Nucleolar And Spindle-associated Protein 1 Research Articles

Alternatively activated NUSAP1 promotes tumor growth and indicates poor prognosis in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies with high mortality. The key genes involved in initiation and development of HCC is not entirely clear. Methods: We performed a meta-analysis of available transcriptome data from 6 independent HCC datasets [5 datasets from the Gene Expression Omnibus (GEO) and 1 dataset from The Cancer Genome Atlas (TCGA)]. The associations of the nucleolar and spindle-associated protein 1 (NUSAP1) expression level with clinicopathological factors and survival times were analyzed. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when NUSAP1 expression was inhibited by shNUSAP1. Results: Based on the transcriptome and survival data in the GEO and TCGA databases, NUSAP1 gene was markedly upregulated in HCC. High expression of NUSAP1 in HCC is related to the iCluster1 molecular subgroup, poor survival, poor tumor differentiation and TNM stage. Additionally, pathway analysis based on RNAseq data suggested that NUSAP1 could activate the expression of genes involves in cell proliferation. Furthermore, downregulation of NUSAP1 expression could significantly inhibit the proliferation of SMMC-7721 and Huh7 cells in vitro . Conclusions: Our study provides evidence that NUSAP1 may serve as a candidate prognostic marker and a target for future therapeutic intervention in HCC.

Alternatively activated NUSAP1 promotes tumor growth and indicates poor prognosis in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies with high mortality. The key genes involved in initiation and development of HCC is not entirely clear.MethodsWe performed a meta-analysis of available transcriptome data from 6 independent HCC datasets [5 datasets from the Gene Expression Omnibus (GEO) and 1 dataset from The Cancer Genome Atlas (TCGA)]. The associations of the nucleolar and spindle-associated protein 1 (NUSAP1) expression level with clinicopathological factors and survival times were analyzed. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when NUSAP1 expression was inhibited by shNUSAP1.ResultsBased on the transcriptome and survival data in the GEO and TCGA databases, NUSAP1 gene was markedly upregulated in HCC. High expression of NUSAP1 in HCC is related to the iCluster1 molecular subgroup, poor survival, poor tumor differentiation and TNM stage. Additionally, pathway analysis based on RNAseq data suggested that NUSAP1 could activate the expression of genes involves in cell proliferation. Furthermore, downregulation of NUSAP1 expression could significantly inhibit the proliferation of SMMC-7721 and Huh7 cells in vitro.ConclusionsOur study provides evidence that NUSAP1 may serve as a candidate prognostic marker and a target for future therapeutic intervention in HCC.

Nucleolar and spindle associated protein 1 promotes metastasis of cervical carcinoma cells by activating Wnt/\u03b2-catenin signaling

BackgroundThe primary obstacle to treat cervical cancer is its high prevalence of metastasis, which severely affects patients’ quality of life and survival time. Nucleolar and spindle associated protein 1 (NUSAP1) has been implicated in the development, progression, and metastasis in several types of cancer. However, its oncogenic role in cervical cancer remains unclear.MethodsWestern blot assay and immunohistochemistry were used to determine the expression of NUSAP1 in 21 clinical fresh Cervical cancer tissues and 233 clinicopathologically characterized cervical cancer specimens. The biological roles of NUSAP1 in the metastasis of cervical cancer were investigated both in vitro by EMT, Side population analysis and Transwell assays and so on, and in vivo using a mouse 4w model of hematogenous metastasis and lymph node metastasis. Bioinformatics analysis, luciferase reporter analysis, immunoprecipitation and immunoblotting of nuclear and cytoplasmic cellular fractions were applied to discern and examine the relationshipbetween NUSAP1 and its potential targets.ResultsThe results demonstrated that NUSAP1 was upregulated in cervical cancer cells and tissues, correlated positively with metastasis and poor clinical outcome of patients. High expression of NUSAP1 promoted metastasis by enhancing cancer stem cell (CSC) traits and epithelial-mesenchyme transition (EMT) progression, while silencing of NUSAP1 reduced CSC traits and EMT progression. Mechanistically, upregulation of NUSAP1 induced SUMOylation of TCF4 via interacting with SUMO E3 ligase Ran-binding protein 2 (RanBP2) and hyperactivated Wnt/β-catenin signaling in cervical cancer cells. Additionally, NUSAP1-induced cervical cancer cells metastasis and the cancer stem cell phenotype were abrogated with the Wnt/β-catenin signaling inhibitor XAV-939 treatment. Importantly, co-therapy of conventional treatment and XAV-939 will provide a novel and effective treatment for NUSAP1-ovexpressed cervical cancer patients.ConclusionsOur results demonstrate thatNUSAP1 upregulation contributes to metastasis of cervical cancer by promoting CSC properties and EMT via Wnt/β-catenin signaling and XAV-939 might serve as a potential tailored therapeutic option for patients with NUSAP1-ovexpressed cervical cancer.

