LRP6 Transgenic Mice: Implication for Breast Cancer Tumorigenesis

Oncogene (2010) 29, 539–549 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Wnt signaling activation and mammary gland hyperplasia in MMTV– LRP6 transgenic mice: implication for breast cancer tumorigenesis

J Zhang1,4,YLi1,3,4, Q Liu1,WLu1,3 and G Bu1,2

1Department of Pediatrics, Washington University School of Medicine, St Louis, MO, USA; 2Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, USA and 3Department of Biochemistry and Molecule Biology, Drug Discovery Division, Southern Research Institute, Birmingham, AL, USA

Although Wnt signaling activation is frequently observed et al., 2004). In the absence of Wnt ligands, b-catenin in human breast cancer, mutations in genes encoding is phosphorylated by a multi-protein complex that intracellular components of the Wnt signaling pathway marks it for ubiquitination and degradation by the are rare. We found that the expression of Wnt signaling proteasome. This b-catenin degradation complex con- co-receptor, LRP6, is upregulated in a subset of human tains the adenomatous polyposis coli (APC) tumor breast cancer tissues and cell lines. To examine whether suppressor, scaffold protein Axin, glycogen synthase the overexpression of LRP6 in mammary epithelial cells is kinase 3b and casein kinase 1. The action of this sufficient to activate Wnt signaling and promote cell complex is inhibited upon binding of Wnt to its proliferation, we generated transgenic mice overexpres- receptors. Experiments carried out in Drosophila sing LRP6 in mammary epithelial cells driven by the (Wehrli et al., 2000), Xenopus (Tamai et al., 2000) and mouse mammary tumor virus (MMTV) promoter. We mice (Pinson et al., 2000) demonstrated that the low- found that mammary glands from MMTV–LRP6 mice density lipoprotein receptor-related protein 5 (LRP5)/ exhibit significant Wnt activation evidenced by the LRP6 (termed Arrow in Drosophila) acts as a co- translocation of b-catenin from membrane to cytoplas- receptor for Wnts that interact with both the mic/nuclear fractions. The expression of several Wnt seven transmembrane receptor of the Frizzled family target genes including Axin2, Cyclin D1 and c-Myc was and LRP5/6 to activate the canonical Wnt signaling also increased in MMTV–LRP6 mice. More importantly, pathway. mammary glands from virgin MMTV–LRP6 mice exhibit The role of Wnt/b-catenin signaling in cell prolifera- significant hyperplasia, a precursor to breast cancer, when tion indicates that the dysregulation of this pathway compared with wild-type littermate controls. Several may result in cancer. Indeed, several components of matrix metalloproteinases are upregulated in MMTV– the Wnt/b-catenin signaling pathway have been identi- LRP6 mice that could contribute to the hyperplasia fied as oncogenes or tumor suppressors (showing gain- phenotype. Our results suggest that Wnt signaling of-function or loss-of-function mutations, respectively) activation at the cell-surface receptor level can contribute in human cancers (Giles et al., 2003; Moon et al., 2004). to breast cancer tumorigenesis. Mutations in these genes are most evident in colorectal Oncogene (2010) 29, 539–549; doi:10.1038/onc.2009.339; cancer. About 85% of all colorectal cancers contain published online 2 November 2009 mutations in the tumor suppressor gene APC. Muta- tions in the oncogene encoding b-catenin (CTNNB1) Keywords: LRP6; Wnt signaling; mammary gland; are present in approximately 10% of the colorectal breast cancer cancers. The consequence of either APC inactivation or b-catenin mutation is similar: failure of proper b-catenin degradation leads to its cytosolic accumulation, nuclear translocation and constitutive activation of b-catenin- Introduction responsive genes (Giles et al., 2003; Moon et al., 2004). Although genetic mutations of APC or CTNNB1 are The defining feature of the canonical Wnt pathway is the rarely observed in breast cancer, compelling evidence stabilization of cytosolic b-catenin, which enters the has indicated abnormal regulation of Wnt/b-catenin nucleus and activates Wnt target genes by binding to signaling in breast cancer tumorigenesis (Turashvili transcription factors of the T-cell factor/lymphoid et al., 2006; Lindvall et al., 2007). Wnt1, the founding enhancing factor family (Giles et al., 2003; Moon member of the Wnt gene family, was initially identified as a mammary oncogene insertionally activated by Correspondence: Dr G Bu, Department of Pediatrics, Washington mouse mammary tumor virus (Nusse and Varmus, University School of Medicine, 660 South Euclid Ave., St Louis, 1982; Peters et al., 1983; Nusse et al., 1984). Over- MO 63110, USA. expression of Wnt1, Wnt10b or an activated form of b- E-mail: [email protected] 4These authors contributed equally to this work. catenin in vivo results in mammary tumorigenesis Received 25 January 2009; revised 10 September 2009; accepted 17 (Tsukamoto et al., 1988; Lane and Leder, 1997), September 2009; published online 2 November 2009 whereas mice deficient in LRP5 are resistant to Wnt1- LRP6 promotes mammary gland hyperplasia J Zhang et al 540 induced mammary tumors (Lindvall et al., 2006). b-catenin distribution such that the cytosolic b-catenin Mammary tumors were also observed in heterozygous level is significantly increased. This is accompanied by a APCMin mice (Moser et al., 1993). In human breast significant increase in Wnt/b-catenin signaling, and cell cancer, secreted Frizzled-related protein1, a member of proliferation in vitro and tumor growth in vivo (Li et al., the secreted Wnt antagonist family, is downregulated in 2004). To investigate the role of LRP6 in mammary malignant tissues (Ugolini et al., 2001; Klopocki et al., tumorigenesis, we generated mouse mammary tumor 2004). More importantly, b-catenin levels are signifi- virus (MMTV)–LRP6 transgenic mice and found that cantly upregulated and correlate with poor prognosis, overexpression of LRP6 in the mouse mammary gland is acting as a strong and independent prognostic factor in sufficient to induce mammary hyperplasia. human breast cancer patients (Lin et al., 2000). Low-density lipoprotein receptor-related protein 6 is expressed in human cancer cell lines and human Results malignant tissues (Li et al., 2004), and is elevated in testicular germ cell tumors (Rodriguez et al., 2003). Generation of MMTV–LRP6 transgenic mice Bafico et al. (2004) reported that there is an autocrine To assess the potential role of LRP6 in mammary mechanism for constitutive Wnt-pathway activation in tumorigenesis, we generated a mouse model in which human cancer cells, and that the autocrine Wnt human LRP6 is overexpressed in the mammary gland. signaling can be inhibited by siRNA directed against The transgenic construct consists of a MMTV promoter LRP6. This is the first demonstration that Wnt signaling placed upstream of human LRP6 cDNA followed by an may be activated in cancerous cells by cell surface SV40 polyadenylation site. MMTV–LRP6 mice were Wnt receptors and not because of mutations in one of generated (named Founder 1–5) and three founders the downstream signaling components. Our previous (Founder 1, 4 and 5) carried germline transmission of studies demonstrated that the stable expression of LRP6 the LRP6 transgene were identified by RT–PCR in human fibrosarcoma HT1080 cells alters subcellular (Figure 1a). Real-time quantitative PCR analysis

