Targeting mitotic exit with hyperthermia or APC/C inhibition to increase paclitaxel efficacy

Microtubule-poisoning drugs, such as Paclitaxel (or Taxol, PTX), are powerful and commonly used anti-neoplastic agents for the treatment of several malignancies. PTX triggers cell death, mainly through a mitotic arrest following the activation of the spindle assembly checkpoint (SAC). Cells treated with PTX slowly slip from this mitotic block and die by mitotic catastrophe. However, cancer cells can acquire or are intrinsically resistant to this drug, posing one of the main obstacles for PTX clinical effectiveness. In order to override PTX resistance and increase its efficacy, we investigated both the enhancement of mitotic slippage and the block of mitotic exit.

To test these opposing strategies, we used physiological hyperthermia (HT) to force exit from PTX-induced mitotic block and the anaphase-promoting complex/cyclosome (APC/C) inhibitor, proTAME, to block mitotic exit. We observed that application of HT on PTX-treated cells forced mitotic slippage, as shown by the rapid decline of cyclin B levels and by microscopy analysis. Similarly, HT induced mitotic exit in cells blocked in mitosis by other antimitotic drugs, such as Nocodazole and the Aurora A inhibitor MLN8054, indicating a common effect of HT on mitotic cells. On the other hand, proTAME prevented mitotic exit of PTX and MLN8054 arrested cells, prolonged mitosis, and induced apoptosis. In addition, we showed that proTAME prevented HT-mediated mitotic exit, indicating that stress-induced APC/C activation is necessary for HT-induced mitotic slippage.

Finally, HT significantly increased PTX cytotoxicity, regardless of cancer cells’ sensitivity to PTX, and this activity was superior to the combination of PTX with pro-TAME. Our data suggested that forced mitotic exit of cells arrested in mitosis by anti-mitotic drugs, such as PTX, can be a more successful anticancer strategy than blocking mitotic exit by inactivation of the APC/C.     

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

Emodin enhances gefitinib-induced cytotoxicity via Rad51 downregulation and ERK1/2 inactivation

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It reportedly exhibits an anticancer effect on lung cancer. Gefitinib (Iressa) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). However, the molecular mechanism of how emodin combined with gefitinib decreases NSCLC cell viability is unclear. The recombinase protein Rad51 is essential for homologous recombination repair, and Rad51 overexpression is resistant to DNA double-strand break-inducing cancer therapies. In this study, we found that emodin enhanced the cytotoxicity induced by gefitinib in two NSCLC cells lines, A549 and H1650. Emodin at low doses of 2-10 μM did not affect ERK1/2 activation, mRNA, and Rad51 protein levels; however, it enhanced a gefitinib-induced decrease in phospho-ERK1/2 and Rad51 protein levels by enhancing Rad51 protein instability. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescued the reduced phospho-ERK1/2 and Rad51 protein levels as well as cell viability on gefitinib and emodin cotreatment. Blocking of ERK1/2 activation by U0126 (an MKK1/2 inhibitor) lowered Rad51 protein levels and cell viability in emodin-treated H1650 and A549 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA enhanced emodin cytotoxicity. In contrast, Rad51 overexpression protected the cells from the cytotoxic effects induced by emodin and gefitinib. Consequently, emodin-gefitinib cotreatment may serve as the basis for a novel and better therapeutic modality in the management of advanced lung cancer.     

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. Knockdown of Cdc20 and Cdh1 prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.     

Suppression of v-Src transformation by andrographolide via degradation of the v-Src protein and attenuation of the Erk signaling pathway

Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.     

RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells

Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.     

