Migration Signaling Research Articles

Receptor tyrosine kinase c-Met controls the cytoskeleton from different endosomes via different pathways.

Receptor tyrosine kinases (RTKs) are increasingly recognized as having the capacity to signal post-internalization. Signalling outputs and/or duration, and subsequent cellular outcome, are thought to be distinct when emanating from endosomes compared with those from the plasma membrane. Here we show, in invasive, basal-like human breast cell models, that different mechanisms are engaged by the RTK c-Met in two different endosomes to control the actin cytoskeleton via the key migratory signal output Rac1. Despite an acute activation of Rac1 from peripheral endosomes (PEs), c-Met needs to traffic to a perinuclear endosome (PNE) to sustain Rac1 signalling, trigger optimal membrane ruffling, cell migration and invasion. Unexpectedly, in the PNE but not in the PE, PI3K and the Rac-GEF Vav2 are required. Thus we describe a novel endosomal signalling mechanism whereby one signal output, Rac1, is stimulated through distinct pathways by the same RTK depending on which endosome it is localized to in the cell.

544 Comprehensive Profiling of Plasma MicroRNAs Reveals Potential Biomarkers for Barrett's Esophagus and Esophageal Adenocarcinoma

Aim: To quantitatively assess complex cancer cell phenotypes after gene inhibition by RNAi interference (RNAi), and assess the effect of these distinct morphologies on cell viability and cell motility using semi-automated image-based high-throughput screening. Method: GOhTRT cells were seeded and treated with the siRNA Human Druggable Genome Library (Dharmacon) by reverse transfection. Cells immunostained for DNA, tubulin and actin were imaged with the InCell Analyzer 1000 and processed using the InCell Analyzer software, CellHTS2 and RNAither. Statistical z-score analysis was performed on the combined A-T metric (F-actin area-α-tubulin area). The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results:127 high confidence hits (Z-factor>2) was refined to six genes (RRM2, ITGB8, GPS1, SPRY1, NOL1, MYO9B) on the basis of distinct morphologies, reproducible metrics, and functional pathway analysis. In siRRM2 cells nuclear displacement (ND) and A-T area was 1.598±0.076; p <0.0001 and 285.70±35.48; p<0.05, respectively. siGPS1 cells had an ND and A-T area of 0.845±0.036; p =0.0369 and 266.201±25.629; p =0.0326, respectively. Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98%±3.097%, 75.78%±5.454% and 72.97%±5.463% (p =0.0022) respectively. No significant difference in cell viability existed for siGPS1 (0.9037 ± 0.06575; p = 0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), suggesting that wound closure is by virtue of migratory signalling and not proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492±0.02798; p <0.0001) and siMYO9B (0.4048±0.04663; p <0.0001) in comparison to siNT cells (1.046±0.07712). Discussion:In summary, this approach successfully identified genes regulating oesophageal cancer cell cytoskeletal remodelling and metastasis using in-vitro assays, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in oesophageal adenocarcinoma patients.

546 Genome-Wide Gene-Environment Interaction Analysis of Aspirin and Non-Steroidal Anti-Inflammatory Drug Use and Colorectal Cancer Identifies Loci At Chromosomes 12p12.3 and 15q25.2

Aim: To quantitatively assess complex cancer cell phenotypes after gene inhibition by RNAi interference (RNAi), and assess the effect of these distinct morphologies on cell viability and cell motility using semi-automated image-based high-throughput screening. Method: GOhTRT cells were seeded and treated with the siRNA Human Druggable Genome Library (Dharmacon) by reverse transfection. Cells immunostained for DNA, tubulin and actin were imaged with the InCell Analyzer 1000 and processed using the InCell Analyzer software, CellHTS2 and RNAither. Statistical z-score analysis was performed on the combined A-T metric (F-actin area-α-tubulin area). The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results:127 high confidence hits (Z-factor>2) was refined to six genes (RRM2, ITGB8, GPS1, SPRY1, NOL1, MYO9B) on the basis of distinct morphologies, reproducible metrics, and functional pathway analysis. In siRRM2 cells nuclear displacement (ND) and A-T area was 1.598±0.076; p <0.0001 and 285.70±35.48; p<0.05, respectively. siGPS1 cells had an ND and A-T area of 0.845±0.036; p =0.0369 and 266.201±25.629; p =0.0326, respectively. Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98%±3.097%, 75.78%±5.454% and 72.97%±5.463% (p =0.0022) respectively. No significant difference in cell viability existed for siGPS1 (0.9037 ± 0.06575; p = 0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), suggesting that wound closure is by virtue of migratory signalling and not proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492±0.02798; p <0.0001) and siMYO9B (0.4048±0.04663; p <0.0001) in comparison to siNT cells (1.046±0.07712). Discussion:In summary, this approach successfully identified genes regulating oesophageal cancer cell cytoskeletal remodelling and metastasis using in-vitro assays, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in oesophageal adenocarcinoma patients.

