NIMA-related Kinase 2 Research Articles

Circular RNA circPITX1 knockdown inhibits glycolysis to enhance radiosensitivity of glioma cells by miR-329-3p/NEK2 axis

BackgroundNumerous circular RNAs (circRNAs) have been recognized as vital modulators of human malignancies, including glioma. Whereas, the functional role of circRNA Pituitary Homeo Box 1 (circPITX1) in the radioresistance of glioma cells remains largely uncertain.MethodsQuantitative real-time PCR (qRT-PCR) or western blot analysis was employed to examine the expression of circPITX1, microRNA (miR)-329-3p and NIMA-related kinase 2 (NEK2). 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to determine cell viability. Glycolysis was assessed by commercial kits and western blot analysis. Colony formation assay was conducted to analyze cell survival and clonogenicity capacity. The relationship among circPITX1, miR-329-3p and NEK2 was confirmed via dual-luciferase reporter assay. The in vivo function of circPITX1 was evaluated by tumor xenograft assay.ResultsExpression of circPITX1 and NEK2 was up-regulated in glioma tissues and cells, while miR-329-3p exhibited reverse trend. CircPITX1 knockdown repressed viability, glycolysis and colony formation, but promoted radiosensitivity of glioma cells, as well as inhibited tumor growth in vivo. MiR-329-3p was a target miRNA of circPITX1 and miR-329-3p deficiency reversed knockdown of circPITX1-mediated glycolysis inhibition and radioresistance reduction. MiR-329-3p exerted inhibitory effects on glycolysis and radioresistance of glioma cells by targeting NEK2. CircPITX1 facilitated NEK2 expression by sponging miR-329-3p. Glycolytic inhibitor 2-deoxy-d-glucose (2-DG) disposition weakened the promoted impact on glycolysis caused by circPITX1.ConclusionCircPITX1 knockdown reduced glycolysis to contribute to radiosensitivity in glioma through miR-329-3p/NEK2 axis, providing a possible mechanism of circPITX1 in the development of glioma.

Molecular characterization and expressional modulation of IRAK1 as downstream signaling adaptor molecule of TLR-signaling pathways in Labeo rohita following PAMPs stimulation and bacterial infections

Interleukin-1 receptor associated kinase (IRAK1) is one of the crucial signal transduction mediators in TLR/IL-1R signaling pathways in host immune system. To investigate about it in rohu (Labeo rohita), one of the economically important freshwater fish species in the Indian subcontinent, we cloned, characterized and analyzed its expression following bacterial infection and pathogens associated molecular patterns (PAMPs) stimulation. The full-length cDNA of rohu IRAK1 (LrIRAK1) consisted of 2765 nucleotide (nt) having an ORF of 2115 nt encoding a polypeptide of 704 amino acids (aa) with a molecular mass of 70.4 kDa. Structurally, LrIRAK1 consisted of twenty-nine helix, twelve strands and forty one coils making one N-terminal death domain (19–94 aa) and a central serine threonine kinase catalytic domain (or kinase domain) (188-489aa). In addition to these two prominent domains, LrIRAK1 also contained highly conserved amino acids viz., lysine 215 and aspartic acid 314 and threonine 185, 361 which were reported to be important for kinase and phosphorylation activity respectively in other animals. Similar to higher vertebrates, LrIRAK1 also consisted of CDK1 (cyclin-dependent kinase1) at 338–352 aa; NEK2 (NIMA-related kinase 2) at 47–61 aa; NEK6 (NIMA-related kinase 6) at 581–595 aa and AMPK (AMP- activated protein kinase) motif at 518–538 aa. Phylogenetically, LrIRAK1 is closely related to cave fish, common carp exhibiting high similarity (~95%) and identity (~90%). In the uninfected fish, the LrIRAK1 expression was highest in liver (~11.5 fold) and lowest in blood. In response to Aeromonas hydrophila, Edwardsiella tarda and Bacillus subtilis infection and various TLR and NLR-ligands stimulation, the expression of LrIRAK1 was markedly enhanced at various time points in almost all the tested tissues. These results together suggest the key role of LrIRAK1 in pattern recognition receptors (PRRs)-mediated host defense against pathogenic insults.

