Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice

Abstract

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer.

Epigenetic Modifications of Nrf2 by 3,3′-diindolylmethane In Vitro in TRAMP C1 Cell Line and In Vivo TRAMP Prostate Tumors

“…We have also reported that there is a progressive loss of Nrf2 expression and Nrf2 downstream target antioxidative genes, such as NQO1, HO-1, GSTm1, GCLC, and UGT1a1, during prostate cancer progresses in transgenic adenocarcinoma of mouse prostate (TRAMP) mice (5,9,10). Hypermethylated CpGs in the promoter region of Nrf2 gene was further found in TRAMP prostate tumors and TRAMP-C1 cells (11). The enhanced Nrf2 CpG methylation during TRAMP prostate tumor progresses was associated with the attenuated expressions of Nrf2 and its target genes (12).…”

“…The genomic DNAs were extracted and subjected to bisulfite conversion carried out using 750 ng of genomic DNA and applying to EZ DNA Methylation Gold Kits (Zymo Research Corp., Orange, CA) following the manufacturer's instructions. The converted DNA was amplified by PCR-utilizing Platinum PCR SuperMix (Invitrogen, Grand Island, NY) with a set of specific primers, forward: 5′-AGT TAT GAA GTA GTA GTA AAA A-3′ and reverse: 5′-AAT ATA ATC TCA TAA AAC CCC AC-3′, amplifying the first five CpGs located between −1,266 and −1,086 of the promoter region of Nrf2 with the translation initiation site defined as +1 (11,14). Gel extraction using Qiaquick™ gel extraction kit (Qiagen, Valencia, CA) were used to purify the PCR products, then cloned into pCR4 TOPO vector using a TOPO™ TA Cloning kit (Invitrogen, Grand Island, NY).…”

“…Gel extraction using Qiaquick™ gel extraction kit (Qiagen, Valencia, CA) were used to purify the PCR products, then cloned into pCR4 TOPO vector using a TOPO™ TA Cloning kit (Invitrogen, Grand Island, NY). Plasmids DNA from at least 20 colonies of each treatment were prepared using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) and sequenced (Genwiz, Piscataway, NJ) as we have previously reported (11,14).…”

The Keap1–Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases

“…Therefore, this leads to the expression of Nrf2 (Wang et al 2008;Muscarella et al 2011). Conversely, DNA methylation by DNA methyltransferases (DNMTs) appears to downregulate the Nrf2 expression indirectly (Khor et al 2014;Yu et al 2010;Rajabi et al 2016;Tagde et al 2016b). (c) Accumulation of p21 and p62 disrupts the Nrf2-Keap1 complex: it has been demonstrated that p53 negatively regulates Nrf2 and specifically suppresses the transcription of target genes of Nrf2 (Faraonio et al 2006).…”

Nuclear Factor–Erythroid-2–Related Factor 2 in Aging and Lung Fibrosis

“…Nrf2 gene promoter methylation associated with methyl-CpG-binding protein 2 and histone modifications epigenetically down-regulated the expression of Nrf2 in murine tumor models. 99 Genetic disruption of molecular clocks alters the rhythmic activation of Nrf2 and potentiates fibrosis in a lung injury model. 35 Although age-related declines in circadian rhythms are known to occur, 100 it is not known whether changes in the expression of clock genes and altered circadian rhythms lead to increased susceptibility to age-related lung fibrosis through impaired activation of Nrf2.…”