Abstract
Abstract We have developed an in vitro model of breast cancer progression that consists of four derived ER-negative PR-negative cell lines: (a) the normal-like breast epithelial cell line MCF-10F; (b) the transformed trMCF cells; (c) the invasive bsMCF cells and; (d) the tumorigenic caMCFs cells which shown all characteristics of a fully malignant breast cancer cells [1,2]. Briefly, the normal human breast epithelial MCF-10F cells were treated with high concentration of 17β-estradiol (70 nM) giving origin to trMCF cells; trMCF cells were seeded in Boyden chambers and the cells that crossed the membrane were selected and expanded giving origin to bsMCF. The bsMCF cells were injected in the mammary fat pad of SCID mice and they induced tumors that were poorly differentiated adenocarcinomas that were ESR, progesterone receptor (PR) and ERBB2 negatives. Cells were isolated from 4 different tumors growth in four animals and they gave origin to the tumorigenic caMCFs cells (caMCF1, caMCF2, caMCF3 and caMCF4) [1,2]. This in vitro model of breast cancer progression was used to study DNA-methylation changes at the different stages using sensitive methylation specific PCR (MSP) and, we found that NRG1 and RARβ showed progressive changes in their methylation pattern. NRG1 and RARβ were unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. The clinical relevance of these findings were pursued by studying NRG1 and RARβ methylation in 17 cases of invasive ductal carcinomas (IDC) and 11 normal breast tissue samples (controls). NRG1 was hypermethylated in 14 out of 17 IDC (82.4%) vs. 2 out of 10 (20%) controls. All the ER-negative PR-negative tumors (10/10) showed NRG1 hypermethylated. From the seven ER-positive IDC, only 4 (57%) showed NRG1 hypermethylated. RARβ was methylated in 9 out of 16 IDC (56.3%) vs. 2 out of 11 (18.2%) controls. Most of the ER-negative PR-negative invasive ductal tumors showed RARβ hypermethylated (9/10). None of the ER-positive IDC shown RARβ hypermethylated. Altogether our data shown that the majority (90%) of the ER-negative PR-negative invasive ductal carcinomas shown hypermethylation of both NRG1 and RARβ. None of the ER-positive invasive ductal carcinomas showed RARβ hypermethylation and, only 57% showed hypermethylation of NRG1. (This work was supported by The Pennsylvania Breast Cancer Coalition). REFERENCES [1] S.V. Fernandez, K.E. Snider, Y.Z. Wu, I.H. Russo, C. Plass and J. Russo DNA methylation changes in a human cell model of breast cancer progression, Mutat Res 688 (2010) 28-35. [2] Y. Huang, S.V. Fernandez, S. Goodwin, P.A. Russo, I.H. Russo, T.R. Sutter and J. Russo Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol, Cancer Res 67 (2007) 11147-11157. 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 75. doi:10.1158/1538-7445.AM2011-75
Published Version
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