Nek6 Expression Research Articles

Serum-Derived Exosomal TBX2-AS1 Exacerbates COPD by Altering the M1/M2 Ratio of Macrophages through Regulating the miR-423-5p/miR-23b-3p Axis

ABSTRACT Objective To investigate the mechanism of serum exosomes in chronic obstructive pulmonary disease (COPD), especially the effect of lncRNA TBX2-AS1 on macrophage polarization. Methods Screen differentially expressed genes through bioinformatics analysis, detect the expression of related molecules in clinical samples and cell experiments, construct a mouse model and conduct functional rescue experiments, using various experimental techniques such as RT - qPCR, Western Blot, flow cytometry, ELISA, and luciferase reporter assay. Results TBX2-AS1 is highly expressed in the serum and serum exosomes of COPD patients, and it can promote macrophage M1 polarization and inhibit M2 polarization; it exerts its role by negatively regulating the miR-423-5p/miR-23b - 3p axis, where miR-423-5p inhibits CELSR2 expression to prevent M1 polarization, and miR-23b-3p inhibits NEK6 expression to promote M2 polarization; in vivo experiments, down-regulation of CELSR2/NEK6 can reverse the promoting effect of COPD serum exosomes on lung injury and inflammation. Conclusion COPD serum exosomes deliver TBX2-AS1 to macrophages, regulate the miR-423-5p-CELSR2/miR-23b-3p-NEK6 pathway, affect macrophage polarization, and exacerbate the progression of COPD, providing new directions and potential targets for the diagnosis and treatment of COPD.

NEK6 dampens FOXO3 nuclear translocation to stabilize C-MYC and promotes subsequent de novo purine synthesis to support ovarian cancer chemoresistance

De novo purine synthesis metabolism plays a crucial role in tumor cell survival and malignant progression. However, the specific impact of this metabolic pathway on chemoresistance in ovarian cancer remains unclear. This study aims to elucidate the influence of de novo purine synthesis on chemoresistance in ovarian cancer and its underlying regulatory mechanisms. We analyzed metabolic differences between chemosensitive and chemoresistant ovarian cancer tissues using mass spectrometry-based metabolomics. Cell growth, metabolism, chemoresistance, and DNA damage repair characteristics were assessed in vitro using cell line models. Tumor growth and chemoresistance were assessed in vivo using ovarian cancer xenograft tumors. Intervention of purines and NEK6-mediated purine metabolism on chemoresistance was investigated at multiple levels. Chemoresistant ovarian cancers exhibited higher purine abundance and NEK6 expression. Inhibiting NEK6 led to decreased de novo purine synthesis, resulting in diminished chemoresistance in ovarian cancer cells. Mechanistically, NEK6 directly interacted with FOXO3, contributing to the phosphorylation of FOXO3 at S7 through its kinase activity, thereby inhibiting its nuclear translocation. Nuclear FOXO3 promoted FBXW7 transcription, leading to c-MYC ubiquitination and suppression of de novo purine synthesis. Paeonol, by inhibiting NEK6, suppressed de novo purine synthesis and enhanced chemosensitivity. The NEK6-mediated reprogramming of de novo purine synthesis emerges as a critical pathway influencing chemoresistance in ovarian cancer. Paeonol exhibits the potential to interfere with NEK6, thereby inhibiting chemoresistance.

Nek6 knockdown polarized macrophages into a pro-inflammatory phenotype via inhibiting STAT3 expression.

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.

NEK6 Promotes the Progression of Osteosarcoma Through Activating STAT3 Signaling Pathway by Down-Regulation of miR-26a-5p.

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. 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. 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. 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.

