Abstract 5559: Long-term exposure of breast cell lines to ethanol affects the transcriptional signature for some oncogenic gene families, but has little effect on this phenotype in mammospheres or on the expression of stem cell markers
Authors: Vernet Dolores , Gelfand Robert , Sarkissyan Suren , Heber David , Vadgama Jay , Gonzalez-Cadavid Nestor F. 
Published: June 2012

Abstract
Objectives. Chronic alcoholism is a risk factor for breast cancer but the mechanism of its potential oncogenic effects is unknown. Stem cells have been found in the epithelium of normal breast tissue and are postulated to convert into cancer stem cells driving tumor generation and metastasis. Breast stem cells are assumed to be enriched within multiple-cell spheroids named mammospheres. Here we aimed to determine whether ethanol, or its metabolite, acetaldehyde, stimulate the in vitro proliferation and malignant transformation of human breast stem cells within epithelial cell cultures and mammospheres, and whether this process is defined by specific gene expression signatures denoting oncogenesis, stemness and/or alcohol-regulation.
Methods. Cultures of monolayer attached cells and mammospheres from MCF-12 (“normal”) and MCF-7 (“malignant”) cell lines were incubated +/- 25 mM ethanol and subjected to DNA microarray analysis, or in triplicate with increasing concentrations of ethanol (0-25 mM) or acetaldehyde (0-12.5 mM) for 6 days, followed by immunocytofluorescence, quantitative western blot for stem cell markers Oct4 and nanog, and by RT-PCR, the formazan assay and growth in soft agar.
Results. Low levels of mRNAs for stem cell markers were seen by DNA microarrays in all cultures, but except for Sox4 and Jag1 were not responsive to ethanol. Oct4 and nanog proteins were detected in the nuclei of MCF-7 attached cells, and expressed respectively as 45 (Oct4a) and 38 kDa bands for the active isoforms, whereas in MCF-12 attached cells they were detected as the variant 33 (Oct4b) and 55 kDa proteins. Ethanol reduced Oct4a moderately in MCF-7 attached cells and acetaldehyde increased it, but no changes occurred in mammospheres. Ethanol increased Oct4b and reduced nanog in MCF-12 attached cells, with no change (Oct4) or an increase (nanog) in mammospheres. In both MCF-7 and MCF-12 attached cells, but not in mammospheres, ethanol upregulated by &gt;2 several alcohol-responsive metallothioneins (mainly 1F, 1X, and 2A), and related trans-membrane proteases serine (TMPRSS), as well as centromer-associated protein and PAI 1, while reducing other serpines and caspase 4, all malignancy-associated genes.
Conclusions. Although stem cell markers were expressed in MCF-7 and MCF-12 attached cells, they were not increased in mammospheres, raising doubts about the putative stem cell enrichment in these spheroids. The inconsistent modulation of stem cell number by ethanol and acetaldehyde requires confirmation by cell sorting. The identification of several ethanol-responsive, malignancy-associated genes in both MCF-7 and MCF-12 attached cells may support an oncogenic risk for high alcohol levels.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5559. doi:10.1158/1538-7445.AM2011-5559