Nucleolar And Spindle-associated Protein 1 Expression Research Articles

NUSAP1 promotes pancreatic ductal adenocarcinoma progression by drives the epithelial-mesenchymal transition and reduces AMPK phosphorylation

BackgroundPancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, and its molecular mechanisms are unclear. Nucleolar and spindle-associated protein 1 (NUSAP1), an indispensable mitotic regulator, has been reported to be involved in the development of several types of tumors. The biological function and molecular mechanism of NUSAP1 in PDAC remain controversial. This study explored the effects and mechanism of NUSAP1 in PDAC.MethodsDifferentially expressed genes (DEGs) were screened. A protein‒protein interaction (PPI) network was constructed to identify hub genes. Experimental studies and tissue microarray (TMA) analysis were performed to investigate the effects of NUSAP1 in PDAC and explore its mechanism.ResultsNetwork analysis revealed that NUSAP1 is an essential hub gene in the PDAC transcriptome. Genome heterogeneity analysis revealed that NUSAP1 is related to tumor mutation burden (TMB), loss of heterozygosity (LOH) and homologous recombination deficiency (HRD) in PDAC. NUSAP1 is correlated with the levels of infiltrating immune cells, such as B cells and CD8 T cells. High NUSAP1 expression was found in PDAC tissues and was associated with a poor patient prognosis. NUSAP1 promoted cancer cell proliferation, migration and invasion, drives the epithelial-mesenchymal transition and reduces AMPK phosphorylation.ConclusionsNUSAP1 is an essential hub gene that promotes PDAC progression and leads to a dismal prognosis by drives the epithelial-mesenchymal transition and reduces AMPK phosphorylation.

Expression Profile and Gene Regulation Network of NUSAP1 in Pan Cancers Based on Integrated Bioinformatics Analysis.

Nucleolar and spindle-associated protein 1 (NUSAP1) plays key roles in microtubules and chromosomes in normal cells both structurally and functionally. In malignancies, NUSAP1 is frequently dysregulated and mutated. However, the expression profiles and biological functions of NUSAP1 in tumors remain unclear. NUSAP1 expression in BALB/c mice and human normal or tumor tissues was examined using immunohistochemistry. Kaplan-Meier survival analysis was utilized to assess the prognostic significance of NUSAP1 in tumors, and principal component analysis and co-expression analysis were performed to explore the unique roles of NUSAP1. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed with DAVID. The relevance between NUSAP1 and tumor-infiltrating immune cells was investigated using TIMER. A transcriptional regulation network was constructed using data from The Cancer Genome Atlas. NUSAP1 expression levels in various mice tissues were different. Compared with normal tissues, NUSAP1 was strongly expressed in several human tumor tissues. We believe that NUSAP1 distinctly impacts the prognosis of several cancers and plays various roles in thymoma and testicular germ cell tumors. Further, NUSAP1 expression levels were significantly positively associated with diverse infiltrating levels of immune cells, including B cells, CD4+ and CD8+ T cells, dendritic cells, and macrophages, in thymoma. The expression level of NUSAP1 demonstrated strong relevance with various immune markers in thymoma. Finally, the miR-1236-5p-NUSAP1 and TCF3-NUSAP1 network revealed the tumor-promoting role of NUSAP1 and pertinent underlying mechanisms in human liver hepatocellular carcinoma. NUSAP1 may be regarded as a therapeutic target or potential prognostic biomarker for various cancer types.

NUSAP1-LDHA-Glycolysis-Lactate feedforward loop promotes Warburg effect and metastasis in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia and hypovascular tumor microenvironment. Nucleolar and spindle associated protein 1 (NUSAP1) is a microtubule-associated protein that is known to be involved in cancer biology. Our study aimed to investigate the role of NUSAP1 in glycolytic metabolism and metastasis in PDAC. Expression and prognostic value of NUSAP1 in PDAC and common gastrointestinal tumors was evaluated. The function of NUSAP1 in PDAC progression was clarified by single-cell RNA-seq and further experiments in vitro, xenograft mouse model, spontaneous PDAC mice model and human tissue microarray. The downstream genes and signaling pathways regulated by NUSAP1 were explored by RNA-Seq. And the regulation of NUSAP1 on Lactate dehydrogenase A (LDHA)-mediated glycolysis and its underlying mechanism was further clarified by CHIP-seq. NUSAP1 was an independent unfavorable predictor of PDAC prognosis that playing a critical role in metastasis of PDAC by regulating LDHA-mediated glycolysis. Mechanically, NUSAP1 could bind to c-Myc and HIF-1α that forming a transcription regulatory complex localized to LDHA promoter region and enhanced its expression. Intriguingly, lactate upregulated NUSAP1 expression by inhibiting NUSAP1 protein degradation through lysine lactylated (Kla) modification, thus forming a NUSAP1-LDHA-glycolysis-lactate feedforward loop. The NUSAP1-LDHA-glycolysis-lactate feedforward loop is one of the underlying mechanisms to explain the metastasis and glycolytic metabolic potential in PDAC, which also provides a novel insights to understand the Warburg effect in cancer. Targeting NUSAP1 would be an attractive paradigm for PDAC treatment.