Integrative Bioinformatics Analysis Identifies NEK2 as a Potential Biomarker in Head and Neck Squamous Cell Carcinoma.

The purpose of this study is to explore the function and regulatory networks of differentially expressed genes (DEGs) in head and neck squamous cell carcinoma (HNSC) by bioinformatics analysis using bioinformatics methods. The gene expression profile associated with HNSC was upregulated from the Cancer RNA-Seq Nexus (CRN) database and Oncomine. Functional analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology was performed on DEGs. The protein-protein interaction (PPI) network of DEGs was constructed using STRING and Cytoscape databases. A PPI network contained 101 genes. According to the comprehensive analysis of Degree and MCODE score, there are 12 genes. Based on the screened protein network and differential genes, further exploration was conducted according to The Cancer Genome Atlas (TCGA) normal and the genotype-tissue expression (GTEx) data. This investigation involved observing the expression of the first eight hub genes in tissues and the expression of NIMA-related kinase 2 (NEK2) in different stages and then survival analysis of HNSC. Results showed that NEK2 has important value in HNSC, which provides a reference for the molecular mechanism of HNSC.

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

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

NIMA related kinase 2 promotes gastric cancer cell proliferation via ERK/MAPK signaling.

BACKGROUNDNIMA related kinase 2 (NEK2) is closely related to mitosis, and it is currently considered to be over-expressed frequently in many poorly prognostic cancers. However, the effect of the up-regulated NEK2 on cellular signaling in tumors, such as gastric cancer (GC), is con-fusing.AIMTo determine the role of the up-regulation of NEK2 in GC.METHODSTo investigate the pathological significance of NEK2 in GC, the expression pattern of NEK2 in GC was investigated based on the “Oncomain” database and compared between 30 pairs of cancer samples and adjacent tissues. The co-expression of NEK2 and ERK in GC was analyzed using The Cancer Genome Atlas (TCGA) database and confirmed in clinical samples by quantitative real-time PCR (qRT-PCR), and the survival curve was also plotted. Western blot or qRT-PCR was used to analyze the effect of NEK2 on the phosphorylation levels of ERK and c-JUN in two GC cell lines (BGC823 and SGC7901) with NEK2 overexpression, and the expression of the downstream effector cyclin D1. Furthermore, CCK8, EdU incorporation assay, and flow cytometry were used to detect the proliferative ability of BGC823 and SGC7901 cells with stably silenced ERK.RESULTSNEK2 was significantly up-regulated in human GC tissues. ERK was significantly associated with NEK2 expression in human clinical specimens, and combined overexpression of NEK2 and ERK potentially forecasted a poor prognosis and survival in GC patients. NEK2 knockdown in GC cells inhibited ERK and c-JUN phosphory-lation and reduced the transcription of cyclin D1. More interestingly, NEK2 can rescue the inhibition of cellular viability, proliferation, and cell cycle progression due to ERK knockdown.CONCLUSIONOur results indicate that NEK2 plays a carcinogenic role in the malignant proliferation of GC cells via the ERK/MAPK signaling, which may be important for treatment and improving patient survival.

MicroRNA-138 induces cell survival and reduces WNT/β-catenin signaling of osteoarthritis chondrocytes through NEK2.

Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain, stiffness, and function degeneration with high incidence. Recent studies have been inspired based on the association between microRNAs (miRs) and therapeutic research of OA. Hence, the present study evaluates the effects of miR-138 on chondrocyte proliferation, differentiation, and apoptosis through the WNT/β-catenin signaling pathway in mice with OA by binding to NIMA-related kinase 2 (NEK2). Appropriate dataset was selected from the Gene Expression Omnibus database, and differentially expressed genes and potential miRNAs that could regulate NEK2 were explored. A mouse model of OA was established. The expressions of miR-138, NEK2, β-catenin, GSK3β, Bcl-2, Bcl-2-associated X protein (Bax), p53, MMP-13, Col2, and Aggrecan and the phosphorylation levels of β-catenin were determined by the reverse transcription quantitative polymerase chain reaction and Western blot analysis. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry were employed to detect cell proliferation and apoptosis, respectively. The potential functional role of NEK2 was revealed to be related to the WNT/β-catenin signaling pathway, and miR-138 was the putative regulator of NEK2. miR-138 expression was downregulated while expressions of NEK2 and β-catenin as well as the phosphorylation levels of β-catenin were upregulated in mice with OA. The chondrocytes treated with miR-138 mimic and siRNA-NEK2 exhibited reduced expressions of NEK2, β-catenin, MMP-13, Bax, and p53 and elevated expressions of Col2, Aggrecan, and Bcl-2 as well as phosphorylation levels of β-catenin along with enhanced chondrocytes' proliferation and suppressed cell apoptosis. Overexpression of miR-138 induces cell survival and reduces WNT/β-catenin signaling of OA chondrocytes through NEK2. © 2019 IUBMB Life, 71(9):1355-1366, 2019.

Abstract P5-03-01: LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer

Abstract Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. TNBC initially respond well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Paclitaxel causes defects in mitotic spindle formation and centrosome function, as well as improper chromosome segregation leading to cell death. While being some of the most effective drugs for this disease, taxanes are associated with high toxicity. Thus discovering new therapeutic targets that are selective for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and associated with poor survival. We further found that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across nine breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, enforced overexpression of LIN9 increases multi- and micronucleation, indicators of chromosomal instability. Conversely, LIN9 silencing also results in multi- and micronucleation, and supernumerary centrosomes. Most importantly, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and MDA-MB-468) resistance. We have previously reported that treating TNBC cells with Bromodomain and ExtraTerminal protein inhibitors (BETi) reduces LIN9 expression, thus we determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, and paclitaxel caused a greater induction of apoptosis compared to either drug alone. Dual treatment also resulted in a potentiation of abnormal centrosomes, multinucleation, and micronucleation compared to that caused by either BETi or JQ1 alone. To identify the mechanism(s) by which genetic or therapeutic suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs in MDA-MB-231 and HCC70 cell lines. We further narrowed the list of candidate LIN9 targets by identifying genes that were bound by LIN9 in a published ChIP-Seq dataset from HeLa cells whose expression is also correlated with LIN9 expression and associated with reduced breast cancer patient survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis as a potential mediator of the effects of LIN9 suppression. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor survival. In addition, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by contributing to centrosome dysfunction through upregulation of NEK2. They also indicate that targeting LIN9 expression in addition to paclitaxel treatment may be a viable therapeutic approach for TNBC patients. Citation Format: Shively MS, Gayle SS, Sahni JM, Keri RA. LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-01.

Prognostic significance of NEK2 in human solid tumors: a systematic review and meta-analysis.

A consensus about the prognostic role of NIMA-related kinase 2 (NEK2) expression in various solid tumors has not been made yet. Thus, this meta-analysis aimed to systematically assess the prognostic role of NEK2 expression in patients with solid tumors. The eligible studies were identified through searching PubMed, Web of Science, and EMBASE. The hazard ratios (HRs) with their corresponding 95% confidence intervals (CIs) were used to evaluate the link between NEK2 overexpression and overall survival (OS) and disease-free survival/recurrence-free survival (DFS/RFS) of patients with solid tumors. A total of 17 studies with 4897 patients were included in this meta-analysis. Among these studies, all of them explored the association between NEK2 expression and OS of patients with solid tumors. Our pooled analysis indicated that NEK2 overexpression was significantly related to adverse OS (HR = 1.66; 95% CI: 1.38–2.00; P = 0.001). Additionally, there were six studies with 854 patients that investigated the association between NEK2 expression and DFS/RFS. Our pooled result indicated that there was a substantial relationship between NEK2 overexpression and poorer DFS/RFS (HR = 2.00; 95% CI: 1.61–2.48; P = 0.003). In conclusion, our meta-analysis indicated that NEK2 may be a useful predictor of prognosis and an effective therapeutic target in solid tumors. Nevertheless, more high-quality studies are warranted to further support our conclusions because of several limitations in our meta-analysis.

