Ras-related C3 Botulinum Toxin Substrate Research Articles

The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic β-cells

Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in β-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic β-cells. β-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic β-cells.

ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a member of the Rho family of GTPases that functions as a molecular switch to regulate many important cellular events including actin cytoskeleton remodeling during neurite outgrowth. Engulfment and cell motility 1 (ELMO1)-dedicator of cytokinesis 1 (DOCK180) is a bipartite guanine nucleotide exchange factor (GEF) complex that has been reported to activate Rac1 on the plasma membrane (PM). Emerging evidence suggests that the small GTPase ADP ribosylation factor 6 (ARF6) activates Rac1 via the ELMO1/DOCK180 complex. However, the exact mechanism by which ARF6 triggers ELMO1/DOCK180-mediated Rac1 signaling remains unclear. Here, we report that the neuronal scaffold protein FE65 serves as a functional link between ARF6 and ELMO1, allowing the formation of a multimeric signaling complex. Interfering with formation of this complex by transfecting either FE65-binding-defective mutants or FE65 siRNA attenuates both ARF6-ELMO1-mediated Rac1 activation and neurite elongation. Notably, the PM trafficking of ELMO1 is markedly decreased in cells with suppressed expression of either FE65 or ARF6. Likewise, this process is attenuated in the FE65-binding-defective mutants transfected cells. Moreover, overexpression of FE65 increases the amount of ELMO1 in the recycling endosome, an organelle responsible for returning proteins to the PM, whereas knockout of FE65 shows opposite effect. Together, our data indicates that FE65 potentiates ARF6-Rac1 signaling by orchestrating ARF6 and ELMO1 to promote the PM trafficking of ELMO1 via the endosomal recycling pathway, and thus, promotes Rac1-mediated neurite outgrowth.

Effects of maternal protein restriction on central and peripheral renin-angiotensin systems in male rat offspring

AimsWe investigated the involvement of the renin angiotensin system (RAS) on the cardiorespiratory control in rats from dams fed with a low-protein diet. Main methodsMale offspring were obtained from dams fed a normoprotein diet (NP, 17% casein) and low-protein diet (LP, 8% casein) during pregnancy and lactation. Direct measurements of arterial pressure (AP), heart rate (HR) and respiratory frequency (RF) were recorded in awake 90-day-old at resting and after losartan potassium through either intracerebroventricular (ICV) microinjections or intravenous (IV) administration. Cardiovascular variability was evaluated by spectral analysis. Peripheral chemoreflex sensitivity was assessed through the potassium cyanide (KCN; 40 μg/0.1 ml/rat, IV). Gene expression was evaluated by qPCR, and MAPK (Mitogen Activated Protein Kinase) expression was evaluated by western blot. Key findingsThe LP offspring had higher mean AP (MAP) and RF than NP offspring. In the spectral analysis, the LP rats also showed higher low frequency of systolic AP (NP: 2.7 ± 0.3 vs. LP: 5.0 ± 1.0 mmHg). After ICV losartan, MAP and RF in LP rats remained higher than those in NP rats, but without changes in HR. The peripheral chemoreflex was similar between the groups. LP group had lower gene expression of Rac1 (Ras-related C3 botulinum toxin substrate 1) (NP: 1.13 ± 0.06 vs. LP: 0.88 ± 0.08). Peripherally, LP rats had larger delta of MAP after IV losartan (NP: −9.8 ± 2 vs. LP: −23 ± 6 mmHg), without changes in HR and RF. SignificanceIn rats, the RAS participates peripherally, but not centrally, in the maintenance of arterial hypertension in male offspring induced by maternal protein restriction.

Activation of neuronal Ras-related C3 botulinum toxin substrate 1 (Rac1) improves post-stroke recovery and axonal plasticity in mice.

