Bladder Cancer Invasion Is Mediated by Mammalian Target of Rapamycin Complex 2-Driven Regulation of Nitric Oxide and Invadopodia Formation

Bladder cancer invasion depends on mammalian target of rapamycin complex 2 (mTORC2) activity, although the downstream mTORC2 effectors that mediate this effect have not been fully defined. One potential downstream effector is the arginine derivative nitric oxide (NO). This study identified a stageassociated increase in the expression of the NO-generating enzymes endothelial NO synthase (eNOS) and inducible NOS (iNOS) in human bladder cancer. Reduction of NOS activity by pharmacologic inhibition or silencing of NOS enzymes reduced cancer cell invasion, with similar effects observed using the NO scavenger cobinamide. By contrast, enhanced invasion was seen with the NO donor DetaNONOate and an analog of the downstream NO second messenger cGMP. Next, NOS expression was evaluated in invadopodia, which are cellular protrusions that form the invasive tips of cancer cells. Invadopodia were enriched in both iNOS protein and mTORC2 activity, and invadopodia formation was increased by Deta-NONOate and decreased by cobinamide and ablation of mTORC2 activity. Additionally, mTORC2 increased expression of iNOS. Using a zebrafish model, injection of iNOS- or rictor-silenced cells reduced the frequency of bladder cancer cell metastasis in zebrafish. These results indicate that mTORC2 can mediate bladder cancer cell invasion through increased iNOS expression, resulting in increased NO and cGMP production in invadopodia and further propagation of invadopodia formation.