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[Cancer Research 59, 1572-1577, April 1, 1999]
© 1999 American Association for Cancer Research

Molecular Biology and Genetics

Transcriptional Activation of Cyclooxygenase-2in Wnt-1-transformed Mouse Mammary Epithelial Cells1

Louise R. Howe, Kotha Subbaramaiah, Wen Jing Chung, Andrew J. Dannenberg and Anthony M. C. Brown2

Strang Cancer Research Laboratory, The Rockefeller University, New York, New York 10021 [L. R. H., K. S., W. J. C., A. J. D., A. M. C. B.], and Departments of Cell Biology and Anatomy [L. R. H., A. M. C. B.] and Medicine [K. S., W. J. C., A. J. D.], Weill Medical College of Cornell University, New York, New York 10021


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Wnt-1 acts as a mammary oncogene when ectopically expressed in the mouse mammary gland. APC is a tumor suppressor gene, mutations in which cause intestinal tumorigenesis in humans and rodents. Both Wnt-1 expression and APC mutation activate a common signaling pathway involving transcriptional activation mediated by ß-catenin/Tcf complexes, but few targets relevant to carcinogenesis have yet been identified. Expression of the inducible prostaglandin synthase cyclooxygenase-2 appears critical for intestinal tumorigenesis resulting from APC mutation, suggesting that cyclooxygenase-2might be a transcriptional target for ß-catenin/Tcf complexes. Here, we have investigated the effect of Wnt-1 on cyclooxygenase-2expression. Wnt-1 expression in the mouse mammary epithelial cell lines RAC311 and C57MG induces stabilization of cytosolic ß-catenin and morphological transformation. Expression of Wnt-1 in these cells caused transcriptional up-regulation of the cyclooxygenase-2 gene, resulting in increased levels of cyclooxygenase-2 mRNA and protein. Prostaglandin E2 production was increased as a consequence of the elevated cyclooxygenase-2 activity and could be decreased by treatment with a selective cyclooxygenase-2 inhibitor. Cyclooxygenase-2 thus appears to be a common downstream target for APC mutation and Wnt-1 expression. In view of the critical role of cyclooxygenase-2 in intestinal tumorigenesis, cyclooxygenase-2up-regulation in response to Wnt signaling may contribute to Wnt-induced mammary carcinogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Wnt-1 was originally identified as a mammary oncogene activated by proviral insertions of mouse mammary tumor virus (1, 2, 3) . Ectopic expression of Wnt-1 under the control of a mouse mammary tumor virus promoter leads to extensive mammary hyperplasia and subsequent generation of adenocarcinomas in mice (4) . Cell culture experiments demonstrate that multiple Wnt gene family members including Wnt-1 can cause partial cellular transformation of some epithelial and fibroblastic cell lines (5, 6, 7, 8, 9, 10, 11, 12) . Collectively, these data implicate Wnt-1 as an oncogene when inappropriately expressed. Several WNT gene family members have been found to be overexpressed in a proportion of human breast cancers and may therefore contribute to carcinogenesis in humans (13, 14, 15, 16, 17) .

The Wnt-1 gene encodes a secreted protein that functions as an extracellular ligand capable of promoting mitogenesis (18, 19, 20, 21) . Wnt-1 appears to signal via a unique pathway, thought to be initiated by interaction of Wnt-1 with a member of the Frizzled family of seven-transmembrane receptors, leading to stabilization of a cytosolic pool of ß-catenin (22) . Accumulated ß-catenin can translocate to the nucleus, interact with Tcf transcription factors, and thereby mediate transcriptional activation (11 , 23, 24, 25, 26, 27, 28, 29, 30, 31) .

ß-Catenin/Tcf-mediated transcriptional activation has recently been implicated in human carcinogenesis. Elevated cytosolic ß-catenin and transcriptionally active ß-catenin/Tcf complexes have been detected in both colon carcinomas and melanomas (32, 33, 34) . ß-Catenin accumulation can occur as a consequence of mutation of either the ß-catenin gene itself or the tumor suppressor gene APC, because wild-type APC protein contributes to ß-catenin destabilization (32, 33, 34, 35, 36) . Mutations in APC cause intestinal tumorigenesis in humans and mice. Although the molecular mechanism by which APC mutation induces tumorigenesis is unclear, many data implicate cyclooxygenase enzymes in this process (37) . Cox-13 and Cox-2 are constitutively expressed and inducible isoforms of prostaglandin synthase, respectively (gene symbols, Ptgs1 and Ptgs2; Ref. 38) . COX-2 expression has been detected in intestinal tumors of both mice and humans with APC mutations (39, 40, 41) . Genetic ablation of the Cox-2 gene or pharmacological inhibition of Cox-2 activity dramatically reduces the incidence of intestinal tumors in Apc mutant mice (42) .

