Yes / Ion channels regulate many aspects of cell physiology, including cell proliferation, motility, and migration, and aberrant expression and activity of ion channels is associated with various stages of tumor development, with K+ and Cl- channels now being considered the most active during tumorigenesis. Accordingly, emerging in vitro and preclinical studies have revealed that pharmacological manipulation of ion channel activity offers protection against several cancers. Merkel cell polyomavirus (MCPyV) is a major cause of Merkel cell carcinoma (MCC), primarily due to the expression of two early regulatory proteins termed small and large tumour antigens (ST and LT, respectively). Several molecular mechanisms have been attributed to MCPyVmediated cancer formation but thus far, no studies have investigated any potential link to cellular ion channels. Here we demonstrate that Cl- channel modulation can reduce MCPyV STinduced cell motility and invasiveness. Proteomic analysis revealed that MCPyV ST upregulates two Cl- channels; CLIC1 and CLIC4, which when silenced, inhibit MCPyV STinduced motility and invasiveness, implicating their function as critical to MCPyV-induced metastatic processes. Consistent with these data, we confirmed that CLIC1 and CLIC4 are upregulated in primary MCPyV-positive MCC patient samples. We therefore, for the first time, implicate cellular ion channels as a key host cell factor contributing to virus-mediated cellular transformation. Given the intense interest in ion channel modulating drugs for human disease, this highlights CLIC1 and CLIC4 activity as potential targets for MCPyV-induced MCC. / BBSRC DTP studentship (BB/J014443/1) and Royal Society University Research Fellowship to JM (UF100419)

Yes / Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated posttranslational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (Ki = 10 μM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers. / Yorkshire Cancer Research; EPSRC; Association for International Cancer Research; Jordanian Government PhD scholarship

Cells interact with their immediate fibrous extracellular matrix (ECM); alignment of which has been shown influence metastasis. Specifically, intra-vital imaging studies on cell invasion from tumor-matrix interface and wounds along aligned fibers describe invasion to occur as singular leader (tip) cells, or as collective mass of a few chain or multiple tip cells. Recapitulation of these behaviors in vitro promises to provide new insights in how, when and where cells get the stimulus to break cell-cell junctions and ensue invasion by migrating along aligned tracks. Using Spinneret based Tunable Engineered Parameters (STEP) technique, we fabricated precise layout of suspended fibers of varying diameters (300, 500 and 1000 nm) mimicking ECM dimensions, which were interfaced with cell monolayers to study invasion. We demonstrated that nanofiber diameter and their spacing were key determinants in cells to invade either as singularly, chains of few cells or multiple-chains collectively. Through time-lapse microscopy, we reported that singular cells exhibited a peculiar invasive behavior of recoiling analogous to release of a stretched rubber band; detachment speed of which was influenced with fiber diameter (250, 425 and 400 µm/hr on small, medium and large diameter fibers respectively). We found that cells initiated invasion by putting protrusion on fibers; dynamics of which we captured using a contrasting network of mismatched diameters deposited orthogonally. We found that vimentin, a key intermediate filament upregulated in cancer invasion localized within a protrusion only when the protrusion had widened at the base, signifying maturation. To develop a comprehensive picture of invasion, we also developed strategies to quantify migratory speeds and the forces exerted by cells on fibers. Finally, we extended our findings of cell invasion to report a new wound healing assay to examine gap closure. We found that gaps spanned by crosshatch network of fibers closed faster than those on parallel fibers and importantly, we reported that gaps of 375 µm or larger did not close over a 45-day period. In summary, the methods and novel findings detailed from this study can be extended to ask multiple sophisticated hypotheses in physiologically relevant phenomenon like wound healing, morphogenesis, and cancer metastasis. / Ph. D. / Disease phenomenon like cancer invasion and wound healing have a myriad of things in common including cell migration and the ability of cells to remodel their immediate environment. Often times these singular or group migratory events of cells are initiated and directed by peculiar cells called tip/leader cells that explore cellular environment and make room for migration. While research in the last few decades has yielded a tremendous wealth of information as to how biochemical factors influence their behavior, our understanding of how the biophysical properties of the environment affect their behavior is in its infancy. This lack of understanding can somewhat be attributed to the difficulty in fabricating mechanically welldefined substrate systems that can be tailored to recapture these invasive episodes in a controlled setting outside of a living organism. In this study, we utilize a novel Spinneret based Tunable Engineered Parameters (STEP) technique to fabricate mechanically tunable nanofibers that show close resemble to native cellular environment. Cells were made to interact with these fibers and it was shown for the first time that factors like fiber spacing, diameter and topography can significantly affect the types of leader cells and their trajectory. Furthermore, once these cells come out of the simulated tumors/wounds, their migratory behaviors were still affected by mechanical properties of the fibers. Similarly, we also showed for the first time that the ability of the gaps to close in simulated wound healing settings could be significantly dependent on size, shape, and properties of fibers. These findings offer a novel outlook to our current understanding of single and collective cell behavior and how the biophysical properties of the native cellular environment can affect these behaviors. This can not only expand our understanding of how this invasive episodes occur, but also help us come up with preventive measures to inhibit such episodes for a better prognosis of diseases like cancer and chronic wounds.

