mTORC2 Regulates hnRNPE1 Phosphorylation and Cytoskeletal Organization in Bladder Cancer Cells
Sounak Gupta, George Hussey, Philip H Howe, Donna E Hansel. Cleveland Clinic, Cleveland; Medical University of South Carolina, Charleston
Background: Aggressive urothelial carcinomas (UCCs) are known to exploit Mammalian Target of Rapamycin (mTOR) signaling. The therapeutic benefit derived from inhibiting mTOR in bladder cancer has, in part, been attributed to reduced cancer-cell migration. Herein, we investigated the role of the mTOR Complex2 (mTORC2) in regulating cytoskeletal organization and cell migration.
Specifically, heterogenous nuclear-ribonucleoprotein E1 (hnRNPE1) forms a ribonucleoprotein (mRNP) complex that binds to the 3'-untranslated region (UTR) of target genes. This complex silences the translation of select genes that promote the transformation of epithelial cells into a more invasive phenotype, a process referred to as epithelial to mesenchymal transition (EMT).
Firstly, we evaluated whether relieving translation inhibition by this mRNP complex and the associated pro-invasive phenotype led to cytoskeletal reorganization. Akt2, a known target of mTORC2, has been demonstrated to phosphorylate hnRNPE1 and thereby trigger a release of this mRNP leading to increased translation of target transcripts that promote EMT. Therefore, we hypothesized that disrupting mTORC2 should conversely reduce AKT2 dependant hnRNPE1 phosphorylation, stabilize the mRNP complex and reduce the invasive behavior of bladder cancer cells.
Design: Aggressive UCC-derived J82 cells were subjected to shRNA mediated knockdowns of hnRNPE1. The resultant changes in Vimentin intermediate filament (IF) organization was evaluated by immunostaining. siRNA directed against RICTOR was used to disrupt mTORC2. Consequent changes in hnRNPE1 phosphorylation, cell migration and Vimentin IF assembly were assessed.
Results: J82 cells exhibited constitutively high levels of hnRNPE1 phosphorylation, which was correlated with a pro-invasive (mesenchymal) phenotype. Knocking down hnRNPE1 to further promote EMT led to Vimentin IF reorganization into higher order filaments extending to the cell periphery. This is similar to what has been reported for the stabilization of microtentacles in circulating tumor cells (CTCs), which are critical for CTC aggregation and capillary endothelial reattachment. Conversely, disrupting mTORC2 led to a significant reduction in hnRNPE1 phosphorylation status. This was correlated with reduced cell migration and a perinuclear collapse of the Vimentin IFs.
Conclusions: Our results suggest a novel mechanism by which mTORC2 regulates cytoskeletal architecture and migration in metastatic bladder cancer cells and further validates mTORC2 as an important target for the treatment of UCCs.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 271, Wednesday Morning