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  • br Furthermore we analyzed multiple

    2020-08-18


    Furthermore, we analyzed multiple additional samples from recurrent ovarian cancer disease (Figure S8). The triple treatment showed again a significant reduction of cell viability, 3D-colony formation in short- and long-term experiments, and apoptosis measurements by Annexin staining (Figure S8, A-E). Although the triple combination induced the most pronounced growth inhibitory effects, the IC50 for paclitaxel treating primary tumor samples was lower compared to samples derived from recurrent disease (Figure 6, Supplementary Figure S8, A-E). Our observations support the clinical relevance of this combinatorial approach in primary ovarian cancer.
    Discussion
    Despite two trials [International Collaborative Ovarian Neoplasm (ICON) 1 and ACTION] that were conducted to address the uncertain survival benefit of immediate adjuvant chemotherapy in early-stage disease [51,52], there is no clear consensus for systemic treatment of early stage patients yet. Since the choice of the optimal adjuvant chemotherapy regimen and the duration of treatment in early-stage EOC are subjects of continuing debate, additional markers for an improved stratification of early-stage EOC patients are urgently needed for a more precise definition of optimal chemotherapeutic regimens. Here, we could demonstrate that ovarian cancer patients in stages I and
    II with high PLK1 expression (62 months) had a shorter progression-free survival compared to patients with low PLK1 expression (76 months), suggesting that PLK1 is a novel marker for the stratification of early-stage ovarian cancer patients to maximize therapeutic efforts. In addition, PLK1 is a promising target in ovarian cancer Pronase for the induction of synthetic lethality [53] and in clinical trials for ovarian cancer patients [39].
    While the microtubule-targeting drug paclitaxel has impressive clinical efficacy in ovarian tumors and other types of cancer, the exact mechanisms of how this drug yields patient benefit and why chemoresistance is still a major issue for many paclitaxel-treated ovarian cancer patients remain to be elucidated [54]. This is in part because construction of the microtubule apparatus that constitutes the mitotic spindle responsible for chromosome segregation and cell division
    encompasses a plethora of regulatory events. Perturbating the superstructure of the spindle apparatus activates the SAC, leading to a prolonged mitotic arrest [55]. After a prolonged mitotic arrest, cells either die in mitosis or exit with still incomplete cell division and return to interphase, which is called slippage [9–11]. In response to paclitaxel, failure to undergo death in mitosis and/or failure to efficiently engage postmitotic responses can lead to proliferation of cells with highly abnormal genomes [56]. In this study, we used a “two-punch strategy” to intensify the mitotic arrest and block mitotic exit, aiming to increase the percentage of cells that undergo death in mitosis, which could lower the number of surviving aneuploid cells. Treatment with paclitaxel and BI6727 induced a strong mitotic arrest in ovarian cancer cells (Figure 4, Supplementary Figure S3). Experiments focusing on cell growth, cell cycle distribution, and apoptosis revealed that a pretreatment with paclitaxel and BI6727 followed by proTAME (triple combination) is very efficient to induce cells death (Figures 3 and 6, Supplementary Figure S5), i.e., combining BI6727 and proTAME is not only a promising strategy to reduce the inhibitory concentration of paclitaxel efficiently (Supplementary Figures S1 and S2), but this “two-punch strategy” increases also the percentage of mitotic cell death significantly as demonstrated in our time-lapse experiments (Figure 4). Increased cell death could be further substantiated in long-term and in 3D cultures (Figure 2, Supplementary Figure S4). Based on our knowledge about apoptotic signaling during an extended mitotic arrest induced by agents that deregulate microtubule dynamics, two approaches to increase the efficacy of microtubule-targeting drugs like paclitaxel are 1) prolonging mitotic arrest and 2) speeding up the decay/degradation of the apoptotic timer MCL-1, i.e., to shift the balance from cell survival to apoptosis, the rate of Cyclin B1 degradation needs to be slowed and the rate of MCL-1 degradation must be increased. In our study, we could show that proTAME prevents binding of CDC20 to CDC27, preventing efficient activation of the APC/C in ovarian cancer cells. According to this strategy, we could stabilize the pool of mitotic Cyclin B1 (Figures 2A and 3D), which extends the mitotic arrest, giving cells sufficient time to activate the apoptotic machinery.