Aberrant PTPRO methylation in tumor tissues as a potential biomarker that predicts clinical outcomes in breast cancer patients

Abstract

Background: Aberrant hypermethylation of gene promoter regions is a primary mechanism by which tumor suppressor genes become inactivated in breast cancer. Epigenetic inactivation of the protein tyrosine phosphatase receptor-type O gene (PTPRO) has been described in several types of cancer.

Results: We screened primary breast cancer tissues for PTPRO promoter hypermethylation and assessed potential associations with pathological features and patient outcome. We also evaluated its potential as a breast cancer biomarker. PTPRO methylation was observed in 53 of 98 (54%) breast cancer tissues but not in adjacent normal tissue. Among matched peripheral blood samples from breast cancer patients, 33 of 98 (34%) exhibited methylated PTPRO in plasma. In contrast, no methylated PTPRO was observed in normal peripheral blood from 30 healthy individuals. PTPRO methylation was positively associated with lymph node involvement (P = 0.014), poorly differentiated histology (P = 0.037), depth of invasion (P = 0.004), and HER2 amplification (P = 0.001). Multivariate analysis indicated that aberrant PTPRO methylation could serve as an independent predictor for overall survival hazard ratio (HR): 2.7; 95% CI: 1.1-6.2; P = 0.023), especially for patients with HER2-positive (hazard ratio (HR): 7.5; 95% CI: 1.8-31.3; P = 0.006), but not in ER + and PR + subpopulation. In addition, demethylation induced by 5-azacytidine led to gene reactivation in PTPRO-methylated and -silenced breast cancer cell lines.

Conclusions: Here, we report that tumor PTPRO methylation is a strong prognostic factor in breast cancer. Methylation of PTPRO silences its expression and plays an important role in breast carcinogenesis. The data we present here may provide insight into the development of novel therapies for breast cancer treatment. Additionally, detection of PTPRO methylation in peripheral blood of breast cancer patients may provide a noninvasive means to diagnose and monitor the disease.

Figure 1

Representative MSP results for methylation of the PTPRO gene. (a) primary breast tumors; (b) matched peripheral blood samples. Numbers indicate the sample number. B, blank (no DNA); N, negative control; P, positive control; M, methylated; U, unmethylated.

Figure 2

Kaplan–Meier survival analysis for breast cancer patients with (solid line) PTPRO tumor methylation or without (dotted line). (a) overall group; (b) ER+; (c) PR+; (d) HER2-amplified subgroup.

Figure 3

PTPRO is methylated in breast cancer cell lines but not in normal breast epithelial cells. (a) Expression of PTPRO in normal human mammary epithelial cells (48R) and human breast cancer cell lines Hs578t, MCF-7, and MDA-MB-231. Total RNA isolated from cell lines was subjected to RT-PCR analysis using PTPRO-specific primers. 18S rRNA was used as an internal loading control. (b) MSP analysis of PTPRO methylation status in breast cancer cell lines. HMESC48R was used as a normal control. M, methylated; U, unmethylated. (c) PTPRO CpG island from randomly selected breast tumor tissue and its matched normal tissue; also shown are HMEC 48R and MCF-7 cells, all of which were subjected to BS genomic sequencing. Each solid square represents a methylated cytosine and an open square represents unmethylated cytosine in a CpG dinucleotide. Each row corresponds to a single clone. N, normal corresponding adjacent non-cancerous tissue; T, tumor tissue.

Figure 4

Re-expression of PTPRO following treatment with 5-AzaC. (a) Breast cancer cell lines MCF-7 and MDA-MB-231 were treated with 1 μM 5-AzaC for 72 h and 2.5 μM 5-AzaC for 96 h, respectively. Total RNA from cells was subjected to RT-PCR to amplify PTPRO mRNA. 18S rRNA was used for normalization; (b) MSP analysis of PTPRO methylation status in breast cancer cell lines with or without 5-AzaC treatment. M, methylated; U, unmethylated.