Abstract LB-219: ApoE and its' receptors (LRP8, VLDLR) function as growth signals for triple-negative breast cancer and represents novel therapeutic targets
Authors: Shiang Christine , Qi Yuan , Wang Bailiang , Broom Bradley , Pusztai Lajos 
Published: June 2012

Abstract
Background: Triple-negative breast cancers (TNBC) lack the expression of estrogen, progesterone, and the human epidermal growth factor 2 receptors and have limited treatment options. We hypothesized that genes that are frequently amplified or overexpressed in these cancers are functionally important for the growth and survival of triple-negative breast cancer.
Materials/Methods: We identified genes overexpressed in TNBC in 3 different human breast cancer gene expression data sets (n=294, n=286, n=198). We assessed the functional importance of the consistently overexpressed genes using siRNA screen on 18 breast cancer cell lines (10 ER−, 8 ER+). We targeted each gene with 4 different siRNAs separately and each experiment was performed in triplicate. The genes with the greatest TNBC-specific inhibitory effect after siRNA down regulation were selected for further mechanistic studies.
Results: 684 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in all 3 data sets (11% of these mapped to DNA segments with frequent copy number gains). 27 genes showed similar effect with 2 or more siRNAs and for 2 genes 3 of the 4 siRNAs showed preferential growth inhibition in ER-negative cells. These two genes were VLDLR (very low-density lipoprotein receptor) and LRP8 (low density lipoprotein receptor-related protein 8). We validated the siRNA screen results and confirmed down regulation of the mRNA and protein levels for VLDLR and LRP8 in 4 different ER-negative cell lines and showed that siRNA inhibition can be rescued by co-transfection of the receptor genes. Reanalysis of gene expression data also indicated expression of the LRP8 and VLDLR ligands, Reelin or ApoE, in both breast cancer tissues and in cell lines. We next demonstrated that exposure to Reelin and ApoE stimulates the growth of ER-negative cells in vitro. The stimulatory effect of ApoE was isoform dependent; ApoE2 (Cys112/Cys158) has the lowest receptor binding affinity and showed no growth stimulation, ApoE3 (Cys112/Arg158) had modest 50–60% growth stimulation and ApoE4 (Arg112/Arg158) had the greatest stimulatory effect (300–400%). Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation.
Conclusions: We show that the ApoE -LRP8/VLDLR ligand receptor system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro. Interestingly, allelic imbalance favoring ApoE4 expression (E3/E4 or E4/E4) has been linked to higher risk of developing early onset breast cancer, that is primarily TNBC. We propose that inhibitors of LRP8/VLDLR signaling may be clinically useful therapeutic agents for TNBC.
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 LB-219. doi:10.1158/1538-7445.AM2011-LB-219