Rapp J, Kiss E, Meggyes M, Szabo-Meleg E, Feller D, Smuk G, Laszlo T, Sarosi V, Molnar T F, Kvell K, Pongracz J E
Department of Pharmaceutical Biotechnology, School of Pharmacy, University of Pecs, 2 Rokus Str, Pecs, 7624, Hungary.
János Szentágothai Research Centre, University of Pécs, 20 Ifjúság Str, Pecs, 7622, Hungary.
BMC Cancer. 2016 Nov 23;16(1):915. doi: 10.1186/s12885-016-2943-4.
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.
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.
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.
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.
血管生成在正常组织功能和疾病中均具有重要意义，是肺癌（LC）治疗的关键靶点。不幸的是，非小细胞肺癌（NSCLC）的两种主要亚型，即腺癌（AC）和鳞状细胞癌（SCC），对抗血管生成治疗（如抗血管内皮生长因子（VEGF）-A治疗）的反应不同，且会出现危及生命的副作用，在SCC中常表现为肺出血。尽管过氧化物酶体增殖物激活受体（PPAR）γ以及Wnt信号通路已被认为是该过程的分子调节因子，但这些不良反应背后的机制仍 largely 未知。由于NSCLC亚型中的Wnt微环境差异巨大，我们推测SCC中特别高的非经典Wnt5a水平可能是血管生长改变的原因，并导致严重的不良反应。
通过qRT-PCR和TaqMan microRNA检测法测定切除的腺癌和鳞状细胞癌样本中PPARγ、VEGF-A、Wnt5a、miR-27b和miR-200b的水平。使用PPARγ基因敲除小鼠、癌细胞系以及完全人源的体外三维（3D）远端肺组织聚集体，研究PPARγ在VEGF-A表达中的作用以及Wnt信号通路在整体调节中的作用。分别通过qRT-PCR和免疫组织化学检测PPARγ的mRNA和蛋白水平。通过PPRE报告系统测量PPARγ活性。用重组Wnt蛋白、化学Wnt信号通路调节剂处理表达基础水平和经慢病毒递送VEGF-A的组织工程肺组织，并进行PPARγ激动剂和拮抗剂处理。
PPARγ下调和VEGF-A上调是AC和SCC的共同特征。VEGF-A水平的升高直接受PPARγ的控制。然而，PPARγ的水平和活性受Wnt信号通路的控制。经典Wnt信号通路（在AC中）和非经典Wnt信号通路（在SCC中）的失衡均导致PPARγ下调。经典Wnt信号通路直接下调PPARγ，而非经典Wnt5a增加已知的PPARγ调节因子miR27b。
在致癌过程中，Wnt微环境发生改变，可下调PPARγ，导致VEGF-A表达增加。NSCLC的AC和SCC中Wnt微环境的差异通过不同程度地改变内皮细胞运动性和分支的机制导致PPARγ降低，进而可能影响治疗反应。
