WASP Family Verprolin-homologous Protein Research Articles

The mutated cytoplasmic fragile X mental retardation 1 (FMR1)-interacting protein 2 (CYFIP2 S968F) regulates cocaine-induced reward behaviour and plasticity in the nucleus accumbens.

Cytoplasmic fragile X mental retardation 1 (FMR1)-interacting protein 2 (CYFIP2), as a component of the Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) regulatory complex, is involved in actin polymerization, contributing to neuronal development and structural plasticity. Mutating serine-968 to phenylalanine (S968F) in CYFIP2 causes an altered cocaine response in mice. The neuronal mechanisms underlying this response remain unknown. We performed cocaine reward-related behavioural tests and examined changes in synaptic protein phenotypes and neuronal morphology in the nucleus accumbens (NAc), using CYFIP2 S968F knock-in mice to investigate the role of CYFIP2 in regulating cocaine reward. CYFIP2 S968F mutation attenuated cocaine-induced behavioural sensitization and conditioned place preference. Cocaine-induced c-Fos was not observed in the NAc of CYFIP2 S968F knock-in mice. However, c-Fos induction was still evident in the medial prefrontal cortex (mPFC). CYFIP2 S968F mutation altered cocaine-associated CYFIP2 signalling, glutamatergic protein expression and synaptic density in the NAc following cocaine exposure. To further determine the role of CYFIP2 in NAc neuronal activity and the mPFC projecting to the NAc activity-mediating reward response, we used optogenetic tools to stimulate the NAc or mPFC-NAc pathway and observed that optogenetic activation of the NAc or mPFC-NAc pathway induced reward-related behaviours. This effect was not observed in the S968F mutation in CYFIP2. These results suggest that CYFIP2 plays a role in controlling cocaine-mediated neuronal function and structural plasticity in the NAc, and that CYFIP2 could serve as a target for regulating cocaine reward.

TRANSPARENT TESTA GLABRA2 defines trichome cell shape by modulating actin cytoskeleton in Arabidopsis thaliana.

The Arabidopsis (Arabidopsis thaliana) TRANSPARENT TESTA GLABRA2 (TTG2) gene encodes a WRKY transcription factor that regulates a range of development events like trichome, seed coat, and atrichoblast formation. Loss-of-function of TTG2 was previously shown to reduce or eliminate trichome specification and branching. Here, we report the identification of an allele of TTG2, ttg2-6. In contrast to the ttg2 mutants described before, ttg2-6 displayed unique trichome phenotypes. Some ttg2-6 mutant trichomes were hyper-branched, whereas others were hypo-branched, distorted, or clustered. Further, we found that in addition to specifically activating R3 MYB transcription factor TRIPTYCHON (TRY) to modulate trichome specification, TTG2 also integrated cytoskeletal signaling to regulate trichome morphogenesis. The ttg2-6 trichomes displayed aberrant cortical microtubules (cMTs) and actin filaments (F-actin) configurations. Moreover, genetic and biochemical analyses showed that TTG2 could directly bind to the promoter and regulate the expression of BRICK1 (BRK1), which encodes a subunit of the actin nucleation promoting complex suppressor of cyclic AMP repressor (SCAR)/Wiskott-Aldrich syndrome protein family verprolin homologous protein (WAVE). Collectively, taking advantage of ttg2-6, we uncovered a function for TTG2 in facilitating cMTs and F-actin cytoskeleton-dependent trichome development, providing insight into cellular signaling events downstream of the core transcriptional regulation during trichome development in Arabidopsis.

Differential Role of the RAC1-Binding Proteins FAM49b (CYRI-B) and CYFIP1 in Platelets.

