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Naked cuticle homolog 1 (NKD1), which is expressed at low levels in many tumors, is considered an inhibitor of the Wnt/β‐catenin pathway, but it is highly expressed in colon cancer and can promote colon cancer cell proliferation. miRNAs are involved in the occurrence and progression of many tumors. However, miRNAs that can regulate NKD1 and the mechanisms by which NKD1 regulates tumor progression remain ambiguous. This research aims to reveal the potential regulatory network of NKD1 in colon cancer. miRNA data downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were analyzed by bioinformatics to screen for potential miRNAs targeting NKD1. Let‐7b‐5p was found to inhibit proliferation, migration, and invasion of colon cancer cells targeting NKD1. Further studies suggested that let‐7b‐5p can modulate Wnt signaling activity, and the nuclear accumulation of β‐catenin was significantly restrained by let‐7b‐5p through targeting NKD1. Moreover, NKD1 could prohibit the expression of the APC protein. Further studies manifested that NKD1 bound to APC and promoted the ubiquitination degradation of APC through restraining the expression of the deubiquitinating enzyme USP15 and blocking the combination between USP15 and APC. Functionally, NKD1 enhanced the proliferation and migration of colon cancer cells by inhibiting APC expression. This research revealed a novel mechanism by which the let‐7b‐5p‐NKD1‐APC‐β‐catenin signaling pathway inhibited colon cancer cell progression. [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancer is an important death‐related disease in the worldwide. However, specific colon cancer tumor markers currently used for diagnosis and treatment are few. The purpose of this study is to screen the potential colon cancer markers by bioinformatics and verify the results with experiments. Methods Gene expression data were downloaded from two different databases: TCGA database and GEO datasets, which were then analyzed by two different methods (difference analysis and WGCNA method). Venn and PPI analysis obtained the potential core genes, which were then performed the GO enrichment and KEGG pathway analysis. Expressions levels of NKD1 in colon carcinoma tissues were further confirmed by immunohistochemical staining and western blot assays. Moreover, the function was measured by MTT, clone formation, and tumor transplantation experiments. Importantly, co‐immunoprecipitation, immunofluorescence, and protein stability assays were further performed to explore the underlying mechanism of NKD1 promoting cell proliferation. Results Nine potential core genes highly expressed in colon cancer samples were screened out by bioinformatics analysis. NKD1, one of the hub genes, highly expressed in the colon carcinoma tissues could enhance the proliferation of colon cancer cells. Mechanism research demonstrated that NKD1 was essential for the combination between Wnt signalosome (DVL) and β‐catenin, and that NKD1 knockout remarkably decreased the β‐catenin expression. Immunofluorescence assays further implied that NKD1 knockout significantly inhibited β‐catenin nuclear accumulation. Importantly, the stability of β‐catenin proteins was maintained by NKD1 in the colon cancer cells. Conclusion We believe that NKD1 well expressed in the colorectal carcinoma tissues can enhance the proliferation of colon cancer cells. Furthermore, the functions that NKD1 may have in colon cancer cells should be different from that NKD1 has played in the zebrafish. Thus, NKD1 could be a specific colorectal cancer marker.

Background: Colorectal cancer is an important death‐related disease in the worldwide. However, specific colon cancer tumor markers currently used for diagnosis and treatment are few. The purpose of this study is to screen the potential colon cancer markers by bioinformatics and verify the results with experiments. Methods: Gene expression data were downloaded from two different databases: TCGA database and GEO datasets, which were then analyzed by two different methods (difference analysis and WGCNA method). Venn and PPI analysis obtained the potential core genes, which were then performed the GO enrichment and KEGG pathway analysis. Expressions levels of NKD1 in colon carcinoma tissues were further confirmed by immunohistochemical staining and western blot assays. Moreover, the function was measured by MTT, clone formation, and tumor transplantation experiments. Importantly, co‐immunoprecipitation, immunofluorescence, and protein stability assays were further performed to explore the underlying mechanism of NKD1 promoting cell proliferation. Results: Nine potential core genes highly expressed in colon cancer samples were screened out by bioinformatics analysis. NKD1, one of the hub genes, highly expressed in the colon carcinoma tissues could enhance the proliferation of colon cancer cells. Mechanism research demonstrated that NKD1 was essential for the combination between Wnt signalosome (DVL) and β‐catenin, and that NKD1 knockout remarkably decreased the β‐catenin expression. Immunofluorescence assays further implied that NKD1 knockout significantly inhibited β‐catenin nuclear accumulation. Importantly, the stability of β‐catenin proteins was maintained by NKD1 in the colon cancer cells. Conclusion: We believe that NKD1 well expressed in the colorectal carcinoma tissues can enhance the proliferation of colon cancer cells. Furthermore, the functions that NKD1 may have in colon cancer cells should be different from that NKD1 has played in the zebrafish. Thus, NKD1 could be a specific colorectal cancer marker. [ABSTRACT FROM AUTHOR]

