靶向ErbB2抗体H2-18与小分子抑制剂联合应用在Trastuzumab耐药乳腺癌中的抗肿瘤作用及其机理

发布时间：2017-12-26 16:41

本文关键词：靶向ErbB2抗体H2-18与小分子抑制剂联合应用在Trastuzumab耐药乳腺癌中的抗肿瘤作用及其机理　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：ErbB2(human epidermal growth factor receptor 2,HER2)是人表皮生长因子受体家族成员。25-30%的乳腺癌细胞及4-50%的胃癌细胞存在着ErbB2过表达。ErbB2的过表达与肿瘤的侵袭及不良预后有着密切关联。曲妥珠单抗(Trastuzumab)是一个靶向ErbB2的人源化抗体,已获批用于ErbB2高表达转移性乳腺癌和胃癌的临床治疗。然而,临床研究表明大部分ErbB2高表达的乳腺癌患者对Trastuzumab治疗不产生反应,即使对Trastuzumab产生反应的患者有半数也会在1年内产生耐药。因此,发展新的乳腺癌靶向治疗策略已成为基础研究和临床治疗的迫切需求。在前期研究中,我们通过筛选噬菌体抗体库获得了一株全新的抗ErbB2全人源单克隆抗体H2-18,其能够强有力诱导Trastuzumab耐药乳腺癌细胞发生程序化死亡(Programmed cell death,PCD),而Trastuzumab则不能有效诱导乳腺癌细胞发生PCD。我们通过乳腺癌荷瘤小鼠模型对H2-18的体内抗肿瘤活性进行了评价,结果表明H2-18能有效抑制Trastuzumab耐药乳腺癌生长。我们的研究进一步提示H2-18的PCD诱导活性是其对Trastuzumab耐药乳腺癌产生抗肿瘤活性的重要原因。ErbB2过表达乳腺癌对Trastuzumab产生耐药的机制有多种,其中一个非常重要的原因是PI3K/AKT信号通路的异常激活。PTEN缺失以及PIK3CA基因突变都能够导致PI3K/AKT信号通路异常激活。但这种异常激活可被PI3K抑制剂所抑制。GDC-0941是一个pan-PI3K的小分子抑制剂,因其在肿瘤细胞中表现出理想的抗肿瘤活性而备受关注。Src是一个属于Src激酶家族的非受体酪氨酸激酶,是多条与Trastuzumab耐药产生相关的细胞信号通路共同的关键结点。Saracatinib是Src的抑制剂,能够在一定程度上使耐药乳腺癌细胞对Trastuzumab的作用重新变得敏感。为了进一步提高H2-18抗体在耐药乳腺癌中的治疗效果,在本研究中我们将探讨H2-18与GDC-0941或Saracatinib的联合抗肿瘤作用。首先,我们通过三维细胞生长实验检测了H2-18与GDC-0941或Saracatinib联用对ErbB2过表达的Trastuzumab敏感的乳腺癌细胞BT-474、SKBR-3和Trastuzumab耐药的乳腺癌细胞HCC-1954、HCC-1419的体外生长抑制活性。实验结果显示,联用H2-18与GDC-0941在以上乳腺癌细胞中均能发挥比单药更强的抗肿瘤作用。用CompuSynsoware软件拟合之后发现H2-18与GDC-0941具有协同抗肿瘤活性。联用H2-18与Saracatinib在这几株乳腺癌细胞中也展现出类似的比单药明显增强的抗肿瘤作用。为了探讨以上药物联用产生协同抗肿瘤活性的原因,我们采用了蛋白印迹法检测不同药物处理下BT-474和HCC-1954细胞内ErbB2下游信号通路的改变。我们发现,GDC-0941能够有效抑制Akt的磷酸化,对ErK磷酸化影响较弱,且不影响p-JNK和p-c-jun。H2-18能够明显抑制Erk的磷酸化,促进JNK/c-jun的磷酸化,对AKT磷酸化的抑制作用较弱。与H2-18单用相比,H2-18/GDC-0941联用在抑制Erk活性或激活JNK/c-jun的能力上没有明显差别,但抑制p-Akt的能力有所增强。接下来我们用pAkt(S473)的Elisa试剂盒检测细胞内pAkt的含量。结果显示,联用H2-18和GDC-0941组与单用GDC-0941组在抑制p-Akt的作用上并没有统计学意义的差异。相似的,联用H2-18与Saracatinib在ErbB2下游信号通路上也没能显示出比单药更强的作用。下一步,我们采用Annexin V/PI双染法进行细胞染色,并通过流式细胞术检测药物处理后乳腺癌细胞BT-474、SKBR-3、HCC-1954和HCC-1419的死亡情况。结果发现,与单药相比,H2-18与GDC-0941联用组诱导以上乳腺癌细胞发生PCD的能力显著增强。而联用H2-18与Saracatinib也显示了比单药更强的诱导细胞PCD的能力。我们还通过PI染色用流式细胞技术来检测药物处理后细胞周期分布的改变。结果表明,在BT-474细胞中,与单药相比,联用H2-18与GDC-0941时发生G1期阻滞的细胞比例更高。而在HCC-1954细胞中,联用H2-18与GDC-0941并未比单用GDC-0941显现出更强的G1期阻滞作用。H2-18与Saracatinib两药联用在Trastuzumab敏感的细胞株BT-474与SKBR-3细胞中较单药组引起更明显的G1期阻滞。而在Trastuzumab耐药的细胞HCC-1954和HCC-1419中,联用H2-18与Saracatinib也并未显示出比单用Saracatinib更强的诱导G1期阻滞的作用。此外,我们用流式细胞技术检测了药物处理后BT-474和HCC-1954细胞内的活性氧含量。结果显示,在这两株细胞中,单用H2-18与GDC-0941都能增高活性氧水平。而与单用组相比,H2-18/GDC-0941联用组不能进一步提高活性氧水平。类似的,H2-18/Saracatinib联用组细胞内活性氧水平并不比H2-18单用组高。我们利用HCC-1954乳腺癌荷瘤小鼠模型对以上药物进行了体内抗肿瘤作用研究,结果表明单用H2-18与单用GDC-0941都能有效抑制移植瘤的生长,而两者联用后比单药组具有显著增强的抑制肿瘤生长的作用。联用H2-18与Saracatinib的情况与H2-18/GCD-0941相似。因此,我们推断无论是联用H2-18与GDC-0941还是联用H2-18与Saracatinib,它们较单药组表现出更强抗肿瘤活性的原因可能主要由于药物联用可协同诱导肿瘤细胞发生PCD。除此之外,两药联用后能同时抑制ErbB2下游关键的PI3K/AKT与RAS/MAPK两条通路也发挥了一定的作用。综上所述,H2-18与GDC-0941或Saracatinib联合应用在Trastuzumab耐药的乳腺癌中均展现出显著增强的体内外抗肿瘤活性,提示这样的药物组合有望成为克服Trastuzumab耐药的新策略。
