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Originally published In Press as doi:10.1074/jbc.M607571200 on September 13, 2006

J. Biol. Chem., Vol. 281, Issue 46, 35081-35087, November 17, 2006
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The Wnt Signaling Receptor Lrp5 Is Required for Mammary Ductal Stem Cell Activity and Wnt1-induced Tumorigenesis*Formula

Charlotta Lindvall{ddagger}, Nicole C. Evans{ddagger}, Cassandra R. Zylstra{ddagger}, Yi Li§, Caroline M. Alexander1, and Bart O. Williams{ddagger}2

From the {ddagger}Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, Michigan 49503, the §Breast Center, Baylor College of Medicine, Houston, Texas 77030, and the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, August 9, 2006 , and in revised form, September 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors, which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype, loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds, which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently, the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore, Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally, we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signaling by the Wnt family of secreted lipoproteins plays a central role in development and disease (1). At the cellular level, Wnt proteins regulate a broad range of functions, including the self-renewal and differentiation of stem cells (2). Activation of the canonical Wnt cascade is initiated by the binding of Wnt proteins to cell surface receptors composed of a member of the Frizzled protein family and one of the low density lipoprotein receptor-related proteins, LRP53 or LRP6 (3, 4). Signaling from Wnt receptors increases cytoplasmic levels of beta-catenin, which binds to transcription factors such as those of the LEF-1/TCF family and modulates the transcription of specific target genes. Whereas Wnt-Frizzled interactions may also be involved in non-canonical Wnt signaling events, the LRP5/6 moiety appears to be specifically required for the canonical pathway (5).

Studies in mice suggest that canonical Wnt signaling plays a significant role during normal mammary gland development (6-11), which begins at about embryonic day 10.5 with the formation of two "mammary lines" (12). In response to signals from the underlying mesenchyme, the mammary lines give rise to five pairs of lens-shaped mammary placodes that grow and invaginate downwards into the dermis to colonize the rudimentary fat pad. Activation of the canonical Wnt signaling pathway along the mammary lines coincides with the initiation of mammary morphogenesis and subsequently localizes to mammary placodes and buds (13, 14). Several Wnt ligands and receptor genes, including Lrp5, are expressed during embryonic mammary morphogenesis (13). Embryos ectopically expressing the canonical Wnt inhibitor Dkk1 display a complete block in the formation of mammary placodes, and mice deficient for Lef-1 fail to maintain their mammary buds (6, 7), showing that Wnt signals are necessary for embryonic mammary development.

By birth, the mammary gland is composed of a few rudimentary ducts, containing an outer layer of myoepithelial and an inner layer of luminal epithelial cells, surrounded by the fat pad. During pre-pubertal and pubertal development, the ductal epithelium proliferates until the fat pad is fully colonized with a sparse ductal tree. Lobuloalveolar precursor cells respond to endocrine signals during pregnancy to colonize all the interductal spaces, increasing cell number at least 10-fold (15). The expansion of mammary epithelium during juvenile growth, estrous, and pregnancy, together with the remarkable regenerative capacity apparent during successive reproductive cycles, imply the existence of a mammary stem cell. In fact, stem-like cells from mature mammary glands have been isolated, and their ability to reconstitute the different epithelial lineages in vitro and functional ductal trees through limiting dilution transplants in vivo has been demonstrated (16, 17). However, the signals that regulate mammary stem cells have yet not been defined.

A connection between mammary stem/progenitor cells and Wnt1- or beta-catenin-induced tumorigenesis has recently been established. Transgenic expression of these genes results in widespread mammary hyperplasia and rapid tumor formation (11, 18). The hyperplastic tissue contains an increased fraction of mammary stem/progenitor cells that are thought to directly give rise to transformed cells (17, 19, 20). Tumors arising from stem/progenitor cells often show mixed lineage differentiation (21), and tumors induced by Wnt effectors indeed contain cells from both epithelial lineages (19, 20). Here we have shown that the Lrp5 receptor is required both during normal mammary development and for Wnt1-induced tumorigenesis. In MMTV-Wnt1 transgenic mice loss of Lrp5 markedly reduces both the early proliferation of the progenitor cell population and the subsequent formation of mammary tumors. Furthermore, Lrp5-/- mammary cells are unable to reconstitute ductal trees through limiting dilution transplants. Taken together, these results suggest that canonical signaling through the Lrp5 receptor is critical for mammary stem cell activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mouse Crosses—The Lrp5-/- mice (22) (maintained on a B6 background) carry a mutation in the first exon, eliminating the initiating ATG and the sequence encoding the signal peptide. MMTV-Wnt1 transgenic mice (18) (maintained on a FVB/N background) and BAT-gal transgenic mice (23) (maintained on a B6D2F1 background) were crossed with Lrp5-/- mice to generate Lrp5+/+, Lrp5+/-, or Lrp5-/- female mice that either carried or lacked the MMTV-Wnt1 or the BAT-gal transgene. PCR-based strategies were then used to genotype these mice (details available upon request). All experiments performed were in compliance with the guiding principles of the "Care and Use of Animals" available at www.nap.edu/books/0309053773/html and were approved in advance by the Van Andel Research Institute Institutional Animal Care and Use Committee. To assay the appearance of mammary tumors, the mice were inspected weekly and were euthanized when tumors appeared.

