Nemo-like Kinase Expression Research Articles

The Mechanism of Nemo-Like Kinase (NLK) in Non-Small Cell Lung Cancer (NSCLC) Cells by Regulating Vascular Endothelial Growth Factor (VEGF)

Nemo-like kinase (NLK) is abnormally expressed in several tumors, but its role in NSCLC have not been reported. Real time PCR and Western blot were used to assess NLK level in tumor tissues and adjacent tissues of NSCLC. NSCLC cell line A549 cells were divided into three groups; NC group and si-NLK group which was transfected with NLK negative control or NLK siRNA respectively followed by analysis of NLK expression by real time PCR and Western blot, cell proliferation by MTT assay, cell migration by cell wound healing assay, cell invasion by transwell chamber and MMP-9 and VEGF expression by Western blot. The expression of NLK in NSCLC tumor tissue was increased, and the difference was statistically significant compared with adjacent tissues (P <0.05), and it was related to tumor size, degree of differentiation, metastasis and survival time (P <0.05). A549 cells showed significantly increased NLK. Transfection of NLK siRNA could significantly inhibit tumor cell proliferation, migration and invasion, and decrease the expression of MMP-9 and VEGF proteins (P <0.05). Elevated NLK level in NSCLC tumor tissues is related to clinicopathological characteristics. Decreased the expression of NLK can inhibit VEGF and MMP-9 expression, and inhibit cell function.

The effect of TLR-4 on the proliferation and differentiation of bone mesenchymal stem cells and its relationship with the Wnt signal transduction pathway during bone nonunion.

BackgroundBone nonunion is a special fracture complication that occurs in about 5% to 10% of cases. This type of fracture is difficult to heal, and causes great pain to patients and affects their quality of life. The mechanism of bone nonunion is not clear. In our study, we investigated the influence of Toll-like receptor (TLR)-3, TLR-4, and Wnt signaling pathways on the occurrence of bone nonunion.MethodsFirstly, we established a Sprague Dawley (SD) rat model of femoral nonunion, and detected the expression levels of TLR-3, TLR-4, β-catenin, nemo-like kinase (NLK), c-Jun N-terminal kinase (JNK), and other proteins during model construction. For in vitro experiments, primary cultured bone mesenchymal stem cells (BMSCs) were divided into 4 groups: lipopolysaccharide (LPS, agonist of TLR-4) group, LPS + CLI095 (inhibitor of TLR-4) group, control group, and LPS + substance P (SP) group. The expression of β-catenin, NLK, JNK, and ALP and the osteogenic differentiation ability of cells were detected during culture.ResultsX-ray and hematoxylin and eosin (HE) staining results confirmed the successful modeling of bone nonunion. During the formation of the bone nonunion model, the expression of TLR-4 showed an upward trend. In vitro experiment results showed that inhibition of TLR-4 expression could enhance the proliferation and differentiation ability of BMSCs. The expression of β-catenin, the core protein of the canonical Wnt signaling pathway, increased rapidly in the first 2 weeks of bone nonunion construction, and decreased after 2 weeks. Non-canonical Wnt signaling pathway proteins NLK and JNK had no change in the first 2 weeks, and showed an upward trend after 2 weeks. In vitro experiment results showed that the expression of β-catenin was dominant in BMSCs with strong proliferation and differentiation ability, while the expression of NLK and JNK was dominant in BMSCs with weak proliferation and differentiation ability. These results suggest that the Wnt signaling pathway may regulate the occurrence of bone nonunion.ConclusionsTLR-4 inhibits the proliferation and differentiation of BMSCs, and the transformation of the canonical Wnt signaling pathway to the non-canonical Wnt signaling pathway may lead to bone nonunion. Our study may provide new insights into the treatment of bone nonunion.