Transcription Factor E2F8 Promotes Cisplatin Resistance in Hepatocellular Carcinoma by Regulating DNA Damage via NUSAP1.

DNA damage repair has been the key mechanism of cisplatin resistance in hepatocellular carcinoma (HCC). The present study elucidated the molecular mechanism by which nucleolar and spindle-associated protein 1 (NUSAP1) influenced cisplatin tolerance in HCC by regulating DNA damage. First, high mRNA expression of E2F8 and NUSAP1 in HCC was detected by real-time quantitative PCR in cells and tumor tissue. The interaction between E2F8 and NUSAP1 was confirmed by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays that E2F8 bound to the promoter region of NUSAP1 and regulated its transcriptional activity. The effects of the E2F8/NUSAP1 axis on cell viability, cell cycle, DNA damage protein γ-H2AX, and cisplatin resistance were investigated by CCK-8, flow cytometry, comet detection, and western blot. The results showed that NUSAP1 knockdown blocked the cell cycle in G0/G1 phase, promoted cisplatin-induced DNA damage, and enhanced cisplatin sensitivity in HCC. Overexpressed E2F8 promoted cell cycle arrest by silencing NUSAP1 in HCC, and promoting DNA damage as well as cisplatin sensitivity. In conclusion, our results suggested that E2F8 enhanced the chemoresistance of HCC cells to cisplatin by activating NUSAP1 to inhibit DNA damage, which provides a basis for describing new therapeutic targets that effectively exacerbate DNA damage and improve the chemical sensitivity of HCC to cisplatin.

NUSAP1 regulates basal cell carcinoma migration, invasion and DNA damage through activation ofthe Hedgehog signaling pathway.

Basal cell carcinoma (BCC) is a prevalent cutaneous cancer with an increasing incidence. Nucleolar and spindle associated protein 1 (NUSAP1) is a cell proliferation-related protein that participates in the development of various cancers. However, its role and mechanism in BCC remain elusive. The expression of NUSAP1 was detected by western blot. Gain- and loss-of-function assays were performedthrough the transfection of overexpression plasmid of NUSAP1 and si NUSAP1 into TE354.T cells. The role and mechanism of action of NUSAP1 in BCC were explored by cell counting kit-8 (CCK-8), colony formation, transwell, flow cytometry and western blot assays. NUSAP1 was highly expressed in TE354.T cells. Overexpression of NUSAP1 enhanced cell viability, colony forming numbers, numbers of migrated and invasive cells and the relative protein expression of RAD51, but reduced the apoptosis rate and the relative protein expression of γH2AX in TE354.T cells. Inverse results were obtained in these indicators after TE354.T cells were downregulated with NUSAP1. Moreover, the relative expression of proteins involved in the Hedgehog signaling pathway was increased by transfection of the overexpression plasmid of NUSAP1 into TE354.T cells, but decreased by the transfection of si NUSAP1 into TE354.T cells. Both gain- and loss-of-function results revealed that NUSAP1 promoted proliferation, migration and invasion but attenuated apoptosis and DNA damage in BCC, which was involved in the activation of the Hedgehog signaling pathway.

Comprehensive pan-cancer analysis reveals NUSAP1 is a novel predictive biomarker for prognosis and immunotherapy response.

