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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 inhibits cancer cell migration and invasion, and modulates expression of epithelial-to-mesenchymal transition markers and MMPs.(a) Quantitative analysis of A549 lung cancer cell migration. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (b) Invasion of tumor cells was quantified as number of cells in five fields of view per filter. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (c) Expression of EMT markers in H322 cells after 24 hr treatment with PBS, dE1-k35/LacZ, or dE1-k35/sLRP6E1E2 in the presence and absence of Wnt3a (100 ng/ml). Cells were stained with DAPI (blue), TRITC-labeled actin (red), or anti E-cadherin (green). Original magnification, ×630. (d) Expression of EMT-related markers in H322 cell lines. Expression levels of mesenchymal markers (N-cadherin & vimentin) as well as transcriptional factor (Snail) was determined by Western blotting. (e, f) A549 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 with or without Wnt3a (100 ng/ml). The enzyme activity of MMP-2 and MMP-9 was measured in supernatants collected from transduced cells at 48 hr using the Sensolyte 520 MMP-2 and MMP-9 assay kit. Experiments were performed in triplicate, and results are expressed as mean ± SEM. (e) *P<0.05, (f) #P<0.01 versus PBS- or dE1-k35/LacZ-treated controls; (e) #P<0.01, (f) **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a.
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pone-0036520-g007: Decoy Wnt receptor sLRP6E1E2 inhibits cancer cell migration and invasion, and modulates expression of epithelial-to-mesenchymal transition markers and MMPs.(a) Quantitative analysis of A549 lung cancer cell migration. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (b) Invasion of tumor cells was quantified as number of cells in five fields of view per filter. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (c) Expression of EMT markers in H322 cells after 24 hr treatment with PBS, dE1-k35/LacZ, or dE1-k35/sLRP6E1E2 in the presence and absence of Wnt3a (100 ng/ml). Cells were stained with DAPI (blue), TRITC-labeled actin (red), or anti E-cadherin (green). Original magnification, ×630. (d) Expression of EMT-related markers in H322 cell lines. Expression levels of mesenchymal markers (N-cadherin & vimentin) as well as transcriptional factor (Snail) was determined by Western blotting. (e, f) A549 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 with or without Wnt3a (100 ng/ml). The enzyme activity of MMP-2 and MMP-9 was measured in supernatants collected from transduced cells at 48 hr using the Sensolyte 520 MMP-2 and MMP-9 assay kit. Experiments were performed in triplicate, and results are expressed as mean ± SEM. (e) *P<0.05, (f) #P<0.01 versus PBS- or dE1-k35/LacZ-treated controls; (e) #P<0.01, (f) **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a.

Mentions: Acquisition of migratory properties by cancer cells is important for metastatic tumor cell spread [28]. Because increasing Wnt3a appeared to enhance motility and invasiveness, we asked whether interfering with the Wnt signaling pathway by expressing sLRP6E1E2 would inhibit in vitro motility and invasion. We examined the effect of sLRP6E1E2 on A549 cells using transwell motility and matrigel invasion assays. We collected conditioned medium from PBS-treated, dE1-k35/LacZ-transduced, and dE1-k35/sLRP6E1E2-transduced cells after treatment with or without Wnt3a. Conditioned medium from dE1-k35/sLRP6E1E2-transduced cells inhibited migration by 12.4% (without Wnt3a) and 23.8% (with Wnt3a) compared with conditioned medium from dE1-k35/LacZ-transduced cells (P<0.001) (Fig. 7A). Similarly, conditioned medium from dE1-k35/sLRP6E1E2-transduced cells inhibited invasion by 34.2% (without Wnt3a) and 56.2% (with Wnt3a) compared with conditioned medium from dE1-k35/LacZ-transduced cells (Fig. 7B).


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 inhibits cancer cell migration and invasion, and modulates expression of epithelial-to-mesenchymal transition markers and MMPs.(a) Quantitative analysis of A549 lung cancer cell migration. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (b) Invasion of tumor cells was quantified as number of cells in five fields of view per filter. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (c) Expression of EMT markers in H322 cells after 24 hr treatment with PBS, dE1-k35/LacZ, or dE1-k35/sLRP6E1E2 in the presence and absence of Wnt3a (100 ng/ml). Cells were stained with DAPI (blue), TRITC-labeled actin (red), or anti E-cadherin (green). Original magnification, ×630. (d) Expression of EMT-related markers in H322 cell lines. Expression levels of mesenchymal markers (N-cadherin & vimentin) as well as transcriptional factor (Snail) was determined by Western blotting. (e, f) A549 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 with or without Wnt3a (100 ng/ml). The enzyme activity of MMP-2 and MMP-9 was measured in supernatants collected from transduced cells at 48 hr using the Sensolyte 520 MMP-2 and MMP-9 assay kit. Experiments were performed in triplicate, and results are expressed as mean ± SEM. (e) *P<0.05, (f) #P<0.01 versus PBS- or dE1-k35/LacZ-treated controls; (e) #P<0.01, (f) **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a.
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pone-0036520-g007: Decoy Wnt receptor sLRP6E1E2 inhibits cancer cell migration and invasion, and modulates expression of epithelial-to-mesenchymal transition markers and MMPs.(a) Quantitative analysis of A549 lung cancer cell migration. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (b) Invasion of tumor cells was quantified as number of cells in five fields of view per filter. Experiments were performed in triplicate, and results are expressed as mean ± SEM. *P<0.05 versus PBS- or dE1-k35/LacZ-treated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. (c) Expression of EMT markers in H322 cells after 24 hr treatment with PBS, dE1-k35/LacZ, or dE1-k35/sLRP6E1E2 in the presence and absence of Wnt3a (100 ng/ml). Cells were stained with DAPI (blue), TRITC-labeled actin (red), or anti E-cadherin (green). Original magnification, ×630. (d) Expression of EMT-related markers in H322 cell lines. Expression levels of mesenchymal markers (N-cadherin & vimentin) as well as transcriptional factor (Snail) was determined by Western blotting. (e, f) A549 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 with or without Wnt3a (100 ng/ml). The enzyme activity of MMP-2 and MMP-9 was measured in supernatants collected from transduced cells at 48 hr using the Sensolyte 520 MMP-2 and MMP-9 assay kit. Experiments were performed in triplicate, and results are expressed as mean ± SEM. (e) *P<0.05, (f) #P<0.01 versus PBS- or dE1-k35/LacZ-treated controls; (e) #P<0.01, (f) **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a.
Mentions: Acquisition of migratory properties by cancer cells is important for metastatic tumor cell spread [28]. Because increasing Wnt3a appeared to enhance motility and invasiveness, we asked whether interfering with the Wnt signaling pathway by expressing sLRP6E1E2 would inhibit in vitro motility and invasion. We examined the effect of sLRP6E1E2 on A549 cells using transwell motility and matrigel invasion assays. We collected conditioned medium from PBS-treated, dE1-k35/LacZ-transduced, and dE1-k35/sLRP6E1E2-transduced cells after treatment with or without Wnt3a. Conditioned medium from dE1-k35/sLRP6E1E2-transduced cells inhibited migration by 12.4% (without Wnt3a) and 23.8% (with Wnt3a) compared with conditioned medium from dE1-k35/LacZ-transduced cells (P<0.001) (Fig. 7A). Similarly, conditioned medium from dE1-k35/sLRP6E1E2-transduced cells inhibited invasion by 34.2% (without Wnt3a) and 56.2% (with Wnt3a) compared with conditioned medium from dE1-k35/LacZ-transduced cells (Fig. 7B).

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