Centrosome Separation Research Articles

NEK9 depletion induces catastrophic mitosis by impairment of mitotic checkpoint control and spindle dynamics

NEK9 is known to play a role in spindle assembly and in the control of centrosome separation, but the consequences of NEK9 targeting in cancer cells remain to be elucidated. In this study, we used siRNA to investigate the consequences of targeting NEK9 in glioblastoma and kidney cancer cells as a first step in assessing its potential as an anti-cancer therapeutic target. Live cell imaging revealed that NEK9 depletion of U1242 glioblastoma and Caki2 kidney carcinoma cells resulted in failure of cytokinesis. Interestingly, NEK9-depleted Caki2 cells overrode mitosis under incorrect chromosome alignment and were converted to a micronucleated phenotype, leading to cell death. Whereas, the RPE1 normal epithelium cell line was refractory to abnormal mitosis upon NEK9 knockdown. Nocodazole-induced mitotic arrest was compromised after NEK9 depletion, indicating that NEK9 has an important role in mitotic checkpoint system. Taken together, we propose that NEK9 inhibition represents a novel anti-cancer strategy by induction of mitotic catastrophe via impairment of spindle dynamics, cytokinesis and mitotic checkpoint control.

The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A

Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2-depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.

EGF-Induced Centrosome Separation Promotes Mitotic Progression and Cell Survival

Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5.

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes

Loss of the tumor suppressor PTEN is observed in many human cancers that display increased chromosome instability and aneuploidy. The subcellular fractions of PTEN are associated with different functions that regulate cell growth, invasion and chromosome stability. In this study, we show a novel role for PTEN in regulating mitotic centrosomes. PTEN localization at mitotic centrosomes peaks between prophase and metaphase, paralleling the centrosomal localization of PLK-1 and γ-tubulin and coinciding with the time frame of centrosome maturation. In primary keratinocytes, knockdown of PTEN increased whole-cell levels of γ-tubulin and PLK-1 in an Akt-dependent manner and had little effect on recruitment of either protein to mitotic centrosomes. Conversely, knockdown of PTEN reduced centrosomal levels of pericentrin in an Akt-independent manner. Inhibition of Akt activation with MK2206 reduced the whole-cell and centrosome levels of PLK-1 and γ-tubulin and also prevented the recruitment of PTEN to mitotic centrosomes. This reduction in centrosome-associated proteins upon inhibition of Akt activity may contribute to the increase in defects in centrosome number and separation observed in metaphase cells. Concomitant PTEN knockdown and Akt inhibition reduced the frequency of metaphase cells with centrosome defects when compared with MK2206 treatment alone, indicating that both PTEN and pAkt are required to properly regulate centrosome composition during mitosis. The findings presented in this study demonstrate a novel role for PTEN and Akt in controlling centrosome composition and integrity during mitosis and provide insight into how PTEN functions as a multifaceted tumor suppressor.

Abstract 3391: Overexpression of Nek2 promotes bortezomib resistance in multiple myeloma cells.

Abstract Drug resistance is a major complication in cancer therapy but the molecular mechanisms driving both innate and acquired drug resistance still remain largely unknown for many anti-cancer agents. Microarray analyses on paired primary multiple myeloma samples at baseline and after therapy or at relapse showed that the mitotic kinase NEK2 was one of the most up-regulated genes in myeloma cells after high-dose chemotherapy or at relapse. Nek2 is a Serine/Threonine kinase that is activated during mitosis and is associated centrosome function and structure at the G2/M transition. A role for Nek2 has been suggested in the separation of centrosomes as well as promoting cell cycle from G2 phase to M phase but no role for Nek2 in mediating drug resistance has been described. By analyzing the published (> 2,500) microarrays and clinical datasets, we found that NEK2 expression is increased in many malignancies, and that high expression of NEK2 was associated with a shorter event-free and overall survival. Moreover, NEK2 expression was typically increased in tumors with aggressive subtype and advanced stage. Our studies in myeloma cells indicate that over-expressing NEK2 results in enhanced cell proliferation and drug resistance, whereas knockdown of NEK2 induced significant cancer cell death and growth inhibition. Nek2 transfected myeloma cell lines ARP1, H929 and KMS28PE were used to investigate Nek2’s function relative to bortezomib resistance. We found that Nek2 over-expression significantly elevated the proteasome activity of transfected cells leading to decreased activity of Bortezomib in these cells. Using novel small molecule Nek2 inhibitors developed in our lab, we demonstrated efficient inhibition of proteasome activity in Nek2 over-expressing cancer cell lines. The treatment of cell with our lead compounds significantly inhibited the degradation of some proteasome targeted cell cycle regulators. Further, the combinations of these Nek2 inhibitors and bortezomib, greatly elevated the efficiency of bortezomib in decreasing the proteasome activity in vitro. The treatment with these novel Nek2 inhibitors successfully rescued the drug resistance in Nek2 over-expressed cells, indicating a potential therapy for bortezomib resistant myeloma patients. We conclude that NEK2 represents a predictor for drug resistance and poor prognosis in cancers and could be a potential target for cancer therapy. Citation Format: Jessica Flory, Rachel Brog, Ling Yao Meng, Lee Call, Hariprasad Vankayalapati, Guido Tricot, Fenghuang Zhan, David J. Bearss. Overexpression of Nek2 promotes bortezomib resistance in multiple myeloma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3391. doi:10.1158/1538-7445.AM2013-3391

