阿司匹林协同拉帕替尼治疗HER2阳性乳腺癌的作用及分子机制研究

Approximately 25% of breast cancers were characterized by the amplification of HER2, which was strongly associated with increased disease recurrence and worse prognosis. Lapatinib, a new FDA approved HER2 targeted drug, showed a remarkable effectiveness on advanced HER2-positive breast cancer or metastatic breast cancer. However, drug resistance had seriously restricted its clinical responsiveness. Therefore, a reasonable combination medication therapy to improve the treatment effect is of great importance. Aspirin, one of the classical Non-steroidal Anti-inflammatory Drugs(NSAIDs), had getting particular attention because of its broad spectrum antitumor activity. Recent clinical trials showed that low doses of Aspirin every day effectively reduced the incidence of breast cancer. Based on our preliminary findings, we found that not only Aspirin alone could inhibit cell proliferation and induced apoptosis, but it had synergistic effect when in combination with Lapatinib on killing HER2-positive breast cancer cells BT474 and AU565 as well. Therefore, in this project, we attempt to identify the synergistic effect of Aspirin in combination with Lapatinib on treatment of HER2-positive breast cancer both in vivo and in vitro. We also plan to study the possible synergistic mechanism especially reveal the interactive function between COX-2 and HER2, then evaluate the biological function about the combination medication therapy. The development of this project will provide the most direct evidence for clinical treatment that Aspirin in combination with Lapatinib can be more effective on HER2-positive breast cancer. It also will provide new ideas and useful theoretical basis for breast cancer molecular targeted therapy.

HER2阳性乳腺癌约占乳腺癌总数的25%左右，预后非常差。拉帕替尼作为新的HER2靶向治疗药物，对复发性晚期或转移性乳腺癌具有显著疗效，但耐药性严重制约了它的临床反应性。因此寻找能协同拉帕替尼治疗的药物，降低其耐药发生率至关重要。近年来，非甾抗炎药阿司匹林的抗肿瘤作用备受重视，临床研究表明它对乳腺癌具有防治作用。我们的前期研究发现，阿司匹林不仅自身可以抑制HER2阳性乳腺癌细胞增殖，而且可以协同拉帕替尼提高其杀伤HER2阳性乳腺癌细胞的能力。本项目拟进一步利用体内外实验，明确阿司匹林协同拉帕替尼杀伤HER2阳性乳腺癌细胞的效果，解析阿司匹林的经典下游信号通路分子COX-2等与HER2下游信号分子之间的交互调控作用机制，并揭示联合用药的生物学功能。本课题的开展将为阿司匹林协同拉帕替尼在HER2阳性乳腺癌的治疗中发挥作用提供最直接的证据，为乳腺癌分子靶向治疗提供新的治疗思路和有益的理论依据。

拉帕替尼作为新的HER2靶向治疗药物，对复发性晚期或转移性乳腺癌具有显著疗效，但耐药性严重制约了它的临床反应性。因此寻找能协同拉帕替尼治疗的药物，降低其耐药发生率至关重要。基于前期研究发现，阿司匹林不仅自身可以抑制HER2阳性乳腺癌细胞增殖，而且可以协同拉帕替尼提高其杀伤HER2阳性乳腺癌细胞的能力。本项目首先利用浓度不断增加的Lapatinib处理HER2高表达的乳腺癌细胞系BT-474和AU-565，维持筛选12个月，最终获得具有Lapatinib耐药性的细胞；并利用MTT、平板克隆和软琼脂克隆形成能力等方法证实所筛选的耐药细胞具有稳定的耐药性。逆转录定量 PCR（qRT-PCR）分析表明HER2阳性乳腺癌患者体内HER2与COX-2均高表达。MTT实验表明阿司匹林可以抑制HER2阳性乳腺癌细胞增殖，其作用与mTOR抑制剂雷帕霉素相似，相反选择性COX-2抑制剂塞来昔布不能抑制其增殖。为解析阿司匹林抑制HER2阳性乳腺癌增殖的机制，本课题成功构建COX-2低表达的稳转细胞系，经MTT实验表明COX-2沉默后不影响细胞的增殖能力。因此，阿司匹林抑制BT474增殖并不依赖于COX-2。Western-blotting分析表明阿司匹林剂量依赖型抑制HER2阳性乳腺癌是通过激活AMPK，同时抑制mTORC1作用。此过程中AKT也被激活，但是塞来昔布没有这样的作用。而AMPK激动剂（ALCAR）表现出相似的性质，可以降低70S6k的活性，同时上调AKT，其作用与雷帕霉素相似。MTT试验进一步证实阿司匹林联合雷帕霉素或者ALCAR均表现出抑制细胞增殖作用增强。Western-blotting分析表明阿司匹林联合拉帕替尼可以激活自噬通路，增加LC3-Ⅱ表达量，这种协同作用可以被自噬抑制剂3-MA削弱。本课题亦通过MTT、EdU、流式细胞术等技术证实阿司匹林对拉帕替尼耐药株的逆转耐药作用。另一方面，MTT、EDU、平板克隆和流式细胞术等方法证实细胞自噬可以促进HER2阳性细胞株获得Lapatinib耐药性，抑制细胞自噬可以显著增强耐药细胞对Lapatinib的治疗敏感性。Western-blotting分析提示AMPK在耐药细胞中处于活化状态，Compound C阻断AMPK下游信号通路后，LC3-Ⅰ向LC3-Ⅱ的转化受到明显的阻断，细胞水平的Lapatinib治疗敏感性显著增