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1.  CyclinD1, a prominent prognostic marker for endometrial diseases 
Diagnostic Pathology  2013;8:138.
Purpose
Alteration of CyclinD1 was suggested to relate with development of endometrial carcinogenesis before, however CyclinD1 expression is not well defined in endometrial hyperplasia lesions. We checked the relationship between its expression and clinic-pathological variables of endometrial lesions to explore the possibility for CyclinD1 as a potential diagnostic and prognostic marker.
Methods
Cyclin D1 immunohistochemical analysis (IHC) was used to evaluate 201 fixed, paraffin-embedded endometrial samples which included simple hyperplasia (n = 27), atypical complex hyperplasia (ACH) (n = 41), endometrioid carcinoma (n = 103), endometrial serous carcinoma (ESC) (n = 21) and clear cell carcinoma (CCC) (n = 9). A breast cancer with known CyclinD1 expression was selected as a positive control in each immunohistochemistry run. We also performed follow-up study to estimate patients’ prognosis.
Results
CyclinD1 was significantly overexpressed in atypical complex hyperplasia (ACH), endometrioid carcinoma and clear cell carcinoma (CCC). The positive signaling of CyclinD1 was showed less than 40% in simple hyperplasia and endometrial serous carcinoma (ESC). The high expression of CyclinD1 was observed in metastasis carcinoma group more significantly than non-metastasis carcinoma group. Kaplan Meier analysis demonstrated that patients with high CyclinD1 expression had an obviously poor prognosis than patients without CyclinD1 staining (p < 0.05). Moreover, according to multivariate Cox regression analysis, CyclinD1 expression, as crucial as metastasis, was a risk marker for overall survival rate.
Conclusion
CyclinD1 exhibited a promising potential to predict the prognosis of patients with endometrial carcinoma. However, the statistical analysis demonstrated that CyclinD1 exhibited a poor ability to differentiate neoplastic lesions from non-neoplastic lesions; thus, the application of CyclinD1 only is not so credible for differentiation between benign and malignant lesions.
Virtual slides
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1871063048950173.
doi:10.1186/1746-1596-8-138
PMCID: PMC3846687  PMID: 23947899
Endometrial cancer; CyclinD1; Prognostic markers; Survival analysis
2.  High levels of Nrf2 determine chemoresistance in type II endometrial cancer 
Cancer research  2010;70(13):5486-5496.
Type II endometrial cancer, which mainly presents as serous and clear cell types, has proved to be the most malignant and recurrent carcinoma among various female genital malignancies. The transcription factor, Nrf2, was first described as having chemopreventive activity. Activation of the Nrf2-mediated cellular defense response protects cells against the toxic and carcinogenic effects of environmental insults by upregulating an array of genes that detoxify reactive oxygen species (ROS) and restore cellular redox homeostasis. However, the cancer-promoting role of Nrf2 has recently been revealed. Nrf2 is constitutively upregulated in several types of human cancer tissues and cancer cell lines. Furthermore, inhibition of Nrf2 expression sensitizes cancer cells to chemotherapeutic drugs. In this study, the constitutive level of Nrf2 was compared in different types of human endometrial tumors. It was found that Nrf2 was highly expressed in endometrial serous carcinoma (ESC), whereas complex hyperplasia (CH) and endometrial endometrioid carcinoma (EEC) had no or marginal expression of Nrf2. Likewise, the ESC derived SPEC-2 cell line had a higher level of Nrf2 expression and was more resistant to the toxic effects of cisplatin and paclitaxel than that of the Ishikawa cell line, which was generated from EEC. Silencing of Nrf2 rendered SPEC-2 cells more susceptible to chemotherapeutic drugs while it had a limited effect on Ishikawa cells. Inhibition of Nrf2 expression by overexpressing Keap1 sensitized SPEC-2 cells or SPEC-2-derived xenografts to chemotherapeutic treatments using both cell culture and SCID mouse models. Collectively, we provide a molecular basis for the use of Nrf2 inhibitors to increase the efficacy of chemotherapeutic drugs and to combat chemoresistance, the biggest obstacle in chemotherapy.
doi:10.1158/0008-5472.CAN-10-0713
PMCID: PMC2896449  PMID: 20530669
Nrf2; chemoresistance; and endometrial cancer
3.  Insights into Endometrial Serous Carcinogenesis and Progression 
Endometrial serous carcinomas (ESC) constitute only approximately 10% of endometrial cancers, but have a substantially higher case-fatality rate than their more common endometrioid counterparts. The precise composite of factors driving endometrial serous carcinogenesis and progression remain largely unknown, but we attempt to review the current state of knowledge in this report. ESC probably do not evolve through a single pathway, and their underlying molecular events probably occur early in their evolution. TP53 gene mutations occur in 22.7 to 96% of cases, and p53 protein overexpression is seen in approximately 76%. By gene expression profiling, p16 is upregulated in ESC significantly above both normal endometrial cells and endometrioid carcinomas, and 92–100% of cases display diffuse expression of the p16 protein by immunohistochemistry (IHC). Together, these findings suggest dysregulation of both the p16INKA/Cyclin D-CDK/pRb-E2F and the ARF-MDM2-p53 cell cycle pathways in ESC. By IHC, HER2/neu is overexpressed (2+ or 3+) in approximately 32.1% of ESC, and approximately 54.5% of cases scored as 2+ or 3+ by IHC display c-erbB2 gene amplification as assessed by fluorescent in situ hybridization. Genetic instability, typically manifested as loss of heterozygosity in multiple chromosomes, is a common feature of ESC, and one study found loss of heterozygosity at 1p32-33 in 63% of cases. A subset of ESC display protein expression patterns that are characteristic of high grade endometrial carcinomas, including loss of the metastasis suppressor CD82 (KAI-1) and epithelial-to-mesenchymal transformation, the latter manifested as E-cadherin downregulation, P-cadherin upregulation, and expression of epithelial-to-mesenchymal transformation-related molecules such as zinc-finger E-box-binding homeobox 1 (ZEB1) and focal adhesion kinase. Preliminary data suggests differential patterns of expression in ESC of some isoforms of claudins, proteases, the tumor invasiveness and progression-associated oncofetal protein insulin-like growth factor II mRNA-binding protein 3 (IMP3), as well as a variety of other molecules. At the morphologic level, evidence that indicates that endometrial glandular dysplasia (EmGD) is the most likely morphologically recognizable precursor lesion to ESC is presented. We advocate use of the term endometrial intraepithelial carcinoma (EIC, or its other appellations) only as a morphologic descriptor and never as a diagnostic/pathologic statement of biologic potential. Given its potential for extrauterine extension, we consider the lesions described as EIC, when present in isolation, as examples of localized ESC, and patients should be managed as such. Morphologically normal, p53 immunoreactive endometrial cells (the so-called “p53 signatures”), show a statistically significant association with ESC, display p53 mutations in a significant subset, and form the start of a progression model, outlined herein, from p53 signatures to EmGD to localized ESC to the more conventionally invasive neoplasm. The identification of a morphologically-recognizable precursor holds the promise of early detection of ESC, with the attendant reduction in its overall associated mortality rate. Deciphering the molecular basis for endometrial serous carcinogenesis should uncover potential targets for diagnosis, therapy, and/or disease surveillance.
PMCID: PMC2655156  PMID: 19294001
Endometrial serous carcinoma; endometrial glandular dysplasia; endometrial intraepithelial carcinoma; p53; cadherins; claudins; CDKs; MDM2 and HER2/neu (erb-B2)
4.  The Involvement of NRF2 in Lung Cancer 
Nuclear factor, erythroid-derived 2, like 2 (NRF2) is a key regulator of antioxidants and cellular stress responses. The role of NRF2 in pulmonary neoplasia, a diverse disease for which few biomarkers exist, is complicated and appears to depend on several main factors including the existence of activating mutations in NRF2 and/or loss of function mutations in KEAP1 and the stage of carcinogenesis studied, particularly in the mouse models tested. Therapeutic strategies for lung cancer targeting NRF2 have observed mixed results, both anti- and protumorigenic effects; however, these differences seem to reflect the mutation status of NRF2 or KEAP1. In this paper, we will discuss the studies on human NRF2 and the mechanisms proposed, several mouse models using various mice deficient in NRF2, as well as xenograft models, and the chemotherapeutic strategies using the NRF2 pathway.
doi:10.1155/2013/746432
PMCID: PMC3614183  PMID: 23577226
5.  Nrf Transcription Factors in Keratinocytes Are Essential for Skin Tumor Prevention but Not for Wound Healing†  
Molecular and Cellular Biology  2006;26(10):3773-3784.
The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.
doi:10.1128/MCB.26.10.3773-3784.2006
PMCID: PMC1489009  PMID: 16648473
6.  Heightened Endoplasmic Reticulum Stress in the Lungs of Patients with Chronic Obstructive Pulmonary Disease 
Rationale: Nuclear factor erythroid 2–related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD.
Objectives: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD.
Methods: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD.
Measurements and Main Results: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death.
Conclusions: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.
doi:10.1164/rccm.200903-0324OC
PMCID: PMC2796732  PMID: 19797762
Nrf2; proteasome system; endoplasmic reticulum stress; unfolded protein response; chronic obstructive pulmonary disease lungs
7.  NRF2 Mutation Confers Malignant Potential and Resistance to Chemoradiation Therapy in Advanced Esophageal Squamous Cancer1 
Neoplasia (New York, N.Y.)  2011;13(9):864-873.
Esophageal squamous cancer (ESC) is one of the most aggressive tumors of the gastrointestinal tract. A combination of chemotherapy and radiation therapy (CRT) has improved the clinical outcome, but the molecular background determining the effectiveness of therapy remains unknown. NRF2 is a master transcriptional regulator of stress adaptation, and gain of-function mutation of NRF2 in cancer confers resistance to stressors including anticancer therapy. Direct resequencing analysis revealed that Nrf2 gain-of-function mutation occurred recurrently (18/82, 22%) in advanced ESC tumors and ESC cell lines (3/10). The presence of Nrf2 mutation was associated with tumor recurrence and poor prognosis. Short hairpin RNA-mediated down-regulation of NRF2 in ESC cells that harbor only mutated Nrf2 allele revealed that themutant NRF2 conferred increased cell proliferation, attachment-independent survival, and resistance to 5-fluorouracil and γ-irradiation. Based on the Nrf2 mutation status, gene expression signatures associated with NRF2 mutation were extracted from ESC cell lines, and their potential utility for monitoring and prognosis was examined in a cohort of 33 pre-CRT cases of ESC. The molecular signatures of NRF2 mutation were significantly predictive and prognostic for CRT response. In conclusion, recurrent NRF2 mutation confers malignant potential and resistance to therapy in advanced ESC, resulting in a poorer outcome. Molecular signatures of NRF2 mutation can be applied as predictive markers of response to CRT, and efficient inhibition of aberrant NRF2 activation could be a promising approach in combination with CRT.
