Nek2 Overexpression Research Articles

Comprehensive proteomics analysis reveals novel Nek2-regulated pathways and therapeutic targets in cancer

The mitotic kinase Nek2, often overexpressed in various cancers, plays a pivotal role in key cellular processes like the cell cycle, proliferation, and drug resistance. As a result, targeting Nek2 has become an appealing strategy for cancer therapy. To gain a comprehensive understanding of the cellular changes associated with Nek2 activity modulation, we performed a global proteomics analysis using LC-MS/MS. Through bioinformatics tools, we identified molecular pathways that are differentially regulated in cancer cells with Nek2 overexpression or depletion. Of the 1815 proteins identified, 358 exceeded the 20 % significance threshold. By integrating LC-MS/MS data with cancer patient datasets, we observed a strong correlation between Nek2 expression and the levels of KIF20B and RRM1. Silencing Nek2 led to a significant reduction in KIF20B and RRM1 protein levels, and potential phosphorylation sites for these proteins by Nek2 were identified. In summary, our data suggests that KIF20B and RRM1 are promising therapeutic targets, either independently or alongside Nek2 inhibitors, to improve clinical outcomes. Further analyses are necessary to fully understand Nek2's interactions with these proteins and their clinical relevance.

Function of NEK2 in clear cell renal cell carcinoma and its effect on the tumor microenvironment.

Previous studies have revealed the critical functions of NEK2 in controlling the cell cycle which is linked to poor prognosis in multiple tumor types, but less research has been devoted to clear cell renal cell carcinoma (ccRCC). We downloaded clinical data from the gene expression omnibus (GEO) and TCGA databases together with transcriptional and mutational datasets. Strongly coexpressed genes with NEK2 were extracted from TCGA-KIRC cohort, and were submitted to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analyses. According to NEK2 levels, the survival status, mutational characteristics, response to immunotherapy and sensitivity to drugs of the patients were studied. The potential correlations between NEK2 levels and immune cell state as well as immune cell infiltration were examined using the GEPIA, TIMER and TISIDB databases. Double immunofluorescence (IF) was performed to identify the NEK2 overexpression and relationship with CD8 in ccRCC. The NEK2 gene was overexpressed and would enhance the nuclear division and cell cycle activities in ccRCC. ccRCC patients with high NEK2 expression had worse clinical outcomes, higher mutation burden and better therapeutic response. Moreover, NEK2 gene overexpression was positively related to various immune cell marker sets, which was also proved by validation cohort, and more infiltration of various immune cells. ccRCC patients with NEK2 high expression have a poorer prognosis than those with NEK2 low expression, resulting from its function of promoting proliferation, accompanied by increased infiltration of CD8 + T cells and Tregs and T-cell exhaustion and will respond better to proper treatments.

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

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. 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. 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. 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.

Anti‐cancer Effects of Dual‐action Inhibitors of Nek2 and EGFR Kinases: Investigation of Synergy

While lung cancer is the leading cause of mortality among all cancer types for both men and women, breast cancer dominates cancer diagnosis among women patients. Never in mitosis gene A (NIMA)‐related kinase 2 (Nek2) is a member of NEK family, a Ser/Thr kinase, that is overabundant in both cancer types. Studies from our laboratory have shown that abnormal expression of Nek2 kinase can promote aggressive tumor behavior and metastasis. Nek2 overexpression also drives drug resistance. These tumors also overexpress another key oncogene, epidermal growth factor receptor (EGFR), that has shown to induce cell proliferation, angiogenesis, and tumorigenesis. Hence, concomitant inhibition of Nek2 and EGFR kinases can potentially be a promising therapeutic strategy for both lung and breast cancers. Our goal is to develop anticancer agents that targets inhibition of both Nek2 and EGFR activities. Our hypothesis is such dual‐action inhibitory agents will provide superior efficacy due to a more predictable PK/PD profile in vivo. In this work, we first test our hypothesis that dual inhibition of Nek2 and EGFR kinases by small molecules will indeed yield net signaling output that results in synergistic cancer cell death in A549 (lung) and MDA‐MB‐231 (breast) cells. Using a novel drug‐like pharmacophore identified in our laboratory, we have also developed small molecule inhibitors that inhibits both Nek2 and EGFR activities. We anticipate that these findings will render new opportunities toward development of targeted small molecule therapeutics for both lung and breast cancers.

Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma.

