Actin-bundling Protein Research Articles

Immunohistochemical expression of Fascin-1 in colorectal cancer in relation to clinical and pathological parameters

Fascins are a group of proteins taking part in the maintenance of a proper structure of the cellular cytoskeleton. Fascin-1 is an actin-bundling protein present in neurons, fibroblasts, endothelial, smooth muscle, dendritic and mesenchymal cells whereas lack of its expression is characteristic of epithelial cells. Fascin-1 overexpression can be observed in neoplastic cells. Therefore, the aim of this study was to assess the expression of Fascin-1 protein in patients with colorectal cancer (CRC) and to analyze associations between Fascin-1 ex-pression and clinical-pathological parameters. The study material included postoperative samples (tumor and unchanged colon tissue) ob-tained from 51 CRC patients. Fascin-1 expression was assessed in the paraffin sections by immunohistochemistry. A statistically significant correlation was found between the histological type of cancer and the expres-sion of Fascin-1 (p = 0.012). Increased expression of Fascin-1 in CRC was more frequent in adenocarcinoma type without the mucosal component with a better prognosis and decreased expression of this protein correlated with infiltration of cancer cells to blood and lymphatic vessels (p = 0.038). Our findings indicate a potential role of Fascin-1 in the pathogenesis of colon cancer; however, further studies will show whether this protein plays a role in the infiltration of colorectal cancer cells.

SM22 is required for the maintenance of actin-rich structures generated during bacterial infections

The host actin cytoskeleton is utilized by an assortment of pathogenic bacteria to colonize and cause disease in their hosts. Two prominently studied actin-hijacking bacteria are enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes. EPEC form actin-rich pedestals atop its host cells to move across the intestinal epithelia, while Listeria monocytogenes generate branched actin networks arranged as actin clouds around the bacteria and as comet tails for propulsion within and amongst their host cells. Previous mass spectrometry analysis revealed that a member of the calponin family of actin-bundling proteins, transgelin/SM22 was enriched in EPEC pedestals. To validate that finding and examine the role of SM22 during infections, we initially immunolocalized SM22 in EPEC and L. monocytogenes infected cells, used siRNA to deplete SM22 and EGFP-SM22 to overexpress SM22, then quantified the alterations to the bacterially generated actin structures. SM22 concentrated at all bacterially-generated actin structures. Depletion of SM22 resulted in fewer pedestals and comet tails and caused comet tails to shorten. The decrease in comet tail abundance caused a proportional increase in actin clouds whereas overexpression of SM22 reversed the actin cloud to comet tail proportions and increased comet tail length, while not influencing EPEC pedestal abundance. Thus, we demonstrate that SM22 plays a role in regulating the transitions and morphological appearance of bacterially generated actin-rich structures during infections.

Fascin1 suppresses RIG-I–like receptor signaling and interferon-β production by associating with IκB kinase Ε (IKKΕ) in colon cancer

Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-β expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane- and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I·C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-β, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IκB kinase ϵ (IKKϵ) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IκB kinase ϵ in DLD-1 colon cancer cells to suppress immune responses to viral infection.

Inframe deletion of human ESPN is associated with deafness, vestibulopathy and vision impairment

BackgroundUsher syndrome (USH) is a neurosensory disorder characterised by deafness, variable vestibular areflexia and vision loss. The aim of the study was to identify the genetic defect in a Pakistani...

Structural Insights into the Induced-fit Inhibition of Fascin by a Small-Molecule Inhibitor

Tumor metastasis is responsible for ~90% of all cancer deaths. One of the key steps of tumor metastasis is tumor cell migration and invasion. Filopodia are cell surface extensions that are critical for tumor cell migration. Fascin protein is the main actin-bundling protein in filopodia. Small-molecule fascin inhibitors block tumor cell migration, invasion, and metastasis. Here we present the structural basis for the mechanism of action of these small-molecule fascin inhibitors. X-ray crystal structural analysis of a complex of fascin and a fascin inhibitor shows that binding of the fascin inhibitor to the hydrophobic cleft between the domains 1 and 2 of fascin induces a ~35o rotation of domain 1, leading to the distortion of both the actin-binding sites 1 and 2 on fascin. Furthermore, the crystal structures of an inhibitor alone indicate that the conformations of the small-molecule inhibitors are dynamic. Mutations of the inhibitor-interacting residues decrease the sensitivity of fascin to the inhibitors. Our studies provide structural insights into the molecular mechanism of fascin protein function as well as the action of small-molecule fascin inhibitors.

