Migrastatics-Anti-metastatic and Anti-invasion Drugs: Promises and Challenges

Opinion Migrastatics— Anti-metastatic and Anti-invasion Drugs: Promises and Challenges Aneta Gandalovi9cová, 1,2 Daniel Rosel, 1,2 Michael Fernandes, 3 Pavel Veselý, 4 Petr Heneberg, 5 Vladimír  Cermák, 1,2 LubošPetruželka, 6 Sunil Kumar, 7 Victoria Sanz-Moreno, 8, *and Jan Brábek 1,2, * In solid cancers, invasion and metastasis account for more than 90% of mortality. However, in the current armory of anticancer therapies, a specific category of anti-invasion and antimetastatic drugs is missing. Here, we coin the term ‘migrastatics’for drugs interfering withall modesof cancer cell invasion and metastasis, to distinguish this class from conventional cytostatic drugs, which are mainly directed against cell proliferation. We define actin polymerization and contractility as target mechanisms for migrastatics, and review candidate migrastatic drugs. Critical assessment of these antimetastatic agents is warranted, because they may define new options for the treatment of solid cancers. Migrastatics [418_TD$DIFF]As Antimetastatic Drugs Cancer is characterized byabnormal cellular proliferationand thepotential tospread to other parts of the body. Hematologic malignancies involve the blood, bone marrow, and lymphatic system, andapredominantfeatureisuncontrolledclonalproliferation[1,2].Forthisreason,cytotoxicdrugs haveproventobeaneffectivetreatment(reviewedin[3]).Bycontrast,solidcancerisaccompanied by local invasion and metastasis [4]. Treatment of solid cancer should be complemented with drugs that inhibit the ability of cancer cells to invade through the extracellular matrix (ECM) and establish secondary tumors. Since mechanisms determining clonal proliferation, cell migration, and invasion are distinct, it is evident that drug discovery efforts should be dichotomized into antiproliferative strategies and those directed towards mechanisms related to motility, migration and/or invasion, and metastasis. This is important and relevant to translational therapies in solid cancer. Candidate drugs for solid tumors are still evaluated predominantly by their ability to induce tumorshrinkage.Progressioninsolidcanceris conventionallydefinedasanincreaseintumorsize, and, in a superficial sense, the equating of therapeutic efficacy with tumor shrinkage is understandable. However, tumor shrinkage is rarely absolute or sustained, and is not predictive of an antimetastaticeffect.Moreover,afocuson dimensiondetractsfromattentiontolocalinvasionand metastasis, which account for more than 90% of mortality [5]. The ability to invade and metastasize is a cancer hallmark, as defined by Weinberg and Hanahan [6]. According to Lazebnik [7], the gain of an invasive phenotype is the most important cancer feature and the one that distinguishes malignant from benign tumors. Most morbidity and mortality in solid cancer stem from metastases. Strikingly, this is not reflected in funding Trends Local invasion and metastasis, rather than clonal proliferation, are the dominant features of solid cancer. However, a specific category of antiinvasion and antimetastatic drugs is missing for [417_TD$DIFF]treatment of solid cancer We propose the term ‘migrastatics’for drugs interfering with all modes of cancer cell invasiveness and, consequently, with their ability to metastasize (e.g., inhibiting not only local invasion, but also extravasation and metastatic colonization). In solid cancer, drug resistance is the main cause of treatment failure, and is attributed to mutations of the target. Since targeting the cause, although academically desirable, may be futile, a pragmatic and near-term option is to move downstream, to common denominators of cell migration and/or invasion, such as actin polymerization and actomyosin-mediated contractility. 