Various deletion mutants of Flag-tagged NOX5 or Pyk2 and HA-tagged NOX5 or Pyk2 were cloned into a pcDNA3.1 vector. poor prognosis. NOX5 induced the malignant progression of ESCC by activating Src, especially under hypoxic condition. Mechanistically, we showed that hypoxia promoted the interaction between NOX5 and Pyk2 on cell membrane via facilitating Ca2+-mediated Pyk2 Tyr402 site phosphorylation. Subsequently, Pyk2 acted as a scaffold for c-Abl phosphorylating the catalytic domain of NOX5 Tyr476/478 sites, which in turn upregulated hydrogen peroxide (H2O2) inside the Pyk2/NOX5 complex to oxidize and activate local Src. These findings provide insights into the biological significance of NOX5 in the development of ESCC. Subject terms: Gastrointestinal cancer, Gastrointestinal cancer Introduction Reactive oxygen species (ROS) are diffusible and short-lived signaling molecules, which induce various biological events. ROS at the specific subcellular compartment are critical for regulating redox-dependent signaling pathways under PHT-427 environmental stresses. Among these environmental stresses, hypoxia is an important prognostic and predictive factor owing to its multiple contributions to proliferation, invasiveness, metastasis, angiogenesis, resistance to cell death and altered metabolism in the PHT-427 process of tumor progression.1,2 Hypoxia activates many of the known oncogenic signaling proteins through stimulating the production of intracellular ROS to induce tumor malignant progression.3,4 Gastrointestinal epithelial cells are sensitive to environment stress to produce ROS, which gradually induce the transformation of these cells and lead to gastrointestinal tumors.5C7 However, the exact mechanisms by which gastrointestinal tumor cells, especially ESCC, sense hypoxia, integrate and activate components of oncogenic signaling pathways via local ROS are still unclear. NADPH oxidases (nicotinamide adenine dinucleotide phosphate oxidase, NOXs) are a family of enzymes with the primary function to generate ROS. They consist of seven members, represented by different catalytic subunits: NADPH oxidase 1 (NOX1) to NOX5, dual oxidase 1 (DUOX1), and DUOX2. NOXs use different regulatory subunits to produce ROS.8,9 PHT-427 NOXs in specific cellular microdomains, such as lamellipodia, membrane ruffles or endosomes, can interact with signaling platforms to provide a redox-dependent effect and resultantly achieve localized ROS production. For example, tyrosine kinase substrates (TKS) proteins, TKS4 and TKS5, interact with NoxA1 proline rich region (PRR) to enhance NoxA1-NOX1 binding and subsequently cause NOX1 localization PHT-427 to invapodia and increased ROS production.10 Pyk2/Grb2 recruits NOX4 to the scaffold protein SHPS-1, and NOX4 locates to cell membrane to activate Src in human vascular smooth muscle cells (VSMCs) treated with IGF-1.11 Increasing studies have shown that NOXs promote cancer progression via stimulating oncogenic signalings.12C17 Nevertheless, the cofactors that facilitate NOXs function in specific subcellular compartments to activate signaling pathways and promote cancer progression remain a huge puzzle. In this study, we intended to evaluate the expression of all members of the NOXs family in ESCC and establish the correlation between NOX5 expression and tumor malignancy. Importantly, we studied the role of NOX5 in regulating the malignant progression of ESCC cells and explored the underlying mechanisms. Results NOX5 is frequently upregulated in human ESCC cells To assess the expression of NOXs family in patients with ESCC, we conducted the analysis of the protein expression of NOX1-5, DUOX1, 2 in 92 pairs of ESCC and adjacent normal tissues (cohort I) using immunohistochemistry (IHC) assay. The protein levels of NOX5 were significantly upregulated in these ESCC samples in contrast to their adjacent normal tissues (Fig. ?(Fig.1a,1a, and Supplementary Table 1). Negative control staining of tumor slide was shown in Supplementary Fig. 1. Immunoblotting analysis clearly showed that the protein expression of NOX5 was Angpt2 significantly higher in primary ESCC cells or ESCC cell lines, compared with normal epithelial esophageal cells (NEECs) (Fig. ?(Fig.1b).1b). Importantly, the staining of carbonic anhydrase IX (CA IX), a marker for tumor hypoxia18,19 was positively correlated with NOX5 in ESCC samples (cohort I; Fig. ?Fig.1c1c). Open in a separate window Fig. 1 NOX5 PHT-427 positively correlates with the progression of ESCC. a Immunohistochemical staining evaluated the expression of NOX1-5, or DUOX1, 2 in 92 pairs of ESCC, and their respective adjacent noncancerous tissues (cohort I). Representative results of immunohistochemical staining for NOX1-5, or DUOX1, 2 in the same set of consecutive tumor tissue and their respective adjacent noncancerous tissue slices. Magnification, 10 as indicated. The chi-square test was employed to analyze correlations between tumors and their respective adjacent normal tissues. b Immunoblotting analysis of NOX5 in primary.