The Putative Ovarian Tumour Marker Gene HE4 (WFDC2), Is Expressed in Normal Tissues and Undergoes Complex Alternative Splicing to Yield Multiple Protein Isoforms
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Oncogene (2002) 21, 2768 ± 2773 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc The putative ovarian tumour marker gene HE4 (WFDC2), is expressed in normal tissues and undergoes complex alternative splicing to yield multiple protein isoforms Lynne Bingle1, Vanessa Singleton1 and Colin D Bingle*,1 1Respiratory Medicine Unit, Division of Genomic Medicine, University of Sheeld Medical School, Sheeld S10 2RX, UK The whey acidic protein (WAP) domain is a conserved 50 amino acids and includes eight cysteines in a motif, containing eight cysteines found in a characteristic conserved arrangement, hence the term 4-DSC (Ran- 4-disulphide core arrangement, that is present in a ganathan et al., 1999). The WAP domain is not number of otherwise unrelated proteins. WAP motifs however exclusive to WAP proteins but is found in are present in SLPI and ela®n, two antiproteinases numerous other proteins, where it may be present as located on chromosome 20q12-13, in a locus rich in multiple domains. WAP domain proteins are typically poorly characterized WAP domain proteins. One of small secretory proteins, which exhibit a variety of these proteins, which contains two WAP domains, is functions including those which eect growth and HE4 (also known as WFDC2), originally described as an dierentiation (Ranganathan et al., 1999; Schalkwijk et epididymis speci®c protein but more recently suggested al., 1999). From a genomic point of view, multiple to be a putative serum tumour marker for ovarian studies have shown that WAP domains are encoded on cancer. We have shown that HE4 is expressed in a single exons and it has been suggested that the modular number of normal human tissues outside of the male nature of such WAP containing proteins may have reproductive system, including regions of the respiratory arisen by exon shuing (Schalkwijk et al., 1999). A tract and nasopharynx, as well as in a subset of lung number of the well characterized members of the WAP tumour cell lines. Comparison of multiple HE4 cDNAs domain containing family have been shown to exhibit and RT ± PCR products with genomic sequence allowed antiproteinase function. Two such WAP proteins, the elucidation of the genomic organization. These ela®n and SLPI are of major importance in the defence studies revealed that HE4 can undergo a complex series of the lung and skin against release of unregulated of alternative splicing events that can potentially yield proteolytic enzymes by in¯ammatory cells in disease ®ve distinct WAP domain containing protein isoforms. (Schalkwijk et al., 1999). Ela®n contains a single WAP These results cast doubt on the potential role of HE4 as domain whereas SLPI contains two. The genes for a serum tumour marker speci®c for ovarian cancer and ela®n and SLPI are co-localized on chromosome 20 open the door to understanding the function of multiple and share a degree of co-regulation in as much as their WAP domain containing protein isoforms arising from a cellular expression pattern overlaps and they are both single gene. induced by many pro-in¯ammatory stimuli (Bingle et Oncogene (2002) 21, 2768 ± 2773. DOI: 10.1038/sj/ al., 2001; Sallenave et al., 1994). Analysis of the region onc/1205363 of chromosome 20 surrounding the ela®n and SLPI genes has shown that further WAP domain proteins Keywords: WAP domain; 4-disulphide core; gene are located within close proximity to these genes. A expression; alternative splicing; chromosome 20; ela®n recently identi®ed gene, Eppin, that appears to be expressed exclusively within the male reproductive tract, has been shown to contain both a WAP domain and a Kunitz-type domain (Richardson et al., 2001). The four disulphide core (4-DSC), containing Whey On the basis of this structure, it has been proposed that Acidic Proteins (WAP) are the major whey proteins in Eppin, which was also shown to undergo alternative the milk of many mammals and are considered to be splicing, will function as a protease inhibitor (Richard- the prototypic members of this family (Ranganathan et son et al., 2001). A number of other novel WAP and al., 1999). The WAP domain comprises approximately Kunitz-domain containing proteins are also predicted to be present in this region of Chromosome 20 (Perry et al., 1999; Trexler et al., 2001), suggesting that this *Correspondence: CD Bingle, Respiratory Cell and Molecular locus may be a `hot spot' for antiproteinase genes. Biology Laboratory, Respiratory Medicine Unit, Division of Close to the Eppin gene is HE4 (also known as Genomic Medicine, University of Sheeld Medical School, M128, WFDC2), a 2 WAP domain containing protein, Royal Hallamshire Hospital, Glossop Road, Sheeld S10 2RX, UK; initially identi®ed as a transcript exclusively