Targeting CIZ1 protein levels via CDK inhibition in Ovarian cancer cells

Trenholme, Mark and Copeland, Nikki and Fielding, Andrew (2025) Targeting CIZ1 protein levels via CDK inhibition in Ovarian cancer cells. Masters thesis, Lancaster University.

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Abstract

High grade serous ovarian cancer (HGSOC) is the most severe and deadly subtype of the disease accounting for around 75% of all epithelial-based ovarian cancer diagnoses. A common feature of HGSOC is the dysregulation of CDK activity through overexpression of cyclin E1 (CCNE1). The Cip1-interacting Zinc finger protein 1 (CIZ1) is a nuclear matrix protein which functions in the initiation of DNA replication to coordinate the activities of cyclin E and cyclin A -CDK2 complexes to origins of replication. Interestingly, the overexpression of CIZ1 is associated with tumourigenesis in several common cancers (although not ovarian cancer) including breast, bladder and prostate bringing to light the possibility that CIZ1 may be potential therapeutic target. There is currently no evidence for CIZ1 playing a role in tumour development or prognosis in ovarian cancer. Here, the prognostic value of CIZ1 is determined, showing that there is a link between high CIZ1 expression and poor patient prognosis. This suggests that CIZ1 may be a potential drug target in HGSOC. This project investigates the potential to modulate CIZ1 levels using a hypothetical model that states that CIZ1 levels are controlled by opposing CDK activities, which protects and stabilises CIZ1 and UPS mediated degradation that controls CIZ1 proteostasis. In ovarian cancer this model suggests that dysregulated CDK activity prevents CIZ1 degradation possibly resulting in overexpression. Therefore, CDK inhibitors (CDKIs) were used to investigate if inhibition of CDK activity could reduce CIZ1 levels and potentially reduce proliferation and HGSOC cell viability. The efficacy of a panel of CDKIs in reducing CIZ1 levels was tested in a range of ovarian cancer cell lines. In addition to this analysis, localisation of CIZ1 in ovarian cancer cells was also tracked. Ovarian cancer cells were found to have three CIZ1 isoforms of different molecular weight (150, 120 and 110 kDa). Of these isoforms the high molecular proteins (150 and 120 kDa) were significantly reduced by CDKI’s, especially dinaciclib, BLU-222 and PHA-767491 (PHA) (all of which inhibit CDK2). However, 110 kDa CIZ1 protein levels were relatively unaffected by CDKIs. Interestingly, it was also shown that in ovarian cancer cells the majority of CIZ1 was mislocalised to the cytoplasm. Furthermore, importantly this work also demonstrated that CDKI’s were effective in reducing cellular viability in all ovarian cancer cells in both 2D and 3D culture which aligned with the significantly reduced CIZ1 levels. However, some of the CDKIs such as dinaciclib and BMS-265246 (BMS) showed a high activity against the healthy human ovarian surface epithelium (HOSE) cell line viability, indicating possible toxicity in using these drugs for treatment. Together the findings of this study suggest that reducing CIZ1 levels are a marker for efficacy of CDKIs in ovarian cancer patients, leading to the proposal that CIZ1 may have a role as a therapeutic marker in this cancer type.