1 Department of Cell Biology, Charles University, Vini9cná 7, Prague, Czech Republic 2 Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (BIOCEV), Pru myslová 595, 25242, Vestec u Prahy, Czech Republic 3 Medbase, Chapel Hill, NC, USA Trends in Cancer, June 2017, Vol. 3, No. 6 http://dx.doi.org/10.1016/j.trecan.2017.04.008 391 © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). and efforts towards antimetastatic research (reviewed in [5]). To date, medicinal chemists continue to focus on antiproliferative agents because tumor shrinkage is a regulatory requirement for approval. However, this approach underestimates the effect on cancer invasion and, as a result, patients and oncologists bemoan the lack of antimetastatic drugs [4]. Here, we introduce the term ‘migrastatics’(from Latin ‘migrare’and Greek ‘statikos’) for drugs interfering with all modes of the invasion of cancer cells and, consequently, with their ability to metastasize. The term is used to emphasize a focus on the inhibition of local invasion and metastasis, and to define and distinguish this class from conventional cytostatic drugs that are mainly directed against cell proliferation. Here, we review mechanisms related to early steps in the process leading to cancer metastasis, namely motility, directed migration, and invasion of the transformed cancer cell. Furthermore, we provide examples of relevant natural products and a rationale for their role as migrastatic candidates. Recently identified synthetic migrastatics candidates are also discussed. To finish, we discuss toxicity and clinical implications of migrastatics. Requirements for the Implementation of Migrastatics For the successful establishment of migrastatics, two main requirements need to be considered: (i) fine-tuning regulations for the approval of anticancer drugs. An emphasis on antimetastatic effects (related mainly to the inhibition of cancer cell motility and invasiveness) will allow clinical evaluation of candidate drugs even in the absence of tumor shrinkage (a point addressed elsewhere [4,8]). A precedent has already been set with checkpoint inhibitors [9]; and (ii) large-scale testing of compound libraries as well as a search for new compounds to select drugs that display low toxicity and interfere with all modes of cancer cell motility in 3D systems and animal models. Although we propose here migrastatics as an independent class of drugs, it should be noted that there is ‘nothing new under the sun’. In broad evolutionary terms, antimigratory and/or antiinvasive mechanisms are likely to have evolved as defensive measures, and migrastatics may be produced by several species of animals, plants, and microorganisms. Understandably, toxicity is a key concern with botanical product-derived candidates, and bioassay-guided fractionation of promising natural products has been helpful to identify promising pharmacophores [10]. Recent medicinal chemistry efforts based on cell biology have now defined attractive candidates for drug development [11]. Cancer Cell Invasion: A Target in Antimetastatic Intervention During dissemination from a primary tumor, cancer cells invade the ECM most commonly in clusters or as sheets [12], which is referred to as ‘collective migration’. This requires proteolytic degradation at the leading edge of the invasive front and cell contractility in the following cells [13]. Alternatively, single cancer cells can detach and invade using protease-dependent mesenchymal migration or protease-independent amoeboid migration, or a combination of both (Figure 1). Furthermore, many cancer cells can actively switch between these invasion modes in response to changes in the surrounding environment and/or to escape therapy (reviewed in [14–16]). For example, the use of matrix metalloprotease inhibitors can arrest mesenchymal migration, but does not halt invasion in general, because cells can undergo the mesenchymal-amoeboid transition (MAT) and switch to protease-independent invasion [17]. Furthermore, MAT was observed after enhancing cell contractility or in loose cell ECM [18,19]. The opposite process, the amoeboid-mesenchymal transition (AMT), can be induced by upregulating Rac activity, which decreases contractility [20]. 