Mammary Development and Breast Cancer: A Wnt Perspective

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Abstract

The Wnt pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis. In this review, we will summarize the current knowledge of how the pathway regulates stem cells and normal development of the mammary gland, and discuss how its various components contribute to breast carcinoma pathology.

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Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors

Jason I. Herschkowitz, Karl Simin, Victor J Weigman … (2007)

Background Global gene expression analyses of human breast cancers have identified at least three major tumor subtypes and a normal breast tissue group [1]. Two subtypes are estrogen receptor (ER)-negative with poor patient outcomes [2,3]; one of these two subtypes is defined by the high expression of HER2/ERBB2/NEU (HER2+/ER-) and the other shows characteristics of basal/myoepithelial cells (basal-like). The third major subtype is ER-positive and Keratin 8/18-positive, and designated the 'luminal' subtype. This subtype has been subdivided into good outcome 'luminal A' tumors and poor outcome 'luminal B' tumors [2,3]. These studies emphasize that human breast cancers are multiple distinct diseases, with each of the major subtypes likely harboring different genetic alterations and responding distinctly to therapy [4,5]. Further similar investigations may well identify additional subtypes useful in diagnosis and treatment; however, such research would be accelerated if the relevant disease properties could be accurately modeled in experimental animals. Signatures associated with specific genetic lesions and biologies can be causally assigned in such models, potentially allowing for refinement of human data. Significant progress in the ability to genetically engineer mice has led to the generation of models that recapitulate many properties of human cancers [6]. Mouse mammary tumor models have been designed to emulate genetic alterations found in human breast cancers, including inactivation of TP53, BRCA1, and RB, and overexpression of MYC and HER2/ERBB2/NEU. Such models have been generated through several strategies, including transgenic overexpression of oncogenes, expression of dominant interfering proteins, targeted disruption of tumor suppressor genes, and by treatment with chemical carcinogens [7]. While there are many advantages to using the mouse as a surrogate, there are also potential caveats, including differences in mammary physiologies and the possibility of unknown species-specific pathway differences. Furthermore, it is not always clear which features of a human cancer are most relevant for disease comparisons (for example, genetic aberrations, histological features, tumor biology). Genomic profiling provides a tool for comparative cancer analysis and offers a powerful means of cross-species comparison. Recent studies applying microarray technology to human lung, liver, or prostate carcinomas and their respective murine counterparts have reported commonalities [8-10]. In general, each of these studies focused on a single or few mouse models. Here, we used gene expression analysis to classify a large set of mouse mammary tumor models and human breast tumors. The results provide biological insights among and across the mouse models, and comparisons with human data identify biologically and clinically significant shared features. Results Murine tumor analysis To characterize the diversity of biological phenotypes present within murine mammary carcinoma models, we performed microarray-based gene expression analyses on tumors from 13 different murine models (Table 1) using Agilent microarrays and a common reference design [1]. We performed 122 microarrays consisting of 108 unique mammary tumors and 10 normal mammary gland samples (Additional data file 1). Using an unsupervised hierarchical cluster analysis of the data (Additional data file 2), murine tumor profiles indicated the presence of gene sets characteristic of endothelial cells, fibroblasts, adipocytes, lymphocytes, and two distinct epithelial cell types (basal/myoepithelial and luminal). Grouping of the murine tumors in this unsupervised cluster showed that some models developed tumors with consistent, model-specific patterns of expression, while other models showed greater diversity and did not necessarily group together. Specifically, the TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3 (Notch4), TgWAP-Tag and TgC3(1)-Tag tumors had high within-model correlations. In contrast, tumors from the TgWAP-T 121 , TgMMTV-Wnt1, Brca1 Co/Co ;TgMMTV-Cre;p53 +/-, and DMBA-induced models showed diverse expression patterns. The p53 -/- transplant model tended to be homogenous, with 4/5 tumors grouping together, while the Brca1 +/-;p53 +/- ionizing radiation (IR) and p53 +/- IR models showed somewhat heterogeneous features between tumors; yet, 6/7 Brca1 +/-;p53 +/- IR and 5/7 p53 +/- IR were all present within a single dendrogram branch. Table 1 Summary of mouse mammary tumor models Tumor model No. of tumors Specificity of lesions Experimental oncogenic lesion(s) Strain Reference TgWAP-Myc 13 WAP* cMyc overexpression FVB [60] TgWAP-Int3 7 WAP Notch4 overexpression FVB [61] TgWAP-T 121 5 WAP pRb, p107, p130 inactivation B6D2 [37] TgWAP-T 121 2 WAP pRb, p107, p130 inactivation BALB/cJ [37] TgWAP-Tag 5 WAP SV40 L-T (pRb, p107, p130, p53, p300 inactivation, others); SV40 s-t C57Bl/6 [62] TgC3(1)-Tag 8 C3(1)† SV40 L-T (pRb, p107, p130, p53, p300 inactivation, others); SV40 s-t FVB [63] TgMMTV-Neu 10 MMTV‡ Unactivated rat Her2 overexpression FVB [64] TgMMTV-Wnt1 11 MMTV Wnt 1 overexpression FVB [65] TgMMTV-PyMT 7 MMTV Py-MT (activation of Src, PI-3' kinase, and Shc) FVB [66] TgMMTV-Cre;Brca1 Co/Co ;p53+/- 10 MMTV Brca1 truncation mutant; p53 heterozygous null C57Bl/6 [67] p53-/-transplanted 5 None p53 inactivation BALB/cJ [68] Medroxyprogesterone-DMBA-induced 11 None Random DMBA-induced FVB [69] p53+/-irradiated 7 None p53 heterozygous null, random IR induced BALB/cJ [70] Brca1 +/-;p53+/-irradiated 7 None Brca1 and p53 heterozygous null, random IR induced BALB/cJ [1] *WAP, whey acidic protein promoter, commonly restricted to lactating mammary gland luminal cells. †C3(1), 5' flanking region of the C3(1) component of the rat prostate steroid binding protein, expressed in mammary ductal cells. ‡MMTV, mouse mammary tumor virus promoter, often expressed in virgin mammary gland epithelium, induced with lactation; often expressed at ectopic sites (for example, lymphoid cells, salivary gland, others). As with previous human tumor studies [1,3], we performed an 'intrinsic' analysis to select genes consistently representative of groups/classes of murine samples. In the human studies, expression variation for each gene was determined using biological replicates from the same patient, and the 'intrinsic genes' identified by the algorithm had relatively low variation within biological replicates and high variation across individuals. In contrast, in this mouse study we applied the algorithm to groups of murine samples defined by an empirically determined correlation threshold of > 0.65 using the dendrogram from Additional data file 2. This 'intrinsic' analysis yielded 866 genes that we then used in a hierarchical cluster analysis (Figure 1 and Additional data file 3 for the complete cluster diagram). This analysis identified ten potential groups containing five or more samples each, including a normal mammary gland group (Group I) and nine tumor groups (designated Groups II-X). Figure 1 Mouse models intrinsic gene set cluster analysis. (a) Overview of the complete 866 gene cluster diagram. (b) Experimental sample associated dendrogram colored to indicate ten groups. (c) Luminal epithelial gene expression pattern that is highly expressed in TgMMTV-PyMT, TgMMTV-Neu, and TgWAP-myc tumors. (d) Genes encoding components of the basal lamina. (e) A second basal epithelial cluster of genes, including Keratin 5. (f) Genes expressed in fibroblast cells and implicated in epithelial to mesenchymal transition, including snail homolog 1. (g) A second mesenchymal cluster that is expressed in normals. See Additional data file 2 for the complete cluster diagram with all gene names. In general, these ten groups were contained within four main categories that included (Figure 1b, left to right): the normal mammary gland samples (Group I) and tumors with mesenchymal characteristics (Group II); tumors with basal/myoepithelial features (Groups III-V); tumors with luminal characteristics (Groups VI-VIII); and tumors containing mixed characteristics (Groups IX and X). Group I contained all normal mammary gland samples, which showed a high level of similarity regardless of strain, and was characterized by the high expression of basal/myoepithelial (Figure 1e) and mesenchymal features, including vimentin (Figure 1g). Group II samples were derived from several models (2/10 Brca1 Co/Co ;TgMMTV-Cre;p53 +/-, 3/11 DMBA-induced, 1/5 p53 -/- transplant, 1/7 p53 +/- IR, 1/10 TgMMTV-Neu and 1/7 TgWAP-T 121 ) and also showed high expression of mesenchymal features (Figure 1g) that were shared with the normal samples in addition to a second highly expressed mesenchymal-like cluster that contained snail homolog 1 (a gene implicated in epithelial-mesenchymal transition [11]), the latter of which was not expressed in the normal samples (Figure 1f). Two TgWAP-Myc tumors at the extreme left of the dendrogram, which showed a distinct spindloid histology, also expressed these mesenchymal-like gene features. Further evidence for a mesenchymal phenotype for Group II tumors came from Keratin 8/18 (K8/18) and smooth muscle actin (SMA) immunofluorescence (IF) analyses, which showed that most spindloid tumors were K8/18-negative and SMA-positive (Figure 2l). Figure 2 Immunofluorescence staining of mouse samples for basal/myoepithelial and luminal cytokeratins. (a) Wild-type (wt) mammary gland stained for Keratins 8/18 (red) and Keratin 5 (green) shows K8/18 expression in luminal epithelial cells and K5 expression in basal/myoepithelial cells. (b-f) Mouse models that show luminal-like gene expression patterns stained with K8/18 (red) and K5 (green). (g-k) Tumor samples that show basal-like, or mixed luminal and basal characteristics by gene expression, stained for K8/18 (red) and K5 (green). (j) A subset of Brca1 Co/Co ;TgMMTV-Cre;p53+/-tumors showing nodules of K5/K8/18 double positive cells. (l) A splindloid tumor stained for K8/18 (red) and smooth muscle actin (green). The second large category contained Groups III-V, with Group III (4/11 DMBA-induced and 5/11 Wnt1), Group IV (7/7 Brca1+/-;p53 +/- IR, 4/10 Brca1 Co/Co ;TgMMTV-Cre;p53 +/-, 4/6 p53 +/- IR and 3/11 Wnt1) and Group V (4/5 p53 -/- transplant and 1/6 p53 +/- IR), showing characteristics of basal/myoepithelial cells (Figure 1d, e). These features were encompassed within two expression patterns. One cluster included Keratin 14, 17 and LY6D (Figure 1d); Keratin 17 is a known human basal-like tumor marker [1,12], while LY6D is a member of the Ly6 family of glycosylphosphatidylinositol (GPI)-anchored proteins that is highly expressed in head and neck squamous cell carcinomas [13]. This cluster also contained components of the basement membrane (for example, Laminins) and hemidesmosomes (for example, Envoplakin and Desmoplakin), which link the basement membrane to cytoplasmic keratin filaments. A second basal/myoepithelial cluster highly expressed in Group III and IV tumors and a subset of DMBA tumors with squamous morphology was characterized by high expression of ID4, TRIM29, and Keratin 5 (Figure 1e), the latter of which is another human basal-like tumor marker [1,12]. This gene set is expressed in a smaller subset of models compared to the set described above (Figure 1d), and is lower or absent in most Group V tumors. As predicted by gene expression data, most of these tumors stained positive for Keratin 5 (K5) by IF (Figure 2g-k). The third category of tumors (Groups VI-VIII) contained many of the 'homogenous' models, all of which showed a potential 'luminal' cell phenotype: Group VI contained the majority of the TgMMTV-Neu (9/10) and TgMMTV-PyMT (6/7) tumors, while Groups VII and VIII contained most of the TgWAP-Myc tumors (11/13) and TgWAP-Int3 samples (6/7), respectively. A distinguishing feature of these tumors (in particular Group VI) was the high expression of XBP1 (Figure 1c), which is a human luminal tumor-defining gene [14-17]. These tumors also expressed tight junction structural component genes, including Occludin, Tight Junction Protein 2 and 3, and the luminal cell K8/18 (Additional data file 2). IF for K8/18 and K5 confirmed that these tumors all exclusively expressed K8/18 (Figure 2b-f). Finally, Group IX (1/10 Brca1 Co/Co ;TgMMTV-Cre;p53 +/-, 4/7 TgWAP-T 121 tumors and 5/5 TgWAP-Tag tumors) and Group X (8/8 TgC3(1)-Tag) tumors were present at the far right and showed 'mixed' characteristics; in particular, the Group IX tumors showed some expression of luminal (Figure 1c), basal (Figure 1d) and mesenchymal genes (Figure 1f), while Group X tumors expressed basal (Figure 1e,f) and mesenchymal genes (Figure 1f,g). IF analyses showed that, as in humans [12,18], the murine basal-like models tended to express K5 while the murine luminal models expressed only K8/18. However, some of the murine basal-like models developed tumors that harbored nests of cells of both basal (K5+) and luminal (K8/18+) cell lineages. For example, in some TgMMTV-Wnt1 [19], DMBA-induced (Figure 2g,i), and Brca1-deficient strain tumors, distinct regions of single positive K5 and K8/18 cells were observed within the same tumor. Intriguingly, in some Brca1 Co/Co ;TgMMTV-Cre;p53 +/- samples, nodules of double-positive K5 and K8/18 cells were identified, suggestive of a potential transition state or precursor/stem cell population (Figure 2j), while in some TgMMTV-Wnt1 (Figure 2h) [19] and Brca1-deficient tumors, large