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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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Characterization of the decoy Wnt receptor sLRP6E1E2.(a) Schematic representation of the genomic structure of Ad vectors used. (b) Endogenous Wnt3a (left panel) and LRP6 (right panel) expression in several human lung cancer cell lines. (c) Secretion and expression of sLRP6E1E2. Cell culture supernatants were assessed with FLAG specific Ab (Upper panel). Ponceau staining is shown as loading control (Bottom panel). (d) H322 and H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) for 48 hr. Cell lysates were immunoprecipitated with antisera against Wnt3a (IP: Wnt3a) or LRP6 (IP: LRP6) followed by western blot analysis with the same antibodies.
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pone-0036520-g001: Characterization of the decoy Wnt receptor sLRP6E1E2.(a) Schematic representation of the genomic structure of Ad vectors used. (b) Endogenous Wnt3a (left panel) and LRP6 (right panel) expression in several human lung cancer cell lines. (c) Secretion and expression of sLRP6E1E2. Cell culture supernatants were assessed with FLAG specific Ab (Upper panel). Ponceau staining is shown as loading control (Bottom panel). (d) H322 and H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) for 48 hr. Cell lysates were immunoprecipitated with antisera against Wnt3a (IP: Wnt3a) or LRP6 (IP: LRP6) followed by western blot analysis with the same antibodies.

Mentions: To study the biochemical function of soluble LRP6 receptor (sLRP6E1E2), we generated constructs of the E1 and E2 extracellular domains (Wnt-binding sites) of LRP6 [15] and FLAG-tagged sLRP6E1E2 was subcloned into a pCA14 shuttle vector [16]. This pCA14-sLRP6E1E2 vector was co-transformed with a replication-incompetent adenovirus 5/35 chimeric vector (dE1-k35) or replication-competent chimeric oncolytic adenovirus vector (RdB-k35) [17], generating pdE1-k35/sLRP6E1E2 and pRdB-k35/sLRP6E1E2, respectively. These recombinant plasmids were transfected into HEK293 cells to generate dE1-k35/sLRP6E1E2 and RdB-k35/sLRP6E1E2. The replication-incompetent dE1-k35/LacZ and replication-competent oncolytic RdB-k35 vectors were used as negative controls [18] (Fig. 1A). All viruses were obtained as previously described [19].


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)

Characterization of the decoy Wnt receptor sLRP6E1E2.(a) Schematic representation of the genomic structure of Ad vectors used. (b) Endogenous Wnt3a (left panel) and LRP6 (right panel) expression in several human lung cancer cell lines. (c) Secretion and expression of sLRP6E1E2. Cell culture supernatants were assessed with FLAG specific Ab (Upper panel). Ponceau staining is shown as loading control (Bottom panel). (d) H322 and H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) for 48 hr. Cell lysates were immunoprecipitated with antisera against Wnt3a (IP: Wnt3a) or LRP6 (IP: LRP6) followed by western blot analysis with the same antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351461&req=5

pone-0036520-g001: Characterization of the decoy Wnt receptor sLRP6E1E2.(a) Schematic representation of the genomic structure of Ad vectors used. (b) Endogenous Wnt3a (left panel) and LRP6 (right panel) expression in several human lung cancer cell lines. (c) Secretion and expression of sLRP6E1E2. Cell culture supernatants were assessed with FLAG specific Ab (Upper panel). Ponceau staining is shown as loading control (Bottom panel). (d) H322 and H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) for 48 hr. Cell lysates were immunoprecipitated with antisera against Wnt3a (IP: Wnt3a) or LRP6 (IP: LRP6) followed by western blot analysis with the same antibodies.
Mentions: To study the biochemical function of soluble LRP6 receptor (sLRP6E1E2), we generated constructs of the E1 and E2 extracellular domains (Wnt-binding sites) of LRP6 [15] and FLAG-tagged sLRP6E1E2 was subcloned into a pCA14 shuttle vector [16]. This pCA14-sLRP6E1E2 vector was co-transformed with a replication-incompetent adenovirus 5/35 chimeric vector (dE1-k35) or replication-competent chimeric oncolytic adenovirus vector (RdB-k35) [17], generating pdE1-k35/sLRP6E1E2 and pRdB-k35/sLRP6E1E2, respectively. These recombinant plasmids were transfected into HEK293 cells to generate dE1-k35/sLRP6E1E2 and RdB-k35/sLRP6E1E2. The replication-incompetent dE1-k35/LacZ and replication-competent oncolytic RdB-k35 vectors were used as negative controls [18] (Fig. 1A). All viruses were obtained as previously described [19].

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