It has been previously shown that (never in mitosis gene A)‐related kinase 2 (NEK2) is upregulated in multiple myeloma (MM) and contributes to drug resistance. However, the mechanisms behind this upregulation remain poorly understood. In this study, it is found that amplification of NEK2 and hypermethylation of distal CpG islands in its promoter correlate strongly with increased NEK2 expression. Patients with NEK2 amplification have a poor rate of survival and often exhibit TP53 deletion, which is an independent prognostic factor in MM. This combination of TP53 knockout and NEK2 overexpression induces asymmetric mitosis, proliferation, drug resistance, and tumorigenic behaviors in MM in vitro and in vivo. In contrast, delivery of wild type p53 and suppression of NEK2 in TP53−/− MM cell lines inhibit tumor formation and enhance the effect of Bortezomib against MM. It is also discovered that inactivating p53 elevates NEK2 expression genetically by inducing NEK2 amplification, transcriptionally by increased activity of cell cycle‐related genes like E2F8 and epigenetically by upregulating DNA methyltransferases. Dual defects of TP53 and NEK2 may define patients with the poorest outcomes in MM with p53 inactivation, and NEK2 may serve as a novel therapeutic target in aggressive MM with p53 abnormalities.

NEK2 enhances malignancies of glioblastoma via NIK/NF-\u03baB pathway

Glioblastoma (GBM) is one of the most lethal primary brain tumor with a poor median survival less than 15 months. Despite the development of the clinical strategies over the decades, the outcomes for GBM patients remain dismal due to the strong proliferation and invasion ability and the acquired resistance to radiotherapy and chemotherapy. Therefore, developing new biomarkers and therapeutic strategies targeting GBM is in urgent need. In this study, gene expression datasets and relevant clinical information were extracted from public cancers/glioma datasets, including TCGA, GRAVENDEEL, REMBRANDT, and GILL datasets. Differentially expressed genes were analyzed and NEK2 was picked as a candidate gene for subsequent validation. Human tissue samples and corresponding data were collected from our center and detected by immunohistochemistry analysis. Molecular biological assays and in vivo xenograft transplantation were performed to confirm the bioinformatic findings. High-throughput RNA sequencing, followed by KEGG analysis, GSEA analysis and GO analysis were conducted to identify potential signaling pathways related to NEK2 expression. Subsequent mechanism assays were used to verify the relationship between NEK2 and NF-κB signaling. Overall, we identified that NEK2 is significantly upregulated in GBM and the higher expression of NEK2 exhibited a poorer prognosis. Functionally, NEK2 knockdown attenuated cell proliferation, migration, invasion, and tumorigenesis of GBM while NEK2 overexpression promoted the GBM progression. Furthermore, High-throughput RNA sequencing and bioinformatics analysis indicated that NEK2 was positively related to the NF-κB signaling pathway in GBM. Mechanically, NEK2 activated the noncanonical NF-κB signaling pathway by phosphorylating NIK and increasing the activity and stability of NIK. In conclusion, NEK2 promoted the progression of GBM through activation of noncanonical NF-κB signaling, indicating that NEK2- NF-κB axis could be a potential drug target for GBM.

Study of mangal based naphthoquinone derivatives anticancer potential towards chemo-resistance related Never in mitosis gene A-related kinase 2-Insilico approach

ABSTRACT Never in mitosis gene A-related kinase 2 (NEK2) is a member of serine-threonine kinase mainly involved in cell cycle process. Clinical studies revealed that NEK2 overexpression in many tumour types, also reported for their association with genetic abnormalities like mitotic machinery deregulation and chromosomal instability. Besides NEK2 plays a key role in maintaining the transmuted phenotype of tumour cells and induction of chemo-resistance in tumour types. Our studies involved NEK2 transcriptional profile search, screening of druggable cavities in NEK2, Drug likeliness of mangrove derived naphthoquinone derivatives avicennone, avicennone A, avicennone B, avicennone C, avicennone D, avicenone E, avicennone F and avicennone G, avicequinone A, stenocarpoquinone B, avicequinone C, avicenol A, avicenol C, brugine, apigenin, chrysin and molecular docking studies to assess compounds binding efficacy towards NEK2. Irreversible inhibitors have several advantages over noncovalent inhibitors, comprising sustained pharmacodynamics with high potency practical application, and specificity. Thus Targeting the cysteine 22 (Cys-22) residue near the catalytic site of NEK2 kinase will result in irreversible inhibition. Avicenol A, Avicennone G, Chrysin and Brugine formed the irreversible covalent binding with NEK2 through Cys 22, thus they can be considered as potential kinase inhibitors with limited off-target response. But proposed compounds need to be tested further through in vitro and in vivo studies to propose as potent NEK2 inhibitors.