MiR-1299 functions as a tumor suppressor to inhibit the proliferation and metastasis of prostate cancer by targeting NEK2.

The aim of this study was to investigate the inhibitory role of microRNA-1299 (miR-1299) in prostate cancer, and to explore the possible underlying mechanism. The expression of miR-1299 in 35 PCa tissues and para-carcinoma tissues, as well as PCa cell lines (PC-3) and prostatic epithelial cell line (RWPE-1), was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Then, we explored the possible targets of miR-1299 by searching online databases. NIMA-related kinase 2 (NEK2) was identified as a direct target gene of miR-1299. Subsequently, qRT-PCR, Western blot (WB), and luciferase reporter gene assay were used to further verify the correlation between miR-1299 and NEK2. To better characterize the role of miR-1299 and NEK2 in PCa, we conducted functional experiments (MTT, flow cytometry, scratch-wound, and transwell assay) by transfecting PC-3 cells with miR-1299 mimics and si-NEK2 in different groups. The expression level of miR-1299 in PCa tissues was significantly lower than that of para-carcinoma tissues. Meanwhile, the expression of miR-1299 in PC-3 cells was also significantly downregulated when compared with RWPE-1 cells. Subsequent qRT-PCR, WB, and luciferase reporter gene assay verified that miR-1299 transcriptionally repressed NEK2 by interacting with the essential binding sequence in 3'-UTR. Also, functional experiments demonstrated that decreased expression of NEK2 resulting from miR-1299 up-regulation could remarkably inhibit the proliferation, invasion, and migration of PCa cells. Our study indicated that miR-1299 was a novel suppressor in PCa through its negative regulation of NEK2. Moreover, our findings revealed that miR-1299/NEK2 axis might be a potential therapeutic target for the treatment of PCa.

NIMA-related kinase 2 overexpression is associated with poor survival in cancer patients: a systematic review and meta-analysis.

ObjectiveNIMA-related kinase 2 (NEK2) has been reported to be overexpressed in various types of cancer and correlated with poor prognosis. The role(s) of NEK2 in cancer, however, is still uncertain. The aim of this study was to evaluate the prognostic value of NEK2 in human tumors.MethodsA comprehensive literature search was performed for PubMed, Embase, Web of Science, and CNKI databases, and eligible studies were included based on the inclusion and exclusion criteria. A meta-analysis of the included studies was then carried out.ResultsFifteen studies with 3,280 cancer patients were included in the present meta-analysis. All publications were of moderate to high quality, and had no significant heterogeneity (I2=46%, P=0.03) or publication bias was discovered. The results showed that a high NEK2 level was associated with shorter overall survival (OS) in patients with various types of cancers (pooled HR=1.72, 95% CI 1.49–2.00, P<0.00001). However, the disease-free survival (DFS) had no significant association with NEK2 level (HR=1.13, 95% CI: 0.29–4.38, P=0.85). In the subgroup analyses, high NEK2 level was correlated with an increased risk of poor OS in patients with hepatocellular carcinoma (HR=1.62, 95% CI: 1.25–2.10, P=0.02) and lung cancer (HR=2.18, 95% CI: 1.40–3.38, P=0.0005). However, other factors, including sample size, follow-up period, HR estimation method, and country, also affect the association between NEK2 expression and OS. Analysis of clinicopathological parameters further showed that increased NEK2 level was correlated with younger age, male gender, better tumor differentiation, and lower number of tumor nodules.ConclusionThe results of this study indicated that increased expression of NEK2 was associated with unfavorable survival of cancer patients and that NEK2 could be used as a prognostic predictor for cancers.

MiR-128 promotes the apoptosis of glioma cells via binding to NEK2.

