Expression Of Arp2 Research Articles

Sulforaphane suppresses bladder cancer metastasis via blocking actin nucleation-mediated pseudopodia formation

Metastasis is the primary stumbling block to the treatment of bladder cancer (BC). In order to spread, tumor cells must acquire increased migratory and invasive capacity, which is tightly linked with pseudopodia formation. Here, we unravel the effects of sulforaphane (SFN), an isothiocyanate in cruciferous vegetables, on the assembly of pseudopodia and BC metastasis, and its molecular mechanism in the process. Our database analysis revealed that in bladder tumor, pseudopodia-associated genes, CTTN, WASL and ACTR2/ARP2 are upregulated. SFN caused lamellipodia to collapse in BC cells by blocking the CTTN-ARP2 axis. SFN inhibited invadopodia formation and cell invasion by reducing WASL in different invasive BC cell lines. The production of ATP, essential for the assembly of pseudopodia, was significantly increased in bladder tumors and strongly inhibited by SFN. Overexpressing AKT1 reversed the downregulation of ATP in SFN-treated bladder cancer cells and restored filopodia and lamellipodia morphology and function. Bioluminescent imaging showed that SFN suppressed BC metastases to the lung of nude mice while downregulating Cttn and Arp2 expression. Our study thus reveals mechanisms of SFN action in inhibiting pseudopodia formation and highlights potential targeting options for the therapy of metastatic bladder cancer.

Radiation-induced eCIRP impairs macrophage bacterial phagocytosis.

Macrophages are essential immune cells for host defense against bacterial pathogens after radiation injury. However, the role of macrophage phagocytosis in infection following radiation injury remains poorly examined. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that dysregulates host immune system responses such as phagocytosis. We hypothesized that radiation-induced eCIRP release impairs macrophage phagocytosis of bacteria. Adult healthy mice were exposed to 6.5-Gy total body irradiation (TBI). Primary peritoneal macrophages isolated from adult healthy mice were exposed to 6.5-Gy radiation. eCIRP-neutralizing monoclonal antibody (mAb) was added to the cell culture prior to irradiation. Bacterial phagocytosis by peritoneal macrophages was assessed using pHrodo Green-labeled E. coli 7 days after irradiation ex vivo and in vitro. Bacterial phagocytosis was also assessed after treatment with recombinant murine CIRP (rmCIRP). Rac1 and ARP2 protein expression in cell lysates and eCIRP levels in the peritoneal lavage were assessed by Western blotting. Bacterial phagocytosis by peritoneal macrophages was significantly decreased after irradiation compared to controls ex vivo and in vitro. Rac1 and ARP2 expression in the peritoneal macrophages were downregulated after TBI. TBI significantly increased eCIRP levels in the peritoneal cavity. rmCIRP significantly decreased bacterial phagocytosis in a dose-dependent manner. eCIRP mAb restored bacterial phagocytosis by peritoneal macrophages after irradiation. Ionizing radiation exposure impairs bacterial phagocytosis by macrophages after irradiation. Neutralization of eCIRP restores the phagocytic ability of macrophages after irradiation. Our findings elucidate a novel mechanism of immune dysfunction and provide a potential new therapeutic approach for limiting infection after radiation injury.

ERVWE1 Reduces Hippocampal Neuron Density and Impairs Dendritic Spine Morphology through Inhibiting Wnt/JNK Non-Canonical Pathway via miR-141-3p in Schizophrenia.