Long-term disability after stroke is common but the mechanisms of post-stroke recovery remain unclear. Cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 contributes to functional recovery after ischemic stroke in mice. As Rac1 plays divergent roles in individual cell types after central neural system injury, we herein examined the specific role of neuronal Rac1 in post-stroke recovery and axonal regeneration. Young male mice were subjected to 60-min of middle cerebral artery occlusion (MCAO). Inducible deletion of neuronal Rac1 by daily intraperitoneal injection of tamoxifen (2mg/40g) into Thy1-creER/Rac1-floxed mice day 7-11 after MCAO worsened cognitive (assayed by novel object recognition test) and sensorimotor (assayed by adhesive removal and pellet reaching tests) recovery day 14-28 accompanied with the reduction of neurofilament-L (NFL) and myelin basic protein (MBP) and the elevation of glial fibrillary acidic protein (GFAP) in the peri-infarct zone assessed by immunostaining. Whereas the brain tissue loss was not altered assayed by cresyl violet staining. In another approach, delayed overexpression of neuronal Rac1 by injection of lentivirus encoding Rac1 with neuronal promotor into both the cortex and striatum (total 4μl at 1×109 transducing units/mL) of stroke side in C57BL/6J mice day 7 promoted stroke outcome, NFL and MBP regrowth and alleviated GFAP invasion. Furthermore, neuronal Rac1 over-expression led to the activation of p21 activating kinases (PAK) 1, mitogen-activated protein kinase kinase (MEK) 1/2 and extracellular signal-regulated kinase (ERK) 1/2, and the elevation of brain-derived neurotrophic factor (BDNF) day 14 after stroke. Finally, we observed higher counts of neuronal Rac1 in the peri-infarct zone of subacute/old ischemic stroke subjects. This work identified a neuronal Rac1 signaling in improving functional recovery and axonal regeneration after stroke, suggesting a potential therapeutic target in the recovery stage of stroke.

MiR-142-3p Reduces the Size, Migration, and Contractility of Endometrial and Endometriotic Stromal Cells by Targeting Integrin- and Rho GTPase-Related Pathways That Regulate Cytoskeletal Function.

Downregulated microRNA-142-3p signaling contributes to the pathogenesis of endometriosis, an invasive disease where the lining of the uterus grows at ectopic locations, by yet incompletely understood mechanisms. Using bioinformatics and in vitro assays, this study identifies cytoskeletal regulation and integrin signaling as two relevant categories of miR-142-3p targets. qPCR revealed that miR-142-3p upregulation in St-T1b cells downregulates Rho-associated protein kinase 2 (ROCK2), cofilin 2 (CFL2), Ras-related C3 botulinum toxin substrate 1 (RAC1), neural Wiskott-Aldrich syndrome protein (WASL), and integrin α-V (ITGAV). qPCR and Western-blotting showed miR-142-3p effect on WASL and ITGAV was significant also in primary endometriotic stroma cells. Luciferase reporter assays in ST-T1b cells then confirmed direct regulation of ITGAV and WASL. On the functional side, miR-142-3p upregulation significantly reduced ST-T1b cell size, the size of vinculin plaques, migration through fibronectin-coated transwell filters, and the ability of ST-T1b and primary endometriotic stroma cells to contract collagen I gels. These results suggest that miR-142-3p has a strong mechanoregulatory effect on endometrial stroma cells and its external administration reduces the invasive endometrial phenotype. Within the limits of an in vitro investigation, our study provides new mechanistic insights into the pathogenesis of endometriosis and provides a perspective for the development of miR-142-3p based drugs for inhibiting invasive growth of endometriotic cells.

Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases.

Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

Enforced C-Src Activation Causes Compartmental Dysregulation of PI3K and PTEN Molecules in Lipid Rafts of Tongue Squamous Carcinoma Cells by Attenuating Rac1-Akt-GLUT-1-Mediated Sphingolipid Synthesis.

Pharmacologic intervention to affect the membrane lipid homeostasis of lipid rafts is a potent therapeutic strategy for cancer. Here we showed that gallic acid (GA) caused the complex formation of inactive Ras-related C3 botulinum toxin substrate 1 (Rac1)-phospho (p)-casein kinase 2 α (CK2α) (Tyr 255) in human tongue squamous carcinoma (TSC) cells, which disturbed the lipid raft membrane-targeting of phosphatidylinositol 3-kinase (PI3K)-Rac1-protein kinase B (Akt) signal molecules by inducing the association of p110α-free p85α with unphosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN) in lipid rafts. The effects on induction of inactive Rac1-p-CK2α (Tyr 255) complex formation and attenuation of p-Akt (Ser 473), GTP-Rac1, glucose transporter-1 (GLUT-1) lipid raft membrane-targeting, and cell invasive activity by GA were counteracted either by CK2α short hairpin RNA or cellular-Src (c-Src) inhibitor PP1. PP1 treatment, GLUT-1 or constitutively active Rac1 ectopic-expression blocked GA-induced decreases in cellular glucose, sphingolipid and cholesterol of lipid raft membranes, p85α-p110α-GTP-Rac1 complexes, glucosylceramide synthase activity and increase in ceramide and p110α-free p85α-PTEN complex levels of lipid raft membranes, which reversed the inhibition on matrix metalloproteinase (MMP)-2/-9-mediated cell invasion induced by GA. Using transient ectopic expression of nuclear factor-kappa B (NF-κB) p65, MMP-2/-9 promoter-driven luciferase, and NF-κB-dependent luciferase reporter genes and NF-κB specific inhibitors or Rac1 specific inhibitor NSC23766, we confirmed that an attenuation of Rac1 activity by GA confers inhibition of NF-κB-mediated MMP-2/-9 expression and cell invasion. In conclusion, GA-induced c-Src activation is a key inductive event for the formation of inactive Rac1-p-CK2α (Tyr 255) complexes, which disturbed lipid raft compartment of PI3K and PTEN molecules by impairing Akt-regulated GLUT-1-mediated sphingolipid synthesis, and finally resulting in inhibition of TSC cell invasion.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Abstract A32: The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases

Abstract Activating mutations in the K-Ras gene are the earliest and most common genetic alterations detected in human pancreatic cancer (PC) specimens. In genetically engineered mouse models of PC, oncogenic K-Ras drives the formation of PanIN precursor lesions and their progression to invasive PC. Oncogenic K-Ras signals through at least four effector pathways that play distinct roles in malignant transformation: MAPK pathway, PI3K pathway, Ral-GDS, and the Rac1 GTPase (Ras-related C3 botulinum toxin substrate 1). Among these effectors, the least well understood is the Rac1 pathway. Yet, this pathway has now been shown to be critical for Ras-driven PC development and malignant transformation. However, the specific mechanisms by which Rac1 promotes anchorage-independent growth and tumorigenicity are still unclear. In recent studies, we have discovered that the Rac1 pathway regulates the stability of the Yes-Associated Protein (YAP). YAP is a transcriptional coactivator that controls the transcription of genes involved in proliferation and survival. In mouse models of Ras-driven PC, YAP is required for the formation of PanIN precursor lesions and their progression to invasive PC. In our telomerase-immortalized human pancreatic ductal cells, overexpressed YAP alone is sufficient to allow anchorage-independent growth. Our results show that in PC cells treated with Rac1 inhibitors (NSC23766, EHT-1864, AZA1, and Rac1T17N), YAP is rapidly and almost completely degraded within 24 hours. Importantly, this degradation of YAP is blocked by the preincubation of the cells with proteasome inhibitor MG132, thereby implying the involvement of a ubiquitin ligase complex. YAP contains a phosphodegron that controls its ubiqutination and subsequent degradation by the proteasome. Activation of this degron recognized by SCF-βTRCP ubiquitin ligase complexes requires its phosphorylation by the LATS1/2 kinases. To identify the molecular signals that control the degradation of YAP after Rac1 inhibition, Flag-tagged mutants of YAP were tested that were missing either a functional degron (ΔβTRCP mutant) or all of their LATS1/2 phosphorylation sites (5SA mutant). Our results show that while the functionality of the degron is required for YAP degradation after Rac1 inhibition, the phosphorylation of this degron by the LATS1/2 kinases is needed. Not only was the 5SA YAP mutant still degraded after Rac1 inhibition, but the knockdown of the LATS1/2 kinases also did not prevent the degradation of YAP. These findings suggest the existence of a novel SCF ubiquitin ligase complex that regulates YAP stability independently of the LATS1/2 kinases. The screening of a siRNA library has now been undertaken to identify the F-box protein involved. The proposed work is expected to shed light on novel mechanisms underlying the development of PC and of other Ras-driven malignancies. Citation Format: Chitra Palanivel, Bailey M. Gabler, Ying Yan, Surinder K. Batra, Michel M. Ouellette. The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A32.

Inhibition of Rac1 reverses enzalutamide resistance in castration-resistant prostate cancer.

