Doxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed. Effects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox. The results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK—including Fyn, Yes, and Src—partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells. Overall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib.

中文翻译：

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亚致死阿霉素促进乳腺癌细胞迁移和侵袭：Src家族非受体酪氨酸激酶的作用


阿霉素 (Dox) 是一种广泛使用的化疗药物，但其有效性受到剂量依赖性副作用的限制。尽管较低剂量的 Dox 可以降低这种风险，但研究报告称，接受低剂量 Dox 的患者局部疾病复发率较高，且生存率没有改善。为了有效减轻这种情况，需要更好地了解次优剂量的阿霉素的副作用。使用光学和共聚焦显微镜评估亚致死剂量的 Dox 对表型变化的影响。通过伤口愈合和跨孔迁移测定来评估迁移和侵袭行为。 MTT 和 LDH 释放测定用于分析细胞生长和细胞毒性。采用流式细胞术检测癌症干细胞群的细胞表面标志物。通过 qRT-PCR 和酶原测定检测基质金属蛋白酶的表达和活性。为了确定受亚致死剂量 Dox 影响的途径，进行了探索性 RNAseq，并通过 qRT-PCR 在多个细胞系（MCF7、ZR75-1 和 U-2OS）中验证了结果。通过 siRNA 敲除以及蛋白质印迹和 qRT-PCR 评估 DNA 损伤反应中关键参与者对 Src 家族激酶 (SFK) 的调节。使用达沙替尼和 Fyn 和 Yes 的 siRNA 来抑制 SFK，并验证它们在用亚致死剂量的 Dox 处理的 MCF7 细胞中增加迁移和侵袭的作用。结果表明，亚致死 Dox 治疗导致非侵袭性 MCF7 乳腺癌细胞的迁移和侵袭增加。从机制上讲，这些效应独立于上皮间质转化，不是由于癌症干细胞群增加所致，并且在其他化疗中未观察到。 相反，亚致死 Dox 会部分以 p53 和 ATR 依赖性方式诱导多种 SFK（包括 Fyn、Yes 和 Src）的表达。这些效果在多种细胞系中得到了验证。从功能上讲，用达沙替尼抑制 SFK 和 Fyn 的特异性下调可抑制 Dox 诱导的 MCF7 细胞迁移和侵袭。总体而言，这项研究表明，亚致死剂量的 Dox 会激活癌细胞中的促侵袭、促迁移程序。此外，通过将 SFK 确定为这些效应的关键介质，我们的结果定义了一种潜在的治疗策略，通过与达沙替尼联合治疗来减轻局部侵袭。