Platelet function at vascular injury sites is tightly regulated through the actin cytoskeleton. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex (WRC) activates lamellipodia formation via ARP2/3, initiated by GTP-bound RAC1 interacting with the WRC subunit CYFIP1. The protein FAM49b (Family of Unknown Function 49b), also known as CYRI-B (CYFIP-Related RAC Interactor B), has been found to interact with activated RAC1, leading to the negative regulation of the WRC in mammalian cells. To investigate the role of FAM49b in platelet function, we studied platelet-specific Fam49b-/--, Cyfip1-/--, and Cyfip1/Fam49b-/--mice. Platelet counts and activation of Fam49b-/- mice were comparable to those of control mice. On fully fibrinogen-coated surfaces, Fam49b-/--platelets spread faster with an increased mean projected cell area than control platelets, whereas Cyfip1/Fam49b-/--platelets did not form lamellipodia, phenocopying the Cyfip1-/--platelets. However, Fam49b-/--platelets often assumed a polarized shape and were more prone to migrate on fibrinogen-coated surfaces. On 2D structured micropatterns, however, Fam49b-/--platelets displayed reduced spreading, whereas spreading of Cyfip1-/-- and Cyfip1/Fam49b-/--platelets was enhanced. In summary, FAM49b contributes to the regulation of morphology and migration of spread platelets, but to exert its inhibitory effect on actin polymerization, the functional WAVE complex must be present.

FXR1 regulates vascular smooth muscle cell cytoskeleton, VSMC contractility, and blood pressure by multiple mechanisms

Appropriate cytoskeletal organization is essential for vascular smooth muscle cell (VSMC) conditions such as hypertension. This study identifies FXR1 as a key protein linking cytoskeletal dynamics with mRNA stability. RNA immunoprecipitation sequencing (RIP-seq) in human VSMCs identifies that FXR1 binds to mRNA associated with cytoskeletal dynamics, and FXR1 depletion decreases their mRNA stability. FXR1 binds and regulates actin polymerization. Mass spectrometry identifies that FXR1 interacts with cytoskeletal proteins, particularly Arp2, a protein crucial for VSMC contraction, and CYFIP1, a WASP family verprolin-homologous protein (WAVE) regulatory complex (WRC) protein that links mRNA processing with actin polymerization. Depletion of FXR1 decreases the cytoskeletal processes of adhesion, migration, contraction, and GTPase activation. Using telemetry, conditional FXR1SMC/SMC mice have decreased blood pressure and an abundance of cytoskeletal-associated transcripts. This indicates that FXR1 is a muscle-enhanced WRC modulatory protein that regulates VSMC cytoskeletal dynamics by regulation of cytoskeletal mRNA stability and actin polymerization and cytoskeletal protein-protein interactions, which can regulate blood pressure.

Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.

Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Rac-mediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP–VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3.

Abstract 5986: WAVE2/EGF signaling and miR29 regulates WAVE2 to promote tumor growth and metastasis in triple negative breast cancer

Abstract Breast cancer is the most frequently diagnosed malignancy in women and is one of the leading causes of death due to cancer invasion, metastasis, and resistance to therapies. Among its variants, triple-negative breast cancer (TNBC) is considered the most aggressive due to its early age of diagnosis, invasive and metastatic properties with poor prognosis. TNBC is a type of breast cancer that lacks expression of the receptors that are commonly found in this type of cancer (estrogen, progesterone, and HER2), which makes it a more difficult target for treatment. Cancer invasiveness and metastasis are known to be promoted by increased cell motility and upregulation of the Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) - WAVE1, WAVE2, and WAVE3. While the contribution of WAVE2 to cancer progression is well documented, the WAVE2-mediated regulation of TNBC oncogenic properties is still under investigated. In this study, we show that WAVE2 plays a significant role in TNBC development, progression, and metastasis. To investigate this notion, we used CRISPR/Cas9 gene editing to successfully inhibit WAVE2 expression in several TNBC cell lines. In vitro, loss of WAVE2 expression inhibited 3D tumorsphere formation and invasion of TNBC cells. In vivo, in mouse models for TNBC, loss of WAVE2 inhibited tumor growth and metastasis. Mechanistically, we show that the WAVE2-mediated modulation of the oncogenic behavior of TNBC cells is in part due to its phosphorylation downstream of EGF. At the post transcriptional level, we found WAVE2 expression to be regulated by miR29. Together, our findings identify a novel post-translational WAVE2/EGF oncogenic signaling axis, along with a post-transcriptional regulatory axis downstream of miR29, which together regulate the oncogenic activity of WAVE2 in TNBC and open a new therapeutic strategy for the treatment of TNBC. Citation Format: Priyanka S. Rana, Wei Wang, Akram Alkrekshi, Vesna Markovic, Khalid Sossey-Alaoui. WAVE2/EGF signaling and miR29 regulates WAVE2 to promote tumor growth and metastasis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5986.

