Aggressive mammary carcinoma progression in Nrf2 knockout mice treated with 7,12-dimethylbenz[a]anthracene

Breast cancer is the most common cancer among American women, except for skin cancers. The chance of developing invasive breast cancer at some time in a woman's life is about 1 in 8 (12%). In 2009, an estimated 192,370 new cases of invasive breast cancer will be diagnosed among women in the United States [1]. Many risk factors have been attributed to breast cancer occurrence. Genetic susceptibility accounts for only ~10% of human breast cancer [1, 2]. Known environmental risk factors include radiation, obesity, and alcohol use [1, 2].

Nuclear factor erythroid 2-related factor (Nrf2) is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases (GSTs), heme oxygenase-1 (HO-1) and peroxiredoxin 1, by activating the antioxidant response element (ARE) [3]. Reactive oxygen species (ROS) are known to activate oncogenic transcription factors such as AP-1 and NF-κB that have been shown in mouse models to be required for carcinogenesis [3]. It has also been noted that Nrf2 deficiency in mice shows an increased risk of chemical carcinogenesis and Nrf2 loss may contribute to tumorigenesis [3]. However, the effects of Nrf2 deficiency in breast cancer have not yet been explored.

Many cancer chemopreventive agents, in particular those that are naturally occurring, boost cellular antioxidant defenses [4]. Evidence is mounting that many of these phytochemicals activate the ARE through Nrf-2. Orally administered AUR induces GST activities via activation of Nrf2, since these effects were significantly attenuated in Nrf2(-/-) knockout (KO) mice [5]. Based on these studies, we hypothesized that AUR could suppress mammary carcinogenesis via activation of Nrf2/ARE. In summary although many chemopreventive phytochemicals are known to activate cellular antioxidant defenses through the ARE, direct evidence that their chemopreventive effects against mammary carcinogenesis are due to this effect is limited.

Oxidative stress is a condition of increased oxidant production in animal cells characterized by the release of free radicals, resulting in cellular degradation. Oxidative stress resulting from excess ROS and/or deficiencies in antioxidant capabilities may play a role in breast cancer etiology [6]. For example, a growing body of evidence suggests that natural and synthetic estrogens, which are oxidized to form quinones, are involved in breast cancer [7]. In a population-based, case-control study (654 cases, 605 controls), African American women harboring the mitochondrial DNA G10398 polymorphism exhibited an increased risk of invasive breast cancer (OR 1.60; 95% CI, 1.10-2.31, P = 0.013) [8]. MtDNA G10398A may be involved in altered structure of Complex I, which could lead to increased ROS [8]. Recently, a nested case-control study of postmenopausal women reported that women with genetic polymorphisms of Nrf2, NAD(P)H quinone oxidoreductase (NQO1), and HO-1 that favored iron-generated oxidative stress were at higher risk of breast cancer [6] An epidemiological study by Santella and colleagues found that women with higher plasma levels of oxidative protein damage (i.e. protein carbonyls) were at higher risk for breast cancer [9]. These studies suggest a role of oxidative stress in breast cancer that may be attenuated by chemopreventive agents.

In order determine whether phytochemicals can prevent mammary carcinogenesis via activation of Nrf2/ARE, first it must be demonstrated that Nrf2/ARE is involved in mammary carcinogenesis. To our knowledge, no mammary carcinogenesis studies have ever been done in Nrf2 knockout mice. Thus, to address this critical gap in knowledge, we conducted a mammary carcinogenesis study in Nrf2 knockout mice. We report, for the first time, that there was no apparent difference in the formation of premalignant lesions, but rather, the KO mice exhibited rapid, aggressive mammary carcinoma progression. Many questions remain as to the mechanism, which we will explore in future studies.

The role of Nrf2 activation in the chemoprevention of many types of cancer has been well-established [21]. However, little is known about the role of Nrf2 in the prevention of mammary carcinogenesis. The current study demonstrated that targeted deletion of Nrf2 in mice treated with DMBA resulted in rapid, aggressive mammary carcinoma growth rate, characterized by significantly larger tumor volume and tumor weight. In contrast, Nrf2 activation (through dietary exposure to AUR) or deletion (KO mice) did not appear to affect the formation of premalignant lesions. Induction of the so-called "phase II detoxifying enzymes" would be expected to block tumor initiation [22], so these results were surprising. On the other hand, we have previously shown that the linear furanocoumarins, such as imperatorin and isopimpinellin, are more effective at suppressing DMBA-DNA adduct formation compared to the simple coumarins, limettin and coumarin [13]. Linear furanocoumarins are much more potent inhibitors of the cytochrome P4501 family of enzymes that bioactivate DMBA [13]. In contrast, AUR and other simple coumarins do not exhibit this effect. Thus, the apparent lack of effect of AUR on premalignant lesions in the current study should not be discouraging. We recently reported that dietary administration of AUR delayed onset of N-methylnitrosourea-induced mammary carcinomas in rats, which corresponded to a decrease in cyclin D1 protein expression [23]. This suggests that AUR affects later stages of mammary carcinogenesis, such as promotion and/or progression.

