Search

Summary Centrosomes [ 1, 2 ] play a central role during spindle assembly in most animal cells [ 3 ]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [ 4, 5 ], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [ 6 ]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [ 7, 8 ]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [ 9 ] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [ 10–17 ]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [ 18 ], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [ 19 ] and the kinase Nek9 [ 20 ], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system. [ABSTRACT FROM AUTHOR]

Summary Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin ∼4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins. [ABSTRACT FROM AUTHOR]

Background: Aurora A (AurA) kinase is a key mitotic protein implicated in cancer. Several small molecule inhibitors targeting the ATP binding site of this enzyme are in various stages of clinical development. However, these inhibitors can result in selectivity and drug resistance problems. Allosteric inhibition of kinases using small molecules is an alternative strategy to target these enzymes selectively and these could serve as the seeds for next generation medicines. This review discusses the developments in the non-ATP site binding small molecule inhibitors of AurA and their prospect as future therapeutics., Discussion: Allosteric targeting of AurA kinase using small molecules is relatively a new strategy, and only a handful of research work has been reported. Two patents and three papers pertaining to allosteric targeting of AurA kinase using small molecules were covered in this review. Topics discussed include, identification of small molecule inhibitors targeting AurA- Targeting Protein for Xenopus kinesin-like protein 2 (TPX2) interaction, anacardic acid - a natural product ligand that selectively modulates AurA activity in the presence of Aurora B kinase, and identification of felodipine as an uncompetitive inhibitor of AurA using Surface Enhanced Raman Spectroscopy (SERS) technique., Conclusion: Allosteric targeting of therapeutically relevant enzymes using small molecules is a burgeoning research area. New techniques such as fragment-based ligand discovery, SERS methods, etc., are expanding to identify the allosteric site binding ligands. Research in this area is expected to deliver fruitful outcome in terms of novel therapeutics against AurA kinase as well as other therapeutically relevant enzymes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein required for mitosis and spindle assembly. Previous studies showed that TPX2 is overexpressed in various human cancers and promotes cancer progression. In this study, the differentially expressed genes including TPX2 were screened in GEO database for gene expression microarray of breast cancer. The TPX2 expression level was significantly increased in breast cancer cells and the breast malignant tissues compared with those controls. In vitro experiment further confirmed that knockdown of TPX2 by small hairpin RNA inhibited breast cancer cell proliferatio, migration, and induced cell apoptosis. TPX2 silencing decreased the expression of PI3K and extent of AKT phosphorylation, as well as increased expression of p53 and p21. Taken together, our findings indicate that TPX2 silencing negatively regulates the PI3K/AKT and activates p53 signaling pathway by which breast cancer cells proliferation were inhibited whereas cellulars apoptosis were accelerated, suggesting that TPX2 may be a potential target for anticancer therapy in breast cancer., (Copyright © 2018. Published by Elsevier Inc.)

The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis., (© 2018 Wiley Periodicals, Inc.)

