How Microtubules Build the Spindle Branch by Branch.

The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.

The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin.

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.

Microtubule nucleation for spindle assembly: one molecule at a time.

The cell orchestrates the dance of chromosome segregation with remarkable speed and fidelity. The mitotic spindle is built from scratch after interphase through microtubule (MT) nucleation, which is dependent on the γ-tubulin ring complex (γ-TuRC), the universal MT template. Although several MT nucleation pathways build the spindle framework, the question of when and how γ-TuRC is targeted to these nucleation sites in the spindle and subsequently activated remains an active area of investigation. Recent advances facilitated the discovery of new MT nucleation effectors and their mechanisms of action. In this review, we illuminate each spindle assembly pathway and subsequently consider how the pathways are merged to build a spindle.

Augmin is a Ran-regulated spindle assembly factor.

Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αß and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. In Xenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is nonessential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. Here, we use in vitro pulldowns and total internal reflection fluorescence microscopy to show that augmin is a Ran-regulated SAF. We demonstrate that augmin directly interacts with both importin-α and importin-ß through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT-binding site. Moreover, we show that Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. Our results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2.

Discovery of N-benzylbenzamide-based allosteric inhibitors of Aurora kinase A.

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 µM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure-activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.

Integrated Bioinformatic Analysis Reveals the Oncogenic, Survival, and Prognostic Characteristics of TPX2 in Hepatocellular Carcinoma.

Targeting protein for Xenopus kinesin-like protein 2 (TPX2), a well-known mitotic protein, has been linked to carcinogenesis in several cancers. This study investigated the role of TPX2 in hepatocellular carcinoma (HCC) from various aspects using bioinformatic analyses. TPX2 expression and its prognostic value in pan-cancers were analyzed using SangerBox. TPX2 expression and its association with prognosis, immune infiltration, tumor mutations, and signaling pathways in HCC were analyzed using UALCAN, BoxKaplan-Meier Plotter, GEPIA, Human Protein Atlas, TIMER 2.0, and SangerBox. Genes co-expressed with TPX2 in HCC were analyzed using the HCCDB database, followed by functional enrichment using SangerBox. Clinical predictive models were established based on TPX2 and its co-expressed genes using the ACLBI database. TPX2 expression significantly increased in pan-cancers and was associated with survival in nearly half of the cancer types. High TPX2 expression has been linked to poor survival outcomes in patients with HCC. TPX2 expression was positively correlated with abundant infiltration of immune cells (including B cells, CD4 + /CD8 + T cells, macrophages, neutrophils, and dendritic cells), TP53 mutation, and carcinogenesis-related pathways, such as the PI3K/AKT/mTOR pathway, cellular response to hypoxia, and tumor proliferation signature. Nineteen genes were found to be co-expressed with TPX2 in HCC, and these genes showed close positive correlations and were mainly implicated in cell cycle-related functions. A prognostic model established using TPX2 and its expressed genes could stratify HCC patients into high- and low-risk groups, with a significantly shorter survival time in high-risk groups. The prognostic model performed well in predicting 1-, 3-, and 5-year survival of patients with HCC, with areas under the curve of 0.801, 0.725, and 0.711, respectively. TPX2 functions as an oncogene in HCC, and its high expression is detrimental to the survival of patients with HCC. Thus, TPX2 may be a prognostic biomarker and potential therapeutic target for HCC.

Importin α phosphorylation promotes TPX2 activation by GM130 to control astral microtubules and spindle orientation.

Spindle orientation is important in multiple developmental processes as it determines cell fate and function. The orientation of the spindle depends on the assembly of a proper astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates astral microtubules. TPX2 in the spindle pole area is activated by GM130 (GOLGA2) on Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by competing for importin α1 (KPNA2) binding. Mitotic phosphorylation of importin α at serine 62 (S62) by CDK1 switches its substrate preference from TPX2 to GM130, thereby enabling competition-based activation. Importin α S62A mutation impedes local TPX2 activation and compromises astral microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130-importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for TPX2 in the organization of astral microtubules. Furthermore, we show that the substrate preference of the important mitotic modulator importin α is regulated by CDK1-mediated phosphorylation.

KIF4A promotes genomic stability and progression of endometrial cancer through regulation of TPX2 protein degradation.

