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Proc Natl Acad Sci U S A. Mar 2, 2004; 101(9): 3118–3123.
Published online Feb 18, 2004. doi:  10.1073/pnas.0308648100
PMCID: PMC365753
Medical Sciences

Activation of the Wnt signaling pathway in chronic lymphocytic leukemia

Abstract

B cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, functionally incompetent B cells. Wnts are a large family of secreted glycoproteins involved in cell proliferation, differentiation, and oncogenesis. The classical Wnt signaling cascade inhibits the activity of the enzyme glycogen synthase kinase-3β, augmenting β-catenin translocation to the nucleus, and the transcription of target genes. Little is known about the potential roles of Wnt signaling in CLL. In this study, we quantified the gene expression profiles of the Wnt family, and their cognate frizzled (Fzd) receptors in primary CLL cells, and determined the role of Wnt signaling in promoting CLL cell survival. Wnt3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16, as well as the Wnt receptor Fzd3, were highly expressed in CLL, compared with normal B cells. Three lines of evidence suggested that the Wnt signaling pathway was active in CLL. First, the Wnt/β-catenin-regulated transcription factor lymphoid-enhancing factor-1, and its downstream target cyclin D1, were overexpressed in CLL. Second, a pharmacological inhibitor of glycogen synthase kinase-3β, SB-216763, activated β-catenin-mediated transcription, and enhanced the survival of CLL lymphocytes. Third, Wnt/β-catenin signaling was diminished by an analog of a nonsteroidal antiinflammatory drug (R-etodolac), at concentrations that increased apoptosis of CLL cells. Taken together, these results indicate that Wnt signaling genes are overexpressed and are active in CLL. Uncontrolled Wnt signaling may contribute to the defect in apoptosis that characterizes this malignancy.

B cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. Characterized by a progressive expansion of apparently quiescent B cells, it generally follows an indolent course (1). However, the subset of CLL that has germ-line IgV genes, and expresses the ZAP70 tyrosine kinase, has a more rapid course, usually leading to death within 5 years (2-6). The fundamental defect in CLL is thought to be abnormal regulation of apoptosis, because the circulating leukemia cells have a low growth faction. However, very little is known about the molecular mechanisms responsible for the prolonged survival of these malignant B cells.

Recent microarray analyses have demonstrated that the Wnt3 gene is overexpressed in CLL, compared with normal B and T cells (7). The Wnt family of secreted glycoproteins regulate early B cell growth and survival (8, 9). Aberrant activation of the Wnt signaling pathway has major oncogenic effects (10, 11). Binding of Wnt to its receptor complex, consisting of a member of the Frizzled (Fzd) family (12-15), and in some instances of the low-density lipoprotein-receptor-related proteins (LRP)5 or LRP6 (16-18), leads to stabilization of β-catenin by inhibiting the phosphorylating activity of the glycogen synthase kinase (GSK)-3β. Unphosphorylated β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it activates target gene expression through interacting with T cell (TCF) and lymphoid-enhancing (LEF) transcription factors (19, 20). This Wnt signaling cascade has been termed classical or canonical, in contrast to the recently described Wnt-induced alternative effects on Ca2+ uptake and protein kinase C activation (21).

The goal of this study was to determine whether the canonical Wnt signaling pathway is active in CLL cells, especially in the aggressive CLL subgroup. We hypothesized that some of the molecular heterogeneity leading to different clinical outcomes in CLL cells might be mediated by enhanced expression of Wnt and Fzd family members and augmented signaling through the β-catenin signaling pathway. Hence, we surveyed CLL, normal peripheral blood leukocytes, and B cells for their expression patterns of all of the known Wnt and Fzd family members by real-time PCR. The CLL samples were further subdivided into groups that expressed mutated and germ-line IgV genes. The gene expression analyses revealed that several Wnt genes from the classical and alternate groups were overexpressed in the leukemia cells, compared with normal B cells. Activation of Wnt/β-catenin signaling with a small-molecule GSK-3β inhibitor enhanced the survival of CLL cells, whereas antagonism of this pathway with an analog of a nonsteroidal antiinflammatory drug had the exact opposite effect. These data suggest that the Wnt pathway is not only active in CLL but also can be manipulated pharmacologically. Thus, Wnt signaling may be an attractive therapeutic target in CLL.

