Phosphorylation Of Paxillin Research Articles

IGF1R and Src inhibition induce synergistic cytotoxicity in HNSCC through inhibition of FAK

Head and neck cancer is the sixth most common cancer worldwide with a 5-year survival of only 65%. Targeting compensatory signaling pathways may improve therapeutic responses and combat resistance. Utilizing reverse phase protein arrays (RPPA) to assess the proteome and explore mechanisms of synergistic growth inhibition in HNSCC cell lines treated with IGF1R and Src inhibitors, BMS754807 and dasatinib, respectively, we identified focal adhesion signaling as a critical node. Focal Adhesion Kinase (FAK) and Paxillin phosphorylation were decreased as early as 15 min after treatment, and treatment with a FAK inhibitor, PF-562,271, was sufficient to decrease viability in vitro. Treatment of 3D spheroids demonstrated robust cytotoxicity suggesting that the combination of BMS754807 and dasatinib is effective in multiple experimental models. Furthermore, treatment with BMS754807 and dasatinib significantly decreased cell motility, migration, and invasion in multiple HNSCC cell lines. Most strikingly, treatment with BMS754807 and dasatinib, or a FAK inhibitor alone, significantly increased cleaved-PARP in human ex-vivo HNSCC patient tissues demonstrating a potential clinical utility for targeting FAK or the combined targeting of the IGF1R with Src. This ex-vivo result further confirms FAK as a vital signaling node of this combinatorial treatment and demonstrates therapeutic potential for targeting FAK in HNSCC patients.

Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbβ3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.

Molecular Basis of LH Action on Breast Cancer Cell Migration and Invasion via Kinase and Scaffold Proteins.

Breast cancer (BC) is a major public health problem affecting women worldwide. Approximately 80% of diagnosed cases are hormone-dependent breast cancers. These hormones are known to stimulate tumor development and progression. In this setting, tentative evidence suggests that luteinizing hormone (LH) may also play a role in tumors. In BC cells that express functional LH receptors (LHR), this hormone regulates cell migration and invasion by controlling several kinases that activate actin cytoskeletal proteins. In this article, we show that LH induces phosphorylation of paxillin and its translocation toward the plasmatic membrane, where focal adhesion complexes are assembled. This process is triggered via a rapid extra-gonadal LHR signaling to Src/FAK/paxillin, which results in the phosphorylation/activation of the nucleation promoter factors cortactin and N-WASP. As a consequence, Arp2/3 complexes induce actin polymerization, essential to promote cell adhesion, migration, and invasion, thus enhancing metastatic spread of tumoral cells. Our findings provide relevant information about how gonadotrophins exert their action in BC. This information helps us understand the extragonadal effects of LH on BC metastasis. It may provide new perspectives for therapeutic treatment, especially for women with high serum levels of gonadotrophins.

YYB-101, a Humanized Antihepatocyte Growth Factor Monoclonal Antibody, Inhibits Ovarian Cancer Cell Motility and Proliferation.

Hepatocyte growth factor (HGF) acts as a key regulator in promoting ovarian cancer metastasis. Previously, we observed that YYB-101, a humanized anti-HGF antibody, effectively inhibits ovarian cancer cell migration, invasion, and progression. Here, we evaluated the signaling mechanisms affected by YYB-101 that are important in ovarian cancer cell progression. Using cell migration, invasion and proliferation assays, we evaluated the effects of YYB-101 on A2780/luc and SKOV3 cells. The effects of YYB-101 on signaling molecules were determined by immunocytochemistry and immunoblot analysis. YYB-101 inhibited HGF-induced ovarian cancer cell motility by down-regulating paxillin phosphorylation and actin-cytoskeleton rearrangement. Also, YYB-101 inhibited ovarian cancer cell proliferation by reducing c-MET phosphorylation and activating apoptosis in vitro and in vivo. These effects were significantly enhanced by combining YYB-101 treatment with paclitaxel, a standard chemotherapy drug. YYB-101 can be examined as a new therapeutic agent for the treatment of patients with ovarian cancer.

Homeopathic Rhus toxicodendron Induces Cell Adhesions in the Mouse Pre-osteoblast Cell Line MC3T3-e1.

