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Objective: Cerebral ischemia–reperfusion injury (CIRI) is a major obstacle to neurological recovery after clinical treatment of ischemic stroke. The aim of this study was to investigate the molecular mechanism of Nek6 alleviating CIRI through autophagy after cerebral ischemia. Materials and methods: A mouse model of CIRI was constructed by middle cerebral artery occlusion (MCAO). TUNEL staining was used to observe the apoptosis of neuronal cells. The oxygen glucose deprivation/reoxygenation (OGD/R) model was established by hypoxia and reoxygenation. The cell apoptosis and activity was detected. Western blot was performed to detect the expression of autophagy-related proteins, protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine 5'-monophosphate-activated protein kinase (AMPK)/mTOR signaling pathway-related proteins. Cellular autophagy flux was observed by fluorometric method. NIMA-related kinase 6 (Nek6) mRNA stability was detected by actinomycin D treatment. Methylation RNA immunoprecipitation technique was used to detect Nek6 methylation level. Results: Nek6 expression was increased in both MCAO and OGD/R models. Overexpression of Nek6 in OGD/R inhibited apoptosis, decreased LC3II and Beclin-1 expression, increased p62 expression, and occurred lysosome dysfunction. Interference with Nek6 has opposite results. Nek6 overexpression promoted p-Akt and p-mTOR protein expressions, inhibited p-AMPK and p-UNC-51-like kinase 1 protein expressions and cell apoptosis, while LY294002, Rapamycin or RSVA405 treatment reversed this effect. Abnormal methyltransferase·like protein 3 (METTL3) expression in CIRI enhanced m6A modification and promoted Nek6 expression level. Conclusion: This study confirmed that Nek6 regulates autophagy and alleviates CIRI through the mTOR signaling pathway, which provides a novel therapeutic strategy for patients with ischemic stroke in the future. [ABSTRACT FROM AUTHOR]

The NEK6 (NIMA-related kinase 6) serine/threonine kinase is a pivotal player in a multitude of cellular processes, including the regulation of the cell cycle and the response to DNA damage. Its significance extends to disease pathogenesis, as changes in NEK6 activity have been linked to the development of cancer. Non-synonymous single nucleotide polymorphisms (nsSNPs) in NEK6 have been linked to cancer as they alter the protein's native structure and function. The association between NEK6 activity and cancer development has prompted researchers to explore the effects of genetic variations within the NEK6 gene. Therefore, we utilized advanced computational tools to analyze 155 high-confidence nsSNPs in the NEK6 gene. From this analysis, 21 nsSNPs were identified as potentially harmful, raising concerns about their impact on NEK6 activity and cancer risk. These 21 mutations were then examined for structural alterations, and eight of nsSNPs (I51M, V76A, I134N, Y152D, R171Q, V186G, L237R, and C285S) were found to destabilize the protein. Among the destabilizing mutations screened, a specific mutation, R171Q, stood out due to its conserved nature. To understand its impact on the protein and conformation, all-atom molecular dynamics simulations (MDS) for 100 ns were performed for both Wildtype NEK6 (WT-NEK6) and R171Q. The simulations revealed that the R171Q variant was unstable and led to significant conformational changes in NEK6. This study provides valuable insights into NEK6 dysfunction caused by single amino acid alterations, offering a novel understanding of the molecular mechanisms underlying NEK6-related cancer progression. [ABSTRACT FROM AUTHOR]

Min Zhu,1 Yuyu Sun,1 Huawei Xue,1 Gang Wu,1 Zhen Wang,1 Junfeng Shi,1 Jiye Ma,1 Baorong Gu,1 Xiaoling Yan2 1Department of Spine Surgery, Nantong Third People’s Hospital, Affiliated Nantong Hospital 3 of Nantong University, Nantong, People’s Republic of China; 2Department of Chemotherapy, Affiliated Hospital of Nantong University, Nantong, People’s Republic of ChinaCorrespondence: Xiaoling Yan, Department of Chemotherapy, Affiliated Hospital of Nantong University, Nantong, 226001, People’s Republic of China, Email 5200007@ntu.edu.cnBackground: Osteosarcoma is a malignant tumor originating from the skeletal system. There is no effective treatment other than surgery and chemotherapy, which seriously endangers the health of children and adolescents. NEK6 is a novel discovered Serine/Threonine protein kinase that can regulate cell cycle and activate several oncogenic pathways.Methods: NEK6 expression in pan-cancer including sarcoma was evaluated using analysis tools of TIMER, UALCNA and GEPIA with TCGA database, and its association with overall survival in patients with sarcoma was also analyzed. TargetScan, tarbase, microT-CDS and Starbase online software were used to predict NEK6-targeted miRNAs, including miR-26a-5p. Tumor tissues from patients with osteosarcoma were collected for NEK6 and miRNA detection using RT-qPCR. NEK6 down-regulated by siRNAs or miR-26a-5p in osteosarcoma cells was detected by RT-qPCR, Western blot and Immunofluorescence staining assays. Effects of NEK6 knockdown on proliferation, migration, invasion and apoptosis of osteosarcoma cells were detected by CCK-8, wound healing, transwell and flow cytometry, respectively. The expressions of STAT3, metastasis and apoptosis-related genes were detected by Western blot.Results: High expression of NEK6 and low expression of miR-26a-5p were lowly expressed in osteosarcoma and they were negative correlation. NEK6 has been confirmed as a direct target for miR-26a-5p. In addition, NEK6 down-regulated by siRNAs or miR-26a-5p led to inhibition of cell proliferation, migration and invasion while promoting cell apoptosis. The levels of phosphorylated STAT3 and metastasis genes (MMP-2, MMP-9) were inhibited, while apoptotic gene Bax was promoted and Bcl2 was inhibited by miR-26a-5p upregulation.Conclusion: NEK6 can promote osteosarcoma progression via activating STAT3 signaling pathway, which is inhibited by miR-26a-5p, suggesting that NEK6 is a potential oncogene and miR-26a-5p is a suppressor of osteosarcoma. The strategy of inhibiting of NEK6 by miR-26a-5p may be an effective approach for osteosarcoma therapy.Keywords: osteosarcoma, NEK6, miR-26a-5p, STAT3 signaling pathway

Recently macrophage polarization has emerged as playing an essential role in the oathogenesis of atherosclerosis, which is the most important underlying process in many types of cardiovascular diseases. Although Nek6 has been reported to be involved in various cellular processes, the effect of Nek6 on macrophage polarization remains unknown. Macrophages exposed to lipopolysaccharide (LPS) or IL‐4 were used to establish an in vitro model for the study of regulation of classically (M1) or alternatively (M2) activated macrophage. Bone marrow‐derived macrophages (BMDMs) transfected with short hairpin RNA‐targeting Nek6 were then in functional studies. We observed that Nek6 expression was decreased in both peritoneal macrophages (PMs) and BMDMs stimulated by LPS. This effect was seen at both mRNA and protein level. The opposite results were obtained after administration of IL‐4. Macrophage‐specific Nek6 knockdown significantly exacerbated pro‐inflammatory M1 polarized macrophage gene expression in response to LPS challenge, but the anti‐inflammatory response gene expression that is related to M2 macrophages was attenuated by Nek6 silencing followed by treatment with IL‐4. Mechanistic studies exhibited that Nek6 knockdown inhibited the phosphorylated STAT3 expression that mediated the effect on macrophage polarization regulated by AdshNek6. Moreover, decreased Nek6 expression was also observed in atherosclerotic plaques. Collectively, these evidences suggested that Nek6 acts as a crucial site in macrophage polarization, and that this operates in a STAT3‐dependent manner. [ABSTRACT FROM AUTHOR]

