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Triple negative breast cancer (TNBC) represents a therapeutic challenge in which standard chemotherapy is limited to paclitaxel. MBQ167, a clinical stage small molecule inhibitor that targets Rac and Cdc42, inhibits tumor growth and metastasis in mouse models of TNBC. Herein, we investigated the efficacy of MBQ167 in combination with paclitaxel in TNBC preclinical models, as a prelude to safety trials of this combination in patients with advanced breast cancer. Individual MBQ167 or combination therapy with paclitaxel was more effective at reducing TNBC cell viability and increasing apoptosis compared with paclitaxel alone. In orthotopic mouse models of human TNBC (MDA-MB231 and MDA-MB468), individual MBQ167, paclitaxel, or the combination reduced mammary tumor growth with similar efficacy, with no apparent liver toxicity. However, paclitaxel single agent treatment significantly increased lung metastasis, whereas MBQ167, single or combined, reduced lung metastasis. In the syngeneic 4T1/BALB/c model, combined MBQ167 and paclitaxel decreased established lung metastases by ∼80%. To determine the molecular basis for the improved efficacy of the combined treatment on metastasis, 4T1 tumor extracts from BALB/c mice treated with MBQ167, paclitaxel, or the combination were subjected to transcriptomic analysis. Gene set enrichment identified specific downregulation of central carbon metabolic pathways by the combination of MBQ167 and paclitaxel but not individual compounds. Biochemical validation, by immunoblotting and metabolic Seahorse analysis, shows that combined MBQ167 and paclitaxel reduces glycolysis. This study provides a strong rationale for the clinical testing of MBQ167 in combination with paclitaxel as a potential therapeutic for TNBC and identifies a unique mechanism of action., (©2024 American Association for Cancer Research.)

Metformin is currently a strong candidate anti-tumor agent in multiple cancers. However, its anti-tumor effectiveness varies among different cancers or subpopulations, potentially due to tumor heterogeneity. It thus remains unclear which hepatocellular carcinoma (HCC) patient subpopulation(s) can benefit from metformin treatment. Here, through a genome-wide CRISPR-Cas9-based knockout screen, we find that DOCK1 levels determine the anti-tumor effects of metformin and that DOCK1 is a synthetic lethal target of metformin in HCC. Mechanistically, metformin promotes DOCK1 phosphorylation, which activates RAC1 to facilitate cell survival, leading to metformin resistance. The DOCK1-selective inhibitor, TBOPP, potentiates anti-tumor activity by metformin in vitro in liver cancer cell lines and patient-derived HCC organoids, and in vivo in xenografted liver cancer cells and immunocompetent mouse liver cancer models. Notably, metformin improves overall survival of HCC patients with low DOCK1 levels but not among patients with high DOCK1 expression. This study shows that metformin effectiveness depends on DOCK1 levels and that combining metformin with DOCK1 inhibition may provide a promising personalized therapeutic strategy for metformin-resistant HCC patients., (© 2022. The Author(s).)

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis., (© 2021. The Author(s).)

Background: Even though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy., Methods: To determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins., Results: Gefitinib and lapatinib treatments reduced mammosphere formation in the sensitive cells, but not in the therapy resistant variants, indicating enhanced mesenchymal and cancer stem cell-like characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in known therapy resistant pathways of AKT and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested, and found to inhibit viability and induce apoptosis of therapy resistant cells., Conclusions: Rac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.

This study aimed to explore the role of LINC00665, miR-4458 and DOCK1 and their interactions in the development of acute myeloid leukemia (AML). The relative expression of LINC00665, miR-4458 and DOCK1 in AML samples was measured using qRT-PCR, and the protein level of DOCK1 in AML cell lines was examined using western blot. CCK8, BrdU, transwell, cell adhesion, and caspase-3 activity assays were carried out to evaluate the viability, proliferation, migration, adhesion, and apoptosis of AML cells, respectively. Luciferase reporter, RIP, and RNA pull-down assays were also performed to confirm the target relationship among LINC00665, miR-4458 and DOCK1. Findings revealed that LINC00665 and DOCK1 were aberrantly overexpressed in AML tissues and that the expression of miR-4458 was low in AML tissues. Silencing LINC00665 or DOCK1 presented significant restriction to the proliferation, migration and adhesion of AML cells. Apart from that, it was found that inhibiting miR-4458 could enhance the proliferation, migration and adhesion of AML cells but suppress the apoptosis of AML cells. Experimental results also indicated that LINC00665 exerted its positive function on AML cells by sponging miR-4458 and that miR-4458 influenced the progression of AML cells by targeting DOCK1 directly. Overall, this finding not only provided a novel molecular pathway for the diagnosis and treatment of AML but also showed that LINC00665 could enhance the progression of AML by regulating the miR-4458/DOCK1 pathway.

Purpose: Among the members of the DOCK family, DOCK1-5 function as guanine-nucleotide exchange factors for small GTPase Rac1, which regulates the actin cytoskeleton. It has been reported that in model organisms the Dock-Rac axis is required for myoblast fusion. We examined the role of DOCK1-5 in trophoblast fusion herein., Methods: We used a quantitative polymerase chain reaction (qPCR) to examine the mRNA expressions of DOCK1-5 and differentiation-related genes, i.e., fusogenic genes, in human trophoblastic cell lines, BeWo and JEG-3. We treated BeWo cells with TBOPP and C21 to inhibit DOCK1 and DOCK5. Cell dynamics and cell fusion were assessed by live imaging and immunostaining. The signaling pathways induced by DOCK1/5 inhibition were examined by western blotting., Results: DOCK1 and DOCK5 were expressed in BeWo cells. The inhibition of DOCK1 or DOCK5 did not prevent the cell fusion induced by forskolin (a common reagent for cell fusion); it induced cell fusion. DOCK1 inhibition induced cell death, as did forskolin. DOCK1 and DOCK5 inhibition for 24 and 48 h increased the expression of the genes ASCT2 and SYNCYTIN2, which code responsive proteins of trophoblast cell fusion, respectively., Conclusion: DOCK1 and DOCK5 inhibition participates in BeWo cell fusion, probably via pathways independent from forskolin-mediated pathways., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. All of the authors contributed significantly and are in agreement with the contents of the manuscript. This study did not involve human participants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: There is a need to develop novel therapies which could be beneficial to patients with prostate cancer (CaP) including those who are predisposed to poor outcome, such as African-Americans. This study investigates the role of ROBO1-pathway in predicting outcome and race-based disparity in patients with CaP., Methods and Results: Aided by RNA sequencing-based DECIPHER-testing and immunohistochemical (IHC) analysis of tumors we show that ROBO1 is lost during the progressive stages of CaP, a prevalent feature in African-Americans. We show that the loss of ROBO1 predicts high-risk of recurrence, metastasis and poor outcome of androgen-deprivation therapy in radical prostatectomy-treated patients. These data identified an aggressive ROBO1 deficient /DOCK1 +ve sub-class of CaP. Combined genetic and IHC data showed that ROBO1 loss is accompanied by DOCK1/Rac1 elevation in grade-III/IV primary-tumors and Mets. We observed that the hypermethylation of ROBO1-promoter contributes to loss of expression that is highly prevalent in African-Americans. Because of limitations in restoring ROBO1 function, we asked if targeting the DOCK1 could be an ideal strategy to inhibit progression or treat ROBO1 deficient metastatic-CaP. We tested the pharmacological efficacy of CPYPP, a selective inhibitor of DOCK1 under in vitro and in vivo conditions. Using ROBO1 -ve and ROBO1 +ve CaP models, we determined the median effective concentration of CPYPP for growth. DOCK1-inhibitor treatment significantly decreased the (a) Rac1-GTP/β-catenin activity, (b) transmigration of ROBO1 deficient cells across endothelial lining, and (c) metastatic spread of ROBO1 deficient cells through the vasculature of transgenic fl Zebrafish model., Conclusion: We suggest that ROBO1 status forms as predictive biomarker of outcome in high-risk populations such as African-Americans and DOCK1-targeting therapy has a clinical potential for treating metastatic-CaP., (© 2020 Wiley Periodicals LLC.)