[Abstract]:ErbB2 (human epidermal growth factor receptor 2, HER2) is a member of the human epidermal growth factor receptor family. The ErbB2 overexpression exists in the breast cancer cells of 25-30% and the gastric cancer cells of 4-50%. The overexpression of ErbB2 is closely related to the invasion and poor prognosis of the tumor. Trastuzumab (Trastuzumab) is a humanized antibody targeting ErbB2, and has been approved for the clinical treatment of ErbB2 high expression of metastatic breast cancer and gastric cancer. However, clinical studies show that most ErbB2 patients with high expression of breast cancer do not respond to Trastuzumab treatment. Even half of patients who respond to Trastuzumab respond to drug resistance within 1 years. Therefore, the development of new targeted treatment strategies for breast cancer has become an urgent need for basic research and clinical treatment. In our previous study, we have obtained a new anti ErbB2 humanized monoclonal antibody H2-18 by screening phage antibody library, which can strongly induce PCD Trastuzumab resistant breast cancer cells (Programmed cell death, PCD), but not Trastuzumab can effectively induce the occurrence of breast cancer cell PCD. We evaluated the in vivo antitumor activity of H2-18 through a breast cancer bearing mice model. The results showed that H2-18 could effectively inhibit the growth of Trastuzumab resistant breast cancer. Our study further suggests that the PCD induced activity of H2-18 is an important reason for its antitumor activity in Trastuzumab resistant breast cancer. There are many mechanisms for ErbB2 over expression of Trastuzumab resistance to breast cancer, and one of the most important reasons is the abnormal activation of the PI3K/AKT signaling pathway. The deletion of PTEN and the mutation of PIK3CA gene can cause abnormal activation of the PI3K/AKT signaling pathway. However, this abnormal activation can be suppressed by PI3K inhibitors. GDC-0941 is a small molecule inhibitor of pan-PI3K, which has attracted much attention because of its ideal antitumor activity in tumor cells. Src is a non receptor tyrosine kinase belonging to the Src kinase family, which is a key node of a number of cell signaling pathways associated with Trastuzumab resistance. Saracatinib is an inhibitor of Src, which can, to a certain extent, make drug resistant breast cancer cells more sensitive to the role of Trastuzumab. In order to further improve the therapeutic effect of H2-18 antibody in drug-resistant breast cancer, we will explore the combined anti-tumor effect of H2-18 and GDC-0941 or Saracatinib in this study. First, we used three-dimensional cell growth assay to detect the inhibitory effect of H2-18 combined with GDC-0941 or Saracatinib on ErbB2 overexpressing Trastuzumab sensitive breast cancer cells BT-474, SKBR-3 and Trastuzumab resistant breast cancer cells HCC-1954 and HCC-1419 in vitro. The results showed that both H2-18 and GDC-0941 could play a stronger anti-tumor effect than single drug in the breast cancer cells. After fitting with CompuSynsoware software, it was found that H2-18 and GDC-0941 have synergistic antitumor activity. The combined use of H2-18 and Saracatinib in these breast cancer cells also showed a similar antitumor effect that was significantly enhanced by a single drug. In order to explore the cause of synergistic antitumor activity of the above drug