Inc.; Brea,

CA).

Microarray Analysis- The total RNA was isolated from B16-F10 cells infected with Ad-GFP or

Ad-POMC at MOI of 1000 for 24 h using RNAzol (TEL-TEST Inc., Friendswoods, TX). The

integrity and quality of RNA samples were confirmed by using agarose electrophoresis and the

Lab-on-chip system. Afterward, 0.5 μg RNA was amplified and labeled with Cy3 (the FC sample)

and Cy5 (the MC sample) respectively as previous described (30). A Qiagen RNeasy Mini Kit

(Qiagen Inc., Valencia, CA) was used to purify fluorescent cRNA probes. An equal amount (2 μg) of

Cy3 and Cy5 labeled probes were mixed and used for hybridization on one Agilent Mouse V2 Oligo

Microarray (array kit serial number US00030099; Agilent Techonologies Inc., Santa Clara, CA)

following the protocol provided by the manufacturer. The hybridization signals were acquired by

using Agilent 2100 Bioanalyzer (G2938B) and analyzed using Agilent G2567AA Feature Extraction

Software (v7.5).

Real-time PCR - RNA was isolated from B16-F10 cells using RNAzol (TEL-TEST, Inc.,

Friendswoods, TX). For reverse transcription, 5 μg of total RNA was used for reverse transcription

with SuperscriptTM III (Invitrogen Co.) using olio-dT and random primers. One-twentieth of

reverse-transcription products were used as template for real-time PCR in Lightcycler (Roche) using

a SYBR green I assay. PCR reaction was performed in 20 μl SYBR Green PCR Master Mix (Roche)

containing 100 μM forward primers and reverse primers, and approximately 30 ng cDNA.




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Amplification and detection were performed by: 1 cycle of 95 °C for 10 minutes, 40 cycles of 95 °C

for 5 seconds, and 62 °C for 5 seconds, and 72 °C for 10 seconds. The primer sequences for

mouse HDGF were: forward primer 5’-CCGGATTGATGAGATGCCTGA-3’, reverse primer 5’-

TTGTTGGGCTTGCCAAACT-3’, which amplified a 150-bp HDGF cDNA fragment. The β-actin

mRNA level was determined using: forward primer 5’-TCACCCACACTGTGC

CTATCTACGA-3’and reverse primer 5’-CAGCGGAACCGCTCATTGCCAATGG-3’, which

amplified a 295-bp β-actin cDNA fragment.

Invasion assay - Invasion assay was assessed using a Boyden chamber as previously described (31).

Transfection and luciferase measurement - For transient transfection, adenovirus infected B16-F10

cells (in a six-well plate) at 80% confluence were transfected with plasmid DNA by using the

lipofectamine plus method (Invitrogen) according to the manufacturer’s instructions. For promoter

activity assay, cells were co-transfected with 1 μg HDGF-driven luciferase vector (32) or

E-cadherin-driven luciferase vector (generous gift from Dr. Yu-Sun Chang, Chang Gen University,

Taiwan) (33) and the 0.2 μg Renilla reniformis luciferase reporter vector (Promega, Madison, WI).

The luciferase activities in cells were determined using a Dual-Light kit (Promega, Madison, WI) in

a luminometer (Microlumat Plus LB96V; Berthold Technologies, Bad Wildbad, Germany) and

normalized with that of R. reniformis luciferase according to manufacturer’s instructions.

Statistic analysis - Differences between the groups were statistically evaluated using the one-way

ANOVA. The results are presented as mean ± SEM. All P values were two-tailed, and a P value of




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less than 0.05 was considered to be statistically significant.




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Results

POMC gene delivery inhibited the invasiveness of melanoma cells in vitro and lung metastasis of

B16-F10 melanoma in vivo

To evaluate the anti-metastatic potential of POMC gene delivery, we investigated the invasiveness of

murine (B16-F0 and B16-F10) and human (A2058) melanoma cells. It was found that POMC

overexpression significantly retarded the matrix-penetrating capability in these melanoma cells (Fig.

1A). Subsequently, we investigated the anti-metastatic efficacy of POMC therapy on lung metastasis

of B16-F10 melanoma cells in mice. For quantification analysis of metastatic events, mice were

administrated with B16-F10 cells which expressing firefly luciferase (Luc-B16-F10) at day 0 and

treated with adenovirus vectors on day 1, then subjected to bioluminescence analysis at various time

intervals (Fig. S1A). It was shown that the bioluminescence intensities in lungs of Ad-POMC-treated

mice were significantly weaker than those of control groups on day 14 (Fig. 1B). Microscopic

analysis revealed that lung tissues in Ad-POMC-treated mice were relatively intact with few visible

foci, whereas it was grossly damaged with numerous colonies in control groups (Fig. 1C).

