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CtBP-interacting protein (CtIP) plays a critical role in controlling the homologous recombination-mediated DNA double-stranded break (DSB) repair pathway through DNA end resection, and recent studies suggest that it also plays a role in mitosis. However, the mechanism by which CtIP contributes to mitosis regulation remains elusive. Here, we show that depletion of CtIP leads to a delay in anaphase progression resulting in misaligned chromosomes, an aberrant number of centrosomes, and defects in chromosome segregation. Additionally, we demonstrate that CtIP binds and colocalizes with Targeting protein for Xklp2 (TPX2) during mitosis to regulate the recruitment of TPX2 to the spindle poles. Furthermore, depletion of CtIP resulted in both a lower concentration of Aurora A, its downstream target, and very low microtubule intensity at the spindle poles, suggesting an important role for the CtIP-TPX2-Auroa A complex in microtubule dynamics at the centrosomal spindles. Our findings reveal a novel function of CtIP in regulating spindle dynamics through interactions with TPX2 and indicate that CtIP is involved in the proper execution of the mitotic program, where deregulation may lead to chromosomal instability.

RepoMan is a chromosome-associated scaffold protein that integrates signaling of multiple kinases and phosphatases to coordinate spindle-kinetochore interactions, chromosome (de)condensation and nuclear envelope (dis)assembly during mitosis. Another key mitotic event is the assembly of a microtubule-based spindle, which involves redundant pathways emanating from the centrosomes, microtubules and chromosomes. Here we describe a novel mitotic function of RepoMan in regulating chromosome-dependent microtubule assembly. At limiting concentrations of microtubule-destabilizing agents, RepoMan-depleted cells showed enhanced chromosome clustering. This clustering was completely dependent on the partial inhibition of microtubule growth originating from the chromosome-dependent pathway. We also demonstrated that RepoMan interacts with prime regulators of the chromosome-dependent spindle assembly such as NuSAP1, NuMA, and TPX2. In addition, RepoMan was required to enable or maintain phosphorylation of NuSAP1 at CDK sites, thereby enabling activation of NuSAP1 through dissociation of inhibitory importin β/7. Our data identify RepoMan as an enhancer of microtubule assembly at chromosomes.

Chronic rhinosinusitis (CRS) is featured with chronic symptoms of inflammation or infection in the nasal and sinus tissues. MicroRNAs (miRNAs/miRs), such as dysregulated expression of miR-125b and miR-26a, has been previously demonstrated to be related to CRS. The present study is intended to define the role of miR-335-5p in inflammation and the related mechanism in a mouse model of CRS. The differentially expressed genes associated with CRS were screened by microarray analysis. The targeting relationship between miR-335-5p and TPX2 was analyzed by target prediction program and dual luciferase reporter gene assay. The mouse model of CRS was established, and mice were introduced with miR-335-5p mimics, miR-335-5p inhibitors, or siRNA against TPX2 to explore the regulatory functions of miR-335-5p. The regulatory effect of miR-335-5p on inflammation with the involvement of the AKT signaling pathway was also analyzed with the expression of inflammatory cytokines and AKT signaling pathway-related factors measured. It was indicated that miR-335-5p regulated the TPX2 gene-mediated AKT signaling pathway. TPX2 was identified as a target gene of miR-335-5p, and miR-335-5p elevation inhibited the activation of the AKT signaling pathway. In mice with CRS, up-regulation of miR-335-5p or silence of TPX2 inhibited the inflammation, as evidenced by decreased levels of TNF-α, IL-6 and IL-8, and higher levels of GSK3β and IL-10. Collectively, miR-335-5p inhibits the activation of AKT signaling pathway by negatively mediating TPX2, which may confer anti-inflammatory protection in CRS., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.)

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.)

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.)

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.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.)

