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ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells.

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.

SUBMITTER: Bhandary L

PROVIDER: S-EPMC4467435 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells.

Bhandary Lekhana L Whipple Rebecca A RA Vitolo Michele I MI Charpentier Monica S MS Boggs Amanda E AE Chakrabarti Kristi R KR Thompson Keyata N KN Martin Stuart S SS

Oncotarget 20150301 8

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-associa ...[more]

PMID: 25749040

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Project description:Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

Project description:Aromatase inhibitors (AIs), such as letrozole, are considered as first-line treatment for estrogen receptor-positive breast cancer in postmenopausal women. Despite the successful use of letrozole, resistance to therapy, tumor relapse and metastasis remain principal causes of patient mortality. Although there is no therapy currently available for AI-resistant breast cancer, previous reports have demonstrated that AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and epithelial to mesenchymal transition (EMT). This suggests a convergence of EMT and cancer stem cells (CSCs) in endocrine resistance. The present study evaluated the contribution of mammospheres in letrozole-resistant breast cancer by characterizing mammospheres and their potential impact on cellular motility. Ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either letrozole-resistant MCF-7 cells (LTLT-Ca) or letrozole-sensitive MCF-7 cells (AC-1). Subsequently, intratumoral CSC marker expression was assessed by immunohistochemistry. The results indicated that LTLT-Ca tumors were CD44+/CD24+, while AC-1 tumors presented low CD44/CD24 expression. Since mammosphere formation depends on CSCs, both cell lines were cultured either adherently (2D) or as mammospheres (3D) to assess the CD44/CD24 protein expression profile. When 3D culturing both cell lines, higher expression levels of CD44 and CD24 were observed when compared with their adherent counterparts, with the most robust change observed in the LTLT-Ca cell line. To quantitate the breast cancer stem cell activity, mammosphere formation assays were performed, and the LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared with AC-1 cells. Additionally, targeted gene expression arrays were conducted to compare the LTLT-Ca 3D and 2D cells, revealing that LTLT-Ca 3D cells displayed decreased expression levels of genes involved in cell adhesion and tumor suppression (e. g., E-cadherin, caveolin 1 and β-catenin). To validate this finding, wound healing assays were performed, and LTLT-Ca mammospheres exhibited a 70% wound closure, whereas AC-1 mammospheres exhibited a 39% wound closure. Collectively, the present findings demonstrated a strong association between AI-resistant mammospheres and an increased propensity for migration, which may be indicative of a poor prognosis.

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ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells.

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ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells.

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