Nucleolar and spindle-associated protein 1 (NUSAP1) is a microtubule-associated protein that plays a crucial role in mitosis. Despite initial reports suggesting a potential involvement of NUSAP1 in tumor progression and malignant cell regulation, there has been no systematic analysis of its role in the tumor immune microenvironment, nor its predictive value for prognosis and immunotherapy response across different cancer types. In this study, we analyze NUSAP1 mRNA and protein expression levels in various human normal and tumor tissues, using data from TCGA, GTEx, CPTAC, HPA databases, and clinical samples. Our findings reveal that NUSAP1 is highly expressed in multiple tumor tissues across most cancer types and is primarily expressed in malignant and immune cells, according to single-cell sequencing data from the TISCH database. Prognostic analysis based on curated survival data from the TCGA database indicates that NUSAP1 expression levels can predict clinical outcomes for 26 cancer types. Furthermore, Gene Set Enrichment Analysis (GSEA) suggests that NUSAP1 promotes cell proliferation, tumor cell invasion, and regulation of anti-tumor response. Analysis of immune score, immune cell infiltration, and anti-cancer immunity cycle using ESTIMATE, TIMER, and TIP databases show that high NUSAP1 levels are associated with low CD4+T and NKT cell infiltration but high Th2 and MDSC infiltration, inversely correlated with antigen-presenting molecules and positively correlated with a variety of immune negative regulatory molecules. Notably, patients with melanoma, lung, and kidney cancer with high NUSAP1 expression levels have shorter survival times and lower immunotherapy response rates. Using Cmap analysis, we identify Entinostat and AACOCF3 as potential inhibitors of NUSAP1-mediated pro-oncogenic effects. In vitro and in vivo experiments further confirm that NUSAP1 knockdown significantly reduces the proliferation ability of A549 and MCF-7 cells. Overall, our study highlights the potential of NUSAP1 expression as a novel biomarker for predicting prognosis and immuno-therapeutic efficacy across different human cancers and suggests its potential for developing novel antitumor drugs or improving immunotherapy.

The Effect of Coumestrol on Hub Genes in Lung Squamous Cell Carcinoma Based on Bioinformatic Strategy

Purpose There is limited treatment for lung squamous cell carcinoma (LUSC), so there is an urgent need to find new antitumor drugs. Materials and Methods We downloaded datasets from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas databases. We used GEO2R and the “limma” package to screen differentially expressed genes. We used the Cytoscape software to screen out hub genes. We screened herbs that act on hub genes on the Chinese medicine website. We then studied the effect of coumestrol (CM) on the hub genes in the H226 cell line. Results Seven hub genes were screened, namely CCNB2, CENPF, KIF11, MELK, nucleolar and spindle-associated protein 1 (NUSAP1), PBK, and RRM2. We observed that CM had a tumor-inhibiting effect on H226 cells by inhibiting the expression of CCNB2, KIF11, and NUSAP1. Conclusion CM, screened by bioinformatics and network pharmacology, can inhibit H226 cells by downregulating CCNB2, KIF11, and NUSAP1.

NUSAP1, a novel stemness-related protein, promotes early recurrence of hepatocellular carcinoma.

Early recurrence (within 2 years after resection) is the primary cause of poor outcomes among hepatocellular carcinoma (HCC) patients, and liver cancer stem cells are the main contributors to postsurgical HCC recurrence. Nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in tumor progression. We investigated the function and clinical value of NUSAP1 in early recurrence of HCC. Data from public datasets and our cohort were used to assess the association between NUSAP1 expression and early HCC recurrence. Gain- and loss-of-function experiments were carried out in vivo and in vitro. The predictive effect of NUSAP1 on early HCC recurrence was further evaluated by a validation cohort. We found that elevated NUSAP1 expression in HCC specimens was correlated with poor outcome, especially in cases with postoperative early recurrence. Functional studies indicated that NUSAP1 significantly promotes HCC progression. A postsurgical recurrence murine model further revealed that upregulated NUSAP1 dramatically increased the likelihood of HCC early recurrence. RNA sequencing data revealed that the gene sets of cancer stemness and the signal transducer and activator of transcription 3 (STAT3) pathway were enriched by NUSAP1 overexpression. Mechanistically, NUSAP1 enhanced cancer stemness through stimulating STAT3 nuclear translocation and activation through receptor of activated protein C kinase 1 (RACK1). In a validation cohort with 112 HCC patients, NUSAP1 effectively predicted HCC early recurrence. Our results indicated that NUSAP1 promotes early recurrence of HCC by sustaining cancer stemness and could serve as a valuable predictive indicator for postsurgical intervention in HCC patients.

