Exogenous DNA enhances DUOX2 expression and function in human pancreatic cancer cells by activating the cGAS-STING signaling pathway.

Pro-inflammatory cytokines upregulate the expression of the H2O2-producing NADPH oxidase dual oxidase 2 (DUOX2)2 which, when elevated, adversely affects survival from pancreatic ductal adenocarcinoma (PDAC). Because the cGAS-STING pathway is known to initiate pro-inflammatory cytokine expression following uptake of exogenous DNA, we examined whether activation of cGAS-STING could play a role in the generation of reactive oxygen species by PDAC cells. Here, we found that a variety of exogenous DNA species markedly increased the production of cGAMP, the phosphorylation of TBK1 and IRF3, and the translocation of phosphorylated IRF3 into the nucleus, leading to a significant, IRF3-dependent enhancement of DUOX2 expression, and a significant flux of H2O2 in PDAC cells. However, unlike the canonical cGAS-STING pathway, DNA-related DUOX2 upregulation was not mediated by NF-κB. Although exogenous IFN-ß significantly increased Stat1/2-associated DUOX2 expression, intracellular IFN-ß signaling that followed cGAMP or DNA exposure did not itself increase DUOX2 levels. Finally, DUOX2 upregulation subsequent to cGAS-STING activation was accompanied by the enhanced, normoxic expression of HIF-1α and VEGF-A as well as DNA double strand cleavage, suggesting that cGAS-STING signaling may support the development of an oxidative, pro-angiogenic microenvironment that could contribute to the inflammation-related genetic instability of pancreatic cancer.

NADPH oxidase 1 is highly expressed in human large and small bowel cancers.

To facilitate functional investigation of the role of NADPH oxidase 1 (NOX1) and associated reactive oxygen species in cancer cell signaling, we report herein the development and characterization of a novel mouse monoclonal antibody that specifically recognizes the C-terminal region of the NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human colorectal cancer cell lines, and correlated protein expression with NOX1 mRNA expression and superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1 mRNA levels could not be demonstrated in colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200 formalin-fixed, paraffin-embedded tissue cores from human epithelial tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal adenocarcinomas, as well as adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the protein level in tumors versus histologically normal tissues in prostate, lung, ovarian, or breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of colon cancer and in normal and malignant human tissues using a thoroughly evaluated monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small intestinal cancer.

IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide Production, Oxidative DNA Damage, and Upregulation of Dual Oxidase 2 in Human Colon and Pancreatic Cancer Cells.

Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-γ and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-κB‒mediated signaling in human pancreatic cancer cells. Using a panel of colon and pancreatic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-κB. The upregulation of DUOX2 was associated with a significant increase in the production of extracellular H2O2 and DNA damage-as indicated by the accumulation of 8-oxo-dG and γH2AX-which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-specific small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic tumors demonstrating significantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies.

Hydrogen peroxide-producing NADPH oxidases and the promotion of migratory phenotypes in cancer.

The cellular microenvironment plays a critical role in cancer initiation and progression. Exposure to oxidative stress, specifically hydrogen peroxide (H2O2), has been linked to aberrant cellular signaling through which the development of cancer may be promoted. Three members of the NADPH oxidase family (NOX4, DUOX1 and DUOX2) explicitly generate this non-radical oxidant in a wide range of tissues, often in support of the inflammatory response. This review summarizes the contributions of each H2O2-producing NOX to the invasive behaviors of tumors and/or the epithelial-mesenchymal transition (EMT) in cancer that plays an essential role in metastasis. Tissue localization in tumorigenesis is also highlighted, with patient-derived TCGA microarray data profiled across 31 cancer cohorts to provide a comprehensive guide to the relevance of NOX4/DUOX1/DUOX2 in cancer studies.

Purification and Characterization of DUOX Peroxidase Homology Domains (PHDs).

The dual oxidase (DUOX) enzymes (DUOX1 and DUOX2) are unique hydrogen peroxide (H2O2)-producing members of the NADPH oxidase (NOX) family, structurally distinguished from their related NOX isoforms by the presence of an additional N-terminal extracellular domain. This region has significant sequence and predicted structural homology to mammalian peroxidases, including myeloperoxidase (MPO) and lactoperoxidase (LPO), therefore justifying the nomenclature of the peroxidase homology domain (PHD). Obtaining detailed structural information and defining a function for this appended region are both critical for elucidation of the uncharacterized mechanism of H2O2 production by DUOX proteins. Purification strategies focused on isolated sections of each DUOX enzyme are a logical means to further characterization, particularly as isolation of the complete membrane-bound enzyme in significant quantities remains unachievable. In this chapter, a reproducible method for production of the homology domain applicable to both human DUOX isoforms is described. The approach utilizes a baculovirus expression vector in insect cell culture to produce secreted recombinant PHD; an appended C-terminal His6 affinity tag was found to be crucial for structural stability. Finally, initial characterization of the activity of the purified PHDs is also described.

Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR.

The identification of NADPH oxidase (NOX) isoforms in tissues is essential for interpreting experiments and for next step decisions regarding cell lines, animal models, and targeted drug design. Two basic methods, immunoblotting and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), are important to monitor NOX protein and messenger RNA (mRNA) levels, respectively, for a range of investigations from understanding cell signaling events to judging NOX inhibitor efficacies. For many other genes that are expressed in high abundance, these methods may seem rather simple. However, detecting the low expression levels of endogenous NOX/DUOX is difficult and can be frustrating, so some guidelines would be helpful to those who are facing difficulties. One reason why detection is so difficult is the limited availability of vetted NOX/DUOX antibodies. Many of the commercial antibodies do not perform well in our hands, and dependable antibodies, often generated by academic laboratories, are in limited supply. Another problem is the growing trend in the NOX literature to omit end-user validation of antibodies by not providing appropriate positive and negative controls. With regard to NOX mRNA levels, knockdown of NOX/DUOX has been reported in cell lines with very low endogenous expression (C q values ≥30) or in cell lines devoid of the targeted NOX isoform (e.g., NOX4 expression in NCI-60 cancer cell panel cell line 786-0). These publications propagate misinformation and hinder progress in understanding NOX/DUOX function. This chapter provides overdue guidelines on how to validate a NOX antibody and provides general methodologies to prepare samples for optimal detection. It also includes validated methodology to perform RT-qPCR for the measurement of NOX mRNA levels, and we suggest that RT-qPCR should be performed prior to embarking on NOX protein detection.

The soluble domains of Gpi8 and Gaa1, two subunits of glycosylphosphatidylinositol transamidase (GPI-T), assemble into a complex.

Glycosylphosphatidylinositol transamidase (GPI-T) catalyzes the post-translational addition of the GPI anchor to the C-terminus of some proteins. In most eukaryotes, Gpi8, the active site subunit of GPI-T, is part of a hetero-pentameric complex containing Gpi16, Gaa1, Gpi17, and Gab1. Gpi8, Gaa1, and Gpi16 co-purify as a heterotrimer from Saccharomyces cerevisiae, suggesting that they form the core of the GPI-T. Details about the assembly and organization of these subunits have been slow to emerge. We have previously shown that the soluble domain of S. cerevisiae Gpi8 (Gpi823-306) assembles as a homodimer, similar to the caspases with which it shares weak sequence homology (Meitzler, J. L. et al., 2007). Here we present the characterization of a complex between the soluble domains of Gpi8 and Gaa1. The complex between GST-Gpi823-306 (α) and His6-Gaa150-343 (ß) was characterized by native gel analysis and size exclusion chromatography (SEC) and results are most consistent with an α2ß2 stoichiometry. These results demonstrate that Gpi8 and Gaa1 interact specifically without a requirement for other subunits, bring us closer to determining the stoichiometry of the core subunits of GPI-T, and lend further credence to the hypothesis that these three subunits assemble into a dimer of a trimer.

NADPH oxidase 5 (NOX5)-induced reactive oxygen signaling modulates normoxic HIF-1α and p27Kip1 expression in malignant melanoma and other human tumors.

NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC-257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased γ-H2AX levels. Additionally, NOX5-overexpressing (stable and inducible) UACC-257 cells demonstrated increased normoxic HIF-1α expression and decreased p27Kip1 expression. Similarly, increased normoxic HIF-1α expression and decreased p27Kip1 expression were observed in stable NOX5-overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC-3. Conversely, knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased cell growth, decreased HIF-1α expression, and increased p27Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC-3 cells, transient knockdown of endogenous NOX5 resulted in increased p27Kip1 and decreased HIF-1α expression. Knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased Akt and GSK3ß phosphorylation, signaling pathways known to modulate p27Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC-257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF-1α and networks that signal through Akt/GSK3ß/p27Kip1 .

Characterization of potent and selective iodonium-class inhibitors of NADPH oxidases.

The NADPH oxidases (NOXs) play a recognized role in the development and progression of inflammation-associated disorders, as well as cancer. To date, several NOX inhibitors have been developed, through either high throughput screening or targeted disruption of NOX interaction partners, although only a few have reached clinical trials. To improve the efficacy and bioavailability of the iodonium class NOX inhibitor diphenylene iodonium (DPI), we synthesized 36 analogs of DPI, focusing on improved solubility and functionalization. The inhibitory activity of the analogs was interrogated through cell viability and clonogenic studies with a colon cancer cell line (HT-29) that depends on NOX for its proliferative potential. Lack of altered cellular respiration at relevant iodonium analog concentrations was also demonstrated. Additionally, inhibition of ROS generation was evaluated with a luminescence assay for superoxide, or by Amplex Red® assay for H2O2 production, in cell models expressing specific NOX isoforms. DPI and four analogs (NSCs 740104, 751140, 734428, 737392) strongly inhibited HT-29 cell growth and ROS production with nanomolar potency in a concentration-dependent manner. NSC 737392 and 734428, which both feature nitro functional groups at the meta position, had >10-fold higher activity against ROS production by cells that overexpress dual oxidase 2 (DUOX2) than the other compounds examined (IC50≈200-400nM). Based on these results, we synthesized and tested NSC 780521 with optimized potency against DUOX2. Iodonium analogs with anticancer activity, including the first generation of targeted agents with improved specificity against DUOX2, may provide a novel therapeutic approach to NOX-driven tumors.

Decoding NADPH oxidase 4 expression in human tumors.

NADPH oxidase 4 (NOX4) is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients), esophagus (12/18 patients), bladder (10/19 patients), ovary (6/17 patients), and prostate (7/19 patients), as well as malignant melanoma (7/15 patients) when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-ß1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.

Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells.

Human colon cancers express higher levels of NADPH oxidase 1 [NOX1] than adjacent normal epithelium. It has been suggested that reactive oxygen species [ROS] derived from NOX1 contribute to DNA damage and neoplastic transformation in the colon, particularly during chronic inflammatory stress. However, the mechanism(s) underlying increased NOX1 expression in malignant tumors or chronic inflammatory states involving the intestine are poorly characterized. We examined the effects of two pro-inflammatory cytokines, IL-4 and IL-13, on the regulation of NOX1. NOX1 expression was increased 4- to 5-fold in a time- and concentration-dependent manner by both cytokines in human colon cancer cell lines when a functional Type II IL-4 receptor was present. Increased NOX1 transcription following IL-4/IL-13 exposure was mediated by JAK1/STAT6 signaling, was associated with a ROS-related inhibition of protein tyrosine phosphatase activity, and was dependent upon activation and specific binding of GATA3 to the NOX1 promoter. NOX1-mediated ROS production increased cell cycle progression through S-phase leading to a significant increase in cellular proliferation. Evaluation of twenty pairs of surgically-resected colon cancers and their associated uninvolved adjacent colonic epithelium demonstrated a significant increase in the active form of NOX1, NOX1-L, in tumors compared to normal tissues, and a significant correlation between the expression levels of NOX1 and the Type II IL-4 receptor in tumor and the uninvolved colon. These studies imply that NOX1 expression, mediated by IL-4/IL-13, could contribute to an oxidant milieu capable of supporting the initiation or progression of colonic cancer, suggesting a role for NOX1 as a therapeutic target.

NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction.

Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2̇̄, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80-90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2-3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.

Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H2O2-induced, ERK-associated up-regulation of HIF-1α and VEGF-A.

Several NADPH oxidase family members, including dual oxidase 2 [DUOX2], are expressed in human tumors, particularly gastrointestinal cancers associated with long-standing chronic inflammation. We found previously that exposure of pancreatic ductal adenocarcinoma cells to the pro-inflammatory cytokine IFN-γ increased DUOX2 expression (but not other NADPH oxidases) leading to long-lived H2O2 production. To elucidate the pathophysiology of DUOX2-mediated H2O2 formation in the pancreas further, we demonstrate here that IFN-γ-treated BxPC-3 and CFPAC-1 pancreatic cancer cells (known to increase DUOX2 expression) produce significant levels of intracellular oxidants and extracellular H2O2 which correlate with concomitant up-regulation of VEGF-A and HIF-1α transcription. These changes are not observed in the PANC-1 line that does not increase DUOX2 expression following IFN-γ treatment. DUOX2 knockdown with short interfering RNA significantly decreased IFN-γ-induced VEGF-A or HIF-1α up-regulation, as did treatment of pancreatic cancer cells with the NADPH oxidase inhibitor diphenylene iodonium, the multifunctional reduced thiol N-acetylcysteine, and the polyethylene glycol-modified form of the hydrogen peroxide detoxifying enzyme catalase. Increased DUOX2-related VEGF-A expression appears to result from reactive oxygen-mediated activation of ERK signaling that is responsible for AP-1-related transcriptional effects on the VEGF-A promoter. To clarify the relevance of these observations in vivo, we demonstrate that many human pre-malignant pancreatic intraepithelial neoplasms and frank pancreatic cancers express substantial levels of DUOX protein compared to histologically normal pancreatic tissues, and that expression of both DUOX2 and VEGF-A mRNAs is significantly increased in surgically-resected pancreatic cancers compared to the adjacent normal pancreas.

NADPH oxidases and cancer.

The mechanism by which reactive oxygen species (ROS) are produced by tumour cells remained incompletely understood until the discovery over the last 15 years of the family of NADPH oxidases (NOXs 1-5 and dual oxidases DUOX1/2) which are structural homologues of gp91phox, the major membrane-bound component of the respiratory burst oxidase of leucocytes. Knowledge of the roles of the NOX isoforms in cancer is rapidly expanding. Recent evidence suggests that both NOX1 and DUOX2 species produce ROS in the gastrointestinal tract as a result of chronic inflammatory stress; cytokine induction (by interferon-γ, tumour necrosis factor α, and interleukins IL-4 and IL-13) of NOX1 and DUOX2 may contribute to the development of colorectal and pancreatic carcinomas in patients with inflammatory bowel disease and chronic pancreatitis, respectively. NOX4 expression is increased in pre-malignant fibrotic states which may lead to carcinomas of the lung and liver. NOX5 is highly expressed in malignant melanomas, prostate cancer and Barrett's oesophagus-associated adenocarcinomas, and in the last it is related to chronic gastro-oesophageal reflux and inflammation. Over-expression of functional NOX proteins in many tissues helps to explain tissue injury and DNA damage from ROS that accompany pre-malignant conditions, as well as elucidating the potential mechanisms of NOX-related damage that contribute to both the initiation and the progression of a wide range of solid and haematopoietic malignancies.

NADPH oxidases: a perspective on reactive oxygen species production in tumor biology.

