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Background: Accumulating data suggest a role of chemokines in tumor cell metastasis. CCR4 has been implicated in hematologic malignancies and recently also in solid tumors. Herein, we hypothesized that CCR4 might be expressed and support
migration of colon cancer cells. We used quantitative RT-PCR and flowcytometry and found clear-cut mRNA levels of CCR4 and surface expression of CCR4 on a colon cancer cell line (HT-29) and on tumor cells (AZ-97) isolated from a patient with colon
cancer. Moreover, we found that the CCR4 ligand CCL17 (TARC) was a potent stimulator of colon cancer cell migration. This CCL17-induced colon cancer cell migration was inhibited by pre-incubation of the colon cancer cells with an antibody directed
against CCR4 or an antagonist against CCR4. Next, we examined CCL17-induced signalling in colon cancer cells and found that CCL17 increased mRNA formation of RhoA-C in colon cancer cells. By use of ELISA, it was found that CCL17 increased total
RhoA and active RhoA protein levels in colon cancer cells. The Rho-kinase inhibitor Y-27632 abolished CCL17-induced colon cancer cell chemotaxis. In addition, inhibition of isoprenylation by GGTI-2133 markedly reduced colon cancer cell migration
triggered by CCL17. Taken together, our novel findings suggest that colon cancer cells express CCR4, which mediates CCL17-induced migration of colon cancer cells in a RhoA/Rho-kinase-dependent manner. Thus, these data indicate for the first time that
the CCL17-CCR4 axis might be involved in the regulation of colon cancer cell metastasis.
Colorectal cancer (CRC) is the fourth leading cause of cancer-related death in Europe (1).
Distant organ metastasis is the major cause of mortality in patients with CRC. The mechanisms
behind cancer cell metastasis are not fully understood but convincing data suggest that increased
expression of adhesion molecules and capacity to respond to chemotactic stimuli are necessary
for the spread of tumor cells(2). Accumulating data indicate that chemokines regulates multiple
aspects of tumor cell biology, including proliferation, survival, angiogenesis and migration (3).
Chemokines belong to a superfamily of small molecules (8–14 kDa) that were initially discovered
because their interaction with chemokine receptors was found to regulate trafficking of leukocytes
to sites of inflammation (4, 5). Colon cancer cells can also express chemokine receptors,
including CXCR3, CXCR4 and CCR6 and respond to specific chemokines (5). For example, it
has been demonstrated that CXCR3 is expressed on colon cancer cells and mediate lymph node
and lung metastasis (6). These findings underline the importance of studying the expression and
function of chemokine receptors in colon cancer cells to better understand the molecular
mechanisms controlling CRC metastasis.
CCR4 is a key receptor for regulating chemokine-dependent immune homeostasis and is
selectively expressed on regulatory T cells and Th2 cells (7). One recent study forwarded that
CCR4 is involved in tumor cell evasion by stimulating accumulation of regulatory T-cells in the
tumor microenvironment (8). Several investigations have reported that CCR4 plays an important
role in hematologic malignancies, such as adult T-cell leukaemia, acute myeloid lymphoblastic
leukaemia and chronic lymphocytic leukaemia (9). Recent studies have reported that gastric and
breast cancer cells express functional CCR4 receptors. Lee et al. (10) showed that CCR4 is
expressed in 17% primary gastric cancers and is associated with a poor prognosis in patients
with gastric cancer. Another investigation reported that only CCR4-positive cancer cells had the
capacity to metastasize to the lung in the metastatic 4T1 mouse mammary carcinoma model (11).
Although these emerging evidences suggest a role of CCR4 in gastric and mammary
carcinogenesis, the role of CCR4 in colon cancer biology remains elusive.
Based on the considerations above, we hypothesized that CCR4 might be expressed and
mediate migration of colon cancer cells. For this purpose, we used an established colon cancer
cell line (HT-29) and a primary colon cancer cell line (AZ-97) isolated from a patient with colon
cancer.
