The Wnt signaling pathway plays key roles in both embryogenesis and tumorigenesis. The low-density lipoprotein (LDL) receptor-related protein-6 (LRP6), a novel member of the expanding LDL receptor family, functions as an indispensable co-receptor for the Wnt signaling pathway. Although the role of LRP6 in embryonic development is now well established, its role in tumorigenesis is unclear. We report that LRP6 is readily expressed at the transcript level in several human cancer cell lines and human malignant tissues. Furthermore, using a retroviral gene transfer system, we find that stable expression of LRP6 in human fibrosarcoma HT1080 cells alters subcellular β-catenin distribution such that the cytosolic β-catenin level is significantly increased. This is accompanied by a significant increase in Wnt/β-catenin signaling and cell proliferation. Finally, we demonstrate that LRP6 expression promotes tumorigenesis in vivo. These results thus indicate that LRP6 may function as a potential oncogenic protein by modulating Wnt/β-catenin signaling.
The Wnt canonical signaling pathway is involved in various differentiation events during embryonic development and can lead to tumor formation when aberrantly activated (Orford et al., 1997; Wodarz and Nusse, 1998; Giles et al., 2003; Lustig and Behrens, 2003). Wnts are secreted glycoproteins that bind to the seven transmembrane receptors frizzled. A key component of the Wnt signaling pathway is β-catenin. In the absence of Wnts, β-catenin is phosphorylated by a multiprotein complex, a modification that is required for its ubiquitination and subsequent proteasomal degradation. β-catenin phosphorylation involves the sequential actions of casein kinase I and glycogen synthase kinase 3, and is regulated by the adenomatous polyposis coli (APC) tumor suppressor protein and the scaffold protein axin. Upon Wnt stimulation, β-catenin phosphorylation and subsequent ubiquitination and degradation are inhibited by a largely unknown mechanism. Wnt signaling thus stabilizes β-catenin, which enters the cell nucleus and associates with the T-cell factor/lymphoid-enhancing factor (TCF/LEF) family of transcription factors, leading to the transcription of Wnt target genes, including regulators of cell proliferation, developmental control genes, and genes implicated in tumor progression (Orford et al., 1997; Wodarz and Nusse, 1998; Giles et al., 2003; Lustig and Behrens, 2003; He et al., 2004).
The low-density lipoprotein receptor-related protein-5 (LRP5) and LRP6 are two members of the expanding low-density lipoprotein receptor family (Herz and Bock, 2002; Schneider and Nimpf, 2003). Recent studies have demonstrated that these two receptors are indispensable elements of the canonical Wnt pathway by interacting with several components of the Wnt signaling pathway. First, LRP5 and LRP6 act as co-receptors for Wnts, which interact with both frizzled and LRP5/LRP6 in order to initiate canonical Wnt signaling (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000). Second, LRP5, when activated by Wnt proteins, recruits the scaffold protein axin to the membrane, and therefore prevents it from participation in the degradation of β-catenin (Mao et al., 2001). Third, individual members of the Dickkopf (Dkk) family of secreted proteins can either antagonize or stimulate Wnt signaling through interaction with LRP6 (Wu et al., 2000; Bafico et al., 2001; Bao et al., 2001, 2002; Semenov et al., 2001; Brott and Sokol, 2002). Fourth, a context-dependent activator and inhibitor of Wnt signaling, WISE, is able to compete with Wnt for binding to LRP6 (Itasaki et al., 2003).
LRP6 is widely expressed in many tissues including heart, brain, placenta, lung, kidney, pancreas, spleen, testis, ovary, and the mucosal lining of the colon (Brown et al., 1998). Despite extensive studies on the expression and alterations of other components of the Wnt/β-catenin signaling pathway in human cancer, little is known regarding the expression and function of LRP6 in human cancer. By using a retroviral gene transfer system, we show herein that overexpression of LRP6 alters β-catenin subcellular distribution, enhances cancer cell Wnt/β-catenin signaling, and promotes cell proliferation in vitro and tumorigenesis in nude mice.
LRP6 expression in human cancer cell lines and tissues
To define a potential role for LRP6 in human cancer, LRP6 expression in a variety of human cancer cell lines and human normal and malignant tissues was examined. We used semiquantitative RT–PCR techniques to determine LRP6 transcript level in human colon cancer cell lines SW620 and DLD-1, breast cancer cell lines MDA-MB-231 and MDA-MB-468, lung cancer cell lines H441 and H520, and fibrosarcoma cell line HT1080. In order to confirm the absence of a genomic DNA contamination from the RNA preparations, a negative control with PCR amplification but without reverse transcription was included in each assay (data not shown). Human normal tissues including lung, colon, kidney, and small intestine, and human malignant tissues including lung tumor, colon tumor, kidney tumor, and breast tumor were also analysed for LRP6 expression. As seen in Figure 1, all cancer cell lines, as well as normal and malignant tissues, express LRP6. These results indicate that LRP6 is readily expressed in human cancers.