Figure 1 Generation and characterization of mouse mammary tumor virus (MMTV)–low-density lipoprotein receptor-related protein (LRP6) mice. (a) RNA isolated from mammary glands of MMTV–LRP6 virgin mice and wild-type littermate controls (12-weeks old) was used for RT–PCR to detect myc-tagged human LRP6 transgene in Founder 4. (b) Quantification of LRP6 transgene expression by quantitative PCR. Founder 4 has the highest level of LRP6 transgene expression, whereas Founder 5 has the lowest. (c) Mammary glands from MMTV–LRP6 mice or wild-type littermate controls (12 weeks old, n ¼ 4) were dissected and lysates were prepared. The levels of LRP6 in crude membrane fraction were analyzed by western blotting with a specific LRP6 antibody. Samples were also probed with an anti-actin antibody to verify equal loading. Lower panel, quantification of the western blot signals of LRP6 from mammary glands, which were normalized to actin levels. Error bars represent s.d. *Po0.05 indicates a significant difference compared with mammary glands from wild-type littermate controls.

Oncogene LRP6 promotes mammary gland hyperplasia J Zhang et al 541 showed that Founder 4 has the highest level of LRP6 mammary gland taken from a wild-type littermate transgene expression, whereas Founder 5 has the control female mouse revealed a branching ductal lowest level (Figure 1b). structure typical of a virgin female. In contrast, To confirm LRP6 expression at the protein level, inspection of the MMTV–LRP6 mammary gland western blot analysis of mammary gland lysates revealed an unusual number of secondary and tertiary prepared from 10-week old MMTV–LRP6 virgin mice branches and small, spiculated side buds. Quantification was carried out. An antibody to detect both the of terminal end buds (TEBs) from mammary glands endogenous mouse LRP6 and the transgenic human revealed that terminal end buds from MMTV–LRP6 LRP6 was used. We found that mammary glands from virgin mice (Founder 4) at 14 weeks (n ¼ 4) and 21 weeks the transgenic MMTV–LRP6 Founder 4 and 1 dis- (n ¼ 4) of age are 3.3- and 6-folds higher, respectively, played 2.6-fold and 1.7-fold greater LRP6 expression than those from wild-type littermate control glands levels, respectively, compared with mammary glands (Figure 3b). Both the Founder 1 and 4 lines displayed from wild-type littermate controls (Figure 1c). Further- similar mammary epithelial abnormalities with the more, immunohistochemical staining confirmed that the Founder 4 line having more significant hyperplasia. LRP6 expression in transgenic MMTV–LRP6 virgin Furthermore, histological sections of virgin glands glands was higher than wild-type virgin glands showed more individual ducts lined with cuboidal (Figures 2a and b). epithelium in transgenic mammary glands than wild- type littermate control glands (Figures 2c and d). These findings indicate that overexpression of LRP6 in the Mammary gland hyperplasia in MMTV–LRP6 mouse mammary gland is sufficient to induce mammary transgenic mice gland hyperplasia. Whole-mount preparations are a well-established meth- od to identify early premalignant lesions of the mammary epithelium (Cardiff et al., 2000). Thus, we Wnt signaling activation in MMTV–LRP6 transgenic carried out whole-mount staining of virgin glands to mice examine the ductal structure of mammary glands in The ability of LRP6 overexpression to induce mammary MMTV–LRP6 transgenic mice and wild-type littermate gland hyperplasia is likely the result of activation of the controls. As shown in Figure 3a, examination of a Wnt/b-catenin signaling pathway. Transactivation of

