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A novel sLRP6E1E2 inhibits canonical Wnt signaling, epithelial-to-mesenchymal transition, and induces mitochondria-dependent apoptosis in lung cancer.

Lee JS, Hur MW, Lee SK, Choi WI, Kwon YG, Yun CO - PLoS ONE (2012)

Bottom Line: We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells.In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes.Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Project for Medical Sciences, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT
Aberrant activation of the Wnt pathway contributes to human cancer progression. Antagonists that interfere with Wnt ligand/receptor interactions can be useful in cancer treatments. In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in cancer gene therapy. We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells. In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes. Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

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Decoy Wnt receptor sLRP6E1E2 reduces cytosolic β-catenin and T-cell factor transcriptional activity.(a) TCF/LEF luciferase reporter assay in A549 cells. To characterize the sLRP6E1E2 effects on the Wnt3a/β-catenin signaling, cells were transfected with TOPflash (containing wild-type TCF binding sites) or FOPflash (containing mutated TCF binding sites) luciferase vector. *P<0.05 versus dE1-k35/LacZ-transduced or PBS-treated cells. (b) TCF/LEF luciferase reporter assay in H460 and H322 cells. *P<0.05 versus PBS or dE1-k35/LacZ-transduced cells with or without Wnt3a. (c) H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) with or without Wnt3a. Cells were labeled with anti-β-catenin. Original magnification, ×630. (d) Semi-quantitative analysis of panel (c) results using MetaMorph® imaging analysis software. Each data point indicates mean ± SEM (each group, n = 5). **P<0.001 versus PBS or dE1-k35/LacZ-transduced cells with Wnt3a.
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pone-0036520-g002: Decoy Wnt receptor sLRP6E1E2 reduces cytosolic β-catenin and T-cell factor transcriptional activity.(a) TCF/LEF luciferase reporter assay in A549 cells. To characterize the sLRP6E1E2 effects on the Wnt3a/β-catenin signaling, cells were transfected with TOPflash (containing wild-type TCF binding sites) or FOPflash (containing mutated TCF binding sites) luciferase vector. *P<0.05 versus dE1-k35/LacZ-transduced or PBS-treated cells. (b) TCF/LEF luciferase reporter assay in H460 and H322 cells. *P<0.05 versus PBS or dE1-k35/LacZ-transduced cells with or without Wnt3a. (c) H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) with or without Wnt3a. Cells were labeled with anti-β-catenin. Original magnification, ×630. (d) Semi-quantitative analysis of panel (c) results using MetaMorph® imaging analysis software. Each data point indicates mean ± SEM (each group, n = 5). **P<0.001 versus PBS or dE1-k35/LacZ-transduced cells with Wnt3a.

Mentions: We next hypotheses that secreted sLRP6E1E2 protein inhibit Wnt signaling by direct binding to Wnt. Therefore, to characterize the sLRP6E1E2 effects on the Wnt3a/β-catenin signaling, we determined its effect on β-catenin using a luciferase reporter system activated by β-catenin/TCF [23]. As shown in Fig. 2A, luciferase activity was low in A549 cells transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 in the absence of Wnt3a, since the endogenous expression level of Wnt3a in A549 is very minimal (Fig. 1B). Wnt3a treatment increased luciferase expression approximately 7- to 8-fold in control cells, but not in dE1-k35/sLRP6E1E2-transduced cells, suggesting that secreted sLRP6E1E2 could block the signaling effect of exogenously treated Wnt3a. In the absence of Wnt3a, luciferase activity was reduced by dE1-k35/sLRP6E1E2 in H460 (48%) and H322 (12%) cells compared with dE1-k35/LacZ controls (Fig. 2B; P<0.05). Wnt3a stimulation increased luciferase activity in H460 (53%) and H322 (102%) cells transduced with dE1-k35/LacZ, but luciferase activity was significantly lower in dE1-k35/sLRP6E1E2-transduced H460 (48%) and H322 (52%) cells compared with dE1-k35/LacZ (P<0.05). In order to make this result more compelling, we investigated the effect of LRP6-specific siRNA (si-LRP6) on the Wnt3a/β-catenin signaling. As shown in Figure S2, luciferase activity was significantly reduced by the treatment of si-LRP6 in both presence and absence of Wnt3a, in agreement with result of above (Fig. 2).


A novel sLRP6E1E2 inhibits canonical Wnt signaling, epithelial-to-mesenchymal transition, and induces mitochondria-dependent apoptosis in lung cancer.

Lee JS, Hur MW, Lee SK, Choi WI, Kwon YG, Yun CO - PLoS ONE (2012)

Decoy Wnt receptor sLRP6E1E2 reduces cytosolic β-catenin and T-cell factor transcriptional activity.(a) TCF/LEF luciferase reporter assay in A549 cells. To characterize the sLRP6E1E2 effects on the Wnt3a/β-catenin signaling, cells were transfected with TOPflash (containing wild-type TCF binding sites) or FOPflash (containing mutated TCF binding sites) luciferase vector. *P<0.05 versus dE1-k35/LacZ-transduced or PBS-treated cells. (b) TCF/LEF luciferase reporter assay in H460 and H322 cells. *P<0.05 versus PBS or dE1-k35/LacZ-transduced cells with or without Wnt3a. (c) H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) with or without Wnt3a. Cells were labeled with anti-β-catenin. Original magnification, ×630. (d) Semi-quantitative analysis of panel (c) results using MetaMorph® imaging analysis software. Each data point indicates mean ± SEM (each group, n = 5). **P<0.001 versus PBS or dE1-k35/LacZ-transduced cells with Wnt3a.
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Related In: Results  -  Collection

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pone-0036520-g002: Decoy Wnt receptor sLRP6E1E2 reduces cytosolic β-catenin and T-cell factor transcriptional activity.(a) TCF/LEF luciferase reporter assay in A549 cells. To characterize the sLRP6E1E2 effects on the Wnt3a/β-catenin signaling, cells were transfected with TOPflash (containing wild-type TCF binding sites) or FOPflash (containing mutated TCF binding sites) luciferase vector. *P<0.05 versus dE1-k35/LacZ-transduced or PBS-treated cells. (b) TCF/LEF luciferase reporter assay in H460 and H322 cells. *P<0.05 versus PBS or dE1-k35/LacZ-transduced cells with or without Wnt3a. (c) H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) with or without Wnt3a. Cells were labeled with anti-β-catenin. Original magnification, ×630. (d) Semi-quantitative analysis of panel (c) results using MetaMorph® imaging analysis software. Each data point indicates mean ± SEM (each group, n = 5). **P<0.001 versus PBS or dE1-k35/LacZ-transduced cells with Wnt3a.
Mentions: We next hypotheses that secreted sLRP6E1E2 protein inhibit Wnt signaling by direct binding to Wnt. Therefore, to characterize the sLRP6E1E2 effects on the Wnt3a/β-catenin signaling, we determined its effect on β-catenin using a luciferase reporter system activated by β-catenin/TCF [23]. As shown in Fig. 2A, luciferase activity was low in A549 cells transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 in the absence of Wnt3a, since the endogenous expression level of Wnt3a in A549 is very minimal (Fig. 1B). Wnt3a treatment increased luciferase expression approximately 7- to 8-fold in control cells, but not in dE1-k35/sLRP6E1E2-transduced cells, suggesting that secreted sLRP6E1E2 could block the signaling effect of exogenously treated Wnt3a. In the absence of Wnt3a, luciferase activity was reduced by dE1-k35/sLRP6E1E2 in H460 (48%) and H322 (12%) cells compared with dE1-k35/LacZ controls (Fig. 2B; P<0.05). Wnt3a stimulation increased luciferase activity in H460 (53%) and H322 (102%) cells transduced with dE1-k35/LacZ, but luciferase activity was significantly lower in dE1-k35/sLRP6E1E2-transduced H460 (48%) and H322 (52%) cells compared with dE1-k35/LacZ (P<0.05). In order to make this result more compelling, we investigated the effect of LRP6-specific siRNA (si-LRP6) on the Wnt3a/β-catenin signaling. As shown in Figure S2, luciferase activity was significantly reduced by the treatment of si-LRP6 in both presence and absence of Wnt3a, in agreement with result of above (Fig. 2).

Bottom Line: We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells.In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes.Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Project for Medical Sciences, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT
Aberrant activation of the Wnt pathway contributes to human cancer progression. Antagonists that interfere with Wnt ligand/receptor interactions can be useful in cancer treatments. In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in cancer gene therapy. We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells. In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes. Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

Show MeSH
Related in: MedlinePlus