Human endogenous retroviruses (HERVs) are remnants of ancestral germline infections by exogenous retroviruses. Human endogenous retroviruses W family envelope gene (HERV-W env, also called ERVWE1), located on chromosome 7q21-22, encodes an envelope glycoprotein from the HERV-W family. Mounting evidence suggests that aberrant expression of ERVWE1 involves the etiology of schizophrenia. Moreover, the genetic and morphological studies indicate that dendritic spine deficits may contribute to the onset of schizophrenia. Here, we reported that ERVWE1 changed the density and morphology of the dendritic spine through inhibiting Wingless-type (Wnt)/c-Jun N-terminal kinases (JNK) non-canonical pathway via miR-141-3p in schizophrenia. In this paper, we found elevated levels of miR-141-3p and a significant positive correlation with ERVWE1 in schizophrenia. Moreover, serum Wnt5a and actin-related protein 2 (Arp2) levels decreased and demonstrated a significant negative correlation with ERVWE1 in schizophrenia. In vitro experiments disclosed that ERVWE1 up-regulated miR-141-3p expression by interacting with transcription factor (TF) Yin Yang 1 (YY1). YY1 modulated miR-141-3p expression by binding to its promoter. The luciferase assay revealed that YY1 enhanced the promoter activity of miR-141-3p. Using the miRNA target prediction databases and luciferase reporter assays, we demonstrated that miR-141-3p targeted Wnt5a at its 3' untranslated region (3' UTR). Furthermore, ERVWE1 suppressed the expression of Arp2 through non-canonical pathway, Wnt5a/JNK signaling pathway. In addition, ERVWE1 inhibited Wnt5a/JNK/Arp2 signal pathway through miR-141-3p. Finally, functional assays showed that ERVWE1 induced the abnormalities in hippocampal neuron morphology and spine density through inhibiting Wnt/JNK non-canonical pathway via miR-141-3p in schizophrenia. Our findings indicated that miR-141-3p, Wnt5a, and Arp2 might be potential clinical blood-based biomarkers or therapeutic targets for schizophrenia. Our work also provided new insight into the role of ERVWE1 in schizophrenia pathogenesis.

SKAP2 is downregulated in the villous tissues of patients with missed abortion and regulates growth and migration in trophoblasts through the WAVE2-ARP2/3 signaling pathway

IntroductionAbnormal placental trophoblast function is the main cause of missed abortion (MA). Src kinase-associated phosphoprotein 2 (SKAP2) indirectly affects actin reunion, which is significantly associated with cell migration. MethodsTwenty women with MA and 20 healthy women who underwent voluntarily induced abortion were included in this study. Immunohistochemistry, qRT-PCR, and western blotting were used to determine SKAP2, WAVE2, and ARP2 expression in the villous tissues. We investigated the effects of SKAP2 and the W336K mutant (blocked SKAP2 Src homology 3 function) on growth and migration in HTR8/SVneo cells using the CCK8 assay, flow cytometry, and transwell assay. The effects of SKAP2 on the WAVE2-ARP2/3 signaling pathway in HTR8/SVneo cells were evaluated by western blotting and immunofluorescence. ResultsCompared to the women in the voluntary abortion group, SKAP2 and WAVE2 expression levels were downregulated in the villous of patients with MA. In HTR8/SVneo cells, SKAP2 siRNA silencing regulated the growth and migration, while SKAP2 overexpression promoted growth and migration, and inhibited apoptosis. Additionally, SKAP2 regulated the expression of WAVE2 and ARP2, as well as the colocalization of actin with WAVE2. The SKAP2 W336K mutant could not alter WAVE2 and ARP2 expression, nor HTR8/SVneo cell growth and migration, with or without SKAP2 siRNA transfection. DiscussionSKAP2 could activate the WAVE2-ARP2/3 pathway resulting in an increase of growth and migration in trophoblasts. SKAP2 probably played an important role in MA by affecting the growth and migration of trophoblasts.

MiR-195 regulates intestinal epithelial restitution after wounding by altering actin-related protein-2 translation.

Early gut epithelial restitution reseals superficial wounds after acute injury, but the exact mechanism underlying this rapid mucosal repair remains largely unknown. MicroRNA-195 (miR-195) is highly expressed in the gut epithelium and involved in many aspects of mucosal pathobiology. Actin-related proteins (ARPs) are key components essential for stimulation of actin polymerization and regulate cell motility. Here, we reported that miR-195 modulates early intestinal epithelial restitution by altering ARP-2 expression at the translation level. miR-195 directly interacted with the ARP-2 mRNA, and ectopically expressed miR-195 decreased ARP-2 protein without effect on its mRNA content. In contrast, miR-195 silencing by transfection with anti-miR-195 oligo increased ARP-2 expression. Decreased ARP-2 levels by miR-195 overexpression were associated with an inhibition of early epithelial restitution, as indicated by a decrease in cell migration over the wounded area. Elevation of cellular ARP-2 levels by transfection with its transgene restored cell migration after wounding in cells overexpressing miR-195. Polyamines were found to decrease miR-195 abundance and enhanced ARP-2 translation, thus promoting epithelial restitution after wounding. Moreover, increasing the levels of miR-195 disrupted F-actin cytoskeleton organization, which was prevented by ARP2 overexpression. These results indicate that miR-195 inhibits early epithelial restitution by decreasing ARP-2 translation and that miR-195 expression is negatively regulated by cellular polyamines.

MFG-E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose-Dependent Mediation of Actin Polymerization.

BackgroundMigration of vascular smooth muscle cells (VSMCs) is the main contributor to neointimal formation. The Arp2/3 (actin‐related proteins 2 and 3) complex activates actin polymerization and is involved in lamellipodia formation during VSMC migration. Milk fat globule‐epidermal growth factor 8 (MFG‐E8) is a glycoprotein expressed in VSMCs. We hypothesized that MFG‐E8 regulates VSMC migration through modulation of Arp2/3‐mediated actin polymerization.Methods and ResultsTo determine whether MFG‐E8 is essential for VSMC migration, a model of neointimal hyperplasia was induced in the common carotid artery of wild‐type and MFG‐E8 knockout mice, and the extent of neointimal formation was evaluated. Genetic deletion of MFG‐E8 in mice attenuated injury‐induced neointimal hyperplasia. Cultured VSMCs deficient in MFG‐E8 exhibited decreased cell migration. Immunofluorescence and immunoblotting revealed decreased Arp2 but not Arp3 expression in the common carotid arteries and VSMCs deficient in MFG‐E8. Exogenous administration of recombinant MFG‐E8 biphasically and dose‐dependently regulated the cultured VSMCs. At a low concentration, MFG‐E8 upregulated Arp2 expression. By contrast, MFG‐E8 at a high concentration reduced the Arp2 level and significantly attenuated actin assembly. Arp2 upregulation mediated by low‐dose MFG‐E8 was abolished by treating cultured VSMCs with β1 integrin function‐blocking antibody and Rac1 inhibitors. Moreover, treatment of the artery with a high dose of recombinant MFG‐E8 diminished injury‐induced neointimal hyperplasia and reduced VSMC migration.ConclusionsMFG‐E8 plays a critical role in VSMC migration through dose‐dependent regulation of Arp2‐mediated actin polymerization. These findings suggest that high doses of MFG‐E8 may have therapeutic potential for treating vascular occlusive diseases.

Arp2/3 Complex Is Expressed in Oral Squamous Cell Carcinoma: An Immunohistochemical Study of 88 Cases

Background: We investigated the immunohistochemical expression of Arp2/3 complex in 88 oral squamous cell carcinomas (OSCCs) and correlated it with the tumor type and grade. Materials and methods: This is a retrospective study of 88 cases of OSCCs (9 verrucous carcinomas, 4 carcinomas in situ, 37 well differentiated OSCCs, 32 moderately differentiated OSCCs, and 6 poorly differentiated OSCCs), stained with mouse monoclonal antibodies for Arp2 and Arp3. Staining was graded according to the extent of positivity by two observers. Results: Arp2 positivity was seen in 79 (89.77%) cases and Arp3 positivity in 78 (88.63%) cases. In positive cases, Arp2 expression was graded as (+) in 12.5%, (++) in 28.4% and (+++) in 48.86% of the cases; Arp3 was graded as (+) in 36.36%, (++) in 30.68% and (+++) in 21.59% of the cases. Conclusion: Expression of Arp2 and Arp3, indicative of the formation of the Arp2/3 complex, was seen in most OSCCs examined. Although no statistically significant correlation was found between the immunohistochemical expression of Arp2/Arp3 and tumor type or grade, the more common expression in invasive OSCCs than in non-invasive verrucous carcinomas and carcinomas in situ is indicative of its involvement in the biological behaviour of the lesions. The availability of Arp2/3 inhibitors that could be used in clinical practice warrants further study of the expression of Arp2/3 in OSCC.