4 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic 5 Charles University, Department of Internal Medicine, Third Faculty of Medicine, Prague, Czech Republic 6 Department of Oncology, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic 7 Ayurveda Molecular Modeling, Hyderabad, Telangana, India 8 Tumor Plasticity Laboratory, Randall Division of Cell and Molecular Biophysics, Guy’s Campus, King’s College London, London, UK *Correspondence: [email protected] (V. Sanz-Moreno) and [email protected] (J. Brábek). 392 Trends in Cancer, June 2017, Vol. 3, No. 6 The plasticity of cancer cell invasion is further promoted by interactions within the tumor stroma, where noncancer cells contribute to signaling circuits regulating invasion. For example, tumorassociated macrophages (TAMs) produce proinvasive cytokines that not only affect invasion directly, but also sustain the cancer-associated phenotype of proximal fibroblasts (reviewed in [21]), which realign fibers of the ECM to facilitate cancer cell invasion (Figure 1). An obvious follow-up question is which molecular mechanisms should be targeted by migrastatics? Ideally, it should be those mechanisms that are common and essential for the motility of all migrating cancer cells derived from solid tumors (Figure 2, Key Figure). Signaling pathways regulating cell migration are highly redundant and inhibition of a single pathway leads almost inevitably to resistance (reviewed in [22]). In fact, resistance itself may explain failures in targeting key, genetically stable mechanisms, since many intracellular signaling processes are redundant. Thus, while precise targeting of suspect pathways is possible, it is unlikely to be successful (Figure 2). Accordingly, we propose that migrastatics should target the ultimate downstream effector mechanisms of cell migration, such as actin polymerization and contractility, which are difficult to bypass. It is unlikely that cancer cells will be able to substitute actin polymerization or develop an alternative contractile apparatus. Actin polymerization and contractility satisfy the Fiber alignment CAF TAM MAT AMT Proinflammatory signaling Cancer cell invasion Reduced fiber alignment + Migrastacs – Migrastacs RhoA/ROCK RhoA/ROCK RhoA/ROCK Collecve invasion Amoeboid Mesenchymal Mesenchymal Fibroblast Figure 1. The Plasticity of Cancer Cell Invasion. Cancer cells can invade either collectively or as individual cells when utilizing the amoeboid or mesenchymal invasion mode. Cells invading in one mode can undergo the mesenchymalamoeboid, or amoeboid-mesenchymal mode (MAT and AMT, respectively) in response to current conditions and signaling within the extracellular matrix (ECM). The plasticity of invasion is further regulated by interactions with noncancer cells that contribute to signaling circuits. Tumor-associated macrophages (TAMs) produce proinvasive cytokines that affect invasion directly and sustain the cancer-associated phenotype of proximal fibroblasts. These cancer-associated fibroblasts (CAFs) realign fibers of the ECM, which facilitates cancer cell invasion. The Rho/Rho-kinase (ROCK) pathway is crucial for many of these interactions and, thus, its inhibition downregulates cancer cell invasion (for more details, see the main text). Trends in Cancer, June 2017, Vol. 3, No. 6 393 requirement for ideal migrastatics targets because these processes are required by all invasion and/or 3D migration mechanisms irrespective of their protease dependence. In general, the migrating cancer cell is characterized by cellular shape rearrangements involving the formation of actin-based protrusions and new adhesions to surfaces, as well as cellular contractility, which is required for rear retraction and cell body translocation [23,24]. The actin cytoskeleton has a crucial role and undergoes constant reassembly during all these processes [25]. Actin also participates in the formation of specialized invasive structures, such as invadosomes, which are adhesive structures with proteolytic activity formed by mesenchymally migrating cells at the cell–ECM interface [26]. In cooperation with myosin motors, actin is the key resource for cellular contraction. Together, they form a meshwork that assembles into various structures, such as the contractile ring in the case of cytokinesis, sarcomeres in muscle cells, stress fibers and/or blebs in migrating cells, or bundles found at the cell cortex [27–29]. The main event regulating actomyosin contractility is the Rho-driven activation of Rho-kinase (ROCK), which directly phosphorylates myosin light chain (MLC) [30]. Furthermore, ROCK as well as myotonic dystrophy kinase-related CDC42binding kinase (MRCK), phosphorylate myosin light chain phosphatase (MLCP), which leads to its inactivation [31,32]. Phosphorylation of both MLC and MLCP results in increased levels of phosphorylated MLC, which promotes its ATPase activity, resulting in actomyosin contractility (Figure 3). Key Figure Target Mechanisms of Migrastatics. Migrastacs should target ulmate effector mechanisms to avoid resistance Extracellular smuli Integrins Glycoproteins RTKs GPCRs Others SFKs, MAPKs, PI3Ks, and others Small RHO GTPases: RhoA, Rac1, Cdc42, and others Contraclity acvators (rock, MRCK, and others) Actomyosin contraclity Acn Polymerizaon Invasion Acn polymerizaon regulators (wasp, limk, cofilin, formin, and others) ECM–cell signal transducon Intracellular pathways Small RHO GTPases Effectors Essenal mechanisms Signaling driving cell molity is highly redundant Inhibion of any pathway is bypassable, resulng in resistance Ulmate effector mechanisms are non-bypassable Migrastacs intervenon zone Figure 2. Cell invasion is affected by various extracellular stimuli and encompasses many signaling pathways that ultimately regulate actomyosin contractility and actin polymerization, which are two essential mechanisms driving cell migration. Since the signaling pathways regulating cell invasion are highly redundant, inhibition of any of these pathways can be overcome and will result in resistance, stemming from another signaling circuit by-passing the inhibited pathway. Thus, migrastatics should target the essential mechanisms (cell contractility and actin polymerization) to efficiently inhibit cell invasion. 394 Trends in Cancer, June 2017, Vol. 3, No. 6 The importance of the actin cytoskeleton during metastasis is reflected at the level of actinbinding proteins because many of these are deregulated in metastatic cells [33,34]. So far, the use of compounds targeting cytoskeletal dynamics has been neglected due to the abundance and importance of cytoskeletal components and possible adverse effects. However, the successful clinical use of microtubule-binding agents as anticancer drugs weakens this argument [35]. While the evaluation of some microtubule-binding agents has been discontinued because of significant toxicity, others have become drugs with crucial importance for cancer treatment, particularly vinca domain-binding agents (vincristine, vinblastine, vinorelbine, vindesine, and vinflunine) and taxol domain-binding agents (paclitaxel, docetaxel, and cabazitaxel) [35]. Moreover, natural products targeting the cytoskeleton as well as synthetic drugs deemed too potent to elicit therapeutic benefits can now be conjugated to an appropriate protein delivery system, thereby delivering highly cytotoxic and specific treatments to neoplastic tissue. Candidate Migrastatic Drugs Drugs Targeting Actin Polymerization and Function Whereas the actin cytoskeleton is a crucial component involved in cancer cell migration, agents targeting actin dynamics have been relatively poorly investigated (reviewed in [36]; see also [37,38]). Consequently, in vitro pharmacological tools are needed to selectively identify this type of agent [39]. These drugs can be categorized as compounds that destabilize the actin cytoskeleton (e.g., cytochalasins, geodiamolides, and latrunculins) and compounds that stabilize actin filaments, initiate deregulated polymerization, monomer depletion, and formation of large actin aggregates (e.g., jasplakinolide, chondramide, and cucurbitacin E) (Figure 4). Migrastatic drug candidates targeting actin polymerization and function, including evidence that these drugs effectively inhibit cancer cell invasion and/or metastasis, are discussed further below and in Table 1. Drugs Destabilizing Actin Cytoskeleton Cytochalasins are drugs interfering with actin polymerization characterized by a highly substituted perhydro-isoindolone structure that is attached to a macrocyclic ring. More than 60 HN N NH NH HN NH NH CI CI CI OH NH2 H2N N N N N N NN N N N O O O O S N H N H N HN H H N DJ4 BDP5290 RKI-18 CCT129254 AT13148 MRCK Rock MLCP-P MLCP-P Actomyosin contraclity MLCP MLC MLC Figure 3. Regulators of Actomyosin Contractility Are Targets for Migrastatics. Rho-kinase (ROCK) mediates the phosphorylation of myosin light chain (MLC) to directly enhance contractility. In addition, ROCK and myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) phosphorylate and, thus, inhibit MLC phosphatase (MLCP), which counteracts MLC phosphorylation. Thus, drugs targeting ROCK or MRCK are candidates for efficient migrastatics because they act to inhibit actomyosin contractility, which is necessary for of all cell invasion modes. Candidate drugs are depicted in blue, whereas enhancers of actomyosin contractility are in red. Trends in Cancer, June 2017, Vol. 3, No. 6 395 different cytochalasins from several species of fungi have been classified into various subgroups based on the size of the macrocyclic ring and the substituent of the perhydroisoindolyl1-one residue at the C-3 position [40]. Despite this diversity, only cytochalasins B and D have been extensively studied for their chemotherapeutic potential. Cytochalasin D was shown to not only inhibit invasion of AGS gastric cells, particularly after induction with LPA [41], and MDAMB-231 breast carcinoma cells [42], but also to promote pulmonary metastasis of B16 melanoma through the expression of tissue factor [43]. Many studies that have examined the anticancer activity of cytochalasins concentrated their efforts on cytochalasin B because it appears to be a safer and less toxic alternative to the more potent cytochalasin D [44]. The antimetastatic effects of Cytochalasin B have been well known since the late 1970s [45]. It was shown to inhibit the metastasis of mouse B16-F10 mouse melanoma cells [46] and Madison 109 mouse lung carcinoma cells [47]. In the latter, an immunosuppressive effect of cytochalasin B was observed, although the same group later showed that this immunosuppression could be completely abolished through the introduction of human recombinant interleukin-2 [48]. Geodiamolides are actin-targeting drugs that disrupt actin filaments and are derived from marine sponges. These compounds are cyclodepsipeptides and have the ability to potently stabilize actin fibers in a manner comparable with phalloidin; however, in contrast to phalloidin, they are freely cell permeable, rendering them exciting targets for drug development (reviewed in [49]). Geodiamolide H was shown to inhibit invasiveness of human breast cancer Hs578T cells when tested in vitro at concentrations of 60–120 nM [50]. Myosin inhibitor: blebbistan Amoeboid invasion Primary tumor Mesenchymal invasion Tropomyosin inhibitor: TR100 +TR100 Acn-destabilizing drugs: cytochalasins geodiamolide H lantrunculins Acn-stabilizing drugs: jasplakinolide chondramide cucurbitacin Kinase inhibitors: Y-27632; BDP5290 CCT129254; AT13148 +P Rock/MRCK PKB/PKA Figure 4. Potential Candidates for