Identification and characterisation of novel therapeutic targets in breast cancer metastasis

Supervisor(s): Professor Clare Isake

Section: Breakthrough Breast Cancer Research Centre

Team: Molecular Cell Biology

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Summary

The majority of breast cancer therapeutic targets have been identified on the basis of their ability to affect cell survival. However, as most breast cancer deaths are due to disease dissemination and treatment failure, there is an urgent need to identify therapeutic targets that specifically block one or most aspects of the metastatic cascade. The process of breast cancer metastasis can be described as having the following steps: (a) invasion of tumour cells into the surrounding tissue, (b) intravasation either directly into the vascular system or via the lymphatics, allowing access to secondary sites, (c) extravasation into secondary sites, (d) metastatic colonisation in which tumour cells from micrometastases (often after a period of dormancy) and progressively growth into vascularised macrometastases. Each of these steps requires complex interactions with the microenvironment and activation of non-epithelial stromal cells. As a consequence, modelling even a single step of this metastatic cascade in vitro is problematic, as is establishing the in vivo relevance of in vitro findings.

In this PhD project, we are proposing to take a multistep approach to the identification of potential therapeutic targets for advance disease. The aims are to (a) develop and implement a chemical screen designed to identify targets which when inhibited impair the invasion of breast cancer cells in a three-dimensional matrix. For this the student will use a combination of commercially available drug libraries comprising ~2,500 compounds and employ a collagen invasion assay using the InCell3000 microscope. 

The project will be initiated using the 4T1 mouse mammary carcinoma cell line [1,2]. This part of the project will be undertaken in collaboration with Professor Chris Marshall whose laboratory has extensive experience in such assays [3,4] and with Professor Alan Ashworth and Dr Chris Lord who have experience in handling the chemical libraries and screen analysis [5]. (b) to validate hits and undertake further characterisation using a range of in vitro 3D assays and cell lines. For this the student will repeat the assay with a range of different human and mouse breast cancer cell lines. Validated hits will be tested in independent assays (e.g. Transwell migration assays across matrix-coated filters and organotypic assays in which tumour cells and stromal cells are cultured together in 3D matrices) routinely employed in our laboratory [6,7]. Where known drug targets have been previously identified, RNAi approaches will be taken to assess the effects of gene downregulation on cell invasion. (c) where appropriate to investigate the expression of target genes in clinical samples. For this the student will work with the Cancer Informatics Team to use bioinformatics approaches to identify potential targets of prognostic significance in patient outcome or which effect treatment response. Where appropriate, the student will investigate expression of potential targets in breast cancer samples as part of our ongoing collaboration with the Molecular Pathology Team [6,8,9], and (d) to take candidate inhibitors into in vivo assays of invasion and metastasis.

The purpose of this investigation is to identify potential therapeutic targets which when inhibited impact upon one or more steps in the metastatic cascade. This project will complement well ongoing work in our laboratory that aim to identify (a) key aspects of tumour: stroma interactions in breast cancer tumourigenesis, and (b) breast cancer invasion suppressor genes.

References

1. Lou Y., et al (2008) Epithelial-mesenchymal transition (EMT) is not sufficient for spontaneous murine breast cancer metastasis. Dev Dyn Vol 237, No 10, p2755-2768
2. Yang J., et al (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell Vol 117, No 7, p927-939
3. Sanz-Moreno V., et al (2008) Rac activation and inactivation control plasticity of tumor cell movement. Cell Vol 135, No 3, p510-523
4. Smith HW., et al (2008) uPAR promotes formation of the p130Cas-Crk complex to activate Rac through DOCK180. J Cell Biol Vol 182, No 4, p777-790
5. Iorns E., et al (2009) Parallel RNAi and compound screens identify the PDK1 pathway as a target for tamoxifen sensitization. Biochem J Vol 417, No 1, p361-370
6. Esseghir S., et al (2007) A role for glial cell derived neurotrophic factor induced expression by inflammatory cytokines and RET/GFR alpha 1 receptor up-regulation in breast cancer. Cancer Res Vol 67, No 24, p11732-11741
7. Sturge J., et al (2003) GPI-anchored uPAR requires Endo180 for rapid directional sensing during chemotaxis. J Cell Biol Vol 162, No 5, p789-794
8. Robertson D., et al (2008) Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol Vol 19, No 9, p13 
9. Wienke D., et al (2007) The collagen receptor Endo180 (CD280) Is expressed on basal-like breast tumor cells and promotes tumor growth in vivo. Cancer Res Vol 67, No 21, p10230-10240

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