Aurora kinases are a family of cell division regulators that govern the correct assembly of a bipolar mitotic spindle and the fidelity of chromosome segregation. Their overexpression is associated with genomic instability and aneuploidy, and is frequently observed in cancer. Accordingly, competitive inhibitors targeting Aurora kinase activity at the ATP-binding site are being investigated for therapeutic purposes. Despite promising pre-clinical data, these molecules display moderate effects in clinical trials and incomplete selectivity, either against distinct family members, or other kinases. As an alternative approach, protein-protein interaction inhibitors targeting mitotic kinases and their activators can be exploited to achieve increased specificity of action. In this study, a virtual screening of small molecules led to the identification of 25 potential inhibitors of the interaction between Aurora-A and its activator TPX2. In vitro experiments confirmed that 4 hits bind Aurora-A in the low micromolar range and compete for TPX2 binding. Immunofluorescence assays showed that 2 compounds also yield lowered Aurora-A activity and spindle pole defects in cultured osteosarcoma cells. The identified protein-protein interaction inhibitors of the Aurora-A/TPX2 complex might represent lead compounds for further development towards pioneering anti-cancer drugs and provide the proof-of-concept for a new exploitable strategy to target mitotic kinases.

Objectives: Our aims were to determine if targeting protein for Xklp2 (TPX2) is correlated with clear cell renal cell carcinoma (ccRCC) histology and oncologic outcomes using The Cancer Genome Atlas (TCGA) and an institutional tissue microarray (TMA)., Methods: Clinicopathological data obtained from the TCGA consisted of 415 samples diagnosed with ccRCC. A TMA was constructed from tumors of 207 patients who underwent radical nephrectomy for ccRCC. TPX2 expression by immunohistochemistry on TMA was assessed by a genitourinary pathologist. Clinical data were extracted and linked to TMA cores. TPX2 and Aurora-A mRNA coexpression were evaluated in the TCGA cohort. Overall survival (OS), cancer-specific survival, and recurrence-free survival (RFS) were analyzed using the Kaplan-Meier method and log-rank statistics. Univariate and multivariate analyses were conducted using Cox proportional hazard models., Results: Median follow-up time for the TCGA cohort was 3.07 years. Aurora-A and TPX2 mRNA coexpression were significantly correlated (Pearson correlation = 0.918). High TPX2 mRNA expression was associated with advanced stage, metastasis, poor OS, and RFS. Median follow-up time for the TMA cohort was 5.3 years. Elevated TPX2 protein expression, defined as greater than 75th percentile staining intensity, was identified in 47/207 (22.7%) patients. Increased TPX2 immunostaining was associated with poor OS (P = 0.0327, 53% 5-year mortality), cancer-specific survival (P<0.01, 47.8% 5-year cancer-specific mortality), RFS (P = 0.0313, 73.6%, 5-year recurrence rate), grade, T stage, and metastasis. Multivariate analysis demonstrated elevated expression served as an independent predictor of RFS (hazard ratio = 3.62 (1.13-11.55), P = 0.029)., Conclusions: We show TPX2, a regulator of Aurora-A, is associated with high grade and stage of ccRCC, and is an independent predictor of recurrence. Future studies are warranted testing its role in ccRCC biology, and its potential as a therapeutic target., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein required for microtubule formation in human cells. Several studies have demonstrated that TPX2 is overexpressed in multiple tumor types and promotes tumor growth and metastasis. However, there have been few reports regarding its role in gastric cancer. In this study, we evaluated TPX2 expression and investigated its correlations with gastric cancer clinicopathological features and prognosis., Methods: Tumor samples were obtained from 290 patients with gastric adenocarcinoma who had undergone gastrectomy. The expression of TPX2 protein was examined using immunohistochemical staining. TPX2 messenger RNA (mRNA) levels were evaluated using real-time quantitative reverse transcription PCR in 19 of the gastric cancer tumors and adjacent normal tissues., Results: The mRNA levels of TPX2 were significantly higher in gastric cancer tissues than in matched adjacent normal tissues (p = 0.004). In the immunohistochemical analysis, TPX2 overexpression was found in 123 (42.4%) of 290 patients. High TPX2 expression was positively associated with age, type of histology, depth of tumor, lymph node metastasis, stage, and remote metastasis or recurrence. High TPX2 expression was significantly associated with poorer disease-specific survival (p = 0.004) and relapse-free interval (p = 0.013)., Conclusions: Our results indicated that high TPX2 expression was associated with tumor progression and poor survival in gastric cancer.

Previously, we demonstrated that Bcl-2-like 10 (Bcl2l10) is associated with meiotic spindle assembly and that the gene that is most strongly down-regulated by Bcl2l10 RNAi is targeting protein for Xklp2 (Tpx2). Tpx2 is a well-known cofactor that controls the activity and localization of Aurora kinase A (Aurka) during mitotic spindle assembly. Therefore, this study was conducted (1) to identify the associations among Bcl2l10, Tpx2, and Aurka and (2) to understand how Bcl2l10 regulates meiotic spindle assembly in mouse oocytes. Bcl2l10, Tpx2, and Aurka co-localized on the meiotic spindles, and Bcl2l10 was present in the same complex with Tpx2. Tpx2 and Aurka expression decreased whereas phospho-Aurka increased in Bcl2l10 RNAi-treated oocytes. Counterbalancing changes in the levels of these 2 activators, Tpx2 and phospho-Aurka, resulted in decreased Aurka catalytic activity after Bcl2l10 RNAi treatment. Bcl2l10 RNAi decreased the expression of microtubule organizing center (MTOC)-related proteins, disturbed MTOC formation and disrupted meiotic spindle assembly. Our data demonstrate that Bcl2l10 is a binding partner of Tpx2 and a new regulator of the complex controlling the organization of microtubules and MTOC biogenesis in meiotic spindle assembly. The discovery of Bcl2l10 as a new effector of Aurka suggests that Bcl2l10 may have diverse functions in mitotic cells.

Background: TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains., Methods: We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements., Results: Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila)., Conclusions: Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.

The targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a putative oncogene in different human cancers. This study assessed TPX2 expression in gastric cancer tissue samples and then determined the effects of TPX2 knockdown on the regulation of gastric cancer cell malignant behaviors in vitro. Tissue samples from 115 gastric cancer patients were analyzed for TPX2 expression. The effects of TPX2 siRNA on gastric cancer cells were assessed in vitro, including cell viability, cell cycle distribution, apoptosis, migration, and invasion. The data showed that TPX2 was overexpressed in gastric cancer tissues compared to that in the adjacent normal epithelia. Moreover, TPX2 overexpression was associated with a poor overall survival and was an independent prognostic predictor of gastric cancer. In addition, the in vitro study further confirmed the ex vivo data, i.e., knockdown of TPX2 expression reduced gastric cancer cell viability but induced apoptosis and arrested cells at the G2/M phase of the cell cycle. Knockdown of TPX2 expression also inhibited the tumor cell migration and invasion capacity in vitro. At the gene level, knockdown of TPX2 expression upregulated the levels of cyclin B1, cdk4, p53, Bax, caspase-3, and E-cadherin, but downregulated the levels of cyclin D1, cdk2, N-cadherin, slug, matrix metalloprotease (MMP)-2, and MMP-9, suggesting that knockdown of TPX2 expression suppressed tumor cell epithelial-mesenchymal transition (EMT). This study demonstrated that detection of TPX2 overexpression could serve as a prognostic marker and therapeutic target for gastric cancer.

The targeting protein for Xklp2 (TPX2) is a microtubule- and, cell cycle-associated protein who's overexpression has been reported in various malignancies. In this study, we verified the overexpression of TPX2 in both surgically resected specimens of pancreatic cancer and multiple pancreatic cancer cell lines. Subsequently, we found that TPX2 siRNA effectively suppressed the proliferation of pancreatic cancer cells in culture, and the direct injection of TPX2 siRNA into subcutaneously implanted pancreatic cancer cells in nude mice revealed antiproliferative effects. These results implied a therapeutic potential of TPX2 siRNA in pancreatic cancer. Among 56 angiogenesis-related factors examined using angiogenesis arrays, the average protein levels of insulin-like growth factor-binding protein-3 (IGFBP-3) were significantly higher in TPX2 siRNA-treated tumors than in the Control siRNA-treated tumors. Moreover, we demonstrated that CD34-positive microvessels were significantly reduced in tumors treated with TPX2 siRNA compared to tumors that treated with Control siRNA. The attenuated expression of CD34 in TPX2 siRNA-treated tumors coincided with the overexpression of IGFBP-3. These results indicated that TPX2 has an impact on tumor angiogenesis in pancreatic cancer. The results also implied that the antiangiogenic effect observed in TPX2 siRNA-treated pancreatic cancer cells may be partly explained by the upregulation of IGFBP-3., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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.)

Background: The MYC oncogene has long been established as a central driver in many types of human cancers including colorectal cancer. However, the realization of MYC-targeting therapies remains elusive; as a result, synthetic lethal therapeutic approaches are alternatively being explored. A synthetic lethal therapeutic approach aims to kill MYC-driven tumors by targeting a certain co-regulator on the MYC pathway., Patients and Methods: We analyzed copy number and expression profiles from 130 colorectal cancer tumors together with publicly available datasets to identify co-regulators on the MYC pathway. Candidates were functionally tested by in vitro assays using colorectal cancer and normal fibroblast cell lines. Additionally, survival analyses were carried out on another 159 colorectal cancer patients and public datasets., Results: Our in silico screening identified two MYC co-regulator candidates, AURKA and TPX2, which are interacting mitotic regulators located on chromosome 20q. We found the two candidates showed frequent co-amplification with the MYC locus while expression levels of MYC and the two genes were positively correlated with those of MYC downstream target genes across multiple cancer types. In vitro, the aberrant expression of MYC, AURKA and TPX2 resulted in more aggressive anchorage-independent growth in normal fibroblast cells. Furthermore, knockdown of AURKA or TPX2, or treatment with an AURKA-specific inhibitor effectively suppressed the proliferation of MYC-expressing colorectal cancer cells. Additionally, combined high expression of MYC, AURKA and TPX2 proved to be a poor prognostic indicator of colorectal cancer patient survival., Conclusions: Through bioinformatic analyses and experiments, we proposed TPX2 and AURKA as novel co-regulators on the MYC pathway. Inhibiting the AURKA/TPX2 axis would be a novel synthetic lethal therapeutic approach for MYC-driven cancers., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: Cancer is characterized by uncontrolled cell growth and genetic instabilities. The human Aurora-A kinase protein plays a crucial role in spindle assembly during mitosis and is activated by another candidate oncogene, targeting protein for Xklp2 (TPX2). It has been proposed that dissociation of Aurora A-TPX2 complex leads to disruption of mitotic spindle apparatus, thereby preventing cell division and further tumor growth., Materials and Methods: A large natural compound library was docked against the active site of Aurora A-TPX2 complex. The protein-ligand complexes were subjected to molecular dynamics simulation to ascertain their binding stability. The drug properties of the compounds were analyzed to observe their drug-like properties., Results: The virtual screening of natural compound library yielded two high scoring compounds, the first compound CTOM [ZINC ID: 38143674] (Glide score: -9.49) was stable for 17 ns while the second TTOM (Glide score: -9.07) was stable for 15 ns. While CTOM interacted with His280, Thr288 of Aurora A and Tyr34, Lys38 of TPX2, TTOM interacted with Arg285 and Arg286 in addition to the residues involved with CTOM., Conclusions: We report two natural compounds as potential drugs leads for the disruption of this complex. These ligands show a preferable docking score and have many drugs like properties within in the range of 95% of known drugs. The study provides evidence that CTOM and TTOM can efficiently inhibit the TPX2-mediated activation of Aurora A. Thus, it paves way for an elaborate investigation and establishes the importance of computational approaches as time- and cost-effective techniques.

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM-TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.

Purpose: To explore the expression of TPX2 and its significance in esophageal squamous cell carcinoma (ESCC) tissue and approach relationship between the TPX2 and clinicopathological characteristic of esophageal squamous cell carcinoma., Method: RT-PCR and immunohistochemical staining were used to compare the expression of TPX2 in 62 esophageal squamous cell carcinoma, 31 atypical hyperplasia and 62 normal esophageal mucosa., Results: In ESCC, atypical hyperplasia and in normal mucous membrane tissues, the positive rate of TPX2 protein expression was 85.5% (53/62), 51.6% (16/31) and 4.8% (3/62); the positive rate of TPX2 mRNA expression was 65.5% (40/62), 35.5 (11/31) and 4.83% (3/62). The expression of TPX2 protein and mRNA were correlated with invasive depth and lymphatic metastasis of ESCC (P<0.01)., Conclusions: Overexpression of TPX2 may be risk factor of lymph node in esophageal carcinoma, and maybe a potential biomarker for early diagnosis and prognosis of esophageal squamous cell carcinoma., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

BACKGROUND: Colorectal cancer (CRC), is the third most common cancer type. MicroRNAs and their roles in cancer progression have gained considerable attention in the scientific community. miR-485-3p has been identified to be abnormally expressed in different types of cancer, but its expression level, biological function, and underlying pathways are still unclear in CRC. Targeting Protein for Xenopus kinesin-like protein 2 (TPX2) is a nuclear protein which plays vital roles in cancer progression and mitotic spindle assembly. TPX2 is overexpressed in various malignancies and has been predicted as an indirect target of miR-485-3p. This study aims to investigate the miR-485-3p and TPX2 expression level, their potential correlation, and underlying molecules like P53 and P21 in forty-one pairs of colorectal cancer tissues compared to matched non-cancerous ones. MATERIALS AND METHODS: We used forty-one pairs of CRC fresh tissue samples and their adjacent normal ones for RNA extraction. After cDNA synthesis, the expression level of miR-485-3p, TPX2, P53 and P21 were determined by Real-time PCR. RESULTS AND CONCLUSIONS: The results revealed that miR-485-3p was significantly downregulated and TPX2 was highly upregulated in CRC tissues. Moreover, miR-485-3p was negatively correlated with TPX2 expression and positively correlated with P21 expression. We present miR-485-3p as a suppressor for colorectal cancer (Tab. 2, Fig. 8, Ref. 44). Text in PDF www.elis.sk. [ABSTRACT FROM AUTHOR]

Aurora A has been extensively characterized as a centrosomal kinase with essential functions during cell division including centrosome maturation and separation and spindle assembly. However, Aurora A localization is not restricted to the centrosomes and compelling evidence support the existence of speciic mechanisms of activation and functions for non-centrosomal Aurora A in the dividing cell. It has been now well established that spindle assembly involves an acentrosomal RanGTP-dependent pathway that triggers microtubule assembly and organization in the proximity of the chromosomes whether centrosomes are present or not. The mechanism involves the regulation of a number of NLS-containing proteins, generically called SAFS (Spindle Assembly Factors) that exert their functions upon release from karyopherins by RanGTP. One of them, the nuclear protein TPX2 interacts with and activates Aurora A upon release from importins by RanGTP. This basic mechanism triggers the activation of Aurora A in the proximity of the chromosomes potentially translating the RanGTP signaling gradient centered on the chromosome into an Aurora A phosphorylation network. Here, we will review our current knowledge on the RanGTP-dependent TPX2 activation of Aurora A away from centrosomes: from the mechanism of activation and its functional consequences on the kinase stability and regulation to its roles in spindle assembly and cell division. We will then focus on the substrates of the TPX2-activated Aurora A having a role in microtubule nucleation, stabilization, and organization. Finally, we will briely discuss the implications of the use of Aurora A inhibitors in anti-tumor therapies in the light of its functional interaction with TPX2. [ABSTRACT FROM AUTHOR]