regions of epithelioid cells were present that had little to no detectable K5 or K8/18 staining (data not shown). The reproducibility of these groups was evaluated using 'consensus clustering' (CC) [20]. CC using the intrinsic gene list showed strong concordance with the results sown in Figure 1 and supports the existence of most of the groups identified using hierarchical clustering analysis (Additional data file 4). However, our further division of some of the CC-defined groups appears justified based upon biological knowledge. For instance, hierarchical clustering separated the normal mammary gland samples (Group I) and the histologically distinct spindloid tumors (Group II), which were combined into a single group by CC. Groups VI (TgMMTV-Neu and PyMT) and VII (TgWAP-Myc) were likewise separated by hierarchical clustering, but CC placed them into a single category. CC was also performed using all genes that were expressed and varied in expression (taken from Additional data file 2), which showed far less concordance with the intrinsic list-based classifications, and which often separated tumors from individual models into different groups (Figure 3c, bottom most panel); for example, the TgMMTV-Neu tumors were separated into two or three different groups, whereas these were distinct and single groups when analyzed using the intrinsic list. This is likely due to the presence or absence of gene expression patterns coming from other cell types (that is, lymphocytes, fibroblasts, and so on) in the 'all genes' list, which causes tumors to be grouped based upon qualities not coming from the tumor cells [1]. Figure 3 Unsupervised cluster analysis of the combined gene expression data for 232 human breast tumor samples and 122 mouse mammary tumor samples. (a) A color-coded matrix below the dendrogram identifies each sample; the first two rows show clinical ER and HER2 status, respectively, with red = positive, green = negative, and gray = not tested; the third row includes all human samples colored by intrinsic subtype as determined from Additional data file 6; red = basal-like, blue = luminal, pink = HER2+/ER-, yellow = claudin-low and green = normal breast-like. The remaining rows correspond to murine models indicated at the right. (b) A gene cluster containing basal epithelial genes. (c) A luminal epithelial gene cluster that includes XBP1 and GATA3. (d) A second luminal cluster containing Keratins 8 and 18. (e) Proliferation gene cluster. (f) Interferon-regulated genes. (g) Fibroblast/mesenchymal enriched gene cluster. (h) The Kras2 amplicon cluster. See Additional data file 5 for the complete cluster diagram. Mouse-human combined unsupervised analysis The murine gene clusters were reminiscent of gene clusters identified previously in human breast tumor samples. To more directly evaluate these potential shared characteristics, we performed an integrated analysis of the mouse data presented here with an expanded version of our previously reported human breast tumor data. The human data were derived from 232 microarrays representing 184 primary breast tumors and 9 normal breast samples also assayed on Agilent microarrays and using a common reference strategy (combined human datasets of [21-23] plus 58 new patients/arrays). To combine the human and mouse datasets, we first used the Mouse Genome Informatics database to identify well-annotated mouse and human orthologous genes. We then performed a distance weighted discrimination correction, which is a supervised analysis method that identifies systematic differences present between two datasets and makes a global correction to compensate for these global biases [24]. Finally, we created an unsupervised hierarchical cluster of the mouse and human combined data (Figure 3 and Additional data file 5 for the complete cluster diagram). This analysis identified many shared features, including clusters that resemble the cell-lineage clusters described above. Specifically, human basal-like tumors and murine Brca1 +/-;p53 +/-;IR, Brca1 Co/Co ;TgMMTV-Cre;p53 +/-, TgMMTV-Wnt1, and some DMBA-induced tumors were characterized by the high expression of Laminin gamma 2, Keratins 5, 6B, 13, 14, 15, TRIM29, c-KIT and CRYAB (Figure 3b), the last of which is a human basal-like tumor marker possibly involved in resistance to chemotherapy [25]. As described above, the Brca1 +/-;p53 +/-;IR, some Brca1 Co/Co ;TgMMTV-Cre;p53 +/, DMBA-induced, and TgMMTV-Wnt1 tumors stained positive for K5 by IF, and human basal-like tumors tend to stain positive using a K5/6 antibody [1,12,18,26], thus showing that basal-like tumors from both species share K5 protein expression as a distinguishing feature. The murine and human 'luminal tumor' shared profile was not as similar as the shared basal profile, but did include the high expression of SPDEF, XBP1 and GATA3 (Figure 3c), and both species' luminal tumors also stained positive for K8/18 (Figure 2 and see [18]). For many genes in this luminal cluster, however, the relative level of expression differed between the two species. For example, some genes were consistently high across both species' tumors (for example, XBP1, SPDEF and GATA3), while others, including TFF, SLC39A6, and FOXA1, were high in human luminal tumors and showed lower expression in murine tumors. Of note is that the human luminal epithelial gene cluster always contains the Estrogen-Receptor (ER) and many estrogen-regulated genes, including TFF1 and SLC39A6 [22]; since most murine mammary tumors, including those profiled here, are ER-negative, the apparent lack of involvement of ER and most ER-regulated genes could explain the difference in expression for some of the human luminal epithelial genes that show discordant expression in mice. Several other prominent and noteworthy features were also identified across species, including a 'proliferation' signature that includes the well documented proliferation marker Ki-67 (Figure 3e) [1,27,28] and an interferon-regulated pattern (Figure 3f) [27]. The proliferation signature was highest in human basal-like tumors and in the murine models with impaired pRb function (that is, Group IX and X tumors). Currently, the growth regulatory impact of interferon-signaling in human breast tumors is not understood, and murine models that share this expression feature (TgMMTV-Neu, TgWAP-Tag, p53 -/- transplants, and spindloid tumors) may provide a model for future studies of this pathway. A fibroblast profile (Figure 3g) that was highly expressed in murine samples with spindloid morphology and in the TgWAP-Myc 'spindloid' tumors was also observed in many human luminal and basal-like tumors; however, on average, this profile was expressed at lower levels in the murine tumors, which is consistent with the relative epithelial to stromal cell proportions seen histologically. Through these analyses we also discovered a potential new human subtype (Figure 3, top line-yellow group, and Additional data file 6). This subtype, which was apparent in both the human only and mouse-human combined dataset, is referred to as the 'claudin-low' subtype and is characterized by the low expression of genes involved in tight junctions and cell-cell adhesion, including Claudins 3, 4, 7, Occludin, and E-cadherin (Figure 3d). These human tumors (n = 13) also showed low expression of luminal genes, inconsistent basal gene expression, and high expression of lymphocyte and endothelial cell markers. All but one tumor in this group was clinically ER-negative, and all were diagnosed as grade II or III infiltrating ductal carcinomas (Additional data file 7 for representative hematoxylin and eosin images); thus, these tumors do not appear to be lobular carcinomas as might be predicted by their low expression of E-cadherin. The uniqueness of this group was supported by shared mesenchymal expression features with the murine spindloid tumors (Figure 3g), which cluster near these human tumors and also lack expression of the Claudin gene cluster (Figure 3d). Further analyses will be required to determine the cellular origins of these human tumors. A common region of amplification across species The murine C3(1)-Tag tumors and a subset of human basal-like tumors showed high expression of a cluster of genes, including Kras2, Ipo8, Ppfibp1, Surb, and Cmas, that are all located in a syntenic region corresponding to human chromosome 12p12 and mouse chromosome 6 (Figure 3h). Kras2 amplification is associated with tumor progression in the C3(1)-Tag model [29], and haplo-insufficiency of Kras2 delays tumor progression [30]. High co-expression of Kras2-linked genes prompted us to test whether DNA copy number changes might also account for the high expression of Kras2 among a subset of the human tumors. Indeed, 9 of 16 human basal-like tumors tested by quantitative PCR had increased genomic DNA copy numbers at the KRAS2 locus; however, no mutations were detected in KRAS2 in any of these 16 basal-like tumors. In addition, van Beers et al. [31] reported that this region of human chromosome 12 is amplified in 47% of BRCA1-associated tumors by comparative genomic hybridization analysis; BRCA1-associated tumors are known to exhibit a basal-like molecular profile [3,32]. In cultured human mammary epithelial cells, which show basal/myoepithelial characteristics [1,33], both high oncogenic H-ras and SV40 Large T-antigen expression are necessary for transformation [34]. Taken together, these findings suggest that amplification of KRAS2 may either influence the cellular phenotype or define a susceptible target cell type for basal-like tumors. Mouse-human shared intrinsic features To simultaneously classify mouse and human tumors, we identified the gene set that was in common between a human breast tumor intrinsic list (1,300 genes described in Hu et al. [21]) and the mouse intrinsic list developed here (866 genes). The overlap of these two lists totaled 106 genes, which when used in a hierarchical clustering analysis (Figure 4) identifies four main groups: the leftmost group contains all the human basal-like, 'claudin-low', and 5/44 HER2+/ER- tumors, and the murine C3(1)-Tag, TgWAP-Tag, and spindloid tumors. The second group (left to right) contains the normal samples from both humans and mice, a small subset (6/44) of human HER2+/ER- and 10/92 luminal tumors, and a significant portion of the remaining murine basal-like models. By clinical criteria, nearly all human tumors in these two groups were clinically classified as ER-negative. Figure 4 Cluster analysis of mouse and human tumors using the subset of genes common to both species intrinsic lists (106 total genes). (a) Experimental sample associated dendrogram color coded according to human tumor subtype and with a matrix below showing murine tumor origins. (b) The complete 106 gene cluster diagram. (c) Close-up of genes known to be important for human basal-like tumors. (d) Close-up of genes known to be important for human luminal tumors, including ER. (e) Expression pattern of HER2/ERBB2/NEU. The third group contains 33/44 human HER2+/ER- tumors and the murine TgMMTV-Neu, MMTV-PyMT and TgWAP-Myc samples. Although the human HER2+/ER- tumors are predominantly ER-negative, this comparative genomic analysis and their keratin expression profiles as assessed by immunohistochemistry, suggests that the HER2+/ER- human tumors are 'luminal' in origin as opposed to showing basal-like features [18]. The fourth and right-most group is composed of ER-positive human luminal tumors and, lastly, the mouse TgWAP-Int3 (Notch4) tumors were in a group by themselves. These data show that although many mouse and human tumors were located on a large dendrogram branch that contained most murine luminal models and human HER2+/ER- tumors, none of the murine models we tested showed a strong human 'luminal' phenotype that is characterized by the high expression of ER, GATA3, XBP1 and FOXA1. These analyses suggest that the murine luminal models like MMTV-Neu showed their own unique profile that was a relatively weak human luminal phenotype that is missing the ER-signature. Presented at the bottom of Figure 4 are biologically important genes discussed here, genes previously shown to be human basal-like tumor markers (Figure 4c), human luminal tumor markers, including ER (Figure 4d), and HER2/ERBB2/NEU (Figure 4e). A comparison of gene sets defining human tumors and murine models We used a second analysis method called gene set enrichment analysis (GSEA) [35] to search for shared relationships between human tumor subtypes and murine models. For this analysis, we first performed a two-class unpaired significance analysis of microarray (SAM) [36] analysis for each of the ten murine groups defined in Figure 1, and obtained a list of highly expressed genes that defined each group. Next, we performed similar analyses using each human subtype versus all other human tumors. Lastly, the murine lists were compared to each human subtype list using GSEA, which utilizes both gene list overlap and gene rank (Table 2). We found that the murine Groups IX (p = 0.004) and X (p = 0.001), which comprised tumors from pRb-deficient/p53-deficient models, shared significant overlap with the human basal-like subtype and tended to be anti-correlated with human luminal tumors (p = 0.083 and 0.006, respectively). Group III murine tumors (TgMMTV-Wnt1 mostly) significantly overlapped human normal breast samples (p = 0.008), possibly due to the expression of both luminal and basal/myoepithelial gene clusters in both groups. Group IV (Brca1-deficient and Wnt1) showed a significant association (p = 0.058) with the human basal-like profile. The murine Group VI (TgMMTV-Neu and TgMMTV-PyMT) showed a near significant association (p = 0.078) with the human luminal profile and were anti-correlated with the human basal-like subtype (p = 0.04). Finally, the murine Group II spindloid tumors showed significant overlap with human 'claudin-low' tumors (p = 0.001), which further suggests that this may be a distinct and novel human tumor subtype. Table 2 Gene set enrichment analysis of the ten murine groups versus five human subtypes Basal-like Luminal HER2+/ER- Normal Claudin-low Mouse class No. of genes p value p value p value p value p value p value p value p value p value p value Is class I 1,882 - - 0.4625 0.8755 0.5388 0.9137 0.1659 0.5628 0.0048 0.1028 II 912 - - - - 0.5867 0.9609 - - 0.0021 0.001 III 143 0.5289 0.9048 - - 0.5285 0.9047 0 0.008 - - IV 1,019 0 0.0581 - - - - - - - - V 34 - - 0.8492 0.998 0.9324 0.999 - - 0.0427 0.09274 VI 820 - - 0.0062 0.0783 0.3536 0.7864 0.8653 0.9769 - - VII 851 0.1258 0.3768 - - 0.5616 0.9137 - - - - VIII 236 0.1449 0.6098 0.3483 0.8205 - - 0.01878 0.2349 - - IX 462 0.0019 0.004 - - 0.56 0.9509 - - - - X 338 0 0.001 - - 0.9275 0.998 - - - - Is not class I 1,882 0.0128 0.1662 - - - - - - - - II 912 0.3996 0.8348 0.8601 0.999 - - 0.3602 0.7655 - - III 143 - - 0.3178 0.7259 - - - - 0.7628 0.991 IV 1,019 - - 0.1833 0.6516 0.398 0.8427 0.2241 0.7255 0.1453 0.6116 V 34 0.86 1 - - - - 0.0656 0.1653 - - VI 820 0 0.04 - - - - - - 0.1043 0.4444 VII 851 - - 0.1733 0.5151 - - 0.5403 0.9128 0.1628 0.5215 VIII 236 - - - - 0.1131 0.5305 - - 0.6427 0.961 IX 462 - - 0.04305 0.0833 - - 0.022 0.037 0.2612 0.5936 X 338 - - 0.02236 0.0682 - - 0.1313 0.3717 0.5437 0.9489 Statistically significant findings are highlighted in bold. NOM = nominal. We also performed a two-class unpaired SAM analysis using each mouse model as a representative of a pathway perturbation using the transgenic 'event' as a means of defining groups. Models that yielded a significant gene list (false discovery rate (FDR) = 1%) were compared to each human subtype as described above (Additional data file 8). The models based upon SV40 T-antigen (all C3(1)-Tag and WAP-Tag tumors) shared significant overlap with the human basal-like tumors (p = 0.002) and were marginally anti-correlated with the human luminal class. The BRCA1 deficient models (all Brca1+/-;p53 +/- IR and Brca1 Co/Co ;TgMMTV-Cre;p53 +/- tumors) were marginally significant with human basal-like tumors (p = 0.088). The TgMMTV-Neu tumors were nominally significant (before correction for multiple comparisons) with human luminal tumors (p = 0.006) and anti-correlated with human basal-like tumors (p = 0.027). The two most important human breast tumor biomarkers are ER and HER2; therefore, we also analyzed these data relative to these two markers. Of the 232 human tumors assayed here, 137 had ER and HER2 data assessed by immunohistochemistry and microarray data. As has been noted before [3,18,21], there is a very high correlation between tumor intrinsic subtype and ER and HER2 clinical status (p 30. The log2 ratio of Cy5/Cy3 was then reported for each gene. In the final dataset, only genes that reported values in 70% or more of the samples were included. The genes were median centered and then hierarchical clustering was performed using Cluster v2.12 [56]. For the murine unsupervised analysis, and human-mouse unsupervised cluster analyses, we filtered for genes that varied at least three-fold or more, in at least three or more samples. Average linkage clustering was performed on genes and arrays and cluster viewing and display was performed using JavaTreeview v1.0.8 [57]. Mouse Intrinsic gene set analysis Intrinsic 'groups' of experimental samples were chosen based upon having a Pearson correlation value of 0.65 or greater from the unsupervised clustering analysis of the 122 murine samples. The analysis was performed using the Intrinsic Gene Identifier v1.0 by Max Diehn/Stanford University [1]. Technical replicates were removed from the file and the members of every highly correlated node were given identical class numbers, giving every sample that fell outside the 0.65 correlation cut-off a class of their own. Using these criteria, 16 groups of samples were identified (see Additional data file 1 for these groups) and a list of 866 'intrinsic' genes was selected using the criteria of one standard deviation below the mean intrinsic gene value. A human intrinsic list of 1,300 genes was created using a subset of 146 of the 232 samples used here, and is described in Hu et al. [21]. Consensus clustering CC [20] was performed locally using Gene Pattern 1.3.1 (built Jan 6, 2005), which was downloaded from the Broad Institute distribution website [58]. Analyses were performed on the mouse dataset with all genes, and just with intrinsic genes separately. Ranges for the number of K clusters (or the focused number of classes) were from 2 to 15 to evaluate a wide range of possible groups. Using a Euclidian distance measure with average linkage, we re-sampled 1,000 times with both column and row normalization. Combining murine and human expression datasets Orthologous genes were reported by Mouse Genome Informatics (MGI 3.1) of The Jackson Laboratory. For both the human and murine datasets, Locus Link IDs assigned to Agilent oligo probe ID numbers were used to assign to MGI ID numbers. In cases where a single gene was represented by multiple probes, the median value of the redundant probes was used. This led to a total of orthologous pairings of 14,680 Agilent probes. Prior to combining the two datasets, each was column standardized to N(0,1), row median centered, and probe identifiers were converted to MGI IDs. The intersection of mouse and human MGI identifiers from genes that passed filters (same as used above) in both datasets yielded 7,907 orthologous genes in the total combined dataset. This dataset was next corrected for systemic biases using distance weighted discrimination [24]. Finally, the combined dataset was used for an average linkage hierarchical clustering analysis. Gene set enrichment analysis We took the 232 human samples and classified them as basal-like, luminal, HER2+/ER-, claudin-low, and normal breast-like according to a clustering analysis of the human dataset only (Additional data file 6), using the new intrinsic/UNC human gene list developed in Hu et al. [21]. Second, the murine samples were also classified based upon their clustering pattern in Figure 1 that used the mouse intrinsic gene list, and were assigned to Groups I-X. Two-class unpaired SAM analysis was performed for each murine class separately versus all other classes using an FDR of 1% [36], resulting in 10 class-specific gene lists. Using only the set of highly expressed genes that were associated with each analysis (and ignoring the genes whose low expression correlated with a given class), GSEA [35] was performed in R (v. 2.0.1) using the GSEA R package [59]. The ten murine gene sets were then compared to each human subtype-ranked gene set and significant enrichments reported. For statistical strength of these enrichments, GSEA uses family wise error rate (FWER) to correct for multiple testing and FDR to reduce false positive reporting. The parameters used for all GSEA were: nperm = 1,000, weighted.score.type = 1, nom.p.val.threshold = -1, fwer.p.val.threshold = -1, fdr.q.val.threshold = 0.25, topgs = 12, adjust.FDR.q.val = FALSE, gs.size.threshold.min = 25, gs.size.threshold.max = 2,000, reverse.sign = FALSE, preproc.type = 0, random.seed = 3,338, perm.type = 0, fraction = 1, replace = FALSE. Immunofluorescence Paraffin-embedded sections (5 μm thick) were processed using standard immunostaining methods. The antibodies and their dilution were α-cytokeratin 5 (K5, 1:8,000, PRB-160P, Covance, Berkeley, CA, USA), and α-cytokeratins 8/18 (Ker8/18, 1:450, GP11, Progen Biotecknik, Heidelberg, Germany). Briefly, slides were deparaffinized and hydrated through a series of xylenes and graded ethanol steps. Heat-mediated epitope retrieval was performed in boiling citrate buffer (pH 6.0) for 15 minutes, then samples cooled to room temperature for 30 minutes. Secondary antibodies for immunofluorescence were conjugated with Alexa Fluor-488 or -594 fluorophores (1:200, Molecular Probes, Invitrogen, Carlsbad, CA, USA). IF samples were mounted with VectaShield Hardset with DAPI mounting media (Vector, Burlingame, CA, USA). Human KRAS2 amplification assay We performed real-time quantitative PCR and fluorescent melting curve analyses using genomic DNAs from 16 basal-like tumors, a normal breast tissue sample, 2 leukocyte DNA, and 3 luminal tumors. DNA was extracted using the DNAeasy kit (Qiagen) and amplification was performed on the LightCycler using the following temperature parameters: 95°C, 8 minutes; 50 cycles of 57°C, 6 s; 72°C, 6 s; 95°C, 2 s; followed by cooling to 60°C and a 0.1°C/s ramp to 97°C. Each PCR reaction contained 7.5 ng template DNA in a 10 μl reaction using the LightCycler Faststart DNA Master SYBR Green I kit (Roche Applied Science, Indianapolis, IN, USA). Relative DNA copy number for each gene was determined by importing an external efficiency curve and using a 'normal' breast sample for a within-run calibrator. For each sample, the copy number for KRAS2 was divided by the average copy number of ACTB and G1P3. Amplification in any tumor was called if the relative fold change was greater than three standard deviations above the average of five control samples (two normal leukocyte samples and three luminal tumors). Additional data files The following additional data are available with the online version of this paper. Additional data file 1 is a table listing mouse tumor and normal sample associated data, including source, transgene and promoter information. Additional data file 2 is a complete unsupervised cluster diagram of all mouse tumors. Samples are colored according to mouse model from which they were derived, and the genes were selected using a variation filter of three-fold or more on three or more samples. Additional data file 3 ia a complete mouse models cluster diagram using the 866 gene murine intrinsic gene list. Additional data file 4 provides CC analyses applied to the mouse models. (a) CC matrices generated using the 866 gene mouse intrinsic list, by cluster numbers K = 2 through K = 15. (b) Empirical cumulative distribution (CDF) plot corresponding to the consensus matrices in the range K = 2 to 15. (c) CC directly compared to the hierarchical clustering-based results. The dendrogram from Figure 1 (using the intrinsic gene set) is shown and immediately below is a colored matrix showing sample assignments based upon the various number of K clusters from the CC. By comparison, the analysis performed on the mouse dataset using all genes (bottom matrix) is presented. Additional data file 5 is a complete unsupervised cluster diagram of the combined gene expression patterns of 232 human breast tumor samples and 122 mouse mammary tumor samples. This unsupervised cluster analysis is based upon the orthologous gene overlap between the human and mouse microarrays, and then we selected for the subset of genes that varied three-fold or more on three or more arrays. Additional data file 6 shows a cluster analysis of the 232 human samples using the human intrinsic/UNC gene set from Hu et al. [21]. This analysis was used to determine a human samples subtype (basal-like, luminal, HER2+/ER-, and so on), which was then used in the various SAM and GSEA analyses. Samples are colored according to their subtype: red = basal-like, blue = luminal, pink = HER2+/ER-, yellow = claudin-low and green = normal breast-like. Additional data file 7 shows a histological characterization of six different human 'claudin-low' tumors using hematoxylin and eosin sections. Additional data file 8 shows GSEA of murine pathway models versus five human subtypes. Additional data file 9 shows GSEA of ten murine classes versus clinical ER status and HER2 status in ER negative patients. Additional data file 10 shows GSEA of murine pathway models versus clinical ER status and HER2 status in ER negative patients. Supplementary Material Additional data file 1 Mouse tumor and normal sample associated data including source, transgene and promoter information. Click here for file Additional data file 2 Samples are colored according to mouse model from which they were derived, and the genes were selected using a variation filter of three-fold or more on three or more samples. Click here for file Additional data file 3 Complete mouse models cluster diagram using the 866 gene murine intrinsic gene list. Click here for file Additional data file 4 (a) CC matrices generated using the 866 gene mouse intrinsic list, by cluster numbers K = 2 through K = 15. (b) Empirical cumulative distribution (CDF) plot corresponding to the consensus matrices in the range K = 2 to 15. (c) CC directly compared to the hierarchical clustering-based results. The dendrogram from Figure 1 (using the intrinsic gene set) is shown and immediately below is a colored matrix showing sample assignments based upon the various number of K clusters from the CC. By comparison, the analysis performed on the mouse dataset using all genes (bottom matrix) is presented. Click here for file Additional data file 5 This unsupervised cluster analysis is based upon the orthologous gene overlap between the human and mouse microarrays, and then we selected for the subset of genes that varied three-fold or more on three or more arrays. Click here for file Additional data file 6 This analysis was used to determine a human samples subtype (basal-like, luminal, HER2+/ER-, and so on), which was then used the various SAM and GSEA analyses. Samples are colored according to their subtype: red = basal-like, blue = luminal, pink = HER2+/ER-, yellow = claudin-low and green = normal breast-like. Click here for file Additional data file 7 Histological characterization of six different human 'claudin-low' tumors using hematoxylin and eosin sections. Click here for file Additional data file 8 GSEA of murine pathway models versus five human subtypes. Click here for file Additional data file 9 GSEA of ten murine classes versus clinical ER status and HER2 status in ER negative patients. Click here for file Additional data file 10 GSEA of murine pathway models versus clinical ER status and HER2 status in ER negative patients. Click here for file

