Abstract A59: MDA-MB-231 stem cell subpopulation upregulates KRAB domain-containing zinc-finger gene family to hold undifferentiated ground state

Introduction: Breast cancer cell lines frequently contain a sub-population of stem cells (SC). MDA-MB-231 is a model of basal-like breast cancer triple negative (TN) lineage widespread used in laboratories across the world. It contains a stem cell subpopulation, however little is known about the molecular mechanisms underlying undifferentiated ground state regulation in this type of cell. Aberrations in epigenetic processes such as histone (de)methylation may cause cancer. The largest family of zinc-finger transcription factors is composed of the Krüpell-associated box (KRAB) domain containing proteins. They do not have intrinsic enzymatic activity, instead, this transcription factor family recruits chromatin remodelers and histone-modifying enzymes to regulate gene transcription, generally acting as repressor proteins. Underlying molecular mechanisms for aggressiveness of basal-like/TN breast cancer stem cell remains to be elucidated. Objective: To investigate the molecular basis of MDA-MB-231 sub-populations (progenitor and differentiated cells), in order to obtain and compare the genome expression profile of stem cell and of differentiated subpopulations and identify gene networks regulating distinct differentiation states. Experimental Procedures: Cells were cultured in T75 flasks, suspended in Aldefluor assay buffer and incubated for 30 minutes for staining. Heterogeneity of aldehyde dehydrogenase expression in MDA-MB-231 sub-populations was revealed by Aldefluor flow cytometry-based assay. Separated cell sub-populations were harvested, had their RNA extracted (TRIzol), purified (silica columns plus DNase), measured (Nanodrop) and analyzed (Bioanalyzer). RNA samples were submitted to Agilent whole genome (8X60K) microarray experiments (one-color), according to manufacturer's instructions. cDNA samples from each experimental group (Aldefluor-positive cells, Aldefluor-negative cells and overall population - control) were loaded into a genome array. Experiments were performed in triplicates. The analysis of differential expression profiles were performed by GX11.5 GeneSpring software. Results: Fluorescence activated cell sorting (FACS) assay detected 4% of the overall MDA-MB-231 cell population as Aldefluor positive and 50% as non-Aldefluor positive. Microarray results analyzed with GeneSpring software demonstrate that among 337 genes upregulated (fold change ≥ 2.0, p≤ 0.001) exclusively in the stem cell-like subpopulation. Gene Ontology (GO) terms allow