Knockdown of NUSAP1 inhibits cell proliferation and invasion through downregulation of TOP2A in human glioblastoma

ABSTRACT Nucleolar and spindle associated protein 1 (NUSAP1), an indispensable mitotic regulator, has been reported to be involved in the development, progression, and metastasis of several types of cancer. Here, we investigated the expression and biological function of NUSAP1 in human glioblastoma (GBM), an aggressive brain tumor type with largely ineffective treatment options. Analysis of the molecular data in CGGA, TCGA and Rembrandt datasets demonstrated that NUSAP1 was significantly upregulated in GBM relative to low grade gliomas and non-neoplastic brain tissue samples. Kaplan-Meier analysis indicated that patients with tumors showing high NUSAP1 expression exhibited significantly poorer survival in both CGGA (P = 0.002) and Rembrandt cohorts (P = 0.017). Analysis of RNA sequencing data from P3-cells with stable knockdown of NUSAP1 revealed topoisomerase 2A (TOP2A) as a possible molecule downregulated by the loss of NUSAP1. Molecular analysis of the CGGA data revealed a strong correlation between NUSAP1 and TOP2A expression in primary gliomas and recurrent gliomas samples. SiRNA knockdown of either NUSAP1 or TOP2A in U251, T98 and GBM derived patient P3 cells inhibited GBM cell proliferation and invasion, and induced cell apoptosis. Finally, stable knockdown of NUSAP1 with shRNA led to decreased tumor growth in an orthotopic xenograft model of GBM in mice. Taken together, NUSAP1 gene silencing induced apoptosis possibly through the downregulation of the candidate downstream molecule TOP2A. Interference with the expression of NUSAP1 might therefore inhibit malignant progression in GBM, and NUSAP1 might thus serve as a promising molecular target for GBM treatment.

Increased NUSAP1 expression is associated with lymph node metastasis and survival prognosis in bladder urothelial carcinoma

The main route of metastasis of bladder urothelial carcinoma is through lymph nodes; however, its exact mechanism remains unclear. In this study, we found an association of nucleolar and spindle associated protein 1 (NUSAP1) expression with BUC tissues along with lymph node metastasis and the survival prognosis. A total of 178 pathological specimens following radical bladder cancer resection were obtained. NUSAP1 expression was analyzed by immunohistochemistry. We evaluated the correlation between clinicopathological characteristics and NUSAP1 expression. Logistic regression was used to determine the independent variables that influenced lymph node metastasis. Uni- and multi-factorial Cox regression methods were used to determine the prognostic value of NUSAP1 expression in urothelial carcinoma of the bladder. High expression of NUSAP1 in BUC was not significantly related to the patient’s gender, age, or tumor number (p > 0.05), however was significantly associated with pathological grade, tumor diameter, pathological stage, and lymph node metastasis (p < 0.05). Lymph node metastasis was significantly correlated with pathological stage, pathological grade, tumor number, tumor diameter, and NUSAP1 expression (p < 0.05); only NUSAP1 expression was an independent predictor of lymph node metastasis in BUC (OR:1.786, 95% CI 1.229–2.596, p = 0.002). In addition, high NUSAP1 expression was an independent prognostic predictor for BUC. In BUC, NUSAP1 showed high expression and was significantly associated with lymph node metastasis, pathological stage, pathological grade, and tumor diameter. NUSAP1 was an independent predictor of lymph node metastasis and prognosis in BUC; higher expression indicated poorer prognosis of BUC patients.

Transcription factor HOXB2 upregulates NUSAP1 to promote the proliferation, invasion and migration of nephroblastoma cells via the PI3K/Akt signaling pathway.

The transcription factor homeobox protein Hox-B2 (HOXB2) and its downstream factor nucleolar and spindle-associated protein 1 (NUSAP1) play important regulatory roles in cell proliferation, invasion and migration. However, their effects and specific mechanisms in nephroblastoma have not been previously investigated, to the best of our knowledge. Therefore, in the present study, the mRNA and protein expression levels of HOXB2 and NUSAP1 were determined in nephroblastoma cells using reverse transcription-quantitative PCR and western blot analyses, respectively. Furthermore, cell transfection experiments were carried out to knock down NUSAP1 and overexpress HOXB2 in nephroblastoma cell lines. The proliferative, invasive and migratory abilities of nephroblastoma cells were assessed by MTT, EdU, colony formation, wound healing and Transwell assays. In addition, the JASPAR website was used to predict the association between HOXB2 and NUSAP1, which was further verified by dual-luciferase reporter and chromatin immunoprecipitation assays. Finally, the expression levels of the PI3K/Akt signaling pathway-related proteins were measured by western blot analysis. The results showed that the expression of NUSAP1 was abnormally upregulated in nephroblastoma cell lines. However, NUSAP1 silencing attenuated the proliferation, invasion and migration abilities of nephroblastoma cells. The results also suggested that HOXB2 could transcriptionally activate NUSAP1. Therefore, HOXB2 overexpression abrogated the inhibitory effect of NUSAP1 silencing on the proliferation and metastasis of nephroblastoma cells, possibly via the PI3K/Akt signaling pathway. The aforementioned findings indicated that HOXB2 may upregulate NUSAP1 to promote the proliferation, invasion and migration of nephroblastoma cells via the PI3K/Akt signaling pathway.

