The nrf1 and nrf2 balance in oxidative stress regulation and androgen signaling in prostate cancer cells

Abstract

Reactive oxygen species (ROS) signaling has recently sparked a surge of interest as being the molecular underpinning for cancer cell survival, but the precise mechanisms involved have not been completely elucidated. This review covers the possible roles of two ROS-induced transcription factors, Nrf1 and Nrf2, and the antioxidant proteins peroxiredoxin-1 (Prx-1) and Thioredoxin-1 (Txn-1) in modulating AR expression and signaling in aggressive prostate cancer (PCa) cells. In androgen independent (AI) C4-2B cells, in comparison to the parental androgen dependent (AD) LNCaP cells, we present evidence of high Nrf1 and Prx-1 expression and low Nrf2 expression in these aggressive PCa cells. Furthermore, in DHT treated C4-2B cells, increased expression of the p65 (active) isoform of Nrf1 correlated with enhanced AR transactivation. Our findings implicate a crucial balance of Nrf1 and Nrf2 signaling in regulating AR activity in AI-PCa cells. Here we will discuss how understanding the mechanisms by which oxidative stress may affect AR signaling may aid in developing novel therapies for AI-PCa.

Figure 1

NOX4 and NOX5 mRNA Expression in a Prostate Cancer Progression Model. (A) RNA was isolated from PCa cell lines (RWPE1, RWPE2, LNCaP, and C4-2B) and NOX4 and NOX5 gene expression was evaluated by RT-PCR. GAPDH was used as an internal control. Fold changes in Nox4 and Nox5 mRNA expression were normalized to GAPDH. Numbers represent fold-changes as compared to normalized RWPE-1 expression (n = 3); (B) Quantitative RT-PCR (qRT-PCR) was done to evaluate NOX5 mRNA expression in RWPE1, RWPE2, LNCaP, and C4-2B cells (n = 2). GAPDH was used as the internal control.

Figure 2

Nrf1 and Nrf2 Expression in Prostate Cancer Cell Lines. (A) RT-PCR for Nrf1 and Nrf2 mRNA expression in RWPE-1, RWPE-2, LNCaP, and C4-2B PCa cells are shown. GAPDH was used as the internal control (n = 3); (B) Nuclear Nrf1 and Nrf2 protein expression was measured in RWPE-1, LNCaP, and C4-2B PCa cells by western immunoblotting (n = 3). TATA Binding Protein (TBP) was used as internal control. Values are normalized to TBP and fold changes were calculated as compared to RWPE-1 cells.

Figure 3

Expression of the p120 and p65 Isoforms of Nrf1 in PCa Cells. Westerns were carried out to measure levels of the p120 and p65 isoforms of Nrf1 in nuclear extracts from LNCaP and C4-2B cells. TBP was used as an internal control and Nrf1 protein levels were normalized to TBP and fold changes were calculated as compared to LNCaP cells (n = 2).

Figure 4

Differential Transcriptional Activation through the EpRE. In LNCaP and C4-2B cells. (A) an NQO-1 Luciferase reporter plasmid was transfected and (B) a GSTA Luciferase reporter plasmid (containing 6 repeats of the GSTA EpRE sequence) was transfected. After 24 h, cell extracts were obtained luc-activities were measured in a Dual Luciferase assay (Promega). A Renilla based reporter plasmid was used as the internal control (n = 2).

Figure 5

Prx-1 Expression in a Model of PCa Progression. (A) qRT-PCR and (B) total lysate westerns for Prx-1 in our panel of PCa progression. Data are normalized to GAPDH and α- tubulin respectively. (C) Nuclear Prx-1 protein levels in LNCaP and C4-2B cells are shown. Data are normalized to nuclear TBP levels.

Figure 6

A Putative Model for Prx-1 and Txn-1 Mediated Signaling in PCa. In PCa cells, hormones, growth factors, inflammatory cytokines, and hypoxia can increase ROS levels, triggering Prx-1 chaperone activity and Txn-1 nuclear localization. The increased chaperone activity of Prx-1 allows enhancement of AR transactivation function in the nucleus. In addition, functional nuclear Prx-1 and Txn-1 may also activate NF-κB transactivation function via reducing its DNA binding domain [82,83,91]. Such a model may facilitate our understanding of how tumor growth may occur under circumstances of high oxidative stress leading towards an aggressive and androgen independent (AI) phenotype in PCa cell.

Figure 7

Differential regulation of AR-directed transcription in DHT stimulated cells. Lipofectamine was used to transfect the PSA Luc reporter plasmid into LNCaP and C4-2B cells. After transfection, cells were cultured in charcoal stripped FBS (CSFBS), either in the absence of DHT or in the presence of 1 nM or 10 nM DHT. Protein was harvested after 24 h of DHT stimulation then luciferase assays were carried out. Renilla was used as the internal control.

Figure 8

An Integrated Model for Nrf1/Nrf2 Balance in Regulating EpRE-Mediated Gene Expression in Androgen Independent PCa Cells. Androgen deprivation, through disruption of tumor vasculature, enables a state of hypoxia [7]. Hypoxia activates NOX activity, which in turn increases oxidative stress through increased production of ^-O₂,which is converted into H₂O₂ by superoxide dismutases [21,34]. Both O₂^- and H₂O₂ can activate Nrf1 or Nrf2 signaling, which results in increased EpRE mediated gene expression (Prx-1, Txn-1, etc.) [49,56,79,92]. With upregulation of these antioxidant enzymes, aggressive cancer cells can protect themselves from ADT and radiation treatment [50,85,93,94]. This correlates with increased activation of AR regulated genes in aggressive PCa cells.

Figure 9

Possible Nrf1 and AR Binding Sites in the AR gene. The Genomatix MatInspector was used to search for transcription factor binding sites within the AR gene. Consensus sequences for the TCF11/MafG (pink) and ARE (teal) are provided at below.