Immunohistochemistry and Western Blotting—Mammary tissues were fixed for 2 h in 4% paraformaldehyde at 4 °C and then embedded in paraffin and sectioned (5 µm). Immunohistochemistry was performed by using Vector ABC and DAB kits according to the manufacturer's recommendations (Vector Laboratories). The following primary antibodies were used: rabbit polyclonal antibody against keratin 6 (1:100; Covance), Lrp5 (1:5,000; kindly provided by John Robinson (24)), Lrp6 (1:250; Zymed Laboratories Inc.), and p21CIP1 (1:250; Santa Cruz). Western blotting using goat polyclonal antibodies against Wnt1 (1:500; Santa Cruz) was performed as previously described (19).

Mammary Whole Mounts and Analysis of BAT-Gal Expression—Inguinal mammary glands were fixed in 4% paraformaldehyde, washed in phosphate-buffered saline and stained with carmine alum, dehydrated, and cleared in xylene. For analysis of BAT-gal expression whole mount embryos were fixed (0.2% glutaraldehyde, 1.5% formaldehyde, 5 mM EGTA, 2 mM MgCl2 in phosphate-buffered saline) and stained with X-gal (1 mg/ml X-gal, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, 5 mM Fe3(CN)6, 5 mM Fe4(CN)6 in phosphate-buffered saline), photographed and paraffin-embedded, sectioned (5 µm), and counterstained with eosin. Embryonic stage was confirmed by analysis of limb morphology.

Preparation of Mammary Epithelial Cells—Mammary epithelial cells were isolated as described (16). Briefly, mammary glands were digested for 8 h at 37°C in EpiCult-B with 5% fetal bovine serum, 300 units/ml collagenase, and 100 units/ml hyaluronidase. After vortexing and lysis of the red blood cells in NH4Cl, a single-cell suspension was obtained by sequential dissociation of the fragments by gentle pipetting for 1-2 min in 0.25% trypsin and then for 2 min in 5 mg/ml Dispase II plus 0.1 mg/ml DNase I, followed by filtration through a 40-mm mesh. All reagents were from StemCell Technologies Inc.

Transplantation of Cleared Mammary Fat Pads—Mammary glands of 3-week-old female B6 mice were cleared of endogenous epithelium as previously described (25). Viable mammary epithelial cells from 2- to 3-month-old Lrp5+/+ or Lrp5-/- virgin female B6 mice were counted and suspended in Dulbecco's modified Eagle's medium plus 2% fetal bovine serum with 5 µg/ml Matrigel (BD Biosciences) at 4 °C together with loading dye (final concentration, 5% glycerol/0.5% trypan blue/25 mM HEPES), and inoculated in a 1-µl volume containing 500-50,000 cells/µl. Three to five months after transplantation, the fat pads were dissected, processed, and stained with carmine as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Lrp5 Deficiency Inhibits MMTV-Wnt1-induced Carcinogenesis—Female mice expressing the Wnt1 gene under the control of the mouse mammary tumor virus (MMTV)-long terminal repeat enhancer reproducibly develop adenocarcinomas within one year (18). To test whether Lrp5 is the principal signaling receptor for Wnt ligands in mammary epithelial cells, we crossed Lrp5-/- mice to MMTV-Wnt1 transgenic mice. These crosses gave rise to females of approximately the same genetic background that were hemizygous for the Wnt1 transgene in the context of Lrp5+/+, Lrp5+/-, and Lrp5-/- genotypes. Wnt1 transgenic mammary tissue and tumors normally express Lrp5 and Lrp6 (Fig. 1, A-F). The presence or absence of Lrp5 did not affect expression of the Wnt1 transgene (Fig. 1G).