Stable expression of LRP6 in HT1080 cells
Compared to the other cell lines examined, fibrosarcoma HT1080 cells express LRP6 at the lowest level (Figure 1). Thus, we transduced LRP6 complementary DNA (cDNA) into HT1080 cells in order to study the roles of LRP6 in Wnt/β-catenin signaling. Transduced HT1080 cells were propagated in medium containing G418. After selection for 10 days, resistant colonies (over 200) were pooled. Expression of LRP6 in transformants was examined by immunoblotting with the hemagglutinin (HA) antibody or a monoclonal antibody that recognizes both LRP5 and LRP6. As seen in Figure 2a, immunoblot analysis of LRP6-transduced cells demonstrated that the transduced LRP6 was expressed and recognized by both monoclonal LRP5/6 antibody and anti-HA antibody. Flow cytometric analysis of nonpermeabilized cells demonstrated that all the G418-resistant colonies were transduced with LRP6, and that abundant LRP6 expression was observed at the cell surface (Figure 2b).
LRP6 expression increases cytosolic β-catenin level and TCF/LEF transcriptional activity
As β-catenin is a key molecule in the Wnt/β-catenin signaling pathway, we hypothesized that LRP6 expression would increase the cytosolic β-catenin level and enhance Wnt/β-catenin signaling activity in human cancer cells. To test this, LRP6-transduced HT1080 cells and control cells were fractionated into membrane and cytosolic fractions, and the β-catenin levels in these fractions, as well as in the whole-cell extract, were examined by Western blotting. It was interesting to note that LRP6-transduced cells expressed a much higher level of cytosolic β-catenin, but a lower level of membrane-associated β-catenin than control cells (Figure 3a). The level of β-catenin in whole-cell extract was unchanged. Fluorescence microscopy revealed that overexpression of LRP6 reduced β-catenin localization at the plasma membrane, especially at the junctions between cells (Figure 3b). Taken together, these results indicate that LRP6 expression in HT1080 cells induces β-catenin disassociation from the plasma membrane and accumulation in the cytoplasm.
The altered intracellular distribution of β-catenin in LRP6-tranduced HT1080 cells was also found to be associated with a corresponding increase in endogenous TCF/LEF transcriptional activity. The TOP-FLASH luciferase reporter contains TCF-binding sites and can be directly activated by the β-catenin/TCF complex (Korinek et al., 1997). We found that LRP6-transduced cells exhibited 3.9-fold greater activity than control cells (Figure 3c).
To further examine the roles of LRP6 in β-catenin localization and activity, we transiently transfected Wnt1 cDNA or β-catenin cDNA into HT1080 cells stably transduced with LRP6 cDNA or control vector. After transfection with Wnt1 cDNA, the level of cytosolic β-catenin in LRP6-transduced cells was much higher than that in control cells, while the level of membrane-associated β-catenin in LRP6-transduced cells was lower than that of control cells. Similarly, after transfection with β-catenin cDNA, the level of cytosolic β-catenin in LRP6-transduced cells was significantly higher than that in control cells, although the level of membrane-associated β-catenin in LRP6-transduced cells was not changed (Figure 4a). As expected, LRP6-transduced HT1080 cells display greater Wnt1 or β-catenin-induced TCF/LEF transcriptional activity than control cells. After transfection with Wnt1 or β-catenin cDNA, LRP6-transduced cells exhibit 2.6- and 7.9-fold increases in TCF/LEF transcriptional activity, respectively, whereas the corresponding control HT1080 cells exhibit only 2.0- and 3.2-fold increases in TCF/LEF transcriptional activity (Figure 4b).
