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.
Nrf2; chemoresistance; and endometrial cancer
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.
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.
Endometrial serous carcinoma; endometrial glandular dysplasia; endometrial intraepithelial carcinoma; p53; cadherins; claudins; CDKs; MDM2 and HER2/neu (erb-B2)
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.
Nuclear factor (erythroid-derived 2)-like 2; Oxidative stress; Insulin resistance; Metabolism; Diabetic drug
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.
stress response; redox homeostasis; transcription; purine nucleotide; glutathione
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.
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.
Uterine serous papillary endometrial cancer; Claudin-3; Claudin-4; lapatinib; endometrium
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.
Arsenic; Carcinogenesis; Nrf2; Oxidative stress; CK2
Uterine carcinosarcoma (UCS) is a rare but very aggressive cancer of the female reproductive tract with an extremely poor prognosis. With the goal of understanding the role of microRNA (miRNA) dysregulation in these tumors, we profiled the expression of 667 human miRNAs in a panel of eight UCS patients and five benign control primary tissue samples. These expression profiles revealed two important characteristics of UCS. First, compared with the two most common uterine cancers, endometrial endometrioid adenocarcinoma and endometrial serous adenocarcinoma, UCS samples display a virtually unique pattern of miRNA dysregulation with an overlap of only 5% among the three tumor types. In addition, nearly one-third of the miRNAs significantly dysregulated in UCS tissues compared with benign endometrium (32 of 114) lie in a single small (250-kb) imprinted region of chromosome 14q32. These data suggest that the presence of such a global, region-specific disruption substantially contributes to the unique histology and poor outcome of this type of cancer.
microRNA; uterine carcinosarcoma; chromosome 14q32
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.
PAX2; PTEN; endometrium; carcinoma; latent precancer
Uterine serous papillary carcinoma (USPC) is a biologically aggressive variant of endometrial cancer. We investigated the expression of Serum Amyloid A (SAA) and evaluated its potential as a serum biomarker in USPC patients.
SAA gene and protein expression levels were evaluated in USPC and normal endometrial tissues (NEC) by real-time PCR, immunohistochemistry (IHC), flow cytometry and by a sensitive bead-based immunoassay. SAA concentration in 123 serum samples from 51 healthy women, 42 women with benign diseases, and 30 USPC patients were also studied.
SAA gene expression levels were significantly higher in USPC when compared with NEC (mean copy number by RT–PCR=162 vs 2.21; P=0.0002). IHC revealed diffuse cytoplasmic SAA protein staining in USPC tissues. High intracellular levels of SAA were identified in primary USPC cell lines evaluated by flow cytometry and SAA was found to be actively secreted in vitro. SAA concentrations (μg ml−1) had a median (95% CIs) of 6.0 (4.0–8.9) in normal healthy females and 6.0 (4.2–8.1) in patients with benign disease (P=0.92). In contrast, SAA values in the serum of USPC patients had a median (95% CI) of 15.6 (9.2–56.2), significantly higher than those in the healthy group (P=0.0005) and benign group (P=0.0006). Receiver operating characteristics (ROC) analysis of serum SAA to classify advanced- and early-stage USPC yielded an area under the ROC curve of 0.837 (P=0.0024).
SAA is not only a liver-secreted protein but is also a USPC cell product. SAA may represent a novel biomarker for USPC to assist in staging patients preoperatively, and to monitor early-disease recurrence and response to therapy.
uterine serous papillary cancer; serum amyloid A; biomarkers; endometrial carcinoma; tumour markers
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.
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.
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.
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.
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.
Nrf2; proteasome system; endoplasmic reticulum stress; unfolded protein response; chronic obstructive pulmonary disease lungs
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.
PKCδ; endometrial cancer; immunohistochemistry; expression; nucleus
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.
Nrf2; NF-κB; DSS; AOM; apoptosis
Triple-negative breast cancer (TNBC) and basal-like breast cancer (BLBC) are breast cancer subtypes with an especially poor prognosis. 8-Hydroxydeoxyguanosine (8-OHdG) is a widely used marker of oxidative stress and the redox-state-regulating enzymes peroxiredoxins (PRDXs) are efficient at depressing excessive reactive oxygen species. NF-E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) are redox-sensitive transcription factors that regulate PRDX expression. This is the first study to assess oxidative stress and or cell redox state-regulating enzymes in TNBC and BLBC.
We assessed immunohistochemical expression of 8-OHdG, Nrf2, Keap1, PRDX III and PRDX IV in 79 women with invasive ductal breast carcinomas. Of these tumors, 37 represented TNBC (grade II-III tumors with total lack of ER, PR and human epidermal growth factor receptor 2 [HER2] expression). Control cases (n = 42) were ER-positive, PR-positive and HER2-negative. Of the 37 TNBCs, 31 had BLBC phenotype (TNBC with expression of cytokeratin 5/6 or epidermal growth factor receptor 1).