FSCN‑1 increases doxorubicin resistance in hepatocellular carcinoma through promotion of epithelial-mesenchymal transition.

Resistance to chemotherapy drugs remains a significant problem for the treatment of many types of cancer. Fascin-1 (FSCN-1) is an actin-bundling protein involved in the invasion and metastasis of a variety of tumors. However, its involvement in drug resistance in hepatocellular carcinoma (HCC) remains unclear. The present study aimed to investigate the function of FSCN-1 in HCC resistance to doxorubicin (DOX). FSCN-1 expression was increased in DOX-resistant HCC cell lines (SNU449 and SNU387) compared with DOX-sensitive cell lines (Huh7 and Hep3B). The resistance of HCC cells to DOX was decreased following FSCN-1 knockdown with small interfering RNA. FSCN-1 knockdown also significantly altered the expression of key markers of epithelial-mesenchymal transition (EMT). Notably, vimentin expression was reduced and epithelial-cadherin expression was increased. Furthermore, when EMT was suppressed through knockdown of Twist, an essential pathway of DOX-induced EMT, the viability of HCC cells following treatment with DOX was not affected by FSCN-1 expression. Furthermore, FSCN-1 knockdown eliminated hypoxia-induced doxorubicin resistance and EMT. The results of the present study indicated that FSCN-1 expression increased DOX resistance in HCC cells via the promotion of EMT, and this phenomenon was maintained in a hypoxic environment. FSCN-1 potentially represents a novel target to overcome resistance to DOX in HCC.

Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1).

Regulation of the cytoskeleton is essential for cell migration in health and disease. Lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) is a hematopoietic-specific actin-bundling protein that is highly conserved in zebrafish, mice and humans. In addition, L-plastin expression is documented as both a genetic marker and a cellular mechanism contributing to the invasiveness of tumors and transformed cell lines. Despite L-plastin’s role in both immunity and cancer, in zebrafish there are no direct studies of its function, and no mutant, knockout or reporter lines available. Using CRISPR-Cas9 genome editing, we generated null alleles of zebrafish lcp1 and examined the phenotypes of these fish throughout the life cycle. Our editing strategy used gRNA to target the second exon of lcp1, producing F0 mosaic fish that were outcrossed to wild types to confirm germline transmission. F1 heterozygotes were then sequenced to identify three unique null alleles, here called ‘Charlie’, ‘Foxtrot’ and ‘Lima’. In silico, each allele truncates the endogenous protein to less than 5% normal size and removes both essential actin-binding domains (ABD1 and ABD2). Although none of the null lines express detectable LCP1 protein, homozygous mutant zebrafish (-/-) can develop and reproduce normally, a finding consistent with that of the L-plastin null mouse (LPL -/-). However, such mice do have a profound immune defect when challenged by lung bacteria. Interestingly, we observed reduced long-term survival of zebrafish lcp1 -/- homozygotes (~30% below the expected numbers) in all three of our knockout lines, with greatest mortality corresponding to the period (4–6 weeks post-fertilization) when the innate immune system is functional, but the adaptive immune system is not yet mature. This suggests that null zebrafish may have reduced capacity to combat opportunistic infections, which are more easily transmissible in the aquatic environment. Overall, our novel mutant lines establish a sound genetic model and an enhanced platform for further studies of L-plastin gene function in hematopoiesis and cancer.