Prognostic Value of NUSAP1 and Its Correlation with Immune Infiltrates in Human Breast Cancer.

Nucleolar and Spindle Associated Protein 1 (NUSAP1), a microtubule-associated protein, plays a critical role in maintaining spindle assembly and function. However, its clinical value and biological function in breast cancer have yet to be fully clarified. In the current study, the expression profile, prognostic value, genetic alterations of NUSAP1 were analyzed using Oncomine, UALCAN, HPA, bc-GenExMiner, Kaplan-Meier Plotter, and cBioPortal, besides, its correlation with tumor immune cell infiltration was explored via TIMER. Furthermore, enrichment analyses, protein-protein interaction, co-expression genes, and hub genes (KIF20A, BUB1, CDC20, CCNB2, BIRC5, MELK, KIF11, KIF23, TTK, MKI67) were performed using DAVID, STRING, LinkedOmics, and Cytoscape. Notably, NUSAP1 expression was upregulated in breast cancer, and was significantly correlated with clinicopathological features. High expression of NUSAP1 predicted a poor overall survival, relapse-free survival, distant metastases-free survival, post-progression survival, and disease-free survival. NUSAP1 was correlated with the infiltration of B cells, CD8+ T cells, neutrophil and dendritic cells, and the marker sets of monocytes, tumor-associated macrophages, M1 macrophages, M2 macrophages, dendritic cells, T cell exhaustion, regulatory T cells. Enrichment analyses showed NUSAP1 played an important role in the mitotic nuclear division, microtubule binding, nucleoplasm, and cell cycle. These findings confirmed NUSAP1 as a promising diagnostic biomarker and therapeutic target in human breast cancer.

Nucleolar and spindle associated protein 1 enhances chemoresistance through DNA damage repair pathway in chronic lymphocytic leukemia by binding with RAD51

Nucleolar and spindle-associated protein 1 (NUSAP1) is an essential regulator of mitotic progression, spindle assembly, and chromosome attachment. Although NUSAP1 acts as an oncogene involved in the progression of several cancers, the exact role of chronic lymphocytic leukemia (CLL) remains elusive. Herein, we first discovered obvious overexpression of NUSAP1 in CLL associated with poor prognosis. Next, the NUSAP1 level was modulated by transfecting CLL cells with lentivirus. Silencing NUSAP1 inhibited the cell proliferation, promoted cell apoptosis and G0/G1 phase arrest. Mechanistically, high expression of NUSAP1 strengthened DNA damage repairing with RAD51 engagement. Our results also indicated that NUSAP1 knockdown suppressed the growth CLL cells in vivo. We further confirmed that NUSAP1 reduction enhanced the sensitivity of CLL cells to fludarabine or ibrutinib. Overall, our research investigates the mechanism by which NUSAP1 enhances chemoresistance via DNA damage repair (DDR) signaling by stabilizing RAD51 in CLL cells. Hence, NUSAP1 may be expected to be a perspective target for the treatment of CLL with chemotherapy resistance.

Prognostic and Clinicopathological Implications of NUSAP1, MELK and L1CAM Immunohistochemical Expression in Cervical Cancer

Background: Evaluation of cancer cervix prognosis is highly needed for novel targeted therapy and improved outcomes. Nucleolar and spindle associated protein 1 (NUSAP1) is a novel biomarker that has roles in spindle formation and mitotic progression. Maternal embryonic leucine zipper kinase (MELK) is involved in cell cycle control and carcinogenesis. The L1 cell adhesion molecule (L1-CAM) plays an essential role in cell migration. This study aimed to investigate NUSAP1, MELK, and L1CAM immunohistochemical expression in cancer cervix tissues and detect their prognostic roles. Method: In this prospective cohort study, we evaluated NUSAP1, MELK, and L1CAM expressions of sections from 62 cervical carcinoma cases using immunohistochemistry. Results: NUSAP1, MELK, and L1CAM expression correlated with tumor high grade, advanced FIGO stage, poor survival rates, and higher recurrence rate after successful therapy (p <0.001). Conclusion: Expression of NUSAP1, MELK, and L1CAM in cancer cervix was associated with poor prognosis.