The treatment of renal cell carcinoma (RCC) with chemotherapy remains a challenge; therefore, improving the knowledge of the molecular mechanisms underlying RCC chemoresistance and developing novel therapeutic strategies is important. Dedicator of cytokinesis 1 (DOCK1), the first member of the DOCK family to be discovered, displays various roles during tumorigenesis; however, its role during RCC progression is not completely understood. Therefore, the present study aimed to clarify the function of DOCK1 and 1‑[2‑(3'‑(trifluoromethyl)‑(1,1'‑biphenyl)‑4‑yl)‑2‑oxoethyl]‑5‑pyrrolidinylsulfonyl‑2 (1H)‑pyridone (TBOPP), a DOCK1‑sensitive inhibitor, during RCC development and chemoresistance. The results of CCK‑8 and EdU assay indicated that TBOPP decreased RCC cell viability and proliferation compared with the control group, and sensitized RCC cells to cisplatin. Moreover, RCC cells with high DOCK1 expression levels displayed increased resistance to cisplatin, whereas DOCK1 knockdown enhanced the lethal effects of cisplatin on RCC cells. Furthermore, the results determined by western blotting, CCK‑8 and cell apoptosis assay indicated that TBOPP effectively reduced DOCK1 expression levels compared with the control group, and the TBOPP‑mediated cisplatin sensitizing effect was mediated by DOCK1 inhibition. The present study suggests that DOCK1 plays a vital role in RCC cell chemoresistance to cisplatin; therefore, TBOPP may serve as a novel therapeutic agent for RCC chemoresistance.

Recently, Rho GTPases substrates include Rac (Rac1 and Rac2) and Cdc42 that have been reported to exert multiple cellular functions in osteoclasts, the most prominent of which includes regulating the dynamic actin cytoskeleton rearrangements. In addition, natural products and their molecular frameworks have a long tradition as valuable starting points for medicinal chemistry and drug discovery. Although currently, there are reports about the natural product, which could play a therapeutic role in bone loss diseases (osteoporosis and osteolysis) through the regulation of Rac1/2 and Cdc42 during osteoclasts cytoskeletal structuring. There have been several excellent studies for exploring the therapeutic potentials of various natural products for their role in inhibiting cancer cells migration and function via regulating the Rac1/2 and Cdc42. Herein in this review, we try to focus on recent advancement studies for extensively understanding the role of Rho GTPases substrates Rac1, Rac2 and Cdc42 in osteoclastogenesis, as well as therapeutic potentials of natural medicinal products for their properties on the regulation of Rac1, and/or Rac2 and Cdc42, which is in order to inspire drug discovery in regulating osteoclastogenesis., (© 2020 The Author(s).)

HACE1 is an E3 ubiquitin ligase with important roles in tumor biology and tissue homeostasis. Loss or mutation of HACE1 has been associated with the occurrence of a variety of neoplasms, but the underlying mechanisms have not been defined yet. Here, we report that HACE1 is frequently mutated in human lung cancer. In mice, loss of Hace1 led to enhanced progression of KRas G12D -driven lung tumors. Additional ablation of the oncogenic GTPase Rac1 partially reduced progression of Hace1 -/- lung tumors. RAC2, a novel ubiquitylation target of HACE1, could compensate for the absence of its homolog RAC1 in Hace1 -deficient, but not in HACE1-sufficient tumors. Accordingly, ablation of both Rac1 and Rac2 fully averted the increased progression of KRas G12D -driven lung tumors in Hace1 -/- mice. In patients with lung cancer, increased expression of HACE1 correlated with reduced levels of RAC1 and RAC2 and prolonged survival, whereas elevated expression of RAC1 and RAC2 was associated with poor prognosis. This work defines HACE1 as a crucial regulator of the oncogenic activity of RAC-family GTPases in lung cancer development. SIGNIFICANCE: These findings reveal that mutation of the tumor suppressor HACE1 disrupts its role as a regulator of the oncogenic activity of RAC-family GTPases in human and murine lung cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/3009/F1.large.jpg., (©2020 American Association for Cancer Research.)

Tendons and ligaments are crucial components of the musculoskeletal system, yet the pathways specifying these fates remain poorly defined. Through a screen of known bioactive chemicals in zebrafish, we identified a new pathway regulating tendon cell induction. We established that statin, through inhibition of the mevalonate pathway, causes an expansion of the tendon progenitor population. Co-expression and live imaging studies indicate that the expansion does not involve an increase in cell proliferation, but rather results from re-specification of cells from the neural crest-derived sox9a + /sox10 + skeletal lineage. The effect on tendon cell expansion is specific to the geranylgeranylation branch of the mevalonate pathway and is mediated by inhibition of Rac activity. This work establishes a novel role for the mevalonate pathway and Rac activity in regulating specification of the tendon lineage., Competing Interests: Competing interestsJ.W.C. is currently a full-time employee at Genentech and holds stocks in Roche., (© 2020. Published by The Company of Biologists Ltd.)