Introduction Results
Abstract
This study was supported by the Swedish Medical Research Council (2009-4872),
Crafoordska stiftelsen, Einar och Inga Nilssons stiftelse, Greta och Johan Kocks
stiftelser, Fröken Agnes Nilssons stiftelse, Magnus Bergvalls stiftelse, Mossfelts stiftelse,
Nanna Svartz stiftelse, Ruth och Richard Julins stiftelse, Dir. A. Påhlsson’s Foundation,
Swedish Cancer Foundation, Malmö University Hospital Cancer Foundation, Lundgren’s
Foundation, Gunnar Nilsson’s Foundation and Apotekaren Hedberg’s Fond, Malmö
University Hospital and Lund University.
Department of Clinical Sciences, Surgery Research Unit, Malmö University Hospital,
Lund University, Malmö, Sweden
Conclusion Our data demonstrate for the first time increased expression of functional CCR4 in colon
cancer cells, suggesting that its chemokine ligand might be involved in the regulation of
colon cancer metastasis.
References
Methods Cell lines and Cell culture The human epithelial colon adenocarcinoma cell lines HT29 was obtained from American Type
collection Culture ATCC. AZ-97, new constructed cell line by surgery department in Malmö
University Hospital, were cultured in Dulbecco's Modified Eagle Medium (DMEM); (Sigma–Aldrich
Co.), supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mmol/L L-
glutamine at 37ºC and 5%CO2
Protein expression Flowcytometry Surface expression of chemokine receptors were assessed
using flowcytometry FACScalibur (Becton Dickinson).
Chemotaxis assay Chemotactic responses of colon cancer cells were evaluated by using 24-well cell migration
chambers with 8µm pore size inserts (Corning Coster Corporation) for HT-29 and AZ-97 cell lines.
For transmigration inhibition, cells were treated the same way as in migration assay except they
were preincubated for 30min with different concentrations of anti CCR4 antibody, 1G1, santacruz
biotechnology, and CCR4 antagonist, 2-[1,4'-Bipiperidin]-1'-yl-N-cycloheptyl-6,7-dimethoxy-4-
quinazolinamine dihydrochloride, Tocris bioscience
Proliferation assay Cell proliferation was evaluated in triplicates using Cell Counting Kit-8 assay (CCK-8; Sigma-
Aldrich,).
RhoA activation assay RhoA-GTP activity was measured using the G-LISA RhoA Activation Assay for measuring Total
and Active RhoA, Biochem Kit (Cytoskeleton Inc., Denver, CO)
Statistical Analysis All statistical analyses were performed using SigmaPlot® 10 software. Statistical comparisons
between more than two datasets were made with Kruskal-Wallis One Way Analysis otherwise
Mann–Whitney rank-sum test was used and P value < 0.05 considered as significant.
Figure 2. Cell surface chemokine receptors expression histograms, A. AZ-
97 cell line, and B. HT-29 cell line, show that CXCR3 and CCR4
are the highest receptors expressed on the cell surface of both
cell lines. Unstained cells were used as negative control and
single tube staining used for each receptor. Data were analyzed
in duplicate at least three times.
Figure 1.
Gel electrophoresis of QRT-PCR
products indicates that both cell
lines expressed increased
chemokine receptors mRNAs of
CCR4, CXCR3, and CCR8. Beta
actin was used as housekeeping
gene. The Data shown represent a
typical one of three independent
experiments.