Nucleolar and Spindle Associated Protein 1 (NUSAP1) Inhibits Cell Proliferation and Enhances Susceptibility to Epirubicin In Invasive Breast Cancer Cells by Regulating Cyclin D Kinase (CDK1) and DLGAP5 Expression.

BackgroundDifferentially expressed genes (DEGs) of IBC were selected from the Gene Expression Omnibus (GEO) chip data: GSE21422 and GSE21974. Network analysis of the DEGs and IBC-related genes was performed in STRING database to find the core gene. Thus, this study aimed to determine the role of NUSAP1 in invasive breast cancer (IBC) and to investigate its effect on drug susceptibility to epirubicin (E-ADM).Material/MethodsThe mRNA expression of NUSAP1 was determined by quantitative polymerase chain reaction (q-PCR). The protein expression was detected by Western blotting. Cell growth and growth cycle were detected by MTT assay and flow cytometry, respectively. Cell migration and invasion were tested by Transwell assay.ResultsThrough use of gene network analysis, we found that NUSAP1 interacts with IBC-related genes. NUSAP1 presented high expression in IBC tissue samples and MCF-7 cells. NUSAP1 overexpression promoted the growth, migration, and invasion of MCF-7 cells. While NUSAP1 gene silencing downregulated the expression of genes associated with cell cycle progression in G2/M phase, cyclin D kinase (CDK1) and DLGAP5 arrested cells in G2/M phase and significantly inhibited the growth, migration, and invasion of MCF-7 cells. si-NUSAP1 increased the susceptibility of MCF-7 cells to E-ADM-induced apoptosis.ConclusionsOur study provides evidence that downregulation of NUSAP1 can inhibit the proliferation, migration, and invasion of IBC cells by regulating CDK1 and DLGAP5 expression and enhances the drug susceptibility to E-ADM.

SWATH proteomic profiling of prostate cancer cells identifies NUSAP1 as a potential molecular target for Galiellalactone

Galiellalactone (GL) is a fungal metabolite that presents antitumor and antiinflammatory activities in vitro and in vivo. Previous studies have shown that GL targets NF-κB and STAT3 pathways and induces G2/M cell cycle arrest in androgen-insensitive prostate cancer cells. In this study, we show that GL-induced cell cycle arrest is independent of the NF-κB and STAT3 pathways in DU145 and PC-3 cells, and also that GL did not affect cell cycling in androgen-sensitive prostate cancer cells such as LNCaP and 22Rv1 cells. In addition, we showed confluence resistance to GL in DU145 cells. Using a SWATH proteomic approach we identified a down-regulation of Nucleolar and spindle associated protein 1 (NUSAP1) under DU145 confluence. Moreover the expression of NUSAP1 in LNCaP cells is low compared to DU145 cells. The inhibition of NUSAP1 by siRNAs induced resistance to GL in DU145 cells, suggesting that NUSAP1 may be a target for GL and could be useful as a biomarker for the responsiveness of the antitumor activity of GL. Altogether, our finding shed light to the potential of GL to be developed as a novel treatment of castration resistance prostate cancer.

Candidate Biomarkers and Molecular Mechanism Investigation for Glioblastoma Multiforme Utilizing WGCNA.

To reveal the potential molecular mechanism of glioblastoma multiforme (GBM) and provide the candidate biomarkers for GBM gene therapy. Microarray dataset GSE50161 was obtained from GEO database. The differentially expressed genes (DEGs) were identified between GBM samples and control samples, followed by the module partition analysis based on WGCNA. Then, the pathway and functional enrichment analyses of DEGs were performed. The hub genes were further investigated, followed by the survival analysis and data validation. A total of 1913 DEGs were investigated between two groups, followed by analysis of 5 modules using WGCNA. These DEGs were mainly enriched in functions like inflammatory response. The hub genes including upregulated N-Myc and STAT Interactor (NMI), Capping Actin Protein-Gelsolin Like (CAPG), and Proteasome Subunit Beta 8 (PSMB8) were revealed as potential liquid biopsy molecules for GBM diagnose. Moreover, Nucleolar and Spindle Associated Protein 1 (NUSAP1) and G Protein-Coupled Receptor 65 (GPR65) were outstanding genes in survival analysis. Our results suggested that CPNE6, HAPLN2, CMTM3, NMI, CAPG, and PSMB8 might be used as potential molecules for liquid biopsy of GBM. NUSAP1 and GPR65 might be novel prognostic targets for GBM gene therapy. Furthermore, the upregulated NMI might play an important role in GBM progression via inflammatory response.