Nucleolar and spindle‑associated protein 1 promotes non‑small cell lung cancer progression and serves as an effector of myocyte enhancer factor 2D.

As a potential oncogene, nucleolar and spindle-associated protein 1 (NUSAP1) is involved in the regulation of tumor cell proliferation, metastasis and drug resistance. However, the role of NUSAP1 in non-small cell lung cancer (NSCLC) remains unclear. The present study aimed to investigate the biological function and underlying molecular mechanisms of NUSAP1 in NSCLC. NUSAP1 expression was measured in NSCLC tissues and cell lines via immunohistochemistry and western blotting, respectively. NSCLC cell lines stably inhibiting NUSAP1 were established to investigate its effects on cell proliferation, colony formation and invasion, and on in vivo tumorigenicity. Additionally, the upstream and downstream mechanisms of NUSAP1 in regulating NSCLC progression were investigated. The results indicated that NUSAP1 expression was upregulated in NSCLC tissues and cell lines. High NUSAP1 expression was associated with tumor size, TNM stage, lymph node metastasis and poor patient survival, whereas knockdown of NUSAP1 inhibited NSCLC cell proliferation, colony formation and invasion. Furthermore, downregulation of NUSAP1 decreased the growth of NSCLC xenografts in vivo. In addition, myocyte enhancer factor 2D (MEF2D) directly targeted the NUSAP1 promoter, thereby enhancing the mRNA and protein expression levels of NUSAP1. Moreover, the results demonstrated that MEF2D expression was upregulated in NSCLC tissues and was positively correlated with NUSAP1 expression. MEF2D-knockdown decreased NSCLC cell proliferation, colony formation and invasion. NUSAP1 upregulation reversed the effects of MEF2D-knockdown on NSCLC progression. Furthermore, it was observed that MEF2D-knockdown inhibited the accumulation and nuclear translocation of β-catenin, thereby repressing the activation of the Wnt/β-catenin signaling pathway in NSCLC cells, whereas NUSAP1 upregulation rescued the effects of MEF2D-knockdown on the activation of the Wnt/β-catenin signaling pathway. In conclusion, the findings of the present study indicated that the MEF2D/NUSAP1 signaling pathway promoted NSCLC progression by inducing the activation of Wnt/β-catenin signaling, and this novel mechanism may represent a potential treatment target for patients with NSCLC.

Nucleolar and spindle-associated protein 1 accelerates cellular proliferation and invasion in nasopharyngeal carcinoma by potentiating Wnt/β-catenin signaling via modulation of GSK-3β.

Nucleolar and spindle-associated protein 1 (NUSAP1) is a pivotal tumor-related protein that has been implicated in the progression of broad spectrum of tumors. However, no detailed study of the role of NUSAP1 in nasopharyngeal carcinoma (NPC) has been reported. The aim of this work is to enhance our understanding of NUSAP1 in the progression of NPC. By analyzing data available within the Oncomine database, we found that NUSAP1 expression was elevated in NPC relative to normal tissues. Further, we showed that NUSAP1 expression in clinical specimens of NPC and several NPC cell lines was elevated. Down-regulation of NUSAP1 by gene silencing markedly depleted the capacity of NPC cells to proliferate and invade. Contrastingly, overexpression of NUSAP1 potentiated the proliferative and invasive abilities of NPC cells. Further mechanistic research revealed that NUSAP1 knockdown decreased levels of Wnt/β-catenin signaling in NPC cells via a mechanism associated with downregulation of glycogen synthase kinase-3β (GSK-3β) phosphorylation. However, suppression of GSK-3β markedly abolished the inhibitory effect of NUSAP1 knockdown on Wnt/β-catenin signaling. Further, inhibition of Wnt/β-catenin signaling partially reversed NUSAP1-mediated tumor growth in NPC cells. In addition, NUSAP1 knockdown restrained tumorigenesis of NPC in vivo, and was associated with down-regulation of Wnt/β-catenin signaling. In conclusion, these findings demonstrate that NUSAP1 is capable of accelerating proliferation and invasion in NPC cells by potentiating Wnt/β-catenin signaling. Our study unveils a potential role of NUSAP1 in promoting NPC tumors and suggests that the protein is an attractive antitumor target for NPC treatment.