PMCID: PMC3182278  PMID: 21969819
8.  Activation of Nrf2-antioxidant signaling attenuates NF-κB-inflammatory response and elicits apoptosis 
Biochemical pharmacology  2008;76(11):1485-1489.
Oxidative stress has been implicated in the etiology of neurodegenerative disease, cancer and aging. Indeed, the reactive oxygen and nitrogen species generated by inflammatory cells that created oxidative stress is thought to be one of the major factor by which chronic inflammation contributes to neoplastic transformation as well as many other diseases. We have recently reported that mice lacking nuclear factor-erythroid 2-related factor 2 (Nrf2) are more susceptibility to dextran sulfate sodium (DSS)-induced colitis and colorectal carcinogenesis. Nrf2 is a basic leucine zipper redox-sensitive transcriptional factor that plays a center role in ARE (antioxidant response element)-mediated induction of phase II detoxifying and antioxidant enzymes. We found that increased susceptibility of Nrf2 deficient mice to DSS-induced colitis and colorectal cancer was associated with decreased expression of antioxidant/phase II detoxifying enzymes in parallel with upregulation of pro-inflammatory cytokines/biomarkers. These findings suggest that Nrf2 may play an important role in defense against oxidative stress possibly by activation of cellular antioxidant machinery as well as suppression of pro-inflammatory signaling pathways. In addition, in vivo and in vitro data generated from our laboratory suggest that many dietary compounds can differentially regulate Nrf2-mediated antioxidant/anti-inflammatory signaling pathways as the first line defense or induce apoptosis once the cells have been damaged. In this review, we will summarize our thoughts on the potential cross-talks between Nrf2 and NF-κB pathways. Although the mechanisms involved in the cross-talk between these signaling pathways are still illusive, targeting Nrf2-antioxidative stress signaling is an ideal strategy to prevent or treat oxidative-stress related diseases.
doi:10.1016/j.bcp.2008.07.017
PMCID: PMC2610259  PMID: 18694732
Nrf2; NF-κB; DSS; AOM; apoptosis
9.  Competition of nuclear factor-erythroid 2 factors related transcription factor isoforms, Nrf1 and Nrf2, in antioxidant enzyme induction☆ 
Redox Biology  2013;1(1):183-189.
Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.
Highlights
► Nrf1 knockout increased GCLC and GCLM expression in human bronchial epithelial cells. ► Overexpressed Nrf1 forms p120 or p65 increased GCLC- and GLCM-EpRE luciferase activity. ► Overexpressed Nrf2 competes with Nrf1 forms. ► Nrf1 forms are unchanged in the lungs of mice by age. ► Nrf1 forms are unchanged in exposure to nanoparticulate air pollution.
doi:10.1016/j.redox.2013.01.005
PMCID: PMC3757680  PMID: 24024152
EpRE, electrophile response element; ER, endoplasmic reticulum; GCL, glutamate cysteine ligase; GCLC, catalytic subunit of GCL; GCLM, modifier subunit of GCL; HBE1, human bronchial epithelial cells; HO-1, heme oxygenase; Nrf1, nuclear factor-erythroid 2 p45 subunit-related factor 1; Nrf2, nuclear factor-erythroid 2 p45 subunit-related factor 2; nPM, nanoparticulate air pollution; Nrf1; Nrf2; Glutamate cysteine ligase; Electrophile response element; Air pollution; Phase II genes
10.  Nrf2-Mediated Transcriptional Induction of Antioxidant Response in Mouse Embryos Exposed to Ethanol in vivo: Implications for the Prevention of Fetal Alcohol Spectrum Disorders 
Antioxidants & Redox Signaling  2008;10(12):2023-2033.
Abstract
Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor that is important in protection against oxidative stress. This study was designed to determine the role of Nrf2 signaling in transcriptional activation of detoxifying and antioxidant genes in an in vivo mouse fetal alcohol syndrome model. Maternal ethanol treatment was found to increase both Nrf2 protein levels and Nrf2-ARE binding in mouse embryos. It also resulted in a moderate increase in the mRNA expression of Nrf2 downstream target detoxifying and antioxidant genes as well as an increase in the expression of antioxidant proteins. Pretreatment with the Nrf2 inducer, 3H-1,2 dithiole-3-thione (D3T), significantly increased Nrf2 protein levels and Nrf2-ARE binding, and strongly induced the mRNA expression of Nrf2 downstream target genes. It also increased the expression of antioxidant proteins and the activities of the antioxidant enzymes. Additionally, D3T pretreatment resulted in a significant decrease in ethanol-induced reactive oxygen species generation and apoptosis in mouse embryos. These results demonstrate that Nrf2 signaling is involved in the induction of antioxidant response in ethanol-exposed embryos. In addition, the potency of D3T in inducing antioxidants as well as in diminishing ethanol-induced apoptosis suggests that further exploration of the antiteratogenic effect of this compound will be fruitful. Antioxid. Redox Signal. 11, 2023–2033.
doi:10.1089/ars.2007.2019
PMCID: PMC2933156  PMID: 18759561
11.  Role of nuclear factor (erythroid-derived 2)-like 2 in metabolic homeostasis and insulin action: A novel opportunity for diabetes treatment? 
World Journal of Diabetes  2012;3(1):19-28.
Redox balance is fundamentally important for physiological homeostasis. Pathological factors that disturb this dedicated balance may result in oxidative stress, leading to the development or aggravation of a variety of diseases, including diabetes mellitus, cardiovascular diseases, metabolic syndrome as well as inflammation, aging and cancer. Thus, the capacity of endogenous free radical clearance can be of patho-physiological importance; in this regard, the major reactive oxygen species defense machinery, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system needs to be precisely modulated in response to pathological alterations. While oxidative stress is among the early events that lead to the development of insulin resistance, the activation of Nrf2 scavenging capacity leads to insulin sensitization. Furthermore, Nrf2 is evidently involved in regulating lipid metabolism. Here we summarize recent findings that link the Nrf2 system to metabolic homeostasis and insulin action and present our view that Nrf2 may serve as a novel drug target for diabetes and its complications.
doi:10.4239/wjd.v3.i1.19
PMCID: PMC3258535  PMID: 22253942
Nuclear factor (erythroid-derived 2)-like 2; Oxidative stress; Insulin resistance; Metabolism; Diabetic drug
12.  Expression of metallothionein and Nrf2 pathway genes in lung cancer and cancer-surrounding tissues 
Background
Nuclear factor (erythroid-derived 2)-like (Nrf)2 and metallothionein have been implicated in carcinogenesis. This study investigated the expression of Nrf2 and of Nrf2-targeted genes (NQO1 and GCLC) and the genes for the metallothionein (MT) isoforms (MT-1A and MT-2A) in human lung cancer and cancer-surrounding tissues.
Methods
Surgically removed lung cancer samples (n = 80) and cancer-surrounding tissues (n = 38) were collected from Zunyi Medical College Hospital, China. Total RNA was extracted, purified, and used for real-time reverse transcription-PCR analysis of interested genes.
Results
Expression of the Nrf2-targed genes NQO1 and GCLC tended to be higher (30 to 60%) in lung cancers, but was not significantly different from that in peri-cancer tissues. By contrast, expression of the genes for M)-1A, MT-2A, and the metal transcription factor MTF-1 were three-fold to four-fold lower in lung cancers.
Conclusion
In surgical samples of lung cancer, MT expression was generally downregulated, whereas Nrf2 expression tended to be upregulated. These changes could play an integral role in lung carcinogenesis.
doi:10.1186/1477-7819-11-199
PMCID: PMC3765715  PMID: 23947958
Lung cancers; Cancer-surrounding tissue; Nrf2; Metallothionein
13.  The Keap1–Nrf2 system in cancers: stress response and anabolic metabolism 
Frontiers in Oncology  2012;2:200.
The Keap1–Nrf2 [Kelch-like ECH-associated protein 1–nuclear factor (erythroid-derived 2)-like 2] pathway plays a central role in the protection of cells against oxidative and xenobiotic stresses. Nrf2 is a potent transcription activator that recognizes a unique DNA sequence known as the antioxidant response element (ARE). Under normal conditions, Nrf2 binds to Keap1 in the cytoplasm, resulting in proteasomal degradation. Following exposure to electrophiles or reactive oxygen species, Nrf2 becomes stabilized, translocates into the nucleus, and activates the transcription of various cytoprotective genes. Increasing attention has been paid to the role of Nrf2 in cancer cells because the constitutive stabilization of Nrf2 has been observed in many human cancers with poor prognosis. Recent studies have shown that the antioxidant and detoxification activities of Nrf2 confer chemo- and radio-resistance to cancer cells. In this review, we provide an overview of the Keap1–Nrf2 system and discuss its role under physiological and pathological conditions, including cancers. We also introduce the results of our recent study describing Nrf2 function in the metabolism of cancer cells. Nrf2 likely confers a growth advantage to cancer cells through enhancing cytoprotection and anabolism. Finally, we discuss the possible impact of Nrf2 inhibitors on cancer therapy.
doi:10.3389/fonc.2012.00200
PMCID: PMC3530133  PMID: 23272301
stress response; redox homeostasis; transcription; purine nucleotide; glutathione
14.  Increased Susceptibility of Nrf2 Knockout Mice to Colitis-Associated Colorectal Cancer 
The nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in protecting various tissues against inflammation, which is a potential risk factor for colorectal and other cancers. Our previously published mouse model work showed that Nrf2 helps protect against dextran sulfate sodium (DSS)–induced colitis/inflammation, and others have shown that Nrf2 helps protect against inflammation-associated colorectal carcinogenesis (aberrant crypt foci). The present study extended these important earlier findings by exploring the role of Nrf2 in colitis-associated colorectal cancer in a mouse model involving azoxymethane/DSS–induced colorectal carcinogenesis in Nrf2 knockout mice. Azoxymethane/DSS–treated Nrf2 knockout mice had increased incidence, multiplicity, and size of all colorectal tumors, including adenomas, versus treated wild-type (WT) mice, and the proportion of tumors that were adenocarcinoma was much higher in knockout (80%) versus WT (29%) mice. Compared with WT mice, knockout mice also had increased markers of inflammation in tumor tissue (cyclooxygenase-2 and 5-lipoxygenase expressions and prostaglandin E2 and leukotriene B4 levels) and in inflamed colonic mucosa (nitrotyrosine expression), supporting the association of knockout mouse tumor formation with inflammation. The phase II detoxifying/antioxidant enzymes NAD(P)H-quinone reductase 1 and UDP-glucurosyltransferase 1A1 were elevated in the normal mucosa of WT, but not Nrf 2 knockout, mice treated with azoxymethane/DSS. Our findings show that Nrf2 plays a critical role in protecting against inflammation-associated colorectal cancer.
doi:10.1158/1940-6207.CAPR-08-0028
PMCID: PMC3580177  PMID: 19138955
15.  Claudin-3 and Claudin-4 expression in serous papillary, clear cell, and endometrioid endometrial cancer 
Gynecologic oncology  2008;109(2):263-269.
The tight junction (TJ) proteins claudin-3 and claudin-4 have been reported to be differentially expressed in uterine serous papillary carcinoma (USPC), a rare form of endometrial cancer characterized by a particularly high recurrence rate and poor prognosis. Preclinical experiments suggest that increased expression of both TJ proteins may in part mediate the biologically aggressive phenotype of USPC. Our aim was to determine claudin-3 and claudin-4 expression in a large cohort of surgically staged patients with USPC and clear cell endometrial cancer (n=137), and to compare the expression pattern and prognostic relevance of both claudins with that seen in patients with endometrioid endometrial cancer (n=150). The rate of claudin-3 and claudin-4 expression was significantly higher in USPC and clear cell endometrial cancer compared to endometrioid endometrial cancer (claudin-3: 78% and 61% versus 38%, p <.0001; claudin-4: 56% and 44% versus 9%, p <.0001). Furthermore, expression of both tight junction proteins was significantly associated with poor clinical outcome (claudin-3, DFS: Risk ratio (RR) 1.70, p=.0087, OS RR 1.62, p=.0247; claudin-4, DFS RR 2.66, p<0.0001, and OS RR 2.50, p<0.0001). However, claudin-3 and claudin-4 expression did not maintain prognostic independence in multivariate analyses, as their expression was tightly associated with more advanced disease stages (p <.0001 for both), and higher nuclear grade (p <.0001 for both). These clinical observations confirm the hypothesis based on preclinical evidence that increased expression of claudin-3 and claudin-4 may contribute to the aggressive phenotype of endometrial cancer of serous papillary or clear cell histology and suggest their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.
doi:10.1016/j.ygyno.2008.01.024
PMCID: PMC2453046  PMID: 18313739
Uterine serous papillary endometrial cancer; Claudin-3; Claudin-4; lapatinib; endometrium
16.  Joint loss of PAX2 and PTEN expression in endometrial precancers and cancer 
Cancer research  2010;70(15):6225-6232.
Latent endometrial carcinoma precancers are normal appearing endometrial glands with sporadic loss of tumor suppressor gene function such as PTEN. Progression to carcinoma is inefficient and requires additional genetic damage that creates a histologic precursor lesion called endometrial intraepithelial neoplasia (EIN). In this study, we examined loss of PAX2 expression, a gene required for embryonic uterine development, during endometrial carcinogenesis. Normal proliferative, EIN, and malignant (endometrial adenocarcinoma) endometrial tissues were immunostained for PTEN and PAX2. Proliferative samples with loss of protein in at least one gland were scored as latent precancers. EIN and cancer lesions were scored by the majority pattern. Overall prevalence and topography of joint PAX2-PTEN expression loss was examined. The prevalence of PAX2 protein loss in the sequence of normal to precancer to cancer was 36%, 71%, and 77% respectively, and for PTEN 49%, 44%, and 68%. Normal endometrial prevalence of PAX2 or PTEN deficient latent precancers was unaffected by biopsy indication, but increased significantly with age. Coincident loss of PAX2 and PTEN expression in an individual normal endometrium was seen in 21% of patients, but usually involved different glands. Coincident loss was more common in precancers (31%) and carcinoma (55%), in which case both markers were protein null in an overlapping clonal distribution. PAX2 and PTEN protein loss occur independently and accumulate with increasing age in latent precancers of normal premenopausal endometrium. Loss of function of both genes in an overlapping distribution characterizes clinical emergence of a premalignant lesion which is carried forward to carcinoma.
doi:10.1158/0008-5472.CAN-10-0149
PMCID: PMC2912978  PMID: 20631067
PAX2; PTEN; endometrium; carcinoma; latent precancer
17.  Possible involvement of nuclear factor erythroid 2-related factor 2 in the gene expression of Cyp2b10 and Cyp2a5☆ 
Redox Biology  2014;2:284-288.
