PARP抑制剂生物标志物和耐药特性与机制研究

发布时间：2018-06-05 12:27

本文选题：PARP抑制剂 + HR　；参考：《南昌大学》2017年硕士论文

【摘要】：BRCA1/2是目前临床应用最为广泛的聚腺苷二磷酸核糖聚合酶(poly(ADP-ribose)polymerase,PARP)抑制剂生物标志物。然而对BRCA1/2突变的肿瘤患者,PARP抑制剂的的临床有效率仅为30%~50%,意味着过半携带BRCA缺陷的患者仍面临着该类药物的无效治疗。探索发现新的疗效预测标志物或标志物组合,可用以增加PARP抑制剂临床治疗的成功率。本研究发现53BP1联合BRCA1可作为预测PARP抑制剂敏感性的生物标志物。在比较四种同源重组(homologous recombination,HR)相关基因的mRNA水平和PARP抑制剂敏感性的基础上,我们选取BRCA1缺陷的乳腺癌细胞MDA-MB-436作为工具细胞株,进行RNA干扰实验。发现只有降低53BP1表达,才影响细胞对PARP抑制剂的敏感性。接着我们构建了53BP1-/-/BRCA1-/-双缺陷细胞株,发现其Simmiparib的IC50是亲本细胞的36.7倍,表明敲除53BP1会导致PARP抑制剂耐药。53BP1缺失也减少了PARP抑制剂诱导的细胞周期阻滞和凋亡,并部分恢复了HR修复功能。更重要的是,敲除53BP1减弱了PARP抑制剂对裸鼠移植瘤的生长抑制作用。在双缺陷的细胞中再表达53BP1,可以恢复PARP抑制剂的敏感性,并恢复HR相关因子至正常水平。由于超过10%的BRCA1突变的乳腺癌和卵巢癌病人不表达53BP1,因此上述发现提示将53BP1和BRCA1作为生物标志物组合用于患者选择中,有可能减少PARP抑制剂的无效治疗。BRCA1/2缺陷的细胞因HR修复功能异常,对PARP抑制剂敏感。虽然PARP抑制剂对BRCA1/2突变的肿瘤可能有效,但最终仍会面临耐药的产生,从而限制其应用。由于PARP抑制剂临床应用时间短,对其耐药特性和机制了解还十分有限。为了深入研究PARP抑制剂的耐药特点和机制,我们以MDA-MB-436细胞为模型,采用梯度递增法构建了四种PARP抑制剂耐药单克隆细胞株。这些耐药株对同类PARP抑制剂明显耐药,也对DNA交联剂和拓扑异构酶Ι抑制剂交叉耐药。另外,PARP抑制剂几乎不影响耐药株的周期分布和细胞凋亡。我们采用特异性抗体研究发现耐药株表达截短型的N-端BRCA1蛋白,提示PARP抑制剂的耐药机制与BRCA1发生继发性突变导致HR修复功能部分恢复相关。
[Abstract]:BRCA1/2 is the most widely used biomarker of polyadenosine diphosphate polymerase (BRCA1/2) inhibitor. However, the clinical efficacy of BRCA1/2 inhibitor in patients with BRCA1/2 mutation is only 30%, which means that more than half of the patients with BRCA deficiency still face the ineffective treatment of the drug. To explore new prognostic markers or combination of markers, which can be used to increase the success rate of clinical treatment of PARP inhibitors. In this study, we found that 53BP1 combined with BRCA1 can be used as a biomarker for predicting the sensitivity of PARP inhibitors. On the basis of comparing the mRNA level and the sensitivity of PARP inhibitor of four homologous recombination genes, we selected BRCA1 deficient breast cancer cell line MDA-MB-436 as the tool cell line and carried out RNA interference test. It was found that the sensitivity of cells to PARP inhibitors was affected only when the expression of 53BP1 was reduced. Then we constructed 53BP1-r BRCA1-r-double defective cell line and found that the IC50 of Simmiparib was 36.7 times that of parent cells, suggesting that knockout of 53BP1 led to the deletion of PARP inhibitor resistance. 53BP1 also reduced cell cycle arrest and apoptosis induced by PARP inhibitor. HR repair function was partially restored. More importantly, knockout of 53BP1 attenuates the growth inhibition of PARP inhibitors on xenografts in nude mice. The expression of 53BP1 in double deficient cells could restore the sensitivity of PARP inhibitor and restore HR related factors to normal level. Since more than 10% of breast and ovarian cancer patients with BRCA1 mutations do not express 53BP1, the findings suggest that 53BP1 and BRCA1 are combined as biomarkers for patient selection. Cells that may reduce ineffective treatment of PARP inhibitors. BRCA 1 / 2 defect are sensitive to PARP inhibitors because of abnormal HR repair function. Although PARP inhibitors may be effective against BRCA1/2 mutated tumors, they may eventually face the production of drug resistance, limiting their use. Due to the short clinical application time of PARP inhibitors, their resistance characteristics and mechanisms are still very limited. In order to study the characteristics and mechanism of drug resistance of PARP inhibitors, four PARP inhibitor resistant monoclonal cell lines were constructed using MDA-MB-436 cell model and gradient increasing method. These resistant strains were significantly resistant to similar PARP inhibitors and cross-resistant to DNA crosslinking agents and topoisomerase I inhibitors. In addition, PARP inhibitors have little effect on cell cycle distribution and apoptosis. The expression of truncated N- terminal BRCA1 protein was detected by specific antibody analysis, suggesting that the mechanism of drug resistance of PARP inhibitor is related to the partial recovery of HR repair function due to secondary mutation of BRCA1.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R979.1

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