Ibrahim, Suad Lateef (2019) Exploration of the mechanism of synergy between paclitaxel and BCKDK inhibitors in breast and ovarian cancer cells. Doctoral thesis, Keele University.

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Abstract

Breast cancer is considered the most frequently diagnosed cancer in women and the second most common type of cancer causing deaths among women. It is accounts 25% of all female cancers. It is expected about 1 in 8 women will develop this disease during their lifetimes. However, the overall 5-year survival rate of breast cancer reaches 89.4%, according to the statistics organized from 2005 to 2011 by Surveillance, Epidemiology, and End Results Program (SEER) at National Cancer Institute. Despite this progress in patients’ survival, there is still a problem treating triple-negative breast cancer (TNBC), a subtype of breast cancers that lacks certain cancer drivers. Corrspondingly, among all breast cancers the treatment and the overall survival is worst for this subtype. Understanding the causes of drug resistance and having appropriate therapies to overcome these is one of the major problems that limits patients from benefiting from chemotherapy.
Work from our group previously reported a RNAi screen which identified genes which are over-expressed in chemosensitive or chemo resistant ovarian cancer. In the current study one of these hits, branched-chain keto acid dehydrogenase kinase (BCKDK) was validated as a potential target to increase the sensitivity of breast caner cells to paclitaxel.
Firstly the expression of three main BCAA catabolism pathway components, branched-chain keto acid dehydrogenase BCKDK was assed in MCF-7, MDA-MB-468 and MDA-MB-231 breast cells. Levels of BCKDK were highest in MCF-7 cells. Commercially available inhibitors chloromethylvaleric acid (CMVA) and 3,6-Dichloro-1-Benzothiophene-2-Carboxylic acid (DCBC) inhibited phosphorylation of BCKDH by BCKDK. These inhibitors combined with paclitaxel also synergistically inhibited cell growth in breast cancer cells as well as potentiated apoptosis induced by paclitaxel. Addition of branched chain amino acids to the culture media reduced the synergy confirming that the inhibitors worked through promoting branched chain amino-acid metabolism. Knockdown of BCKDK with siRNAs also potentiated the activity of paclitaxel, confirming the role of BCKDK in resistance to paclitaxel.
Next the mechanism by which BCKDK inhibitors potentiate the activity of paclitaxel was investigated. CMVA and DCBC reduced BCAA levels, decreased phosphorylation of mTORC1 and that of its substrate 4E-BP-1. Phosphorylation of Aurora A, a mitotic regulator which is known to be a substrate of mTORC1, was also reduced. Levels of c-Myc, whose expression is regulated by both mTORC1 and Aurora A were also diminised. Underwent mitotic slippage – cells exited mitosis without dividing leading to >4N DNA content with multiple nuclei. The involvement of BCKDK in these processes results was confirmed by using BCKDK siRNAs in place of the drugs.
Finally, compounds identified by a virtual screen for novel BCKDK inhibitors were evaluated and two compounds identified which inhibited phosphorylation of BCKDH in cells with low nanoMolar potency. These compounds were also synergistic with paclitaxel and they provide a starting point for subsequent drug discovery efforts.
This work points, for the first time, to BCKDK as a potential target for increasing the sensitivity of cancer cells to paclitaxel.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Medicine and Health Sciences > Institute for Science and Technology in Medicine
Depositing User: Lisa Bailey
Date Deposited: 07 Jun 2019 15:16
Last Modified: 07 Jun 2019 15:16
URI: http://eprints.keele.ac.uk/id/eprint/6464

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