Differential Expression of Human Wnt Genes 2, 3, 4, and 7B in Human Breast Cell Lines and Normal and Disease States of Human Breast Tissue'

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ICANCERRESEARCH54,2615-2621,May15,19941 Differential Expression of Human Wnt Genes 2, 3, 4, and 7B in Human Breast Cell Lines and Normal and Disease States of Human Breast Tissue'

Emmanuel L. Huguet, Jill A. McMahon, Andrew P. McMahon, Roy Bicknell, and Adrian L Harris2

Growth Factors Group, Imperial Cancer Research Fun4 University of Oxford, Institute ofMolecular Medicine, John Radcl(fte Hospita4 Headington, Oxford, 013 9DU, United Kingdom fE. L. H., R. B., A. L. H.], and Departments ofBiochemisny and Molecular Biology and Cellular and Developmental Biology, Harvard University, Massachusetts 02138 (I. A. M., A. P. M.J

ABSTRACT the cell surface. However, relatively little is known about the precise molecular structure and function of the Wnts because the strong Wit: gene expression was investigated by ribonuclease protection anal association of the proteins with the extracellular matrix has to date ysis in human breast cancer, nontumorous breast tissue, and a variety of human breast cell lines. We report the expression of Wnt3, Wn14,and prevented isolation of active protein. Wnt7b In human breast cell lines and Wnt2, Wnt3, Wn14, and Wni7b in At a cellularlevel, Wntgenes show specific spatiotemporalexpres human breast tissues. Wnt3a and Wnt7a were absent in the cell lines and sion in both embryonic and adult tissues of species in which they have tissues tested. The level ofexpression of WntZand Wnt4 was 10. to 20-fold been investigated. Studies in the mouse (14), Xenopus (15), and other higher In fibroadenomas than It was in normal or malignant breast tissue, systems all suggest a role for Wnt genes as signal transducers func and in 10% oftumors Wnt7b expression was 30-fold hiajier than in normal tioning locally to produce biological effects in cells adjacent to the orbenign breast tissues. In contrast to the mouse, in which Wail and Wnt3 source of secretion. Thus, Wnt genes play a part in tissue organization are involved in tumorigenesis, our results suggest that Wn12,Wn14,and events such as central nervous system development in the mouse and Wnt7b may be assodated with abnormal proliferation in human breast segmentation in Drosophila. tissue. Interest in the role of Wnt genes in breast biology stems from studies in the mouse. Gavin and McMahon (16) observed that INTRODUCTION mouse breast tissue showed differential expression of Wnt4, Wnt genes (reviewed in Refs. 1 and 2) form a large family, the first Wnt5a, Wnt5b, Wnt6, and Wnt7b which correlated with mammary member of which was discovered as a result of its role in mouse changes associated with pregnancy and lactation. This and the mammary cancer (3). In murine breast tumors, mouse mammary work of Buhler et a!. (17), demonstrating the expression of Wnt2 tumor virus was found consistently at preferential loci in the tumor in the mouse mammary gland, suggest a role for Wnt genes in DNA. The gene activated at the commonest of these integration sites normal breast development. In addition, a number of lines of (initially called mt 1) was later shown to be homologous to the evidence show that some Wnt genes have growth-promoting prop Drosophila â€œwinglessâ€•gene(4), and the combination of these two erties in breast epithelium. Thus, Wntl and Wnt3 are two of the terms gave rise to the name â€œWntâ€•(5). genes, which, when activated by mouse mammary tumor virus, Numerous new Wnt genes have subsequently been identified in cause mouse mammary tumors. Also, Writi (18) and Wnt2 (19) manyorganismsincludingnot only Drosophila(6), Xenopus(7), mice transform some mouse