Dysregulation of NKD1 has been identified in several solid tumors. However, the status of NKD1 expression and its clinical implication in acute myeloid leukemia remain largely elusive. NKD1 transcript level in bone marrow mononuclear cells was detected by real-time quantitative polymerase chain reaction in 126 de novo acute myeloid leukemia patients and 30 controls. Clinical significance of NKD1 expression was obtained by the comparison between the patients with low and high NKD1 expression. NKD1 messenger RNA level was significantly decreased in acute myeloid leukemia patients compared with controls (p = 0.019). There were no significant differences between patients with low and high NKD1 expression in sex, age, peripheral blood cells, bone marrow blasts, French-American-British/World Health Organization subtypes, and karyotypes/karyotypic classifications (p > 0.05). Although no significant difference was observed in complete remission rate between NKD1low and NKD1high patients (p > 0.05), Kaplan-Meier analysis revealed that NKD1low patients showed shorter overall survival time than NKD1high patients in whole-cohort acute myeloid leukemia, non-M3 acute myeloid leukemia, and cytogenetically normal acute myeloid leukemia (p = 0.014, 0.063, and 0.020). Multivariate analyses disclosed the low NKD1 expression was an independent risk factor in cytogenetically normal acute myeloid leukemia patients (hazard ratio = 0.397, p = 0.017). Moreover, the prognostic value of NKD1 expression was confirmed by gene expression profile data in cytogenetically normal acute myeloid leukemia patients (p = 0.028 and 0.011). NKD1 showed significantly increased level after induction chemotherapy achieved complete remission in follow-up paired acute myeloid leukemia patients (p < 0.001). These findings indicated that reduced NKD1 expression is associated with unfavorable clinical outcome in cytogenetically normal acute myeloid leukemia. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a poorly prognostic cardiopulmonary disease characterized by abnormal contraction and remodeling of pulmonary artery (PA). Excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) are considered as the major etiology of PA remodeling. As a negative regulator of Wnt/β-catenin pathway, naked cuticle homolog 1 (NKD1) is originally involved in the tumor growth and metastasis via affecting the proliferation and migration of different types of cancer cells. However, the effect of NKD1 on PAH development has not been investigated. In the current study, downregulated NKD1 was identified in hypoxia-challenged PASMCs. NKD1 overexpression by adenovirus carrying vector encoding Nkd1 (Ad- Nkd1 ) repressed hypoxia-induced proliferation and migration of PASMCs. Mechanistically, upregulating NKD1 inhibited excessive reactive oxygen species (ROS) generation and β-catenin expression in PASMCs after hypoxia stimulus. Both inducing ROS and recovering β-catenin expression abolished NKD1-mediated suppression of proliferation and migration in PASMCs. In vivo , we also observed decreased expression of NKD1 in dissected PAs of monocrotaline (MCT)-induced PAH model. Upregulating NKD1 by Ad- Nkd1 transfection attenuated the increase in right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), pulmonary vascular wall thickening, and vascular β-catenin expression after MCT treatment. After recovering β-catenin expression by SKL2001, the vascular protection of external expression of NKD1 was also abolished. Taken together, our data suggest that NKD1 inhibits the proliferation, migration of PASMC, and PAH via inhibition of β-catenin and oxidative stress. Thus, targeting NKD1 may provide novel insights into the prevention and treatment of PAH.

