EGFR信号通路调控肿瘤相关巨噬细胞极化的机制及其在细胞恶性转化中的作用

Tumor microenvironment is an important aspect of gastric cancer and other cancer development. Tumor-associated macrophages (TAMs) produce mediators in the tumor microenvironment to promote cell malignant transformation,tumor angiogenesis, immunosuppression, and metastasis. TAMs are predominantly polarized to M2 macrophage phenotype. Epidermal growth factor receptor (EGFR) activation in tumor cells accelerates tumorigenesis. However, therapy to inhibit EGFR activity is not universally efficacious, suggesting possible widespread resistance of tumor cells to EGFR inhibition or EGFR in other cell types exerts different functions. Our preliminary data show that activation of EGFR in macrophages down-regulates IL-10 production and up-regulates TNF in response to inflammatory stimuli. EGFR activation level in TAM is higher in low-grade than that in high-grade gastric cancer tissues from patients. Furthermore, we find that IL-4 transactivates EGFR in macrophages, which is required for inhibiting IL-4-induced Arg1, a M2 marker expression and suppressing production of mediators for gastric cancer cell growth. Therefore, our overall objective is to understand the effects and mechanisms of by which EGFR in TAMs regulates gastric tumor development. We hypothesize that EGFR activation in TAMs inhibits TAM's effects on gastric cancer development. First, will further confirm that EGFR is activated in TAMs in gastric cancer tissues isolated from patients, and determine the relationship between the level of EGFR activation in TAMs and gastric tumor grade, expression of gastric cancer related genes, and activation status of signaling involved in promoting gastric cancer progress. Second,we will determine the role of EGFR in macrophages in induction of M2 macrophages. We will define the role of EGFR in induction of M2 macrophages by IL-4 or IL-13 and STAT6 activation using induced human and mouse macrophages.EGFR expression in human macrophages will be knocked down using a siRNA transfection method. We will use peritoneal macrophages isolated from mice with EGFR specifically deleted from macrophages. Third, we will determine the role of EGFR in macrophages in regulation of gastric cancer cell growth and epithelial-to-mesenchymal transition (EMT). We will use supernatants from induced M2 macrophageswith or without EGF expression to culture a novel ex vivo mouse gastric organoid system and human gastric cancer cells, respectively. Factors which are produced by IL-4 or IL-13 induced M2 macrophahes in an EGFR-dependent manner will be analyzed using shotgun proteomics and confirmed by their gene and protein expression levels. Results from this project will provide information to elucidate how cell specific EGF receptor activation regulates tumor microenvironment for tumorigenesis. These studies will be a critical step towards translating these observations into the next generation predictors of disease or therapeutics for gastric cancer.

肿瘤相关巨噬细胞（TAMs）呈M2型巨噬细胞表型，具有诱导细胞恶性转化和促进肿瘤转移等作用，但诱导其M2极化及功能变化的分子机制尚未明确。我们在前期工作中证实高分级胃癌TAMs中EGFR活化水平较低分级肿瘤降低；特异性敲除巨噬细胞的EGFR，可促进IL-4诱导的M2极化；提示EGFR信号具有调控巨噬细胞分化及功能的作用。因此提出研究假设：巨噬细胞中EGFR信号活化，可抑制其M2极化,下调其促肿瘤生长和转移的作用。本研究将检测胃癌患者TAMs中EGFR的活化状态，分析与临床指标、胃癌细胞IL-11,ptger4,TGF-β表达和Wnt信号活化的关系；下调巨噬细胞EGFR表达及活化水平，观察对巨噬细胞M2极化及STAT6活化的影响；调控巨噬细胞EGFR信号，观察其影响胃癌细胞生长及上皮间质转化（EMT）作用的变化。为探讨TAMs在细胞恶性转化以及肿瘤生长和转移中的作用及调控机制奠定基础。

肿瘤相关巨噬细胞（TAMs）是一类具有M2表型特点的巨噬细胞，对肿瘤细胞的增殖、侵袭、转移及血管生成均发挥了促进作用，但诱导其M2极化及功能变化的分子机制尚未明确。课题组在前期工作中发现EGFR活化在炎症模型中可以调控巨噬细胞的极化及细胞因子的合成，提示巨噬细胞表面EGFR活性对其极化及功能具有潜在的调控作用。因此，本项目深入探讨肿瘤微环境中EGFR信号通路对巨噬细胞M2型极化的影响及分子机制，进而描述其对肿瘤细胞增殖及EMT的作用，为阐释EGFR通路在肿瘤发生发展过程中所发挥的作用提供新的视角。首先利用小鼠巨噬细胞系及Egfrfl/flLysM-Cre、Egfrwa-5两种不同EGFR背景的转基因小鼠，明确了巨噬细胞EGFR在体外可被IL-4激活，并抑制其诱导的STAT-6磷酸化及细胞的M2型极化，活化的EGFR可通过内吞等方式降解；之后利用小鼠腹腔注射Chitin，在体内验证上述结果。其次，通过将不同EGFR背景巨噬细胞条件培养基与上皮细胞共孵育，发现抑制巨噬细胞表面EGFR活性后，其条件培养基对上皮细胞增殖及EMT的促进作用显著增强，且上述过程是通过巨噬细胞分泌的HB-EGF与上皮细胞表面EGFR所介导的。之后，通过小鼠荷瘤模型进行体内验证，发现EGFR缺陷的巨噬细胞条件培养基，对肿瘤细胞的增殖及EMT的促进作用明显高于野生型巨噬细胞，通过对小样本胃癌及胃炎标本的检测，发现巨噬细胞多聚集于癌与癌旁交界部位，且其表面EGFR表达水平随肿瘤进展而逐步下降。这与体外实验中EGFR被激活后降解相一致。综上所述，本研究探讨了巨噬细胞EGFR活化在IL-4介导的细胞M2型极化及功能中的负向调控作用；研究发现巨噬细胞EGFR可以通过影响HB-EGF的表达，最终影响巨噬细胞对上皮细胞增殖及EMT的调节作用。本研究同时揭示出EGFR信号通路在不同细胞内，特别是巨噬细胞及上皮细胞中可以产生截然相反的效果，这一结果为肿瘤抗 EGFR治疗失败提供新的分析思路，为新治疗靶点的开发提供部分理论支撑。