Abstract
The oncogene amplified in breast cancer 1 (<i>AIB1</i>) is a nuclear receptor coactivator that plays a major role in the progression of various cancers. We previously identified a splice variant of AIB1 called AIB1-Δ4 that is overexpressed in breast cancer. Using mass spectrometry, we define the translation initiation of AIB1-Δ4 at Met<sup>224</sup> of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N terminus. We show that AIB1-Δ4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-Δ4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP. We report that the endogenously and exogenously expressed AIB1-Δ4 is recruited as efficiently as full-length AIB1 to estrogen-response elements of genes, and it enhances estrogen-dependent transcription more effectively than AIB1. Expression of an N-terminal AIB1 protein fragment, which is lost in the AIB1-Δ4 isoform, potentiates AIB1 as a coactivator. This suggests a model whereby the transcriptional activity of AIB1 is squelched by a repressive mechanism utilizing the N-terminal domain and that the increased coactivator function of AIB1-Δ4 is due to the loss of this inhibitory domain. Finally, we show, using Scorpion primer technology, that <i>AIB1</i>-Δ<i>4</i> expression is correlated with metastatic capability of human cancer cell lines.
Highlights
Gene transcription in eukaryotes is a complex and highly regulated process
amplified in breast cancer 1 (AIB1)-⌬4 was shown to act as a molecular bridge between epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in the cytoplasm, and its overexpression increased the invasiveness of the MDA-MB231 metastatic breast cancer cell line [21]
In accordance with previous data that the nuclear localization sequence (NLS) is contained in the N terminus of AIB1 (26 –28), we found that the majority of AIB1-⌬4, which lacks an NLS, is predominantly in the cytoplasm at steady state levels
Summary
ER␣, estrogen receptor-␣; EGFR, epidermal growth factor receptor; NLS, nuclear localization sequence; bHLH, basic helix loop helix; FAK, focal adhesion kinase; ANOVA, analysis of variance; IP, immunoprecipitation; IMEM, Iscove’s modified Eagle’s medium; CCS, charcoal-stripped serum; ERE, estrogen-responsive element; S, sense; AS, antisense; HMEC, human mammary epithelial cell; MMTV, mouse mammary tumor virus; PAS, Per Arnt Sim domain. AIB1-⌬4 is recruited to estrogen-response elements of endogenous estrogen-regulated genes and increases their expression. Through the use of Scorpion primer technology, we have created the first quantitative assay for the AIB1-⌬4 transcript and found a correlation between AIB1-⌬4 expression and the metastatic phenotype of human cancer cell lines. These data suggest that the nuclear activities of AIB1-⌬4 can contribute to its function in malignancy
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