Thus, both APC mutation and ectopic Wnt-1 expression can cause tumorigenesis, and this may be, at least in part, via a common signaling pathway involving ß-catenin/Tcf complexes. Furthermore, Cox-2 appears critical for tumor formation resulting from APC mutation. Consequently, we reasoned that Cox-2 might also be a target for Wnt-1 signaling and might potentially contribute to Wnt-1-induced mammary tumorigenesis. We therefore tested the effect of Wnt-1 expression on Cox-2 in mouse mammary epithelial cells. Here we show that Wnt-1 expression in RAC311 and C57MG cells causes increased transcription of Cox-2, resulting in elevated Cox-2 protein levels. An increase in PGE2 synthesis is also observed in Wnt-1-expressing cells, which can be reversed by treatment with a selective Cox-2 inhibitor. These data may be significant not only in terms of Wnt-mediated carcinogenesis in the mouse but also in relation to human cancers in which components of the Wnt signaling pathway are activated.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Cell Culture.
Two mouse mammary epithelial cell lines were used, C57MG (43) and RAC311, a clonal subline derived from RAC311c (44 , 45) . RAC311 cells were grown in DMEM (4.5 g/l D-glucose) containing 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin. C57MG growth medium was supplemented with 10 µg/ml insulin (Sigma). Cells were infected with MV7 or MVWnt-1 retrovirus using helper-free virus stocks as described previously (21) . Approximately 50–100 G418-resistant colonies were pooled to generate the pooled populations designated RAC/MV7, RAC/Wnt-1, C57/MV7, and C57/Wnt-1. A clonal subline of RAC/Wnt-1 was derived by limiting dilution, selected on the basis of highly transformed morphology, and designated RAC/Wnt-1 #9. For cell lysate and RNA preparation, cells were plated at 1 x 106 cells per 10-cm dish and grown until MV7-infected control cells were confluent (5 days for RAC/MV7; 4 days for C57/MV7). DFU was a generous gift of the Merck Frosst Center for Therapeutic Research (Quebec, Canada).

Cell Lysate Preparation and Analysis.
For Wnt-1 protein analysis, ECM fractions were prepared after removing the cells from the dishes by incubation with Dulbecco’s PBS (Life Technologies, Inc.) containing 2 mM EDTA. ECM remaining on the plates was solubilized in boiling Laemmli SDS sample buffer and stored at -20°C. For analysis of cytosolic ß-catenin levels, lysates were prepared as described (46) , and total protein was assayed using Bio-Rad Protein Assay reagent. For Cox-2 protein analysis, lysates were prepared essentially as described (47) . Cells were washed twice with PBS and harvested in lysis buffer containing 150 mM NaCl, 100 mM Tris-Cl (pH 8.0), 1% Tween 20, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM diethyldithiocarbamic acid. After one cycle of freeze-thawing at -20°C, cells were sonicated (three times for 15 s each time) on ice, then debris was pelleted by centrifugation at 10,000 x g for 10 min at 4°C. Supernatants were stored at -80°C, and proteins were assayed using a Lowry-based protein assay kit (Sigma).

For Western analysis, samples were subjected to SDS-PAGE as follows: Wnt-1 ECM fractions, 10% gel; ß-catenin samples, 8% gel, 5 µg of protein; Cox-2 lysates, 10% gel, 50 µg of protein. Proteins were transferred to polyvinylidene fluoride membrane (Immobilon; Millipore), blotted with anti-Wnt-1 antibody (MC123; Ref. 21 ), anti-ß-catenin antibody (Transduction Laboratories; Ref. 46 ), or anti-Cox-2 antibody (715; Ref. 47 ), and developed with Amersham enhanced chemiluminescence reagents. The anti-Cox-2 antibody 715 was a rabbit polyclonal antibody, raised against the unique 18-amino acid sequence from the COOH-terminal region of human Cox-2, which does not react with Cox-1.

RNA Preparation and Northern Blotting.
RNA was prepared from confluent cells using RNAzol B (Tel-Test, Inc.) according to the manufacturer’s instructions. Twenty µg of RNA were subjected to electrophoresis in 1% agarose/formaldehyde/3-[N-morpholino]propanesulfonic acid gels and transferred to Zeta-Probe membrane (Bio-Rad). Radiolabeled random-primed probes were prepared using the Rediprime DNA labeling system (Amersham), and hybridization was undertaken at 65°C in 0.5 M Na2HPO4 (pH 7.2), 7% SDS, and 1 mM EDTA (48) . Washes were performed in 40 mM Na2HPO4 (pH 7.2), 1% SDS at 65°C. Probes used were murine Cox-2 (TIS-10; a gift from H. R. Herschman, University of California at Los Angeles, Los Angeles, CA), murine Cox-1 (a gift from W. L. Smith, Michigan State University, East Lansing, MI), and murine GAPDH (obtained from A. Ashworth, Institute of Cancer Research, London, England). GAPDH was used to demonstrate equal loading of each lane.

Nuclear Run-Ons.
Nuclei were prepared, and nuclear run-ons were performed as described (47) .

Autoradiographic exposures of both Northern blots and nuclear run-ons were quantitated by analysis on a Macintosh computer using the public domain NIH Image program (developed at the United States NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/). Values obtained were normalized to those obtained for GAPDH and 18S rRNA for Northern blots and nuclear run-ons, respectively.

PGE2 Assays.
Cells were plated in 12-well plates at 4 x 104 cells/well and grown to confluence. Culture medium was collected and assayed for PGE2 by enzyme immunoassay (Cayman Co., Ann Arbor, MI). To assay PGE2 production in the presence of excess arachidonic acid (AA "spiked"), cells were incubated with fresh medium containing 10 µM arachidonic acid for 30 min, and then this medium was harvested and assayed as above. For experiments assaying the effect of DFU on PGE2 production, cells were plated in 6-cm dishes at 3 x 105 cells/dish. DFU was added in fresh medium 72 h after plating and readded at 96 h. PGE2 production was assayed at 120 h after plating, at which time control cells were confluent.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
To examine the effect of Wnt-1 on Cox-2 expression, we generated fresh cell populations expressing Wnt-1 by infection of the mouse mammary epithelial cell lines C57MG and RAC311 with retrovirus encoding Wnt-1 (MVWnt-1) or control retrovirus (MV7). As observed previously (5 , 6) , both C57/Wnt-1 and RAC/Wnt-1 cells appeared morphologically transformed and grew to higher cell densities than control cells (C57/MV7 and RAC/MV7, respectively). An additional clonal subline, RAC/Wnt-1 #9, was generated from RAC/Wnt-1 by limiting dilution and selected because of its high degree of morphological transformation. Western blot analysis using an anti-Wnt-1 antibody revealed that RAC/Wnt-1 #9 produced more Wnt-1 protein than the pooled RAC/Wnt-1 population (Fig. 1A)Citation . Therefore, we included both RAC/Wnt-1 and RAC/Wnt-1 #9 in subsequent analyses. As demonstrated previously, expression of Wnt-1 in C57MG led to accumulation of uncomplexed cytosolic ß-catenin (Refs. 46 and 49 ; data not shown). In addition, cytosolic ß-catenin was elevated in RAC311 cells expressing Wnt-1 (Fig. 1B)Citation , and higher levels were detected in RAC/Wnt-1 #9 relative to RAC/Wnt-1, correlating with relative Wnt-1 protein production.



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  		Fig. 1. Characterization of RAC311 cells expressing Wnt-1. RAC/MV7, RAC/Wnt-1, and RAC/Wnt-1 #9 cells were generated by retroviral infection as described in "Materials and Methods." Cells were analyzed by Western blotting for Wnt-1 protein and cytosolic ß-catenin levels. A,Wnt-1 expression. ECM fractions were prepared and assayed for Wnt-1 protein as described in "Materials and Methods." Anti-Wnt-1 antibody MC123 detected two bands of Mr 42,000 and Mr 44,000 in Wnt-1-expressing cells, as observed previously (71) . These represent differentially glycosylated forms of Wnt-1 protein (20) . No Wnt-1 protein was detected in ECM from control RAC/MV7 cells. B, cytosolic ß-catenin. Cytosol fractions were prepared from cells and assayed for ß-catenin as described in " Materials and Methods." The position of a Mr 97,000 molecular weight marker is shown.

