Abstract A59: MDA-MB-231 stem cell subpopulation upregulates KRAB domain-containing zinc-finger gene family to hold undifferentiated ground state
Authors: Noronha Samuel Marcos Ribeiro de , Baptista Carlos Fernandes , Bernardo Werica , Silva Ismael Dale Cotrim Guerreiro , Correa-Noronha Silvana Aparecida Alves 
Published: August 2014

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 allowed annotation of these 337 upregulated genes as, molecular function (61.7.0%), cellular component (46.2) and biological process (2.1%). Among sixty-four molecular function related genes, twety-two (34.4%) are zinc-finger proteins and eighteen of them are genes coding for KRAB domain-containing zinc-finger proteins (ZNF688, ZNF419, ZNF397, ZNF821, ZNF12, ZNF280C, ZNF821, ZNF239, ZNF468, ZNF702P, ZNF496, ZNF205, ZNF829, ZNF345, ZNF526, ZNF329, ZNF561 and ZNF562). HDAC5 is downregulated 3.13 times in stem cell-like subpopulation when compared to Aldefluor negative sub-population.

Conclusions: Our results show that the upregulation of the Krüpell family of zinc finger proteins might be part of the genome signature that distincts MDA-MB-231 stem cell sub-population from its progeny. It is known that HDACs have an important role in mammary tumor cell growth control. KRAB-containing zinc-finger proteins are involved in transcriptional regulation by recruiting histone modifiers and chromatin remodelers like, DNMT3a, HDAC5, JARID2 and Activation Protein 1 (AP1), in order to repress gene transcription. This being the case, zinc-finger proteins are linked to epigenetic processes and can be selectively targeted to regulate gene expression and determine cell fate.

Citation Format: Samuel Marcos Ribeiro de Noronha, Carlos Fernandes Baptista, Werica Bernardo, Ismael Dale Cotrim Guerreiro Silva, Silvana Aparecida Alves Correa-Noronha. MDA-MB-231 stem cell subpopulation upregulates KRAB domain-containing zinc-finger gene family to hold undifferentiated ground state. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A59.