Lamellipodia Of Cells Research Articles

Abstract 1276: Kindlin-2: a novel target of Parkin regulating cancer metastasis

Abstract Although targeted therapies in prostate cancer have clinically shown successful outcomes, resistance to these therapies and acquisition of lineage plasticity pose challenges for fully treating prostate cancer. Among several microscopic perspectives to solve these obstacles, much evidence has emerged indicating that mitochondria dynamics are closely associated with tumor metastasis and tumor progression in various types of cancer. The E3 ubiquitin ligase Parkin is associated with PINK1-mediated mitophagy, allowing cells to control mitochondria damages and generate the needed energy to survive in harsh physiological conditions. Interestingly, we found that Parkin potentially ubiquitinates proteins independently of PINK1-associated mitophagy. By SALIC analysis, we found that K681 and K582 of Kindlin-2 is one of the potential ubiquitination sites by Parkin in prostate cancer cells. Kindlin-2 is a cytoskeletal membrane protein and is a component of the focal adhesion complex. Computational modeling suggested that N-terminal region of Kindlin-2 binds to Parkin, with K581 and K582 of Kindlin-2 exposed outside for ubiquitination. Our studies showed that Kindlin-2 is ubiquitinated and degraded by wild-type Parkin but not by the catalytic site, C431S, mutant Parkin. Furthermore, K581A and K582A mutant Kindlin-2 did not exhibit poly-ubiquitination under conditions of Parkin overexpression. Surprisingly, in our immunofluorescence study, Kindlin-2 was recruited to the mitochondria and co-localized with Parkin. From a mitochondrial functional perspective, wild-type Kindlin-2 reduced mitochondria dynamics, whereas mutant Kindlin-2 didn’t in the presence of Parkin. This suggests that the cytoskeletal protein Kindlin-2 might play a role in mitochondrial dynamics that mediates tumor metastasis. Wild-type Kindlin-2 reduced both focal adhesion generation and decay, unlike mutant Kindlin-2 under Parkin-induced condition. Additionally, this pattern was observed in the single cell lamellipodia dynamics through live cell imaging. 2D and 3D cell invasive assays supported the evidence that Parkin decreases tumor metastatic potential by ubiquitinating Kindlin-2 and regulating mitochondria and focal adhesion dynamics. Our findings indicate that Parkin reduces tumor metastatic potential by ubiquitinating Kindlin-2 and demonstrate the possibility as a therapeutic agent for advanced and immune suppressive tumors. Furthermore, Parkin could be a promising agent for Parkin suppressed tumor patients in line with personalized cancer therapy development. Citation Format: Minjeong Yeon, Irene Bertolini, Dario C. Altieri. Kindlin-2: a novel target of Parkin regulating cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1276.

Protease-activated receptor 2 drives migration in a colon cancer cell line but not in noncancerous human epithelial cells.

The inflamed mucosa contains a complex assortment of proteases that may participate in wound healing or the development of inflammation-associated colon cancer. We sought to determine the role of protease-activated receptor 2 (PAR2) in epithelial wound healing in both untransformed and transformed colonic epithelial cells. Monolayers of primary epithelial cells derived from organoids cultivated from patient colonic biopsies and of the T84 colon cancer cell line were grown to confluence, wounded in the presence of a selective PAR2-activating peptide, and healing was visualized by live cell microscopy. Inhibitors of various signaling molecules were used to assess the relevant pathways responsible for wound healing. Activation of PAR2 induced an enhanced wound-healing response in T84 cells but not primary cells. The PAR2-enhanced wound-healing response was associated with the development of lamellipodia in cells at the wound edge, consistent with sheet migration. The response to PAR2 activation in T84 cells was completely dependent on Src kinase activity and partially dependent on Rac1 activity. The Src-associated signaling molecules, focal adhesion kinase, and epidermal growth factor receptor, which typically mediate wound-healing responses, were not involved in the PAR2 response. Experiments repeated in the presence of the inflammatory cytokines TNF and IFNγ revealed a synergistically enhanced PAR2 wound-healing response in T84s but not primary cells. The epithelial response to proteases may be different between primary and cancer cells and is accentuated in the presence of inflammatory cytokines. Our findings have implications for understanding epithelial restitution in the context of inflammatory bowel disease (IBD) and inflammation-associated colon cancer.NEW & NOTEWORTHY Protease-activated receptor 2 enhances wound healing in the T84 colon cancer cell line, but not in primary cells derived from patient biopsies, an effect that is synergistically enhanced in the presence of the inflammatory cytokines TNF and IFNγ.