Figure 5. Inhibition of cell migration using anti -CCR4 antibody (1G1) and
CCR4-antagonist;2-[1,4'-Bipiperidin]-1'-yl-N-cycloheptyl-6,7-
dimethoxy-4-quinazolinamine hydrochloride. Cell migration was
significantly inhibited (*P= 0.006, ##P= 0.003) in A, B; HT-29 cell
line, and (**P= 0.008) in C,D; AZ-97 cell line, (n=4). Cells were
starved for 24 hours and checked for viability by trypan blue
exclusion assay and they were > 99% viable cells. 100 ng/ml
CCL17/TARC was added to the lower chambers and different
concentrations of antibody1, 5, 20 µg/ml and 100, 140, 200 ng/ml
antagonist were added to the upper chambers and cells were
seeded at density of 8×105 cells/ml and incubated for 24 hours at
37ºC, 5% CO2. Non migrated cells were washed three times by
PBS under constant vacuum and the inserts stained by 0.5%
crystal violet in 25% methanol and then counted microscopically
using 40 × high power fields for at least 5 different fields. Data
shown represent %( mean ± SEM) of three independent
experiments.
A
Anti-CCR4 Ab (µg/ml)
Cell
mig
ra
tio
n %
0
20
40
60
80
100
120
__ 1 5 20
*
*
*
B
CCR4 Antagonist (ng/ml)
Cell
mig
rati
on
%
0
20
40
60
80
100
120
__ 100 140 200
##
C
Anti- CCR4 Ab (µg/ml)
Cell
mig
ra
tio
n %
0
20
40
60
80
100
120
__ 1 5 20
**
**
__ 100 140 200
CCR4 Antagonist (ng/ml)
Cell
mig
ra
tio
n %
0
20
40
60
80
100
120
D
**
Figure 7.
Invitro effect of isoprenylation inhibition on HT-29 Cell migration in response to CCL17/ TARC 100ng/ml stimulation for
24hr. A, Geranylgeranyl transferase inhibitor ,GGTI-2133, 1 and 10 µM .B, simvastatin 25, 50, and 100 µM. C, Farnesyl
transferase inhibitor, FTI 277, 10 and 20 µM. D, Rho kinase inhibitor, Y-27632, 10 and 50 µM. Cells were serum starved
and seeded at density of 8×105 cells/ml for 24hr. complete media with or without 1, 10, and 100 ng/ml CCL17/TARC
were added to the lower chambers. Cells were preincubated with the respective inhibitors for 30 min at 37ºC, 5% CO2
and placed in the lower chambers and then incubated at 37ºC, 5% CO2. after 24hr the cells on the lower sides of the
inserts were fixed by cold 100% methanol and stained with 0.5% crystal violet and counted microscopically using 100×
HPF at least in 5 different fields. GGTI 1 and 10µM, Simvastatin 50 and 100µM, FTI 20 µM, Y-27632 10 and 50µM, all
induce significant inhibition of cell migration (**P= 0.008 vs control cells, with CCL17/TARC stimulation) n=8. Data shown
represent %( mean ± SEM) of at least two independent experiments.
Figure 4. Expression of CCR4 in human colon carcinoma.
Comparative RT–PCR analysis of CCR4 in surgical
resection pieces of human colon carcinoma (n=37)
compared with healthy colon tissues (10cm adjacent to
the tumor).
Re
lati
ve
leve
l o
f C
CR
4 e
xp
ress
ion
0
2
4
6
8
10
12
14
16
Healthy colon Tumor colon
P= 0.001
Gene expression Quantitative RT- PCR:
Quantitative Real time polymerase chain reaction (qRT – PCR) was done for
chemokine receptors gene expression in a final volume of 25 µl using syber green
dye for absolute gene quantification (Stratagene’s Mx Real-Time PCR Platform)
Acknowledgements
Figure 3. Cell migration in response to CCL17/ TARC stimulation, indicates cell migration with or without 1, 10, and 100 ng/ml
CCL17/TARC in A.12 hrs, B. 24 hrs, and C. 48hrs in HT-29 and AZ-97 cell lines. Cells were serum starved and seeded at
density of 8×105 cells/ml. complete media containing the respective ligand were added to the lower chambers and incubated
at 37ºC, 5% CO2 for the indicated time set points. The cells on the lower sides of the inserts were fixed by ice cold 100%
methanol and stained with 0.5% crystal violet and counted microscopically using 10× HPF at least in 5 different fields.