Cytochrome P450 gene expression is altered by various chemical compounds. In this study, we used nuclear factor erythroid 2-related factor 2 (Nrf2)–deficient (Nrf2−⧸−) mice to investigate the involvement of Nrf2 in Cyp2b10 and Cyp2a5 gene expression. Phorone, an Nrf2 activator, strongly increased Cyp2b10 and Cyp2a5 mRNA as well as Nrf2 target genes, including NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1, in wild-type mouse livers 8 h after treatment. The phorone-induced mRNA levels in Nrf2−⧸− mouse livers were lower than that in wild-type mouse livers. Nrf2−⧸− mice showed attenuated Cyp2b10 and Cyp2a5 induction by phenobarbital, a classical Cyp2b inducer. These findings suggest that the Nrf2 pathway is involved in Cyp2b10 and Cyp2a5 gene expression.
Graphical abstract
Highlights
•Phorone, an α,β-unsaturated carbonyl compound, induced Cyp2b10 and Cyp2a5.•Phorone suppressed the induction of Cyp2b10 and Cyp2a5 in Nrf2−⧸− mouse livers.•Nrf2−⧸− mice showed suppressed phenobarbital-induced Cyp2b10 and Cyp2a5.
doi:10.1016/j.redox.2013.12.025
PMCID: PMC3909825  PMID: 24494203
ARE, antioxidant response element; CAR, constitutive androstane receptor; P450, cytochrome P450; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Hmox1, heme oxygenase-1; Nqo1, NAD(P)H-quinone oxidoreductase-1; Maf, musculoaponeurotic fibrosarcoma oncogene homolog; Nrf2, nuclear-factor erythroid 2-related factor 2; PBREM, phenobarbital responsive element module; PCR, polymerase chain reaction; Nuclear-factor erythroid 2-related factor 2; Cyp2b10; Cyp2a5; Phorone; Phenobarbital; Mouse
18.  Increased Nrf2 Activation in Livers from Keap1-Knockdown Mice Increases Expression of Cytoprotective Genes that Detoxify Electrophiles more than those that Detoxify Reactive Oxygen Species 
Toxicological Sciences  2009;108(1):35-47.
Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor critical for protection against electrophilic and oxidative stress. In a recently engineered mouse with knockdown of kelch-like ECH associated protein 1 (Keap1-kd mice), the cytosolic repressor of Nrf2, there is a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the hepatic phenotype of Keap1-kd mice, as well as the hepatic mRNA expression of cytoprotective genes were compared among wild-type, Nrf2-null, and Keap1-kd mice. Three distinct patterns of hepatic gene expression were identified among wild-type, Nrf2-null, and Keap1-kd mice. The first pattern encompassed genes that were lower in Nrf2-null mice and considerably higher in Keap1-kd mice than wild-type mice, which included genes mainly responsible for the detoxification and elimination of electrophiles, such as NAD(P)H:quinone oxidoreductase 1 and glutathione-S-transferases (Gst), and multidrug resistance–associated proteins. The second pattern encompassed genes that were lower in Nrf2-null mice but not increased in Keap1-kd mice, and included genes, such as epoxide hydrolase-1, UDP-glucuronosyltransferases, aldehyde dehydrogenases, as well as genes important in the detoxification of reactive oxygen species, such as superoxide dismutase 1 and 2, catalase, and peroxiredoxin 1. The third pattern encompassed genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and included genes such as glutathione peroxidase, microsomal Gsts, and uptake transporters. In conclusion, the present study suggests that increased activation of hepatic Nrf2 is more important for the detoxification and elimination of electrophiles than reactive oxygen species.
doi:10.1093/toxsci/kfn267
PMCID: PMC2644398  PMID: 19129213
Nrf2; electrophilic stress; oxidative stress; cytoprotection
19.  Clinical Significance of Keap1 and Nrf2 in Oral Squamous Cell Carcinoma 
PLoS ONE  2013;8(12):e83479.
Oxidative stress has been reported to play an important role in progression and prognostication in various kinds of cancers. However, the role and clinical significance of oxidative stress markers Keap1 and Nrf2 in oral squamous cell carcinoma (OSCC) has not been elucidated. This study aimed to investigate the correlation of oxidative stress markers Keap1 and Nrf2 expression and pathological features in OSCC by using tissue microarray. Tissue microarrays containing 17 normal oral mucosa, 7 oral epithelial dysplasia and 43 OSCC specimens were studied by immunohistochemistry. The association among these proteins and pathological features were analyzed. Expression of oxidative stress markers Keap1, Nrf2, and antioxidants PPIA, Prdx6, as well as CD147 was found to increase consecutively from normal oral mucosa to OSCC, and the Keap1, Nrf2, PPIA, Prdx6, CD147 expression in OSCC were significantly higher when compared to normal oral mucosa. Expression of Keap1, Nrf2 in tumors was not found to be significantly associated with T category, lymph node metastases, and pathological grade. Furthermore, we checked the relationship among these oxidative stress markers and found that Keap1 was significantly correlated with Nrf2, Prdx6 and CD147. Significant relationship between Nrf2 and Prdx6 was also detected. Finally, we found patients with overexpression of Keap1 and Nrf2 had not significantly worse overall survival by Kaplan–Meier analysis. These findings suggest that ROS markers are associated with carcinogenesis and progression of OSCC, which may have prognostic value and could be regarded as potential therapeutic targets in OSCC.
doi:10.1371/journal.pone.0083479
PMCID: PMC3873935  PMID: 24386210
20.  Analysis of Protein Kinase C Delta (PKCδ) Expression in Endometrial Tumors 
Human pathology  2007;39(1):21-29.