Nutritional Supplements, Ginseng and Leucine, Increase Erythropoiesis in Diamond Blackfan Anemia Models through Inhibition of Nemo-like Kinase

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is typically associated with mutations in one of at least 20 ribosomal genes and is associated with anemia, congenital abnormalities, and cancer predisposition. The current treatment for DBA is associated with toxicities including iron overload from repeated transfusions, immune suppression from chronic corticosteroid therapy or consequences from stem cell transplantation. Developing new therapies for DBA remains a challenge, since it is a rare disease and the connection between faulty ribosomes and defects in erythropoiesis is still being explored. As such, repurposing existing, approved drugs is one approach to find new ways to treat this disease.We recently identified that Nemo-like Kinase (NLK) is hyper-activated and contributes to disease pathogenesis in the erythroid progenitors of patients with DBA, irrespective of the genetic mutation. NLK is an atypical member of the MAPK family of kinases. Due to a high degree of conservation, kinase inhibitors that specifically target NLK are not currently available, although several small molecules inhibit NLK as an off-target. We are actively pursuing a number of these compounds as potential therapies for DBA. In addition to known kinase inhibitors, we have examined the active components of widely available nutritional supplements. Although rigorous clinical evaluation is required, these supplements are well tolerated and offer an alternative or a complement to conventional drug therapy for DBA.Ginsenoside Rb1, an active component of ginseng, increases erythropoiesis in in vitro models of DBA (2.6-fold) at 50mM (p=0.048) with an EC 50 of 2.3mM. Importantly, ginsenoside Rb1 does not impact healthy erythropoiesis or other hematopoietic lineages and is nontoxic to normal cells. Our results demonstrate that ginsenoside Rb1 does not inhibit NLK kinase activity directly, but rather induces a microRNA (miR-208) that binds to NLK mRNA leading to degradation of transcript by 35.9% (p=0.007) before the protein can be translated.Another nutritional supplement that has been indicated to improve erythropoiesis (in DBA and non-diseased models) and is currently in clinical trials is the amino acid leucine. In DBA progenitors, 5mM leucine increases erythropoiesis from 8.8 to 16.3% of healthy controls. Leucine acts by stimulating the activity of mTORC1 but similar to erythropoiesis, mTORC1 activity is only stimulated from 26.4 to 57.2% of controls in DBA progenitors.Our results demonstrate that leucine does not impact NLK expression or activity directly. Aberrantly activated NLK phosphorylates and inhibits the activation of mTORC1 (target of leucine). The suppression of NLK by 50mM ginsenoside Rb1 restored mTORC1 activity to basal (94.7% of non-diseased control) but also restored leucine sensitivity (from 57.2 to 88.1% of non-diseased controls). Importantly, combining 5mM leucine and 50mM ginsenoside Rb1 increased erythropoiesis from 1.9-fold and 2.6-fold when used alone, to 8.9-fold (or 78.3% of healthy controls) together in our in vitro models of DBA (CI=0.31).Ginseng and leucine offer promising alternatives to steroids and other immunosuppressive drugs for DBA patients. The goal of these studies is to raise the hemoglobin in DBA patients to avoid the need for red cell transfusions. As nutritional supplements are widely available and well tolerated, this class of compounds provides alternatives to currently approved drugs to treat DBA. DisclosuresGlader: Agios: Consultancy.

The active component of ginseng, ginsenoside Rb1, improves erythropoiesis in models of Diamond–Blackfan anemia by targeting Nemo-like kinase

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.

Pan-cancer analysis of nemo-like kinase (NLK) expression convergence to pancreatic adenocarcinoma that has diagnostic, prognostic and treatment value

BackgroundNemo-like kinase (NLK) is an evolutionarily conserved mitogen-activated protein (MAP) kinase-related kinase. Nemo-like kinase (NLK) can not only undergo autophosphorylation but is also involved in many critical signaling pathways as a regulator of tumorigenesis. In recent years, accumulating shreds of evidence have suggested that NLK is a crucial molecule regulating the occurrence and development of various tumors. Abnormal expression of NLK not only participates in cancer cell growth, proliferation, migration, and apoptosis, but also has significant reference value for disease prognosis. Moreover, in different types of cancer, the effects of NLK over- or under-expression are not consistent. Despite these advances, no studies comprehensively describe the significance of Nemo-like kinase expression in all 33 kinds of cancer, or have evaluated its prognostic value. Materials & methodsA total of 10,201 samples were obtained from the GDC data portal and four groups of GEO datasets were collected for verification. A variety of bioinformatics analysis methods were used to reveal the roles of NLK in the diagnosis, treatment, and prognosis of tumor patients. ConclusionNLK is significantly overexpressed in pancreatic adenocarcinoma (PAAD) tissues than normal controls. The NLK expression in PAAD is also different in different pathological stages. NLK is a prognostic biomarker and is correlated with immune infiltrates in PAAD. The abnormal expression of NLK in PAAD will influence the expression of a variety of genes, whose functions are enriched in multiple metabolism, hormone secretion, and even neuron regulation pathways. More importantly, the IHC slides for PAAD and corresponding normal tissues were significantly different, which can be used in clinical diagnosis.

