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

Abstract

Background: Pancreatic 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.

Methods: Differentially 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.

Results: Network 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.

Conclusions: NUSAP1 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.

Keywords: Differentially expressed genes; Functional enrichment analysis; Pancreatic ductal adenocarcinoma; Protein‒protein interaction; Survival analysis.

Fig. 1

NUSAP1 was among the top 10 key genes in PDAC. a Volcano plots of DEGs in each GEO dataset were drawn using GEO2R. Red represents the genes that were significantly upregulated, and blue represents the downregulated genes in PDAC samples. Black dots represent the genes that were not significantly upregulated or downregulated in PDAC samples. b The Venn diagram shows the number of common upregulated and downregulated DEGs shared by the three GEO datasets. c STRING PPI network of common DEGs identified from three GEO datasets. d Subnetwork of the top 10 hub genes from the PPI network using Cytoscape software. e Perturbation effects of the hub genes on 44 PDAC cell lines. f Information on the genetic alterations of the hub genes

Fig. 2

Correlations between NUSAP1 expression and genome heterogeneity in PDAC. a Pan -cancer analysis of the correlation between NUSAP1 and tumor genome heterogeneity. b Homologous recombination deficiency (HRD). c Purity. d Ploidy. e Neoantigen load. f Microsatellite instability (MSI). g Mutant-allele tumor heterogeneity (MATH). h Loss of heterozygosity (LOH). i Tumor mutation burden (TMB). Pearson correlation analysis was used to analyze correlations and obtain P and R values

Fig. 3

Correlations between the expression of NUSAP1 and the infiltration levels of immune cells in PDAC. Student’s t test was used for comparisons between groups. *P < 0.05

Fig. 4

DEGs between PDAC samples with high and low NUSAP1 expression in the TCGA database. a Heatmap of the 96 DEGs between the two groups in the TCGA. b GO enrichment results for the 96 DEGs. c KEGG pathway enrichment results for the 96 DEGs.

Fig. 5

NUSAP1 expression is increased in PDAC tissues. a Representative images of IHC staining for NUSAP1 in TMAs (scale bar, 200 μm; inset scale bar, 40 μm). b NUSAP1 expression in PDAC and adjacent normal tissues, as determined by the IHC score (n = 102, Student’s t test, P < 0.001). c Box plot analyses comparing the expression levels of NUSAP1 in patient PDAC tissues (red, n = 179) and normal tissues (gray, n = 171) in the GEPIA database (Student’s t test, P < 0.05). d The OS of patients with PDAC from our center was assessed using Kaplan–Meier analysis based on NUSAP1 expression (n = 160, log-rank test, P = 0.0086). (e) OS analyses of PDAC patients according to the expression of NUSAP1 (high versus low) in the GEPIA database (n = 178, log-rank test, P = 0.0046)

Fig. 6

Decreasing NUSAP1 expression inhibits PDAC cell proliferation. a Relative expression levels of NUSAP1 among the PDAC cell lines. b mRNA expression levels of NUSAP1 in PANC-1 and CAPAN-1 cells were detected by reverse transcription quantitative PCR after transfection of si-NUSAP1. c The protein expression level of NUSAP1 decreased significantly after transfection of cells with si-NUSAP1. d The proliferation of PDAC cells was measured using CCK-8 assays. e Visualization of DNA replication by EdU incorporation. Cell nuclei stained red represent DNA replication. f A significant reduction in colony formation ability was observed after transfection with si-NUSAP1. g Apoptosis rates of the si-NUSAP1 cell lines. Student’s t test was used for comparisons between groups. All experiments were performed in triplicate. *P < 0.05. NC, negative control; si, small interfering RNA.

Fig. 7

Decreasing the expression of NUSAP1 inhibits the migration and invasion of PDAC cells. a Migration of PANC-1 cells in the si-NC and si-NUSAP1 groups according to Wound healing assays. b Migration of PANC-1 and CAPAN-1 cells in the si-NC and si-NUSAP1 groups according to Transwell assays. c Invasion of PANC-1 and CAPAN-1 cells in the si-NC and si-NUSAP1 groups according to Transwell assays. Student’s t test was used for comparisons between groups. All experiments were performed in triplicate. *P < 0.05. NC, negative control; si, small interfering RNA.

Fig. 8

NUSAP1 overexpression promotes the proliferation, migration and invasion of PDAC cells. a mRNA expression levels of NUSAP1 in PANC-1 and CAPAN-1 cells were detected by reverse transcription quantitative PCR after transfection of lentiviral vectors containing NUSAP1 OE plasmids. b The protein expression level of NUSAP1 increased significantly after transfection with OE-NUSAP1. c The proliferation of PDAC cells was measured using CCK-8 assays. d Cell migration of CAPAN-1 cells in the NC and OE groups according to Wound healing assays. e Cell migration of PANC-1 and CAPAN-1 cells in the NC and OE groups according to Transwell assays. f Cell invasion of PANC-1 and CAPAN-1 cells in the NC and OE groups according to Transwell assays. Student’s t test was used for comparisons between groups. All experiments were performed in triplicate. *P < 0.05. NC, negative control; OE, overexpression

Fig. 9

NUSAP1 promoted progression of PDAC in vivo. a We randomly divided the mice into the NC and the KD groups (n = 5), PANC-1 cells stably transfected with an empty vector or the NUSAP1 shRNA were subcutaneously inoculated in them respectively. Then we treated the mice as described in the Methods. b The tumor sizes were tested using Vernier calipers. Tumor growth curves were constructed based on the tumor volumes measured in the mice. c Quantification of the average weights of collected tumors from the above experiments. d The expression of NUSAP1, Ki-67, BAX and Cleaved caspase-3 were determined in tumor tissue sections from the xenografts using IHC (scale bar, 40 μm, n = 5). e The expression of NUSAP1, Ki-67, BAX, and Cleaved caspase-3 in tumor tissue between the NC and the KD groups, as determined by the IHC score. Student’s t test was used for comparisons between groups. *P < 0.05, **P < 0.01. NC, negative control; KD, knockdown

Fig. 10

NUSAP1 drives the EMT and reduces AMPK phosphorylation. a The expression of epithelial and mesenchymal cell phenotype markers was determined by western blot analysis. b Western blot analysis of AMPKα protein expression in NC and NUSAP1-KD cell lines, as well as phospho-AMPKα protein expression in NC, NUSAP1-KD, and NUSAP1-OE cell lines. NC, negative control; KD, knockdown; OE, overexpression