Decreased Nek9 expression correlates with aggressive behaviour and predicts unfavourable prognosis in breast cancer.

As a new member of Neks family, Nek9 regulates spindle assembly and controls chromosome alignment and centrosome separation. In the current study we aimed to investigate the expression of Nek9 in breast cancer and its clinical significance. We evaluated the expression of Nek9 in invasive ductal carcinoma (IDC, n=316), ductal carcinoma in situ (DCIS), usual ductal hyperplasia, atypical ductal hyperplasia, fibroadenoma and normal breast tissues using immunohistochemistry. The results revealed significantly reduced Nek9 in IDCs (41.8%) compared to benign breast lesions. Moreover, gradually reduced Nek9 was found from DCIS to invasive carcinoma and metastatic tumour within the same tumours. The decrease in Nek9 expression was associated with larger tumour size (p=0.0087), high grade (p<0.0001) and high Ki-67 index (p<0.0020). TCGA and GEO datasets analysis revealed low level of Nek9 mRNA was more frequent in triple negative breast cancers, and associated with poor overall survival and distant metastasis-free survival. These findings suggest an important role of Nek9 in the progression of breast cancer, and aberrantly expressed Nek9 correlates with more aggressive clinicopathological variables and predicts poor clinical prognosis. Nek9 may serve as a potential predictive factor for patients with breast cancer.

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.

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase.

BACKGROUND: Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP. METHODS: Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP. RESULTS: In this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors  to validate this new luminescence method. CONCLUSION: With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

NEK2 serves as a prognostic biomarker for hepatocellular carcinoma.

Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is a microtubule-associated protein that regulates spindle assembly in human cells and is overexpressed in various malignancies. However, the role of NEK2 in hepatocellular carcinoma (HCC) remains undetermined. We performed RNA-seq of the HCC cell line SMMC-7721 and the normal liver cell line HL-7702 using the Ion Proton System. NEK2 expression was detected using quantitative reverse transcription polymerase chain reaction in two cell lines and 5 matched HCC and adjacent non-tumorous liver tissues. The correlation between survival and NEK2 expression was analyzed in 359 patients with HCC using RNASeqV2 data available from The Cancer Genome Atlas (TCGA) website (https://tcga-data.nci.nih.gov/tcga/). The expression of NEK2, phospho-AKT and MMP-2 was evaluated by immunohistochemistry in 63 cases of HCC and matched adjacent non-tumorous liver tissues. Relationships between protein expression and clinicopathological parameters were assessed, and the correlations between NEK2 with phospho-AKT and MMP-2 expressions were evaluated. A total of 610 differentially expressed genes (DEGs) were revealed in the transcriptome comparison, 297 of which were upregulated and 313 were downregulated in HCC. NEK2, as the most obviously different DEG in cells and tissues from the RNA-seq data, was listed as an HCC candidate biomarker for further verification. NEK2 was overexpressed in HCC cells and tissues (P=0.002, P=0.013) and HCC patients with a high expression of NEK2 had a poor prognosis (P=0.0145). Clinical analysis indicated that the overexpression of NEK2 in HCC was significantly correlated with diolame complete (P<0.001), tumor nodule number (P=0.012) and recurrence (P=0.004). NEK2 expression was positively correlated with the expression of phospho-AKT (r=0.883, P<0.01) and MMP-2 (r=0.781, P<0.01). Overexpression of NEK2 was associated with clinicopathological characteristics and poor patient outcomes, suggesting that NEK2 serves as a prognostic biomarker for HCC. Alteration of NEK2 protein levels may contribute to invasion and metastasis of HCC, which may occur through activation of AKT signaling and promotion of MMP-2 expression.

Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes.

Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes.