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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 inhibits tumor growth and characterization.(a) Tumors were injected with PBS (▪), dE1-k35/LacZ (♦), RdB-k35 (▴), dE1-k35/sLRP6E1E2 (×), or RdB-k35/sLRP6E1E2 (•) on days 1, 3, and 5. Results are expressed as mean ± SEM (n = 7). *P<0.05 versus PBS-treated or dE1-k35-treated controls and versus dE1-k35/sLRP6E1E2. #P<0.01 versus PBS-treated or dE1-k35-treated controls. (b) Tumor sections from each group were immunostained against E1A or FLAG (original magnification, ×40 and ×100). (c) Tumor tissues from each group were stained with DAPI (blue), anti-Ki67 (red), and TdT-mediated TUNEL (green). Original magnification: ×100. (d) Blood vessels were visualized by staining for CD31. Original magnification, ×100. (e) Mean microvessel density for each treatment group (CD31 positive cells/field). Results are expressed as mean ± SEM (each group, n = 3 tumors). *P<0.05 versus PBS, dE1-k35, or dE1-k35/sLRP6E1E2. n.s.  =  not significant. (f) Cells were stained with DAPI (blue), anti-Wnt3a (red), or anti-β-catenin (green). Original magnification: ×100. (g, h) The expression levels of Wnt3a (g) and β-catenin (h) were assessed semi-quantitatively using MetaMorph® imaging analysis software. Results are expressed as mean ± SEM (each group, n = 5 tumors). #P<0.01 versus dE1-k35, *P<0.05 versus RdB-k35.
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pone-0036520-g005: Decoy Wnt receptor sLRP6E1E2 inhibits tumor growth and characterization.(a) Tumors were injected with PBS (▪), dE1-k35/LacZ (♦), RdB-k35 (▴), dE1-k35/sLRP6E1E2 (×), or RdB-k35/sLRP6E1E2 (•) on days 1, 3, and 5. Results are expressed as mean ± SEM (n = 7). *P<0.05 versus PBS-treated or dE1-k35-treated controls and versus dE1-k35/sLRP6E1E2. #P<0.01 versus PBS-treated or dE1-k35-treated controls. (b) Tumor sections from each group were immunostained against E1A or FLAG (original magnification, ×40 and ×100). (c) Tumor tissues from each group were stained with DAPI (blue), anti-Ki67 (red), and TdT-mediated TUNEL (green). Original magnification: ×100. (d) Blood vessels were visualized by staining for CD31. Original magnification, ×100. (e) Mean microvessel density for each treatment group (CD31 positive cells/field). Results are expressed as mean ± SEM (each group, n = 3 tumors). *P<0.05 versus PBS, dE1-k35, or dE1-k35/sLRP6E1E2. n.s.  =  not significant. (f) Cells were stained with DAPI (blue), anti-Wnt3a (red), or anti-β-catenin (green). Original magnification: ×100. (g, h) The expression levels of Wnt3a (g) and β-catenin (h) were assessed semi-quantitatively using MetaMorph® imaging analysis software. Results are expressed as mean ± SEM (each group, n = 5 tumors). #P<0.01 versus dE1-k35, *P<0.05 versus RdB-k35.

Mentions: We next evaluated the ability of sLRP6E1E2 to inhibit tumor growth in a mouse xenograft model. Tumors were generated by subcutaneous injection of H460 cells into the abdominal region of nude mice. When tumors reached a mean size of 80–100 mm3, they were injected with PBS, dE1-k35, RdB-k35, dE1-k35/sLRP6E1E2, or RdB-k35/sLRP6E1E2 on days 1, 3, and 5. Fig. 5A shows that the volume of tumors injected with sLRP6E1E2-expressing vectors was significantly lower than that of corresponding controls. After 25 days, tumors treated with PBS reached a mean volume of 3883.1±418.08 mm3, and tumors treated with dE1-k35 and RdB-k35 reached 3388.1±226.9 mm3 and 1991±311.8 mm3, respectively. In contrast, tumor growth was strongly suppressed in mice injected with dE1-k35/sLRP6E1E2 (1645.3±353.6 mm3; P<0.05 compared with PBS or dE1-k35 groups) or RdB-k35/sLRP6E1E2 (923.3±180.4 mm3; P<0.01 compared with PBS or RdB-k35 groups).


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 tumor growth and characterization.(a) Tumors were injected with PBS (▪), dE1-k35/LacZ (♦), RdB-k35 (▴), dE1-k35/sLRP6E1E2 (×), or RdB-k35/sLRP6E1E2 (•) on days 1, 3, and 5. Results are expressed as mean ± SEM (n = 7). *P<0.05 versus PBS-treated or dE1-k35-treated controls and versus dE1-k35/sLRP6E1E2. #P<0.01 versus PBS-treated or dE1-k35-treated controls. (b) Tumor sections from each group were immunostained against E1A or FLAG (original magnification, ×40 and ×100). (c) Tumor tissues from each group were stained with DAPI (blue), anti-Ki67 (red), and TdT-mediated TUNEL (green). Original magnification: ×100. (d) Blood vessels were visualized by staining for CD31. Original magnification, ×100. (e) Mean microvessel density for each treatment group (CD31 positive cells/field). Results are expressed as mean ± SEM (each group, n = 3 tumors). *P<0.05 versus PBS, dE1-k35, or dE1-k35/sLRP6E1E2. n.s.  =  not significant. (f) Cells were stained with DAPI (blue), anti-Wnt3a (red), or anti-β-catenin (green). Original magnification: ×100. (g, h) The expression levels of Wnt3a (g) and β-catenin (h) were assessed semi-quantitatively using MetaMorph® imaging analysis software. Results are expressed as mean ± SEM (each group, n = 5 tumors). #P<0.01 versus dE1-k35, *P<0.05 versus RdB-k35.
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pone-0036520-g005: Decoy Wnt receptor sLRP6E1E2 inhibits tumor growth and characterization.(a) Tumors were injected with PBS (▪), dE1-k35/LacZ (♦), RdB-k35 (▴), dE1-k35/sLRP6E1E2 (×), or RdB-k35/sLRP6E1E2 (•) on days 1, 3, and 5. Results are expressed as mean ± SEM (n = 7). *P<0.05 versus PBS-treated or dE1-k35-treated controls and versus dE1-k35/sLRP6E1E2. #P<0.01 versus PBS-treated or dE1-k35-treated controls. (b) Tumor sections from each group were immunostained against E1A or FLAG (original magnification, ×40 and ×100). (c) Tumor tissues from each group were stained with DAPI (blue), anti-Ki67 (red), and TdT-mediated TUNEL (green). Original magnification: ×100. (d) Blood vessels were visualized by staining for CD31. Original magnification, ×100. (e) Mean microvessel density for each treatment group (CD31 positive cells/field). Results are expressed as mean ± SEM (each group, n = 3 tumors). *P<0.05 versus PBS, dE1-k35, or dE1-k35/sLRP6E1E2. n.s.  =  not significant. (f) Cells were stained with DAPI (blue), anti-Wnt3a (red), or anti-β-catenin (green). Original magnification: ×100. (g, h) The expression levels of Wnt3a (g) and β-catenin (h) were assessed semi-quantitatively using MetaMorph® imaging analysis software. Results are expressed as mean ± SEM (each group, n = 5 tumors). #P<0.01 versus dE1-k35, *P<0.05 versus RdB-k35.
Mentions: We next evaluated the ability of sLRP6E1E2 to inhibit tumor growth in a mouse xenograft model. Tumors were generated by subcutaneous injection of H460 cells into the abdominal region of nude mice. When tumors reached a mean size of 80–100 mm3, they were injected with PBS, dE1-k35, RdB-k35, dE1-k35/sLRP6E1E2, or RdB-k35/sLRP6E1E2 on days 1, 3, and 5. Fig. 5A shows that the volume of tumors injected with sLRP6E1E2-expressing vectors was significantly lower than that of corresponding controls. After 25 days, tumors treated with PBS reached a mean volume of 3883.1±418.08 mm3, and tumors treated with dE1-k35 and RdB-k35 reached 3388.1±226.9 mm3 and 1991±311.8 mm3, respectively. In contrast, tumor growth was strongly suppressed in mice injected with dE1-k35/sLRP6E1E2 (1645.3±353.6 mm3; P<0.05 compared with PBS or dE1-k35 groups) or RdB-k35/sLRP6E1E2 (923.3±180.4 mm3; P<0.01 compared with PBS or RdB-k35 groups).

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