Double-hit (DH) or double-expresser (DE) lymphomas are high-grade diffuse large B-cell lymphomas (DLBCL) that are mostly incurable with standard chemo-immunotherapy due to treatment resistance. The generation of drug-induced aneuploid/polyploid (DIAP) cells is a common effect of anti-DLBCL therapies (e.g. vincristine, doxorubicin). DIAP cells are thought to be responsible for treatment resistance, as they are capable of re-entering the cell cycle during off-therapy periods. Previously we have shown that combination of alisertib plus ibrutinib plus rituximab can partially abrogate DIAP cells and induce cell death. Here, we provide evidence that DIAP cells can re-enter the cell cycle and escape cell death during anti-DLBCL treatment. We also discuss MYC/BCL2 mediated molecular mechanism that underlie treatment resistance. We isolated aneuploid/polyploid populations of DH/DE-DLBCL cells after treatment with the aurora kinase (AK) inhibitor alisertib. Time-lapse microscopy of single polyploid cells revealed that following drug removal, a subset of these DIAP cells divide and proliferate by reductive cell divisions, including multipolar mitosis, meiosis-like nuclear fission and budding. Genomic, proteomic, and kinomic profiling demonstrated that alisertib-induced aneuploid/polyploid cells up-regulate DNA damage, DNA replication and immune evasion pathways. In addition, we identified amplified receptor tyrosine kinase and T-cell receptor signaling, as well as MYC-mediated dysregulation of the spindle assembly checkpoints RanGAP1, TPX2 and KPNA2 . We infer that these factors contribute to treatment resistance of DIAP cells. These findings provide opportunities to develop novel DH/DE-DLBCL therapies, specifically targeting DIAP cells., Key Points: ● MYC mediated upregulation of TPX2, KPNA2 and RanGAP1 dysregulate the spindle assembly checkpoint in drug-induced polyploid cells.● Drug-induced polyploid cells re-enter the cell cycle via multipolar mitosis, fission or budding, a mechanism of disease relapse., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing financial interests.

Purpose: The molecular signatures of cholangiocarcinoma are not well characterized. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) has been shown to promote oncogenesis in the context of several cancers; however, its' role in cholangiocarcinoma has not been studied. We evaluated the role of TPX2 in cholangiocarcinoma., Methods: Expression levels of TPX2 in cholangiocarcinoma were assessed by immunohistochemistry. Potential correlations were assessed by Chi-squared test. Impact of TPX2 expression on cell proliferation, cell cycle, apoptosis, cell invasion and migration was investigated by CCK-8, flow cytometric analysis, and transwell assay, respectively. The expressions of cell-cycle, cell-apoptosis and EMT related target proteins were detected by immunoblotting., Results: TPX2 expression in cholangiocarcinoma tissues was significantly higher than that paracancerous tissue (44.3% vs. 5.7%; P<0.01). Overexpression of TPX2 showed a positive correlation with TNM stage, lymph node metastasis, and prognosis of patients. Knockdown of TPX2 expression induced G2-M arrest, apoptosis and inhibited invasion and migration of cholangiocarcinoma cells. Treatment of cholangiocarcinoma cells with TPX2 siRNA resulted in upregulation of cyclin A1, cyclin B1, p53, Bax, and E-cadherin; while downregulation of cyclin D1, CDK2, Bcl-2, N-cadherin, β-cadherin MMP-2, MMP-9, Slug, and Twist1., Conclusions: Collectively, these results indicate that TPX2 may serve as a potential biomarker of prognostic relevance and a potential therapeutic target for cholangiocarcinoma., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Objective: This study is intended to identify the key gene from gene expression profile and validate its role and regulatory mechanism in melanoma., Methods: Gene expression profile of GSE3189 from GEO database was selected among which 7 are normal skin samples, 18 are benign skin lesion samples, and 45 are melanoma samples. The present study examined the 7 normal skin samples and the 45 melanoma samples. Differentially expressed genes (DEGs) between melanoma patients and health people were performed using Morpheus online tool. The 100 most differentially expressed genes (50 upregulated genes and 50 downregulated genes) were selected as hub genes. Then, expression levels and survival analysis of hub genes were conducted via GEPIA tool to choose target gene. The expression of target gene in melanoma cell lines was examined by RT-qPCR and western blotting. The biological function of target gene on cell proliferation in melanoma was measured in vitro. The predicted target of target gene was validated by dual-luciferase reporter assay and rescue experiment. The gene expression in clinical samples were determined by RT-qPCR, immunohistochemistry (IHC) and in situ hybridization (ISH). The tumor formation study was conducted in vivo., Results: Targeting protein for Xklp2 (TPX2) was identified as key gene in melanoma. TPX2 could promote the proliferation of melanoma cells. The dual luciferase reporter assay confirmed that miR-330-3p targets TPX2. In rescue experiment, it was proved that miR-330-3p inhibits the proliferation of melanoma cells by negatively regulating the expression of TPX2. The results in vitro were also confirmed in vivo. miR-330-3p/TPX2 pathway expressed differently between melanoma patients and health people. These differences were statistically significant (P < 0.05)., Conclusion: Inhibiting TPX2 by miR-330-3p suppresses the proliferation of melanoma cell lines. miR-330-3p/TPX2 pathway could be a potential therapeutic target in melanoma., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: The current TNM staging system is far from perfect in predicting the survival of individual non-small cell lung cancer (NSCLC) patients. In this study, we aim to combine clinical variables and molecular biomarkers to develop a prognostic model for patients with NSCLC., Methods: Candidate molecular biomarkers were extracted from the Gene Expression Omnibus (GEO), and Cox regression analysis was performed to determine significant prognostic factors. The survival prediction model was constructed based on multivariable Cox regression analysis in a cohort of 152 NSCLC patients. The predictive performance of the model was assessed by the Area under the Receiver Operating Characteristic Curve (AUC) and Kaplan-Meier survival analysis., Results: The survival prediction model consisting of two genes (TPX2 and MMP12) and two clinicopathological factors (tumor stage and grade) was developed. The patients could be divided into either high-risk group or low-risk group. Both disease-free survival and overall survival were significantly different among the diverse groups (P < 0.05). The AUC of the prognostic model was higher than that of the TNM staging system for predicting survival., Conclusions: We developed a novel prognostic model which can accurately predict outcomes for patients with NSCLC after surgery.