Background/aims: We showed that patho-physiological concentrations of either 7-keto-cholesterol (7-KC), or cholestane-3beta, 5alpha, 6beta-triol (TRIOL) caused the eryptotic death of human red blood cells (RBC), strictly dependent on the early production of reactive oxygen species (ROS). The goal of the current study was to assess the contribution of the erythrocyte ROS-generating enzymes, NADPH oxidase (RBC-NOX), nitric oxide synthase (RBC-NOS) and xanthine oxido-reductase (XOR) to the oxysterol-dependent eryptosis and pertinent activation pathways., Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, reactive oxygen/nitrogen species (RONS) and nitric oxide formation from 2',7'-dichloro-dihydrofluorescein (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) -dependent fluorescence, respectively; Akt1, phospho-NOS3 Ser 1177 , and PKCζ from Western blot analysis. The activity of individual 7-KC (7 μM) and TRIOL (2, μM) on ROS-generating enzymes and relevant activation pathways was assayed in the presence of Diphenylene iodonium chloride (DPI), N-nitro-L-arginine methyl ester (L-NAME), allopurinol, NSC23766 and LY294002, inhibitors in this order of RBC-NOX, RBC-NOS, XOR and upstream regulatory proteins Rac GTPase and phosphoinositide3 Kinase (PI3K); hemoglobin oxidation from spectrophotometric analysis., Results: RBC-NOX was the target of 7-KC, through a signaling including Rac GTPase and PKCζ, whereas TRIOL caused activation of RBC-NOS according to the pathway PI3K/Akt, with the concurrent activity of a Rac-GTPase. In concomitance with the TRIOL-induced . NO production, formation of methemoglobin with global loss of heme were observed, ascribable to nitrosative stress. XOR, activated after modification of the redox environment by either RBC-NOX or RBC-NOS activity, concurred to the overall oxidative/nitrosative stress by either oxysterols. When 7-KC and TRIOL were combined, they acted independently and their effect on ROS/RONS production and PS exposure appeared the result of the effects of the oxysterols on RBC-NOX and RBC-NOS., Conclusion: Eryptosis of human RBCs may be caused by either 7-KC or TRIOL by oxidative/nitrosative stress through distinct signaling cascades activating RBC-NOX and RBC-NOS, respectively, with the complementary activity of XOR; when combined, the oxysterols act independently and both concur to the final eryptotic effect., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The MET proto-oncogene-encoded receptor tyrosine kinase (MET) and AXL receptor tyrosine kinase (AXL) are independently operating receptor tyrosine kinases (RTKs) that are functionally associated with aggressive and invasive cancer cell growth. However, how MET and AXL regulate the migratory properties of cancer cells remains largely unclear. We report here that the addition of hepatocyte growth factor (HGF), the natural ligand of MET, to serum-starved human glioblastoma cells induces the rapid activation of both MET and AXL and formation of highly polarized MET-AXL clusters on the plasma membrane. HGF also promoted the formation of the MET and AXL protein complexes and phosphorylation of AXL, independent of AXL's ligand, growth arrest-specific 6 (GAS6). The HGF-induced MET-AXL complex stimulated rapid and dynamic cytoskeleton reorganization by activating the small GTPase RAC1, a process requiring both MET and AXL kinase activities. We further found that HGF also promotes the recruitment of ELMO2 and DOCK180, a bipartite guanine nucleotide exchange factor for RAC1, to the MET-AXL complex and thereby stimulates the RAC1-dependent cytoskeleton reorganization. We also demonstrated that the MET-AXL-ELMO2-DOCK180 complex is critical for HGF-induced cell migration and invasion in glioblastoma or other cancer cells. Our findings uncover a critical HGF-dependent signaling pathway that involves the assembly of a large protein complex consisting of MET, AXL, ELMO2, and DOCK180 on the plasma membrane, leading to RAC1-dependent cell migration and invasion in various cancer cells., (© 2018 Li et al.)

The migration and invasion of cells through tissues in the body is facilitated by a dynamic actin cytoskeleton. The actin-associating protein, tropomyosin Tpm3.1 has emerged to play important roles in cell migration and invasion. To date, investigations have focused on single cell migration and invasion where Tpm3.1 expression is inversely associated with Rac GTPase-mediated cell invasion. While single cell and collective cell invasion have many features in common, collective invasion is additionally impacted by cell-cell adhesion, and the role of Tpm3.1 in collective invasion has not been established. In the present study we have modelled multicellular invasion using neuroblastoma spheroids embedded in 3D collagen and analysed the function of Tpm3.1 using recently established compounds that target the Tpm3.1 C-terminus. The major findings from our study reveal that combined Rac inhibition and Tpm3.1 targeting result in greater inhibition of multicellular invasion than either treatment alone. Together, the data suggest that Tpm3.1 disruption sensitises neuroblastoma cells to inhibition of Rac-mediated multicellular invasion., (© 2018 Wiley Periodicals, Inc.)

Rac and Cdc42 are small GTPases that have been linked to multiple human cancers and are implicated in epithelial to mesenchymal transition, cell-cycle progression, migration/invasion, tumor growth, angiogenesis, and oncogenic transformation. With the exception of the P29S driver mutation in melanoma, Rac and Cdc42 are not generally mutated in cancer, but are overexpressed (gene amplification and mRNA upregulation) or hyperactivated. Rac and Cdc42 are hyperactivated via signaling through oncogenic cell surface receptors, such as growth factor receptors, which converge on the guanine nucleotide exchange factors that regulate their GDP/GTP exchange. Hence, targeting Rac and Cdc42 represents a promising strategy for precise cancer therapy, as well as for inhibition of bypass signaling that promotes resistance to cell surface receptor-targeted therapies. Therefore, an understanding of the regulatory mechanisms of these pivotal signaling intermediates is key for the development of effective inhibitors. In this review, we focus on the role of Rac and Cdc42 in cancer and summarize the regulatory mechanisms, inhibitory efficacy, and the anticancer potential of Rac- and Cdc42-targeting agents. Cancer Res; 78(12); 3101-11. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Rac1 is a member of the Rho family of small GTPases that regulates cytoskeletal reorganization, membrane polarization, cell migration and proliferation. Recently, a self-activating mutation of Rac1, Rac1 P29S , has been identified as a recurrent somatic mutation frequently found in sun-exposed melanomas, which possesses increased inherent GDP/GTP exchange activity and cell transforming ability. However, the role of cellular Rac1-interacting proteins in the transforming potential of Rac1 P29S remains unclear. We found that the catalytic domain of DOCK1, a Rac-specific guanine nucleotide exchange factor (GEF) implicated in malignancy of a variety of cancers, can greatly accelerate the GDP/GTP exchange of Rac1 P29S . Enforced expression of Rac1 P29S induced matrix invasion and macropinocytosis in wild-type (WT) mouse embryonic fibroblasts (MEFs), but not in DOCK1-deficient MEFs. Consistently, a selective inhibitor of DOCK1 that blocks its GEF function suppressed the invasion and macropinocytosis in WT MEFs expressing Rac1 P29S . Human melanoma IGR-1 and breast cancer MDA-MB-157 cells harbor Rac1 P29S mutation and express DOCK1 endogenously. Genetic inactivation and pharmacological inhibition of DOCK1 suppressed their invasion and macropinocytosis. Taken together, these results indicate that DOCK1 is a critical regulator of the malignant phenotypes induced by Rac1 P29S , and suggest that targeting DOCK1 might be an effective approach to treat cancers associated with Rac1 P29S mutation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/aims: Thrombin induces the activation of human platelets through protease-activated receptor (PAR) 1 and PAR4, and Rac, a member of the Rho family of small GTPases, is implicated in PAR activation. We previously reported that phosphorylated-heat shock protein 27 (HSP27) is released from the thrombin receptor-activating peptide (TRAP)-stimulated platelets of diabetic patients. In the present study, we investigated the role of Rac in the TRAP-elicited release of phosphorylated-HSP27 from human platelets., Methods: Platelet aggregation was measured using an aggregometer with laser scattering. Protein phosphorylation was analyzed by Western blotting. The levels of phosphorylated-HSP27 and platelet-derived growth factor-AB (PDGF-AB) were measured by enzyme-linked immunosorbent assays., Results: NSC23766, an inhibitor of Rac-guanine nucleotide exchange factor interaction, suppressed the TRAP-elicited release of phosphorylated-HSP27 as well as platelet aggregation. The TRAP-induced phosphorylation of HSP27, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was attenuated by NSC23766. SB203580, a p38 MAPK inhibitor, but not SP600125, a JNK inhibitor, suppressed the release of phosphorylated-HSP27 in addition to HSP27 phosphorylation. On the other hand, both SB203580 and SP600125 reduced the TRAP-stimulated secretion of PDGF-AB., Conclusion: Our results strongly suggest that Rac acts as a positive regulator of the PAR-elicited release of phosphorylated-HSP27 from human platelets via p38 MAPK but not JNK., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The polarization of leukocytes toward chemoattractants is essential for the directed migration (chemotaxis) of leukocytes. How leukocytes acquire polarity after encountering chemical gradients is not well understood. We found here that leukocyte polarity was generated by TIPE2 (TNFAIP8L2), a transfer protein for phosphoinositide second messengers. TIPE2 functioned as a local enhancer of phosphoinositide-dependent signaling and cytoskeleton remodeling, which promoted leading-edge formation. Conversely, TIPE2 acted as an inhibitor of the GTPase Rac, which promoted trailing-edge polarization. Consequently, TIPE2-deficient leukocytes were defective in polarization and chemotaxis, and TIPE2-deficient mice were resistant to leukocyte-mediated neural inflammation. Thus, the leukocyte polarizer is a dual-role phosphoinositide-transfer protein and represents a potential therapeutic target for the treatment of inflammatory diseases.

