Dynamic Mitotic Localization of the Centrosomal Kinases CDK1, Plk, AurK, and Nek2 in Dictyostelium amoebae.

The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed.

Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin.

Sister chromatid cohesion is mediated by the cohesin complex. In mitotic prophase cohesin is removed from chromosome arms in a Wapl- and phosphorylation-dependent manner. Sgo1-PP2A protects pericentromeric cohesion by dephosphorylation of cohesin and its associated Wapl antagonist sororin. However, Sgo1-PP2A relocates to inner kinetochores well before sister chromatids are separated by separase, leaving pericentromeric regions unprotected. Why deprotected cohesin is not removed by Wapl remains enigmatic. By reconstituting Wapl-dependent cohesin removal from chromatin in vitro, we discovered a requirement for Nek2a and Cdk1/2-cyclin A2. These kinases phosphorylate cohesin-bound Pds5b, thereby converting it from a sororin- to a Wapl-interactor. Replacement of endogenous Pds5b by a phosphorylation mimetic variant causes premature sister chromatid separation (PCS). Conversely, phosphorylation-resistant Pds5b impairs chromosome arm separation in prometaphase-arrested cells and suppresses PCS in the absence of Sgo1. Early mitotic degradation of Nek2a and cyclin A2 may therefore explain why only separase, but not Wapl, can trigger anaphase.

Role of NEK2 in tumorigenesis and tumor progression.

Never in mitosis A (NIMA)-related kinase 2 (NEK2) is a serine/threonine kinase found in the nucleus and cytoplasm throughout the cell cycle. NEK2 is overexpressed in many cancers and is a biomarker of poor prognosis. Factors contributing to NEK2 elevation in cancer cells include oncogenic transcription factors, decreased ubiquitination, DNA methylation, and the circular RNA (circRNA)/long noncoding RNA (lncRNA)-miRNA axis. NEK2 overexpression produces chromosomal instability and aneuploidy, thereby enhancing cancer progression and suppressing antitumor immunity, which highlights the prominence of NEK2 in tumorigenesis and tumor progression. Small-molecule inhibitors targeting NEK2 have demonstrated promising therapeutic potential in vitro and in vivo across various cancer types. This review outlines the regulatory mechanisms of NEK2 expression, emphasizes its functional roles in cancer initiation and progression, and highlights the anticancer properties of NEK2 inhibitors.

In silico analysis unveiling potential biomarkers in gallbladder carcinogenesis.

Gallbladder cancer (GBC) is a rare but very aggressive most common digestive tract cancer with a high mortality rate due to delayed diagnosis at the advanced stage. Moreover, GBC progression shows asymptomatic characteristics making it impossible to detect at an early stage. In these circumstances, conventional therapy like surgery, chemotherapy, and radiotherapy becomes refractive. However, few studies reported some molecular markers like KRAS (Kirsten Rat Sarcoma) mutation, upregulation of HER2/neu, EGFR (Epidermal Growth Factor Receptor), and microRNAs in GBC. However, the absence of some specific early diagnostic and prognostic markers is the biggest hurdle for the therapy of GBC to date. The present study has been designed to identify some specific molecular markers for precise diagnosis, and prognosis, for successful treatment of the GBC. By In Silico a network-centric analysis of two microarray datasets; (GSE202479) and (GSE13222) from the Gene Expression Omnibus (GEO) database, shows 50 differentially expressed genes (DEGs) associated with GBC. Further network analysis revealed that 12 genes are highly interconnected based on the highest MCODE (Molecular Complex Detection) value, among all three genes; TRIP13 (Thyroid Receptor Interacting Protein), NEK2 (Never in Mitosis gene-A related Kinase 2), and TPX2 (Targeting Protein for Xklp2) having highest network interaction with transcription factors and miRNA suggesting critically associated with GBC. Further survival analysis data corroborate the association of these genes; TRIP13, NEK2, and TPX2 with GBC. Thus, TRIP13, NEK2, and TPX2 genes are significantly correlated with a greater risk of mortality, transforming them from mere biomarkers of the GBC for early detections and may emerge as prognostic markers for treatment.

Mitotic kinases are emerging therapeutic targets against metastatic breast cancer.

