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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.
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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: minghongtai@gmail.com
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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21
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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23
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*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*
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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)
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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
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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
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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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