Centrosome Separation Research Articles

O-GlcNAcylation of myosin phosphatase targeting subunit 1 (MYPT1) dictates timely disjunction of centrosomes

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)-protein phosphatase 1cβ (PP1cβ) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc-profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions.

Cell Cycle-Dependent Dynamics of the Golgi-Centrosome Association in Motile Cells.

Here, we characterize spatial distribution of the Golgi complex in human cells. In contrast to the prevailing view that the Golgi compactly surrounds the centrosome throughout interphase, we observe characteristic differences in the morphology of Golgi ribbons and their association with the centrosome during various periods of the cell cycle. The compact Golgi complex is typical in G1; during S-phase, Golgi ribbons lose their association with the centrosome and extend along the nuclear envelope to largely encircle the nucleus in G2. Interestingly, pre-mitotic separation of duplicated centrosomes always occurs after dissociation from the Golgi. Shortly before the nuclear envelope breakdown, scattered Golgi ribbons reassociate with the separated centrosomes restoring two compact Golgi complexes. Transitions between the compact and distributed Golgi morphologies are microtubule-dependent. However, they occur even in the absence of centrosomes, which implies that Golgi reorganization is not driven by the centrosomal microtubule asters. Cells with different Golgi morphology exhibit distinct differences in the directional persistence and velocity of migration. These data suggest that changes in the radial distribution of the Golgi around the nucleus define the extent of cell polarization and regulate cell motility in a cell cycle-dependent manner.

LIN9 and NEK2 Are Core Regulators of Mitotic Fidelity That Can Be Therapeutically Targeted to Overcome Taxane Resistance.

A significant therapeutic challenge for patients with cancer is resistance to chemotherapies such as taxanes. Overexpression of LIN9, a transcriptional regulator of cell-cycle progression, occurs in 65% of patients with triple-negative breast cancer (TNBC), a disease commonly treated with these drugs. Here, we report that LIN9 is further elevated with acquisition of taxane resistance. Inhibiting LIN9 genetically or by suppressing its expression with a global BET inhibitor restored taxane sensitivity by inducing mitotic progression errors and apoptosis. While sustained LIN9 is necessary to maintain taxane resistance, there are no inhibitors that directly repress its function. Hence, we sought to discover a druggable downstream transcriptional target of LIN9. Using a computational approach, we identified NIMA-related kinase 2 (NEK2), a regulator of centrosome separation that is also elevated in taxane-resistant cells. High expression of NEK2 was predictive of low survival rates in patients who had residual disease following treatment with taxanes plus an anthracycline, suggesting a role for this kinase in modulating taxane sensitivity. Like LIN9, genetic or pharmacologic blockade of NEK2 activity in the presence of paclitaxel synergistically induced mitotic abnormalities in nearly 100% of cells and completely restored sensitivity to paclitaxel, in vitro. In addition, suppressing NEK2 activity with two distinct small molecules potentiated taxane response in multiple in vivo models of TNBC, including a patient-derived xenograft, without inducing toxicity. These data demonstrate that the LIN9/NEK2 pathway is a therapeutically targetable mediator of taxane resistance that can be leveraged to improve response to this core chemotherapy. SIGNIFICANCE: Resistance to chemotherapy is a major hurdle for treating patients with cancer. Combining NEK2 inhibitors with taxanes may be a viable approach for improving patient outcomes by enhancing mitotic defects induced by taxanes alone.