Enzalutamide, an androgen receptor inhibitor, has been clinically approved for the treatment of metastatic castration-resistant prostate cancer (CRPC) in the United States. However, patients only benefit from enzalutamide for a short period of time as resistance may develop. Therefore, it is vital to develop a novel strategy to overcome enzalutamide resistance. Ras-related C3 botulinum toxin substrate 1 (Rac1), which is commonly upregulated in human cancer types, has been recognized as a key molecular component in tumorigenesis, invasion and metastasis. However, the role of Rac1 in enzalutamide-resistance in prostate cancer (PCa) remains unknown. In the present study, Rac1 was demonstrated to be upregulated in enzalutamide-resistant PCa cells, and Rac1 knockdown inhibited enzalutamide-resistant cell proliferation and colony formation. Western blotting results indicated that enzalutamide treatment downregulated the expression levels of JNK and activated transcription factor 2, as well as enhanced the Bax/Bcl-2 ratio and induced cleavage of poly-ADP ribose polymerase. Moreover, knockdown of Rac1 in MR49F cells significantly inhibited cell migration and invasion via the downregulation of Snail and the upregulation of E-cadherin. The results of a nude mouse xenograft tumor model using 22RV1 cells demonstrated that enzalutamide inhibited tumor growth after Rac1 knockdown dramatically, compared to vehicle and single treatment groups. Therefore, the present study provided novel evidence that Rac1 may serve a crucial role in enzalutamide resistance, and that targeting Rac1 may be a potential approach for the treatment of CRPC.

Abstract B59: Rac1 overexpression promotes epithelial-to-mesenchymal transition in ovarian cancer cells

Abstract The small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) is commonly overexpressed in cancer and high Rac1 levels are associated with poor patient outcomes in ovarian cancer. Rac1 drives multiple hallmarks of cancer such as proliferation, quiescence, epithelial-to-mesenchymal transition (EMT), and transcriptional programs relevant to tumor development, progression, and chemoresistance. EMT promotes metastatic dissemination, chemoresistance, and cancer stem cell properties. We find that modest overexpression of Rac1 in ovarian tumor cells causes morphologic changes consistent with EMT. Elevated Rac1 (4- fold) expression decreases colony number and increases colony area when compared to vector control cells. In addition, cell invasion through an artificial basement membrane is increased by 10-fold in cells overexpressing Rac1. An increase in mesenchymal markers (e.g., N-cadherin) and decrease in epithelial markers (e.g., E-cadherin) was observed as a consequence of Rac1 expression. Rac1 overexpression was associated with selective upregulation of Slug/Snai2 (8-fold) and Zeb2 (100-fold) mRNA levels with little impact on Snai1, Twist, or Zeb1. Slug/Snai2 expression is reportedly induced by oxidative stress and treatment of six ovarian tumor cell lines with tert-butyl hydroperoxide (TBHP) to generate oxidative stress increased the expression Slug/Snai2, Snai1, and heme oxygenase-1, an indicator of oxidative stress response. This finding confirms that elevated oxidative stress regulates certain EMT transcription factors in various ovarian cancer cells. Rac1 is an essential factor for activation of NADPH oxidases 1, 2, and 3 and generation of reactive oxygen species (ROS). Basal level of ROS was elevated in cells overexpressing Rac1 compared to vector control cells as detected by the fluorescent probe Dihydroethidium (DHE). Similarly, elevated Rac1 increased expression of the oxidative stress response genes heme oxygenase-1 (17-fold) and superoxide dismutase-1 (12-fold), further supporting that Rac1 stimulated ROS signaling in these cells. We further tested the impact of Rac1 inhibition in vivo using R-ketorolac. Analysis of tumor tissue from mice showed decreased expression of heme-oxygenase-1 and Slug/Snai2 in mice treated with R-ketorolac compared to placebo controls. R-ketorolac also decreased invasive implantation and expansion of ovarian cancer xenografts. Taken together, these findings illustrate the functional impact of Rac1 overexpression in ovarian cancer cells and suggest that Rac1 may be a viable target for further drug development to attenuate EMT in ovarian tumors. Citation Format: Martha M. Grimes, Dayna R. Dominquez, Michaela L. Granados, Alejandra Rosales, Melanie Rivera, Angela Wandinger-Ness, Laurie G. Hudson. Rac1 overexpression promotes epithelial-to-mesenchymal transition in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B59.

Hydroxytyrosol modifies skeletal muscle GLUT4/AKT/Rac1 axis in trained rats.

Training induces a number of healthy effects including a rise in skeletal muscle (SKM) glucose uptake. These adaptations are at least in part due to the reactive oxygen species produced within SKM, which is in agreement with the notion that antioxidant supplementation blunts some training-induced adaptations. Here, we tested whether hydroxytyrosol (HT), the main polyphenol of olive oil, would modify the molecular regulators of glucose uptake when HT is supplemented during exercise. Rats were included into sedentary and exercised (EXE) groups. EXE group was further divided into a group consuming a low HT dose (0.31 mg·kg·d; EXElow), a moderate HT dose (4.61 mg·kg·d; EXEmid), and a control group (EXE). EXE raised glucose transporter type 4 (GLUT4) protein content, Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, andprotein kinase b (AKT) phosphorylation in SKM. Furthermore, EXElow blunted GLUT4 protein content and AKT phosphorylation while EXEmid showed a downregulation of the GLUT4/AKT/Rac1 axis. Hence, a low-to-moderate dose of HT, when it is supplemented as an isolated compound, might alter the beneficial effect of training on basal AKT phosphorylation and Rac1 activity in rats.

