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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 in PPARgamma KO mice and human lung tumors. a, Hematoxylin eosin staining and immunofluorescent staining of the lung of wild type C57BL/6 and PPARgamma knock-out (KO) mice show a significantly increased level of mVEGF-A in KO lung tissues. Scale bars, 200 μm and 50 μm. Intensity data are representation of three independent experiments as mean ± SEM. b, Resected human lung AC and SCC revealed significant PPARgamma decrease and VEGF-A expression increase. Data are presented as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
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Fig1: VEGF-A expression in PPARgamma KO mice and human lung tumors. a, Hematoxylin eosin staining and immunofluorescent staining of the lung of wild type C57BL/6 and PPARgamma knock-out (KO) mice show a significantly increased level of mVEGF-A in KO lung tissues. Scale bars, 200 μm and 50 μm. Intensity data are representation of three independent experiments as mean ± SEM. b, Resected human lung AC and SCC revealed significant PPARgamma decrease and VEGF-A expression increase. Data are presented as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Mentions: To clarify the role of PPARgamma in regulation of VEGF-A expression, lungs of PPARgamma knock-out mice were studied. Fluorescent immunohistochemistry of VEGF-A protein revealed a significantly higher expression of the VEGF-A protein in the lungs of PPARgamma KO mice than in their wild-type litter mates (Fig. 1a), indicating that PPARgamma inactivation is required for VEGF-A production. Emphasizing the initial observation, significantly (p < 0.05) increased VEGF-A expression was detected in primary clinical samples (Additional file 2: Table S1) of both AC and SCC (Fig. 1b), while PPARgamma levels were reduced (p < 0.05 and p < 0.01, respectively) in both tumor types compared to normal, non-diseased, primary lung controls. Additionally, comparative analysis of primary AC and SCC samples highlighted existing differences in the two NSCLC subtypes. While lower PPARgamma mRNA levels characterized significantly higher VEGF-A expression in AC, higher PPARgamma and lower VEGF-A mRNA described SCC (Fig. 1b).Fig. 1


Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility
VEGF-A expression in PPARgamma KO mice and human lung tumors. a, Hematoxylin eosin staining and immunofluorescent staining of the lung of wild type C57BL/6 and PPARgamma knock-out (KO) mice show a significantly increased level of mVEGF-A in KO lung tissues. Scale bars, 200 μm and 50 μm. Intensity data are representation of three independent experiments as mean ± SEM. b, Resected human lung AC and SCC revealed significant PPARgamma decrease and VEGF-A expression increase. Data are presented as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
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Related In: Results  -  Collection

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Fig1: VEGF-A expression in PPARgamma KO mice and human lung tumors. a, Hematoxylin eosin staining and immunofluorescent staining of the lung of wild type C57BL/6 and PPARgamma knock-out (KO) mice show a significantly increased level of mVEGF-A in KO lung tissues. Scale bars, 200 μm and 50 μm. Intensity data are representation of three independent experiments as mean ± SEM. b, Resected human lung AC and SCC revealed significant PPARgamma decrease and VEGF-A expression increase. Data are presented as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
Mentions: To clarify the role of PPARgamma in regulation of VEGF-A expression, lungs of PPARgamma knock-out mice were studied. Fluorescent immunohistochemistry of VEGF-A protein revealed a significantly higher expression of the VEGF-A protein in the lungs of PPARgamma KO mice than in their wild-type litter mates (Fig. 1a), indicating that PPARgamma inactivation is required for VEGF-A production. Emphasizing the initial observation, significantly (p < 0.05) increased VEGF-A expression was detected in primary clinical samples (Additional file 2: Table S1) of both AC and SCC (Fig. 1b), while PPARgamma levels were reduced (p < 0.05 and p < 0.01, respectively) in both tumor types compared to normal, non-diseased, primary lung controls. Additionally, comparative analysis of primary AC and SCC samples highlighted existing differences in the two NSCLC subtypes. While lower PPARgamma mRNA levels characterized significantly higher VEGF-A expression in AC, higher PPARgamma and lower VEGF-A mRNA described SCC (Fig. 1b).Fig. 1

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