NIMA (never in mitosis, gene A)-related kinase-6 (NEK6), a cell cycle regulatory gene, was found to regulate cardiac hypertrophy. However, its role in diabetes-induced cardiomyopathy has not been fully elucidated. This research was designed to illustrate the effect of NEK6 involved in diabetic cardiomyopathy. Here we used a streptozotocin (STZ)-induced mice diabetic cardiomyopathy model and NEK6 knockout mice to explore the role and mechanism of NEK6 in diabetic-induced cardiomyopathy. NEK6 knockout mice and wild-type littermates were subjected to STZ injection (50 mg/kg/day for 5 days) to induce a diabetic cardiomyopathy model. As a result, 4 months after final STZ injection, DCM mice revealed cardiac hypertrophy, fibrosis, and systolic and diastolic dysfunction. NEK6 deficiency causes deteriorated cardiac hypertrophy, fibrosis, and cardiac dysfunction. Furthermore, we observed inflammation and oxidative stress in the hearts of NEK6 deficiency mice under diabetic cardiomyopathy pathology. Adenovirus was used to upregulate NEK6 in neonatal rat cardiomyocytes, and it was found that NEK6 ameliorated high glucose-induced inflammation and oxidative stress. Our findings revealed that NEK6 increased the phosphorylation of heat shock protein 72 (HSP72) and increased the protein level of PGC-1α and NRF2. Co-IP assay experiment confirmed that NEK6 interacted with HSP72. When HSP72 was silenced, the anti-inflammation and anti-oxidative stress effects of NEK6 were blurred. In summary, NEK6 may protect diabetic-induced cardiomyopathy by interacting with HSP72 and promoting the HSP72/PGC-1α/NRF2 signaling. Key messages: NEK6 knockout deteriorated cardiac dysfunction, cardiac hypertrophy, fibrosis as well as inflammation response, and oxidative stress. NEK6 overexpression attenuated high glucose induced inflammation and oxidative stress. The underlying mechanisms of the protective role of NEK6 in the development of diabetic cardiomyopathy seem to involve the regulation of HSP72-NRF2- PGC-1α pathway. NEK6 may become a new therapeutic target for diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Introduction: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. Methods: We performed a kinome‐wide CRISPR/Cas9 knock‐out screen in human induced pluripotent stem cell (iPSC) ‐derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex‐vivo studies. Results: Knock‐down of NIMA‐related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock‐down of nek6 also ameliorated the poly(PR)‐induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53‐related DNA damage. Discussion: We identified NEK6, which regulates poly(PR)‐mediated p53‐related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS. [ABSTRACT FROM AUTHOR]

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma (HNSCC), as one of the common malignant tumors, seriously threatens human health. NEK6 (Never in Mitosis A (NIMA) related kinases 6), as a cyclin, promotes cancer cell proliferation and cancer progression. However, the prognostic value of NEK6 and its correlation with immune cell infiltration in HNSCC remain unclear. In this study, we comprehensively elucidated the prognostic role and potential function of NEK6 expression in HNSCC. The expression of NEK6 was significantly up-regulated by immunohistochemistry in HNSCC. Upregulation of NEK6 expression in gene expression studies predicts poor prognosis in HNSCC patients. The results of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene set variation analysis indicated that NEK6 is mainly involved in extracellular matrix metabolism and EMT processes. The expression of NEK6 increased with the level of immune cell infiltration and the expression of various immune checkpoints. In conclusion, NEK6 may serve as a candidate prognostic predictor and may predict the response of HNSCC patients to immunotherapy. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs are a diverse catalog of RNAs that have been implicated in various aspects of tumorigenesis. Emerging evidence indicates that they play crucial roles in tumor growth, disease progression, and drug resistance. However, the clinical significance of lncRNAs in tumor behavior prediction and disease prognosis as well as the underlying mechanism in renal cell carcinoma (RCC) remains elusive. By analyzing the gene expression profiles of 539 RCC patients from the TCGA cohort and 40 RCC patients from an independent cohort, we identified FAM13A-AS1, a poorly studied lncRNA, upregulated in RCC patients. Knockdown experiments revealed that FAM13A-AS1 promotes cell proliferation, migration, and invasion by interacting with miR-141-3p. FAM13A-AS1 regulates the expression of NEK6 by decoying miR-141-3p. In addition, there was a strong positive correlation between the expression of FAM13A-AS1 and NEK6 in RCC patients. In summary, our results demonstrate the oncogenic role of FAM13A-AS1 in RCC and suggest that it promotes tumorigenesis by upregulating the expression of NEK6 by competitively binding to miR-141-3p.