The aim of this study was to explore whether miR-128 could promote the apoptosis of glioma cells by targeting NIMA related kinase-2 (NEK2), thus participating in the occurrence and progression of glioma. Expression levels of miR-128 and NEK2 in glioma tissues and normal brain tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between miR-128 expression, tumor size and stage of glioma was analyzed. The effect of miR-128 on the apoptosis of glioma cells was detected by flow cytometry and Western blot, respectively. Dual-luciferase reporter gene assay was applied to verify the binding condition of miR-128 and NEK2. Meanwhile, rescue experiments were conducted to determine whether miR-128 could promote the apoptosis of glioma cells by targeting NEK2. The expression level of miR-128 in glioma tissues was significantly lower than that of normal brain tissues. However, NEK2 was highly expressed in glioma tissues. MiR-128 expression was correlated to tumor size and malignant level of glioma, whereas not related to age and gender of glioma patients. Meanwhile, overexpression of miR-128 promoted the apoptosis of U87 cells, upregulated protein levels of cleaved Caspase-3 and BCL2-associated X (Bax), and downregulated B-cell lymphoma-2 (Bcl-2). Dual-luciferase reporter gene assay indicated that miR-128 directly bound to NEK2. Further rescue experiments suggested that NEK2 overexpression partially reversed the effect of miR-128 on the apoptosis of glioma cells. Downregulated miR-128 inhibited the apoptosis of glioma cells via targeting NEK2.

Upregulation of FOXM1 leads to diminished drug sensitivity in myeloma

BackgroundFollowing up on previous work demonstrating the involvement of the transcription factor forkhead box M1 (FOXM1) in the biology and outcome of a high-risk subset of newly diagnosed multiple myeloma (nMM), this study evaluated whether FOXM1 gene expression may be further upregulated upon tumor recurrence in patients with relapsed multiple myeloma (rMM). Also assessed was the hypothesis that increased levels of FOXM1 diminish the sensitivity of myeloma cells to commonly used myeloma drugs, such as the proteasome inhibitor bortezomib (Bz) and the DNA intercalator doxorubicin (Dox).MethodsFOXM1 message was evaluated in 88 paired myeloma samples from patients with nMM and rMM, using gene expression microarrays as measurement tool. Sources of differential gene expression were identified and outlier analyses were performed using statistical methods. Two independent human myeloma cell lines (HMCLs) containing normal levels of FOXM1 (FOXM1N) or elevated levels of lentivirus-encoded FOXM1 (FOXM1Hi) were employed to determine FOXM1-dependent changes in cell proliferation, survival, efflux-pump activity, and drug sensitivity. Levels of retinoblastoma (Rb) protein were determined with the assistance of Western blotting.ResultsUpregulation of FOXM1 occurred in 61 of 88 (69%) patients with rMM, including 4 patients that exhibited > 20-fold elevated expression peaks. Increased FOXM1 levels in FOXM1Hi myeloma cells caused partial resistance to Bz (1.9–5.6 fold) and Dox (1.5–2.9 fold) in vitro, using FOXM1N myeloma as control. Reduced sensitivity of FOXM1Hi cells to Bz was confirmed in vivo using myeloma-in-mouse xenografts. FOXM1-dependent regulation of total and phosphorylated Rb agreed with a working model of myeloma suggesting that FOXM1 governs both chromosomal instability (CIN) and E2F-dependent proliferation, using a mechanism that involves interaction with NIMA related kinase 2 (NEK2) and cyclin dependent kinase 6 (CDK6), respectively.ConclusionsThese findings enhanced our understanding of the emerging FOXM1 genetic network in myeloma and provided preclinical support for the therapeutic targeting of the FOXM1-NEK2 and CDK4/6-Rb-E2F pathways using small-drug CDK and NEK2 inhibitors. Clinical research is warranted to assess whether this approach may overcome drug resistance in FOXM1Hi myeloma and, thereby, improve the outcome of patients in which the transcription factor is expressed at high levels.

NEK2 Promotes Hepatoma Metastasis and Serves as Biomarker for High Recurrence Risk after Hepatic Resection