Nanoscale visualization of the anti-tumor effect of a plasma-activated Ringer's lactate solution.

Plasma-activated Ringer's lactate solutions (PALs), which are Ringer's lactate solutions treated with non-thermal atmospheric-pressure plasma, have an anti-tumor effect and can be used for chemotherapy. As the anti-tumor effect of the PAL is influenced by the cell-treatment time, it is necessary to monitor the structural changes of the cell surface with non-invasive, nanoscale, and time-lapse imaging to understand the anti-tumor effect. In this study, to characterize the anti-tumor effect of the PAL, we used scanning ion conductance microscopy (SICM), using glass nanopipettes as probes, to visualize the structural changes of the cell surface. SICM time-lapse topographic imaging visualized a decrease in the movement of lamellipodia in normal cells and cancer cells after the PAL treatment. Furthermore, in normal cells, protrusive structures were observed on the cell surface. Time-lapse imaging using SICM allowed us to characterize the differences in the morphological changes between the normal and cancer cells upon exposure to the PAL.

Microtubules under mechanical pressure can breach dense actin networks.

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.

Role of Na+/Ca2+ Exchanger (NCX) in Glioblastoma Cell Migration (In Vitro).

Glioblastoma (GBM) is the most malignant form of primary brain tumor. It is characterized by the presence of highly invasive cancer cells infiltrating the brain by hijacking neuronal mechanisms and interacting with non-neuronal cell types, such as astrocytes and endothelial cells. To enter the interstitial space of the brain parenchyma, GBM cells significantly shrink their volume and extend the invadopodia and lamellipodia by modulating their membrane conductance repertoire. However, the changes in the compartment-specific ionic dynamics involved in this process are still not fully understood. Here, using noninvasive perforated patch-clamp and live imaging approaches on various GBM cell lines during a wound-healing assay, we demonstrate that the sodium-calcium exchanger (NCX) is highly expressed in the lamellipodia compartment, is functionally active during GBM cell migration, and correlates with the overexpression of large conductance K+ channel (BK) potassium channels. Furthermore, a NCX blockade impairs lamellipodia formation and maintenance, as well as GBM cell migration. In conclusion, the functional expression of the NCX in the lamellipodia of GBM cells at the migrating front is a conditio sine qua non for the invasion strategy of these malignant cells and thus represents a potential target for brain tumor treatment.

Cell shape characterization, alignment and comparison using FlowShape.

The shape of a cell is tightly controlled, and reflects important processes including actomyosin activity, adhesion properties, cell differentiation and polarization. Hence, it is informative to link cell shape to genetic and other perturbations. However, most currently used cell shape descriptors capture only simple geometric features such as volume and sphericity. We propose FlowShape, a new framework to study cell shapes in a complete and generic way. In our framework a cell shape is represented by measuring the curvature of the shape and mapping it onto a sphere in a conformal manner. This single function on the sphere is next approximated by a series expansion: the spherical harmonics decomposition. The decomposition facilitates many analyses, including shape alignment and statistical cell shape comparison. The new tool is applied to perform a complete, generic analysis of cell shapes, using the early Caenorhabditis elegans embryo as a model case. We distinguish and characterize the cells at the seven-cell stage. Next, a filter is designed to identify protrusions on the cell shape to highlight lamellipodia in cells. Further, the framework is used to identify any shape changes following a gene knockdown of the Wnt pathway. Cells are first optimally aligned using the fast Fourier transform, followed by calculating an average shape. Shape differences between conditions are next quantified and compared to an empirical distribution. Finally, we put forward a highly performant implementation of the core algorithm, as well as routines to characterize, align and compare cell shapes, through the open-source software package FlowShape. The data and code needed to recreate the results are freely available at https://doi.org/10.5281/zenodo.7778752. The most recent version of the software is maintained at https://bitbucket.org/pgmsembryogenesis/flowshape/. Supplementary data are available at Bioinformatics online.

