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Oncotarget. 2017 Jun 20;8(25):40434-40453. doi: 10.18632/oncotarget.17107.

Epigenetic silencing of triple negative breast cancer hallmarks by Withaferin A.

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Laboratory of Protein Chemistry, Proteomics and Epigenetic Signaling (PPES), Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
Current address: Division of Hematology, Oncology and Stem Cell Transplantation, Center for Translational Cell Research, The University Medical Center Freiburg, Freiburg, Germany.
Current address: Laboratory for GPCR Expression and Signal Transduction (L-GEST), Department of Biochemistry and Microbiology, University of Ghent, Ghent, Belgium.
Workgroup Cancer Chemoprevention and Epigenomics, Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany.


Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. Withaferin A (WA) is a plant derived steroidal lactone which holds promise as a therapeutic agent for treatment of breast cancer (BC). We determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines, following 72h treatment to a sub-cytotoxic concentration of WA. In contrast to the DNA demethylating agent 5-aza-2'-deoxycytidine (DAC), WA treatment of MDA-MB-231 cells rather tackles an epigenetic cancer network through gene-specific DNA hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 (ADAM8), urokinase-type plasminogen activator (PLAU), tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1), or mitochondrial metabolism (malic enzyme 3, ME3). Gene expression and pathway enrichment analysis further reveals epigenetic suppression of multiple cancer hallmarks associated with cell cycle regulation, cell death, cancer cell metabolism, cell motility and metastasis. Remarkably, DNA hypermethylation of corresponding CpG sites in PLAU, ADAM8, TNSF12, GSTM1 and ME3 genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ESR)/progesterone receptor (PR) status in primary BC tumors. Moreover, upon comparing differentially methylated WA responsive target genes with DNA methylation changes in different clinical subtypes of breast cancer patients in the cancer genome atlas (TCGA), we found that WA silences HER2/PR/ESR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks, with an improved therapeutic sensitivity. In this respect, WA may represent a novel and attractive phyto-pharmaceutical for TNBC treatment.


DNA methylation; epigenetics; luminal breast cancer; triple negative breast cancer; withaferin A

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