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Purpose: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved.Experimental Design: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model.Results: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell–cell contacts, altered cell–matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II.Conclusions: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions. Clin Cancer Res; 23(21); 6697–707. ©2017 AACR.

Wound healing in control conditions.

Evans Blue endothelial permeability assay shows leakage in the intestine of mice treated with dasatinib.

We introduce and use Wavelet Imaging on Multiple Scales (WIMS) as an improvement to fluorescence correlation spectroscopy to measure physical processes and features that occur across multiple length scales. In this study, wavelet transforms of cell images are used to characterize molecular dynamics at the cellular and subcellular levels (i.e. focal adhesions). We show the usefulness of the technique by applying WIMS to an image time series of a migrating osteosarcoma cell expressing fluorescently labelled adhesion proteins, which allows us to characterize different components of the cell ranging from optical resolution scale through to focal adhesion and whole cell size scales. Using WIMS we measured focal adhesion numbers, orientation and cell boundary velocities for retraction and protrusion. We also determine the internal dynamics of individual focal adhesions undergoing assembly, disassembly or elongation. Thus confirming as previously shown, WIMS reveals that the number of adhesions and the area of the protruding region of the cell are strongly correlated, establishing a correlation between protrusion size and adhesion dynamics. We also apply this technique to characterize the behavior of adhesions, actin and myosin in Chinese hamster ovary cells expressing a mutant form of myosin IIB (1935D) that displays decreased filament stability and impairs front-back cell polarity. We find separate populations of actin and myosin at each adhesion pole for both the mutant and wild type form. However, we find these populations move rapidly inwards toward one another in the mutant case in contrast to the cells that express wild type myosin IIB where those populations remain stationary. Results obtained with these two systems demonstrate how WIMS has the potential to reveal novel correlations between chosen parameters that belong to different scales, This work was supported by Natural Sciences and Engineering Research Council of Canada (www.nserc.ca) and Fonds de recherche du Québec ± Nature et technologies (www.frqnt.gouv.qc.ca)

et al., The orphan G protein-coupled receptor GPR55 has been directly or indirectly related to basic alterations that drive malignant growth: uncontrolled cancer cell proliferation, sustained angiogenesis, and cancer cell adhesion and migration. However, little is known about the involvement of this receptor in metastasis. Here, we show that elevated GPR55 expression in human tumors is associated with the aggressive basal/triple-negative breast cancer population, higher probability to develop metastases, and therefore poor patient prognosis. Activation of GPR55 by its proposed endogenous ligand lysophosphatidylinositol confers pro-invasive features on breast cancer cells both in vitro and in vivo. Specifically, this effect is elicited by coupling to G heterotrimeric proteins and the subsequent activation, through ERK, of the transcription factor ETV4/PEA3. Together, these data show that GPR55 promotes breast cancer metastasis, and supports the notion that this orphan receptor may constitute a new therapeutic target and potential biomarker in the highly aggressive triple-negative subtype., This work was supported by grants from Spanish Ministry of Economy and Competitiveness [PI11/00295 to CS, PI14/01101 to CS and EP-G, SAF2013-46183-R to MQ, and SAF2014-54705-R to MV-M, supported with European Regional Development (FEDER) funds] and Madrid Regional Government (S2010/BMD-2308 to MG, and 2010/ BMD-2359 to MQ). EPG was a recipient of a Postdoctoral Research Contract from Fundación Científica Asociación Española Contra el Cáncer and a Federation of the Societies of Biochemistry and Molecular Biology (FEBS) Short-term Fellowship. SB-B and SC-L are recipients of a Formación de Profesorado Universitario (FPU) fellowship and a Ramón y Cajal research contract, respectively, from the Spanish Ministry of Economy and Competitiveness.

[Purpose]: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved. [Experimental Design]: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model. [Results]: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell–cell contacts, altered cell–matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II. [Conclusions]: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions., A. Kreutzman is a Sigrid Jusélius postdoctoral fellow. Grants PI12/00494P and PI15/02085 from the Fondo de Investigaciones Sanitarias to C. Muñoz-Calleja supported this work. C. Muñoz-Calleja was co-financed by FEDER funds. M. Vicente-Manzanares is an investigator from the Ramon y Cajal Program (RYC-2010-06094) and is supported by grants SAF2014-54705-R from MINECO, Marie Curie CIG-293719 from the EU, CIVP16A1831 from Ramon Areces Foundation and the BBVA Foundation. S. Mustjoki is supported by the Finnish Cancer Institute, Academy of Finland, Sigrid Juselius Foundation and Finnish Cancer Associations.

The discovery of nonmuscle myosin II (NMII) is linked to the story of muscle contraction (reviewed in Szent-Gyorgyi 2004). It started in 1864,..., This work was funded by grant SAF2014-54705-R from MINECO. Miguel Vicente-Manzanares is a Ramon y Cajal Assistant Professor (RYC2010-06094).