We found that within 10 months, 100% of Lrp5+/+ mice developed tumors with a median time of onset of 25 weeks, and 68% of Lrp5+/- mice formed tumors with a median time of onset of 35 weeks (Fig. 1H). Thus, tumor appearance was delayed several weeks in Lrp5+/- mice (p = 3 x 10-5), indicating that the gene dose of Lrp5 affects the onset of tumorigenesis. In sharp contrast, 100% of Lrp5-/- mice were tumor free at 10 months of age (Fig. 1H), demonstrating that absence of Lrp5 suppressed tumor formation. We extended this analysis over two years and found that Lrp5-/- mice formed tumors with a median time of 90 weeks. Three Lrp5-/- mice (12%) failed to develop palpable tumors and were sacrificed at the end of the study (124 weeks old). All mammary glands from these animals exhibited epithelial hyperplasia, but no foci of mammary tumors were found (data not shown).


Figure 1
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  		FIGURE 1.
Lrp5 and Lrp6 are expressed in MMTV-Wnt1 transgenic mammary glands and tumors (A-F). Shown in panels A-C are immunohistochemical stainings using an Lrp5-specific polyclonal antibody. Lrp5 is expressed in Wnt1-induced tumors (A) and in a fraction of mammary ductal cells from hyperplastic Wnt1 transgenic mammary gland (B). The arrow indicates a representative cell with positive staining. No staining was observed in Wnt1;Lrp5-/- mammary ductal cells (C). Shown in panels D-F are immunohistochemical stainings using an Lrp6-specific polyclonal antibody. Lrp6 is expressed in Wnt1-induced tumors (D) and in a fraction of mammary ductal cells from hyperplastic Wnt1 transgenic mammary gland (E). The arrow indicates a representative cell with positive staining. No staining was observed in Lrp6-/- embryos, which were used as negative controls because Lrp6-/- pups die shortly after birth (F). Western blot of total protein from MMTV-Wnt1 transgenic Lrp5+/+ or Lrp5-/- mammary glands show that the expression of the Wnt1 transgene is not affected by the Lrp5 genotype (G). Protein from a normal mammary gland was used as a negative control (left lane). The arrow indicates a nonspecific band we routinely use to monitor equal loading. Emergence of Wnt1-induced mammary tumors is delayed in Lrp5-/- mice (H). Thirty-seven Lrp5+/+, 31 Lrp5+/-, and 26 Lrp5-/- Wnt1 transgenic female mice were palpated weekly and dates of tumor appearance recorded. Data are plotted as the proportion of mice in each of the three genotypes remaining tumor free as a function of days of age. Standard histopathological evaluation showed that all Lrp5+/+ mammary tumors and the majority of Lrp5-/- mammary tumors are moderately differentiated alveolar adenocarcinomas. A subset of Lrp5-/- mammary tumors exhibits a papillary growth pattern. Shown are representative haematoxylin and eosin sections of Lrp5+/+ (I) and Lrp5-/- (J) Wnt1-induced alveolar adenocarcinoma and Wnt1;Lrp5-/- papillary adenocarcinoma (K).

 
Histopathological examination of the tumors in this study revealed that all Lrp5+/+ and Lrp5+/- tumors, as well as 18/26 Lrp5-/- tumors, were moderately differentiated alveolar mammary adenocarcinomas (Fig. 1, I and J). Alveolar adenocarcinoma is the most common type of mammary tumor reported in Wnt1 transgenic mice. Five Lrp5-/- tumors were papillary adenocarcinomas (Fig. 1K), a morphological variant that is more differentiated and less aggressive than alveolar adenocarcinoma and normally occurs at a low frequency in Wnt1 transgenic mice (26). Hence, even though we could only detect the papillary growth pattern in Lrp5-/- tumors, the Lrp5-/- tumors were not of a different tumor type than previously described for Wnt1-induced tumors. Furthermore, all tumors regardless of Lrp5 genotype expressed cell markers from both the myoepithelial and the luminal epithelial cell lineages, and all tumors contained cells positive for the putative mammary progenitor cell marker keratin 6 (19) (supplemental Fig. S1, A-F). Taken together, these findings suggest that the tumor precursor cell is likely the same regardless of Lrp5 genotype.

