Biochemical heterogeneity and developmental varieties in T-cell leukemia

During mitosis, chromosomes undergo dynamic structural changes that include condensation of chromosomes – the formation of individual compact chromosomes necessary for faithful segregation of sister chromatids in anaphase. Polo-like kinase 1 (Plk1) regulates multiple mitotic events by binding to targeting factors at different mitotic structures in a phosphorylation dependent manner. In this study, we report the identification of a putative ATPase that targets Plk1 to chromosome arms during mitosis. PICH (Plk1-interacting checkpoint “helicase”) displays a temporal localization on chromosome arms and kinetochores during early mitosis. Interaction with PICH recruits Plk1 to chromosome arms and disruption of this interaction abolishes Plk1 localization on chromosome arms. Moreover, depletion of PICH or overexpression of PICH mutant that is defective in Plk1 binding or ATP binding causes defects in mitotic chromosome compaction, formation of anaphase bridge and cytokinesis failure. We provide data to show that both PICH phosphorylation and its ATPase activity are required for mitotic chromosome compaction. Our study provides a mechanism for targeting Plk1 to chromosome arms and suggests that the PICH ATPase activity is important for the regulation of mitotic chromosome architecture.     

MiR-210 disturbs mitotic progression through regulating a group of mitosis-related genes

MiR-210 is up-regulated in multiple cancer types but its function is disputable and further investigation is necessary. Using a bioinformatics approach, we identified the putative target genes of miR-210 in hypoxia-induced CNE cells from genome-wide scale. Two functional gene groups related to cell cycle and RNA processing were recognized as the major targets of miR-210. Here, we investigated the molecular mechanism and biological consequence of miR-210 in cell cycle regulation, particularly mitosis. Hypoxia-induced up-regulation of miR-210 was highly correlated with the down-regulation of a group of mitosis-related genes, including Plk1, Cdc25B, Cyclin F, Bub1B and Fam83D. MiR-210 suppressed the expression of these genes by directly targeting their 3′-UTRs. Over-expression of exogenous miR-210 disturbed mitotic progression and caused aberrant mitosis. Furthermore, miR-210 mimic with pharmacological doses reduced tumor formation in a mouse metastatic tumor model. Taken together, these results implicate that miR-210 disturbs mitosis through targeting multi-genes involved in mitotic progression, which may contribute to its inhibitory role on tumor formation.     

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.     

Non-mitotic functions of polo-like kinases in cancer cells

Inhibitors of mitotic protein kinases are currently being developed as non-neurotoxic alternatives of microtubule-targeting agents (taxanes, vinca alkaloids) which provide a substantial survival benefit for patients afflicted with different types of solid tumors. Among the mitotic kinases, the cyclin-dependent kinases, the Aurora kinases, the kinesin spindle protein and Polo-like kinases (PLKs) have emerged as attractive targets of cancer therapeutics.The functions of mammalian PLK1-5 are traditionally linked to the regulation of the cell cycle and to the stress response. Especially the key role of PLK1 and PLK4 in cellular growth and proliferation, their overexpression in multiple types of human cancer and their druggability, make them appealing targets for cancer therapy. Inhibitors for PLK1 and PLK4 are currently being tested in multiple cancer trials. The clinical success of microtubule-targeting agents is attributed not solely to the induction of a mitotic arrest in cancer cells, but also to non-mitotic effects like targeting intracellular trafficking on microtubules. This raises the question whether new cancer targets like PLK1 and PLK4 regulate critical non-mitotic functions in tumor cells. In this article we summarize the important roles of PLK1-5 for the regulation of non-mitotic signaling. Due to these functions it is conceivable that inhibitors for PLK1 or PLK4 can target interphase cells, which underscores their attractive potential as cancer drug targets. Moreover, we also describe the contribution of the tumor-suppressors PLK2, PLK3 and PLK5 to cancer cell signaling outside of mitosis. These observations highlight the urgent need to develop highly specific ATP-competitive inhibitors for PLK4 and for PLK1 like the 3^rd generation PLK-inhibitor Onvansertib to prevent the inhibition of tumor-suppressor PLKs in- and outside of mitosis. The remarkable feature of PLKs to encompass a unique druggable domain, the polo-box-domain (PBD) that can be found only in PLKs offers the opportunity for the development of inhibitors that target PLKs exclusively. Beyond the development of mono-specific ATP-competitive PLK inhibitors, the PBD as drug target will support the design of new drugs that eradicate cancer cells based on the mitotic and non-mitotic function of PLK1 and PLK4.     

