EZH2 mutation is associated with the development of visceral metastasis by enhancing proliferation and invasion and inhibiting apoptosis in breast cancer cells

Abstract

Abstract Background The prognosis of breast cancer patients with visceral metastasis (VM) is significantly worse than that of patients without VM. We aimed to evaluate EZH2 (enhancer of zeste homolog 2) mutation as a biomarker associated with VM. Methods Data from forty-nine patients with metastatic breast cancer (MBC) pathologically confirmed at our hospital between March 2016 and September 2018 were collected. Metastatic tissue samples were obtained via ultrasound-guided needle biopsy, and paired peripheral blood samples were also collected. Tissue and blood samples were subjected to targeted next-generation sequencing via a 247-gene panel. Stably transfected MDA-MB-231 cells expressing wild-type EZH2 (EZH2WT) or a mutant form of EZH2 (EZH2K515R) were generated. Cell proliferation, colony formation ability, migration and invasion abilities and apoptosis were assessed using CCK-8 assays, plate colony formation assays, Transwell chamber assays and flow cytometry. Results The incidence of EZH2 mutations in the VM subgroup was greater than that in the non-VM subgroup in the entire cohort (n = 49, 42.3% vs. 13.0%, p = 0.024) and in the triple-negative breast cancer (TNBC) subgroup (n = 20, 50.0% vs. 10.0%, p = 0.05). Patients carrying EZH2 mutations had a significantly greater risk of developing VM than did those in the non-EZH2 mutation group in the entire cohort (HR 2.9) and in the TNBC subgroup (HR 6.45). Multivariate analysis revealed that EZH2 mutation was an independent prognostic factor for VM (HR 2.99, p = 0.009) in the entire cohort and in the TNBC subgroup (HR 10.1, p = 0.006). Data from cBioPortal also showed that patients with EZH2 mutations had a significantly greater risk of developing VM (HR 3.1), and the time to develop VM was significantly earlier in the EZH2 mutation group (31.5 months vs. 109.7 months, p = 0.008). Multivariate analysis revealed that EZH2 mutation (HR 2.73, p = 0.026) was an independent factor for VM after breast cancer surgery. There was no correlation between EZH2 mutations and BRCA1/2 mutations. Most of the patients (81.8%) in our cohort who developed VM carried the “c.1544A > G (p.K515R)” mutation. Compared with EZH2WT MDA-MB-231 cells, EZH2K515R MDA-MB-231 cells had greater colony formation rates (p < 0.01), greater migration and invasion rates (p < 0.001), and lower apoptosis rates (p < 0.01). The proportion of S + G2/M phase cells in the EZH2K515R group was significantly greater than that in the EZH2WT group. Conclusions EZH2 mutation is associated with VM development in breast cancer patients. The EZH2K515R mutation leads to VM and a poor prognosis by enhancing proliferation and invasion and inhibiting apoptosis in breast cancer cells.

Keywords

• EZH2 mutation
• Visceral metastasis
• Breast cancer
• Targeted next-generation sequencing