Strong Association of ARK5 with Tumor Invasion and Metastasis

J. Exp. Clin. Cancer Res., 23, 2, 2004
Strong Association of ARK5 with Tumor Invasion and Metastasis
G. Kusakai 1,2* , A. Suzuki 1* , T. Ogura 1 , M. Kaminishi 2 , H. Esumi 1
Investigative Treatment Division 1 , National Cancer Center Research Institute East, Chiba; Dept. of Gastrointestinal Surgery 2 , University
of Tokyo, Tokyo; Japan
We recently identified a novel human AMPK family member, ARK5, and discovered that is a major factor in Aktdependent
cancer cell survival (1) and migration activity through activation of MT1-MMPs in vitro. The mRNA
expression of other AMPK family members and ARK5 was measured using RT-PCR in human colorectal carcinoma
cell lines DLD-1, WiDr, HCT-15, SW620, LoVo, SW480, and mRNA expression of AMPK-α1, SNARK,
MELK and ARK5, but not AMPK-α2, was detected in every line. Quantitative-PCR (Q-PCR) to estimate the
amount of ARK5 mRNA expression in the cell lines showed that there is a variety of ARK5 expressions among
the cell lines and high expression was observed in a cell line derived from the metastatic lesion, LoVo. To determine
the effect of ARK5 overexpression on metastasis in vivo, we established human pancreas cancer cell line
PANC-1 stably transfected with ARK5 full-length expression vector (P/ARK) and DLD-1 stably transfected with
the same vector (D/ARK). Migration assay showed a remarkable increase in the activity both in P/ARK and
D/ARK, and an in vivo metastasis assay showed a marked increase of P/ARK in liver metastasis. Based on these
observations, it is suggested that ARK5 expression is involved in cancer invasion and metastasis.
Key Words: ARK5, Metastasis, Invasion, Glucose starvation, Migration
Blood vessel formation of tumors is often insufficient
to sustain its excessive growth and proliferation,
and the resulting poor blood supply, in the microenvironment
of tumor, usually exposes cancer cells to nutrient
starvation and hypoxia. The major activator of
angiogenic factors and glycolysis in response to hypoxia
is HIF-1 (hypoxia-inducible factor-1), which modulates
the transcriptional activity of vascular endothelial
growth factor (VEGF), glycolytic enzymes, nitric oxide
(NO), and heme-oxygenase type I genes as a cellular
defensive response or an adaptation under hypoxic conditions
(2,3). In addition to the hypoxic response, some
cancer cells have a high tolerance to nutrient starvation,
and we termed this tolerance austerity (4).
We previously demonstrated that the austerity and
hypoxic responses are essential for cancer cell survival
and tumor formation, and that they are dependent on
the activity of Akt and 5'-AMP activated protein kinase
(AMPK) (5,6). AMPKs are a family of metabolitesensing
serine/threonine protein kinases that are activated
by various cellular stresses. AMPK plays a major
role in protecting cells through mediating changes in
energy metabolism and phosphorylates and inhibits
acetyl-coenzyme A (CoA) carboxylase (ACC) and
activation of glucose transporters (7,8). We recently
identified a novel AMPK family member, ARK5,
which is activated by glucose starvation through Akt
activity, and demonstrated that it is a mediator of Aktdependent
cancer cell survival during glucose starvation
and of migration activity in cancer cell lines (1).
The results of our recent in vitro study suggested that
ARK5 is directly involved in cancer metastasis in an
Akt-dependent manner (9).
D/ARK which was stably transfected with ARK5
full-length expression vector into DLD-1 showed higher
migration activity than DLD-1. We also established
a PANC-1 cells derivative, P/ARK cells, by transfecting
ARK5 full-length expression vector into PANC-1
cells, and comparison of the ability of PANC-1 and
P/ARK cells to give rise to liver metastasis in SCID
mice revealed that overexpression of ARK5 resulted in
increased metastasis. Based on these findings, we concluded
that ARK5 is one of the factors associated with
cancer metastasis.
263

G. Kusakai et al.
Materials and Methods
Homology Search for Subunit Structures of ARK5
and AMPK Catalytic Subunits. Based on their reported
amino acid sequences, a BLAST search analysis was
used to detect a putative consensus catalytic peptide
sequence of the AMPK family members AMPK-α1,
AMPK-2, MELK, SNARK, and ARK5. A homology
search was performed by computer analysis using
GENETYX 10.1 software.
Cell Lines and Culture. All human carcinoma cells
were cultured in Dulbecco's modified Eagle Medium
(DMEM: NISSUI, Japan) supplemented with 10%
fetal bovine serum, 2 mM L-glutamine and 25 mM
HEPES-NaOH (pH 7.3) at 37°C under an atmosphere
of 5% CO 2 in air.
Reverse Transcription-PCR (RT-PCR) and Quantitative-RT-PCR
(Q-RT-PCR). Total RNA was isolated
from cancer cells by the AGPC method using ISOGEN
(NIPPONGENE, Japan), and 1 µg of total RNA was
reverse transcripted into cDNA with oligo-dT primers
and AMV-reverse transcriptase (TaKaRa, Japan)
according to the manufacturer's instructions. The
cDNA was subjected to 25 cycles of PCR amplification
on a PCR Thermal Cycler 480 (TaKaRa, Japan), and the
PCR primers used for ARK5 detection were: forward,
5'-ATGCTAAGTACCCTCTGAATG-3', and reverse,
5'-GCAACAAGCAGTCAGTCGATC-3'. PCR products
were fractionated by agarose electrophoresis,
stained with ethidium bromide, and visualized under
UV light. GAPDH served as a control for PCR.
A Light-Cycler (Roche, Germany) was used to
detect the number of ARK5 and GAPDH gene copies.
The amplification mixture contained cDNA derived
from 100 ng of total RNA. The PCR primers used for
GAPDH detection were: forward, 5'-AGGGCTG-
GTTTTAACTCTGGT-3', and reverse, 5'-CCC-
CACTTGATTTTGGAGGGA-3'. Q-PCR was performed
under cycling conditions of 95°C for 10 min,
followed by 40 cycles of denaturation at 95°C for 10
sec, annealing at 60°C for 10 sec, extension at 72°C for
20 sec.
Transfection and Selection. For transfection, 1˘10 5
cells were seeded into each well of a 6-well plate, and
ARK5-full length expression vector was transfected
into DLD-1 and PANC-1 cells with TransFast transfection
reagent (Promega Corp., USA) according to the
manufacturer's instructions with some modifications.
Stable transfectants were selected with G418.
264
Immunoblotting. Cells were washed with PBS containing
1mM sodium ortho-vanadate, 10mM sodium
fluoride and 25 mM sodium glycerophosphate. Cells
were collected, and lyzed with a buffer containing 1%
sodium dodecyl sulfate (SDS), 1 mM SOV4 and 10
mM Tris-HC1 (pH 7.4). Proteins were separated on a
10% SDS polyacrylamide gel, and transferred onto a
polyvinylidene difluoride membrane. A polyclonal
antibody to human SNARK was made by immunizing
rabbits with a peptide based on the sequence of
SNARK (KKPRQRESGYYSSPEPS) and it was found
to crossreact with ARK5 (1). Membranes were incubated
with the anti-SNARK antibody at dilution of
1:1000 and anti-rabbit antibody conjugated with horseradish
peroxidase (HRP) was used as a second antibody.
For visualisation, Western blotting detection
reagent (ECL: Amersham Pharmacia Biotech, UK) and
Hyperfilm ECL (Amersham Pharmacia Biotech, UK)
were used.
Migration Assay. In vitro migration assay was performed
with a matrigel-coated invasion chamber (Becton-DickinsoN,
USA). DLD-1 cells (5˘10 4 /chamber)
were seeded into each chamber, and the media in the
inner and outer chamber were changed after 6 hrs. The
cells that had invaded were counted under a phase-contrast
microscope after 48 hrs.
In vivo Liver Metastasis Assay in SCID Mice. SCID
mice (CB-17/Icr Crj-scid, 5-week-old males) were
anesthetized with pentobarbital-Na (Nembutal:
ABBOTT, USA). Ventrotomy was performed, the
spleen was exposed, and 200 µl of serum free DMEM
containing 1˘10 6 PANC-1 or P/ARK cells was injected
under the splenic capsule of ten mice each. The mice
were sacrificed 5 weeks later, and liver metastasis was
assessed by counting the number microscopically. The
liver metastases were stained with hematoxylin and
eosin (H.E.) and confirmed pathologically.
Results
Subunit Structures of ARK5 and AMPK Catalytic
Subunits. The AMPK family consists of five members,
AMPK-α1, AMPK-α2, MELK, SNARK, and ARK5
recently identified (Fig.1). As shown in Fig. 1, all
AMPK members have a putative consensus catalytic
domain structure. ARK5 contained the putative consensus
catalytic peptide sequence at amino acids 55 -
305, and an Akt-phosphorylation site (RXRXXS) was
found near the C-terminal (Fig.1). The C-terminal sub-

Fig. 1 - Peptide structure of the AMPK catalytic subunits of
AMPK-α1, AMPK-α2, MELK, SNARK, and ARK5.