A kinome screen reveals that Nemo-like kinase is a key suppressor of hepatic gluconeogenesis

Antihyperglycemic therapy is an important priority for the treatment of type 2 diabetes (T2D). Excessive hepatic glucose production (HGP) is a major cause of fasting hyperglycemia. Therefore, a better understanding of its regulation would be important to develop effective antihyperglycemic therapies. Using a gluconeogenesis-targeted kinome screening approach combined with transcriptome analyses, we uncovered Nemo-like kinase (NLK) as a potent suppressor of HGP. Mechanistically, NLK phosphorylates and promotes nuclear export of CRTC2 and FOXO1, two key regulators of hepatic gluconeogenesis, resulting in the proteasome-dependent degradation of the former and the inhibition of the self-transcriptional activity and expression of the latter. Importantly, the expression of NLK is downregulated in the liver of individuals with diabetes and in diabetic rodent models and restoring NLK expression in the mouse model ameliorates hyperglycemia. Therefore, our findings uncover NLK as a critical player in the gluconeogenic regulatory network and as a potential therapeutic target for T2D.

Nemo-Like Kinase in Development and Diseases: Insights from Mouse Studies.

The Wnt signalling pathway is a central communication cascade between cells to orchestrate polarity and fate during development and adult tissue homeostasis in various organisms. This pathway can be regulated by different signalling molecules in several steps. One of the coordinators in this pathway is Nemo-like kinase (NLK), which is an atypical proline-directed serine/threonine mitogen-activated protein (MAP) kinase. Very recently, NLK was established as an essential regulator in different cellular processes and abnormal NLK expression was highlighted to affect the development and progression of various diseases. In this review, we focused on the recent discoveries by using NLK-deficient mice, which show a phenotype in the development and function of organs such as the lung, heart and skeleton. Furthermore, NLK could conduct the function and differentiation of cells from the immune system, in addition to regulating neurodegenerative diseases, such as Huntington’s disease and spinocerebellar ataxias. Overall, generations of NLK-deficient mice have taught us valuable lessons about the role of this kinase in certain diseases and development.

Metformin Upregulates Mir-26a to Improve Erythropoiesis in Preclinical Models of Diamond Blackfan Anemia through Suppression of Nlk Expression

Nemo-like Kinase (NLK) is an atypical member of the Mitogen-activated protein (MAP) kinase family and shares a highly conserved kinase domain with other MAP kinases. For this reason it is challenging to design small molecules that specifically inhibit NLK. In hematopoiesis, NLK expression is suppressed in all lineages except erythroid progenitors. In non-erythroid progenitors, micro-ribonucleic acid (miR)-181 is upregulated and binds to the 3' untranslated region (UTR) of NLK transcripts, resulting in dramatic degradation of NLK transcripts. NLK is not activated or required for healthy erythropoiesis, but in Diamond Blackfan Anemia (DBA), NLK is chronically hyper-activated, which contributes to failed erythroid expansion and disease pathogenesis. Given the sensitivity of NLK transcripts to 3'UTR binding-mediated degradation, we hypothesized that targeting NLK degradation by upregulating miRNAs that bind the NLK 3'-UTR could provide an alternative approach to directly inhibiting the kinase to suppress aberrant NLK activity in bone marrow progenitors from DBA patients. One miRNA binding site that is present in the NLK-3'UTR is miR-26a. We determined that miR-26a is upregulated 2.4-fold (p=0.009) in CD235+ erythrocytes by the Type 2 diabetes drug metformin. Metformin suppressed NLK expression 62.4% (p=0.006) and improved erythroid expansion 4-fold (p=0.001) in human and 6-fold (p=0.042) in zebrafish models of DBA. This effect required an intact miR-26a binding site within the NLK 3'UTR. However, metformin failed to induce miR-26a in murine models of DBA, however the exogenous expression of miR-26a suppressed NLK 36.4% (p=0.028) and improved erythropoiesis 2-fold (p=0.017). In summary, targeted suppression of NLK through metformin-mediated upregulation of NLK transcript-binding miRNAs effectively improved red blood cell production from 5.6% up to 33.3% of controls in a preclinical model of DBA. These results suggest that metformin is a potential drug for treatment of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy. OffLabel Disclosure: metformin. This drug is given to differentiating HSPCs in vitro to examine effect on specific kinases.

