Withaferin A (WA) isolated from Withania somnifera (
Ashwagandha) has recently become an attractive
phytochemical under investigation in various preclinical studies for treatment of different
cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231
breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple
cancer hallmarks, the withanolide analogue
Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific
cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell
collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading
proteases (uPA, PLAT, ADAM8),
cell adhesion molecules (
integrins, laminins), pro-inflammatory mediators of the
metastasis-promoting tumor microenvironment (TNFSF12,
IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated
breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased
protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84
chromatin writer-reader-eraser
enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in
therapy-resistant
triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic
breast cancer.