Breast cancer is the most prevalent cancer in women in the UK with over 50,000 new cases diagnosed each year. Almost all breast cancer deaths are due to metastatic disease. The RUNX1-CBFbeta transcription factor complex has been implicated in the development of human breast cancer and recent evidence from our laboratory indicated that it might have a role in metastasis. The aim of this project was therefore to determine the role of the RUNX1 transcription factor in breast cancer metastasis. Initial experiments to knockdown RUNX1 by shRNA also decreased the expression of RUNX2. Therefore, due to the off-target effect of shRUNX1, CRISPR-Cas9n was used to establish a RUNX1-negative cell line by targeting the first exon of the RUNX1 gene. Migration and invasion capacity of the cells decreased in the absence of RUNX1 and it was comparable to the absence of RUNX2 and CBFbeta respectively, which are known to play roles in migration and invasion of MDA-MB-231 cells. The cells also formed spherical clusters in 3D culture which was associated with the changes in cell morphology from stellate to round shape in the absence of RUNX1. The expression of the metastasis-related genes MMP13, MMP9, OPN and SLUG also decreased in parallel with the loss of the mesenchymal-like phenotype whilst the expression of the epithelial markers cytokeratin, desmoplakin and E-cadherin increased concomitantly. Importantly, re-expression of RUNX1 in the RUNX1-negative cell lines using an inducible expression system rescued migration and invasion. Therefore, RUNX1 is required to maintain the mesenchymal-like phenotype of MDA-MB-231 cells and hence is important for the epithelial to mesenchyme transition (EMT), a key characteristic of metastatic cells. The transcription factor SLUG is a known regulator of EMT. Data obtained shows that RUNX1 down-regulates the expression of SLUG. ChIP analysis demonstrated that RUNX1 was bound to the SLUG promoter and RUNX1 was subsequently shown to activate the promoter activity. Finally, experiments to inhibit the activity of the RUNX transcription factors pharmacologically showed changes in cell differentiation and also affected cell viability, possibly by off-target effects. Taken together, data presented in this work demonstrates that RUNX1 is required for EMT in the metastatic breast cancer cells and it is therefore a potential therapeutic target to prevent breast cancer metastasis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764525 |
| Date | January 2017 |
| Creators | Ariffin, Nur Syamimi |
| Contributors | Shore, Paul ; Tournier, Cathy |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-mesenchymallike-phenotype-of-metastatic-breast-cancer-is-maintained-by-the-transcription-factor-runx1(9b759a61-925a-4e0b-8faa-b74c2271eb18).html |
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