NOX4 promotes tumor progression through the MAPK-MEK1/2-ERK1/2 axis in colorectal cancer

Figure 1 Identification of dysregulated metabolic genes in tumor tissues. A: Multi-step analysis of metabolic genes in this study; B: Volcano plot showing metabolic differentially expressed genes (DEGs) between tumor and adjacent tissues; C: Box plots showing the top ten metabolic DEGs based on the adjusted P-value; D: Bar plot showing top ten upregulated and downregulated Kyoto Encyclopedia of Genes and Genomes items; E: Pheatmap showing the consensus matrix (k = 3); F and G: Kaplan-Meier curves showing overall survival and progression free interval across three clusters. ^bP < 0.01. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4; FC: Fold change; OS: Overall survival; PFI: Progression free interval.

Figure 2 Construction and validation of the prediction model. A-C: Kaplan-Meier curves showing the survival differences between high and low-risk groups in training, test datasets and our clinical cohort; D-F: Receiver operating characteristic curves showing the area under curve in training, test datasets and our clinical cohort. AUC: Area under curve.

Figure 3 Validation of the risk score and survival prediction model. A and B: Forest plots showing the univariate and multivariate analysis of risk scores and other clinical parameters; C: Receiver operating characteristic analysis showing the area under curve of each factor for predicting colorectal cancer overall survival; D: Construction of nomogram using the independent risk score. ^aP < 0.05, ^bP < 0.01. AUC: Area under curve.

Figure 4 Nicotinamide adenine dinucleotide phosphate oxidase 4 induces colorectal cancer progression. A: Venn diagram showing the overlap of metabolic differentially expressed genes and genes correlated with poor prognosis; B: Quantitative real-time polymerase chain reaction analysis of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) expression in tumor and adjacent normal tissues; C: Immunohistochemical representative images of NOX4 expression in colorectal cancer patient samples; D: Quantification of NOX4 expression in tumor and adjacent normal tissues in tumor samples; E: Kaplan-Meier curves showing the overall survival between high and low-NOX4 expression groups in our dataset; F and G: Clone and sphere formation was evaluated in NOX4-overexpressing RKO (RKO-OE) and wild-type (RKO-Ctrl) cells; H-J: Cell invasion and migration of RKO-OE and RKO-Ctrl cells were measured using the transwell chamber assay and scratch assay. ^aP < 0.05, ^bP < 0.01. DEG: Differentially expressed gene; NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4.

Figure 5 Nicotinamide adenine dinucleotide phosphate oxidase 4 promotes activation of mitogen-activated protein kinase/MEK1/2/ERK1/2 signaling. A: Principal component analysis of the transcription patterns in nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)-overexpressing RKO (RKO-OE) and wild-type (RKO-Ctrl) cells; B: Volcano map showing the differentially expressed genes between RKO-OE and RKO-Ctrl cells; C: Bar plot showing top ten Kyoto Encyclopedia of Genes and Genomes; D: Pattern diagram showing the RAS-RAF-MEK1/2-ERK1/2 axis; E: Western blot analysis showing the expression of NOX4, MEK, pMEK, ERK, pERK in RKO-OE and RKO-Ctrl cells, cultured with or without trametinib; F-J: Clone formation, invasion, migration, sphere formation in RKO-OE and RKO-Ctrl cells, cultured with or without trametinib. ^aP < 0.05, ^bP < 0.01. NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4; FC: Fold change; MAPK: Mitogen-activated protein kinase.

Figure 6 Nicotinamide adenine dinucleotide phosphate oxidase 4 promotes tumorigenesis and metastasis in vivo. A: Volume of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)-overexpressing RKO and wild-type tumor and measured at indicated time points; B: The representative image of subcutaneous tumors at the end of the experiment (day 28); C: Immunohistochemical staining analysis showing NOX4 and pERK expression in two groups; D: The representative image of metastatic nodes in the mouse lung tissue; E: Hematoxylin-eosin staining of tumor metastasis in mouse lung tissues; F: Quantification of tumor metastasis in lung tissues. ^aP < 0.05, ^bP < 0.01. NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4.