NIMA-related kinase 6 as an effective target inhibits the hepatocarcinogenesis and progression of hepatocellular carcinoma

BackgroundNIMA-related kinase 6 (NEK 6) is over-expressed in some tumor cell lines and tissues. However, its expression in hepatocellular carcinoma (HCC) and its correlation with clinical features remain unclear. MethodsTotal RNA from HCC liver tissues, other liver specimens, and hepatic cell lines was extracted and QPCR was adopted to detect NEK6 expression. The correlation between NEK6 expression and the clinical characteristics of HCC was analyzed. Scratch assay, Transwell assay, and tumor-formation assay were used to evaluate the effects of NEK6 on the HCC progression in vitro and in vivo. ResultsThe expression of NEK6 was up-regulated in HCC tissues and HCC cell lines: Li-7 and HepG2. The overexpression of NEK6 was correlated with hepatitis B virus infection and tumor diameter (P = 0.045). When down-regulated the expression of NEK6, both the migration and invasion capabilities of Li-7 and HepG2 cells and the growth of xenograft tumors were suppressed. (P < 0.05). ConclusionsNEK6 expression was up-regulated in HCC and correlated with the progression, suggesting it might be a valuable biomarker and a potential therapeutic target for HCC.

Predictive Role of NEK6 in Prognosis and Immune Infiltration in Head and Neck Squamous Cell Carcinoma.

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.

NEK6 gene silencing using siRNA for overcome multidrug resistance in chronic myeloid leukemia cells

Tumor cells become resistant to structurally or functionally unrelated chemotherapeutics which is called multidrug resistance (MDR). There are several mechanisms including the impairment of apoptotic pathway resulting in MDR development. NEK6 is a member of NIMA-related kinase family and it is an important mitotic kinase for proper cell cycle progression. Recent studies showed that NEK6 gene expression, protein level, and its kinase activity are increased in variety of cancer cells. We aimed to search the involvement of NEK6 in multidrug resistance and apoptosis in chronic myeloid leukemia. The expression levels of NEK6 and some of the apoptotic pathway genes such as BAX, BCL-2 and SURVIVIN were determined in sensitive and drug resistant subtypes of K652 chronic myeloid leukemia cell lines by RT-PCR method. siRNA silencing studies were performed to examine the effect of expression of NEK6 on apoptotic behavior in parental K-562 cell line. Cell viability assay was performed by XTT method in order to investigate whether NEK6 silencing leads to resistance in parental K-562 cells. NEK6 expression is significantly reduced in imatinib resistant K562 cells. After NEK6 gene is silenced by specific siRNA in parental K562 cell line, the expression levels of some apoptotic genes, such as BAX and SURVIVIN were found similar to drug resistant K562 cells. NEK6 may have potential role in imatinib resistance which may be through apoptotic pathway in chronic myeloid leukemia.

LncRNA FAM13A-AS1 Promotes Renal Carcinoma Tumorigenesis Through Sponging miR-141-3p to Upregulate NEK6 Expression.

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.

Circular RNA NEK6 contributes to the development of non-small-cell lung cancer by competitively binding with miR-382-5p to elevate BCAS2 expression at post-transcriptional level

BackgroundNon-small-cell lung cancer (NSCLC) is the commonest type of lung cancer, which is one of most deadly cancers that possess high morbidity and mortality all over the world. The function of circular RNA NIMA related kinase 6 (circ_NEK6) in NSCLC is still unknown. Therefore, circ_NEK6 is worth studying in detail.MethodsRT-qPCR and western blot assays were employed to detect gene expression. Colony formation, EdU, JC-1, flow cytometry, and Transwell assays were implemented to explore the function of circ_NEK6 on biological activities of NSCLC cells. Mechanism experiments were conducted to unveil the relationship among molecules.ResultsCirc_NEK6 expression was highly expressed in NSCLC tissues and cells. Functionally, the silencing of circ_NEK6 could effectively suppress NSCLC cell proliferation, migration and invasion. Circ_NEK6 sequestered miR-382-5p to fortify the expression of breast carcinoma amplified sequence 2 (BCAS2) in NSCLC. Besides, BCAS2 had tumor-promoting function in NSCLC. Furthermore, the effects of down-regulated circ_NEK6 on the malignant behaviors of NSCLC cells were totally recovered by miR-382-5p inhibition or BCAS2 overexpression.ConclusionsCirc_NEK6 served as a competing endogenous RNA (ceRNA) of BCAS2 by absorbing miR-382-5p, which may be treated as a novel promising target for the treatment of NSCLC.

NEK6 is an injury-responsive kinase cooperating with STAT3 in regulation of reactive astrogliosis.