The Role of WAVE2 Signaling in Cancer.

The Wiskott–Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE)—WAVE1, WAVE2 and WAVE3 regulate rapid reorganization of cortical actin filaments and have been shown to form a key link between small GTPases and the actin cytoskeleton. Upon receiving upstream signals from Rho-family GTPases, the WASP and WAVE family proteins play a significant role in polymerization of actin cytoskeleton through activation of actin-related protein 2/3 complex (Arp2/3). The Arp2/3 complex, once activated, forms actin-based membrane protrusions essential for cell migration and cancer cell invasion. Thus, by activation of Arp2/3 complex, the WAVE and WASP family proteins, as part of the WAVE regulatory complex (WRC), have been shown to play a critical role in cancer cell invasion and metastasis, drawing significant research interest over recent years. Several studies have highlighted the potential for targeting the genes encoding either part of or a complete protein from the WASP/WAVE family as therapeutic strategies for preventing the invasion and metastasis of cancer cells. WAVE2 is well documented to be associated with the pathogenesis of several human cancers, including lung, liver, pancreatic, prostate, colorectal and breast cancer, as well as other hematologic malignancies. This review focuses mainly on the role of WAVE2 in the development, invasion and metastasis of different types of cancer. This review also summarizes the molecular mechanisms that regulate the activity of WAVE2, as well as those oncogenic pathways that are regulated by WAVE2 to promote the cancer phenotype. Finally, we discuss potential therapeutic strategies that target WAVE2 or the WAVE regulatory complex, aimed at preventing or inhibiting cancer invasion and metastasis.

Mechanism of WASP and WAVE family proteins in the progression of prostate cancer.

Prostate cancer (PCa) is the second most commonly diagnosed and third lethal cause of death from cancer in men worldwide. Despite the availability of vast treatment procedures, still the high occurrence of invasion and metastasis of PCa are reported in cancer patients. The WASP (Wiskott-Aldrich syndrome protein) and WAVE (WASP family verprolin homologous protein) family of proteins are actin cytoskeleton regulatory proteins, reported to enhance cancer cell invasion and migration in prostate cancer. Hence, this review sheds light on the studies that explored the potential role of WASP and WAVE family of proteins in invasion and metastasis of prostate cancer. The research articles explored for the completion of this review were mostly from PubMed and Google Scholar by using the appropriate keywords for indexing. The conserved function of WASP and WAVE protein family is to receive the upstream signals from the Rho GTPase family and transmit them to activate the Arp2/3 complex that leads to rapid actin polymerization at leading edge of cells, which is crucial for PCa metastasis. Therefore, targeting these proteins could reflect a very interesting therapeutic opportunity to combat prostate cancer.

