Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, 11794- 8430, USA.
Stony Brook University Cancer Center, MART Level 9, Stony Brook University, Stony Brook, NY, 11794-8430, USA.
Breast Cancer Res. 2021 Jul 27;23(1):76. doi: 10.1186/s13058-021-01452-5.
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.
阿霉素（Dox）是一种广泛应用的化疗药物，但由于剂量依赖性的副作用，其疗效受到限制。尽管降低 Dox 剂量可以降低这种风险，但研究报告表明，接受低剂量 Dox 治疗的患者局部疾病复发率更高，生存率没有提高。为了有效缓解这一问题，需要更好地了解亚致死剂量 Dox 的不良影响。
通过光镜和共聚焦显微镜评估亚致死剂量 Dox 对表型变化的影响。通过划痕愈合和 Transwell 迁移实验评估迁移和侵袭行为。MTT 和 LDH 释放实验用于分析细胞生长和细胞毒性。流式细胞术用于检测癌症干细胞群体的细胞表面标志物。通过 qRT-PCR 和酶原测定法探测基质金属蛋白酶的表达和活性。为了确定亚致死剂量 Dox 影响的途径，进行了探索性 RNAseq 实验，并在多个细胞系（MCF7、ZR75-1 和 U-2OS）中通过 qRT-PCR 进行了验证。通过 siRNA 敲低以及 Western blot 和 qRT-PCR 评估 DNA 损伤反应关键因子对 Src 家族激酶（SFK）的调节作用。使用 Dasatinib 和针对 Fyn 和 Yes 的 siRNA 抑制 SFK，并验证它们在 MCF7 细胞中对亚致死剂量 Dox 处理后迁移和侵袭增加的作用。
结果表明，亚致死剂量的 Dox 处理导致原本非侵袭性的 MCF7 乳腺癌细胞迁移和侵袭增加。从机制上讲，这些作用独立于上皮-间充质转化，不是由于癌症干细胞群体增加，也不会发生在其他化疗药物中。相反，亚致死剂量的 Dox 诱导包括 Fyn、Yes 和 Src 在内的多个 SFK 的表达，部分依赖于 p53 和 ATR。这些作用在多个细胞系中得到了验证。从功能上讲，使用 Dasatinib 抑制 SFK 和特异性下调 Fyn 可抑制 Dox 诱导的 MCF7 细胞迁移和侵袭。
总的来说，这项研究表明，亚致死剂量的 Dox 激活了癌细胞中的促侵袭、促迁移程序。此外，通过确定 SFK 作为这些作用的关键介质，我们的结果定义了一种潜在的治疗策略，通过与 Dasatinib 联合治疗来减轻局部侵袭。
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