NOX family NADPH oxidases: roles in innate immunity and inflammatory disease

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This program explores innate immune, pro-inflammatory, and signaling functions of NOX family NADPH oxidases. Current research focuses on non-phagocytic NADPH oxidases (NOX1, NOX4, DUOX1, DUOX2) expressed primarily in epithelial cells, as well as NOX2 in hematopoietic cells. Reactive oxygen species (ROS) production by NOX enzymes relays redox signals in responses to cytokines, chemokines, growth factors, hormones, and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In addition to serving direct microbicidal roles, NOX-derived ROS regulate cell migration, proliferation, differentiation, senescence, apoptosis, tumor invasiveness and metastasis. In 2022, we have explored functions of several NOX family NADPH oxidase components in two areas of investigation: 1) studies on genetic variants of NOX and DUOX components associated with innate antimicrobial defense and barrier function defects or inflammatory disease, 2) studies on roles of NADPH oxidases in cancer progression. Our interests in rare NOX and DUOX genetic variants in patients with innate immune defects and inflammatory disease syndromes originated with identification of NOX2 component defects in patients with chronic granulomatous disease (CGD). As whole exome sequencing (WES) data has become widely available from patients of our clinical collaborators, we have explored functional consequences of oxidase defects using several heterologous NOX and DUOX expression systems. Collaborative studies with investigators in the IPS/LCIM (A. Hsu, S. Holland) characterized several DUOX1 and DUOXA1 loss-of-function variants linked to pulmonary and disseminated Coccidiodomycosis. We reconstituted signaling pathways linking DUOX1/DUOXA1 activation with Dectin1 fungal pathogen receptor detection and signaling. We also showed rare NOX1 variants detected in patients with inflammatory bowel disease exhibit variable protein stabilities, diminished NADPH oxidase activity, and lower NOX1-dependent cell migration in our reconstituted model of colon epithelial wound closure and barrier function (NHGRI collab.). We also characterized several partially functional CYBA, CYBB, and NCF1 variants linked to enhanced susceptibility to microbial infection and inflammation in several atypical CGD cases. Our interests in NOX4 function in cancer originated with work in lung, breast, liver, and pancreatic tumor cell lines showing that NOX4 is induced in a TGF-beta- and SMAD3-dependent manner in tumors bearing TP53 hot spot mutations (PMID: 22728268; PMID: 28574838). We recently conducted an in-depth pan-cancer informatic analysis of primary human tumors of 23 cancer types in The Cancer Genome Atlas (TCGA) and validated our in vitro observations demonstrating roles of NOX4 in promoting programs of cancer progression in tumors with TP53 hotspot mutations, regardless of tumor tissue origin (PMID: 33557266). NOX4 expression correlates positively with gene programs associated with tumor cell proliferation, invasiveness, and angiogenesis and negatively with programs of cell apoptosis in tumors with mutant TP53; the opposite was observed in tumors with wild-type TP53. Thus, TP53 mutations switch NOX4 from acting as a protective oxidase predicting a favorable prognosis to one with deleterious effects on cancer progression and clinical outcome in late-stage cancers. In this last year, related follow-up cell culture experiments have explored NOX4-dependent chemotactic and inflammatory signaling between macrophages and tumor cells as a model of the tumor microenvironment (TME). We investigated the roles of mutant p53-induced NOX4 on the cancer cell secretome and the effects of NOX4-based signaling in the TME. We found the secretome from p53-null H1299 lung epithelial cells stably expressing mutant p53 proteins (R248Q or R273H) promotes the migration and invasion of naive H1299, as well as chemotactic recruitment of THP-1 monocytes. These effects were diminished when the secreting tumor cells were transfected with dominant negative NOX4 (P437H). Immunoblot-based cytokine array analysis revealed tumor cell secretion of CCL5 was mutant p53 and NOX4-dependent, promoting autocrine and paracrine-mediated cell migration and invasion, whereas neutralization of CCL5 reduced autocrine-mediated H1299 cell mobility. Furthermore, we showed neutralization of CCL5 and TGF-beta released by M2-polarized macrophages have a significant role in this TME crosstalk by promoting H1299 cell migration and invasion. Our findings provide further insight into NOX4-based communication in the tumor microenvironment and its potential as a therapeutic target affecting metastatic disease progression. Finally, in related long-term studies in a mouse model of pancreatic ductal adenocarcinoma (PDAC), with targeted mutations in TP53 and K-Ras, we observed 10-15-fold enhanced NOX4 and NOX2 levels relative to normal pancreatic tissue, consistent with our TCGA informatics analysis of human PDAC datasets. Highest NOX4 expression was detected in ductal epithelial tumor cells, as well as in adjacent fibrotic stomal tissue, whereas highest NOX2 (Cybb) expression was detected in tumor associated macrophages. Current work is exploring whether the absence of functional NOX4 or NOX2 genes affects tumor cell escape from primary tumor sites or modifies the access or immunophenotype of inflammatory cells within the tumor microenvironment.