Limits...
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

Summary of the role of Wnt dependent regulation of PPARgamma in tumor angiogenesis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5120464&req=5

Fig5: Summary of the role of Wnt dependent regulation of PPARgamma in tumor angiogenesis

Mentions: For better understanding of the process of PPARgamma down-regulation we considered that down-regulation of PPARgamma transcription is a beta-catenin-dependent and therefore canonical Wnt pathway dependent process [44]. As lung ACs are characterized by increased canonical and therefore beta-catenin dependent Wnt signal activity [58] such molecular basis can explain PPARgamma reduction in AC [44]. The similarly high VEGF-A and reduced PPARgamma levels in SCC samples seem counter-intuitive as in SCC the non-canonical, beta-catenin independent Wnts play the dominant role [59]. Our initial data, however, provide some explanations. Wnt5a is the characteristically highly expressed Wnt in cancers with squamous histology and increased Wnt5a levels are also a trademark of lung SCC. Interestingly, Wnt5a can up-regulate miR-27b which miRNA has been reported to bind to the highly conserved binding site in the 3′UTR of PPARgamma [48] and suppress its expression [49]. Consequently, up-regulation of VEGF-A [50] can use this particular route (Fig. 5). Wnt5a did not induce VEGF-A directly at the time-points measured in this study, indicating that other mechanisms are also needed to achieve inhibition of PPARgamma. Inhibition of PPARgamma in the presence of Wnt5a, however, modified VEGF-A levels supporting our theory that Wnt5a plays a modulatory role in the angiogenic process. Certainly, there are limitations to the study as not all VEGF-A isoforms [60] nor pericytes were investigated in the above experiments therefore the full effect of Wnt5a on the angiogenic process could not be assessed. However, since Cox-2 (an enzyme responsible for pericyte recruitment) is inhibited by PPARgamma [61, 62], we can hypothesize that lower PPARgamma levels and/or activity could reduce pericyte coverage. As SCC has higher PPARgamma levels than AC, perhaps high Wnt5a can not only reduce endothelial cell but also pericyte mobility resulting in immature vessel formation that makes SCC more prone to hemorrhage. Further studies are needed to investigate the above hypothesis.Fig. 5


Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility
Summary of the role of Wnt dependent regulation of PPARgamma in tumor angiogenesis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120464&req=5

Fig5: Summary of the role of Wnt dependent regulation of PPARgamma in tumor angiogenesis
Mentions: For better understanding of the process of PPARgamma down-regulation we considered that down-regulation of PPARgamma transcription is a beta-catenin-dependent and therefore canonical Wnt pathway dependent process [44]. As lung ACs are characterized by increased canonical and therefore beta-catenin dependent Wnt signal activity [58] such molecular basis can explain PPARgamma reduction in AC [44]. The similarly high VEGF-A and reduced PPARgamma levels in SCC samples seem counter-intuitive as in SCC the non-canonical, beta-catenin independent Wnts play the dominant role [59]. Our initial data, however, provide some explanations. Wnt5a is the characteristically highly expressed Wnt in cancers with squamous histology and increased Wnt5a levels are also a trademark of lung SCC. Interestingly, Wnt5a can up-regulate miR-27b which miRNA has been reported to bind to the highly conserved binding site in the 3′UTR of PPARgamma [48] and suppress its expression [49]. Consequently, up-regulation of VEGF-A [50] can use this particular route (Fig. 5). Wnt5a did not induce VEGF-A directly at the time-points measured in this study, indicating that other mechanisms are also needed to achieve inhibition of PPARgamma. Inhibition of PPARgamma in the presence of Wnt5a, however, modified VEGF-A levels supporting our theory that Wnt5a plays a modulatory role in the angiogenic process. Certainly, there are limitations to the study as not all VEGF-A isoforms [60] nor pericytes were investigated in the above experiments therefore the full effect of Wnt5a on the angiogenic process could not be assessed. However, since Cox-2 (an enzyme responsible for pericyte recruitment) is inhibited by PPARgamma [61, 62], we can hypothesize that lower PPARgamma levels and/or activity could reduce pericyte coverage. As SCC has higher PPARgamma levels than AC, perhaps high Wnt5a can not only reduce endothelial cell but also pericyte mobility resulting in immature vessel formation that makes SCC more prone to hemorrhage. Further studies are needed to investigate the above hypothesis.Fig. 5

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