SIGNIFICANCE: Reactive oxygen species (ROS) promote genomic instability, altered signal transduction, and an environment that can sustain tumor formation and growth. The NOX family of NADPH oxidases, membrane-bound epithelial superoxide and hydrogen peroxide producers, plays a critical role in the maintenance of immune function, cell growth, and apoptosis. The impact of NOX enzymes in carcinogenesis is currently being defined and may directly link chronic inflammation and NOX ROS-mediated tumor formation. RECENT ADVANCES: Increased interest in the function of NOX enzymes in tumor biology has spurred a surge of investigative effort to understand the variability of NOX expression levels in tumors and the effect of NOX activity on tumor cell proliferation. These initial efforts have demonstrated a wide variance in NOX distribution and expression levels across numerous cancers as well as in common tumor cell lines, suggesting that much remains to be discovered about the unique role of NOX-related ROS production within each system. Progression from in vitro cell line studies toward in vivo tumor tissue screening and xenograft models has begun to provide evidence supporting the importance of NOX expression in carcinogenesis. CRITICAL ISSUES: A lack of universally available, isoform-specific antibodies and animal tumor models of inducible knockout or over-expression of NOX isoforms has hindered progress toward the completion of in vivo studies. FUTURE DIRECTIONS: In vivo validation experiments and the use of large, existing gene expression data sets should help define the best model systems for studying the NOX homologues in the context of cancer.

Molecular mechanisms underlying chronic inflammation-associated cancers.

Although it is now accepted that chronic inflammation plays an essential role in tumorigenesis, the underlying molecular mechanisms linking inflammation and cancer remain to be fully explored. Inflammatory mediators present in the tumor microenvironment, including cytokines and growth factors, as well as reactive oxygen species (ROS) and reactive nitrogen species (RNS), have been implicated in the etiology of inflammation-associated cancers. Epithelial NADPH oxidase (Nox) family proteins, which generate ROS regulated by cytokines, are upregulated during chronic inflammation and cancer. ROS serve as effector molecules participating in host defense or as chemo-attractants recruiting leukocytes to wounds, thereby influencing the inflammatory reaction in damaged tissues. ROS can alter chromosomal DNA, leading to genomic instability, and may serve as signaling molecules that affect tumor cell proliferation, survival, metabolism, angiogenesis, and metastasis. Targeting Noxs and their downstream signaling components may be a promising approach to pre-empting inflammation-related malignancies.

Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody.

Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600-746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.

Functional activity and tumor-specific expression of dual oxidase 2 in pancreatic cancer cells and human malignancies characterized with a novel monoclonal antibody.

Dual oxidase 2 (Duox2), one of the seven members of the NADPH oxidase gene family, plays a critical role in generating H2O2 for thyroid hormone biosynthesis and as an integral part of the host defense system of the respiratory epithelium and the gastrointestinal tract. Recent evidence suggests that the regulation of Duox2 expression is under the control of pro-inflammatory cytokines and that Duox2-induced reactive oxygen species (ROS) contribute to the inflammation-related tissue injury that occurs in two pre-malignant, inflammatory conditions: chronic pancreatitis and inflammatory bowel disease. Because no reliable Duox antibodies are commercially available, we report the development of a murine monoclonal antibody (MAb) to Duox2 (clone Duox S-12) and its use for the characterization of Duox2 expression in human tumors, tumor cell lines and normal tissues. Duox S-12 specifically detected both endogenously- and ectopically-expressed Duox2 protein by immunoblotting, immunofluorescence microscopy and immunohistochemistry (where both membranous and cytoplasmic staining were present). Duox2 expression detected by Duox S-12 was functionally coupled to the generation of H(2)O(2) in pancreatic cancer cells that expressed Duox2 and its cognate maturation factor DuoxA2. Although Duox S-12 recognizes ectopically expressed Duox1 protein because of the extensive amino acid homology between Duox1 and Duox2, the lack of substantial Duox1 mRNA expression in human tumors (except thyroid cancer) allowed us to evaluate Duox2 expression across a wide range of normal and malignant tissues by immuno-histochemistry. Duox2 was expressed at elevated levels in many human cancers, most notably tumors of the prostate, lung, colon and breast while brain tumors and lymphomas demonstrated the lowest frequency of expression. The Duox-specific monoclonal antibody described here provides a promising tool for the further examination of the role of Duox-dependent reactive oxygen production in inflammation-related carcinogenesis, where alterations in oxidant tone play a critical role in cell growth and proliferation.

Activation of TLR4 is required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by IFN-γ and lipopolysaccharide in human pancreatic cancer cell lines.

Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1 acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.

Conserved cysteine residues provide a protein-protein interaction surface in dual oxidase (DUOX) proteins.

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1.