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Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

View Article: PubMed Central - PubMed

ABSTRACT

Background: Angiogenesis is important both in normal tissue function and disease and represents a key target in lung cancer (LC) therapy. Unfortunately, the two main subtypes of non-small-cell lung cancers (NSCLC) namely, adenocarcinoma (AC) and squamous cell carcinoma (SCC) respond differently to anti-angiogenic e.g. anti-vascular endothelial growth factor (VEGF)-A treatment with life-threatening side effects, often pulmonary hemorrhage in SCC. The mechanisms behind such adverse reactions are still largely unknown, although peroxisome proliferator activator receptor (PPAR) gamma as well as Wnt-s have been named as molecular regulators of the process. As the Wnt microenvironments in NSCLC subtypes are drastically different, we hypothesized that the particularly high levels of non-canonical Wnt5a in SCC might be responsible for alterations in blood vessel growth and result in serious adverse reactions.

Methods: PPARgamma, VEGF-A, Wnt5a, miR-27b and miR-200b levels were determined in resected adenocarcinoma and squamous cell carcinoma samples by qRT-PCR and TaqMan microRNA assay. The role of PPARgamma in VEGF-A expression, and the role of Wnts in overall regulation was investigated using PPARgamma knock-out mice, cancer cell lines and fully human, in vitro 3 dimensional (3D), distal lung tissue aggregates. PPARgamma mRNA and protein levels were tested by qRT-PCR and immunohistochemistry, respectively. PPARgamma activity was measured by a PPRE reporter system. The tissue engineered lung tissues expressing basal level and lentivirally delivered VEGF-A were treated with recombinant Wnts, chemical Wnt pathway modifiers, and were subjected to PPARgamma agonist and antagonist treatment.

Results: PPARgamma down-regulation and VEGF-A up-regulation are characteristic to both AC and SCC. Increased VEGF-A levels are under direct control of PPARgamma. PPARgamma levels and activity, however, are under Wnt control. Imbalance of both canonical (in AC) and non-canonical (in SCC) Wnts leads to PPARgamma down-regulation. While canonical Wnts down-regulate PPARgamma directly, non-canonical Wnt5a increases miR27b that is known regulator of PPARgamma.

Conclusion: During carcinogenesis the Wnt microenvironment alters, which can downregulate PPARgamma leading to increased VEGF-A expression. Differences in the Wnt microenvironment in AC and SCC of NSCLC lead to PPARgamma decrease via mechanisms that differentially alter endothelial cell motility and branching which in turn can influence therapeutic response.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-016-2943-4) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

VEGF-A expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A mRNA expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t-test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
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Fig2: VEGF-A expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A mRNA expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t-test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Mentions: To prove that canonical Wnt signaling induced downregulation of PPARgamma triggers VEGF-A expression, A549 lung adenocarcinoma cell line was treated with the chemical activator of the canonical Wnt signaling pathway, the beta-catenin activator LiCl [41]. Inhibition of PPAR reporter activity (Fig. 2a) by LiCl (10 mM) lead to increased VEGF-A mRNA (Fig. 2b) and protein expression (Fig. 2c). Inhibition of PPARgamma by LiCl did not affect PPARgamma mRNA expression suggesting that PPARgamma activity is the key factor in VEGF-A regulation.Fig. 2


Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility
VEGF-A expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A mRNA expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t-test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
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Fig2: VEGF-A expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A mRNA expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t-test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
Mentions: To prove that canonical Wnt signaling induced downregulation of PPARgamma triggers VEGF-A expression, A549 lung adenocarcinoma cell line was treated with the chemical activator of the canonical Wnt signaling pathway, the beta-catenin activator LiCl [41]. Inhibition of PPAR reporter activity (Fig. 2a) by LiCl (10 mM) lead to increased VEGF-A mRNA (Fig. 2b) and protein expression (Fig. 2c). Inhibition of PPARgamma by LiCl did not affect PPARgamma mRNA expression suggesting that PPARgamma activity is the key factor in VEGF-A regulation.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Angiogenesis is important both in normal tissue function and disease and represents a key target in lung cancer (LC) therapy. Unfortunately, the two main subtypes of non-small-cell lung cancers (NSCLC) namely, adenocarcinoma (AC) and squamous cell carcinoma (SCC) respond differently to anti-angiogenic e.g. anti-vascular endothelial growth factor (VEGF)-A treatment with life-threatening side effects, often pulmonary hemorrhage in SCC. The mechanisms behind such adverse reactions are still largely unknown, although peroxisome proliferator activator receptor (PPAR) gamma as well as Wnt-s have been named as molecular regulators of the process. As the Wnt microenvironments in NSCLC subtypes are drastically different, we hypothesized that the particularly high levels of non-canonical Wnt5a in SCC might be responsible for alterations in blood vessel growth and result in serious adverse reactions.

Methods: PPARgamma, VEGF-A, Wnt5a, miR-27b and miR-200b levels were determined in resected adenocarcinoma and squamous cell carcinoma samples by qRT-PCR and TaqMan microRNA assay. The role of PPARgamma in VEGF-A expression, and the role of Wnts in overall regulation was investigated using PPARgamma knock-out mice, cancer cell lines and fully human, in vitro 3 dimensional (3D), distal lung tissue aggregates. PPARgamma mRNA and protein levels were tested by qRT-PCR and immunohistochemistry, respectively. PPARgamma activity was measured by a PPRE reporter system. The tissue engineered lung tissues expressing basal level and lentivirally delivered VEGF-A were treated with recombinant Wnts, chemical Wnt pathway modifiers, and were subjected to PPARgamma agonist and antagonist treatment.

Results: PPARgamma down-regulation and VEGF-A up-regulation are characteristic to both AC and SCC. Increased VEGF-A levels are under direct control of PPARgamma. PPARgamma levels and activity, however, are under Wnt control. Imbalance of both canonical (in AC) and non-canonical (in SCC) Wnts leads to PPARgamma down-regulation. While canonical Wnts down-regulate PPARgamma directly, non-canonical Wnt5a increases miR27b that is known regulator of PPARgamma.

Conclusion: During carcinogenesis the Wnt microenvironment alters, which can downregulate PPARgamma leading to increased VEGF-A expression. Differences in the Wnt microenvironment in AC and SCC of NSCLC lead to PPARgamma decrease via mechanisms that differentially alter endothelial cell motility and branching which in turn can influence therapeutic response.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-016-2943-4) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus