Protocadherin-10 (PCDH10) is a tumor suppressor gene. Its expression level is downregulated by promoter methylation in certain types of human tumors. The aim of the present study was to examine the expression level and promoter methylation status of PCDH10 in breast cancer cells and to evaluate the association of PCDH10 methylation and tumor progression and prognosis. MethyLight was used to detect the methylation status of PCDH10 in breast cancer tissues and healthy breast tissues. Reverse transcription-quantitative polymerase chain reaction was used to assess the mRNA expression level of PCDH10, as well as to evaluate the association between PCDH10 methylation and clinicopathological features, along with patients' overall survival (OS). PCDH10 5′-C-phosphate-G-3′ (CpG) methylated sites were identified in tumor tissues and matched healthy tissues (n=392). Tumor tissues and matched healthy tissues exhibited identifiable PCR results, with PCDH10 gene promoter methylation identified in ductal carcinoma
Breast cancer is a variable and complex disease, and is a leading causes of mortality (
Epigenetic maps serve an important role in identifying specific functional variations that cause or contribute to a number of diseases. Previous studies have reported that epigenetic abnormalities may serve an important role in the initial stages of a number of cancer types, including colorectal cancer and prostate cancer (
Numerous studies have used methylation patterns as diagnostic and prognostic biomarkers for breast cancer (
The aim of the present study was to investigate a novel methylation-based diagnostic tool for breast cancer and to resolve the difficulties associated with the implementation of methylation genes as biomarkers. It was demonstrated that PCDH10 could be an effective prognostic biomarker for patients with various types of breast cancer, including ductal carcinoma
A total of 392 samples of flash-frozen cancerous and paired healthy breast tissues (≥5 cm distant from the tumor tissue) were collected from patients with breast cancer, who underwent mastectomy at the Harbin Medical University Cancer Hospital (Heilongjiang, China) between May 2009 and October 2012. Serum samples (1 ml) were obtained from 300 patients (47±18 years old) with breast cancer, as well as from healthy subjects (45±12 years old) at the Second Affiliated Hospital of Harbin Medical University (Heilongjiang, China) between May 2009 and October 2012. The breast cancer patients with other diseases were excluded from this study according to clinical detection. The healthy subjects were from patients who received physical examination and were identified as healthy. The types of benign breast diseases included fibroadenoma, desmoid tumors, benign phyllodes tumors, mastopathy, papilloma, duct ectasia and hamartoma. The healthy serum sample (n=300) was acquired from the Affiliated Tumor Prevention and Treatment Institution of the Harbin Medical University (Heilongjiang, China) between May 2009 and October 2012. All patients provided written informed consent for tissue and serum collection, in consistence with regulations of the institutional review board of the Harbin Medical University (Heilongjiang, China). The present study was completed in compliance with the Declaration of Helsinki and was approved by the ethics committee of the Harbin Medical University (Heilongjiang, China).
Tissue sections (8 µm) were obtained from breast tissues and stored at −25°C. These sections were stained with 10% hematoxylin for 5 min and 0.5% eosin for 1 min at room temperature, and then were examined by two independent pathologists from Harbin Medical University Cancer Hospital (Harbin, China), who were blind to the study, to ensure the integrity of the tumor sample (tumor content >70%), and to verify that healthy tissue blocks contained no tumor cells under light microscopy at ×100 and ×400 magnifications. Malignant samples were categorized into four groups based on histopathology: i) DCIS; ii) IDC; iii) IDC-L; or iv) HpBC, included patients with a first-degree relative with breast cancer, patients with bilateral breast tumors, and <35-year-old patients with early-onset breast cancer (
The estrogen receptor (ER) mouse monoclonal antibody was obtained from Ventana Medical Systems, Inc. (1:200 dilution; cat. no. 760-2596; Tucson, AZ, USA) and progesterone receptor (PR) mouse monoclonal antibody from Dako (1:200 dilution; cat. no. M3569; Agilent Technologies, Inc., Santa Clara, CA, USA). The sections were incubated with antibodies at 4°C overnight. The bound antibodies were detected using peroxidase-conjugated goat anti-mouse IgG (ready-to-use secondary antibody; cat. no. TA130004; OriGene Technologies, Inc., Beijing, China) at 37°C for 2 h, and the final staining was completed with DAB (OriGene Technologies, Inc.) at room temperature for 3 min. Nuclear labeling revealed that >1% of cells were ER- or PR-positive (
Genomic DNA was extracted from the fresh-frozen primary breast tumor tissues and the matched healthy breast tissues. Samples were pre-treated with 20 mg/ml proteinase K (Promega Corporation, Madison, WI, USA) at 55°C overnight. DNA was extracted using the AxyPrep™ Multisource Genomic DNA Miniprep kit (Axygen; Corning Incorporated, Corning, NY, USA), according to manufacturer's protocol, and ~5 ml of peripheral blood was collected prior to the physical examination or surgery. All samples were analyzed in the laboratory within 4 h. Circulating free DNA was obtained from 1 ml of serum using the QIAamp UltraSens Virus kit (cat. no. 53706; Qiagen GmbH, Hilden, Germany).
