Filopodia Formation Research Articles

Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end-directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end-directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.

Improved in vitro angiogenic behavior on anodized titanium dioxide nanotubes

BackgroundNeovascularization over dental implants is an imperative requisite to achieve successful osseointegration onto implanted materials. The aim of this study was to investigate the effects on in vitro angiogenesis of anodized 70 nm diameter TiO2 nanotubes (NTs) on Ti6Al4V alloy synthesized and disinfected by means of a novel, facile, antibacterial and cost-effective method using super oxidized water (SOW). We also evaluated the role of the surface roughness and chemical composition of materials of materials on angiogenesis.MethodsThe Ti6Al4V alloy and a commercially pure Ti were anodized using a solution constituted by SOW and fluoride as electrolyte. An acid-etched Ti6Al4V was evaluated to compare the effect of micro-surface roughness. Mirror-polished materials were used as control. Morphology, roughness, chemistry and wettability were assessed by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy, atomic force microscopy, energy dispersive X-ray spectroscopy (EDX) and using a professional digital camera. Bovine coronary artery endothelial cells (BCAECs) were seeded over the experimental surfaces for several incubation times. Cellular adhesion, proliferation and monolayer formation were evaluated by means of SEM. BCAEC viability, actin stress fibers and vinculin cellular organization, as well as the angiogenic receptors vascular endothelial growth factor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) were measured using fluorescence microscopy.ResultsThe anodization process significantly increased the roughness, wettability and thickness of the oxidized coating. EDX analysis demonstrated an increased oxygen (O) and decreased carbon (C) content on the NTs of both materials. Endothelial behavior was solidly supported and improved by the NTs (without significant differences between Ti and alloy), showing that endothelial viability, adhesion, proliferation, actin arrangement with vinculin expression and monolayer development were evidently stimulated on the nanostructured surface, also leading to increased activation of VEGFR2 and eNOS on Ti6Al4V-NTs compared to the control Ti6Al4V alloy. Although the rougher alloy promoted BCAECs viability and proliferation, filopodia formation was poor.ConclusionThe in vitro results suggest that 70 nm diameter NTs manufactured by anodization and cleaned using SOW promotes in vitro endothelial activity, which may improve in vivo angiogenesis supporting a faster clinical osseointegration process.

A novel role for the DNA repair gene Rad51 in Netrin-1 signalling

Mutations in RAD51 have recently been linked to human Congenital Mirror Movements (CMM), a developmental disorder of the motor system. The only gene previously linked to CMM encodes the Netrin-1 receptor DCC, which is important for formation of corticospinal and callosal axon tracts. Thus, we hypothesised that Rad51 has a novel role in Netrin-1-mediated axon development. In mouse primary motor cortex neurons, Rad51 protein was redistributed distally down the axon in response to Netrin-1, further suggesting a functional link between the two. We next manipulated Rad51 expression, and assessed Netrin-1 responsiveness. Rad51 siRNA knockdown exaggerated Netrin-1-mediated neurite branching and filopodia formation. RAD51 overexpression inhibited these responses, whereas overexpression of the CMM-linked R250Q mutation, a predicted loss-of-function, had no effect. Thus, Rad51 appears to negatively regulate Netrin-1 signalling. Finally, we examined whether Rad51 might operate by modulating the expression of the Unc5 family, known negative regulators of Netrin-1-responsiveness. Unc5b and Unc5c transcripts were downregulated in response to Rad51 knockdown, and upregulated with RAD51 overexpression, but not R250Q. Thus, Rad51 negatively regulates Netrin-1 signalling, at least in part, by modulating the expression of Unc5s. Imbalance of positive and negative influences is likely to lead to aberrant motor system development resulting in CMMs.

Myosin-X knockout is semi-lethal and demonstrates that myosin-X functions in neural tube closure, pigmentation, hyaloid vasculature regression, and filopodia formation

Myosin-X knockout is semi-lethal and demonstrates that myosin-X functions in neural tube closure, pigmentation, hyaloid vasculature regression, and filopodia formation

Distinct and redundant functions of Esama and VE-cadherin during vascular morphogenesis.

