Functional Investigation Of MTORC2/Rictor In Metastasis Of Non-Small Cell Lung Cancer

ObjectivesThe incidence of lung cancer has increased as the most frequently diagnosed cancer and the leading cause of cancer death in China. Metastasis is one of main causes of treatment failure and death in cancer patients, most evidences have demonstrated that chemotaxis and Epithelial-Mesenchymal Transition (EMT) play critical roles in promoting cancer cell metastasis. However, the detailed mechanism remains largely unknown. Our previous studies have found that Rictor, a key component of mTORC2, was up-regulated in several cancer tissues, and its elevated expression correlated with metastasis of cancer patients. However, how Rictor regulates cancer cell metastasis is unclear. In the present study, we aim to investigate the role of Rictor in EGF induced chemotaxis of non small cell lung cancer cells, and to explore the potential function of Rictor/mTORC2in the mediation of TGFβ induced EMT of NSCLC cells. We also examine the effect of Rictor deletion on metastasis of lung cancer cells in an in vivo SCID mouse model.Methods1) Lentivirus mediated shRNA delivery system was used to knockdown the expression of Rictor in lung cancer cells, then chemotaxis assay, cell polarization and directed cell migration assay, cell adhesion assay and F-actin polymerization assay was used to examine the effect of Rictor deletion on EGF induced chemotaxis and cell migration of NSCLC cells.2) Immunofluresence staining and western blotting methods were used to investigate the effect of Rictor or β-catenin deletion on TGFP induced EMT and activation of canonical Smads and non-canonical pathways in lung cancer cells.3) Immunofluresence staining and western blotting methods were used to exmine the effect of GSK3β inhibition by LiCl on TGFβ induced EMT in Rictor knockdown cells.4) Immunohistochemistry method was used to detect the expression status of Rictor in lung cancer tissues; A SCID mouse model was used to evaluate the effect of Rictor knockdown on the metastatic ability of lung cancer cells.Results1) Rictor knockdown impaired EGF induced phoshorylation of Akt, and inhibited EGF induced chemotaxis of lung cancer cells along with defects in cell polarization, directed cell migration, cell adherion and F-actin polymerization.2) Loss of Rictor expression in A549cells results in an up-regulation of epithelial maker E-cadherin and a decrease of mesenchymal protein vimentin, which indicating a mesenchymal to epithelial transtion, meanwhile, Rictor knockdown also impaired TGFβ induced EMT of lung cancer cells.3) Knockdown of Rictor in A549cells has no significant effect on TGFβ induced activation of canonical Smad2/3and Erkl/2, but phosphorylation of Akt at473sites and GSK3β at ser9sites were decreased, along with a reduction of nuclear translocation of β-catenin.4) Inhibition of GSK3β activity by LiCl in Rictor depletion cells rescued TGFβ induced EMT; knockdown of β-catenin impaired TGFβ induced EMT of lung cancer cells without affecting canonical Smads and non-canonical pathways;5) Elevated expression of Rictor was observed in lung cancer tissues compared with tumor-adjacent normal tissues, and high expression of Rictor correlated with distant metatasis of cancer patients.6) Rictor depletion inhibited lung cancer cell metastsis in an in vivo mouse model.ConclusionsOur results support Rictor as a key regulator for lung cancer cell migration and metastasis. Rictor mediates cancer cell metastasis by regulating chemotaxis and Epithelial-Mesenchymal Transition of cancer cells. Rictor knockdown impaired EGF induced chemotaxis and phosphorylation of Akt, pivitol melecules in chemotaxis signaling, in parallel, cell polarization and directed cell migration was also decreased. Further, Rictor/mTORC2is required for TGFβinduced EMT of NSCLC cells through cross activation of GSK3β/β catenin pathway.