Homing of peripheral stem cells is regulated by one of the most representative homing factors, stromal cell-derived factor 1 alpha (SDF-1α), which specifically binds to the plasma membrane receptor CXCR4 of mesenchymal stem cells (MSCs) in order to initiate the signaling pathways that lead to directional migration and homing of stem cells. This complex homing process and directional migration of stem cells have been mimicked on a microfluidic device that is capable of generating a chemokine gradient within the collagen matrix and embedding endothelial cell (EC) monolayers to mimic blood vessels. On the microfluidic device, stem cells showed directional migration toward the higher concentration of SDF-1α, whereas treatment with the CXCR4 antagonist AMD3100 caused loss of directionality of stem cells. Furthermore, inhibition of stem cell's main migratory signaling pathways, Rho-ROCK and Rac pathways, caused blockage of actomyosin and lamellipodia formation, decreasing the migration distance but maintaining directionality. Stem cell homing regulated by SDF-1α caused directional migration of stem cells, while the migratory ability was affected by the activation of migration-related signaling pathways.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of skeletal muscle origin in children and adolescents. Among RMS subtypes, alveolar rhabdomyosarcoma (ARMS), which is characterized by the presence of the PAX3-FOXO1A or PAX7-FOXO1A chimeric oncogenic transcription factor, is associated with poor prognosis and a strong risk of metastasis compared with the embryonal subtype (ERMS). To identify molecular pathways involved in ARMS aggressiveness, we first characterized the migratory behavior of cell lines derived from ARMS and ERMS biopsies using a three-dimensional spheroid cell invasion assay. ARMS cells were more invasive than ERMS cells and adopted an ellipsoidal morphology to efficiently invade the extracellular matrix. Moreover, the invasive potential of ARMS cells depended on ROCK activity, which is regulated by the GTPase RhoE. Specifically, RhoE expression was low in ARMS biopsies, and its overexpression in ARMS cells reduced their invasion potential. Conversely, ARHGAP25, a GTPase-activating protein for Rac, was up-regulated in ARMS biopsies. Moreover, we found that ARHGAP25 inhibits Rac activity downstream of ROCKII and is required for ARMS cell invasion. Our results indicate that the RhoE/ROCK/ARHGAP25 signaling pathway promotes ARMS invasive potential and identify these proteins as potential therapeutic targets for ARMS treatment., (© 2016 Thuault et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Signaling via tumor necrosis factor receptor (TNFR) superfamily members regulates cellular life and death decisions. A subset of mammalian TNFR proteins, most notably the p75 neurotrophin receptor (p75NTR), induces cell death through a pathway that requires activation of c-Jun N-terminal kinases (JNKs). However the receptor-proximal signaling events that mediate this remain unclear. Drosophila express a single tumor necrosis factor (TNF) ligand termed Eiger (Egr) that activates JNK-dependent cell death. We have exploited this model to identify phylogenetically conserved signaling events that allow Egr to induce JNK activation and cell death in vivo. Here we report that Rac1, a small GTPase, is specifically required in Egr-mediated cell death. rac1 loss of function blocks Egr-induced cell death, whereas Rac1 overexpression enhances Egr-induced killing. We identify Vav as a GEF for Rac1 in this pathway and demonstrate that dLRRK functions as a negative regulator of Rac1 that normally acts to constrain Egr-induced death. Thus dLRRK loss of function increases Egr-induced cell death in the fly. We further show that Rac1-dependent entry of Egr into early endosomes is a crucial prerequisite for JNK activation and for cell death and show that this entry requires the activity of Rab21 and Rab7. These findings reveal novel regulatory mechanisms that allow Rac1 to contribute to Egr-induced JNK activation and cell death.

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network assembly. Overall, this work defines novel key regulators and their functional roles during human EC tubulogenesis.

The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1-10 and GFP11 cells). GFP1-10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1-10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1-10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, or prostate growth. However, current therapeutic options are insufficient. Here, we investigated the role of Rac in the control of smooth muscle tone in human prostates and growth of prostate stromal cells., Experimental Approach: Experiments were performed using human prostate tissues from radical prostatectomy and cultured stromal cells (WPMY-1). Expression of Rac was examined by Western blot and fluorescence staining. Effects of Rac inhibitors (NSC23766 and EHT1864) on contractility were assessed in the organ bath. The effects of Rac inhibitors were assessed by pull-down, cytotoxicity using a cell counting kit, cytoskeletal organization by phalloidin staining and cell growth using an 5-ethynyl-2'-deoxyuridine assay., Key Results: Expression of Rac1-3 was observed in prostate samples from each patient. Immunoreactivity for Rac1-3 was observed in the stroma, where it colocalized with the smooth muscle marker, calponin. NSC23766 and EHT1864 significantly reduced contractions of prostate strips induced by noradrenaline, phenylephrine or electrical field stimulation. NSC23766 and EHT1864 inhibited Rac activity in WPMY-1 cells. Survival of WPMY-1 cells ranged between 64 and 81% after incubation with NSC23766 (50 or 100 μM) or EHT1864 (25 μM) for 24 h. NSC23766 and EHT1864 induced cytoskeletal disorganization in WPMY-1 cells. Both inhibitors impaired the growth of WPMY-1 cells., Conclusions and Implications: Rac may be a link connecting the control of prostate smooth muscle tone with proliferation of smooth muscle cells. Improvements in LUTS suggestive of BPH by Rac inhibitors appears possible., (© 2015 The British Pharmacological Society.)

Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Rho GTPase Rac is an important regulator of cancer cell migration and invasion; processes required for metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in metastatic breast cancer cells and recently found that EHop-016 was effective at reducing tumor growth in nude mice at 25 mg/kg bodyweight (BW). The purpose of this study was to compare the pharmacokinetics and bioavailability of EHop-016 at different dosages in a single dose input scheme (10, 20 and 40 mg/kg BW) following intraperitoneal (IP) and oral gavage (PO) administration to nude mice. We developed and validated a rapid and sensitive method for the quantitation of EHop-016 in mouse plasma by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/MS/MS). Separation was carried out on an Agilent Poroshell 120 EC-C18 column (3.0 mm × 50 mm) using organic and aqueous mobile phases. EHop-016 was identified from its accurate mass and retention time from the acquired full-scan chromatogram and quantified by its peak area. The validated method was linear (R(2)>0.995) over the range of 5-1000 ng/mL (1/x(2) weighting). Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin. The area under the curve (AUC₀-∞) ranged from 328 to 1869 ng h/mL and 133-487 ng h/mL for IP and PO dosing, respectively. The elimination half-life (t₁/₂) ranged from 3.8-5.7 h to 3.4-26.8 h for IP and PO dosing, respectively. For both IP and PO administration, the AUC₀-∞values were proportional to the tested doses demonstrating linear PK profiles. The relative bioavailability of EHop-016 after oral gavage administration ranged from 26% to 40%. These results support further preclinical evaluation of EHop-016 as a new anti-cancer therapy., (Published by Elsevier B.V.)

Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria. How neutrophils establish and precisely orient this polarity toward pathogens remains unclear. Here we report that in resting neutrophils, the ERM (ezrin, radixin, and moesin) protein moesin in its active form (phosphorylated and membrane bound) prevented cell polarization by inhibiting the small GTPases Rac, Rho, and Cdc42. Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity. Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions. Therefore, both moesin-mediated inhibition and its localized deactivation by myosin phosphatase are essential for neutrophil polarization and effective neutrophil tracking of pathogens., (© 2015 Liu et al.)

Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower" cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin β1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin β1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin β1, and PI3K are upregulated in leader cells and drive collective cell migration.

Gefitinib is widely used for the treatment of lung cancer in patients with sensitizing epidermal growth factor receptor mutations, but patients tend to develop resistance after an average of 10 months. Low molecular weight heparins, such as enoxaparin, potently inhibit experimental metastasis. This study aimed to determine the potential of combined enoxaparin and gefitinib (enoxaparin + gefitinib) treatment to inhibit tumor resistance to gefitinib both in vitro and in vivo. A549 and H1975 cell migration was analyzed in wound closure and Transwell assays. Akt and extracellular signal-related kinase 1/2 signaling pathways were identified, and a proteomics analysis was conducted using SDS-PAGE/liquid chromatography-tandem mass spectrometry analysis. Molecular interaction networks were visualized using the Cytoscape bioinformatics platform. Protein expression of dedicator of cytokinesis 1 (DOCK1) and cytoskeleton intermediate filament vimentin were identified using an enzyme-linked immunosorbent assay, Western blot, and small interfering RNA transfection of A549 cells. In xenograft A549-luc-C8 tumors in nude mice, enoxaparin + gefitinib inhibited tumor growth and reduced lung colony formation compared with gefitinib alone. Furthermore, the combination had stronger inhibitory effects on cell migration than either agent used individually. Additional enoxaparin administration resulted in better effective inhibition of Akt activity compared with gefitinib alone. Proteomics and network analysis implicated DOCK1 as the key node molecule. Western blot verified the effective inhibition of the expression of DOCK1 and vimentin phosphorylation by enoxaparin + gefitinib compared with gefitinib alone. DOCK1 knockdown confirmed its role in cell migration, Akt expression, and vimentin phosphorylation. Our data indicate that enoxaparin sensitizes gefitinib antitumor and antimigration activity in lung cancer by suppressing DOCK1 expression, Akt activity, and vimentin phosphorylation., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Vav proteins are guanine nucleotide exchange factors (GEFs) that activate a group of small G proteins (GTPases). Vav1 is predominantly expressed in hematopoietic cells, whereas Vav2 and Vav3 are ubiquitously distributed in almost all human tissues. All three Vav proteins contain conserved structural motifs and associate with a variety of cellular activities including proliferation, migration, and survival. Previous observation with Jurkat leukemia T cells showed that Vav1 possessed anti-apoptotic activity by enhancing Bcl-2 transcription. However the mechanism has not been unveiled. Here, we explored the effectors of Vav1 in promoting Bcl-2 expression in Jurkat cells and revealed that Rac2-Akt was specifically evoked by the expression of Vav1, but not Vav2 or Vav3. Although all three Vav isoforms existed in Jurkat cells, Rac2 was distinguishably activated by Vav1 and that led to enhanced Bcl-2 expression and cell survival. Akt was modulated downstream of Vav1-Rac2, and the activation of Akt was indispensable in the enhanced transcription of Bcl-2. Intriguingly, neither Vav2 nor Vav3 was able to activate Rac2-Akt pathway as determined by gene silencing approach. Our data illustrated a unique role of Vav1 in T leukemia survival by selectively triggering Rac2-Akt axis and elevating the expression of anti-apoptotic Bcl-2., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Throughout life, the tight equilibrium between cell death and the prompt clearance of dead corpses is required to maintain a proper tissue homeostasis and prevent inflammation. Following lactation, mammary gland involution is triggered and results in the death of excessive epithelial cells that are rapidly cleared by phagocytes to ensure that the gland returns to its prepregnant state. Orthologs of Dock1 (dedicator of cytokinesis 1), Elmo and Rac1 (ras-related C3 botulinum toxin substrate 1) in Caenorhabditis elegans are part of a signaling module in phagocytes that is linking apoptotic cell recognition to cytoskeletal reorganization required for engulfment. In mammals, Elmo1 was shown to interact with the phosphatidylserine receptor Bai1 and relay signals to promote phagocytosis of apoptotic cells. Still, the role of the RacGEF Dock1 in the clearance of dying cells in mammals was never directly addressed. We generated two mouse models with conditional inactivation of Dock1 and Rac1 and revealed that the expression of these genes is not essential in the mammary gland during puberty, pregnancy and lactation. We induced mammary gland involution in these mice to investigate the role of Dock1/Rac1 signaling in the engulfment of cell corpses. Unpredictably, activation of Stat3 (signal transducer and activator of transcription 3), a key regulator of mammary gland involution, was impaired in the absence of Rac1 and Dock1 expression. Likewise, failure to activate properly Stat3 was coinciding with a significant delay in the initiation and progression of mammary gland involution in mutant animals. By using an in vitro phagocytosis assay, we observed that Dock1 and Rac1 are essential to mediate engulfment in epithelial phagocytes. In vivo, cell corpses accumulated at late time points of involution in Dock1 and Rac1 mutant mammary glands. Overall, our study demonstrated an unsuspected role for Dock1/Rac1 signaling in the initiation of mammary gland involution, and also suggested a role for this pathway in the clearance of dead cells by epithelial phagocytes.

