【摘要】：曲妥珠單抗是一個(gè)抗人表皮生長(zhǎng)因子受體(Human Epidermal Growth Factor receptor;HER2)的單克隆抗體藥,已被歐盟及美國(guó)食品藥品管理局(FDA)先后批準(zhǔn)用于治療HER2陽性轉(zhuǎn)移性乳腺癌和胃癌患者。然而,部分腫瘤患者在接受治療時(shí)會(huì)出現(xiàn)原發(fā)性或者獲得性耐藥,從而使得曲妥珠單抗療效甚微。因此,探索曲妥珠單抗耐藥的具體機(jī)制以期尋求更好的用藥策略是臨床上亟待解決的重要問題。本次研究揭示了以信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子3(Signal transducer and activator of transcription 3;STAT3)為中心的正反饋環(huán)介導(dǎo)曲妥株單抗耐藥的具體機(jī)制。我們課題組已經(jīng)在前期成功建立了乳腺癌及胃癌曲妥珠單抗耐藥細(xì)胞株,兩種耐藥細(xì)胞株在體外和體內(nèi)實(shí)驗(yàn)中,均對(duì)曲妥珠單抗不敏感。進(jìn)一步研究發(fā)現(xiàn),STAT3在構(gòu)建的耐藥細(xì)胞株和PTEN缺失所致的原發(fā)性耐藥模型中均呈現(xiàn)出高度活化的狀態(tài)。用小干擾RNA(small interfering RNA)抑制STAT3基因表達(dá)后,耐藥的腫瘤細(xì)胞重新恢復(fù)了對(duì)曲妥珠單抗的敏感性。我們同時(shí)發(fā)現(xiàn)長(zhǎng)期暴露于曲妥珠單抗環(huán)境中可以使乳腺癌和胃癌敏感細(xì)胞株中的纖連蛋白(Fbronectin;FN)、表皮生長(zhǎng)因子(Epidermal Growth Factor;EGF)和白介素6(Interleukin-6;IL-6)等分子的表達(dá)顯著增加,這些分子作為STAT3的上游激活分子,進(jìn)一步引起STAT3信號(hào)通路的超活化。此外,活化的STAT3信號(hào)也能反饋性地上調(diào)FN、EGF和IL6的轉(zhuǎn)錄及表達(dá),從而形成一個(gè)正反饋環(huán),增強(qiáng)和維持STAT3的磷酸化水平。我們發(fā)現(xiàn)相比于親本細(xì)胞,在耐藥細(xì)胞中黏蛋白1(mucin 1;MUC1)和黏蛋白4(mucin 4;MUC4表達(dá)上調(diào),且其表達(dá)受STAT3的調(diào)節(jié);進(jìn)一步發(fā)現(xiàn)由FN、EGF和IL-6誘導(dǎo)的STAT3活化可上調(diào)其下游的靶基因MUC1和MUC4的轉(zhuǎn)錄和表達(dá)。MUC1和MUC4分別通過持續(xù)活化HER2信號(hào)和阻礙單抗與HER2結(jié)合,維持HER2下游信號(hào)的持續(xù)活化,從而導(dǎo)致對(duì)曲妥珠單抗耐藥。在體外,通過基因水平或藥物靶向抑制STAT3,可以打破以STAT3為中心的正反饋環(huán),下調(diào)了MUC1和MUC4的表達(dá),解除了對(duì)HER2活性的維持作用和與抗體結(jié)合的空間位阻,恢復(fù)了HER2單抗藥物的結(jié)合,重新發(fā)揮有效的抗腫瘤效應(yīng)。在乳腺癌和胃癌的耐藥裸鼠移植瘤模型中,曲妥珠單抗和STAT3小分子抑制劑S3I-201的聯(lián)合療法也顯著抑制了耐藥細(xì)胞移植瘤的生長(zhǎng),顯示出協(xié)同的抗腫瘤作用。綜上所述,我們的研究揭示了STAT3信號(hào)的正反饋活化是介導(dǎo)曲妥珠單抗耐藥的關(guān)鍵機(jī)制。在HER2陽性的乳腺癌和胃癌中,聯(lián)合應(yīng)用靶向HER2和STAT3的藥物可以增強(qiáng)曲妥珠單抗或其他HER2靶向藥的臨床療效。
[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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號(hào)：2275612