Marotta, Valeria Elisa 
ORCID: 0009-0008-5085-0325
(2024)
Translating discovery science:
DUBs that regulate centrosome clustering, an Achilles’ heel of cancer
PhD thesis, University of Liverpool.
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Abstract
Triple negative breast cancer (TNBC) is an aggressive subtype that lacks targeted treatments. TNBC displays higher oncogenic centrosome amplification (CA) levels than non-TNBC. Whilst CA can facilitate tumorigenesis, it may also result in multipolar spindles that if uncorrected lead to mitotic catastrophe and cell death. To complete mitosis, cancer cells cluster their supernumerary centrosomes to form a pseudo-bipolar spindle, for which they require the kinesin KIFC1/HSET. TNBC is highly dependent on KIFC1, but normal cells do not require KIFC1 to complete division, making it a cancer-specific therapeutic target. However, small molecule KIFC1 inhibitors have toxic effects in cells without CA, and improved knowledge of the KIFC1 regulatory network may suggest alternative strategies. KIFC1 is regulated by the APC/C through ubiquitin-mediated proteasomal degradation at mitotic exit, while it is phosphorylated and stabilised by ATM/ATR upon DNA damage. However, it was unknown whether these mechanisms may be antagonised by specific deubiquitylases (DUBs). Systematic siRNA screens in our laboratory, targeting 94 DUBs in BT549 TNBC cells, had previously identified JOSD2 and OTUD6B as positive regulators of both KIFC1 expression and centrosome clustering. I found that, of these two DUBs, OTUD6B was more clinically relevant. Analysis of TCGA data revealed that OTUD6B is often amplified and overexpressed in breast cancer tissue, correlating with worse overall survival in patients. TNBC showed higher OTUD6B mRNA levels compared to other subtypes. A positive correlation was evident between OTUD6B and KIFC1 proteins, but not their transcripts, consistent with the hypothesis that OTUD6B regulates KIFC1 protein stability. I confirmed that depletion of OTUD6B with multiple siRNAs reduced KIFC1 protein levels and increased multipolar spindles in a second TNBC cell line, MDA-MB-231. In vitro, I also showed that OTUD6B depletion does not alter cell-cycle progression but impairs the proliferation of TNBC cells with CA. Thus, like KIFC1, they may rely upon OTUD6B to divide safely. Importantly, I demonstrated that GFP-OTUD6B is enriched at the centrosomes and colocalises with KIFC1 at mitosis on the nascent spindle, in cells with normal and altered centrosome number. In addition, OTUD6B interacts with KIFC1 and both interaction and centrosome localisation are reliant on the N-terminal domain of OTUD6B. These findings raise the possibility that OTUD6B might directly regulate KIFC1 during mitosis. Indeed, I showed that OTUD6B regulates K11-linked KIFC1 polyubiquitylation, most likely counteracting the activity of the APC/C towards KIFC1 3 early in mitosis. Consistent with this hypothesis, I found that KIFC1 protein and ubiquitination levels are both dependent on OTUD6B catalytic activity. DNA damage is known to induce KIFC1 protein levels, and I discovered that OTUD6B depletion also limits KIFC1 accumulation in this context. The mechanism remains poorly understood, but my preliminary data suggest that OTUD6B might be an ATM/ATR substrate. As a first step towards translating these in vitro findings, an in ovo CAM xenograft model of MDA-MB-231 was generated. In this model, systemic treatment with the DNA-damaging agents etoposide or cisplatin increases KIFC1 protein levels. Furthermore, a combination of PET/CT, tumour weight and immunohistochemistry confirmed reduced tumour size and viability following cisplatin treatment. This in ovo assay can in future be used to evaluate how OTUD6B manipulation impacts cancer growth, KIFC1 levels and response to cisplatin, with the long-term goal of therapeutic intervention. In summary, my data support a model whereby the deubiquitylase OTUD6B regulates KIFC1 ubiquitination and stability during mitosis and the DNA damage response to support centrosome clustering. Therefore, OTUD6B may be a potential surrogate target for KIFC1 in cancers with high CA, such as TNBC, which currently lack a common targeted therapy.
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health & Life Sciences Faculty of Health & Life Sciences > Inst. Systems, Molec & Integrative Biology > Inst. Systems, Molec & Integrative Biology |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 16 Jan 2025 10:30 |
| Last Modified: | 08 Feb 2025 03:08 |
| DOI: | 10.17638/03185193 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3185193 |
| Disclaimer: | The University of Liverpool is not responsible for content contained on other websites from links within repository metadata. Please contact us if you notice anything that appears incorrect or inappropriate. |
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