CD147 regulates cancer migration via direct interaction with Annexin A2 and DOCK3-ß-catenin-WAVE2 signaling.

The acquisition of inappropriate migratory feature is crucial for tumor metastasis. It has been suggested that CD147 and Annexin A2 are involved in regulating tumor cell movement, while the regulatory mechanisms are far from clear. In this study, we demonstrated that CD147 physically interacted with the N-terminal domain of Annexin A2 and decreased Annexin A2 phosphorylation on tyrosine 23. In vitro kinase assay showed that the I domain of CD147 was indispensable for CD147-mediated downregulation of Annexin A2 phosphorylation by Src. Furthermore, we determined that p-Annexin A2 promoted the expression of dedicator of cytokinesis 3 (DOCK3) and DOCK3 blocked ß-catenin nuclear translocation, resulting in inhibition of ß-catenin signaling. In addition, DOCK3 inhibited lamellipodium dynamics and tumor cell movement. Also, we found that ß-catenin signaling increased WAVE2 expression. Therefore, DOCK3 was characterized as a negative regulator of WAVE2 expression via inhibiting ß-catenin signaling. Our study provides the first evidence that CD147 promotes tumor cell movement and metastasis via direct interaction with Annexin A2 and DOCK3-ß-catenin-WAVE2 signaling axis.

Involvement of the Arp2/3 complex and WASP proteins in the effect of glutoxim and molixan on intracellular Ca(2+) concentration in macrophages.

The Fura-2AM fluorescent Ca(2+) probe was used to study the possibility that the Arp2/3 complex and WASP proteins are involved in the effects of glutoxim and molixan on the intracellular Ca(2+) concentration in macrophages. It has been demonstrated that preincubation of macrophages with inhibitors of the Arp2/3 complex or WASP proteins (CK-0944666 or wiskostatin, respectively) results in a significant suppression of Ca(2+)-responses induced by glutoxim or molixan. This suggests that polymerization of actin filaments is a process involved in the effect of glutoxim or molixan on intracellular Ca(2+) concentration in macrophages.

Inhibition of invasion by glycogen synthase kinase-3 beta inhibitors through dysregulation of actin re-organisation via down-regulation of WAVE2.

Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3ß (GSK-3ß) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3ß on invasion is unclear. In this report, we showed that GSK-3ß inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3ß inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3ß inhibitors in cancer cell invasion.

MiR-200b modulates the properties of human monocyte-derived dendritic cells by targeting WASF3.

AIMS: The aim of the study was to explore the effect of miR-200b on the development of human peripheral blood monocyte-deriveddendritic-cell (DC) and its mechanisms. MAIN METHODS: Expression levels of miR-200b and its predicted targets were measured by real time-PCR. Protein expression of WASF3 was determined by Western blot and immunohistochemistry. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation from the buffy coat fraction of anticoagulated blood. Monocytes were purified from PBMCs using anti-CD14 microbeads. The immunophenotypes of DCs were tested by flow cytometry. KEY FINDINGS: A strong reduction in miR-200b expression was associated with human peripheral blood monocyte-derivedDC differentiation. The overexpression of miR-200b significantly reduced the numbers of protruding veils in mature DCs (mDCs) that are critical for promoting antigen-specificT-cell activation. Further experiments showed that miR-200b could regulate the function of DCs by targeting WASF3, a protein involved in cell movement and invasion. SIGNIFICANCE: Our results define an important function of miR-200b in the negative regulation of DC development and provide a potential form of miRNA-mediated cell therapy for diseases that range from auto-immunity to graft-versus-host disease.

Structure and role of WASP and WAVE in Rho GTPase signalling in cancer.

A major factor controlling the metastatic nature of cancer cells is their motility. Alterations in the signalling pathways controlling its regulation can lead to tumor cell invasion and metastasis. Directional motility involves protrusion of the cell's leading edge, via formation of filopodia and lamellipodia, adhesion to the substrate followed by tail retraction and de-adhesion. Rho GTPase binding proteins function as activators of the actin cytoskeleton and are key players in the transendothelial migration of cancer cells. Activation of the specific GTPases Rho, Rac1 and Cdc42 results in formation of actin stress fibres, membrane ruffles, lamellipodia and filopodia respectively and in cortical actin assembly. Pathways through which Rho GTPases elicit these effects are through direct interaction with members of the Wiskott-Alrich Syndrome Protein (WASP) family which stimulates structures such as lamellipodia and filopodia. The present review explores the role and function of Rho GTPases, WASP and WAVE in cancer metastasis.

Urokinase-type plasminogen activator expression and Rac1/WAVE-2/Arp2/3 pathway are blocked by pterostilbene to suppress cell migration and invasion in MDA-MB-231 cells.