LRP6 expression enhances HT1080 cell proliferation in culture
Having established that LRP6 expression results in increases in the cytosolic β-catenin level, and TCF/LEF transcriptional activity in HT1080 cells, we then investigated the role of LRP6 in cell proliferation. The effect of LRP6 expression on HT1080 cell proliferation was examined in a 7-day proliferation assay. Figure 5a shows that there was a significant difference between LRP6-transduced cells and control cells in their proliferation rates (P<0.01). After 7 days of culture, an approximately 1.6-fold increase in the number of cells was found in LRP6-transduced cells compared to control cells. The population doubling time of LRP6-transduced cells was 17.8±0.7 h (i.e. the standard deviation; n=3), while that of control cells was 21.3±0.8 h (P<0.05 when compared to LRP6-transduced cells). These results indicate that LRP6 expression significantly increases anchorage-dependent growth of HT1080 cells.
As anchorage-independent growth correlates well with tumorigenicity in vivo (Freedman and Shin, 1974), we examined this parameter using a soft agar colony assay. As seen in Figure 5b, the soft agar assay demonstrated that LRP6-transduced cells form greater numbers of colonies compared to control cells.
LRP6 expression promotes HT1080 cell growth in vivo
Results from the in vitro assays described above suggest that tumorigenicity in vivo may also be enhanced by LRP6 expression. To examine this possibility, we performed tumorigenesis assays by comparing tumor volume in nude mice after 7, 14, 21, or 28 days following tumor cell injection. We found that LRP6-transduced cells are more tumorigenic than control cells (Figure 6). The difference in tumor size between LRP6-transduced cells and control cells increased as tumor size increased. By the termination of the experiment (28 days following cell injection), LRP6-transduced cells had formed tumors approximately twofold larger than those derived from control cells (Figure 6).
Genetic and biochemical studies have demonstrated that LRP6, by interacting with several components of the pathway, is an indispensable element of the Wnt signaling pathway (Herz and Bock, 2002; Schneider and Nimpf, 2003; He et al., 2004). Our present study provides the first direct evidence that genetic manipulation of LRP6 expression enhances Wnt/β-catenin signaling and the malignant phenotype of tumor cells. We found that increased LRP6 expression elevated the cytosolic β-catenin level and TCF/LEF transcriptional activity, and promoted cell proliferation in culture, colony formation in soft agar, and tumor formation in nude mice.
β-Catenin is an intracellular multifunctional protein. In complex with the transmembrane adhesive receptor E-cadherin, it becomes plasma membrane-associated and mediates intercellular adhesion. A cytosolic pool of β-catenin interacts with DNA-binding proteins and participates in Wnt signal transduction (Hinck et al., 1994; Gottardi et al., 2001; Klingelhofer et al., 2003). At the heart of the canonical Wnt pathway is the stabilization of cytosolic β-catenin, which activates target genes by binding to the TCF/LEF family of transcription factors. In the present study, we found that overexpression of LRP6 in HT1080 cells resulted in an increase of cytosolic β-catenin and a decrease of membrane-associated β-catenin. These results suggest that LRP6 modulate Wnt/β-catenin signaling primarily by altering intracellular distribution of β-catenin with an unidentified mechanism. Currently, we do not rule out the possibility that part of the cytosolic increase of β-catenin level in LRP6-transduced cells might result from inhibition of β-catenin degradation by the ubiquitin/proteasome pathway. This possibility will be examined in future studies.
Previous studies have provided strong evidence that the single transmembrane LRP5/6 receptors and the seven transmembrane frizzled receptors cooperate in Wnt/β-catenin signaling (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000; Mao et al., 2001). Recent studies also showed that LRP5 or LRP6 truncated from the amino-terminal ends (i.e. lacking the extracellular domain) are capable of mediating Wnt/β-catenin signaling independent of either a Wnt ligand or frizzled receptor (Mao et al., 2001; Liu et al., 2003; Brennan et al., 2004). Furthermore, it was demonstrated that a PPPSP motif, which is reiterated five times in the LRP6 intracellular domain, is necessary and sufficient to trigger Wnt/β-catenin signaling (Brennan et al., 2004; Tamai et al., 2004). In the present study, we demonstrated that LRP6 modulates Wnt signaling by altering intracellular distribution of β-catenin. Thus, the mechanisms by which LRP5/6 modulates the Wnt signal are complex. It was proposed that, under normal conditions when Wnt ligand and receptors are at the physiological levels, both frizzled and LRP5/6 receptors are required to achieve efficient intracellular coupling to Wnt/β-catenin signaling (Liu et al., 2003). However, under certain pathological conditions such as in malignancy, high levels of LRP5/6 alone might be able to induce Wnt/β-catenin signaling by modulating subcellular β-catenin distribution.