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Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

Amanda Mikels, Roel Nusse (2006)

Introduction Wnt signaling controls a variety of adult and developmental processes, largely by modulating gene transcription [ 1]. The necessity of precise regulation to prevent the inappropriate activation of Wnt signaling is underscored by the fact that misregulation of several components of the canonical Wnt signal transduction pathway leads to tumorigenesis [ 2]. Wnt signaling is also thought to play a key role in controlling stem cell fate [ 3]. Thus, understanding the mechanisms that regulate Wnt signaling is of critical importance. Wnt proteins are found in all metazoan organisms and as many as 19 mammalian homologs are known (Wnt home page: http://www.stanford.edu/~rnusse/wntwindow.html). While homologs have a high degree of sequence similarity, expression of different Wnt proteins can lead to vastly different developmental outcomes. In the most well-understood “canonical” Wnt/β-catenin signaling pathway, in the absence of a Wnt ligand, the main mediator of the signal relay, β-catenin, is bound in a cytosolic protein complex containing Axin, the adenomatous polyposis coli gene product (APC), glycogen synthase kinase-3β (GSK-3β), and other proteins. Axin and APC serve as scaffolding proteins that enable GSK-3β to phosphorylate β-catenin, thereby targeting it for ubiquitination by βTrCP (beta-transducin repeat–containing homologue protein) and subsequent degradation in the proteasome. Cytosolic β-catenin protein levels are thus kept low in the absence of ligand stimulation. Wnt protein binding to cognate Frizzled (Fz) and low-density lipoprotein (LDL) receptor–related protein (LRP)5/6 coreceptors leads to the activation of the Dishevelled (Dvl) protein, which then inhibits GSK-3β–mediated phosphorylation of β-catenin. Cytosolic β-catenin protein becomes stabilized and newly synthesized β-catenin is able to accumulate and then translocate to the nucleus where binding to T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors leads to the activation of target gene expression [ 1]. Prior to the discovery of the Fz and LRP coreceptors, Wnt proteins were classified into two functional groups based on the observation that ectopic expression of some Wnts, such as Wnt1 and Wnt3a, is sufficient to induce a secondary dorsal-ventral axis in Xenopus embryos and morphologically transform C57MG mouse mammary epithelial cells, whereas expression of “Wnt5a class” Wnts, including Wnts 4, 5a, and 11, is not sufficient [ 4– 8]. One hypothesis for how the structurally similar, although functionally distinct, extracellular Wnt ligands trigger different developmental outcomes is that the two classes of Wnts signal via different intracellular pathways. Indeed, experiments in zebrafish and Xenopus embryos using mRNA injection to activate Wnt signaling have suggested that expression of Wnt5a stimulates intracellular calcium (Ca 2+) flux leading to the activation of Ca 2+–dependent effector molecules such as calcium/calmodulin–dependent kinase II (CamKII), nuclear factor associated with T cells (NFAT), and protein kinase C (PKC) in a pertussis toxin (PTX)–sensitive manner [ 9– 12]. However, due to the lack of active soluble Wnt5a protein, direct activation of “Wnt/Ca 2+” pathway signaling in mammalian cell culture systems has not been fully investigated. In addition to activating alternative signaling pathways, Wnt5a may also inhibit Wnt/β-catenin signaling. Early experiments in Xenopus embryos showed that coexpression of XWnt5a with XWnt8 abrogates the ability of XWnt8 to induce a secondary axis [ 13– 15]. Wnt5a knockout mice show increased β-catenin signaling in the distal limb, indicating that Wnt5a may inhibit β-catenin stabilization [ 16]. In contrast, experiments from Ishitani et al. have shown that Wnt5a-induced Ca 2+ flux blocks canonical signaling downstream of β-catenin stabilization by inhibiting TCF-mediated transcription in a PTX-sensitive manner [ 16– 18]. As Wnt5a heterozygous mice develop myeloid leukemias and B-cell lymphomas, an intriguing hypothesis is that Wnt5a serves as a tumor suppressor in part by preventing excess Wnt/β-catenin signaling [ 19]. While most Wnt signaling has been attributed to the activation of Fz receptors, an alternative possibility is that Wnts carry out their diverse roles by signaling via different receptors. One such receptor is Ror2, an orphan tyrosine kinase possessing an extracellular cysteine-rich Wnt binding domain (CRD) [ 20– 23]. Wnt5a and mRor2 have overlapping expression patterns, their knockout phenotypes are similar, and recently mRor2 has been shown to act synergistically with Wnt5a to activate Jun kinase [ 23– 26]. Thus, Wnt5a may be mediating its inhibitory role through the activation of this novel receptor. There are conflicting data in the literature regarding the mechanism by which Wnt5a and other so-called noncanonical Wnt protein signals. To understand how signals like Wnts influence cells, one must discriminate between early and late effects and measure those effects in a concentration-dependent manner. To that end, we have purified active Wnt5a protein and established quantitative assays for signaling. We show for the first time that soluble Wnt5a protein is able to directly inhibit canonical Wnt signaling. Treatment of cells with purified protein over short time intervals is sufficient to inhibit the activation of the TCF/LEF-driven luciferase reporter SuperTopFlash (STF). The inhibition observed is dose responsive, occurs downstream of β-catenin stabilization, and is PTX insensitive. We also show that an alternative Wnt receptor, Ror2, is required for Wnt5a-mediated inhibition of canonical signaling and that the extracellular, cysteine-rich Wnt binding domain and intracellular, cytoplasmic domain are crucial for the receptor's inhibitory function. Whereas Wnt5a protein is usually associated with noncanonical Wnt signaling, we show that Wnt5a can activate Wnt/β-catenin signaling in the presence of Fz 4 and LRP5. Based on these and other observations, we propose a model wherein receptor context dictates Wnt signaling output, suggesting a new layer of regulatory complexity in the Wnt signaling cascade with important implications in development and disease. Results Purification of Wnt5a Protein: Evidence that It Is Cleaved from a Precursor We purified the Wnt5a protein from cells overexpressing the mouse Wnt5a gene using methods derived from those developed for other members of the Wnt family with several modifications (Figure1A) [ 27]. Throughout the purification, we followed the Wnt5a protein using an antibody that detects Wnt5a on a Western blot. Later steps in the purification were also monitored by activity assays (see below). To unambiguously identify the purified protein as the product of the Wnt5a gene, we determined the amino-terminal sequence. We found that the mature protein starts with a sequence IIGAQPLCSQLAGLSQGQKKL, a sequence beginning 62 amino acids downstream from the predicted initiator methionine of Wnt5a and 24 amino acids from the predicted signal cleavage site between amino acids 37 and 38 [ 28]. While all Wnt proteins contain signal sequences required for secretion from cells, generally they are not internally cleaved at a site downstream from the signal sequence. The exception, interestingly, is the Drosophila ortholog of Wnt5a; DWnt5 protein is made as a precursor of more than 1,000 amino acids weighing 140 kDa and cleaved into a smaller mature protein of 80 kDa [ 29]. It is not known whether the processing of the Wnt5a molecule involves specific proteases. We find that, similar to other Wnt proteins that we have purified, the Wnt5a protein is hydrophobic as it requires detergent to stay in solution and partitions in a detergent phase when subjected to extraction (unpublished data) [ 27]. We presume that the protein, like Wnt3a, is modified by the covalent attachment of a palmitate, but we have not examined this further. Significantly, the cysteine residue that is modified by palmitoylation in Wnt3a is conserved in Wnt5a, even after the internal cleavage of the protein. Purified Wnt5a Protein Inhibits Canonical Wnt Reporter Activation Standard readouts for the activity of various Wnt family members include an increase in the level of the β-catenin protein and the activation of reporter genes, in particular, the Topflash reporter that contains TCF binding sites upstream of a luciferase transgene. We found that when transiently transfected into 293 cells, the SuperTopflash (STF) luciferase reporter variant [ 30] is robustly activated in response to Wnt3a treatment and exhibits dose-dependency. As shown in Figure1B, Wnt3a protein treatment induces a 100-fold increase in reporter activation and a linear dose response when the concentration is lowered. In accordance with other groups, we observe that Wnt5a protein by itself does not lead to the activation of the STF reporter in 293 cells, nor does it alter levels of β-catenin protein ( Figures 1C and 2A) [ 16, 31]. Based on reports from Ishitani et al. and others [ 14, 17, 32, 33], we then tested whether the addition of Wnt5a protein could inhibit Wnt3a-induced reporter activity. Using a concentration of Wnt3a protein within the linear range of reporter activation (50 ng/ml), Wnt5a protein was added to cells concomitantly with Wnt3a. As shown in Figure 1C, Wnt5a elicits a dose-responsive decrease in Wnt3a-mediated reporter activation. Wnt5a protein inhibits Wnt3a signaling at the earliest time points following reporter activation. When cells are pretreated with Wnt3a protein for 1 or 3 h prior to Wnt5a treatment, Wnt5a can inhibit Wnt3a-induced reporter activation within 1 h of exposure ( Figure 1D). In addition, Wnt5a pretreatment of cells for 8 h followed by washing has no effect on either Wnt3a-induced STF reporter activation or Wnt5a-mediated inhibition, suggesting that Wnt5a does not induce the accumulation of an inhibitory factor ( Figure 1E). These data taken together indicate that purified Wnt5a protein is sufficient to rapidly inhibit canonical Wnt signaling in a potentially post-translational fashion. Wnt5a Does Not Affect β-Catenin Protein Stabilization One possible explanation for the observed abrogation of reporter activation is that Wnt5a protein competes with Wnt3a for Fz receptor binding sites. Alternatively, Wnt5a has been proposed to inhibit canonical Wnt signaling via upregulation of Siah-2, which targets β-catenin for βTrCP-independent proteasomal degradation [ 16]. In both of these models, β-catenin protein levels should be reduced following concomitant Wnt5a treatment compared to Wnt3a protein treatment alone; in the former case, signal transduction is blocked at the level of the receptor, whereas in the latter, any β-catenin protein stabilized due to Wnt3a signal activation should be appreciably