The mig-10 gene in C. elegans is required for proper axon guidance and/or cell migration of certain neurons during development. In mig-10 (ct41) mutant worms, there is incomplete migration of the anterior lateral microtubule cells (ALMs), hermaphrodite specific neuron (HSN), left coelomocyte cells (ccL), and canal associated neuron (CAN) (Manser and Wood, 1990). The mig-10 (ct41) mutation also causes axon guidance defects in the IL2 neurons, and it enhances unc-6 defects in the axon guidance of the anterior ventral microtubule cell (AVM) (Rusiecki, 1999; C. Quinn, personal communication). mig-10's function in axon guidance and neuronal migration is unknown, but is believed to be involved in a signal transduction pathway that uses a G-protein, such as ras. The two mig-10 transcripts discussed in this thesis, mig-10 A and mig-10 B, encode proteins that are similar to Grb-7 and Grb-10 proteins, which are also believed to function in a signal transduction pathway (Manser et al., 1997). One of these similarities is the presence of a proline-rich region, which may be used to bind another protein (Manser et al., 1997). The MIG-10 A protein has an additional proline region, compared to MIG-10 B, which may indicate that the MIG-10 A and B proteins are utilized in different cells, or at different developmental stages. As a first step in learning where MIG-10 is expressed, mig-10 (ct41) mutant worms containing a wild-type mig-10 B::GFP fusion were constructed. Rescue of the mutant phenotype would indicate that the expression pattern of the transgene was similar to that of the endogenous gene. As this experiment did not allow for rescue, even after integration of the construct, a strain of worms containing a mig-10 promoter::GFP transgene was used. Preliminary observations of this strain indicated that mig-10 is expressed in neuronal tissue. The AIY neurons were observed in wild-type and mig-10 (ct41) worms to determine if they are affected by the mig-10 mutation as previously reported (O. Hobert, personal communication). As no difference was detected, the AIYs were not used in any further experiments. In order to determine which cells require functional MIG-10 protein for the proper development/migration of neurons to occur, mig-10 (ct41) worms containing mec-3 promoter::mig-10 A or B cDNA transgenes were constructed. The mec-3 promoter drives expression of the mig-10 cDNA in the ALM neurons and other touch cells early in the development of the embryo. If these transgenes rescued the ALM migration defect, then mig-10 would be acting cell autonomously in ALM. Partial rescue was obtained, which may be due to the need for both of the mig-10 transcripts to be expressed in the same cell; alternatively, one or both transcripts may need to be expressed in a cell nonautonomous fashion in addition to being expressed cell autonomously. Low production of the rescuing protein, or expression of the protein at a later developmental stage than is needed for rescue to occur, may also have been the cause of the partial rescue. Future work in this area includes putting mig-10 promoter::mig-10 A or B cDNA in mig-10 (ct41) background to investigate if the different transcripts rescue different aspects of the mig-10 phenotype. The mig-10 A and mig-10 B cDNA constructs could also be expressed in the same worm in an attempt to correct for partial rescue that may be due to the lack of both MIG-10 proteins.

No description available.