Abstract 1746: Coordination of overexpressed NIMA-related kinase 2 and TRF1 results in mitotic abnormalities in breast cancer cells.

Abstract NIMA-related kinase 2 (NEK2), a serine-threonine protein kinase, plays a role in features of mitotic progression including timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Kinase death NEK2 mutant expression or NEK2 depleted cells lead to failure of centrosome separation while NEK2 overexpression results in premature centrosome separation. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with NEK2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between NEK2 and TRF1 are far from clear. Here, we show that mitotic aberrations through NEK2 overexpression are likely to require TRF1. Our results demonstrate that NEK2 directly binds and phosphorylates TRF1 in vitro and in vivo through multiple sites on TRF1. NEK2 overexpression in breast cancer cells, MCF 7 and MDA-MB-231, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the occurrence of unaligned chromosomes by NEK2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1 depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA transfected cells. Therefore, we propose that TRF1 is required for overexpressed NEK2 to trigger abnormal mitosis and chromosomal instability. Citation Format: Jaehyung Lee. Coordination of overexpressed NIMA-related kinase 2 and TRF1 results in mitotic abnormalities in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1746. doi:10.1158/1538-7445.AM2013-1746

Abstract 4501: Diverse microtubule stabilizers cause differential expression of mitotic kinases leading to distinct centrosomal and mitotic defects.

Abstract Microtubule stabilizers are some of the most useful and successful drugs used to treat adult solid tumors. However, the molecular events responsible for their antimitotic actions are not yet fully understood. In this study we evaluated the mitotic defects and signaling events initiated by three structurally and biologically different microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel. These microtubule stabilizers cause the formation of aberrant, but morphologically distinct mitotic spindles leading to the hypothesis that they initiate distinct mitotic signaling events. Each microtubule stabilizer caused different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ caused centrosome separation failure to a much greater extent than paclitaxel or laulimalide. Taccalonolide AJ is also unique in that it was the only stabilizer to cause centrosome disjunction failure. These observations were consistent with the different defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer, which suggests a common mechanism of aster formation. However, taccalonolide AJ was the only stabilizer evaluated that caused pericentrin accumulation on every spindle aster. Pericentrin's localization to every spindle aster could facilitate the maintenance and stability of the highly focused asters formed by taccalonolide AJ. Laulimalide and paclitaxel caused completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Elucidating how chemically distinct microtubule stabilizers disturb mitotic signaling could identify key proteins involved in controlling sensitivity and resistance to the antimitotic actions of these agents. Citation Format: Cristina C. Rohena, Jiangnan Peng, Tyler A. Johnson, Phillip Crews, Susan L. Mooberry. Diverse microtubule stabilizers cause differential expression of mitotic kinases leading to distinct centrosomal and mitotic defects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2013-4501

Abstract 3407: A novel CENP-E-selective inhibitor exhibits potent anti-tumor efficacy by two distinct mechanisms of action dependent on spindle assembly checkpoint activity.

