Abstract A32: Identification of biomarkers in normal human breast cells regulated by the cancer preventive rexinoid LG100268

Abstract

Abstract Background: Drugs targeting estrogen signaling or production have become useful in preventing estrogen receptor-positive breast cancers, but not estrogen receptor-negative (ER-negative) breast cancers. Our laboratory has demonstrated that rexinoids prevent ER-negative mammary tumors in transgenic mice by 90%. However, since these agents are not 100% effective, we are investigating the mechanism by which rexinoids prevent cancer to develop more efficacious prevention strategies. We hypothesized that by identifying molecules regulated by the rexinoid LG100268, future chemopreventive agents could be developed to target these molecules alone or in combination with rexinoids to totally prevent breast cancer development. To investigate this hypothesis we utilized an Affymetrix genome array to identify biomarkers regulated by LG100268. Methods: Cell counts were used to confirm rexinoid-induced growth suppression in normal human mammary epithelial cells (HMECs). TUNEL analysis was conducted to determine whether the growth suppression was a result of induction of apoptosis. We conducted Affymetrix analysis using 10ug total RNA from HMECs treated with 0.1% vehicle or 1uM LG100268 for 1 hour or 24 hours. cRNA was hybridized with the GeneChip® Expression Analysis Array (U133) from Affymetrix. Results were analyzed using Microarray Suite 5.0 (Affymetrix). Genes to be considered for further confirmation had a fold change of > 1.5 or < 0.8 (p < 0.05) for up- or down-regulated genes, respectively. Geneontology analysis identified candidate genes involved in proliferation, cell cycle and metabolism as genes to consider for further analysis. Quantitative RT-PCR (qRT-PCR) and standard western blot techniques were used to confirm regulation of selected up- and down-regulated genes at the mRNA and protein levels, respectively. Results: Cell count analysis confirmed that LGD1069 greatly suppressed growth of HMEC cells, while LG100268 slightly suppressed HMEC growth. TUNEL analysis indicated that neither rexinoid treatment induced a significant amount of apoptosis in HMEC cells, indicating that the induction of growth suppression was not a result of apoptosis. A full list of genes up- and down-regulated by LGD100268 will be presented. Of those genes selected for further study, qRT-PCR analysis confirmed that LG100268 up-regulated stanniocalcin 1 (STC1), carbonic anhydrase IX (CA9), angiopoietin-like 4 (ANGPTL4), transglutaminase 2 (TGM2), and acyl-CoA synthetase long-chain family member 3 (ACSL3). Confirmed down-regulated genes at the mRNA level included cyclin B1 (CCNB1). Additional validation studies are ongoing. These up- and down-regulated genes may be useful targets for chemopreventive drug development. Conclusions: These experiments identified a list of biomarkers that are regulated by LG100268. Such rexinoid-regulated molecules represent possible targets for breast cancer preventative therapy, alone or in combination with rexinoids. Support: NCI R01 CA78480 (PHB and IPU), and NIH/NCI T32 CA90221 (JMR). Citation Information: Cancer Prev Res 2010;3(1 Suppl):A32.