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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

Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t-test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t-test n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
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Fig3: Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t-test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t-test n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Mentions: As only the beta-catenin dependent canonical Wnt signaling has been reported repeatedly to down-regulate PPARs [30, 44] and consequently up-regulate VEGF-A [47], it was not clear how SCC samples with high levels of non-canonical Wnt ligands (Fig. 3a and b) can trigger the same mechanism? In the literature PPARgamma has been described to have a highly conserved binding site in its 3′UTR that is a direct target of miR-27b [48]. Binding of miR-27b to its target sequence can suppress PPARgamma expression [49] and augment VEGF-A induced angiogenesis [50]. Importantly, both miR-27b as well as another miRNA, miR-200b can inhibit blood vessel branching during angiogenesis [51, 52]. Based on the above studies we theorized that differences in formation of blood vessel networks in AC and SCC are due to variations in microRNAs and VEGF-A levels. To investigate, both miR-27b and miR-200b levels were measured in primary human AC and SCC samples. Significantly higher expression levels were determined for both miRNA in primary SCC tissues compared to AC (Fig. 3c) indicating existence of miRNA dependent differential regulation of angiogenesis in the two NSCLC subtypes. To test whether the different molecular microenvironment characterized by increased Wnt5a levels in SCC would explain variations in miRNA expression, 3D lung aggregate cultures were exposed to non-canonical Wnt-s; rhWnt5a and rhWnt11. Wnt5a and Wnt11 were selected in the experiments as they were both reported to have higher levels in SCC than in AC [37]. Interestingly, while rhWnt11 had no effect on either miRNAs (Fig. 3d), miR-27b expression was significantly increased by rhWnt5a treatment (Fig. 3d), while miR-200b levels were unaffected. Remarkably, rhWnt5a could not up-regulate miR-27b in the presence of a PPARgamma agonist, only if the antagonist was present (Fig. 3e). The actual VEGF-A expression was, however, lower (Fig. 3e) than induced by the PPARgamma antagonist on its own. Additionally, while rhWnt5a treatment on its own did not increase VEGF-A levels at the time point analyzed (48 h treatment, data not shown) implicating a parallel mechanism that is required for PPARgamma inhibition even in the presence of the non-canonical Wnt pathway dominated microenvironment.Fig. 3


Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility
Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t-test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t-test n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
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Fig3: Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t-test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t-test n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001
Mentions: As only the beta-catenin dependent canonical Wnt signaling has been reported repeatedly to down-regulate PPARs [30, 44] and consequently up-regulate VEGF-A [47], it was not clear how SCC samples with high levels of non-canonical Wnt ligands (Fig. 3a and b) can trigger the same mechanism? In the literature PPARgamma has been described to have a highly conserved binding site in its 3′UTR that is a direct target of miR-27b [48]. Binding of miR-27b to its target sequence can suppress PPARgamma expression [49] and augment VEGF-A induced angiogenesis [50]. Importantly, both miR-27b as well as another miRNA, miR-200b can inhibit blood vessel branching during angiogenesis [51, 52]. Based on the above studies we theorized that differences in formation of blood vessel networks in AC and SCC are due to variations in microRNAs and VEGF-A levels. To investigate, both miR-27b and miR-200b levels were measured in primary human AC and SCC samples. Significantly higher expression levels were determined for both miRNA in primary SCC tissues compared to AC (Fig. 3c) indicating existence of miRNA dependent differential regulation of angiogenesis in the two NSCLC subtypes. To test whether the different molecular microenvironment characterized by increased Wnt5a levels in SCC would explain variations in miRNA expression, 3D lung aggregate cultures were exposed to non-canonical Wnt-s; rhWnt5a and rhWnt11. Wnt5a and Wnt11 were selected in the experiments as they were both reported to have higher levels in SCC than in AC [37]. Interestingly, while rhWnt11 had no effect on either miRNAs (Fig. 3d), miR-27b expression was significantly increased by rhWnt5a treatment (Fig. 3d), while miR-200b levels were unaffected. Remarkably, rhWnt5a could not up-regulate miR-27b in the presence of a PPARgamma agonist, only if the antagonist was present (Fig. 3e). The actual VEGF-A expression was, however, lower (Fig. 3e) than induced by the PPARgamma antagonist on its own. Additionally, while rhWnt5a treatment on its own did not increase VEGF-A levels at the time point analyzed (48 h treatment, data not shown) implicating a parallel mechanism that is required for PPARgamma inhibition even in the presence of the non-canonical Wnt pathway dominated microenvironment.Fig. 3

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