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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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Related in: MedlinePlus

Decoy Wnt receptor sLRP6E1E2 decreases proliferation in human lung cancer cells.(a) A549 and H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The next day, these cells were incubated with or without Wnt3a (100 ng/ml). After 3 days, cell proliferation was assessed by the MTT assay (mean ± SEM). *P<0.05, #P<0.01 versus untreated control for each group; **P<0.001 versus dE1-k35/LacZ-transduced or PBS-treated cells. n.s.  =  not significant. (b) A549 cells were treated as indicated above (Fig. 3a). Western blot using antibodies specific to LRP6, Dvl2, Axin, Cyclin D1, or GSK-3β. (c) A549 cells were harvested at 6 hr after Wnt3a treatment. The p-Erk1/2, Erk1/2, p-PI3K, PI3K, p-Akt, and Akt proteins were detected by western blot analysis.
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pone-0036520-g003: Decoy Wnt receptor sLRP6E1E2 decreases proliferation in human lung cancer cells.(a) A549 and H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The next day, these cells were incubated with or without Wnt3a (100 ng/ml). After 3 days, cell proliferation was assessed by the MTT assay (mean ± SEM). *P<0.05, #P<0.01 versus untreated control for each group; **P<0.001 versus dE1-k35/LacZ-transduced or PBS-treated cells. n.s.  =  not significant. (b) A549 cells were treated as indicated above (Fig. 3a). Western blot using antibodies specific to LRP6, Dvl2, Axin, Cyclin D1, or GSK-3β. (c) A549 cells were harvested at 6 hr after Wnt3a treatment. The p-Erk1/2, Erk1/2, p-PI3K, PI3K, p-Akt, and Akt proteins were detected by western blot analysis.

Mentions: The Wnt pathway regulates a wide range of cellular functions including proliferation [24]. To test the effects of sLRP6E1E2 on proliferation of A549 and H322 cells in vitro, cells were treated with PBS or transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2. At 72 hr after transduction with dE1-k35/sLRP6E1E2 (20 MOI), cell proliferation was reduced by 39% in A549 cells and 51% in H322 cells compared with dE1-k35/LacZ-transduced controls. Wnt3a stimulation increased proliferation approximately 10–20% in control cells, but had no apparent effect on dE1-k35/sLRP6E1E2-transduced cells. Proliferation was 54% lower in A549 cells and 61% lower in H322 dE1-k35/sLRP6E1E2-transduced cells than dE1-k35/LacZ-transduced cells (P<0.001; Fig. 3A).


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 decreases proliferation in human lung cancer cells.(a) A549 and H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The next day, these cells were incubated with or without Wnt3a (100 ng/ml). After 3 days, cell proliferation was assessed by the MTT assay (mean ± SEM). *P<0.05, #P<0.01 versus untreated control for each group; **P<0.001 versus dE1-k35/LacZ-transduced or PBS-treated cells. n.s.  =  not significant. (b) A549 cells were treated as indicated above (Fig. 3a). Western blot using antibodies specific to LRP6, Dvl2, Axin, Cyclin D1, or GSK-3β. (c) A549 cells were harvested at 6 hr after Wnt3a treatment. The p-Erk1/2, Erk1/2, p-PI3K, PI3K, p-Akt, and Akt proteins were detected by western blot analysis.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351461&req=5

pone-0036520-g003: Decoy Wnt receptor sLRP6E1E2 decreases proliferation in human lung cancer cells.(a) A549 and H322 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The next day, these cells were incubated with or without Wnt3a (100 ng/ml). After 3 days, cell proliferation was assessed by the MTT assay (mean ± SEM). *P<0.05, #P<0.01 versus untreated control for each group; **P<0.001 versus dE1-k35/LacZ-transduced or PBS-treated cells. n.s.  =  not significant. (b) A549 cells were treated as indicated above (Fig. 3a). Western blot using antibodies specific to LRP6, Dvl2, Axin, Cyclin D1, or GSK-3β. (c) A549 cells were harvested at 6 hr after Wnt3a treatment. The p-Erk1/2, Erk1/2, p-PI3K, PI3K, p-Akt, and Akt proteins were detected by western blot analysis.
Mentions: The Wnt pathway regulates a wide range of cellular functions including proliferation [24]. To test the effects of sLRP6E1E2 on proliferation of A549 and H322 cells in vitro, cells were treated with PBS or transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2. At 72 hr after transduction with dE1-k35/sLRP6E1E2 (20 MOI), cell proliferation was reduced by 39% in A549 cells and 51% in H322 cells compared with dE1-k35/LacZ-transduced controls. Wnt3a stimulation increased proliferation approximately 10–20% in control cells, but had no apparent effect on dE1-k35/sLRP6E1E2-transduced cells. Proliferation was 54% lower in A549 cells and 61% lower in H322 dE1-k35/sLRP6E1E2-transduced cells than dE1-k35/LacZ-transduced cells (P<0.001; Fig. 3A).

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