Loss of Lrp5 Delays Wnt1-induced Mammary Hyperplasia— Tumors induced by Wnt effectors ultimately arise within a context of widespread mammary hyperplasia that is noticeable as early in development as embryonic day 18 (18). To determine the contribution of Lrp5 to Wnt1-induced mammary gland hyperplasia, ductal development was analyzed in virgin MMTV-Wnt1;Lrp5-/- mice. Inguinal mammary glands were isolated, whole mounted, and compared in the juvenile (5-week) and mature (3 and 15-month) mammary glands from MMTV-Wnt1;Lrp5-/- and control female mice. In the absence of Lrp5, we found that the hyperplastic response to Wnt1 was dramatically delayed (Fig. 2A). Morphometric analysis showed that hyperplasia was inhibited by 80% in mammary glands from Lrp5-/- mice relative to Lrp5+/+ matched controls (p = 6.5 x 10-6) (Fig. 2B).

The hyperplastic mammary tissue of Wnt1 transgenic mice contains an increased ratio of mammary progenitor cells (17, 19, 20). These progenitor cells are thought to directly give rise to transformed cells. To test whether the delay in tumorigenesis could be due to a reduced accumulation of mammary progenitor cells, we immunostained mammary sections from MMTV-Wnt1;Lrp5-/- and control female mice using keratin 6 antibody. We found that Wnt1 transgenic mammary ducts from Lrp5-/- females contained less than half the number of keratin 6-positive cells detected in littermate controls (Fig. 2, C and D). This was further confirmed by staining for another putative mammary progenitor cell marker, p21CIP1 (27), which also showed a significant reduction of positive cells in Lrp5- ducts (Fig. 2C). Taken together, these findings suggest that Lrp5 deficiency reduces the normal accumulation of mammary progenitor cells in MMTV-Wnt1 transgenic mice.

Impaired Mammary Gland Development in Lrp5-/- Mice—To determine the contribution of Lrp5 to normal mammary gland function, ductal development was analyzed in virgin Lrp5-/- mice. Whole mount preparations of inguinal mammary glands are shown for juvenile (5-week) and mature (11-week) mammary glands from Lrp5-/- and control female littermates (Fig. 3A). At 5 weeks the ductal network extends away from the nipple through the fat pad, past the lymph node. The mammary ducts of Lrp5-/- mice were clearly shorter than those of littermate controls. Whereas the Lrp5-/- ductal tree ended right around the lymph node, the wild type had extended considerably further. Terminal end buds (TEBs) are club-shaped epithelial thickenings at the distal ends of growing ducts and are the sites of most rapid cell proliferation and ductal elongation. TEBs are presumed to be rich in mammary stem cells (28, 29). We found that the number of TEBs was reduced by 42% in juvenile Lrp5-/- mice compared with littermate wild-type mice (p = 0.0003) (Fig. 3B). In both control and Lrp5-/- mice, the histology of the TEBs appeared normal (data not shown).


Figure 2
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  		FIGURE 2.
The absence of Lrp5 delays Wnt1-induced mammary hyperplasia and reduces the accumulation of keratin 6- and p21CIP1-positive cells. A, hyperplastic side branching characteristic of MMTV-Wnt1 transgenic mice throughout development is inhibited in Lrp5-/- mammary glands. Representative whole mount preparations (stained with carmine to reveal the mammary ductal tree) are shown for juvenile mice (5-week) and mature virgin female mice. LN, lymph node. B, morphometric analysis of carmine-stained mammary glands from 3-month-old MMTV-Wnt1 transgenic females. The same area of each inguinal mammary gland was scored for the number of tips (ends of branches and lateral buds) from four MMTV-Wnt1 mice of each Lrp5 genotype. The number of tips is reduced by 80% in Wnt1;Lrp5-/- mammary glands compared with Wnt1;Lrp5+/+ control glands (p = 6.5 x 10-6, 2-tailed t test assuming unequal variances). Immunohistochemical staining of Lrp5+/+ (6) and Lrp5-/- (7) MMTV-Wnt1 mammary samples from 11-week-old females was used to determine the number and distribution of cells positive for mammary progenitor cell marker keratin 6. The average number of keratin 6-positive cells per total number of ductal cells is shown in panel C. Wnt1;Lrp5-/- ducts contained 58% fewer keratin 6-positive cells (p = 2.8 x 10-6, 2-tailed t test assuming unequal variances). The same counting strategy was used for p21CIP1. C, Lrp5+/- (3) and Lrp5-/- (3) MMTV-Wnt1 mammary samples from 5-week-old females were used for the morphometric analysis of p21CIP1. Wnt1;Lrp5-/- ducts contained 30% fewer p21CIP1-positive cells (p = 0.0044, 2-tailed t test assuming unequal variances). D, representative MMTV-Wnt1 transgenic mammary sections of keratin 6 immunohistochemical staining. Inserts show a higher magnification of representative ducts. Keratin 6-positive cells stain dark brown.