Objective: Engulfment and cell motility 1 (Elmo1) has been reported to cooperate with dedicator of cytokinesis 1 (Dock180) and to be linked to the invasive phenotype of cancer cells through activating small G-protein Rac. We aimed to study the role of Elmo1 in the malignant migration of ovarian cancer., Methods: Engulfment and cell motility 1 expression was evaluated in specimens from 93 patients with serous ovarian cancer (SOC) by immunohistochemical staining. Next, Elmo1-RNAi cells were established by validated small interference RNAs. Cell proliferation and cell motility were observed and compared with Dock180-RNAi cells. To confirm their synergetic contribution to forming focal adhesion and activating Rac1, Rac1-GTP level was measured by GST pull-down assay and immunofluorescence was used to observe focal adhesion formation both in Elmo1-RNAi and Dock180-RNAi cells., Results: Engulfment and cell motility 1 was mainly overexpressed in high-grade SOC tissues. Western blot analysis demonstrated that both Elmo1 and Dock180 expressions were hampered in Elmo1-RNAi cells. Compared with the negative control, decreased colony formation and cell invasion were observed in Elmo1-RNAi cells and Dock180-RNAi cells. Consistently, both exhibited reduced Rac1-GTP level and inhibited focal adhesion formation., Conclusions: Engulfment and cell motility 1 presents with synergetic action in helping Dock180 to activate Rac1 and promote cell motility, and thus promote untoward expansion and aggressiveness of SOC.

Here, we investigated the influence of Rac family small GTPases on mechanisms of the DNA damage response (DDR) stimulated by topoisomerase II poisons. To this end, we examined the influence of the Rac-specific small molecule inhibitor EHT1864 on Ser139 phosphorylation of histone H2AX, a widely used marker of the DDR triggered by DNA double-strand breaks. EHT1864 attenuated the doxorubicin-stimulated DDR in a subset of cell lines tested, including HepG2 hepatoma cells. EHT1864 reduced the level of DNA strand breaks and increased viability following treatment of HepG2 cells with topo II poisons. Protection by EHT1864 was observed in both p53 wildtype (HepG2) and p53 deficient (Hep3B) human hepatoma cells and, furthermore, remained unaffected upon pharmacological inhibition of p53 in HepG2. Apparently, the impact of Rac on the DDR is independent of p53. Protection from doxorubicin-induced DNA damage by EHT1864 comprises both S and G2 phase cells. The inhibitory effect of EHT1864 on doxorubicin-stimulated DDR was mimicked by pharmacological inhibition of various protein kinases, including JNK, ERK, PI3K, PAK and CK1. EHT1864 and protein kinase inhibitors also attenuated the formation of the topo II-DNA cleavable complex. Moreover, EHT1864 mitigated the constitutive phosphorylation of topoisomerase IIα at positions S1106, S1213 and S1247. Doxorubicin transport, nuclear import/export of topoisomerase II and Hsp90-related mechanisms are likely not of relevance for doxorubicin-stimulated DDR impaired by EHT1864. We suggest that multiple kinase-dependent but p53- and heat shock protein-independent Rac-regulated nuclear mechanisms are required for activation of the DDR following treatment with topo II poisons., (© 2013.)

Efferocytosis, which is the homeostatic phagocytosis of apoptotic cells, prevents the release of toxic intracellular contents and subsequent tissue damage. Impairment of efferocytosis was reported in alveolar macrophages (AMs) of patients with chronic obstructive pulmonary disease (COPD), a common disease caused by smoking. In COPD, histone deacetylase (HDAC) activity is reduced in AMs. We investigated whether the reduction of HDAC activity is associated with the impairment of efferocytosis. Murine AMs were collected by bronchoalveolar lavage and their ability to efferocytose apoptotic human polymorphonuclear leukocytes was assessed. Pre-treatment of AMs with cigarette smoke extract (CSE) or trichostatin A (TSA), an HDAC inhibitor, suppressed efferocytosis and CSE reduced HDAC activity. TSA inhibited the activity of Rac, a key mediator of efferocytosis. These TSA-induced impairments were restored by treatment of AMs with aminophylline, a potent activator of HDAC. To further elucidate the underlying mechanism, we explored a role of CD9 in TSA-induced impairment of efferocytosis. CD9 is a transmembrane protein of the tetraspanin family that facilitates the uptake of several pathogens and other material. TSA profoundly down-regulated the expression of CD9 on AMs. The expression of CD9 was partly down-regulated by the Rac inhibitor. Pretreatment with an anti-CD9 mAb or CD9 small interfering RNA inhibited efferocytosis, which was attributable to the reduced binding of AMs to apoptotic cells. These results suggest that smoking impairs efferocytosis via inhibition of HDAC/Rac/CD9 pathways. Aminophylline/theophylline is effective in restoring the impairment of efferocytosis and might have benefit for the treatment of patients with COPD.

'Oxidative stress' is a term defining states of elevated reactive oxygen species (ROS) levels. Normally, ROS control several physiological processes, such as host defence, biosynthesis of hormones, fertilization and cellular signalling. However, oxidative stress has been involved in different pathologies, including metabolic syndrome and numerous cardiovascular diseases. A major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology is the NADPH oxidase (NOX) family of enzymes. NOX is a multi-component enzyme complex that consists of membrane-bound cytochrome b-558, which is a heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP-binding protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1 has been reported to be a key regulator of oxidative stress through its co-regulatory effects on both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a brief outline about the important role of the Rac1-NOX axis in the pathophysiology of both metabolic syndrome and cardiovascular disease., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

Rationale and Objectives: Drug reinforcement and the reinstatement of drug seeking are associated with the pathological processing of drug-associated cue memories that can be disrupted by manipulating memory consolidation and reconsolidation. Ras-related C3 botulinum toxin substrate (Rac) is involved in memory processing by regulating actin dynamics and neural structure plasticity. The nucleus accumbens (NAc) and amygdala have been implicated in the consolidation and reconsolidation of emotional memories. Therefore, we hypothesized that Rac in the NAc and amygdala plays a role in the consolidation and reconsolidation of cocaine-associated cue memory., Methods: Conditioned place preference (CPP) and microinjection of Rac inhibitor NSC23766 were used to determine the role of Rac in the NAc and amygdala in the consolidation and reconsolidation of cocaine-associated cue memory in rats., Results: Microinjections of NSC23766 into the NAc core but not shell, basolateral (BLA), or central amygdala (CeA) after each cocaine-conditioning session inhibited the consolidation of cocaine-induced CPP. A microinjection of NSC23766 into the BLA but not CeA, NAc core, or NAc shell immediately after memory reactivation induced by exposure to a previously cocaine-paired context disrupted the reconsolidation of cocaine-induced CPP. The effect of memory disruption on cocaine reconsolidation was specific to reactivated memory, persisted at least 2 weeks, and was not reinstated by a cocaine-priming injection., Conclusions: Our findings indicate that Rac in the NAc core and BLA are required for the consolidation and reconsolidation of cocaine-associated cue memory, respectively.

