Propolis, and its active component, CAPE, is a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown that CAPE, inhibits 1) growth of ER+/PR+, ER-/PR- and Her2 + breast cancer cells, 2) the tumorigenic potential of breast cancer stem cells, and 3) mediates its effects through epigenetic modifications via HDAC inhibition, resulting among others, in increased ER in ER- cells. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers (TNBC). The cell-of-origin hypothesis states that these cancers arise from transformation of mammary stem cells (SC).
We tested the ability of CAPE to inhibit SC self-renewal in murine mammary epithelial cells (MEC) and a human breast epithelial cell line (MCF10A). MEC isolated from BALB/c mice were cultured as mammospheres (MMS) for 7 days in ULA 96 well plates using media containing 5% FBS and 5% Matrigel. MMS were dissociated into single cells, recultured in the presence of CAPE for 7 days and passaged in secondary and tertiary passages in media alone. Alternatively, MCF10A transfected with a Let7c-miRNA reporter were sorted for Let7c-negative cells, and similarly cultured with CAPE. Murine MMS were analyzed for luminal marker, K18, basal marker K14, or progesterone receptor (PR) using immunofluorescence. Open chromatin states were identified using ATAC-seq and peaks were independently called using PeakDeck. Open chromatin areas unique to CAPE treated murine MMS were used for pathway analysis performed by Ingenuity Pathway Analysis and confirmed by Integrative Genome Viewer.
CAPE treatment resulted in a dose dependent decrease in mammosphere forming efficiency that persisted in secondary and tertiary passages, indicating reduced self-renewal. CAPE shifted the cells within murine MMS from predominantly K14 to K18-positive and increased PR expression. Interestingly, ATAC-seq showed enrichment of open chromatin of the SMARCA4 gene in the exonic region. SMARCA4 is a chromatin remodeling gene that is involved in SWI/SNF complex and alters the positioning of the nucleosome, it is thought to regulate transcription of certain genes by altering the chromatin structure around those genes. In addition, SMARCA4 can bind BRCA1, and regulate the expression of the tumorigenic breast cancer SC marker, CD44.
These data indicate that CAPE both inhibited mammary SC self-renewal and shifted the lineage commitment to a luminal lineage. ATAC-seq demonstrated effects at the chromatin level that may regulate the SMARCA4 gene, which controls the transcription of many genes involved in stem cell renewal, pluripotency, and embryonic development and is differentially expressed in the luminal phenotype of human MEC. Thus, CAPE may have an effect on lineage commitment in agreement with our chemoprevention strategy to reduce TNBC breast cancer development.
Citation Format: Coral O. Omene, Manan Patel, Kasthuri Kannan, Adriana Heguy, Mary Helen Barcellos-Hoff. CAPE (caffeic acid phenethyl ester) induces a mammary stem cell lineage restriction to a luminal phenotype via chromatin remodeling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4232. doi:10.1158/1538-7445.AM2015-4232