Oncogene (1997) 14, 1249 ± 1253  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

SHORT REPORT A novel human Wnt , WNT10B, maps to 12q13 and is expressed in human breast carcinomas

Thuan D Bui1, Julia Rankin2, Kenneth Smith1, Emmanuel L Huguet1, Steve Ruben3, Tom Strachan2, Adrian L Harris1 and Susan Lindsay2

1Growth Factors Group, Imperial Cancer Research Fund, University of Oxford, Institute of Molecular Medicine, John Radcli€e Hospital, Headington, Oxford OX3 9DU; 2University of Newcastle upon Tyne, Department of Human Genetics, Ridley Building, Newcastle upon Tyne NE1 7RU, UK; 3Human Genome Sciences, Inc., 9620 Medical Center Dr, Suite 300, Rockville, MD 20850 3338, USA

Several members of the Wnt gene family have been silent or expressed at low levels in this tissue causes shown to cause mammary tumors in mouse. Using mammary carcinomas. Thus mouse Wnt1, Wnt3, and degenerate primer polymerase chain reaction (PCR) on recently Wnt10b, has been shown to be some of the human genomic DNA, and speci®c PCR of cDNA oncogenes insertionally activated in the process of libraries, we have isolated a WNT gene which has not MMTV induced (Nusse and Varmus, previously been described in human. The gene is the 1982; Roelink et al., 1990; Lee et al., 1995). human homologue of mouse Wnt10b, recently shown to Furthermore, murine Wnt1, Wnt2, Wnt3a, Wnt5b, be one of the oncogenes cooperating with FGF3 in the Wnt7a and Wnt7b but not Wnt4, Wnt5a and Wnt6 development of mouse mammary tumour virus (MMTV) have been shown to transform mouse epithelial cells in induced mouse mammary carcinomas. The human vitro (Wong et al., 1994). WNT10B sequence was 88% and 95% identical to the To date the known members of the Wnt family in murine gene at nucleotide and levels, humans are WNT1, WNT2, WNT3, WNT3A, WNT4, respectively. YAC FISH mapping localises the gene to WNT5A, WNT7A, WNT7B and WNT8B (van Ooyen 12q13, a chromosomal region frequently rearranged in et al., 1985; Wainwright et al., 1988; Roelink et al., human tumours and also containing the WNT1 gene. In 1993; Huguet et al., 1994; Clark et al., 1993; Lejeune et normal and benign proliferations of human breast tissue, al., 1995; Lako et al., 1996). In studying the role of WNT10B expression was not detected by ribonuclease Wnt in human breast biology, we have previously protection assays but was found at low levels in RT ± shown that WNT2, WNT3, WNT4, WNT5A and PCR experiments. In contrast, using both methods, WNT7B genes are expressed in normal human tissue WNT10B expression was found to be elevated in 3 of 50 and that elevated levels of WNT2, WNT5A and primary breast carcinomas. Southern blot analysis of the WNT7B genes are found in proliferative lesions of carcinoma expressing the highest levels of WNT10B the breast (Huguet et al., 1994; Lejeune et al., 1995). showed no ampli®cation or rearrangement of the gene. Although these studies have established the expression The WNT10B gene was also expressed in some cancer pro®les of several WNT genes in breast tissue, human and non cancerous breast cell lines. These ®ndings homologues of a number of Wnt genes in lower suggest that the WNT10B gene may be involved in organisms have not yet been isolated, and therefore human breast cancer, and show that there is di€erential their possible role in breast biology has not been expression of the WNT10B gene in benign and malignant investigated. disease. Here we described the cloning and characterisation of a novel human WNT gene (WNT10B). We mapped Keywords: WNT10B; cloning; breast carcinoma WNT10B gene to 12q13 and analysed its expression in normal human breast tissues, benign breast proliferations and breast carcinomas.

The Wnt genes are a family of highly conserved genes, Cloning of the complete WNT10B coding sequence present in species ranging from invertebrates to mammals, which have a role in the morphological Degenerate primers designed from conserved regions G G development of tissues in both embryonic and adult of exon 4 (DEG-F: 5'-GGGGAATTCCA AGA AT- C G C C - contexts (Nusse and Varmus, 1992; McMahon, 1992). G TAA ATG TCA T-3', DEG-R: 5'-AAAATCTAGA G G G G Previous studies have suggested a role for Wnt genes in ACA ACACCA ATG AAA-3') (Gavin et al., 1990), mammary tissue biology (Gavin and McMahon, 1992; were used to amplify a 420 bp band from human Buhler et al., 1994; Edwards et al., 1992; Lin et al., genomic DNA. The PCR conditions were: 1 cycle of 1992). In the mouse, a subset of Wnt genes are 948C for 4 min, 35 cycles of 948C for 1 min, 608C involved in the development of the mammary gland for 2 min and 728C for 3 min, and followed by 1 and aberrant expression of those Wnt genes normally cycle of 728C for 10 min. The PCR product was cloned into pCR vector (Invitrogen) and colonies which did not hybridise to known human WNT Correspondence: AL Harris probes were further sequenced using an ABI TDB and JR contributed equally to this work Received 31 July 1996; revised 4 November 1996; accepted 5 automated sequencer. One novel clone (G81) had a November 1996 sequence that exhibited 94.5% identity over 130 A novel Wnt gene in human breast carcinomas TD Bui et al 1250