Bipolar spindle assembly is necessary to ensure the proper progression of cell division. Loss of spindle pole integrity leads to multipolar spindles and aberrant chromosomal segregation. However, the mechanism underlying the maintenance of spindle pole integrity remains unclear. In this study, we show that the actin-binding protein adducin-1 (ADD1) is phosphorylated at S726 during mitosis. S726-phosphorylated ADD1 localizes to centrosomes, wherein it organizes into a rosette-like structure at the pericentriolar material. ADD1 depletion causes centriole splitting and therefore results in multipolar spindles during mitosis, which can be restored by re-expression of ADD1 and the phosphomimetic S726D mutant but not by the S726A mutant. Moreover, the phosphorylation of ADD1 at S726 is crucial for its interaction with TPX2, which is essential for spindle pole integrity. Together, our findings unveil a novel function of ADD1 in maintaining spindle pole integrity through its interaction with TPX2., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation., (© 2018 Wiley Periodicals, Inc.)

Background: Hepatocellular carcinoma (HCC) has an increasing incidence and high mortality. Surgical operation is not a comprehensive strategy for liver cancer. Moreover, tolerating systemic chemotherapy is difficult for patients with HCC because hepatic function is often impaired due to underlying cirrhosis. Therefore, a comprehensive strategy for cancer treatment should be developed. DTL (Cdc10-dependent transcript 2) is a critical regulator of cell cycle progression and genomic stability. In our previous study, the upregulation of DTL expression in aggressive HCC correlated positively with tumor grade and poor patient survival. We hypothesize that targeting DTL may provide a novel therapeutic strategy for liver cancer. DTL small interference RNAs were used to knock down DTL protein expression., Methods: A clonogenic assay, immunostaining, double thymidine block, imaging flow cytometry analysis, and a tumor spheroid formation assay were used to analyze the role of DTL in tumor cell growth, cell cycle progression, micronucleation, ploidy, and tumorigenicity., Results: Our results demonstrated that targeting DTL reduced cell cycle regulators and chromosome segregation genes, resulting in increased cell micronucleation. DTL depletion inhibited liver cancer cell growth, increased senescence, and reduced tumorigenesis. DTL depletion resulted in the disruption of the mitotic proteins cyclin B, CDK1, securin, seprase, Aurora A, and Aurora B as well as the upregulation of the cell cycle arrest gene p21 . A rescue assay indicated that DTL should be targeted through TPX2 downregulation for cancer cell growth inhibition. Moreover, DTL silencing inhibited the growth of patient-derived primary cultured HCC cells., Conclusion: Our study results indicate that DTL is a potential novel target gene for treating liver cancer through liver cancer cell senescence induction. Furthermore, our results provide insights into molecular mechanisms for targeting DTL in liver cancer cells. The results also indicate several other starting points for future preclinical and clinical studies on liver cancer treatment., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein that plays an important role in spindle assembly and dynamics. However, the clinical and prognostic value of TPX2 in the digestive system cancers remains unclear. The objective of this review was to evaluate the association of TPX2 expression with disease-free survival (DFS), overall survival (OS), and clinicopathological features of digestive system cancers. The software Stata 12.0 was used to analyze the outcomes, including OS, disease-free survival (DFS), and clinicopathological characteristics. A total of 10 eligible studies with 906 patients were included. Elevated TPX2 expression was significantly associated with poor DFS (pooled hazard ratio [HR] =2.48, 95% confidence interval [CI]: 1.96-3.13) and OS (pooled HR =2.66, 95% CI: 2.04-3.48) of digestive system malignancies. Subgroup analyses showed that cancer type, sample size, study quality, and laboratory detection methods did not alter the significant prognostic value of TPX2. Additionally, TPX2 expression was found to be an independent predictive factor for DFS (HR =2.31, 95% CI: 1.78-3.01). TPX2 expression might be associated with TNM stage and pathological grade in digestive system cancer. In conclusion, TPX2 is an independent prognostic factor for survival of patients with digestive system cancer. Furthermore, its overexpression is associated with TNM stage and pathological grade in digestive system cancer., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The targeting protein for Xenopus kinesin-like protein 2 (TPX2) is associated with the metastasis and prognosis of bladder cancer. p53 is closely related to the progression of bladder cancer. Human glioma pathogenesis-related protein 1 (GLIPR1) is a p53 target gene with antitumor activity. This study aims to explore the interplay between TPX2, p53, and GLIPR1 and its correlation with cell proliferation, invasion, and tumor growth in bladder cancer. Here, Western blot and qRT-PCR analysis revealed that TPX2 at both mRNA and protein levels was up-regulated in bladder carcinoma tissues compared to their paired adjacent normal tissues. Additionally, tissues expressing high TPX2 level exhibited high p53 level and low GLIPR1 level. The expressions of TPX2 and p53 in non-muscle-invasive bladder cancer cells (KK47 and RT4) were lower than those in muscle-invasive bladder cancer cells (T24, 5637, and UM-UC-3), while GLIPR1 showed the converse expression pattern. Further investigation revealed that TPX2 activated the synthesis of p53; and GLIPR1 is up-regulated by wild-type (wt)-p53 but not affected by mutated p53; Additionally, GLIPR1 inhibited TPX2. These data suggested a TPX2-p53-GLIPR1 regulatory circuitry. Meanwhile, TPX2 overexpression promoted while overexpression of GLIPR1 or p53 inhibited bladder cancer growth. Interestingly, in T24 cells with mutated p53, p53 silence suppressed bladder cancer growth. This study identified a novel TPX2-p53-GLIPR1 regulatory circuitry which modulated cell proliferation, migration, invasion, and tumorigenicity of bladder cancer. Our findings provide new insight into underlying mechanisms of tumorigenesis and novel therapeutic options in bladder cancer., (© 2017 Wiley Periodicals, Inc.)

