分子影像监测乳腺癌吉西他滨耐药的多重逆转

Breast cancer is the highest incidence of malignant tumors in women. Gemcitabine (GEM) is the first-line treatment of metastatic and recurrent breast cancer. Drug resistance is the biggest bottleneck in the treatment of breast cancer. It has been shown that human equilibrative nucleoside transporter 1 (hENT1) can promote GEM influx, while hexokinase 2 (HK2) can activate multidrug resistance protein (MRP) and hypoxia inducible factor-1α (HIF-1α) pathway, resulting in drug-resistance of tumor cells. Our previous study found that miR-143 targets HK2 in breast cancer cells. We aim to explore the best effect of combination multi-joint of reversal MDA-MB-231/GEM resistant breast cancer. Meanwhile, we use 99mTc-MIBI, 18F-FDG, 18F-FLT and 18F-FMISO micro SPECT/CT and PET/CT imaging to monitor efficacy of multi-step reversal drug resistance in vivo, including using miR-143 that targeting down regulation of HK2 in the Warburg effect of tumor cells, increasing the inflow of GEM and then reducing ATP production, using tumor-specific promoter TERT driving lentivirus that carrying hENT1 to increase the inflow of GEM. And it can also prevent HIF-1α activation to enhance the DNA damage. The results of the project will provide a new strategy for the reversal and monitoring of GEM resistance in breast cancer.

乳腺癌是女性发病率最高的恶性肿瘤。吉西他滨(GEM)是复发转移乳腺癌的一线治疗用药，但其耐药是其治疗瓶颈。已有证据表明，人平衡型核苷转运蛋白1（hENT1）可促进GEM内流，而己糖激酶2（HK2）可激活多药耐药蛋白(MRP)及乏氧诱导因子-1α（HIF-1α）通路，引起肿瘤细胞耐药。我们的前期研究发现miR-143靶向调控乳腺癌细胞的HK2。本申请项目拟利用99mTc-MIBI、18F-FDG、18F-FLT和18F-FMISO micro SPECT/CT和PET/CT显像等分子影像技术，监测GEM耐药性乳腺癌的多重逆转治疗，包括：miR-143靶向抑制HK2，抑制MRP表达及活性，减少GEM流出，并联合以肿瘤特异性启动子TERT驱动hENT1表达，增加GEM内流；协同抑制HIF-1α活性，增强GEM对DNA合成的影响并激活凋亡通路。项目研究结果将为乳腺癌GEM耐药逆转及监测提供新策略。

三阴性乳腺癌（Triple negative breast cancer, TNBC）是乳腺癌中最具侵袭性和致命性的亚型，比其他乳腺癌亚型更容易产生其化疗药物吉西他滨（Gemcitabine, GEM）耐药性。人类平衡核苷转运蛋白1（hENT1）低表达与GEM耐药性密切相关。此外，化学耐药性伴随着高的糖酵解率，糖酵解促进化学耐药。MiR-143模拟物通过靶向糖酵解中的己糖激酶2（HK2）来抑制TNBC发展。在这项研究中，我们评估了hENT1上调和miR-143联合给药对TNBC的GEM耐药逆转。本研究分为体外验证和体内验证两部分。在体外建立了吉西他滨耐药的MDA-MB-231细胞系（GEM-R）和过表达hENT1的GEM-R细胞系（GEM-R-hENT1）。采用CCK8和流式细胞术分析了GEM处理及GEM和miR-143共同处理下不同细胞的IC50和凋亡百分比。分别通过RT-PCR和蛋白质印迹法测定不同细胞系中hENT1和HK2在mRNA和蛋白水平的表达。通过质谱多反应监测分析确定不同细胞对GEM摄取率。通过葡萄糖测定和18F-FDG摄取实验来评估细胞糖酵解水平。在裸鼠体内建立了肿瘤异种移植模型，通过计算肿瘤体积生长率和18F-FDG micro PET/CT显像测量最大标准化摄取值（SUV max）评估不同治疗方案的抗肿瘤作用。研究结果表明，hENT1的过表达降低了GEM-R细胞对GEM的IC50，提高了细胞凋亡率，一定程度上逆转了GEM-R细胞对GEM的耐药性。MiR-143抑制了GEM-R细胞中的糖酵解，并增强了GEM-R-hENT1细胞中逆转GEM耐药性的作用。异种移植小鼠模型的治疗效果表明，联合治疗更有效地降低了肿瘤生长速率和肿瘤的SUVmax。外源性上调hENT1表达和注射miR-143模拟物的联合治疗可有效逆转TNBC的GEM耐药性，这为治疗GEM耐药性TNBC提供了一种有效策略。上述耐药治疗及监测研究也在胰腺癌中得到了证实。