Correlation between circulating and tumour tissue CCL5 and TGFβ1 in breast cancer patients
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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
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Correlation between circulating and tumour tissue CCL5 and TGFβ1 in breast cancer patients
- 1. M.C. Hartmann, R.M. Dwyer and M.J. Kerin Division of Surgery, School of Medicine National University of Ireland Galway
- 2. 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
- 3. 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
- 4. 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.
- 5. Investigation of CCL5 and TGFβ1 in breast cancer at circulating, tumour tissue and cellular level
- 6. 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
- 7. 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 Extraction cDNA synthesis Real time quantitative PCR
- 8. 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-)
- 9. CCL5 TGFβ1 * Breast Cancer (n=102)Control (n=66)Breast Cancer (n=102)Control (n=66) 100 80 60 40 20 0 CCL5andTGFß1(ng/ml) p< 0.0001
- 10. (n=24)(n=22)(n=52) 120 100 80 60 40 20 0 CCL5(ng/ml) 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 0 TGFß1(ng/ml)
- 11. 0 20 40 60 80 100 120 0 10 20 30 40 50 60 70 CCL5(ng/ml) TGFβ1 (ng/ml) r=0.43 p<0.0001
- 12. 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 Log10RelativeQuantity
- 13. 0 0.5 1 1.5 2 0 0.5 1 1.5 2 Log10RelativeQuantity Log 10 Relative Quantity TGFβ1 r=0.435 p<0.001
- 14. 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 Log10RelativeQuantity *p< 0.0001 CCR5 TGFβRII
- 15. Isolated Primary Stromal Cells (n=22) Epithelial Cell Lines TGFßRIITGFß1CCR5CCL5 3 2 1 0 -1 0 Log10RelativeQuantity 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 Log10RelativeQuantity
- 16. 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
- 17. 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
Editor's Notes
- Good afternoon Mr. Chairperson, Ladies and Gentlemen
- Chronic inflammation is known to predispose to cancer formation and progression and the chemokine system plays a pivotal role within this environment. Tumours seem to seize the molecular pathways present in sites of tissue damage and as a consequence have been referred to as “wounds that do not heal”. Chemokines are chemotactic cytokines that play a central role in the inflammatory response through regulation of leukocyte motility. Interestingly, tumour cell motility is thought to be regulated in a similar fashion.
- Chemokines facilitate stromal-epithelial interactions within primary breast tumours and contribute to tumour progression. Initial data from the Department of Surgery highlighted a potential relationship between two factors within this microenvironment, CCL5 also known as RANTES and transforming growth factor beta one. CCL5 and its principle receptor CCR5 have an important role in the immune response and have been implicated in breast cancer. Transforming growth factor beta one (TGFβ1) and its principle receptor TGFβRII play a key role in maintaining tissue homeostasis, cell differentiation, proliferation and chemotaxis.
- While there have been conflicting reports regarding a potential role for CCL5 in breast cancer, TGFb1 has a more well established role in the disease. Elevated systemic levels of both factors have been detected in breast cancer patients compared to healthy controls. TGFβ1 acts as a tumour suppressor in early stage breast cancer and is thought to promote tumourigenesis as the disease progresses. In terms of in vivo models of breast cancer, stromal cell secretion of CCL5 within the tumour microenvironment has been shown to stimulate increased formation of lung metastasis, with no role for CCL5 reported when epithelial cells alone were used for tumour establishment. This highlights the importance of cellular interactions in tumour progression. In the tumour microenvironment, loss of stromal cell expression of TGFB1 has been shown to support epithelial cell metastasis
- It was the aim of this study to investigate CCL5 and TGFb1 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. As shown on the table on the right, there was an equal proportion of pre and post menopausal subjects in each group. The tumour characteristics were representative of a typical breast cancer cohort, with the majority of ductal histology and Luminal A epithelial subtype. ELISA was used for detection of levels of both factors.
- In order to analyse gene expression, tumour tissue from a subset of the same patients and normal tissue harvested at reduction mammoplasty was homogenised and RNA extracted. Real time quantitative PCR was performed targeting CCL5, CCR5 ,TGFb1 and TGFbRII.
- To analyse individual tumour cell populations, stromal cells were isolated from tumour and normal breast tissue specimens harvested in theatre. The cells were cultured in selective media. To analyse epithelial cell gene expression, a range of commercially available breast cancer cell lines were used.
- Now I would like to present our results, starting with systemic circulating levels of CCL5 and TGFB1 measured in serum samples of 102 breast cancer patients and 66 healthy, age-matched controls. The x-axis represents the individual groups, with the y-axis showing the levels of the factors in ng/mL. No significant difference in CCL5 levels was detected as shown on the left of the graph, while TGFβ1, shown on the right, was found to be significantly elevated in breast cancer patients relative to controls.
- When stratified on the basis of lymph node status CCL5 levels, shown in the graph on the left, were found to decrease in the transition from node negative to node positive disease and rise again as the number of positive lymph nodes increased. This pattern has previously been reported for circulating TGFβ1 and was also observed in this study cohort as shown on the right.
- Further investigation revealed a significant positive correlation between circulating CCL5 and TGFβ1 across all samples examined. Here TGFB is shown on the x-axis with CCL5 on the y-axis, and a linear relationship between the two factors was observed. Based on this data I then went on to analyse CCL5 and TGFβ1 in whole tumour tissue from a subset of the same breast cancer patients.
- This graph shows gene expression of the two factors in tumour tissue from 43 breast cancer patients compared to 16 normal tissues harvested at reduction mammoplasty shown in green. This revealed significantly elevated expression of both CCL5 and TGFβ1 in breast tumour compared to normal tissue.
- Indeed, further investigation revealed that similar to the correlation found in systemic levels of the factors, a significant positive correlation between CCL5 and TGFβ1 gene expression in whole tissues was detected.
- In the case of tissues it was also possible to analyze expression of the principle receptor for each ligand. Expression of CCR5 shown on the left panel, was found to be significantly higher in breast cancer compared to normal tissue. However, no change in expression of TGFβRII between the two groups was detected.
- Analysis of expression of the ligands and their principle receptors was then performed on individual cell populations. Focusing first on the tumour stromal cells shown on the left. CCL5 was found to be significantly elevated in tumour compared to normal stromal cells which are represented by the baseline, while its receptor, CCR5 was not detected in any of the samples analysed. In the case of the epithelial cells lines on the right, results are expressed relative to the non-tumorigenic epithelial cell line, MCF10-2A. In this case, all targets were found to be elevated in the tumour cell lines. It is of interest that CCR5 was detected in all the epithelial populations, while undetected in the stromal cells, suggesting a paracrine action of the chemokine.
- Levels of CCL5 and TGFb1 decreased in the switch from node negative to node positive disease and increased again as the number of positive lymph nodes increased. There was a significant positive correlation observed between circulating and tissue gene expression levels of the two factors. CCL5, TGFβ1 and CCR5 gene expression was significantly higher in tumour compared to normal tissue. On a cellular level, an upregulation of CCL5 in tumour compared to normal stromal cells was detected. The principle CCL5 receptor CCR5 was not detected in stromal cells while epithelial cell lines expressed the receptor.
- In conclusion Ladies and Gentlemen, 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 might open avenues for therapeutic manipulation in selected patients