mammary epithelial cell lines in vitro. Wntl (8), and humans (9) but also a variety of fish, birds, and reptiles (2). transgenic mice (20) and mice bearing reconstituted mammary Of the above, the profile of Wnt expression and function has been glands transfected with Wntl (21) show hormone-independent studied in most detail in the mouse. Ten murine genes have been hyperplasia and an increased incidence of breast tumors. Wntl may identified, which show homology ranging from 50 to 90% in amino produce these effects in both autocrine and paracrine mechanisms acid content (8). (22). It is pertinent that those Wnt genes which are associated with The factors which control Wnt gene expression either as individual growth deregulation are those which are absent in the normal adult genes or as an interacting group have not yet been identified. Wnt breast. Thus, an overall hypothesis consistent with the above data proteins contain 350â€”380amino acids, including 24 highly conserved is that, in the mouse, Wnt genes are involved in the developmental cysteines and a number of glycosylation sites which, with one excep processes of normal breast but aberrant Wnt gene expression can tion, are poorly conserved. In Wntl, with the exception of some contribute to the development of mammary cancers. temperature-sensitive mutants, mutations affecting cysteine residues In view of this we have investigated whether a similar mecha abolish biological activity, whereas those affecting glycosylation sites nism might operate in some human breast cancers. The only human do not (10, 11). When in the cell, the protein is bound to the Writ genes so far described are Wntl, Wnt2 and Wnt3. Using PCR3 chaperonin-binding protein (12) and is associated with the endoplas techniques, we have generated fragments specific to several human mic reticulum and the Golgi apparatus. The N terminus of the protein Wnt genes for use as templates in ribonuclease protection analysis. carries a signal peptide as well as an adjacent signal peptidase recog We have examined Wntgene expression in benign and malignant nition site. This, together with the observations that mutants lacking diseases of the breast and compared their expression profile to that the signal peptide are inactive (10) and that there are no apparent of normal breast tissues. An immortalized human breast epithelial transmembrane domains, suggests that Wnt protein is secreted. In cell line and human breast cancer cell lines were examined as a deed, Papkoff and Schryver (13) have shown that Wntl is present on pure epithelial population. Our results show that, as in the mouse (16), only a subset of Win gene are expressed in normal human Received 11/16/93; accepted 3/15/94. breast tissue. Interestingly, quantitative differences in expression Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage chargesThisarticlemustthereforebeherebymarkedadvertisementinaccordancewith are seen between normal tissue and benign and malignant tumors 18 U.S.C. Section 1734 solely to indicate this fact. of the human breast. I This work was funded by the Imperial Cancer Research Fund, the Oxford District ResearchCommittee[E.L H., R. B., A. L H.], and HoffmanLa Roche[J. A. M., A. P. M.J. E. L H. and J. A. M. contributed equally to this study. 3 â€˜l'heabbreviations used are: PCR, polymerase chain reaction; DMEM, Dulbecco's 2 To whom requests for reprints should be addressed, at Imperial Cancer Research modified Eagle's medium; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phos Fund, University of Oxford, John Radcliffe Hospital, Headington, Oxford 0X3 9DU, phate dehydrogenase; EGFR, epidermal growth factor receptor; MCF7adr, Adryami UnitedKingdom;ER,estrogenreceptor. cm-resistant MCF7; ER, estrogen receptor. 2615