Active RAC1 Promotes Tumorigenic Phenotypes and Therapy Resistance in Solid Tumors.

Acting as molecular switches, all three members of the Guanosine triphosphate (GTP)-ase-family, Ras-related C3 botulinum toxin substrate (RAC), Rho, and Cdc42 contribute to various processes of oncogenic transformations in several solid tumors. We have reviewed the distribution of patterns regarding the frequency of Ras-related C3 botulinum toxin substrate 1 (RAC1)-alteration(s) and their modes of actions in various cancers. The RAC1 hyperactivation/copy-number gain is one of the frequently observed features in various solid tumors. We argued that RAC1 plays a critical role in the progression of tumors and the development of resistance to various therapeutic modalities applied in the clinic. With this perspective, here we interrogated multiple functions of RAC1 in solid tumors pertaining to the progression of tumors and the development of resistance with a special emphasis on different tumor cell phenotypes, including the inhibition of apoptosis and increase in the proliferation, epithelial-to-mesenchymal transition (EMT), stemness, pro-angiogenic, and metastatic phenotypes. Our review focuses on the role of RAC1 in adult solid-tumors and summarizes the contextual mechanisms of RAC1 involvement in the development of resistance to cancer therapies.

Overexpression of MicroRNA-133a Inhibits Apoptosis and Autophagy in a Cell Model of Parkinson's Disease by Downregulating Ras-Related C3 Botulinum Toxin Substrate 1 (RAC1).

BackgroundParkinson’s disease (PD) is a movement disorder. microRNA (miR)-133 expression is reduced in PD patients and in mice with a dopamine neuron deficiency. We aimed to identify the mechanism of miR-133a in apoptosis and autophagy in PD.Material/MethodsThe optimal concentration of MPP+ (1-methyl-4-phenylpyridinium ion) was initially determined to construct a PD cell model. Gain-of function experiments were carried out to evaluate the role of miR-133a in PD. The levels of miR-133a, RAC1 (Ras-related C3 botulinum toxin substrate 1), apoptosis-related factors, and autophagy-related factors were detected after detection of cell proliferation, cell cycle, and apoptosis. Transmission electron microscopy was applied to observe autophagosomes, and immunofluorescence staining was performed to detect LC3 and further analyze the effect of miR-133a on autophagy in a PD cell model.ResultsLow miR-133a expression was detected in a cell model of MPP+-induced PD. After overexpressing miR-133a, cell proliferation increased, and apoptosis (cleaved caspase-3 and Bax levels decreased, while Bcl2 levels increased) and autophagy was inhibited (LC3II/I and Beclin-1 levels decreased, while p62 levels increased). MiR-133a targeted RAC1. RACY upregulation attenuated the inhibitory effects of miR-133a on PC12 cell apoptosis and autophagy.ConclusionsOur data highlighted that miR-133a overexpression prevented apoptosis and autophagy in a cell model of MPP+-induced PD by inhibiting RAC1 expression.

Rac1: A potential radiosensitization target of human nasopharyngeal carcinoma CNE2 cells

Radiotherapy has a high cure rate for early nasopharyngeal carcinoma(NPC). However, the radiation resistance of poorly differentiated NPC cells impacts the effectiveness of treatment of early-stage NPC patients. Here, we explored the relationship between Ras-related C3 botulinum toxin substrate 1(Rac1) expression and NPC radiosensitivity. In vitro and in vivo studies revealed that upregulation of Rac1, when combined with X-ray treatment, increased growth inhibition and induced remarkable morphological changes and apoptosis in CNE2 cells. Furthermore, rupturing of the cell and nuclear membranes, degeneration of the cristae and significant swelling of the mitochondria were observed, which were consistent with the high apoptotic rate. The Rac1(+) cells exhibited approximately 50% more migration compared with that of the NC and Rac1(-) cells. The overexpression of Rac1 can increase the radiation sensitivity of NPC CNE2 cells, and the mechanism may be closely related to the oxidative damage of mitochondria. Rac1 might be a potential target for radiosensitization in poorly differentiated NPC.

RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer.

Radiation therapy is a common and acceptable approach for lung cancer. Although the benefit of ionizing radiation (IR) is well-established, cancer cells can still survive via pro-survival and metastasis signaling pathways. Ras related C3 botulinum toxin substrate1 (RAC1), a member of Rho family GTPases, plays important roles in cell migration and survival. In the present study, we investigated the effects of RAC1 on the survival of lung cancer cells treated with irradiation. The results showed RAC1 is overexpressed in lung cancer cells and promoted cell proliferation and survival. Furthermore, IR induced RAC1 expression and activity via the activation of PI3K/AKT signaling pathway, and then enhancing cell proliferation, survival, migration and metastasis and increasing levels of epithelial-to-mesenchymal transition (EMT) markers, which facilitated the cell survival and invasive phenotypes. In addition, overexpression of RAC1 attenuated the efficacy of irradiation, while inhibition of RAC1 enhanced sensitivity of irradiation in xenograft tumors in vivo. Collectively, we further found that RAC1 enhanced radioresistance by promoting EMT via targeting the PAK1-LIMK1-Cofilins signaling in lung cancer. Our finding provides the evidences to explore RAC1 as a therapeutic target for radioresistant lung cancer cells.

Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno.

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

MTORC2/Rac1 Pathway Predisposes Cancer Aggressiveness in IDH1-Mutated Glioma.

Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in lower grade gliomas. The neomorphic enzyme activity of IDH mutants leads to tumor formation through epigenetic alteration, dysfunction of dioxygenases, and metabolic reprogramming. However, it remains elusive as to how IDH mutants regulate the pathways associated with oncogenic transformation and aggressiveness. In the present study, by using unbiased transcriptomic profiling, we showed that IDH1 mutations result in substantial changes in the gene sets that govern cellular motility, chemotaxis, and invasion. Mechanistically, rapamycin-insensitive companion of mammalian target of rapamycin (Rictor)/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling plays an essential role in the motility and proliferation of IDH1-mutated cells by prompting cytoskeleton reorganization, lamellipodia formation, and enhanced endocytosis. Targeting the Rictor/Rac1 pathway suppresses IDH1-mutated cells by limiting endocytosis and cell proliferation. Overall, our findings indicate a novel metabolic reprogramming mechanism of IDH1-mutated cells by exploiting metabolites from the extracellular milieu. Targeting the Rictor/Rac1 pathway could be an alternative therapeutic strategy for IDH1-mutated malignancies.

Effect of increased expression of both ras-related C3 botulinum toxin substrate 1 and p21-activated kinase 1 in patients with N0M0 upper urinary tract urothelial carcinoma and cancer-free surgical margins.

As a member of the Rho small guanosine triphosphatase family, ras-related C3 botulinum toxin substrate 1 (RAC1) interacts with various specific effectors, and p21-activated kinase 1 (PAK1), which has a role in both carcinogenesis and cellular invasion, binds to RAC1, after which activated PAK1 regulates cellular functions. There have been few reports about the simultaneous analysis of RAC1 and its downstream effector PAK1 in upper urinary tract urothelial carcinoma (UTUC). We assessed the expressions of both RAC1 and PAK1 and evaluated their association with clinicopathological parameters. Immunohistochemical studies of RAC1 or PAK1 were performed with specimens from 104 patients with N0M0 UTUC and cancer-free surgical margins. Correlation of the positive expression of RAC1 or PAK1 or both with clinicopathological parameters was evaluated. A hazard model showed that the presence of mixed histologic features and moderate or strong positive expression of both RAC1 and PAK1 were independent factors for shortened disease-specific survival time (Ps=0.041 and 0.016, respectively), and another hazard model revealed that only moderate or strong positive expression of both RAC1 and PAK1 was an independent factor for shortened recurrence-free survival time in the multivariate analysis (P=0.036). Neither moderate or strong positive expression of RAC1 alone nor moderate or strong positive expression of PAK1 alone was an independent factor for a worse rate of disease-specific or recurrence-free survival in multivariate analysis. Patients with N0M0 UTUC, cancer-free surgical margins and moderate or strong positive expression of both RAC1 and PAK1 should be carefully monitored after surgery.

Inhibition of chemotherapy-related breast tumor EMT by application of redox-sensitive siRNA delivery system CSO-ss-SA/siRNA along with doxorubicin treatment.

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) downregulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.