In response to brain injury, resident astrocytes become reactive and play dynamic roles in neural repair and regeneration. The signaling pathways underlying such reactive astrogliosis remain largely unclear. We here show that NEK6, a NIMA-related serine/threonine protein kinase, is rapidly induced following pathological stimulations and plays critical roles in reactive astrogliosis. Enhanced NEK6 expression promotes reactive astrogliosis and exacerbates brain lesions; and conversely, NEK6 downregulation dampens injury-induced astrocyte reactivity and reduces lesion size. Mechanistically, NEK6 associates with and phosphorylates STAT3. Kinase activity of NEK6 is required for induction of GFAP and PCNA, markers of reactive astrogliosis. Interestingly, NEK6 is also localized in the nucleus and binds to STAT3-responsive genomic elements in astrocytes. These results indicate that NEK6 constitutes a molecular target for the regulation of reactive astrogliosis.

Knockdown of circ_NEK6 Decreased 131I Resistance of Differentiated Thyroid Carcinoma via Regulating miR-370-3p/MYH9 Axis.

Radioresistance is a crucial factor for the failure of iodine 131 (131I)-based radiotherapy for differentiated thyroid carcinoma (DTC). This study aimed to explore the effect of circ_NEK6 on the development of 131I resistance in DTC and its potential mechanism. In this study, we demonstrated that circ_NEK6 expression was significantly elevated in 131I-resistant DTC tissues and cell lines. Knockdown of circ_NEK6 significantly repressed 131I resistance via inhibiting cell proliferation, migration, invasion abilities, and inducing cell apoptosis and DNA damage in 131I-resistant DTC cells. Mechanistically, knockdown of circ_NEK6 suppressed 131I resistance in DTC by upregulating the inhibitory effect of miR-370-3p on the expression of myosin heavy chain 9 (MYH9). In vivo experiments showed that circ_NEK6 inhibition aggravated 131I radiation-induced inhibition of xenograft tumor growth. Taken together, knockdown of circ_NEK6 repressed 131I resistance in DTC cells by regulating the miR-370-3p/MYH9 axis, indicating that circ_NEK6 may act as a potential biomarker and therapeutic target for DTC patients with 131I resistance.

Overexpression of NIMA-related kinase 6 (NEK6) contributes to malignant growth and dismal prognosis in Human Breast Cancer

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.

Cis-Regulatory Circuits Regulating NEK6 Kinase Overexpression in Transformed B Cells Are Super-Enhancer Independent

Alterations in distal regulatory elements that controlgene 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 prioritizetranscriptional control elements as therapeutic targets.

Expression of NEK-6 in gastric cancer and its clinical significance

To explore the NEK-6 expression in gastric cancer tissue and its relationship with clinicopathological features. Fluorescent quantification PCR and Western blotting were used to examine the NEK-6 expression in 36 samples of fresh gastric cancer tissues and para-cancer gastric mucosal tissues, human gastric cancer cell lines(BGC-823, MKN-28, SGC-7901, MGC-803, HGC-27, AGS), and human normal gastric epithelial cell line (GES-1). Gastric cancer cell lines with the highest expression level were selected to perform the invasion and migration tests, and the effect of down-regulated NEK-6 expression by siRNA transfection on above invasion and migration tests were observed. Meanwhile NEK-6 expression in 94 paraffin samples of gastric cancer tissues was examined by immunohistochemistry and its positivity was compared among different clinicopathologic features. Fluorescent quantification PCR revealed gastric cancer tissues had significantly higher NEK-6 expression than para-cancer tissues(0.002 80±0.001 36 vs. 0.001 91±0.001 48, P<0.05), NEK-6 expression was up-regulated in 31 gastric cancer tissues (86.1%), and human gastric cancer cell lines had significantly higher NEK-6 expression than GES-1 cells, among whom BGC-823 and AGS cell lines were the highest. Invasion and migration tests showed that as compared to negative siRNA control group, ability of invasion and migration in BGC-823 and AGS cells after siRNA transfection was obviously decreased. In 94 paraffin samples, positive expression rate of NEK-6 was 60.6%(57/94), and NEK-6 expression was significantly associated with gastric cancer distant metastasis, lymph nodes metastasis and TNM staging(all P<0.05). NEK-6 expression is up-regulated in gastric cancer tissues, which is significantly associated with distant metastasis, lymph nodes metastasis and TNM staging. Down-regulation of NEK-6 expression can inhibit the ability of invasion and migration in gastric cancer cells.