Fasudil inhibits actin polymerization and collagen synthesis and induces apoptosis in human urethral scar fibroblasts via the Rho/ROCK pathway.

PurposeTo examine the effects and mechanism of action of fasudil on cytoskeletal polymerization, collagen synthesis, and apoptosis in fibroblasts derived from human urethral scar tissue.Materials and methodsFibroblasts treated with or without transforming growth factor β1 (TGF-β1, 10 ng/mL) were incubated with fasudil (12.5, 25, 50 μmol/L) for 24 hours. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression of Arp2, Arp3, WASP, and WAVE2. Collagen I and III protein levels were also evaluated by Western blotting. The filamentous actin cytoskeleton was examined by immunofluorescence and epifluorescence microscopy. An Annexin V-FITC/PI staining assay was used to investigate apoptosis.ResultsTGF-β1-dependent induction of actin polymerization and collagen synthesis and promotion of apoptosis were dose dependent. When compared with untreated controls, fasudil significantly decreased the expression of Arp2, Arp3, WASP, WAVE2, Collagen I, and Collagen III in cells treated with or without TGF-β1. Fasudil also promoted apoptosis in cells, irrespective of TGF-β1 treatment.ConclusionIrrespective of TGF-β1 activation status, fasudil suppressed actin polymerization and collagen synthesis and induced apoptosis in human urethral scar fibroblasts via the Rho/ROCK signaling pathway.

Upregulation of Arp2 expression is associated with the prognosis and prediction of lymph node metastasis in bladder urothelial carcinoma.

ObjectiveUpregulation of actin-related protein 2/3 complex subunit 2 (Arp2) is observed in various tumors, but its expression pattern has not been revealed in bladder urothelial carcinoma (BUC). The purpose of this study was to investigate the role of Arp2 expression in the pathological features and the prognosis as well as lymph node metastasis of BUC.MethodsA total of 228 tissue specimens from BUC patients who underwent a radical cystectomy were collected. In addition, 40 lymph node specimens and 40 normal bladder tissue specimens as controls were used. All of the specimens were used to construct a tissue microarray and were subsequently subjected to immunohistochemical staining for Arp2 expression. Logistic and Cox regression analyses and Kaplan–Meier curve analysis were applied to analyze the relation between Arp2 expression and multiple clinical features of patients with BUC.ResultsImmunohistochemical staining observation showed that Arp2 was mainly expressed in the cytoplasm and nucleus of positive cells and that Arp2 expression in BUC was significantly higher than that in normal bladder tissues. Arp2 expression in BUC tissues was associated with tumor size, tumor multiplicity, tumor stage, tumor grade, and lymph node metastasis (P < 0.05). Logistic regression analysis demonstrated that Arp2 expression was an independent risk factor for lymph node metastasis of BUC (P < 0.05). Kaplan–Meier curve analysis showed that increased Arp2 expression was associated with a shortened recurrence-free survival (RFS) and overall survival (OS) of BUC patients after radical cystectomy (P < 0.05).ConclusionThese findings suggest that Arp2 is significantly upregulated in BUC tissues when compared with normal bladder tissues, and that Arp2 expression is an independent predictor for lymph node metastasis, RFS, and OS.

Citrinin exposure affects oocyte maturation and embryo development by inducing oxidative stress-mediated apoptosis.