RBBP6 is a multidomain protein, with four splice variants translated into four functional isoforms. RBBP6 isoform 1 has been reported to interact with TP53 and pRb as well as with proteins that regulate transcriptional response to tumorigenesis such as HDM2, ZBTB38, YBX1 and NEK6. Experimental validation of isoforms 2 and 4 is yet to be conducted. The third isoform, consisting of only the DWNN domain and a short unordered c terminus, has been shown to be down-regulated in several human cancers and demonstrated as a regulator of G2/M cell cycle arrest. A number of studies have supported the role of DWNN in cell cycle regulation, however, its mechanism in these processes remains obscure. Posttranslational modification of DWNN could be critical for its function and this study was formulated to understand how the DWNN regulates the cell cycle. Our study identified 12 cell cycle-related proteins interacting with DWNN using various bioinformatics tools. We also identified 10 ubiquitin ligases that interact with DWNN. The most relevant interacting partner, the cell cycle regulator Nek6, has been reported to interact with DWNN during the cell cycle. It was therefore critical to interrogate the interaction between Nek6 and DWNN by homology modelling and docking. The DWNN mutants had a reduced affinity for NEK6 with at least one of the mutants having changes that affect at least one phosphorylation site. It is likely that NEK6 promotes cell proliferation by phosphorylating DWNN. This work suggests that DWNN co-regulates RNA splicing, ubiquitination, and cell cycle control. DWNN may therefore, be targeted for novel anticancer therapies through cell cycle regulation.

Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses.

Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses. [ABSTRACT FROM AUTHOR]

Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis-regulatory circuits remain untested. Here, we dissect cis-regulatory circuits that lead to overexpression of NEK6, a mitosis-associated kinase, in human B cell lymphoma. We find that only a minor subset of predicted enhancers is required for NEK6 expression. Indeed, an annotated super-enhancer is dispensable for NEK6 overexpression and for maintaining the architecture of a B cell-specific regulatory hub. A CTCF cluster serves as a chromatin and architectural boundary to block communication of the NEK6 regulatory hub with neighboring genes. Our findings emphasize that validation of predicted cis-regulatory circuits and super-enhancers is needed to prioritize transcriptional control elements as therapeutic targets.

Abstract NIMA Related Kinase 6 (Nek6) is a protein kinase involved in various cellular processes, including cell cycle regulation, apopotosis, senescence, telomere maintainance and chemoresistance. In the present study, we investigated the role of Nek6 in breast tumorigenesis and the prognostic merit of Nek6 expression in breast cancer. Immunohistochemistry and Western blot analyses was conducted to determine the expression profile of Nek6 in 133 breast cancer specimens. Nek6 was overexpressed in a majority of breast cancer specimens, compared with the adjacent non-tumorous tissues. Furthermore, we revealed that high expression of Nek6 was associated with histologic grade, tumor size and TNM stage in breast cancer. Liner regression analysis showed a significant association between the levels of Nek6 and Ki-67. Cox regression analysis indicated that Nek6 expression was an independent prognostic predictor for breast cancer. Moreover, intracellular level of Nek6 was remarkably increased following the release from serum starvation in MCF-7 cells. EdU incorporation assay indicated that depletion of Nek6 remarkably impaired the proliferation of MCF-7 cells. Finally, spheroid formation assay revealed that interference of Nek6 led to diminished oncospheroid-forming capacity of breast cancer cells. In conclusion, our results imply that Nek6 plays a facilitating role in breast cancer cell proliferation and may serve as a promising therapeutic target for breast cancer. [ABSTRACT FROM AUTHOR]

Summary Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis -regulatory circuits remain untested. Here, we dissect cis -regulatory circuits that lead to overexpression of NEK6, a mitosis-associated kinase, in human B cell lymphoma. We find that only a minor subset of predicted enhancers is required for NEK6 expression. Indeed, an annotated super-enhancer is dispensable for NEK6 overexpression and for maintaining the architecture of a B cell-specific regulatory hub. A CTCF cluster serves as a chromatin and architectural boundary to block communication of the NEK6 regulatory hub with neighboring genes. Our findings emphasize that validation of predicted cis -regulatory circuits and super-enhancers is needed to prioritize transcriptional control elements as therapeutic targets. [ABSTRACT FROM AUTHOR]