Materials and Methods

Cell Isolation and Culture. Protocols for the use of human samples were reviewed and approved by the University of California at San Diego Institutional Review Board. Blood samples were collected by the Chronic Lymphocytic Leukemia Research Consortium, after obtaining informed consent from patients fulfilling diagnostic criteria for CLL, at all disease stages. Mononuclear cells were isolated by density-gradient centrifugation over Ficoll/Hypaque and contained at least 85% B cells as determined by fluorescent CD19 staining. The cells were suspended in RPMI medium 1640, with 10% FBS, and antibiotics at 37°C, 5% CO2. The human embryonic kidney cell line HEK293 (American Type Culture Collection) was maintained in DMEM with high glucose supplemented with 10% FBS and antibiotics.

RNA Isolation and Real-Time PCR. Total RNA was isolated from 1 × 106 cells by TRIzol reagent (Invitrogen). The RNA samples were further purified by using a Qiagen RNeasy Protect kit (Qiagen, Valencia, CA). The mRNA levels were quantified in duplicate by real-time RT-PCR on the ABI Prism 7700 sequence detection system (Applied Biosystems) by using the primer sets and reaction conditions in Supporting Methods and Table 2, which are published as supporting information on the PNAS web site. The data were analyzed by using the comparative Ct method, where Ct is the cycle number at which fluorescence first exceeds the threshold. The ΔCt values from each tissue were obtained by subtracting the values for 18S Ct from the sample Ct. One difference of Ct value represents a 2-fold difference in the level of mRNA.

RT-PCR Analyses. Different pairs of gene-specific primers, based on GenBank sequences of cloned human LEF and TCF genes, were used for amplification of cDNA from CLL cells and normal lymphocytes (Table 3, which is published as supporting information on the PNAS web site). Reverse transcription was performed with a SuperScript preamplification kit (Invitrogen). One microgram of RNA and 30 cycles of amplification were used. After electrophoresis, the amplicons were visualized under UV light.

Plasmids. The β-catenin expression plasmid and the TCF/LEF TOPflash reporter plasmid were gifts from H. Clevers (Hubrecht Laboratory, Utrecht, The Netherlands). An expression plasmid for LRP6 (phLRP6-V5) was the kind gift of B. O. Williams (Van Andel Research Institute, Grand Rapids, MI) (22). pCMXβgal was used as a control plasmid (23). Expression plasmids for human Fzd3 (Origene Technologies, Rockville, MD), Wnt1, Wnt3, Wnt5a, and Wnt5b (Upstate Biotechnology, Lake Placid, NY) were purchased. The cDNA for Wnt16 and Fzd5 were amplified from human blood and Jurkat cells, respectively, and were then cloned into the pcDNA3 expression vector (Invitrogen). The expression of the Wnt16 and Fzd5 vectors was confirmed by transfection of HEK293 cells, followed by immunoblotting or RT-PCR, respectively.

Cell Transfection. HEK293 cells were transfected in 12-well plates by using FuGENE (Roche, Mannheim, Germany), and 0.5 μg of reporter plasmid, 0.1-0.2 μg of the control plasmid pCMXβgal, 100-200 ng of the various expression plasmids, and carrier DNA pBluescriptKSII, for a total of 1 μg per well. After overnight incubation, the cells were washed and given fresh medium containing the different drugs or vehicle. For luciferase assays, cells were lysed, and light emission was detected in the presence of luciferin by using a microtiter plate luminometer (MicroBeta TriLux, Gaithersburg, MD). The luciferase values were normalized for variations in transfection efficiency by using the β-galactosidase internal control, and are expressed as fold stimulation of luciferase activity, compared with the designated control cultures. All of the transfection results are representative of a minimum of three independent transfections.

Drugs. R-etodolac was prepared by fractional crystallization, using a modification of a literature procedure (ref. 24 and Supporting Methods). SB-216763 was obtained from Sigma (25).

Flow Cytometry. CLL cells were plated at a density of 1 × 107 cells per ml and treated with IL-4 (10 ng/ml, from R & D Systems), R-etodolac (300-500 μM), SB-216763 (5 μM), or vehicle for 48 h. The cells were removed from the plate and incubated for 15 min in medium with 5 μg/ml propidium iodide and 40 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC6), and were then analyzed with a FACSCalibur fluorescence-activated cell sorter (Becton Dickinson). Viable cells had high DiOC6 (FL-1) and low propidium iodide (FL-3) fluorescence, whereas apoptotic cells had low DiOC6 (FL-1) and low propidium iodide (FL-3) fluorescence.

3-[4,5-Dimethylthiazol-2-yl]-2,5-dipheyl Tetrazolium Bromide (MTT)-Based Cell Survival Assay. Briefly, 0.5 × 106 cells CLL cells in 96-well plates were treated with graded concentrations of R-etodolac, SB-216763, or vehicle. After 72 h, 20 μl of MTT solution was added to each well. Six hours later, the cells were lysed, and absorbances at 590 and 650 nM were measured. The assays were performed in triplicate, and the results represent the mean values ± SD.

Immunoblotting. Cells were disrupted in lysis buffer and blotted as described in Supporting Methods. Primary antibodies against GSK-3β (BD Biosciences), pY216 GSK-3β (BD Biosciences), β-catenin (Cell Signaling Technology, Beverly, MA), and β-actin (Chemicon International) were used. Horseradish peroxidase-conjugated anti-IgG (Santa Cruz Biotechnology) was used as the secondary antibody. Antibody binding was visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Life Science, Aylesbury, U.K.).

Statistical Analysis. The gene expression levels in CLL, CLL subsets, and normal B cells were compared by ANOVA. Only a P value ≤0.001 was considered significant because of the number of genes compared (≈40).

Results

Wnt and Fzd Genes Expressed by Normal and CLL B Cells. Several Wnt family member transcripts were detectable in normal and malignant B cells (Table 1). In the CLL cells, Wnt16 and Wnt10a were most abundant, with ΔCt values of 10 or below. Compared with normal B cells, the CLL samples had significantly higher levels of Wnt3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16 (Fig. 1). Notably, the aggressive CLL subset with germ-line IgV genes expressed higher levels of Wnt3, Wnt5b, and Wnt14 than did the mutated samples. Both Fzd3 and Fzd6 were prominent in normal and malignant B cells. Fzd3 levels were significantly higher in CLL than in normal B cells (Fig. 1). In addition, LRP5 and LRP6 mRNA transcripts were readily detectable in the CLL cells (mean ΔCt values of 13.2 ± 2.5 and 16 ± 1.6, respectively).

Fig. 1.
Relative expression of Wnts and Fzd3 in CLL cells. The relative mRNA levels of Wnt3 (A), Wnt5b (B), Wnt6 (C), Wnt10a (D), Wnt14 (E), Wnt16 (F), and Fzd3 (G) were detected by using real-time PCR in normal peripheral blood B cells (n = 3), unfractionated ...
Table 1.
Expression profile of Wnt and Fzd family in CLL vs. B cells and PBLs

Wnt3 Activates the TCF/LEF Promoter. The HEK293 cell line was transfected with the TCF and β-catenin dependent TOPflash reporter plasmid, and with various combinations of Wnt, Fzd, and LRP expression plasmids (Fig. 2). Transfection with a Wnt3 plasmid resulted in a small increase in reporter gene activity, which was augmented by the transfection with the LRP6 coreceptor (Fig. 2 A). Further synergy was seen after cotransfection with Fzd3 or Fzd5 expression plasmids (Fig. 2B). The activation of the TOPflash reporter was specific to only certain Wnt genes, as reported (26-28). Thus, the Wnt1 and Wnt3 plasmids stimulated the reporter, whereas Wnt16, Wnt5a, and Wnt5b did not, under the conditions tested (Fig. 2C).

Fig. 2.
Role of different Wnts and Fzds in activating β-catenin/TCF-mediated transcription. HEK293 cells were cotransfected with the TOPflash reporter construct and a β-galactosidase vector, along with the indicated plasmids. All cells were harvested ...

