The objective of this study is to investigate the role of microtubule based organelle, the primary cilia in lung adenocarcinoma by i) Quantifying the presence of primary cilia in several Non Small Cell Lung Cancer (NSCLC) cell lines in response to mechanical stimuli, ii) Attempting to determine the role of primary cilia in cell migration, iii) Investigating the effects of Paclitaxel(Taxol) resistance in lung cancer cells, iv) Analyzing the response of lung cancer cells to Smoothened Inhibitors and v) Determining the effects of Transforming Growth Factor Beta-1(TGF-β1) induced Epithelial to Mesenchymal Transition(EMT) in lung cancer cells. To ascertain the effects of primary cilia in the hall marks of tumor progression, several experiments involved prohibition of primary cilia formation by silencing IFT88, the gene responsible using small interfering RNA. Three out of the five cell lines tested, showed increased expression of primary cilia under mechanical stretch. IFT88 inhibition of H460 cells decreased their migration rate to the injury site under stretch conditions. Smoothened (SMO) Inhibitors decreased proliferation and migration rates in human lung adenocarcinoma cell lines (A549luc) similar to the effects observed in IFT88 silenced cells. IFT88 silenced A549luc cells showed a partial reversal of TGF-beta1 induced up-regulation of a mesenchymal marker. These results indicate that primary cilia play a role in the progression and metastasis of lung cancer by aiding the adhesion, proliferation, migration and EMT of lung cancer cells.

Les cellules musculaires lisses (CML) vasculaires les composants cellulaires principaux de la paroi artérielle, sont exposées constamment aux contraintes mécaniques. Les contraintes mécaniques cycliques régulent de nombreuses fonctions des CML vasculaires via les intégrines. Parmi les intégrines, l'[alpha]v[gamma]3 est non seulement un mécano-transducteur mais aussi le récepteur de la prothrombine à la surface des CML. L?activation de l'intégrine [alpha]v[gamma]3 par les contraintes mécaniques pourrait favoriser l'adhésion des CML à la prothrombine et aussi accélérer la génération de thrombine à la surface des CML. Pour vérifier cette hypothèse, nous avons étudié l'effet des contraintes mécaniques sur la génération de thrombine par les CML et identifié les voies de la signalisation impliquées. Nous avons utilisé un modèle de Flexcell utilisant les CML aortiques de rat, soumises à un étirement cyclique (10%, 1Hz). L'exposition à l'étirement cyclique pendant 1h et 6h induit un phénotype de différenciation et non-apoptotique des CML et une augmentation de l'expression de l'intégrine [alpha]v[gamma]3. Il y a aussi une augmentation de la phosphorylation de Src, FAK, AKT de façon temps dépendant et une augmentation de la phosphorylation de l'ILK à 15 min et du clivage de taline de 5 à 60 min. L'étirement cyclique augmente l'adhésion des CML à la prothrombine et la génération de thrombine avec un effet maximum à 6h de 67% et 30% respectivement. Le peptide mimétique de l'intégrine [alpha]v[gamma]3 (cRGDPV) et le siARN [alpha]v bloquent tous les effets de l'étirement cyclique sur les CML. Le siARN taline inhibe l'expression de la sous-unité [alpha]v et également la phosphorylation de Src, AKT et ILK. Le siARN ILK n'a pas d'effet sur l'expression de l'[alpha]v mais inhibe la phosphorylation d'AKT et le clivage de taline à 6h de l'étirement cyclique. Ainsi, l'étirement cyclique induit une plus forte génération de thrombine par les CML vasculaires via l'activation des voies de signalisation dépendante de l'[alpha]v[gamma]3. Cette étude suggère que la génération de thrombine intravasculaire peut être régulée par des antagonistes de l'intégrine [alpha]v[gamma]3 et peut devenir une nouvelle cible thérapeutique chez les patients avec une pression pulsée élevée / Vascular smooth muscle cells (SMC), the main cellular components of the arterial wall, are constantly exposed to mechanical stretch. Cyclic mechanical stress regulates many functions of vascular SMC via integrins. Among the integrins, [alpha]v[gamma]3 is not only a mechanotransducer but also the receptor of prothrombin in the vascular SMC. Activation of integrin [alpha]v[gamma]3 by mechanical stretch may promote SMC adhesion to prothrombin and also accelerate thrombin generation on the surface of SMC. To test this hypothesis, we have studied the effect of mechanical stretch on the generation of thrombin by SMC and identified possible signaling pathway involved. We used a Flexcell model using rat aortic SMC subjected to cyclic stretch (10%, 1Hz). Exposure to cyclic stretch for 1h and 6h induced a phenotype of differentiation and non-apoptosis of SMC and an increased expression of integrin [alpha]v[gamma]3. There was also an increase in phosphorylation of Src, FAK, and AKT in a time dependent manner, increased phosphorylation of ILK at 15min and the cleavage of talin from 5 to 60min. Cyclic stretch increased the adhesion of prothrombin to the SMC, and thrombin generation with a maximum effect of 67% and 30% respectively. A peptide mimetic of integrin [alpha]v[gamma]3 (cRGDPV) and [alpha]v siRNA both blocked all the effects of cyclic stretch on SMC. A talin siRNA inhibited the expression of [alpha]v and the phosphorylation of Src, AKT and ILK. An ILK siRNA has no effect on the expression of [alpha]v but inhibited the phosphorylation of AKT and the cleavage of talin at 6h of stretch. Thus, cyclic stretch induced a higher thrombin generation by vascular SMC via activation of signaling pathways dependant on [alpha]v[gamma]3. This study suggests that intravascular thrombin generation can be regulated by antagonists of integrin [alpha]v[gamma]3 and can become a new therapeutic target for the patients with a high pulse pressure