Project description:Withaferin A 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 concentrations of Withaferin A (WA) which can be detected systemically in in vivo experiments. Whereas WA revealed attenuation of multiple cancer hallmarks the withanolide analogue Withanone (WN), did not exert any of the described effects in these cells at comparable concentrations. Pathway enrichment analysis identified that WA targeted relevant cancer programs related to cell death, cell cycle and proliferation, which could functionally be validated flow cytometrically and by real-time proliferation xCELLigence assay. With respect to cell motility, invasion and metastasis processes, WA was found to strongly decrease MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This is further supported by gene expression changes which demonstrate increased expression of a validated breast cancer metastasis suppressor gene (BRMS1) and concomitant decreased expression of extracellular matrix-degrading proteases (PLAU, PLAT, ADAM8), cell adhesion molecules (integrins, laminins) as well as pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R). Subsequent in silico network analysis listed key node transcription factors regulating these processes (p53, E2F1, CDKN2A, NRF2) and revealed the role of chromatin modifying enzymes (p300, JARID1B) as central master switches, linking multiple anticancer activities of WA. Furthermore, 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 in account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy resistant triple negative breast cancer, WA based therapeutic strategies hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer. Total RNA from 2 different cell lines treated with WA, are compared to their untreated states

Project description:Withaferin A 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 concentrations of Withaferin A (WA) which can be detected systemically in in vivo experiments. Whereas WA revealed attenuation of multiple cancer hallmarks the withanolide analogue Withanone (WN), did not exert any of the described effects in these cells at comparable concentrations. Pathway enrichment analysis identified that WA targeted relevant cancer programs related to cell death, cell cycle and proliferation, which could functionally be validated flow cytometrically and by real-time proliferation xCELLigence assay. With respect to cell motility, invasion and metastasis processes, WA was found to strongly decrease MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This is further supported by gene expression changes which demonstrate increased expression of a validated breast cancer metastasis suppressor gene (BRMS1) and concomitant decreased expression of extracellular matrix-degrading proteases (PLAU, PLAT, ADAM8), cell adhesion molecules (integrins, laminins) as well as pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R). Subsequent in silico network analysis listed key node transcription factors regulating these processes (p53, E2F1, CDKN2A, NRF2) and revealed the role of chromatin modifying enzymes (p300, JARID1B) as central master switches, linking multiple anticancer activities of WA. Furthermore, 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 in account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy resistant triple negative breast cancer, WA based therapeutic strategies hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer.

Project description:Triple negative breast cancer (TNBC) is a highly heterogeneous disease representing the most aggressive breast cancer (BC) subtype. Lack of Estrogen Receptor alpha (ERα), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu) expression makes TNBC immune to common therapies, significantly limiting the treatment options and suggesting the need to identify novel therapeutic targets. It was previously reported that Estrogen Receptor beta (ERβ) is expressed in a fraction of TNBC patients, where its presence correlates with improved patient outcome. Recently, we demonstrated an oncosuppressive ERβ effect in TNBC cell models expressing exogenous ERβ. On the other hand, it was shown that ERβ is involved in miRNA-mediated gene regulation in hormone-responsive BC cells, suggesting similar effect also in TNBC. To verify this hypothesis, we performed small non-coding RNA (sncRNA) sequencing on three engineered cell lines belonging to different TNBC molecular subtypes. ERβ-specific changes of sncRNA profile revealed that the major part of deregulated molecules are subtype specific, with only few commonly regulated ones. In order to validate the obtained results, we performed sncRNA profiling of 12 ERβ positive and 32 ERβ negative TNBC tissues, whose receptor status was assessed by immunohistochemistry in our previous research. Also here, ERβ-specific group of deregulated sncRNAs was identified. Interestingly, comparison of obtained in vitro and in vivo results revealed 2 differentially expressed miRNAs, displaying the same behavior in all three analyzed cell lines and tissues. In concordance with our previous results, IPA signaling pathway analysis performed on genes targeted by deregulated miRNAs highlighted downregulation of cholesterol biosynthesis pathway and upregulation of several signaling processes. Taken together, these findings suggest that ERβ is able to exert its oncosuppressive role in TNBC through miRNA-mediated regulation of gene expression.

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Epigenetic silencing of triple negative breast cancer hallmarks by Withaferin A

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