184AA3: a xenograft model of ER+ breast adenocarcinoma.

Despite the prevalence and significant morbidity resulting from estrogen receptor positive (ER(+)) breast adenocarcinomas, there are only a few models of this cancer subtype available for drug development and arguably none for studying etiology. Those models that do exist have questionable clinical relevance. Given our goal of developing luminal models, we focused on six cell lines derived by minimal mutagenesis from normal human breast cells, and asked if any could generate clinically relevant xenografts, which we then extensively characterized. Xenografts of one cell line, 184AA3, consistently formed ER(+) adenocarcinomas that had a high proliferative rate and other features consistent with "luminal B" intrinsic subtype. Squamous and spindle cell/mesenchymal differentiation was absent, in stark contrast to other cell lines that we examined or others have reported. We explored intratumoral heterogeneity produced by 184AA3 by immunophenotyping xenograft tumors and cultured cells, and characterized marker expression by immunofluorescence and flow cytometry. A CD44(High) subpopulation was discovered, yet their tumor forming ability was far less than CD44(Low) cells. Single cell cloning revealed the phenotypic plasticity of 184AA3, consistent with the intratumoral heterogeneity observed in xenografts. Characterization of ER expression in cultures revealed ER protein and signaling is intact, yet when estrogen was depleted in culture, and in vivo, it did not impact cell or tumor growth, analogous to therapeutically resistant ER(+) cancers. This model is appropriate for studies of the etiology of ovarian hormone independent adenocarcinomas, for identification of therapeutic targets, predictive testing, and drug development.

Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1.

Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44-65) and 10 (amino acids 220-241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209-219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic alpha-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic alpha-helices is required to mediate cholesterol efflux and stabilize ABCA1.