Introduction and aim. Developing reliable biomarkers for hepatocellular carcinoma (HCC) patients who are at a high risk of recurrence after curative hepatic resection is very important for determining subsequent therapeutic strategies. We investigated the role of the cell cycle factor NIMA-related kinase 2 (NEK2) in HCC progression in hepatoma cells and post-surgery patients.Material and methods. The effects of NEK2 on proliferation, invasion and migration of hepatoma HuH7 and SK-Hep1 cells were evaluated. In a post-surgery HCC cohort (N = 97), the Nek2 induction levels in the tumors were examined with real-time RT-PCR analysis, and the results were analyzed for their correlations with recurrence.Results. NEK2 promoted G1 to S phase cell cycle progression by causing increases in cyclin D1 and AKT phosphorylation and decreases in the cyclin-dependent kinase inhibitor p27, indicating that NEK2 plays an important role during interphase in addition to its previously identified role in M phase. NEK2 also enhanced the proliferation, migration and invasion of hepatoma cells and regulated the expression of E-cadherin and MMP9. The Nek2 mRNA levels in the tumors were highly correlated with recurrence rates in the post-surgery HCC patients. Combined evaluation of the tumor AJCC stage and the Nek2level can serve as a reliable method for predicting the relative risk of HCC recurrence in these patients. Conclusions. NEK2 plays a significant role in cell cycle progression in the inter- and M-phases. NEK2 enhances HCC metastasis and is correlated with recurrence and thus can potentially serve a promising high-risk biomarker for HCC.

The Prognostic Significance of NEK2 in Hepatocellular Carcinoma: Evidence from a Meta-Analysis and Retrospective Cohort Study

Background/Aims: Numerous studies have shown that NIMA-related kinase 2 (NEK2) expression in hepatocellular carcinoma (HCC) tissue is associated with survival and clinicopathological features; however, the evidence remains inconclusive. Thus, we aimed to further explore the prognostic and clinicopathological significance of NEK2 expression in HCC using a two-part study consisting of a retrospective cohort study and a meta-analysis. Methods: In the cohort study, NEK2 expression in 206 HCC samples and adjacent normal liver tissues was detected by immunohistochemistry (IHC). Patients were divided into a high NEK2 expression group and a low NEK2 expression group by the median value of the immunohistochemical scores. The Kaplan–Meier method with the log-rank test was used to analyze survival outcomes in the two groups, and multivariate analysis based on Cox proportional hazard regression models was applied to identify independent prognostic factors. In the meta-analysis, eligible studies were searched in PubMed, EMBASE, Web of Science, and CNKI databases. STATA version 12.0 (Stata Corporation, College Station, TX) was used for statistical analyses. Results: The IHC results of our cohort study showed higher NEK2 expression in HCC tissues compared with adjacent normal liver tissues. Multivariate analysis revealed that high NEK2 expression was an independent risk factor for poor overall survival (OS) [hazard ratio (HR) = 1.763; 95% CI, 1.060–2.935; P = 0.029] and disease-free survival (DFS) [hazard ratio (HR) = 1.687; 95% CI, 1.102–2.584; P = 0.016] in HCC patients. A total of 11 studies with 1,698 patients were enrolled in the meta-analysis, consisting of 10 studies from the database search and our cohort study. The pooled results revealed that high NEK2 expression correlated closely with poor OS among HCC patients (HR = 1.47; 95% CI, 1.21–1.80; P < 0.01), and DFS/recurrence-free survival (RFS) (HR = 1.92; 95% CI, 1.41–2.63; P < 0.01). Additionally, our meta-analysis also showed that the proportion of HCC patients with high NEK2 expression was greater in the group with larger tumors (> 5 cm) than in the group with smaller tumors (≤ 5 cm) [odds ratio (OR) = 2.02; 95% CI, 1.13–3.64; P < 0.01). Conclusion: Our study demonstrated that high NEK2 expression is a risk factor for poor survival in HCC patients. More prospective, homogeneous, and multiethnic studies are required to validate our findings.

Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2.

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity-dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Low expression of NEK2 is associated with hepatocellular carcinoma progression and poor prognosis.

