Tumoral NOX4 recruits M2 tumor-associated macrophages via ROS/PI3K signaling-dependent various cytokine production to promote NSCLC growth

Abstract

M2-type tumor-associated macrophages (TAMs) infiltration contributes to cancer malignant progression. However, the mechanisms for controlling recruitment and M2 polarization of macrophages by cancer cells are largely unclear. NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and mediates cancer progression. NOXs are in close relation with cancer-related inflammation, nevertheless, whether tumoral NOXs influence microenvironmental macrophages remains undentified. This study found that there was a close association between NOX4 expression and macrophage chemotaxis in patients with NSCLC analyzed using TCGA RNA-sequencing data. NOX4 in NSCLC cells (A549 and Calu-1 cell lines) efficiently enhanced murine peritoneal macrophage migration and induces M2 polarization. Immunohistochemical analysis of clinical specimens confirmed the positive correlation of NOX4 and CD68 or CD206. The mechanical study revealed that tumoral NOX4-induced reactive oxygen species (ROS) stimulated various cytokine production, including CCL7, IL8, CSF-1 and VEGF-C, via PI3K/Akt signaling-dependent manner. Blockade of the function of these cytokines reversed NOX4 effect on macrophages. Specifically, the results showed that tumoral NOX4-educated M2 macrophages exhibited elevated JNK activity, expressed and released HB-EGF, thus facilitating NSCLC proliferation in vitro. Pretreatment of macrophages with JNK inhibitor blocked tumoral NOX4-induced HB-EGF production in M2 macrophages. Finally, in a xenograft mouse model, overexpression of NOX4 in A549 cells enhanced the tumor growth. Elimination of ROS by NAC or inhibition of NOX4 activity by GKT137831 suppressed tumor growth accompanied by reduction in macrophage infiltration and the percentage of M2 macrophages. In conclusion, our study indicates that tumoral NOX4 recruits M2 TAMs via ROS/PI3K signaling-dependent various cytokine production, thus contributing NSCLC cell growth.

Transcriptional, Epigenetic and Metabolic Programming of Tumor-Associated Macrophages

“…ROS produced by cancer cells stimulates various cytokine production, including CCL7, IL-8, CSF-1 and VEGF-C, that all contribute to enhanced NSCLC cell growth. IHC analysis of clinical specimens confirmed the positive correlation of NOX4 and CD68 or CD206 [253]. ROS accumulation in BMDMs that was reached by ROS inducer, glutathione synthesis inhibitor buthionine sulphoximine (BSO), results in increased expression of programmed death ligand-1 (PD-L1) and production of IL-10, IL-17, IL-4, IL-1b, insulin-like growth factor-binding protein 3 (IGFBP-3), and chemokine (C-X-C motif) ligand 1 (CXCL1) that are associated with an immune-suppressive phenotype of macrophages [271].…”

“…ROS production is regulated by NADPH oxidases. NADPH oxidase 4 (NOX4)-overexpressed lung cancer cell lines A549 and Calu-1, induced the recruitment of murine M2-like TAMs via the ROS/PI3K signaling-dependent pathway [253]. ROS produced by cancer cells stimulates various cytokine production, including CCL7, IL-8, CSF-1 and VEGF-C, that all contribute to enhanced NSCLC cell growth.…”

Gallbladder cancer-associated fibroblasts promote vasculogenic mimicry formation and tumor growth of gallbladder cancers via upregulating the expression of NOX4, a poor prognosis factor, through activating IL-6-JAK-STAT3 signal pathway

“…Among them, the expression of NOX4 was signi cantly increased.At the same time, the result of lncRNAs microarray showed that the expression of NOX4, JAK and STAT3 was signi cantly increased in 154 VM-related genes of GBC. As mentioned above, NOX4, as a member of the NADPH oxidase family, plays vital roles in proliferation, metastasis and angiogenesis of tumors through the production of ROS [29,43,44]. Then, we detected NOX4 expression in vitro and in vivo using qRT-PCR, western blot and IHC.…”

Gallbladder cancer-associated fibroblasts promote vasculogenic mimicry formation and tumor growth in gallbladder cancer via upregulating the expression of NOX4, a poor prognosis factor, through IL-6-JAK-STAT3 signal pathway

“…Then, we detected NOX4 expression in vitro and in vivo, and con rmed that NOX4 was not only highly expressed in GCAFs, but also in human GBC tissue and stroma. As mentioned above, NOX4, as a member of the NADPH oxidase family, plays vital roles in proliferation, metastasis and angiogenesis of tumors through the production of ROS [42,44,45]. It has been con rmed that NOX4 is highly expressed in many types of tumor cells such as pancreatic cancer [46], renal cell carcinoma [47] and gastric cancer [48].…”