Potential Role of Monocyte Chemotactic Protein 1 (MCP-1) in the Breast Cancer Microenvironment
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MCP-1 has a potentially important role in the breast tumour microenvironment. The results presented suggest it may exert its effect by promotion of tumour cell survival through up-regulation of anti-apoptotic factors and pro-migratory factors and thus may be a target for therapeutic intervention.
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Potential Role of Monocyte Chemotactic Protein 1 (MCP-1) in the Breast Cancer Microenvironment
- 1. Potential Role of Monocyte ChemotacticPotential Role of Monocyte Chemotactic Protein 1 (MCP-1) in the Breast CancerProtein 1 (MCP-1) in the Breast Cancer MicroenvironmentMicroenvironment MC Hartmann, RM Dwyer, MP Boyle, SM Potter and MJ Kerin Department of Surgery, National University of Ireland, Galway
- 2. Introduction Breast cancer is the leading type of female cancer in women in Ireland Despite advances in diagnosis and therapy, there are few options available for treatment of advanced disease Further understanding of the mechanisms involved in cancer spread from the primary tumour site is important to develop novel therapies The primary tumour microenvironment plays an important part in tumour progression
- 3. Breast tumour microenvironment Neoplastic epithelial cells co-exist in carcinomas with a biologically complex stroma, composed of various types of stromal cells and extracellular matrix Cross talk between epithelial and stromal cells is mediated by chemokine signalling
- 4. Monocyte Chemotactic Protein 1 (MCP-1)Tumour stromal cells Tumour epithelial cells MCP-1/CCL2 Tumour stromal cells Tumour epithelial cells MCP-1/CCL2 MCP-1 In Breast Cancer:MCP-1 In Breast Cancer: MCP-1 is a chemotactic cytokine, primary role in inflammation.MCP-1 is a chemotactic cytokine, primary role in inflammation. Whole tumour explants secrete high levels of MCP-1.Whole tumour explants secrete high levels of MCP-1. Tumour stromal cells secrete significantly higher levels thanTumour stromal cells secrete significantly higher levels than normal stromal cells.normal stromal cells. Epithelial cells express its receptor, CCR2Epithelial cells express its receptor, CCR2 Effect of MCP-1 on epithelial cell gene expression is unknownEffect of MCP-1 on epithelial cell gene expression is unknown
- 5. Aim Investigate the effect of Monocyte ChemotacticInvestigate the effect of Monocyte Chemotactic Protein-1 on breast cancer cellProtein-1 on breast cancer cell proliferationproliferation and geneand gene expressionexpression
- 6. Methods Breast cancer cell lines SK-BR-3 PR-, ER-, HER2/neu ++ T47 D ER+, PR+, HER2/neu low MDA-MB 231 ER-, PR-, HER2/neu – Breast cancer cell lines incubated with recombinant human MCP-1 for 72hrs Cells harvested and RNA extracted Changes in proliferation analysed T47D
- 7. Cells incubated with MCP-1 Real Time Quantitative PCR ELK-1, VIL-2 MKi67, BAG-1, Bcl-2 PPIA, MRPL19 RNA Extraction Methods cDNA synthesis Oxyluciferin+ AMP + PPi + CO2 ATP+Luciferin+O2 Luciferase Mg++ LIGHT Cell Proliferation Gene expression →
- 8. MCP-1 mediated changes in gene expression in T47D cells 0 1 2 3 4 5 6 BC L2 BAG 1 VIL-2 E LK-1 FoldChangeinGeneExpression * p<0.05 * *
- 9. MCP-1 mediated changes in gene expression in SK-BR-3 cells 0 1 2 3 4 BCL2 BAG1 VIL-2 ELK-1 FoldChangeinGeneExpression * p<0.05 * *
- 10. MCP-1 mediated change in gene expression of MDA-MB-231 cells 0 1 2 3 4 BCL2 BAG1 VIL-2 ELK-1 FoldChangeinGeneExpression * p<0.05 *
- 11. Effect of MCP-1 on Cell ProliferationRelativeProliferation(%) 0 10 20 30 40 50 60 70 80 90 100 T47D T47D MDA-MB-231 MDA-MB-231 + MCP-1 Well Content + MCP-1 SK-BR-3 SK-BR-3 + MCP-1
- 12. Summary Up regulation of BAG-1 and Bcl-2 in T47D andUp regulation of BAG-1 and Bcl-2 in T47D and SK-BR-3 cellsSK-BR-3 cells Up regulation of VIL 2 in MDA-MB-231Up regulation of VIL 2 in MDA-MB-231 No significant change in breast cancer cellNo significant change in breast cancer cell proliferation in response to MCP-1proliferation in response to MCP-1
- 13. Conclusion MCP-1 has a potentially important role in the breastMCP-1 has a potentially important role in the breast tumour microenvironment.tumour microenvironment. The results presented suggest it may exert its effectThe results presented suggest it may exert its effect by promotion of tumour cell survival throughby promotion of tumour cell survival through upregulation of anti-apoptotic factors and pro-upregulation of anti-apoptotic factors and pro- migratory factors and thus may be a target formigratory factors and thus may be a target for therapeutic interventiontherapeutic intervention..
Editor's Notes
- Breast cancer is the most common female cancer with ~1700 cases in Ireland each year and a mortality rate of about a third of that figure. Over the past decade mortality rates have begun to decrease as a result of improved therapy and also due to increased awareness of the disease which leads to earlier detection. Despite this positive trend, &gt;80% of patients with advanced disease will develop bone metastases, for which there is no cure. Clearly there is an urgent need for improved therapy for this cohort of patients. The bone marrow provides an ideal environment for engraftment, migration and neovascularization of metastatic breast cancer cells, through it’s ability to produce cytokines and extracellular matrix molecules, but the pathways involved in metastasis to bone, and interactions between the cells types is poorly understood. Understanding the pathways involved in metastasis to bone is fundamental to the development of these therapies. PET scan in patient with breast cancer that has spread to bones. Thin arrow shows abnormal sites of uptake in spine and lower right pelvis.
- Tumour stromal cells have distinct properties that set them apart from stromal cells found in normal tissue Tumour stromal cells have distinct properties that set them apart from stromal cells found in normal tissue
- Sequence of methods included: incubation of cell lines with varying concentrations of CCL2 in a 6 well plate. RNA extraction using Qiagen RNeasy MinElute clean-up kit. Measurement of the concentration of RNA using Nanodrop spectrophotometer was preformed. We then proceeded to reverse transcription to cDNA using. RQ-PCR was used to determine using primers targeting the chemokines MCP-1, SDF-1α, their receptors CCR2 and CXCR4, and the anti-apoptotic genes Bcl2 and BAG1. Results were normalised to endogenous control genes MRPL19 and PPIA and expressed relative to cells cultured without MCP-1.