Abstract Centromere-associated protein-E (CENP-E) is a mitotic spindle motor protein belonging to the kinesin superfamily and controls chromosome alignment during metaphase by capturing the microtubule plus end at the kinetochore. Loss of CENP-E function has been reported to result in misaligned chromosomes at metaphase leading to spindle assembly checkpoint (SAC) activation. Here, we developed a novel small-molecule inhibitor of CENP-E (Compound-A), which targets an ATPase domain at the CENP-E N-terminal motor region. Enzymatic kinetics reveals that Compound-A is a slow-off rate type of ATP-competitive CENP-E inhibitor. Treatment of Hela cells with Compound-A induced chromosome misalignment during the SAC-dependent mitotic arrest, resulting in potent growth suppression by apoptosis. Furthermore, intraperitoneal administration of Compound-A displayed potent anti-tumor efficacy in colo205-xenograft mouse models (T/C=11 % at day 8). Given that SAC activation by CENP-E inhibition induced mitotic death, we next investigated whether SAC attenuation by BubR1 knockdown recovers cell viability in these cells. However, CENP-E inhibition was able to cause apoptosis after mitotic slippage in the BubR1-knockdown Hela cells, while inhibition of another mitotic kinesin Eg5, which controls centrosome separation during mitosis, induced polyploidy instead of apoptosis in these cells. Our data suggest that asymmetric chromosome segregation accelerated by CENP-E inhibition, but not polyploidy by Eg5 inhibition, is responsible for apoptosis after mitotic slippage. A comprehensive gene expression analysis of microarray comparisons revealed that p53 pathways are activated by CENP-E inhibition after mitotic slippage, and p53 knockdown suppressed post-mitotic caspase-3/7 activation by CENP-E inhibition. Furthermore, we found that both phosphorylation of at Ser-15 by ATM and ATR kinases and accumulation of p53 protein by unfolding protein response (UPR) were involved in post-mitotic p53 activation after mitotic slippage. In conclusion, we developed the novel CENP-E inhibitor, Compound-A, and CENP-E inhibition by Compound-A induced potent growth inhibition in both SAC-intact and SAC-defective cancer cells. In the latter case, p53 pathways play important roles in the induction of apoptosis. Our data demonstrate that SAC and p53 pathways complementally function to eliminate aberrant chromosome segregation accelerated by CENP-E inhibition. Thus, SAC activation and p53 protein accumulation are available for complementary PD biomarkers of the CENP-E inhibitors, and several molecules in the SAC and p53 pathways could be potential biomarkers to select sensitive tumors to the CENP-E inhibitors. Citation Format: Akihiro Ohashi, Momoko Ohori, Kenichi Iwai, Yusuke Nakayama, Tadahiro Nambu, Daisuke Morishita, Tomohiro Kawamoto, Maki Miyamoto, Takaharu Hirayama, Masanori Okaniwa, Hiroshi Banno, Tomoyasu Ishikawa, Hitoshi Kandori, Kentaro Iwata. A novel CENP-E-selective inhibitor exhibits potent anti-tumor efficacy by two distinct mechanisms of action dependent on spindle assembly checkpoint activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3407. doi:10.1158/1538-7445.AM2013-3407

G Protein‐Coupled Receptor Kinase 2 (GRK2) is Localized to Centrosomes and Mediates Epidermal Growth Factor‐Promoted Separation of Duplicated Centrosomes

G protein‐coupled receptor kinases (GRKs) play a central role in regulating receptor signaling. Recent studies have suggested a broader role for these enzymes in regulating normal cellular functions. For example, GRK5 has been shown to localize in centrosomes and modulate cell cycle progression. In this study, we demonstrate that GRK2 is also localized to centrosomes. Similar to GRK5, the centrosomal localization of GRK2 is microtubule‐independent and it does not affect normal centrosome duplication. However, in contrast to GRK5, GRK2 is not involved in centrosome nucleation. Interestingly, knockdown of GRK2 inhibits epidermal growth factor (EGF)‐initiated separation of duplicated centrosomes during S phase in both HeLa cells and retinal pigment epithelial 1 (RPE1) cells. This EGFR‐GRK2 mediated process is dependent on the protein kinases MST‐2 and Nek2a but does not involve Polo‐like kinase 1 (PLK1). This separation during S phase results in enhanced centrosome amplification, resulting in a rise in the number of cells demonstrating aberrant duplicated centrosomes. Collectively, these findings demonstrate that GRK2 is localized in centrosomes and plays a central role in mitogen‐promoted centrosome separation.

Phosphorylation of NuMA by Aurora‐A kinase in PC‐3 prostate cancer cells affects proliferation, survival, and interphase NuMA localization