NUSAP1 Enhances Drug Resistance By Binding to RAD51 through DNA Damage Repair Pathway in Chronic Lymphocytic Leukemia

Introduction: Enhanced DNA damage repair effect is an important mechanism for drug-resistance in chronic lymphocytic leukemia (CLL). Moreover, the ability of cancer cells to repair under radiation or chemotherapy drug induced DNA damage also serves as one of the mechanisms for therapy resistance. It is reported that nucleolar and spindle associated protein 1 (NUSAP1), a microtubule binding protein, has been involved in DNA damage repair process and plays important roles in the development, progression, and metastasis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Expression levels of NUSAP1 mRNA and protein in CLL cell lines and patient specimens were detected by qRT-PCR and Western blot, and Kaplan-Meier survival curve and overall survival were analyzed by log-rank test. Peripheral blood samples from de novo CLL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE22762 were obtained from Gene Expression Omnibus. With altering NUSAP1 expression by lentivirus-transfected cells in vitro, the effects of NUSAP1 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE /7AAD staining and PI/RNase staining respectively. Bioinformatics analysis, luciferase reporter analysis, immunoprecipitation and were applied to discern and examine the relationship between NUSAP1 and its potential targets. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of CLL patients was significantly increased than that of healthy donors (P&amp;lt;0.05) (Figure A). Besides, the results indicated that the OS of patients with highly expressed NUSAP1 was significantly worse than in patients with low expression with the statistical analysis database GSE22762. mRNA and protein expression levels of NUSAP1 were significantly higher in CLL cell lines than in PBMCs from healthy donors (Figure C). Our findings indicated that NUSAP1 knockdown notably inhibited cell proliferation when compared with the Scramble group (Figure D). Moreover, the amounts of DNA fragmentation of the apoptotic cells were remarkably increased by NUSAP1 shRNA in MEC-1 and EHEB cells when compared with the Scramble group (Figure E). In addition, after knocking down NUSAP1, MEC-1 and EHEB cells were blocked in G0/G1 phase (Figure F). Moreover, addition to fludarabine or ibrutinib with shNUSAP1 group showed enhanced cytotoxicity in CLL cells (Figure G). The differential genes were analyzed via RNA-seq between Scramble and ShNUSAP1 group. Intriguingly, annotations of gene ontology (GO) analysis indicated that NUSAP1 was closely related to biological processes including cell cycle and response to drug. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that NUSAP1 were enriched in pathways in cancer, DNA replication and cell cycle. Gene set enrichment analysis (GSEA) implicated that NUSAP1 was functionally enriched in DNA replication, cell cycle and proteasome (Figure H). Immunofluorescence showed that NUSAP1 was mainly distributed in the cell nucleus, and the expression level of RAD51 was positively correlated with the change of NUSAP1 expression (Figure I). Surppression of NUSAP1 inhibited the action of proteins in DNA damage repair pathway (Figure J). Through COIP, NUSAP1 was identified to bind with RAD51 and play an important role in DNA damage repair pathway (Figure K). Hence, NUSAP1 participates in the DNA damage repair process and enhances the drug resistance in CLL. Conclusions: This study first demonstrated that the high expression of NUSAP1 in CLL patients is associated with poor prognosis through database analysis and experiments in vitro. Interference of NUSAP1 expression led to a slower CLL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest. Collectively, our findings demonstrated that NUSAP1 contributes to DNA damage repairing by binding to RAD51 and enhances drug resistance in CLL. Therefore, NUSAP1 is expected to be a potential target for the treatment of CLL with drug-resistance. Figure 1 Disclosures No relevant conflicts of interest to declare.

ANKRD22 enhances breast cancer cell malignancy by activating the Wnt/β-catenin pathway via modulating NuSAP1 expression.