Figure 2 Low-density lipoprotein receptor-related protein (LRP6) immunohistochemical staining in the mammary glands of mouse mammary tumor virus (MMTV)–LRP6 mice. (a and b) LRP6 expression in mammary glands of MMTV–LRP6 virgin mice and wild- type littermate controls was shown through immunohistochemical staining using human LRP6 polyclonal antibody. Bar, 200 mm. (c and d) Hematoxylin and eosin staining of mammary glands from MMTV–LRP6 virgin mice and wild-type littermate controls. Bar, 400 mm, inset, 200 mm. A full colour version of this figure is available at the Oncogene journal online.

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Figure 3 Mammary gland hyperplasia in mouse mammary tumor virus (MMTV)–low-density lipoprotein receptor-related protein (LRP6) mice. (a) Mammary glands from MMTV–LRP6 virgin mice or wild-type littermate controls (Founder 4 line) at 14 or 21 weeks of age were analyzed by whole mount staining. Inset: high magnification of a selected area. Note mammary hyperplasia in MMTV– LRP6 mice. Bar, 500 mm, inset, 400 mm. (b) Quantification of terminal end buds (TEBs) from wild-type and MMTV–LRP6 mice (Founder 4, n ¼ 4) at 14 or 21 weeks of age. Error bars represent s.d. *Po0.05 indicates a significant difference compared with mammary glands from wild-type littermate controls. A full colour version of this figure is available at the Oncogene journal online.

gene expression by the b-catenin–T-cell factor/lymphoid In addition, Wnt3A treatment greatly enhanced the enhancing factor complex represents the nuclear target effect of LRP6 on TOPFlash luciferase activity in of the Wnt/b-catenin signaling pathway. To measure T- human mammary epithelial cells (Figure 4a). cell factor/lymphoid enhancing factor-dependent tran- Uncomplexed cytosolic b-catenin (free b-catenin) is scriptional activation we carried out reporter-gene the active form of b-catenin that is translocated to the expression analysis with the Wnt/b-catenin signaling cell nucleus, which activates transcription factors of the reporter TOPFlash in primary human mammary epithe- T-cell factor/lymphoid enhancing factor family, leading lial cells (Figure 4a). A vector with mutated copies of the to the transcription of Wnt target genes (Bafico et al., T-cell factor/lymphoid enhancing factor (FOPFlash) 1998, 2004). We then examined the extent of Wnt/ was used for measuring non-specific transactivation b-catenin signaling in mammary glands from MMTV– (Figure 4b). We found that transient transfection of LRP6 mice or wild-type littermate controls by determin- LRP6 into human mammary epithelial cells results in a ing the levels of free b-catenin present in the cytoplasma significant increase of TOPFlash luciferase activity, an of mammary tissues. Although there was no significant effect blocked by co-transfection with Dkk1 (Figure 4a). difference of total cellular b-catenin in mammary glands

