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The Role of the Runx2/CBFβ complex in Breast Cancer
[Thesis]. Manchester, UK: The University of Manchester; 2014.
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Abstract
Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction. Effective treatment of bone metastasis remains a considerable clinical challenge. In the UK around 70% of the 12,000 patients that die from breast cancer annually have bone metastasis. Whilst existing therapies provide some pain relief, by limiting the tumour-mediated bone degradation, bone metastases are presently incurable. There is therefore an urgent need to develop therapies to prevent bone metastatic breast cancer. The transcription factor complex Runx2/CBFβ is a key regulator of bone development and is aberrantly expressed in breast cancer, leading to up-regulation of bone metastasis-associated genes. Previous work has demonstrated that Runx2/CBFβ determines the invasive phenotype of metastatic breast cancer cells and is required for the expression of metastatic genes. The Runx2/CBFβ complex also has a role in normal breast gene expression, activating expression of the milk protein β-casein in response to hormones. However, little is known about the normal role of Runx2/CBFβ in breast cells. The overarching aim of this project was to determine the role of Runx2/CBFβ in metastasis and identify the target genes that determine the metastatic phenotype. In order to understand the role of Runx2/CBFβ in breast cancer, initial experiments were performed to determine the role of Runx2/CBFβ in normal breast cells. A 3D culture system was established to examine the role of Runx2/CBFβ in regulating gene expression in non-cancerous differentiated epithelial breast cells. Attempts were also made to determine the Runx2/CBFβ target genes after lactogenic hormone stimulation. Unfortunately siRNA knockdown of Runx2 was incompatible with hormonal stimulation. However, 3D cell culture of normal mammary gland cell line HC11 showed Runx2 was expressed throughout the development of mammary acini structures. In addition the expression of CBFβ was confirmed in these cells.Having established the 3D culture system, experiments were subsequently performed to examine the role of CBFβ in the metastatic breast cancer cell line MDA-MB-231. These experiments demonstrated that depletion of CBF has a remarkable effect on the phenotype of the cells, leading to the development of mammary acini structures normally formed by non-cancerous breast cancer cell lines. Thus, depletion of CBF results in a reversion to an epithelial phenotype, suggesting that CBF is required to maintain the epithelial to mesenchymal transition (EMT). RT-PCR analysis also revealed changes in the expression of EMT marker genes. We also demonstrated that the EMT reversion could be rescued by re-expressing an inducible form of CBFβ. These data suggest that CBFβ is required to maintain the mesenchymal phenotype of metastatic breast cancer cells. Finally, a microarray analysis of MDA-MB-231 cells was performed to identify Runx2/CBFβ target genes that might contribute to the mesenchymal phenotype. Cells depleted of CBFβ and grown in 3D revealed reduced expression of IL11. This is known to be involved in bone remodelling. Inspection of the IL11 promoter revealed potential DNA binding sites which confirmed binding to Runx2 using EMSA.