Fascin in tooth germs: an immunohistochemical analysis

Development of a tooth is a complex process that involves three stages namely bud, cap and bell which results from the interaction of cells of oral mucosa and neural crest. During this process, oral and neural crest cells undergo various changes resulting in proliferation, apoptosis, morphological, and cyto-differentiation, which in turn would have changes in the cytoskeletal framework of the cell that includes actin and tubulin. Fascin is an actin-bundling protein that cross-links individual actin filaments into straight, compact, stiff bundles, which are crucial for the formation of finger-like membrane protrusions and aids in cell migration but is less explored in tooth germs. The aim of this study was analyze the expression and distribution of fascin in various stages of human tooth germs. Twenty human tooth germs were stained with hematoxylin and eosin and with immunohistochemical staining for fascin. Strong membranous expression for fascin was observed in various stages with decreased expression in stellate reticulum from cap stage to bell stage. Also, outer enamel epithelial cells had a weak fascin expression. Although cells of dental papilla did not express fascin in early cap stage of tooth development, strong expression of fascin was observed in later bell stages. It was also observed the inner enamel epithelium close to areas of calcification showed a decreased expression. Thus, the expression of fascin in various stages of tooth development being site and time specific may play an important role in cell remodulation.

TAGLN2 is a candidate prognostic biomarker promoting tumorigenesis in human gliomas

BackgroundTransgelin-2 (TAGLN2) is a member of the calponin family of actin-bundling proteins that is involved in the regulation of cell morphology, motility, and cell transformation. Here, the clinical significance and potential function of TAGLN2 in malignant gliomas were investigated.MethodsMolecular and clinical data was obtained from The Cancer Genome Atlas (TCGA) database. Gene ontology and pathway analysis was used to predict potential functions of TAGLN2. RNA knockdown was performed using siRNA or lentiviral contructs in U87MG and U251 glioma cell lines. Cells were characterized in vitro or implanted in vivo to generate orthotopic xenografts in order to assess molecular status, cell proliferation/survival, and invasion by Western blotting, flow cytometry, and 3D tumor spheroid invasion assay, respectively.ResultsIncreased TAGLN2 expression was associated with increasing tumor grade (P < 0.001), the mesenchymal molecular glioma subtype and worse prognosis in patients (P < 0.001). Immunohistochemistry performed with anti-TAGLN2 on an independent cohort of patients (n = 46) confirmed these results. Gene silencing of TAGLN2 in U87MG and U251 significantly inhibited invasion and tumor growth in vitro and in vivo. Western blot analysis revealed that epithelial-mesenchymal transition (EMT) molecular markers, such as N-cadherin, E-cadherin, and Snail, were regulated in a manner corresponding to suppression of the EMT phenotype in knockdown experiments. Finally, TAGLN2 was induced ~ 2 to 3-fold in U87MG and U251 cells by TGFβ2, which was also elevated in GBM and highly correlated with TAGLN2 mRNA levels (P < 0.001).ConclusionsOur findings indicate that TAGLN2 exerts a role in promoting the development of human glioma. The regulation and function of TAGLN2 therefore renders it as a candidate molecular target for the treatment of GBM.

Fascin Is Associated With Aggressive Behavior and Poor Outcome in Uterine Carcinosarcoma.

The mechanisms underlying the histogenesis and aggressiveness of uterine carcinosarcoma (UCS) are poorly understood; however, previous studies implicate epithelial-mesenchymal transition (EMT). Fascin is a proinvasive, actin-bundling protein and an important component of EMT. It is associated with poor outcomes in human carcinoma, especially in estrogen receptor (ER)-negative tumors arising in organs normally expressing ER. We sought to evaluate fascin expression in UCS and its relationship to ER status, clinicopathologic indicators of tumor aggressiveness, and survival outcomes. Forty-four surgically staged cases of UCS were immunohistochemically evaluated for fascin and estrogen receptor-α expression and correlated with clinicopathologic parameters derived from electronic medical records and pathology reports. Fascin was only expressed in malignant epithelium and mesenchyma and was uniformly absent in background benign counterparts. Increased expression was associated with extrapelvic disease (P = 0.028), higher stage (P = 0.021), larger tumor size (P = 0.032), shorter progression-free interval (P = 0.035), and reduced estrogen receptor-α expression (P = 0.04). Fascin is aberrantly expressed in both elements of UCS and is associated with aggressive behavior and worse outcome. As a component of EMT and mediator of invasion, fascin may serve as a target in future therapies.