Metformin-induced suppression of Nemo-like kinase improves erythropoiesis in preclinical models of Diamond–Blackfan anemia through induction of miR-26a

Diamond-Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3' untranslated region (3'UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.

Overexpression of Nemo-like Kinase Promotes the Proliferation and Invasion of Lung Cancer Cells and Indicates Poor Prognosis.

Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinaserelated kinase involved in the pathogenesis of several human cancers. The aim of this study was to investigate the expression and role of NLK in lung cancers, and its underlying mechanisms. We examined the expression of NLK in lung cancer tissues through western blot analysis. We enhanced or knocked down NLK expression by gene transfection or RNA interference, respectively, in lung cancer cells, and examined expression alterations of key proteins in the Wnt signaling pathway and in epithelial-mesenchymal transition (EMT). We also examined the roles of NLK in the proliferation and invasiveness of lung cancer cells by cell proliferation, colony formation, and Matrigel invasion assays. NLK expression was found to be significantly higher in lung cancer tissue samples than in corresponding healthy lung tissue samples. Overexpression of NLK correlated with poor prognosis of patients with lung cancer. Overexpression of NLK upregulated β-catenin, TCF4, and Wnt target genes such as cyclin D1, c-Myc, and MMP7. N-cadherin and TWIST, the key proteins in EMT, were upregulated, while E-cadherin expression was reduced. Additionally, proliferation, colony formation, and invasion turned out to be enhanced in NLK-overexpressing cells. After NLK knockdown in lung cancer cells, we obtained the opposite results. NLK is overexpressed in lung cancers and indicates poor prognosis. Overexpression of NLK activates the Wnt signaling pathway and EMT and promotes the proliferation and invasiveness of lung cancer cells.

Decreased expression of nemo-like kinase in melanoma is correlated with increased vascularity and metastasis.

Melanoma is a highly metastatic cancer, and its incidence has increased over the past several decades. Angiogenesis is associated with melanoma metastasis and a poor prognosis. Many genetic and epigenetic factors affecting tumour vascularization and metastasis have been investigated, despite the heterogeneity of cancer cells and the complicated mechanisms involved in melanoma. Nemo-like kinase (NLK) is a serine/threonine kinase regulating the transcription factor by negatively regulating Wnt and downstream vascular endothelial growth factor receptor 2 (VEGFR2) signalling. This study aimed to investigate whether NLK expression in melanoma correlates with VEGFR2-related angiogenesis and melanoma metastasis. Immunohistochemistry analysis using 175 biopsied tissues of melanoma patients showed that NLK is expressed in 73.7% of melanoma tissues, whereas 26.3% of the samples showed absent expression of NLK. In metastatic melanoma, the expression of NLK was significantly lower than that in primary melanoma (P = 0.002). Furthermore, tissues with a lower expression of NLK showed a higher microvessel density as detected by VEGFR2 expression compared with tissues showing higher NLK expression. These data suggest that reduced expression of NLK in melanoma correlates with VEGFR2-related microvessel formation and melanoma metastasis. This study showed that NLK may serve as a novel prognosis marker and revealed new mechanisms in melanoma metastasis.