Mechanical forces play fundamental roles in cellular processes related to movement. The major generator of mechanical forces in living systems is myosin II, which lies inside cells. Myosin II propels actin filament sliding, converting chemical energy into movement in various scales, from stress fibre formation and sliding to muscle contraction. In non-muscle cells, such forces are exerted at a cellular/subcellular scale and drive motility processes, e.g. cell migration, division, etc. There are several isoforms of myosin II, divided into muscle, smooth muscle and non-muscle. They all consist of a hexamer made of two heavy chains (MHCII) and four light chains, two structural (ELC) and two regulatory (RLC). The heavy chain displays three domains: a globular, actin- and ATP-binding domain, a supercoiled long domain that mediates dimerization of the MHCII; and a non-helical tail domain. Non-muscle myosin II (NMII) appears as three isoforms, II-A, II-B and II-C depending on the MHCII. Their ability to generate mechanical forces is context-dependent, thereby each isoform regulates specific cellular functions. In addition, every cell type displays a specific pattern of expression that may evolve over time, e.g. during embryogenesis, being crucial for proper development. Of the three isoforms, NMII-A is the fastest moving, although it can bear considerably less load than the other two. It displays fast assembly immediately behind the leading edge of migrating cells. Conversely, NMII-B and II-C are slower, but bear more load [1]. NMII-B assembles slowly in central, lateral and back regions of the cell and use pre-formed NMII-A-based filaments for their elongation. Selective depletion experiments have revealed specific cellular roles for these isoforms. NMII-A depletion impairs focal adhesion formation and retraction of the rear, whereas NMII-B depletion causes a marked loss of front-rear polarity in 2-D [2]. When not forming filaments, NMII is folded in a compact, assembly-incompetent conformation (10S) that extends by phosphorylation of the RLC on Ser19. The same phosphorylation triggers the conformational movement of the head of the heavy chain that enables actin filament movement [3]. Another level of regulation resides in the stability of the filaments that is controlled by the 10S-6S balance and the ability of the hexamers to form oligomers (mini-filaments) of 6-25 hexamers arrayed in an anti-parallel fashion [2]. A regulatory hotspot of the stability of the oligomers is the phosphorylation of the heavy chain. Unlike RLC phosphorylation, this is an isoform-specific mechanism. For example, the stability of the NMII-A filaments is controlled by phosphorylation of MHCII-A on S1943. Phospho-S1943 NMII-Amini-filaments are unstable, thus forming very small and weak filaments, and overall loss of cellular contraction [4]. Regarding NMII-B, S1935 is a novel phospho-regulatory residue that controls the dynamics of NMII-B filaments. When phosphorylated, NMII-B forms smaller mini-filaments that bear less load. In fact, assembled phospho-Ser1935 NMII-B behaves like assembled NMII-A, including its ability to nucleate mini-filaments de novo [5]. This motifis crucially important for the speed and load of the NMII isoforms, since its “transplantation” into the same spot in NMII-A endows it with NMII-B-like properties (larger filaments, higher load and decreased de novo mini-filament formation). S1935 is much closer to the domain-breaking Pro in NMII-B than S1943 is in NMII-A, suggesting different mechanisms that nevertheless have a similar outcome, i.e. decreased filament stability. In the case of NMII-A, S1943 phosphorylation regulates its interaction with S100A4/Mts1, which may control NMII-A assembly dynamics and stability [4]. We speculate that addition of a strong negative charge due to phosphorylation of S1935 next to the domain-breaking Pro (1933) compromises the stability of the bundled coiled-coil + non-helical domain pieces of NMII-B. As a consequence, filaments are less stably packed which may cause higher filament turnover disassembly. At a cellular level, a phosphomimetic mutation of S1935 impairs formation of the large actomyosin budles that define the backof the cell and a concomitant loss of directional polarity in 2-D, indicating that S1935 controls the formation of a stable directional rear that supports polarized migration [2]. The physiological implications of these mechanisms of regulation are just beginning to be elucidated. Phosphorylation of S1943 controls CD34+ hematopoietic stem cell fate [6]. Also, an inhibitory peptide from Met aminopeptidase-2 blocks the interaction of Mts1 with NMII-A, increasing NMII-A assembly and preventing tumor angiogenesis [7]. Whether S1935 phosphorylation directly controls the assembly of NMII-B or it does it by managing the interaction of NMII-B with accessory proteins remains to be shown. These studies suggest that the selective control of NMII assembly in an isoform-dependent manner is a central issue in processes involving symmetry breaking, motility and cell division.

Mechanotransduction encompasses the role of mechanical forces in controlling cell behavior by activating signal transduction pathways. Most forces at a cellular level are caused by myosin II, which contracts and cross-links actin. Myosin II-dependent forces are transmitted through the actin cytoskeleton to molecular endpoints that promote specific cellular outcomes, e.g., cell proliferation, adhesion, or migration. For example, most adhesive and migratory phenomena are mechanically linked by a molecular clutch comprised of mechanosensitive scaffolds. Myosin II activation and mechanosensitive molecular mechanisms are finely tuned and spatiotemporally integrated to coordinate morphogenetic events during development. Mechanical events dependent on myosin II also participate in tumor cell proliferation, invasion, and metastatic dissemination. Specifically, tumor cells alter the mechanical properties of the microenvironment to create favorable conditions for proliferation and/or dissemination. These observations position myosin II-dependent force generation and mechanotransduction at the crossroads between normal development and cancer.