反馈激活的STAT3通过上调MUC1和MUC4的表达介导曲妥株单抗耐药
发布时间：2018-10-16 20:58
【摘要】：曲妥珠单抗是一个抗人表皮生长因子受体(Human Epidermal Growth Factor receptor;HER2)的单克隆抗体药,已被欧盟及美国食品药品管理局(FDA)先后批准用于治疗HER2阳性转移性乳腺癌和胃癌患者。然而,部分肿瘤患者在接受治疗时会出现原发性或者获得性耐药,从而使得曲妥珠单抗疗效甚微。因此,探索曲妥珠单抗耐药的具体机制以期寻求更好的用药策略是临床上亟待解决的重要问题。本次研究揭示了以信号转导与转录激活因子3(Signal transducer and activator of transcription 3;STAT3)为中心的正反馈环介导曲妥株单抗耐药的具体机制。我们课题组已经在前期成功建立了乳腺癌及胃癌曲妥珠单抗耐药细胞株,两种耐药细胞株在体外和体内实验中,均对曲妥珠单抗不敏感。进一步研究发现,STAT3在构建的耐药细胞株和PTEN缺失所致的原发性耐药模型中均呈现出高度活化的状态。用小干扰RNA(small interfering RNA)抑制STAT3基因表达后,耐药的肿瘤细胞重新恢复了对曲妥珠单抗的敏感性。我们同时发现长期暴露于曲妥珠单抗环境中可以使乳腺癌和胃癌敏感细胞株中的纤连蛋白(Fbronectin;FN)、表皮生长因子(Epidermal Growth Factor;EGF)和白介素6(Interleukin-6;IL-6)等分子的表达显著增加,这些分子作为STAT3的上游激活分子,进一步引起STAT3信号通路的超活化。此外,活化的STAT3信号也能反馈性地上调FN、EGF和IL6的转录及表达,从而形成一个正反馈环,增强和维持STAT3的磷酸化水平。我们发现相比于亲本细胞,在耐药细胞中黏蛋白1(mucin 1;MUC1)和黏蛋白4(mucin 4;MUC4表达上调,且其表达受STAT3的调节;进一步发现由FN、EGF和IL-6诱导的STAT3活化可上调其下游的靶基因MUC1和MUC4的转录和表达。MUC1和MUC4分别通过持续活化HER2信号和阻碍单抗与HER2结合,维持HER2下游信号的持续活化,从而导致对曲妥珠单抗耐药。在体外,通过基因水平或药物靶向抑制STAT3,可以打破以STAT3为中心的正反馈环,下调了MUC1和MUC4的表达,解除了对HER2活性的维持作用和与抗体结合的空间位阻,恢复了HER2单抗药物的结合,重新发挥有效的抗肿瘤效应。在乳腺癌和胃癌的耐药裸鼠移植瘤模型中,曲妥珠单抗和STAT3小分子抑制剂S3I-201的联合疗法也显著抑制了耐药细胞移植瘤的生长,显示出协同的抗肿瘤作用。综上所述,我们的研究揭示了STAT3信号的正反馈活化是介导曲妥珠单抗耐药的关键机制。在HER2阳性的乳腺癌和胃癌中,联合应用靶向HER2和STAT3的药物可以增强曲妥珠单抗或其他HER2靶向药的临床疗效。
[Abstract]:Trotozumab is a monoclonal antibody against human epidermal growth factor receptor (Human Epidermal Growth Factor receptor;HER2). It has been approved by the European Union and the United States Food and Drug Administration (FDA) for the treatment of HER2 positive metastatic breast cancer and gastric cancer. However, some cancer patients may develop primary or acquired drug resistance during treatment, which makes trotozumab ineffective. Therefore, it is an important problem to explore the mechanism of drug resistance in order to find a better drug use strategy. This study revealed the specific mechanism of positive feedback loop mediated drug resistance of tricot strain with signal transduction and transcription activator 3 (Signal transducer and activator of transcription 3 / STAT3 as the center. Our team has successfully established the tumor cell lines of breast cancer and gastric cancer, both of which are insensitive to tratuzumab in vitro and in vivo. Furthermore, it was found that STAT3 was highly activated in the drug-resistant cell line and the model of primary drug resistance induced by PTEN deletion. After inhibition of STAT3 gene expression by small interfering RNA (small interfering RNA), the drug resistant tumor cells recovered their sensitivity to trotozumab. We also found that prolonged exposure to trotozumab significantly increased the expression of fibronectin (Fbronectin;FN), epidermal growth factor (Epidermal Growth Factor;EGF (EGF) and interleukin 6 (Interleukin-6;IL-6) in breast and gastric cancer sensitive cell lines. These molecules act as upstream activators of STAT3, which further induce superactivation of STAT3 signaling pathway. In addition, activated STAT3 signals can up-regulate the transcription and expression of FN,EGF and IL6, thus forming a positive feedback loop to enhance and maintain the phosphorylation level of STAT3. We found that the expression of mucin 1 (mucin 1) and mucin 4 (mucin 4) were up-regulated in drug-resistant cells compared with parental cells, and their expression was regulated by STAT3. It was further found that the activation of STAT3 induced by FN,EGF and IL-6 could up-regulate the transcription and expression of the downstream target genes MUC1 and MUC4. MUC1 and MUC4 maintained the continuous activation of HER2 downstream signals by continuously activating HER2 signals and blocking the binding of McAbs to HER2, respectively. This led to drug resistance to tratozuz McAb. In vitro, inhibition of STAT3, by gene level or drug targeting can break the positive feedback loop centered on STAT3, down-regulate the expression of MUC1 and MUC4, and relieve the maintenance of HER2 activity and the steric blocking of antibody binding. The combination of HER2 monoclonal antibody was restored and the effective anti-tumor effect was replayed. In nude mice model of breast cancer and gastric cancer, the combination therapy of trotozumab and STAT3 small molecule inhibitor S3I-201 also significantly inhibited the growth of drug-resistant tumor, and showed synergistic antitumor effect. In conclusion, our study revealed that the positive feedback activation of STAT3 signal is the key mechanism to mediate the drug resistance of tratuzumab. In HER2 positive breast cancer and gastric cancer, combined use of targeted HER2 and STAT3 drugs can enhance the clinical efficacy of tratozumab or other HER2 targeting drugs.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96
