This program explores innate immune, pro-inflammatory, and signaling functions of NOX family NADPH oxidases. The current research focuses on non-phagocytic oxidases (NOX1, NOX4, NOX5, DUOX1, DUOX2) expressed primarily in epithelial cells, on mucosal surfaces (lung and gastrointestinal tract), in liver, kidney, thyroid and exocrine glands (salivary, mammary), and in vascular and tumor tissues. Deliberate reactive oxygen species (ROS) production by these enzymes relays signals participating in cell migration, proliferation, tumor invasiveness and metastasis, cell differentiation, senescence, apoptosis, microbial killing, and responses to cytokines, growth factors, hormones and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In 2018, we explored functions of several NOX family NADPH oxidases in three areas of investigation: 1) studies on the roles of NOX4 in cancer progression in vitro, using established tumor lines, in silico, using primary human tumor genetic database repositories, and in mice with inducible tumors, 2) studies on subcellular targeting and functions of p22phox-dependent NOX enzymes, and 3) studies on genetic variants of NOX components linked to immunodeficiencies or inflammatory disease. We previously described opposing effects of wild-type (WT) and mutant (mut-) p53 on the TGF- beta/SMAD3/NOX4 pathway and how NOX4 promotes TGF-mediated focal adhesion kinase (FAK) activation, cell migration and invasiveness of human breast and lung epithelial tumor cells. We provided new insight into epigenetic regulation of the NOX4 gene promoter by both WT- and mut-p53 and SMAD3 in response to TGF-beta stimulation. Histone deacetylase (HDAC) inhibitors relieved repressive effects of WT-p53, whereas histone acetyltransferase (HAT) activity of p300 enhanced mut-p53-mediated induction of NOX4 promoter activity, as well as NOX4-dependent cell migration. Current work investigates roles of mutant p53-induced NOX4 on the cancer cell secretome and effects of NOX4-derived ROS on the tumor microenvironment. Our data suggest NOX4 regulates tumor cell production of inflammatory cytokines that promote cell motility under inflammatory conditions. These studies provide further insight on NOX4 as a potential therapeutic target in the inflammatory tumor microenvironment. This year's efforts aimed at exploring the broad significance of NOX4 induction by common p53 'hot spot' mutations have investigated correlations of p53 mutational status with NOX4 expression using data from >3250 primary human tumors in The Cancer Genome Atlas (TCGA). In these studies, we constructed a Pan-Cancer dataset from 23 types of primary tumors and employed non-parametric correlation analyses, multiple-testing corrections, and Kaplan-Meier survival estimates in R. We found evidence suggesting NOX4 plays a role in different transcriptional programs that represent signatures of cancer progression, including the epithelial-to-mesenchymal transition (EMT), extracellular matrix (ECM) production and angiogenesis, and showed these relationships are differentially modulated by WT and Mut-p53. The clinical outcomes associated with increased NOX4 expression are different in patients with tumors bearing WT- versus mut-p53, in that increased NOX4 in patients with mut-p53 is deleterious, whereas increased NOX4 is protective in those with WT-p53. Furthermore, we describe molecular correlates that provide insight on the role of NOX4 in cancer progression. We found that tumor macrophages also appear to be a source of enhanced NOX2; here, the association of NOX2 with genetic programs of cancer progression emulate those of NOX4. Most notably, increased NOX4 expression is linked to poorer survival in patients with mut-p53, but to better survival in patients with WT-p53. NOX4 is negatively associated with markers of apoptosis and positively with markers of proliferation in patients with mut-p53, which may explain their poorer survival. Collectively, our pan-cancer study identified several pathways by which NOX4 can contribute to cancer progression, and how p53 mutations could switch NOX4 from being protective and an indicator of good prognosis to deleterious outcomes. Thus, decisions on the therapeutic targeting of NOX4 or NOX4-derived ROS must consider tumor p53 mutation status, as these molecules may provide survival benefits in the presence of WT-p53, but would be worthwhile inhibiting in advanced cancers in which mut-p53 is detected. In 2018 we also generated a Nox4 knockout mouse strain using CRISPR/Cas9 methodology. We characterized Nox4 KO animals by measuring TGF-beta stimulated Nox4 H2O2 production, which was absent in Nox4-/- and reduced in Nox4+/- fibroblasts. Using the Nox4 KO mice, we plan to extend our in vitro observations on Nox4 regulation in epithelial tumors, by examining critical functions of Nox4 in vivo in mutant p53-mediated tumor metastasis and cancer progression, as well as overall survival. In mut-p53 pancreatic tumor samples of 60-day old mice we detected significantly elevated Nox2 and Nox4 transcript levels. Immunohistochemical analysis detected Nox2 staining not only in tumor-infiltrating macrophages but also in tumor and fibroblast cells. In related work in collaboration with Dr. Geiszt, we generated 2 transgenic (Tg) knock-in mouse models of the CYBA gene to engineer epitope tags on endogenous p22phox proteins. Our goal with these transgenic p22phox-tagged mice is to track Nox4, Nox1 and Nox2-based NADPH oxidases in healthy and diseased tissues, which was previously not possible due to lack of antibodies recognizing murine Nox proteins. We analyzed effects of epitope tagging by measuring Nox2 and Nox4 generated ROS production and showed no effects on PMA-induced Nox2 superoxide production by mouse bone marrow cells nor on TGF-beta1-induced Nox4 hydrogen peroxide release from mouse tail fibroblasts. Using Nox4 and p22phox knockout kidney lysates, we showed p22phox is necessary for Nox4 protein stabilization, as was shown earlier with other p22phox-dependent oxidases, Nox1-3. Nox4 mRNA is detected in kidney proximal tubules, but subcellular localization of Nox4 protein has never been described. We detected p22phox epitope staining in mouse kidney proximal tubule plasma membrane and resident macrophages, and in colon epithelial and myeloid cells. In collaboration with the laboratory of Dr. S. Holland, we studied a novel mono-allelic RAC2E62K mutation associated with combined immune deficiency, profound T and B cell lymphopenia, and absent newborn TRECs. Cos7 and CHO-K1 cell lines transfected with RAC2E62K demonstrated exuberant NOX2 ROS generation, increased membrane ruffling, enhanced RAC2-PAK1 binding and increased phospho-AKT, characteristics of a dominant active RAC2 molecule that are consistent with the patient's aberrant myeloid and lymphoid phenotypes. Other studies explored functional consequences of NOX components genetic variants linked to immunodeficiencies or inflammatory disease. In collaboration with Dr. R. Abraham, we characterized a p22phoxS98L variant in NOX2 reconstituted cell models. This was detected in a patient with recurrent pulmonary infections, who lacked other classical features of chronic granulomatous disease. We showed p22phoxS98L supports 20-25% less ROS production by PMA-stimulated NOX2 when compared with WT p22phox and showed this variant has reduced stability and is prone to degradation to a 15kDa protein band.
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