Breast cancer is one of the most prevalent malignancies in women worldwide. Although great advancements have been achieved in the diagnosis and treatment of breast cancer, the prognosis of patients with breast cancer is still poor due to distal recurrence and metastasis after surgery. This study aimed to assess the role of ankyrin repeat domain 22 (ANKRD22) in the progression of breast cancer and investigate the molecular mechanism. Using immunohistochemistry, we demonstrated that the expression level of ANKRD22 in human breast cancer tissues was significantly higher than that in normal breast tissues. ANKRD22 knockdown inhibited the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of breast cancer cells, as confirmed by BrdU, colony formation, transwell, and immunoblot assays. Immunoblot assays further indicated that ANKRD22 regulated the expression of nucleolar and spindle-associated protein 1 (NuSAP1) and then caused the activation of Wnt/β-catenin signaling pathway. Moreover, overexpression of NUSAP1 reversed the inhibitory effects of ANKRD22 knockdown on the proliferation, invasion, and EMT of breast cancer cells. In summary, this study demonstrated that ANKRD22 enhanced breast cancer cell malignancy by activating the Wnt/β-catenin pathway via modulating NuSAP1 expression, which might shed light on new therapeutic approaches for breast cancer.

Nusap1, Melk and L1cam Expression in Cervical Cancer Tissues; Prognostic and Clinicopathological Implications

Background: It is important to identify of novel therapeutic targets for cervical carcinoma. Nucleolar and spindle associated protein 1 (NUSAP1) could be able to bind microtubules, and has important role in spindle formation and mitotic progression. Maternal embryonic leucine zipper kinase (MELK) is a cell cycle related conserved protein kinase that is involved in control and regulation of the cell cycle, apoptosis, and carcinogenesis. The L1 cell adhesion molecule (L1-CAM) was discovered as a protein which has an essential role in cell migration and axon guidance in the nervous system. The Aim of This Study Is to investigate the tissue protein expression of NUSAP1, MELK and L1CAM in tissues of cervical carcinoma and to detect their prognostic roles. Methods: Expression of NUSAP1, MELK and L1CAM was evaluated in sections from 62 cases of cervical carcinoma using immunohistochemistry we followed our patients for 3 years correlate their expression with clinicopathological parameters and patients outcome. Results: High levels of NUSAP1, MELK and L1CAM expression was positively related to older age of the patients, high grade of the tumor, lympho-vascular invasion, advanced FIGO stage, poor survival rates and higher recurrence rate after successful therapy (p&lt;0.001). L.N metastases (p=0.002), distant metastases (p=0.003). There are statistically significant positive association between NUSAP1, MELK and L1CAM Phi correlation coefficient= + 0.87 and + 0.721 respectively (p&lt;0.001). Conclusion: NUSAP1, MELK and L1CAM are considered markers of poor prognosis in cervical carcinoma cells.

Decreased Expression of NUSAP1 Predicts Poor Overall Survival in Cervical Cancer.

Background: Nucleolar and spindle-associated protein 1 (NUSAP1) was previously reported to be associated with poor prognosis in multiple cancers. In the present study, we comprehensively investigated the clinicopathological features and potential prognostic value of NUSAP1 in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC).Methods: The expression profiles of the genes were extracted from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), Cancer Cell Line Encyclopedia (CCLE), Gene Expression Profiling Interactive Analysis (GEPIA), and The Human Protein Atlas databases. The association between clinicopathological characteristics and NUSAP1 was analyzed using logistic regression in TCGA patients and receiver operating characteristic (ROC) curve analysis for GSE7803, GSE9750, and GSE63514 datasets. The prognostic value of NUSAP1 in TCGA patients was evaluated using the Kaplan-Meier method and Cox regression. Gene set enrichment analysis (GSEA) was conducted using TCGA dataset.Results: A total of 68 differentially expressed genes (DEGs) were identified in CESC. ROC analysis of NUSAP1 suggested that the area under the ROC curve was 0.968. Kaplan-Meier survival analysis indicated that CESC with low expression of NUSAP1 has a worse prognosis than CESC with high NUSAP1 expression (P = 0.005). The logistic regression revealed that low NUSAP1 expression in CESC was related to advanced tumor stage in TCGA database. Moreover, Cox regression analysis showed that NUSAP1 expression correlated significantly with prognosis in the case of patients in TCGA database. GSEA demonstrated that CESC patients with high expression of NUSAP1 were enriched in the G2M checkpoint, MYC targets, and breast cancer ZNF217.Conclusion: The results suggest that identification of DEGs might enhance our understanding of the causes and molecular mechanisms underlying the development of CESC. Moreover, NUSAP1 may play an important role in CESC progression and prognosis and may serve as a valuable indicator of poor survival in CESC.