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.     

Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest

Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy.     

The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly

During cell division, cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule copurifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential antimitotic target.     

Deficiency in SNM1 abolishes an early mitotic checkpoint induced by spindle stress

Spindle poisons represent an important class of anticancer drugs that act by interfering with microtubule polymerization and dynamics and thereby induce mitotic checkpoints and apoptosis. Here we show that mammalian SNM1 functions in an early mitotic stress checkpoint that is distinct from the well-characterized spindle checkpoint that regulates the metaphase-to-anaphase transition. Specifically, we found that compared to wild-type cells, Snm1-deficient mouse embryonic fibroblasts exposed to spindle poisons exhibited elevated levels of micronucleus formation, decreased mitotic delay, a failure to arrest in mitosis prior to chromosome condensation, supernumerary centrosomes, and decreased viability. In addition, we show that both Snm1 and 53BP1, previously shown to interact, coimmunoprecipitate with components of the anaphase-promoting complex (APC)/cyclosome. These findings suggest that Snm1 is a component of a mitotic stress checkpoint that negatively targets the APC prior to chromosome condensation.     

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.     

Roles of Aurora kinases in mitosis and tumorigenesis

Aurora kinases, which have been implicated in several vital events in mitosis, represent a protein kinase family highly conserved during evolution. The activity of Aurora kinases is delicately regulated, mainly by phosphorylation and degradation. Deregulation of Aurora kinase activity can result in mitotic abnormality and genetic instability, leading to defects in centrosome function, spindle assembly, chromosome alignment, and cytokinesis. Both the expression level and the kinase activity of Aurora kinases are found to be up-regulated in many human cancers, indicating that these kinases might serve as useful targets for the development of anticancer drugs. This review focuses on recent progress on the roles of Aurora kinases in mitosis and tumorigenesis.     

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     

Spindle poisons and cell fate: a tale of two pathways

Spindle poisons, such as paclitaxel and vinblastine, exert their potent anti-neoplastic effects through activation of the spindle assembly checkpoint (SAC), thereby arresting cells in mitosis. Unfortunately, only certain cancers are susceptible to these drugs, and many patients fail to respond to treatment. We review the pathways that are triggered by spindle poisons and highlight recent studies that describe the great variability of tumor cells in responding to these drugs. We also describe the recent identification of an apoptotic pathway that is activated by mitotic arrest in response to spindle poisons. Emerging from these studies is not only a greater understanding of how these classic antimitotic agents bring about cell death, but also a wealth of potential new targets of anticancer therapeutics.     

Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe

Disturbing mitotic progression via targeted anti-mitotic therapy is an attractive strategy for cancer treatment. Therefore, the exploration and elucidation of molecular targets and pathways in mitosis are critical for the development of anti-mitotic drugs. Here, we show that cell division cycle 5-like (Cdc5L), a pre-mRNA splicing factor, is a regulator of mitotic progression. Depletion of Cdc5L causes dramatic mitotic arrest, chromosome misalignments and sustained activation of spindle assembly checkpoint, eventually leading to mitotic catastrophe. Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage. Genome-wide gene expression analysis reveals that Cdc5L modulates the expression of a set of genes involved in the mitosis and the DNA damage response. We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down. Interestingly, Cdc5L is highly expressed in cervical tumors and osteosarcoma. Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells. These results suggest that Cdc5L is a key regulator of mitotic progression and highlight the potential of Cdc5L as a target for cancer therapy.     

NUSAP1与恶性肿瘤

核仁纺锤体相关蛋白1(nucleolar and spindle-associated protein 1,NUSAP1)是一种微管结合蛋白,它与微管结合后稳定微管并参与细胞分裂,与染色体结合后,促进有丝分裂纺锤体微管的形成;因此NUSAP1与细胞有丝分裂进程、纺锤体的形成有密切关系.NUSAP1的一个关键功能是在有丝...     

Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.     

Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)–dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.     

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-x_L by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.     

Ran is required before metaphase for spindle assembly and chromosome alignment and after metaphase for chromosome segregation and spindle midbody organization

The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.