Abstract
The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated 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, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy.
Highlights
Cytoskeletal rearrangements within tumor cells play important roles during cancer metastasis, which is the leading cause of patient mortality [1]
In order to assess the effects of inhibiting Rho-associated kinase (ROCK) in metastatic breast cancer cell lines, BT549 and Hs578T, the cells were treated with the compound Y-27632, an ATP competitive inhibitor of ROCK activity [32]
Cell migration and invasion are steps that are critical for metastasis and rely on Rho/ROCK-mediated cytoskeletal modifications and actomyosin contraction [41]
Summary
Cytoskeletal rearrangements within tumor cells play important roles during cancer metastasis, which is the leading cause of patient mortality [1]. Growth factor binding and/or integrin clustering activates Rho and ROCK resulting in increased cellular contraction and actin filament stabilization through a series of downstream phosphorylation events [4]. Increased bundling of actin by myosin as a result of Rho/ROCK activation leads to cellular contraction and formation of actomyosin stress fibers in the cytosol that terminate at the plasma membrane in focal adhesions [11]. In addition to modulating the actin cortex, the Rho/ ROCK signaling pathway has bifurcating roles in the regulation of microtubule stability [13]. While activation of Rho GTPase can increase stable detyrosinated microtubules through its downstream effector mDia [14], it can destabilize microtubules through ROCK mediated inactivation of tubulin-stabilizing protein tau [15] and inhibition of tubulin stabilizing posttranslational modification acetylation [16]. The microtubule cytoskeleton can in turn regulate Rho signaling since depolymerization of microtubules releases the microtubule-bound RhoGEF-H1, resulting in an increase in Rho-mediated stress fiber formation and actomyosin contractility [17, 18]
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