The model chosen for initiating carcinogenesis was selected based on a report by Aldaz and colleagues that pre-treatment with depot medroxyprogesterone acetate increased DMBA-induced mammary tumor incidence, and significantly shortened the latency [12]. Also there were fewer non-mammary tumor related deaths (8%) in their study compared to the DMBA only groups (51%-81%). In our study, we observed a high level of morbidity and mortality in the WT group (46% due to lymphomas). However, the background strains of the mice were different between our study and theirs (ICR vs. CD2F1, respectively).

Understanding the molecular basis for the drastic differences between mammary carcinogenesis in WT vs. Nrf2 KO mice is critical. The 2-fold increase in NF-κB activation in Nrf2 KO mammary carcinomas was intruiguing and suggests possible cross-talk between Nrf2 and NF-κB. This concept was explored recently by Tusi and colleagues [24], who demonstrated that triazine derivatives, which possess anti-cancer activities, inhibited the activation of NF-κB by H₂O₂ in PC12 cells. The triazine derivatives also induced HO-1, induced Nrf2 protein expression, and increased GSH content. The conclusion was that the Nrf2 mediated neuroprotective effects of these triazine derivatives may be due to suppression of NF-κB. Thus, the exact role of NF-κB activation in mammary carcinogenesis in the Nrf2 KO mice remains to be determined.

The increased expression of activated β-catenin in the Nrf2 KO mammary carcinomas was also interesting. It has been previously reported that the heterogeneity of DMBA-induced mouse mammary carcinomas could be due to changes in the Wnt pathway in mammary progenitor cells [18]. Nucleoredoxin is a member of the redox-catalyic family including thioredoxin, which is regulated by Nrf2 activation [25]. Nucleoredoxin acts as a redox-sensor that negatively regulates signaling of Wnt/β-catenin by forming a complex with Dissheveled (DVL) [26]. In the "on state", Wnt binds to its receptor, frizzled (FZD). DVL inhibits the β-catenin destruction complex, thus β-catenin accumulates, translocates to the nucleus, where it activates transcription factors for target genes [26]. Therefore, we hypothesized that the active form of β-catenin would be elevated in the KO mammary carcinomas, which was supported by our data.

In regards to the quantitative real time pcr array, we consider these results to be leads for future studies. We will highlight a few of the top leads herein. The downregulation of Casp3, a pro-apoptotic protein [27], in the KO mammary carcinomas might suggest that the tumors are more resistant to apoptosis, which could explain why the rapid growth and size. However, upregulation of Cdh1 and downregulation of Mmp9 are counter-intuitive to the observed results. Cdh1 regulates cellular adhesion, and loss of Cdh1 is associated with invasion and metastasis [28]. Mmp9 is a gelatinase also involved in tumor invasion [29]. Also, the striking increase in thioredoxin reductase 2 (Txnrd2) and the selenoproteins glutathione peroxidase 3 and 7 (Gpx3, GPX7) may explain why there were no differences in oxidized proteins in WT vs. KO mammary carcinomas. This would then suggest that these genes are regulated by factors other than or in addition to Nrf2. Txnrd2 is a mitochondrial defense against oxidative stress [30]. Gpx3 is expressed in plasma and normal kidney where it counteracts oxidative stress [31]. It can be increased at various stages of cancer, but also possesses protective effects. Gpx are regulated by Nrf2 and p63, which is a member of the p53 family, but not p53 itself [31]. Overexpression of Gpx inhibited p53-dependent H₂O₂ induced apoptosis in human MCF-7 breast carcinoma cells. Gpx7 is silenced in Barrett's oesophagus and adenocarcinoma via epigenetic mechanisms [32]. Again at this point we can only speculate that these are compensatory mechanisms as the KO carcinomas adapt to their environment.