 


Figure 3
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  		FIGURE 3.
The absence of Lrp5 delays normal mammary development. A, representative whole mount preparations (stained with carmine to reveal the mammary ductal tree) are shown for juvenile (5-week) and mature (11-week) virgin female mice. The arrows indicate typical terminal end buds. LN, lymph node. The result of morphometric analysis of the average number of TEBs at 5 weeks (B) and branches per gland at 11 weeks (C). In the absence of Lrp5 the number of TEBs is reduced by 42% (p = 0.0003, 2-tailed t test assuming unequal variances), and the number of branches per gland is reduced by 46% (p = 0.001, 2-tailed t test assuming unequal variances) compared with Lrp5+/+ littermate controls.

 
The branching complexity in adult mice is a function of terminal end bud activity during juvenile ductal extension. TEBs normally disappear when the ductal tree is fully branched and fills the fat pad. In contrast, the ductal tree of adult Lrp5-/- mice still contained TEBs and did not completely fill the mammary fat pad (Fig. 3A). Morphometric analysis showed that the branching complexity of adult Lrp5-/- glands was decreased by 46% compared with littermate wild-type mice (p = 0.001) (Fig. 3C). On the histological level, the adult Lrp5-/- mammary glands looked normal except for the reduction of mammary ducts as seen on whole mounts (data not shown).

Epithelial Transplants from Lrp5-/- Mice Lack Stem Cell Activity—The outgrowth of a full mammary branching tree from limiting dilutions of mammary epithelial cell transplants is considered to be an assay of clonal stem cell function (25). Surgical removal of the area between the nipple and the fat pad at 3 weeks of age leaves a fat pad free of the endogenous mammary epithelium. Mammary cells from another syngenic animal can be implanted and will develop an epithelial tree if the transplant contains cells with stem cell activity. One benefit of this technique is that the transplanted cells are exposed to normal circulating hormone levels and wild-type stroma. To test whether the reduction in terminal end bud numbers and branching complexity of Lrp5-/- mammary glands could be due to compromised mammary stem cell activity, we transferred cells from 12- to 15-week-old Lrp5-/- and wild-type glands by limiting dilutions (500-50,000) into cleared fat pads of 3-week-old congenic or isogenic recipients. Transplants were harvested after 3-5 months, and whole mounts were prepared to evaluate the extent of epithelial outgrowth. Half and 32% of host glands were colonized after the transfer of 5,000 and 500 wild-type mammary cells, respectively (Fig. 4A). Only one of 46 fat pads hosting Lrp5-/- mammary cells contained a mammary tree (Fig. 4A); an additional three host glands contained an epithelial rudiment (data not shown). In fact, transfers of 50,000 Lrp5-/- mammary cells still failed to reconstitute a mammary tree (Fig. 4, A and B), suggesting a loss of stem cell activity in the context of Lrp5 deficiency.