Fascin-1 Contributes to Neuropathic Pain by Promoting Inflammation in Rat Spinal Cord.

Neuropathic pain is a complicated clinical syndrome caused by heterogeneous etiology. Despite the fact that the underlying mechanisms remain elusive, it is well accepted that neuroinflammation plays a critical role in the development of neuropathic pain. Fascin-1, an actin-bundling protein, has been proved to be involved in the processing of diverse biological events including cellular development, immunity, and tumor invasion etc. Recent studies have shown that Fascin-1 participates in antigen presentation and the regulation of pro-inflammatory agents. However, whether Fascin-1 is involved in neuropathic pain has not been reported. In the present study we examined the potential role of Fascin-1 by using a rodent model of chronic constriction injury (CCI). Our results showed that Fascin-1 increased rapidly in dorsal root ganglions (DRG) and spinal cord (SC) after CCI. The increased Fascin-1 widely expressed in DRG, however, it localized predominantly in microglia, seldom in neuron, and hardly in astrocyte in the SC. Intrathecal injection of Fascin-1 siRNA not only suppressed the activation of microglia and the release of pro-inflammatory mediators, but also attenuated the mechanical allodynia and thermal hyperalgesia induced by CCI.

Fimbrins 4 and 5 Act Synergistically During Polarized Pollen Tube Growth to Ensure Fertility in Arabidopsis.

The germination and polar growth of pollen are prerequisite for double fertilization in plants. The actin cytoskeleton and its binding proteins play pivotal roles in pollen germination and pollen tube growth. Two homologs of the actin-bundling protein fimbrin, AtFIM4 and AtFIM5, are highly expressed in pollen in Arabidopsis and can form distinct actin architectures in vitro, but how they co-operatively regulate pollen germination and pollen tube growth in vivo is largely unknown. In this study, we explored their functions during pollen germination and polar growth. Histochemical analysis demonstrated that AtFIM4 was expressed only after pollen grain hydration and, in the early stage of pollen tube growth, the expression level of AtFIM4 was low, indicating that it functions mainly during polarized tube growth, whereas AtFIM5 had high expression levels in both pollen grains and pollen tubes. Atfim4/atfim5 double mutant plants had fertility defects including reduced silique length and seed number, which were caused by severe defects in pollen germination and pollen tube growth. When the atfim4/atfim5 double mutant was complemented with the AtFIM5 protein, the polar growth of pollen tubes was fully rescued; however, AtFIM4 could only partially restore these defects. Fluorescence labeling showed that loss of function of both AtFIM4 and AtFIM5 decreased the extent of actin filament bundling throughout pollen tubes. Collectively, our results revealed that AtFIM4 acts co-ordinately with AtFIM5 to organize and maintain normal actin architecture in pollen grains and pollen tubes to fulfill double fertilization in plants.

Studying the role of fascin-1 in mechanically stressed podocytes

Glomerular hypertension causes glomerulosclerosis via the loss of podocytes, which are challenged by increased mechanical load. We have demonstrated that podocytes are mechanosensitive. However, the response of podocytes to mechanical stretching remains incompletely understood. Here we demonstrate that the actin-bundling protein fascin-1 plays an important role in podocytes that are exposed to mechanical stress. Immunofluorescence staining revealed colocalization of fascin-1 and nephrin in mouse kidney sections. In cultured mouse podocytes fascin-1 was localized along actin fibers and filopodia in stretched and unstretched podocytes. The mRNA and protein levels of fascin-1 were not affected by mechanical stress. By Western blot and 2D-gelelectrophoresis we observed that phospho-fascin-1 was significantly downregulated after mechanical stretching. It is known that phosphorylation at serine 39 (S39) regulates the bundling activity of fascin-1, e.g. required for filopodia formation. Podocytes expressing wild type GFP-fascin-1 and non-phosphorylatable GFP-fascin-1-S39A showed marked filopodia formation, being absent in podocytes expressing phosphomimetic GFP-fascin-1-S39D. Finally, the immunofluorescence signal of phosphorylated fascin-1 was strongly reduced in glomeruli of patients with diabetic nephropathy compared to healthy controls. In summary, mechanical stress dephosphorylates fascin-1 in podocytes in vitro and in vivo thereby fascin-1 may play an important role in the adaptation of podocytes to mechanical forces.

Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

Within the kidney, angiotensin II (AngII) targets different cell types in the vasculature, tubuli, and glomeruli. An important part of the renal filtration barrier is composed of podocytes with their actin-rich foot processes. In this study, we used stable isotope labeling with amino acids in cell culture coupled to mass spectrometry to characterize relative changes in the phosphoproteome of human podocytes in response to short-term treatment with AngII. In 4 replicates, we identified a total of 17,956 peptides that were traceable to 2081 distinct proteins. Bioinformatic analyses revealed that among the increasingly phosphorylated peptides are predominantly peptides that are related to actin filaments, cytoskeleton, lamellipodia, mammalian target of rapamycin, and MAPK signaling. Among others, this screening approach highlighted the increased phosphorylation of actin-bundling protein, l-plastin (LCP1). AngII-dependent phosphorylation of LCP1 in cultured podocytes was mediated by the kinases ERK, p90 ribosomal S6 kinase, PKA, or PKC. LCP1 phosphorylation increased filopodia formation. In addition, treatment with AngII led to LCP1 redistribution to the cell margins, membrane ruffling, and formation of lamellipodia. Our data highlight the importance of AngII-triggered actin cytoskeleton-associated signal transduction in podocytes.-Schenk, L. K., Möller-Kerutt, A., Klosowski, R., Wolters, D., Schaffner-Reckinger, E., Weide, T., Pavenstädt, H., Vollenbröker, B. Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

Abstract LB-228: Identifying small molecule inhibitors of Fascin 1 using fragment-based drug discovery

Abstract The actin-bundling protein fascin 1 is markedly overexpressed in a range of invasive tumors and is believed to play a critical role in cancer cell metastasis. Targeting fascin is however very challenging owing to its mechanism of protein-protein interaction and a lack of knowledge regarding the crucial actin-binding sites. By combining a fragment-based approach, biophysical assay screening and X-ray crystallography, we have been able to identify and optimize novel fascin 1 inhibitors that show nanomolar affinity in biochemical binding and bundling assays. We now show the development of functional cell assays in which the lead fascin inhibitor compounds have been tested. For the first time we disclose the structures of the most advanced compounds and show their effects in 2D and 3D cell culture. Fascin 1 binds and cross-links filamentous actin (F-actin) into parallel bundles that are used in the formation of dynamic cellular protrusions (such as lamellipodia and filopodia) used during cell migration, and in the formation of invadopodia used by tumor cell lines to degrade the tumor extracellular matrix (ECM). Whereas fascin 1 expression is low or absent in normal epithelia, its expression is dramatically increased in a variety of invasive tumor types including colon, lung, ovarian and pancreas, with increased expression being an independent prognostic indicator of poor clinical outcome in the most aggressive and metastatic tumor types. Knockdown of fascin expression has been shown to reduce tumor cell invasion both in vitro and in vivo, thereby highlighting fascin as an important drug discovery target. Small molecule inhibitors of fascin 1 were identified by screening our fragment library (~1000 compounds) using surface plasmon resonance (SPR). X-ray co-crystallography with these hit compounds showed four distinct ligand binding sites within the fascin protein. Optimized site 2 binders induce a substantial conformational change, with the deeply enclosed pocket between fascin domains 1 and 2 opening a channel that accesses the surface of the protein. A virtual screen identified the compound BDP-00010834, which binds in this pocket with an SPR Kd of 29.7µM and also inhibits (IC50=50µM) the functional activity of fascin measured in an F-actin bundling assay. Structure-based optimization in combination with X-ray co-crystallography has enabled the generation of compounds with more than 300-fold increase in both binding affinity and functional activity over this screening hit. Our current best-in-series compounds include BDP-00013544 (Kd=29nM, IC50=185nM). These lead compounds have now been tested in a number of cell based invasion assays including both 2D and 3D cultures. Citation Format: Daniel Croft, Stuart Francis, Justin Bower, Andrea Gohlke, Gillian Goodwin, Christopher Gray, Jennifer Konczal, Sophie Macconnachie, Patricia McConnell, Laura McDonald, Heather McKinnon, Mokdad Mezna, Charles Parry, Nikki Paul, Angelo Pugliese, Alexander Schuettelkopf, Laura Machesky, Martin Drysdale. Identifying small molecule inhibitors of Fascin 1 using fragment-based drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-228. doi:10.1158/1538-7445.AM2017-LB-228

