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 HE4 (WFDC2), is expressed in normal tissues and undergoes complex alternative splicing to yield multiple isoforms

Lynne Bingle1, Vanessa Singleton1 and Colin D Bingle*,1

1Respiratory Medicine Unit, Division of Genomic Medicine, University of Sheeld Medical School, Sheeld 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 . 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 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 e€ect growth and HE4 (also known as WFDC2), originally described as an di€erentiation (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 shu‚ing (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 for a serum tumour marker speci®c for ovarian cancer and ela®n and SLPI are co-localized on 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 Sheeld Medical School, M128, WFDC2), a 2 WAP domain containing protein, Royal Hallamshire Hospital, Glossop Road, Sheeld S10 2RX, UK; initially identi®ed as a transcript exclusively expressed E-mail: c.d.bingle@sheeld.ac.uk 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 di€erent 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). In further cDNAs. Consistent with previous studies, ela®n

Oncogene Alternative splicing of the human HE4 gene L Bingle et al 2770 expression was more restricted than that of SLPI (Sallenave et al., 1994; Bingle et al., 2001). Abundant ela®n mRNA was seen in NCI-H647 cells (Figure 1a, lane 2) with lower levels being found in A549 and BEAS-2B cells (Figure 1a, lanes 1 and 5). In contrast to the restricted expression of HE4 and ela®n, SLPI mRNA was found to be expressed in all six lines tested, with the highest level found in the NCI-H322 cells (Figure 1a, lane 6). In light of the observed expression of HE4 in a number of lung cancer cell lines and to determine if HE4 was expressed in normal human lung and a wider range of tissues, we probed a multiple tissue Poly(A)+ Figure 2 Human HE4 is highly conserved with that from other RNA dot blot, containing RNA from 50 di€erent species. The derived amino acid sequence of human HE4, is human tissues, with the same HE4 probe. HE4 was aligned with dog, pig, rabbit, mouse and rat proteins. Rabbit and most abundantly expressed in trachea (Figure 1b) with dog sequences were from the cloned genes whereas the pig, mouse signi®cant expression also being found in salivary and rat sequences were derived from conceptual translations of clones assembled from the EST databases. The rat sequence gland, kidney and lung (Figure 1b). Readily detectable, represents a partial sequence. The human HE4 sequence is (but weak) expression was also found in a variety of amended from that presented in X63187 due to errors in the other tissues. Interestingly, signi®cant expression was primary sequence submission (see text section). Identical amino not noted in whole testes RNA on the dot blot. acids are indicated by white on black and conserved amino acids Abundant HE4 expression was also found in RNA by white on grey. A (7) indicates where gaps are inserted for maximum alignment. The position of the conserved C residues in isolated from nasal epithelium (Figure 1c). Both SLPI the two WAP domains are indicated by * and ela®n are also expressed within the nasal septal epithelium (results not shown). These results suggest that HE4 is expressed in a number of regions of the pulmonary system as well as in multiple other tissues resulting protein is therefore one amino acid shorter and counter the suggestion that HE4 is a epidydimal than that previously published. The C-terminal WAP speci®c transcript. The co-expression of HE4, SLPI domain was more highly conserved than the N- and ela®n in these tissues lends further support to our terminal domain although two fewer amino acids were hypothesis that HE4 may be part of the host defence found between Cysteines C2 and C3 in the rabbit, shield of the airways and within the oral-nasal mouse and rat sequences compared to the equivalent environment. The widespread distribution of HE4 sequence in the other proteins. This observation has expression is also con®rmed by the existence of a large previously been noted in other multiple WAP domain number of ESTs (148) from multiple tissues present containing proteins (Schalkwijk et al., 1999; Simpson et within the Genebank database. These ESTs are derived al., 2000). Both WAP domains conserve what are from 13 normal tissues as well as from tumours derived considered to be functionally important regions, from ®ve organs, with tumours of the female designated blocks 1, 2 and 3. Block 1, which has the reproductive tract being most prominent. sequence KXGXCP, is strikingly conserved (Ranga- Previous studies have shown the existence of HE4 nathan et al., 1999). homologues in rabbit (BE-20, U26725) (Xu et al., Unexpectedly, the alignment also revealed a sig- 1996) and dog (CE4, S77395) (Ellerbrock et al., 1994). ni®cant di€erence in the deduced size and presumed By searching the public EST databases we were able to structure of the mouse and rat HE4 proteins compared identify further homologues from pig (represented by to that of the human, pig, rabbit and dog homologues BE234395), mouse (AF334269) and rat (represented by (Figure 2). In the latter proteins the two WAP domains AI411527). Translation of such EST sequences allowed directly join whereas in the mouse and rat proteins the the deduction of the completed sequence of the pig and two domains are distant from each other by 54 and 48 mouse proteins and the almost complete sequence of amino acids respectively. Such a structure is unique the rat protein. Alignment of the deduced amino acid amongst previously identi®ed two WAP domain sequence of all of these proteins (Figure 2) revealed a containing proteins. In WAP proteins and SLPI the high degree of particularly within two domains are directly repeated with no intervening the two WAP domains. The sequence of the human linker region (Ranganathan et al., 1999). In all HE4 protein used in this alignment is not identical to previously characterized WAP domain containing that found in the original submission (X63187) genes, individual WAP domains are encoded by single (Kirchho€ et al., 1991). Our sequence analysis, exons (Schalkwijk et al., 1999). It might be expected combined with that of multiple ESTs in the database, therefore that in the rat and mouse HE4 genes the suggests that the original cDNA contains some linker regions would be encoded by additional exons. sequencing errors. In the amended HE4 peptide The functional signi®cance of such regions is unclear sequence an S replaces an L immediately C-terminal but it might be expected that this sequence would to the eighth C in the ®rst WAP domain and a C two result in the two WAP domains being positioned a amino acids C-terminal to this is removed. The signi®cant distance from each other.