Kinesin family member 4A (KIF4A) belongs to the kinesin superfamily proteins, which are closely associated with mitophagy. Nonetheless, the role of KIF4A in endometrial cancer (EC) remains poorly characterized. The present study showed that KIF4A not only was upregulated but also predicted poor prognosis in patients with EC. KIF4A knockdown in EC cells resulted in attenuated proliferative capacity in vitro and in vivo. Transcriptome sequencing and gene function analysis revealed that KIF4A contributed to the maintenance of EC cells' genomic stability and that KIF4A knockdown induced the DNA damage response, cell cycle arrest, and apoptosis. Mechanistically, KIF4A interacted with TPX2 (a protein involved in DNA damage repair to cope with the replication pressure) to enhance its stability via inhibition of TPX2 ubiquitination and eventually ensured the genomic stability of EC cells during mitosis. Taken together, our results indicated that KIF4A functions as a tumor oncogene that facilitates EC progression via the maintenance of genomic stability. Therefore, targeting the KIF4A/TPX2 axis may provide new concepts and strategies for the treatment of patients with EC.

Therapeutic targeting of the TPX2/TTK network in colorectal cancer.

BACKGROUND: While the increased screening, changes in lifestyle, and recent advances in treatment regimen have decreased colorectal cancer (CRC) mortality, metastatic disease and recurrence remains a major clinical challenge. In the era of precision medicine, the identification of actionable novel therapeutic targets could ultimately offer an alternative treatment strategy for CRC. METHODS: RNA-Seq was conducted using the illumina platform, while bioinformatics analyses were conducted using CLC genomics workbench and iDEP.951. Colony forming unit, flow cytometry, and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. The growth potential of CRC cells under 3-dimensional (3D) conditions was assessed using Matrigel. STRING database (v11.5) and Ingenuity Pathway Analysis (IPA) tool were used for network and pathway analyses. CRISPR-Cas9 perturbational effects database was used to identify potential therapeutic targets for CRC, through integration with gene-drug interaction database. Structural modeling and molecular docking were used to assess the interaction between candidate drugs and their targets. RESULTS: In the current study, we investigated the therapeutic potential of targeting TPX2, TTK, DDX39A, and LRP8, commonly upregulated genes in CRC identified through differential expression analysis in CRC and adjacent non-cancerous tissue. Targeted depletion of TPX2 and TTK impaired CRC proliferation, cell cycle progression, and organoid formation under 3D culture conditions, while suppression of DDX39A and LRP8 had modest effects on CRC colony formation. Differential expression analysis and bioinformatics on TPX2 and TTK-deficient cells identified cell cycle regulation as the hallmark associated with loss of TPX2 and TTK. Elevated expression of TPX2 and TTK correlated with an oncogenic state in tumor tissue from patients with colon adenocarcinoma, thus corroborating an oncogenic role for the TPX2/TTK network in the pathogenesis of CRC. Gene set enrichment and pathway analysis of TPX2high/TTKhigh CRC identified numerous additional gene targets as integral components of the TPX2/TTK network. Integration of TPX2/TTK enriched network with CRISPR-Cas9 functional screen data identified numerous novel dependencies for CRC. Additionally, gene-drug interaction analysis identified several druggable gene targets enriched in the TPX2/TTK network, including AURKA, TOP2A, CDK1, BIRC5, and many others. CONCLUSIONS: Our data has implicated an essential role for TPX2 and TTK in CRC pathogenesis and identified numerous potential therapeutic targets and their drug interactions, suggesting their potential clinical use as a novel therapeutic strategy for patients with CRC. Video Abstract.

miR-29c Suppresses the Malignant Phenotype of Hepatocellular Carcinoma Cells In Vitro by Mediating TPX2 Associated with Immune Infiltration.

BACKGROUND: miR-29-3p, an important tumor suppressor, with inhibitory effects in multiple cancers that have been studied. Its exact molecular function is in HCC, however, still not been explored clearly. The purpose of our study is to make certain how miR-29c-3p affects HCC through TPX2. MATERIALS AND METHODS: Expression profile data of miR-29c-3p and TPX2 were acquired and downloaded from the TCGA database, and the respective differential expression was verified by qPCR and immunohistochemistry. The StarBase and dual luciferase reporter confirmed TPX2 targeting miR-29c-3p. Their effects on the biological functions of Hep3B and HepG2 were investigated by cellular assays. RESULTS: miR-29-3p was found to be significantly down-regulated in HCC, and the miR-29-3p low expression group had a poor prognosis. Overexpression of miR-29-3p was detrimental to invasion and migration ability of HCC cells and promoted their apoptosis. We identified miR-29c-3p targeting TPX2 by predictive analysis. TPX2 was significantly upregulated in HCC, and patients with high TPX2 expression had a poor prognosis. TPX2 knockdown partially counteracted the promoting effect of miR-29-3p inhibition on hepatocellular carcinoma cells, and its effect on hepatocellular carcinoma cell biology was similar to miR-29c-3p overexpression. CONCLUSION: miR-29c, a key gene regulating HCC, is lowly expressed in HCC, its overexpression can remarkably inhibit the biological function of tumor cells. miR-29c can perform this function by regulating the expression of TPX2.

