PCDHB15 as a potential tumor suppressor and epigenetic biomarker for breast cancer

Abstract

Breast cancer is among the most frequently diagnosed cancer types and the leading cause of cancer-related death in women. The mortality rate of patients with breast cancer is currently increasing, perhaps due to a lack of early screening tools. In the present study, using The Cancer Genome Atlas (TCGA) breast cancer dataset (n=883), it was determined that methylation of the protocadherin β15 (PCDHB15) promoter was higher in breast cancer samples than that in normal tissues. A negative association between promoter methylation and expression of PCDHB15 was observed in the TCGA dataset and breast cancer cell lines. In TCGA cohort, lower PCDHB15 expression was associated with shorter relapse-free survival times. Treatment with the DNA methyltransferase inhibitor restored PCDHB15 expression in a breast cancer cell line; however, overexpression of PCDHB15 was shown to suppress colony formation. PCDHB15 methylation detected in circulating cell-free DNA (cfDNA) isolated from serum samples was higher in patients with breast cancer (40.8%) compared with that in patients with benign tumors (22.4%). PCDHB15 methylation was not correlated with any clinical parameters. Taken together, PCDHB15 is a potential tumor suppressor in cases of breast cancer, which can be epigenetically silenced via promoter methylation. PCDHB15 methylation using cfDNA is a novel minimally invasive epigenetic biomarker for the diagnosis and prognosis of breast cancer.

Keywords: DNA methylation; biomarker; breast cancer; cell-free DNA; protocadherin β15.

Figure 1.

PCDHB15 may be a tumor suppressor gene that is epigenetically silenced in in breast cancer. (A) Schematic diagram depicting the genomic structure and position of the CG sites (vertical dashes) in the PCDHB15 promoter region (from-1,000 to +400 with respect to the transcriptional start site). The location of the microarray probes (red vertical dashes), bisulphite pyrosequencing (yellow horizontal line) and qMSP primers (blue solid arrows) are indicated. (B) DNA methylation level (β-value from Illumina Infinium 450 K microarray) of the PCDHB15 CpG island from-278 (cg03572772) to +2252 (cg15006101) in solid tissue normal (white) vs. primary tumor (red) in TCGA breast cancer dataset. Primary tumor tissues (n=785) had higher methylation levels than solid tissue normal tissues (n=98). The x-axis indicates the name of the probe on the microarray. (C) Scatter plot showing correlation between PCDHB15 promoter methylation (cg17023770; x-axis) and expression (y-axis) in TCGA breast cancer dataset (n=873). A negative association between promoter methylation and expression was observed. (D) Kaplan-Meier analysis of PCDHB15 mRNA expression in tumor tissues for relapse-free survival of patients with breast cancer. Patients with breast cancer with lower PCDHB15 expression demonstrated shorter relapse-free survival times than patients with higher PCDHB15 expression (log-rank test, P=0.00011). (E) Relative expression level of PCDHB15 mRNA in MCF7 and MDA-MB-231 breast cancer cells. (F) Methylation analysis of PCDHB15 promoter in breast cancer cell lines using bisulphite pyrosequencing. (G) Relative expression level of PCDHB15 in 0.1 µM 5aza-treated MDA-MB-231 breast cancer cells, compared with DMSO control. (H) Ectopic expression of PCDHB15 inhibited tumor proliferation by colony formation assay. MDA-MB-231 breast cancer cells were transfected with empty (control) or PCDHB15 expression vector. Left panel, reverse transcription-quantitative PCR confirmed overexpression of PCDHB15 in MDA-MB-231 cells transiently transfected with PCDHB15 expression vector. Medium panel, MDA-MB-231 breast cancer cells overexpressing PCDHB15 had significantly fewer colonies than the control. Right panel, quantitative analysis of the colony formation assay. Colony formation assay were performed in duplicate and in two independent experiments (mean ± SD). *P<0.05 and **P<0.001. PCDHB15, protocadherin β15; TCGA, The Cancer Genome Atlas; 5aza, 5′-aza-2′-deoxycytidine; qMSP, quantitative methylation-specific PCR.

Figure 2.

Methylated PCDHB15 level is higher in cfDNA of serum samples of patients with breast cancer. (A) Gel electrophoresis image of PCDHB15 (upper panel) and COL2A1 (lower panel) MSP in cfDNA isolated from serum samples. IVD was a positive control for methylation and H₂O was a negative control for PCR. (B) qMSP was performed to determine the amount of methylated PCDHB15 in cfDNA of breast cancer (n=49) and benign tumor samples (n=49). Compared with the benign tumor samples, higher amounts of PCDHB15 were detected in cancer samples. (C) A receiver operating characteristic curve of PCDHB15 methylation in serum samples from 49 patients with breast cancer and 49 patients with benign tumors. The original uncropped gel electrophoresis images can be found in supplementary Fig. S1. *P<0.05. PCDHB15, protocadherin β15; qMSP, quantitative methylation-specific PCR; cfDNA, cell free DNA; IVD, in vitro methylated DNA; AUC, area under the curve; COL21A, collagen type II α1 chain.

Figure 3.

PCDHB15 promoter methylation and the clinicopathological features of breast cancer are not associated. (A and B) Scatter plot showing the correlation between PCDHB15 promoter methylation in cfDNA and expression level of (A) Ki67 and (B) PR in breast tumor tissues. (C and D) Dot plot showing the amount of methylated PCDHB15 in cfDNA with expression status of ER (ER⁺, ER⁻) and HER (HER⁺, HER⁻). PCDHB15, protocadherin β15; PR, progesterone receptor; cfDNA, cell free DNA; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2.