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and re-establish endothelial contacts, while allowing cell motility. We have compared the contribution of two endothelial cell-specific adhesion proteins, VE-cadherin (VE-cad/Cdh5) and Esama (endothelial cell-selective adhesion molecule a), during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appears similar to wild type, whereas esama-/-; ve-cad+/- and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, and display intrajunctional gaps, dislocalization of Zo-1 and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis, and both adhesion proteins are central to endothelial cell recognition during anastomosis.

Inhibitory Effects and Anti-Invasive Activities of Trehalose Liposomes on the Proliferation of Lung Carcinoma Cells

Inhibitory effects and anti-invasive activities of trehalose liposomes (DMTreC14) composed of L-α-dimyristoylphosphatidylcholine and α-D-glycopyranosyl-α-D-glucopiranoside monomyristate (TreC14) on the proliferation of human non-small cell lung carcinoma (A549) cells were examined in vitro. DMTreC14 with a hydrodynamic diameter less than 100 nm, were preserved for 4 weeks. The inhibitory effects of DMTreC14 on the proliferation of A549 cells accompanied with apoptosis were obtained. Enhancement of the accumulation of DMTreC14 into A549 membranes was observed. An increase in cellular membrane fluidity of A549 cell treated with DMTreC14 was observed on the basis of fluorescence depolarization method. DMTreC14 caused apoptosis for A549 cells through the activation of caspases and mitochondrial pathway. The anti-migration effects of DMTreC14 for A549 cells were observed through the inhibition of filopodia formation. The anti-invasion effects with the reduction of MMP-14 of DMTreC14 for A549 cells were obtained.

Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation.

Apoptosis Might Contribute to the Thrombocytopenia in Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a prothrombotic autoimmune complication of heparin therapy. Thrombocytopenia and thrombosis in HIT patients are caused by immune complexes containing pathogenic antibodies against platelet factor 4 (PF4)/glycosaminoglycan complexes. Mechanisms of platelet activation and/or destruction in HIT are not fully understood. Phosphatidylserine expression is a marker of platelet activation that contributes to the procoagulant function. On the other hand, phosphatidylserine expression is generally an early marker of cell apoptosis, which, similarly to other cells, controls platelet life span. The aim of this study was to investigate if apoptosis might play a role in HIT.Gel-filtered normal platelets were incubated for 15 or 60 min with recombinant PF4 (10 µg/ml) and KKO antibodies (50 µg/ml) and studied by electron microscopy and flow cytometry using fluorescently labeled markers of cell activation and apoptosis, such as annexin V, antibodies to CD62P (P-selectin) and MitoTracker DeepRed FM. Platelets and platelet-derived microparticles were identified by flow cytometry using labeled antibodies to CD41 and electron microscopy. Calcium ionophore A23187 (10 µM) was used as a positive control.Incubation of platelets with PF4+KKO caused fast expression of P-selectin on platelets comparable with calcium ionophore A23187 stimulation, suggesting that platelets were fully activated by PF4+KKO within 15 min, when they also started to produce CD41 and annexin-positive microparticles. Activation of platelets with PF4+KKO for 60 minutes led to a further increase in phosphatidylserine expression on their surface, with a time-dependent reduction of mitochondrial membrane potential, which reflects a disturbance of energy metabolism and is characteristic of cell apoptosis. Scanning electron microscopy showed that platelets treated with PF4+KKO or A23187, unlike untreated cells, displayed dramatic morphological changes with a loss of discoid shape, formation of filopodia, and microvesiculation. By transmission electron microscopy, the PF4+KKO-treated platelets had an irregular shape due to formation of plasma membrane invaginations and pseudopodia. Formation of an increasing number of intracellular vacuoles and enlargement of the lumen of the open canalicular system were observed. Some vacuoles contained various inclusions, such as secretory granules, membrane components, and grainy particles. The number of secretory granules in the PF4+KKO-treated cells was dramatically reduced. In all cases, formation of microparticles of various shapes and sizes was observed.These results indicate that the PF4-containing pathogenic immune complexes induce strong and time-dependent platelet activation leading to procoagulant microparticle formation that may contribute to thrombosis. At the same time, the results strongly suggest that the HIT-like immune complexes likely induce platelet apoptosis that can be an important mechanism of thrombocytopenia. DisclosuresNo relevant conflicts of interest to declare.