Endometrial cancer is the most common gynecological malignancy in the US, however, its underlying molecular mechanisms are poorly understood and few prognostic indicators have been identified. The Protein Kinase C (PKC) family have been shown to regulate pathways critical to malignant transformation, and in endometrial tumors, changes in PKC expression and activity have been linked to a more aggressive phenotype and poor prognosis. We have recently shown that PKCδ is a critical regulator of apoptosis and cell survival in endometrial cancer cells; however, PKCδ levels in endometrial tumors had not been determined. We used immunohistochemistry to examine PKCδ protein levels in normal endometrium and endometrioid carcinomas of increasing grade. Normal endometrium exhibited abundant nuclear and cytoplasmic staining of PKCδ, confined to glandular epithelium. In endometrial tumors, decreased PKCδ expression, both in intensity and fraction of epithelial cells stained, was observed with increasing tumor grade, with PKCδ being preferentially lost from the nucleus. Consistent with these observations, endometrial cancer cell lines derived from poorly differentiated tumors exhibited reduced PKCδ levels relative to well-differentiated lines. Treatment of endometrial cancer cells with etoposide resulted in a translocation of PKCδ from cytoplasm to nucleus concomitant with induction of apoptosis. Decreased PKCδ expression, particularly in the nucleus, may compromise the ability of cells to undergo apoptosis, perhaps conferring resistance to chemotherapy. Our results indicate that loss of PKCδ is an indicator of endometrial malignancy and increasing grade of cancer. Thus, PKCδ may function as a tumor suppressor in endometrial cancer.
doi:10.1016/j.humpath.2007.05.023
PMCID: PMC2180423  PMID: 17959229
PKCδ; endometrial cancer; immunohistochemistry; expression; nucleus
21.  Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines 
Oncogene  2010;29(25):3703-3714.
Nuclear Factor (erythroid-derived 2)-like 2 (Nrf2) expression is deregulated in many cancers. Genetic and biochemical approaches coupled with functional assays in cultured cells were used to explore the consequence of Nrf2 repression. Nrf2 suppression by Keap1-directed ubiquitylation or expression of independent shRNA/siRNA sequences enhanced cellular ROS, Smad-dependent tumor cell motility, and growth in soft agar. Loss of Nrf2 was accompanied by concomitant Smad linker region/C-terminus phosphorylation, induction of the E-Cadherin transcriptional repressor Slug, and suppression of the cell-cell adhesion protein E-Cadherin. Ectopic expression of wildtype Nrf2, but not dominant negative Nrf2, suppressed the activity of a synthetic TGF-β1 responsive CAGA-directed luciferase reporter. shRNA knock-down of Nrf2 enhanced the activity of the synthetic CAGA-reporter, as well as the expression of the endogenous Smad target gene plasminogen activator inhibitor-1. Finally, we found that Nrf2/Smad3/Smad4 formed an immunoprecipitable nuclear complex. Thus, loss of Nrf2 increased R-Smad phosphorylation and R-Smad signaling, supporting the hypothesis that loss of Nrf2 in an oncogenic context-dependent manner can enhance cellular plasticity and motility, in part by using TGF-β/Smad signaling.
doi:10.1038/onc.2010.118
PMCID: PMC2892014  PMID: 20440267
Nrf2; TGF-β; Smad; Cadherin; motility; oncogenesis; ROS
22.  Arsenic-induced malignant transformation of human keratinocytes: Involvement of Nrf2 
Free radical biology & medicine  2008;45(5):651-658.
Arsenic is a well-known human skin carcinogen but the underlying mechanisms of carcinogenesis are unclear. Transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism, and emerging data suggest that constitutive activation of Nrf2 contributes to malignant phenotype. In the present study when an immortalized, non-tumorigenic human keratinocyte cell line (HaCaT) was continuously exposed to environmentally relevant level of inorganic arsenite (100 nM) for 28 weeks, malignant transformation occurred as evidenced by the formation of highly aggressive squamous cell carcinoma after inoculation into nude mice. To investigate the mechanisms involved, a broad array of biomarkers for transformation were assessed in these arsenic-transformed cells (termed As-TM). In addition to increased secretion of matrix metalloproteinase-9 (MMP-9), a set of markers for squamous differentiation and skin keratinization, including keratin-1, keratin-10, involucrin, and loricrin, were significantly elevated in As-TM cells. Furthermore, As-TM cells showed increased intracellular glutathione, elevated expression of Nrf2 and its target genes, as well as generalized apoptotic resistance. In contrast to increased basal Nrf2 activity in As-TM cells, a diminished Nrf2-mediated antioxidant response induced by acute exposure to high dose of arsenite or tert-butyl hydroxyquinone occurred. The findings that multiple biomarkers for malignant transformation observed in As-TM cells, including MMP-9 and cytokeratins, are potentially regulated by Nrf2 suggest constitutive Nrf2 activation may be involved in arsenic carcinogenesis of skin. The weakened Nrf2 activation in response to oxidative stressors observed in As-TM cells, coupled with acquired apoptotic resistance, would potentially have increased the likelihood of transmittable oxidative DNA damage and fixation of mutational/DNA damage events.
doi:10.1016/j.freeradbiomed.2008.05.020
PMCID: PMC2658531  PMID: 18572023
Arsenic; Carcinogenesis; Nrf2; Oxidative stress; CK2
23.  Aggressive mammary carcinoma progression in Nrf2 knockout mice treated with 7,12-dimethylbenz[a]anthracene 
BMC Cancer  2010;10:540.