The Nek2 centrosome-mitotic kinase contributes to the mesenchymal state, cell invasion, and migration of triple-negative breast cancer cells

Nek2 (NIMA‐related kinase 2) is a serine/threonine-protein kinase that localizes to centrosomes and kinetochores, controlling centrosome separation, chromosome attachments to kinetochores, and the spindle assembly checkpoint. These processes prevent centrosome amplification (CA), mitotic dysfunction, and chromosome instability (CIN). Our group and others have suggested that Nek2 maintains high levels of CA/CIN, tumor growth, and drug resistance. We identified that Nek2 overexpression correlates with poor survival of breast cancer. However, the mechanisms driving these phenotypes are unknown. We now report that overexpression of Nek2 in MCF10A cells drives CA/CIN and aneuploidy. Besides, enhanced levels of Nek2 results in larger 3D acinar structures, but could not initiate tumors in a p53+/+ or a p53−/− xenograft model. Nek2 overexpression induced the epithelial-to-mesenchymal transition (EMT) while its downregulation reduced the expression of the mesenchymal marker vimentin. Furthermore, either siRNA-mediated downregulation or INH6’s chemical inhibition of Nek2 in MDA-MB-231 and Hs578t cells showed important EMT changes and decreased invasion and migration. We also showed that Slug and Zeb1 are involved in Nek2 mediated EMT, invasion, and migration. Besides its role in CA/CIN, Nek2 contributes to breast cancer progression through a novel EMT mediated mechanism.

NEK2 regulates cellular proliferation and cabergoline sensitivity in pituitary adenomas.

Objective: To identify critical roles played by NEK2 in prolactinomas and to clarify the corresponding underlying mechanisms.Methods: We performed RNA-seq on MMQ cell lines treated with the dopamine receptor agonist cabergoline (CAB) to identify genes involved in prolactinoma progression and dopamine receptor-agonist (DA) sensitivity. NEK2 was then selected for further study. The expression of NEK2 was examined using quantitative real-time PCR, western immunoblotting, and immunohistochemistry - both in pituitary adenomas (PA) and in normal pituitary tissue. We used gain-of-function and loss-of-function assays to explore the biologic roles of NEK2 in cell growth in vivo and in vitro. Co-immunoprecipitation was also used to detect the binding between NEK2 and USP7.Results: Herein, we reported that NEK2 was upregulated in prolactinomas, particularly dopamine-resistant prolactinomas. NEK2 overexpression significantly promoted pituitary tumor GH3 and MMQ cell proliferation, and it impaired cellular sensitivity to CAB. Conversely, knockdown of NEK2 inhibited GH3 and MMQ cell growth, and sensitized the cells to CAB. Mechanistically, NEK2 regulated cell proliferation via the Wnt-signaling pathway; and in addition, we demonstrated that USP7 interacted with, deubiquitylated, and stabilized NEK2.Conclusions: Collectively, our results suggest that NEK2 might be a potential therapeutic target for prolactinoma.

High expression of NEK2 promotes gastric cancer progression via activating AKT signaling.

Never in mitosis gene A-related kinase 2 (NEK2) has been recognized as an oncogene involved in the initiation and progression of various human cancers. However, our knowledge is still lacking in regard to the function of NEK2 in gastric cancer, the most common cancer in Eastern Asia associated with poor prognosis. Therefore, in the present study, we investigated the association of NEK2 with gastric cancer. We found that the development of gastric cancer is associated with NEK2 overexpression, particularly in patients with large tumor size and lymph node metastasis. We also provided evidence that NEK2 overexpression binds to and inhibits protein phosphatase 1 (PP1), which subsequently activates AKT and the downstream oncogenic pathways. As a result, via AKT/HIF1α axis, the glucose metabolism is reprogrammed towards aerobic glycolysis to provide rapid energy for the growth of gastric cancer cells. Moreover, the autophagic activity is suppressed via AKT/mTOR axis, leading to impaired response to cancer treatment and enhanced cell survival. In contrast, inactivating AKT by NEK2 silencing decreases aerobic glycolysis and promotes autophagic cell death, which eventually inhibits the growth of gastric cancer cell. All these results revealed that NEK2 promotes gastric cancer progression via activating AKT-mediated signaling pathways, which expanded our knowledge on gastric cancer pathogenesis and also provided novel target for clinical treatment.

High expression of NEK2 promotes lung cancer progression and drug resistance and is regulated by mutant EGFR.

