The role of Nek2 and TRF1 in mitotic perturbations: Potential implications for breast cancer treatment

Although the anti-cancer drugs paclitaxel and doxorubicin are commonly used to treat many solid tumors, their effectiveness is highly variable due to tumor cell resistance. Therefore, it is important to find mechanisms that can be targeted to increase the sensitivity of cancer cells to current chemotherapy agents. NIMA related kinase 2 (Nek2), a serine/threonine kinase, is emerging as an important oncogene because of its regulatory role in multiple mitosis-related events including timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Thus, regulation of the Nek2 expression levels may prove important as a target for cancer treatment. In order to determine the potential of Nek2 as a viable anti-cancer target that increases drug sensitivity, the triple negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were used. The approach design utilized was to pretreat the cells with small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) against Nek2, and then expose them to various concentrations of paclitaxel and doxorubicin. Results demonstrated that drug susceptibility in these pre-treated cells was dramatically increased compared with either agent alone. FACS results showed that apoptosis was induced in siRNA or ASO pretreated. Furthermore, Nek2 knockdown worked synergistically with paclitaxel and doxorubicin by inhibition of cell proliferation. Hence, results suggest that these drugs in combination with Nek2 depletion may improve the sensitivity of breast cancer cells to chemotherapy treatments. To understand how Nek2 affects cancer development caused by chromosomal instability, the relationship between Nek2 and telomeric repeat binding factor 1 (TRF1) was investigated. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. Nek2 depleted cells lead to centrosome separation failure while Nek2 overexpression results in premature centrosome separation. While it has been shown in mice that TRF1 interacts directly with Nek2, the interactions and correlations between Nek2 and TRF1 in human cells are far from clear. In this study, the results showed that mitotic aberrations through Nek2 overexpression require TRF1. Kinase assay results demonstrate that Nek2 directly binds to and phosphorylates TRF1 in vitro and in vivo at multiple sites. Nek2 overexpression MCF 7 and MDA-MB-231 breast cancer cells resulted in increased numbers of centrosomes and multinucleated cells, ultimately leading to cytokinetic failure and aneuploidization. Furthermore, TRF1 depletion by siRNA prevented Nek2-induced unaligned chromosomes during metaphase. Concurrent Nek2 overexpression and TRF1 depletion showed that cells with  2 centrosomes restored cytokinetic failure and chromosome instability similar to controls. Therefore, I propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability. Taken together, overexpression of the Nek2 in breast cancer suggests that inhibition of Nek2 beneficially interferes with the cancer proliferation. Furthermore, combinational treatments involving Nek2 depletion in conjunction with anti-cancer drugs could potentially be developed as a cancer inhibitor. Therefore, Nek2’s contribution to cancer development may serve as a new starting point to exploit as a therapeutic target.