NIMA-related kinase 2 (NEK2), a serine/threonine kinase, is located in the centrosome and is a member of cell cycle regulation related protein kinase (CCRK) family. Aberrant expression of NEK2 is linked with carcinogenesis and progression of various tumors. To investigate the expression level of NEK2 and its relationship with clinicopathological factors in hepatocellular carcinoma (HCC). Immunohistochemistry was used to measure the expression of NEK2 in 310 patients' specimen tissues and 197 adjacent normal liver tissues of HCC cases, and the subsequent prognostic value for each sample was estimated. NEK2 expression levels in HCC were lower than in adjacent tissues (49.7% vs. 72.6%, P< 0.001). First, patients with relatively low NEK2 expression had increased cancer progression and poorer prognosis than those with high expression. Second, NEK2 expression was significantly reduced in patients with large tumors (P= 0.025), with stage III Edmondson-Steiner Grading (P= 0.015). Third, patients' tumor size positively correlated with high AFP concentration (P= 0.017). Fourth, using the Kaplan-Meier survival curve, we found a lower survival rate in patients with decreased expression of NEK2 than those with high NEK2 expression in HCC (P= 0.029, Log-rank test). Low NEK2 expression might be a useful predictor in HCC as a poor prognostic factor, and could serve as a potential therapeutic target for HCC.

Pre-clinical analysis of the CDK4/6 inhibitor palbociclib in HER2-positive breast cancer.

e12520 Background: There has been substantial progress in treatment of metastatic HER2+ breast cancer, unfortunately, treatment progression of the disease is universal. Palbociclib is an oral cyclin-dependent kinase (CDK) 4/6 inhibitor that is FDA approved in combination with endocrine therapy for treatment of patients with ER+ metastatic breast cancer. Here we present pre-clinical studies in HER2+ breast cancer cell lines investigating whether addition of palbociclib to the combination of trastuzumab and pertuzumab (TP) is an effective therapy, and whether we could identify predictive biomarkers for palbociclib in this setting. Methods: Crystal violet assays to measure growth of BT-474 (HER2+, ER+), SKBr3 (HER2+, ER-) and MDA-MB-361 (HER2+, ER+ but resistant to TP) were performed in the presence and absence of TP (5 µg/ml each) with increasing concentrations of palbociclib (25-500 µg/ml). Western blots were performed in the presence and absence of TP with and without palbociclib to demonstrate efficacy of TP (blocking phosphorylation of HER2), palbociclib (blocking phosphorylation of Rb), and expression of NIMA-related kinase 2 (NEK2), an established downstream target of CDK4 in HER2+ breast cancer cells. Results: We found growth inhibition in response to palbociclib as single agent in a dose-dependent manner in BT-474, SKBr3, and MDA-MB-361 cells (P &lt; 0.05). Treatment with TP prevented growth in BT-474 and SKBr3 cells to such a great extent that an additive effect between TP and palbociclib was not detectable. However, treatment with palbociclib maintained a dose-dependent decrease in growth in the presence of TP in MDA-MB-361 cells (P &lt; 0.05). Western blots showed decreased expression of pHER2 in response to TP in BT-474 and SKBr3, but not in MDA-MB-361. Treatment with palbociclib decreased pRB in BT474, SKBr3, and MDA-MB-361 with or without TP. NEK2 expression, however, was not affected in any of these cell lines. Conclusions: Palbociclib has an inhibitory effect in resistant and non-resistant HER2-positive cell lines suggesting palbociclib represents an alternate targeting pathway for growth inhibition. Our initial work did not show reliable biomarkers to predict palbociclib effects.

MicroRNA-128 promotes apoptosis in lung cancer by directly targeting NIMA-related kinase 2.

BackgroundMicroRNA‐128 (miR‐128) serves as a regulator by inducing cancer cell apoptosis, differentiation, the epithelial‐to‐mesenchymal transition process, and tumor growth by mediating different targets. NIMA‐related kinase 2 (NEK2) is aberrantly expressed in lung cancer. The miR‐128/NEK2 pathway has been reported to predict prognosis in colorectal cancer; however, the determination of a relationship between miR‐128 and NEK2 in lung cancer has remained elusive. We explored the association between miR‐128 and NEK2 in lung cancer.MethodsMiR‐128 and NEK2 expression were examined in 15 lung cancer tissues by real time‐PCR. Lung cancer SK‐MES‐1 cells were transfected with miR‐128 mimic, an inhibitor or a negative control. MiR‐128 and NEK2 expression levels were detected using quantitative real time‐PCR and Western blot. SK‐MES‐1 cell apoptosis was performed by flow cytometry.ResultsCompared to adjacent non‐tumor tissues, miR‐128 was downregulated and NEK2 was upregulated in 15 lung cancer tissues. Lung cancer SK‐MES‐1 cells transfected with miR‐128 mimic induced a higher apoptotic rate than those transfected with the negative control. Dual luciferase assay further confirmed that NEK2 was a direct target of miR‐128 in lung cancer, and transfection with miR‐128 mimic could decrease the NEK2 protein level while the miR‐128 inhibitor increased NEK2 expression. Finally, the apoptotic effect of lung cancer cells induced by miR‐128 mimic could be reversed by NEK2 overexpression.Conclusions NEK2 was regulated by miR‐128 in lung cancer and miR‐128 induced lung cancer cell apoptosis by mediating NEK2 expression.

