Validating the Nima-related kinase Nek2 as a novel chemotherapeutic target

Mitotic division in human cells prompts a dramatic rearrangement of cellular architecture chromosome condensation, nuclear envelope breakdown and bipolar spindle formation facilitates segregation of sister chromatids into daughter cells. Nek2 is a cell cycle-regulated serine/threonine kinase which is implicated in mitotic progression and establishment of a bipolar spindle. Overexpression of Nek2A in cultured human cells has resulted in an increased incidence of spindle abnormalities and aneuploidy, suggestive of defects in chromosome segregation. Currently there is considerable interest in determining whether centrosomal protein kinases that promote mitotic progression, such as Cdk1, Aurora-A, Plk1 and Nek2, are altered in tumour cells, are tumorigenic when upregulated and hence are viable chemotherapeutic targets. In this thesis I demonstrate that levels of Nek2 protein are upregulated in cell lines derived from leukaemia, breast and ovarian cancers. Importantly, I extended this analysis to primary human tumours and found Nek2 protein expression was increased in sixteen of twenty breast tumours studied, the first report of increased Nek2 protein in human neoplastic disease. I next established that the ablation of Nek2 by siRNA in cultured cervical carcinoma cells induced apoptosis. Lastly I present a series of small molecules that inhibit Nek2A in vitro and show the development of two cell-based assays of Nek2A activity, centrosomal disjunction and displacement of the inter-centrosomal component rootletin. Using stable cell lines inducibly expressing Nek2A I demonstrate ablation of Nek2 dependent phenotypes following treatment with a small molecule inhibitor. The ability to inhibit Nek2 in cells coupled with the observation that Nek2 depletion reduces the viability of cultured cells means Nek2 should now be considered a candidate for therapeutic intervention.