BACKGROUND: Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration - some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces. RESULTS: Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour. CONCLUSION: For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

Surface interactions and reactivity are of critical importance in current biomedical technologies, for example, satisfactory cell attachment and long term viability are essential for optimal in vitro tissue culture and for successful implantation and stability of cardiovascular medical implants such as stents and grafts. To achieve this, the control of fundamental forces and the resulting molecular interactions between the relevant surface and absorbing or adhering species in the physiological system is compulsory. This work utilised the surface modification technique of Ultra-Violet/ Ozone to improve the polystyrene biocompatibility by oxidising the surface with additional polar oxygen functional groups without damaging the surface bulk property. UV/Ozone treatment utilised throughout this study produced controllable oxygen functional groups and led to an increase in surface atomic oxygen level to 41% on unwashed and 35% on washed polystyrene surfaces, washing resulted in the removal of low molecular weight oxidised materials. Surface energy was increased by the addition of oxygen functional groups with the combination of alcohol (C-OR), carbonyl (C=O) and carboxyl (O-C=O); Saturation state was reached after 300s of UV/Ozone treatment where no more oxygen functionalities were incorporated to the surface. Moreover, UV/ozone treatment did not show an effect on the surface roughness studied by atomic force microscopy. The biological responses of human endothelial umbilical vein cells (HUVECs) were studied at the different level of UV/Ozone treated surfaces. HUVEC adhesion, proliferation and migration were significantly improved by the treatment compared to untreated and tissue cultures plastics (TCPs). Among the levels of UV/ozone treatment studied, 120s and 180s were found to be the most effective and HUVEC proliferation did not seem to be affected by the high level of oxygen. Similarly, the surface oxygen level did not affect the migration over UV/Ozone treated over 60s. Hypoxic condition significantly increased HUVECs migration on UV/Ozone treated, TCPs and untreated surfaces compared to normoxia, the oxygen rich surface did not favour to HUVECs that underwent regulatory process to enable the cells to increase migration. Under laminar flow conditions, HUVECs did not only grow, proliferate and migrate but also showed standard responses on UV/Ozone treated polystyrene surface. A decrease in cell size was observed at all shear stress intensities studied (1 dyn/cm2, 9 dyn/cm2 and 25 dyn/cm2) and the decrease was more obvious at higher shear stress. High shear stress intensity also induced high cell turnovers, which may be related to air bubbles induced at high flow rate. The overall findings of this study clearly illustrate that UV/Ozone surface treatment can be applied on polystyrene to improve human endothelial cells functionalities in term of adhesion, proliferation and migration in both static and laminar flow environment.

Surgical intervention can result in endothelial denudation, driving growth factor-stimulated vascular smooth muscle cell (VSMC) migration towards the intima, leading to luminal narrowing and restenosis. Clinically approved PGI₂ analogues, including beraprost, activate the cyclic adenosine monophosphate (cAMP) signaling pathway to inhibit VSMC migration in vitro. This pathway is a potential therapeutic target, however the downstream proteins involved in the inhibitory effects of cAMP on migration remain unknown. The aims of this study were to determine the signalling pathways involved in inhibiting VSMC migration through cAMP downstream mediators, protein kinase A (PKA) and the more recently characterised exchange protein activated by cAMP (Epac), and delineate the mechanisms involved. In human saphenous vein VSMCs, Epac activation using an Epac analogue inhibited VSMC migration. Therapeutic concentrations of beraprost (1 nM) also resulted in an inhibition of VSMC migration. The use of fluorescence resonance energy transfer (FRET) confirmed 1 nM beraprost activated Epac, but not PKA. Epac is a guanine nucleotide exchange factor (GEF) for Rap1 thus Rap1 siRNA was used to inhibit the Epac pathway. This blocked the inhibitory effects of beraprost on VSMC migration. Epac1 was localised to the leading edge of migrating VSMCs. Another G-protein, RhoA, was investigated since it is essential for cell migration and is involved in several processes including actin regulation. Epac signaling inhibited PDGF-induced RhoA activation and disassembled F-actin at the leading edge, where Epac1 was previously located. This indicates that beraprost activated the Epac pathway, which inhibited RhoA to decrease VSMC migration. The clinical relevance of this study has discovered the mechanisms of Epac's inhibitory action on VSMC migration and this pathway could be targeted therapeutically to reduce restenosis. In the future the potential use of beraprost on a drug eluting stent might be beneficial to prevent restenosis formation following surgical intervention.