Central role of PI3K-SYK interaction in fibrinogen-induced lamellipodia and filopodia formation in platelets.

The WAVE complex‐1, a complex of WAVE, Abi1, NAP1, PIR121, HSPC300, RacGTP and Arp2/3 proteins, and WASP complex‐1, a complex of WASP, Cdc42, PIP2, and Arp2/3 proteins, are involved in lamellipodia and filopodia formation, respectively. It is known that the two complexes have opposite dynamics. Furthermore, Rac has two guanine nucleotide exchange factors, Vav and Sos, whose role in activating Rac is not well understood. In this work, by the construction of signaling network, analysis, and mathematical modeling, I show that Sos generates a pulse of WAVE complex‐1, decreasing the response time of WAVE complex‐1 formation upon the stimulation of platelets by fibrinogen. Furthermore, I also show that the dynamics of WAVE and WASP complexes depends on PI3K–SYK interaction. In the absence of this interaction, the WAVE complex‐1 does not form and the WASP complex‐1 remains at the initial, sustained level. Thus, I show the significance of the two protein/protein complexes: Sos and PI3K–SYK interaction, in fibrinogen‐induced lamellipodia and filopodia formation in platelets.

Effect of Polyelectrolyte Multilayers Assembled on Ordered Nanostructures on Adhesion of Human Fibroblasts.

Nanosphere lithography (NSL) and the layer-by-layer (LbL) technique are combined here for the first time to design a flexible system to achieve nanotopographical control of cell adhesion. NSL is used to generate regular patterns of tetrahedral gold nanodots of different size and distance. Besides the change in topography, LbL is used to generate a polyelectrolyte multilayer (PEM) system consisting of heparin (HEP) and poly(ethylene imine) (PEI) on top of the gold dots. The localized formation of PEM on gold dots is achieved by prior passivation of the surrounding silicon or glass surface. Properties of PEM are changed by adjusting the pH value of HEP solution to either acidic or alkaline values. Studies with human dermal fibroblasts (HDF) reveal that cells spread to a higher extent on PEM formed at pH 5.0 in dependence on the structure dimension. Further, filopodia formation is highly increased in cells on nanostructures exhibiting HEP as a terminal layer. The new system offers a great potential to guide stem cell differentiation in the future owing to its high degree of chemical and topographical heterogeneity.

Consequences of excessive plasticity in the hippocampus induced by perinatal asphyxia

Perinatal asphyxia (PA) is one of the most frequent risk factors for several neurodevelopmental disorders (NDDs) of presumed multifactorial etiology. Dysfunction of neuronal connectivity is thought to play a central role in the pathophysiology of NDDs. Because underlying causes of some NDDs begin before/during birth, we asked whether this clinical condition might affect accurate establishment of neural circuits in the hippocampus as a consequence of disturbed brain plasticity. We used a murine model that mimics the pathophysiological processes of perinatal asphyxia. Histological analyses of neurons (NeuN), dendrites (MAP-2), neurofilaments (NF-M/Hp) and correlative electron microscopy studies of dendritic spines were performed in Stratum radiatum of the hippocampal CA1 area after postnatal ontogenesis. Protein and mRNA analyses were achieved by Western blot and RT-qPCR. Behavioral tests were also carried out. NeuN abnormal staining and spine density were increased. RT-qPCR assays revealed a β-actin mRNA over-expression, while Western blot analysis showed higher β-actin protein levels in synaptosomal fractions in experimental group. M6a expression, protein involved in filopodium formation and synaptogenesis, was also increased. Furthermore, we found that PI3K/Akt/GSK3 pathway signaling, which is involved in synaptogenesis, was activated. Moreover, asphyctic animals showed habituation memory changes in the open field test. Our results suggest that abnormal synaptogenesis induced by PA as a consequence of excessive brain plasticity during brain development may contribute to the etiology of the NDDs. Consequences of this altered synaptic maturation can underlie some of the later behavioral deficits observed in NDDs.