expressed E-mail: [email protected] Received 31 October 2001; revised 29 January 2002; accepted 29 in the epididymis and suggested to be a marker for this January 2002 tissue (Kirchho et al., 1991, 1998). On the basis of its Alternative splicing of the human HE4 gene L Bingle et al 2769 similarity to ela®n and SLPI it has been suggested a that HE4 functions as an anti-proteinase within the male reproductive tract and is important in the process of sperm maturation (Kirchho, 1998). No studies have been performed on the HE4 protein to con®rm these functions. More recently, however, a number of independent studies have reported that HE4 is over expressed in ovarian tumours (Wang et al., 1999; Schummer et al., 1999; Hough et al., 2000). These observations have led to the proposal that, due to its small size and secreted nature, HE4 may serve as a potential serum marker for these types of cancers (Schummer et al., 1999). Analysis of data deposited on the Stanford Genomics Breast Cancer b Consortium Portal (http://genome.www.stanford.edu/ breast_cancer/) also reveals that HE4 expression is increased in some breast tumours (Perou et al., 2000). Additionally analysis of the data set generated by Ross et al. (2000) has shown that HE4 is highly expressed in a number of tumour cell lines, including Ovcar-3 and Ovcar-4 (ovarian), HT-29, HCT-116 and COL0205 (colon), MALME-3M (melanoma), MCF-7 (breast) and A498 and 786-0 (renal) found in the NCI 60 panel. These results further suggest that HE4 may have some utility as a cancer marker. The mechanism underlying this disregulated expression is unresolved. Previous studies have shown that expression of both ela®n and c SLPI is altered in a number of cancers (Robinson et al., 1996; Zhang et al., 1995; Yamamoto et al., 1997). In the case of ela®n expression in breast cancers, it has been shown that abnormal expression is the result of a transcriptional event (Zhang et al., 1997). Multiple cytogenetic studies have shown that the q12-q13.1 region of chromosome 20, in which all of these WAP domain containing genes are located, is abnormal in a number of tumours. For example, ampli®cation of this Figure 1 Constitutive expression of the human HE4 gene is region has been reported in both ovarian and breast limited to a subset of pulmonary epithelial derived tumour cell cancer (Larramendy et al., 2000; Tanner et al., 2000). lines and is found in multiple tissues outside of the male Deletions of this region have also been reported in oral reproductive tract. (a) A549 and BEAS-2B cells were obtained squamous cell carcinoma (Imai et al., 2001). These from American Tissue Culture Collection. NCI-H226, NCI-H358, NCI-H322 and NCI-H647 cells were a gift of Professor J studies suggest that gene present within this region of Carmichael, University of Nottingham. Total RNA were resolved chromosome 20 may play unde®ned roles in carcinogen- on denaturing agarose gels, Northern blotted and hybridized with esis and or tumour progression. random primed cDNA probes as previously described (Bingle and In view of the fact that both ela®n and SLPI are Bingle, 2000). The cell lines used are: A549 (lane 1), NCI-H647 expressed in multiple epithelium including that in the (lane 2), NCI-H226 (lane 3), NCI-H358 (lane 4), BEAS-2B (lane 5) and NCI-H322 (lane 6). Replicate blots were hybridized with reproductive tract and airways, we hypothesized that 32P labeled cDNA probes corresponding to HE4, ela®n and SLPI HE4 may also be expressed in cells of the pulmonary (Bingle et al., 2001). (b) A commercial multiple tissue poly(A)+ system where it may contribute to the host defence dot blot containing samples from 50 dierent human tissues, was function of the lung. Blast searches of the public EST hybridized with a random primed full length HE4 cDNA probe. Positive signals clearly above background are indicated by the databases with the published full length HE4 sequence grey arrows. (c) A Northern blot containing total RNA from (X63187) identi®ed multiple clones several of which normal peripheral lung and nasal septal epithelium, was were derived from lung tumour libraries. These clones hybridized with a random primed full length HE4 cDNA probe were obtained from the MRC HGMP, Cambridge, UK (http://www.hgmp.mrc.ac.uk/) and sequenced for ver- i®cation. We used one of these fully sequenced ESTs experiments HE4 expression was also found in (accession number BE674284), as a probe on Northern CORL23 cells but not in CORL279 or NCI-H841 blots of total RNA isolated from a variety of lung cells (results not shown). No consistent pattern of derived cell lines. Expression of HE4 was found in expression in certain tumour types was noted. To look NCI-H226, NCI-H358 and BEAS-2B cells (Figure 1a, at the overlap of expression with ela®n and SLPI, lanes 3 ± 5) but not in A549, NCI-H447 and NCI-H322 replicate blots were also probed with these two cells (Figure 1a, lanes 1, 2 and 6).