AURKA destruction is decoupled from its activity at mitotic exit but is essential to suppress interphase activity.

Activity of AURKA is controlled through multiple mechanisms including phosphorylation, ubiquitin-mediated degradation and allosteric interaction with TPX2. Activity peaks at mitosis, before AURKA is degraded during and after mitotic exit in a process strictly dependent on the APC/C coactivator FZR1. We used FZR1 knockout cells (FZR1KO) and a novel FRET-based AURKA biosensor to investigate how AURKA activity is regulated in the absence of destruction. We found that AURKA activity in FZR1KO cells dropped at mitotic exit as rapidly as in parental cells, despite absence of AURKA destruction. Unexpectedly, TPX2 was degraded normally in FZR1KO cells. Overexpression of an N-terminal TPX2 fragment sufficient for AURKA binding, but that is not degraded at mitotic exit, caused delay in AURKA inactivation. We conclude that inactivation of AURKA at mitotic exit is determined not by AURKA degradation but by degradation of TPX2 and therefore is dependent on CDC20 rather than FZR1. The biosensor revealed that FZR1 instead suppresses AURKA activity in interphase and is critically required for assembly of the interphase mitochondrial network after mitosis.This article has an associated First Person interview with the first authors of the paper.

SPI1 mediates transcriptional activation of TPX2 and RNF2 to regulate the radiosensitivity of lung squamous cell carcinoma.

Radiotherapy acts by damaging DNA and hindering cancer cell proliferation. H2AX is phosphorylated to produce γH2AX that accumulates in a response to DNA double-strand breaks. Non-coding RNA can influence DNA damage response and enhance DNA repair, which show potential for cancer treatment. The study aimed to observe the influence of SPI1 on the radiosensitivity of lung squamous cell carcinoma (LUSC) and to investigate the mechanisms. SPI1, TPX2, and RNF2 were overexpressed in LUSC tissues and radioresistant cells comspared with adjacent tissues and parental cells, respectively. The binding between SPI1 and TPX2 or RNF2 promoter was investigated using ChIP-qPCR and dual-luciferase assays. SPI1 bound to TPX2 and RNF2 promoters and activated their transcription. SPI1 downregulation increased the radiosensitivity of LUSC cells, which was compromised by TPX2 or RNF2 overexpression. Meanwhile, SPI1 downregulation elevated the protein expression of γH2AX at the late stage of DNA damage response and suppressed DNA damage repair in LUSC cells, which were compromised by TPX2 or RNF2. These results indicate that SPI1 silencing potentiates radiosensitivity in LUSC cells by downregulating the transcription of TPX2 and RNF2, which provides a potential target for the radiotherapy in LUSC.

Precious Gene: The Application of RET-Altered Inhibitors.

The well-known proto-oncogene rearrangement during transfection (RET), also known as ret proto-oncogene Homo sapiens (human), is a rare gene that is involved in the physiological development of some organ systems and can activate various cancers, such as non-small cell lung cancer, thyroid cancer, and papillary thyroid cancer. In the past few years, cancers with RET alterations have been treated with multikinase inhibitors (MKIs). However, because of off-target effects, these MKIs have developed drug resistance and some unacceptable adverse effects. Therefore, these MKIs are limited in their clinical application. Thus, the novel highly potent and RET-specific inhibitors selpercatinib and pralsetinib have been accelerated for approval by the Food and Drug Administration (FDA), and clinical trials of TPX-0046 and zetletinib are underway. It is well tolerated and a potential therapeutic for RET-altered cancers. Thus, we will focus on current state-of-the-art therapeutics with these novel RET inhibitors and show their efficacy and safety in therapy.

TPX2 mediates prostate cancer epithelial-mesenchymal transition through CDK1 regulated phosphorylation of ERK/GSK3ß/SNAIL pathway.

