M.C. Hartmann, R.M. Dwyer and M.J. Kerin

Division of Surgery, School of Medicine

National University of Ireland Galway

Malignancies develop at sites of chronic

inflammation - tumours considered

“wounds that do not heal”

Chemokines are chemotactic cytokines

that play a central role in the inflammatory

response through regulation of leukocyte

motility

Barrett J Rollins, European Journal of Cancer 2006

Stromal-epithelial interaction

Stromal cell

Chemokines

Chemokines facilitate stromal-epithelial interactions within primary breast tumours

Initial data highlighted potential relationship

between two factors within this microenvironment

CCL5 (RANTES) /CCR5 interactions have a

principle role in inflammation and have

been implicated in tumourigenesis

Transforming Growth Factor β 1 (TGFβ1) and its

principle receptor TGFβRII play a key role in

maintaining tissue homeostasis

cell differentiation

proliferation

chemotaxis

Tumour cell

Elevated systemic levels of both factors reported in breast cancer

patients compared to healthy controls

TGFβ1 is thought to act as a tumour suppressor in early stage breast

cancer and to promote tumourigenesis as the disease progresses

Stromal cell secretion of CCL5 within the tumour microenvironment

stimulates increased formation of lung metastasis, with no role for CCL5

reported when epithelial cells alone were used for tumour establishment

In the tumour microenvironment, loss of stromal cell expression of

TGFβ1 has been shown to support epithelial cell metastasis.

Investigation of CCL5 and TGFβ1 in breast cancer

at circulating, tumour tissue and cellular level

CCL5 and TGFβ1 levels were

measured in serum samples

from 102 breast cancer

patients and 66 age-matched

controls using ELISA

Breast cancer

patients

Control group

Number of Patients n (%) n(%)

Total 102 66

premenopausal 40 (39) 26 (40)

postmenopausal 62 (61) 40 (60)

Tumour Characteristics n

Histology

Ductal 66

Lobular 15

Other 10

Unknown 11

Epithelial subtype

Luminal A 68

Luminal B 9

Her-2/neu 6

Basal 8

Unknown 11

Targets of Interest

• CCL5

• Principle CCL5 receptor (CCR5)

• Transforming Growth Factor β 1 (TGF1)

• Transforming Growth Factor β Receptor II (TGF RII)

Homogenisation of

corresponding tumour tissue

(n=43) and normal tissue

(n=16) harvested at reduction

mammoplasty

RNA ExtractioncDNA synthesis

Real time

quantitative

PCR

Primary Culture Epithelial Breast Cancer Cell Lines

Breast tumour specimen

Finely minced with scalpels

Digested overnight in collagenase

Differential centrifugation

Stromal cell fraction

T47 D (ER+, PR+, Her+)

Sk-BR-3 (ER-, PR-, Her+)

MDA-MB-231 (ER-, PR-, Her-)

BT – 474 (ER+, Pr-, Her-)

CCL5 TGFβ1

*

Breast Cancer (n=102)Control (n=66)Breast Cancer (n=102)Control (n=66)

100

80

60

40

20

0

CC

L5 a

nd

TG

Fß

1 (

ng

/ml)

p< 0.0001

(n=24)(n=22)(n=52)

120

100

80

60

40

20

0

CC

L5

(ng/m

l)

LN=0 LN=1-3 LN>3 LN=0 LN=1-3 LN>4

CCL5 and Lymph Node Status TGFβ1 and Lymph Node Status

(n=24)(n=22)(n=52)

70

60

50

40

30

20

10

0T

GF

ß1

(ng/m

l)

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

CC

L5 (

ng

/ml)

TGFβ1 (ng/ml)

r=0.43

p<0.0001

Breast Cancer (n=43) Normal (n=16)Breast Cancer (n=43)Normal (n=16)

2.0

1.5

1.0

0.5

0.0

* p <0.001 * p <0.0001

CCL5 TGFβ1

Lo

g1

0R

ela

tive Q

ua

nti

ty

0

0.5

1

1.5

2

0 0.5 1 1.5 2

Lo

g1

0R

ela

tive Q

uan

tity

Log 10 Relative Quantity TGFβ1

r=0.435

p<0.001

Cancer (n=43)Normal (n=16)Cancer (n=43)Normal (n=16)

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Lo

g1

0R

ela

tive Q

uan

tity

*p< 0.0001

CCR5 TGFβRII

Isolated Primary Stromal Cells (n=22) Epithelial Cell Lines

TGFßRIITGFß1CCR5CCL5

3

2

1

0

-1

0

Lo

g10

Re

lati

ve

Qu

an

tity

TGFßRIITGFß1CCR5CCL5

4

3

2

1

0

-1

0

Analysis of isolated tumour stromal cell populations

(n=22) expressed relative to normal stromal cells

harvested at reduction mammoplasty (n=4)

Analysis of breast cancer epithelial cell lines

Results expressed relative to the non-

tumourigenic cell line MCF10-2A

Lo

g10

Re

lati

ve

Qu

an

tity

CCL5 and TGFβ1 levels dropped in the switch from node negative to node

positive disease and increased again as lymph node burden increased

Significant positive correlation between CCL5 and TGFβ1 at both circulating and

tissue gene expression level

CCL5, TGFβ1 and CCR5 gene expression significantly higher in tumour

compared to normal tissue

Increased expression of CCL5 in tumour compared to normal stromal cells. CCR5

was not detected in stromal cells while epithelial cell lines expressed the receptor

This study demonstrates a novel correlation between

CCL5 and TGFβ1 in breast cancer which is maintained

in early and advanced disease

The mechanisms and controlling influences warrant

further investigation and may open avenues for

therapeutic manipulation in selected patients