曲妥珠单抗刺激HER2+乳腺癌细胞分泌CLIC3调控抗体依赖细胞吞噬的临床和机制研究

Trastuzumab-based regimen is the standard treatment for HER2 + breast cancer patients, but still about 20% of patients will experience recurrence and metastasis. Studying the mechanism of trastuzumab resistance is of great significance to improve its efficacy and precision medicine. The RNA sequence data showed that chloride channel protein 3 (CLIC3) was highly expressed in trastuzumab-sensitive HER2 + breast cancer cells and in patients with HER2 + breast cancer who achieved pathological complete response (pCR). Experiments have shown that over-expression of CLIC3 increases the sensitivity of trastuzumab and inhibits the PI3K-AKT pathway, but does not affect the cell cycle and apoptosis. RNA sequence data showed that CLIC3 might be associated with macrophage activation, chemotaxis and Fc-gamma receptor signaling pathway involved in phagocytosis. In a mouse human tumor xenograft model, the expression of CLIC3 in the tumor stroma enriched with trastuzumab was significantly higher. Co-culture of HER2 + breast cancer cells and macrophages, trastuzumab can increase the expression of CLIC3 in macrophages. All of these suggested that CLIC3 might be involved in trastuzumab-mediated tumor cell killing in the tumor microenvironment Therefore, this study hypothesized that trastuzumab may cause HER2 + breast cancer cells to secrete CLIC3 and then recruit macrophages to exert tumor cell killing effects.

以曲妥珠单抗为基础的方案是HER2+乳腺癌患者的标准治疗方案，但仍约有20%的患者会出现复发转移。研究曲妥珠单抗耐药机制对提高其疗效和精准治疗意义重大。测序数据发现氯离子通道蛋白3（CLIC3）在曲妥珠单抗敏感的HER2+乳腺癌细胞和达到病理完全缓解（pCR）的HER2+乳腺癌患者中高表达。实验证实，过表达CLIC3增加曲妥珠单抗的敏感性，抑制PI3K-AKT通路，并不影响生长周期和凋亡。测序数据显示，CLIC3过表达与巨噬细胞活化、趋化、Fcγ信号通路相关。鼠人源肿瘤异种移植模型中，曲妥珠单抗富集的肿瘤间质中CLIC3表达水平显著增高。HER2+乳腺癌细胞和巨噬细胞共培养，曲妥珠单抗可以增加巨噬细胞中CLIC3的表达水平。这提示CLIC3在肿瘤微环境中参与曲妥珠单抗介导的肿瘤细胞杀伤作用。因此我们假设曲妥珠单抗可能导致HER2+乳腺癌细胞分泌CLIC3进而招募巨噬细胞发挥肿瘤杀伤作用。

国内外乳腺癌治疗指南均推荐曲妥珠单抗作为HER2阳性乳腺癌的标准治疗方案。曲妥珠单抗可以降低高达40%的复发风险和34%的死亡风险。但仍有15-24%的患者出现复发、转移。这主要是由于HER2阳性乳腺癌患者中存在曲妥珠单抗耐药。我们分析曲妥珠单抗耐药的HER2+乳腺癌细胞和组织的测序数据，发现氯离子通道蛋白3（CLIC3）在曲妥珠单抗敏感株的HER2乳腺癌中高表达，且在达到病理完全缓解（pCR）的新辅助患者乳腺癌标本中高表达。我们构建CLIC3过表达的稳转细胞系，CLIC3过表达导致曲妥珠单抗敏感，且影响PI3K-AKT信号通路来发挥作用。免疫荧光试验显示CLIC3主要位于细胞核和细胞质，也可以分泌到肿瘤间质和外周血。乳腺癌患者外周血中CLIC3的表达水平显著高于健康人外周血中的表达水平。我们分离了健康人和乳腺癌患者外周血中的外泌体，发现CLIC3在乳腺癌外周血中高表达。我们分析乳腺癌患者外周血外泌体中CLIC3对HER2+乳腺癌新辅助治疗靶向治疗病理完全缓解的预测价值，外泌体中 CLIC3高表达的患者更容易达到pCR。免疫共沉淀结果显示，CLIC3与人程序性细胞死亡因子10（PDCD10）之间存在相互作用。正向验证显示过表达CLIC3的细胞系中可以看到PDCD10的富集，反向使用PDCD10抗体验证CLIC3的互作，PDCD10可以与CLIC3相互作用。这提示CLIC3与PDCD10相互作用。在CLIC3过表达的细胞系中可以发现，CLIC3过表达可以维持PDCD10的表达稳定，敲低CLIC3可以促进PDCD10的表达降解。数据分析发现，曲妥珠单抗可能导致HER2+乳腺癌细胞分泌CLIC3进而招募巨噬细胞发挥肿瘤细胞杀伤作用。HER2+乳腺癌细胞NF639与巨噬细胞RAW2647共培养且加入曲妥珠单抗处理后可以显著诱导巨噬细胞中CLIC3表达上调。共培养实验发现，曲妥珠单抗处理后的BT474可以上调巨噬细胞中CLIC3的表达水平，MAPK信号通路在ADCP过程中发挥着重要作用。本研究对CLIC3在HER2+乳腺癌中临床价值进行了深入的分析和评估，组织中和外周血外泌体中CLIC3的表达水平对HER2+乳腺癌患者的曲妥珠单抗的治疗疗效有预测价值。基于CLIC3的单克隆抗体将有助于克服HER2+乳腺癌曲妥珠单抗耐药。

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