Microglia contact induces synapse formation in developing somatosensory cortex.

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8–10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca2+ transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

The effect of the NMDA receptor-dependent signaling pathway on cell morphology and melanosome transfer in melanocytes

BackgroundThe pigmentation of skin and hair in mammals is driven by the intercellular transfer of melanosome from the melanocyte to surrounding keratinocytes However, the detailed molecular mechanism is still a subject of investigation. ObjectiveTo investigate the effects of N-methyl-d-aspartate (NMDA) receptor-dependent signaling pathway on melanocyte morphologic change and melanosome transfer between melanocytes and keratinocytes. MethodsThe expression and the intracellular distribution of NMDA receptor in human melanocyte were analyzed by Western blot and immunofluorescence staining. Melanocytes were treated with 100μM NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate] and 100μM NMDA receptor agonist NMDA, after which the morphological change of melanocyte dendrites and filopodias were observed by scanning electron microscope. The β-tubulin distribution and intracellular calcium concentration ([Ca2+]i) were observed by immunofluorescence staining and flow cytometry under the same treatment respectively. In addition, melanocytes and keratinocytes were co-cultured with or without treatment of MK-801, and the melanosome transfer efficacy were analyzed by flow cytometry. ResultsWe show that human epidermal melanocytes expresses NMDA receptor 1, one subtype of the ionotropic glutamate receptors (iGluRs). Stimulation with agonist of NMDA receptor increased the number of melanocyte filopodia. In contrast, blockage of NMDA receptor with antagonist decreased the number of melanocyte filopodia and this morphological change was accompanied by the disorganization of β-tubulin microfilaments in the intracellular cytoskeleton. In melanocyte-keratinocyte co-cultures, numerous melanocyte filopodia connect to keratinocyte plasma membranes; agonist of NMDA receptor exhibited an increased number of melanocyte filopodia attachments to keratinocyte, while antagonist of NMDA receptor led to a decreased. Moreover, antagonist of NMDA receptor decreased the intracellular calcium concentration in melanocytes and reduced the efficacy of melanosome transfer. ConclusionOur data suggest that filopodia delivery is the major mode of melanosome transfer between melanocytes and keratinocytes. NMDA drives melanosome transfer by promoting filopodia delivery and direct morphological effects on melanocytes, while MK-801 affects the intracellular β-tubulin redistribution and the filopodia delivery between melanocytes and keratinocytes. We hypothesize that NMDA receptor-dependent signaling is involved in melanosome transfer, which is associated with calcium influx, cytoskeleton protein redistribution, dendrites and filopodia formation. A thorough understanding of melanosome transfer is crucial for designing treatments for hyper- and hypo-pigmentary disorders of the skin.

Filopodia Formation and Proliferation of Mammalian Cells Are Increased on Closely Packed Silica Nanobeads

Filopodia Formation and Proliferation of Mammalian Cells Are Increased on Closely Packed Silica Nanobeads

Regulation of the actin cytoskeleton by the Ndel1-Tara complex is critical for cell migration.

Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement.

