IQGAP1 and NWASP promote human cancer cell dissemination and metastasis by regulating ß1-integrin via FAK and MRTF/SRF.

Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that ß1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of ß1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase ß1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.

Small GTPase Cdc42, WASP, and scaffold proteins for higher-order assembly of the F-BAR domain protein.

The higher-order assembly of Bin-amphiphysin-Rvs (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, into lattice on the membrane is essential for the formation of subcellular structures. However, the regulation of their ordered assembly has not been elucidated. Here, we show that the higher ordered assembly of growth-arrested specific 7 (GAS7), an F-BAR domain protein, is regulated by the multivalent scaffold proteins of Wiskott-Aldrich syndrome protein (WASP)/neural WASP, that commonly binds to the BAR domain superfamily proteins, together with WISH, Nck, the activated small guanosine triphosphatase Cdc42, and a membrane-anchored phagocytic receptor. The assembly kinetics by fluorescence resonance energy transfer monitoring indicated that the GAS7 assembly on liposomes started within seconds and was further increased by the presence of these proteins. The regulated GAS7 assembly was abolished by Wiskott-Aldrich syndrome mutations both in vitro and in cellular phagocytosis. Therefore, Cdc42 and the scaffold proteins that commonly bind to the BAR domain superfamily proteins promoted GAS7 assembly.

N-WASP-Arp2/3 signaling controls multiple steps of dendrite maturation in Purkinje cells in vivo.

During neural development, the actin filament network must be precisely regulated to form elaborate neurite structures. N-WASP tightly controls actin polymerization dynamics by activating an actin nucleator Arp2/3. However, the importance of N-WASP-Arp2/3 signaling in the assembly of neurite architecture in vivo has not been clarified. Here, we demonstrate that N-WASP-Arp2/3 signaling plays a crucial role in the maturation of cerebellar Purkinje cell (PC) dendrites in vivo in mice. N-WASP was expressed and activated in developing PCs. Inhibition of Arp2/3 and N-WASP from the beginning of dendrite formation severely disrupted the establishment of a single stem dendrite, which is a characteristic basic structure of PC dendrites. Inhibition of Arp2/3 after stem dendrite formation resulted in hypoplasia of the PC dendritic tree. Cdc42, an upstream activator of N-WASP, is required for N-WASP-Arp2/3 signaling-mediated PC dendrite maturation. In addition, overactivation of N-WASP is also detrimental to dendrite formation in PCs. These findings reveal that proper activation of N-WASP-Arp2/3 signaling is crucial for multiple steps of PC dendrite maturation in vivo.

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.

Compound heterozygous variants in Wiskott-Aldrich syndrome like (WASL) gene segregating in a family with early onset Parkinson's disease.

BACKGROUND: Knowledge of genetic determinants in Parkinson's disease is still limited. Familial forms of the disease continue to provide a rich resource to capture the genetic spectrum in disease pathogenesis, and this approach is exploited in this study. METHODS: Informative members from a three-generation family of Indian ethnicity manifesting a likely autosomal recessive mode of inheritance of Parkinson's disease were used for whole exome sequencing. Variant data analysis and in vitro functional characterisation of variant(s) segregating with the phenotype were carried out in HEK-293 and SH-SY5Y cells using gene constructs of interest. RESULTS: Two compound heterozygous variants, a rare missense (c.1139C > T:p.P380L) and a novel splice variant (c.1456 + 2 delTAGA, intron10) in Wiskott-Aldrich syndrome like gene (WASL, 7q31), both predicted to be deleterious were shared among the proband and two affected siblings. WASL, a gene not previously linked to a human Mendelian disorder is known to regulate actin polymerisation via Arp2/3 complex. Based on exon trapping assay using pSPL3 vector in HEK-293 cells, the splice variant showed skipping of exon10. Characterisation of the missense variant in SH-SY5Y cells demonstrated: i) significant alterations in neurite length and number; ii) decreased reactive oxygen species tolerance in mutation carrying cells on Tetrabutylphosphonium hydroxide induction and iii) increase in alpha-synuclein protein. Screening for WASL variants in two independent PD cohorts identified four individuals with heterozygous but none with biallelic variants. CONCLUSION: WASL, with demonstrated functional relevance in neurons may be yet another strong candidate gene for autosomal recessive PD encouraging assessment of its contribution across populations.