Activation of Wnt/β-catenin signaling is now recognized as an important event that leads to tumorigenesis in several malignancies, especially colorectal cancer (Giles et al., 2003; Lustig and Behrens, 2003). Activation has been shown to occur through mutations of either the APC tumor-suppressor gene or β-catenin itself. The consequence of either APC inactivation or β-catenin mutation is similar: failure of proper β-catenin degradation leads to its cytosolic accumulation, nuclear translocation, and constitutive activation of β-catenin-responsive genes (Giles et al., 2003; Lustig and Behrens, 2003). In the current study, we show that human cancer cell lines and malignant tissues readily express LRP6, and that LRP6 overexpression in human fibrosarcoma cells promotes Wnt/β-catenin signaling and tumorigenesis. These results suggest that LRP6 is a potential oncogenic protein by modulating Wnt/β-catenin signaling. LRP6 expression in all the examined normal tissues, malignant tissues, and cultured cell lines may suggest that LRP6 is either ubiquitously expressed or essential for cell survival. Although our data do not prove that LRP6 is associated with tumorigenesis, they are consistent with the notion that LRP6 is an obligate receptor for the canonical Wnt signaling pathway. Recently, it has been demonstrated that expression of LRP5, a sister receptor of LRP6, is a common event in osteosarcoma, and may serve as a potential novel marker for disease progression in high-grade osteosarcoma (Hoang et al., 2004a). Furthermore, overexpression of a dominant-negative LRP5 mutant in osteosarcoma Saos-2 cells significantly reduces cell invasion capacity and cell motility (Hoang et al., 2004b). Future studies on LRP6 expression in malignant tissues should allow us to determine whether alteration of LRP6 expression occurs in primary tumors, and, if so, whether LRP6 expression correlates with the malignant state.
Materials and methods
Human cancer cell lines HT1080, HCT116, DLD-1, MDA-MB-231, MDA-MB-468, H441, and H520 were obtained from the American Type Culture Collection (Manassas, VA, USA), and cultured in DMEM with 10% fetal calf serum. LRP6 transcript level in human cancer cell lines and malignant tissues was analysed by RT–PCR as described by Qiang et al. (2003) with minor modifications. Briefly, total RNA was isolated from cell cultures using RNA-Bee (Tel-Test, Friendwood, TX 77546). Total RNA of human normal tissues (lung, colon, kidney, and small intestine) and malignant tissues (lung, colon, kidney, and breast) was from Clontech (Palo Alto, CA, USA). First-strand cDNA synthesis was performed using ProSTARTM Ultro HF RT–PCR Kit (Strategene) primed with oligo(dT) primer in a 10 μl reaction mixture containing 0.3 μg total RNA. For semiquantitative analysis of LRP6 expression, the PCR was carried out using 1 μl of cDNA in a total volume of 50 μl over 32 cycles according to the manufacturer's instructions, and GAPDH was used as a control. The forward and reverse primers for LRP6 are 5′-IndexTermGATTATCCAGAAGGCATGGCAG-3′ (+2113 to + 2134) and 5′-IndexTermTCCCATCACCATCTTCCA-3′ (+2827 to +2848), respectively. The PCR product was loaded onto a 1.2% Agarose gel and stained with ethidium bromide.
Stable expression of LRP6
Human LRP6 cDNA was subcloned into a retroviral vector pLNCX2 (Clontech Laboratories, Inc., Palo Alto, CA, USA) using standard procedures. A HA epitope was inserted into the construct, located at the amino terminus after the signal peptide, to facilitate immunodetection of the LRP6 protein in infected cells. The HA tag does not interfere with LRP6 processing and its function, as we found that after transfection HA-LRP6 is properly processed to the cell surface, and can bind and internalize several LRP6 ligands (data not shown). The integrity of the subcloned DNA sequence was confirmed by DNA sequencing. RetroPack PT67 packaging cells (Clontech Laboratories, Inc.) were transfected with pLNCX2-LRP6 or pLNCX2 alone using FUGENE 6 (Roche Molecular Biochemicals). Supernatants from transfected PT67 cells were incubated with 50% confluent HT1080 cells in the presence of Polybrene (4 μg/ml, final concentration, Sigma Chemical Co., St Louis, MO, USA). LRP6-transduced HT1080 cells and control cells were propagated in medium containing G418 (Life Technologies, Inc.) at 700 μg/ml. After G418 selection for 10 days, resistant colonies were pooled. The expression of total cellular LRP6 was determined by Western blotting using both anti-HA antibody and monoclonal anti-LRP5/6 antibody (BioVision, Mountain View, CA, USA). Proper folding and trafficking of the receptor to the cell surface were confirmed by examining cell surface LRP6 levels via flow cytometry as described before (Li et al., 2000, 2002).