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure.

Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.

Rapid lamellipodial responses by neighbor cells drive epithelial sealing in response to pyroptotic cell death.

Cell injury poses a substantial challenge for epithelia homeostasis. Several cellular processes preserve epithelial barriers in response to apoptosis, but less is known about other forms of cell death, such as pyroptosis. Here we use an inducible caspase-1 system to analyze how colon epithelial monolayers respond to pyroptosis. We confirm that sporadic pyroptotic cells are physically eliminated from confluent monolayers by apical extrusion. This is accompanied by a transient defect in barrier function at the site of the pyroptotic cells. By visualizing cell shape changes and traction patterns in combination with cytoskeletal inhibitors, we show that rapid lamellipodial responses in the neighbor cells are responsible for correcting the leakage and resealing the barrier. Cell contractility is not required for this resealing response, in contrast to the response to apoptosis. Therefore, pyroptosis elicits a distinct homeostatic response from the epithelium that is driven by the stimulation of lamellipodia in neighbor cells.

ROBO1 Regulates the Junctional Actin Cytoskeleton of Pancreatic Tumor Cells Through the RhoA/ABI/Diaph1/NMII Pathway and Promotes Vasculogenic Mimicry

SLIT ligand-ROBO receptor signaling, originally identified as a pathway involved in axon guidance, reportedly has a metastasis-regulating function in several solid tumors including pancreatic ductal adenocarcinoma (PDAC). However, its exact role and the mechanism of action are poorly understood. Immunofluorescent analysis of the subcellular distribution revealed that ROBO1 localized at filopodia, lamellipodia, ruffles, and adherens junctions of PDAC cells, overlapping with dynamic F-actin structures. ROBO1 expression levels impacted on the formation of actin-rich protrusions and ROBO1 loss resulted in disorganized cell junction–associated F-actin and disturbed adherens junctions with immature morphology. ROBO1 acted through the RhoA/ABI/Diaph1/Non-muscle Myosin II axis. Furthermore, ROBO1 supported adherens junctions and filopodial alignment accompanying vasculogenic mimicry (VM), a pro-metastatic competence of PDAC cells. Accordingly, expression of a VM gene signature (which includes RhoA) positively correlated with ROBO1 expression in tumor cells isolated from PDAC patients, validating its potential relevance in vivo. These results demonstrate that ROBO1 regulates actin cytoskeletal organization at tumor cell peripheries, thereby modulating cell-cell contacts and VM formation in pancreatic cancer.

Regulation of Ras homolog family member G by microRNA-124 regulates proliferation and migration of human retinal pigment epithelial cells

Uncontrolled retinal pigment epithelial (RPE) cell proliferation/migration contribute to the pathological tractional membrane development in proliferative vitreoretinopathy. Recent studies reported that microRNA (miR)-124 controls various cellular functions via the direct targeting of small Ras homolog family member G (RHOG). Therefore, we investigated the role of the neuron-specific miR-124 and RHOG in RPE cell proliferation/migration. Alterations in miR-124 and RhoG expression, as per cell confluence were evaluated through quantitative real-time PCR and western blotting, respectively. After transfection with miR-124, we quantified RPE cell viability and migration and observed cell polarization and lamellipodia protrusions. We evaluated the expression of RHOG/RAC1 pathway molecules in miR-124-transfected RPE cells. Endogenous miR-124 expression increased proportionally to RPE cell density, but decreased after 100% confluence. Overexpression of miR-124 decreased cell viability and migration, BrdU incorporation, and Ki-67 expression. Inhibition of endogenous miR-124 expression promoted RPE cell migration. Transfection with miR-124 reduced cell polarization, lamellipodia protrusion, and RHOG mRNA 3′ untranslated region luciferase activity. Like miR-124 overexpression, RhoG knockdown decreased RPE cell viability, wound healing, and migration, and altered the expression of cell cycle regulators. These results suggest that miR-124 could be a therapeutic target to alleviate fibrovascular proliferation in retinal diseases by regulating RPE proliferation/migration via RHOG.

