Sabat-Pospiech, Dorota Kaja
(2020)
Regulation of kinesin KIFC1 and centrosome clustering by OTUD6B deubiquitylase.
PhD thesis, University of Liverpool.
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Abstract
Centrosome amplification is a hallmark of human cancers and is especially common in breast cancer, including triple-negative (TNBC) subtype. Centrosome amplification may lead to multipolar spindle formation and lethal multipolar mitosis. To avoid this, cancer cells often cluster their centrosomes to form a pseudo-bipolar spindle. This process relies on the kinesin KIFC1, which can be regulated by ubiquitin-mediated degradation. Deubiquitylases (DUBs) are a family of druggable enzymes, often dysregulated in cancer, which can remove ubiquitin from protein targets and rescue them from proteasomal degradation. Disruption of centrosome-clustering represents an attractive therapeutic approach for cancers with high centrosome amplification status, and one potential strategy may be targeting a KIFC1 regulatory DUB. 94 human DUBs were systematically tested for their effect on KIFC1 expression and centrosome clustering by two parallel siRNA screens. Co-analysis of two screens identified OTUD6B and JOSD2 as positive regulators of KIFC1 protein expression and centrosome clustering in the TNBC cell line BT549. Analysis of the TCGA breast cancer dataset demonstrates OTUD6B transcript is often overexpressed in breast cancer and correlates with worse survival which suggests a potential clinical relevance; thus, OTUD6B was selected for follow-up experiments. We show that OTUD6B depletion increases multipolar mitosis frequency in BT549, but its re-overexpression rescues bipolar spindle phenotype. OUTD6B localises to the centrosome and interacts with KIFC1 but is not affecting KIFC1 ubiquitylation in asynchronous cells. KIFC1 levels peaks at mitosis and is rapidly degraded during mitosis exit, OTUD6B follows a similar expression pattern. Preliminary data suggest that OTUD6B may stabilise KIFC1 specifically at mitosis. Furthermore, we establish KIF2A, a kinesin previously associated with the centrosome and bipolar spindle formation, as a common interactor of OTUD6B and KIFC1, potentially also regulated by OTUD6B. Finally, we show that BT549 cells with a high degree of centrosome amplification, but not healthy breast epithelial cells, are dependent on KIFC1 and OTUD6B for their survival. We also performed a pilot study to examine OTUD6B and KIFC1 expression, and centrosome amplification in uveal melanoma tissue samples. The research demonstrated that centrosome amplification is a frequent phenomenon in a subgroup of uveal melanomas and hence may be an attractive therapeutic target. In summary, our data suggest that, together with KIFC1, OTUD6B may be a promising novel target for anti-cancer specific therapies applicable for cancers with high centrosome amplification status such as TNCB and uveal melanoma.
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences > School of Medicine |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 15 Jan 2021 14:35 |
| Last Modified: | 18 Jan 2023 23:22 |
| DOI: | 10.17638/03106581 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3106581 |
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