非小细胞肺癌中NOX4诱导肿瘤相关巨噬细胞募集及M2型极化介导肿瘤侵袭转移

M2-polarized tumor-associated macrophages (TAMs) are key regulators of invasion and metastasis of non-small cell lung cancer (NSCLC). However, the mechanisms underlying that cancer cells induce macrophage infiltration and M2 polarization remain unclear. NADPH oxidases (NOX) have been confirmed to play important roles in cancer invasion and metastasis. Nevertheless, the studies only focused on that NOX influence biological characteristics of cancer cells themselves. Our previous works indicate that NOX4 is closely correlated with malignant progression of NSCLC. Recently, we found that NOX4 in NSCLC cells could promote macrophage migration and M2 polarization, which was independent of the direct action of NOX4-derived reactive oxygen species (ROS) on macrophages or the paracrine effect of NOX4-upregulated IL-6. Interestingly, we found that NOX4 knockdown could substantially inhibit CCL2 expression in NSCLC cells based on RNA-seq and Western blotting techniques. CCL2 has been widely accepted to be one of the most important factors inducing chemotactic movement and M2 polarization of macrophages. Taken together, we put forward the following assumption that NOX4 could promote invasion and metastasis of NSCLC through inducing infiltration and M2 polarization of macrophages via stimulating CCL2 expression and secretion. This study will provide new experimental evidences for clarifying the regulatory networks of NOX in cancer cells and contribute to development of effective therapeutical strategies for NSCLC treatment based on inflammatory microenvironment.

M2型巨噬细胞的富集促进非小细胞肺癌（NSCLC）侵袭转移，但癌细胞招募巨噬细胞并诱导其M2极化的机制至今未明。NOX在肿瘤侵袭转移中发挥重要作用，但相关研究均只关注NOX影响肿瘤细胞本身的生物学特性。我们以往研究表明NOX4与NSCLC恶性进展密切相关。最近我们发现NSCLC中NOX4可诱导巨噬细胞迁移及M2极化，这一效应不是通过其介导产生ROS直接作用于巨噬细胞或是促进IL-6分泌而产生。基于RNA-seq及免疫印迹技术，我们发现干扰NOX4可显著抑制NSCLC细胞中CCL2的表达。目前已知：CCL2是介导NSCLC诱导巨噬细胞趋化运动及M2极化的最重要因子之一。综上，我们提出如下假设：NSCLC中NOX4可通过促进CCL2表达分泌招募巨噬细胞并诱导其M2极化，进而促进NSCLC侵袭转移。本研究为全面了解肿瘤细胞中NOX的调控网络，并为基于炎性微环境的NSCLC靶向治疗提供实验依据。

M2型巨噬细胞的富集促进非小细胞肺癌（NSCLC）侵袭转移，但癌细胞招募巨噬细胞并诱导其M2极化的机制至今未明。NOX在肿瘤侵袭转移中发挥重要作用，但相关研究均只关注NOX影响肿瘤细胞本身的生物学特性。我们以往研究表明NOX4与NSCLC恶性进展密切相关。本研究发现NOX4的表达水平与巨噬细胞趋化信号通路密切相关。肿瘤NOX4表达水平与巨噬细胞标志物CD68、巨噬细胞M2极化标志物CD206的表达水平密切相关。NOX4的表达水平升高可促进非小细胞肺癌细胞产生IL-10，还能促进巨噬细胞表达CD206，即肿瘤NOX4与巨噬细胞M2型极化相关。NOX4的高表达显著增加巨噬细胞的迁移。抑制NOX4的表达能逆转上述的效应。NOX4的表达水平升高主要刺激肿瘤细胞产生和释放巨噬细胞趋化性和M2型极化的经典调节剂CCL7，IL8，CSF-1，VEGF-C。抑制PI3K/ Akt信号通路活性可阻断NOX4刺激非小细胞肺癌细胞产生CCL7，IL8，CSF-1和VEGF-C的效应。NOX4抑制剂GKT137831通过抑制NOX4活性减少ROS以及相关细胞因子的生成，从而抑制巨噬细胞的趋化和M2型极化，减少体外非小细胞肺癌细胞的增殖。肿瘤NOX4诱导M2型极化巨噬细胞JNK通路活性升高且表达释放HB-EGF。抑制JNK通路可减少HB-EGF的产生。使用GKT137831抑制NOX4活性可抑制体内肿瘤增殖，并减少肿瘤组织中M2型巨噬细胞的比例。结论：本研究首次发现NOX4通过ROS/PI3K/Akt信号依赖的多种细胞因子的产生募集巨噬细胞并诱导巨噬细胞M2型极化，从而通过活化巨噬细胞中的JNK通路和由此产生的HB-EGF的释放促进非小细胞肺癌细胞增殖。因此，GKT137831作为目前唯一进入临床试验的NOX4抑制剂，对于NSCLC的微环境干预治疗可能具有重要的临床价值。