 
Cox-2 protein levels in control and Wnt-1-expressing cell lines were analyzed by Western blotting (Fig. 2)Citation . C57/MV7 exhibited a markedly higher basal amount of Cox-2 than RAC/MV7, in which Cox-2 protein was virtually undetectable. In both C57MG and RAC311 cell lines, however, expression of Wnt-1 led to an increase in Cox-2 protein, and Cox-2 protein was more abundant in RAC/Wnt-1 #9 than in RAC/Wnt-1, correlating with Wnt-1 expression levels. Analysis of Cox-2 RNA by Northern blotting demonstrated that Cox-2 mRNA levels closely reflected the changes observed in Cox-2 protein (Fig. 3)Citation , suggesting the effect of Wnt-1 on Cox-2 was likely to be due to transcriptional activation of the Cox-2 gene. To test this directly, nuclear run-on assays were performed. These and subsequent assays were performed in the RAC311-derived cell lines in preference to C57MG-derived lines, because the latter tend to lose Wnt-1 expression during continuous culture.4 The rates of transcription from the Cox-2 gene in RAC/Wnt-1 and RAC/Wnt-1 #9 were increased to 270 and 400%, respectively, relative to that in RAC/MV7 (Fig. 4)Citation , mirroring the differences observed in Cox-2 RNA and protein. Thus, expression of Wnt-1 in RAC311, and most likely C57MG, causes transcriptional activation of the Cox-2 gene.



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  		Fig. 2. Cox-2 protein is increased by Wnt-1 expression. Lysates were prepared from C57MG-derived cells (A) and RAC311-derived cells (B). Fifty µg of lysate were analyzed by Western blotting for Cox-2 as described in "Materials and Methods," using rabbit polyclonal anti-Cox-2 antibody 715. Data shown in A and B are from separate experiments. The position of a Mr 69,000 molecular weight marker is shown.

 


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  		Fig. 3. Cox-2 mRNA is increased in cells expressing Wnt-1. Total RNA was prepared from cells, and 20 µg of each RNA sample were analyzed by Northern blotting as described in "Materials and Methods." The blot was probed sequentially with a murine Cox-2 probe and a murine GAPDH probe. Cox-2 signals were quantitated using the program NIH Image and normalized to those obtained from GAPDH probing. Values obtained are expressed relative to the control MV7-infected cell line in each case. C57/MV7, 100%; C57/Wnt-1, 500%; RAC/MV7, 100%; RAC/Wnt-1, 174%; RAC/Wnt-1 #9, 327%.

 


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  		Fig. 4. Cox-2 transcription is up-regulated in RAC311 cells expressing Wnt-1. Nuclei were prepared, and nuclear run-on assays were performed as described in "Materials and Methods." Labeled nascent transcripts were hybridized to 18S rRNA and Cox-2 cDNAs, which were immobilized on nitrocellulose. Signals were quantitated using the program NIH Image, and Cox-2 was normalized to 18S rRNA. Values obtained are expressed relative to RAC/MV7. RAC/MV7, 100%; RAC/Wnt-1, 267%; RAC/Wnt-1 #9, 399%.

 
Cox-2 is an inducible isoform of prostaglandin synthase (38) . Thus, one predicted functional consequence of Cox-2 up-regulation would be an increase in prostaglandin synthesis, of which PGE2 is the predominant eicosanoid produced by most epithelial cells. We therefore assayed PGE2 production in RAC311-derived cell lines. Spontaneous production of PGE2 in RAC/Wnt-1 and RAC-Wnt/1 #9 was increased by 240 and 420%, respectively, over that in RAC/MV7 (Fig. 5Citation , "spontaneous"). Spontaneous PGE2 production was also measured in C57MG-derived lines and was increased ~100% in C57/Wnt-1 cells relative to C57/MV7 (data not shown). Given that arachidonic acid is the substrate from which cyclooxygenases synthesize prostaglandins, incubation of cells with excess arachidonic acid can increase PGE2 production. In our experiments, treatment of the cells with arachidonic acid increased the absolute amounts of PGE2 synthesis (Fig. 5Citation , "AA spiked"), but PGE2 production was still elevated in Wnt-1-expressing cells relative to RAC/MV7. This suggests that the measured spontaneous synthesis rates reflected relative levels of Cox-2 activity in the cells, rather than differential availability of arachidonic acid.