combination, we used Western blot to detect the changes of downstream ErbB2 signaling pathway in BT-474 and HCC-1954 cells treated with different drugs. We found that GDC-0941 can effectively inhibit the phosphorylation of Akt, which has a weak effect on the phosphorylation of ErK, and does not affect p-JNK and p-c-jun. H2-18 can significantly inhibit the phosphorylation of Erk, promote the phosphorylation of JNK/c-jun, and the inhibition of AKT phosphorylation is weak. Compared with H2-18 alone, there was no significant difference in the ability of H2-18/GDC-0941 to inhibit Erk activity or to activate JNK/c-jun, but the ability to inhibit p-Akt was enhanced. Then we used the pAkt (S473) Elisa kit to detect the content of pAkt in the cells. The results showed that there was no significant difference in the inhibition of p-Akt between the combined H2-18 and GDC-0941 group and the single GDC-0941 group. Similarly, the use of H2-18 and Saracatinib in the downstream signal pathway of ErbB2 also did not show a stronger effect than the single drug. Next, we used Annexin V/PI double staining to detect cell death and detect the death of BT-474, SKBR-3, HCC-1954 and HCC-1419 after treatment with flow cytometry. The results showed that compared with the single drug, the ability to induce PCD in the breast cancer cells was significantly enhanced by the combination of H2-18 and GDC-0941. The combined use of H2-18 and Saracatinib also showed a stronger ability to induce cell PCD than single drug. We also used PI staining to detect the changes in cell cycle distribution after treatment with flow cytometry. The results showed that in BT-474 cells, compared with the single drug, the proportion of cells with G1 block at G1 was higher than that of GDC-0941. In HCC-1954 cells, the combination of H2-18 and GDC-0941 did not show a stronger G1 phase blocking effect than the single use of GDC-0941. The combined use of H2-18 and Saracatinib two drugs in Trastuzumab sensitive cell lines, BT-474 and SKBR-3 cells, resulted in a more pronounced G1 phase block than in the single drug group. In Trastuzumab resistant cells HCC-1954 and HCC-1419, H2-18 and Saracatinib also did not show a stronger effect than G1 alone on induction of G1 phase arrest. In addition, we used flow cytometry to detect the content of reactive oxygen species in BT-474 and HCC-1954 cells after treatment. The results showed that both H2-18 and GDC-0941 could increase the level of reactive oxygen species in both of the two cells. Compared with the single use group, the H2-18/GDC-0941 combined group could not further improve the activity of reactive oxygen species. Similarly, the level of intracellular reactive oxygen species in the H2-18/Saracatinib combined group was not higher than that of the H2-18 single use group. We used the HCC-1954 breast cancer bearing mice model to study the antitumor effect of the above drugs. The results showed that H2-18 alone and GDC-0941 alone can effectively inhibit the growth of transplanted tumor.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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