Excessive neutrophil recruitment is a major feature in septic lung damage although the signaling mechanisms behind pulmonary infiltration of neutrophils in sepsis remain elusive. In the present study, we hypothesized that Rac1 might play an important role in pulmonary neutrophil accumulation and tissue injury in abdominal sepsis. Male C57BL/6 mice were treated with Rac1 inhibitor NSC23766 (5 mg/kg) before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid and lung tissue were collected for the quantification of neutrophil recruitment and edema and CXC chemokine formation. Blood was collected for the determination of Mac-1 on neutrophils and proinflammatory compounds in plasma. Gene expression of CXC chemokines and tumor necrosis factor alpha was determined by quantitative reverse transcription-polymerase chain reaction in alveolar macrophages. Rac1 activity was increased in lungs from septic animals, and NSC23766 significantly decreased pulmonary activity of Rac1 induced by CLP. Administration of NSC23766 markedly reduced CLP-triggered neutrophil infiltration, edema formation, and tissue damage in the lung. Inhibition of Rac1 decreased CLP-induced neutrophil expression of Mac-1 and pulmonary formation of CXC chemokines. Moreover, NSC23766 abolished the sepsis-evoked elevation of messenger RNA levels of CXC chemokines and tumor necrosis factor alpha in alveolar macrophages. Rac1 inhibition decreased the CLP-induced increase in plasma levels of high mobility group protein B1 and interleukin 6, indicating a role of Rac1 in systemic inflammation. In conclusion, our results demonstrate that Rac1 signaling plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis. Thus, targeting Rac1 activity might be a useful strategy to protect the lung in abdominal sepsis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Signaling from small GTPases is a tightly regulated process. In this work we used a protein microarray screen to identify the Rac-specific GAP, ArhGAP15, as a substrate of the Rac effectors Pak1 and Pak2. In addition to serving as a substrate of Pak1/2, we found that ArhGAP15, via its PH domain, bound to these kinases. The association of ArhGAP15 to Pak1/2 resulted in mutual inhibition of GAP and kinase catalytic activity, respectively. Knock-down of ArhGAP15 resulted in activation of Pak1/2, both indirectly, as a result of Rac activation, and directly, as a result of disruption of the ArhGAP15/Pak complex. Our data suggest that ArhGAP15 plays a dual negative role in regulating small GTPase signaling, by acting at the level of the GTPase itself, as well interacting with its effector, Pak kinase.

: Development of cardiac hypertrophy after thyroxin (T4) treatment is well recognized. Recently, we observed that T4-induced cardiac hypertrophy is associated with increased cardiac Rac1 expression and activity. Whether this Rac1 increase has a role in inducing this cardiac phenotype is, however, still unknown. Here, we showed that T4 treatment (500 µg/kg/d) for 2 weeks resulted in increased myocardial Rac1 activity with subsequent hypertension, cardiac hypertrophy, and left ventricular systolic dysfunction in vivo. Isolated right ventricular papillary muscles of T4-treated mice maintained their peak isometric active developed tension but exhibited significant decreases in their corresponding time to peak and in relaxation times. Positive inotropic responses to increasing pacing rate and β-adrenergic stimulation were also depressed in these muscles. Pravastatin (10 mg/kg/d), a Rac1 inhibitor, significantly decreased myocardial Rac1 activity, hypertension, and cardiomyocyte size in T4-treated mice but could not attenuate gross heart weight or functional cardiac changes in these mice. Our data showed that T4 could activate different signaling pathways with distinct cardiovascular outcomes. We also provide the first mechanistic evidence for the partial involvement of Rac1 activation in T4-induced cardiomyocyte hypertrophy and reveal a putative role for Rac1 in the development of T4-induced hypertension.

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (~60-100%) and humans (~40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20-58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

The chemokine receptors CXCR3 and CXCR4 are primarily involved in memory Th1 cell-driven autoimmune diseases. Although recent studies in chronic inflammatory disease showed therapeutic success using combined blockade, details of CXCR3 and CXCR4 synergism are not understood. In this investigation, we intended to unravel the interaction of these chemokine receptors in static and dynamic cell-migration assays at both the cellular and molecular levels. Effects of combined stimulation by murine CXCL9 and CXCL12, ligands of CXCR3 and CXCR4, respectively, were analyzed using a murine central memory Th1 cell clone. Costimulation with CXCL9 desensitized the chemotaxis of Th1 cells toward CXCL12 by up to 54%. This effect was found in murine EL-4 cells, as well as in primary human T cells. Furthermore, under dynamic flow conditions CXCL12-induced crawling and endothelial transmigration of Th1 cells was desensitized by CXCL9. Subsequent experiments uncovered several molecular mechanisms underlying the heterologous cross-regulation of CXCR4 signaling by the CXCR3 ligand. CXCR4 surface expression was reduced, whereas CXCL12-induced Akt phosphorylation and intracellular Ca(2+) signals were modulated. Moreover, blockade of Rac by NSC23766 revealed differential effects on CXCL12 and CXCL9 chemotaxis and abolished the desensitizing effect of CXCL9. The desensitization of CXCR4 via CXCR3 in memory Th1 cells suggests that their in vivo homeostasis, widely regulated by CXCL12, seemed to be significantly altered by CXCR3 ligands. Our data provide a more detailed understanding for the continuing extravasation and recruitment of Th1 lymphocytes into sites of persistent inflammation.

Aims: Doxorubicin causes damage to the heart, often leading to irreversible cardiomyopathy, which is fatal. Reactive oxygen species (ROS) or oxidative stress is involved in cardiomyocyte death, contributing to doxorubicin-induced cardiotoxicity. This study investigated the role of Rac1, an important subunit of NADPH oxidase, in doxorubicin-induced cardiotoxicity and the underlying mechanisms., Methods and Results: In a mouse model of acute doxorubicin-induced cardiotoxicity, cardiomyocyte-specific deletion of Rac1 inhibited NADPH oxidase activation and ROS production, prevented cardiac cell death, and improved myocardial function in Rac1 knockout mice. Therapeutic administration of the specific Rac1 inhibitor NSC23766 achieved similar cardio-protective effects in doxorubicin-stimulated mice. In rat cardiomyoblasts (H9c2 cells) and cultured neonatal mouse cardiomyocytes, Rac1 inhibition attenuated apoptosis as evidenced by decreases in caspase-3 activity and DNA fragmentation in response to doxorubicin, which correlated with a reduction in ROS production and down-regulation of p53 acetylation and histone H2AX phosphorylation. In contrast, overexpression of Rac1 enhanced apoptosis. Doxorubicin also inhibited the activity of classical histone deacetylases (HDAC), which was preserved by Rac1 inhibition and further decreased by Rac1 overexpression. Interestingly, scavenging ROS mitigated apoptosis but did not change HDAC activity and p53 acetylation stimulated by doxorubicin, suggesting both ROS-dependent and -independent pathways are involved in Rac1-mediated cardiotoxicity. Furthermore, the HDAC inhibitor trichostatin A enhanced apoptosis, p53 acetylation and H2AX phosphorylation in doxorubicin-treated cardiomyocytes., Conclusions: Rac1 signalling contributes to doxorubicin-induced cardiotoxicity through both a ROS-dependent mechanism and ROS-independent HDAC/p53 signalling in cardiomyocytes. Thus, inhibition of Rac1 may be a useful therapy for doxorubicin-induced cardiotoxicity.

Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications.

The Rac inhibitor EHop-016 was developed as a compound with the potential to inhibit cancer metastasis. Inhibition of the first step of metastasis, migration, is an important strategy for metastasis prevention. The small GTPase Rac acts as a pivotal binary switch that is turned "on" by guanine nucleotide exchange factors (GEFs) via a myriad of cell surface receptors, to regulate cancer cell migration, survival, and proliferation. Unlike the related GTPase Ras, Racs are not usually mutated, but overexpressed or overactivated in cancer. Therefore, a rational Rac inhibitor should block the activation of Rac by its upstream effectors, GEFs, and the Rac inhibitor NSC23766 was developed using this rationale. However, this compound is ineffective at inhibiting the elevated Rac activity of metastatic breast cancer cells. Therefore, a panel of small molecule compounds were derived from NSC23766 and screened for Rac activity inhibition in metastatic cancer cells. EHop-016 was identified as a compound that blocks the interaction of Rac with the GEF Vav in metastatic human breast cancer cells with an IC50 of ~1μM. At higher concentrations (10μM), EHop-016 inhibits the related Rho GTPase Cdc42, but not Rho, and also reduces cell viability. Moreover, EHop-016 inhibits the activation of the Rac downstream effector p21-activated kinase, extension of motile actin-based structures, and cell migration. Future goals are to develop EHop-016 as a therapeutic to inhibit cancer metastasis, either individually or in combination with current anticancer compounds. The next generation of EHop-016-based Rac inhibitors is also being developed., (© 2013 Elsevier Inc. All rights reserved.)

Objective: The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, mediates platelet adhesion at sites of vascular injury and transmits signals leading to platelet activation. von Willebrand factor/GPIb-IX interaction sequentially activates the Src family kinase Lyn (SFK), phosphoinositide 3-kinase (PI3K), and Akt, leading to activation of integrin α(IIb)β(3) and integrin-dependent stable platelet adhesion and aggregation. It remains unclear how Lyn activates the PI3K/Akt pathway after ligand binding to GPIb-IX., Methods and Results: Using platelet-specific Rac1(-/-) mice and the Rac1 inhibitor NSC23766, we examined the role of Rac1 in GPIb-IX-dependent platelet activation. Rac1(-/-) mouse platelets and NSC23766-treated human platelets were defective in GPIb-dependent stable adhesion to von Willebrand factor under shear stress, integrin activation, thromboxane A(2) synthesis, and platelet aggregation. Interestingly, GPIb-induced activation of Rac1 and the guanine nucleotide exchange factor for Rac1, Vav, was abolished in both Lyn(-/-) and SFK inhibitor-treated platelets but was unaffected by the PI3K inhibitor LY294002, indicating that Lyn mediates activation of Vav and Rac1 independently of PI3K. Furthermore, GPIb-induced activation of Akt was abolished in Rac1-deficient platelets, suggesting that Rac1 is upstream of the PI3K/Akt pathway., Conclusions: A Lyn-Vav-Rac1-PI3K-Akt pathway mediates von Willebrand factor-induced activation of integrin α(IIb)β(3) to promote GPIb-IX-dependent platelet activation.

RNA receptors such as TLR3 and retinoid acid-inducible gene I/melanoma differentiation-associated gene 5 play essential roles in innate immunity to RNA viruses. However, how innate immunity to RNAs is controlled at the molecular level is not well understood. We describe in this study a new regulatory pathway of anti-RNA immunity that is composed of PI3K, its target GTPase Rac, and the newly described immune regulator TNF-α-induced protein 8 like-2 (TIPE2, or TNFAIP8L2). Polyinosinic-polycytidylic acid [Poly (I:C)], a dsRNA receptor ligand, activates Rac via its guanine nucleotide exchange factor Tiam; this leads to the activation of cytokine genes and, paradoxically, downregulation of the Tipe2 gene. TIPE2 is a negative regulator of immunity; its deficiency leads to hyperactivation of the PI3K-Rac pathway as exemplified by enhanced AKT, Rac, P21-activated kinase, and IFN regulatory factor 3 activities. As a consequence, TIPE2 knockout myeloid cells are hyperreactive to Poly (I:C) stimulation, and TIPE2 knockout mice are hypersensitive to Poly (I:C)-induced lethality. These results indicate that TIPE2 controls innate immunity to RNA by targeting the PI3K-Rac pathway. Therefore, manipulating TIPE2 or Rac functions can be effective for controlling RNA viral infections.

Cell polarization is essential for migration and the exploratory function of leukocytes. However, the mechanism by which cells maintain polarity or how cells revert to the immobilized state by gaining cellular symmetry is not clear. Previously we showed that interaction between oxidized low-density lipoprotein (oxLDL) and CD36 inhibits macrophage migration; in the current study we tested the hypothesis that oxLDL/CD36-induced inhibition of migration is the result of intracellular signals that regulate cell polarity. Live cell imaging of macrophages showed that oxLDL actuated retraction of macrophage front end lamellipodia and induced loss of cell polarity. Cd36 null and macrophages null for Vav, a guanine nucleotide exchange factor (GEF), did not show this effect. These findings were caused by Rac-mediated inhibition of nonmuscle myosin II, a cell polarity determinant. OxLDL induced dephosphorylation of myosin regulatory light chain (MRLC) by increasing the activity of Rac. Six-thioguanine triphosphate (6-thio-GTP), which inhibits Vav-mediated activation of Rac, abrogated the effect of oxLDL. Activation of the Vav-Rac-myosin II pathway by oxidant stress may induce trapping of macrophages at sites of chronic inflammation such as atherosclerotic plaque.