MATERIALS AND METHODS breast tissue. Details of the samples of breast tissue from which RNA was extracted are given in the legend to Fig. 2. Normal breast RNA was CellCulture.Celllinesusedinthisstudywereobtainedfromthefollow obtained either from breast reduction tissue samples (n = 4) or from normal ing sources: MTSV1â€”7(23)from Dr Joyce Taylor, Imperial Cancer Research breast tissue adjacent to diseased breast excised at operation (n = 4). For Fund,London;T47D, MDA231, MDA361, BT2O,MDA415, MDA453, and normal tissues the age range of the patients was 18â€”78years (mean, 42 MDA157 from the American Type Culture Collection, Bethesda, MD; years; median, 46 years). The tissues representing benign breast disease ZR9BI1 and ZR75 from Dr. Eva Valvenus, University Hospital, 5-75185, Uppsala,Sweden;MCF7from Dr. BarbaraDurkacz,CancerResearchUnit, consisted of fibroadenomas (n = 5), benign phylloides tumors (n = 2), and fibrocystic disease (n 2). For benign tissues the age range of the patients University of Newcastle upon Tyne; and MCF7adr from Dr. Ken Cowen, National Institutes of Health. MTSV17 were cultured in DMEM-10% FCS, was 25â€”52years (mean, 39 years; median, 38 years). The tumor tissues with 5 g.@g/mlhydrocortisone(Sigma) and 20 gig/mIbovine insulin (Sigma). were obtained postoperatively and were chosen to represent different MDAMB157 was cultured in RPMI medium-10% FCS. All other cell lines subgroups (patient age range, 32â€”86years; mean, 56 years; median, 57 were cultured in DMEM-10% FfS. All cells were cultured on plastic culture years. By receptor status the tumors consisted of 40% ER positive, EGFR plates (Becton Dickinson) at 37Â°C,5%C02, 95% air, in a humidified incu positive; 30% ER positive, EGFR negative; 30% ER negative, EGFR bator. All cultures were free of Mycoplasma. DMEM and RPMI were obtained positive. The size of the tumors at the time of resection ranged from 8â€”40 from the Imperial Cancer Research Fund Clare Hall laboratories. FCS was mm, 40% of the tumors were lymph node positive, 19 of the tumors were obtained from Globepharm. ductal and one was lobular in type). Receptor analysis and tumor tissue Tissue Selection. Human breast tissue samples were selected as follows. handling was performed as previously described by Lejeune et al. (24). Wnt expression was examined in normal, benign disease, and malignant Tumors were considered ER positive if analysis showed at least 10 fmol

H Wnt-3a GLSGSCEVXT CWWSQPDFRA IGDFLKDKYD SASEMVVEKH H Wnt-3a GLSGSCEVKT CWWSQPDFRT IGDFLXDKYD SASEMVVEXH

H Wnt-3a RESRGWVETL RPRYTYFKVP TERDLVYYEA SPNFCEPNPE M Wnt-3a RESRGWVETL RPRYTYFXVP TERDLVYYEA SPNFCEPNPE

H Wnt-3a TGSFGTRDRT CNVSSHGIDG CDLLCCGRGH NARAERRREK M Wnt-3a TGSFGTRDRT CNVSSHGIDG CDLLCCGRGH NARTERRREK

H Wnt-3a CRCV M Wnt-3a CHCV

H Wnt-4 GVSGSCEVKT CWRAVPPFRQ VGHALKEKPD GATEVEPRRV M Wnt-4 GVSGSCEVKT CWRAVPPFRQ VGHALXEKFD GATE VEPRRV

H Wnt-4 GSSRALVPRN AQFXPHTDED LVYLEPSPDF CEQDMRSGVL N Wnt-4 GSSRALVPRN AQFKPHTDED LVYLEPSPDF CEQDIRSGVL

H Wnt-4 GTRGRTCNXT SXAIDGCELL CCGRGFHTAQ VELAERCSCK M Wnt-4 GTRGRTCNXT SXAIDGCELL CCGRGFHTAQ VELAERCGCR

Fig. 1. Amino acid sequences of PCR-generated fragments of human Wnt3a, Wnt4, Wnt7a, and Wnt7b, aligned with the corresponding mouse se quences. H, human sequences; M, mouse sequences. H Wnt-7a GVSGSCTTXT CWTTLPQFRE LGYVLKDKYN EAVHVEPVRA M Wnt-7a GVSGSCTTKT CWTTLPQFRE LGYVLKDKYN EAVHVEPVRA

