Relationship between CCL5 and TGFβ1 in breast cancer

patients at both systemic and cellular levels Hartmann MC, Dwyer RM, Costello M and Kerin MJ

Division of Surgery, National University of Ireland Galway, Ireland

Background Results

Discussion

Aim

Methods

Chemokines are chemotactic cytokines that play an important role in inflammation through promotion of leukocyte motility. Studies have shown that expression and activation of chemokine receptors promotes growth and migration of primary tumour cells which leads to metastatic spread. Stromal-epithelial crosstalk in the tumour microenvironment is facilitated through chemokines and their receptors. The chemokine (C-C motif) ligand 5 (CCL5) and its principle receptor CCR5 has a primary role in inflammation and has been implicated in breast cancer progression. Previous studies linked elevated CCL5 serum levels with late stage breast cancer, similarly to what has been described for transforming growth factor beta 1 (TGFβ1), a well established factor in tumourigenesis. TGFβ1 is thought to act as a tumour suppressor in early stage disease and switch to being potentially tumour promoting in later stage breast cancer.

It was the aim of this study to investigate CCL5 and TGFβ1 in breast cancer at circulating, tumour tissue and cellular level

>3 (n=28)1-3 (n=22)0 (n=52)

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>3 (n=24)1-3 (n=22)0 (n=52)

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(A) Circulating CCL5 and lymph node status (B) Circulating TGFβ1 and lymph node status

Fig.1 While there was no significant difference between circulating CCL5 in breast cancer patients compared to controls overall, within the breast cancer cohort, CCL5 was found to decrease in the transition from node negative to node positive disease and increase again as number of positive lymph nodes increased (A). A similar pattern has previously been described for TGFβ1 and was also seen in this study cohort (B).

D) Epithelial Cell Lines

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

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

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

BT – 474 (ER+, Pr-, Her-)

MCF 10-2A

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

C) Primary stromal cell culture

Breast tumour specimen

Finely minced with scalpels

Differential centrifugation

Digested 18-20hrs at 37ºC in 0.1% collagenase type III

Stromal cell fraction

Breast Cancer (n=44)Control (n=16)Breast Cancer (n=44)Control (n=16)

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(A) Gene expression of CCL5 and TGFβ1 (B) Gene expression of CCR5 and TGFβRII

Fig.2 Gene expression of CCL5, CCR5 and TGFβ1 was significantly elevated in breast cancer compared to normal tissue. TGFβRII gene expression remained unchanged.

CCR5 TGFβRII CCL5 TGFβ1

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,

suggesting a paracrine action of the chemokine

This study demonstrates a novel correlation between CCL5 and TGFβ1 in breast

cancer which warrants further investigation

B) Breast cancer cohort A) Enzyme linked immunosorbent assay

* p <0.001

* p <0.0001

Number of positive lymph nodes Number of positive lymph nodes

E) Gene expression analysis

TGFßRIITGFß1CCR5CCL5

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(A) Gene expression analysis in primary stromal cells (B) Gene expression in epithelial cell lines

Fig.4 (A) Analysis of isolated tumour stromal cell populations (n=22) expressed relative to normal stromal cells harvested at reduction mammoplasty (n=4), represented by the baseline. CCL5 gene expression was increased in tumour compared to normal stromal cells while TGFβ1 and TGFβRII gene expression was slightly decreased. CCR5 was not expressed in the stromal cells. (B) Analysis of gene expression in epithelial cell lines, baseline represents the non-tumourigenic cell line MCF10-2A. All targets were elevated in the breast cancer epithelial cells.

Primary stromal cell population (n=22) Epithelial cell population (n=4)

For analysis of epithelial cell gene expression commercially available breast epithelial cell lines were cultured

Fig.3 A significant positive Pearson correlation was found between circulating CCL5 and TGFβ 1 across all serum samples examined (A). Gene expression of CCL5 and TGFβ 1 in whole tissue samples (B).

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L5

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Log10 Relative Quantity TGFß1

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CL5

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(A) Correlation between circulating CCL5 and TGFβ1 (B) Correlation between gene expression of CCL5 and TGFβ1

Pearson Correlation Coefficient

r=0.43, p<0.0001

Pearson Correlation Coefficient

r= 0.435, p<0.001

Targets

CCL5

Principle CCL5 receptor (CCR5)

Transforming Growth Factor β 1 (TGF1)

Transforming Growth Factor β Receptor II (TGFRII)

Homogenisation of corresponding tumour tissue (n=44) and normal tissue (n=16) harvested at reduction mammoplasty

RNA Extraction

cDNA synthesis

& Real Time Quantitative PCR

CCR5 TGFβRII Breast Cancer (n=43)Normal (n=16)Breast Cancer (n=43)Normal (n=16)

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* p <0.0001

CC

R5 a

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Fβ

RII (

ng

/ml)

CCR5 TGFβRII