The actin-bundling protein plastin increases cortical connectivity to promote robust polarization and timely cytokinesis in early C. elegans embryogenesis

The actin-bundling protein plastin increases cortical connectivity to promote robust polarization and timely cytokinesis in early C. elegans embryogenesis

Abstract NTOC-085: PPARγ AGONISTS AUGMENT ANTICANCER EFFECTS OF XIAP INHIBITION ON HUMAN GRANULOSA CELL TUMOR–DERIVED CELLS

Abstract BACKGROUND: Ovarian granulosa cell tumors (GCT) are hormonally active cancers characterized by indolent growth and late, invasive relapse. GCT are unusual in that they have an unexplained propensity for late recurrence. ~80% of patients with aggressive or recurrent tumors die from their disease. Aside from surgery the therapeutic options are very limited. We have identified the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) and the X-linked inhibitor of apoptosis protein (XIAP) as potential specific therapeutic targets. PPARγ is a transcription factor that impedes proliferation and promotes terminal differentiation in granulosa cells (GC), is highly expressed in GCT. Its activity however is transrepressed by constitutive activity of the critical pro-survival NF-κB signaling pathway. Constitutive NF-κB signaling is potentially a consequence of a positive feed-forward loop involving XIAP, which is also highly expressed in GCT. As a potent inhibitor of apoptosis, XIAP is an attractive therapeutic target. We have shown in vitro that inhibiting XIAP releases NF-κB transrepression, and together with PPARγ activation, induces apoptosis. We thus hypothesize that XIAP antagonism sensitizes GCT to pro-apoptotic strategies such as PPARγ activation. METHODS AND RESULTS: We have used stable isotope labeling with amino acids in cell culture (SILAC), a proteomic approach to identify differentially expressed proteins after combined PPARγ activation (rosiglitazone) and XIAP inhibition (Smac mimetic). Using two GCT-derived cell lines, KGN and COV434, we identified 52 proteins which were differentially expressed (by 1.5 fold) with the combined treatment. Upregulated proteins included several involved in metabolic processes including acyl-CoA desaturase (4.50-fold), phosphoglycerate kinase 1 (2.87-fold) and α-enolase (1.75-fold). As PPARγ plays a pivotal role in lipid and glucose metabolism, upregulation of these proteins is a consequence of PPARγ activity being restored. Additionally, we observed downregulation of fascin (-1.65 fold), the main actin-bundling protein associated with cell motility and migration. Overexpression has been observed in metastatic tumors and correlated with clinically aggressive phenotypes, poor prognosis and shorter survival. We used the xCELLigence RTCA system to monitor invasion of KGN cells through a Matrigel® basement membrane in real-time. KGN cells treated either with Vehicle, RGZ/RA or SM alone or combined RGZ/RA/SM were plated in the upper chamber of a CIM-Plate 16 and cell invasion through the Matrigel® membrane was monitored. When compared to vehicle treated cells, we observed that the RGZ/RA/SM-treated KGN cells were (i) 30% less invasive and (ii) demonstrated a delayed onset of invasion by 17 hours towards serum-containing media with and without drugs in the lower chamber, respectively. CONCLUSION: We are currently investigating the mechanism involved in XIAP inhibition abrogating transrepression of PPARγ in GCT-derived cells. This study will improve our understanding of the molecular mechanisms in GCT pathophysiology as well as enable identification of new targets for therapeutic strategies. These observations may also translate to ovarian epithelial cell cancers (EOC) as increased apoptosis was observed in serous EOC-derived cell lines expressing both XIAP and PPARγ. We propose that a combination therapy involving the abrogation of XIAP may be of greater efficacy for the treatment of GCT and potentially other ovarian cancer subtypes. Citation Format: Simon Chu, Dilys TH Leung, Maria Alexiadis and Peter J Fuller. PPARγ AGONISTS AUGMENT ANTICANCER EFFECTS OF XIAP INHIBITION ON HUMAN GRANULOSA CELL TUMOR–DERIVED CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-085.