Knockdown of Nemo‑like kinase promotes metastasis in non‑small‑cell lung cancer

The evolutionarily conserved serine/threonine kinase Nemo-like kinase (NLK) serves an important role in cell proliferation, migration, invasion and apoptosis by regulating transcription factors among various cancers. In the present study, the function of NLK in human non-small cell lung cancer (NSCLC) was investigated. Immunohistochemical analysis and western blotting demonstrated that NLK expression was significantly reduced in NSCLC tissues compared with corresponding peritumoral tissues. Statistical analysis revealed that decreased NLK expression was associated with the presence of primary tumors, tumor node metastasis (TNM) staging, differentiation, lymph node metastasis, and E-cadherin and vimentin expression. Univariate analysis indicated that NLK expression, differentiation, lymph node metastasis, TNM stage, and E-cadherin and vimentin expression affected the prognosis of NSCLC. Cox regression analyses revealed NLK expression and TNM as independent factors that affected prognosis. Kaplan-Meier survival analysis revealed that patients with NSCLC and low NLK expression had relatively shorter durations of overall survival. In vitro, NLK overexpression inhibited A549 ncell migration and invasion as determined by wound healing and Transwell migration assays, respectively. Additionally, immunofluorescence staining indicated that downregulation of NLK expression could induce epithelial-mesenchymal transition in NSCLC. NLK knockdown significantly decreased the expression of the epithelial marker E-cadherin, and markedly increased that of β-catenin and the mesenchymal marker vimentin. Furthermore, NLK was reported to directly interact with β-catenin as determined by a co-immunoprecipitation assay. Collectively, the results of the present study indicated that decreased NLK expression could promote tumor metastasis in NSCLC.

Effect of NLK on the proliferation and invasion of laryngeal carcinoma cells by regulating CDCP1.

The morbidity and mortality of laryngeal cancer are increasing rapidly, seriously threatening human health. There are several causes of laryngeal cancer, but the exact molecular mechanism is unclear. Finding the molecular targets of laryngeal cancer has become an emerging hot spot. Nemo-like kinase (NLK) is abnormally expressed in tumors, but its role in laryngeal cancer has not been reported. Real Time PCR and Western blot were used to detect NLK mRNA and protein expression in cancer tissues and adjacent tissues of laryngeal cancer patients. The laryngeal carcinoma cell line Hep-2 cells were cultured in vitro and randomly divided into three groups: control group, NC group, and NLK siRNA group followed by the analysis of cell proliferation by MTT assay, Caspase3 activity, and cell invasion by the transwell chamber. MMP-9 and CDCP1 expression was measured by Western blot. NLK mRNA and protein expression was significantly increased in laryngeal carcinoma tissues compared with those in adjacent tissues (p<0.05). NLK siRNA transfection into Hep-2 cells significantly down-regulated NLK expression, inhibited Hep-2 cell proliferation and invasion, increased Caspase-3 activity with statistical differences compared to control group (p<0.05). Down-regulation of NLK expression in Hep-2 cells inhibited MMP-9 expression and decreased CDCP1 expression. NLK is expressed in tumor tissues of patients with laryngeal cancer. The down-regulation of NLK expression may play a role in the proliferation, apoptosis, and invasion of laryngeal carcinoma cells and it is possible by regulating MMP-9 and CDCP1 expression.

Expression of Nemo-like kinase in cervical squamous cell carcinoma: a clinicopathological study.

ObjectiveNemo-like kinase (NLK) has been reported to play different roles in tumors. However, the role of NLK in cervical squamous cell carcinoma (CSCC) remains unknown. In this study, we explored the clinical significance including survival of NLK protein expression in CSCCs.Patients and methodsImmunohistochemical method was performed using tissues from 130 patients with CSCC. The associations between NLK expression and the clinicopathological factors and prognosis of CSCCs were evaluated. Statistical analyses were performed using the chi-square test, the multivariate Cox proportional hazard model, and the Kaplan–Meier method.ResultsImmunohistochemical staining analysis showed that NLK was localized predominately in the nucleus of the tumor cells, and increased NLK expression was detected in 71 (54.6%) of 130 patients. NLK overexpression significantly correlated with higher histological grade (P=0.001), vascular/lymphatic invasion (P=0.010), lymph node metastasis (P=0.012), and recurrence (P=0.022). Patients with elevated NLK expression had poorer overall survival (OS) and disease-free survival (DFS) (P=0.006 and P=0.004, respectively) compared with patients with decreased NLK expression. Multivariate Cox analysis demonstrated that NLK overexpression was an independent factor for OS and DFS (P=0.034 and P=0.025, respectively).ConclusionNLK may be a valuable biomarker for predicting the prognosis of CSCC patients and may serve as a potential target for cancer therapy.