Prostate cancer with high Gleason grade is prone to metastasis, which is one of the factors that seriously threaten the survival of patients, and it is also a treatment difficulty. In this study, we first revealed the potential connection between TPX2 and prostate cancer metastasis. We found that TPX2 is highly expressed in high-grade prostate cancer and is significantly related to poor prognosis. Depletion of TPX2 can significantly inhibit cell activity and migration, and in vivo experiments show that knockdown of TPX2 can significantly inhibit tumor growth. In terms of mechanism, we found that knocking down TPX2 can inhibit the expression of CDK1, repress the phosphorylation of ERK/GSK3ß/SNAIL signaling pathway, and thereby inhibit tumor epithelial-mesenchymal transition. Subsequently, we found that after rescuing TPX2, all related proteins and phenotype changes were restored, and this effect can be inhibited by CDK1 inhibitor, RO-3306. Our findings suggest the potential of TPX2 as an important target in anti-tumor metastasis therapy, which is conducive to precision medicine for prostate cancer.

A guide to plant TPX2-like and WAVE-DAMPENED2-like proteins.

TPX2 proteins were first identified in vertebrates as a key mitotic spindle assembly factor. Subsequent studies demonstrated that TPX2 is an intricate protein, with functionally and structurally distinct domains and motifs including Aurora kinase-binding, importin-binding, central microtubule-binding, and C-terminal TPX2 conserved domain, among others. The first plant TPX2-like protein, WAVE-DAMPENED2, was identified in Arabidopsis as a dominant mutation responsible for reducing the waviness of roots grown on slanted agar plates. Each plant genome encodes at least one 'canonical' protein with all TPX2 domains and a family of proteins (20 in Arabidopsis) that diversified to contain only some of the domains. Although all plant TPX2-family proteins to date bind microtubules, they function in distinct processes such as cell division, regulation of hypocotyl cell elongation by hormones and light signals, vascular development, or abiotic stress tolerance. Consequently, their expression patterns, regulation, and functions have diverged considerably. Here we summarize the current body of knowledge surrounding plant TPX2-family proteins.

High nuclear TPX2 expression correlates with TP53 mutation and poor clinical behavior in a large breast cancer cohort, but is not an independent predictor of chromosomal instability.

BACKGROUND: Targeting Protein for Xenopus Kinesin Like Protein 2 (TPX2) is a microtubule associated protein that functions in mitotic spindle assembly. TPX2 also localizes to the nucleus where it functions in DNA damage repair during S-phase. We and others have previously shown that TPX2 RNA levels are strongly associated with chromosomal instability (CIN) in breast and other cancers, and TPX2 RNA levels have been demonstrated to correlate with aggressive behavior and poor clinical outcome across a range of solid malignancies, including breast cancer. METHODS: We perform TPX2 IHC on a cohort of 253 primary breast cancers and adopt a clinically amenable scoring system to separate tumors into low, intermediate, or high TPX2 expression. We then correlate TPX2 expression against diverse pathologic parameters and important measures of clinical outcome, including disease-specific and overall survival. We link TPX2 expression to TP53 mutation and evaluate whether TPX2 is an independent predictor of chromosomal instability (CIN). RESULTS: We find that TPX2 nuclear expression strongly correlates with high grade morphology, elevated clinical stage, negative ER and PR status, and both disease-specific and overall survival. We also show that increased TPX2 nuclear expression correlates with elevated ploidy, supernumerary centrosomes, and TP53 mutation. TPX2 nuclear expression correlates with CIN via univariate analyses but is not independently predictive when compared to ploidy, Ki67, TP53 mutational status, centrosome number, and patient age. CONCLUSIONS: Our findings demonstrate a strong correlation between TPX2 nuclear expression and aggressive tumor behavior, and show that TPX2 overexpression frequently occurs in the setting of TP53 mutation and elevated ploidy. However, TPX2 expression is not an independent predictor of CIN where it fails to outperform existing clinical and pathologic metrics.

Knockdown of circ_0003340 induces cell apoptosis, inhibits invasion and proliferation through miR-564/TPX2 in esophageal cancer cells.

Circular RNA (circRNA) is a promising biomarker of cancer occurrence and development. The different expression levels of circRNAs in various cancers also make them possible therapeutic targets. In this work, we researched the function and underlying mechanisms of circ_0003340 (circ3340) in esophageal cancer EC1 and EC9706 cells. Firstly, we found the expression levels of circ3340 are higher in ESCC and two esophageal cancer cells than in adjacent normal tissues and Het-1a cells. Bioinformatics analysis showed circ3340 has a binding site with miR-564. This was verified by luciferase assay, which revealed that miR-564 can be sponged by circ3340, and that the TPX2 3'UTR is a direct target of miR-564. Upregulation of miR-564 decreased TPX2 protein levels, as shown by Western blot. Moreover, knockdown of circ3340 or enhancement of miR-564 expression had similar effects in EC1 and EC9706 cells, i.e., inducing cell apoptosis, inhibiting cell proliferation, and arresting cell invasion. Downregulation of circ3340 had a negative influence on EC1 and EC9706 cells by affecting the miR-564/TPX2 pathway. Additionally, animal experiments revealed that downregulation of circ3340 inhibited tumor growth in vivo, making circ3340 a potential therapeutic target for patients with esophageal squamous cell cancer.