degraded by the opposing effects of Wnt5a. To assess the levels of accumulated β-catenin protein, we prepared cytosolic extracts from 293 cells treated for 3 h with Wnt3a protein, Wnt5a protein, or both. Cellular fractionation is necessary to ensure that only cytosolic β-catenin protein stabilized due to active Wnt signaling is observed as opposed to the relatively stable membrane-associated pool of β-catenin protein. Although a particularly low dose of Wnt3a protein (10 ng/ml) was used in this analysis to ensure that subtle differences in β-catenin protein levels could be discerned, no appreciable reduction in β-catenin accumulation was observed ( Figure 2A), even when concentrations of Wnt5a protein (200 ng/ml) sufficient to inhibit the STF reporter 5-fold were used. These data show that the Wnt3a signal is initiated in the presence of Wnt5a protein, indicating that Wnt5a does not efficiently compete with Wnt3a for receptor binding sites. To determine whether Wnt5a treatment inhibits β-catenin nuclear entry, cell-staining experiments were performed ( Figure 2B). The 293 cells treated with vehicle or Wnt5a protein alone display a membrane-associated staining pattern (arrowheads) consistent with β-catenin's role in cell adhesion. By contrast, cells treated with Wnt3a protein alone appeared brighter than vehicle-treated cells, with β-catenin observed in the cytoplasm and nuclei (arrows). No significant difference in β-catenin signal intensity or localization was observed when cells were treated with Wnt3a in combination with Wnt5a protein ( Figure 2B). The observation that Wnt3a-mediated β-catenin stabilization and nuclear entry appear unaffected by Wnt5a treatment suggests that upregulation of Siah-2, and subsequent degradation of β-catenin, is not the primary mechanism of Wnt5a protein-mediated inhibition of gene transcription in these cells. Wnt5a-Mediated Inhibition of Canonical Signaling Is Not Associated with Wnt-Stimulated Ca 2+ Flux Past studies in zebrafish and Xenopus embryos have suggested that overexpression of Wnt5a can trigger intracellular Ca 2+ flux, leading to the activation of Ca 2+-dependent effector molecules such as CamKII [ 9, 34, 35]. It has been proposed that active CamKII protein can then initiate the mammalian TGF-β–activated kinase 1(TAK)/Nemo-like kinase (NLK) mitogen-activated protein kinase signaling cascade, resulting in NLK-mediated phosphorylation of TCF/LEF transcription factors. This phosphorylation of TCF/LEF prevents the β-catenin–TCF/LEF transcriptional complex from binding to DNA [ 17, 18, 36]. In this model, inhibition of Wnt/β-catenin signaling due to Wnt5a-stimulated Ca 2+ flux occurs downstream of β-catenin stabilization and at the level of TCF-mediated transcription. In a variety of organisms and assays, Ca 2+ signaling induced by Wnts has consistently been shown to be PTX sensitive, as the Fz receptor described as transmitting the inhibitory Wnt signal (RFz2) is thought to be coupled to a PTX-sensitive G protein [ 8, 37, 38]. To determine whether Wnt5a protein inhibits β-catenin signaling via the direct activation of intracellular Ca 2+ flux downstream of heterotrimeric G proteins, 293 cells transiently transfected with the STF reporter were treated with 50 ng/ml W3a protein and increasing doses of Wnt5a protein following 24-h pretreatment with vehicle or 100 ng/ml (0.1 μM) PTX. Figure 3A shows that Wnt5a protein-mediated inhibition of the STF reporter was not affected by PTX treatment. The PTX used was active in these cells, as lysophosphatidic acid (LPA) treatment of cells following pretreatment with PTX was unable to stimulate the phosphorylation of the mitogen-activated protein kinases Erk1 and Erk2, a known PTX-sensitive process in 293 cells [ 39] ( Figure 3B). These data are consistent with Topol et al.'s [ 16] findings that overexpression of a dominant negative version of CamKII, and treatment of cells with a specific inhibitor of CamKII, does not perturb Wnt5a-mediated inhibition of Wnt/β-catenin signaling in 293 cells. Wnt5a protein was also assayed for its ability to directly stimulate intracellular calcium flux. Figure 3C shows that Wnt5a protein treatment at high doses does not alter the intracellular concentration of Ca 2+ in 293 cells, although subsequent ionomycin treatment of the same cells promotes robust Ca 2+ flux. Similar results were observed in 293 cells stably expressing mouse Fz4 (mFz4); a Wnt receptor thought to be involved in Wnt-stimulated calcium flux ( Figure 3C) [ 40]. In addition, transient treatment with Wnt5a protein does not activate other Ca 2+-sensitive reporter constructs such as an NFAT-responsive luciferase reporter (unpublished data). These data taken together strongly suggest that Wnt-stimulated Ca 2+ flux is not the direct mechanism utilized by Wnt5a to inhibit canonical Wnt signaling. Wnt5a Protein Activates β-Catenin Signaling Depending on Receptor Context Although we show that Wnt5a protein can inhibit canonical Wnt signaling, the question remains of whether Wnt5a can activate Wnt/β-catenin signaling in mammalian systems. Multiple Fz receptors, including mFz4, mFz6, mFz7, and mFz8, were assayed for their ability to transduce Wnt5a-mediated canonical Wnt signaling (unpublished data). We found that Wnt5a could indeed activate Wnt/β-catenin signaling when cells overexpressed mFz4; none of the other overexpressed receptors could transduce the Wnt5a signal. Western blot analysis of cytosolic extracts probed for β-catenin ( Figure 4A) shows that while Wnt5a has no effect on cytosolic β-catenin levels in the parental 293 cell line, Wnt5a can induce modest β-catenin accumulation in cells stably expressing a FLAG-tagged mFz4 construct (293Fz4). These results are in accordance with observations by Umbhauer et al. [ 41] that coinjection of Xwnt5a RNA with Xfz4 RNA leads to the synergistic activation of Wnt/β-catenin target genes in Xenopus animal caps. We next tested whether Wnt5a could activate the STF reporter in 293Fz4 cells. Neither Wnt5a protein treatment at high doses nor transfection of Wnt5a DNA into cells expressing mFz4 could activate the STF reporter. However, as shown in Figure 4B, when LRP5 is coexpressed with mFz4, Wnt5a protein is able to activate the luciferase reporter. Thus, Wnt5a can activate TCF/β-catenin signaling given the expression of the appropriate receptors. Interestingly, Wnt5a is unable to activate the STF reporter in 293Fz4 cells transiently transfected with LRP6, indicating the specificity of the Wnt5a/Fz4/LRP5 signaling complex (unpublished data). To determine whether Wnt5a protein can inhibit Wnt3a-mediated STF reporter activation in 293Fz4 cells, 293 and 293Fz4 cells were transiently transfected with the STF reporter and then treated with Wnt proteins over the course of 24 h. Whereas Wnt5a protein potently inhibits the STF reporter in parental 293 cells, Wnt5a no longer inhibits Wnt3a-induced reporter activation in 293Fz4 cells ( Figure 4C). This effect was specific to mFz4, as stable expression of mFz8 was not sufficient to abrogate Wnt5a-mediated inhibition of STF reporter activation ( Figure 4D). Additionally, Wnt5a does not stabilize β-catenin protein in the presence of mFz8 (unpublished data). Thus, specifically in the context of mFz4 and LRP5 expression, Wnt5a is unable to inhibit Wnt3a-mediated β-catenin signaling and instead induces β-catenin accumulation and STF reporter activation. An Alternative Wnt Receptor, mRor2, Mediates Wnt5a's Inhibitory Activity One explanation for why Wnt5a is unable to inhibit Wnt3a-mediated β-catenin signaling when mFz4 is overexpressed is that in 293Fz4 cells, increased mFz4 receptor binding sites may titrate available Wnt5a protein away from an alternative receptor carrying out its inhibitory function. To investigate this hypothesis, we turned to the orphan receptor mRor2, a single-pass transmembrane tyrosine kinase with proposed kinase-dependent and -independent activities [ 25, 42]. mRor2 is an appealing candidate receptor mediating Wnt5a's inhibitory activity because Wnt5a and mRor2 have overlapping expression patterns, their knockout phenotypes are similar, and mRor2 has been shown previously to act synergistically with Wnt5a to activate Jun kinase [ 22– 26]. In addition, CAM-1, the C. elegans Ror2 homolog, has been shown to inhibit canonical Wnt signaling–mediated cell migration, although the Wnt ligand mediating this inhibition remains to be determined [ 21]. To determine whether Wnt5a mediates its inhibitory effects through the mRor2 receptor, 293 cells stably expressing the full-length mRor2 receptor (293mRor2) were treated with Wnt proteins over the course of 24 h. As shown in Figure 5A, Wnt3a protein treatment robustly activates the STF reporter in the presence of mRor2. However, at every time point measured, Wnt5a protein-mediated inhibition of Wnt/β-catenin signaling is synergistically enhanced by mRor2 overexpression as compared to the parental cell line (compare to Figure 4C and see Figure S1). We next assayed whether mRor2 was carrying out its inhibitory function through direct binding to Wnt5a. Two variants of the extracellular cysteine-rich Wnt binding domain (CRD) of mRor2 were cloned in frame amino-terminal to an immunoglobulin heavy chain domain (IgG) as previously described and expressed in 293 cells [ 43]. One variant is comprised of the Ror2 CRD alone cloned downstream of an exogenous signal sequence (Ror2 CRD-IgG); the other variant utilizes the endogenous Ror2 signal sequence and possesses the full amino terminus of the Ror2 protein ending at the carboxyl terminus of the CRD (Ror2 NT CRD-IgG). The secreted mRor2 CRD-IgG fusion proteins were then purified from conditioned media, bound to protein A beads, and tested for their ability to bind to purified Wnt proteins. The Smoothened receptor is a Fz family member that possesses a CRD domain but, rather than playing a role in Wnt signaling, it functions exclusively Hedgehog signal transmission. As a negative control, purified Smoothened (Smo) CRD-IgG protein was also bound to beads and assayed for Wnt binding. Figure 5B shows that Wnt5a protein specifically binds to the purified Ror2 and Fz4 CRD domains, whereas only background binding to the Smoothened CRD occurs. Wnt3a protein shows specific binding only to the Fz4 CRD-IgG protein (unpublished data). We next utilized a Ror2 deletion construct that lacks the extracellular cysteine-rich Wnt-binding domain (mRor2ΔCRD) to determine whether the CRD domain was necessary for Wnt5a signal transmission. When transiently transfected into 293 cells in conjunction with the STF reporter, mRor2ΔCRD does not enhance Wnt5a-mediated inhibition of the STF reporter ( Figure 5C). Thus, the CRD domain binds directly to Wnt5a and is required for Ror2 to transduce Wnt5a's inhibitory activity. Expression of mRor2 Is Required for Wnt5a-Mediated Inhibition of Canonical Signaling We next tested whether increased mRor2 expression was necessary to shift the balance of Wnt5a-mediated activation versus inhibition of Wnt/β-catenin signaling in cells overexpressing mFz4. 