P5-07-01: Novel Alternative Splice Variants of HER2 in Invasive Breast Cancer.
Authors: EE Assam, A Rhodes, M Ladomery, L Harries, M Sohail
Journal: Cancer Research (2011)

Abstract Background: The Human Epidermal Growth Factor Receptor 2 (HER2) is an oncogene expressed in 25–30% of invasive breast cancers. HER2 shares extensive homology with other members of the HER family (HER1, HER3 and HER4), and is constitutively active as an homo- and heterodimer. The HER2 gene encodes an 185kDa transmembrane protein with tyrosine kinase activity. Gene amplification or protein expression of HER2 is a predictor of poor clinical outcome and decreased survival in women with breast cancer, and also indicates a favourable response to Trastuzumab (Herceptin) therapy, or a combinational therapy comprising Herceptin plus chemotherapy. However, resistance to Trastuzumab remains the case in approximately 50% of HER2 amplified/overexpressing tumours. Understanding the molecular mechanisms of Trastuzumab resistance and identifying more effective therapies, is critical in the treatment of patients whose breast cancers express this aggressive disease phenotype. In this study, it is postulated that the abnormal generation of mRNA splice variants may be responsible for the continued tumour growth and progression. Aims: The aim of this study is to increase our understanding of the role of HER-2 splice variants in the development and progression of breast cancer. This will inform the development of more sophisticated and effective therapies that target specific HER-2 isoforms, rather than Herceptin, which targets just the generic wild type HER-2 protein. Materials and Methods: The entire coding region of HER-2 cDNA was PCR amplified using 12 sets of HER-2 specific primers in HER-2 positive cell lines (SKOV-3, SKBR-3, MDA-MB-453 and MDA-MB-361). Results: RT-PCR results showed multiple bands in various regions of the HER2 mRNA. Sequencing of these bands revealed novel alternative splice variants with deletions in exons 13 and 18 of the HER2 gene expressed in addition to the wild type HER2. Bioinformatic analysis of the deletions revealed a cassette exon deletion in exon 13, and a loss of 42 base pairs in the 3’ end of exon 18 compared to the full length HER2. Both the full length HER2 sequence and the sequence containing the deletions were translated using the ExPASy Translate Tool. This revealed an in-frame deletion of 14 amino acids and revealed a novel splice isoform with a deletion in the HER2 protein which encompasses the entire ATP binding pocket. This was determined by analysis using UniProtKB to identify the composition of amino acids for each domain of HER2. Discussion: Our studies have identified novel splice variants in the tyrosine-kinase domain of the HER-2 gene using HER-2 positive cell lines. The loss of an ATP binding site in the HER2 gene may lead to a less active HER2 isoform which may play a significant role in prognosis. Current work is being carried out to study the regulation of these splice variants and to study the role of splice factors ASF/SF2 and SRPK1 in the regulation of HER2 splicing, and to elucidate any significant changes in the HER2 signalling pathways. In addition, the expression of these isoforms are currently being investigated in tissues from formalin fixed, paraffin embedded and frozen breast tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-01.