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  		Fig. 5. PGE2 production is increased by expression of Wnt-1. Cells were grown for 5 days after plating to achieve confluence, during which time no medium change was performed. Growth medium from the cells was harvested and assayed using enzyme immunoassay to measure spontaneous PGE2 production (SPONTANEOUS, light hatching). Cells were incubated for an additional 30 min in fresh medium containing 10 µM sodium arachidonate. The medium was then collected and assayed for PGE2 (AA SPIKED, dark hatching). Results were normalized to µg of protein obtained from the cells after harvesting of medium. Results shown are mean values of six replicates; bars, SD. PGE2 production from both Wnt-1-expressing cell lines was significantly greater than that from RAC/MV7 control cells (*, P < 0.001; +, P < 0.003).

 
The observed increases in PGE2 synthesis in Wnt-1-expressing cells could also be a consequence of changes in the level of Cox-1. Although Cox-1 is constitutively and ubiquitously expressed, there have been reports of ligand-induced Cox-1 up-regulation (50, 51, 52, 53) . Therefore, we addressed the involvement of Cox-1 using two assays: (a) we measured Cox-1 mRNA by Northern blotting and found little or no increase in Cox-1 mRNA in RAC/Wnt-1 and C57MG/Wnt-1, respectively, relative to control cells (Fig. 6)Citation ; and (b) we tested the relative contribution of Cox-1 and Cox-2 to PGE2 production in the RAC311-derived cell lines by using DFU, a selective Cox-2 inhibitor. DFU has at least a 1000-fold specificity for Cox-2 relative to Cox-1 in tissue culture cells (54) . RAC/MV7, RAC/Wnt-1. and RAC/Wnt-1 #9 were treated with varying concentrations of DFU for 48 h, and culture supernatants were then assayed for PGE2. A dose-dependent inhibition of PGE2 production was observed, with 1 µM DFU being sufficient to reduce PGE2 production to approximately the same basal level in all three cell lines (Fig. 7)Citation . Higher concentrations of DFU did not cause any additional inhibition of PGE2 synthesis. The residual PGE2 production observed in all cell lines after inhibition of Cox-2 with DFU is presumed to reflect Cox-1 activity. Because the amount of Cox-1-mediated PGE2 synthesis is apparently constant in all three cell lines, we conclude that the enhanced production of PGE2 in Wnt-1-transformed cells is attributable to increased Cox-2 activity, consistent with the observed differences in Cox-2 RNA and protein levels. Cell morphology was unaffected by treatment with DFU (data not shown). The failure of DFU to affect morphological transformation of Wnt-1-expressing cells suggests that elevated prostanoid production by Cox-2 is not necessary for maintenance of the transformed phenotype of these cells in vitro.



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  		Fig. 6. Effect of Wnt-1 expression on Cox-1 mRNA. RNA was prepared, and a Northern blot was generated as described in "Materials and Methods" using 20 µg of each RNA. The blot was probed sequentially with a murine Cox-1 probe and a murine GAPDH probe. Cox-1 signals were quantitated using the program NIH Image and normalized to those obtained from GAPDH probing. Values obtained are expressed relative to the control MV7-infected cell line in each case. C57/MV7, 100%; C57/Wnt-1, 70%; RAC/MV7, 100%; RAC/Wnt-1, 118%; RAC/Wnt-1 #9, 163%.

 


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  		Fig. 7. Inhibition of PGE2 production by a selective Cox-2 inhibitor DFU. Cells were treated with the indicated concentrations of DFU for 48 h. Culture medium was harvested and assayed for PGE2 by enzyme immunoassay to determine spontaneous release of PGE2. Results were normalized to µg of protein obtained from the cells after harvesting of the medium. Mean values of two replicates are shown; bars, spread.

 
We have shown here that expression of Wnt-1 in two mammary epithelial cell lines causes elevated expression and activity of Cox-2, via transcriptional activation, resulting in increased PGE2 synthesis. The Cox-2 gene was initially identified as an early response gene up-regulated in response to phorbol ester and serum and was subsequently found to be induced by multiple agents, particularly during inflammatory responses (38) . A large body of evidence has accumulated implicating Cox-2 in intestinal carcinogenesis. COX-2 expression is frequently detected in tumor tissue (39 , 40 , 55, 56, 57, 58) , and the incidence of intestinal tumorigenesis in both mice and humans can be reduced by pharmacological agents that inhibit Cox activity (41 , 42 , 59, 60, 61, 62, 63, 64) . A crucial role for Cox-2 in tumorigenesis has been demonstrated by Oshima et al. (1996; Ref. 42 ), who found that intestinal polyposis in Apc mutant mice was markedly reduced by genetic ablation of Cox-2. However, Cox-2 induction in response to Wnt proteins has not been demonstrated previously.