MicroRNA-1294 (miR-1294) has been reported to be involved in the progression of esophageal squamous cell carcinoma. However, the function and the mechanisms of miR-1294 in glioma remain unclear. In this study, we explore the potential biological roles of miR-1294 in glioma cell lines. First, we detected the aberrant down-regulation of miR-1294 in glioma tissues and cell lines. Second, we determined that miR-1294 suppresses the proliferation, migration and invasiveness and enhances the chemosensitivity of glioma cells lines to temozolomide. Third, we found that the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is the functional target of miR-1294; miR-1294 acts through TPX2 to exert an important biological effect in glioma. Importantly, TPX2 knockdown had the same effect on glioma cell lines as miR-1294 overexpression. In addition, when TPX2 was up-regulated in these cells, the effects of miR-1294 on glioma cell lines were suppressed. Moreover, the effect of miR-1294 on glioma was verified using a xenograft model. These findings demonstrated that miR-1294 inhibits the development of glioma by targeting TPX2. These findings provide a new potential therapeutic target for glioma treatment., Competing Interests: None.

Background: Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated proteinrequired for mitosis and spindle assembly. It has been revealed that TPX2 is overexpressedin various human cancers and promotes cancer progression., Methods: The expression of TPX2 was examined in ovarian cancer (OC) tissues and by Western blotting, quantitative real-time reverse transcription PCR (qRT-PCR) and immunohistochemistry. The effects of TPX2 on proliferation and migration of two OC cell lines SKOV3and RMG1 were analyzed using the methylthiazol tetrazolium (MTT) assay, flow cytometry and transwell assay. The mechanisms underlying the effects of TPX2 on OC cells were explored by qRT-PCR and Western blot., Results: In this study, we found that TPX2 was upregulated in OC tissues. We observed knockdown of TPX2 inhibited the expression of Polo-like kinase 1 (PLK1), which has an important role in the regulation of M phase of the cell cycle, and the activity of Cdc2, induced cell arrested at the G2/M phase and decreased proliferation. Moreover, our data revealed that the levels of PLK1, β-catenin, MMP7 and MMP9 were inhibited following TPX2 knockdown, leading to decrease of cell migration. Finally, we showed that the restoration of PLK1 expression attenuated the anti-proliferation and anti-migration effects of TPX2 knockdown in OC cells., Conclusions: TPX2 promotes the proliferation and migration of human OC cells by regulating PLK1 expression.

This study was aimed to explore the effects of miR-29a-5p expression and its target gene TPX2 (target protein for Xenopus kinesin-like protein 2) on endometrial cancer (EC) devel on EC development and to assess the prognostic impacts of TPX2. Microarray-based GEO and TCGA (the Cancer Genome Atlas) EC expression data were used to identify differentially expressed miRNAs and mRNAs. The observed potential target relationship between miR-29a-5p and TPX2 was verified using TargetScan and luciferase reporter assays. The mRNA and protein expression levels of miR-29a-5p and TPX2 were confirmed by qRT-PCR and western blot, respectively. Associations between TPX2 expression and patient prognosis were assessed using Kaplan-Meier and log-rank assays. Changes in EC-derived cell proliferation, invasion and apoptosis after exogenous miR-29a-5p and TPX2 over-expression and/or silencing were assessed using CCK-8 (cell counting kit-8), colony formation, Transwell and flow cytometry assays, respectively. We found that in primary EC tissues the expression of miR-29a-5p was down-regulated and the expression of TPX2 was up-regulated. We also found that low expression of TPX2 were associated with a better prognosis, and vice versa. Subsequent exogenous miR-29a-5p over-expression and TPX2 silencing could inhibit EC-derived cell proliferation and invasion, and to induce apoptosis. We also found that miR-29a-5p might target and repress TPX2, thereby inhibiting EC-derived cell proliferation and invasion and enhancing apoptosis. We conclude that miR-29a-5p could inhibit the proliferation and invasion of EC-derived cells and enhance the apoptosis of EC-derived cells via TPX2 down-regulation. A high TPX2 expression in primary EC tissues was found to be associated with a poor prognosis. As such, these biomarkers may serve as promising prognostic indicators., Abbreviations: EC: Endometrial cancer; 3'-UTR: 3'-untranslated regions; TPX2: target protein for Xenopus kinesin-like protein 2; TCGA: the Cancer Genome Atlas; UCEC: uterine corpus endometrial carcinoma; CCK-8: cell counting kit-8; OD: optical density; FCM: flow cytometry; EMT: epithelial-mesenchymal transition.