Subcellular fractionation and β-catenin Western blotting
To examine the role of LRP6 in β-catenin subcellular distribution, cytosolic and membrane distributions of β-catenin were compared in cells transduced with either LRP6 cDNA or vector alone. Cells were seeded in six-well plates, and used at ∼80–90% confluence. After washing in ice-cold PBS, cells were collected and homogenized in a glass Dounce homogenizer in buffer consisting of 100 mM Tris–HCl, pH 7.4, 140 mM NaCl, 2 mM DTT, 2 mM PMSF, and 1 × Complete™ protease inhibitors (500 μl/well). The homogenate was centrifuged for 10 min at 500 g, and the resulting supernatant was designated as the whole-cell extract. This whole-cell extract was then centrifuged at 100 000 g at 4°C for 90 min, after which the supernatant was designated as the cytosolic fraction, while the pellet was dissolved in 1 × Laemmli sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, and 5% β-mercaptoethanol), and designated as the membrane fraction. The levels of β-catenin in the whole-cell extract, cytosolic fraction, and membrane fraction were then examined by quantitative Western blotting using β-catenin-specific antibody from Cell Signaling Techologies.
LRP6 transduced HT1080 cells and control cells were fixed in 4% paraformaldehyde, labeled with mouse monoclonal β-catenin antibody, and detected with Alexafluor488 goat anti-mouse IgG. The immunofluorescence was detected by a laser-scan confocal microscope (Olympus Fluoview 500).
Luciferase reporter assay
HT1080 cells were plated into six-well plates. For each well, 0.5 μg of the TOP-FLASH TCF luciferase construct (Upstate Biotechnology) was cotransfected with 0.5 μg of β-catenin-expressing vector, 0.5 μg of Wnt1-expressing vector, or empty pcDNA3 vector. A β-galactosidase-expressing vector (Promega, Madison, WI, USA) was included as an internal control for transfection efficiency. After 48 h, cells were lysed and both luciferase and β-galactosidase activities were determined with enzyme assay kits (Promega). The luciferase activity was determined with a luminometer using the Dual Luciferase Assay system (Promega). Luciferase activity was normalized to the activity of the β-galactosidase.
Cell proliferation assays
Cells were seeded into six-well plates (5 × 104 cells per well). Media were changed every other day, and cells were harvested and counted every day from day 1 to 7 using the trypan blue exclusion assay. Doubling times (DT) for LRP6-transduced cells and control cells during periods of logarithmic growth were determined using the formula DT=ln2/(growth rate).
Soft agar colony assays
Cells were cultured in six-well plates covered with an agar layer (DMEM medium with 0.5% agar and 5% FBS). The middle layer contained 2 × 103 cells in DMEM with 0.33% agar and 5% FBS, and this cell layer was overlaid with medium only to prevent drying of the agar gels. Triplicate plates were prepared for each cell line. After 3 weeks of incubation, colonies larger than 0.1 mm in diameter were scored.
Female athymic nude mice (4–5 weeks old) were obtained from Harlan Sprague–Dawley Inc. (Indianapolis, IN, USA). HT1080 cells (6 × 106 cells) were suspended in 0.2 ml of serum-free DMEM with 50% matrigel matrix, and injected s.c. into one flank of the mice. Tumors were measured every 7 days, and tumor volumes were calculated using width (a) and length (b) measurements (a2b/2, where a<b).
Bafico A, Liu G, Yaniv A, Gazit A and Aaronson SA . (2001). Nat. Cell. Biol., 3, 683–686.
Bao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, Delius H, Hoppe D, Stannek P, Walter C, Glinka A and Niehrs C . (2002). Nature, 417, 664–667.
Bao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A and Niehrs C . (2001). Nature, 411, 321–325.
Brennan K, Gonzalez-Sancho JM, Castelo-Soccio LA, Howe LR and Brown AM . (2004). Oncogene (Epub ahead of print).
Brott BK and Sokol SY . (2002). Mol. Cell. Biol., 22, 6100–6110.
Brown SD, Twells RC, Hey PJ, Cox RD, Levy ER, Soderman AR, Metzker ML, Caskey CT, Todd JA and Hess JF . (1998). Biochem. Biophys. Res. Commun., 248, 879–888.
Freedman VH and Shin S . (1974). Cell, 3, 355–359.
Giles RH, van Es JH and Clevers H . (2003). Biochim. Biophys. Acta, 1653, 1–24.