CircPOSTN/miR-361-5p/TPX2 axis regulates cell growth, apoptosis and aerobic glycolysis in glioma cells.

BACKGROUND: Glioma is the most primary central nervous system tumor in adults. The 5 year survival rate for glioma patients remains poor, although treatment strategies had improved in the past few decades. The cumulative studies have shown that circular RNA (circRNA) is associated with glioma process, so the purpose of this study is to clarify the function of circPOSTN in glioma. METHODS: The expression levels of circPOSTN, miR-361-5p, and targeting protein for Xenopus kinesin-like protein 2 (TPX2) were assessed with real-time quantitative polymerase chain reaction (RT-qPCR). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays were executed to examine proliferation and apoptosis of glioma cells, respectively. Western blot was applied to assess protein expression. The glucose metabolism of glioma cells was analyzed by testing the glucose consumption, lactate production, ATP level, reactive oxygen species (ROS) accumulation and performing Seahorse XF assay. The interaction relationship between miR-361-5p and circPOSTN or TPX2 was analyzed by bioinformatics database and dual-luciferase reporter assay. The influences of circPOSTN silencing in vivo were observed by a xenograft experiment. RESULTS: CircPOSTN was overexpressed in glioma tissues and cells. Absence of circPOSTN in glioma cells promoted apoptosis while impeded proliferation and aerobic glycolysis, which were mitigated by silencing miR-361-5p. What's more, loss-of-functional experiment suggested that knockdown of TPX2 repressed proliferation and aerobic glycolysis, while induced apoptosis in glioma cells. In addition, circPOSTN targetedly regulated TPX2 expression in glioma cells via sponging miR-361-5p. In vivo study revealed that deficiency of circPOSTN restrained tumor growth. CONCLUSION: Mechanistically, circPOSTN regulated cell growth, apoptosis, and aerobic glycolysis in glioma through miR-361-5p/TPX2 axis.

The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation.

BACKGROUND: Aberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), nevertheless, the precise molecular mechanism remains unclear. Forkhead box M1 (FOXM1), a target of the Hh pathway, is a key oncofetal transcription factor and a master cell cycle regulator. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is an oncogene critical for mitosis. However, how these molecular events affect HCC progression remains unclear. METHODS: Realtime PCR, immunohistochemistry, western blotting, and analyses of datasets TCGA and Gene Expression Omnibus (GEO) were conducted to assess the expression of TPX2 and FOXM1 at the mRNA and protein levels in HCC samples or HCC cells. Expression and knockdown of TPX2 and FOXM1 were performed to assess their role in regulating HCC cell proliferation in vitro and in vivo. Dual luciferase report assay and chromosome immunoprecipitation (ChIP) were investigated to seek the FOXM1 binding sites in the promoter of TPX2. RESULTS: Specific antagonists (cyclopamine and GANT61) of the Hh pathway down-regulated TPX2, whereas activation of Hh signaling stimulated TPX2 expression. Furthermore, TPX2 over-expression accelerated HCC cell proliferation when upstream events of Hh signaling were inhibited, and TPX2 knockdown significantly alleviated Sonic Hh ligand (Shh)-induced HCC cell proliferation. Reporter assays and ChIP showed that FOXM1 bound to the TPX2 promoter, confirming that TPX2 is a direct downstream target of FOXM1. Xenograft model further verified the cell function and expression regulation of TPX2 and FOXM1 in vivo. Furthermore, FOXM1 regulated TPX2 activity to drive HCC proliferation. Immunohistochemical (IHC) analysis indicated that FOXM1 and TPX2 were highly-expressed in HCC samples and cohort study revealed that FOXM1 and TPX2 may act as negative predictors for the prognosis of patients with HCC. CONCLUSIONS: TPX2 acts as a novel downstream target and effector of the Hh pathway, and Hh signaling contributes to HCC proliferation via regulating the FOXM1-TPX2 cascade, suggesting that this signaling axis may be a novel therapeutic target for HCC.

Adducin-1 is essential for spindle pole integrity through its interaction with TPX2.

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.