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.

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4.Murdoch C, Finn A. Chemokine receptors and their role in inflammation

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8.Ishida T, Ueda R. Immunopathogenesis of lymphoma: focus on CCR4.

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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