Nemo-like Kinase Hyper-Activated in Erythroid Progenitors in Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life. Approximately 70% of DBA patients possess a mutation in one of at least 12 ribosomal proteins, with RPS19 being the most prevalent, accounting for over 25% of cases. In mice, complete loss of RPS19 is not viable, yet mutations that result in haploinsufficiency result in failure of erythropoiesis due to a blockage during differentiation of early erythroblast progenitors. An increase in p53 stability has been attributed to the disease, but the downstream signaling molecules mediating aberrant erythropoiesis have not been completely characterized.Here, we report that Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19 insufficient CD34+ HSPCs derived from fetal liver, cord blood and mobilized peripheral blood differentiating toward the erythroblast lineage, as well as DBA-patient derived EBV-transformed Human B lymphoblastic cell lines In in vitro kinase assays, significant increases in the extent of phosphorylation of three (3) NLK kinase substrates - NLK (3.86 fold), c-Myb (5.22 fold) and Raptor (9.82 fold) was observed in the presence of immunopurified NLK during RPS19 insufficiency. This activation is due to p53-mediated upregulation of TNFα that activates NLK in neighboring cells in a paracrine fashion. In differentiating HSPCs, RPS19 insufficiency results in increased phosphorylation of the critical erythroid transcription factor c-Myb (9.6 fold) and autophagy-regulating Raptor (5.3 fold), both of which are restored to basal levels upon expression of siRNA against NLK.Phosphorylation of c-Myb by NLK results in increased ubiquitination and proteasomal degradation with subsequent loss of c-Myb-induced genes critical for transcriptional regulation of erythropoiesis (including LMO2 and KLF1). Raptor phosphorylation by NLK inhibits mTOR activation, resulting in premature autophagy in erythroblast progenitors. Knockdown of NLK using siRNAs significantly increased c-Myb downstream gene expression (p<0.0001), reduced autophagy (p<0.001) and increased erythrocyte maturation (p<0.001) in RPS19 insufficient human HSPCs. NLK expression is high in HSPCs and committed erythroid progenitors but is significantly downregulated in lymphoid, myeloid and megakaryocyte committed progenitors. In contrast, TNFα receptors are expressed at low levels in stem cells but increases during differentiation. Collectively, we propose the synergy of high NLK and TNFα receptor expression in erythroid progenitors contributes to selective erythroid progenitor hyper-activation of NLK in the TNFα-rich bone marrow niche DBA patients that contributes to red blood cell aplasia.Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise as potential druggable targets in the clinical management of DBA. DisclosuresGlader:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

The effect of EBV on WIF1, NLK, and APC gene methylation and expression in gastric carcinoma and nasopharyngeal cancer.

Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p.

Lentivirus-mediated knockdown of NLK inhibits small-cell lung cancer growth and metastasis.

Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, has been recognized as a critical regulator of various cancers. In this study, we investigated the role of NLK in human small-cell lung cancer (SCLC), which is the most aggressive form of lung cancer. NLK expression was evaluated by quantitative real-time polymerase chain reaction in 20 paired fresh SCLC tissue samples and found to be noticeably elevated in tumor tissues. Lentivirus-mediated RNAi efficiently suppressed NLK expression in NCI-H446 cells, resulting in a significant reduction in cell viability and proliferation in vitro. Moreover, knockdown of NLK led to cell cycle arrest at the S-phase via suppression of Cyclin A, CDK2, and CDC25A, which could contribute to cell growth inhibition. Furthermore, knockdown of NLK decreased the migration of NCI-H446 cells and downregulated matrix metalloproteinase 9. Treatment with NLK short hairpin RNA significantly reduced SCLC tumor growth in vivo. In conclusion, this study suggests that NLK plays an important role in the growth and metastasis of SCLC and may serve as a potential therapeutic target for the treatment of SCLC.

MicroRNA-208a Potentiates Angiotensin II-triggered Cardiac Myoblasts Apoptosis via Inhibiting Nemo-like Kinase (NLK).

MicroRNA-208a (miR-208a) exacerbated cardiomyocyte apoptosis via inhibiting nemo-like kinase (NLK). miR-208a is a crucial molecule in the regulation of heart diseases, however, the biological function and underlying mechanism of miR-208a in the progression of cardiomyocyte apoptosis is not clearly elucidated. We hypothesized that miR-208a might potentiate cardiomyocyte apoptosis through inhibiting NLK. Male Sprague-Dawley rats were underwent permanent coronary artery ligation to establish myocardial infarction (MI) model. The quantitative real-time RT-PCR (qRT-PCR) was used to evaluate the expression of miR-208a and NLK mRNA. Western blot was applied to detect NLK and Bcl-2 proteins expression. Luciferase reporter assay was performed to indentify NLK as a target of miR-208a. The apoptosis of H9C2 cells was assessed by flow cytometry (FCM). miR-208a was upregulated accompanying with a significant decrease of NLK in response to MI, and stronger miR-208a staining was detected by in situ hybridization in the cytoplasm of cardiomyocytes in MI group compared to the sham group. In vitro, overexpression of miR-208a greatly enhance Ang II-induced the apoptosis of H9C2 cells through downregulating of NLK and the anti-apoptosis protein Bcl-2 expression, whereas these effects were reversed when miR-208a was downregulated. Dual luciferase reporter assay and western blot results demonstrated that NLK was a direct target of miR-208a. Interestingly, upregulation of NLK obviously increased Bcl-2 expression and reduced the percentage of apoptotic cells, while attenuation of NLK reduced the level of Bcl-2 and cells apoptosis after treatment with Ang II. miR-208a can promote Ang II-induced cardiomyocyte apoptosis via negatively regulating NLK expression, and inhibition of miR-208a may provide a novel therapeutic target for cardiomyocyte apoptosis.

Nemo-like kinase as a negative regulator of nuclear receptor Nurr1 gene transcription in prostate cancer.

BackgroundNurr1, a member of the orphan receptor family, plays an important role in several types of cancer. Our previous work demonstrated that increased expression of Nurr1 plays a significant role in the initiation and progression of prostate cancer (PCa), though the mechanisms for regulation of Nurr1 expression remain unknown. In this study, we investigated the hypothesis that Nemo-like kinase (NLK) is a key regulator of Nurr1 expression in PCa.MethodsImmunohistochemistry and Western blot analysis were used to evaluate levels of NLK and Nurr1 in prostatic tissues and cell lines. The effects of overexpression or knockdown of Nurr1 were evaluated in PCa cells through use of PCR, Western blots and promoter reporter assays. The role of Nurr1 promoter cis element was studied by creation of two mutant Nurr1 promoter luciferase constructs, one with a mutated NF-κB binding site and one with a mutated CREB binding site. In addition, three specific inhibitors were used to investigate the roles of these proteins in transcriptional activation of Nurr1, including BAY 11–7082 (NF-κB inhibitor), KG-501 (CREB inhibitor) and ICG-001 (CREB binding protein, CBP, inhibitor). The function of CBP in NLK-mediated regulation of Nurr1 expression was investigated using immunofluorescence, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation assays (ChIPs).ResultsNLK expression was inversely correlated with Nurr1 expression in prostate cancer tissues and cell lines. Overexpression of NLK suppressed Nurr1 promoter activity, leading to downregulation of Nurr1 expression. In contrast, knockdown of NLK demonstrated opposite results, leading to upregulation of Nurr1. When compared with the wild-type Nurr1 promoter, mutation of NF-κB- and CREB-binding sites of the Nurr1 promoter region significantly reduced the upregulation of Nurr1 induced by knockdown of NLK in LNCaP cells; treatment with inhibitors of CREB, CBP and NF-κB led to similar results. We also found that NLK directly interacts with CBP, that knockdown of NLK significantly increases the recruitment of CBP to both NF-κB- and CREB-binding sites, and that regulation of NLK on Nurr1 expression is abrogated by knockdown of CBP.ConclusionsOur results suggest that NLK inhibits transcriptional activation of Nurr1 gene by impeding CBP’s role as a co-activator of NF-κB and CREB in prostate cancer.

The emerging role of Nemo-like kinase (NLK) in the regulation of cancers.

Nemo-like kinase (NLK) is an evolutionarily conserved mitogen-activated protein (MAP) kinase-related kinase that is highly expressed in neural tissues and minimally detected in others. Accumulating evidence demonstrates that NLK exerts a pivotal role in cell proliferation, migration, invasion, and apoptosis via regulation of a variety of transcriptional molecules. The results of recent studies have shown that aberrant expression of NLK is significantly associated with the initiation and progression of various types of human cancers, as well as clinicopathologic features and survival rate. NLK is gradually considered as a potential tumor suppressor or an oncogene depending on the tumor system, and silencing or upregulating of NLK may provide an effective therapeutic approach against tumors. In this review, we will make a summary on the comprehensive roles of NLK in the regulation of various cancers.