Aurora-A is a serine/threonine kinase that has oncogenic properties in vivo. The expression and kinase activity of Aurora-A are up-regulated in multiple malignancies. Aurora-A is a key regulator of mitosis that localizes to the centrosome from the G2 phase through mitotic exit and regulates mitotic spindle formation as well as centrosome separation. Overexpression of Aurora-A in multiple malignancies has been linked to higher tumor grade and poor prognosis through mechanisms that remain to be defined. Using an unbiased proteomics approach, we identified the protein nuclear mitotic apparatus (NuMA) as a robust substrate of Aurora-A kinase. Using a small molecule Aurora-A inhibitor in conjunction with a reverse in-gel kinase assay (RIKA), we demonstrate that NuMA becomes hypo-phosphorylated in vivo upon Aurora-A inhibition. Using an alanine substitution strategy, we identified multiple Aurora-A phospho-acceptor sites in the C-terminal tail of NuMA. Functional analyses demonstrate that mutation of three of these phospho-acceptor sites significantly diminished cell proliferation. In addition, alanine mutation at these sites significantly increased the rate of apoptosis. Using confocal immunofluorescence microscopy, we show that the NuMA T1804A mutant mis-localizes to the cytoplasm in interphase nuclei in a punctate pattern. The identification of Aurora-A phosphorylation sites in NuMA that are important for cell cycle progression and apoptosis provides new insights into Aurora-A function.

Chemically diverse microtubule stabilizing agents initiate distinct mitotic defects and dysregulated expression of key mitotic kinases

Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent than paclitaxel or laulimalide, which is consistent with the distinct defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer. This suggests a common mechanism of aster formation. However, taccalonolide AJ also causes pericentrin accumulation on every spindle aster. The presence of pericentrin at every spindle aster initiated by taccalonolide AJ might facilitate the maintenance and stability of the highly focused asters formed by this stabilizer. Laulimalide and paclitaxel cause completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Delineating how diverse microtubule stabilizers affect mitotic signaling pathways could identify key proteins involved in modulating sensitivity and resistance to the antimitotic actions of these compounds.

POPX2 phosphatase regulates the KIF3 kinesin motor complex

The kinesin motors are important in the regulation of cellular functions such as protein trafficking, spindle organization and centrosome separation. In this study, we have identified POPX2, a serine-threonine phosphatase, as an interacting partner of the KAP3 subunit of the kinesin-2 motor. The kinesin-2 motor is a heterotrimeric complex composed of KIF3A, KIF3B motor subunits and KAP3, the non-motor subunit, which binds the cargo. Here we report that the phosphatase POPX2 is a negative regulator of the trafficking of N-cadherin and other cargoes; consequently, it markedly influences cell-cell adhesion. POPX2 affects trafficking by determining the phosphorylation status of KIF3A at serine 690. This is consistent with the observation that the KIF3A-S690A mutant is defective in cargo trafficking. Our studies also implicate CaMKII as the kinase that phosphorylates KIF3A at serine 690. These results strongly suggest that POPX2 and CaMKII are a phosphatase-kinase pair that regulates kinesin-mediated transport and cell-cell adhesion.

Mitosis-Coupled, Microtubule-Dependent Clustering of Endosomal Vesicles around Centrosomes

Upon cell division, not only cells themselves but also their organelles undergo drastic shape changes, although the behaviors of organelles other than the Golgi apparatus remain poorly understood. We followed the spatiotemporal changes in the localization of transferrin receptor (TfnR) and other proteins. In early mitotic phases, a population of proteins cycling through the endocytic recycling compartment (ERC) exhibits a distinct spatiotemporal change from that of Golgi proteins. In prophase/prometaphase, when the cell surface-to-volume ratio is reaching its minimum, the ERC proteins are transiently assembled around the centrated centrosome in a microtubule- and dynein-dependent manner, and soon separated polewards into two clusters concomitant with separation of duplicated centrosomes. Electron microscopic analysis revealed that endosomal vesicles containing endocytosed transferrin cluster tightly around centrosomes without fusing with one another. As cytokinesis proceeds, the clusters gradually collapse, and the ERC proteins reassemble around the furrowing equatorial region. FRAP (fluorescence recovery after photobleaching) analyses of EGFP-TfnR-expressing cells revealed minimal membrane exchange between the endosomal clusters and other cellular compartments until anaphase/telophase, when membrane traffic resumes. Our observations indicate that ERC clustering around centrosomes plays a fundamental role in restricting membrane delivery to the plasma membrane during early mitotic phases, when the cell surface-to-volume ratio reaches its minimum.

Evaluation of the Aurora Kinase Inhibitor, ZM447439, in Canine Malignant Lymphoid Cells <i>in Vitro</i>

Aurora kinases play an important role in the cell cycle. These enzymes help establish mitotic spindles by directing centrosome duplication and separation and by regulating the spindle assembly checkpoint thereby helping control cytokinesis. An over-expression of aurora kinases has been reported in a variety of human tumors. In this study, we identified the expression of aurora-A and aurora-B kinases in canine malignant lymphoid cells. We also evaluated the effects of the aurora kinase inhibitor (ZM447439), and found that this inhibitor decreases cell viability, increases DNA content change, and leads to apoptosis in canine B- and T-cell lymphoid cell lines. The lymphotoxicity induced by ZM447439 in these canine lymphoid cell lines suggests that further in vivo evaluation of aurora kinase inhibitors as a potential treatment for canine malignant lymphoid tumors is warranted.

In silico Screening for Identification of Novel Aurora Kinase Inhibitors by Molecular Docking, Dynamics Simulations and Ligand-Based Hypothesis Approaches

Aurora kinase family members are involved in a wide variety of cell cycle events including centrosome separation, cytokinesis, kinetochore formation, spindle assembly, chromosomal segregation and microtubule dynamics. Typically, dysfunction of Aurora proteins is associated with aneuploidy, cell death and mitotic arrest, leading to tumorigenesis. This spurred a vast interest to identify pharmacologically active small-molecule inhibitors of Aurora proteins. In this study, we isolated four novel inhibitors through virtual screening and docking analyses. These hits were subsequently characterized by molecular dynamic simulations to monitor their binding stabilities at ATP binding site. To aid characterizing novel and more potent inhibitors for Aurora kinases, we explored selective features of our ligand dataset by ligand-based pharmacophore modeling approach. The best pharmacophore model was then employed for performing virtual screenings of libraries isolated from Princeton and Uorsy databases. On the basis of common pharmacophore features, Lipinski’s rule of five, absorption, distribution, metabolism and elimination properties, hits were short listed and refined by molecular dockings. Finally, the selected compounds were validated on the basis of binding capabilities, consensus scoring and activity values. We propose that the novel inhibitors described in this study may warrant characterization in designing active lead for clinical studies that may serve as anticancer drugs in future.

APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation

APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation

USP44 regulates centrosome positioning to prevent aneuploidy and suppress tumorigenesis

Most human tumors have abnormal numbers of chromosomes, a condition known as aneuploidy. The mitotic checkpoint is an important mechanism that prevents aneuploidy by restraining the activity of the anaphase-promoting complex (APC). The deubiquitinase USP44 was identified as a key regulator of APC activation; however, the physiological importance of USP44 and its impact on cancer biology are unknown. To clarify the role of USP44 in mitosis, we engineered a mouse lacking Usp44. We found that USP44 regulated the mitotic checkpoint and prevented chromosome lagging. Mice lacking Usp44 were prone to the development of spontaneous tumors, particularly in the lungs. Additionally, USP44 was frequently downregulated in human lung cancer, and low expression correlated with a poor prognosis. USP44 inhibited chromosome segregation errors independent of its role in the mitotic checkpoint by regulating centrosome separation, positioning, and mitotic spindle geometry. These functions required direct binding to the centriole protein centrin. Our data reveal a new role for the ubiquitin system in mitotic spindle regulation and underscore the importance of USP44 in the pathogenesis of human cancer.

Kinesin spindle protein SiRNA slows tumor progression

The kinesin spindle protein (KSP), a member of the kinesin superfamily of microtubule-based motors, plays a critical role in mitosis as it mediates centrosome separation and bipolar spindle assembly and maintenance. Inhibition of KSP function leads to cell cycle arrest at mitosis with the formation of monoastral microtubule arrays, and ultimately, to cell death. Several KSP inhibitors are currently being studied in clinical trials and provide new opportunities for the development of novel anticancer therapeutics. RNA interference (RNAi) may represent a powerful strategy to interfere with key molecular pathways involved in cancer. In this study, we have established an efficient method for intratumoral delivery of siRNA. We evaluated short interfering RNA (siRNA) duplexes targeting luciferase as surrogate marker or KSP sequence. To examine the potential feasibility of RNAi therapy, the siRNA was transfected into pre-established lesions by means of intratumor electro-transfer of RNA therapeutics (IERT). This technology allowed cell permeation of the nucleic acids and to efficiently knock down gene expression, albeit transiently. The KSP-specific siRNA drastically reduced outgrowth of subcutaneous melanoma and ovarian cancer lesions. Our results show that intratumoral electro-transfer of siRNA is feasible and KSP-specific siRNA may provide a novel strategy for therapeutic intervention.

Nuclear envelope-associated dynein drives prophase centrosome separation and enables Eg5-independent bipolar spindle formation

The microtubule motor protein kinesin-5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti-cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5-independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)-associated dynein that drives centrosome separation in prophase. This NE-dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5-dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin-5 inhibitors.

Functional analysis of centrosomal kinase substrates in Drosophila melanogaster reveals a new function of the nuclear envelope component otefin in cell cycle progression.

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.