Gottardi CJ, Wong E and Gumbiner BM . (2001). J. Cell Biol., 153, 1049–1060.
Herz J and Bock HH . (2002). Annu. Rev. Biochem., 71, 405–434.
He X, Semenov M, Tamai K and Zeng X . (2004). Development, 131, 1663–1677.
Hinck L, Nathke IS, Papkoff J and Nelson WJ . (1994). J. Cell. Biol., 125, 1327–1340.
Hoang BH, Kubo T, Healey JH, Yang R, Nathan SS, Kolb EA, Mazza B, Meyers PA and Gorlick R . (2004a). Int. J. Cancer, 109, 106–111.
Hoang BH, Kubo T, Healey JH, Sowers R, Mazza B, Yang R, Huvos AG, Meyers PA and Gorlick R . (2004b). Cancer Res., 64, 2734–2739.
Itasaki N, Jones CM, Mercurio S, Rowe A, Domingos PM, Smith JC and Krumlauf R . (2003). Development, 130, 4295–4305.
Klingelhofer J, Troyanovsky RB, Laur OY and Troyanovsky S . (2003). Oncogene, 22, 1181–1188.
Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B and Clevers H . (1997). Science, 275, 1784–1787.
Liu G, Bafico A, Harris VK and Aaronson SA . (2003). Mol. Cell. Biol., 23, 5825–5835.
Li Y, Knisely JM, Lu W, McCormick LM, Wang J, Henkin J, Schwartz AL and Bu G . (2002). J. Biol. Chem., 277, 42366–42371.
Li Y, Marzolo MP, van Kerkhof P, Strous GJ and Bu G . (2000). J. Biol. Chem., 275, 17187–17194.
Lustig B and Behrens J . (2003). J. Cancer Res. Clin. Oncol., 129, 199–221.
Mao J, Wang J, Liu B, Pan W, Farr III GH, Flynn C, Yuan H, Takada S, Kimelman D, Li L and Wu D . (2001). Mol. Cell, 7, 801–809.
Orford K, Crockett C, Jensen JP, Weissman AM and Byers SW . (1997). J. Biol. Chem., 272, 24735–24738.
Pinson KI, Brennan J, Monkley S, Avery BJ and Skarnes WC . (2000). Nature, 407, 535–538.
Qiang YW, Endo Y, Rubin JS and Rudikoff S . (2003). Oncogene, 22, 1536–1545.
Schneider WJ and Nimpf J . (2003). Cell Mol. Life Sci., 60, 892–903.
Semenov MV, Tamai K, Brott BK, Kuhl M, Sokol S and He X . (2001). Curr. Biol., 11, 951–961.
Tamai K, Semenov M, Kato Y, Spokony R, Liu C, Katsuyama Y, Hess F, Saint-Jeannet JP and He X . (2000). Nature, 407, 530–535.
Tamai K, Zeng X, Liu C, Zhang X, Harada Y, Chang Z and He X . (2004). Mol. Cell, 13, 149–156.
Wehrli M, Dougan ST, Caldwell K, O'Keefe L, Schwartz S, Vaizel-Ohayon D, Schejter E, Tomlinson A and DiNardo S . (2000). Nature, 407, 527–530.
Wodarz A and Nusse R . (1998). Annu. Rev. Cell. Dev. Biol., 14, 59–88.
Wu W, Glinka A, Delius H and Niehrs C . (2000). Curr. Biol., 10, 1611–1614.
This work was supported in part by grant from the American Heart Association (0330118N) to YL, and grants from the National Institutes of Health to GB. GB is an Established Investigator of the American Heart Association. We are grateful to Dr Christof Niehrs for providing the LRP6 cDNA, and Dr Theodore C Simon for providing the cDNAs for human Wnt1 and β-catenin, and for the helpful discussion during the course of this study.
About this article
MALAT1 regulates the transcriptional and translational levels of proto-oncogene RUNX2 in colorectal cancer metastasis
Cell Death & Disease (2019)
Hypoxic ER stress suppresses β-catenin expression and promotes cooperation between the transcription factors XBP1 and HIF1α for cell survival
Journal of Biological Chemistry (2019)
(Pro)renin receptor promotes colorectal cancer through the Wnt/beta-catenin signalling pathway despite constitutive pathway component mutations
British Journal of Cancer (2019)
Scientific Reports (2019)
Computationally Design of Inhibitory Peptides Against Wnt Signaling Pathway: In Silico Insight on Complex of DKK1 and LRP6
International Journal of Peptide Research and Therapeutics (2018)