Transcriptional regulation of the p73 gene by Nrf-2 and promoter CpG methylation in human breast cancer.

Jing Lai,Peng Cao,Jing Xu,Fang Yang,Yanru Wang,Wei Song,Guichun Huang,Jun Gu,Weiwei Nie,Xiaoxiang Guan,Yucai Wang,Ruilian Xie,Wenwen Zhang

doi:10.18632/oncotarget.2230

Abstract

To understand the transcriptional regulation of p73 by promoter methylation and Nrf-2 in breast carcinogenesis, ChIP assay indicated that Nrf-2 can bind to both promoters and can activate the transcription of TAp73 and ΔNp73 in MCF-7 cell line, knockdown of Nrf-2 gene resulted in an abrogation of TAp73 and ΔNp73 expression in the cells transfected with sh-Nrf-2 as well as Nrf-2 knock out mouse model. However, we found Nrf-2 induced ΔNp73 expression was abolished with 5-aza-dC treatment, thus lead to a down-regulated ΔNp73 and an up-regulated TAp73 expression in breast cancer cells lines. Consistent with this model, we detected decreased TAp73 and increased ΔNp73 expression in breast cancer tissue, along with increased TAp73 but decreased ΔNp73 expression in corresponding surrounding noncancerous tissues (NCTs) in a breast cancer tissue assay. A significant inverse correlation was found between TAp73 and ΔNp73 expression in the above tissue-array (P = 0.047) and validated in another set consisting of 128 breast cancer tumor tissue (P = 0.034). Taken together, our findings suggest that Nrf-2 and promoter methylation cooperatively govern the transcriptional regulation of p73, and unbalanced expression of TAp73 and ΔNp73 expression plays a critical role in breast cancer development.

Highlights

The p73 gene has two distinct promoters coding for two major isoforms, full-length TAp73 and the aminoterminally truncated ΔNp73, respectively [1,2,3]
After transiently transfected MCF-7 cells with p-nuclear factor erythroid 2-related factor 2 (Nrf-2) and sh-Nrf-2, we found that Nrf-2 transfection induced TAp73 and ΔΝp73 expression in MCF-7 cell line, knockdown of Nrf-2 gene resulted in an abrogation of TAp73 and ΔΝp73 expression in the cells transfected with sh-Nrf-2 suggesting a positive regulation of P1 and P2 by Nrf-2 transcriptional factor (Figure 5A)
We observed that the DNA Methyltransferase (DNMTs) activity in the three cell lines was detected a significant dose-dependent decrease after treatment with 5-aza-dC (Figure 2A), we detected that both P1 and P2 promoters were methylated, which could be reversed by 5-aza-dC treatment (Figure 2B)

Summary

INTRODUCTION

The p73 gene has two distinct promoters coding for two major isoforms, full-length TAp73 and the aminoterminally truncated ΔNp73, respectively [1,2,3]. It had been reported that the P1 promoter contains functional E2F1-binding sites [26], through which the E2F1 transcription factor can induce TAp73 overexpression and led to apoptosis [27]. We demonstrated that Nrf-2 can regulate the transcription of the p73 gene by bind to the P1 and P2 promoter and 5-aza-dC treatment can led to an increased binding with P1 and decreased binding with P2 promoter. To the best of our knowledge, this is the first report revealing that Nrf-2 takes opposite effect in the demethylation-induced p73 isoforms transcriptional regulation in human breast cancer. This finding would provide insights into the potential target for the future therapy of breast cancer

RESULTS

DISCUSSION

Findings

MATERIALS AND METHODS