RNA-seq reveals novel cancer-selective and disease subtype-independent mechanistic targets of withaferin A in human breast cancer cells

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 beta-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.

Automated summary

The study revealed that Withaferin A (WA) may prevent breast cancer by downregulating genes associated with cellular senescence, upregulating p21, affecting cellular response to stress, and decreasing glycolysis-related proteins. Additionally, NRF2 knockout cells were found to be more resistant to WA-induced apoptosis, suggesting a potential mechanism for WA's chemopreventive efficacy in breast cancer.