The Role and Regulation of the Mechanistic Target of Rapamycin Complex 2 and Ras in Non-Small Cell Lung Cancer Cell Migration

Abstract

Cell migration is vital in normal cellular functions, such as development and immune responses. However, in the case of cancer cells, abnormal regulation of the signaling pathways that govern cell migration can lead to cancer metastasis, the cause of 90% of cancer-related deaths. The mechanistic Target of Rapamycin Complex 2 (mTORC2) is increasingly implicated as a regulator of cell migration, however its activation and potential signaling mechanisms are incompletely understood. Lung cancer is the leading cause of cancer-related death in both the US and the world and 70% of lung cancers are metastatic at diagnosis. Therefore, we utilized the A549 Non-Small Cell Lung Cancer (NSCLC) cell line to investigate mTORC2 signaling and mTORC2-mediated migration in lung cancer cells. We investigated two potentially implicated membrane receptors, CXC-motif Receptor 4 (CXCR4) and Epidermal Growth Factor Receptor (EGFR) and found that both could activate mTORC2 and mTORC2-mediated migration. Interestingly, we found that when the receptors were activated in combination, the mTORC2 downstream substrate AKT was robustly phosphorylated, but migration was not significantly increased. Therefore, we propose a feedback mechanism between mTORC1 and mTORC2, modulated through AKT activity. We also identified PI3K and Src as signaling partners of mTORC2 leading to AKT activation. Furthermore, Ras GTPases and their oncogenic mutations have recently been linked directly to mTORC2 activation. Therefore, we used a CRISPR/Cas9-based gene knock-in approach to determine a role for oncogenic Ras versus wild-type Ras proteins in mTORC2 activity and cell migration. Interestingly, we found that in A549 cells, neither oncogenic Ras nor wild-type Ras contributes to mTORC2 or mTORC2-mediated cell migration, but we do highlight a role for wild-type Ras proteins in the proliferation of A549 cells. Taken together, our work defines novel mTORC2 signaling mechanisms in A549 NSCLC cells.