293 and 293Fz4 cells were transiently transfected with the STF reporter and mRor2 and then treated with the indicated Wnt proteins. Figure 6A shows that Wnt5a-induced reporter activation in the presence of mFz4 and LRP5 is abrogated when mRor2 is expressed. In addition, while Wnt5a protein is unable to inhibit Wnt/β-catenin signaling in the presence of mFz4, as compared to parental 293 cells, mRor2 overexpression synergistically enhances Wnt5a's ability to inhibit the reporter gene in both cell lines. Once again, inhibition of Wnt3a-mediated reporter activation was observed only in the presence of Wnt5a; mRor2 expression alone did not reduce Wnt3a-mediated reporter activation, indicating that the inhibitory activity is dependent on the presence of Wnt5a. Wnt5a's inhibitory activity was also tested in another cell line, mouse L cells. Whereas Wnt5a protein treatment in parental 293 cells robustly inhibits the Wnt3a-mediated activation of the STF reporter, Wnt5a treatment has little to no inhibitory affect in L cells ( Figure 6B). To assess whether this is due to reduced expression of mRor2, L cells were transfected with mRor2 and then treated with Wnt proteins. As shown in Figure 6B, when mRor2 is overexpressed, Wnt5a protein potently inhibits the Wnt3a-induced reporter activation in L cells. Again, mRor2 expression has no inhibitory effect on Wnt3a-stimulated reporter activation in the absence of Wnt5a. Although Wnt5a is able to activate the luciferase reporter in L cells stably expressing mFz4 and transiently transfected with LRP5, mRor2 cotransfection effectively inhibits Wnt5a's canonical signaling ability ( Figure 6B). Quantitative RT-PCR analysis was performed to ascertain the expression of Ror2 in 293 and L cells. A specific Ror2 transcript in the L cell sample did not emerge until after 40 cycles of amplification, indicating that Ror2 is expressed in almost immeasurably low abundance in L cells. Quantification of the real-time data revealed that Ror2 transcripts were over 700 times more abundant in 293 cells than in L cells ( Figure 6C). mRor2 expression thus appears to be required for Wnt5a-mediated inhibition of Wnt/β-catenin signaling in mouse L cells. It has been proposed that the C. elegans Ror protein CAM-1 has tyrosine kinase–independent functions [ 42, 44] and that Xenopus XRor2 can function in the absence of its cytoplasmic domain [ 45]. We thus created a membrane-tethered variant of mRor2 (mRor2-GPI) to assay whether mRor2 could still function in the absence of its transmembrane and cytoplasmic domains. Expression of mRor2-GPI reduced Wnt5a's ability to inhibit Wnt3a-induced STF reporter activation when transfected into 293 cells as compared to cells transfected with the reporters alone ( Figure 6D). In addition, when Ror2-GPI was transfected into cells in conjunction with wild-type mRor2, mRor2-GPI blocked wild-type mRor2′s enhancement of Wnt5a-mediated inhibition, suggesting that the membrane-tethered variant of mRor2 serves as a dominant negative. The fact that expression of mRor2-GPI alone did not enhance Wnt5a-mediated inhibition indicates that the cytoplasmic, and potentially the transmembrane, domain is required for mRor2 to carry out its inhibitory function. To assess the role the mRor2 transmembrane domain plays in mediating the Wnt5a signal, we created a mRor2 construct that contains the endogenous extracellular and transmembrane domains of the protein but lacks the cytoplasmic domain (Ror2-TM). We find that similar to the mRor2-GPI construct, expression of Ror2-TM was sufficient to inhibit wild-type mRor2′s ability to enhance Wnt5a signaling ( Figure 6E). Unlike the mRor2-GPI construct, however, the mRor2-TM construct did not reduce Wnt5a's ability to inhibit Wnt3a-induced STF reporter activation when transfected into 293 cells alone. Western blot analysis indicates that wild-type mRor2 expression is unaffected by cotransfection with mRor2-GPI or mRor2-TM, further strengthening the idea that these constructs serve as dominant negative variants of mRor2. These data taken together suggest that mRor2 does not simply present Wnt5a to another receptor but rather that Wnt5a binding initiates Ror2-induced cytoplasmic signaling events. Discussion Due to early overexpression studies in Xenopus embryos, the Wnts were grouped into various classes based on their canonical Wnt signaling ability without regard for the cellular context in which they were overexpressed. Receptor expression, however, clearly plays a role in determining signaling output; when coexpressed with the appropriate Fz receptor, the prototypical “noncanonical” Wnt, XWnt5a, can signal in a canonical fashion to induce the formation of a secondary axis [ 46]. Whereas some have argued that the lack of functional interaction with LRP distinguishes Wnt5a from so-called canonical Wnts in mammalian cells [ 31], previous studies and our present work confirm that Wnt signaling output is not intrinsically related to the Wnt protein itself but rather due to a combination of factors including receptor availability [ 41, 46]. Several controversies exist in the literature today regarding the mechanisms by which Wnt proteins signal. Much of the conflicting data, however, can be attributed to the variety of cellular and organismal systems studied coupled with the previous lack of soluble, active Wnt proteins. In the case of Wnt5a, for example, one report has shown that overexpression of Wnt5a inhibits canonical signaling by promoting the degradation of β-catenin protein [ 16]. By contrast, we found that, in accordance with other groups, Wnt5a protein treatment has no affect on β-catenin protein levels but rather inhibits canonical Wnt signaling at the level of TCF transcription [ 17, 18, 33]. Although our data are in agreement with Wnt5a overexpression studies by Ishitani et al. [ 17] with respect to the inhibition occurring downstream of β-catenin protein stabilization, we found that Wnt5a does not stimulate Ca 2+ flux and that Wnt5a-mediated inhibition is not sensitive to pertussis toxin treatment. These data effectively eliminate G protein–mediated activation of calcium signaling as the primary mechanism of Wnt5a-mediated inhibition. Fz receptor signaling capabilities have also often been debated. Previous studies pertaining to Fz4′s role in the disease familial exudative vitreoretinopathy have suggested that the Fz4 receptor does not mediate Wnt/β-catenin signaling but rather elicits intracellular calcium flux that subsequently activates downstream calcium effector molecules [ 34, 40]. By contrast, we show here that in multiple cell lines, Fz4 allows Wnt5a to specifically activate TCF/Lef transcription in the presence of LRP5 and that mFz4 expression does not enhance calcium flux ( Figure 3C). The true signaling capabilities of the Fz4 receptor may have been previously overlooked due to the use of an inappropriate Wnt ligand in the analysis, Wnt1 as opposed to Wnt5a. Additionally, as calcium signaling capabilities were attributed to Fz4 in the absence of exogenous ligand stimulation, it remains to be seen whether Wnt ligands are required for Fz-mediated Ca 2+ flux. We observe that when mFz4 is overexpressed alone, Wnt5a protein treatment is sufficient to induce β-catenin protein stabilization but not STF reporter activation. It is possible that Wnt5a does not activate a transcriptional response when mFz4 is expressed alone in 293 cells due to the endogenous expression of mRor2. In addition, coreceptor expression and heterodimerization following ligand stimulation have previously been shown to enhance ligand binding and signal transduction in other systems [ 47, 48]. As Wnt5a-induced β-catenin stabilization in cells expressing mFz4 is less robust than Wnt3a-induced stabilization, overexpression of both mFz4 and LRP5 may be necessary for full potentiation of the canonical signal resulting in transcriptional activation. Furthermore, previous reports have suggested that the accumulation of β-catenin and Wnt signal transduction are separable events by showing that β-catenin protein levels alone do not dictate signal output but rather the phosphorylation state of β-catenin [ 49, 50]. Coexpression of LRP5 may be necessary for the dephosphorylation, and hence full activation, of β-catenin resulting in optimal signal transduction. The disparities between previous reports and our current observations may be due to the fact that in this study, the effects of Wnt5a could be monitored immediately following Wnt5a protein addition as opposed to several hours or cell divisions following cellular expression, thereby allowing us to separate early and late effects of Wnt5a treatment. Activation of downstream transcriptional targets of Wnt5a, such as Siah-2, may contribute to the overall Wnt5a-mediated inhibition of Wnt/β-catenin signaling subsequent to the initial signaling events that we observed [ 16]. Additionally, in the proposed Wnt/Ca 2+ signaling model, Wnt5a-stimulated Ca 2+ flux leading to NLK activation is required for transcriptional inhibition [ 17]. However, Wnt1 has recently been shown to inhibit β-catenin–TCF–mediated transcription via activation of NLK, although Wnt1 has never been shown to induce Ca 2+ flux [ 33]. Thus, Wnt5a may exert its inhibitory effects through the activation of NLK in a similar Ca 2+-independent manner. Further experimentation into the mechanism by which Wnt5a inhibits canonical signaling is therefore necessary and will be addressed in subsequent reports. In this study, we show that one Wnt ligand can function in two discreet pathways based on receptor availability ( Figure 7). Data in the literature suggest that this may also apply to other Wnts. For example, Wnt1 is known to activate canonical Wnt signaling through β-catenin and TCF and act as an oncogene [ 51]. However, using overexpression in various cell lines, Smit et al. presented evidence that Wnt1 inhibits TCF activity [ 33]. Another example is Wnt11, which has been implicated in the noncanonical convergence-extension pathway in zebrafish [ 52]. Recent work, by contrast, demonstrates that Wnt11 is the long-sought ligand that activates the β-catenin pathway in the early Xenopus embryo, showing that one Wnt can activate two different pathways, possibly also by activating different receptors [ 53]. Although the ability of one Wnt ligand to function in two distinct pathways based on receptor context is novel for the Wnt field, we note that in an entirely different system, opposing effects brought about by a single ligand have also been explained by the use of different receptor classes. This example is formed by the Netrins, ligands that can either attract or repel axons depending on whether they interact with the DCC (deleted in colorectal carcinoma) or UNC5 (UNCoordinated family member 