Withaferin A (WA) exhibits cancer chemopreventive efficacy in preclinical models representative of two different subtypes of breast cancer. However, the mechanism(s) underlying breast cancer chemoprevention by WA is not fully elucidated. We performed RNA‐seq analyses using a non‐tumorigenic mammary epithelial cell line (MCF‐10A) and human breast cancer cells (BCC) belonging to the luminal‐type (MCF‐7), HER2‐enriched (SK‐BR‐3), and basal‐like subtype (MDA‐MB‐231) to identify novel cancer‐selective mechanistic targets of WA. The WA‐regulated transcriptome was strikingly different between MCF‐10A versus BCC. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulation of genes associated with cellular senescence in WA‐treated BCC. Consequently, the number of senescence‐associated β‐galactosidase positive cells was decreased significantly in WA‐treated BCC but not in the MCF‐10A cells. WA treatment caused upregulation of senescence marker p21 more robustly in BCC than in MCF‐10A. Breast cancer prevention by WA in rats was also associated with upregulation of p21 protein expression. The Reactome pathway analyses indicated upregulation of genes associated with cellular response to stress/external stimuli in WA‐treated BCC but not in MCF‐10A. Two proteins represented in these pathways (HSPA6 and NRF2) were further investigated. While HSPA6 was dispensable for WA‐mediated apoptosis and autophagy or inhibition of cell migration, the NRF2 knockout cells were more resistant to apoptosis resulting from WA treatment than control cells. Finally, expression of some glycolysis‐related proteins was decreased by WA treatment both in vitro and in vivo. In summary, this study provides novel insights into cancer‐selective pathways affected by WA that may contribute to its chemopreventive efficacy in breast cancer.

中文翻译：

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RNA-seq揭示了人乳腺癌细胞中withaferin A的新型癌症选择性和疾病亚型独立机制靶点

Withaferin A (WA) 在代表两种不同乳腺癌亚型的临床前模型中表现出癌症化学预防功效。然而，WA 对乳腺癌化学预防的潜在机制尚未完全阐明。我们使用非致瘤性乳腺上皮细胞系 (MCF-10A) 和属于管腔型 (MCF-7)、富含 HER2 (SK-BR-3) 的人乳腺癌细胞 (BCC) 进行了 RNA-seq 分析和基底样亚型 (MDA-MB-231) 来鉴定 WA 的新型癌症选择性机制目标。WA 调节的转录组在 MCF-10A 与 BCC 之间存在显着差异。京都基因和基因组学百科全书通路分析揭示了 WA 治疗的 BCC 中与细胞衰老相关的基因下调。最后，在 WA 处理的 BCC 中，衰老相关 β-半乳糖苷酶阳性细胞的数量显着减少，但在 MCF-10A 细胞中没有。与 MCF-10A 相比，WA 处理导致 BCC 中衰老标志物 p21 的上调更强烈。WA 在大鼠中预防乳腺癌也与 p21 蛋白表达的上调有关。Reactome 通路分析表明，在 WA 处理的 BCC 中，与细胞对应激/外部刺激的反应相关的基因上调，但在 MCF-10A 中没有。进一步研究了这些途径中代表的两种蛋白质（HSPA6 和 NRF2）。虽然 HSPA6 对于 WA 介导的细胞凋亡和自噬或抑制细胞迁移是可有可无的，但与对照细胞相比，NRF2 敲除细胞对 WA 处理导致的细胞凋亡具有更强的抵抗力。最后，WA 治疗在体外和体内都降低了一些糖酵解相关蛋白的表达。总之，这项研究提供了对受 WA 影响的癌症选择性途径的新见解，这些途径可能有助于其在乳腺癌中的化学预防功效。