Oncogene Alternative splicing of the human HE4 gene L Bingle et al 2771 When we compared the sequences of two of the lung a derived HE4 ESTs (AI298658 and AI680769) with those of the published HE4 sequence it was clear that these two clones contained regions of novel sequence. By alignment of these sequences with the region of genomic DNA containing the HE4 gene (AL031663) we could determine that all three contained di€erent exonic arrangements suggesting that the HE4 gene could undergo alternative splicing. We used a combination of approaches to elucidate the full genomic structure of the HE4 gene. Firstly, we compared the sequences of all of the human HE4 containing ESTs (http://www.ncbi.nlm.nih.gov/Uni- Gene/clust.cgi?ORG=Hs&CID=2719) with AL031663 and secondly, we used RT ± PCR analysis with multiple primer pairs and human lung and lung cell line RNA as template. This analysis resulted in the establishment of the gene structure as outlined in Figure 3a. The human HE4 gene contains ®ve exons and spans approximately 12 Kb. Within this structure the third intron is over 8 kb b long. The WAP domains are encoded on single exons, designated 2 and 4. This structure shows that full length HE4 is the results of splicing of exons 1, 2, 4 and 5. Exons 3 and 4 can exist in three forms, two of which can be c spliced. The 5' portion of each of these exons was not found (by the use of repeated RT ± PCR analysis with multiple primer pairs) to be spliced to the preceding exons and may therefore, represent exons which use novel promoter regions. Such formal designation will require additional studies. The complex nature of the HE4 gene was con®rmed by the isolation of multiple individual mRNA species. These are represented schematically in Figure 3a and examples of di€erent RT ± PCR products are shown in Figure 3b. Such analysis also suggests that the levels of these mRNAs may be di€erentially regulated. Figure 3 The genomic organization of the human HE4 gene By conceptual translation of these mRNAs we were reveals that multiple transcripts can generate unique peptide able to deduce that HE4 protein can potentially exist as products with di€erent domain structures. The complete human at least ®ve isoforms. These are represented in Figure 3c. HE4 locus is contained with 12 Kb on the PAC clone RP3- As described above, full length HE4 contains two WAP 461P17 (Accession number AL031663). (a) A schematic repre- sentation of the HE4 gene structure based on sequences of EST domains whereas all of the additional protein isoforms cDNAs and RT ± PCR products. The number above represent the contain only a single WAP domain. The two N-terminal exons and those below represent the exon sizes in bps. The shaded WAP domain containing proteins, HE4-V1 and HE4-V4 boxes represent the exons containing the two WAP domains. The have di€erent C-terminal sequences. One C-terminal exons used to generate the HE4 isoforms are shown. Only WAP isoform (HE4-V2) is the result of splicing of exon 1 isoforms which generate open reading frames containing WAP domains are indicated. Exons 3 and 3b are not found in any of into exon 4 and retains the same signal peptide region as the cDNA products that generate WAP domain containing full length HE4. The further C-terminal WAP domain reading frames. Small arrows mark the putative translation start containing isoform (HE4-V3) is the product of the sites and the (*) indicate positions of the inframe stop codons. The position of the primers used for the RT ± PCR studies shown below are indicated. (b) Reverse transcription (RT) PCR was performed using 1 mg of total RNA and oligo dT priming. Primers were designed with Primer-3 (http://www-genome.wi.mi- F6 (5' GCA AGA TTT TCC CCA ATT CC 3')/R3 (5' CTC TCC t.edu/cgi-bin/primer/primer3_www.cgi). The annealing tempera- TCA CTG CTC AGC CT 3') (lanes 2 ± 6), F1 (5' CGG CTT ture of all primers was 608C. Reactions were performed for CAC CCT AGT CTC AG 3')/R1 (5' AAA GGG AGA AGC between 30 ± 40 cycles and the resulting products were resolved on TGT GGT CA 3') (lanes 7 ± 11) and F1/R4 (5' TGG CTG CTG 1% Metaphore (Seakem) TAE gels. Individual products, GAG TCA TAA TG 3') (lanes 12 ± 16). (c) A schematic identi®ed by Southern blotting, were excised from the gels and representation of full length human HE4 (FL) is aligned with cloned using the TOPO PCRII system (Invitrogen). All products that of the four possible protein isoforms (HE4-V4) (AF330262), were sequenced in both directions. RT ± PCR analysis was HE4-V1 (AF330259), HE4-V2 (AF330260) and HE4-V3 performed with multiple primer pairs on total RNA isolated (AF330261) derived from sequence analysis of cloned RT ± PCR from human lung (lanes 2, 7, 12), BEAS-2B cells (3, 8, 13), H226 products and EST clones. The putative signal sequence, the cells (lanes 4, 9, 14) and H358 cells (lanes 5, 10, 15). RT negative position of the two WAP domains and the unique peptide controls are shown in lanes 6, 11 and 16. The primer pairs were sequences are indicated