Histological analysis further validated that the metastatic colonies were prominently reduced in the

lung tissue of Ad-POMC-treated mice (Fig. 1C).

To further investigate the time required for systemic POMC therapy to elicit metastatic

suppression, bioluminescence analysis was performed on day 0, day 7 and day 14 after induction of

lung metastasis. It was found that POMC therapy significantly reduced the lung bioluminescence




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intensities on day 7 and day 14 (Fig. S2), suggesting POMC therapy effectively perturbed the lung

metastasis within 7 days of treatment.

POMC gene delivery suppressed the neovascularization and colonization of metastatic melanoma

in lungs

Since angiogenesis plays a pivotal role in tumor metastasis and colonization, we then investigated

the influence of POMC therapy on lung neovascularization and colonization of metastatic melanoma.

After received the intravenous injection of GFP-B16-F10 cells for 14 days, mice were sacrificed

after injection of the fluorescent dye (rhodamine label-dextran) for vessels tracking and histological

analysis (Fig. S1B). Simultaneous analysis of rhodamine label-dextran and GFP fluorescence

revealed that both metastatic foci and neovascularization in lungs were prominently reduced in

Ad-POMC-treated compared with Ad-GFP-treated mice (Fig. S3A). Moreover, CD31

immunostaining also unveiled a significant reduction in the number and size of CD31-positive blood

vessels in Ad-POMC-treated foci compared with control (Fig. S3B). Thus, these findings indicate

that POMC gene delivery attenuates the lung metastasis of B16-F10 melanoma cells through

inhibition of neovascularization, thereby perturbing the colonization of metastatic melanoma in

lungs.




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POMC gene delivery reversed the epithelial-mesenchymal transition of melanoma through

up-regulation of E-cadherin and down-regulation of vimentin and α-SMA

Since the EMT is involved in tumor metastasis, we evaluated whether POMC therapy affected the

EMT of melanoma cells by examining the expression of EMT marker molecules. By using western

blot and flow cytometry analysis, it was showed that POMC gene delivery significantly elevated the

protein level of E-cadherin, an epithelial marker, while reduced the protein levels of mesenchymal

markers, including vimentin and α-SMA in melanoma cells (Fig. 2A). Flow cytometry analysis also

confirmed that POMC gene delivery enhanced the cell surface expression of E-cadherin in B16-F10

melanoma cells (Fig. 2B). Histological analysis demonstrated that E-cadherin immunostaining was

increased whereas both vimentin and α-SMA immunostaining intensities were decreased in

Ad-POMC-treated melanoma tissues (Fig. 2C). These observations strongly suggest POMC therapy

attenuates melanoma metastasis through EMT modulation in vitro and in vivo.

POMC gene delivery elicited HDGF downregulation in B16-F10 cells and metastatic melanoma

In addition to regulating EMT genes expression, we searched for the potential genes that contributed

to the anti-metastatic function of POMC therapy by microarray analysis. As showed in Table 1,

POMC gene delivery significantly decreased the gene expression of fibronectin, thymosin β4 (Tβ4),

connective tissue growth factor (CTGF) and HDGF. Since the role of fibronectin, Tβ4 and CTGF

during melanoma metastasis has been reported (34), we delineated whether HDGF down-regulation




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was involved in POMC-induced metastatic inhibition. By using RT-PCR and promoter assay, we

found that POMC gene delivery significantly decreased the gene expression of HDGF in melanoma

cells (Fig. 3A&B). Similarly, we also demonstrated that POMC gene delivery decreased the protein

levels of HDGF by western blot analysis (Fig. 3C). Moreover, immunohistochemical study revealed

the prominent reduction in HDGF immunostaining in Ad-POMC-treated metastatic melanoma in

vivo, but not in Ad-GFP-treated groups (Fig. 3D).

Exogenous HDGF supply failed to reverse the POMC-induced suppression of invasiveness and

EMT change in melanoma cells

Although elevated HDGF is associated with the tumor progression in melanoma (11), the function of

HDGF in invasiveness and EMT in melanoma cells has not been validated. Since POMC gene

delivery reduced HDGF expression in melanoma cells, we investigated whether exogenous HDGF

supply affected the invasiveness and EMT of B16-F10 melanoma cells. It was found that exogenous

HDGF significantly stimulated the invasion (Fig. 4A) and EMT in melanoma cells (Fig. 4B).

However, even in the presence of excessive HDGF, POMC gene delivery still potently suppressed

the invasion of melanoma cells (Fig. 4A). Moreover, HDGF treatment failed to abrogate the

POMC-mediated downregulation of vimentin and α-SMA as well as E-cadherin upregulation (Fig.

4B). Therefore, exogenous HDGF supply was not sufficient to abolish the anti-metastatic function of

POMC therapy in melanoma cells.




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Mimicking POMC gene delivery by POMC-derived peptides perturbed the invasiveness and EMT

in melanoma cells

To identify the neuropeptide(s) that contributed to the inhibition of invasion and reduction of EMT

change by POMC gene delivery, B16-F10 were treated with various POMC peptides (ACTH, and

α-, β-, γ-MSH) to recapitulate the effect of POMC gene delivery. For invasion capacity, it was

shown that application of ACTH, α-MSH, and β-MSH significantly retarded the matrix-penetrating

capability in these melanoma cells (Fig. 5A). In contrast, γ-MSH treatment had no effect. Similarly,

treatment with ACTH, α-MSH and β-MSH significantly decreased the HDGF expression in

B16-F10 cells whereas γ-MSH had no such effect (Fig. 5B). Moreover, by evaluating the effect of

POMC-derived peptides on EMT change of melanoma, it was found that application of ACTH,

α-MSH, or β-MSH significantly increased the E-Cadherin expression, whereas γ-MSH had no such

effect (Fig. 5C). Therefore, the POMC-derived melanocortins, including ACTH, α-MSH and β-MSH

but not γ-MSH, participated in POMC-mediated inhibition of tumor invasion and EMT change in

melanoma cells.




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Discussion

The novel findings of the present study are, (i) that systemic POMC gene delivery by post

treatment shows that significant retards lung metastasis via reduced tumor invasion, colonization and

angiogenesis in established melanoma. The systemic POMC expression seems tolerable given no

obvious changes in body weight and feeding behavior was observed in mice receiving POMC

therapy. (ii) Our findings also demonstrate that POMC gene delivery potentially inhibits EMT

change by E-cadherin up-regulation and vimentin, α-SMA down-regulation, and which contributes to

tumor cell invasion and metastasis in melanoma in vitro and in vivo. (iii) Through the microarray

analysis, our data suggests POMC gene delivery decreased the expression of HDGF, which

contribute to inhibition of tumor invasion via reducing EMT change. Moreover, exogenously

supplied HDGF studies confirm that HDGF expression directly regulates the invasion and EMT of

melanoma cells. Together with our early studies demonstrating POMC therapy suppresses melanoma

through MC1R/NFκB signaling and angiogenesis inhibition (20, 21), we herewith provide a

hypothetical model for POMC-induced metastasis suppression through HDGF depletion, which leads

to EMT perturbation, and angiogenesis inhibition (Fig. 5D).

Our studies demonstrated that POMC gene delivery inhibited the motility and invasiveness in

human and mice melanoma cells, that suggests attenuation of invasion is involved in the

POMC-mediated suppression of metastasis. In lung-metastasis model, B16-F10 melanoma cells are

directly injected into the bloodstream and metastatic colonies form more rapidly than that in the




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primary melanoma model, thereby allowing the rapid screening of anti-metastatic efficacy of

therapeutic agents. Current study, POMC therapy was initiated 24 hours after melanoma cells

entered the circulation. Based on our previous studies (21), an additional 12-24 hours was required

for the production of anti-inflammatory peptides in circulation after POMC transgene expression and

processing in the liver. Despite the delayed therapy, a significant reduction in lung by

bioluminescence image was observed in Ad-POMC-treated mice at as early as day 7 after

implantation. Thus, systemic POMC therapy was robust even when the tumor cells took a 24 hours

head start entering the circulation before the treatment began.

