目的:随着曲妥珠单抗deruxtecan(T-DXd)的广泛应用,HER2低的乳腺癌患者的生存率显著提高.然而,对T-DXd的耐药性仍然存在于一部分患者中,分子机制尚不清楚。
方法:进行体内shRNA慢病毒文库功能筛选以鉴定介导T-DXd抗性的潜在环状RNA(crRNA)。RNA下拉,质谱,RNA免疫沉淀,并进行了免疫共沉淀测定以研究其分子机制。使用C11-BODIPY检测铁凋亡,Liperfluo,铁橙染色,谷胱甘肽定量,丙二醛定量,和透射电子显微镜。分子对接,虚拟筛选,和患者来源的异种移植(PDX)模型用于验证治疗药物.
结果:VDAC3衍生的crRNA(crVDAC3)在功能性shRNA文库筛选中排名第一。敲除crVDAC3增加了低HER2乳腺癌细胞对T-DXd治疗的敏感性。进一步的机制研究表明,crVDAC3特异性结合HSPB1蛋白并抑制其泛素化降解,导致细胞内积累和HSPB1蛋白水平升高。值得注意的是,抑制crVDAC3可显着增加过量的ROS水平和不稳定的铁池积累。抑制crVDAC3通过降低HSPB1表达诱导乳腺癌细胞铁凋亡,从而介导T-DXd抗性。通过虚拟筛选和实验验证,我们确定paritaprevir可以有效地结合crVDAC3并阻止其与HSPB1蛋白的相互作用,从而增加HSPB1蛋白的泛素化降解以克服T-DXd抗性。最后,在HER2低PDX模型中,我们验证了paritaprevir增强T-DXd的治疗效果.
结论:这一发现揭示了低HER2乳腺癌T-DXd耐药的分子机制。我们的研究提供了一种通过抑制crVDAC3与HSPB1蛋白之间的相互作用来克服T-DXd抗性的新策略。
OBJECTIVE: With the wide application of trastuzumab deruxtecan (T-DXd), the survival of HER2-low breast cancer patients is dramatically improved. However, resistance to T-DXd still exists in a subset of patients, and the molecular mechanism remains unclear.
METHODS: An in vivo shRNA lentiviral library functional screening was performed to identify potential circular RNA (crRNA) that mediates T-DXd resistance. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and co-immunoprecipitation assays were conducted to investigate the molecular mechanism. Ferroptosis was detected using C11-BODIPY, Liperfluo, FerroOrange staining, glutathione quantification, malondialdehyde quantification, and transmission electron microscopy. Molecular docking, virtual screening, and patient-derived xenograft (PDX) models were used to validate therapeutic agents.
RESULTS: VDAC3-derived crRNA (crVDAC3) ranked first in functional shRNA library screening. Knockdown of crVDAC3 increased the sensitivity of HER2-low breast cancer cells to T-DXd treatment. Further mechanistic research revealed that crVDAC3 specifically binds to HSPB1 protein and inhibits its ubiquitination degradation, leading to intracellular accumulation and increased levels of HSPB1 protein. Notably, suppression of crVDAC3 dramatically increases excessive ROS levels and labile iron pool accumulation. Inhibition of crVDAC3 induces ferroptosis in breast cancer cells by reducing HSPB1 expression, thereby mediating T-DXd resistance. Through virtual screening and experimental validation, we identified that paritaprevir could effectively bind to crVDAC3 and prevent its interaction with HSPB1 protein, thereby increasing ubiquitination degradation of HSPB1 protein to overcome T-DXd resistance. Finally, we validated the enhanced therapeutic efficacy of T-DXd by paritaprevir in a HER2-low PDX model.
CONCLUSIONS: This finding reveals the molecular mechanisms underlying T-DXd resistance in HER2-low breast cancer. Our study provides a new strategy to overcome T-DXd resistance by inhibiting the interaction between crVDAC3 and HSPB1 protein.