Oncogene Alternative splicing of the human HE4 gene L Bingle et al 2772 putative alternative promoter region 5' of exon 4b and analysis of mouse HE4 is less extensive than that has a novel N-terminal sequence. The existence of this performed with the human gene we conclude that the number of putative HE4 proteins suggests that multiple mouse gene is able to undergo similar alternative antibodies may be needed to di€erentiate between these splicing to that seen in the human gene. This isoforms in analysis and as a tool for potential tumour observation supports our contention that such events diagnosis. It also remains to be seen if these di€erent may be of biological signi®cance. isoforms are regulated independently. A number of ESTs In summary we have shown that far from being (BE515017, AW102571, AW003747, AI680769 and restricted in its expression to the epididymis, HE4 is BF111273) and RT ± PCR products were found to expressed in a number of tissues including those of the contain exons 3b and 3a spliced into exon 4 or exons oral, nasal and upper respiratory regions. HE4 3b, 3a and 3 spliced into exon 4. No ATGs in frame with expression is found in a subset of lung derived tumour the C-terminal WAP domain are produced by these cell lines. Analysis of the HE4 gene reveals that the transcripts. It may be that HE4 transcripts containing genomic structure is more complicated than previously this exonic arrangement produce unrelated proteins. A described WAP proteins and show that the gene can similar observation has been reported for the HE2 gene, undergo complex alternative splicing that can yield at which can yield a b-defensin molecule when spliced in least ®ve putative protein isoforms. In light of these one isoform but not when spliced in another ®ve observations and the previously published data show- isoforms (Jia et al., 2001). ing HE4 overexpression in both ovarian and breast To add weight to our ®nding of multiple alterna- tumours, we suggest that understanding the regulation tively spliced HE4 gene products arising from the and expression of these isoforms in both normal and human gene we performed a search for potential tumour tissue may con®rm HE4 as a novel, useful, alternatively spliced products of mouse HE4. We were tumour marker. able to identify a single EST in the public database (BB565306, obtained from mouse tongue) which represents an N-terminal single WAP domain contain- ing protein. We then performed RT ± PCR using Acknowledgements primers corresponding to the 5' and 3' ends of the We are grateful to Dr RC Read who kindly provided mouse cDNA. Using these primers we were able to samples of nasal septal tissue. The UK-HGMP at Cam- clone a cDNA corresponding to a single C-terminal bridge provided IMAGE clones used in this study. The WAP domain containing protein. Although this study was supported in part by The Wellcome Trust.

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