6-Gingerol suppresses tumor cell metastasis by increasing YAPser127 phosphorylation in renal cell carcinoma.

According to the World Health Organization, the incidence and mortality rates of renal cell carcinoma (RCC) are rapidly increasing worldwide. Serious side effects caused by immune therapy and resistance to targeted drug therapy are urgent clinical problems facing kidney treatment. There is increasing global interest in developing natural products with a reduced number of side effects as adjunctive therapeutic options for RCC. Ginger is a spice and herbal remedy used worldwide, and 6-gingerol is a major pharmacologically active ingredient in ginger. In our study, we found that 6-gingerol suppressed RCC cell migration and metastasis in vitro and in vivo. Moreover, reduction in MMP2, Slug, and Vimentin protein levels was observed following 6-gingerol treatment of 786-O and ACHN cells. Furthermore, we revealed the mechanisms underlying the ability of 6-gingerol to inhibit RCC cell migration and metastasis. 6-Gingerol increased yes-associated protein (YAP)ser127 phosphorylation and reduced YAP levels in cell nuclei. We also used a series of loss-of-function and gain-of-function experiments to support our results. Western blot results showed that MMP2, Slug, and Vimentin protein expression was downregulated in YAP-silenced cells and upregulated in YAP-overexpressing cells. Transwell data demonstrated that YAP suppressed RCC migration ability. Immunofluorescence images showed that 6-gingerol decreased YAP levels, leading to disordered F-actin and a reduction in cell lamellipodia. Overall, our results indicated that 6-gingerol is a potential antimetastatic compound for use in kidney therapy.

Abstract 4002: Adipose tissue supernatant of overweight ER positive breast cancer patients promotes migration of MCF-7 breast cancer cells

Abstract Introduction: Adipose tissue is a major component of the breast cancer microenvironment, and preclinical studies have suggested that adipose tissue might play a pivotal role in breast cancer progression. In this study we investigated the effect of adipose tissue from patients with ER positive breast cancer on migration of ER positive MCF-7 breast cancer cells. Materials and Methods: Adipose tissue samples were collected from patients with ER positive breast cancer undergoing a mastectomy. In total, adipose tissue samples from 3 normal weight (BMI: 18.5-24.9) and 7 overweight breast cancer patients (BMI: 25-29.9) were collected. Samples from the mastectomy specimens were minced in small pieces, incubated in RPMI-1640 and after 48 hours (h) the supernatant was harvested. The effect of adipose tissue supernatant on the migration of MCF-7 cells was monitored in real-time for 48 h with the xCELLigence cell invasion and migration plates expressed in delta cell index (DCI). In addition, effect of adipose tissue supernatant on migration was assessed by quantification of F-actin positive lamellipodia of MCF-7 cells in a scratch assay after 24 h. In all experiments, RPMI-1640 with 1% or 20% FCS was used as a negative or positive control respectively. Results: Supernatant of overweight patients significantly induced MCF-7 migration compared to supernatant of normal weight patients after 24 h (xCELLigence DCI: 1.22 ± 0.15 vs. 0.95 ± 0.19 (P=0.038)), 36 h (DCI 1.40 ± 0.21 vs. 0.91 ± 0.31 (P=0.019)) and 48 h (DCI 1.72 ± 0.23 vs. 0.98 ± 0.43 (P=0.007)). In addition, there was a significant difference between the overweight group and negative control at 36 h, and 48 h (DCI 1.40 ± 0.21 vs. 1.06 ± 0.12, P=0.038 and DCI 1.72 ± 0.23 vs. 1.11 ± 0.21, P=0.004 respectively). No significant difference between normal weight group and negative control was found at any of the time points. After 24 h MCF-7 cells incubated with supernatant of overweight patients developed significantly more F-actin positive lamellipodia compared to MCF-7 cells incubated with supernatant of normal weight patients (44.26 ± 3.26% vs. 25.47 ± 1.88% P<0.001). Conclusion: In this study, we showed that adipose tissue supernatant of patients with ER positive breast cancer can promote migration of MCF-7 breast cancer cells in a weight dependent manner. Whether this is the case in other breast cancer subtypes as well, and the potential clinical implication, is of interest for future studies. Citation Format: Jie Ma, Hetty Timmer-Bosscha, Paul M. Werker, Carolina P. Schröder, Marlous Arjaans. Adipose tissue supernatant of overweight ER positive breast cancer patients promotes migration of MCF-7 breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4002.

