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

ABSTRACT: 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.

SUBMITTER: Szarc Vel Szic K

PROVIDER: S-EPMC5522326 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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

Szarc Vel Szic Katarzyna K Declerck Ken K Crans René A J RAJ Diddens Jolien J Scherf David B DB Gerhäuser Clarissa C Vanden Berghe Wim W

Oncotarget 20170601 25

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 treat ...[more]

PMID: 28467815

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Project description:TGFβ signaling plays crucial role during development and cancer, however the role for TGFβ signaling in regulating the noncoding part of the human genome in triple negative breast cancer (TNBC) is still being unraveled. Herein, we provide the transcriptional landscape of TNBC in response to TGFβ activation and subsequent inhibition employing SB431542, selective TGFβ1 Receptor ALK5 Inhibitor. Our data revealed 72 commonly upregulated [fold change (FC) ≥ 2.0], including PLAU, TPM1, TAGLN, COL1A1, TGFBI, and SNAI1, and 53 downregulated (FC ≤ 2.0) protein coding genes in BT-549 and MDA-MB-231 models in response to TGFβ1 activation. Alignment to the geocode (V33) identified 41 upregulated (FC ≥ 2.0) and 22 downregulated (FC ≤ 2.0) long non-coding RNA (lncRNA) in response to TGFβ1 activation, which were inhibited by concurrent treatment with SB431542. To place our data from the in vitro models into their clinical context, we identified AC015909.1, AC013451.1, CYP1B1-AS1, AC004862.1, LINC01824, AL138828.1, B4GALT1-AS1, AL353751.1, AC090826.3, AC104695.4, ADORA2A-AS1, PTPRG-AS1, LINC01943, AC026954.3, TPM1-AS, ZFPM2-AS1, AC007362.1, AC112721.2, MALAT1, AL513314.2, AC112721.1, AC010343.3, LINC01711, and MAP3K2-DT lncRNA expression to positively correlate with TGFβ1 expression in a cohort of 360 TNBC patients. To provide mechanistic insight into lncRNA regulation by TGFβ signaling, SMAD2/3 ChIp-Seq data from BT-549 TNBC model retrieved from Gene Expression Omnibus (GEO) revealed direct binding of SMAD2/SMAD3 to the promoter of AC112721.1, AC112721.2, MALAT1, HHIP-AS1, LINC00472, and SLC7A11, suggesting their direct regulation by TGFβ1/SMAD2/SMAD3 pathway. Interestingly, AC112721.1, AC112721.2 exhibited higher expression in TNBC compared to normal breast tissue suggesting a possible role for those lncRNA in TNBC biology. Our miRNA analysis in the BT-549 model in response to exogenous TGFB1 revealed several affected miRNAs (2.0 ≤ FC ≤ 2.0), whose expression pattern was reversed in the presence of SB431542, suggesting those miRNA as plausible targets for TGFβ regulation. In particular, we observed hsa-miR-1275 to be downregulated in response to TGFB1 which was highly predicted to regulate PCDH1, FIBCD1, FXYD7, GDNF, STC1, EDN1, ZSWIM4, FGF1, PPP1R9B, NUAK1, PALM2AKAP2, IGFL3, and SPOCK1 whose expression were upregulated in response to TGFβ1 stimulus. On the other hand, hsa-miR-181b-5p was among the top upregulated miRNAs in response to TGFB1, which is also predicted to regulate CDKN1B, TNFRSF11B, SIM1, and ARSJ in the BT-549 model. Taken together, our data is the first to provide such in depth analysis of lncRNA and miRNA epigenetic changes in response to TGFβ signaling in TNBC.

Project description: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.

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

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

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