Activating mutations within the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) gene are observed in 10 ~ 30% of the patients diagnosed with non-small cell lung cancer (NSCLC), and are causally related to NSCLC initiation and progression. Treatments with tyrosine kinase inhibitors (TKIs) targeting EGFR significantly improve the outcome of NSCLC patients with EGFR mutation, but are often associated with drug resistance, which is the main cause of treatment failure and cancer relapse. In the present study, by screening the transcriptome of NSCLC patients, we found that EGFR activation is highly correlated with the up-regulation of mitotic regulator, never in mitosis gene A-related kinase 2 (NEK2). NEK2 overexpression is associated with the poor survival of EGFR-mutant patients but not the wild-type patients. Further functional validation revealed that EGFR mutation induces NEK2 expression by activating ERK signaling pathway. Elevated NEK2 level promotes the rapid cell cycle progression and favors the rapid proliferation of EGFR-mutant NSCLC cells. Of note, NEK2 overexpression also impairs the efficacy of TKI treatment via inhibiting apoptosis, while depleting NEK2 suppresses cell growth and restored the sensitivity of TKI in NSCLC cells. Taken together, our study revealed that NEK2 is an oncogene regulated by EGFR mutation and is involved in disease progression and treatment response in NSCLC with EGFR mutation. These findings will pave the road for optimizing personalized treatment strategies to overcome drug resistance and improve the prognosis of lung cancer patients with EGFR mutation.

Abstract 2545: Nek2 and other mitotic kinases are downstream of the E2F activators, and may modulate the epithelial-to-mesenchymal transition in breast cancer

Abstract The E2F transcription factors have been extensively studied in cancer since they are known for their role in the regulation of cell cycle progression, centrosome duplication, cell survival, and other functions. We recently demonstrated that the E2F activators (E2F1, E2F2, and E2F3) regulate mitosis through the elevated expression of multiple mitotic proteins including Sgo1, Nek2, Hec1, BubR1, and Mps1. We have shown that Mps1 triggers EMT through multiple mechanisms. NIMA-related kinase 2 (Nek2) is a centrosome-associated protein kinase particularly abundant in cells during the G2-M phase, which primary function is to phosphorylate centrosome linker proteins to cause centrosome disjunction and the formation of the bipolar mitotic spindle. Previous results from our lab showed that overexpression of Nek2 in HCC1954 (Her2+ breast cancer cell line) with a knock down for E2F3 rescued a high centrosome amplification pattern and enhances the formation of invasive protrusions. cBioportal analysis using the TCGA database showed Nek2 mRNA alterations in 9.1% of breast cancer patients and is predominantly present in tumors that are ER-, PR-, and Her2- (25%). Similarly, analysis using the METABRIC database showed Nek2 mRNA alterations in 6.1% of breast cancer patients and is predominantly present in tumors from basal (24%), Her2+ (10%), and luminal B (13.9%) subtypes. Based on these results, we hypothesize that Nek2 overexpression has a role in the epithelial-to-mesenchymal (EMT) pathway leading to cell migration and invasion as prelude to metastasis in breast cancer. To address this, we used MCF10A non-tumorigenic mammary cells with or without the overexpression of Nek2 (MCF10A/GFP and MCF10A/GFP-Nek2). Our preliminary data indicates that overexpression of GFP-Nek2 in mammary epithelial cells enhances levels of vimentin, whereas decreased the levels of E-cadherin, suggesting a role of Nek2 in a mesenchymal state. Likewise, siRNA-mediated silencing of Nek2 in mesenchymal breast cancer cells modulates markers of EMT. Because inhibitors are available for Nek2, it would be feasible to target this kinase to combat metastatic HR- breast cancers. Our work will allow the identification of mechanistic molecular pathways influenced by Nek2 role in driving invasion and metastasis to highlight this mitotic kinase as a potential prognostic marker for high-risk breast tumors. Citation Format: Yainyrette Rivera-Rivera, Mihaela Marina, Harold I. Saavedra. Nek2 and other mitotic kinases are downstream of the E2F activators, and may modulate the epithelial-to-mesenchymal transition in 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 2545.