Kinesin-5 Eg5 is essential for spindle assembly and chromosome alignment of mouse spermatocytes

BackgroundMicrotubule organization is essential for bipolar spindle assembly and chromosome segregation, which contribute to genome stability. Kinesin-5 Eg5 is known to be a crucial regulator in centrosome separation and spindle assembly in mammalian somatic cells, however, the functions and mechanisms of Eg5 in male meiotic cell division remain largely unknown.ResultsIn this study, we have found that Eg5 proteins are expressed in mouse spermatogonia, spermatocytes and spermatids. After Eg5 inhibition by specific inhibitors Monastrol, STLC and Dimethylenastron, the meiotic spindles of dividing spermatocytes show spindle collapse and the defects in bipolar spindle formation. We demonstrate that Eg5 regulates spindle bipolarity and the maintenance of meiotic spindles in meiosis. Eg5 inhibition leads to monopolar spindles, spindle abnormalities and chromosome misalignment in cultured GC-2 spd cells. Furthermore, Eg5 inhibition results in the decrease of the spermatids and the abnormalities in mature sperms.ConclusionsOur results have revealed an important role of kinesin-5 Eg5 in male meiosis and the maintenance of male fertility. We demonstrate that Eg5 is crucial for bipolar spindle assembly and chromosome alignment in dividing spermatocytes. Our data provide insights into the functions of Eg5 in meiotic spindle assembly of dividing spermatocytes.

Decreased Nek9 expression correlates with aggressive behaviour and predicts unfavourable prognosis in breast cancer

As a new member of Neks family, Nek9 regulates spindle assembly and controls chromosome alignment and centrosome separation. In the current study we aimed to investigate the expression of Nek9 in breast cancer and its clinical significance. We evaluated the expression of Nek9 in invasive ductal carcinoma (IDC, n=316), ductal carcinoma in situ (DCIS), usual ductal hyperplasia, atypical ductal hyperplasia, fibroadenoma and normal breast tissues using immunohistochemistry. The results revealed significantly reduced Nek9 in IDCs (41.8%) compared to benign breast lesions. Moreover, gradually reduced Nek9 was found from DCIS to invasive carcinoma and metastatic tumour within the same tumours. The decrease in Nek9 expression was associated with larger tumour size (p=0.0087), high grade (p<0.0001) and high Ki-67 index (p<0.0020). TCGA and GEO datasets analysis revealed low level of Nek9 mRNA was more frequent in triple negative breast cancers, and associated with poor overall survival and distant metastasis-free survival. These findings suggest an important role of Nek9 in the progression of breast cancer, and aberrantly expressed Nek9 correlates with more aggressive clinicopathological variables and predicts poor clinical prognosis. Nek9 may serve as a potential predictive factor for patients with breast cancer.

MiR-1285-5p/TMEM194A axis affects cell proliferation in breast cancer.

The onset of breast cancer among young patients is a major issue in cancer etiology. Our previous study has shown that poor prognosis in young women with breast cancer is associated with lower expression of the microRNA miR‐1285‐5p. In this study, we showed that the expression of miR‐1285‐5p is lower in tumor tissues than in normal tissues. Accumulating evidence suggests that miR‐1285‐5p plays critical roles in various types of cancers. However, the functional role of miR‐1285‐5p in breast cancer remains to be elucidated. Here, we showed the tumor‐suppressive role of miR‐1285‐5p and detailed its mechanism of action in breast cancer. Overexpression of miR‐1285‐5p significantly inhibited cell proliferation in breast cancer cells regardless of the tumor subtype. Among the target genes of miR‐1285‐5p, we found that transmembrane protein 194A (TMEM194A) was directly regulated by miR‐1285‐5p. Notably, separation of centrosomes from the nuclear envelope was observed upon knockdown of TMEM194A or overexpression of miR‐1285‐5p. In conclusion, our findings show that miR‐1285‐5p is a tumor suppressor via TMEM194A inhibition in breast cancer.

TRIM8 interacts with KIF11 and KIFC1 and controls bipolar spindle formation and chromosomal stability

The faithful inheritance of chromosomes is essential for the propagation of organisms. In eukaryotes, central to this process is the mitotic spindle. Recently, we have identified TRIM8 as a gene aberrantly expressed in gliomas whose expression reduces the clonogenic potential in the patients' glioma cells. TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. To gain insights into the TRIM8 functions, we characterized the TRIM8 interactome in primary mouse embryonic neural stem cells using proteomics. We found that TRIM8 interacts with KIFC1, and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. By exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability.