Patients with TNBC had worse breast cancer-specific survival (BCSS) than the control group (p = 0.015). Expression of 8-OHdG was significantly lower in TNBC than in the non-TNBC group (p < 0.005). 8-OHdG immunostaining was associated with better BCSS (p = 0.01), small tumor size (p < 0.0001) and low grade (p < 0.0005). Keap1 overexpression was observed in the TNBC cohort (p = 0.001) and Keap1-positive patients had worse BCSS than Keap1-negative women (p = 0.014). PRDX IV was overexpressed in the TNBC vs. the non-TNBC group (p = 0.022).
Cellular redox state markers may be promising targets when elucidating the pathogenesis of TNBC.
Both serous intraepithelial carcinoma and endometrial glandular dysplasia are associated with uterine serous carcinoma. Recently a candidate serous cancer precursor containing p53 mutations (p53 signature) was described in the fallopian tube. We analyzed normal and neoplastic endometrium for a similar entity. Ten endometrial polyps involved by intraepithelial and/or invasive carcinoma and 137 benign polyps were studied. All were stained for p53 and MIB-1. A subset of p53 signatures and carcinomas were analyzed for γ-H2AX and p53 mutations. p53 signatures were identified in 7 of 10 cases intraepithelial carcinoma and were multicentric in 2. In one case, the signature was in continuity with intraepithelial carcinoma. Six of 137 benign polyps (4%) contained p53 signatures. The MIB-1 fraction in most signatures was less than 5%, and ranged from 50-90% in carcinomas. DNA damage (γ-H2AX) was demonstrated in both p53 signatures and adjacent carcinomas but not in benign polyps. Shared identical p53 mutations were found in paired signatures and carcinomas in two of three cases analyzed, including one case with multiple signatures. In one, a co-existent invasive serous cancer was not found to contain a p53 mutation. In a third, a p53 signature and an invasive cancer harbored two different p53 mutations. This is the first description of p53 signatures adjacent to carcinoma, suggesting a role for this entity in the genesis of serous malignancy. The significance of p53 signatures in benign conditions (polyps) remains to be determined.
Peroxisome proliferator-activated receptors (PPARs) belong to a family of nuclear hormone receptors acting as transcriptional factors, recently involved also in carcinogenesis. Present study was undertaken to evaluate the presence and subcellular localization of different PPAR isoforms (α, β, γ) in healthy endometrial tissue (n = 10) and endometrial carcinoma (FIGO I, endometrioides type, G1, n = 35). We sought to analyze PPARs mRNA content as well as protein immunohistochemical expression that was further quantified by Western Blot technique. For both PPARα and PPARβ, protein expression was significantly higher in endometrial cancers compared to normal endometrial mucosa. In opposite, PPARγ protein expression was lower in endometrial cancer cells. In each case, immunohistochemical reaction was confined to the perinuclear and/or nuclear region. At the transcriptional level, the content of mRNA of all PPAR subunits did not follow the protein pattern of changes. These results provide evidence for altered PPAR's protein expression and disregulation of posttranslational processes in endometrial cancers.
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.
microRNAs (miRNAs) control a multitude of pathways in human cancers. Differential expression of miRNAs among different histological types of tumors within the same type of tissue offers insight into the mechanism of pathogenesis and may help to direct treatment to improve prognosis. We assessed expression of 667 miRNAs in endometrial endometrioid and serous adenocarcinomas using RNA extracted from benign endometrium as well as from primary endometrial tumors. Quantitative miRNA profiling of endometrial adenocarcinomas revealed four overlapping groups of significantly overexpressed and underexpressed miRNAs. The first group was composed of 20 miRNAs significantly dysregulated in both adenocarcinoma types compared with benign endometrium, two groups were composed of miRNAs significantly dysregulated in either endometrioid adenocarcinomas or in serous adenocarcinomas compared with benign endometrium, and the fourth group was composed of 17 miRNAs that significantly distinguished between endometrioid adenocarcinomas and serous adenocarcinomas themselves. Validation of the expression levels of the selected miRNAs was carried out in a second panel composed of ten endometrioid and five serous tumors. Experimentally validated mRNA targets of these dysregulated miRNAs were identified using published sources, whereas TargetScan was used to predict targets of miRNAs in the first and fourth profile groups. These validated and potential miRNA target lists were filtered using published lists of genes displaying significant overexpression or underexpression in endometrial cancers compared to benign endometrium. Our results revealed a number of dysregulated miRNAs that are commonly found in endometrial (and other) cancers as well as several dysregulated miRNAs not previously identified in endometrial cancers. Understanding these differences may permit the development of both prognostic and diagnostic biomarkers.
microRNA; endometrial cancer; endometrioid adenocarcinoma; serous adenocarcinoma
Endometrial cancer (EC) is the most common female genital malignancy in the USA. Most carcinomas arising from the uterus are estrogen dependent and are associated with obesity and hypertension. They are designated type I ECs and typically, due to their early diagnosis secondary to postmenopausal bleeding, have a good prognosis. By contrast, type II ECs develop in older patients, are not hormone dependent and are responsible for most recurrences and deaths from EC. Uterine serous cancer constitutes up to 10% of all endometrial tumors, and represents the most biologically aggressive variant of type II EC. This article will describe the most salient molecular markers that have been identified in uterine serous cancer, thus far with emphasis on the use of erbB2 (HER2/neu) as the first of a series of therapeutic markers for the treatment of this highly-aggressive subset of ECs.
endometrial cancer; erbB2; hRS7; molecular markers; MT-201; patupilone; pertuzumab; targeted therapy; trastuzumab; uterine serous cancer
NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation.
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.
Inactivation of the PTEN suppressor gene occurs in the majority of endometrial cancer cases. Somatic PTEN inactivation by deletion and/or mutation, the first detectible change of endometrial carcinogenesis, occurs at a high frequency in the endometrium of normal premenopausal women, though few of these progress to cancer. We hypothesized that the 50–60% reduced cancer risk of oral contraceptives (OCP) and intrauterine devices (IUD) occurs in part through their activity as negative selection factors for these subclinical mutated glands.
71 women with a history of oral contraceptive use and 80 with a history of IUD use were age matched with 191 and 119 controls, respectively. Endometrial biopsies were immunostained for PTEN and each scored for presence or absence of PTEN null glands (latent precancer).
The frequency of latent precancers was significantly reduced in OCP (13%, OR 0.19, p<0.001) and IUD (18%, OR 0.42, p=0.015) exposed women compared to respective matched controls (43 and 34%). Presence or absence of endometritis did not significantly correlate with PTEN status within the IUD exposed group (p=0.24).
Normal appearing PTEN mutated endometrial glands, which are highly prevalent in the normal population, may be targets of endometrial cancer risk modulating exposures. Some exposures known to diminish endometrial cancer occurrences in epidemiologic outcome studies, including OCP and IUD use, are associated with a proportionate decline in the frequency of latent precancers. Involution of pre-existing endometrial latent precancers, as evaluated by PTEN analysis, may provide an accessible surrogate marker for long term endometrial cancer risk.
endometrium; latent precancer; PTEN; oral contraceptive; intrauterine device
Uterine Serous Papillary Carcinoma (USPC), is an aggressive and chemotherapy resistant variant of endometrial cancer. We evaluated the expression of human –trophoblast-cell-surface-marker (Trop-2) and the potential of hRS7, a humanized anti-Trop-2 monoclonal antibody, as a novel therapeutic strategy against USPC.
Trop-2 expression was evaluated by immunohistochemistry (IHC) in a total of 23 USPC. Six primary USPC cell lines were assessed by flow cytometry and real-time-PCR for Trop-2 expression. Sensitivity to hRS7 (Immunomedics, Inc.) antibody-dependent-cellular-cytotoxicity (ADCC) and complement-dependent-cytotoxicity (CDC) was tested in standard 5-hrs-51Cr-release-assays against primary USPC cell lines.
Expression of Trop-2 was found in 15 out of 23 (65%) of the tumor tissues tested by IHC and in 50% (3/6) of the USPC cell lines tested by real-time-PCR and flow-cytometry [Trop-2 expression in USPC versus normal-endometrial-cells (NEC)(p < 0.005)]. USPC cell lines overexpressing Trop-2, regardless of their intrinsic resistance to natural killer cytotoxicity, were highly sensitive to hRS7-mediated ADCC in vitro (range of killing 28.2% to 64.4%) (p< 0.001). Negligible cytotoxicity against USPC was seen in the absence of hRS7 or in the presence of Rituximab control-antibody (range of killing 1.1% to 12.4%). Incubation with interleukin-2 (50 IU/ml) in addition to hRS7 further increased the cytotoxic activity against USPC cell lines overexpressing Trop-2 (p= 0.008).
Trop-2 is highly expressed in uterine serous carcinoma at mRNA and protein levels. Primary USPC cell lines are highly sensitivity to hRS7-mediated-cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for USPC refractory to standard treatment modalities.
Endometrial neoplasms; Uterine Serous Papillary Carcinoma; Trop-2; trophoblast cell-surface marker; hRS7; antibody dependent cellular cytotoxicity