The targeting protein for Xenopus kinesin-like protein 2 (TPX2) has been demonstrated to be associated with the tumourigenesis of many cancers. In the present study, we investigated the role and preliminary mechanism of TPX2 in the resistance of lung squamous carcinoma to radiation therapy. The results showed that SK-MES-1R and NCI-H226R cells were more resistant to X-ray irradiation than the parental cells (SK-MES-1 and NCI-H226). Moreover, TPX2 was upregulated in the radioresistant cells compared with the parental cells. TPX2 knockdown significantly decreased TPX2 expression in SK-MES-1 cells, while TPX2 overexpression increased TPX2 expression in NCI-H226 cells compared with the corresponding control cells. TPX2 knockdown enhanced the radiosensitivity of SK-MES-1 and promoted cell apoptosis following exposure to irradiation, whereas TPX2 overexpression decreased the radiosensitivity of NCI-H226 and inhibited cell apoptosis. In in vivo studies, the combination of TPX2 knockdown and irradiation significantly inhibited tumour growth, decreased tumour weight, downregulated TPX2 expression in tumour tissue and induced cell apoptosis in nude mice, while TPX2 overexpression exerted an opposite effect. Our results indicated that TPX2 was correlated with cell radioresistance and it might be served as a therapeutic target to enhance cell radiosensitivity in the radiation therapy of lung squamous carcinoma., (© 2017 John Wiley & Sons Australia, Ltd.)

FAM83D , a microtubule-associated protein (MAP), is overexpressed in diverse types of human cancer. The expression and critical role of FAM83D in human gastric cancer (GC), however, remains largely unknown. Here, we conducted molecular, cellular and clinical analyses to evaluate the functional link of FAM83D to GC. FAM83D expression was elevated in gastric tumors, and its expression strongly correlated with lymph node metastasis and TNM stage. In addition, over-expression of FAM83D in GC cell lines enhanced cell proliferation, cycle progression, migration, invasion, as well as tumor growth and metastatic dissemination in vivo . Furthermore, FAM83D exhibited a strong cell cycle correlated expression. The knockdown of FAM83D inhibited the regrowth of microtubules in GC cells. FAM83D was co-immunoprecipitated with HMMR, TPX2, and AURKA, a set of drivers of mitosis progression. Taken together, our results demonstrate FAM83D as an important player in the development of human gastric cancer, and as a potential therapeutic target for the treatment of cancer., Competing Interests: CONFLICTS OF INTERESTS None declared.

Hepatocellular carcinoma (HCC) remains one of the most difficult cancers to treat, with chemotherapies being relatively ineffective. Therefore, a better knowledge of molecular hepatocarcinogenesis will provide opportunities for designing targeted therapies. TPX2 (targeting protein for Xklp2) is overexpressed as a consequence of oncogenic alterations and is likely to alter the proper regulation of chromosome segregation in cancer cells. Disrupting the machinery which is responsible for mitosis and chromosome instability in cancer cells can be one of the most successful strategies for cancer therapy. Therefore, we consider the targeting TPX2 could provide novel therapeutic strategies for cancer. In this study, increased TPX2 protein expression was present in 16 (42%) of 38 primary HCCs and was associated with advanced stage, distant metastatic HCCs and poor prognosis. Knockdown of TPX2 inhibited cancer cell growth and downregulation of cyclin A, cyclin E and CDK2 proteins. However, over-expressed EGFP-TPX2 protein enhanced the in vitro tumor spheroid formation and rescued the TPX2 depleted cell growth. Targeting TPX2 caused a rising impaired chromosomal instability resulting in multinuclearity, cell cycle progression arrest, apotosis, senescence and an increased polyploidy in cells. An image-cytometry analysis revealed cell cycle progression arrest after TPX2 inhibition. A correlation was observed between the downregulation of the protein levels of genes related to chromosomal segregation and spindle assembly checkpoint (securin, seprase, Aurora A, Aurora B, Cyclin B1, Cyclin B2, MPS1, BUB1, BUB3, MAD1 and MAD2) and increased cell ploidy, indicating mitotic progression failure and the loss of the balance of genomic instability. In vitro tumor spheroid assay and in vivo xenografts mouse model showed a therapeutic opportunity. Our findings indicate that targeting TPX2 lead to suppress tumorigenicity in liver cancer cells, suggesting that TPX2 is a potential target for anticancer therapy in HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼ 200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

TPX2 (targeting protein for Xklp2) is a multifunctional mitotic spindle assembly factor that in mammalian cells localizes and regulates mitotic motor protein kinesin-5 (also called Eg5 or kif11). We previously showed that upon depletion or inhibition of kinesin-5 in cultured neurons, microtubule movements increase, resulting in faster growing axons and thinner dendrites. Here, we show that depletion of TPX2 from cultured neurons speeds their rate of process outgrowth, similarly to kinesin-5 inhibition. The phenotype is rescued by TPX2 re-expression, but not if TPX2's kinesin-5-interacting domain is deleted. These results, together with studies showing a spike in TPX2 expression during dendritic differentiation, suggest that the levels and distribution of TPX2 are likely to be determinants of when and where kinesin-5 acts in neurons., (© 2015 Wiley Periodicals, Inc.)

Segregation of chromosomes during cell division requires correct formation of mitotic spindles. Here, we show that a scaffold attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein-U, contributes to the attachment of spindle microtubules (MTs) to kinetochores and spindle organization. During mitosis, SAF-A was localized at the spindles, spindle midzone and cytoplasmic bridge. Depletion of SAF-A by RNA interference induced mitotic delay and defects in chromosome alignment and spindle assembly. We found that SAF-A specifically co-immunoprecipitated with the chromosome peripheral protein nucleolin and the spindle regulators Aurora-A and TPX2, indicating that SAF-A is associated with nucleolin and the Aurora-A-TPX2 complex. SAF-A was colocalized with TPX2 and Aurora-A in spindle poles and MTs. Elimination of TPX2 or Aurora-A from cells abolished the association of SAF-A with the mitotic spindle. Interestingly, SAF-A can bind to MTs and contributes to the targeting of Aurora-A to mitotic spindle MTs. Our finding indicates that SAF-A is a novel spindle regulator that plays an essential role in kinetochore-MT attachment and mitotic spindle organization. [ABSTRACT FROM AUTHOR]

The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a peak in G2 and M phases, followed by regulated proteolysis at the end of mitosis. The microtubule-binding protein TPX2 plays a major role in regulating the activity and localisation of Aurora-A in mitotic cells. Here, we report a novel regulatory role of TPX2 and show that it protects Aurora-A from degradation both in interphase and in mitosis in human cells. Specifically, Aurora-A levels decrease in G2 and prometaphase cells silenced for TPX2, whereas degradation of Aurora-A is impaired in telophase cells overexpressing the Aurora-A-binding region of TPX2. The decrease in Aurora-A in TPX2-silenced prometaphases requires proteasome activity and the Cdh1 activator of the APC/C ubiquitin ligase. Reintroducing either full-length TPX2, or the Aurora-A-binding region of TPX2, but not a truncated TPX2 mutant lacking the Aurora-A-interaction domain, restores Aurora-A levels in TPX2-silenced prometaphases. The control by TPX2 of Aurora-A stability is independent of its ability to activate Aurora-A and to localise it to the spindle. These results highlight a novel regulatory level impinging on Aurora-A and provide further evidence for the central role of TPX2 in regulation of Aurora-A. [ABSTRACT FROM AUTHOR]