STEM-03. WAVE1 KNOCKDOWN ENHANCES THE ANTITUMOR EFFICACY IN PRIMARY GLIOBLASTOMA NEUROSPHERES

Abstract INTRODUCTION Despite multi-model therapies that include maximal surgical resection, radiation, chemotherapy, and tumor treating fields, the median survival of Glioblastoma (GBM) patients is around 15 months. WASP-family verprolin homologous protein 1 (WAVE1) is a downstream effector that receives signals from small GTPases to regulate the actin cytoskeleton. WAVE1’s interaction with arp2/3 modulates critical roles, such as cell motility and morphologic changes. Expression of WAVE1 has been implicated in leukemia, ovarian, and prostate cancer. In this study, we tested the role of WAVE1 in GBM tumor biology. METHODS Expression of WAVE1 in normal brain and GBM tumor specimens was assessed by immunohistochemistry (IHC). The relevance of targeting WAVE1 for GBM therapy was evaluated in vitro by western blot, proliferation assay, cell cycle analysis, apoptosis assay, migration assay, and neurosphere formation assay using scrambled and target specific WAVE1-siRNA in patient-derived primary GBM neurospheres (GBMNS). RESULTS IHC data shows that the expression of WAVE1 is higher in GBM tumor samples than in low-grade gliomas and normal brain tissues. WAVE1 expression is upregulated in GBMNS compared to normal human astrocytes. WAVE1 knockdown significantly decreased the proliferation, migration, and self-renewal of GBMNS without affecting its cell cycle progression. Furthermore, WAVE1 depletion did not show any alterations in the apoptotic cell population, indicating that WAVE1 knockdown has a cytostatic effect on GBMNS. CONCLUSION Expression of WAVE1 positively correlates with GBM, and its knockdown imparts antitumor efficacy through cytostasis.

PTEN dephosphorylates Abi1 to promote epithelial morphogenesis.

The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study, we demonstrate that PTEN's protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN's lipid phosphatase activity or both PTEN's lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3-regulated actin cytoskeletal dynamics in epithelial morphogenesis.

Platelet lamellipodium formation is not required for thrombus formation and stability

AbstractDuring platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.

Cell confinement reveals a branched-actin independent circuit for neutrophil polarity.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.

Conditional disruption of Hematopoietic protein-1 in mice reveals an essential role for Hem-1 in myeloid cell functions

Abstract Hematopoietic protein1 (Hem-1) is a hematopoietic cell-specific subunit of WAVE (WASP-family verprolin homologous protein) complex, which acts downstream immune receptors (including BCR, TCR, TLR, and cytokine receptors) to stimulate filamentous actin (F-actin) polymerization. Inactivating mutations in NCKAP1L, the gene encoding Hem-1, have been recently associated with Primary Immunodeficiency Disease in humans (unpublished), and high NCKAP1L expression has been associated with a poor prognosis in Chronic Lymphocytic Leukemia. Using constitutive Hem-1 null mice, we previously published that Hem-1 is critical for normal lymphopoiesis and innate immunity. However, the cell autonomous functions of Hem-1 in individual immune cell types remain an enigma. The objective of this study was to define the cell autonomous roles of Hem-1 in myeloid cells. We created conditional Nckap1lfl/flmice, which were bred to LyzMCre mice to delete Hem-1 specifically in myeloid cells. We found that neutrophils from myeloid cell specific Hem-1 null mice exhibited defective F-actin polymerization and impaired migration in response to the chemoattractant fMLP. Hem-1 deficient macrophages were unable to efficiently phagocytose live bacteria. Myeloid specific Hem-1 null mice were much more susceptible to influenza virus (H1N1/PR8) infection based on significantly increased body weight loss, increased peribronchial inflammation, reduced alveolar macrophage number, and increased pro-inflammatory cytokines in bronchiolar lavage fluid. Collectively, our results reveal previously uncharacterized cell autonomous roles for Hem-1 in primary myeloid cells, and suggest that Hem-1 is critically important for effective anti-viral immunity.

De novo hotspot variants in CYFIP2 cause early-onset epileptic encephalopathy.

The cytoplasmic fragile X mental retardation 1 interacting proteins 2 (CYFIP2) is a component of the WASP-family verprolin-homologous protein (WAVE) regulatory complex, which is involved in actin dynamics. An obvious association of CYFIP2 variants with human neurological disorders has never been reported. Here, we identified de novo hotspot CYFIP2 variants in neurodevelopmental disorders and explore the possible involvement of the CYFIP2 mutants in the WAVE signaling pathway. We performed trio-based whole-exome sequencing (WES) in 210 families and case-only WES in 489 individuals with epileptic encephalopathies. The functional effect of CYFIP2 variants on WAVE signaling was evaluated by computational structural analysis and in vitro transfection experiments. We identified three de novo CYFIP2 variants at the Arg87 residue in 4 unrelated individuals with early-onset epileptic encephalopathy. Structural analysis indicated that the Arg87 residue is buried at an interface between CYFIP2 and WAVE1, and the Arg87 variant may disrupt hydrogen bonding, leading to structural instability and aberrant activation of the WAVE regulatory complex. All mutant CYFIP2 showed comparatively weaker interactions to the VCA domain than wild-type CYFIP2. Immunofluorescence revealed that ectopic speckled accumulation of actin and CYFIP2 was significantly increased in cells transfected with mutant CYFIP2. Our findings suggest that de novo Arg87 variants in CYFIP2 have gain-of-function effects on the WAVE signaling pathway and are associated with severe neurological disorders. Ann Neurol 2018;83:794-806.

Mangiferin inhibits cell migration and invasion through Rac1/WAVE2 signalling in breast cancer.

Breast tumour progression results from the advancement of the disease to a metastatic phenotype. Rac1 and Cdc42 belong to the Rho family of genes that, together with their downstream effectors, Wiskott-Aldrich Syndrome protein-family verprolin-homologous protein 2 (WAVE2) and Arp2/3, assume a vital part in cytoskeletal rearrangement and the arrangement of film projections that advance malignant cell relocation and invasion. Mangiferin is a characteristic polyphenolic compound from Mangifera indica L. (Anacardiaceae), ordinarily referred to as mango, that is consumed worldwide as a natural product, including culinary and seasoning applications. Mangiferin delays breast malignancy development and progression by inhibiting different signalling pathways required in mitogenic signalling and metastatic progression. Studies were performed to analyse the impact of mangiferin on Rac1/WAVE2 flagging, relocation and invasion in highly metastatic human MDA-MB-231 mammary cells. Additional studies led to the observation that comparative treatment with mangiferin caused marked reduction in tumour cell movement and invasion. Taken together, these discoveries demonstrate that mangiferin treatment adequately hinders Rac1/WAVE2 flagging and diminishes metastatic phenotypic expression in malignant mammary cells, indicating that mangiferin may provide a benefit as a novel restorative approach in the treatment of metastatic breast cancer.

Abstract 1053: Synergistic anticancer effects of combined γ-tocotrienol and pterostilbene is associated with a suppression in Rac1/WAVE 2 signaling in highly malignant breast cancer cells

Abstract Breast cancer is the most common cancer and the second leading cause of cancer death in American women. Rho GTPases play a crucial role in regulating different cellular functions, including cell proliferation, gene expression, cell morphology, actin polymerization, cell motility, metastasis and apoptosis. Wiskott-Aldrich syndrome protein family verprolin-homologous protein2 (WAVE2) is a member within the Wiskott-Aldrich syndrome protein (WASP) family displays a significant role in actin polymerization and cytoskeletal organization which, are essential for cellular migration and invasion. γ-Tocotrienol is a natural isoform within the vitamin E family that displays anticancer activity against a variety of cancer cell types. Pterostilbene is a natural dimethyl ether analog of resveratrol that has several beneficial effects such as anticancer activity. Studies were conducted to examine the effects of combined γ-tocotrienol and pterostilbene on Rac1/WAVE 2 signaling in mouse +SA and human MDA-MB231 breast cancer cells and cellular migration. Results show that combined treatment of γ-tocotrienol and pterostilbene caused a synergistic inhibition of both mouse +SA and human MDA-MB231 breast cancer cells growth, and corresponding reduction in Rac1/WAVE2 signaling as characterized by a significant inhibition in the levels of phospho-Rac1/cdc42, WAVE2, Arp2, and Arp3 expression. Additional studies indicated that this combination resulted in a significant inhibition in mammary cancer cell migration. In summary, these findings strongly suggest that combined treatment of γ-tocotrienol with pterostilbene may provide great benefit as a therapeutic approach in the treatment of metastatic breast cancer. Supported by a grant from the Louisiana Cancer Foundation. Citation Format: Ibrahim G. Algayadh, Paul William Sylvester. Synergistic anticancer effects of combined γ-tocotrienol and pterostilbene is associated with a suppression in Rac1/WAVE 2 signaling in highly malignant breast cancer cells [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 1053. doi:10.1158/1538-7445.AM2017-1053

A conformational change within the WAVE2 complex regulates its degradation following cellular activation

WASp family Verprolin-homologous protein-2 (WAVE2), a member of the Wiskott-Aldrich syndrome protein (WASp) family of actin nucleation promoting factors, is a central regulator of actin cytoskeleton polymerization and dynamics. Multiple signaling pathways operate via WAVE2 to promote the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. WAVE2 exists as a part of a pentameric protein complex known as the WAVE regulatory complex (WRC), which is unstable in the absence of its individual proteins. While the involvement of WAVE2 in actin polymerization has been well documented, its negative regulation mechanism is poorly characterized to date. Here, we demonstrate that WAVE2 undergoes ubiquitylation in a T-cell activation dependent manner, followed by proteasomal degradation. The WAVE2 ubiquitylation site was mapped to lysine 45, located at the N-terminus where WAVE2 binds to the WRC. Using Förster resonance energy transfer (FRET), we reveal that the autoinhibitory conformation of the WRC maintains the stability of WAVE2 in resting cells; the release of autoinhibition following T-cell activation facilitates the exposure of WAVE2 to ubiquitylation, leading to its degradation. The dynamic conformational structures of WAVE2 during cellular activation dictate its degradation.

Mir-181c Modulates T Cell Function By Regulating the Expression of BRK1

IntroductionMicroRNAs (miRNAs) are short endogenous non-coding RNAs consisting of 18-25 nucleotides in length which influence gene expression and play pivotal roles in a diverse range of cellular processes. Aberrant miRNA expression has been implicated in a variety of cancers, including haematological malignancies. The miR-181 family plays a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation and both B and T cell development and differentiation. We therefore focused our study on validating novel downstream targets of miR-181.MethodsA novel functional assay utilising an optimised 3’UTR enriched library and a dual selection strategy (Gäken et al., 2012) was performed to identify biologically relevant targets of miR-181c. BRK1 (BRICK1, SCAR/WAVE Actin Nucleating Complex Subunit) was identified as a potential target and validation was performed by quantitative real time PCR and western blot analysis. Given the potential role of BRK1 in the Wiskott-Aldrich Syndrome Protein Family Verprolin-Homologous Protein-2 (WAVE2) complex and actin polymerisation in T cells, we investigated the influence of the miR-181c-BRK1 axis on T cell function. Knockdown of BRK1, using short hairpin RNA (shRNA) lentiviral vectors, and overexpression of miR-181c, via transfection with miR-181c expression vectors, were performed in Jurkat and primary T cells. T cell activation was examined by measurement of CD69 and CD154 expression and actin polymerisation was quantified by total cellular F-actin content. Immune synapse formation was studied by conjugate formation between T cells and antigen-pulsed B cells. Lastly, lamellipodia formation was investigated by assessing the ability of T cells to spread on anti-CD3 coated slides.ResultsTarget genes downregulated by miR-181c were identified. One such target was BRK1, a component of the WAVE2 complex that has been shown to play a pivotal role in actin polymerisation. Validation experiments showed that overexpression and inhibition of miR-181c had no impact on BRK1 mRNA expression but did in fact modulate protein expression, suggesting that miR-181c regulates BRK1 at the translational level.We demonstrated that primary T cell activation resulted in downregulation of miR-181c and upregulation of BRK1 protein expression, further strengthening our hypothesis that the miR-181c-BRK1 axis may play an important role in T cell activation. Next, we found that loss of BRK1 resulted in reduced T cell activation as shown by decreased expression of CD69 and CD154. Furthermore, we showed that downregulation of BRK1 expression by shRNA resulted in reduced actin polymerisation after T cell stimulation. Reduced expression of BRK1 led to a marked reduction in the total area (in square micrometers) of F-actin accumulation at T cell contact sites and synapses with B cells indicating defective immune synapse formation. Moreover, reduced BRK1 expression resulted in defect in lamellipodia formation in response to T cell receptor stimulation. Similarly, ectopic expression of miR-181c in Jurkat T cells also led to a reduction in T cell activation and actin polymerisation coupled with defects in immune synapse and lamellipodia formation, hence confirming the important role of the miR-181c-BRK1 axis in T cell activation. Lastly, we demonstrated that suppression of BRK1 induced reduced expression of other pivotal proteins in the WAVE2 complex including WAVE2, Abi1 and Sra1. This suggests that impairment of actin polymerisation-dependent T cell functions were a result of instability of the WAVE2 complex following BRK1 suppression.ConclusionFor the first time, we hereby demonstrate that BRK1 is a target of miR-181c. Moreover, we have highlighted the potential role of the miR-181c-BRK1 axis in impaired actin polymerisation-dependent T cell function and immune synapse formation. Deregulation of the miR-181c-BRK1 axis requires further evaluation in haematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

