A Novel Human Wnt Gene, WNT10B, Maps to 12Q13 and Is Expressed in Human Breast Carcinomas
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Oncogene (1997) 14, 1249 ± 1253 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 SHORT REPORT A novel human Wnt gene, 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 Radclie 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 carcinogenesis (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 amino acid 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 genes 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 dierential 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 chromosome 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 Human Genome 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 le I ionulese protetion nlysis of xIHf mxe expression in humn rest ell lines xIHf gell line ype mxe fenign rest omponent PFSFP ± RH immortlised luminl ell wRFI C RH immortlised luminl ell wRFP C smmortl non mlignnt wgpEIHe ± RH immortlised luminl ell rfP CC grinom in situ omponent SFQFI ± grinom in situ omponent QFRFI ± i±ve rest ner wheEwfEISU CC i±ve rest ner wheEwfEPQI C i±ve rest ner wheEwfERQS CC i±ve rest ner wheEwfERSQ ± i±ve rest ner wheEwfERTV CC i±ve rest nerD ufrQ ± mplified erfP iCve rest ner wgpEU wt Figure 2 Hybridisation of a probe made from ICI YAC 28AD4 ± r hrug resistnt wgpU wgpEU edr (containing WNT10B) to metaphase chromosome spreads CC RUh iCve rest ner showing signal on 12q13 (arrowheads). Digital Scienti®c C rUSFI iCve rest ner Smartcapture software was used to show G-banding of the CC chromosomes he level of xIHf mxe expression ws qulittively ssessed fter U dys exposure sXCCahigh expressionY Calow expressionY ±ano expression nd iaestrogen reeptorF IH mg of totl ellulr xe extrted from on¯uent ells ws inuted in oktil S QP FpFmF of Elelled pfntIH ntisense riopE ontining IH R QP FpFmF of humn Elelled glyerldehydeEQE roes nd STIH Chromosomal assignment of the human WNT10B gene phosphte dehydrogense @qehrA ntisense rioproes Vg s desried @eusuel et PCR screening of a human/hamster or mouse @wgrthy nd fiknellD IWWPA t RP lFD IWWHAF pfntIH ontined QHW p mpli®ed frgment of from rsfggQU using I nd P primers @see pigure IAF monochromosomal somatic cell hybrid panel (UK xIHf io site of pfluesript HGMP Resource Centre) was used to amplify a he g frgment ws loned into the xIHf ntisense rioproes were generted using 204 bp fragment from exon 4 of the WNT10B gene uC in whih rindsss linerised pfntIHF eferE using human speci®c primer H10BF (5'- U xe polymerse with et lF @IWWQA TGGGCCGGGCCATCTTATT-3') and primer enes of the ell lines re ited in ylorEpdimitriou H10BR under the same PCR conditions described above.