Objective: Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation., Methods: HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1 -/- cells), and SW620-nkd1 -/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells., Results: In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells ( P < 0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter ( P < 0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells ( P < 0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells ( P < 0.05)., Conclusion: NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.

Background: Naked cuticle Drosophila 1 (NKD1) has been related to non-small cell lung cancer in that decreased NKD1 levels have been associated with both poor prognosis and increased invasive quality. Methods: Forty cases of lung adenocarcinoma staged as Tis or T1a were selected. Cases were subclassified into adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and small adenocarcinoma (SAD). Immunohistochemical studies for NKD1 were performed. Results: Forty samples comprised five cases of AIS (12.5%), eight of MIA (20.0%), and 27 of SAD (67.5%). AIS and MIA showed no lymph node metastasis and 100% disease-free survival, whereas among 27 patients with SAD, 2 (7.4%) had lymph node metastasis, and 3 (11.1%) died from the disease. Among the 40 cases, NKD1-reduced expression was detected in 8 (20%) samples, whereas normal expression was found in 15 (37.5%) and overexpression in 17 (42.5%). Loss of NKD1 expression was significantly associated with lymph node metastasis (p=0.001). All cases with predominant papillary pattern showed overexpression of NKD1 (p=0.026). Conclusions: Among MIA and SAD, MIA had better outcomes than SAD. Down-regulated NKD1 expression was closely associated with nodal metastasis, and overexpression was associated with papillary predominant adenocarcinoma. [ABSTRACT FROM AUTHOR]

In most colorectal tumours, APC mutation stabilises beta-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of beta-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca(2+) receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours. [ABSTRACT FROM AUTHOR]

The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor characterized by an inevitably dismal prognosis, impel for investigation. The importance of Long noncoding RNAs as regulators of gene expression has recently become evident. Among them, H19 has a recognized oncogenic role in several types of human tumors and was shown to correlate to some oncogenic aspects of glioblastoma cells. Here we, hypothesyze that in glioblastoma H19 exerts its function through the interaction with the catalytic subunit of the PRC2 complex, EZH2. By employing a factor analysis on a SAGE dataset of 12 glioblastoma samples, we show that H19 expression in glioblastoma tissues correlates with that of several genes involved in glioblastoma growth and progression. H19 knock-down reduces viability, migration and invasiveness of two distinct human glioblastoma cell lines. Most importantly, we provide a mechanistic perspective about the role of H19 in glioblastoma cells, by showing that its expression is inversely linked to that of NKD1, a negative regulator of Wnt pathway, suggesting that H19 might regulate NKD1 transcription via EZH2-induced H3K27 trimethylation of its promoter. Indeed, we showed that H19 binds EZH2 in glioblastoma cells, and that EZH2 binding to NKD1 and other promoters is impaired by H19 silencing. In this work we describe H19 as part of an epigenetic modulation program executed by EZH2, that results in the repression of Nkd1. We believe that our results can provide a new piece to the complex puzzle of H19 function in glioblastoma., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Dysregulation of NKD1 has been identified in several solid tumors. However, the status of NKD1 expression and its clinical implication in acute myeloid leukemia remain largely elusive. NKD1 transcript level in bone marrow mononuclear cells was detected by real-time quantitative polymerase chain reaction in 126 de novo acute myeloid leukemia patients and 30 controls. Clinical significance of NKD1 expression was obtained by the comparison between the patients with low and high NKD1 expression. NKD1 messenger RNA level was significantly decreased in acute myeloid leukemia patients compared with controls ( p = 0.019). There were no significant differences between patients with low and high NKD1 expression in sex, age, peripheral blood cells, bone marrow blasts, French-American-British/World Health Organization subtypes, and karyotypes/karyotypic classifications ( p > 0.05). Although no significant difference was observed in complete remission rate between NKD1 low and NKD1 high patients ( p > 0.05), Kaplan-Meier analysis revealed that NKD1 low patients showed shorter overall survival time than NKD1 high patients in whole-cohort acute myeloid leukemia, non-M3 acute myeloid leukemia, and cytogenetically normal acute myeloid leukemia ( p = 0.014, 0.063, and 0.020). Multivariate analyses disclosed the low NKD1 expression was an independent risk factor in cytogenetically normal acute myeloid leukemia patients (hazard ratio = 0.397, p = 0.017). Moreover, the prognostic value of NKD1 expression was confirmed by gene expression profile data in cytogenetically normal acute myeloid leukemia patients ( p = 0.028 and 0.011). NKD1 showed significantly increased level after induction chemotherapy achieved complete remission in follow-up paired acute myeloid leukemia patients ( p < 0.001). These findings indicated that reduced NKD1 expression is associated with unfavorable clinical outcome in cytogenetically normal acute myeloid leukemia.

