The Nuclear Function of the Splice Variant Amplified in Breast Cancer 1-Delta4 Contributes to its Oncogenicity

Abstract

The oncogene amplified in breast cancer 1 (AIB1) 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-Delta4 that is overexpressed in breast cancer. In this same report AIB1-Delta4 was found to be a more potent coactivator of steroid hormone transcription that full-length AIB1. The underlying mechanism to explain this potent coactivation had yet to be explored. The AIB1-Delta4 protein is a N-terminally truncated isoform of AIB1 and we propose that loss of this N-terminal region is the reason why AIB1-Delta4 is a more potent coactivator. In this study we used mass spectrometry to define the translation initiation of AIB1-Delta4 at Met224 of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N-terminus. We determined that AIB1-Delta4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-Delta4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP and AIB1. We report that the endogenously and exogenously expressed AIB1-Delta4 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-Delta4 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-Delta4 is due to the loss of this inhibitory domain. We observed that this N-terminal region of AIB1 is a region of negative phosphorylation and possibly a domain of protein protein interaction. Using Scorpion primer technology, we show that AIB1-Delta4 expression is correlated with metastatic capability of human cancer cell lines. And lastly, we do not see an effect on in vitro proliferation of cells or invasiveness due to expression of AIB1-Delta4 protein.