Tumor neovascularization plays critical roles for the development, progression and metastasis

of cancers (35) and novel therapeutic approaches have been developed for treatment of malignancies

by controlling angiogenic activities (36). Our previous studies demonstrated that POMC gene

transfer disrupted the angiogenic processes of cultured endothelial cells (31, 37) and primary tumor

neovascularization through α-MSH signaling pathway (21). In the present study, we further

demonstrated that systemic POMC therapy elicited the neovascularization blockade in metastatic

melanoma by using fluorescent dye tracking and CD31 immunostaining. The striking correlation

between neovascularized vessels and metastatic melanoma nodules strongly supports the dependence

of newly colonized melanoma cells on neovascularization. Because HDGF is an angiogenic factor

(12, 38), the decreased HDGF expression would likely contribute to the POMC-mediated




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angiogenesis inhibition and metastasis suppression. Together, these findings support that

neovascularization blockade is involved in the anti-metastatic mechanism of POMC therapy.

EMT was originally described by embryologists and occurs in many developmental processes,

however it is also a key step in cancer progression when cells acquire invasive behavior and

disseminate (39, 40). Down-regulation or loss of E-cadherin results in de-differentiation, gain of

invasiveness, and promotion of EMT in carcinoma cells including malignant melanoma (41). Our

finding reveals that E-cadherin protein level is up-regulated, whereas vimentin and α-SMA are

down-regulated in Ad-POMC-treated melanoma cells and lung colonies. Microarray analysis

unveiled that POMC is a potent anti-metastatic suppressor by simultaneous repression of multiple

pro-metastatic genes such as fibronectin, CTGF (42, 43), Tβ4 (27, 44) and HDGF (45). Our recent

study demonstrated that HDGF up-regulation is correlated with recurrence, lymph node metastasis

and EMT in breast cancer patients (15). Moreover, HDGF protein or gene delivery also regulates the

EMT in breast cancer cells through modulation of E-cadherin and vimentin expression. Here, our

findings demonstrate that inhibition of HDGF might contribute in POMC-mediated inhibition of

metastatic and tumor angiogenesis in melanoma progression. However, a recent study has

demonstrated that melanocyte-specific HDGF overexpression did not lead to oncogenic

transformation of melanocytes (46). The discrepancy between these studies remains to be resolved.

In summary, our study provides the proof-of-principle evidence supporting POMC therapy for

control of metastatic melanoma. Moreover, POMC therapy elicited down-regulation of HDGF




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expression, which may subsequently perturb the EMT process in melanoma. Future studies are

warranted to systematically evaluate the POMC-based therapy for management of metastatic

melanoma.

Author Disclosure Statement

The authors who have taken part in this study declared that they do not have anything to disclose

regarding funding from industry or conflict of interest with respect to this manuscript.

Acknowledgements

We thank Ming-Ru Chuang and Dr. Hsiao-Mei Kuo for technical assistance and National Sun

Yat-Sen University for support. Centre for Eye Research Australia acknowledges the Victorian State

Government’s Department of Innovation, Industry and Regional Development’s Operational

Infrastructure Support Program.

Grant Support

This work was supported by grants from the National Science Council, Taiwan (NSC 100-2325-B-

110-002-MY3 and NSC-99-2321- B-110-005) to MH Tai, the National Health and Medical Research

Council of Australia (NHMRC 09007G) to GS Liu, Kaohsiung Veterans General Hospital, Taiwan

(VGHKS-99-036) to CS Wu.




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Authors' Contributions

Conception and design: HE Tsai, GS Liu, LF Liu, JC Wu, CH Huang, HC Lam, CH Wu, MH Tai

Development of methodology: HE Tsai, GS Liu, JC Wu, SC Chen, MH Tai

Acquisition of data (provided animals, acquired and managed patients, provided facilities,

etc.): HE Tsai, GS Liu, ML Kung, CH Tang, MH Tai

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational

analysis): HE Tsai, GS Liu, MH Tai

Writing, review, and/or revision of the manuscript: HE Tsai, GS Liu, LF Liu, SC Chen, MH Tai

Administrative, technical, or material support (i.e., reporting or organizing data, constructing

databases): HE Tsai, ML Kung, CH Huang, SC Chen, MH Tai

Study supervision: HE Tsai, GS Liu, CH Huang, HC Lam, CH Wu, MH Tai




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30

*Statistically significance (P < 0.05)

Table 1. Genes downregulated in Ad-POMC-infected B16-F10 melanoma cells by microarray analysis

Gene Name Accession

Number

Normalized Ratio

(versus Ad-GFP) P value

qRT-PCR Ratio

(versus Ad-GFP)

hepatoma-derived growth

factor (Hdgf) NM_008231 0.749 0.012 0.57 ± 0.048*

thymosin, beta 4,

X chromosome (Tmsb4x) NM_021278 0.547 0.001 0.51 ± 0.026*

fibronectin 1

(Fn1) NM_010233 0.556 0.021 0.65 ± 0.028*

connective tissue growth factor

(Ctgf) NM_010217 0.688 0.003 0.57 ± 0.075*




31

Figure legends

Figure 1. Effects of POMC gene transfer on metastatic capability of melanoma cells in vitro

and in vivo.