Chlamydia trachomatis TmeA Directly Activates N-WASP To Promote Actin Polymerization and Functions Synergistically with TarP during Invasion.

Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis, such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process.IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion.

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.

N-Wasp Regulates Oligodendrocyte Myelination.

Oligodendrocyte myelination depends on actin cytoskeleton rearrangement. Neural Wiskott-Aldrich syndrome protein(N-Wasp) is an actin nucleation factor that promotes polymerization of branched actin filaments. N-Wasp activity is essential for myelin membrane wrapping by Schwann cells, but its role in oligodendrocytes and CNS myelination remains unknown. Here we report that oligodendrocytes-specific deletion of N-Wasp in mice of both sexes resulted in hypomyelination (i.e., reduced number of myelinated axons and thinner myelin profiles), as well as substantial focal hypermyelination reflected by the formation of remarkably long myelin outfolds. These myelin outfolds surrounded unmyelinated axons, neuronal cell bodies, and other myelin profiles. The latter configuration resulted in pseudo-multimyelin profiles that were often associated with axonal detachment and degeneration throughout the CNS, including in the optic nerve, corpus callosum, and the spinal cord. Furthermore, developmental analysis revealed that myelin abnormalities were already observed during the onset of myelination, suggesting that they are formed by aberrant and misguided elongation of the oligodendrocyte inner lip membrane. Our results demonstrate that N-Wasp is required for the formation of normal myelin in the CNS. They also reveal that N-Wasp plays a distinct role in oligodendrocytes compared with Schwann cells, highlighting a difference in the regulation of actin dynamics during CNS and PNS myelination.SIGNIFICANCE STATEMENT Myelin is critical for the normal function of the nervous system by facilitating fast conduction of action potentials. During the process of myelination in the CNS, oligodendrocytes undergo extensive morphological changes that involve cellular process extension and retraction, axonal ensheathment, and myelin membrane wrapping. Here we present evidence that N-Wasp, a protein regulating actin filament assembly through Arp2/3 complex-dependent actin nucleation, plays a critical role in CNS myelination, and its absence leads to several myelin abnormalities. Our data provide an important step into the understanding of the molecular mechanisms underlying CNS myelination.

WASP family proteins regulate the mobility of the B cell receptor during signaling activation.

Regulation of membrane receptor mobility tunes cellular response to external signals, such as in binding of B cell receptors (BCR) to antigen, which initiates signaling. However, whether BCR signaling is regulated by BCR mobility, and what factors mediate this regulation, are not well understood. Here we use single molecule imaging to examine BCR movement during signaling activation and a novel machine learning method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors, Arp2/3 and formin, decreases BCR mobility. Constitutive loss or acute inhibition of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, increases the proportion of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces the diffusivity of CD19, a stimulatory co-receptor, but not that of FcγRIIB, an inhibitory co-receptor. Our results implicate a dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions for modulating B cell signaling.

Inhibition of N-WASP affects actin-mediated cytokinesis during porcine oocyte maturation.

N-WASP is the mammalian ortholog of WASP which is an actin nucleation promoting factor and has been reported to regulate actin nucleation and polymerization for multiple cell activities. However, the expression and functions of N-WASP in porcine oocytes are still unclear. In this study, we showed that N-WASP expressed at all stages during porcine oocyte maturation, and immunofluorescence staining indicated that N-WASP mainly accumulated at the cortex in different stages of meiosis. Inhibition of N-WASP activity by Wiskostatin significantly decreased the rate of first polar body extrusion and disturbed the cell cycle progression of porcine oocytes. Further analysis indicated that cortical actin distribution was interfered by N-WASP inhibition, and this might be through its regulatory roles on the expression and localization of ARP2, a key component of actin nucleator Arp2/3 complex. Moreover, the expression of N-WASP decreased after ROCK activity inhibition, indicating a ROCK-N-WASP-ARP2/3 pathway for actin assembly in porcine oocytes. Taken together, these results suggest that N-WASP is critical for the regulation of actin filaments for cytokinesis during porcine oocyte maturation.

