Abstract
Histone demethylases are involved in transcriptional regulation and have recently been implicated in human diseases including cancer. KDM5B belongs to the KDM5 family of histone demethylases, which catalyse the removal of the methyl group from the active tri-methyl mark, H3K4me3, thereby regulating transcription. KDM5B was first identified in our lab as being downregulated when HER2 was inhibited by Trastuzumab (Herceptin). KDM5B is widely expressed in breast cancer and other cancers, where it may act by repressing genes such as Caveolin1 (CAV1). Recent studies have implicated KDM5 proteins in resistance to targeted therapies and so considerable effort is being employed to develop KDM5 small molecule inhibitors. A role for KDM5B in mammary gland development has also been demonstrated. In the mouse model lacking demethylase activity (DARID mouse), KDM5B has been shown to repress CAV1 in the mid-pregnant mammary gland. Thus, suggesting similar pathways operate in the normal and malignant mammary gland.This PhD aimed to further understand the role of KDM5B in breast cancer and in the normal mammary gland. The first aim was to generate KDM5B knockout (KO) breast cancer cell line models using CRISPR-Cas9 technology and investigate the effect of this modulation on gene expression, proliferation and drug resistance. The second aim was to investigate the extent of CAV1 downregulation by KDM5B in breast cancer and, identify the cell types where this downregulation may occur in the developing mammary gland.
KDM5B KO cell lines were generated in two HER2+ breast cancer cell lines, BT-474 and SKBr3. KDM5B KO significantly reduced cell proliferation of SKBr3, but not BT-474 cells in cell viability assays. Treatment of KDM5B KO cells with Herceptin and Lapatinib, showed increased sensitivity to Herceptin in BT-474. There was no significant increase in sensitivity to Lapatinib in the KDM5B KO cells. Microarray gene expression analysis in the SKBr3 KDM5B KO was used to elucidate targets of KDM5B and revealed the early response gene EGR1, in addition to CAV1 and MYC, to be amongst the top upregulated genes. Surprisingly, CAV1 was not upregulated in BT-474 KO cells. Immunohistochemistry analysis showed co-localisation of CAV1 and KDM5B in the myoepithelial and stromal cells, in the mid-pregnant mouse mammary gland.
The data demonstrate a cell phenotype dependent role for the function of KDM5B and the importance of pre-clinical investigations for potential therapeutic targets. These cell line models can be used to identify novel mechanisms through which KDM5B promotes cancer progression and drug resistance in breast cancer. This will in turn enable interrogation of these mechanisms for the development of novel therapies.
| Date of Award | 2018 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Joy Burchell (Supervisor) & Elinor Sawyer (Supervisor) |