The mechanism by which Wnt-1 activates Cox-2 transcription is unclear. Given that COX-2 induction occurs in response to Wnt-1 expression and APC mutation, both of which result in cytosolic ß-catenin accumulation, our initial expectation was that the Cox-2 promoter might be subject to direct regulation by ß-catenin/Tcf complexes. The human COX-2 promoter contains two potential Tcf-binding sites, although their overlap with the canonical TCF binding motif is only partial (ACTTTGATC and TCTTTGTAG compared with CCTTTGA/TA/TC; Ref. 27 ). One of these sites is not conserved in the murine Cox-2 promoter, and the other lies outside the sequence presently reported for the mouse promoter. To investigate the mechanism of regulation, we have performed transient transfection assays using a human COX-2 promoter-luciferase reporter construct but thus far have been unable to detect increased reporter activity as a result of ß-catenin overexpression. Thus, it is possible that Cox-2 transcription may not be directly regulated by ß-catenin/Tcf complexes but may be activated in Wnt-1-expressing cells by alternative transcription factors. We also cannot exclude the possibility that Cox-2 induction is a more downstream or indirect consequence of Wnt signaling.

Because Wnt-1 is a mammary oncogene, our data suggest that Cox-2 up-regulation might also contribute to mammary tumorigenesis. Consistent with this idea, Cox-2 is expressed in ras- and virally transformed mammary cells, as well as in some human breast cancers and breast cancer cell lines (47 , 65 , 66) . Interestingly, despite abundant evidence of the importance of Cox-2 during intestinal tumorigenesis, the precise mechanism by which Cox-2 contributes is unclear. Prostaglandin overproduction is likely to have multiple consequences. Prostaglandins can exert local immunosuppressive effects that could facilitate tumorigenesis (37 , 67) . Additionally changes in gene expression can occur because selected prostaglandins are ligands of the peroxisome proliferator-activated receptor {gamma} (68) . Cox-2 induction in tumors may promote survival of cells otherwise destined to undergo apoptotic cell death; negative regulation of apoptosis by Cox-2 overexpression has been demonstrated in intestinal epithelial cells (69) . Recent data also demonstrate a role for Cox-2 in angiogenesis. Selective inhibition of Cox-2 reduces secretion of angiogenic factors from colon cancer cells, thereby suppressing de novo formation of endothelial tubules in vitro (70) . Because of the pleiotropic consequences of Cox-2 overexpression, it is difficult to predict what role Cox-2 might play in Wnt-1-induced mammary tumorigenesis. Transgenic mice that express Wnt-1 ectopically in the mammary gland display extensive mammary hyperplasia at an early age and subsequently develop mammary adenocarcinomas stochastically after a latent period of several months (4) . If Cox-2 contributes to Wnt-1-mediated tumorigenesis in vivo, it could do so either at the initial hyperplastic stage or by affecting progression to carcinoma. Our findings may also be pertinent to human breast cancer. Although expression of WNT-1 itself in human mammary tissue has not been reported, several others members of the WNT gene family are overexpressed in human breast tumors relative to normal tissue (13, 14, 15, 16) . A number of Wnt proteins exhibit functional redundancy with Wnt-1, inducing stabilization of cytosolic ß-catenin and morphological transformation of mammary cells (8 , 10) . Therefore, it is likely that some of the WNTgenes overexpressed in human breast cancers may have transcriptional consequences similar to those of Wnt-1.


   ACKNOWLEDGMENTS
 
We thank A. Ashworth, H. R. Herschman, and W. L. Smith for plasmids. The generous assistance of J. R. Timmer with production of figures is gratefully acknowledged.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was funded by NIH Grants CA47207 (to A. M. C. B.) and CA68136 (to A. J. D.), by Department of the Army Grant BC972610, and by donations to the Strang Cancer Prevention Center from the Fashion Footwear Association of New York. L. R. H. is a Lilith fellow, funded in part by proceeds from the Lilith Fair. Back

2 To whom requests for reprints should be addressed, at Strang Cancer Research Laboratory, The Rockefeller University, Box 231, 1230 York Avenue, New York, NY 10021. Phone: (212) 734-0567, extension 232; Fax: (212) 472-9471; E-mail: amcbrown{at}mail.med.cornell.edu Back

3 The abbreviations used are: Cox, cyclooxygenase; PGE2, prostaglandin E2; DFU, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

4 A. M. C. Brown, unpublished observations. Back

Received 10/22/98. Accepted 1/27/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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