Jue Shi

Evidence that Mitotic Exit Is a Better Cancer Therapeutic Target Than Spindle Assembly

Current antimitotics work by perturbing spindle assembly, which activates the spindle assembly checkpoint, causes mitotic arrest, and triggers apoptosis. Cancer cells can resist such killing by premature exit, before cells initiate apoptosis, due to a weak checkpoint or rapid slippage. We reasoned blocking mitotic exit downstream of the checkpoint might circumvent this resistance. Using single-cell approaches, we showed that blocking mitotic exit by Cdc20 knockdown slowed cyclin B1 proteolysis, thus allowed more time for death initiation. Killing by Cdc20 knockdown did not require checkpoint activity and can occur by intrinsic apoptosis or an alternative death pathway when Bcl2 was overexpressed. We conclude targeting Cdc20, or otherwise blocking mitotic exit, may be a better cancer therapeutic strategy than perturbing spindle assembly.

Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

To improve cancer chemotherapy, we need to understand the mechanisms that determine drug sensitivity in cancer and normal cells. Here, we investigate this question across a panel of 11 cell lines at a phenotypic and molecular level for three antimitotic drugs: paclitaxel, nocodazole, and an inhibitor of kinesin-5 (also known as KSP, Eg5, Kif11). Using automated microscopy with markers for mitosis and apoptosis (high content screening), we find that the mitotic arrest response shows relatively little variation between cell types, whereas the tendency to undergo apoptosis shows large variation. We found no correlation between levels of mitotic arrest and apoptosis. Apoptosis depended on entry into mitosis and occurred both from within mitosis and after exit. Response to the three drugs strongly correlated, although paclitaxel caused more apoptosis in some cell lines at similar levels of mitotic arrest. Molecular investigations showed that sensitivity to apoptosis correlated with loss of an antiapoptotic protein, XIAP, during the drug response, but not its preresponse levels, and to some extent also correlated with activation of the p38 and c-Jun NH(2) kinase pathways. We conclude that variation in sensitivity to antimitotic drugs in drug-naive cell lines is governed more by differences in apoptotic signaling than by differences in mitotic spindle or spindle assembly checkpoint proteins and that antimitotics with different mechanisms trigger very similar, but not identical, responses.

Attractor Landscape Analysis Reveals Feedback Loops in the p53 Network That Control the Cellular Response to DNA Damage

State-space analysis of the p53 network identifies a therapeutic strategy for treating cancer. Charting a Deadly Landscape Signaling pathways exhibit complex regulatory interactions that can make it difficult to predict the outcome of inhibition of components. Choi et al. used a computational approach called attractor landscape analysis to identify how the states of the activity of molecules in the p53 network resulted in specific cellular responses to DNA damage. Conditions that produced a particular type of attractor state, called a cyclic attractor, were associated with pulsatile p53 activity and with the cell entering a state of cell cycle arrest. Furthermore, modeling the combined inhibition of specific components in the pathway suggested a mechanism to enhance the apoptotic response to DNA damage. The p53 dynamics and cellular response of MCF7 cells to the combined treatment verified the computational predictions. Thus, the authors demonstrated the potential application of this method to identify enhanced chemotherapeutic strategies. The protein p53 functions as a tumor suppressor and can trigger either cell cycle arrest or apoptosis in response to DNA damage. We used Boolean network modeling and attractor landscape analysis to analyze the state transition dynamics of a simplified p53 network for which particular combinations of activation states of the molecules corresponded to specific cellular outcomes. Our results identified five critical interactions in the network that determined the cellular response to DNA damage, and simulations lacking any of these interactions produced states associated with sustained p53 activity, which corresponded to a cell death response. Attractor landscape analysis of the cellular response to DNA damage of the breast cancer cell line MCF7 and the effect of the Mdm2 (murine double minute 2) inhibitor nutlin-3 indicated that nutlin-3 would exhibit limited efficacy in triggering cell death, because the cell death state was not induced to a large extent by simulations with nutlin-3 and instead produced a state consistent with oscillatory p53 dynamics and cell cycle arrest. Attractor landscape analysis also suggested that combining nutlin-3 with inhibition of Wip1 would synergize to stimulate a sustained increase in p53 activity and promote p53-mediated cell death. We validated this synergistic effect in stimulating p53 activity and triggering cell death with single-cell imaging of a fluorescent p53 reporter in MCF7 cells. Thus, attractor landscape analysis of p53 network dynamics and its regulation can identify potential therapeutic strategies for treating cancer.

