P3-17-01: ApoE and Its Receptors (LRP8, VLDLR) Function as Growth Signals for Triple-Negative Breast Cancer and Represent a Novel Therapeutic Target.
Authors: Shiang C , Qi Y , Wang B , Broom B , Pusztai L 
Published: June 2012

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+) in vitro. We targeted each gene with 4 different siRNA constructs 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 and signal transduction studies.

Results: 684 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in all 3 data sets. siRNA suppression of 161 of these genes inhibited cell growth significantly more in the ER-negative compared to ER-positive cells by at least 1 of the 4 siRNAs, 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. ApoE4 stimulation results in the transcriptional upregulation of genes involved in proliferation, metabolism, and inflammatory signaling pathways. ApoE4 stimulation increases expression of proteins involved in MAPK/ERK pathway, DNA damage repair, and inflammation.

Conclusions: We show that theApoE - 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, which is primarily TNBC. We propose that inhibitors of LRP8/VLDLR signaling may be clinically useful therapeutic or preventive agents for TNBC.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-01.