Trackosome: a computational toolbox to study the spatiotemporal dynamics of centrosomes, nuclear envelope and cellular membrane.

During the initial stages of mitosis, multiple mechanisms drive centrosome separation and positioning. How they are coordinated to promote centrosome migration to opposite sides of the nucleus remains unclear. Here, we present Trackosome, an open-source image analysis software for tracking centrosomes and reconstructing nuclear and cellular membranes, based on volumetric live-imaging data. The toolbox runs in MATLAB and provides a graphical user interface for easy access to the tracking and analysis algorithms. It provides detailed quantification of the spatiotemporal relationships between centrosomes, nuclear envelope and cellular membrane, and can also be used to measure the dynamic fluctuations of the nuclear envelope. These fluctuations are important because they are related to the mechanical forces exerted on the nucleus by its adjacent cytoskeletal structures. Unlike previous algorithms based on circular or elliptical approximations, Trackosome measures membrane movement in a model-free condition, making it viable for irregularly shaped nuclei. Using Trackosome, we demonstrate significant correlations between the movements of the centrosomes, and identify specific oscillation modes of the nuclear envelope. Overall, Trackosome is a powerful tool that can be used to help unravel new elements in the spatiotemporal dynamics of subcellular structures.

The balance of forces generated by kinesins controls spindle polarity and chromosomal heterogeneity in tetraploid cells.

Chromosomal instability, one of the most prominent features of tumour cells, causes aneuploidy. Tetraploidy is thought to be an intermediate on the path to aneuploidy, but the mechanistic relationship between the two states is poorly understood. Here, we show that spindle polarity (e.g. bipolarity or multipolarity) in tetraploid cells depends on the level of functional phosphorylated Eg5, a mitotic kinesin, localised to the spindle. Multipolar spindles are formed in cells with high levels of phosphorylated Eg5. This process is suppressed by inhibition of Eg5 or expression of a non-phosphorylatable Eg5 mutant, as well as by changing the balance between opposing forces required for centrosome separation. Tetraploid cells with high levels of functional Eg5 give rise to a heterogeneous aneuploid population through multipolar division, whereas cells with low levels of functional Eg5 continue to undergo bipolar division and remain tetraploid. Furthermore, Eg5 protein levels correlate with ploidy status in tumour specimens. We provide a novel explanation for the tetraploid intermediate model, i.e. spindle polarity and subsequent tetraploid cell behaviour are determined by the balance of forces generated by mitotic kinesins at the spindle.

Drosophila pericentrin-like protein promotes the formation of primordial germ cells.

Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin-like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles.

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Cell polarity-dependent centrosome separation in the C. elegans embryo.

In animal cells, faithful chromosome segregation depends on the assembly of a bipolar spindle driven by the timely separation of the two centrosomes. Here we took advantage of the highly stereotypical cell divisions in Caenorhabditis elegans embryos to identify new regulators of centrosome separation. We find that at the two-cell stage, the somatic AB cell initiates centrosome separation later than the germline P1 cell. This difference is strongly exacerbated by the depletion of the kinesin-13 KLP-7/MCAK, resulting in incomplete centrosome separation at NEBD in AB but not P1. Our genetic and cell biology data indicate that this phenotype depends on cell polarity via the enrichment in AB of the mitotic kinase PLK-1, which itself limits the cortical localization of the dynein-binding NuMA orthologue LIN-5. We postulate that the timely separation of centrosomes is regulated in a cell type-dependent manner.

The balance between KIFC3 and EG5 tetrameric kinesins controls the onset of mitotic spindle assembly.

