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. 2024 Sep 19;24(1):1166.
doi: 10.1186/s12885-024-12950-y.

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

Fan Wu #  1   2 , Nani Li #  1 , Xiufeng Wu  3 , Mulan Chen  1 , Weiwei Huang  1   2 , Xinhua Chen  1 , Yi Hong  1 , Lili Wang  1 , Kan Chen  1 , Lin Lin  1 , Minjin You  1 , Jian Liu  4
Affiliations

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

Fan Wu et al. BMC Cancer. .

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: Breast cancer; EZH2 mutation; Targeted next-generation sequencing; Visceral metastasis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The flow chart of patient selection process
Fig. 2
Fig. 2
EZH2 mutation is associated with the development of visceral metastasis. (A) Patients carrying EZH2 mutations had a significantly shorter VMFS (22 months vs. 50 months, p = 0.005) than patients without EZH2 mutations. (B) Patients carrying EZH2 mutations had a significantly shorter VMFS (10 months vs. unreached, p = 0.001) than patients without EZH2 mutations in the TNBC subtype. (C) DFS time of patients with EZH2 mutations was significantly shorter than that of patients without EZH2 mutations (26.9 months vs. 52.7 months, p = 0.029) in 1,392 patients from cBioPortal. (D) VMFS was significantly shorter in the EZH2 mutation group than that of patients without EZH2 mutations (31.5 months vs. 109.7 months, log-rank p = 0.008) in 1,392 patients from cBioPortal
Fig. 3
Fig. 3
*** p < 0.001; ** p < 0.01. The cell functions in stably transduced MDA-MB-231 cells (EZH2WT and EZH2K515R). (A, B.) The cell proliferation rates of the EZH2K515R and EZH2WT groups were decreased at 48 h and 72 h compared with those of the control group (p < 0.001), but there was no difference in the cell proliferation rate between the EZH2K515R and EZH2WT groups. (C, D.) The EZH2K515R group had a greater colony formation rate than the EZH2WT group (p < 0.01). (E, F.) The EZH2K515R group had greater migration rate than the EZH2WT group (p < 0.001). (G, H.) The EZH2K515R group had a greater invasion rate than the EZH2WT group (p < 0.001). (I, J.) The EZH2K515R group had a lower apoptosis rate than the EZH2WT group (p < 0.01). The EZH2K515R group had a greater proportion of cells in the S phase (p < 0.001) and G2/M phase (p < 0.01) and a lower proportion of cells in the G1/G0 phase (p < 0.001) than did the EZH2WT group.
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