T-plastin is essential for basement membrane assembly and epidermal morphogenesis.

The establishment of epithelial architecture is a complex process involving cross-talk between cells and the basement membrane. Basement membrane assembly requires integrin activity but the role of the associated actomyosin cytoskeleton is poorly understood. Here, we identify the actin-bundling protein T-plastin (Pls3) as a regulator of basement membrane assembly and epidermal morphogenesis. In utero depletion of Pls3 transcripts in mouse embryos caused basement membrane and polarity defects in the epidermis but had little effect on cell adhesion and differentiation. Loss-of-function experiments demonstrated that the apicobasal polarity defects were secondary to the disruption of the basement membrane. However, the basement membrane itself was profoundly sensitive to subtle perturbations in the actin cytoskeleton. We further show that Pls3 localized to the cell cortex, where it was essential for the localization and activation of myosin II. Inhibition of myosin II motor activity disrupted basement membrane organization. Our results provide insights into the regulation of cortical actomyosin and its importance for basement membrane assembly and skin morphogenesis.

Hypoxia-stimulated membrane trafficking requires T-plastin.

Traffic between the plasma membrane and the endomembrane compartments is an essential feature of eukaryotic cells. The secretory pathway sends cargoes from biosynthetic compartments to the plasma membrane. This is counterbalanced by a retrograde endocytic route and is essential for cell homoeostasis. Cells need to adapt rapidly to environmental challenges such as the reduction of pO2 which, however, has not been analysed in relation to membrane trafficking in detail. Therefore, we determined changes in the plasma membrane trafficking in normoxia, hypoxia, and after reoxygenation. Membrane trafficking was analysed by using the bulk membrane endocytosis marker FM 1-43, the newly developed membrane probe mCLING, wheat germ agglutinin as well as fluorescently labelled cholera toxin subunit B. Additionally, the uptake of specific membrane proteins was determined. In parallel, a non-biased SILAC screen was performed to analyse the abundance of membrane proteins in normoxia and hypoxia. Membrane trafficking was increased in hypoxia and quickly reversed upon reoxygenation. This effect was independent of the hypoxia-inducible factor (HIF) system. Using SILAC technology, we identified that the actin-bundling protein T-plastin is recruited to the plasma membrane in hypoxia. By the use of T-plastin knockdown cells, we could show that T-plastin mediates the hypoxia-induced membrane trafficking, which was associated with an increased actin density in the cells as determined by electron microscopy. Membrane trafficking is highly dynamic upon hypoxia. This phenotype is quickly reversible upon reoxygenation, which suggests that this mechanism participates in the cellular adaptation to hypoxia.

CD40–CD40L cross-talk drives fascin expression in dendritic cells for efficient antigen presentation to CD4+ T cells

Fascin is an actin-bundling protein that, among immune cells, is restricted to expression in dendritic cells (DCs). Previous reports have suggested that fascin plays an important role in governing DC antigen presentation to CD4+ T cells. However, no report has clearly linked the receptor-ligand engagement that can direct downstream regulation of fascin expression. In this study, bone marrow-derived DCs from wild-type versus CD40-knockout C57BL/6 mice were used to elucidate the mechanisms of fascin expression and activity upon CD40-CD40 ligand (CD40L) engagement. These investigations now show that CD40 engagement governs fascin expression in DCs to promote CD4+ T-cell cytokine production. Absence of CD40 signaling resulted in diminished fascin expression in DCs and was associated with impaired CD4+ T-cell responses. Furthermore, the study found that loss of CD40-CD40L engagement resulted in reduced DC-T-cell contacts. Rescue by ectopic fascin expression in CD40-deficient DCs was able to re-establish sustained contacts with T cells and restore cytokine production. Taken together, these results show that cross-talk through CD40-CD40L signaling drives elevated fascin expression in DCs to support acquisition of full T-cell responses.