MicroRNA 193a-5p Regulates Levels of Nucleolar- and Spindle-Associated Protein 1 to Suppress Hepatocarcinogenesis

We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237HCCs and 5 nontumor liver samples using the TaqMan assay. Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolarand spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels ofMIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice. MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).

NUSAP1 gene silencing inhibits cell proliferation, migration and invasion through inhibiting DNMT1 gene expression in human colorectal cancer

Colorectal cancer (CRC) is one of the most common cause of cancer-related death in both female and male patients, with a high capacity for tumor migration and invasion. Recently, aberrant nucleolar and spindle-associated protein 1 (NUSAP1) expression has been reported in several cancers. However, the biological function and molecular mechanism of NUSAP1 in CRC have not been reported. Here, we demonstrated that NUSAP1 gene expression was notably upregulated in CRC tissues and cell lines (Caco2, LS174T, SW480, and LoVo). Subsequently, SW480 and LoVo cells were transfected with NUSAP1 siRNA, respectively, and the biological function of NUSAP1 was investigated. Results indicated that NUSAP1 silencing by siRNA inhibited CRC cell proliferation, and induces cell apoptosis. Moreover, NUSAP1 knockdown suppressed cell migration, cell invasion, and epithelial-to-mesenchymal transition (EMT). Furthermore, NUSAP1 silencing notably inhibited the mRNA and protein expression level of DNA methyltransferase 1 (DNMT1). DNMT1 overexpression partly rescued the effect of NUSAP1 silencing on colorectal cancer biological function. Taken together, NUSAP1 gene silencing induced cell apoptosis, and inhibited cell proliferation, cell migration, cell invasion, and EMT in colorectal cancer through inhibiting DNMT1 gene expression. These findings indicat that NUSAP1 is a promising molecular target for CRC treatment.

Nucleolar and spindle-associated protein 1 is a tumor grade correlated prognosis marker for glioma patients.

Despite therapeutic advances in glioma management including surgery, radiation, and chemotherapy, the improvement of patient outcome is far from satisfactory. Nucleolar and spindle-associated protein 1 (NUSAP1) is an important functional protein during mitosis, and its abnormal expression is implicated in progression of different types of tumors. However, the role of NUSAP1 in gliomas remains unclear. NUSAP1 expression in gliomas with different grades was investigated based on GEO glioma datasets. Kaplan-Meier survival analysis was used to evaluate its prognostic significance. In vitro assays were also performed to evaluate effects of NUSAP1 on malignant phenotypes of glioma cells by silencing NUSAP1. NUSAP1 expression was correlated not only with glioma grade but also with prognosis of glioma patients. NUSAP1 depletion suppressed proliferation of U251 cells by inducing cell cycle arrest at G2/M phase and apoptosis. NUSAP1 depletion rendered U251 cells impaired migratory ability as well. NUSAP1 is a potential prognosis marker for glioma patients and therapeutic strategies targeting NUSAP1 might hold promise in improving glioma treatment.

Silymarin-mediated regulation of the cell cycle and DNA damage response exerts antitumor activity in human hepatocellular carcinoma.