LEF1 and Cyclin D1 Overexpression in CLL. The canonical Wnt signaling pathway is mediated by transcription factors of the TCF/LEF family (29). Whereas LEF1 transcripts were barely detectable in normal B cells, the CLL samples expressed this gene in abundance (Fig. 3A). TCF7 was not detectable in any of the samples tested. The cyclin D genes are established downstream targets of Wnt and β-catenin signaling (30, 31). The CLL cells had an ≈3-fold higher level of cyclin D1 mRNA than did normal B cells (Fig. 3B).

Fig. 3.
Elevated expression of the LEF1 and cyclin D1 in CLL cells compared with normal B cells. (A) The indicated TCF transcription factor family members were amplified by RT-PCR in normal B cells and CLL specimens. (B) Cyclin D1 expression in normal B cells, ...

GSK-3β Inhibition Enhances CLL Survival. The phosphorylation of β-catenin by GSK-3β marks it for ubiquitination and degradation. Inhibition of this activity allows unbound β-catenin to accumulate in the cytoplasm, translocate into the nucleus, and activate TCF/LEF-dependent transcription. In HEK293 cells that were cotransfected with the TOPflash and β-catenin plasmids, the GSK-3β inhibitor SB-216763 substantially enhanced reporter gene activity (Fig. 4A). CLL cells treated with the same compound had less active GSK-3β, as demonstrated by diminished tyrosine 216-phosphorylation detectable by immunoblotting (Fig. 4B). Moreover, incubation of CLL cells with the GSK-3β inhibitor at concentrations up to 7 μM improved their survival in culture, and reduced apoptotic cell death (Fig. 4C and D).

Fig. 4.
Activation of β-catenin/TCF signaling and enhancement of CLL survival by a GSK-3β inhibitor. (A) SB-216763 activates the TCF-response elements. HEK293 cells were transfected with the TOPflash reporter and with or without the expression ...

Inhibition of β-Catenin Signaling Diminishes Cell Survival. Various nonsteroidal antiinflammatory drugs have been reported to inhibit β-catenin signaling, independent of cyclooxygenase inhibition (32, 33). The R-enantiomer of the nonsteroidal antiinflammatory drug etodolac (R-etodolac), which lacks cyclooxygenase inhibitory activity (34, 35), reduced TOPflash activity in HEK293 cells (Fig. 5A). The same concentrations of R-etodolac that inhibited Wnt/β-catenin signaling also shortened the in vitro survival of the CLL cells (Fig. 5 B-D).

Fig. 5.
R-etodolac partially blocked canonical Wnt signaling and induced apoptosis of CLL cells. (A) R-etodolac partially blocked β-catenin- and Wnt3/LRP6-mediated transcription in a dose-dependent manner. The β-catenin- and TCF/LEF-regulated ...

Discussion

The expansion of a malignantly transformed cell clone is the outcome of an abnormal balance between proliferation and death. In CLL, the abnormal lymphocytes grow slowly, but have a prolonged lifespan. A central aim of CLL research is to discover the factors that both induce the growth and sustain the lifespan of the malignant B cells, and then to develop therapeutic agents that can interfere specifically with their actions. As potential candidates, we investigated the expression and functional role of the Wnt ligand and Fzd receptor families in CLL. The 19 known human Wnt genes are secreted growth factors that bind to 10 or more Fzd receptors, to induce a complex signaling cascade that regulates cell growth and differentiation during embryogenesis. Considering that many cancers arise from immature cells, it seemed logical that one or more Wnt genes could be overexpressed and functionally important in some malignancies.

Our experiments showed that six of 19 Wnt genes (Wnt 3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16) were significantly overexpressed in CLL, compared with normal B cells. Fzd3 and its coreceptors LRP5 and 6 were also abundantly expressed in the malignant lymphocytes, as was the Wnt-regulated transcription factor LEF1. Three CLL-specific Wnt genes (Wnt3, Wnt5b, and Wnt14) were present at higher levels in the CLL subgroup with germ-line IgV genes than in the subgroup with mutated V genes, with Wnt5b showing the greatest disparity.