Oncogene LRP6 promotes mammary gland hyperplasia J Zhang et al 543 wild-type littermate controls. Furthermore, we prepared mammary gland nuclear extracts and found that the levels of both total b-catenin and unphosphorylated, active b-catenin in mammary nuclear extracts from MMTV–LRP6 transgenic mice were higher than those from wild-type littermate controls (Figures 5b and c). All together, these data suggest that the increased LRP6 expression in mammary glands promotes Wnt/b-catenin signaling by altering b-catenin subcellular distribution. Axin2 (Yan et al., 2001; Jho et al., 2002; Leung et al., 2002; Lustig et al., 2002) is a direct and specific transcriptional target of the Wnt/b-catenin signaling pathway. It is well recognized that the expression level of Axin2 is the signature of the activation of Wnt/ b-catenin signaling. To further confirm that Wnt/ b-catenin signaling is upregulated in MMTV–LRP6 transgenic mammary glands, we examined the expres- sion of Axin2 by quantitative PCR. As expected, Axin2 in mammary glands from MMTV–LRP6 transgenic mice were 3.3-folds higher than those from wild-type littermate controls (Figure 6a). Cyclin D1 and c-Myc are key transcriptional targets of the Wnt/b-catenin pathway (He et al., 1998; Shtutman et al., 1999; Tetsu and McCormick, 1999). To identify a potential mechanism by which LRP6 accelerates the development of dysplastic mammary lesions, we exam- ined the expression of c-Myc and Cyclin D1 in mammary tissues. As expected, the virgin glands from MMTV–LRP6 transgenic mice exhibited higher levels of c-Myc and Cyclin D1 expression than wild-type littermate controls (Figure 6b). Quantification of the western blot signals revealed that the expression levels of c-Myc and Cyclin D1 in mammary glands from MMTV-LRP6 transgenic mice are 1.5- and 3.3-fold higher, respectively, than those from wild-type litter- mate controls (Figure 6c). Figure 4 Wnt signaling is upregulated in human mammary Cyclin D1 and c-Myc are two important cell-cycle epithelial cells (HMECs) by low-density lipoprotein receptor- regulators. Having established that the expression levels related protein (LRP6) overexpression. HMECs were cultured in of c-Myc and Cyclin D1 in mammary glands from 24-well plates. For each well, 0.05 mg of Wnt signaling reporter MMTV–LRP6 transgenic mice are upregulated, we then construct TOPFlash (a) or FOPFlash (b), and 0.05 mg of the b-galactosidase-expressing vector were co-transfected with 0.05 mg examined the proliferation status of mammary epithelial of LRP6-, Dkk1-expressing vector or empty pcDNA3 vector. cells. Ki67 is a nuclear protein that is tightly linked to After 24 h, cells were treated with 5% of Wnt3A CM or L cell the cell cycle, and a marker of cell proliferation. Indeed, control CM. After further 24 h incubation, cells were lysed and immunohistochemical staining revealed that Ki67 ex- both luciferase and b-galactosidase activities were determined. pression in transgenic MMTV–LRP6 virgin glands was The luciferase activity was normalized to the activity of the b-galactosidase. All values are the average of triple determinations higher than wild-type virgin glands (Figures 6d and e). with the s.d. indicated by error bar. *Po0.05 indicates a significant In contrast, TUNEL staining revealed that there was no difference compared with HMECs transfected with LRP6 and significant difference in apoptosis status between trans- Dkk1, or with empty vector only; **Po0.01 indicates a significant genic MMTV–LRP6 virgin glands and wild-type virgin difference compared with HMECs transfected with LRP6 or Dkk1 only, or to HMECs transfected with LRP6 and Dkk1 and treated glands (Supplementary Figure 1), suggesting that the with Wnt3A CM. regulation of apoptosis is not involved in mammary gland hyperplasia of MMTV–LRP6 mice. between MMTV–LRP6 transgenic mice and wild-type littermate controls, virgin glands from MMTV–LRP6 Matrix metalloproteinase (MMP) expression transgenic mice (Founder 4) exhibited higher levels of in MMTV–LRP6 mammary glands cytoplasmic free b-catenin than wild-type littermate The MMPs are known to degrade extracellular matrix controls (Figure 5a). Quantification of western blot proteins and to carry out key functions in tissue signals revealed that expression levels of cytoplasmic development and tumor progression (Egeblad and free b-catenin in mammary glands from MMTV–LRP6 Werb, 2002). MMP-2, -3, -7, -9, -13 and -14 are all transgenic mice are 1.9-folds higher than those from known target genes of the Wnt/b-catenin pathway

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Figure 5 Wnt/b-catenin signaling activation in the mammary glands of mouse mammary tumor virus (MMTV)–low-density lipoprotein receptor-related protein (LRP6) mice. (a) Virgin glands from MMTV–LRP6 mice or wild-type littermate controls (14 weeks old, n ¼ 3) were dissected and lysates were prepared. Cytoplasmic free b-catenin was pulled down from 200 mg of cell lysate using pGST–E-cadherin, and then examined by western blotting with a specific b-catenin antibody. The levels of total cellular b-catenin and actin were also analyzed by western blotting. Right panel, quantification of the western blotting signals of cytoplasmic free b-catenin. (b and c) Nuclear extracts from mammary glands of MMTV–LRP6 mice or wild-type littermate controls (14-weeks old, n ¼ 3) were prepared by fractionation, and examined by western blotting with either regular b-catenin antibody (b) or active b-catenin (unphosphorated b-catenin) antibody (c). Lamin B1 was blotted as a loading control. Lower panel, quantification of the western blot signals of b-catenin from mammary gland nuclear extracts. The levels of b-catenin were normalized to the Lamin B1 levels. Error bars represent s.d. *Po0.05 indicates a significant difference compared with mammary glands from wild-type littermate controls.