Extracellular vesicles in diabetes mellitus induce alterations in endothelial cell morphology and migration

BackgroundInflammation-related atherosclerotic peripheral vascular disease is a major end organ complication of diabetes mellitus that results in devastating morbidity and mortality. Extracellular vesicles (EVs) are nano-sized particles that contain molecular cargo and circulate in the blood. Here, we examined EV protein cargo from diabetic individuals and whether these EVs cause functional changes in endothelial cells.MethodsWe quantified inflammatory protein levels in plasma-derived EVs from a longitudinal cohort of euglycemic and diabetic individuals and used in vitro endothelial cell biological assays to assess the functional effects of these EVs with samples from a cross-sectional cohort.ResultsWe found several significant associations between EV inflammatory protein levels and diabetes status. The angiogenic factor, vascular endothelial growth factor A (VEGF-A), was associated with diabetes status in our longitudinal cohort. Those with diabetes mellitus had higher EV VEGF-A levels compared to euglycemic individuals. Additionally, EV levels of VEGF-A were significantly associated with homeostatic model assessment of insulin resistance (HOMA-IR) and β-cell function (HOMA-B). To test whether EVs with different inflammatory cargo can demonstrate different effects on endothelial cells, we performed cell migration and immunofluorescence assays. We observed that EVs from diabetic individuals increased cell lamellipodia formation and migration when compared to EVs from euglycemic individuals.ConclusionsHigher levels of inflammatory proteins were found in EVs from diabetic individuals. Our data implicate EVs as playing important roles in peripheral vascular disease that occur in individuals with diabetes mellitus and suggest that EVs may serve as an informative diagnostic tool for the disease.

Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.

Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin-binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex, an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell-edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.This article has an associated First Person interview with the first author of the paper.

Nε-Carboxymethyl-Lysine Negatively Regulates Foam Cell Migration via the Vav1/Rac1 Pathway.

Background Macrophage-derived foam cells play a central role in atherosclerosis, and their ultimate fate includes apoptosis, promotion of vascular inflammation, or migration to other tissues. Nε-Carboxymethyl-lysine (CML), the key active component of advanced glycation end products, induced foam cell formation and apoptosis. Previous studies have shown that the Vav1/Rac1 pathway affects the macrophage cytoskeleton and cell migration, but its role in the pathogenesis of diabetic atherosclerosis is unknown. Methods and Results In this study, we used anterior tibiofibular vascular samples from diabetic foot amputation patients and accident amputation patients, and histological and cytological tests were performed using a diabetic ApoE−/− mouse model and primary peritoneal macrophages, respectively. The results showed that the atherosclerotic plaques of diabetic foot amputation patients and diabetic ApoE−/− mice were larger than those of the control group. Inhibition of the Vav1/Rac1 pathway reduced vascular plaques and promoted the migration of macrophages to lymph nodes. Transwell and wound healing assays showed that the migratory ability of macrophage-derived foam cells was inhibited by CML. Cytoskeletal staining showed that advanced glycation end products inhibited the formation of lamellipodia in foam cells, and inhibition of the Vav1/Rac1 pathway restored the formation of lamellipodia. Conclusion CML inhibits the migration of foam cells from blood vessels via the Vav1/Rac1 pathway, and this process affects the formation of lamellipodia.

Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility variants modulate cellular plasticity via Ras and Akt signaling.