Breast cancer is the most common malignancy among females, and cancer invasion and metastasis are the leading causes of cancer death in breast cancer patients. Pterostilbene, a naturally occurring dimethylether analogue of resveratrol, has been demonstrated to possess anti-cancer effects. However, inhibitory effects of pterostilbene on cell migration and invasion and its underlying mechanisms are not fully understood. In this study, we investigated the anti-invasive mechanisms of pterostilbene in human breast cancer cell line MDA-MB-231 cells. Pterostilbene effectively inhibited serum-induced migration and invasion without affecting the viability of breast cancer cells. The mRNA expression and activity of urokinase-type plasminogen activator (uPA) were markedly reduced by pterostilbene treatment. Moreover, pterostilbene attenuated nuclear factor κB (NF-κB) transcriptional activity and DNA binding of NF-κB on uPA promoter. In addition, pterostilbene significantly impaired the activity of Rac1 and the expression of WASP-family verprolin-homologous protein-2 (WAVE-2) and actin-related protein 2/3 (Arp2/3). Overall, these results suggest that pterostilbene caused considerable suppression of cell migration and invasion through blocking NF-κB-mediated uPA expression and Rac1/WAVE/Arp2/3 pathway.

Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation.

Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti-Buruli ulcer therapeutic potential.

Role of Bcl-2 in tumour cell survival and implications for pharmacotherapy.

OBJECTIVES: Bcl-2 is a protein that inhibits apoptosis, leading to cell survival. The Bcl-2 family has six different anti-apoptotic proteins, three pro-apoptotic proteins that are similar in structure, and other integrating proteins that function as promotors or inhibitors in the progression of apoptosis. In this discussion paper, we provide an overview of apoptosis, the role of Bcl-2 in normal cellular and molecular processes, and the role of Bcl-2 in tumour cell survival. It focuses primarily on anti-apoptotic Bcl-2, its activation in cancer, the manner in which it regulates the intrinsic and extrinsic mechanisms of apoptosis, and its broad molecular interactions with other critical proteins in the cell. Certain cancer treatments are reviewed and related directions for the future are presented. KEY FINDINGS: Apoptosis is common to all organisms - for eukaryotes it is a normal process of development and regeneration. The rate at which apoptosis occurs is critical to the survival of the organism, as too much can lead to the onset of degenerative diseases such as dementia, and too little may lead to cancer. FKBP-38 is a binding protein that has been discovered to be upregulated in highly aggressive cancers and binds to Bcl-2 rather than the pro-apoptotics to induce a state of hyper-mitosis. A short binding protein (Nur-77) provides new insights into Bcl-2 'masking'. Nurr-77 binds to Bcl-2 and exposes the BH3 domain, transforming it from a cancer promoter to an unorthodox cancer inhibitor. This presents in itself an interesting and exciting opportunity - increasing the rate of apoptosis in neoplastic cells that are usually protected by Bcl-2 activity at the mitochondria. SUMMARY: Development of drugs in the form of BH3-only and BH123 mimetic drugs provide a interesting avenue for cancer therapy for the future. Drugs that can either promote, or mimic anti-IAP activity such as Smac/Diablo would certainly be productive, thereby inducing apoptosis. Medicinal usage which can effectively suppress FKBP38 in Bcl-2-dependent cancers would provide further arsenal to combat apoptotic irregularities, particularly a treatment that is more dominant than kinetin riboside. WAVE-1 inhibitors may effectively suppress the phosphorylation of Bcl-2, thereby potentially reducing hyper-mitosis and increasing apoptosis. Recent findings shed molecular light on PDT, namely ER stress, and potential for anti-cancer therapy via either apoptosis or autophagy. A drug that can effectively upregulate Nurr-77, thereby masking the anti-apoptotic properties of Bcl-2, would indeed be life-saving for cancer patients.

Scar/WAVE3 contributes to motility and plasticity of lamellipodial dynamics but not invasion in three dimensions.

The Scar (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein) verprolin homologous] complex plays a major role in the motility of cells by activating the Arp2/3 complex, which initiates actin branching and drives protrusions. Mammals have three Scar/WAVE isoforms, which show some tissue-specific expression, but their functions have not been differentiated. In the present study we show that depletion of Scar/WAVE3 in the mammalian breast cancer cells MDA-MB-231 results in larger and less dynamic lamellipodia. Scar/WAVE3-depleted cells move more slowly but more persistently on a two-dimensional matrix and they typically only show one lamellipod. However, Scar/WAVE3 appears to have no role in driving invasiveness in a three-dimensional Matrigel™ invasion assay or a three-dimensional collagen invasion assay, suggesting that lamellipodial persistence as seen in two-dimensions is not crucial in three-dimensional environments.

The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

BACKGROUND: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. PRINCIPAL FINDINGS: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. CONCLUSIONS: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

The WAVE regulatory complex is inhibited.

The WAVE regulatory complex (WRC) transmits information from the Rac GTPase to the actin nucleator Arp2/3 complex. We have reconstituted recombinant human and Drosophila WRC in several forms and shown that they are inactive toward Arp2/3 complex but can be activated by Rac in a nucleotide-dependent fashion. Our observations identify core components needed for WAVE inhibition, reconcile contradictory existing mechanisms and reveal common regulatory principles for the WAVE/WASP family of proteins.

N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts.

The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1(+/+) and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP(-/-) fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.