LysoPCs induce Hck- and PKCδ-mediated activation of PKCγ causing p47phox phosphorylation and membrane translocation in neutrophils.

Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKCγ knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET)+ interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47phox Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47phox.

Alzheimer-related decrease in CYFIP2 links amyloid production to tau hyperphosphorylation and memory loss.

Characteristic features of Alzheimer's disease are memory loss, plaques resulting from abnormal processing of amyloid precursor protein (APP), and presence of neurofibrillary tangles and dystrophic neurites containing hyperphosphorylated tau. Currently, it is not known what links these abnormalities together. Cytoplasmic FMR1 interacting protein 2 (CYFIP2) has been suggested to regulate mRNA translation at synapses and this may include local synthesis of APP and alpha-calcium/calmodulin-dependent kinase II, a kinase that can phosphorylate tau. Further, CYFIP2 is part of the Wiskott-Aldrich syndrome protein-family verprolin-homologous protein complex, which has been implicated in actin polymerization at synapses, a process thought to be required for memory formation. Our previous studies on p25 dysregulation put forward the hypothesis that CYFIP2 expression is reduced in Alzheimer's disease and that this contributes to memory impairment, abnormal APP processing and tau hyperphosphorylation. Here, we tested this hypothesis. First, in post-mortem tissue CYFIP2 expression was reduced by ∼50% in severe Alzheimer's hippocampus and superior temporal gyrus when normalized to expression of a neuronal or synaptic marker protein. Interestingly, there was also a trend for decreased expression in mild Alzheimer's disease hippocampus. Second, CYFIP2 expression was reduced in old but not in young Tg2576 mice, a model of familial Alzheimer's disease. Finally, we tested the direct impact of reduced CYFIP2 expression in heterozygous null mutant mice. We found that in hippocampus this reduced expression causes an increase in APP and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) protein, but not mRNA expression, and elevates production of amyloid-β42 Reduced CYFIP2 expression also increases alpha-calcium/calmodulin-dependent kinase II protein expression, and this is associated with hyperphosphorylation of tau at serine-214. The reduced expression also impairs spine maturity without affecting spine density in apical dendrites of CA1 pyramidal neurons. Furthermore, the reduced expression prevents retention of spatial memory in the water maze. Taken together, our findings indicate that reduced CYFIP2 expression triggers a cascade of change towards Alzheimer's disease, including amyloid production, tau hyperphosphorylation and memory loss. We therefore suggest that CYFIP2 could be a potential hub for targeting treatment of the disease.