[Abstract]:Trotozumab is a monoclonal antibody against human epidermal growth factor receptor (Human Epidermal Growth Factor receptor;HER2). It has been approved by the European Union and the United States Food and Drug Administration (FDA) for the treatment of HER2 positive metastatic breast cancer and gastric cancer. However, some cancer patients may develop primary or acquired drug resistance during treatment, which makes trotozumab ineffective. Therefore, it is an important problem to explore the mechanism of drug resistance in order to find a better drug use strategy. This study revealed the specific mechanism of positive feedback loop mediated drug resistance of tricot strain with signal transduction and transcription activator 3 (Signal transducer and activator of transcription 3 / STAT3 as the center. Our team has successfully established the tumor cell lines of breast cancer and gastric cancer, both of which are insensitive to tratuzumab in vitro and in vivo. Furthermore, it was found that STAT3 was highly activated in the drug-resistant cell line and the model of primary drug resistance induced by PTEN deletion. After inhibition of STAT3 gene expression by small interfering RNA (small interfering RNA), the drug resistant tumor cells recovered their sensitivity to trotozumab. We also found that prolonged exposure to trotozumab significantly increased the expression of fibronectin (Fbronectin;FN), epidermal growth factor (Epidermal Growth Factor;EGF (EGF) and interleukin 6 (Interleukin-6;IL-6) in breast and gastric cancer sensitive cell lines. These molecules act as upstream activators of STAT3, which further induce superactivation of STAT3 signaling pathway. In addition, activated STAT3 signals can up-regulate the transcription and expression of FN,EGF and IL6, thus forming a positive feedback loop to enhance and maintain the phosphorylation level of STAT3. We found that the expression of mucin 1 (mucin 1) and mucin 4 (mucin 4) were up-regulated in drug-resistant cells compared with parental cells, and their expression was regulated by STAT3. It was further found that the activation of STAT3 induced by FN,EGF and IL-6 could up-regulate the transcription and expression of the downstream target genes MUC1 and MUC4. MUC1 and MUC4 maintained the continuous activation of HER2 downstream signals by continuously activating HER2 signals and blocking the binding of McAbs to HER2, respectively. This led to drug resistance to tratozuz McAb. In vitro, inhibition of STAT3, by gene level or drug targeting can break the positive feedback loop centered on STAT3, down-regulate the expression of MUC1 and MUC4, and relieve the maintenance of HER2 activity and the steric blocking of antibody binding. The combination of HER2 monoclonal antibody was restored and the effective anti-tumor effect was replayed. In nude mice model of breast cancer and gastric cancer, the combination therapy of trotozumab and STAT3 small molecule inhibitor S3I-201 also significantly inhibited the growth of drug-resistant tumor, and showed synergistic antitumor effect. In conclusion, our study revealed that the positive feedback activation of STAT3 signal is the key mechanism to mediate the drug resistance of tratuzumab. In HER2 positive breast cancer and gastric cancer, combined use of targeted HER2 and STAT3 drugs can enhance the clinical efficacy of tratozumab or other HER2 targeting drugs.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96
【共引文献】
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