Identification and analysis of the signalling networks that regulate Ciz1 levels in normal and cancer cell lines

Pauzaite, Tekle and Copeland, Nikki (2019) Identification and analysis of the signalling networks that regulate Ciz1 levels in normal and cancer cell lines. PhD thesis, Lancaster University.

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Abstract

Ciz1 is a nuclear protein that associates with cyclin A – cyclin dependent kinase 2 (CDK2) and facilitates the initiation of DNA replication. Ciz1 overexpression has been linked to common cancer types, including breast, colon, prostate, lung, and liver cancers. This suggests that identification of mechanisms that regulate Ciz1 levels may represent potential drug targets in cancer. This work identifies that CDK2 and DDK activity are required to maintain Ciz1 levels. Chemical or genetic inhibition of CDK2 or DDK (Cdc7-Dbf4) activity in murine fibroblasts reduced Ciz1 levels. Further analysis demonstrated that CDK and DDK activity promotes Ciz1 accumulation in G1 phase by reducing ubiquitin proteasome system (UPS) mediated degradation. Furthermore, Ciz1 levels are actively controlled by the proteasome, as inhibition of protein translation rapidly reduced Ciz1 levels, and this is reversed by proteasomal inhibition. The data suggest a model where Ciz1 is regulated by opposing kinase and UPS activities, leading to Ciz1 accumulation in response to rising kinase activity in G1 phase, and its degradation later in the cell cycle. Significantly, human prostate adenocarcinoma (PC3) and oestrogen receptor positive breast cancer (MCF7) cell lines require Ciz1 for efficient proliferation. The data demonstrate that Ciz1 levels can be reduced with CDK2/ DDK inhibitors via proteasomally mediated degradation in human cancer cell lines similarly to normal fibroblasts. In PC3 and MCF7 cell lines, repurposing small molecule CDK2 inhibitors efficiently reduce Ciz1 levels, decrease E2F mediated transcription and proliferation. The targeted depletion of Ciz1 via CDK2/ DDK inhibition and UPS mediated degradation requires a functional E3 ligase to be effective. As a first step towards identifying the regulatory E3 ligase(s), a biochemical fractionation and mass spectrometry approach revealed three putative E3 ligases: UBR5, FBXO8 and UBE2O, which require further characterisation. Taken together, this work suggests that deregulation of CDK activity or inactivation of UPS signalling may promote Ciz1 overexpression in specific cancers. Importantly, Ciz1 is required for proliferation of some cancer cell lines, suggesting that approaches, which reduce Ciz1 levels may be of clinical benefit. Therefore, the identification of the regulatory mechanisms that control Ciz1 levels, represent potential targets in Ciz1 dependent cancers.