Abstract 4424: Acetylation of Fascin on the lysine 471 residue produces a change in growth and mobility phenotype of esophageal cancer cells by inducing F-actin rearrangement

Abstract Elevated expressions of fascin mRNA and protein in cancer cells have been correlated with aggressive clinical course, poor prognosis and shorter survival. Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is reported that the lysine 471 residue (K471) of fascin can be acetylated and also involves in its actin binding, but the roles of K471 acetylation in filopodia formation are still uncertain. In this paper, we produced point mutants mimicking the active deacetylated (Lys471Arg, K471R) or inactive acetylated (Lys471Gln, K471Q) states of fascin. The actin-binding assay showed that the fascin K471Q mutation decreased the actin binding activity of fascin, compared with wild-type fascin (WT) and K471R mutant fascin in the esophageal cancer cells. Moreover, live imaging revealed that K471Q mutant fascin reduced the number and length of filopodia, whereas the K471R mutant increased filopodia frequency. The fascin K471Q mutation changed the straight filopodia became forniciform filopodia. The protrusion presented abnormal morphology when cells spread. Further functional experiments showed that the K471Q mutant fascin rendered the tumor cell migration, invasion and tumorigenesis decrease significantly, whereas K471R mutant fascin like WT fascin still maintained a strong ability. Taken together, we propose that the lysine K471 residue may be a key specific actin crosslinking site and also involved in the tumor biological function of fascin. Citation Format: Fa-Min Zeng, Xiao-Ning Wang, Ying-Li Zhang, Lian-Di Liao, En-Min Li, Li-Yan Xu. Acetylation of Fascin on the lysine 471 residue produces a change in growth and mobility phenotype of esophageal cancer cells by inducing F-actin rearrangement. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4424.

Abstract 702: Serglycin promotes cell migration via modulating cytoskeleton reorganization and enhancing focal adhesion turnover

Abstract Serglycin (SRGN), a hematopoietic cell granule proteoglycan, has recently been shown to be overexpressed in several aggressive cancer types. In nasopharyngeal and hepatocellular carcinomas, elevated expression of SRGN was shown to correlate with poor prognosis. However, the molecular mechanisms underlying SRGN-mediated malignancies remain to be explored. In this study, we showed that SRGN is expressed at elevated levels in several lung cancer-derived cell lines. By gain-of-function and loss-of-function approaches, we showed that SRGN promoted NSCLC cell migration. SRGN modulated actin cytoskeleton reorganization and promoted lamellipodia and filopodia formation at the leading edge, facilitating a directional movement during wound closure in NSCLC cells. In consistence, increased levels of activated Rac1, which is required for lamellipodia formation, and CDC42, which is required for filopodia formation, were detected. Increased focal adhesion (FA) turnover, the process of continuous assembly and disassembly of adhesions, would lead to increased cell migration. We further showed that SRGN promoted Src activation, leading to increased phosphorylation of paxillin (PAX) at Y118 as well as reduced PAX/FAK complex formation, which were well-defined indicators of FA turnover. Taken together, these data suggested that SRGN promotes cell migration through inducing cytoskeleton reorganization and focal adhesion turnover. Citation Format: Jeou-Yuan Chen, Jing-You Guo. Serglycin promotes cell migration via modulating cytoskeleton reorganization and enhancing focal adhesion turnover. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 702.

Abstract 3799: The PAK4 allosteric modulator (KPT-9274) attenuates the growth of renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is an increasingly prevalent cancer type that is frequently asymptomatic on presentation and is associated with poor responses and resistance even to the current targeted therapies. Thus, novel therapeutic approaches to treat this disease are urgently needed. P21-activated kinase 4 (PAK4) is a mediator of filopodia formation and stabilizes β-catenin transcriptional activity, both of which are integral to nephrogenesis and cancer. We hypothesized that inhibitors of the PAK4 signaling pathway would result in salutary effects on RCC. To test this, we evaluated the in vitro response of several human RCC cells and normal kidney proximal epithelial cells (NHKs) to the specific PAK4 Allosteric Modulators (PAMs; KPT-8752 and KPT-9274). 786-O (VHL-mutant RCC) and caki-1 (VHL-wt RCC) cells showed decreases in cell viability (MTT), induction of apoptosis and arrest in G2/M phase when incubated with these inhibitors. These responses were diminished in NHK cells which served as a “normal” control cell line. Target and specific pathway proteins (phospho-PAK4, Phospho-β-catenin, c-Myc and cyclin D1) were reduced after RCC, but not NHK, were incubated with KPT-8752 and KPT-9274. To confirm specificity of the inhibitor to PAK4, all these responses were reproduced in RCC cells using specific PAK4 siRNA. Since ∼85% of RCC cases are associated with mutation in vhl we used 786-0 xenograft mouse model to evaluate the clinical candidate KPT-9274. KPT-9274 was orally administered at 100 and 200 mg/kg BIDX5 for 4 weeks, resulting in clear attenuation of tumor growth at both doses. There was no obvious change in the health or weight of any of the animals when compared to vehicle group suggesting manageable tolerability. We are currently evaluating combination therapy and plan to test this inhibitor on a metastatic RCC model. In summary, PAK4 inhibitors show considerable promise as novel treatments of RCC as a single agent and warrants further investigation. Citation Format: Robert H. Weiss, Omran Abu Aboud, Erkan Baloglu, William Senapedis, Sharon Shacham. The PAK4 allosteric modulator (KPT-9274) attenuates the growth of renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3799.