(Crawford et al., 1999; Takahashi et al., 2002; analysis to detect the expression levels of MMP-2, -3, Tamamura et al., 2005; Wu et al., 2007). To further -7, -9, -13 and -14 in virgin glands from 12-week old mice. identify potential mechanisms by which LRP6 As shown in Figure 7, MMP levels in mammary glands accelerates the development of dysplastic mammary from MMTV–LRP6 transgenic mice were 2–10-fold lesions, we carried out real-time quantitative PCR higher than those from wild-type littermate controls.

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Figure 6 Axin2, c-Myc, Cyclin D1 and Ki-67 are up-regulated in mouse mammary tumor virus (MMTV)–low-density lipoprotein receptor-related protein (LRP6) mice. (a) Total RNA was isolated from virgin mammary gland of MMTV–LRP6 virgin mice and wild- type littermate controls (4 weeks, n ¼ 3). The levels of Axin2 were measured by quantitative PCR. Fold changes in expression compared with wild-type littermate controls were plotted. (b) Virgin glands from MMTV–LRP6 mice or wild-type littermate controls (14 weeks old, n ¼ 4) were dissected, and lysates were prepared. The levels of total cellular c-Myc and Cyclin D1 were analyzed by western blotting with a specific c-Myc antibody or Cyclin D1 antibody, and the samples were also probed with an anti-actin antibody to verify equal loading. (c) Quantification of the western blot signals of c-Myc and Cyclin D1 expression described in (b). The levels of c-Myc and Cyclin D1 expression were normalized to the actin levels. (d and e) Ki67 immunostaining of mammary glands from MMTV–LRP6 virgin mice (e) and wild-type littermate controls (d). Bar, 200 mm. Error bars represent s.d. *Po0.05 indicates a significant difference compared with mammary glands from wild-type littermate controls. A full colour version of this figure is available at the Oncogene journal online.

Discussion grade, poor prognosis and younger patient age. Alto- gether, these findings indicate that mammary tumori- Although genetic mutations of certain intracellular genesis can be initiated at the cell surface receptor level components of the Wnt/b-catenin pathway, such as in mammary epithelium and that LRP6 is a potential APC and CTNNB1, are significant contributing factors target for breast cancer therapy. for colorectal cancers, they are typically not the Cyclin D1 and c-Myc are two important cell-cycle predominate mechanism associated with other cancer regulators. Clinically, the Cyclin D1 gene is amplified in types such as breast cancer. Instead, it appears that the upto 20% of human breast cancers and Cyclin D1 dysregulation of cell surface Wnt/b-catenin signaling protein is overexpressed in >50% of human mammary components leads to aberrant activation of this pathway carcinomas (Bartkova et al., 1994; Gillett et al., 1994; in breast cancer (Turashvili et al., 2006; Lindvall et al., McIntosh et al., 1995). For c-Myc, a comprehensive 2007). We found that the expression of the Wnt meta-analysis suggests that at least 15% of breast signaling co-receptor LRP6 is upregulated in a subset cancers present with significant amplification of c-Myc, of human breast cancer tissues and cell lines (unpub- and that c-Myc amplification is significantly associated lished data). In this study, we demonstrated that with a poor prognosis in breast cancer (Deming et al., transgenic mice overexpressing LRP6 in mammary 2000). In animal studies, transgenic mice overexpressing epithelial cells driven by the MMTV promoter is Cyclin D1 in mammary epithelium (MMTV–Cyclin D1) sufficient to induce mammary gland hyperplasia, a develop mammary hyperplasia and mammary carcino- precursor to breast cancer. During the revision process mas (Wang et al., 1994). Similarly, constitutive c-Myc of the paper, Lindvall et al. (2009) reported that expression under the control of MMTV or the whey canonical Wnt signaling through LRP6 is required for acidic protein promoters is oncogenic in transgenic mice normal mouse mammary gland development, and that (reviewed in Amundadottir et al., 1996; Nass and LRP6 expression is increased in basal-like human breast Dickson, 1997). Amplification of c-Myc or Cyclin D1 cancer, a triple-negative phenotype associated with high has been identified as a downstream step at the end of

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Figure 7 Matrix metalloproteinases (MMPs) are upregulated in mouse mammary tumor virus (MMTV)–low-density lipoprotein receptor-related protein (LRP6) mice. RNA isolated from mammary glands of 14-month old MMTV–LRP6 virgin mice and wild-type littermate controls were prepared, and the expression levels of 6 MMPs were measured by quantitative PCR. Fold changes compared with wild-type littermate controls were plotted. All the values are the average of triple determinations with the s.d. indicated by error bars. *Po0.05 indicates a significant difference compared with mammary glands from wild-type littermate controls.

Wnt/b-catenin pathway activation (He et al., 1998; MMTV–Wnt1 mice were crossed with transgenic mice Shtutman et al., 1999; Tetsu and McCormick, 1999). overexpressing a natural MMP inhibitor, tissue inhibi- Therefore, it is expected that LRP6 overexpression tor of MMP-2, in the mammary gland, the double would cause the activation of Wnt/b-catenin signaling, transgenic mice displayed an increase in tumor latency upregulation of Cyclin D1 and c-Myc expression levels, and a reduction in tumor formation. MMP-2, -3, -7, -9, - and increases the expression of the cell proliferation 13 and -14 are all known target genes of the Wnt/b- marker Ki67, all of which we observed experimentally catenin pathway (Crawford et al., 1999; Takahashi in MMTV–LRP6 mice. As such, these events et al., 2002; Tamamura et al., 2005; Wu et al., 2007). In could account for mammary hyperplasia in MMTV– this study, we demonstrated that mammary glands from LRP6 mice. MMTV–LRP6 transgenic mice exhibit significantly Matrix metalloproteinases are multifunctional en- higher levels of MMPs than wild-type littermate zymes capable of targeting the extracellular matrix, controls. Therefore, upregulation of MMPs could also growth factors, cytokines and cell surface-associated account for mammary hyperplasia in MMTV–LRP6 adhesion and signaling receptors. Clinically, MMPs mice. have been associated with advanced-stage cancer and Whole-mount staining of mammary gland is a well- contribute to tumor progression, invasion and metas- established and recommended method to identify early tasis. In ductal breast carcinomas, it has been demon- premalignant lesions of the mammary epithelium strated that MMP-2, -3, -9, -11, -13 and -14 are (Cardiff et al., 2000). In our mammary gland model, synthesized either by stromal fibroblasts, infiltrating we found that the MMTV–LRP6 female mice exhibit macrophages or vascular pericytes (Wolf et al., 1993; mammary gland hyperplasia. However, none of the Okada et al., 1995; Heppner et al., 1996; Chenard et al., MMTV–LRP6 mice developed adenocarcinoma of 1999; Nielsen et al., 1997, 2001). In animal models, it has breast for more than an 18-month span. Therefore, it been found that the expression of an autoactivating is possible that the extent of the Wnt/b-catenin form of MMP-3 under the control of whey acidic activation upon LRP6 overexpression is sufficient to protein promoter induces premalignant and malignant cause mammary gland hyperplasia, but is insufficient to lesions in mammary glands. Moreover overexpression of lead to breast cancer in virgin mice. It will be interesting a natural inhibitor of MMPs, tissue inhibitor of MMP- to examine whether multiparous MMTV–LRP6 mice 1, inhibits tumor formation in MMP-3 transgenic mice develop breast cancer. Crossing MMTV–LRP6 mice (Sternlicht et al., 1999). More interestingly, Blavier et al. with MMTV–Wnt mice will allow us to test whether (2006) recently reported that the expression of several LRP6 and Wnt1 synergistically promote breast cancer MMPs including MMP-2, -3, -9, -13 and -14 was tumorigenesis. Together, these studies should help to increased in hyperplastic glands and mammary tumors define whether LRP6 and Wnt ligands are novel targets of MMTV–Wnt1 transgenic mice. Furthermore, when for breast cancer therapy.

Oncogene LRP6 promotes mammary gland hyperplasia J Zhang et al 547 Materials and methods Digital Science image analysis software. Human LRP6 anti- body was from R&D (Indianapolis, IN, USA). c-Myc and Generation of MMTV–LRP6 mice Cyclin D1 antibodies were from Santa Cruz (Santa Cruz, CA, To generate the MMTV–human LRP6 construct, human USA). b-catenin antibody was from BD Transduction LRP6 cDNA fragment was removed from the pCS–myc– Laboratories (San Jose, CA, USA), active b-catenin antibody human LRP6 vector (kindly provided by Dr Christof Niehrs, was from Millipore (Billerica, MA, USA). b-Actin antibody German Cancer Research Center, Heidelberg, Germany) by was from Sigma (St Louis, MO, USA). digestion with Cla and XbaI, followed by n-fill with Klenow enzyme. The LRP6 fragment was then blunt-end ligated into the EcoRI site (n-filled) of the MMTV–SV40–Bssk vector Luciferase reporter assay (kindly provided by Dr Philip Leder, Harvard Medical School, Wnt3A conditioned medium (Wnt3A CM) and L cell control Boston, MA, USA). Purified MMTV–LRP6 DNA fragment CM were prepared as previously described (Lu et al., 2008). was microinjected into the pronuclei of fertilized mouse eggs, Normal human mammary epithelial cells were purchased and transferred to pseudopregnant mothers by the Transgenic from Lonza. human mammary epithelial cells were plated and ES Cell Core of Washington University School of into 24-well plates. For each well, 0.05 mg of the TOPFlash Medicine. Mice were genotyped for the presence of the TCF luciferase construct (Upstate Biotechnology, Billerica, transgenes by PCR with primers 50-GGCTATACCAG MA, USA) or the negative control FOPFlash TCF luciferase TGACTTGAACTATGATT-30, and 50-GGTTCCTTCAC construct was co-transfected with LRP6-, Dkk1-expressing AAGATCCTCTAGAGTC-30 to identify the MMTV– vector, or empty pcDNA3 vector. A b-galactosidase-expres- LRP6 transgene. sing vector (Promega) was included as an internal control for transfection efficiency. After 24 h incubation, the cells were treated with 5% of Wnt3A CM or L cell control CM. After RNA isolation and quantitative PCR further 24 h incubation, cells were lysed and both luciferase Total RNA from inguinal mammary gland (number 4) of 14- and b-galactosidase activities were determined with enzyme week old mice was extracted by SV total RNA isolation kit assay kits (Promega). Luciferase activity was normalized to the (Promega Madison, WI, USA; Z3100). Total RNA was activity of the b-galactosidase. dissolved in nuclease-free water and stored at À80 1C. Reverse transcription was carried out using a SuperScript II RNase H- reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and the GST–E-cadherin binding assay for cytoplasmic free b-catenin reaction mixture was subjected to either RT–PCR or quantita- The GST–E-cadherin binding assay was carried out as tive real-time PCR to detect levels of human LRP6, MMPs, previously described (Lu et al., 2008). Briefly, recombinant Axin2 and actin, which was used as an internal control. For a GST–E-cadherin protein was expressed, purified and conju- 50 ml of quantitative PCR reaction, 25 ml of SYBR Supermix gated with agarose beads. The GST–E-cadherin beads were incubated with 200 mg of total cell lysates prepared from from BioRad (Hercules, CA, USA), 1 mlof10mM forward mammary gland tissues of MMTV–LRP6 mice and wild-type primer, 1 mlof10mM reverse primer, 2 ml of mixture of reverse 1 transcription reaction and 21 ml of water were included. After 40 littermate controls for 4 h at 4 C. This process allows cycles, the relative levels of gene expression were quantified with uncomplexed cytoplasmic free b-catenin in total cell lysates BioRad iCycler iQ software. The primers used to amplify target to bind to GST–E-cadherin beads. The cytoplasmic free genes by RT–PCR and quantitative PCR were as b-catenin was then eluted from the GST–E-cadherin beads, follows: hLRP6-F (50-TAGCATTGAAAGAGTTCATAAAC- subjected to SDS–PAGE, and detected using a monoclonal GA-30), hLRP6-R (50-CACTCGATGAACATTTGTAGCC antibody to b-catenin. TTT-30); Axin2-F (50-TGACTCTCCTTCCAGATCCCA-30), Axin2-R (50-TGCCCACACTAGGCTGACA-30); MMP2-F Preparation of crude membrane fractions (50-CAAGTTCCCCGGCGATGTC-30), MMP2-R (50-TTCTG Membrane fractionation can enrich membrane proteins to be GTCAAGGTCACCTGTC-30); MMP3-F (50-TGTCCCGT detected. Thoracic and inguinal mammary glands from both TTCCATCTCTCTC-30), MMP3-R (50-TGGTGATGTCTCA MMTV–LRP6 transgenic mice and wild-type littermate GGTTCCAG-30); MMP7-F (50-CTGCCACTGTCCCAGGA controls were harvested and immediately put into prechilled AG-30), MMP7-R (50-GGGAGAGTTTTCCAGTCATGG-30); 20 mM Tris pH 8.0 containing 150 mM NaCl, 1 mM CaCl and MMP9-F (50-GGACCCGAAGCGGACATTG-30), MMP9-R 2 EDTA-free complete proteinase inhibitors. After homogeniza- (50-CGTCGTCGAAATGGGCATCT-30); MMP13-F (50-ACC tion with a dounce homogenizer and low-speed centrifugation TCCACAGTTGACAGGCT-30), MMP13-R (50-AGGCACT at 1500 g for 5 mins, a small portion of homogenate was kept CCACATCTTGGTTT-30); MMP14-F (50-CAGTATGGCT at 4 1C as whole tissue lysate, whereas the majority of ACCTACCTCCAG-30), MMP14-R (50-GCCTTGCCTGTC homogenate was further centrifuged at 100 000 g for 30 min ACTTGTAAA-3 0). at 4 1C to separate the membrane fraction (insoluble pellet) from cytoplasmic fraction (supernatant). The insoluble pellet Western blot analysis was resuspended in 50 mM Tris pH 8.0 containing 80 mM NaCl, Thoracic and inguinal mammary glands from both wild 2mM CaCl2 and EDTA-free complete proteinase inhibitors. type and transgenic mice were lysed with phosphate-buffered Triton X-100 was then added to whole tissue lysate, membrane saline containing 1% Triton X-100, 1 mM phenylmethylsulfo- and cytoplasmic fractions at 1% final concentration. The nyl fluoride and the protease inhibitor cocktail from Roche membrane fraction was passed several times through a 28-G (Indianapolis, IN, USA). Protein concentrations were deter- needle and cleared further by centrifugation at 100 000 g for mined with Protein Assay kit from BioRad. Equal amount of 15 min. Total protein concentrations were determined with proteins from each sample was used for SDS–PAGE. The Protein Assay kit (BioRad). Equal amounts of total protein immunoreactive bands were visualized by enhanced chemilu- were separated by SDS–PAGE under reducing condition, and minescence and exposure to film. For densitometric analyses, analyzed by western blotting. To validate the membrane immunoreactive bands were scanned using a Kodak Digital protein preparation, Western blot analysis was carried out Science DC120 Zoom camera and quantified using Kodak with antibodies against cell surface receptor LRP1 (Bu et al.,