Navitoclax (ABT-263) Accelerates Apoptosis during Drug- Induced Mitotic Arrest by Antagonizing Bcl-xL

Combining microtubule-targeting antimitotic drugs with targeted apoptosis potentiators is a promising new chemotherapeutic strategy to treat cancer. In this study, we investigate the cellular mechanism by which navitoclax (previously called ABT-263), a Bcl-2 family inhibitor, potentiates apoptosis triggered by paclitaxel and an inhibitor of kinesin-5 (K5I, also called a KSP inhibitor), across a panel of epithelial cancer lines. By using time-lapse microscopy, we showed that navitoclax has little effect on cell death during interphase, but strongly accelerates apoptosis during mitotic arrest, and greatly increases the fraction of apoptosis-resistant cells that die. By systematically knocking down individual Bcl-2 proteins, we determined that Mcl-1 and Bcl-xL are the primary negative regulators of apoptosis during prolonged mitotic arrest. Mcl-1 levels decrease during mitotic arrest because of an imbalance between synthesis and turnover, and turnover depends in part on the MULE/HUWE1 E3 ligase. The combination of Mcl-1 loss with inhibition of Bcl-xL by navitoclax causes rapid apoptosis in all lines tested. Variation in expression levels of Mcl-1 and Bcl-xL largely determines variation in response to antimitotics alone, and antimitotics combined with navitoclax, across our panel. We concluded that Bcl-xL is a critical target of Bcl-2 family inhibitors for enhancing the lethality of antimitotic drugs in epithelial cancers, and combination treatment with navitoclax and a spindle specific antimitotic, such as a K5I, might be more effective than paclitaxel alone.

Stochastic competition between mechanistically independent slippage and death pathways determines cell fate during mitotic arrest

Variability in cell-to-cell behavior within clonal populations can be attributed to the inherent stochasticity of biochemical reactions. Most single-cell studies have examined variation in behavior due to randomness in gene transcription. Here we investigate the mechanism of cell fate choice and the origin of cell-to-cell variation during mitotic arrest, when transcription is silenced. Prolonged mitotic arrest is commonly observed in cells treated with anti-mitotic drugs. Cell fate during mitotic arrest is determined by two alternative pathways, one promoting cell death, the other promoting cyclin B1 degradation, which leads to mitotic slippage and survival. It has been unclear whether these pathways are mechanistically coupled or independent. In this study we experimentally uncoupled these two pathways using zVAD-fmk to block cell death or Cdc20 knockdown to block slippage. We then used time-lapse imaging to score the kinetics of single cells adopting the remaining fate. We also used kinetic simulation to test whether the behaviors of death versus slippage in cell populations where both pathways are active can be quantitatively recapitulated by a model that assumes stochastic competition between the pathways. Our data are well fit by a model where the two pathways are mechanistically independent, and cell fate is determined by a stochastic kinetic competition between them that results in cell-to-cell variation.

DNA damage strength modulates a bimodal switch of p53 dynamics for cell-fate control.

BackgroundThe p53 pathway is differentially activated in response to distinct DNA damage, leading to alternative phenotypic outcomes in mammalian cells. Recent evidence suggests that p53 expression dynamics play an important role in the differential regulation of cell fate, but questions remain as to how p53 dynamics and the subsequent cellular response are modulated by variable DNA damage.ResultsWe identified a novel, bimodal switch of p53 dynamics modulated by DNA-damage strength that is crucial for cell-fate control. After low DNA damage, p53 underwent periodic pulsing and cells entered cell-cycle arrest. After high DNA damage, p53 underwent a strong monotonic increase and cells activated apoptosis. We found that the damage dose-dependent bimodal switch was due to differential Mdm2 upregulation, which controlled the alternative cell fates mainly by modulating the induction level and pro-apoptotic activities of p53.ConclusionsOur findings not only uncover a new mode of regulation for p53 dynamics and cell fate, but also suggest that p53 oscillation may function as a suppressor, maintaining a low level of p53 induction and pro-apoptotic activities so as to render cell-cycle arrest that allows damage repair.