Migrastatics. Drugs targeting the actin cytoskeleton are suitable candidates for the inhibition of cell invasion because they impair both amoeboid and mesenchymal invasion. Chosen groups of migrastatic agents are depicted. Drugs interfering with actin dynamics include actin cytoskeletondestabilizing drugs (cytochalasins, latrunculins, and geodiamolide H) and actin filament-stabilizing drugs (jasplakinolide, chondramide, and cucurbitacin). TR100, a tropomyosin inhibitor, disrupts the actin cytoskeleton by affecting its stability. Other drugs target actomyosin contractility, such as blebbistatin (an inhibitor of nonmuscle myosin II) or inhibitors (e.g., Y-27632, BDP5290, CCT129254, or AT13148) that target kinases involved in the regulation of actomyosin contractility. The group of kinase inhibitors is emphasized because they have shown the potential to inhibit cell invasion in in vivo experiments. For more detail on certain drugs, refer to the main text. 396 Trends in Cancer, June 2017, Vol. 3, No. 6 Table 1. Selected [35_TD$DIFF] Q6 Migrastatic Candidates Structure Target Activity Models [36_TD$DIFF]Refs O O OOH N H S HO G-actin; interaction with thymosin b4 [37_TD$DIFF]>95% inhibition of invasiveness at 100 ng/mL[38_TD$DIFF];# invasiveness AMDC-S and AMDC-AS cell lines [53] Latrunculin A G3S1 cells [54] HN O O NO N H O O OH N H Actin #Invasiveness (<50% at 30 nM); #phosphorylation of MLC[39_TD$DIFF]2; #contractility MDA-MB-231 cells [40_TD$DIFF][62] Chondramide N O N N H N Tropomyosin EC 50 = 1.9 uM SK-MEL-28 cell line [68] EC 50 = 4.1 uM Melanoma cell lines [68] EC 50 = 2.8 uM Pediatric tumor cell lines [41_TD$DIFF][68] TR100 #Invasiveness Melanoma cell lines [68] N H N N HN H O ROCK1 IC 50 = 397 nM MDA-MB-231 cells [119] ROCK2 IC 50 = 349 nM RKI-18 #Invasiveness HN N NN H O Cl NH N N ROCK1 IC 50 = 230 nm MDA-MB-231 cells [120] EC 50 = 501 nm at 0–3mM ROCK2 IC 50 = 123 nm EC 50 = 447 nm at 0–3mM MRCKaIC 50 =10nm Ki = 10 nm MRCKbKi = 4 nm EC 50 = 166 nm at 0–3mM Trends in Cancer, June 2017, Vol. 3, No. 6 397 Latrunculins are microfilament-directed agents, also derived from marine sponges, that inhibit actin polymerization through the sequestration of G-actin monomers [51]. The compound structure is a 14or 16-membered macrolide base attached to a 2-thiazolidinone moiety [52]. Latrunculin A was found to inhibit the invasion of the tumorigenic AdoMetDC transformants of murine fibroblasts [53], the human breast cancer G3S1 cell line [54] and HeLa-O3 cells [55]. Latrunculin A and its derivatives, latrunculin A-17-O-carbamates, inhibited the invasiveness of human prostate cancer PC3 cells and T47D breast carcinoma cells [56]. Other semisynthetic derivatives of Latrunculin A (acetylated, esterified, and N-alkylated) exhibited anti-invasive effects against MDA-MB-231 cells [57]. Latrunculin A also inhibited the peritoneal dissemination of human gastric carcinoma MKN45 and NUGC-4 cells [58], making it a good candidate for a migrastatic drug against carcinoma cells. Table 1. (continued) Structure Target Activity Models [36_TD$DIFF]Refs BDP5290 #invasiveness; # phosphorylation of MLC N N H S ON H N [42_TD$DIFF]ROCK1 IC 50 = 5 nM NSCLC cell lines [121] ROCK2 IC 50 = 50 nM H522, MDA-MB231, and PANC1 cell lines [43_TD$DIFF][121] MRCKaIC 50 =10nM MRCKbIC 50 = 100 nM DJ4 Blocked recombinant MYPT1 and MLC phosphorylation at 5[44_TD$DIFF]mM; inhibited migration and invasiveness N NH O Cl H 2 NN N NH [45_TD$DIFF]ROCK I IC 50 = 214 nM Melanoma cell lines, mouse [46_TD$DIFF][112] ROCK II IC 50 = 141 nM AKT2 IC 50 = 2.2 nM #invasiveness; # metastasis; # phosphorylation of MLC2 and AKT [47_TD$DIFF]CCT129254 AGC kinases >70% inhibition at 1mM Cl N H N H2N OH [48_TD$DIFF]ROCK I IC 50 = 6 nM Melanoma cell lines, mouse [49_TD$DIFF][112] ROCK II IC 50 =4nM AKT1 IC 50 =38nM AKT2 IC 50 = 402 nM AKT3 IC 50 =50nM #Invasiveness; # phosphorylation of MLC2 and AKT [50_TD$DIFF]AT13148 AGC kinases >70% inhibition at 1mM 