High Expression of NEK2 Mediated By p53 Contributes to Progression and Relapse of Multiple Myeloma

High expression of NEK2 mediated by p53 contributes to progression and relapse of multiple myelomaXiangling Feng1,2, Jiaojiao Guo1, Bowen Ouyang2, Yinghong Zhu1,Gang An3, Hao Zhen1, Jiliang Xia1, Yongjun Guan1, Xinying Zhao2, Lugui Qiu3, Jiaxi Zhou3, Fenghuang Zhan4,Wen Zhou11, Cancer Research Institute,Central South University, Changsha 410078, China. 2Xiang Ya School of Public Health, Central South University, Changsha, Hunan, China. USA. 3State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China. 4Department of Internal Medicine, Division of Hematology, Oncology, and Blood & Marrow Transplantation, University of Iowa, Iowa City, USA. E-mail: wenzhou@csu.edu.cn.Background: Loss of p53 is an independent prognostic factor in patients with multiple myeloma (MM). Our previous studies found abnormal high expression of NEK2 was closely related to the poor prognosis and drug resistance of myeloma patients. However, it's unclear how NEK2 was up-regulated in MM. Through bioinformatics analysis, the binding site of p53 protein is found in NEK2 promoter, but the relationship and function of p53 and NEK2 in MM are poorly understood.Materials and Methods: In this study, p53-/- MM cell lines (ARP1 and KMS11) and p53 p53+/+ MM cell lines (MM1S and H929) were used for investigating the role of NEK2 in p53-/- MM cell. FISH was performed on interphase nuclei of MM primary cells to detect p53 and NEK2 copy numbers. Chromatin immunoprecipitation and fluorescence reporter system were applied for examining the binding site of p53 protein in the distal NEK2 promoter. CGH and RNA-seq were performed to validate copy number changes and variations in the expression of several transcripts.Results: The top 10% of MM patients with the highest NEK2 expression and lowest p53 had a significantly inferior OS (P<0.001) in TT2 and TT3 patients (GSE2658) and the expression of NEK2 increased significantly in myeloma cells during chemotherapy(GSE19554), while p53 decreased with the disease progression, suggesting a strong relationship with drug resistance. Single cell PCR showed increased NEK2 expression correlated with decreased p53 expression in single CD138+ plasma cell. FISH confirmed the loss of p53 in CD138+ plasma cells with amplification of NEK2 copies. Furthermore, NEK2 was also high expressed in p53 low expressed MM cells by Immunofluorescence (IF) (P<0.01). In addition, NEK2 was upregulated in p53-/- MM cell lines and HEK293 cells by deleted p53 gene with CRISPR technique both on mRNA and protein level (P<0.01), suggesting a negative correlation between the p53 and the expression level of NEK2. Meanwhile, when p53 deletion and NEK2 overexpression occur simultaneously, the phenomena of asymmetric mitosis and multipolar division are more obvious (P<0.001), suggested that the double hit of p53 deletion and NEK2 overexpression increases the chromosomal instability. Further in vivo study indicated the subcutaneous tumorigenesis in p53 deletion and NEK2 overexpression group was significantly greater than that of the single overexpression of NEK2 and deletion of p53 group (P<0.001), suggested that NEK2 overexpression and p53 deletion enhances the tumorigenic ability in vivo. While down-regulation of NEK2 by shRNA in p53 deletion cells, cell growth was inhibited in vitro and in vivo.To explore the relationship between p53 and NEK2, chromatin immunoprecipitation and fluorescence reporter system showed that p53 could bind to the promoter region of NEK2 and regulate its transcriptional expression. Further CGH analysis of the deletion of p53 expression in HEK293 cells can cause 1q21.4-44 amplification of the chromosome region of the NEK2 directly, which further confirmed by FISH. Finally, RNA-seq revealed several chromosome instability genes were abnormal expressed in NEK2 overexpression and p53 deletion double-hit group.Conclusion: In summary, p53 deletion and NEK2 overexpression induced cancer cell drug resistance, proliferation and chromosomal instability. p53 could bind to the promoter region of NEK2 and cause NEK2 amplification. Down-regulation of NEK2 by shRNA in p53 deletion cells inhibited cell growth in vitro and in vivo. Thus,The significance of this study will provide the pre-clinical application of the NEK2 inhibitor to overcome the drug resistance induced by p53 in MM. DisclosuresNo relevant conflicts of interest to declare.

Prognostic value of NEK2 overexpression in digestive system cancers: a meta-analysis and systematic review.