A novel module-search algorithm method was used to screen for potential signatures and investigate the molecular mechanisms of inhibiting hepatocellular carcinoma (HCC) growth following treatment with silymarin (SM). The modules algorithm was used to identify the modules via three major steps: i) Seed gene selection; ii) module search by seed expansion and entropy minimization; and iii) module refinement. The statistical significance of modules was computed to select the differential modules (DMs), followed by the identification of core modules using the attract method. Pathway analysis for core modules was implemented to identify the biological functions associated with the disease. Subsequently, results were verified in an independent sample set using reverse transcription polymerase chain reaction (RT-PCR). In total, 18 seed genes and 12 DMs (modules 1–12) were identified. The core modules were isolated using gene expression data. Overall, there were 4 core modules (modules 11, 5, 6 and 12). Additionally, DNA topoisomerase 2-binding protein 1 (TOPBP1), non-structural maintenance of chromosomes condensing I complex subunit H, nucleolar and spindle associated protein 1 (NUSAP1) and cell division cycle associated 3 (CDCA3) were the initial seed genes of module 11, 5, 6 and 12, respectively. Pathway results revealed that cell cycle signaling pathway was enriched by all core modules simultaneously. RT-PCR results indicated that the level of CDCA3, TOPBP1 and NUSAP1 in SM-treated HCC samples was markedly decreased compared with that in non-SM-treated HCC. No statistically significant difference between the transcriptional levels of CDCA3 in SM-treated and non-treated HCC groups was identified, although CDCA3 expression was increased in the treated group compared with the untreated group. Furthermore, although the expression level of TOPBP1 and NUSAP1 in the SM-treated group was decreased compared with that in the normal group, no significant difference was observed. From the results of the present study it can be inferred that TOPBP1, NUSAP1 and CDCA3 of the core modules may serve notable functions in SM-associated growth suppression of HCC.

Nucleolar and spindle-associated protein 1 (NUSAP1) interacts with a SUMO E3 ligase complex during chromosome segregation

The mitotic spindle is composed of dynamic microtubules and associated proteins that together direct chromosome movement during mitosis. The spindle plays a vital role in accurate chromosome segregation fidelity and is a therapeutic target in cancer. Nevertheless, the molecular mechanisms by which many spindle-associated proteins function remains unknown. The nucleolar and spindle-associated protein NUSAP1 is a microtubule-binding protein implicated in spindle stability and chromosome segregation. We show here that NUSAP1 localizes to dynamic spindle microtubules in a unique chromosome-centric pattern, in the vicinity of overlapping microtubules, during metaphase and anaphase of mitosis. Mass spectrometry-based analysis of endogenous NUSAP1 interacting proteins uncovered a cell cycle-regulated interaction between the RanBP2-RanGAP1-UBC9 SUMO E3 ligase complex and NUSAP1. Like NUSAP1 depletion, RanBP2 depletion impaired the response of cells to the microtubule poison Taxol. NUSAP1 contains a conserved SAP domain (SAF-A/B, Acinus, and PIAS). SAP domains are common among many other SUMO E3s, and are implicated in substrate recognition and ligase activity. We speculate that NUSAP1 contributes to accurate chromosome segregation by acting as a co-factor for RanBP2-RanGAP1-UBC9 during cell division.

Nucleolar and spindle associated protein 1 promotes the aggressiveness of astrocytoma by activating the Hedgehog signaling pathway

BackgroundThe prognosis of human astrocytoma is poor, and the molecular alterations underlying its pathogenesis still needed to be elucidated. Nucleolar and spindle associated protein 1 (NUSAP1) was observed in several types of cancers, but its role in astrocytoma remained unknown.MethodsThe expression of NUSAP1 in astrocytoma cell lines and tissues were measured with western blotting and Real-Time PCR. Two hundred and twenty-one astrocytoma tissue samples were analyzed by immunochemistry to demonstrate the correlation between the NUSAP1 expression and clinicopathological characteristics. 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, colony formation, transwell matrix penetration assay, wound healing assay and anchorage-independent growth assay were used to investigate the biological effect of NUSAP1 in astrocytoma. An intracranial brain xenograft tumor model was used to confirm the oncogenic role of NUSAP1 in human astrocytoma. Luciferase reporter assay was used to investigate the effect of NUSAP1 on Hedgehog signaling pathway.ResultsNUSAP1 was markedly overexpressed in astrocytoma cell lines and tissues compared with normal astrocytes and brain tissues. NUSAP1 was found to be overexpressed in 152 of 221 (68.78%) astrocytoma tissues, and was significantly correlated to poor survival. Further, ectopic expression or knockdown of NUSAP1 significantly promoted or inhibited, respectively, the invasive ability of astrocytoma cells. Moreover, intracranial xenografts of astrocytoma cells engineered to express NUSAP1 were highly invasive compared with the parental cells. With regard to its molecular mechanism, upregulation of NUSAP1 in astrocytoma cells promoted the nuclear translocation of GLI family zinc finger 1 (GLI1) and upregulated the downstream genes of the Hedgehog pathway.ConclusionThese findings indicate that NUSAP1 contributes to the progression of astrocytoma by enhancing tumor cell invasiveness via activation of the Hedgehog signaling pathway, and that NUSAP1 might be a potential prognostic biomarker as well as a target in astrocytoma.

Bioinformatics analysis of gene expression alterations in microRNA‑122 knockout mice with hepatocellular carcinoma.

Reduced microRNA (miR)-122 expression levels are frequently observed in hepatocellular carcinoma (HCC). The present study was conducted to investigate potential targets of miR-122 and determine the underlying regulatory mechanisms of miR-122 in HCC development. The public dataset GSE31731 was utilized, consisting of 8 miR-122 knockout (KO) mice (miR-122 KO) and 8 age-matched wild-type mice (WT group). Following data preprocessing, the differentially expressed genes (DEGs) were selected, followed by enrichment analysis. A protein-protein interaction (PPI) network was established, and a module network was further extracted. Combining the DEGs with microRNA targeting databases permitted the screening of the overlapping targets of miR-122. Furthermore, previously reported genes were screened out by literature mining. Transcription factors (TFs) of the targets were subsequently investigated. DEGs between miR-122 KO and WT groups were selected, including 713 upregulated and 395 downregulated genes. Of these, upregulated genes were enriched in cell cycle-associated processes [including nucleolar and spindle associated protein 1 (NUSAP1)], the cytokine-cytokine receptor interaction pathway [including C-X-C motif chemokine receptor 4 (CXCR4) and C-C motif chemokine receptor 2 (CCR2)], and the extracellular matrix-receptor interaction pathway [including integrin subunit alpha V (ITGAV)]. In addition, multiple overlapping targets were highlighted in the PPI network, including NUSAP1, CXCR4, CCR2 and ITGAV. Notably, CXCR4 and CCR2 were linked in module C, enriched in the cytokine-cytokine receptor interaction pathway. Furthermore, upregulated sex determining region Y-box 4 (SOX4) was identified as a TF. The results of the present study may provide a theoretical basis for further studies on the mechanisms of miR-122 in the development of HCC.

NUSAP1 promotes invasion and metastasis of prostate cancer.

We have previously identified nucleolar and spindle associated protein 1 (NUSAP1) as a prognostic biomarker in early stage prostate cancer. To better understand the role of NUSAP1 in prostate cancer progression, we tested the effects of increased and decreased NUSAP1 expression in cell lines, in vivo models, and patient samples. NUSAP1 promotes invasion, migration, and metastasis, possibly by modulating family with sequence similarity 101 member B (FAM101B), a transforming growth factor beta 1 (TGFβ1) signaling effector involved in the epithelial to mesenchymal transition. Our findings provide insights into the importance of NUSAP1 in prostate cancer progression and provide a rationale for further study of NUSAP1 function, regulation, and clinical utility.

Downregulation of nucleolar and spindle-associated protein1 expression suppresses cell migration, proliferation and invasion in renal cell carcinoma.

Nucleolar and spindle-associated protein1 (NUSAP1) is a microtubule-binding protein that plays an essential role in mitosis and cancer. Previous studies have demonstrated that NUSAP1 expression is relatively elevated in several malignancies. However, the biological roles of NUSAP1 in renal cell carcinoma (RCC) remain unknown. In the present study, we firstly performed reverse transcription‑polymerase chain reaction (RT-PCR) and western blot analysis to reveal that the expression of NUSAP1 was relatively elevated in clear cell RCC (ccRCC) tissue specimens and RCC cell lines. Immunohistochemical analysis showed that upregulation of NUSAP1 was significantly correlated with Fuhrman grade (P<0.001), tumor size (P=0.016), clinical stage (P<0.001) and distant metastasis (P=0.023). Additionally, high expression of NUSAP1 was closely associated with a shorter overall survival time of the ccRCC patients (P=0.006). Furthermore, we investigated the biological behaviors of RCC cells invitro, and we identified that NUSAP1 depletion inhibited RCC cell migration, proliferation and invasion, and apoptosis was induced and the cell cycle was arrested. On the basis of our studies, NUSAP1 was identified as a potential prognostic indicator and a novel therapeutic target for RCC patients.

Down-Regulation of Nucleolar and Spindle-Associated Protein 1 (NUSAP1) Expression Suppresses Tumor and Cell Proliferation and Enhances Anti-Tumor Effect of Paclitaxel in Oral Squamous Cell Carcinoma.

BackgroundNucleolar and spindle-associated protein 1 (NUSAP1) is an important mitotic regulator. In addition to its crucial function in mitosis, NUSAP1 has recently received attention due to the interesting roles in carcinogenesis. The aim of this study was to reveal functional mechanisms of NUSAP1 in oral squamous cell carcinoma (OSCC).MethodsmRNA and protein expression levels of NUSAP1 in 9 OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. The correlation between the NUSAP1 expression profile and the clinicopathological factors was evaluated by immunohistochemistry (IHC) in clinical OSCC samples (n = 70). The NUSAP1 knockdown cells were established with short hairpin RNA (shRNA) in OSCC cells, and functional assays were performed using these cells. In addition to the evaluation of cellular proliferation and cell cycle, we also investigated the potential role of NUSAP1 in paclitaxel (PTX)-induced cellular responses.ResultsmRNA and protein expression of NUSAP1 were significantly up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (P < 0.05). IHC revealed that NUSAP-1 expression is closely associated with primary advanced T stage (P<0.05). Suppression of NUSAP1 expression levels led to significant (P < 0.05) inhibition of cellular proliferation. Furthermore, apoptosis induced by PTX was enhanced in NUSAP1 knockdown OSCC cells.ConclusionsNUSAP1 may be a crucial biomarker for OSCC. Moreover, down-regulated NUSAP1 expression suppresses tumor proliferation and also enhances anti-tumor effect of PTX by activating apoptotic pathways. Thus, the present study strongly suggests that regulating NUSAP1 expression should contribute to the therapy for OSCC.

High EGFR_1 Inside-Out Activated Inflammation-Induced Motility through SLC2A1-CCNB2-HMMR-KIF11-NUSAP1-PRC1-UBE2C.

48 different Pearson mutual-positive-correlation epidermal growth factor receptor (EGFR_1)-activatory molecular feedback, up- and down-stream network was constructed from 171 overlapping of 366 GRNInfer and 223 Pearson under EGFR_1 CC ≥0.25 in high lung adenocarcinoma compared with low human normal adjacent tissues. Our identified EGFR_1 inside-out upstream activated molecular network showed SLC2A1 (solute carrier family 2 (facilitated glucose transporter) member 1), CCNB2 (cyclin B2), HMMR (hyaluronan-mediated motility receptor (RHAMM)), KIF11 (kinesin family member 11), NUSAP1 (nucleolar and spindle associated protein 1), PRC1 (protein regulator of cytokinesis 1), UBE2C (ubiquitin-conjugating enzyme E2C) in high lung adenocarcinoma. EGFR_1 inside-out upstream activated terms network includes intracellular, membrane fraction, cytoplasm, plasma membrane, integral to membrane, basolateral plasma membrane, transmembrane transport, nucleus, cytosol, cell surface; T cell homeostasis, inflammation; microtubule cytoskeleton, embryonic development (sensu Mammalia), cell cycle, mitosis, thymus development, cell division, regulation of cell cycle, Contributed--cellular process--Hs cell cycle KEGG, cytokinesis, M phase, M phase of mitotic cell cycle, estrogen-responsive protein Efp controls cell cycle and breast tumors growth, cell motility, locomotion, locomotory behavior, neoplasm metastasis, spindle pole, spindle microtubule, microtubule motor activity, microtubule-based movement, mitotic spindle organization and biogenesis, mitotic centrosome separation, spindle pole body organization and biogenesis, microtubule-based process, microtubule, cytokinesis after mitosis, mitotic chromosome condensation, establishment of mitotic spindle localization, positive regulation of mitosis, mitotic spindle elongation, spindle organization and biogenesis, positive regulation of exit from mitosis, regulation of cell proliferation, positive regulation of cell proliferation based on integrative GO, KEGG, GenMAPP, BioCarta and disease databases in high lung adenocarcinoma. Therefore, we propose high EGFR_1 inside-out activated inflammation-induced motility through SLC2A1-CCNB2-HMMR-KIF11-NUSAP1-PRC1-UBE2C in lung adenocarcinoma.

