Prolonged mitotic arrest induced by Wee1 inhibition sensitizes breast cancer cells to paclitaxel.

Cody W Lewis,Xuejun Sun,Dawn Macdonald,Xu Jing Qian,Gordon Chan,Ruicen He,Zhigang Jin,Won Shik Choi,Wenya Wei

doi:10.18632/oncotarget.17848

Abstract

Wee1 kinase is a crucial negative regulator of Cdk1/cyclin B1 activity and is required for normal entry into and exit from mitosis. Wee1 activity can be chemically inhibited by the small molecule MK-1775, which is currently being tested in phase I/II clinical trials in combination with other anti-cancer drugs. MK-1775 promotes cancer cells to bypass the cell-cycle checkpoints and prematurely enter mitosis. In our study, we show premature mitotic cells that arise from MK-1775 treatment exhibited centromere fragmentation, a morphological feature of mitotic catastrophe that is characterized by centromeres and kinetochore proteins that co-cluster away from the condensed chromosomes. In addition to stimulating early mitotic entry, MK-1775 treatment also delayed mitotic exit. Specifically, cells treated with MK-1775 following release from G1/S or prometaphase arrested in mitosis. MK-1775 induced arrest occurred at metaphase and thus, cells required 12 times longer to transition into anaphase compared to controls. Consistent with an arrest in mitosis, MK-1775 treated prometaphase cells maintained high cyclin B1 and low phospho-tyrosine 15 Cdk1. Importantly, MK-1775 induced mitotic arrest resulted in cell death regardless the of cell-cycle phase prior to treatment suggesting that Wee1 inhibitors are also anti-mitotic agents. We found that paclitaxel enhances MK-1775 mediated cell killing. HeLa and different breast cancer cell lines (T-47D, MCF7, MDA-MB-468 and MDA-MB-231) treated with different concentrations of MK-1775 and low dose paclitaxel exhibited reduced cell survival compared to mono-treatments. Our data highlight a new potential strategy for enhancing MK-1775 mediated cell killing in breast cancer cells.

Highlights

Cyclin dependent kinase (Cdk)-1/cyclin B1 is the key complex, that when active, initiates mitosis with subsequent inactivation triggering mitotic exit [1]
To examine the role of Wee1 kinase in preventing premature mitosis, HeLa cells were synchronized in G1/S phase by double thymidine block and released into fresh media containing either MK-1775 or a solvent control (DMSO) (Figure 1A)
We observed that MK-1775 treatment increased the percentage of phosphoserine histone H3 (PH3)-positive cells in HeLa (p < 0.005), T-47D (p < 0.005), and MDA-MB-231 (p < 0.05) to a similar level (~20%) compared to DMSO controls; the percent of PH3-positive cells increased for MCF7 cells (p < 0.05), but to a lesser extent (~5%)

Summary

Introduction

Cyclin dependent kinase (Cdk)-1/cyclin B1 is the key complex, that when active, initiates mitosis with subsequent inactivation triggering mitotic exit [1]. Cdk activity is tightly regulated in interphase by Wee and Myt kinases, which add inhibitory phosphates to Cdk on threonine and tyrosine preventing premature mitosis [2, 3]. At the end of mitosis, mitotic exit is characterized by the rephosphorylation and inhibition of Cdk and degradation of cyclin B1 [9]. Both are dependent upon the reactivation of residual Wee during anaphase [6,7,8, 10]. In the absence of Wee activity, mitotic cells maintain high levels of cyclin B1 and low levels of phosphorylated tyrosine 15-Cdk1 [6, 7]

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Discussion

Conclusion