Citrinin is one of the mycotoxins and has been shown to have various toxic effects in animals and humans. Although previous study showed the toxic effects of citrinin on the female reproductive system, especially on oocyte maturation, however, the causes or mechanism of citrinin on oocyte quality is unclear. In present study we deeply investigated this topic. We found thatcitrinin toxin exposure inhibited mouse oocyte maturation and early embryo development. Further investigation showed that the actin distribution in oocytes and embryos was disrupted, and the reduced expression of actin nucleator ARP2 expression in the oocyte cortex further confirmed this. We also found that meiotic spindle morphology was abnormal after citrinin treatment. These results indicated that citrinin toxin exposure could disrupt cytoskeleton dynamics to affect oocyte maturation and early embryo development. We also examined the ROS level and early apoptosis marker Annexin signals, and the results showed that both levels increased, indicating that citrinin induced oxidative stress and further resulted in oocyte early apoptosis. Taken together, our results indicated that citrinin toxin exposure could reduce mouse oocyte maturation and early embryo development capability by affecting cytoskeletal dynamics, which may be due to the oxidative stress induced early apoptosis.

Decreased expression of cortactin in the schizophrenia brain.

Schizophrenia is a severe psychiatric disorder that is characterized by a wide array of symptoms and a complex neuropathology. A well-characterized neurobiological feature of schizophrenia is abnormal synaptic plasticity, although the mechanisms underlying this are not fully understood. Numerous studies have demonstrated a link between proper functioning of the cytoskeleton and synaptic plasticity. The actin-related protein-2/3 (Arp2/3) complex is responsible for the nucleation of new actin filaments and elongation of existing actin filaments and is thus crucial to cytoskeletal dynamics, especially actin polymerization and organization. To determine whether the Arp2/3 complex is abnormally expressed in schizophrenia, we measured the protein expression of Arp2 and Arp3, as well as Arp2/3 complex binding partners and associated proteins including cortactin, neuronal-Wiskott-Aldrich syndrome protein (WASP), WASP-family verprolin homologous protein 1 (WAVE1), and Abelson interactor 1 (Abi1) in the superior temporal gyrus of paired schizophrenia and comparison participants. No changes were found in Arp2, Arp3, neuronal-WASP, WAVE1, or Abi1. However, all three isoforms of cortactin were decreased in schizophrenia. Specifically, the 62 kDa isoform was decreased by 43%; the 71 kDa isoform was decreased by 32%; and the 58 kDa isoform was decreased by 35%. Cortactin regulates branching of filamentous actin through its binding and activation of the Arp2/3 complex, and it is thus critical to the formation of stable actin networks. These findings contribute to a growing body of evidence implicating altered cytoskeletal dynamics in schizophrenia.

Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development.

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.

Coronin3 regulates gastric cancer invasion and metastasis by interacting with Arp2

Coronin3 expression is increased in gastric cancer (GC) tissues and can promote GC invasion and metastasis. However, the mechanisms underlying Coronin3 function in GC remain unclear. In this study, we aimed to explore the interacting molecules essential for the tumor-promoting effects of Coronin3 in GC. Using mass spectrometric analysis, functional studies, and immunohistochemistry, we found that Arp2 interacted with Coronin3, and ectopic expression of Arp2 promoted GC cell migration and invasion, while Arp2 knockdown suppressed whole-cell motility and attenuated the Coronin3-mediated upregulation of cell migration and invasion. In addition, both proteins correlated with the metastatic status of GC patients. Furthermore, survival analyses demonstrated that both Coronin3 and Arp2 correlated with overall GC patient survival, and the combination of Coronin3 and Arp2 most accurately predicted GC patient prognosis. Combined, these data demonstrate that Coronin3 can regulate GC invasion and metastasis through Arp2, and the combination of Coronin3 and Arp2 provides a potential marker for predicting GC prognosis.

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.

Abstract 4598: Silibinin targets fibronectin-integrin interaction and actin remodeling towards inhibiting motility and proliferation of human prostate carcinoma PC3 cells

Abstract Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths in US men. Patients with localized PCA have a very high 5-year survival rate; however, in patients with clinically detectable metastatic disease, median survival is reduced to 12-15 months. Therefore, preventing or inhibiting metastasis through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have shown recently that silibinin possess strong anti-metastatic efficacy against PCA, but mechanism of its action still remains largely unknown. The first major event during PCA metastasis is replacement of cell-cell interaction with integrins based cell-matrix interaction which then controls motility, invasiveness and survival as well as confers chemoresistance to PCA cells. Accordingly, here we examined silibinin effect on cell-matrix interaction using advanced human PCA PC3 cells and extracellular matrix (ECM) component fibronectin. Fibronectin (5-15 microgram/ml) strongly induced motile morphology in PC3 cells within 1 hr of plating and significantly enhanced cell proliferation measured after 24 hrs compared to cells plated on BSA. Silibinin (50-200 microM) significantly inhibited fibronectin-induced motile morphology by 24-83% and cell proliferation by 34-82%. Cell detachment assay clearly showed that PC3 cells plated on fibronectin are attached through strong interactions and silibinin (50-200 microM) significantly inhibits these interactions in a dose-dependent manner. To rule out the possibility that silibinin physically affects cell-fibronectin interaction, we performed studies where PC3 cells were pre-treated with silibinin for 48 hrs, and then cells collected and plated on fibronectin. Under these experimental conditions too, silibinin significantly inhibited fibronectin-induced motile morphology and proliferation of PC3 cells. Next, we performed wound healing assay to study silibinin effect on cell migration, and our results clearly showed that silibinin (50-200 microM, 6 hrs) inhibits fibronectin-induced motility in PC3 cells. Integrins, over-expressed by metastatic PCA cells, primarily interact with fibronectin and transduce signals related to proliferation and motility through actin remodeling. Accordingly, next we studied silibinin effect on these critical signaling molecules and found that it strongly decreases the basal protein levels of integrin beta 3, cortactin and ARP2 (actin-related protein) as well as fibronectin-induced integrin beta 3 and ARP2 expression in PC3 cells. Together, these results, for the first time, showed that silibinin targets PCA cells’ interaction with ECM, thereby inhibiting their motility and survival. These observations further support silibinin use in PCA intervention including metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2011-4598

Aberrant Expression of ARP2/3 in Breast Cancer and the Association with Disease Progression.

Abstract Background: Increased motility is an important characteristics of neoplastic cells, a cell function mediated through actin polymerization. During this process, aside from creation of new branches and lengthening of pre-existing actin molecule, Actin-Related Proteins (ARP)- 2 and ARP-3 work as a complex promotes polymerization through production of new nuclei for actin polymerization. This regulation is orchestrated by other intracellular regulators including and WAVE and WASP proteins, which have been shown to be aberrant in breast cancer (1,2). In this study we determined the differential expression of ARP-2 and ARP-3 and correlated the expression with various prognostic factors.Methods: Expression of ARP-2 and ARP-3 was examined in a cohort of mammary tissues (n=33 normal breast tissue and n=127 primary breast tumor tissue samples). Transcript levels of ARP2 and ARP3 were then determined using quantitative real time PCR (Q-PCR) and protein levels were assessed using immunohistochemical (IHC) staining. Results were analyzed by Mann-Whitney U test.Results: Cytoplasmic staining for both ARP-2 and ARP-3 was noted along with strong epithelial staining as compared to stromal cells. Quantitative real time PCR (Q-PCR) data analysis showed lower expression of both ARP -2 &amp; -3 in tumour tissue as compared to normal but without statistical significance. ARP-2 expression was significantly reduced in tumour samples from patients with poor prognosis (p=0.037) and patients who died of breast cancer (p=0.0265). Primary breast tumor tissue samples from patients classified as TNM stage 3 and 4 showed statistically significant lower expression of ARP-3 as compared to normal tissue (p= 0.019 and 0.020, respectively). ARP-3 expression was also significantly lower in patients who developed local recurrence of breast cancer (p=0.027). Using a Spearman correlation analysis, ARP-3 transcripts were find to be significantly correlated with the WAVE-2 transcript (r=0.42, p&amp;lt;0.01).Conclusions: Breast cancer shows aberrant expression of ARP-2 and ARP-3, a pattern linked to the prognosis of the patients. ARP-3 and WAVE-2 may have an intimate interplay in this association which warrants further investigation.

