结直肠癌肝转移MIC-B基因突变促进TAMs极化导致西妥昔单抗原发耐药的机制

Liver metastasis is significantly associated with prognosis of colorectal cancer. Cetuximab is effect for colorectal cancer liver metastasis (CLM) with wild-type RAS. However, there are still half patients with primary cetuximab resistance. And the mechanism is still unknown. By gene microarray and next generation sequencing of a large sample size, we find that MIC-B mutation at rs1051788 in CLM is significantly associated with primary resistance of cetuximab. In cell line, MIC-B mutation significantly increases the expression of soluble MIC-B, and promotes TAMs polarization. Immunohistochemical stains in FFPE tumor sections also prove that TAMs polarization is significantly associated with MIC-B mutation and cetuximab resistance. At present, NKG2D is the only receptor of MIC-B. Immunofluorescences also prove that macrophages also express NKG2D on the membrane. Thus, we suppose that soluble MIC-B stimulate NKG2D on macrophages, and promotes TAMs polarization, then finally induces cetuximab resistance. In this project, we will concentrate on the mechanism in which NKG2D promotes TAMs polarization. We will detect the NKG2D/DAP10-endosome-ERK pathway and PI3K-Akt pathway to make clear the mechanism. This project is tightly associated with clinical questions. The achievement will help screen the applicable population for cetuximab, and prove evidence for multi-targeted treatment.

肝转移显著影响结直肠癌预后。西妥昔单抗治疗RAS野生型肠癌肝转移有效，但尚有半数患者原发耐药，且机制不明。申请人通过大样本基因芯片二代测序，发现肠癌MIC-B基因rs1051788位点突变（简称MIC-B突变）与西妥昔原发耐药显著相关。细胞实验显示，MIC-B突变导致可溶性MIC-B显著增加，促进巨噬细胞向TAMs极化；临床标本免疫组化也证实，TAMs极化与MIC-B突变、西妥昔耐药显著相关。目前MIC-B已知的唯一受体是NKG2D，免疫荧光也表明巨噬细胞表面表达NKG2D。申请人据此推想，可溶性MIC-B作用于巨噬细胞NKG2D，促进TAMs极化，导致西妥昔耐药。本项目拟重点研究NKG2D激活后促进TAMs极化的精细机制：通过检测NKG2D/DAP10-内吞体-ERK通路及PI3K-Akt通路，明确调控方式。本项目紧密结合临床，有助于筛选西妥昔单抗治疗适应人群，为多靶点联合治疗提供依据。

结直肠癌（colorectal cancer, CRC）是目前世界范围内发病率与死亡率较高的恶性肿瘤之一。肝转移是结直肠癌致死的主要原因，对于大部分的结直肠癌肝转移患者，以化疗联合靶向治疗为主的多学科治疗已成为将肝转移灶由不可切除转化为可切除，改善预后，延长生存的主要手段。西妥昔单抗作为抗表皮生长因子受体（epidermal growth factor receptor，EGFR）的单克隆抗体，是结直肠癌肝转移靶向治疗的重要组成部分。.但即便在全RAS野生型转移性结直肠癌患者中，依旧有约1/3的患者会出现西妥昔单抗的原发耐药且具体机制尚不明确。本课题组前期通过二代测序，发现人类主要组织相容性复合物I类链相关基因B（major histocompatibility complex class I chain-related gene，MICB）rs1051788位点突变（以下简称为MICB突变）与西妥昔单抗原发耐药显著相关，即MICB基因rs1051788位点突变的结直肠癌肝转移患者对西妥昔单抗的疗效显著差于MICB基因rs1051788位点野生型患者，更容易出现西妥昔单抗的原发耐药。.本课题通过临床水平、体外细胞水平、实验动物水平、分子机制水平等多层面发现MICB突变的结直肠癌细胞通过高分泌可溶性MICB的作用于肿瘤微环境中的M0型巨噬细胞表面的NKG2D受体，下调NKG2D在细胞表面的表达，进而通过巨噬细胞内部PI3K-Akt通路诱导M0型巨噬细胞向M2型巨噬细胞极化。