The bisulfite conversion of genomic DNA and MethyLight assay was performed using the EZ DNA Methylation kit (Zymo Research Corp., Irvine, CA, USA), according to the manufacture's protocol. For each bisulfite sequencing PCR (BSP) reaction, the PCR mixture included 1.5 mM MgCl2, 200 µM dNTP, 1 µM of forward and reverse primers (sequences in
The probable promoter CpG island methylated sites were selected to design probes for the PCDH10 gene, in accordance with the results of BSP sequencing. CpG methylation of the PCDH10 gene was detected using methylation-specific primers (
MethyLight is a high-throughput assay for DNA methylation based on real time qPCR. MethyLight requires only minute amounts of modest quality DNA, making it clinically applicable and compatible for use with small biopsies and paraffin-embedded tissues (
The levels of methylation and mRNA expression were analyzed using Fisher's exact test and Kruskal-Wallis test. Receiver operating characteristic (ROC) curve analysis, Mann-Whitney U test, Kaplan-Meier survival curves and Cox proportional hazards regression model were used to assess prognostic associations. All tests were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Using bioinformatics analysis to identify DNA fragments (n=31) of the PCDH family, the proximal promoter and exon 1 of the PCDH10 gene were located in 4q28.3 (
PCDH10 displayed widespread methylation of the aberrant promoter CpG island. The frequency of PCDH10 methylation significantly increased with disease progression from
The association between PCDH10 methylation and various clinicopathological features was also examined (
The results of the present study demonstrated that the frequency of gene methylation was significantly increased in breast cancer tissues compared with control. Methylation was investigated as a possible diagnostic predictor of breast cancer. Identical specific probes were used to assess PCDH10 methylation in matched serum samples using MethyLight. Samples included 300 age-matched healthy controls and 300 age-matched patients with benign breast diseases. ROC analysis of PCDH10 indicated a sensitivity of 75%, a specificity of 62.5%, and an area under the curve of 0.682 (95% confidence interval; 0.645–0.719; P<0.001;
Detection of hypermethylation is an important tool in the diagnosis of cancer and in prognostic and therapeutic guidance (
It has been reported that PCDH10 suppresses proliferation, metastasis and invasion of cancer cells (
MethyLight was used in the present study to identify hypermethylation-silenced genes in tumor tissues. These genes may be candidate tumor suppressor genes (TSGs). PCDH10 is frequently silenced by methylation in a tumor-specific manner (
Breast cancer tissues and paired healthy tissues were examined and PCDH10 methylation was identified in ~72% of breast cancer tissues. It was also determined that the rate of low PCDH10 expression rate was ~70%. DNA methylation can silence TSGs and, therefore, we hypothesized that decreased PCDH10 expression may be attributed to its methylation. Using a large sample size, it was concluded that PCDH10 methylation occurs in the majority of breast cancer cases. The present study demonstrated that PCDH10 methylation is associated with tumor size (P=0.004). In the tumor size ≥2 cm group, methylated PCDH10 tissues were more prevalent than unmethylated PCDH10 tissues. This indicates that DNA methylation may serve an important role in breast cancer. The present study, to the best of our knowledge, is the first to investigate the prognostic value of PCDH10 gene promoter methylation in patients with breast cancer. Patients with PCDH10 methylation exhibited notably reduced OS rates compared with patients exhibiting unmethylated PCDH10 (P=0.005, log-rank test). Therefore, PCDH10 methylation is indicated to be independently associated with poor prognosis in patients with invasive breast cancer. The present study is the first, to the best of our knowledge, to illustrate the association between DNA methylation, clinicopathological characteristics and survival in Chinese patients with breast cancer.
Furthermore, cell-free DNA detected in serum and extracted from cancer cells has been used as a non-invasive biomarker to facilitate diagnosis and prognostic guidance for various types of cancer, including gastric cancer (
In conclusion, the results of the present study suggest that frequency of PCDH10 promoter methylation is increased in human breast cancer tissues, compared with adjacent non-tumor tissues. PCDH10 was concluded to be an important TSG, which restricted the progression of breast cancer. In addition, the methylation status of PCDH10 may be a useful diagnostic and prognostic biomarker for breast cancer. However, it has been reported that environmental and lifestyle factors can alter the status of DNA methylation (
Not applicable.
Not applicable.
The data used and analyzed during the current study are available from the corresponding author on reasonable request.
QZ designed the study and revised the paper. WL and JW performed the studies. GS, XY and DL performed the statistical analysis. All authors read and approved the final manuscript.
This research was completed in compliance with the Helsinki Declaration. The data collection and analysis were conducted without disclosing patients' identities. All patients provided written informed consent for tissue and serum collection, in consistence with regulations of the institutional review board of the Harbin Medical University (Heilongjiang, China).
All patients provided written informed consent for publication, in consistence with regulations of the institutional review board of the Harbin Medical University (Heilongjiang, China).
The authors declare that they have no competing interests.
CpG site analysis of the PCDH10 promoter. CpG, 5′-C-phosphate-G-3′; PCDH10, Protocadherin-10.
Frequency of PCDH10 methylation in DCIS, IDC and IDC-L tissues. PCDH10, Protocadherin-10; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; IDC-L, invasive ductal carcinoma plus lymph-metastasis. *P<0.05.
PCDH10 mRNA expression in breast cancer tissues and matched normal breast tissues. *P<0.05. PCDH10, Protocadherin-10; mRNA, microRNA.
Kaplan-Meier survival curves of the overall survival rate of patients with methylated and unmethylated PCDH10. PCDH10, Protocadherin-10; OS, overall survival.
Receiver operating characteristic analysis of PCDH10 expression in breast cancer tissues. PCDH10, Protocadherin-10; ROC, receiver operating characteristic.
The sequences of probes and primers used polymerase chain reaction and methylation analysis.
Primer | Sequence |
---|---|
Methylation-specific primers | |
Forward | 5′-TCGTTAAATAGATACGTTACGC-3′ |
Reverse | 5′-TAAAAACTAAAAACTTTCCGCG-3′ |
TaqMan MGB probe | 5′-TGGTTAAGGGTTCGGTGGT-3′ |
Globin reference primers | |
Forward | 5′-AGGTAGAAAAGGAGAATGAAGATAAA-3′ |
Reverse | 5′-CTTTCCACTCTTTTCTCATTCTCTC-3′ |
TaqMan MGB probe | 5′-AGGAGGATAAGGAAGAGGGGAAATAGG-3′ |
Frequency of PCDH10 methylation in patients with sporadic and hereditary breast cancer.
Breast cancer tissue methylation frequency (%) | ||||
---|---|---|---|---|
Sporadic (n=296) | ||||
Methylated gene | DCIS | IDC | IDC-L | HpBC (n=96) |
PCDH10 | 66 | 82 | 85.32 | 72.37 |
PCDH10, Protocadherin-10; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; IDC-L, invasive ductal carcinoma plus lymph-metastasis; n, number; HpBC, hereditary breast cancer.
Association between PCDH10 methylation and different clinicopathological parameters.
Unmethylated PCDH10 | Methylated PCDH10 | ||||||
---|---|---|---|---|---|---|---|
Variables | Total, n | n | % | n | % | χ2 | P-value |
Age (years) | 0.798 | 0.372 | |||||
<45 | 128 | 60 | 36.1 | 68 | 30.1 | ||
≥45 | 264 | 106 | 63.9 | 158 | 69.9 | ||
Grade | 0.168 | 0.682 | |||||
I+II | 360 | 154 | 92.8 | 206 | 91.2 | ||
III | 32 | 12 | 7.2 | 20 | 8.8 | ||
Tumor size (cm) | 8.325 | 0.004 |
|||||
<2 | 126 | 72 | 43.4 | 54 | 23.9 | ||
≥2 | 266 | 94 | 56.6 | 172 | 76.1 | ||
LNM | 0.040 | 0.841 | |||||
Negative | 214 | 92 | 55.4 | 122 | 54.0 | ||
Positive | 178 | 74 | 44.6 | 104 | 46.0 | ||
ER | 0.221 | 0.638 | |||||
Negative | 106 | 42 | 25.3 | 64 | 28.3 | ||
Positive | 286 | 124 | 74.7 | 162 | 71.7 | ||
PR | 0.950 | 0.330 | |||||
Negative | 138 | 52 | 31.3 | 86 | 38.1 | ||
Positive | 254 | 114 | 68.7 | 140 | 61.9 | ||
HER-2 | 2.276 | 0.131 | |||||
Negative | 280 | 128 | 77.1 | 152 | 67.3 | ||
Positive | 112 | 38 | 22.9 | 74 | 32.7 | ||
P53 | 0.285 | 0.594 | |||||
Negative | 324 | 140 | 84.3 | 184 | 81.4 | ||
Positive | 68 | 26 | 15.7 | 42 | 18.6 | ||
Ki-67 | 0.791 | 0.374 | |||||
<20% | 198 | 90 | 54.2 | 108 | 47.8 | ||
≥20% | 194 | 76 | 45.8 | 118 | 52.2 | ||
Molecular subtype | 3.399 | 0.334 | |||||
Luminal A | 226 | 102 | 61.4 | 124 | 54.9 | ||
Luminal B | 64 | 24 | 14.5 | 40 | 17.7 | ||
HER-2 | 50 | 14 | 8.4 | 36 | 15.9 | ||
TNBC | 52 | 26 | 15.7 | 26 | 11.5 |
P<0.05. PCDH10, Protocadherin-10; LNM, lymph node metastasis; ER, estrogen receptor; PR, progesterone receptor; TNBC, triple-negative breast cancer; HER-2, human epidermal growth factor receptor 2; P53, tumor protein P53; n, number; Ki-67, antigen Ki-67.
Cox proportional hazards assessment of prognostic factors.
Univariate analysis | Multivariate analysis | ||||||
---|---|---|---|---|---|---|---|
Variables | HR | 95% CI | P-value | HR | 95% CI | P-value | |
Age (≥45 vs. <45 years) | 0.879 | (0.544–1.416) | 0.591 | ||||
Grade (III vs. II+I) | 0.732 | (0.282–1.780) | 0.482 | ||||
Tumor size (≥2 vs. <2 cm) | 3.712 | (2.039–7.569) | <0.00 |
3.115 | (1.597–6.274) | <0.00 |
|
LNM (positive vs. negative) | 2.531 | (1.903–4.181) | <0.001 |
2.337 | (1.939–3.842) | <0.001 |
|
ER (positive vs. negative) | 0.682 | (0.464–0.929) | 0.026 |
||||
PR (positive vs. negative) | 0.744 | (0.539–1.197) | 0.127 | ||||
Her2 (positive vs. negative) | 1.547 | (0.983–2.119) | 0.115 | ||||
p53 (positive vs. negative) | 1.411 | (0.886–2.124) | 0.298 | ||||
Ki-67 (positive vs. negative) | 1.269 | (0.846–1.692) | 0.534 | ||||
PCDH10 (methylated vs. unmethylated) | 1.780 | (1.322–3.117) | 0.006 |
1.798 | (1.231–3.071) | 0.011 |
P<0.05. CI, confidence interval; HR, hazard ratio; PCDH10, Protocadherin-10; LNM, lymph node metastasis; ER, estrogen receptor; PR, progesterone receptor; TNBC, triple-negative breast cancer; HER-2, human epidermal growth factor receptor 2; P53, tumor protein P53; Ki-67, antigen Ki-67.