Canonical Wnt Signaling Is Compromised in Lrp5-/- Mammary Placodes—Mammary development begins at E10.5, and by birth a primitive ductal tree has formed. The stem cells required for its extension during puberty are already present at birth. To test whether Lrp5 is the principal signaling receptor for Wnt ligands during embryonic mammary development, we crossed Lrp5-/- mice to transgenic mice carrying a BAT-gal lacZ reporter gene that is expressed at sites of canonical pathway activity (23). Reporter gene activity, detected by X-gal staining for beta-galactosidase, was significantly reduced in Lrp5-/- embryos relative to littermate controls (Fig. 5, A and B). On the histological level the mammary placodes of Lrp5-/- embryos were significantly smaller and contained fewer cells with reporter gene activity (Fig. 5, C and D). We also performed X-gal staining on mammary whole mounts from newborn, juvenile, and adult virgin females. Reporter gene activity was significantly reduced in the ductal tree of 2-day-old Lrp5-/- female mice relative to littermate controls (Fig. 5, E and F). BAT-gal expression could not be detected after the first week of life (data not shown), which is consistent with previous reports (13, 14).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we describe a requirement for the Wnt co-receptor Lrp5 in mammary morphogenesis and tumor formation mediated by ductal stem cells. Importantly, Lrp5-/- mice are resistant to Wnt1-induced tumors, which have been shown to be derived from the mammary stem/progenitor cell population (Fig. 1). These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced hyperplasia and reduced accumulation of mammary progenitor cells (Fig. 2). In addition to the tumor resistance phenotype, loss of Lrp5 impairs various stem cell activities required for normal mammary development (Fig. 3), and Lrp5-/- ductal cells exhibit little to no stem cell activity in limiting dilution transplants (Fig. 4). Lrp5-/- embryos also exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placode (Fig. 5). Lrp5-/- mice still express Lrp6 throughout mammary development,4 and the Wnt1-induced tumors that finally do arise in Lrp5-/- mice also express Lrp6 (Fig. 1). Therefore, Lrp6 may mediate the residual canonical signaling seen in the absence of Lrp5, but clearly Lrp6 cannot compensate for the loss of Lrp5 in tumor initiation or development. Non-canonical Wnt signaling or mTOR signaling directly induced by Wnt ligands may also play a role in Wnt1-induced tumorigenesis and contribute to tumor development in Lrp5-/- mice (30, 31). These findings have important implications for the characterization of mammary stem cells and tumors induced by Wnt effectors.


Figure 4
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  		FIGURE 4.
Assay of stem cell activity. Mammary epithelial cells were isolated from Lrp5-/- and control mice, and different numbers of cells were transferred to cleared fat pads to test their outgrowth potential. The fraction of cleared fat pads colonized by cells is shown in panel A. Numbers above columns represent the number of glands colonized per total number of glands transplanted. The morphogenesis of a representative outgrowth from 5,000 Lrp5+/+ cell inocula and lack of outgrowth from 5,000 Lrp5-/- cell inocula is shown in panel B.

 
Canonical Wnt signaling has been implicated in the regulation of various stem cells, including hematopoietic, intestinal, and epidermal stem cells (32). For example, soluble Wnt proteins promote growth and inhibit differentiation in hematopoietic stem cells (2). Wnt signaling also inhibits the differentiation of stem cells in the intestinal epithelium and in hair follicles (33, 34). Importantly, in many of the same tissues where the Wnt pathway controls stem cells, deregulation of Wnt signaling leads to tumor formation. Stabilization of beta-catenin in the intestinal epithelium or overexpression of beta-catenin in the epidermis results in the development of intestinal adenomas or hair tumors, respectively (35, 36). This suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Consistently, mice that overexpress Wnt signaling effectors in the mammary epithelium develop tumors with stem/progenitor cell properties, in contrast to mice overexpressing several other oncogenes (19).


Figure 5
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  		FIGURE 5.
Mammary BAT-gal expression in the presence or absence of Lrp5. X-gal-stained BAT-gal embryo whole mounts (A and B) and histology sections of mammary placode (C and D) at E12.5. Cells expressing BAT-gal stain blue. A, at E12.5 X-gal staining reveals that the mammary placodes stain dark blue in embryos that carry at least one copy of Lrp5. B, in Lrp5-/- BAT-gal transgenic embryos the staining of the mammary placodes is significantly fainter. Arrows indicate mammary placodes number 2, 3, 4, and 5. Inserts show a higher magnification of placode number 4. C and D, the mammary placodes of Lrp5-/- embryos are significantly smaller and exhibit fewer cells with BAT-gal expression than mammary placodes from littermates carrying at least one intact copy Lrp5. E and F, the ductal trees of Lrp5-/- neonates exhibit significantly less reporter gene activity both in regard to staining intensity and to the number of BAT-gal-positive cells.

 
The pre-neoplastic hyperplasia contains an increased fraction of cells positive for molecular markers that have been associated with mammary progenitor cells, and the likelihood of progression to carcinoma correlates with the overall number of progenitor cells (17, 19, 20). In addition, mammary ductal cells from pre-neoplastic Wnt1 transgenic mice show an increased frequency of cells with stem cell activity, measured by transferring limiting dilutions of cells to fat pads in vivo (17, 20). This finding again demonstrates the ability of the Wnt pathway to target stem/progenitor cells for transformation, possibly reflecting a role of the Wnt pathway in the self-renewal of normal breast epithelium.