Figure 1 Nucleotide and amino acid sequence of human WNT10B assembled from sequences of genomic and cDNA clones (bases 1 ± 175 from the vectorette clone, 176±349 from 10BLI2, and 350 onwards from HIBCC33) and amino acid sequence of mouse Wnt10b (dashes represent identical amino acids). Conserved cysteine residues are shown in bold and potential N-linked glycosylation sites are double-underlined. Primers are underlined. GenBank accesssion number is X97057

amino acids to the published mouse Wnt10b parallel to these experiments, a clone was identi®ed in sequence (Lee et al., 1995) (also called Wnt12 by the sequence analysis project of Adamson et al., 1994) and was therefore named Sciences Inc. in a human fetal brain cDNA library human WNT10B. (donated by Dr Bento Soares, Columbia University, Since WNT10B cDNA was present at low abundance USA). This clone (designated HIBCC37) contained in the cDNA libraries tested (data not shown), a PCR- 841 bp of the 3' coding sequence and an additional based strategy was adopted. Speci®c primers M5'10bF 146 bp of 3' untranslated sequence. PCR of a vectorette (5'-CCAACACCGTGTGCTTGACC-3') (Lee et al., library constructed from ICI YAC clone 28AD4 using 1995) and H10BR (5'-GGCTGCCACAGCCATC- the universal vectorette primer and H10BR2 (5'- CAAC-3') were used to amplify a 910 bp partial GGTCAAGCACACGGTGTTGG-3') chosen from WNT10B cDNA clone from a 17-week human fetal the 5' end of the 910 bp cDNA clone generated another brain cDNA library in Lambda ZapII (Stratagene) 281 bp of the most 5' region and completed the coding using the PCR conditions as described above. The sequence of human WNT10B (Figure 1). The sequence sequence of this clone (10BLI2) was found to be 96% of WNT10B shows the typical conservation of cysteine identical to mouse Wnt10b over 303 amino acids and residues and N-linked glycosylation site common to all contained the expected conserved cysteine residues and Wnt genes. A number of non cysteine residues which are glycosylation sites (Figure 1). The sequence corre- either absolutely or frequently conserved amongst the sponded to nucleotides 461 to 1371 of the mouse Wnt genes are also present (Figure 1). The human Wnt10b cDNA, where the coding sequence extended WNT10B gene is 95% identical to the mouse Wnt10b from nucleotides 331 to 1500 (Lee et al., 1995). In gene over 389 amino acids. A novel Wnt gene in human breast carcinomas TD Bui et al

1251

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TGGGCCGGGCCATCTTATT-3') and primer en™es of the ™ell lines —re ™ited in „—ylorE€—p—dimitriou H10BR under the same PCR conditions described above. The WNT10B gene was found to be located on chromsome 12 (data not shown). The same PCR approach was used to screen the ICI YAC library (UK 1995). In addition, it was found to be expressed in HGMP Resource Centre) using conventional methods virginal mouse breast tissue and when insertionally (Anand et al., 1990). Two positive YAC clones were activated by MMTV, could cooperate with FGF3 to identi®ed (26BB4, 28AD4) to contain the WNT10B cause mouse mammary cancers (Lee et al., 1995). Table gene and used in the ¯uorescence in situ hybridisation 1 shows the expression of WNT10B in a panel of human (FISH) to metaphase spreads as described (Fantes et breast cell lines as detected using the ribonuclease al., 1992). Both YACs were non chimaeric and mapped protection analysis. WNT10B expression was variable to chromosome 12q13 (Figure 2). In mouse, the with, on average, 10 mg of RNA producing detectable Wnt10b and Wnt1 genes mapped to within 2.3 cM of signals after 48 h exposure. WNT10B expression was each other on chromosome 15 (Adamson et al., 1994), higher in cell lines from invasive breast cancer with the a region syntenic with human chromosome 12q13 exception of HB2. Amongst the breast cancer cell lines, (Mock et al., 1994) which also contained the human no relationship could be observed between hormone WNT1 gene. In addition, one of the YACs identi®ed status and level of WNT10B expression. (28AD4) also gave positive PCR signals with primers WNT10B was not detected in the MCF-7 parental speci®c for both WNT1 and WNT10B (data not breast cancer cell line (MCF-7 wt), but was expressed in shown). This conservation of synteny along with high a multidrug resistant clone which has lost estrogen sequence identity con®rmed that the WNT10B gene receptor expression and is able to grow in a hormone presented is indeed the orthologue of mouse Wnt10b. independent manner in vitro and in vivo (MCF-7 Adrr). The chromosomal localisation of WNT10B is of The variability between cell lines may provide a model interest from the point of view of tumorigenesis such system both for transfection of WNT10B and antisense as chromosomal rearrangements involving 12q13-q15 experiments to further evaluate the role of human were frequently observed in various soft tissue tumours WNT10B in breast cancer. (Nilbert et al., 1995; van den Ven et al., 1995), and Expression of WNT10B was also examined in 12q13 was a common site for human papilloma virus normal breast tissues, benign breast lesions and integration in cervical carcinoma (Lazo et al., 1992). primary breast carcinomas. Of these, this assay only detected expression in three out of 50 carcinomas tested (Figure 3). The elevated level of WNT10B Analysis of WNT10B expression in human breast cell lines and tissues expression in three out of 50 human primary breast carcinomas is consistent with the hypothesis that Recently, mouse Wnt10b was identi®ed at a MMTV WNT10B may contribute to the transformed pheno- insertion site in mouse mammary tumours (Lee et al., type in a minority of breast cancers. A novel Wnt gene in human breast carcinomas TD Bui et al 1252

a 5 7 r HB2 MCF-7 Adr SKBr3 MDA-MB-43 MDA-MB-15

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123456789101112131415161718192021222324252627 28

L LL

WNT10B

GAPDH

Figure 3 (a) Ribonuclease protection analysis of WNT10B mRNA expression in human breast tissues. 10 mg of total cellular RNA was used and performed as described in Table 1. Signals are shown for WNT10B and GAPDH protected fragments after 7 days and 1 day exposure, respectively. Lanes 1 ± 24 represent RNA extract from primary breast cancers, lanes 25 ± 27: RNA from benign breast tissues, and lane 28: (b) RNA from HB2 cells. Carcinomas 9, 22 and 24 expressed elevated levels of WNT10B

12345678910111213 bp and lymph node positive tumours. Of these, one (case LL 22) was estrogen receptor positive and epidermal —872 growth factor receptor negative, whilst the other (case —603 24) was estrogen receptor negative and epidermal growth factor receptor positive. The third carcinoma —310 with a high level of WNT10B expression (case 9) was a —234 small, lymph node negative, estrogen receptor negative —194 and epidermal growth factor receptor negative tumour. —118 Therefore, two of the three cases with elevated levels of WNT10B showed some common clinical features in Figure 4 Southern blot analysis of reverse transcriptase PCR in that they presented with large and locally invasive human breast samples. Reverse transcription was performed at carcinomas. 378C using Moloney murine reverse transcriptase (GIBCO-BRL) Southern blot analysis was performed to investigate with primer P2. The resulting single stranded cDNA was the possibility of genomic ampli®cation or rearrange- ampli®ed using primers P1 and P2 to yield a 309 bp PCR product which was then electrophoresed, blotted onto Hybond- ment of WNT10B in the over-expressing carcinomas. N+ membranes (Amersham) and hybridised to 1.2 kb PstI 10 mg of genomic DNA was digested with EcoRI, fragment of WNT10B cDNA from HIBCC33. Autoradiography HindIII or SalI, electrophoresed on 0.7% agarose gels, was done at 7708C for 12 h with intensifying screens. Lane 1: blotted onto Hybond-N+ membranes (Amersham) HB 2 cells; lane 2: placenta; lanes 3, 4, 7, 9 and 11: primary breast and hybridised to a32P-labeled 1.2 kb PstI WNT10B carcinomas: lanes 5, 6 and 13: normal breast tissue; lanes 8, 10 and 12: benign breast disease. Carcinomas 22 and 24 in Figure 3 cDNA fragment from HIBCC37. No evidence of are highlighted ampli®cation or rearrangement of the gene could be found (data not shown). Therefore, the increased expression of WNT10B in these cancers is presumably related to increased transcription or increased mRNA The WNT10B expression was further examined by stability. RT ± PCR followed by Southern hybridisation. This In summary we report the isolation and chromoso- technique con®rmed the higher levels of WNT10B mal mapping of a novel WNT gene previously expression in the three carcinomas above, and showed undescribed in the human. The expression of this that lower levels of expression of WNT10B were gene in human breast tissue further strengthens the present in all other samples tested (Figure 4). Two of data suggesting a role for WNT genes in human breast the three carcinomas expressing higher levels of biology, and its upregulation parallels recent results in WNT10B (cases 22 and 24) were large, locally invasive mouse experimental models of breast cancer. A novel Wnt gene in human breast carcinomas TD Bui et al 1253 Acknowledgements supported by a fellowship from the UK Medical Research We would like to thank Professor Andrew McMahon for Council. Our research was supported by awards from the the generous gifts of the known human WNT gene clones, Imperial Cancer Research Fund and the Wellcome Trust. and the UK HGMP for the monochromosomal hybrid cell Genbank accession number X97057. panel, ICI YACs and computing facilities. JR was

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