(A) After infection with adenovirus vectors for 12 hours, the cells were subjected invasion assay.

Representative photomicrographs of migrated cells (B16-F0 cells, B16-F10 cells and A2058 cells)

through the Matrigel-coated filter were quantified respectively. (B) Live images and the

quantification of photon units for 14 days were shown. (C) After mice sacrificed, lungs were

harvested and the metastatic lung foci were counted with stereo-microscope. Representative H&E

staining pictures of lung tissue from mice were also shown. Quantification data are given as mean ±

SEM (n = 5) *: P<0.05, **: P<0.01.

Figure 2. POMC gene delivery decreased the EMT in metastatic melanoma by inhibiting the

regulatory protein expression in vitro and in vivo.

(A) EMT relative protein expression in B16-F10 cells were analyzed by western blot. (B) Surface

E-cadherin expression in B16-F10 cells was determined by Flow cytometry analysis. (C)

Immunohistochemical analysis was used to investigate E-cadherin (upper panel), vimentin (middle

panel) and α-SMA (lower panel) expression level in vivo. Quantification data are given as mean ±

SEM (n = 5). *: P<0.05. (Magnification, 10x and Bar, 500 μm; 40x and Bar, 200 μm)




32

Figure 3. The role of HDGF in POMC-mediated metastasis inhibition.

To analyze the HDGF gene expression, mRNA and protein extractions were isolated from B16-F10

cells after infection with adenovirus vectors for 48 hours. (A) HDGF mRNA level in

Ad-POMC-treated B16-F10 cells were evaluated by qRT-PCR. (B) HDGF transcriptional activity

was analysis in Ad-POMC-treated B16-F10 cells by using luciferase assay. (C) Immunoblot analysis

was performed to determine the levels of HDGF protein in Ad-POMC-treated B16-F10 cells. (D)

Histological analysis of HDGF expression in metastasis nodules from different treatment groups.

Quantification data are given as mean ± SEM (n = 5). *: P<0.05, **: P<0.01. (Magnification, 10 x

and bar, 500 μm; Insets, 40 x and bar, 200 μm).

Figure 4. HDGF expression in POMC-mediated EMT inhibition.

(A) Effect of exogenous HDGF supply on POMC-mediated inhibition of invasion in melanoma cells.

Adenovirus-infected-B16-F10 cells were treated with or without HDGF (10 ng/ml) for an additional

24 hours before harvest for invasion assay. (B) To investigate the effects of exogenous HDGF supply

on POMC-mediated inhibition of EMT in melanoma cells, adenovirus-infected-B16-F10 cells were

treated with or without HDGF (10 ng/ml) for an additional 24 hours before harvest for immunoblot

analysis. Quantification data are given as mean ± SEM (n = 6-8 per group). * p<0.05, ** p<0.01




33

Figure 5. Effect of POMC-derived peptides on invasion and EMT gene expression in

melanoma cells. (A) Effect of POMC-derived peptides (α-MSH, β-MSH, γ-MSH, and ACTH)

treatment on the invasion in melanoma cells. Adenovirus-infected-B16-F10 cells were treated with

or without peptides (10-8M) for an additional 24 hours before harvest for invasion assay. (B) To

investigate the effects of peptides treatment on gene expression of HDGF in B16-F10 cells. After

treated with or without peptides (10-8M) for an additional 24 hours, the cells were harvested and

expression level of HDGF was examined by Real-time PCR analysis. (C) The protein level of

E-cadherin was determined by immunoblot analysis. (D) Hypothetical scheme for POMC-mediated

metastasis suppression. Quantification data are given as mean ± SEM (n = 5 per group). * p<0.05, **

p<0.01



















Published OnlineFirst March 6, 2013.Mol Cancer Ther Han-en Tsai, Guei-Sheung Liu, Mei-Lang Kung, et al. Delivery in MelanomaEpithelial-Mesenchymal Transition by Systemic POMC GeneContributes to Retarded Lung Metastasis via inhibition of Downregulation of Hepatoma-derived Growth Factor

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