Colorectal adenomatous polyp (CRAP) is a major risk factor for the development of sporadic colorectal cancer (CRC). Histone modifications are one of the epigenetic mechanisms that may have key roles in the carcinogenesis of CRC. The objective of the present study is to investigate the alternations in the defined histone modification gene expression profiles in patients with CRAP and CRC. Histone modification enzyme key gene expressions of the CRC, CRAP, and control groups were evaluated and compared using the reverse transcription PCR (RT-PCR) array method. Gene expression analysis was performed in the CRAP group after dividing the patients into subgroups according to the polyp diameter, pathological results, and morphological parameters which are risk factors for developing CRC in patients with CRAP. PAK1, NEK6, AURKA, AURKB, HDAC1, and HDAC7 were significantly more overexpressed in CRC subjects compared to the controls ( p < 0.05). PAK1, NEK6, AURKA, AURKB, and HDAC1 were significantly more overexpressed in the CRAP group compared to the controls ( p < 0.005). There were no significant differences between the CRAP and CRC groups with regards to PAK1, NEK6, AURKA, or AURKB gene overexpression. PAK1, NEK6, AURKA, and AURKB were significantly in correlation with the polyp diameter as they were more overexpressed in polyps with larger diameters. In conclusion, overexpressions of NEK6, AURKA, AURKB, and PAK1 genes can be used as predictive markers to decide the colonoscopic surveillance intervals after the polypectomy procedure especially in polyps with larger diameters. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) are a class of highly conserved, small noncoding RNA that emerge as key posttranscriptional regulators in various neoplastic transformations. Our previous study profiling the miRNA transcriptome in Marek's disease virus (MDV)-in-duced lymphoma revealed many novel and differentially expressed miRNA, including gga-miR-26a, which was downregulated in MDV-infected spleens of chickens. In this study, differential expression of gga-miR-26a between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection was analyzed by real-time PCR. The results showed gga-miR-26a were downregulated in MDV-infected spleens at cytolytic infection, latency, and tumor transformation phases. Subsequent cell proliferation assay revealed cell viability was lower in gga-miR,-26a mimic transfection group than that in negative controls. Target genes of gga-miR-26a were identified by luciferase reporter gene assay. The results showed significant interaction between gga-miR-26a and Never In Mitosis Gene A (JV7MA)-related kinase 6 (NEK6) gene. Subsequent gain of function experiment and Western blot assay showed that mRNA and protein levels of NEK6 were downregulated after gga-miR-26 mimic was transfected into MDV-transformed lymphoid cell line (MSB-1), indicating that NEK6 was modulated by gga-miR-26a. The expression of NEK6 showed a higher trend in MDV-infected samples including tumorous spleen and MD lymphoma from liver than that in noninfected controls. The results suggested that gga-miR-26a inhibited MSB-1 cell proliferation. Gga-miR-26a and its direct target, NEK6, might play important roles in MDV infection. [ABSTRACT FROM AUTHOR]

Abstract: The induction of premature senescence in cancer cells was proposed as an effective cancer treatment strategy. In this paper, we show that the inhibition of Nek6 expression by Nek6 siRNA-mediated knockdown or the overexpression of a dominant negative form of Nek6 (Nek6KM) induced premature senescence as well as cell death under reduced serum conditions in multiple cancer cell lines, including both p53 wild-type and p53 mutant/null backgrounds. Moreover, cancer cells expressing Nek6KM exhibited significantly increased premature senescence upon treatment with the anticancer drugs doxorubicin (DOX) and camptothecin (CPT). Significantly, the overexpression of Nek6KM also inhibited tumor growth and promoted premature senescence in vivo in a xenograft mouse model. Taken together, our results further confirm that Nek6 plays an important role in the premature senescence of cancer cells, suggesting that Nek6 may be a potential therapeutic target for human cancers. [Copyright &y& Elsevier]