αII-spectrin controls calcium-regulated exocytosis in neuroendocrine chromaffin cells through neuronal Wiskott-Aldrich Syndrome protein interaction.

Besides a fundamental structural role at the plasma membrane, spectrin- and actin-based skeletons have been proposed to participate in various processes including vesicular trafficking. Neuroendocrine cells release hormones and neuropeptides through calcium-regulated exocytosis, a process that is coordinated by a fine remodeling of the actin cytoskeleton. We describe here that calcium-regulated exocytosis is impaired in chromaffin and PC12 cells with reduced αII-spectrin expression levels. Using yeast two-hybrid screening, we show that neuronal Wiskott-Aldrich Syndrome protein (N-WASP) is a partner of the αII-spectrin SH3 domain and demonstrate that secretagogue-evoked N-WASP recruitment at cell periphery is blocked in the absence of αII-spectrin. Additionally, experiments performed with ectopically expressed αII-spectrin mutant unable to bind N-WASP indicated that the interaction between SH3 domain and N-WASP is pivotal for neuroendocrine secretion. Our results extend the list of spectrin interactors and strengthen the idea that αII-spectrin is an important scaffold protein that gathers crucial actin-related players of the exocytic machinery.

N-WASP knockdown upregulates inflammatory cytokines expression in human gingival fibroblasts.

OBJECTIVE: The neuronal wiskott-aldrich syndrome protein (N-WASP) is a member of the wiskott-aldrich syndrome protein (WASP) family. N-WASP plays a vital role in promoting cell migration, receptor signaling and immune inflammatory responses. This study aimed to observe the changes in the expression of inflammatory factors and involving pathways after N-WASP knockdown in human gingival fibroblasts (HGFs). DESIGN: Gingival inflammatory condition of N-WASP knockout mice was evaluated by H&E staining. N-WASP in HGFs was knockdown by siRNA and the best knockdown efficiency was determined by qRT-PCR and immunofluorescence. The mRNA levels of interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), superoxide dismutase 2 (SOD2) and prostaglandin endoperoxide synthase 2 (PTGS2) were evaluated by qRT-PCR after N-WASP knockdown with or without mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors. The protein levels of IL-6, IL-8 and CCL2 were assessed by ELISA. Western blotting was used to detect the activation of NF-κB and MAPK signaling pathways. RESULTS: Gingival tissue from N-WASP knockout mice exhibited an inflammatory reaction. The expression of IL-6, IL-8, CCL2, SOD2 and PTGS2 was significantly upregulated after N-WASP knockdown in HGFs for 6, 24 and 48 h, except for the SOD2 at 6 h. N-WASP knockdown significantly activated the signaling pathways of NF-κB and MAPK. The inhibitors of p65, p38, ERK and JNK clearly decreased IL-6, IL-8, CCL2, SOD2 and PTGS2 expression after N-WASP knockdown. CONCLUSION: These data indicated that N-WASP deficiency in HGFs increases the production of inflammatory cytokine and is regulated via NF-κB and MAPK signaling pathways.

N-WASP Control of LPAR1 Trafficking Establishes Response to Self-Generated LPA Gradients to Promote Pancreatic Cancer Cell Metastasis.

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.

The prognostic value of CXCR4, α-SMA and WASL in upper lip basal cell carcinomas.

Lip cancers account for 10-12% of the total head and neck cancers and, although squamous cell carcinoma is by far the most common lower lip cancer, the basal cell carcinoma (BCC) seems to be more common for the upper lip. Most BCCs have a clinically indolent behavior, but there are also local aggressive and∕or metastatic cases, with the incidence of such cases being estimated at about 1-10% of all cases of BCC. Many of the molecular mechanisms underlying this aggression are still unknown, which is why our study aimed to investigate the potential prognosis of a few markers, such as C-X-C chemokine receptor type 4 (CXCR4), alpha-smooth muscle actin (α-SMA) and Wiskott-Aldrich syndrome like (WASL) in upper lip BCCs. For this purpose, 24 basocellular cancers with this localization have been investigated immunohistochemically, histopathologically belonging to the next varieties: superficial, nodular, micronodular, adenoid cystic, keratotic, sclerodermiform and mixed. Regardless of the histopathological subtype, for all invasive cases we have recorded an increased reactivity of the three markers especially in the invasion front, reactivity also present at the stroma level, especially at the stroma-parenchyma interface. The most intense immunoreactivity was obtained for the micronodular and sclerodermiform subtypes, confirming their biological behavior to be more aggressive than the rest of the investigated strains. All these results confirm the prognostic value of the CXCR4∕α-SMA∕WASL panel in assessing the biological behavior of the upper lip BCC.

Actin polymerization controls cilia-mediated signaling.

Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42-aPKC-Gli axis in actin-dependent regulation of primary cilia signaling.

[N-WASP regulates cortical neuron migration through its polyPro and VCA domains].

Cortical neuron migration in the developing mouse forebrain is a complex process, which contains several steps related to cytoskeleton dynamics and remodeling. Neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP-WAVE family, regulates actin cytoskeleton reorganization through the binding of its VCA domain to the Arp2/3 complex. Here we report expression patterns of N-WASP gene in the mouse developing embryonic cortex (E12.5~ E18.5) and find its expression levels are decreased during embryonic development. By using in utero electroporation (IUE) method, we find that either N-WASP overexpression or knockdown impairs cortical neuron migration, and the defects of cortical neuron migration caused by N-WASP overexpression are much more severe than that by its knockdown. N-WASP protein contains four domains: WH1, GBD, polyPro, and VCA. We generated a series of dominant negative N-WASP mutants by modifying these domains. Overexpression of N-WASP mutant lacking domain polyPro, VCA, or WH1, impairs cortical neuron migration. However, overexpression of N-WASP with the H208D point mutation, which abolishes the Cdc42 binding to N-WASP, causes only a marginal defect of cortical neuron migration. Finally, overexpression of the individual domain polyPro or VCA, but not WH1, can recapitulate the defects by N-WASP overexpression. However, overexpression of WH1-GBD fragment has no apparent effect on cortical neuron migration. In conclusion, our data demonstrate that N-WASP regulates cortical neuron migration mainly through its polyPro and VCA domains.

Epigenetic control of IL-23 expression in keratinocytes is important for chronic skin inflammation.

The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation.

MicroRNA-214-5p Inhibits the Invasion and Migration of Hepatocellular Carcinoma Cells by Targeting Wiskott-Aldrich Syndrome Like.

BACKGROUND/AIMS: This study aims to explore the effects of microRNA-214-5p (miR-214-5p) on the invasion and migration of Hepatocellular Carcinoma cells (HCC). METHODS: Hepatocellular Carcinoma tissues and adjacent normal tissues from 44 hepatocellular carcinoma patients were prepared for this study. The HepG2 and BEL-7402 cells were transfected with miR-214-5p mimic and inhibitor. qRT-PCR was performed to detect the expressions of miR-214-5p. Transwell assays were used to detect the invasion and migration assays in HepG2 and BEL-7402 cells. A dual-luciferase reporter assay was conducted to examine the effect of miR-214-5p on Wiskott-Aldrich Syndrome Like (WASL/ N-WASP). Western blot and qRT-PCR were used to measure the expressions of the E-cadherin, N-cadherin and Vimentin proteins. Transwell chamber assays were performed to detect cell invasion and migration. RESULTS: Compared with normal tissues, HCC tissues demonstrated significantly lower expression of miR-214-5p. Overexpression of miR-214-5p significantly inhibited the migration and invasion of HCC cells and inhibition of miR-214-5p promoted the migration and invasion. Additionally, miR-214-5p suppressed the epithelial-mesenchymal transition (EMT). Further study showed WASL was a putative target gene of miR-214-5p. Up-regulating the expression of WASL could reverse the inhibition effect of miR-214-5p on invasion and migration. CONCLUSION: Our data suggested that miR-214-5p inhibited the invasion and migration of HepG2 and BEL-7402 by targeting WASL in Hepatocellular carcinoma.

Loss of N-WASP drives early progression in an Apc model of intestinal tumourigenesis.

N-WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N-WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N-WASP in the initiation and progression of colorectal cancer. While deletion of N-wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche, and migration up the crypt-villus axis was enhanced. Loss of N-wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N-WASP in early intestinal carcinogenesis despite its later pro-invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre-neoplastic tissues. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature.

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP-WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.