METHODS: Autophagy levels were compared between CSC-enriched side population (SP) and non-SP cells (NSP) in multiple ovarian cancer cell lines using immunoblotting, immunofluorescence, and transmission electron microscopy. The impact of autophagy modulation on CSC markers and differentiation was assessed by flow cytometry, immunoblotting and qRT-PCR. In silico modeling and co-immunoprecipitation identified ID1 interacting proteins. Pharmacological and genetic approaches along with Annexin-PI assay, ChIP assay, western blotting, qRT-PCR and ICP-MS were used to evaluate effects on cisplatin sensitivity, apoptosis, SLC31A1 expression, promoter binding, and intracellular platinum accumulation in ID1 depleted backdrop. Patient-derived tumor spheroids were analyzed for autophagy and SLC31A1 levels.
RESULTS: Ovarian CSCs exhibited increased basal autophagy compared to non-CSCs. Further autophagy stimulation by serum-starvation and chemical modes triggered proteolysis of the stemness regulator ID1, driving the differentiation of chemo-resistant CSCs into chemo-sensitive non-CSCs. In silico modeling predicted TCF12 as a potent ID1 interactor, which was validated by co-immunoprecipitation. ID1 depletion freed TCF12 to transactivate the cisplatin influx transporter SLC31A1, increasing intracellular cisplatin levels and cytotoxicity. Patient-derived tumor spheroids exhibited a functional association between autophagy, ID1, SLC31A1, and platinum sensitivity.
CONCLUSIONS: This study reveals a novel autophagy-ID1-TCF12-SLC31A1 axis where targeted autophagic degradation of ID1 enables rapid remodeling of CSCs to reverse chemo-resistance. Modulating this pathway could counter drug resistance in ovarian cancer.
方法:使用免疫印迹法比较了多种卵巢癌细胞系中CSC富集侧群(SP)和非SP细胞(NSP)之间的自噬水平,免疫荧光,和透射电子显微镜。通过流式细胞术评估自噬调节对CSC标记和分化的影响,免疫印迹和qRT-PCR。计算机建模和免疫共沉淀鉴定了ID1相互作用蛋白。药理学和遗传学方法以及膜联蛋白-PI测定,ChIP分析,西方印迹,qRT-PCR和ICP-MS用于评估对顺铂敏感性的影响,凋亡,SLC31A1表达式,启动子结合,和细胞内铂积累在ID1耗尽的背景下。分析患者来源的肿瘤球体的自噬和SLC31A1水平。
结果:与非CSC相比,卵巢CSC表现出增加的基础自噬。通过血清饥饿和化学模式的进一步自噬刺激触发了干性调节剂ID1的蛋白水解,从而驱动化学抗性CSC分化为化学敏感性非CSC。在计算机建模中,TCF12被预测为有效的ID1交互因子,通过免疫共沉淀进行验证。ID1消耗释放TCF12以反式激活顺铂流入转运蛋白SLC31A1,从而增加细胞内顺铂水平和细胞毒性。患者来源的肿瘤球体表现出自噬之间的功能关联,ID1、SLC31A1和铂的敏感度。
结论:这项研究揭示了一种新的自噬-ID1-TCF12-SLC31A1轴,其中ID1的靶向自噬降解能够快速重塑CSCs以逆转化疗抗性。调节该途径可以对抗卵巢癌的耐药性。