Rhus toxicodendron (R. tox) has been used as a homeopathic remedy for the treatment of inflammatory conditions. Previously, we reported that R. tox modulated inflammation in the mouse chondrocyte and pre-osteoblastic MC3T3-e1 cell line. During the inflammatory process, cells adhere to the extracellular matrix (ECM) and then migrate to the inflammation site. We examine here the process of cell adhesion in MC3T3-e1 cells after their stimulation with homeopathic R. tox. For the cell-substrate adhesion assay, the cultured MC3T3-e1 cells were trypsinized, starved for 1 h in serum-free media, and plated onto culture plates coated with fibronectin (FN), 30c R. tox or gelatin, respectively. The cells were allowed to adhere for 20 min incubation and unattached cells were washed out. Adherent cells were measured using the water-soluble tetrazolium salt-8 assay. The intracellular signals after stimulation of R. tox were examined by analyzing the tyrosine phosphorylation of focal adhesion kinase (FAK), Src kinase, and Paxillin using immunoblot assay. Formation of focal adhesion (FA, an integrin-containing multi-protein structure that forms between intracellular actin bundles and the ECM) was analyzed by immunocytochemistry using NIH ImageJ software. Cell adhesion increased after stimulation with R. tox (FN, 20.50%; R. tox, 44.80%; and gelatin, 17.11% vs. uncoated cells [control]). Tyrosine phosphorylation of FAK, Paxillin, and Src increased compared with that of gelatin when stimulated with R. tox. Additionally, R. tox-stimulated cells formed many FAs (number of FAs per cell, 35.82 ± 7.68) compared with gelatin-stimulated cells (number of FAs per cell, 19.80 ± 7.18) and exhibited extensive formation of actin stress fibers anchored by FAs formed at the cell periphery. Homeopathic R. tox promotes the formation of cell adhesions in vitro.

MAGI1 localizes to mature focal adhesion and modulates endothelial cell adhesion, migration and angiogenesis

ABSTRACT MAGI1 is an intracellular adaptor protein that stabilizes cell junctions and regulates epithelial and endothelial integrity. Here, we report that that in endothelial cells MAGI1 colocalizes with paxillin, β3-integrin, talin 1, tensin 3 and α-4-actinin at mature focal adhesions and actin stress fibers, and regulates their dynamics. Downregulation of MAGI1 reduces focal adhesion formation and maturation, cell spreading, actin stress fiber formation and RhoA/Rac1 activation. MAGI1 silencing increases phosphorylation of paxillin at Y118, an indicator of focal adhesion turnover. MAGI1 promotes integrin-dependent endothelial cells adhesion to ECM, reduces invasion and tubulogenesisin vitro and suppresses angiogenesis in vivo. Our results identify MAGI1 as anovel component of focal adhesions, and regulator of focal adhesion dynamics, cell adhesion, invasion and angiogenesis.

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166+ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166+ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.

Integrin and autocrine IGF2 pathways control fasting insulin secretion in β-cells

Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2–AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)–dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, β-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, β-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary β-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.

Paxillin mediates ATP-induced activation of P2X7 receptor and NLRP3 inflammasome

BackgroundExtracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K+, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions.ResultsWe show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K+ efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages.ConclusionsWe have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.

Effect of silica-coated magnetic nanoparticles on rigidity sensing of human embryonic kidney cells

BackgroundNanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)), even at 0.1 μg/μL. These protruded structures are involved in a cell’s rigidity sensing, but how these NPs affect rigidity sensing is unknown.ResultsHere, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 μg/μL of MNPs@SiO2(RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 μm in height, 24.21 nN/μm in stiffness k) under MNPs@SiO2(RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO2(RITC).ConclusionsOur results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

Abstract 480: Non-muscle Myosin II Regulates Aortic Stiffness via Tension-dependent Phosphorylation of Specific Focal Adhesion Proteins

Non-muscle myosin II plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in vascular function is not well understood. Here, we investigated the function of non-muscle myosin II in the biomechanical properties of mouse proximal aorta. We found that blebbistatin, a specific inhibitor of non-muscle myosin II decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate, in freshly isolated contractile aortic smooth muscle cells, using deconvolution microscopy that the NM myosin IIA isoform co-localizes with contractile filaments in the core of the cell as well as in the non-muscle cell cortex. However, the NM myosin IIB isoform is only colocalized with contractile filaments, and is excluded from the cell cortex. Furthermore, both the siRNA knockdown of NMIIA and NMIIB isoforms in a differentiated smooth muscle cell line A7r5 and blebbistatin-mediated inhibition of NM myosin II suppresses agonist-activated increases in phosphorylation of FAK Y925 and paxillin Y118. Thus, in the present study, we show, for the first time, that NM myosin II regulates aortic stiffness and that this regulation is mediated at least in part through the tension-dependent phosphorylation of focal adhesion proteins FAK and paxillin.

AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network

Aberrant expression of receptor tyrosine kinase AXL is linked to metastasis. AXL can be activated by its ligand GAS6 or by other kinases, but the signaling pathways conferring its metastatic activity are unknown. Here, we define the AXL-regulated phosphoproteome in breast cancer cells. We reveal that AXL stimulates the phosphorylation of a network of focal adhesion (FA) proteins, culminating in faster FA disassembly. Mechanistically, AXL phosphorylates NEDD9, leading to its binding to CRKII which in turn associates with and orchestrates the phosphorylation of the pseudo-kinase PEAK1. We find that PEAK1 is in complex with the tyrosine kinase CSK to mediate the phosphorylation of PAXILLIN. Uncoupling of PEAK1 from AXL signaling decreases metastasis in vivo, but not tumor growth. Our results uncover a contribution of AXL signaling to FA dynamics, reveal a long sought-after mechanism underlying AXL metastatic activity, and identify PEAK1 as a therapeutic target in AXL positive tumors.

Controlling Fibronectin Fibrillogenesis Using Visible Light.

We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.

Sea cucumber Cucumaria frondosa fucoidan inhibits osteosarcoma adhesion and migration by regulating cytoskeleton remodeling.

Osteosarcoma (OS) has been demonstrated to be difficult to cure due to its potently malignant metastasis. Therefore, new therapeutic approaches blocking the metastatic potential of OS are urgently required to improve the outcomes for OS patients. In the present study, the anti-metastatic capacity of sea cucumber (Cucumaria frondosa) fucoidan (Cf-Fuc) was evaluated on osteosarcoma cells by cell adhesion assay, Transwell assay and U2OS cell migration assay. The underlying mechanism on the dynamic remodeling of the cytoskeleton was also explored. The present data indicated that Cf-Fuc could block the U2OS osteosarcoma cell adhesion to fibronectin and significantly inhibit U2OS cell migration. Cf-Fuc greatly impaired the migration capacity of U2OS cells, and the migrated distance and velocity of Cf-Fuc-treated cells were markedly reduced. Also, Cf-Fuc could impair the dynamic remodeling of the cytoskeleton possibly by suppressing the phosphorylation of focal adhesion kinase and paxillin, as well as the activation of the Rac1/PAK1/LIMK1/cofilin signaling axis. Collectively, the present findings provide a novel therapeutic potential of C. frondosa fucoidan for osteosarcoma metastasis.

Ste20-like Kinase-mediated Control of Actin Polymerization Is a New Mechanism for Thin Filament-associated Regulation of Airway Smooth Muscle Contraction.

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle on contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is underinvestigated. SLK (Ste20-like kinase) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated Plk1 (polo-like kinase 1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the nonphosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.

Phenotype transitions induced by mechanical stimuli in airway smooth muscle are regulated by differential interactions of parvin isoforms with paxillin and Akt.

Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and β-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to β-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or β-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and β-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.

Inhibition of Endoglin Exerts Antitumor Effects through the Regulation of Non-Smad TGF-β Signaling in Angiosarcoma

Angiosarcoma is a rare malignant tumor derived from endothelial cells, and its prognosis is poor because advanced angiosarcoma is often resistant to taxane therapy. Endoglin (CD105) acts as a coreceptor for TGF-β signaling and is overexpressed in tumor-associated endothelial cells and enhances tumor angiogenesis. Numerous clinical trials are testing the effectiveness of anti-endoglin antibodies in various types of malignancies. Here, we investigated the role of endoglin in the pathogenesis of angiosarcoma and whether endoglin inhibition results in antitumor activity. Endoglin was overexpressed in angiosarcoma, and its inhibition was effective in promoting apoptosis and the suppression of migration, invasion, tube formation, and Warburg effect in angiosarcoma cells. Knockdown of endoglin activated caspase 3/7 that is essential for apoptosis, reduced survivin levels, and decreased paxillin and vascular endothelial cadherin phosphorylation and matrix metalloproteinase 2 and matrix metalloproteinase 9 activities in angiosarcoma cells. Although endoglin is a coreceptor that regulates TGF-β signaling, the antitumor effect of endoglin in angiosarcoma was not based on Smad signaling regulation but on non-Smad TGF-β signaling. Taken together, these results indicated that endoglin could be a novel therapeutic target for angiosarcoma.

Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

BackgroundSerglycin (SRGN), previously recognized as an intracellular proteoglycan involved in the storage processes of secretory granules, has recently been shown to be upregulated in several solid tumors. We have previously shown that SRGN in non-small cell lung cancer (NSCLC) promotes malignant phenotypes in a CD44-dependent manner and increased expression of SRGN predicts poor prognosis of primary lung adenocarcinomas. However, the underlying mechanism remains to be defined.MethodsOverexpression, knockdown and knockout approaches were performed to assess the role of SRGN in cell motility using wound healing and Boyden chamber migration assays. SRGN devoid of glycosaminoglycan (GAG) modification was produced by site-directed mutagenesis or chondroitinase treatment. Liquid chromatography/tandem mass spectrometry was applied for quantitative analysis of the disaccharide compositions and sulfation extent of SRGN GAGs. Western blot and co-immunoprecipitation analyses were performed to determine the expression and interaction of proteins of interest. Actin cytoskeleton organization was monitored by immunofluorescence staining.ResultsSRGN expressed by NSCLC cells is readily secreted to the extracellular matrix in a heavily glycosylated form attached with mainly chondroitin sulfate (CS)-GAG chains, and to a lesser extent with heparin sulfate (HS). The CS-GAG moiety serves as the structural motif for SRGN binding to tumor cell surface CD44 and promotes cell migration. SRGN devoid of CS-GAG modification fails to interact with CD44 and has lost the ability to promote cell migration. SRGN/CD44 interaction promotes focal adhesion turnover via Src-mediated paxillin phosphorylation and disassembly of paxillin/FAK adhesion complex, facilitating cell migration. In support, depletion of Src activity or removal of CS-GAGs efficiently blocks SRGN-mediated Src activation and cell migration. SRGN also promotes cell migration via inducing cytoskeleton reorganization mediated through RAC1 and CDC42 activation accompanied with increased lamellipodia and filopodia formation.ConclusionsProteoglycan SRGN promotes NSCLC cell migration via the binding of its GAG motif to CD44. SRGN/CD44 interaction induces Rho-family GTPase-mediated cytoskeleton reorganization and facilitates Src-mediated focal adhesion turnover, leading to increased cell migration. These findings suggest that targeting specific glycans in tumor microenvironment that serve as ligands for oncogenic pathways may be a potential strategy for cancer therapy.

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

ABSTRACT T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule-organizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1 phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1 phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1 re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1 regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization. Abbreviations Ab, antibody; AICD, activation-induced cell death; AIP, average intensity projection; APC, antigen-presenting cell; BCR, B-cell receptor for antigen; C, centre of mass; cent2, centrin 2; cIS, central region of the immune synapse; CMAC, CellTracker™ Blue (7-amino-4-chloromethylcoumarin); cSMAC, central supramolecular activation cluster; CTL, cytotoxic T lymphocytes; DAG, diacylglycerol; DGKα, diacylglycerol kinase α; Dia1, Diaphanous-1; dSMAC, distal supramolecular activation cluster; ECL, enhanced chemiluminescence; ESCRT, endosomal sorting complex required for traffic; F-actin, filamentous actin; Fact-low cIS, F-actin-low region at the centre of the immune synapse; FasL, Fas ligand; FMNL1, formin-like 1; fps, frames per second; GFP, green fluorescent protein; HBSS, Hank’s balanced salt solution; HRP, horseradish peroxidase; ILV, intraluminal vesicles; IS, immune synapse; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MIP, maximal intensity projection; MVB, multivesicular bodies; MTOC, microtubule-organizing centre; NS, not significant; PBL, peripheral blood lymphocytes; PKC, protein kinase C; PKCδ, protein kinase C δ isoform; PLC, phospholipase C; PMA, phorbol myristate acetate; Pol. Index, polarization index; pSMAC, peripheral supramolecular activation cluster; PSF, point spread function; ROI, region of interest; SD, standard deviation; shRNA, short hairpin RNA; SEE, Staphylococcus enterotoxin E; SMAC, supramolecular activation cluster; TCR, T-cell receptor for antigen; T-helper (Th); TRANS, transmittance; WB, Western blot.

Kindlin-3 stokes the life span of podosomes.

Macrophages and other cell types can form podosomes, highly dynamic adhesive structures that mediate the interaction of these cells with the surrounding matrix. In this issue, Klapproth et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201903109) find that kindlin-3 regulates podosome stability by recruiting leupaxin, with concomitant effects on PTP-PEST phosphatase activity and paxillin phosphorylation.