抑制并阻断肺腺癌细胞整合素相关激酶（ILK）相关信号通路的研究

This project seeks to examine the signaling mechanisms that control non small cell lung carcinoma (NSCLC) EGF-R wild type adenocarcinoma subtype cell growth, and to explore novel strategies designed to modulate these signals. It focuses on the actions of peroxisome proliferator activated receptor gamma (PPARγ) ligands. These ligands activate PPARγ, a ligand-inducible nuclear transcription factor that has been implicated in the pathogenesis of lung carcinoma. In several in vitro and in vivo studies reported by this and other groups, PPARγ ligands have been found to exert potent anti-cancer activity. However, the exact role that PPARγ signaling plays in NSCLC and the mechanisms by which PPARγ ligands suppress NSCLC cell growth and induce apoptosis have not been fully elucidated. Interestingly, we reported that PPARγ ligands inhibit NSCLC cell growth by reducing the expression of integrin-linked kinase (ILK), which is involved in the suppression of apoptosis and promotion of tumor cell survival, and that overexpression of ILK overcomes the inhibitory effect of PPARγ ligand on the PI3-K downsteam effector Akt/mTOR, pro-oncogene COX-2 and cyclin D1 protein expression, and subsequent NSCLC cell growth. These observations suggest that ILK is a potential dominant driver of lung cancer growth and novel target for PPARγ ligands. Our hypothesis will be tested in vitro and in vivo in the following three specific aims: Aim 1, To examine the molecular mechanisms by which PPARγ ligands control ILK gene expression in inhibition of human lung carcinoma cell growth in vitro. Aim 2, To examine the role of PPARγ and ILK on tumor progression and metastasis in vivo. These experiments will allow us to investigate the role of ILK and PPARγ in mediating the inhibitory effect of PPARγ ligands on NSCLC cell growth, and to study how PPARγ ligands affect this process. Importantly, the data generated are expected to unveil novel mechanisms by which PPARγ ligands inhibit human lung cancer cell growth, and possibly set the stage for pre-clinical studies aimed at investigating the role of PPARγ ligands as potential novel anti-lung carcinoma therapies.

PPARγ在抑制肺癌细胞生长中的确切作用机制仍未阐明。我们前期工作表明，PPARγ配体抑制非小细胞肺癌（NSCLC）细胞生长的机制可能与抑制整合素相关激酶（ILK）的活性以及表达有关，ILK可促进肿瘤细胞生长。过表达ILK可消除PPARγ配体对PI3K下游Akt/mTOR和COX-2等的抑制作用，促进NSCLC细胞生长。这些现象表明ILK是一种潜在的肺癌生长的关键驱动器，并且有可能是一种新的PPARγ配体靶分子。本项目将开展以下研究：1）采用人肺癌细胞体外培养研究PPARγ配体抑制ILK激酶活性，基因表达，以了解其抑制癌细胞生长的新的信号传递途径和其分子机制。2）体内试验，研究PPARγ和ILK在介导PPARγ配体对肿瘤生长以及转移抑制中的作用和影响。通过此研究将使我们明确ILK以及PPARγ在介导PPARγ配体对NSCLC细胞的抑制效应中的作用，有可能寻找到抑制肺癌细胞生长的新的作机制。

我们研究发现，PPARγ配体（如Rosiglitazone，ciglitazone和GW1929等）能有效的抑制肺癌细胞和组织的生长及转移，且显著抑制ILK和PDK1的激酶活性和蛋白表达水平。过表达ILK可消除PPARγ配体rosiglitazone对COX2和P21等的调控作用，说明ILK可能是一种新的PPARγ配体靶分子；另外，我们发现ILK除了参与PPARγ配体的抑癌作用外，还能有效介导中药单体大黄素（Emodin）对肺癌细胞的生长抑制效应。Emodin下调肺癌细胞ILK启动子活性和蛋白表达，且呈剂量依赖性关系；过表达ILK后，emodin对肺癌细胞的生长抑制效应显著减弱。另外，SiRNA沉默PPARγ后，可有效抵抗emodin对ILK的抑制作用，且PPARγ配体ciglitazone联合emodin对ILK具有协同抑制效应，说明ILK可能是PPARγ的下游靶蛋白。深入研究后我们发现，Emodin通过上调PPARγ磷酸化水平，刺激AMPKα和ERK1/2激酶活性，进一步减少转录调控因子SP1和C-jun的表达，从而下调ILK的激酶活性和蛋白表达。肺癌动物模型实验结果进一步发现，与对照组相比，ILK敲除的肺癌组织的生长速度和瘤体体积显著减小。综上所述，PPARγ以及ILK相关信号通路在肺腺癌发生和发展过程中发挥着重要的作用，PPARγ配体以及emodin可能是通过激活PPARγ活性，影响其他激酶和转录调控因子的表达，进一步抑制ILK的激酶活性和蛋白表达水平，最终抑制肺腺癌细胞和组织的生长及转移。