J Breast Cancer.  2016 Jun;19(2):133-141. 10.4048/jbc.2016.19.2.133.

Aberrant Promoter Methylation at CpG Cytosines Induce the Upregulation of the E2F5 Gene in Breast Cancer

Affiliations
  • 1Cancer Genetics and Epigenetics Lab, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan. muhammad.saeed@comsats.edu.pk
  • 2Department of Biotechnology & Genetic Engineering, Kohat University of Science & Technology, Kohat, Pakistan.
  • 3Department of Surgical C Unit, Post Graduate Medical Institution, Lady Reading Hospital, Peshawar, Pakistan.
  • 4Department of Surgery, Divisional Headquarter Hospital, Kohat, Pakistan.

Abstract

PURPOSE
The promoter methylation status of cell cycle regulatory genes plays a crucial role in the regulation of the eukaryotic cell cycle. CpG cytosines are actively subjected to methylation during tumorigenesis, resulting in gain/loss of function. E2F5 gene has growth repressive activities; various studies suggest its involvement in tumorigenesis. This study aims to investigate the epigenetic regulation of E2F5 in breast cancer to better understand tumor biology.
METHODS
The promoter methylation status of 50 breast tumor tissues and adjacent normal control tissues was analyzed. mRNA expression was determined using SYBR® green quantitative polymerase chain reaction (PCR), and methylation-specific PCR was performed for bisulfite-modified genomic DNA using E2F5-specific primers to assess promoter methylation. Data was statistically analyzed.
RESULTS
Significant (p<0.001) upregulation was observed in E2F5 expression among tumor tissues, relative to the control group. These samples were hypo-methylated at the E2F5 promoter region in the tumor tissues, compared to the control. Change in the methylation status (Δmeth) was significantly lower (p=0.022) in the tumor samples, indicating possible involvement in tumorigenesis. Patients at the postmenopausal stage showed higher methylation (75%) than those at the premenopausal stage (23.1%). Interestingly, methylation levels gradually increased from the early to the advanced stages of the disease (p<0.001), which suggests a putative role of E2F5 methylation in disease progression that can significantly modulate tumor biology at more advanced stage and at postmenopausal age (Pearson's r=0.99 and 0.86, respectively). Among tissues with different histological status, methylation frequency was higher in invasive lobular carcinoma (80.0%), followed by invasive ductal carcinoma (46.7%) and ductal carcinoma in situ (20.0%).
CONCLUSION
Methylation is an important epigenetic factor that might be involved in the upregulation of E2F5 gene in tumor tissues, which can be used as a prognostic marker for breast cancer.

Keyword

Breast neoplasms; E2F5 transcription factor; Methylation; Promoter

MeSH Terms

Biology
Breast Neoplasms*
Breast*
Carcinogenesis
Carcinoma, Ductal
Carcinoma, Intraductal, Noninfiltrating
Carcinoma, Lobular
Cell Cycle
Disease Progression
DNA
E2F5 Transcription Factor
Epigenomics
Eukaryotic Cells
Genes, Regulator
Humans
Methylation*
Polymerase Chain Reaction
Promoter Regions, Genetic
RNA, Messenger
Up-Regulation*
DNA
E2F5 Transcription Factor
RNA, Messenger

Figure

  • Figure 1 Agarose gel (2%) showing promoter methylation pattern of E2F5 gene in tumors and control tissues of breast cancer. "M" and "UM" represent methylation and unmethylation, respectively. "Converted control" means bisulphite treated CpG methylated human genomic DNA, whereas "unconverted control" means bisulphite untreated human genomic DNA. The letter "L" represent 100 bp DNA size ladder.

  • Figure 2 Change in methylation (Δmeth) among control and diseased tissues of breast cancer. Unmeth=unmethylation; Meth=methylation; ΔMeth=unmethylation-methylation.

  • Figure 3 Age wise distribution of promoter methylation status at E2F5 gene in breast cancer patients. ΔMeth=unmethylation-methylation.

  • Figure 4 Promoter methylation of E2F5 gene among various stages of breast cancer. Unmeth=unmethylation; Meth=methylation; ΔMeth=unmethylation-methylation.

  • Figure 5 E2F5 promoter methylation status among different histological grades of breast cancer. ΔMeth=unmethylation-methylation.

  • Figure 6 Change in methylation (Δmeth) of E2F5 promoter among different histopathological type of breast cancer patients. IDC=invasive ductal carcinoma; ILC=invasive lobular carcinoma; DCIS=ductal carcinoma in situ; ΔMeth=unmethylation-methylation.

  • Figure 7 Fold relative mRNA expression of E2F5 gene among tumor and control tissues of breast cancer patients.


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