Nek2A Prevents Centrosome Clustering and Induces Cell Death in Cancer Cells Via KIF2C Interaction

PURPOSE Modern chemotherapeutics frequently suffer from selective targeting of cancer cells. In contrast to normal cells, cancer cells frequently exhibit extra centrosomes, which tend to form multipolar spindles (MPS), triggering cell death. Nevertheless, cancer cells can divide successfully by coalescing their extra centrosomes into two poles. Nek2 is a mitotic kinase regulating several mitotic processes. Our preliminary data showed that Nek2 overexpression unclusters extra centrosomes while its disruption favors centrosome clustering. Thus, revealing the molecular mechanism and targeting Nek2 in cells with extra centrosomes may be an alternative strategy for selective targeting for cancer cells. METHODS IF stainings, cell viability, apoptosis and competition assays were performed to investigate the effect of Nek2 overexpression to induce MPS. To investigate how Nek2 regulates centrosome clustering, we tested known Nek2 targets with relevant function (C-NAP1, Rootlein, Trf1, Hec1, Gas2L1) and assessed their involvement in centrosome clustering by knock out or siRNA mediated silencing. Next, we identified the interactome of Nek2 using TurboID proximity labelling and validated our results with co-IP. RESULTS Cells harboring supernumerary centrosomes were depleted from the population upon Nek2 overexpression. Known Nek2 targets exhibited no effect on centrosome clustering. Furthermore, the effect of Nek2 overexpression on MPS was additive with other known unclustering factors such as HSET, suggesting an independent mechanism. We identified several potential targets of Nek2 using TurboID proximity labelling. NuMA - also previously known to uncluster centrosomes, was significantly enriched in our samples. Interestingly, while NuMA did not co-IP with Nek2, its knockdown reverted the unclustering activity. Another significantly enriched motor protein, Kif2C not only reverted Nek2 activity, but also co-IP'ed with Nek2. Moreover, we identified MAPRE3 a regulator of microtubule dynamics, as interaction partners of both Nek2 and Kif2C. Our data suggests that Nek2 partners with Kif2C and MAPRE3, regulating centrosome clustering. CONCLUSION We assigned a novel function for Nek2 in centrosome clustering. We are currently elucidating the detailed mechanism of action for how target proteins interact with Nek2 regulating its centrosome unclustering activity in cancer cells. Understanding the mechanism will provide new translational approaches for cancer-specific treatment.