5) families of receptors [ 54– 56]. Thus, ligands engaging multiple receptors to effect different signaling outcomes is not unprecedented in nature. With as many as 19 mammalian homologs known, the question of whether all Wnt family members have evolved to signal in the same fashion is an important one. In this report, we address controversies in the literature regarding how one Wnt family member, Wnt5a, functions. Through quantitative kinetic analyses, we provide evidence for the first time that Wnt5a protein can directly inhibit canonical Wnt/β-catenin signaling and that the single-pass transmembrane receptor Ror2 is required to mediate this activity. Although Wnt5a can inhibit canonical Wnt signaling when Ror2 is expressed at detectable levels, Fz 4 and LRP5 coreceptor expression allows Wnt5a to signal in a canonical fashion. This ability of Wnt5a to toggle between two considerably diverse forms of signaling is particularly intriguing when one considers that Wnt5a has been classified as both a tumor suppressor and an oncogene in various cell types [ 57– 59]. While previously somewhat paradoxical, it is now clear that in the former role, Wnt5a expression may inhibit uncontrolled Wnt/β-catenin signaling in the presence of Ror2, whereas in the latter, Wnt5a could promote canonical Wnt signaling when Fz4 and LRP5 are expressed. Future study into specific receptor-ligand interactions will thus contribute to understanding the complexities of Wnt signaling. Materials and Methods Wnt5a purification Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ 27] with the addition of a fourth purification step. Briefly, following a gel filtration sizing step, partially purified Wnt5a protein was bound to a copper-chelated resin and eluted with increasing concentrations of Imidazole (HiTrap Chelating HP; Amersham Biosciences, Little Chalfont, United Kingdom). Peak fractions were then further purified by heparin affinity chromatography as previously described. Typical yields of Wnt5a protein following final heparin affinity step range from 25 to 50 ng/μl as assessed by Coomassie blue staining. CRD protein purification Serum-free media conditioned from 293 cells stably secreting CRD-IgG proteins was collected and bound to a Hitrap Protein A HP 1-ml column (Amersham Biosciences). Bound proteins were eluted with 100 mM citric acid (pH 3.0) and immediately made pH to neutral with 800 mM HEPES, 300 mM NaCl (pH 7.5) buffer supplemented with protease inhibitors. Protein from the peak fraction was incubated with protein A–Sepharose beads for 4 h at 4 °C and then washed 4 times with TNT buffer (see below). CRD-bound beads then incubated overnight at 4 °C with Wnt proteins. Beads were washed 4 times with TNT buffer, brought up in sample buffer, and analyzed for coimmunoprecipitation via Western blotting. cDNA constructs and antibodies pcDNA3-mRor2 and mRor2ΔCRD constructs were obtained from the Minami lab [ 23]. We generated the mRor2 CRD-IgG construct by fusing the Fz8 signal sequence (bases 1 to 100) to the mRor2 CRD (bases 505 to 909 of the complementary DNA of mRor2) in frame with IgG as previously described [ 43]. The mRor2NT CRD-IgG construct corresponds to bases 1 to 909 of mRor2 fused to IgG. pRK5-mRor2-GPI construct was generated by fusing bases 1 to 1221 of mRor2 in frame to DAF-GPI. pRK5-DAF-GPI fusion protein vector was provided by the Nathans lab [ 43]. pEF1-mRor2-TM corresponds to bases 1 to 1303 of mRor2. Human LRP5 and LRP6 constructs in the CS2+ vector were obtained from the He lab [ 60]. pcDNA3.1- Fz 4 (FLAG-tagged) was provided by the Lefkowitz lab [ 15]. Fz 8 complementary DNA was subcloned into the pEF1-myc/his A vector (Invitrogen, Carlsbad, California, United States) and pEF1a-LacZ (Invitrogen) was used for β-galactosidase activity normalization. STF was obtained from the Moon lab [ 30]. All DNA segments generated by PCR were sequenced to rule out spurious mutations. Antibodies were used at the following concentrations: rabbit anti-Wnt5A (1:1,000), rabbit anti-Fz4 (1:1,000), mouse 9E10 (1:200; Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, Iowa, United States), mouse anti-β-catenin (1:500; Santa Cruz Biotechnology, Santa Cruz, California, United States), mouse anti-GSK-3β (1:2,000; Transduction Labs, BD Biosciences Pharmingen, San Diego, California, United States), anti-phospho p44/p42 (1:1,000, Cell Signaling Technology, Beverly, Massachusetts, United States), and anti-total p44/p42 (1:1,000; Cell Signaling Technology). Proteins were detected using HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) with ECL Western blot detection reagents (Amersham Biosciences). Anti-mouse Wnt5a antibody was generated by cloning a 167–base pair fragment corresponding to bases 757 to 924 of the complementary DNA of mWnt5a into the pGEX4T-1 vector. Purified mWnt5a-GST fusion protein was then injected into a rabbit following standard procedures (Josman Laboratories). The final bleed was subsequently affinity purified. Cell culture, mammalian cell transfection, and luciferase assays HEK293 cells, mouse L cells, and their variants were cultured in DMEM, 10% FBS, and antibiotics. To generate cell lines stably expressing Fz receptors, cells grown on 10-cm plates were transfected using LipofectAMINE2000 according to the manufacturer's instructions (Invitrogen). At 24 h post-transfection, cells were plated out in limiting dilution and cultured under neomycin selection (1 mg/ml G418) for 10 to 14 d. At least 20 single cell clones were screened for expression via Western blot analysis. Transient transfections were performed with LipofectAMINE2000 (Invitrogen) on 293 cells, L cells, or stable cell line variants in six-well plates. Empty vector, CS2+-hLrp5, pcDNA3-mRor2, or derivatives were transfected (1 μg/well) together with the STF luciferase reporter (1 μg/well) and β-galactosidase (0.33 μg) (pEF-1α-LacZ; Invitrogen) plasmids. At 24 h post-transfection, cells were replated into a 96-well plate, allowed to recover for 6 to 8 h, and then treated in duplicate or triplicate with Wnt proteins as described in the text. Luciferase assays were performed using Dual-Light reporter gene assay system (Applied Biosystems). Relative luciferase units were measured and normalized against β-galactosidase activity at 48 h post-transfection. Error bars represent standard deviation, and each assay was performed at least two times. Cell extraction and Western blotting For assaying Fz expression, cells were lysed in TNT buffer on ice (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100). After clearing lysates with high-speed spin, protein supernatants were diluted into SDS sample buffer. In Erk1,2 phosphorylation assays, cells were lysed in hot sample buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue) supplemented with protease inhibitors (Complete Inhibitors; Roche, Basel, Switzerland) and then passaged 10 times through a 25-gauge syringe. For visualization of β-Catenin accumulation in 293 cells, cells were lysed by hypotonic lysis: cells were scraped into hypotonic buffer (10 mM Tris-HCl, 0.2 mM MgCl 2 [pH 7.4]) supplemented with protease and phosphatase inhibitors, incubated on ice for 10 min, and homogenized using a tight-fitting glass dounce. Sucrose and EDTA were added to final concentrations of 0.25 M and 1 mM, respectively. Lysates were then centrifuged at 20,000 × g for 1 h at 4 °C. Supernatant representing cytosolic cell fraction was then diluted into SDS sample buffer. For immunoblotting, samples were resolved on SDS-10% polyacrylamide gels and transferred to nitrocellulose. The membranes were incubated for 1 h in blocking solution (1% BSA, 3% nonfat dry milk in Tris-buffered saline containing 0.2% Tween-20) and then overnight at 4 °C with antibody in blocking solution. Quantitative RT-PCR analysis Qiagen RNeasy midi-kit (Valencia, California, United States) was used with on-column DNase step to prepare total RNA from each cell line. Then 1 μg of total RNA was converted to cDNA using random hexamer priming (Thermoscript RT-PCR System; Invitrogen). Quantitative real-time PCR analysis was performed in a Roche LightCycler 2.0 using LightCycler FastStart DNA Master PLUS SYBR Green I kit (Roche) with 0.5 μl of prepared cDNA and 0.5 μM forward and reverse primers per reaction. The primers for real-time PCR were designed using LightCycler Probe Design Software 2.0. Following 45 cycles of amplification, Ror2 expression in each cell line was normalized to HPRT expression following primer efficiency analysis. Reactions were performed in triplicate at least two times. Primers used Primers used were human Ror2 F: 5′- ATGGAACTGTGTGACGTACCC-3′; mouse Ror2 F: 5′- TGGAACTGTGTGACGTACCC-3′; human/mouse Ror2 R: 5′- GCGAGGCCATCAGCTG-3′; human HPRT F: 5′- CAAGTTTGTTGTAGGATATGCCC-3′; human HPRT R: 3′- CGATGTCAATAGGACTCCAGA-3′; mouse HPRT F: 5′- TGTTGTTGGATATGCCCTTG-3′; and mouse HPRT R: 3′- TTGCGCTCATCTTAGGCTTT-3′. Immunofluorescence staining For immunofluorescence microscopy, cells were grown on glass coverslips, fixed with 4% paraformaldehyde at 4 °C for 10 min, and permeabilized in methanol at 20 °C for 20 sec. The coverslips were then exposed to primary antibodies, followed by Cy3-conjugated secondary antibodies. Slides were mounted with Vectashield mounting media with Dapi (Vector Laboratories, Burlingame, California, United States) and fluorescence examined with a Zeiss Axioplan 2 microscope. Calcium flux assays The 293 cells plated onto glass chamber slides (Nalge Nunc International, Rochester, New York, United States) were loaded with 1 μM fura-2 AM (Molecular Probes, Eugene, Oregon, United States) for 20 min prior to imaging. The cells were washed and then imaging was carried out on a previously described microscope system [ 61]. For time-lapse experiments, image pairs were collected at 340- and 380-nm excitation wavelength (510-nm emission) at 15-s intervals for 15 min following the addition of Wnt5a protein (500 ng/ml). Following Wnt protein incubation, cells were treated with ionomycin (1 μM) and immediately imaged for an additional 5 min. The ratio image, a pixel-by-pixel match of both excitation wavelengths, was calculated by computer software and the sequence of ratio images was processed. Microscope control, data acquisition, and image analysis were performed in MetaMorph (Universal Imaging, Molecular Dynamics, Sunnyvale, California, United States). Supporting Information Figure S1 mRor2 Enhances Wnt5a-Mediated Inhibition of Canonical Wnt Signaling (A–C) Although Wnt5a protein shows inhibitory activity in 293 cells, it does not inhibit the STF reporter in 293Fz4 cells. Wnt5a-mediated inhibition is enhanced when mRor2 is stably expressed. At 24 h post-transfection, 293, 293Fz4, and 293Ror2 cells were treated with Wnt proteins for the indicated time points. Data are represented as relative light units (RLUs) ± SD rather than as average fold change. (140 KB PDF) Click here for additional data file.