Plasminogen activator urokinase expression reveals TRAIL responsiveness and supports fractional survival of cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10/Apo2L) holds promise for cancer therapy as it induces apoptosis in a large variety of cancer cells while exerting negligible toxicity in normal ones. However, TRAIL can also induce proliferative and migratory signaling in cancer cells resistant to apoptosis induced by this cytokine. In that regard, the molecular mechanisms underlying the tumor selectivity of TRAIL and those balancing apoptosis versus survival remain largely elusive. We show here that high mRNA levels of PLAU, which encodes urokinase plasminogen activator (uPA), are characteristic of cancer cells with functional TRAIL signaling. Notably, decreasing uPA levels sensitized cancer cells to TRAIL, leading to markedly increased apoptosis. Mechanistic analyses revealed three molecular events taking place in uPA-depleted cells: reduced basal ERK1/2 prosurvival signaling, decreased preligand decoy receptor 2 (DcR2)-death receptor 5 (DR5) interaction and attenuated recruitment of DcR2 to the death-inducing signaling complex upon TRAIL challenge. These phenomena were accompanied by increased FADD and procaspase-8 recruitment and processing, thus guiding cells toward a caspase-dependent cell death that is largely independent of the intrinsic apoptosis pathway. Collectively, our results unveil PLAU mRNA levels as marker for the identification of TRAIL-responsive tumor cells and highlight a key role of uPA signaling in ‘apoptosis versus survival' decision-making processes upon TRAIL challenge.

WSX-1 Signalling Inhibits CD4+ T Cell Migration to the Liver during Malaria Infection by Repressing Chemokine-Independent Pathways

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4+ T cells in the liver of infected IL27R−/− (WSX-1−/−) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4+ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4+ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1−/− mice than infected WT mice, and hepatic CD4+ T cells from WSX-1−/− mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4+ T cells to the liver of WSX-1−/− mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4+ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4+ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.

Abstract A76: EGFR activation signaling cascade leads to phosphorylation on serine 732 of FAK and mitosis in a subset of epithelial ovarian cancer

Abstract The epidermal growth factor receptor (EGFR) is expressed in up to 70% of epithelial ovarian cancer (EOC) where it correlates with poor prognosis. Despite the evidence for EGFR expression in the majority of EOCs, to date a uniform picture of the biological and clinical consequences of EGFR expression and activation has not emerged. Indeed, treatment of EOC patients with anti-EGFR results in a very poor response and is not correlated to EGFR expression. We aimed to characterize EGFR-expressing EOCs in the aim to better define EGFR-expressing EOC subtypes. We have recently showed that co-expression as well as the concomitant presence of IL-6 and PAI-1 in EOC ascites could characterize a subset of EGFR-expressing EOCs with shorter progression-free survival after chemotherapy. In vitro we have also found that the EOC cell line resembling this subset of EOC is more sensitive to anti-EGFR compounds suggesting that the association of anti-EGFR agents with taxol and cisplatin could be more appropriate for treating this subset of EOCs. We are now further investigating EGFR signaling cascade in EOC cell lines not only in traditional 2D cultures but also in 3D culture. Indeed, we found that in EOC E-cadherin (cadh) may be still present at cell-cell contacts during tumor progression and that EOC cells present in the ascites preferentially express E-cadh at cell-cell contacts. Therefore, we have set up a 3D culture system, resembling the multicellular aggregates (MCAs) found in the ascites, by using a commercially available alginate matrix to which human cells do not attach. Immunoblotting on 3D EOC cell line showed a significant increase of E-cadh expression together with that of cytokeratin 8/18 and vimentin. Interestingly, in this system we found a tremendous increase of EGFR phosphorylation and that EGF stimulation induced EGFR phosphorylation and triggered PI3K/AKT and MEK/ERK activations. In line with these results, down-modulation of E-cadh expression by specific siRNA, was associated with a significant decrease of EGFR phosphorylation with consequent decrease of both AKT and MAPK phosphorylations. Interestingly, EGF stimulation led to phosphorylation (P-) of FAK on tyrosine residues and also on FAK at Ser-732 (FakSer732). In vivo, in E-cadh-expressing EOC cells present in the ascites P-FAKser732 was also present. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its level was inversely related to the level of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MT) of the spindle, biochemically associated with MT and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. In conclusion, we have identified that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MT. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling, but even in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of EOC cells in which the EGFR/MEK/ERK/CDK5 pathway is active. Partially supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) Citation Format: Antonella Tomassetti, Katia Rea, Marialuisa Sensi, Silvana Canevari. EGFR activation signaling cascade leads to phosphorylation on serine 732 of FAK and mitosis in a subset of epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A76.

EGFR/MEK/ERK/CDK5-dependent integrin-independent FAK phosphorylated on serine 732 contributes to microtubule depolymerization and mitosis in tumor cells

FAK is a non-receptor tyrosine kinase contributing to migration and proliferation downstream of integrin and/or growth factor receptor signaling of normal and malignant cells. In addition to well-characterized tyrosine phosphorylations, FAK is phosphorylated on several serines, whose role is not yet clarified. We observed that phosphorylated FAK on serine 732 (P-FAKSer732) is present at variable levels in vitro, in several melanoma, ovarian and thyroid tumor cell lines and in vivo, in tumor cells present in fresh ovarian cancer ascites. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its levels were inversely related to the levels of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MTs) of the spindle, biochemically associated with MTs and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. We also identified, for the first time, that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MTs. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling but also in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of tumor cells in which the EGFR/MEK/ERK/CDK5 pathway is active.

Intra-Tumoral Heterogeneity in Metastatic Potential and Survival Signaling between Iso-Clonal HCT116 and HCT116b Human Colon Carcinoma Cell Lines

BackgroundColorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).Materials and MethodsHCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.ResultsBoth HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.ConclusionsWe demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer.

Nox2 is required for macrophage chemotaxis towards CSF-1.

Macrophage migration and infiltration is an important first step in many pathophysiological processes, in particular inflammatory diseases. Redox modulation of the migratory signalling processes has been reported in endothelial cells, vascular smooth muscle cells and fibroblasts. However the redox modulation of the migratory process in macrophages and in particular that from the NADPH oxidase-2 (Nox2) dependent ROS has not been established. To investigate the potential role of Nox2 in the migratory response of macrophages, bone marrow derived macrophages were obtained from WT and NOX2 knockout mice (Nox2KO) and subjected to CSF-1 stimulation. We report here that loss of Nox2 expression in BMM resulted in a significant reduction in the CSF-1 induced spreading response suggesting that Nox2 can modulate cytoskeletal events. Moreover, Nox2KO BMMs were deficient in cellular displacement in the presence of CSF-1. More significantly, when challenged with a gradient of CSF-1, Nox2KO BMMs showed a complete loss of chemotaxis accompanied by a reduction in cell migration speed and directional migration persistence. These results point to a specific role for Nox2KO downstream of CSF-1 during the BMM migratory response. Indeed, we have further found that Nox2KO BMMs display a significant reduction in the levels of ERK1/2 phosphorylation following stimulation with CSF-1.Thus Nox2 is important in BMM cellular motion to CSF-1 stimulation and necessary for their directed migration towards a CSF-1 gradient, highlighting Nox2 dependent signalling as a potential anti-inflammatory target.

Abstract TP433: Free Leptin And Soluble Leptin Receptor: Novel Circulating Markers Of Carotid Plaque - Related Symptomatology And Potential Role In Plaque Stability (from The Opal-lille Carotid Endarterectomy Study)

Leptin, an adipocyte-derived cytokine with various effects on the cardiovascular system, circulates in a free biologically active form and a bound form to a soluble leptin receptor (sObR) involved in modulating leptin activity. The links between free leptin, sObR, human atherosclerotic carotid plaque phenotype remain unknown. In the present study, we investigated the potential links between serum/plaque free leptin, serum sObR, carotid plaque stability and related symptomatology in 146 consecutive patients (71 asymptomatic and 75 symptomatic) scheduled for carotid endarterectomy. Free leptin was assessed in serum (17.05±23.19 ng/mL, range 0.47-191) and carotid plaques (0.204±0.486 ng/mg protein) and sObR in serum (21.48±8.81 ng/ml). Serum free leptin concentrations were correlated negatively with serum sObR concentrations and positively with intra-plaque free leptin concentrations (p&lt;0.0001, r=0.491, p&lt;0.0001, r=0.726, respectively). Serum free leptin and sObR concentrations were independently associated with carotid artery symptomatology (ß=--0.048; 95%CI [-0.093;-0.002]; -0.102, 95% CI [-0.994;-0.026], respectively). Regarding plaque phenotype, histological examination of carotid endarterectomy specimens revealed plaque stability index (collagen to macrophages area ratio) and Vascular Smooth Muscle Cell (VSMC) content to be significantly higher in asymptomatic patients compared to symptomatic ones (2.1 (1.9-2.5) vs. 1.5 (1.1-1.8), p&lt; 0.0001; 39.2 (35.9-55.0) vs. 20.4 (17.8-26.3), p= 0.0003, respectively). When serum or plaque leptin levels were above median value, plaque stability index, plaque VSMC content, plaque VSMC proliferation index and ERK activation were significantly higher (p&lt; 0.02). Interestingly, via ERK pathway activation, at 20 ng/mL, leptin induced a migratory signal and higher collagen synthesis in human VSMC, followed by a proliferative response of VSMC at the concentration of 75 ng/mL. For the first time, we reported free leptin and sObR to be novel circulating markers of carotid plaque - related symptomatology. Free leptin could be an active player in carotid plaque stability via its effects on human VSMC.

PKB/Akt-Dependent Regulation of Cell Motility

The prosurvival activity of phosphoinositide 3 kinase (PI3K)/Akt (also known as protein kinase B, PKB) pathway has been investigated in great detail in human physiology and disease. Accumulating evidence is emerging that this signaling axis also actively engages with the migratory process in motile cells, including metastatic cancer cells. Interference with the role of PI3K/Akt-mediated cell motility impairs cellular development and attenuates malignant progression of cancer metastasis. Because metastasis is responsible for 90% of mortality in cancer patients, the acceleration of cancer cell spreading observed in association with hyperactivation of the PI3K pathway, triggered for example by chemotherapy/radiotherapy in the clinic, has heightened awareness of the conflict between "good drugs" and unfavorable effects. Here, we discuss recent studies on PI3K/Akt-regulated cell motility in both physiological and pathological settings, with the aim of a better understanding of how activities of the PI3K/Akt axis initiate and transmit "migratory signals" that stimulate cell movement. We focus in particular on its direct influence on cell migration and invasion, epithelial-mesenchymal transition, and cancer metastasis.

Pseudopodial and β-arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation

Metastatic cancer cells form pseudopodia (PD) to facilitate their migration. The proteinase-activated receptor-2 (PAR-2) transduces migratory signals from proteases, and it forms protein complexes with β-arrestin and other signalling molecules that are enriched in pseudopodia. More generally, however, pseudopodial regulation is poorly understood. Here, we purified the pseudopodial proteomes of breast cancer cells after activation of the endogenous PAR-2 and we combined gel-based approaches with label-free high-resolution mass spectrometry to identify proteins that accumulate at the pseudopodia upon PAR-2-mediated migration. We identified >410 proteins in the cell body and >380 in the pseudopodia upon PAR2 activation, of which 93 were enriched in the pseudopodia. One of the pathways strongly enriched in the PD was the clathrin-mediated endocytosis signalling pathway, highlighting the importance of the scaffolding function of β-arrestin in PAR-2 signalling via its endocytosis. We therefore immunoprecipitated β-arrestins, and with mass spectrometry we identified 418 novel putative interactors. These data revealed novel β-arrestin functions that specifically control PAR-2-regulated signalling in migrating breast cancer cells but also showed that some β-arrestin functions are universal between GPCRs and cell types. In conclusion, this study reveals novel proteins and signalling pathways potentially important for migration of breast cancer cells.

Phosphorylation of chemoattractant receptors regulates chemotaxis, actin re-organization, and signal-relay

SummaryMigratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.

Learning in motion: pericytes instruct migrating innate leukocytes

During sterile inflammation, emigrating leukocytes sequentially engage subsets of pericytes associated with blood vessels and acquire adhesive, migratory and survival signals.

Lymphocytes subset composition and modulation of sphingosie-1-phosphate mediated migration in multiple sclerosis patients treated with fingolimod or interferon-β

Lymphocytes strongly contribute to the pathology of multiple sclerosis (MS). Thereby, frequently activated or reactivated autoreactive lymphocytes differentiate into effector cells that enter the central nervous system (CNS) causing tissue inflammation and damage. In order to reach the CNS, migratory signals allowing the entry into the circulatory system are vital. Sphingosine-1-phosphate (S1P) is a key mediator of lymphocyte egress that binds to five S1P receptors (S1P1-5). The immunomodulatory drug fingolimod (FTY720, Gilenya) directly targets S1P receptors and inhibits lymphocyte egress, whereas the exact effector function of interferon-β (IFN-β) in MS therapy remains elusive. However, IFN-β is known to induce CD69 expression, which by direct interaction, downmodulates S1P1 surface expression preventing lymphocyte egress. Since the block of lymphocyte egress is not complete, we analyzed subset composition and migratory responsiveness towards S1P of the lymphocytes remaining in the circulation of MS patients treated with fingolimod or IFN-β. Here we show that fingolimod treatment increased the responsiveness of the remaining T-lymphocytes towards S1P and that B cell migration of IFN-β treated patients changed from weak to normal. We found changes in the subset composition and absolute numbers in both treatment groups. While fingolimod reduced the number of almost all analyzed subsets in the circulation IFN-β specifically reduced the numbers of marginal zone and memory B cells. In conclusion, we think that by reducing “memory” type lymphocytes in the circulation fingolimod and IFN-β impair reactivation of autoreactive cells. In addition, treatment might inhibit the egress of newly formed effector cells and selects for stronger S1P responsive cells. Taken together these effects contribute to an overall beneficial outcome.

A unified term for directed and undirected motility in collective cell invasion

In this paper, we develop mathematical models for collective cell motility. Initially we develop a model using a linear diffusion–advection type equation and fit the parameters to data from cell motility assays. This approach is helpful in classifying the results of certain cell motility assay experiments. In particular, this model can determine degrees of directed versus undirected collective cell motility. Next we develop a model using a nonlinear diffusion term that is able to capture in a unified way directed and undirected collective cell motility. One goal of this work is to demonstrate that the forms of collective cell motility seen in the scratch assays and possibly other systems of interest need not reference external and more complicated migratory signals such as chemotaxis, but rather could be based on quorum sensing alone, collectively represented as density-dependent diffusivity. As an application we apply the nonlinear diffusion approach to a problem in tumor cell invasion, noting that neither chemotaxis or haptotaxis are present in the system under consideration in this article.

Multiphoton intravital microscopy analysis of signaling in T cells reveals persistent NFAT activation during dynamic APC scanning. (121.21)

Abstract The Ca2+ dependent activation and nuclear shuttling of the cytoplasmic transcription factor NFAT is a hallmark of productive T cell receptor (TCR) triggering. However, the kinetics of NFAT activation and the relationship to the Ca2+ dependent migratory stop signal have not been explored in vivo. We used Multiphoton Intravital Microscopy (MP-IVM) to visualize the subcellular dynamics of a fluorescent reporter of NFAT signaling (NFAT-eGFP) in cytotoxic T cells during their antigen-dependent interactions with various APC in lymph nodes (LNs) and in tumor tissue in vivo. During interactions with peptide-loaded B cells representing optimal APC in LNs, t1/2 of NFAT nuclear translocation was 1 minute. Relocation to the cytoplasm upon contact cessation occurred with a slower t1/2 of ~20 minutes. During its delayed nuclear export, NFAT continued to drive transcription of IFN-gamma. We therefore hypothesized that NFAT may facilitate the integration of TCR signals received through serial APC contacts. To test this in vivo, we performed MP-IVM to visualize contacts between cytotoxic T cells and tumor cells, representing suboptimal APCs. We observed that among T cells with continuously nuclear NFAT, ~30% remained motile while engaging in transient, serial contacts with multiple tumor cells. Hence, NFAT slow off-kinetics may constitute a mechanism of short-term biochemical memory enabling the integration of intermittent TCR signaling into a continuous transcriptional response.