CCL17/TARC induces significant cell migration in a dose and time dependent manner in both cell lines, **P= 0.008 vs control
cells (without CCL17/TARC stimulation), *P=0.01 vs control cells, #P= 0.03 vs control cells. Data shown represent %( mean
± SEM) of at least three independent experiments. Migration index was calculated as the ratio of the number of migrated
cells on wells – containing potential chemoattractant divided by the number of cells in the control wells.
Amr A. Al-haidari, Ingvar Syk, Karin Jirström and Henrik Thorlacius
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Y, et al. Chemokine receptor CXCR3 promotes colon cancer metastasis
to lymph nodes. Oncogene2007 Jul 12;26(32):4679-88.
7.Campbell DJ, Ziegler SF. FOXP3 modifies the phenotypic and
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8.Ishida T, Ueda R. Immunopathogenesis of lymphoma: focus on CCR4.
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9.Ishida T, Utsunomiya A, Iida S, Inagaki H, Takatsuka Y, Kusumoto S, et
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CCR4 mediates CCL17 (TARC)-induced migration of human colon cancer cells via
RhoA/Rho-kinase signalling
of active RhoA in HT-29 cell line. Confluence of 80% was serum starved overnight and then stimulated for 5min with
TARC/CCL17 100ng/ml, 10% serum as positive control and with serum free media as a negative control. Cells then lysed
and lysates were snap frozen in liquid nitrogen directly and protein determination was made. 1mg/ml protein was used for
quantitative detection of Active and Total RhoA. TARC stimulation increases levels of RhoA-GTP significantly, #, ##P<0.05
vs control, (n=4). Data represent quadruplicates determinations expressed by Mean ± SEM.
Figure 6. mRNA Relative quantity of Rho proteins
after CCL17/TARC stimulation. Confluence
of 80% HT29 was serum starved for 24hr
and then stimulated by 100ng/ml TARC in
serum free media for 24hr. RNAs was
extracted and cDNA was synthesized and
comparative PCR was performed using β-
actin as normalizer and stimulated cells
were calibrated against unstimulated cells.
A, gel electrophoresis show Rho mRNA
gene expression respective bands. Data
shown represent typical one of three
independent experiments. B, fold changes
in Relative quantity of Rho mRNA levels.
C, Total RhoA was measured by ELISA at
absorbance 490nm. D, G-LISA activation
assay was used to quantify levels
CCL17/TARC (ng/ml)
Tra
nsm
igra
tio
n (
% o
f co
ntr
ol)
95
100
105
110
115
120
HT29
AZ-97
1 10 100
A
CCL17/TARC (ng/ml)
Tra
ns
mig
rati
on
(%
of
co
ntr
ol)
100
110
120
130
140
150
160
170
HT29
AZ-97
1 10 100
***
#
**
**
B
CCL17/TARC (ng/ml)
Tra
ns
mig
rati
on
(%
of
co
lon
ca
nc
er)
95
100
105
110
115
120
125
130
HT29
AZ-97
1 10 100
**
*
**
*
**
C
Figure 8.
Cell proliferation was evaluated in triplicates using Cell Counting Kit-8 assay (CCK-
8; Sigma-Aldrich,). Briefly, Cells were seeded in 96 – microplate at 1×105 cells/well
in media either with/without CCL17/TARC (100ng/ml) for 24, 48, and 72 hours. To
assess proliferation, 10µl CCK-8 was added per well, and the cells were incubated
for an additional 4 hours and absorbance at 450 nm was recorded using 96-well
plate ELISA reader. Stimulation of HT-29 and AZ-97 cells with different doses of
CCL17 up to three days had no effect on the proliferation of colon cancer cells A.
HT-29, B. AZ-97. P> 0.05