Caenorhabditis elegans is a powerful tool to study cellular migration and morphogenesis during organ development. During pharynx development, the dorsal gland cell, g1p, is born in the anterior aspect of the pharyngeal primordium and undergoes a form of morphogenesis called retrograde extension. egl-15, the single Fibroblast Growth Factor Receptor (FGFR) in C. elegans and ina-1, one of two α-integrin receptors, are both required for the proper extension or migration of g1p cell. Mutations in either egl-15 or ina-1 show similar gland cell over-migration defects where the gland cell body migrates past the terminal bulb and is located in proximity of the intestine. The kinase domain of EGL-15 was found to be required for migration and transgenic rescue strategies were used to determine the tissue of EGL-15 function. RNA interference was used to determine if egl-15 and ina-1 are functioning in the same pathway to regulate gland cell migration. / February 2015

La polarité planaire ou PCP est un processus dans lequel les cellules et des structures apicales sedéveloppent et s’orientent dans le plan de l’épithélium. Cette polarité est régulée par un ensemble deprotéines incluant Vangl2. Un mutant murin de Vangl2 nommé looptail présente une forme sévèred’absence de fermeture du tube neural. Des pertes de fonctions de Vangl2 sont également retrouvées chezdes enfants atteints de défaut de fermeture du tube neural. Vangl2 possède un motif d’interaction pour desdomaines PDZ et s’organise en protéine à quatre domaines transmembranaires. Malgré de nombreusesdonnées génétiques disponibles concernant la perte de fonction de Vangl2, beaucoup reste à faire pourélucider les mécanismes moléculaires impliqués. Le locus du gène vangl2 humain est localisé dans unerégion fréquemment réarrangée dans différents cancers incluant les cancers du sein. Nous nous sommesintéressés à l’expression de Vangl2 dans les cancers du sein. Nous montrons que Vangl2 est surexprimédans des cancers du sein de sous-type basal (cancers du sein agressifs et de mauvais pronostic) danslesquels le locus génétique de Vangl2 est significativement amplifié et où le niveau d’expression del’ARNm et de la protéine est également élevé. De plus, par des études in vitro et in vivo, nous démontronsle rôle de Vangl2 dans la croissance tumorale et dans les processus de migration cellulaire. Ces effets sontdépendants de son motif d’interaction aux domaines PDZ et sont dépendants de Scrib, une autre protéinede la PCP, étudiée dans l’équipe et impliquée dans les processus de migration cellulaire. Par desexpériences de gain et de perte de fonction dans des cellules de cancers du sein, nous démontrons quel’expression de Vangl2 régule la signalisation JNK via l’activité des GTPases Rac1 et Cdc42 importantespour les processus de réorganisation du cytosquelette d’actine. L’ensemble de nos données indique un rôlerégulateur de Vangl2 dans les processus de tumorigenèse l’impliquant dans la croissance tumorale, laprolifération et migration cellulaires. / Planar cell polarity (PCP) is a process by which cells and apical structures grow in a uniformorientation within the plane of the epithelium. This type of polarity is regulated by a set of “core” proteinsincluding Vangl2. A vangl2 mutant, known as looptail exhibits a severe form of neural tube defect.Mutations of vangl2 associated to neural tube defects were also described in human. Vangl2 possesses aPDZ binding motif and is potentially organized in a four transmembrane domain structure. While geneticdata has very well described the importance of Vangl2 in embryonic development, its molecular functionsare still unknown. The human vangl2 gene is localized in a region with frequent rearrangements involvedin multiple cancers, including breast cancer. We decided to explore the expression of Vangl2 in breastcancer and defined Vangl2 as a PCP “core” protein associated with a signature of poor prognosis basaltypebreast cancer. The Vangl2 genetic locus is amplified in breast cancers, and this amplificationcorrelates with high mRNA and protein levels. Moreover, in in vitro and in vivo studies, we demonstratethe role of Vangl2 in tumor growth and cell migration. These functions are dependent on its PDZ bindingmotif and implicates Scrib, a PCP protein containing PDZ domains and involved in cell migration. Finally,using gain-of-function and loss-of-function approaches in breast cancer cell lines, we demonstrate thatVangl2 modulates the JNK pathway activation via the Rac1 and Cdc42 GTPases which are important forcytoskeleton reorganization. Together, our data reveal that Vangl2 has a role in tumor growth, cellproliferation and cell migration.