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Coexistence of quiescent and active adult stem cells in mammals.

Linheng Li, Hans Clevers (2010)

Adult stem cells are crucial for physiological tissue renewal and regeneration after injury. Prevailing models assume the existence of a single quiescent population of stem cells residing in a specialized niche of a given tissue. Emerging evidence indicates that both quiescent (out of cell cycle and in a lower metabolic state) and active (in cell cycle and not able to retain DNA labels) stem cell subpopulations may coexist in several tissues, in separate yet adjoining locations. Here, we summarize these findings and propose that quiescent and active stem cell populations have separate but cooperative functional roles.

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Renée van Amerongen: Role: Academic Editor

Walter Birchmeier: Role: Academic Editor

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Journal ID (nlm-ta): Cancers (Basel)

Journal ID (iso-abbrev): Cancers (Basel)

Journal ID (publisher-id): cancers

Title: Cancers

Publisher: MDPI

ISSN (Electronic): 2072-6694

Publication date (Electronic): 13 July 2016

Publication date Collection: July 2016

Volume: 8

Issue: 7

Electronic Location Identifier: 65

Affiliations

[1 ]State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; cissyyu@ 123456sibcb.ac.cn

[2 ]Department of Molecular Biology and Biochemistry, Centre for Cell Biology, Development and Disease, Simon Fraser University, Burnaby, BC V5A 1S6, Canada; everheye@ 123456sfu.ca

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[* ]Correspondence: yzeng@ 123456sibcb.ac.cn ; Tel.: +86-21-5492-1433; Fax: +86-21-5492-1225

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Publisher ID: cancers-08-00065

DOI: 10.3390/cancers8070065

PMC ID: 4963807

PubMed ID: 27420097

SO-VID: dce72e1f-3d7b-417d-8ac6-83ac5f9236a8

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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

Mammary Development and Breast Cancer: A Wnt Perspective

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Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors

Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

Coexistence of quiescent and active adult stem cells in mammals.

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