Deguelin inhibits epithelial-to-mesenchymal transition and metastasis of human non-small cell lung cancer cells by regulating NIMA-related kinase 2.

BackgroundNon‐small cell lung cancer is a lethal malignancy with a high mortality rate. Deguelin displays an anti‐tumor effect and inhibits metastasis in various cancers. The aberrant expression of NIMA‐related kinase 2 (NEK2) indicates poor prognosis and induces epithelial‐to‐mesenchymal transition (EMT) and metastasis processes. However, the underlying mechanism between deguelin and NEK2 has remained elusive.Methods NSCLC cell lines were treated with deguelin. Wound‐healing and invasion assays were applied to study the inhibitory effect of deguelin on NSCLC cells. EMT markers, E‐cadherin and Vimentin, were also detected by Western blot. NEK2 protein and messenger RNA expression levels were evaluated when NSCLC cells were treated with different concentrations of deguelin. The effect of NEK2 on NSCLC cell metastasis was evaluated through NEK2 knockdown. To investigate whether deguelin induced EMT by regulating NEK2, we overexpressed NEK2 in both NCI‐H520 and SK‐MES‐1 cell lines, and then used real time‐PCR to study the E‐cadherin and Vimentin messenger RNA expression in both NSCLC cells.ResultsDeguelin inhibited migration and invasion processes in NSCLC cell lines and decreased NEK2 expression in a concentration‐dependent manner. Furthermore, NEK2 knockdown inhibited NSCLC cell migration and invasion. Finally, overexpressing NEK2 in NCI‐H520 and SK‐MES‐1 cells could restore the inhibition of metastasis induced by deguelin.ConclusionsDeguelin could inhibit EMT and metastasis, while overexpression of NEK2 promotes these processes. Deguelin could decrease NEK2 expression, while NEK2 overexpression could restore deguelin‐induced inhibition of metastasis.

Overexpression of NIMA-related kinase 2 is associated with poor prognoses in malignant glioma.

Eleated expression of NIMA-related kinase 2 (NEK2) was frequently observed in a variety of malignant cancers, and it appears to be involved in the initiation, maintenance, progression, metastasis of cancer and is positively associated with poor prognosis. We sought to investigate NEK2 expression and its predictive roles in malignant gliomas, and study the correlation of NEK2 protein expression with proliferation, clinical parameters, overall survival and some other parameters. We investigate NEK2 protein expression in 99 samples of malignant gliomas, including 35 WHO grade II, 22 grade III, and 42 grade IV gliomas, by immunohistochemistry and western blot (n = 50). We then made correlative analysis of protein overexpression using the Kaplan-Meier method, Log rank test, and Cox proportional-hazards model analysis. NEK2 protein was overexpressed in malignant gliomas, but not in normal brain tissues. Overexpression of NEK2 correlated with malignancy, proliferation and adverse overall survival in gliomas. Moreover, chemotherapy, resection extent and WHO grade also correlate with overall survival in gliomas. However, within WHO grade II glioma subgroup, NEK2 overexpression showed no impact on overall survival. The present study firstly reveals that NEK2 protein is widely overexpressed in gliomas. NEK2 overexpression correlates significantly with malignancy (WHO grades), proliferation (Ki-67) and prognosis in malignant gliomas. NEK2 is a potential gene therapy target and prognostic indicator.