Abstract: Cellular senescence plays an important role in tumor suppression. The mitotic kinase Nek6 has recently been shown to be overexpressed in various cancers and has been implicated in tumorigenesis. Previously, we reported that the down-regulation of Nek6 expression was required for p53-induced senescence. In this study, we examined the effect of Nek6 overexpression on the premature senescence of cancer cells induced by the anticancer drugs camptothecin (CPT) and doxorubicin (DOX). We found that CPT- and DOX-induced morphology changes and increases in senescence-associated β-galactosidase staining were significantly inhibited in EJ human bladder cancer cells and H1299 human lung cancer cells overexpressing HA-Nek6. DOX-induced G2/M cell cycle arrest and the reduction in cyclin B and cdc2 levels after DOX treatment were significantly reduced by Nek6 overexpression. In addition, an increase in the intracellular levels of ROS in response to DOX was also inhibited in cells overexpressing Nek6. These results suggest that the increased expression of Nek6 renders cancer cells resistant to premature senescence, and targeting Nek6 could be an efficient strategy for cancer treatment. [Copyright &y& Elsevier]

Abstract: The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we present evidence that human Nek6 is associated with Fe65. Based on the presence of Fe65 WW domain binding motifs (267PPLP270) in the Nek6 catalytic domain, we observed that Nek6 interacts physically with Fe65 both in vivo and in vitro, using a pull-down approach. Additionally, we detected co-localization of Nek6 and Fe65 via confocal microscopy. Co-localization of Nek6 and Fe65 was disrupted by mutation of the WW domain binding motifs (267PPLP270). Finally, when transient transfection assays were performed, interaction of Nek6 (wt) with Fe65 induced substantial cell apoptosis, whereas interaction using the Nek6 pplp mutant (267PPLP270 changes 267APVA270) did not. Thus, our observations indicated that Nek6 binds to Fe65 through its 267PPLP270 motif and that the protein-protein interaction between Nek6 and Fe65 regulates their subcellular localization and cell apoptosis. [Copyright &y& Elsevier]

Abstract: The peptidyl–prolyl isomerase Pin1 is prevalently overexpressed in human cancers and is regarded as a new diagnostic and therapeutic target. Pin1 interacts with several proteins involved in cell cycle events in a phosphorylation-dependent manner. Among them, NIMA (never in mitosis, gene A) was first identified to interact with Pin1. In this report, we found that Pin1 could interact with Nek6, one of the human NIMA-related kinases (Neks). This interaction was confirmed by GST pull-down assay, which was further confirmed by immunoprecipitation experiments, as well as immunofluorescence colocalization. We further studied Pin1 and Nek6 mRNA level in 40 pairs of hepatocellular carcinoma cases, finding significant correlations between Nek6 and Pin1 mRNA expression levels in these samples. [Copyright &y& Elsevier]

Neks (NIMA-related kinases) are mammalian serine/threonine (Ser/Thr) protein kinases structurally related to Aspergillus NIMA (Never in Mitosis, gene A), which plays essential roles in mitotic signaling. Among these kinases, Nek6 and Nek7 are structurally related and constitute a subfamily in the NIMA/Nek family, although their functions still remain almost elusive. In this report, we studied the enzymatic regulation of Nek6 and Nek7 to gain an insight into their cellular functions. Recombinant Nek7 produced in bacteria was active comparably to Nek6; however, the Nek7 activity in mammalian cells was found to be significantly lower than Nek6. Since Nek6 previously has been reported to in vitro phosphorylate p70 ribosomal S6 kinase at Thr412, we examined if Nek6 and Nek7 activities were controlled by the amino acid supplement, which is known to affect the phosphorylation at Thr412, and did not observe any significant effect. However, we unexpectedly found that Nek7 kinase activity was rapidly and efficiently increased by serum deprivation, while Nek6 activity was decreased. This is well consistent with the lower activity of Nek7 in cells under normal growth conditions. In addition, it was suggested that Nek7 activity would be regulated in a cell cycle-dependent manner, although Nek6 was not. These clear differences in enzymatic control between the highly similar kinases, Nek6 and Nek7, suggest their distinct signaling functions in mammalian cells. [Copyright &y& Elsevier]

Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates ~3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5. [ABSTRACT FROM AUTHOR]