Protein kinase C β inhibition by enzastaurin leads to mitotic missegregation and preferential cytotoxicity toward colorectal cancer cells with chromosomal instability (CIN)

Enzastaurin is a selective inhibitor of protein kinase C β and a potent inhibitor of tumor angiogenesis. In addition, enzastaurin shows direct cytotoxic activity toward a subset of tumor cells including colorectal cancer cells (CRC). In spite of promising results in animal models, the clinical activity of enzastaurin in CRC patients has been disappointing although a subset of patients seems to derive benefit. In the present study we investigated the biological and cytotoxic activities of enzastaurin toward a panel of well-characterized CRC cell lines in order to clarify the mechanistic basis for the cytotoxic activity. Our results show that enzastaurin is significantly more cytotoxic toward CRC cells with chromosome instability (CIN) compared to cells with microsatellite instability (MSI). Since CIN is usually attributed to mitotic dysfunction, the influence of enzastaurin on cell cycle progression and mitotic transit was characterized for representative CIN and MSI cell lines. Enzastaurin exposure was accompanied by prolonged metaphase arrest in CIN cells followed by the appearance of tetraploid and micronuclei-containing cells as well as by increased apoptosis, whereas no detectable mitotic dysfunctions were observed in MSI cells exposed to isotoxic doses of enzastaurin. Our study identifies enzastaurin as a new, context dependent member of a heterogeneous group of anticancer compounds that induce “mitotic catastrophe," that is mitotic dysfunction accompanied by cell death. These data provide novel insight into the mechanism of action of enzastaurin and may allow the identification of biomarkers useful to identify CRC patients particularly likely, or not, to benefit from treatment with enzastaurin.     

Targeting mitosis exit: A brake for cancer cell proliferation

The transition from mitosis to interphase, referred to as mitotic exit, is a critical mitotic process which involves activation and inactivation of multiple mitotic kinases and counteracting protein phosphatases. Loss of mitotic exit checkpoints is a common feature of cancer cells, leading to mitotic dysregulation and confers cancer cells with oncogenic characteristics, such as aberrant proliferation and microtubule-targeting agent (MTA) resistance. Since MTA resistance results from cancer cells prematurely exiting mitosis (mitotic slippage), blocking mitotic exit is believed to be a promising anticancer strategy. Moreover, based on this theory, simultaneous inhibition of mitotic exit and additional cell cycle phases would likely achieve synergistic antitumor effects. In this review, we divide the molecular regulators of mitotic exit into four categories based on their different regulatory functions: 1) the anaphase-promoting complex/cyclosome (APC/C, a ubiquitin ligase), 2) cyclin B, 3) mitotic kinases and phosphatases, 4) kinesins and microtubule-binding proteins. We also review the regulators of mitotic exit and propose prospective anticancer strategies targeting mitotic exit, including their strengths and possible challenges to their use.     

CGK733 enhances multinucleated cell formation and cytotoxicity induced by taxol in Chk1-deficient HBV-positive hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most deadly human cancers. Chronic hepatitis B virus (HBV) infection is one of the predominant risk factors associated with the development of HCC and complicates the treatment of HCC. In this study, we demonstrate that a HBV-positive HCC cell line HepG2.2.15, was more resistant to chemotherapy agents than its parental HBV-negative cell line HepG2. HBV-positive HCC cells exhibited defective Chk1 phosphorylation and increased chromosomal instability. CGK733, a small molecule inhibitor reportedly targeting the kinase activities of ATM and ATR, significantly enhanced taxol-induced cytotoxicity in HBV-positive HepG2.2.15 cells. The mechanism lies in CGK733 triggers the formation of multinucleated cells thus promotes the premature mitotic exit of taxol-induced mitotic-damaged cells through multinucleation and mitotic catastrophe in HBV-positive HepG2.2.15 cells. These results suggest that CGK733 could potentially reverse the taxol resistance in HBV-positive HCC cells and may suggest a novel strategy to treat HBV-infected HCC patients.     

Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells

We found that benomyl, a benzimidazole fungicide, strongly suppressed the reassembly of cold-depolymerized spindle microtubules in HeLa cells. Benomyl perturbed microtubule-kinetochore attachment and chromosome alignment at the metaphase plate. Benomyl also significantly decreased the distance between the sister kinetochore pairs in metaphase cells and increased the level of the checkpoint protein BubR1 at the kinetochore region, indicating that benomyl caused loss of tension across the kinetochores. In addition, benomyl decreased the intercentrosomal distance in mitotic HeLa cells and blocked the cells at mitosis. Further, we analyzed the effects of benomyl on the signal transduction pathways in relation to mitotic block, bcl2 phosphorylation and induction of apoptosis. The results suggest that benomyl causes loss of tension across the kinetochores, blocks the cell cycle progression at mitosis and subsequently, induces apoptosis through the bcl2-bax pathway in a manner qualitatively similar to the powerful microtubule targeted anticancer drugs like the vinca alkaloids and paclitaxel. Considering the very high toxicity of the potent anticancer drugs and the low toxicity of benomyl in humans, we suggest that benomyl could be useful as an adjuvant in combination with the powerful anticancer drugs in cancer therapy.     

Modulation of oxidative phosphorylation augments antineoplastic activity of mitotic aurora kinase inhibition

Uncontrolled mitosis is one of the most important features of cancer, and mitotic kinases are thought to be ideal targets for anticancer therapeutics. However, despite numerous clinical attempts spanning decades, clinical trials for mitotic kinase-targeting agents have generally stalled in the late stages due to limited therapeutic effectiveness. Alisertib (MLN8237) is a promising oral mitotic aurora kinase A (AURKA, Aurora-A) selective inhibitor, which is currently under several clinical evaluations but has failed in its first Phase III trial due to inadequate efficacy. In this study, we performed genome-wide CRISPR/Cas9-based screening to identify vulnerable biological processes associated with alisertib in breast cancer MDA-MB-231 cells. The result indicated that alisertib treated cancer cells are more sensitive to the genetic perturbation of oxidative phosphorylation (OXPHOS). Mechanistic investigation indicated that alisertib treatment, as well as other mitotic kinase inhibitors, rapidly reduces the intracellular ATP level to generate a status that is highly addictive to OXPHOS. Furthermore, the combinational inhibition of mitotic kinase and OXPHOS by alisertib, and metformin respectively, generates severe energy exhaustion in mitotic cells that consequently triggers cell death. The combination regimen also enhanced tumor regression significantly in vivo. This suggests that targeting OXPHOS by metformin is a potential strategy for promoting the therapeutic effects of mitotic kinase inhibitors through the joint targeting of mitosis and cellular energy homeostasis.Subject terms: Mitosis, Cancer screening, Preclinical research     

Killing cells by targeting mitosis

Cell cycle deregulation is a common feature of human cancer. Tumor cells accumulate mutations that result in unscheduled proliferation, genomic instability and chromosomal instability. Several therapeutic strategies have been proposed for targeting the cell division cycle in cancer. Whereas inhibiting the initial phases of the cell cycle is likely to generate viable quiescent cells, targeting mitosis offers several possibilities for killing cancer cells. Microtubule poisons have proved efficacy in the clinic against a broad range of malignancies, and novel targeted strategies are now evaluating the inhibition of critical activities, such as cyclin-dependent kinase 1, Aurora or Polo kinases or spindle kinesins. Abrogation of the mitotic checkpoint or targeting the energetic or proteotoxic stress of aneuploid or chromosomally instable cells may also provide further benefits by inducing lethal levels of instability. Although cancer cells may display different responses to these treatments, recent data suggest that targeting mitotic exit by inhibiting the anaphase-promoting complex generates metaphase cells that invariably die in mitosis. As the efficacy of cell-cycle targeting approaches has been limited so far, further understanding of the molecular pathways modulating mitotic cell death will be required to move forward these new proposals to the clinic.     

Src-dependent autophagic degradation of Ret in FAK-signalling-defective cancer cells

We have recently described that autophagic targeting of Src maintains cancer cell viability when FAK signalling is defective. Here, we show that the Ret tyrosine kinase is also degraded by autophagy in cancer cells with altered/reduced FAK signalling, preventing its binding to FAK at integrin adhesions. Inhibition of autophagy restores Ret localization to focal adhesions. Importantly, Src kinase activity is required to target Ret to autophagosomes and enhance Ret degradation. Src is thus a general mediator of selective autophagic targeting of adhesion-linked kinases, and Ret a second FAK-binding tyrosine kinase degraded through autophagy in cancer cells under adhesion stress. Src-by controlling not only its own degradation but also that of other FAK-binding partners-allows cancer cell survival, suggesting a new therapeutic strategy.     

p53 is not directly relevant to the response of Polo-like kinase 1 inhibitors

Polo-like kinase 1 (Plk1) is elementary for cell proliferation and its deregulation is involved in tumorigenesis. Plk1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small molecule inhibitors targeting either the kinase domain or the Polo-box binding domain (PBD) of Plk1 have been identified and intensively investigated. Intriguingly, Plk1 depletion affects more cancer cells than normal cells. It is also reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with defective p53. The data lead to the hypothesis that p53 might be a predictive marker for the response of Plk1 inhibition. In this study, we demonstrate that there is no obvious different cytotoxic response between cancer cells with and without functional p53, including the isogenic colon cancer cell lines HCT116p53(+/+) and HCT116p53(-/-), breast cancer cell line MCF7, lung cancer cell line A549 and cervical carcinoma cell line HeLa, after treatment with either siRNA against Plk1, the kinase domain inhibitors BI 2536 and BI 6727 or the PBD inhibitor Poloxin. We suggest that the p53 status is not a predictor for the response of Plk1 inhibition, at least not directly. Yet, the long-term outcomes of losing p53, such as genome instability, could be associated with the cytotoxicity of Plk1 inhibition. Further studies are required to investigate whether other circumstances of cancer cells, such as DNA replication/damage stress, mitotic stress, and metabolic stress, which make possibly the survival of cancer cells more dependent on Plk1 function, are responsible for the sensitivity of Plk1 inhibition.     

FAK regulates tyrosine phosphorylation of CAS, paxillin, and PYK2 in cells expressing v-Src, but is not a critical determinant of v-Src transformation.

FAK (focal adhesion kinase) is a nonreceptor protein-tyrosine kinase activated by tyrosine phosphorylation following integrin-mediated cell adhesion. Oncogenic Src promotes enhanced and deregulated FAK tyrosine phosphorylation which has been proposed to contribute to altered cell growth and/or morphological properties associated with transformation. In this study, an inducible FAK expression system was used to study the potential role of FAK in v-Src transformation. Our results portray FAK as a major v-Src substrate that also plays a role in recruiting v-Src to phosphorylate substrates CAS (Crk-associated substrate) and paxillin. The FAK Tyr-397 autophosphorylation site was necessary for this scaffolding function, but was not required for v-Src to stably interact with and phosphorylate FAK. FAK was also shown to negatively regulate v-Src mediated phosphorylation of the FAK-related kinase PYK2. Despite these effects, FAK does not play an essential role in targeting v-Src to major cellular substrates including CAS and paxillin. Nor is FAK strictly required to achieve the altered morphological and growth characteristics of v-Src transformed cells.     

Characterization of the cytotoxic mechanism of Mana-Hox, an analog of manzamine alkaloids

Mana-Hox is a synthetic analog of manzamines, which are beta-carboline alkaloids isolated from marine sponges. Mana-Hox exhibited cytotoxicity against various tumor cell lines with the IC(50) range from 1 to 5 microM. Cell cycle synchronization and flow cytometric analysis showed that Mana-Hox delayed cell cycle progression at mitosis. At the concentration that delayed mitotic progression, bipolar spindle with lagged chromosomes and multipolar spindle with disorganized chromosomes were detected. The presence of such aberrant mitotic cells accompanied by the activation of spindle checkpoint that delayed cells exit from mitosis. However, after a short delay, lagged chromosomes were able to display in the abnormal metaphase plates, and subsequent cell division resulting in chromosome missegregation. Furthermore, the aberrant mitotic cells showed lower viability, indicating that Mana-Hox-induced cell death resulting from chromosome missegregation. This study is the first to explore cytotoxic mechanism of a manzamine-related compound and understand its potential as a lead compound for the development of future anticancer agents.     