Background
Activation of nuclear factor erythroid 2-related factor (Nrf2), which belongs to the basic leucine zipper transcription factor family, is a strategy for cancer chemopreventive phytochemicals. It is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1 and peroxiredoxin 1, by activating the antioxidant response element (ARE). We hypothesized that (1) the citrus coumarin auraptene may suppress premalignant mammary lesions via activation of Nrf2/ARE, and (2) that Nrf2 knockout (KO) mice would be more susceptible to mammary carcinogenesis.
Methods
Premalignant lesions and mammary carcinomas were induced by medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene treatment. The 10-week pre-malignant study was performed in which 8 groups of 10 each female wild-type (WT) and KO mice were fed either control diet or diets containing auraptene (500 ppm). A carcinogenesis study was also conducted in KO vs. WT mice (n = 30-34). Comparisons between groups were evaluated using ANOVA and Kaplan-Meier Survival statistics, and the Mann-Whitney U-test.
Results
All mice treated with carcinogen exhibited premalignant lesions but there were no differences by genotype or diet. In the KO mice, there was a dramatic increase in mammary carcinoma growth rate, size, and weight. Although there was no difference in overall survival, the KO mice had significantly lower mammary tumor-free survival. Also, in the KO mammary carcinomas, the active forms of NF-κB and β-catenin were increased ~2-fold whereas no differences in oxidized proteins were observed. Many other tumors were observed, including lymphomas. Interestingly, the incidences of lung adenomas in the KO mice were significantly higher than in the WT mice.
Conclusions
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.
doi:10.1186/1471-2407-10-540
PMCID: PMC2964634  PMID: 20932318
24.  NRF2 and p53: Januses in cancer? 
Oncotarget  2012;3(11):1272-1283.
The transcription factor nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, is a master regulator of the anti-oxidative stress response and positively controls the expression of a battery of anti-oxidative stress response proteins and enzymes implicated in detoxification and glutathione generation. Although its detoxifying activity is important in cancer prevention, it has recently been shown that cancer cells also exploit its protective functions to thrive and resist chemotherapy. NRF2 was also shown to the pentose phosphate pathway and glutaminolysis, which promotes purine synthesis for supporting rapid proliferation and glutathione for providing anti-oxidative stress protection. Evidence obtained from cancer patients and cell lines suggest that NRF2 is highly active in a variety of human cancers and is associated with aggressiveness. p53 is a tumor suppressor that also promotes an anti-oxidative stress metabolic program and glutaminolysis. Here we will discuss the similarities between NRF2 and p53 and review evidence that p53 might be exploited by cancer cells to gain protection against oxidative stress, as is the case for NRF2. We discuss findings of co-regulation between these transcription factors and propose possible therapeutic strategies that can be used for treatment of cancers that harbor WT p53 and express high levels of NRF2.
PMCID: PMC3717791  PMID: 23174755
P53; p73; TIGAR; p21; NQO1; NADPH; ROS; metabolism; proteasome; KEAP1; MDM2; chemotherapy; BSO; piperlongumine; BPTES; brusatol
25.  Correlation of Nrf2, NQO1, MRP1, cmyc and p53 in colorectal cancer and their relationships to clinicopathologic features and survival 
Due to emergence of resistant tumor populations, prognosis for metastatic colorectal cancer (CRC) patients remains poor and five-year survival rate is still very low. To guide clinicians in selecting treatment option for CRC patients, reliable markers predictive of poor clinical outcome are desirable. This study analyzed the correlation of NF-E2-related factor 2 (Nrf2), NAD(P)H quinine oxidoreductase 1 (NQO1), multidrug resistant protein 1 (MRP1), cmyc and p53 in CRC and their relationships to Duke’s stage and clinical prognosis. 76 specimens of CRC tissues were immunohistochemically investigated using Nrf2, NQO1, MRP1, cmyc and p53 antibodies. IHC stain showed that Nrf2, NQO1, MRP1, cmyc and p53 were highly expressed in CRC tissues compared with adjacent non-tumor tissues. Significant positive correlations were found between the expression of Nrf2 and that of NQO1, MRP1, cmyc and p53. Moreover, there was significant correlation between the high level of Nrf2, NQO1, MRP1, p53 expression and Duke’s stage, as well as poor clinical prognosis. We confirmed that Nrf2, NQO1, MRP1, and p53 expression exhibits considerable heterogeneity according to CRC clinical stage and prognosis. Nrf2 is the most promising biomarker in identifying a poor prognostic group of CRC.
PMCID: PMC3971317
Colorectal cancer (CRC); NF-E2-related factor 2 (Nrf2); NAD(P)H quinine oxidoreductase 1 (NQO1); multidrug resistant protein 1 (MRP1); cmyc; p53

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