H Wnt-7a SRNXRPTFLK IKKPLSYRKP MDTDLVYIEK SPNYCEGDPV M Wnt-7a SRNKRPTFLK IKXPLSYRKP MDTDLVYIEL SPNYCEEDPV

H Wnt-7a TGSVGTQGRA CNKTAPQASG CDLMCCGRGY NTHQYARVWQ M Wnt-7a TGSVGTQGRA CNKTAPQASG CDLMCCGRGY NTHQYARVWQ

H Wnt-7a CNCK M Wntâ€”7a CNCX

H Wnt-7b GVSGSCTTKT CWTTLPKFRE VGHLLXEKYN AAVQVEVVRA M Wnt-7b GVSGSCTTKT CWTTLPXFRE VGHLLKEKYN AAVQVEVVRA

H Wnt-7b SRLRQPTFLR IXQLRSYQKP METDLVYIEX SPNYCEEDAA M Wnt-7b SRLRQPTFLR IXQLRSYQKP METDLVYIEK SPNYCEEDAA

H Wntâ€”7b TGSVGTQGRI CNRTSPGADG CDTMCCGRGY NTHQYTKVWQ M Wnt-7b TGSVGTQGRI CNRTSPGADG CDTMCCGRGY NTHQYTKVWQ

H Wnt-7b CNCK M Wnt-7b CNCK 2616

Wag GENE EXPRES5ION IN HUMAN BREAST TISSUE

lineswnt3wnt4wnt7hMTSV1-7+@@(1)b+(50)MCF7â€”â€”+(30)ZR75â€”â€”+(60)T47Dâ€”â€”+(50)MCF7adr+Table 1 Summaryof Wnt expression profile in human breast cell protocol. [a-32P]dCTP (Amersham) labeled probes were generated by random priming. Southern analysis of blotted DNA was performed using standard protocols (26). Construction of Wnt Templates for RNase Protection Analysis. Using reverse (5'-AAAATCFAGAACAACACCAATGAAAA-3') and forward (5'- 0000AATFCCAAGAATGTAAATGTCAT-3') primers, expected frag (1)MDA231â€”â€”+(iOO)MDA361â€”â€”+(10)â€”+ ments of 406 and 424 base pairs were generated by PCR amplification from (50)BT2O+ plasmids pIRP (supplied by Dr. Robert Williamson, St. Mary's Hospital, (1)â€”+(i60)MDA415â€”+ London) and pHPl (27) (supplied by Dr. Rod Nusse, Stanford University, (20)MDA457â€”â€”+ (1)+ (45)MDAMB157â€”â€”+ Palo Alto, CA), which contain full-length human Wnt32 and Wnt3 cDNAs, (4)ZR9B11â€”â€”+ respectively. The Wnt2 fragment was subcloned into the EcoRV site of (50) pBluescript 5K, and the Wnt3 fragment was subcloned into the EcoRI and a @,expression detected; â€”,expression not detected. XbaI sites of pBluescipt KS. Both constructs were sequenced to confirm their b Numbers in parentheses, relative level of Wnt expression in each cell line. identity. The BamHI-linearized Wnt2 construct and the EcoRI-linearized Wnt3 construct were used to generate [a-32P]CFP-labeled antisense probes with â€˜11 specific binding sites per mg cytosolic protein and EGFR positive if RNA polymerase. The full-length Wnt2 and Wnt3 probes are then 487 and 470 analysis showed at least 20 fmol specific binding sites per mg membrane base pairs, respectively, and can be distinguished from the respective protected protein. fragments of 389 and 400 base pairs. RNA Preparation. RNA was prepared from cells in vitro and from tissues Probes for human Wnt3a, Wnt4,Wnt7a, and Wnt7bwere isolated according by acid guanidium thiocyanate phenol chloroform extraction, as described by to previously published procedures (8) by PCR amplification using 100 ng of Chomczinskiand Sacchi (25). human genomic DNA and the degenerate forward (5@@GGGGAATfCCAA/ DNA Preparation. Genomic DNA was prepared from tissue samples GGA1O'l'Â°/C@/GT0/CCAT3) and reverse (5?@AAAATCFAGA@@/0CAA/ by lysis with proteinase K and sodium dodecyl sulfate, followed by 0CACCAA/0TG@@/@AA@3l)primers@PCRproducts for Wnt4(expected size, 360 purification with phenol chloroform isoamyl alcohol using standard base pairs) and Wnt3a, Wnt7a, and Wnt7b (expected size, 372 base pairs) were protocols (26). subcloned into the plasmid pGem 3z (Promega) and their nucleotide sequences Southern Blot and Slot Blot Analysis. Genomic DNA was isolated from determined by dideoxy chain termination sequencing. Antisense [a-32PJCTP tissues as above and digested with EcoRI. Digested DNA (20 pg) was size labeled RNA probes were generated from EcoRI-linearized templates by SP6 selected by electrophoresis on a 1% agarose gel and blotted onto a Hybond polymerase. The amino acid sequences of the human Wnt3a, Wnt4, Wnt7a, and N membrane. Serial dilutions of 1 ng of digested DNA were prepared and Wnt7b fragmentsgeneratedby PCR are shown in Fig. 1 with the corresponding slot blotted onto a Hybond N membrane using a Bio-Rad apparatus and mouse sequences.

