Targeting NOX4 Inhibits Proliferation Promoting Effect Of M2 Macrophages In NSCLC

Object: 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 carcinoma(NSCLC)and mediates cancer progression.NOXs are in close relation with cancer-related inflammation,nevertheless,whether tumoral NOXs influence microenvironmental macrophages remains unidentified.NOX4 inhibitor GKT137831 is currently in clinical trials which used in the treatment of idiopathic pulmonary fibrosis,diabetic nephropathy and primary biliary cholangitis due to its low toxicity.This study will explore its efficacy in the treatment of non-small cell lung carcinoma.Provide a basis for expanding clinical indications of GKT137831.Method:(1)The clinical NSCLC specimens were analyzed by immunohistochemistry(IHC)using indicated antibodies as anti-NOX4,anti-CD206,and anti-CD68.(2)Mice macrophage IL-10,IL-12,and IL-23 were measured using commercial ELISA kits.NSCLC cell CCL7,IL8,CSF-1 and VEGF-C were measured using commercial ELISA kits.The levels of phospho-JNK(T183 / Y185)and total JNK were assayed by the ELISA kit.(3)Extracellular steady-state generation of H2O2 was determined by Amplex Red hydrogen peroxide assay kit.(4)The membranes of Western Blotting were probed with primary antibodies as follows: NOX4,CD206 and ?-Tubulin.(5)Cells were transfected with 100 nM of a control shRNA,two individual shRNA against NOX4 or a pCMV-NOX4 cDNA plasmid with Lipofectamine 2000.(6)The proliferation of NSCLC cell lines were measured by MTT assay.(7)NSCLC cells with or without NOX4 overexpression were subcutaneously inoculated into the right flank of 6-week-old BALB/c nude mice.(8)The expression of F4/80 and CD206 of tissues were measured by flow cytometry technique.Results:(1)This study found that there was a close association between NOX4 expression and macrophage chemotaxis in patients with Non-small-cell lung carcinoma analyzed using TCGA RNA-sequencing data.Immunohistochemical analysis of clinical specimens confirmed the positive correlation of NOX4 and CD68 or CD206.(2)NOX4 in NSCLC cells(A549 and Calu-1 cell lines)efficiently enhanced murine peritoneal macrophage migration and induces M2 polarization.(3)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.Effect of NOX4 on macrophages was blocked by either NOX4 inhibitor GKT137831 or ROS scavenger NAC treatment for 24 h with continuing administration throughout the experiment.(4)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.(5)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.Conclusion: Our study indicates that tumoral NOX4 recruits M2 TAMs via ROS / PI3 K signaling-dependent various cytokine production,thus contributing NSCLC cell growth.GKT137831,as the only specific NOX4 inhibitor entered into clinical trials,has potential clinical significance for the treatment of non-small cell lung carcinoma.