Oncogene LRP6 promotes mammary gland hyperplasia J Zhang et al 548 1995) and nuclear marker Lamin B1 (Abcam, Cambridge, CA, Hematoxylin and eosin staining USA) (Supplementary Figure 2a). Inguinal mammary glands (number 4) were excised, fixed with paraformadehyde and embedded in paraffin. Sections were cut Nuclear extraction at 5 mm, dewaxed, rehydrated and stained with hematoxylin NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo and eosin. Scientific, Waltham, MA, USA) was used for the preparation of nuclear and cytoplasmic fractions from mammary gland TUNEL staining tissues. To validate the purity of the nuclear and cytoplasmic Paraffin sections at 5 mm of mammary glands from MMVT– fractions, western blot analysis was carried out with antibodies LRP6 and wild-type littermate controls were depariffinized. against nuclear marker Lamin B1 (Abcam) and cytoplasmic TUNEL staining was carried out with TUNEL Apop- marker HSP90 (Cell Signaling Technology, Danvers, MA, tosis Detection kit (Upstate) following manufacturer’s instruc- USA) (Supplementary Figure 2b). tions, and nuclei were counterstained with 5 mg/ml DAPI for 5 min. Whole-mount staining of mammary gland Inguinal mammary glands (number 4) were dissected at the Statistical analysis indicated times of development and spread on glass slides. All quantified data represent an average of at least three After fixation in Carnoy’s fixative for 4 h at room temperature, samples. Error bars represent s. d. Statistical significance was the tissues were hydrated and stained in carmine alum as determined by Student’s t-test, and P 0.05 was considered as described on http://mammary.nih.gov. Samples were then o significant. dehydrated, cleared in Histoclear, and photographed. Count- ing of terminal end buds was carried out by defining counting areas first, drawing 1 Â 1 mm squares on whole-mount pictures, and taking the average number of terminal end buds Conflict of interest from 5 randomly picked areas. The authors declare no conflict of interest. Immunohistochemistry Inguinal mammary glands (number 4) were dissected from both MMTV–LRP6 transgenic mice and wild-type littermate controls, fixed in 4% phosphate-buffered paraformadehyde Acknowledgements overnight, transferred to 70% ethanol, embedded in paraffin and sectioned at 5 mm. After deparaffinization, rehydration We are grateful to Christof Niehrs (German Cancer Research and antigen retrieval by heating in antigen unmasking solution Center) for providing LRP6 cDNA, Dr Philip Leder (Harvard (Dako, Carpinteria, CA, USA), tissue slices were blocked with Medical School) for providing the MMTV–SV40–Bssk vector, serum using Vectastain ABC kit (Vector Laboratories, Dr Gail VW Johnson (University of Rochester) for providing Burlingame, CA, USA), incubated with primary anti-human the GST–E-cadherin construct, and Dr Jin-Moo Lee for LRP6 antibody (R&D) or Ki67 antibody (Abcam) at 4 1C providing rat brain sections subjected to hypoxia–ischemia as overnight. After washing with phosphate-buffered saline, the positive controls for apoptosis. We also thank Dr Taj King for slices were incubated with biotinylated secondary antibody for critical reading of the manuscript. This work was supported by 30 min at room temperature, and detected by Histostain Plus grants from the National Institutes of Health R01CA100520 DAB kit (Zymed Laboratories, CA, USA). (to GB) and RO1CA124531 (to YL).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene