Rho-associated protein kinase inhibition promotes microtentacle formation in detached breast tumor cells through destabilization of the actin cortex

Abstract

The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with a higher risk of metastasis. Recent research has shown that microtubule-based cellular protrusions known as microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions that are formed in cells that are detached from the extracellular matrix and occur when physical forces generated by the outwardly expanding microtubule network overcome the contractile force of the actin cortex. Rho-associated protein kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, breast tumor cells in suspension are used to mimic detached CTCs and show that using ROCK inhibitors to destabilize the actin cortex promotes the formation of microtentacles in suspended cells and enhances reattachment of cells from suspension. Furthermore the use of ROCK isoform-specific silencing-RNA shows that inhibiting ROCK1 but not ROCK2 increases microtentacle formation and cell reattachment. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although Rho/ROCK pathway also regulates the microtubule network, this study shows that the increase in microtentacle frequency observed upon ROCK inhibition is not a result of enhanced microtubule stabilization. These results indicate that the Rho/ROCK pathway plays a role in regulating McTN formation through its effect on actin cortical integrity.