Background and objectiveMany studies have reported that NEK2 is overexpressed in digestive system cancers (DSCs) and is also correlated with patient survival. We performed a meta-analysis to comprehensively evaluate the prognostic role of NEK2 expression in DSCs.Materials and methodsA comprehensive literature search was performed using PubMed, EMBASE, and Web of Science. Synthesized hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the influence of NEK2 overexpression on the prognosis and clinicopathological features of patients with DSCs.ResultsA total of 13 studies involving 1,917 patients was included. Overall, patients with high NEK2 expression had poorer overall survival (HR =1.45; 95% CI: 1.15–1.83; P=0.002) and disease-free survival/recurrence-free survival (HR =2.28; 95% CI: 1.54–3.37; P<0.0001). Furthermore, subgroup analysis also suggested that elevated NEK2 expression was associated with poorer overall survival in patients with hepatocellular carcinoma (HR =1.45; 95% CI: 1.05–2.00; P=0.02) and colorectal cancer (HR =2.03; 95% CI: 1.16–3.54; P=0.01). Additionally, NEK2 overexpression was also associated with pretreatment serum AFP level (OR =1.79; 95% CI: 1.23–2.61; P<0.01) and portal vein thrombosis (OR =2.74; 95% CI: 1.22–6.17; P=0.01) in hepatocellular carcinoma.ConclusionNEK2 might act as a useful prognostic predictor and a potential therapeutic target in DSCs. However, multicenter homogeneous studies with larger sample sizes are needed to further confirm our findings owing to some limitations in our meta-analysis.

NEK2 promotes hepatocellular carcinoma migration and invasion through modulation of the epithelial-mesenchymal transition.

Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) has been recently reported to play a role in tumor progression, drug resistance and tumorigenesis. However, little is known about the effects of NEK2 in hepatocellular carcinoma (HCC) metastasis and the underlying mechanism. NEK2 expression levels were examined by immunochemistry, qRT-PCR and western blot analyses in HCC cell lines and HCC tissues. A Transwell assay was used to determine the migration and invasion capacity of NEK2-silenced or NEK2-overexpressing HCC cells. Cell proliferation was investigated by MTT [(3-(4,5)-dimethylthiazol(-z-y1)-3,5-di-phenytetrazolium bromide] assay. The expression levels of epithelial-mesenchymal transition (EMT) markers in NEK2-silenced or NEK2-overexpressing HCC cells were examined by western blot analyses and qRT-PCR. The correlations between NEK2 expression and clinicopathological characteristics were further analyzed. Gene microarray was further used to analyze the effect of NEK2 expression on downstream cell signals. Our study showed that NEK2 was overexpressed in human HCC (37.84%; 98/259). NEK2 overexpression was significantly associated with liver non-capsulation and predicted poor survival outcomes in HCC patients after hepatectomy. In addition, NEK2 significantly enhanced HCC cell invasive ability. Mechanistically, we found that the epithelial-mesenchymal transition (EMT) plays a pivotal role in the NEK2-mediated promotion of HCC cell invasion. Furthermore, we provided evidence that signaling through the Wnt, NF-κB, focal adhesion, VEGF, Hippo and p53 pathways may be downstream of NEK2. Our findings highlight the importance of NEK2 in HCC metastasis and suggest that NEK2 is a reliable prognostic marker for HCC patients after hepatectomy.

Abstract C29: Addressing how the Nek2 mitotic kinase facilitates the epithelial to mesenchymal transition in breast cancer

Abstract Hispanics- (HAs) and African- Americans (AAs) are at higher risk of breast cancer (BC)-related death relative to European-Americans (EAs). Poor survival associates with socioeconomic status, genetics, and higher likelihood of developing hormone receptor-negative (ER-PR-, or HR-) BCs relative to EAs. Thus, it is imperative to find novel strategies to curb progression of these highly-malignant tumors. We propose that the accumulation of high frequencies of chromosome instability (CIN) is a risk factor for BC-related deaths in AA and HAs, since HR- BCs accumulate more CIN than other subtypes. The presence of CA/CIN in pre-malignant breast lesions and in most breast cancers (BCs), and their association with metastasis strongly suggest that CA/CIN are major drivers of multistage breast tumorigenesis. However, the direct involvement of CA/CIN in BCs has to be tested experimentally. TCGA analyses of BCs show that approximately 50% BCs are tetraploid; thus, it is highly significant to investigate whether, how and when CA/CIN contributes to mammary tumorigenesis. Our laboratory studies how unregulated centrosome and mitotic kinases drive CA/CIN in mammary epithelial cells to initiate and sustain HR- BCs. We demonstrated that CA appears in pre-malignant epithelial lesions, and that CA correlated with shorter-times of onset and invasion. In fact, CA in Her2+HR- BC and in MCF10A cells expressing H-RasG12V or H-RasG12V and c-Myc is partly dependent on Nek2. Nek2 regulates centrosome separation, and the spindle assembly checkpoint. Nek2 is overexpressed in 12% BCs and unregulation correlates with relapse-free survival of all BCs, and all subtypes, including basal, Her2+, Luminal A and Luminal B BCs. We have published that overexpression of GFP-Nek2 enhances CA in Her2+HR- cells silenced for E2F3 and results in invasive protrusions, suggestive of involvement in EMT, invasion, and metastasis. To establish the role of CA in mammary tumorigenesis, we overexpressed GFP-Nek2 in MCF10A cells, which are non-transformed human mammary epithelial cells. Nek2 overexpression is sufficient to enhance CA and CIN in MCF10A cells, and increase N-cadherin and vimentin, suggestive of EMT. Overall, our published and preliminary data indicate that Nek2-driven CA influence breast tumorigenesis. Future experiments will test Nek2's role in initiating and sustaining mammary tumors and metastasis. Because inhibitors against Nek2 are available, it is feasible to understand how Nek2 drives mammary tumorigenesis. Citation Format: Yainyrette Rivera-Rivera, Mihaela Marina, Harold I. Saavedra. Addressing how the Nek2 mitotic kinase facilitates the epithelial to mesenchymal transition in breast cancer. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C29.