One of the first steps in mitotic spindle assembly is the dissolution of the centrosome linker followed by centrosome separation driven by EG5, a tetrameric plus-end-directed member of the kinesin-5 family. However, even in the absence of the centrosome linker, the two centrosomes are kept together by an ill-defined microtubule-dependent mechanism. Here we show that KIFC3, a minus-end-directed kinesin-14, provides microtubule-based centrosome cohesion. KIFC3 forms a homotetramer that pulls the two centrosomes together via a specific microtubule network. At mitotic onset, KIFC3 activity becomes the main driving force of centrosome cohesion to prevent premature spindle formation after linker dissolution as it counteracts the increasing EG5-driven pushing forces. KIFC3 is eventually inactivated by NEver in mitosis-related Kinase 2 (NEK2) to enable EG5-driven bipolar spindle assembly. We further show that persistent centrosome cohesion in mitosis leads to chromosome mis-segregation. Our findings reveal a mechanism of spindle assembly that is evolutionary conserved from yeast to humans.

KIFC3 directs a centrosome cohesive force.

Assembly of the mitotic spindle requires timely separation of the centrosomes. Their movement apart is driven by the plus-end-directed kinesin Eg5. A new study demonstrates that the kinesin KIFC3 provides an opposing microtubule-based cohesive force that modulates centrosome separation and ensures accurate chromosome segregation.

Abstract 2097: LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer

Abstract Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. This disease initially responds well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Taxanes cause defects in centrosome function and chromosome segregation leading to cell death. While greatly effective, they are also associated with high toxicity. Thus discovering new therapeutic targets that provide a selective vulnerability for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and is associated with poor survival. We now report that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across eight breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, LIN9 silencing results in multi- and micronucleation, and supernumerary centrosomes. Moreover, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and -468) resistance. We previously reported that Bromodomain and ExtraTerminal protein inhibitors (BETi) treatment reduces LIN9 expression, and thus determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, in conjunction with paclitaxel caused a greater induction of apoptosis, abnormal centrosomes, multi- and micronucleation compared to either drug alone. To identify the mechanism(s) by which suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of MDA-MB-231 and HCC70 TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs. The resulting gene list was then filtered to include only genes whose expression correlates with LIN9 in breast cancer, are bound to LIN9 in a published ChIP-Seq dataset, and are associated with breast cancer survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis, as a potential mediator of LIN9-associated paclitaxel resistance. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor patient outcomes. Additionally, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Importantly, silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by upregulating NEK2 and that loss of LIN9 or NEK2 contributes to centrosome dysfunction, potentiating paclitaxel sensitivity. They also indicate that suppressing LIN9 expression using agents such as BET inhibitors may be a viable therapeutic approach for improving paclitaxel efficacy in TNBC patients. Citation Format: Melyssa S. Shively, Ruth A. Keri. LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2097.

Calcium and phosphorylation double-regulating caltractin initiating target protein XPC function

Human centrin 2 (HsCen2) is a member of the EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. Reversible protein phosphorylation is an important regulatory mechanism for centrin. To obtain insight into the structural basis for the functional effects of phosphorylation, it was verified that the serine residue at position 170 of HsCen2 can be phosphorylated by protein kinase A (PKA) using 31P NMR spectroscopy. While the protein was phosphorylated, the extents of α-helix and the exposed hydrophobic surfaces on HsCen2 were decreased significantly. In the present paper, we reported the regulation and control of peptide p22-XPC binding with HsCen2 by isothermal titration calorimetry (ITC) and spectra. After being phosphorylated, the affinity of p22-XPC with HsCen2 was weakened almost 2 orders of magnitudes. And removing calcium ions from the system, no significant binding between them has been observed. Their binding was reversible. Functions of XPC may be double-regulated and controlled by calcium ions as well as phosphorylation. These results are of significance for understanding the relationship between PTM and metal regulation.

NEK5 interacts with topoisomerase IIβ and is involved in the DNA damage response induced by etoposide.

Cells are daily submitted to high levels of DNA lesions that trigger complex pathways and cellular responses by cell cycle arrest, apoptosis, alterations in transcriptional response, and the onset of DNA repair. Members of the NIMA-related kinase (NEK) family have been related to DNA damage response and repair and the first insight about NEK5 in this context is related to its role in centrosome separation resulting in defects in chromosome integrity. Here we investigate the potential correlation between NEK5 and the DNA damage repair index. The effect of NEK5 in double-strand breaks caused by etoposide was accessed by alkaline comet assay and revealed that NEK5-silenced cells are more sensitive to etoposide treatment. Topoisomerase IIβ (TOPIIβ) is a target of etoposide that leads to the production of DNA breaks. We demonstrate that NEK5 interacts with TOPIIβ, and the dynamics of this interaction is evaluated by proximity ligation assay. The complex NEK5/TOPIIβ is formed immediately after etoposide treatment. Taken together, the results of our study reveal that NEK5 depletion increases DNA damage and impairs proper DNA damage response, pointing out NEK5 as a potential kinase contributor to genomic stability.

Cryo-Electron Tomography and Proteomics studies of centrosomes from differentiated quiescent thymocytes

We have used cryo Electron Tomography, proteomics and immunolabeling to study centrosomes isolated from the young lamb thymus, an efficient source of quiescent differentiated cells. We compared the proteome of thymocyte centrosomes to data published for KE37 cells, focusing on proteins associated with centriole disengagement and centrosome separation. The data obtained enhances our understanding of the protein system joining the centrioles, a system comprised of a branched network of fibers linked to an apparently amorphous density that was partially characterized here. A number of proteins were localized to the amorphous density by immunolabeling (C-NAP1, cohesin SMC1, condensin SMC4 and NCAPD2), yet not DNA. In conjuction, these data not only extend our understanding of centrosomes but they will help refine the model that focus on the protein system associated with the centriolar junction.

Label-free and iTRAQ proteomics analysis in the liver of zebrafish (Danio rerio) following dietary exposure to the organochlorine pesticide dieldrin

The organochlorine dieldrin (DLD) bioaccumulates in lipid-rich tissues and is associated with immunosuppression, altered metabolism, and cancer. The objective of this study was to determine the effect of DLD on the hepatic proteome in zebrafish following dietary treatment as the liver is central to metabolism. Females were fed a control dose or one of three doses of DLD-contaminated food pellets over 21 days. Both label-free and iTRAQ proteomics were conducted as two complementary methods to expand coverage of the proteome. Label-free proteomics quantified 1563 proteins: 6 proteins showed a linear dose-response with DLD. iTRAQ quantified >3500 proteins; 5 proteins were decreased and 34 proteins were increased in abundance within the liver with all three doses. Overall, DLD reduced the abundance of proteins associated with glucose and cholesterol metabolism, lipid oxidation, liver function, and immune-related processes. Few proteins were identified by both methods as being altered (~1%), suggesting that each method detected different subsets of proteins. Protein responses in the liver were largely dependent on dose, however proteins related to liver and organ function, centrosome separation, glucose/energy metabolism, and immune-related pathways were confirmed by each independent technique and were suppressed with DLD exposure. This study identifies proteomic responses that are associated with organochlorine-induced hepatotoxicity. Biological significanceEnvironmental contaminants cause hepatotoxicity because the liver is the major organ for detoxification. The legacy pesticide dieldrin significantly bioaccumulates in tissues, and can affect molecular processes that can lead to liver pathology. LC MS/MS proteomics identified protein networks related to tumors, energy homeostasis, and chromosomal separation as those affected by dietary exposure to dieldrin. We applied two orthogonal mass spectrometry-based methods to more completely survey the liver proteome, strengthening data interpretation. These data improve understanding as to the effects of organochlorine pesticide toxicity in the liver and the study identifies proteome networks that can contribute to adverse outcome pathways for pesticide exposure.

The co-chaperone UNC45A is essential for the expression of mitotic kinase NEK7 and tumorigenesis

Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.