Platelet-rich plasma promotes the migration and invasion of synovial fibroblasts in patients with rheumatoid arthritis

Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets, and the number of platelets is correlated with rheumatoid activity. PRP is a concentrated source of autologous platelets, and contains several different growth factors and cytokines, including platelet‑derived growth factor, transforming growth factor‑β and insulin‑like growth factor‑1, which stimulate healing of bone and soft tissue. Rheumatoid arthritis (RA) is characterized by synovial hyperplasia, cell activation, articular inflammation and invasion of the synovium into the adjacent bone and cartilage. The adhesion of fibroblast‑like synoviocytes (FLSs) onto the extracellular matrix (ECM), migration and invasion are important for the erosion and destruction of the articular cartilage of patients with RA. The aim of the present study was to investigate the effects of PRP on the adhesion, migration and invasion of RA‑FLSs. Scratch and Transwell migration assays determined that PRP at a concentration of 2 and 5% significantly enhanced the migration ability of RA‑FLSs. Treatment of RA‑FLSs with 2and 5% PRP promoted the adhesion and invasion of the cells. Additionally, the immunofluorescence assay revealed that PRP induced a decrease in the number of centrally located stress fibers and led to an increase in the formation of filopodia and lamellipodia in the detectable leading edge protrusions in RA‑FLSs. In addition, reverse transcription‑quantitative polymerase chain reaction and western blot analysis determined that PRP upregulated the protein and mRNA expression levels of matrix metalloproteinase‑1 (MMP‑1). In conclusion, the promotion of RA‑FLS cell migration, invasion and adhesion on the ECM by PRP may be modulated through the upregulation of MMP‑1 expression and the induction of actin cytoskeletal reorganization.

Actin bundling by dynamin2 and cortactin is implicated in cell migration by stabilizing filopodia in human non-small cell lung carcinoma cells.

The endocytic protein dynamin participates in the formation of actin-based membrane protrusions such as podosomes, pseudopodia, and invadopodia, which facilitate cancer cell migration, invasion, and metastasis. However, the role of dynamin in the formation of actin-based membrane protrusions at the leading edge of cancer cells is unclear. In this study, we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F-actin bundles in filopodia of the lung cancer cell line H1299. Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation. Furthermore, dynamin 2 and cortactin mostly colocalized along F-actin bundles in filopodia of serum-stimulated H1299 cells by immunofluorescent and immunoelectron microscopy. Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum-stimulated H1299 cells, concomitant with a loss of F-actin bundles. Expression of wild-type cortactin rescued the punctate-like localization of dynamin 2 and filopodial formation. The incubation of dynamin 2 and cortactin with F-actin induced the formation of long and thick actin bundles, with these proteins colocalizing at F-actin bundles. A depolymerization assay revealed that dynamin 2 and cortactin increased the stability of F-actin bundles. These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F-actin bundles in filopodia. Taken together, these findings suggest that dynamin might be a possible molecular target for anticancer therapy.