Analysis of the putative peptide catalytic domain and Akt
phosphorylation motif was searched by GENETYX 10.1.1
on a computer.
unit-targeting site of AMPK-α1 and AMPK-α2 bound
to β and γ subunits, and AMPK-α1 and AMPK-α2 possessed
an autoregulatory domain, unlike the other three
AMPK members (Fig.1). The overall homology of
ARK5 calculated using GENETYX 10.1 was 47%
compared with AMPK-α1 and 55% compared to
SNARK.
ARK5 Expression in Cancer Cell Lines. We previously
demonstrated that at least AMPK-α1 and 2 contribute
to tumor progression and survival via austerity
(4). RT-PCR to confirm mRNA expression of AMPK
family members, in six human colorectal carcinoma
cell lines, DLD-1, WiDr, HCT-15, SW620, LoVo, and
SW480 (Fig.2A), showed that all six cell lines
expressed AMPK-α1, SNARK, and MELK mRNA,
and that AMPK-α2 was strongly expressed in HCT-15,
SW620, LoVo, and SW480, but not in DLD-1 or WiDr.
The amount of ARK5 mRNA expression was estimated
by Q-PCR in the same human colorectal carcinoma
cell lines, and the results showed that ARK5 was highly
expressed in LoVo and SW480 (Fig.2B).
Correlation between ARK5 Expression and Migration
Activity. In our previous studies, ARK5 expression
is often associated with tumor cell survival and ARK5
is a direct downstream target of Akt1 (1). We asked if
ARK5 might be involved in tumor cell migration and
metastasis. In order to answer this question, an in vivo
Strong Association of ARK5 with Tumor Invasion and Metastasis
Fig. 2 - mRNA expression of other AMPK family members and
ARK5 in human colorectal carcinoma cell lines. A. Expression
of AMPK family member mRNA was estimated
by RT-PCR in DLD-1, WiDr, HCT-15, SW620, LoVo,
andSW480. B. ARK5 mRNA expression in human colorectal
carcinoma cell lines was measured by Q-PCR.
*Significant at p < 0.05
migration assay was performed using DLD-1 and
D/ARK cells. As shown in Fig. 3A, ARK5 overexpression
of D/ARK was confirmed by western blotting. In
comparison with DLD-1, D/ARK showed an exceedingly
higher activity with a significant difference
(p

G. Kusakai et al.
Fig. 3 - The correlation between ARK5 overexpression and migration
activity. A. Confirmation of ARK5 expression in
DLD-1 and D/ARK cells by using western blot. B. Migration
activity induced by ARK5 overexpression. **Significant
at p < 0.01
In vivo Liver Metastasis Assay in SCID Mice. To
evaluate the effect of ARK5 overexpression on in vivo
metastasis, 1˘10 6 PANC-1 or P/ARK cells were transplanted
under the splenic capsule of SCID mice. ARK5
overexpression of PANC-1 and P/ARK was examined
by western blotting, and P/ARK cells were confirmed
to overexpress ARK5 protein (Fig.4A).
Five weeks later, 40% of the mice transplanted with
P/ARK cells showed liver metastasis (Fig.4B,C). The
number and size of tumor nodules of the liver metastases
were counted (Table I). One of them also showed
extensive peritoneal dissemination in the peritoneal
wall, mesentery, omentum, and intra-abdominal lymph
nodes. No liver metastasis or peritoneal dissemination
was found in the SCID mice transplanted with PANC-
1 cells (Fig.4C). In similar experiments in which the
number of cells transplanted was doubled, the incidence
of liver metastasis in the PANC-1 transplanted
SCID mice increased by only 10% (data not shown).
Discussion
Tumors are classified clinically into two types according
to their vascularity: hypervascular and hypovascular.
Since low circulating blood volume is caused
by hypovascularity, and insufficient blood circulation
can cause both hypoxia and nutrient starvation, hypovascular
tumors are presumed to have acquired high
tolerance to nutrient starvation. Indeed, we previously
reported that cell lines isolated from pancreatic cancer,
which is a representative hypovascular tumor showed
extremely strong tolerance to nutrient starvation (4, 5),
266
Fig. 4 - in vivo liver metastasis assay after transplantation of
PANC-1 and P/ARK cells in SCID mice. A. Western blotting
was performed to confirm ARK5 expression in
PANC-1 and P/ARK cells. B. Macroscopic view of liver
metastasis after transplantation under the splenic capsule
of SCID mice and between microscopic view after staining
with hematoxylin and eosin. C. Comparison the incidence
of liver metastasis in SCID mice after transplantation
of PANC-1 and P/ARK cells.
and we termed this tolerance austerity (4). The molecular
mechanism of austerity has not been fully elucidated,
but Akt and AMPK were found to be involved
(1, 4, 5, 10, 11).
