NRG1/HER3通路激活致乳腺癌曲妥珠单抗原发耐药的分子机制

Trastuzumab targeted therapy is a hot spot in the treatment of HER2 positive breast cancer. Its application has achieved a significant clinical effect, but how to predict and overcome the primary resistance is a great challenge to medical researchers. Our preliminary study found that NRG1mRNA was expressed positively in trastuzumab primary resistance cell, recombination NRG1 stimulation could significantly increase pHER3 level in trastuzumab primary resistance cells, trastuzumab had no effect on pHER3 level and cell proliferation, but HER3 antibody 3D4 effectively inhibited pHER3 up-regulation and cell proliferation, and both antibodies had synergistic effect on inhibition of primary resistance cell growth. Therefore, we propose the hypothesis that activated NRG1/HER3 exert pivotal role in trastuzumab primary resistance. In the present study, MDA-MB-453 (trastuzumab primary resistance cell line) and SK-BR3 (trastuzumab sensitive cell line) will be selected as the research object, systematically interventing NRG1 or HER3 by shRNA and blocking HER2 or HER3 by HER3 antibody and/or trastuzumab will be adopted as the research method, the relationship among the changes of NRG1, HER monomer, HER dimer and major downstream signal transduction molecular and trastuzumab primary resistance will be analyzed. And then the BALB/c breast cancer xenograft in nude mice model will be used to verify the sensitivity and resistance of trastuzumab. All the results will help to provide a new idea for the drug prediction and resistance surveillance of the trastuzumab clinical treatment.

曲妥珠单抗是目前HER2阳性乳腺癌的治疗选择热点；其应用已取得显著疗效，但如何预测及克服原发耐药对医学研究者是一项巨大挑战。我们前期研究发现曲妥珠单抗原发耐药细胞NRG1mRNA表达阳性，重组NRG1刺激原发耐药细胞pHER3表达上调，HER3抗体可显著抑制pHER3上调及耐药细胞的增殖，因此，我们提出NRG1/HER3激活致曲妥珠单抗原发耐药的假说。本研究选用MDA-MB-453（曲妥珠单抗原发耐药）与SK-BR3（敏感）HER2阳性乳腺癌细胞为研究对象，利用shRNA沉默NRG1或HER3基因，利用HER3抗体和/或曲妥珠单抗阻断HER2/HER3激活，分析细胞经不同处理后HER单体、二聚体及下游信号转导分子变化规律，阐明NRG1/HER3激活致曲妥珠单抗原发耐药的分子机制，并利用BALB/c移植瘤裸鼠模型进行敏感性和耐药性的验证，以期为曲妥珠单抗临床疗效预测与耐药监测提供新的思路。

曲妥珠单抗是目前HER2阳性乳腺癌患者的治疗选择热点，其应用已取得显著疗效，但如何预测及克服原发耐药对医学工作者是一项巨大挑战。本研究选择HER2阳性乳腺癌曲妥珠单抗敏感细胞株BT474和耐药细胞株MDA-MB-453为研究对象，以NRG1刺激、siRNA干扰及新型NRG1依赖性HER3抗体(3D4)为研究手段，比较敏感与耐药细胞株HER单体、二聚体、下游信号转导分子及细胞凋亡的不同。无NRG1刺激，曲妥珠单抗显著下调BT474细胞pHER2的表达，诱导BT474细胞早期凋亡的增加；NRG1刺激后，曲妥珠单抗的前述作用消失，出现曲妥珠单抗的耐药，而HER3抗体可通过显著下调pHER3的表达而促进BT474细胞的凋亡。无NRG1刺激时，曲妥珠单抗及HER3抗体对MDA-MB-453细胞pHER2、pHER3均无显著下调作用；NRG1刺激后，HER3抗体可显著下调pHER2和pHER3，并促进这株耐药细胞的凋亡。NRG1/HER3激活促曲妥珠单抗耐药及HER3抗体逆转曲妥珠单抗耐药是通过pAKT、pMAPK相关信号通路而发挥作用的。沉默MDA-MB-453细胞HER3基因，NRG1刺激仍可激活HER3及相关下游信号，抵消HER3基因沉默作用。在耐药BALB/C裸鼠移植瘤模型中，HER3抗体与曲妥珠单抗均可抑制肿瘤的生长，以前者效果显著，联合用药显示协同抑制趋势。因此本课题研究得出结论：NRG1/HER3激活是导致HER2阳性乳腺癌曲妥珠单抗原发耐药的关键因素；依赖于NRG1的HER3抗体可在一定程度上逆转曲妥珠单抗耐药，HER3有望成为HER2阳性乳腺癌的分子治疗靶标；NRG1/HER3可能作为预测曲妥珠单抗原发耐药的一个分子标记物。此研究为未来HER2阳性乳腺癌患者更有效、更精准的个体化治疗策略提供实验依据。