Antibodies specifically target AML antigen NuSAP1 after allogeneic bone marrow transplantation

AbstractIdentifying the targets of immune response after allogeneic hematopoietic cell transplantation (HCT) promises to provide relevant immune therapy candidate proteins. We used protein microarrays to serologically identify nucleolar and spindle-associated protein 1 (NuSAP1) and chromatin assembly factor 1, subunit B (p60; CHAF1b) as targets of new antibody responses that developed after allogeneic HCT. Western blots and enzyme-linked immunosorbent assays (ELISA) validated their post-HCT recognition and enabled ELISA testing of 120 other patients with various malignancies who underwent allo-HCT. CHAF1b-specific antibodies were predominantly detected in patients with acute myeloid leukemia (AML), whereas NuSAP1-specific antibodies were exclusively detected in patients with AML 1 year after transplantation (P < .001). Complete genomic exon sequencing failed to identify a nonsynonymous single nucleotide polymorphism (SNP) for NuSAP1 and CHAF1b between the donor and recipient cells. Expression profiles and reverse transcriptase–polymerase chain reaction (RT-PCR) showed NuSAP1 was predominately expressed in the bone marrow CD34+CD90+ hematopoietic stem cells, leukemic cell lines, and B lymphoblasts compared with other tissues or cells. Thus, NuSAP1 is recognized as an immunogenic antigen in 65% of patients with AML following allogeneic HCT and suggests a tumor antigen role.

Allogeneic Antibodies Identify GVL Targets CHAF1b and NuSAP1 in AML Patients.

Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but is limited by graft-versus-host disease (GVHD). Previous studies demonstrate allogeneic antibodies (Ab) develop against minor histocompatibility antigens (mHA) encoded on the Y-chromosome (H-Y antigens) after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize that novel mHA can be serologically identified as targets of allo-Ab responses that develop post-transplant. For this study, plasma was collected from five AML patients one year post-transplant, pre-transplant, and from their donors. The technology used to identify allo-Ab responses was human protein microarrays. ProtoArray™ (Invitrogen) displays 5,000 full-length human proteins with N-terminal GST epitopes expressed in baculovirus and affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. Technical replicates of 10 plasma measured at 1:50, 1:150, 1:500, 1:1500 and 1:4500 provided correlation coefficients of R2=0.94–0.97 confirming technical feasibility. In order to identify targets of allo-Ab, pre-transplant fluorescent signal intensities were subtracted from their one-year plasma results and also screened for the absence in donor sera for all 5000 antigens. While Ab responses were unchanged for 4600 (92%) antigens, new allo-Ab responses targeted 60–75 antigens with fluorescent differences ranging 0.5 to 3 logs. Over 90% of allo-Ab targets have known non-synonymous SNP which when disparate in donor and recipient may elicit alloimmunity. From the index patient, Nucleolar and Spindle Associated Protein 1 (NuSAP1) and Chromatin Assembly Factor 1b (CHAF1b) were two predominant proteins recognized after HCT. These allo-Abs were absent pre and 2 months post-HCT, developed in association with cGVHD at 12 months, and persisted through 18 months. On comparing all the five AML patients using ProtoArrays, we identified two patients of five developed all-ab responses to CHAF1b and NuSAP1. CHAF1b and NuSAP1 were expressed in insect cells and in E.coli, and were validated using ELISA and Western Blotting. We hypothesize alloimmunity in the index patient resulted from disparity between the donor and the recipient. We sequenced all exons for CHAF1b and NuSAP1 but failed to identify disparate non-synonymous coding SNPs between the donor and the index patient. A second hypothesis for immunogeneicity assumes CHAF1b and NuSAP1 are aberrantly expressed as tumor antigens stimulating donor allo immunity after transplantation. We determined if the antibody correlated with disease type. Eight-seven allo-transplant patients after one year post-transplant were screened by ELISA for antibody responses against CHAF1b and NuSAP1. Antibodies against NuSAP1 and CHAF1b are significantly recognized in patients with acute myelogenous leukemia (n=27) as compared to ALL, CLL, CML, MCL, MM and NHL, with a significance of p=0.00028 for CHAF1 and p=0.0011 for NuSAP1 using Wilcox test. Analysis of published AML expression profiles demonstrate CHAF1b and NuSAP1 were mainly present in the bone marrow CD34+ cells and leukemic cell lines and B lymphoblasts as compared to other tissues/cells. We sorted different cell populations and confirmed by RT-PCR results that CHAF1b and NuSAP1 were highly expressed in bone marrow CD34+Thy1+ cells suggesting antibodies specifically target bone marrow stem cells after HCT.