Abstract A51: Chromatin-related genes are upregulated in stem cell Aldefluor-positive subpopulation of MDA-MB-231 triple-negative breast cancer cells
Authors: Correa-Noronha Silvana Aparecida Alves , Ritto Maria de Nazareth Gamboa , Freitas Eduardo Henrique da Silva , Wolgien Maria Del Carmen Molina , Silva Ismael Dale Cotrim Guerreiro da , Noronha Samuel Marcos Ribeiro de 
Published: August 2014

Introduction: Mesenchymal stem cells (MSCs) are progenitor cells that can be isolated from all connective tissues such as bone, adipose, cartilage, blood and muscle. MSCs have recently been described to localize within breast carcinomas where the stem cells integrate into tumor-associated stromal tissues whereby the MSCs promote breast cancer cell invasion and metastasis. The MDA-MB-231 cell line is a triple negative lineage that likely contains more than one type of cell sub-population. It is highly aggressive, invasive and poorly differentiated. Many breast cell lines contain high percentages of the breast cancer stem cells (BSCs), despite the exactly amount be a controversial issue.

Objectives: To investigate genes known to regulate the activation status of chromatin and of chromatin-related proteins and their possible differential expression values in the triple negative breast cancer (TNBC) cells MDA-MB-231 Aldefluor-positive (ALD+) and in the negative (ALD-) subpopulations.

Experimental Procedures: MDA-MB-231 cells cultured in DMEM (10% fetal bovine serum) were separated in both ALD+ (BSCs) and in ALD- (non-BSCs) subpopulations by flow cytometry (Fluorescence Activated Cell Sorter — FACS). Total RNA was extracted of these cell sub-populations using TrizolTM reagent, quantified and qualified by NanodropTM and BioanalyzerTM, respectively. The total RNA was used to generate complementary DNA (cDNA) that was amplified and labeled with Cyanine 3-CTP (Cy3) to hybridization procedure. After that, these samples were submitted to One-Color Microarray-Based gene Expression Analysis. We used the SurePrint G3 slides kit (8x60K human genes) microarray formats (Agilent), scanning, and feature extraction. The analysis of differential expression profiles were performed by GX11.5 GeneSpring software.

Results: Following cell sorting, gene expression values from the different experimental groups (ALD+, ALD- and control) were compared. This comparison showed us a group of upregulated DNA binding related genes. When we used ALD- group as the calibrator sample, the following upregulated fold change values from DNA binding related genes could be observed: CHD4, POLR1A, BRPF1, HIST2H4B, HMGN1, GON4L and KLF14 (respectively: 2.64, 2.94, 3.69, 3.97, 4.24, 4.41, 4.69 and 9.68; for p&lt;0.05). Besides that, when ALD+ sub-population was used as the calibrator against control cells, we observed that chromatin remodeling related genes like HIST2H4B and KLF14 were downregulated (3.46 and 8.94, respectively; for p&lt;0.05).

Conclusions: Results here shown suggest that important genes playing a role in the epigenetic of cancer are upregulated in Aldefluor-positive MDA-MB-231 TNBC cells. Epigenetic aberrations might contribute to the high aggressiveness presented by these cells and overexpression of these genes might play a significant role in cancer development. Self-renewing cancer cells, often called cancer stem cells are of great interest to researchers; they often remain unaffected by currently available drugs. Therefore, these genes can be used as possible tumoral markers and also as specific molecular targets to control BSCs surveillance.

Financial support: FAPESP

Citation Format: Silvana Aparecida Alves Correa-Noronha, Maria de Nazareth Gamboa Ritto, Eduardo Henrique da Silva Freitas, Maria Del Carmen Molina Wolgien, Ismael Dale Cotrim Guerreiro da Silva, Samuel Marcos Ribeiro de Noronha. Chromatin-related genes are upregulated in stem cell Aldefluor-positive subpopulation of MDA-MB-231 triple-negative breast cancer cells. [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 A51.