In our study, the Nrf2 KO mice also displayed significantly higher incidence of lung adenomas. It is clear from the literature that under certain experimental conditions, phytochemicals and other molecules that activate Nrf2 confer protection from the early stages of carcinogenesis (reviewed in [4, 33]). Enzymes that can protect against oxidative stress and detoxify carcinogens are induced, thus protecting the body from harmful chemicals such aflatoxin B1 and benzo[a]pyrene. For example, daily oral dosing of 125 mg oltipraz for a month in humans increased the excretion of aflatoxin-mercapturic acid [34]. In rodents treated with aflatoxin B1, dietary exposure to oltipraz induced phase II carcinogen detoxifying enzymes including GST, and conferred protection against DNA adduct formation and tumorigenicity [35, 36]. In another example, dietary exposure to dibenzoylmethane activated Nrf2/ARE and decreased the formation of benzo[a]pyrene DNA adducts in A/J mouse lungs [37]. With these and other examples in the literature, the chemopreventive agents were administered prior to disease progression. Thus it may not be surprising that there were increased lung adenomas in the Nrf2 KO mice treated with DMBA.

On the other hand, epidemiological evidence and xenograft models with lung carcinoma cells have suggested that Nrf2 is upregulated in cancer and that suppression of Nrf2 may be a therapeutic target [38, 39]. In consideration of our findings with the published literature, the sequence of Nrf2 activation over time during carcinogenesis may be the key factor in determining whether it is protective or stimulatory in cancer. Whereas antioxidant enzymes may be preventive against cancer development, in already transformed cells, these enzymes may confer protective advantage of cancer cells in the hostile tumor microenvironment [40, 41]. In this regard, Nrf2 upregulation of cellular antioxidant defense may protect cancer cells from oxidative stress, and make them more resistant to chemotherapy and radiation. Furthermore, an N-terminal domain mutation of Keap-1 (C23Y) was identified in human breast cancer [42]. Nioi and Nguyen demonstrated that this mutation impairs the ability of Keap-1 to repress Nrf2 activity [43]. However, what is not known is when in the carcinogenic process is Keap-1 is mutated. Carcinogenesis results in compounding mutations, and by end-stage disease, causative factors can be difficult to discern. The potential effects of chemopreventive phytochemicals in cancer patients may not be clear. Recently, a comparison of genetic vs. pharmacologic activation of Nrf2 using Keap-1 knockout mice and a synthetic oleanane triterpenoid, respectively [44]. Distinct but overlapping genetic changes exist, suggests that dietary intervention with phytochemicals may not have the same effect as genetic manipulation. Up to now, most of the Nrf2 manipulations in cancer cells have been done using sh-RNA against already transformed cancer cells. We hope that our future characterization of the stable, immortalized WT and Nrf2 KO mammary carcinoma cell lines we developed will help address the conflicting role of Nrf2, because Nrf2 was either present or knocked-out completely prior to cancer development.

In a broader sense, the current results may offer clues into factors that drive rapid malignant progression. These results are consistent with the idea that ROS can act as signaling molecules to redox sensitive pathways [25]. However, more studies will be necessary to identify whether this phenomenon drives breast cancer progression in humans. If this is found to be the case, then these pathways could be considered for therapeutic intervention.

In conclusion, we have developed a mammary carcinogenesis model in Nrf2 KO mice. Treatment with the carcinogen, DMBA, resulted in the formation of premalignant lesions in mouse mammary glands, but no differences in these premalignant lesions were observed between WT and Nrf2 KO mice. Instead, Nrf2 KO mice developed rapidly growing, aggressive mammary carcinomas that were larger than the WT mammary carcinomas. Although there was no difference in overall survival between WT and KO mice, there was a significant decline in mammary tumor-free survival in the KO mice. The active forms of NF-κB and β-catenin were significantly elevated ~2-fold in the KO mammary carcinomas. Real-time quantitative pcr analysis suggested that Casp3 was downregulated in the KO mammary carcinomas. Many other lesions were observed in the tumor study, particularly lung adenomas, which were more predominant in the Nrf2 KO mice. We conclude that Nrf2 KO mice were more susceptible to DMBA-induced mammary carcinogenesis, particularly in terms of tumor size, rate of growth, and mammary tumor-free survival. Key factors involved in the rapid growth of these carcinomas remain to be determined. Identification of factors responsible for the rapid tumor growth rate may provide insight into human breast cancer.

LB, B.S., is currently a M.S. student. MP, B.S., is the laboratory manager and student in nursing school. HB, CC, and MB were high-school students. KI, M.D., Ph.D., and MY, M.D., Ph.D. are both Professors in Japan. MM, Ph.D., is a Professor. EO, DVM, is the Associate Director of the Department of Veterinary Medicine. RS, Ph.D. and J.M., Ph.D. are both statisticians. KP is an Assistant Professor. SZ, M.D., Ph.D., is a pathologist. HEK is an Associate Professor and the Director of the Breast Cancer Focus Group at the Feist-Weiller Cancer Center.