Abstract 4327: Nek6 and Hif-1α cooperate with the cytoskeletal gateway of drug resistance to drive outcome in serous ovarian cancer

Abstract Hypoxia selects the most aggressive and drug-resistant clones in solid malignancies. One of the pivotal transcription factors induced by hypoxia is Hif-1α. However, in serous ovarian cancer (SEOC), Hif-1α expression is not a prognostic biomarker. This study aims to assess the hypothesis that the serine-threonine kinase Nek6 functions as a downstream effector cooperating with Hif-1α in driving ovarian cancer aggressiveness. Nek6 was overexpressed and Hif-1α was silenced in A2780 cells. Nek6 was also stably silenced in Hey cells. The dependence of Nek6 expression on Hif-1α was assayed as a function of hypoxic growth conditions. Nek6 interaction with the cytoskeletal gateway of drug resistance was investigated with far western blot. The co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts was quantified with qPCR in two cohorts of SEOC patients (346 locally treated patients and 344 from the TCGA dataset). Nek6 expression is induced by hypoxia in a Hif-1α dependent fashion. Nek6 directly interacts with GBP-1, thus being a component of the cytoskeletal gateway of drug resistance. Nek6 overexpression increases and silencing decreases the anchorage-independent growth of cultured cells. In SEOC patients, NEK6 expression is significantly correlated with HIF1A. Co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts identifies a subset of SEOC patients characterized by poor outcome and drug resistance. This study demonstrates the functional relevance of Nek6 in the context of the adaptive response to hypoxia in SEOC. This finding may help identify a sub-population of patients at high risk of relapse to standard first-line chemotherapy. Citation Format: Marta De Donato, Mara Fanelli, Marisa Mariani, Giuseppina Raspaglio, Deep Pandya, Shiquan He, Paul Fiedler, Marco Petrillo, Giovanni Scambia, Cristiano Ferlini. Nek6 and Hif-1α cooperate with the cytoskeletal gateway of drug resistance to drive outcome in serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2015-4327

Gga-miR-26a targets NEK6 and suppresses Marek's disease lymphoma cell proliferation

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)-induced 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 (NIMA)-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.

The inhibition of Nek6 function sensitizes human cancer cells to premature senescence upon serum reduction or anticancer drug treatment

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.

Abstract 222: NEK6 promotes JB6 C141 cell transformation and activates STAT3 through its phosphorylation on Ser727

Abstract NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that ectopic expression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. We also discovered that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser(727), which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser(206) and Thr(210) of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser(727) phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser(727). Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 222. doi:1538-7445.AM2012-222

Clinical and Biological Significance of Never in Mitosis Gene A-Related Kinase 6 (NEK6) Expression in Hepatic Cell Cancer

Nek6 is a cell cycle regulatory gene, which can control cell proliferation and survival. Recent studies suggested that desregulation of Nek6 expression plays a key role in oncogenesis. This study was aimed to investigate the potential roles of Nek6 in hepatocellular carcinoma (HCC) development. Immunohistochemistry and Western blot analysis was performed for Nek6 in 80 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were performed to determine the prognostic significance of Nek6 in HCC. In addition, Nek6 expression vector was used to detect its role in cell cycle control. Nek6 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Nek6 was associated with histological grade and the level of alpha fetal protein, and Nek6 was positively correlated with proliferation marker Ki-67. Univariate analysis showed that Nek6 expression was associated with poor prognosis. Multivariate analysis indicated that Nek6 and Ki-67 protein expression was an independent prognostic marker for HCC. While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Nek6 was upregulated. Overexpression Nek6 in Huh7 cell could promote the cell cycle. In conclusion, Nek6 is involved in the pathogenesis of hepatocellular carcinoma. It may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.

Nek6 suppresses the premature senescence of human cancer cells induced by camptothecin and doxorubicin treatment

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.