This review aims to outline mitotic kinase inhibitors' roles as potential therapeutic targets and assess their suitability as a stand-alone clinical therapy or in combination with standard treatments for advanced-stage solid tumors, including triple-negative breast cancer (TNBC). Breast cancer poses a significant global health risk, with TNBC standing out as the most aggressive subtype. Comprehending the role of mitosis is crucial for understanding how TNBC advances from a solid tumor to metastasis. Chemotherapy is the primary treatment used to treat TNBC. Some types of chemotherapeutic agents target cells in mitosis, thus highlighting the need to comprehend the molecular mechanisms governing mitosis in cancer. This understanding is essential for devising targeted therapies to disrupt these mitotic processes, prevent or treat metastasis, and improve patient outcomes. Mitotic kinases like Aurora kinase A, Aurora Kinase B, never in mitosis gene A-related kinase 2, Threonine-Tyrosine kinase, and Polo-kinase 1 significantly impact cell cycle progression by contributing to chromosome separation and centrosome homeostasis. When these kinases go awry, they can trigger chromosome instability, increase cell proliferation, and activate different molecular pathways that culminate in a transition from epithelial to mesenchymal cells. Ongoing clinical trials investigate various mitotic kinase inhibitors as potential biological treatments against advanced solid tumors. While clinical trials against mitotic kinases have shown some promise in the clinic, more investigation is necessary, since they induce severe adverse effects, particularly affecting the hematopoietic system.

NEK2 contributes to radioresistance in esophageal squamous cell carcinoma by inducing protective autophagy via regulating TRIM21.

BACKGROUND: Radiotherapy (RT) has been identified as a vital treatment for esophageal squamous cell carcinoma (ESCC), while the development of radioresistance remains a major obstacle in ESCC management. The aim of this study was to investigate the effect of NIMA-related kinase 2 (NEK2) on radioresistance in ESCC cells and to reveal potential molecular mechanisms. METHODS: Human esophageal epithelial cells (HEEC) and human ESCC cell lines were obtained from the Research Center of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Cell Counting Kit-8 (CCK-8) and flow cytometry assays were applied to assess the proliferation ability, cell cycle, apoptosis rates, and ROS production of ESCC cells. The colony-forming assay was used to estimate the effect of NEK2 on radiosensitivity. Autophagy was investigated by western blotting analysis, GFP-mRFP-LC3 fluorescence assay, and transmission electron microscopy (TEM). RESULTS: In the present study, our results showed that NEK2 was associated with radioresistance, cell cycle arrest, apoptosis, ROS production, and survival of ESCC. NEK2 knockdown could significantly inhibit growth while enhancing radiosensitivity and ROS production in ESCC cells. Interestingly, NEK2 knockdown inhibited ESCC cell autophagy and reduced autophagic flux, ultimately reversing NEK2-induced radioresistance. Mechanistically, NEK2 bound to and regulated the stability of tripartite motif-containing protein 21 (TRIM21). The accumulation of NEK2-induced light chain 3 beta 2 (LC3B II) can be reversed by the knockdown of TRIM21. CONCLUSION: These results demonstrated that NEK2 activated autophagy through TRIM21, which may provide a promising therapeutic strategy for elucidating NEK2-mediated radioresistance in ESCC.

NEK2 promotes the development of ovarian endometriosis and impairs decidualization by phosphorylating FOXO1.

Ovarian endometriosis is a common gynecological disease, and one of its most significant symptoms is infertility. In patients with endometriosis, defects in endometrial decidualization lead to impaired endometrial receptivity and embryo implantation, thus affecting early pregnancy and women's desire to have children. However, the mechanisms underlying the development of endometriosis and its associated defective decidualization are unclear. We find that NEK2 expression is increased in the ectopic and eutopic endometrium of patients with endometriosis. Meanwhile, NEK2 interacts with FOXO1 and phosphorylates FOXO1 at Ser184, inhibiting the stability of the FOXO1 protein. Importantly, NEK2-mediated phosphorylation of FOXO1 at Ser184 promotes cell proliferation, migration, invasion and impairs decidualization. Furthermore, INH1, an inhibitor of NEK2, inhibits the growth of ectopic lesions in mouse models of endometriosis and promotes endometrial decidualization in mouse models of artificially induced decidualization. Taken together, these findings indicate that NEK2 regulates the development of endometriosis and associated disorders of decidualization through the phosphorylation of FOXO1, providing a new therapeutic target for its treatment.