Post-slippage multinucleation renders cytotoxic variation in anti-mitotic drugs that target the microtubules or mitotic spindle

One common cancer chemotherapeutic strategy is to perturb cell division with anti-mitotic drugs. Paclitaxel, the classic microtubule-targeting anti-mitotic drug, so far still outperforms the newer, more spindle-specific anti-mitotics in the clinic, but the underlying cellular mechanism is poorly understood. In this study we identified post-slippage multinucleation, which triggered extensive DNA damage and apoptosis after drug-induced mitotic slippage, contributes to the extra cytotoxicity of paclitaxel in comparison to the spindle-targeting drug, Kinesin-5 inhibitor. Based on quantitative single-cell microscopy assays, we showed that attenuation of the degree of post-slippage multinucleation significantly reduced DNA damage and apoptosis in response to paclitaxel, and that post-slippage apoptosis was likely mediated by the p53-dependent DNA damage response pathway. Paclitaxel appeared to act as a double-edge sword, capable of killing proliferating cancer cells both during mitotic arrest and after mitotic slippage by inducing DNA damage. Our results thus suggest that to predict drug response to paclitaxel and anti-mitotics in general, 2 distinct sets of bio-markers, which regulate mitotic and post-slippage cytotoxicity, respectively, may need to be considered. Our findings provide important new insight not only for elucidating the cytotoxic mechanisms of paclitaxel, but also for understanding the variable efficacy of different anti-mitotic chemotherapeutics.

Chapter 7 Application of Single-Molecule Spectroscopy in Studying Enzyme Kinetics and Mechanism

This chapter reviews recent developments in the application of single-molecule spectroscopy (SMS) to studies of enzyme kinetics and mechanism. Protocols for conducting single-molecule experiments on enzymes, based largely on the experience in our laboratory, are provided, including methods of sample preparation, instrumentation, and data analysis. We also address general issues related to the design of meaningful single-molecule experiments and include specific examples of the application of SMS to enzyme studies, which reveal new and intriguing aspects of enzyme behavior, including static and dynamic heterogeneity, as well as subunit cooperativity. Finally, we discuss the advantages of employing single-molecule approach in obtaining new information beyond ensemble studies.

Cell death response to anti-mitotic drug treatment in cell culture, mouse tumor model and the clinic

Anti-mitotic cancer drugs include classic microtubule-targeting drugs, such as taxanes and vinca alkaloids, and the newer spindle-targeting drugs, such as inhibitors of the motor protein; Kinesin-5 (aka KSP, Eg5, KIF11); and Aurora-A, Aurora-B and Polo-like kinases. Microtubule-targeting drugs are among the first line of chemotherapies for a wide spectrum of cancers, but patient responses vary greatly. We still lack understanding of how these drugs achieve a favorable therapeutic index, and why individual patient responses vary. Spindle-targeting drugs have so far shown disappointing results in the clinic, but it is possible that certain patients could benefit if we understand their mechanism of action better. Pre-clinical data from both cell culture and mouse tumor models showed that the cell death response is the most variable point of the drug action. Hence, in this review we focus on current mechanistic understanding of the cell death response to anti-mitotics. We first draw on extensive results from cell culture studies, and then cross-examine them with the more limited data from animal tumor models and the clinic. We end by discussing how cell type variation in cell death response might be harnessed to improve anti-mitotic chemotherapy by better patient stratification, new drug combinations and identification of novel targets for drug development.

Alocasia cucullata Exhibits Strong Antitumor Effect In Vivo by Activating Antitumor Immunity

Chinese herbal medicines have long been used to treat various illnesses by modulating the human immune response. In this study, we investigate the immuno-modulating effect and antitumor activity of Alocasia Cucullata (AC), a Chinese herb traditionally used to treat infection and cancer. We found that the whole water extract of AC roots could significantly attenuate tumor growth in mouse tumor models. The median survival time of the AC-treated mice was 43 days, 16 days longer than that of the control group. Moreover, the AC-treated mice showed substantially higher induction of key antitumor cytokines, such as IL-2, IFN-γ, and TNF-α, indicating that AC may exert antitumor effect by activating antitumor immunity. To further pinpoint the cellular and molecular mechanism of AC, we studied the dose response of a human monocytic cell line, THP-1, to the whole water extract of AC. Treatment of the AC extract significantly induced THP-1 differentiation into macrophage-like cells and the differentiated THP-1 showed expression of specific macrophage surface markers, such as CD11b and CD14, as well as productions of antitumor cytokines, e.g. IFN-γ and TNF-α. Our data thus point to AC as potentially a new, alternative immuno-modulating herbal remedy for anticancer treatment.