Multi-Omics Exploration of the Mechanism of Curcumol to Reduce Invasion and Metastasis of Nasopharyngeal Carcinoma by Inhibiting NCL/EBNA1-Mediated UBE2C Upregulation

Abstract

Nasopharyngeal carcinoma (NPC) is closely linked to Epstein-Barr virus (EBV) infection. Curcumae Rhizoma, a traditional Chinese herb, has shown antitumor effects, primarily through its component curcumol (Cur), which has been shown to reduce NPC cell invasion and migration by targeting nucleolin (NCL) and Epstein-Barr Virus Nuclear Antigen 1 (EBNA1). We constructed an EBV-positive NPC cell model using C666-1 cells and performed transcriptomics studies after treatment with curcumol, which revealed a significant enrichment of ubiquitin-mediated proteolysis, the PI3K-AKT and mTOR signaling pathways, cell cycle and apoptosis involved in tumor invasion and migration. To investigate the importance of NCL and EBNA1 in curcumol-resistant EBV-positive NPC, we performed a multi-omics study using short hairpin NCL (shNCL) and shEBNA1 EBV-positive NPC cells, and the proteomics results showed enrichment in complement and coagulation cascades and ubiquitin-mediated proteolysis signaling pathways. Here, we focused on ubiquitin-conjugating enzyme E2C (UBE2C), which plays an important role in the ubiquitin-mediated proteolysis signaling pathway. In addition, metabolomics revealed that UBE2C is highly associated with 4-Aminobutanoic acid (GABA). In vitro studies further validated the function of the key targets, suggesting that UBE2C plays an important role in NCL and EBNA1-mediated curcumol resistance to nasopharyngeal carcinoma invasion and metastasis.

Keywords: EBNA1; NCL; UBE2C; curcumol; metabolomics; nasopharyngeal carcinoma; proteomics; transcriptomics.

Figure 1

Curcumol treatment altered the transcriptomics profile of NPC cells. (A) Heatmap of DEGs after curcumol treatment. (B) Volcano plots of DEGs after curcumol treatment. (C) GO annotation of DEGs, which were divided into BP, CC, and MF. (D) KEGG pathways’ enrichment analysis bubble plot of DEGs.

Figure 2

Proteomics analysis of DEPs in NPC cells of the shNCL or shEBNA1. (A) Principal component analysis (PCA) of the DEPs. (B) Up-regulation and down-regulation of the amount of protein. (C) Volcano plots of the enriched DEPs. (D) Heatmaps of the enriched DEPs.

Figure 3

KEGG analysis of DEPs in NPC cells with shNCL or shEBNA1. (A) Significantly enriched KEGG pathways based on the DEPs in “shNCL vs. Control”. (B) Significantly enriched KEGG pathways based on the DEPs in “shEBNA1 vs. Control”.

Figure 4

Correlation analysis of transcriptomics and proteomics revealed key proteins in the inhibition of NPC by curcumol. (A) Flow chart of key target approaches for curcumol inhibition of NPC. (B) Venn diagram of DEPs for “shNCL vs. Control” and “shEBNA1 vs. Control”, red boxes indicate the number of DEPs regulated in the same direction. (C) Venn diagram of DEGs and DEPs, and common key targets shown, red boxes were same with (B).

Figure 5

Correlation analysis of the key proteins. Red represented a positive correlation and blue represented a negative correlation. Larger red or blue circles indicated a stronger correlation. The correlation coefficients were shown on the diagonal box. (A) The group of “shNCL vs. Control”. (B) The group of “shEBNA1 vs. Control”.

Figure 6

Knockdown of NCL or EBNA1 altered the metabolomic profile of EBV-positive NPC cells. (A) OPLS-DA analysis. (B) PCA analysis. (C) Volcano diagram of “shNCL vs. Control”. (D) Volcano diagram of “shEBNA1 vs. Control”.

Figure 7

Hierarchical clustering heatmap of differential metabolites. (A) The group of “shNCL vs. Control”. (B) The group of “shEBNA1 vs. Control”.

Figure 8

Combined metabolomics and proteomics analyses revealed that 4-Aminobutanoic acid (GABA) was highly negatively correlated with UBE2C. (A) Heatmap of the correlation network of UBE2C, NCL and differential metabolites in “shNCL vs. Control”. (B) Heatmap of correlation network of UBE2C, NCL with differential metabolites in “shEBNA1 vs. Control”.

Figure 9

The expression of UBE2C in the experimental model of EBV-positive NPC both in vitro and in vivo. (A) mRNA expression levels of UBE2C in RNA-seq of NPC cells. (B) Protein abundance levels of UBE2C in proteomics of NPC cells. (C) qRT-PCR to detect the expression of UBE2C in the subcutaneously transplanted tumor tissue of nude mice. (D) WB to detect the expression of UBE2C in the subcutaneously transplanted tumor tissue of nude mice (data are presented as mean ± SEM. n = 3, ** p  <  0.01, *** p  <  0.001, **** p  <  0.0001). Original images can be found in Figure S7.

Figure 10

si-UBE2C inhibited EBV-positive NPC cell invasion and migration. (A) The migration ability of the C666-1 cell line after silencing UBE2C was detected by scratch assay. (B) The invasion ability of the C666-1 cell line after silencing of UBE2C was detected by transwell assay (data are presented as mean ± SEM. n = 3, ** p  <  0.01).

Figure 11

Mechanism of curcumol reducing NPC invasion and metastasis through inhibition of NCL/EBNA1/UBE2C explored by multi-omics.