Accumulated evidence indicate that miR-744 functions as either tumor suppressor or oncogene in the progression of a variety of tumors, with a tumor type-specific way. However, little is known about how miR-744 impacts on the tumorigenesis of human prostate cancer. In this study, employing the analyses of microarray, qRT-PCR and re-analysis of MSKCC data, we found that CRPC tissues expressed much more miR-744 than ADPC tissues did, and the expression level of miR-744 was inversely associated with survival of CRPC patients. In vitro studies revealed that miR-744 promotes PCa cells proliferation, enhances migration, invasion; in vivo results demonstrated that silencing of miR-744 mediated by shRNA dramatically reduces PCa xenograft tumor growth. Importantly, through human gene expression array, pathway enrichment analysis and Western blot, we identified that miR-744 dramatically activated Wnt/β-catenin pathway by targeting multiple negative regulators of Wnt/β-catenin signaling, including SFRP1, GSK3β, TLE3 and NKD1. At molecular level, we further defined that NKD1 is a major functional target of miR-744. Our findings indicate that miR-744 acts as one of oncogenic factor in the progression of CRPC by recruiting a mechanism of aberrant activation of Wnt/β-catenin signaling.

As a negative modulator of the canonical Wnt signaling pathway, Naked1 (NKD1) is widely expressed in many normal tissues. However, the expression and clinicopathological significance of NKD1 in patients with breast cancer is still unclear. The aim of this study was to evaluate NKD1 expression in breast cancer and to investigate the question of whether reduced expression of NKD1 may have any pathological significance in breast cancer development or progression. In this study, we performed western blotting and immunohistochemistry to evaluate the expression of NKD1 and relevance with clinicopathological factors in the breast invasive ductal carcinoma. Reduction of NKD1 was significantly correlated with lymph node metastasis, histological grade and ER expression in breast cancer. Patients with negative NKD1 expression had significantly lower cumulative postoperative 5 year survival rate than those with positive NKD1 expression. This interpretation is in keeping with the results obtained from our in vitro experiments on MDA-MB-231 cells, we demonstrated that upregulation of NKD1 expression by infect with an adenovirus containing a NKD1 vector significantly reduced the migration of breast cancer cells. These data suggest that NKD1 plays an important role in invasion in human breast cancer and it appears to be a potential prognostic marker for patients with breast cancer.

Aim: To examine epigenetic changes and the function of HOXA11 in human gastric cancer (GC)., Materials & Methods: Seven GC cell lines, five cases of normal gastric mucosa and 112 cases primary GC samples were used in this study., Results: Expression of HOXA11 and lack of promoter region methylation were found in NCI-N87, MKN45, BGC823 and HGC27 cells. Loss of expression and complete methylation were found in AGS gastric cancer cells. Reduced expression and partial methylation were found in MGC803 and SGC7901 cells. Restoration of HOXA11 expression was induced by 5-aza-2'-deoxycytidine. HOXA11 was methylated in 81.25% (91/112) of primary GCs. The presence of methylation was associated with male gender, tumor size, tumor differentiation and lymph node metastasis (all p < 0.05). Restoration of HOXA11 expression reduced cell proliferation, invasion, migration and induced apoptosis and G2/M phase arrest. HOXA11 was found to inhibit Wnt signaling by upregulating NKD1 expression., Conclusion: Epigenetic silencing of HOXA11 promotes GC proliferation, migration and invasion through activation of Wnt signaling.