Nek2 Stabilization By Usp7 Leads to Activation of NF-Kb in Multiple Myeloma

NIMA (Never In Mitosis Gene A)-Related Kinase 2 (Nek2), a centrosomal Serine/Threonine kinase, is a key player in numerous malignancies. Overexpression of Nek2 has been related to many cancers including Multiple Myeloma (MM). In MM, Nek2 is one of the chromosomal instability genes associated with drug resistance and disease relapse. However, very little is known about the mechanisms that lead to these Nek2-driven disparities. Here, we show that the Ubiquitin Specific Peptidase 7 (USP7) stabilizes Nek2 leading to activation of NF-kb pathway.Using gene expression profile (GEP) data from patients and cell lines we discovered that Nek2 overexpression leads to increases of several targets of the NF-kb pathway. We, thus, hypothesize that Nek2 is activating NF-kb. To address this, we overexpressed Nek2 and tested the classic canonical NF-kb hallmarks proteins by western blotting. Nek2 overexpression led to an increase in phosphorylation of IKK, activator of NF-kb, and to decrease levels of IKb-alpha, a negative regulator of the pathway. Nek2 overexpression also increased nuclear and phosphorylated p65 on residue S536, known as active transcriptional site. To further confirm that Nek2 is activating canonical NF-kb luciferase assay was performed. The luciferase reporter is driven by a p65 promoter and in cells overexpressing Nek2 luciferase levels were increased.To characterize Nek2 interacting partners a tandem affinity purification/mass spectrometry (TAP/MS) approach was performed. We found that Nek2 binds to Usp7, a deubiquitinase overexpressed in numerous cancers. This led to hypothesize that that Nek2, a known target of the ubiquitin proteasome system, is being stabilized by the Usp7 contributing to its overexpression and the increased activation of the NF-kb pathway. To test our hypothesis, we treated different cancer cell lines with the commercially available Usp7 inhibitor, P5091, or silenced the protein using shRNA. In both case, we found a reduction in Nek2 protein level. Additionally, we overexpressed Usp7 and Nek2 increased confirming that Usp7 stabilizes Nek2. To further show that Usp7 stabilizes Nek2 by de-ubiquitination, we overexpressed Usp7 and analyzed Nek2 ubiquitination after immunoprecipitation. When Usp7 was overexpressed no ubiquitination of Nek2 was detected.Finally, by using GEP data from MM patients, we found that individuals who overexpressed Nek2 along with an active NF-kb signature have worst event free survival as well as overall survival, indicating Nek2 overexpression leading to increased NF-kb signature has clinical significance. DisclosuresNo relevant conflicts of interest to declare.

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry.

