Can inhibition of cyclin B1 destruction in G1 of mitosis be exploited to promote cancer cell death?

点击翻译按钮获取中文摘要

Background; Oscillations in CDK activity drive cell-cycle progression from the outset of the cell cycle in G1. Indeed, from the earliest stages of G1, CDK activity plays a central role in a cells 'decision' to either commit to cell-cycle entry (by initiating DNA replication) or exit into quiescence. Importantly, CDKs are only active when bound to cyclins. There are many cyclin-CDK complexes, however, only cyclin B1-CDK1 activity is essential. Critically, CDK activity levels are highly regulated to drive progression at the correct speed, allowing sufficient time to complete each stage of the cycle accurately. To facilitate this cyclin levels are strictly attenuated in healthy cells.In contrast, cyclin B1 (B1) is overexpressed in numerous human cancers. Further, its upregulation is associated with poor prognosis, treatment resistance, and suggested to act as a signal that initiates communications between cancer cells and their microenvironment. Indeed, highly expressed B1 binds to CDK1 from the outset in G1, contributing to aggressive proliferation in neoplastic tissues. We have revealed a novel motif in B1 that is critical for its regulation where B1 exists in excess in oocyte meiosis (ref). Following this, we have exciting unpublished data in mitosis demonstrating that this same motif is important to stabilise B1 levels G1 if B1 is overexpressed. Significantly, where we inhibit access to this motif, cells overexpressing B1 die. Objectives;1. To identify the molecular mechanism by which excessive levels of B1 are regulated in G1 of mitosis. 2. Following this, to propose candidate proteins which if perturbed, prevent this mechanism of excess B1 regulation, forcing cell death.Novelty and Timeliness; Our data in mitosis are entirely novel and present us with a unique opportunity, to identify a new mechanism by which we may trigger cell death. Significantly, this mechanism of B1 regulation is only essential in cells overexpressing B1, meaning that any future derived treatment programme could differentiate between unstable and healthy cells. This is timely as clinicians search for effective treatments that preserve healthy cells (limiting the often debilitating off target effects of some alternative treatments). Experimental Approach; The student will identify binding partners of the novel cyclin B1 motif in G1 by Bio-ID assay in live mitotic cells, and by IP experiments with lysed cells over expressing B1 with and without this critical motif (lab 1). They will then confirm candidate proteins in in-vivo assays such as BiFc to confirm proximity (lab 1 and in collaboration with VisiTech) and in-vitro (by generating proteins to characterise interactions biochemically; lab 2). Finally, we will identify mutations in our interacting protein(s) of interest that perturbs the regulation excess B1, resulting in cell death.