398 Trends in Cancer, June 2017, Vol. 3, No. 6 Drugs Stabilizing Actin Cytoskeleton Another actin-targeting drug derived from marine sponges is jasplakinolide, which promotes actin polymerization and stabilizes actin filaments. Its binding to F-actin is competitive with phalloidin [59]. Jasplakinolide is a cyclodepsipeptide containing a tripeptide moiety linked to a polypeptide chain [59]. It was found to reduce lung metastases of systemic Lewis lung carcinoma [60]. Chondramides are cyclodepsipeptides isolated from the myxobacterium Chondromycescrocatus crocatus [61]. Their binding to F-actin is competitive with phalloidin. Chondramides inhibit the invasion of human MDA-MB-231 breast carcinoma and inhibit metastasis of 4T1 breast carcinoma cells to the lung without acute toxicity [62], which supports their role as a migrastatic drug. Cucurbitacin E, a natural product of plants from the family Cucurbitaceae, inhibits the depolymerization of actin filaments by specifically binding to filamentous actin, forming a covalent bond at residue Cys257 [63]. In animal experiments, intraperitoneal administrations of cucurbitacin E significantly inhibited breast tumor metastasis to the lung without affecting apoptosis or proliferation of inoculated 4T1 and MDA-MB-231 breast cancer cells [64]. Drugs Targeting Contractility Actomyosin contractility is required for both cell deformability and rear retraction, key mechanisms in amoeboid and mesenchymal invasion, respectively (reviewed in [14,65];Figure 3). Accordingly, there is clear evidence for a role of ROCK/MRCK/MLC activation in enhancing tumor cell invasion and metastasis via direct effects on amoeboid or mesenchymal cancer cell invasion [66] and/or via indirect effects on cancer-associated fibroblasts to increase ECM stiffness and facilitate cancer cell movement [65,67] (Figure 1). As described in detail below, there is increasing evidence that inhibiting contractility chemically decreases cancer cell invasiveness and metastasis. Contractility targeting drugs can be categorized as inhibitors that target actin (chondramides), tropomyosin (TR100), myosin (blebbistatin), MLC kinase (MLCK) (ML-7 and ML-9), ROCK (e.g., fasudil, Y-27632, H-1152, Wf-536, RKI-1447, and RKI-18), MRCK (e.g., BDP5290), ROCK/ MRCK (e.g., DJ4) and ROCK/PKA/PKB (e.g., CCT129254 and AT13148) (Figure 4). Tropomyosin Inhibitors A novel class of anti-tropomyosin compounds has been developed that preferentially disrupt the actin cytoskeleton of tumor cells, thus impairing tumor cell motility. The lead compound, TR100, is effective in vitro and in vivo in reducing melanoma cell invasive outgrowth and tumor cell growth in neuroblastoma and melanoma models at a low micromolar range. Importantly, in testing for potential adverse effects of the treatment, TR100 was shown to have no adverse impact on cardiac structure and function in a mouse xenograft model [68], making it a good candidate for a migrastatic drug. Myosin Inhibitors Blebbistatin is a 1-phenyl-2-pyrrolidinone derivative capable of inhibiting non-muscle myosin II activity. It was shown to inhibit the invasiveness of pancreatic adenocarcinoma [69], mesenchymally invading BE human colon carcinoma cells and MDA-MB-231 human breast carcinoma cells [32], 501mel melanoma cells [70], 4T1 breast cancer cells [71], MCF7/6 breast cancer cells [72], A337/311RP rat and PR9692 avian sarcoma cells [66], and D54 glioblastoma cells [73]. However, no in vivo data are yet available for blebbistatin. Trends in Cancer, June 2017, Vol. 3, No. 6 399 104. de Toledo, M. et al. (2012) Cooperative anti-invasive effect of Cdc42/Rac1 activation and ROCK inhibition in SW620 colorectal cancer cells with elevated blebbing activity. PLoS One 7, e48344 105. An, L. et al. (2013) microRNA-124 inhibits migration and invasion by down-regulating ROCK1 in glioma. PLoS One 8, e69478 106. Zhang, L. et al. 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