Drug Resistant Myeloma Cells Cause More Severe Bone Destruction in Myeloma

Background: Multiple myeloma (MM) is the second most common hematologic malignancy in the US and is characterized by osteolytic disease. More than 80% of MM patients have osteolysis, leading to in fractures, severe bone pain, spinal cord compression, and hypercalcemia, greatly compromising patient quality of life and resulting in mortality. While bone exists in a dynamic state between resorption and deposition, the molecular mechanisms underlying bone destruction in MM remain elusive. One contributing factor is heparanase (HPSE). In our work, we have previously shown that the expression of NEK2 and HPSE are closely correlated. We have also shown that high expression of chromosomal instability-related genes (e.g. NEK2) induces drug resistance and poor prognosis in MM. Though tumor burden is dramatically decreased in remission, bone destruction is poorly repaired, suggesting drug resistant MM cells may play a critical role in inducing bone destruction or in preventing the repair of bone disease in remission.Materials and Methods: Gene expression profiling (GEP) data and bone lytic lesions detected by MRI were collected from 333 newly diagnosed MM patients from the Total Therapy 2 (TT2) clinical trial. MM cell lines with stable overexpression or knockdown of NEK2 and/or HPSE were created using lentiviral delivery. Protein and mRNA expression was assessed by immunoblotting and real-time PCR, respectively. TRAP staining was used to assess osteoclast differentiation. In vivo studies were performed in the KaLwRij 5TGM1 MM mouse model in 3 different groups: empty vector (EV), NEK2 overexpression (NEK2 OE), or NEK2 OE with HPSE shRNA. Bone density and imaging assessment was performed via X-ray and microCT scanning. HPSE, NFκB, and USP7 contributions were examined using the specific small molecule inhibitors SST0001, BSM-345541, and P5091, respectively.Results: In correlating NEK2 expression with osteolytic lesion from 333 newly diagnosed MMs, we found the expression of NEK2 was significantly higher in MM patients with bone lytic lesion than those without lesions. NEK2 expression was also higher in patients with severe bone disease (7 or more lesions) than those fewer bone lytic lesions (< 7).To determine if NEK2 causes bone lytic lesions in MM, we performed in vivo studies injecting NEK2 OE 5TGM1 cells into KaLwRij mice. MicroCT showed clearly that bone volume and thickness of trabecular bone were decreased significantly in mice injected with NEK2 OE cells compared to EV cells. Strikingly, treatment with the HPSE inhibitor SST0001 dramatically rescued bone mass loss induced by NEK2 overexpression. Histological TRAP staining of decalcified bone sections revealed a significant increase in the number of osteoclasts in the bone surface area in mice injected with NEK2 OE cells compared to EV-treated mice, while addition of the SST0001 significantly reduced the number of osteoclast.Because osteoclast causes bone resorption, we examined the effect of applying conditioned media from MM cells with or without NEK2 overexpression to pre-osteoclast cells derived from MM patients, C57BL6 mice, and the pre-osteoclast RAW264.7 cell line. We found significantly increased mature osteoclasts (>3 nuclei) after culture with media collected from NEK2 MM cells compared to conditioned media collected from EV cells. Consistently, osteoclast differentiation-associated genes (i.e. CTSK, NFATc1, RANK, and TRAP) were upregulated in pre-osteoclasts cultured with NEK2 OE conditioned media. SST0001 suppressed osteoclast differentiation induced by NEK2, strongly suggesting that HPSE mediates NEK2 induced osteoclastogensis and bone destruction in MM. Mechanistic studies revealed that NEK2 directly binds to and activates USP7 resulting in deubiquitination of NFκB. Inhibition of NFκB decreased NEK2-induced HPSE expression induced by NEK2 at both the mRNA and protein level.Conclusions: Our findings suggest that high NEK2 expression may be a biomarker for bone disease in MM. We demonstrate that a subpopulation of drug-resistant, NEK2-overexpressing MM cells leads to increased production and activity of HPSE, and increased osteoclast differentiation contributing to severe bone disease. Mechanistically, we show that NEK2 interacts with USP7, stabilizing NFκB and upregulating HPSE. Our results demonstrate drug resistant MM cells may not only promote tumor cell survival but also contribute to bone destruction in MM. DisclosuresNo relevant conflicts of interest to declare.

Silencing of E2F3 suppresses tumor growth of Her2+ breast cancer cells by restricting mitosis

The E2F transcriptional activators E2F1, E2F2 and E2F3a regulate many important cellular processes, including DNA replication, apoptosis and centrosome duplication. Previously, we demonstrated that silencing E2F1 or E2F3 suppresses centrosome amplification (CA) and chromosome instability (CIN) in Her2+ breast cancer cells without markedly altering proliferation. However, it is unknown whether and how silencing a single E2F activator, E2F3, affects malignancy of human breast cancer cells. Thus, we injected HCC1954 Her2+ breast cancer cells silenced for E2F3 into mammary fat pads of immunodeficient mice and demonstrated that loss of E2F3 retards tumor growth. Surprisingly, silencing of E2F3 led to significant reductions in mitotic indices relative to vector controls, while the percentage of cells undergoing S phase were not affected. Nek2 is a mitotic kinase commonly upregulated in breast cancers and a critical regulator of Cdk4- or E2F-mediated CA. In this report, we found that Nek2 overexpression rescued back the CA caused by silencing of shE2F3. However, the effects of Nek2 overexpression in affecting tumor growth rates of shE2F3 and shE2F3; GFP cells were inconclusive. Taken together, our results indicate that E2F3 silencing decreases mammary tumor growth by reducing percentage of cells undergoing mitosis.