Microtubule-targeted agents: when mitochondria become essential to chemotherapy

Microtubule-Targeting Agents (MTAs) constitute a class of drugs largely used for cancer treatment in adults and children. In cancer cells, they suppress microtubule dynamics, and induce cell death via the mitochondrial intrinsic pathway. To date, links between mitochondria and microtubule network disturbance in MTAs mechanism of action are not obvious. The aim of the present contribution is to provide elements that could answer to the question: how far are mitochondria essential to anticancer chemotherapy that targets the microtubule cytoskeleton? We review the main molecular candidates to link microtubule alteration with the apoptotic mitochondrial pathway control. Involvement of direct targeting of mitochondria in MTA efficacy is also discussed. Furthermore, we line up current evidence and emerging concepts on the participation of both mitochondria and microtubule in MTA neurotoxic side effects. To decipher the interconnections between the mitochondrial and the microtubule networks may help to improve cancer cell response to chemotherapy.     

v-Src-Induced Modulation of the Calpain-Calpastatin Proteolytic System Regulates Transformation

v-Src-induced oncogenic transformation is characterized by alterations in cell morphology, adhesion, motility, survival, and proliferation. To further elucidate some of the signaling pathways downstream of v-Src that are responsible for the transformed cell phenotype, we have investigated the role that the calpain-calpastatin proteolytic system plays during oncogenic transformation induced by v-Src. We recently reported that v-Src-induced transformation of chicken embryo fibroblasts is accompanied by calpain-mediated proteolytic cleavage of the focal adhesion kinase (FAK) and disassembly of the focal adhesion complex. In this study we have characterized a positive feedback loop whereby activation of v-Src increases protein synthesis of calpain II, resulting in degradation of its endogenous inhibitor calpastatin. Reconstitution of calpastatin levels by overexpression of exogenous calpastatin suppresses proteolytic cleavage of FAK, morphological transformation, and anchorage-independent growth. Furthermore, calpastatin overexpression represses progression of v-Src-transformed cells through the G(1) stage of the cell cycle, which correlates with decreased pRb phosphorylation and decreased levels of cyclins A and D and cyclin-dependent kinase 2. Calpain 4 knockout fibroblasts also exhibit impaired v-Src-induced morphological transformation and anchorage-independent growth. Thus, modulation of the calpain-calpastatin proteolytic system plays an important role in focal adhesion disassembly, morphological transformation, and cell cycle progression during v-Src-induced cell transformation.     

Inhibitors of phosphatidylinositol 3'-kinases promote mitotic cell death in HeLa cells

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in many biological processes, including cell cycle progression, cell growth, survival, actin rearrangement and migration, and intracellular vesicular transport. However, the involvement of the PI3K pathway in the regulation of mitotic cell death remains unclear. In this study, we treated HeLa cells with the PI3K inhibitors, 3-methyladenine (3-MA, as well as a widely used autophagy inhibitor) and wortmannin to examine their effects on cell fates using live cell imaging. Treatment with 3-MA decreased cell viability in a time- and dose-dependent manner and was associated with caspase-3 activation. Interestingly, 3-MA-induced cell death was not affected by RNA interference-mediated knockdown (KD) of beclin1 (an essential protein for autophagy) in HeLa cells, or by deletion of atg5 (an essential autophagy gene) in mouse embryonic fibroblasts (MEFs). These data indicate that cell death induced by 3-MA occurs independently of its ability to inhibit autophagy. The results from live cell imaging studies showed that the inhibition of PI3Ks increased the occurrence of lagging chromosomes and cell cycle arrest and cell death in prometaphase. Furthermore, PI3K inhibitors promoted nocodazole-induced mitotic cell death and reduced mitotic slippage. Overexpression of Akt (the downstream target of PI3K) antagonized PI3K inhibitor-induced mitotic cell death and promoted nocodazole-induced mitotic slippage. These results suggest a novel role for the PI3K pathway in regulating mitotic progression and preventing mitotic cell death and provide justification for the use of PI3K inhibitors in combination with anti-mitotic drugs to combat cancer.     

PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.