Epigenetic hypomethylation and upregulation of GD3s in triple negative breast cancer

Abstract

Background: Breast cancer remains a major health problem in the world. Triple-negative breast cancer (TNBC) is an aggressive subtype with very poor prognosis. Up to now, the mechanism behind TNBC's activity is still unclear and no candidate drug target has been identified. Thus, it is of critical importance to elucidate the pathways in TNBC and identify the relevant biomarkers. Recent studies showed that ganglioside D3 synthase (GD3s) played a very important role in development of cancers. However, the physiological functions and associated pathways of GD3s in TNBC are still unclear.

Methods: In silico analysis of the expression of GD3s in TNBC was conducted using The Cancer Genome Atlas (TCGA) and Oncomine databases. The proliferation of breast cancer cells was measured by MTT assay, colony formation by the soft agar method, and migration and invasion using Boyden chamber inserts. The methylation level of the gene encoding GD3s, ST8SIA1, in specimens was assessed by qMS-PCR and in silico using the UCSC gene browser. Protein expression was examined via immunohistochemistry (IHC), qRT-PCR and Western immunoblotting.

Results: In silico analysis showed a higher GD3s expression in ER^- than ER⁺ breast cancers and GD3s was also highly expressed in TNBC compared to other types of breast cancers. The elevated GD3s expression in TNBC cells and tissues was associated with hypomethylation of the ST8SIA1 gene. Overexpression of GD3s in human breast cancer cells increased their proliferation, migration, invasion and colony formation ability. GD3s expression in breast cancers was closely associated with relapse-free survival (RFS) and overall survival (OS).

Conclusions: In summary, these results suggest that GD3s may be a potential biomarker and drug target in treatment of TNBC.

Keywords: Ganglioside D3 synthase (GD3s); biomarker; methylation; prognosis; triple-negative breast cancer (TNBC).

Figure 1

GD3s is inversely correlated with signatures of luminal breast cancers, ESR1, FOXA1, GATA3, XBP1 and MYB. GD3s is inversely correlated with (A) ESR1, (B) FOXA1, (C) GATA3, (D) XBP1, and (E) MYB. GD3s is positively correlated with (F) FOXC1, which is a biomarker of BLBC.

Figure 2

GD3s expression is significantly higher in ER⁺ breast cancer than ER⁻. (A,B,C,D,E) GD3s expression is higher in ER⁺ breast cancer than ER⁻. Data and statistics were obtained from www.oncomine.org (TCGA 2011; Richardson et al., 2006; Curtis et al., 2012; Gluck et al., 2011; Zhao et al., 2004).

Figure 3

GD3s is highly expressed in TNBCs. (A,B,C,D,E) GD3s expression is higher in TNBC than other types of breast cancers. Data and statistics were obtained from www.oncomine.org (TCGA 2011; Richardson et al., 2006; Curtis et al., 2012; Zhao et al., 2004; Turashvili et al., 2007); (F) GD3s mRNA expression is higher in TNBC cell lines (MDA-MB-231, MDA-MB-435 and HCC1143) than other types of breast cancer cell lines (MCF-7, T47D, BT47D and ZR75-1); (G) GD3s protein expression is higher in TNBC cell lines (MDA-MB-231, MDA-MB-435 and HCC1143) than other types of breast cancer cell lines (MCF-7, T47D, BT47D and ZR75-1).

Figure 4

GD3s promotes proliferation, migration, invasion and colony formation of breast cancer cells. (A) Overexpression of GD3s promotes proliferation of MCF-7 cells while knockdown of GD3s inhibits proliferation of MDA-MB-468; (B) overexpression of GD3s increases migration of MCF-7 cells while knockdown of GD3s reduces migration of MDA-MB-468; (C) overexpression of GD3s increases invasion of MCF-7 cells while knockdown of GD3s reduces invasion of MDA-MB-468 (stained with 1% crystal violet); (D) overexpression of GD3s promotes colony formation of MCF-7 cells while knockdown of GD3s inhibited colony formation of MDA-MB-468, stained with 5 mg/mL MTT.

Figure 5

GD3s expression is associated with methylation of the ST8SIA1 gene. (A) A 102 bp CpG island is present in the promoter region of the ST8SIA1 gene; (B) expression of GD3s is negatively correlated with methylation of ST8SIA1; (C) survival rate of patients with breast cancers is significantly associated with methylation of ST8SIA1; (D) methylation of ST8SIA1 is significantly lower in TCGA breast cancer tissues; (E) methylation levels of ST8SIA1 gene promoter in eight breast cancer cell lines are significantly lower (P<0.05) than in non-tumor MCF-10A cells; (F) GD3s expression in MCF-7 cells and T47D cells treated with 5-azacytidine are increased compared to controls.

Figure 6

Expression of GD3s protein is much higher in TNBC than luminal breast cancer. (A) Representative pictures of luminal (left) and triple negative breast cancer (right). Staining was done with diaminobenzidine and cells were counterstained with hematoxylin; (B) expression of GD3s is significantly higher in TNBC compared to luminal breast cancer; (C) expression of GD3s is significantly associated with overall survival (OS) in all patients with breast cancer; (D) expression of GD3s is significantly associated with OS in all patients with breast cancer in TCGA data.

Figure S1

ST8SIA1 gene 1,000 bp promoter.

Figure S2

Expression of GD3s is significantly associated with relapse-free survival in patients with breast cancer.

Figure S3

Expression of GD3s is significantly associated with overall survival in patients with grade 3 breast cancer.

Figure S4

Expression of GD3s is significantly associated with relapse-free survival in patients with grade 3 breast cancer.

Figure S5

Expression of GD3s is significantly associated with distant metastasis-free survival in patients with grade 3 breast cancer.