Overexpression of EZH2/NSD2 Histone Methyltransferase Axis Predicts Poor Prognosis and Accelerates Tumor Progression in Triple-Negative Breast Cancer

Abstract

Two histone methyltransferases, enhancer of zeste homolog 2 (EZH2) and nuclear SET domain-containing 2 (NSD2), are aberrantly expressed in several types of human cancers. However, the regulatory relationship between EZH2 and NSD2 and their prognostic values in breast cancer (BC) have not been fully elucidated. In this study, we demonstrated that EZH2 and NSD2 were overexpressed in BC compared with benign lesions and normal tissues using tissue microarray, immunohistochemistry, and bioinformatic databases. Both EZH2 and NSD2 expression were associated with pathological grade of tumor and lymph node metastasis. A comprehensive survival analysis using Kaplan-Meier Plotter database indicated that EZH2 expression was negatively correlated with relapse-free survival (RFS), overall survival (OS), distant metastasis-free survival (DMFS), and postprogression survival (PPS) in 3951 BC patients, and NSD2 expression was negatively correlated with RFS and DMFS. Notably, EZH2 and NSD2 expression were coordinately higher in triple-negative breast cancer (TNBC) than that in other subtypes. Stable knockdown of EZH2 using lentiviral shRNA vector significantly reduced the proliferation, migration and invasion abilities of TNBC cell line MDA-MB-231 and MDA-MB-468, and downregulated NSD2 expression as well as the levels of H3K27me3 and H3K36me2, two histone methylation markers catalyzed by EZH2 and NSD2, respectively. By contrast, overexpression of EZH2 using adenovirus vector displayed an inverse phenotype. Furthermore, knockdown of NSD2 in EZH2-overexpressing cells could dramatically attenuate EZH2-mediated oncogenic effects. Bioinformatic analysis further revealed the function and pathway enrichments of co-expressed genes and interactive genes of EZH2/NSD2 axis, suggesting that EZH2/NSD2 axis was associated with cell division, mitotic nuclear division and transition of mitotic cell cycle in TNBC. Taken together, EZH2/NSD2 axis may act as a predictive marker for poor prognosis and accelerate the progression of TNBC.

Keywords: EZH2; NSD2; prognosis; progression; triple-negative breast cancer.

Figure 1

EZH2 and NSD2 proteins were overexpressed in breast cancer (BC) tissues. (A) HE staining of tissue microarray (TMA) sections containing benign lesion (×400), IDC (×400), DCIS (×40), and ILC (×100). (B) EZH2 and NSD2 expression in benign lesion (×400), IDC (×400), DCIS (×40), and ILC (×100) tissues were analyzed by IHC. (C) EZH2 and NSD2 expression in normal breast tissue, ductal carcinoma and lobular carcinoma were analyzed using Human Protein Atlas database.

Figure 2

EZH2 and NSD2 messenger RNA (mRNA) levels were upregulated in breast cancer (BC) tissues. (A–D) The relative expression of EZH2 mRNA in BC and normal tissues was analyzed using Oncomine database. (E–H) The relative expression of NSD2 mRNA in BC and normal tissues was analyzed using Oncomine database. (I) The correlation between EZH2 and NSD2 mRNA expression in BC was analyzed using UALCAN database.

Figure 3

Survival curves for relapse-free survival (RFS) (A), overall survival (OS) (B), distant metastasis-free survival (DMFS) (C), and postprogression survival (PPS) (D) in patients with BC according to EZH2 expression were analyzed using Kaplan-Meier Plotter database.

Figure 4

Survival curves for relapse-free survival (RFS) (A), overall survival (OS) (B), distant metastasis-free survival (DMFS) (C), and postprogression survival (PPS) (D) in patients with breast cancer (BC) according to NSD2 expression were analyzed using Kaplan-Meier Plotter database.

Figure 5

The relative expression of EZH2 messenger RNA (mRNA) (A) and NSD2 mRNA (B) among different molecular subtypes of breast cancer (BC) were analyzed using UALCAN database. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Stable knockdown of EZH2 inhibited the proliferation, migration and invasion of MDA−MB−231 cells, whereas overexpression of EZH2 displayed an inverse phenotype. Stable knockdown or transient overexpression of EZH2 was performed in MDA-MB-231 cells. (A) The relative messenger RNA (mRNA) levels of EZH2 and NSD2 were determined by qRT-PCR. (B) The protein levels of EZH2, NSD2 and histone methylation markers were determined by Western blot. Left panel showed the representative images of protein expression and right panel showed the fold changes of protein levels. (C) The cell proliferation ability was determined by CCK8 assay. (D) The cell migration ability was determined by wound healing assay. Left panel showed the representative images of cell migration and right panel showed the wound healing rate at 24 and 48 h. (E) The cell invasion ability was determined by transwell assay. Left panel showed the representative images of cell invasion and right panel showed the absorbance of invasive cells stained by crystal violet. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Knockdown of NSD2 attenuated the proliferation, migration and invasion abilities of EZH2-overexpressing MDA-MB-231 cells. Transient overexpression of EZH2 was performed in MDA-MB-231 cells with stable knockdown of NSD2. (A) The relative messenger RNA (mRNA) levels of EZH2 and NSD2 were determined by qRT-PCR. (B) The protein levels of EZH2, NSD2, and histone methylation markers were determined by Western blot. Left panel showed the representative images of protein expression and right panel showed the fold changes of protein levels. (C) The cell proliferation ability was determined by CCK8 assay. (D) The cell migration ability was determined by wound healing assay. Left panel showed the representative images of cell migration and right panel showed the wound healing rate at 24 and 48 h. (E) The cell invasion ability was determined by transwell assay. Left panel showed the representative images of cell invasion and right panel showed the absorbance of invasive cells stained by crystal violet. *P < 0.05, **P < 0.01, NS, non-significant.

Figure 8

The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of co-expressed genes of EZH2/NSD2 axis. (A) The protein-protein interaction (PPI) network of EZH2, NSD2, and 94 co-expressed genes in breast cancer (BC). (B) Top 10 of enriched biological processes. (C) Top 10 of enriched cellular components. (D) Top 10 of enriched molecular functions. (E) Top 10 of enriched KEGG pathways.

Figure 9

The protein-protein interaction (PPI) network and relative expression of interactive genes of EZH2/NSD2 axis in triple-negative breast cancer (TNBC). (A) The PPI network of EZH2, NSD2 and the interactive genes in BC. (B) The expression heatmap of EZH2, NSD2, and the interactive genes in TNBC. (C) The median expression levels of CCNA2, CDK2, KDM2B, KIF11, KIF23, and PCNA in TNBC and normal breast tissues were analyzed using UALCAN database. (D) The relative mRNA levels of CCNA2, CDK2, KDM2B, KIF11, KIF23, and PCNA were analyzed by qRT-PCR in MDA-MB-231 and MDA-MB-468 cells with stable knockdown of EHZ2. (E) The relative mRNA levels of CCNA2, CDK2, KDM2B, KIF11, KIF23, and PCNA were analyzed by qRT-PCR in MDA-MB-231 and MDA-MB-468 cells with stable knockdown of NSD2. *P<0.05, **P<0.01, ***P<0.001.

Figure 10

A schematic diagram displaying the proposed mechanism by which EZH2/NSD2 axis promotes cell proliferation, migration and invasion and mediates poor prognosis in triple-negative breast cancer (TNBC).