Accumulating evidence has recently demonstrated
that activation of AMPK during myocardial and skele-

Table I - Effect of ARK5 overexpression on liver metastasis
Tumor nodules of liver metastasis
Mean number Mean size (mm)
PANC-1 0 0
P/ARK 5.3±10.9 (0-33)* 0.8±1.1 (0-3.0)*
Mean±SD (range), *p

G. Kusakai et al.
new biochemical target for cancer therapy. Cancer Res. 60:
6201-6207, 2000.
6. Chen Z.P., McConell G.K., Michell B.J., Snow R.J., Canny
B.J., Kemp B.E.: AMPK signaling in contracting human
skeletal muscle: acetyl-CoA carboxylase and NO synthase
phosphorylation. Am. J. Physipl. Endocrinol. Metab. 279:
1202-1206, 2000.
7. Dale S., Wilson W.A., Edelman A.M., Hardie D.G.: Similar
substrate recognition motifs for mammalian AMPK-activated
protein kinase, higher plant HMG-CoA reductase kinase-A,
yeast SNF1, and mammalian calmodulin-dependent protein
kinase I. FEBS Lett. 361: 191-195, 1995.
8. Gao G., Widmer J., Stapleton D., Teh T., Cox T., Kemp B. E.,
Witters L.A.: Catalytic subunits of the porcine and rat 5'-
AMP-activated protein kinase members of the SNF1 protein
kinase family. Biochim. Biophys. Acta. 1266: 73-82, 1995.
9. Suzuki A., Lu J., Kusakai G., Kishimoto A., Oguna T., Esumi
H.: ARK5 is a tumor invasion-associated factor downstream
of AKT signalling. Mol. Cell. Biol. 24:3526-3535, 2004.
10. Kato K., Ogra T., Kishimoto A., Minegishi Y., Nakajima N.,
Esumi H.: Critical roles of AMP-activated kinase in constitutive
tolerance cancer cells to nutrient deprivation and tumor
formation. Oncogene 21: 6082-6090, 2002.
11. Xi X., Han J., Zhang J.Z.: Stimulation of glucose transport by
AMP-activated protein kinase via activation of p38 mitogenactivated
protein kinase. J. Biol. Chem. 276: 41029-41034,
2001.
12. Sambandam N., Lopaschuk G.D.: AMP-activated protein kinase
(AMPK) control of fatty acid glucose metabolism in the
ischemic heart. Prog. Lipid Res. 42: 238-256, 2003.
13. Hopkins T.A., Dyck J.R., Lopaschuk G.D.: AMP-activated
protein kinase reglation of fatty acid oxidation in the ischemic
heart. Biochem. Soc. Trans. 31: 207-212, 2003.
268
14. Marsin A.S., Bouzin C., Bertrand L., Hue L.: The stimulation
of glycolysis by hypoxia in activated monocytes is mediated
by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase.
J. Biol. Chem. 277: 30778-30783, 2002.
15. Abbud W., Habinowski S., Zhang J.Z., Kendrew J., Elkairi
F.S., Kemp B.E., Witters L.A., Ismail-Beigi F.: Stimulation of
AMP-activated protein kinase (AMPK) is associated with enhancement
of Glut1-mediated glucose transport. Arch.
Biochem. Biophys. 380: 347-352, 2000.
16. Haas T.L., Davis S.J., Madri J.A.: Three-dimensional type I
collagen lattices induce coordinate expression of matrix metalloproteases
MT1-MMP and MMP-2 in microvascular endotherial
cells. J. Biol. Chem. 273: 3604-3610, 1998.
17. Lei T.C., Vieira W.D., Hearing V.J.: In vitro migration of
melanoblasts requires matrix metalloprotease-2: implications
to vitiligo therapy by photochemotherapy. Pigment Cell Res.
15: 426-432, 2002
18. Tester A.M., Ruangpani N., Anderso R.L., Thompson E.W.:
MMP-9 secretion and MMP-2 activation distinguish invasive
and metastatic sublines of a mouse mammary carcinoma system
showing epithelial-mesenchymal transition traits. Clin.
Exp. Metastasis 18: 553-560, 2000.
A selected article from JSGC
Hiroyasu Esumi, M.D.
Investigative Treatment Division,
National Cancer Center Research Institute
East, 6-5-1 Kashiwanoha, Kashiwa,
Chiba 277-8577, Japan
Tel.: +81-4-7134-6880; Fax: +81-4-7134-6859
E-mail:hesumi@east.ncc.go.jp