WNT2 EXPRESSIONPROFILE

WNT2 SIGNAL @@@@@ @.. -Sâ€”-

normal tissue benign tissue malignant tissue @ . .@ 0 @*

GAPDH SIGNAL

Wnt2: normal tissue 20 Wnt2: benign tissue Wnt2: tumor tissue 20

15 15

10 10 10

5 5

U :@:+:@:++ ++:1::@:++ + ++ + ::::@: :t:

Fig. 2. RNase protection assay showing Wnt2and corresponding GAPDH signals in normal, benign, and malignant human breast tissue samples. Graphs show densitometric analysis ofthedata.Normaltissues:A,normaltissueadjacenttotumor;8, normaltissueobtainedfrombreastreductions.Benigntissues:FA,fibroadenoma;FC,fibrocysticdisease;Ph,benign phylloides tumor. Malignant tissues: first Â±,ER status, second Â±,EGFRstatus. For all samples, 1, premenopausal tissue; 2, postmenopausal tissue. For each tissue sample the Wni signal is shown above the GAPDH signal. The densitometric quantitation of the corrected Wnt signal is shown graphically in bar charts, and the order of the bars corresponds to the order of the tissues above. 2617

WNT3 EXPRESSION PROFILE

WNT3 SIGNAL @ . _.-@

normal tissue benign tissue malignant tissue

@ â€”--... _@. â€” -â€”â€” w

GAPDH SIGNAL

Wnt3: normal tissue Wnt3: benign tissue Wnt3: tumortissue 3 3

2 21 11

1LJ@i c@@@@'-,-csJv- 0@-C@@.1-C%iC%4@- -+++â€¢._,_,â€¢ @ @[email protected] .tfftf@@

Fig. 3. RNase protection assay showing Wnt3and corresponding GAPDH signals in normal, benign, and malignant human breast tissue samples. Graphs show densitometric analysis of the data. See Fig. 2 for additional details.

A 120-base pair fragmentof the human GAPDH gene cloned into the expression of Wnt7b between the lowest and highest expressing cell plasmid P Bluescript SK+ (28) was used to generate antisense GAPDH lines. No consistent relationship was found between levels of expres probes. sion and ER or EGFR status, although the 3 Wnt3-expressing cell lines RNase Protection Analysis. RNase protection analysis was performed were all ER negative, EGFR positive. using the described constructs by standard protocols (26). In all assays 10 p@g Wnt Expression in Human Breast Tissue. Figs. 2â€”5show the of RNA was hybridized to a cocktail containing 10@cpmof each of Wnt and signals obtained for Wnt2, Wnt3, Wnt4, and Wnt7b with the cone GAPDH probes. Protected fragment signals for both Wnt and GAPDH were sponding GAPDH signals and densitometric quantitation of the data. quantified by laser densitometry using a Bio Image analyzer (Millipore). In all assays the GAPDH signal was used as a loading control. Wnt2 expression was 10- to 20-fold higher (P = 0.03) in fibro Statistical Analysis. P values were derived from Mann-Whitney U test adenomas (n = 5) than it was in normal, malignant, or benign tissues calculations, using the â€œstatviewâ€•version4 program (Abacus Concepts Inc.) (taken together n = 30). Wnt2 expression was also found in fibroblasts freshly isolated from a breast reduction sample (data not shown). RESULTS Wnt3 was expressed at similar levels in normal, benign, and ma lignant breast tissue. There was a 20-fold range in expression between Expression of Wnt RNA was examined by RNase protection using the lowest and highest expressing samples. probes for Wnt2, Wnt3, Wnt3a, Wnt4, Wnt7a, and Wnt7b. Expression Like Wnt2, Wnt4 expression was higher (P = 0.001) in fibro was assayed in human cell lines, as well as in normal, benign, and adenomas (n = 5) than it was in other tissues (taken together n = 31). malignant human breast tissue. The increase in expression was approximately 10-fold, with one Wnt Expression in Breast Cell Lines. The panel of cell lines sample showing a 40-fold elevation. (shown in Table 1 with their Wnt expression profile) consisted of Wnt7b was expressed at similar levels in normal and benign tissues MTSV1â€”7(a human immortalized benign mammary epithelial cell but showed an elevation of approximately 30-fold in 10% of tumors. line) (23), three ER-positive breast cancer cell lines (MCF7, ZR75, Southern analysis of genomic DNA extracted from normal tissue with T47D), six ER-negative breast cancer cell lines (Adriamycin-resistant low Wnt7b expression and from the tumor expressing the highest level MCF7, MDA231, MDA361, MDA415, MDA457, MDAMB157), and of Wnt7b showed no obvious rearrangement of the gene, and slot blot the EGFR-transfected breast cancer cell line ZR9B1 1. Wnt3, Wnt4, analysis of the same DNA samples showed no genomic amplification and Wnt7b were expressed in some of the cell lines, but Wnt2, Wnt3a, (slot blot and Southern data not shown). and Wnt7a were not. Wnt3 was expressed in 3 cell lines. MTSV1â€”7and BT2O showed a DISCUSSION similar level of expression, which was 10-fold lower than that found in MCF7adr. Wnt4 was present only in MDA415. Wnt7b was present Our results show that several Wnt genes are expressed in normal in all cell lines examined. There was a 100-fold range in the level of human breast tissue. The specific role of Wnt genes as developmental 2618

WNT4 EXPRESSIONPROFILE

WNT4 SIGNAL @ -ff@T:j@

normal tissue benign tissue malignant tissue @@@ ___-@â€”â€˜I@@ .-. â€”@.

GAPDH SIGNAL

Wnt4: normal tissue Wnt4: benign tissue Wnt4: tumortissue 50 50 40 40

30 30 20 20 10 1@ 10

-,-CsJ@-@ _-esJ.,.-c'JI-,-. @ @[email protected] u_ U. U@Li@U@0 u u@Q. C@-'-C@'-C@JC%JC%J Fig. 4. RNase protection assay showing Wnt4 and corresponding GAPDH signals in normal, benign, and malignant human breast tissue samples. Graphs show densitometric analysis of the data. See Fig. 2 for additional details.

regulators in all systems in which they have been studied supports the Wnt2 mRNA is clearly present in fibroblasts freshly isolated from a hypothesis that they may play a similar role in human breast tissue. breast tissue sample, and it is interesting that Wnt2 is elevated in The fact that not all Wnt genes examined are expressed suggests that tissues from benign breast disease, where there is a large stromal they have different functions. It is interesting that even the most element. In addition, mesenchymal expression of Wnt2 has been homologous Wnt genes, such as pairs 3 and 3a and 7a and 7b, show demonstrated in other systems (29, 30). If the in vivo expression of different expression profiles (3 and 7b are expressed, 3a and 7a are Wnt2 is indeed stromal, this would suggest that the protein can act in not), suggesting a lack of redundancy in Wnt function. paracrine fashion in the human breast and would parallel the in vitro Our data allow a comparisonto be made between the Writgenes demonstration of Wntl paracrine action in coculture assays (22). expressed in normal human breast and those Wnt genes that are If Wnt genes have a role in normal developmental processes of the expressed at some stage of development in the mouse breast (Table 2). human breast, then disease states of the breast in which tissue mor Mouse and human breast resemble each other in their expression of phology is altered may be associated with changes in Writexpression. Wnt2, Wnt4, and Wnt7b and in the absence of Wnil, Wnt3a, and Our results show an increased level of Wnt2 and Wnt4 in fibroade Wnt7a. However, they differ in that Wnt3 (absent in normal mouse nomas and an elevated level of Wnt7b in approximately 10% of breast breast, by Northern analysis at least), is present in the human breast. tumors. The finding that Wnt2 and Wnt4 are overexpressed in fibro When studying the mouse, Gavin and McMahon (16) were able to adenomas does not establish an etiological relationship. However, the show a clear correlation between breast dcye1op@nt during preg facts that Wntl can cause mammary hyperplasia in the mouse and that nancy and lactation and differential Wnt expression. In our experi fibroadenomas are hyperplastic rather than neoplastic (31) support a ments the profile of Wnt expression of postmenopausal breast was no causal role for Wnt genes in the pathology of these lesions. It is different from that of premenopausal breast. Clearly, the fact that generally accepted that the term â€œfibrocysticdiseaseâ€•has no prog breast tissue from pregnant and lactating women is not readily avail nostic significance per se but that some of the histological features able will make it difficult to repeat the experiments of Gavin and sometimes exhibited do correlate with an increased risk of breast McMahon in the human. However, the question of Wnt gene regula cancer. These include the different types of epithelial hyperplasia lion may be partially answered by investigating the effect of pregnan (32). In the mouse, aberrant Wnt expression causes mammary epithe cy-related and other growth factors on the Wnt expression profile of hal cell hyperplasia, and it would, therefore, be of great interest to human mammary epithelial cells in vitro. investigate Wnt expression in human breast epithelial hyperplasia, Cell line expression of Wrngenes mirroredthe tissue expression particularly â€œatypicalepithelial hyperplasiaâ€• in which a 5-fold in with the exception of Wnt2, which was absent in cell lines but present creased risk of breast cancer is recognized (32). in tissues. It is possible that this discrepancy is due to the expression Wnt7b was expressed in all tissues examined but was elevated in of Wnt2by stromalcells, which are presentin tissuesbut not in the 10% of tumors. These tumors could not be distinguished from others cell line cultures. This hypothesis is supported by the finding that in the panel on the basis of clinical criteria such as estrogen receptor 2619

WaS GENE ExPRESSION IN HUMAN BREAST TISSUE

WJ1T7bEXPRESSIONPROFILE

WNT7b SIGNAL

normal tissue benign tissue malignant tissue

@ â€¢.e..I-Iâ€¢ â€¢1..@L1 @.. 0SO.@ . I. â€¢..O@

GAPDH SIGNAL

Wntlb: normal tissue Wnt7b: benign tissues Wntlb: tumortissue 50 50 50 40 40 40 30 30 30 20 20 20 10 10 10 Iâ€” V @@

levels, epidermal growth factor receptor levels, differentiation grade, of Writ expression can be transforming, or at least alter normal histological type, or nodal status. Our analysis of the tumor expressing mammary epithelial cell biology. the highest levels of Wnt7b would not demonstrate a minor translo In summary,ourresultsshow thatseveralhumanWntgenes areexpressed cation of the gene but makes a major rearrangement of Wnt7b in this in normal breast tissue. Diseased breast shows levels of expression of some tumor unlikely. Furthermore, the gene shows no genomic amplifica Wnt genes different from that found in normal breast These data establish a tion; therefore, the increased RNA level may be due to increased role for Writgenes in human breast biology andjustify a further examination transcription or decreased degradation. of apossiblerolein breastpethologj@. In all cases in which Wnt signal was detected, the level of expres sion was nevertheless generally low, in that it was usually necessary REFERENCES to expose the gels for 1 week to obtain signals intense enough for reliable quantitation. This suggests that low quantities of Writmay be 1. Nusse, R., and Varmus, H. E. Wnt genes. Cell, 69: 1073-1087, 1992. 2. McMahon, A. P. The wnt family of developmental regulators. Trends Genet., 8: sufficient to produce biological effects and would fit with the obser 236â€”242,1992. vation that, in the appropriate context, low-level Wnt expression is 3. Nusse, R., and Varmus, H. E. Many tumours induced by the mouse mammary tumour enough to achieve transformation (33). virus contain a provirus integrated in the same region of the host genome. Cell, 31: 99â€”109,1982. An association between disease states and differential Wnt expres 4. Rijsewijk, F., Schuermann, M., wagcnasr, E., Parren, P., Weigel, D., and Nusse, R. sion in the breast does not establish a causal relation between the two. The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the It would be of interest to transfect human Wnt genes into human segment polarity gene wingless. Cell, 50: 649â€”657,1987. 5. Nusse, R., Brown, A., Papkoff, J., Scambler, P., Shackleford, G., McMahon, A, mammary epithelial cells to demonstrate whether or not altered levels Moon, R., and Varmus, H. A new nomenclature for mt-i and related genes: the Wnt gene family (letter). Cell, 64: 231, 1991. 6. Russell, J., Gennissen, A., and Nusse, R. Isolation and expression of two novel Wnt/wingless gene homologues in Drosophila. Development (Camb.), 115: Table2 Wntgeneexpressioninmousemammaryglandand in humanbreastcelllines 475â€”485,1992. and breast tissues 7. Wolda, S. L., and Moon, R. 1. Cloning and developmental expression in Xenopus Wnt233a47a7bMousebreastÃ·a+â€”+(Refs. laevis of seven additional members of the wnt family. Oncogene, 7: 1941â€”1947, 1992. 8. Gavin, B. 1., McMahon, J. A., and McMahon, A. P. Expression of multiple novel Wnt-1/int-1-related genes during fetal and adult mouse development. Genes Dcv., 4: 2319â€”2332, 1990. 16 and 17) 9. Wainwright, B. 1., Scambler, P. J., Stanier, P., Watson, E. K., Bell, 0., Wicking, C., Human celllinesâ€”+â€”+â€”+Humantissue++â€”+â€”+NormalBenign Estivill, X., Courtney, M., Boue, A., and Pedersen, P. S. Isolation of a human gene with proteinsequencesimilarityto humanand murinemt-i and the Drosophila segment polarity mutant wingless. EMBO J., 7: 1743-1748, 1988. 10. McMahon, A. P., and Moon, R. T. Ectopic expression of the proto-oncogene int-1 in +a Malignant+ ++ +â€” -+ +â€” â€”+ Xenopus embryos leads to duplication of the embryonic axis. Cell, 58: 1075-1084, @ expression detected;â€”, expression not detected. 1989. 2620

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. Differential Expression of Human Wnt Genes 2, 3, 4, and 7B in Human Breast Cell Lines and Normal and Disease States of Human Breast Tissue

Emmanuel L. Huguet, Jill A. McMahon, Andrew P. McMahon, et al.

Cancer Res 1994;54:2615-2621.

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