During apoptosis, the interphase microtubule network is dismantled then later replaced by a novel, non-centrosomal microtubule array. These microtubules assist in the peripheral redistribution of nuclear fragments in the apoptotic cell; however, the regulation of apoptotic microtubule assembly is not understood. Here, we demonstrate that microtubule assembly depends upon the release of nuclear RanGTP into the apoptotic cytoplasm because this process is blocked in apoptotic cells overexpressing dominant-negative GDP-locked Ran (T24N). Actin-myosin-II contractility provides the impetus for Ran release and, consequently, microtubule assembly is blocked in blebbistatin- and Y27632-treated apoptotic cells. Importantly, the spindle-assembly factor TPX2 (targeting protein for Xklp2), colocalises with apoptotic microtubules, and siRNA silencing of TPX2, but not of the microtubule motors Mklp1 and Kid, abrogates apoptotic microtubule assembly. These data provide a molecular explanation for the assembly of the apoptotic microtubule network, and suggest important similarities with the process of RanGTP- and TPX2-mediated mitotic spindle formation. [ABSTRACT FROM AUTHOR]

Centrosomes and their fungal equivalents, spindle pole bodies (SPBs), are the main microtubule (MT)-organizing centers in eukaryotic cells. Several proteins have been implicated in microtubule formation by centrosomes and SPBs, including microtubule-minus-end-binding proteins and proteins that bind along the length or stabilize the plus ends of microtubules. Recent work has improved our understanding of the molecular mechanisms of MT formation. In particular, it has shown that γ-tubulin and its associated proteins play key roles in microtubule nucleation and spindle assembly in evolutionarily distant species ranging from fungi to mammals. Other work indicates that γ-tubulin-mediated microtubule nucleation, although necessary, is not sufficient for mitotic spindle assembly but requires additional proteins that regulate microtubule nucleation independently of centrosomes. [ABSTRACT FROM AUTHOR]

Introduction: Based on its role as a mitotic regulatory kinase, overexpressed and associated with aneuploidy in cancer, small-molecule inhibitors have been developed for Aurora-A (AURKA) kinase. In preclinical and clinical assessments, these agents have shown efficacy in inducing stable disease or therapeutic response. In optimizing the use of Aurora-A inhibitors, it is critical to have robust capacity to measure the kinase activity of Aurora-A in tumors., Areas Covered: We provide an overview of molecular mechanisms of mitotic and non-mitotic activation of Aurora-A kinase, and interaction of Aurora-A with its regulatory partners. Typically, Aurora-A activity is measured by use of phospho-antibodies targeting an autophosphorylated T288 epitope. However, recent studies have identified alternative means of Aurora-A activation control, including allosteric regulation by partners, phosphorylation on alternative activating residues (S51, S98), dephosphorylation on inhibitory sites (S342) and T288 phosphorylation by alternative kinases such as Pak enzymes. Additional work has shown that the relative abundance of Aurora-A partners can affect the activity of Aurora-A inhibitors, and that Aurora-A activation also occurs in interphase cells., Expert Opinion: Taken together, this work suggests the need for comprehensive analysis of Aurora-A activity and expression of Aurora-A partners in order to stratify patients for likely therapeutic response.

Cell cycle deregulation is a common motif in human cancer, and multiple therapeutic strategies are aimed to prevent tumor cell proliferation. Whereas most current therapies are designed to arrest cell cycle progression either in G1/S or in mitosis, new proposals include targeting the intrinsic chromosomal instability (CIN, an increased rate of gain or losses of chromosomes during cell division) or aneuploidy (a genomic composition that differs from diploid) that many tumor cells display. Why tumors cells are chromosomally unstable or aneuploid and what are the consequences of these alterations are not completely clear at present. Several mitotic regulators are overexpressed as a consequence of oncogenic alterations, and they are likely to alter the proper regulation of chromosome segregation in cancer cells. In this review, we propose the relevance of TPX2, a mitotic regulator involved in the formation of the mitotic spindle, in oncogene-induced mitotic stress. This protein, as well as its partner Aurora-A, is frequently overexpressed in human cancer, and its deregulation may participate not only in chromosome numeric aberrations but also in other forms of genomic instability in cancer cells.