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. 2004 Mar 2;101(9):3118-23.
doi: 10.1073/pnas.0308648100. Epub 2004 Feb 18.

Activation of the Wnt signaling pathway in chronic lymphocytic leukemia

Desheng Lu  1 , Yandong Zhao, Rommel Tawatao, Howard B Cottam, Malini Sen, Lorenzo M Leoni, Thomas J Kipps, Maripat Corr, Dennis A Carson
Affiliations

Activation of the Wnt signaling pathway in chronic lymphocytic leukemia

Desheng Lu et al. Proc Natl Acad Sci U S A. .

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-3beta, augmenting beta-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/beta-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-3 beta, SB-216763, activated beta-catenin-mediated transcription, and enhanced the survival of CLL lymphocytes. Third, Wnt/beta-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.

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Figures

Fig. 1.
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 PBLs (n = 3), and CLL cells, including mutated (mut) (n = 10), and unmutated (unmut) (n = 13) CLL specimens. The relative expression levels were determined by normalizing the ΔCt values against the average ΔCt values for normal B cells for the specified genes.
Fig. 2.
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 48 h after transfection, and cell extracts were assayed for luciferase and β-galactosidase activities. The β-galactosidase activity was used to confirm consistent transfection efficiencies (data not shown). (A) Wnt3-activated TCF-response elements in the presence of LRP6. HEK293 cells were cotransfected with Wnt3 and LRP6 plasmids. (B) Fzd3 and Fzd5 enhanced Wnt3-mediated transcription. HEK293 cells were cotransfected with expression plasmids for Wnt3, LRP6, Fzd3, and Fzd5, as indicated. (C) Unlike Wnt1 and Wnt3, expression of the Wnt5a, Wnt5b, and Wnt16 genes did not activate the TCF-response element. Vectors expressing Wnt1, Wnt3, Wnt5a, Wnt5b, and Wnt16 were cotransfected into HEK293 cells in the presence of both LRP6 and Fzd5.
Fig. 3.
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, PBLs, and CLL cells. The expression of cyclin D1 was assessed by real-time PCR. The relative expression level of cyclin D1 was determined by normalizing the ΔCt value against the B cell value. The gene expression in B cells was arbitrarily set to 1. Cyclin D1 levels were significantly higher in the CLL cells, compared with normal lymphocytes (P < 0.0005 by ANOVA).
Fig. 4.
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 plasmid for β-catenin. Transfected cells were treated with the GSK-3β inhibitor SB-216763 (5 μM) or with the DMSO vehicle control for 24 h. (B) SB-216763 increased β-catenin levels by inhibiting GSK-3β in CLL cells. CLL cells from four specimens were incubated with 5 μM SB-216763 or with the positive control survival factor IL-4 (10 ng/ml) for 48 h. Whole-cell lysates were immunoblotted with the indicated antibodies. (C) The prosurvival activity of SB-216763 in CLL cells. CLL cells (0.5 × 106 cells per well) were incubated with SB-216763 for 72 h before determinations of viability by MTT assay. The mean incremental survival measured in triplicate and the SD are shown. (D) SB-216763 protected CLL cells from apoptosis. CLL cells were incubated with 5 μM SB-216763 or 10 ng/ml recombinant human IL-4 for 48 h. The mitochondrial transmembrane potential was measured by flow cytometry by using the dye, DiOC6. Three specimens of CLL cells were analyzed.
Fig. 5.
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 TOPflash reporter plasmid was cotransfected into HEK293 cells with expression plasmids for β-catenin, Wnt3, and LRP6. The transfected cells were then treated with R-etodolac. (B) Cytotoxic effects of R-etodolac in CLL cells. CLL cells were incubated with R-etodolac, and viability was measured by the MTT assay after 72 h. Representative results for three different patients are shown. (C) Proapoptotic effects of R-etodolac in CLL cells. CLL cells, PBLs, and B cells were incubated with 250 μM R-etodolac for 24 and 48 h. Apoptosis was measured by flow cytometry by using DiOC6/PI staining. (D) Two representative examples of the proapoptotic effect of R-etodolac are shown.
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