NEK2 promotes the migration, invasion, proliferation of ESCC and mediates ESCC immunotherapy.

Purpose: Esophageal squamous cell carcinoma (ESCC) is a disease with a high incidence rate and high mortality worldwide. The Never in Mitosis A (NIMA) family member NIMA-related kinase 2 (NEK2) plays an important role in mitosis. However, the role of NEK2 in the pathogenesis of ESCC remains unclear. Patients and methods: The expression and function of NEK2 in TCGA and GEO data sets were analyzed by bioinformatics. We verified the expression of NEK2 in ESCC tissues and cell lines by Western blotting and immunohistochemical methods and further explored the relationship between tumor stage and NEK2 expression. The differences in NEK2 expression and survival in patients with EC were verified by bioinformatics analysis. ESCC cell lines with stable knockdown of NEK2 were established by lentivirus-mediated shRNA delivery. The effects of NEK2 on ESCC cells were analyzed on the cytological level with assays including CCK-8, EdU, cell scratch, Transwell migration and invasion, colony formation, flow cytometry and apoptosis assays. Tumor growth was measured in a mouse xenograft model. Results: We found that NEK2 is highly expressed in ESCC tissues and ESCC cells and that the high expression of NEK2 is associated with poor tumor healing. Knockdown of the NEK2 gene inhibits the migration, proliferation, invasion and cell cycle of ESCC cells. Biologic analysis shows that NEK2 is involved in biological processes such as progression and apoptosis of esophageal cancer, and is related to E2F.Mechanistically, NEK2 knockdown decreases the expression levels of E2F1 and IGF2. NEK2 competes with the transcription factor E2F1 to bind CDC20, resulting in decreased degradation and increased expression of E2F1. IGF2 expression is also increased, which promotes the expression of thymidylate synthase, further promoting the drug resistance of ESCC cells. NEK2 is associated with immune infiltration in esophageal cancer. Conclusion: NEK2 is highly expressed in ESCC and can promote the migration, proliferation and invasion of ESCC cells. NEK2 mediates ESCC immunotherapy.

NEK2 affects the ferroptosis sensitivity of gastric cancer cells by regulating the expression of HMOX1 through Keap1/Nrf2.

NEK2 is a serine/threonine protein kinase that is involved in regulating the progression of various tumors. Our previous studies have found that NEK2 is highly expressed in gastric cancer and suggests that patients have a worse prognosis. However, its role and mechanism in gastric cancer are only poorly studied. In this study, we established a model of ferroptosis induced by RSL3 or Erastin in AGS cells in vitro, and konckdown NEK2, HOMX1, Nrf2 by siRNA. The assay kit was used to analyzed cell viability, MDA levels, GSH and GSSG content, and FeRhoNox™-1 fluorescent probe, BODIPY™ 581/591 C11 lipid oxidation probe, CM-H2DCFDA fluorescent probe were used to detected intracellular Fe2+, lipid peroxidation, and ROS levels, respectively. Calcein-AM/PI staining was used to detect the ratio of live and dead cells, qRT-PCR and Western blot were used to identify the mRNA and protein levels of genes in cells, immunofluorescence staining was used to analyze the localization of Nrf2 in cells, RNA-seq was used to analyze changes in mRNA expression profile, and combined with the FerrDb database, ferroptosis-related molecules were screened to elucidate the impact of NEK2 on the sensitivity of gastric cancer cells to ferroptosis. We found that inhibition of NEK2 could enhance the sensitivity of gastric cancer cells to RSL3 and Erastin-induced ferroptosis, which was reflected in the combination of inhibition of NEK2 and ferroptosis induction compared with ferroptosis induction alone: cell viability and GSH level were further decreased, while the proportion of dead cells, Fe2+ level, ROS level, lipid oxidation level, MDA level, GSSG level and GSSG/GSH ratio were further increased. Mechanism studies have found that inhibiting NEK2 could promote the expression of HMOX1, a gene related to ferroptosis, and enhance the sensitivity of gastric cancer cells to ferroptosis by increasing HMOX1. Further mechanism studies have found that inhibiting NEK2 could promote the ubiquitination and proteasome degradation of Keap1, increase the level of Nrf2 in the nucleus, and thus promote the expression of HMOX1. This study confirmed that NEK2 can regulate HMOX1 expression through Keap1/Nrf2 signal, and then affect the sensitivity of gastric cancer cells to ferroptosis, enriching the role and mechanism of NEK2 in gastric cancer.

Dual roles of CCDC102A in governing centrosome duplication and cohesion.

In animal cells, the dysregulation of centrosome duplication and cohesion maintenance leads to abnormal spindle assembly and chromosomal instability, contributing to developmental disorders and tumorigenesis. However, the molecular mechanisms involved in maintaining accurate centrosome number control and tethering are not fully understood. Here, we identified coiled-coil domain-containing 102A (CCDC102A) as a centrosomal protein exhibiting a barrel-like structure in the proximal regions of parent centrioles, where it prevents centrosome overduplication by restricting interactions between Cep192 and Cep152 on centrosomes, thereby ensuring bipolar spindle formation. Additionally, CCDC102A regulates the centrosome linker by recruiting and binding C-Nap1; it is removed from the centrosome after Nek2A-mediated phosphorylation at the onset of mitosis. Overall, our results indicate that CCDC102A participates in controlling centrosome number and maintaining centrosome cohesion, suggesting that a well-tuned system regulates centrosome structure and function throughout the cell cycle.

Behavior of intracellular lipid droplets during cell division in HuH7 hepatoma cells.

Intracellular lipid droplets (LDs) are ubiquitous organelles found in many cell types. During mitosis, membranous organelles, including mitochondria, are divided into small pieces and transferred to daughter cells; however, the process of LD transfer to daughter cells is not fully elucidated. Herein, we investigated the behavior of LDs during mitosis in HuH7 human hepatoma cells. While fragments of the Golgi apparatus were scattered in the cytosol during mitosis, intracellular LDs retained their size and spherical morphology as they translocated to the two daughter cells. LDs were initially distributed throughout the cell during prophase but positioned outside the spindle in metaphase, aligning at the far sides of the centrioles. A similar distribution of LDs during mitosis was observed in another hepatocarcinoma HepG2 cells. When the spindle was disrupted by nocodazole treatment or never in mitosis gene A-related kinase 2A knockdown, LDs were localized in the area outside the chromosomes, suggesting that spindle formation is not necessary for LD localization at metaphase. The amount of major LD protein perilipin 2 reduced while LDs were enriched in perilipin 3 during mitosis, indicating the potential alteration of LD protein composition. Conclusively, the behavior of LDs during mitosis is distinct from that of other organelles in hepatocytes.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma.

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory T cells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated T cell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

Identification of multidrug chemoresistant genes in head and neck squamous cell carcinoma cells.

Multidrug resistance renders treatment failure in a large proportion of head and neck squamous cell carcinoma (HNSCC) patients that require multimodal therapy involving chemotherapy in conjunction with surgery and/or radiotherapy. Molecular events conferring chemoresistance remain unclear. Through transcriptome datamining, 28 genes were subjected to pharmacological and siRNA rescue functional assays on 12 strains of chemoresistant cell lines each against cisplatin, 5-fluorouracil (5FU), paclitaxel (PTX) and docetaxel (DTX). Ten multidrug chemoresistance genes (TOP2A, DNMT1, INHBA, CXCL8, NEK2, FOXO6, VIM, FOXM1B, NR3C1 and BIRC5) were identified. Of these, four genes (TOP2A, DNMT1, INHBA and NEK2) were upregulated in an HNSCC patient cohort (n = 221). Silencing NEK2 abrogated chemoresistance in all drug-resistant cell strains. INHBA and TOP2A were found to confer chemoresistance in majority of the drug-resistant cell strains whereas DNMT1 showed heterogeneous results. Pan-cancer Kaplan-Meier survival analysis on 21 human cancer types revealed significant prognostic values for INHBA and NEK2 in at least 16 cancer types. Drug library screens identified two compounds (Sirodesmin A and Carfilzomib) targeting both INHBA and NEK2 and re-sensitised cisplatin-resistant cells. We have provided the first evidence for NEK2 and INHBA in conferring chemoresistance in HNSCC cells and siRNA gene silencing of either gene abrogated multidrug chemoresistance. The two existing compounds could be repurposed to counteract cisplatin chemoresistance in HNSCC. This finding may lead to novel personalised biomarker-linked therapeutics that can prevent and/or abrogate chemoresistance in HNSCC and other tumour types with elevated NEK2 and INHBA expression. Further investigation is necessary to delineate their signalling mechanisms in tumour chemoresistance.

Protein Expression of NEK2, JMJD4, and REST in Clear Cell Renal Cell Carcinoma (ccRCC): Clinical, Pathological, and Prognostic Findings.

Background & Objective: Cells of renal cell carcinoma (RCC) are resistant to the most currently used chemotherapeutic agents and targeted therapies; hence, we evaluated the expression of NEK2, JMJD4, and REST in cases of clear cell renal cell carcinoma (ccRCC) and benign adjacent tissues of kidney to detect associations between their expression and clinicopathological features, prognostic data, tumor recurrence, and survival rates. Methods: We collected 200 samples including tumoral and adjacent non-neoplastic tissues related to 100 ccRCC patients. All samples were evaluated for the expression of NEK2, JMJD4, and REST, and the patients were followed up for about 5 years. Tumor recurrence and survival data were documented and analyzed. Results: NEK2 and JMJD4 expression showed increase in ccRCC tissues (P=0.002 and 0.006), while REST was downregulated (P<0.001). The elevated expression of NEK2 was positively related ro the tumor size (P=0.015), higher grades (P=0.002), higher stages (P=0.013), distant spread (P=0.004), tumor recurrence, shorter progression-free survival (PFS) rate, and overall survival (OS) rate (P<0.001). Likewise, the high expression of JMJD4 showed positive correlation with the tumor size (P=0.047), higher grades (P=0.003), higher stages (P=0.043), distant spread (P=0.001), tumor recurrence, shorter PFS rate, and OS rate (P<0.001). Conversely, low expression of REST demonstrated positive relationship with the tumor size, higher grades, higher stages, distant spread, tumor recurrence, and shorter PFS and OS rates (P<0.001). Conclusion: Overexpression of NEK2 and JMJD4 and downregulation of REST may be noted in malignant renal tissues compared to benign renal tissues and may be correlated with unfavorable pathological findings, poor clinical parameters, and poor patient outcomes.

Screening of novel biomarkers for breast cancer based on WGCNA and multiple machine learning algorithms.

Background: Breast cancer (BC) ranks first in incidence among women, with approximately 2 million new cases per year. Therefore, it is essential to investigate emerging targets for BC patients' diagnosis and prognosis. Methods: We analyzed gene expression data from 99 normal and 1,081 BC tissues in The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) were identified using "limma" R package, and relevant modules were chosen through Weighted Gene Coexpression Network Analysis (WGCNA). Intersection genes were obtained by matching DEGs to WGCNA module genes. Functional enrichment studies were performed on these genes using Gene Ontology (GO), Disease Ontology (DO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Biomarkers were screened via Protein-Protein Interaction (PPI) networks and multiple machine-learning algorithms. The Gene Expression Profiling Interactive Analysis (GEPIA), The University of ALabama at Birmingham CANcer (UALCAN), and Human Protein Atlas (HPA) databases were employed to examine mRNA and protein expression of eight biomarkers. Kaplan-Meier mapper tool assessed their prognostic capabilities. Key biomarkers were analyzed via single-cell sequencing, and their relationship with immune infiltration was examined using Tumor Immune Estimation Resource (TIMER) database and "xCell" R package. Lastly, drug prediction was conducted based on the identified biomarkers. Results: We identified 1,673 DEGs and 542 important genes through differential analysis and WGCNA, respectively. Intersection analysis revealed 76 genes, which play significant roles in immune-related viral infection and IL-17 signaling pathways. DIX domain containing 1 (DIXDC1), Dual specificity phosphatase 6 (DUSP6), Pyruvate dehydrogenase kinase 4 (PDK4), C-X-C motif chemokine ligand 12 (CXCL12), Interferon regulatory factor 7 (IRF7), Integrin subunit alpha 7 (ITGA7), NIMA related kinase 2 (NEK2), and Nuclear receptor subfamily 3 group C member 1 (NR3C1) were selected as BC biomarkers using machine-learning algorithms. NEK2 was the most critical gene for diagnosis. Prospective drugs targeting NEK2 include etoposide and lukasunone. Conclusions: Our study identified DIXDC1, DUSP6, PDK4, CXCL12, IRF7, ITGA7, NEK2, and NR3C1 as potential diagnostic biomarkers for BC, with NEK2 having the highest potential to aid in diagnosis and prognosis in clinical settings.

NEK2 overexpression aggravates IL-22-induced keratinocyte proliferation and cytokine level increases and IMQ-induced psoriasis-like dermatitis.

BACKGROUND: Psoriasis is a common inflammatory skin disease characterized by the excessive proliferation and abnormal differentiation of keratinocytes. Protein kinases could act on intracellular signaling pathways associated with cell proliferation. OBJECTIVE: Identifying more hub protein kinases affecting cellular and molecular processes in psoriasis, and exploring the dynamic effects of baicalin and NEK2 on the IL-22-induced cellular inflammation and IMQ-induced psoriasis-like mice. METHODS AND RESULTS: In this study, differentially expressed protein kinases playing a hub role in psoriasis initiation and development were identified using integrative bioinformatics analyses, and NEK2 has been chosen. NEK2 was significantly up-regulated in psoriatic samples according to online datasets and experimental analyses. In IL-22-induced cellular inflammation model in HaCaT cells, NEK2 overexpression promoted, whereas NEK2 knockdown partially abolished IL-22-induced alterations in cell viability, DNA synthesis, cytokine levels, as well as STAT3 phosphorylation and p-RB, cyclin D1, CDK4, and CDK6 protein contents. Baicalin treatment partially suppressed IL-22-induced HaCaT cell viability, DNA synthesis, and increases in cytokine levels, whereas NEK2 overexpression significantly abolished Baicalin-induced protection against cellular inflammation. In IMQ-induced psoriasis-like skin inflammation model in mice, baicalin markedly ameliorated IMQ-induced psoriasis-like symptoms and local skin inflammation, whereas NEK2 overexpression partially eliminated the therapeutic effects of baicalin. CONCLUSION: NEK2, up-regulated in psoriatic lesion skin, could aggravate IMQ-induced psoriasis-like dermatitis and attenuate the therapeutic efficiency of baicalin through promoting keratinocyte proliferation and cytokine levels. The STAT3 signaling might be involved.

NEK2 promotes esophageal squamous cell carcinoma cell proliferation, migration and invasion through the Wnt/ß-catenin signaling pathway.

OBJECTIVES: The NEK2 (never in mitosis gene A-related kinase 2), a serine/threonine kinase involved in chromosome instability and tumorigenesis. Hence, this study aimed to explore the molecular function of NEK2 in esophageal squamous cell carcinoma (ESCC). METHODS: By available transcriptome datasets (GSE53625 cohort, GSE38129 cohort, and GSE21293 cohort), we analyzed the differentially expressed genes in invading and non-invading ESCC. Subsequently, we evaluated the association between NEK2 expression level and clinical outcomes through Kaplan-Meier analysis method. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) analyses were performed to determine the expression levels of NEK2 mRNA and protein, respectively. We knocked down the NEK2 expression in ESCC cells (ECA109 and TE1), and evaluated the NEK2 biology function associated with ESCC cell proliferation, migration, invasion, and colony formation abilities. Finally, the downstream pathway of NEK2 was analyzed through Gene Set Enrichment Analysis (GSEA) and validated the regulatory mechanism of NEK2 on the potential pathway through WB. RESULTS: We found that NEK2 was highly expressed in ESCC cells compared with human esophageal epithelial cells (HEEC) (P < 0.0001), and high NEK2 expression was remarkably associated with poor survival (P = 0.019). Knockdown of NEK2 showed the significant inhibitory effect for tumorigenesis, and suppressed the ESCC cells proliferation, migration, invasion, and formation of colonies abilities. Additionally, GSEA revealed that Wnt/ß-catenin pathway was a downstream pathway of NEK2. WB results further validated the regulatory mechanism of NEK2 for Wnt/ß-catenin signaling. CONCLUSIONS: Our results indicated that NEK2 promotes ESCC cell proliferation, migration and invasion by activating the Wnt/ß-catenin pathway. NEK2 could be a promising target for ESCC.

Control of centrosome distal appendages assembly and disassembly.

Centrosomes are microtubule organizing centers involved in chromosome segregation, spindle orientation, cell motility and cilia formation. In recent years, they have also emerged as key modulators of asymmetric cell division. Centrosomes are composed of two centrioles that initiate duplication in S phase. The conservative nature of centriole duplication means that the two centrioles of a G1 cell are of different ages. They are also structurally different as only the older centriole carries appendages, an assembly of a subset of proteins primarily required for cilia formation. In a growing tissue, the non-motile, primary cilium acts as a mechano- and sensory organelle that influences cell behavior via modulation of signaling pathways. Here, we discuss the most recent findings about distal appendage composition and function, as well as cell cycle-specific regulation and their implications in various diseases.

Identification of a novel spirocyclic Nek2 inhibitor using high throughput virtual screening.

NIMA Related Kinase 2 (Nek2) kinase is an attractive target for the development of therapeutic agents for several types of highly invasive cancers. Despite this, no small molecule inhibitor has advanced to the late clinical stages thus far. In this work, we have identified a novel spirocyclic inhibitor (V8) of Nek2 kinase, utilizing a high-throughput virtual screening (HTVS) approach. Using recombinant Nek2 enzyme assays, we show that V8 can inhibit Nek2 kinase activity (IC50 = 2.4 ± 0.2 µM) by binding to the enzyme's ATP pocket. The inhibition is selective, reversible and is not time dependent. To understand the key chemotype features responsible for Nek2 inhibition, a detailed structure-activity relationships (SAR) was performed. Using molecular models of the energy-minimized structures of Nek2-inhibitory complexes, we identify key hydrogen-bonding interactions, including two from the hinge-binding region, likely responsible for the observed affinity. Finally, using cell-based studies, we show that V8 attenuates (a) pAkt/PI3 Kinase signaling in a dose-dependent manner, and (b) proliferative and migratory phenotypes of highly aggressive human MDA-MB-231 breast and A549 lung cancer cell lines. Thus, V8 is an important novel lead compound for the development of highly potent and selective Nek2 inhibitory agents.

Circular forms of dedicator of cytokinesis 1 promotes breast cancer progression by derepressing never in mitosis related kinase 2 via sponging miR-128-3p.

The conjecture of breast cancer is uncertain because of its explosive growth and the complicated molecular mechanisms. Circular RNAs (circRNAs) are regulatory RNA sequences present in the genome and their regulatory mechanism involves the sponging of microRNAs (miRNAs). In this study, we explored the regulation between circular forms of dedicator of cytokinesis 1 (circDOCK1) (hsa_circ_0007142) and miR-128-3p, and its implication on the pathogenesis of breast cancer modulated by never in mitosis (NIMA) related kinase 2 (NEK2). We revealed an increase in circDOCK1 and NEK2 expression, and a decrease in miR-128-3p expression in breast cancer tissues and cell lines. Bioinformatics analysis and experimental validation indicated a positive correlation between circDOCK1 and NEK2 expression but a negative correlation was recorded between miR-128-3p and circDOCK1 or NEK2, respectively. Furthermore, inhibition of circDOCK1 expression was followed by an increase in miR-128-3p and a decrease in NEK2 levels in vitro and in vivo. The luciferase assay concluded that miR-128-3p was a direct target of circDOCK1 while NEK2 was the direct target of miR-128-3p. Furthermore, circDOCK1 inhibition hindered breast cancer development by repressing NEK2 and thus promoting the increased expression of miR-128-3p both in vitro and in vivo. We therefore conclude that circDOCK1 promotes breast cancer progression by targeting miR-128-3p-mediated downregulation of NEK2 and that the circDOCK1/hsa-miR-128-3p/NEK2 axis may be a novel therapeutic target for breast cancer treatment.