PO.ET02.09 · 实验与分子治疗
组合筛选揭示乳腺癌细胞中新的基因-药物协同作用
Combinatorial screening reveals novel gene-drug synergies in breast cancer cells
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：乳腺癌是美国女性中最常见的恶性肿瘤。它是一种分子异质性疾病，治疗反应各异。我们此前的全转录组关联研究（TWAS）和精细定位研究鉴定出可能参与乳腺癌易感性的候选基因。为推进个体化的化学预防和治疗策略，我们针对选定的候选易感基因开展了一项基因-药物协同研究。
方法：基于我们此前的多组学分析、TWAS 和精细定位，我们进行了高通量 siRNA 筛选以评估细胞增殖，并优先选出 25 个候选基因用于组合筛选。使用 ON-TARGETplus SMARTpool siRNA（Dharmacon）进行反向转染，在三种乳腺癌细胞系（MCF-7、T47D 和 MDA-MB-231）中沉默这些基因。敲低后，细胞用 18 种临床相关的癌症治疗药物（包括 PARP 抑制剂、CDK4/6 抑制剂、内分泌治疗药物和化疗药物）以临床相关浓度处理 72 h。通过 Hoechst 33342 和碘化丙啶染色结合自动化图像分析评估细胞活力和细胞毒性。与经 siRNA 沉默的 DMSO 对照相比，细胞死亡增加 >2 倍的基因-药物组合被视为具有协同作用。采用 Student's t 检验确定统计学显著性。
结果：这种组合筛选方法揭示了多种基因-药物协同作用。在 MCF-7 细胞中，沉默 R3HDM2 显著增加了对 CDK4/6 抑制剂的敏感性，abemaciclib 的倍数变化 >8，palbociclib 的倍数变化 >4，提示其在细胞周期调控中的关键作用。SUGP1 敲低增强了对 docetaxel（3.81 倍）和 everolimus（3.37 倍）的反应性。在 T47D 细胞中，敲低 SLC25A12、DNAJC27 和 DYNC1I2 基因使细胞对 everolimus、docetaxel 和 lapatinib 敏感（倍数变化分别 >14、>6 和 >5）。在 MDA-MB-231 细胞中，敲低 CEP192 和 SLC25A12 基因显著增强了对 anastrozole 和 docetaxel 的敏感性（倍数变化分别 >7 和 >4）。
结论：敲低若干新鉴定的推定乳腺癌易感基因深刻改变了乳腺癌细胞的药物敏感性，揭示了机制性见解和乳腺癌的潜在药物靶点。R3HDM2、SUGP1、CEP192 和 SLC25A12 等基因与 CDK4/6 抑制剂、紫杉烷类和 mTOR 靶向药物表现出强协同作用，提示其在细胞周期调控、微管动力学和代谢信号传导中的作用。这些发现支持利用遗传信息指导乳腺癌治疗和个体化诊疗。
查看英文原文 English abstract
Background: Breast cancer is the most common malignancy among women in the United States. It is a molecularly heterogeneous disease with diverse therapeutic responses. Our previous transcriptome-wide association (TWAS) and fine-mapping studies identified candidate genes potentially involved in breast cancer susceptibility. To advance personalized chemo-preventive and therapeutic strategies, we conducted a gene-drug synergy study on selected candidate susceptibility genes.
Methods: Based on our previous multi-omics analyses, TWAS, and fine-mapping, we conducted a high-throughput siRNA screen to assess cell proliferation and prioritized 25 candidate genes for combinatorial screening. Using reverse transfection with ON-TARGETplus SMARTpool siRNAs (Dharmacon), these genes were silenced in three breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). Following knockdown, cells were treated for 72 h with 18 clinically relevant agents for cancer therapy, including PARP inhibitors, CDK4/6 inhibitors, endocrine therapies, and chemotherapeutics, at clinically relevant concentrations. Cell viability and cytotoxicity were assessed by Hoechst 33342 and propidium iodide staining with automated image analysis. Gene-drug combinations showing >2-fold increase in cell death versus siRNA-silenced DMSO controls were considered synergistic. Statistical significance was determined using Student's t-test.
Results: This combinatorial screening approach revealed multiple gene-drug synergies. In MCF-7 cells, silencing R3HDM2 markedly increased sensitivity to CDK4/6 inhibitors, with fold-changes >8 for abemaciclib and >4 for palbociclib, implicating a critical role in cell cycle regulation. SUGP1 knockdown enhanced responsiveness to docetaxel (3.81-fold) and everolimus (3.37-fold). In T47D cells, knockdown of SLC25A12 , DNAJC27 , and DYNC1I2 genes sensitized cells to everolimus, docetaxel, and lapatinib (fold-change >14, >6, and >5). In MDA-MB-231 cells, knockdown of CEP192 and SLC25A12 genes significantly enhanced sensitivity to anastrozole and docetaxel (fold-change >7 and >4).
Conclusions: Knockdown of several newly identified putative breast cancer susceptibility genes profoundly altered drug sensitivity in breast cancer cells, revealing mechanistic insights and potential drug targets for breast cancer. Genes such as R3HDM2, SUGP1, CEP192 , and SLC25A12 exhibited strong synergy with CDK4/6 inhibitors, taxanes, and mTOR-targeted agents, implicating roles in cell cycle regulation, microtubule dynamics, and metabolic signaling. These findings support using genetic information to guide treatment and personalized approaches in breast cancer care.
利益披露 Disclosure
J. Wang, None..
J. Wu, None..
L. Xu, None..
R. Courtney, None..
J. Long, None..
W. Zheng, None..
Q. Cai, None.