Voltage-gated potassium (K+) channel subfamily B member 1 (KCNB1, Kv2.1) and integrin-α5 form macromolecular complexes-named integrin-α5-KCNB1 complexes (IKCs)-in the human brain, but their function was poorly understood. Here we report that membrane depolarization triggered IKC intracellular signals mediated by small GTPases of the Ras subfamily and protein kinase B (Akt) to advance the development of filopodia and lamellipodia in Chinese hamster ovary cells, stimulate their motility, and enhance neurite outgrowth in mouse neuroblastoma Neuro2a cells. Five KCNB1 mutants (L211P, R312H G379R, G381R, and F416L) linked to severe infancy or early-onset epileptic encephalopathy exhibited markedly defective conduction. However, although L211P, G379R, and G381R normally engaged Ras/Akt and stimulated cell migration, R312H and F416L failed to activate Ras/Akt signaling and did not enhance cell migration. Taken together, these data suggest that IKCs modulate cellular plasticity via Ras and Akt signaling. As such, defective IKCs may cause epilepsy through mechanisms other than dysregulated excitability such as, for example, abnormal neuronal development and resulting synaptic connectivity.-Yu, W., Shin, M. R., Sesti, F. Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility variants modulate cellular plasticity via Ras and Akt signaling.

Aquaporin-2 and Na+ /H+ exchanger isoform 1 modulate the efficiency of renal cell migration.

Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin β1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na+ /H+ exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2-expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.

Myricetin inhibits migration and invasion of hepatocellular carcinoma MHCC97H cell line by inhibiting the EMT process.

The recurrence and metastasis of hepatocellular carcinoma (HCC) are a major concern in current research. Epithelial-mesenchymal transition (EMT) is the leading cause underlying the high mobility and invasiveness of tumor cells. Myricetin is a natural flavonol with various pharmacological activities. The effects of myricetin on the migration and invasion of HCC MHCC97H cells were evaluated in the present study. Wound healing, Transwell migration and invasion assays were used to examine cell migration and invasion. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to examine the expression of epithelial (E)-cadherin, neural (N)-cadherin and vimentin. The present study aimed to investigate the effects of myricetin on the migration and invasion of HCC MHCC97H cells. It was indicated that myricetin decreased the viability of MHCC97H cells in a concentration and time-dependent manner, and inhibited MHCC97H cells migration and invasion. As the concentration of myricetin increased, filopodia and lamellipodia in cells weakened and cells were arranged more closely. RT-qPCR and western blotting revealed that myricetin upregulated E-cadherin expression and downregulated N-cadherin. Collectively, the results of the present study demonstrate that myricetin may inhibit the migration and invasion of HCC MHCC97H cells by inhibiting the EMT process.

MA15.01 Cellular Prion Protein Transcriptionally Regulated by NFIL3 Enhances Lung Cancer Cell Lamellipodium Formation and Migration

MA15.01 Cellular Prion Protein Transcriptionally Regulated by NFIL3 Enhances Lung Cancer Cell Lamellipodium Formation and Migration

Cellular prion protein transcriptionally regulated by NFIL3 enhances lung cancer cell lamellipodium formation and migration through JNK signaling.

Tumor invasion and metastasis are the major causes of treatment failure and mortality in lung cancer patients. In this study, we identified a group of genes with differential expression in in situ and invasive lung adenocarcinoma tissues by expression profiling; among these genes we further characterized the association of the upregulation of PRNP, the gene encoding cellular Prion protein (PrPc), with lung adenocarcinoma invasiveness. Immunohistochemistry on clinical specimens showed an association of PrPc expression with invasive but not in situ lung adenocarcinoma. Consistently, the expression of PrPc was higher in the highly invasive than in the lowly invasive lung adenocarcinoma cell lines. Knockdown of PrPc expression in cultured lung adenocarcinoma cells decreased their lamellipodium formation, in vitro migration and invasion, and in vivo experimental lung metastasis. Phosphorylation of JNKs was found to correlate with PrPc expression and the inhibition of JNKs suppressed the PrPc-induced up-regulation of lamellipodium formation, cell migration, and invasion. Moreover, we identified the nuclear factor, interleukin 3 regulated (NFIL3) protein as a transcriptional activator of the PRNP promoter. Accordingly, NFIL3 promoted lung cancer cell migration and invasion in a PrPc-dependent manner. High NFIL3 expression in clinical specimens of lung adenocarcinoma was also associated with tumor invasiveness. Overall, our observations suggest that the NFIL3/PrPc axis, through regulating lamellipodium formation and cell mobility via JNK signaling, plays a critical role in lung cancer invasiveness and metastasis.