A previous microarray study identified Wnt3 as a “signature” gene in CLL (7). The homologous gene, Wnt3a, promoted the proliferation of mouse pro-B cells in the bone marrow by initiating signaling events leading to β-catenin-dependent activation of the transcription factor LEF1 (8). Further tissue analyses suggested that most of the Wnt3a was expressed by bone marrow stromal cells. The fact that mice genetically deficient in LEF1 had defects in pro-B cell proliferation and survival implicated the Wnt pathway in the regulation of immature B cell development. However, there was little or no expression of Wnt3a or LEF1 in mature germinal center B cells (8).

Wnt16 was originally discovered as a gene that is overexpressed in leukemia and normal pro-B cells (36, 37). Our investigations revealed that Wnt16 was similarly overabundant in CLL cells, compared with normal B cells. In transfection studies with HEK293 cells, Wnt3 activated the canonical β-catenin Wnt signaling pathway. However, Wnt16 did not induce TCF/LEF-dependent transcription, even after overexpression of Fzd and LRP6 genes. Thus, whereas Wnt16 appears to be a marker for immature B cells and their malignant counterparts, its function in lymphocyte proliferation and survival remains to be established.

Fzd3 was the predominant Fzd gene in CLL cells. However, both CLL and normal B cells did express other Fzd genes. Recently, Fzd gene expression has been examined in other B cell malignancies (9). Myeloma cells were found to express multiple Fzd genes, whereas B lymphoma cell lines expressed predominantly Fzd3. Unlike the B lymphoma cell lines, our experiments revealed that the LRP5 and LRP6 genes were readily detectable in primary CLL cells.

The synthesis of a growth factor receptor and ligand by the same malignant cell is a well established autocrine pathway in the evolution of cancer. The CLL clones expressed both Wnt growth factors and the major elements of the canonical Wnt signaling pathway. However, the detection of a complete Wnt/Fzd/LRP/β-catenin/LEF1 gene expression signature in CLL cells did not prove that the canonical Wnt signaling pathway was functional. We used pharmacologic modulators of β-catenin-dependent function to study the effects of Wnt signaling on CLL survival. A GSK-3β inhibitor, which would effectively release active β-catenin, and mimic constitutive canonical Wnt signaling, prolonged the survival of the malignant B cells.

Several compounds related to the nonsteroidal antiinflammatory drugs have been reported recently to inhibit β-catenin stability or function (32, 33). The R-enantiomer of etodolac inhibited the transcription of a β-catenin-dependent TCF/LEF reporter gene in HEK293 cells, and at the same concentrations, diminished the in vitro survival of CLL cells.

The expression of all of the components of the canonical Wnt signaling cascade in CLL cells, together with the observed effects of β-catenin activators and inhibitors, implicate the Wnt pathway in CLL pathogenesis, and suggest that it could play a role in extending the lifespan of the malignant B cells. If CLL cell survival is more dependent on Wnt pathway activation than is the survival of normal cells, then Wnt signal transduction antagonists could have therapeutic benefit in the disease. Even if Wnt signaling is necessary for the survival of some normal cells, the Wnt and Fzd genes that are overexpressed in CLL could represent attractive targets for the development of more specific pharmacologic antagonists, including antibodies and cytotoxins (38).

Supplementary Material

Supporting Information:

Acknowledgments

We thank Dr. Laura Rassenti, Michael Rosenbach, and Kathy Pekny for their assistance. This work was supported in part by National Institutes of Health Grants CA23100, AR44850, CA81534, and GM23200 and the University of California Biotechnology Strategic Targets for Alliances in Research Project (BioSTAR). T.J.K. and D.A.C. are members of the Chronic Lymphocytic Leukemia Research Consortium.

Notes

Abbreviations: CLL, chronic lymphocytic leukemia; Fzd, Frizzled; GSK, glycogen synthase kinase; TCF, T cell factor; LEF, lymphoid-enhancing factor; LRP, low-density lipoprotein-receptor-related protein; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheyl tetrazolium bromide; PBL, peripheral blood lymphocyte.

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