We have shown for the first time that loss of Lrp5-mediated canonical Wnt signaling impairs the mammary stem cell compartment. During normal development, the ductal tree fills the mammary fat pad by the end of puberty and TEBs disappear. In the absence of Lrp5, juvenile ductal branching and extension is supported by fewer TEBs and is significantly delayed. TEBs persist several weeks after they disappear in control littermate females, and the ductal tree never fills the fat pad, even after the TEBs disappear. Lrp5-/- ductal cells are unable to reconstitute ductal trees even when transplanted in large numbers (50,000 ductal cells). Shackleton et al. (17) recently showed that a functional mammary gland could be generated from the transplantation of one ductal stem cell. They estimated the fraction of ductal stem cells in the mature mammary gland to be 1/1,400. Thus we conclude that the mammary glands from adult Lrp5-/- females lack functioning somatic stem cells.

There are various ways to explain why Lrp5-/- mammary glands are relatively normal but contain no or very few stem cells: 1) fewer primitive mammary stem cells develop leading to very low stem cell fractions in the adult mammary gland; 2) the proportion of stem cells dividing by self-renewal (and symmetric division) is decreased, leading to progenitor-based organogenesis (either because the stem cell niche is ineffective or the cells differentiate precociously); or 3) canonical Wnt signaling is required for stem/progenitor cell survival (37). We have no data to support or refute the second and third proposals, but in support of the first proposal our analysis of Wnt reporter mice shows that Lrp5-/- embryos develop abnormally small mammary placodes with significantly reduced canonical signaling relative to littermate controls. Previous literature on Wnt reporter mice has shown that canonical Wnt signaling is specifically active during embryonic mammary development (13, 14). Furthermore, Wnt signaling is absolutely required, because mammary placodes fail to develop in transgenic mice overexpressing the Wnt inhibitor Dkk1 (6). Dkk1 inhibits the Wnt signaling pathway by binding to, and presumably inactivating, Lrp5 and Lrp6.

Finally, a growing body of evidence suggests that specific subtypes of the most common human tumors, including breast (38), lung (39), and colon (40), originate in stem cell compartments. Signaling pathways that regulate stem cell activity could therefore be effective drug targets. In fact, several studies have shown that activation of canonical Wnt signaling is common in human breast cancer (41-43). We show that in the absence of Lrp5, the response to ectopically expressed Wnt1 in the mammary epithelium is almost eliminated (Fig. 2), as is tumor development (Fig. 1). The few mammary tumors that arise in Lrp5-/- mice exhibit histology and differentiation markers similar to those of Lrp5-expressing tumors (Fig. 1 and supplemental Fig. S1), suggesting that Lrp5-/- tumors also originate from the mammary progenitor cell population. The delay in tumorigenesis could thus be explained by a reduction in the number of transformable progenitor cells. Taken together, these findings suggest that the Lrp5 receptor may be a highly effective target for inhibiting oncogenic canonical Wnt signaling in human malignancies.


   FOOTNOTES
 
* This work was supported by the Van Andel Research Institute, National Institutes of Health RO1 CA113869-01 (to Y. L.), Susan G. Komen Breast Cancer Foundation Grant BCTR 0202106 (to C. M. A.), and a fellowship from the Wenner-Gren Foundation (to C. L.). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence may be addressed: McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Ave. Madison, WI 53706. Tel.: 608-265-5182; Fax: 608-262-2824; E-mail: alexander{at}oncology.wisc.edu. 2 To whom correspondence may be addressed: Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Inst., 333 Bostwick Ave. NE, Grand Rapids, MI 49504. Tel.: 616-234-5308; Fax: 616-234-5309; E-mail: Bart.Williams{at}vai.org.

3 The abbreviations used are: Lrp5, low density lipoprotein receptor-related protein 5; TEB, terminal end bud; X-gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside; MMTV, murine mammary tumor virus. Back

4 C. Lindvall, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Sara Millar and Stefano Piccolo for providing the BAT-gal transgenic mice and the VARI Vivarium staff for excellent animal husbandry. We thank Kyle Furge and Michael Lewis for helpful discussions and Troy Giambernardi, Sheri Holmen, Susan Kitchen, and Scott Robertson for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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