Background: Chemoresistance in triple-negative breast cancer (TNBC) presents a significant clinical hurdle, limiting the efficacy of treatments like doxorubicin. This study aimed to explore the molecular changes associated with doxorubicin resistance and identify potential therapeutic targets to overcome this resistance, thereby improving treatment outcomes for TNBC patients. Methods: Doxorubicin-resistant (DoxR) TNBC models (MDA-MB-231 and BT-549) were generated by exposing cells to increasing concentrations of doxorubicin. RNA sequencing (RNA-Seq) was performed using the Illumina platform, followed by bioinformatics analysis with CLC Genomics Workbench and iDEP. Functional assays assessed proliferation, sphere formation, migration, and cell cycle changes. Protein expression and phosphorylation were confirmed via Western blotting. Pathway and network analyses were conducted using Ingenuity Pathway Analysis (IPA) and STRING, while survival analysis was performed using Kaplan–Meier Plotter database. Results: DoxR cells exhibited reduced proliferation, sphere formation, and migration, but showed enhanced tolerance to doxorubicin. Increased CHK2 and p53 phosphorylation indicated cellular dormancy as a resistance mechanism. RNA-Seq analysis revealed upregulation of cytokine signaling and stress-response pathways, while cholesterol and lipid biosynthesis were suppressed. Activation of the IL1β cytokine network was prominent in DoxR cells, and CRISPR-Cas9 screens data identified dependencies on genes involved in rRNA biogenesis and metabolism. A 27-gene signature associated with doxorubicin resistance was linked to worse clinical outcomes in a large breast cancer cohort (HR = 1.76, FDRp< 2.0 × 10−13). Conclusions: This study uncovers potential therapeutic strategies for overcoming TNBC resistance, including dormancy reversal and targeting onco-ribosomal pathways and cytokine signaling networks, to improve the efficacy of doxorubicin-based treatments.
背景：三阴性乳腺癌（TNBC）的化疗耐药性构成了显著的临床障碍，限制了阿霉素等治疗方法的疗效。本研究旨在探索与阿霉素耐药相关的分子变化，并识别潜在的治疗靶点以克服这种耐药性，从而改善TNBC患者的治疗结果。方法：通过将细胞暴露于浓度递增的阿霉素中，构建了阿霉素耐药（DoxR）TNBC模型（MDA-MB-231和BT-549细胞系）。使用Illumina平台进行RNA测序（RNA-Seq），随后利用CLC Genomics Workbench和iDEP进行生物信息学分析。功能实验评估了细胞增殖、球体形成、迁移和细胞周期变化。通过蛋白质印迹法确认了蛋白质表达和磷酸化水平。使用Ingenuity Pathway Analysis（IPA）和STRING进行通路和网络分析，同时利用Kaplan–Meier Plotter数据库进行生存分析。结果：DoxR细胞表现出增殖、球体形成和迁移能力下降，但对阿霉素的耐受性增强。CHK2和p53磷酸化水平升高表明细胞休眠是一种耐药机制。RNA-Seq分析揭示了细胞因子信号传导和应激反应通路上调，而胆固醇和脂质生物合成受到抑制。IL1β细胞因子网络的激活在DoxR细胞中尤为显著，CRISPR-Cas9筛选数据识别出对核糖体RNA生物合成和代谢相关基因的依赖性。一个与阿霉素耐药相关的27基因特征在一个大型乳腺癌队列中与较差的临床结果相关（风险比=1.76，错误发现率校正p值< 2.0 × 10−13）。结论：本研究揭示了克服TNBC耐药性的潜在治疗策略，包括逆转细胞休眠状态以及靶向癌性核糖体通路和细胞因子信号网络，以提高基于阿霉素的治疗方案的疗效。
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