Correlation between liver metastasis of the colocalization of actin‐related protein 2 and 3 complex and WAVE2 in colorectal carcinoma

Directed movement of normal cells occurs when actin-related protein 2 and 3 complex (Arp2/3 complex) triggers the actin polymerization that forms lamellipodia immediately after binding to WAVE2. In order to determine whether the same mechanism correlates with liver metastasis from colorectal cancer, paired mirror sections of 154 cancer specimens (29 cases with liver metastasis and 125 cases without liver metastasis in which T factor, gender, primary tumor site, and age at operation were matched) were examined immunohistochemically for the localization of Arp2 and WAVE2. Expression of both Arp2 and WAVE2 was detected in the same cancer cells in 55 (35.7%) of the 154 cases, but not detected in the normal colonic epithelial cells. Univariate analysis showed that the colocalization was significantly predictive of liver metastasis (risk ratio [RR] 8.760. Likewise, histological grade (RR 2.46), lymphatic invasion (RR 9.95), and tumor budding (RR 4.00) were significant predictors. Among these, colocalization and lymphatic invasion were shown to be independent risk factors by multivariate analysis. Another 59 colorectal specimens were examined for mRNA expression of Arp2 by real time polymerase chain reaction. High mRNA levels of Arp2, that in situ hybridization revealed to be expressed by the cancer cells, were significantly associated with liver metastasis. However, its effect was absorbed by the influence of risk of the colocalization that is closely related to high expression of Arp2. These results indicate that the colocalization of Arp2 and WAVE2 is an independent risk factor for liver metastasis of colorectal carcinoma.

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2

Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (P<0.0001). MRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (P<0.0001). However, no difference was observed in the expression of Arp2, Arp3 or other WASPs. A neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (P<0.0001), and reversed the morphological effects of MRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

Involvement of Arp2/3 complex in the process of colorectal carcinogenesis

Increased motility is one of the characteristics of cancer cells, and actin polymerization and disassembly are essential for cellular motility. Since actin-related protein (Arp) 2/3 complex acts as a nucleus for actin polymerization, in this study, we immunohistochemically investigated the expression of Arp2 and Arp3 in 175 colorectal tumors in various stages of neoplastic progression. Arp2 and Arp3 showed identical expression patterns, and both were expressed in the stromal cells around neoplastic tubules or glands and in the tumor cells themselves. The frequency of expression of Arp2 and Arp3 (Arp2&3) by the stromal cells increased with the atypia of the colorectal neoplasms, from 5.5% (3/55) in adenoma with mild or moderate atypia, to 11.8% (2/17) in adenoma with severe atypia, 53.3% (16/30) in intramucosal carcinoma, and 91.8% (67/73) in invasive carcinoma (P<0.0001). The frequency of expression of Arp2&3 in the tumor cells was similar and was 1.8% (1/55) in adenoma with mild or moderate atypia, 23.5% (4/17) in adenoma with severe atypia, 23.5% (7/30) in intramucosal carcinoma, and 32.9% (24/73) in invasive carcinoma. Expression of Arp2&3 by the stromal cells was significantly correlated with nuclear accumulation of p53 in the tumor cells and stromal expression of CD10. These results suggest that formation of Arp2/3 complex by both neoplastic and stromal cells contributes to the increased motility of both cell types and thus provides suitable conditions for invasion.