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

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

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. Deliberate reactive oxygen species (ROS) production by the epithelial 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, ROS generated by several NOX enzymes also regulate cell migration, proliferation, differentiation, senescence, apoptosis, tumor invasiveness and metastasis. In 2021, we explored functions of several NOX family NADPH oxidase components in two general areas of investigation: 1) studies on distinct roles of NADPH oxidases in cancer progression (current focus on NOX4, NOX2, and NOX1), and 2) studies on genetic variants of NOX and DUOX components associated with innate antimicrobial defense defects or inflammatory disease. Our previous work using established tumor cell lines from lung, breast, liver, and pancreas showed that NOX4 is induced in a TGF-beta- and SMAD3-dependent manner in tumors bearing TP53 hot spot mutations. We have now undertaken an in depth bioinformatic analysis of data from thousands of primary human tumors in The Cancer Genome Atlas (TCGA) to validate our previous in vitro observations on the roles of NOX4 in promoting programs of cancer progression in a variety of tumor types and determine the relationship of NOX4 expression to overall survival of cancer patients. The complete analysis, required files, and R scripts can be found at https://github.com/wfma/Ma_2018. Here, we constructed a Pan-Cancer database from 23 different tumor types. We found that high NOX4 tumor expression correlates with poorer survival in patients with mutant TP53 tumors, whereas survival was better in patients with high NOX4 tumor levels in wild type TP53 tumors. Thus, it appears that mutations in TP53 switch NOX4 from acting as a protective oxidase and an indicator of good prognosis to one with deleterious effects on cancer progression and overall clinical outcome in late-stage cancers. Consistent with these findings, NOX4 expression correlates positively with gene programs associated with cell proliferation and negatively with programs of cell apoptosis in tumors with mutant TP53, whereas the opposite trends were observed in tumors with wild-type TP53. Our observations also confirmed that NOX4 expression correlates with critical players in tumor cell migration, invasiveness, and angiogenesis, specifically in tumors where TP53 is mutated regardless of tumor tissue origin. Interestingly, NOX2 expression patterns also correlated with the same cancer promoting gene expression programs, albeit to a lesser extent than NOX4, while also correlating with markers of tumor macrophages. The above informatics analysis did not identify the tumor cell types giving rise to the observed altered gene expression patterns, since the methodology involved a Pan-Cancer database curation of whole tumor tissue transcripts in relation to clinical outcomes. Therefore, we embarked on parallel whole animal studies examining Nox2 and Nox4 expression patterns in the KPC mouse model of pancreatic ductal adenocarcinoma (PDAC), which develops from pancreatic-targeted mutations in TP53 and KRAS. KPC tumors show a 10-15-fold enhancement of Nox4 and Nox2 levels relative to normal pancreatic tissue, consistent with our TCGA informatics analysis of human PDAC datasets (doi:10.18632/oncotarget.17892 and doi: 0.3390/antiox10020235). High Nox4 expression was detected not only in ductal epithelial tumor cells, but also in adjacent fibrotic stomal tissue. In contrast, highest Nox2 (Cybb) expression was detected in tumor associated macrophages. The KPC mice were also crossbred with Nox4 or Nox2 (Cybb) deficient mice to assess the roles of these oxidases in cancer progression and overall survival. Here, ongoing experiments are addressing whether the absence of functional Nox4 or Cybb2 genes affects the access or immunophenotype of inflammatory cells within the tumor microenvironment, and whether fibroblast or tumor cell Nox4 deficiency alters the escape of tumor cells from primary tumor sites based on detection of distal metastases. Related cell culture experiments are exploring NOX-dependent chemotactic and inflammatory signaling between macrophages and tumor cells as a model of the tumor microenvironment. Other collaborative studies with the Univ. of Cordoba are exploring roles of NOX1 and its regulator, PRDX6, in tumor cell proliferation, migration, and metastasis. Thus far, we have shown that NOX1 and PRDX6 together promote migration and expression of EMT markers in both colon adenocarcinoma and hepatoma cells, suggesting novel targets against metastatic disease. Our second major research emphasis concerns studies on the characterization of several rare NOX and DUOX genetic variants detected in patients with innate immune defects and inflammatory disease syndromes. As whole exome sequencing (WES) data has become more widely available from patients of our intramural and extramural clinical collaborators, we are exploring mechanistic consequences of oxidase defects using several heterologous oxidase expression systems. In studies on several loss-of-function (LOF) DUOXA1 and DUOX1 variants linked to pulmonary and disseminated fungal infection (LCIM IPS collab.), we have reconstituted signaling pathways connecting DUOX1 activation with Dectin1 fungal pathogen receptor detection and signaling. Here, several DUOX1 LOF variants were shown to be unstable and degraded. We showed rare NOX1 variants detected in patients with inflammatory bowel disease exhibit low levels of NADPH oxidase activity and support 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, NCF1, and PRDX6 variants linked to enhanced susceptibility to microbial infection and inflammation atypical of chronic granulomatous disease. Finally, we have provided recombinant expression systems and expertise for heterologous reconstitution of NADPH oxidase activities of NOX1, NOX2, NOX5, DUOX1 and DUOX2 enzymes which are being used to assess specificities of several candidate oxidase inhibitors (collab. with CPS, LCIM (Drs. Gallin and Zarember) and NCATS). Such inhibitors have therapeutic potential against a variety of NOX-related inflammatory disease processes, including traumatic brain injury, atherogenesis, acute respiratory distress syndromes (ARDS), and pathways promoting cancer progression.