Decoding NADPH oxidase 4 expression in human tumors

Abstract

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-β1 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.

Redox signals at the ER –mitochondria interface control melanoma progression

“…We could not discount that other sources of ROS were also adding to the oxidative stress during TMX disruption, such as the ER lumen or NOX4 (Appenzeller-Herzog et al, 2016). ER stress was not observed, but our data showed that NOX4, which is upregulated in melanoma (Yamaura et al, 2009;Meitzler et al, 2017), is also a key player in ROS production following TMX knockdown. This is in agreement with a study showing that TMX3 is an interaction partner for NOX4 (Prior et al, 2016).…”

“…An alternative source of ROS, and thereby H 2 O 2 , within cells are the NADPH oxidases. NADPH oxidase 4 (NOX4) is highly upregulated in melanoma and absent in healthy skin (Yamaura et al , ; Meitzler et al , ) and the interaction between TMX proteins and NOX4 was previously reported, proposing the presence of NOX4 within the MAM domains (Prior et al , ). Hence, we examined the impact of NOX4 on cellular ROS production in TMX1‐silenced cells with the H 2 O 2 sensor HyPer.…”

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

“…Secondary antibodies: HRP-conjugated goat anti-rabbit, HRP-goat anti-mouse, or fluorescent-labeled antibodies. 12. ECL reagent such as SuperSignal West Pico Chemiluminescent Substrate.…”

Regulation of mitochondrial ROS production by HIC‐5: a common feature of oncogene‐induced senescence and tumor invasiveness?

“… published in this issue of the FEBS Journal, one of the actions of HIC‐5 is to downregulate NADPH oxidase 4 (NOX4), a membrane‐bound protein reported to localize to various subcellular sites, including the plasma membrane, the endoplasmic reticulum membrane, and the mitochondria. As cellular localization studies with NOX4 are usually performed by immunofluorescence assays, the choice of the appropriate antibodies is crucial, and only a few reliable antibodies are currently available . Downregulation of NOX4 by HIC‐5 resulted in a decrease of mitochondrial ROS levels (mtROS), in accordance with the described function of NOX4 to increase mtROS.…”