Abstract 3252: Divergence of activin and TGFβ-induced SMAD-independent signaling in colon cancer

Abstract Introduction: Activin and TGFβ are members of the TGFβ superfamily with growth suppressive and pro-migratory properties. While growth suppressive SMAD 2/3/4 signaling is shared, we have evidence of diverging SMAD-independent migratory signaling involving the CDK inhibitor p21. Here, we dissect the participation of respective mitogenic pathways in activin and TGFβ-specific SMAD-independent signaling in colon cancer. Methods: Colon cancer cell lines with and without SMAD4 were assessed for SMAD, PI3K and MEK pathway influences on activin or TGFβ signaling and associated effects using pharmacologic inhibition, knock-down, co-immunoprecipitation, Western blotting, and functional assays of migration. Results: Irrespective of SMAD4 status, activin-induced migration was PI3K-dependent and associated with PI3K-induced p21 downregulation, while TGFβ-induced migration was MEK1/2-dependent. Activin but not TGFβ lead to colocalization of ACVR1, ACVR2's primary binding partner, with p85, the regulatory subunit of PI3K, in an ACVR1-dependent SMAD-independent manner. Further, activin-induced p21 downregulation was PI3K dependent and associated with proteasomal degradation, while TGFβ stabilized p21 via MEK/ERK. Inhibition of proteasomal degradation of p21 lead to a decrease in migration with activin implicating p21 downregulation in activin-induced migration but not in TGFβ-induced migration. Conclusion: Activin and TGFβ diverge in their pro-migratory SMAD-independent signaling in colon cancer affecting distinct targets to include p21 and employing diverse mitogenic signals. Dissecting ligand-specific functions in colon cancer may be exploited for risk stratification or therapeutic intervention in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3252. doi:1538-7445.AM2012-3252

Uridine adenosine tetraphosphate (Up4A) is a strong inductor of smooth muscle cell migration via activation of the P2Y2 receptor and cross-communication to the PDGF receptor

The recently discovered dinucleotide uridine adenosine tetraphosphate (Up4A) was found in human plasma and characterized as endothelium-derived vasoconstrictive factor (EDCF). A further study revealed a positive correlation between Up4A and vascular smooth muscle cell (VSMC) proliferation. Due to the dominant role of migration in the formation of atherosclerotic lesions our aim was to investigate the migration stimulating potential of Up4A. Indeed, we found a strong chemoattractant effect of Up4A on VSMC by using a modified Boyden chamber. This migration dramatically depends on osteopontin secretion (OPN) revealed by the reduction of the migration signal down to 23% during simultaneous incubation with an OPN-blocking antibody. Due to inhibitory patterns using specific and unspecific purinoreceptor inhibitors, Up4A mediates it’s migratory signal mainly via the P2Y2. The signaling behind the receptor was investigated with luminex technique and revealed an activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway. By use of the specific PDGF receptor (PDGFR) inhibitor AG1296 and siRNA technique against PDGFR-β we found a strongly reduced migration signal after Up4A stimulation in the PDGFR-β knockdown cells compared to control cells.In this study, we present substantiate data that Up4A exhibits migration stimulating potential probably involving the signaling cascade of MEK1 and ERK1/2 as well as the matrix protein OPN. We further suggest that the initiation of the migration process occurs predominant through direct activation of the P2Y2 by Up4A and via transactivation of the PDGFR.

Sorafenib targets BCR kinases and blocks migratory and microenvironmental survival signals in CLL cells

Sorafenib targets BCR kinases and blocks migratory and microenvironmental survival signals in CLL cells