SHORT REPORT Two erbB-4 transcripts are expressed in normal breast and in most breast cancers Carol Sawyer 1 , Ian Hiles 3 , Martin Page 3 , Mark Crompton 2 and Christopher Dean 1 Sections of 1 Immunology, 2 Cell Biology and Experimental Pathology, Institute of Cancer Research, Sutton, Surrey; and 3 Oncology Research Unit, Glaxo-Wellcome Research Laboratories, Stevenage, Hertfordshire, UK ErbB-4 is a recently described member of the epidermal growth factor receptor (EGFR) family which together with erbB-3 acts as a receptor for a group of ligands known as the neuregulins (NRGs) or heregulins (HRGs). Unlike the EGFR and erbB-2 relatively little is known about the expression of erbB-4 in human tumours. Using RT ± PCR and Southern blotting analysis we have investigated the expression of erbB-4 mRNA in a range of human tumour cell lines and in normal and malignant breast tissue. Using primers which ampli®ed a 658 base pair (bp) region corresponding to part of the cytoplasmic domain of c-erbB-4 we found the receptor was expressed in some but not all breast and ovarian tumour cell lines and also in a glioma cell line. The highest level of erbB-4 expression was found in the ovarian carcinoma OVCAR- 3 and the breast carcinoma T-47D. In all cell lines where the `full-length' erbB-4 was detected, a second previously undescribed c-erbB-4 sequence was also found as a 610 bp PCR product. The alternative PCR product was identical in sequence to c-erbB-4 except for a deletion of 48 bp which encodes a consensus phosphatidylinositol 3- kinase (PI3K) binding site. This suggested that the two forms of erbB-4 might interact with dierent intracel- lular signalling pathways and therefore in¯uence a wider variety of cellular responses to heregulin than previously thought. Expression of both erbB-4 variants was found in 7/7 normal breast tissues but only in 9/12 breast tumours analysed. In line with the terminology of Elenius et al. (1997b) we have designated the two isoforms of the C- terminal transcripts as CT-a (full-length) and CT-b which lacks the PI3K binding motif. These results identify suitable cell lines for the further investigation of erbB-4 expression and function and suggest that the role of erbB-4 in breast cancer warrants further investigation with larger numbers of normal and malignant breast tissues. Keywords: receptor tyrosine kinase; breast cancer; erbB-4; signal transduction; phosphatidylinositol 3- kinase ErbB-4, a transmembrane receptor tyrosine kinase, is a member of the epidermal growth factor receptor (EGFR) family which in addition to the EGFR includes erbB-2 (Coussens et al., 1985) and erbB-3 (Kraus et al., 1989). Over-expression of the epidermal growth factor receptor and p185 erbB-2 has been reported in a wide variety of human malignancies (Modjtahedi and Dean, 1994; Hynes and Stern, 1994) and has been found to correlate with poor prognosis in patients bearing these tumours. Importantly, these molecules have been used as targets for the therapy of human disease (Baselga et al., 1996; Modjtahedi et al., 1996). Relatively little is known about the expression of the recently discovered, related receptor, erbB-4 (Plowman et al., 1993) and its importance in human malignancy but a group of ligands, neuregulins, which bind to and activate this receptor have been described. The neuregulin family (NRG) includes the products of two alternately spliced genes NRG1 (Marchionni et al., 1993) and NRG2 (Chang et al., 1997; Carraway et al., 1997) and a third gene, NRG3 (Zhang et al., 1997). The neuregulins bind to erbB-4 and erbB-3 and can stimulate phosphorylation of all four receptors via the formation of receptor heterodimers (Riese et al., 1995; Cohen et al., 1996b). In addition, there is evidence that ligands thought initially to bind exclusively to the EGFR also bind to and activate erbB-4 including betacellulin (Riese et al., 1996) and HB-EGF (Elenius et al., 1997a). The activated receptors interact with a variety of intracellular signalling pathways which can ultimately lead to either cell division or dierentiation. Clearly, it is important to determine the expression of p180 erbB-4 in cancer cells but preliminary experiments suggested that the level of expressed protein was low in many tumour cell lines. It was decided therefore to investigate initially erbB-4 expression by monitoring erbB-4 mRNA in human tumour cell lines most of which have been characterized previously in terms of their erbB-2 and EGFR expression. RT ± PCR is more sensitive than immunochemical techniques and by optimization of the number of PCR cycles the results obtained were semi-quantitative and re¯ected the dierential levels of mRNA present in the cell lines. Messenger RNA was isolated from 13 tumour cell lines of dierent origin (seven breast carcinoma, three ovarian, one glioma, one squamous cell carcinoma and one synovial cell carcinoma) then cDNAs were synthesized. PCR ampli®cation of cDNA using the erbB-4 speci®c primers, 253 and 256, resulted in the production of a 658 bp band in some cell lines which was the expected size for the product of erbB-4 ampli®cation (Figure 1a, upper panel). In addition, a second band was produced which was not anticipated and this appeared to be approximately 610 bp. Where the 658 bp product was observed then a 610 bp product was always present. The amount of the 658 bp PCR product varied and was absent from Correspondence: C Sawyer, Section of Immunology, McElwain Laboratories, Institute of Cancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, UK Received 22 December 1997; revised 24 March 1998; accepted 30 March 1998 Oncogene (1998) 17, 919 ± 924  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc