Transcriptome analysis of cardiac endothelial cells after myocardial infarction reveals temporal changes and long-term deficits.

Endothelial cells (ECs) have essential roles in cardiac tissue repair after myocardial infarction (MI). To establish stage-specific and long-term effects of the ischemic injury on cardiac ECs, we analyzed their transcriptome at landmark time points after MI in mice. We found that early EC response at Day 2 post-MI centered on metabolic changes, acquisition of proinflammatory phenotypes, initiation of the S phase of cell cycle, and activation of stress-response pathways, followed by progression to mitosis (M/G2 phase) and acquisition of proangiogenic and mesenchymal properties during scar formation at Day 7. In contrast, genes involved in vascular physiology and maintenance of vascular tone were suppressed. Importantly, ECs did not return to pre-injury phenotypes after repair has been completed but maintained inflammatory, fibrotic and thrombotic characteristics and lost circadian rhythmicity. We discovered that the highest induced transcript is the mammalian-specific Sh2d5 gene that promoted migration and invasion of ECs through Rac1 GTPase. Our results revealed a synchronized, temporal activation of disease phenotypes, metabolic pathways, and proliferation in quiescent ECs after MI, indicating that precisely-timed interventions are necessary to optimize cardiac tissue repair and improve outcomes. Furthermore, long-term effects of acute ischemic injury on ECs may contribute to vascular dysfunction and development of heart failure.

Excessive endometrial PlGF- Rac1 signalling underlies endometrial cell stiffness linked to pre-eclampsia.

Cell stiffness is regulated by dynamic interaction between ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1) proteins, besides other biochemical and molecular regulators. In this study, we investigated how the Placental Growth Factor (PlGF) changes endometrial mechanics by modifying the actin cytoskeleton at the maternal interface. We explored the global effects of PlGF in endometrial stromal cells (EnSCs) using the concerted approach of proteomics, atomic force microscopy (AFM), and electrical impedance spectroscopy (EIS). Proteomic analysis shows PlGF upregulated RhoGTPases activating proteins and extracellular matrix organization-associated proteins in EnSCs. Rac1 and PAK1 transcript levels, activity, and actin polymerization were significantly increased with PlGF treatment. AFM further revealed an increase in cell stiffness with PlGF treatment. The additive effect of PlGF on actin polymerization was suppressed with siRNA-mediated inhibition of Rac1, PAK1, and WAVE2. Interestingly, the increase in cell stiffness by PlGF treatment was pharmacologically reversed with pravastatin, resulting in improved trophoblast cell invasion. Taken together, aberrant PlGF levels in the endometrium can contribute to an altered pre-pregnancy maternal microenvironment and offer a unifying explanation for the pathological changes observed in conditions such as pre-eclampsia (PE).

Cardiomyocytes, cardiac endothelial cells and fibroblasts contribute to anthracycline-induced cardiac injury through RAS-homologous small GTPases RAC1 and CDC42.

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.

Modulation of the RAC1/MAPK/ERK signalling pathway by farnesyl diphosphate synthase regulates granulosa cells proliferation in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex gynaecological endocrine disease that occurs in women of childbearing age. The pathogenesis of PCOS is still unclear and further exploration is needed. Here, proteomic analysis indicated that the expression of farnesyl diphosphate synthase (FDPS) protein in ovarian tissue of PCOS mice was significantly decreased. The purpose of this study is to investigate the relationship between potential biomarkers of PCOS and granulosa cells (GCs) function. The mechanisms by which FDPS affected the proliferation of granulosa cells were also explored both in vitro and in vivo. We found that knockdown of FDPS inhibited the proliferation of KGN (human ovarian granulosa cell line), while overexpression of FDPS had the opposite effect. FDPS activated Rac1 (Rac Family Small GTPase 1) activity and regulated MAPK/ERK signalling pathway, which affecting the proliferation of KGN cells significantly. In addition, treatment with the adeno-associated virus (AAV)-FDPS reverses the dehydroepiandrosterone (DHEA)-induced PCOS-phenotype in mice. Our data indicated that FDPS could regulate the proliferation of ovarian GCs by modulating MAPK/ERK (mitogen-activated protein kinase/extracellular regulated protein kinases) pathway via activating Rac1 activity. These findings suggest that FDPS could be of great value for the regulation of ovarian granulosa cell function and the treatment of PCOS.

Tumour-associated macrophage-derived DOCK7-enriched extracellular vesicles drive tumour metastasis in colorectal cancer via the RAC1/ABCA1 axis.

BACKGROUND: Metastasis accounts for the majority of deaths among patients with colorectal cancer (CRC). Here, the regulatory role of tumour-associated macrophages (TAMs) in CRC metastasis was explored. METHODS: Immunohistochemical (IHC) analysis of the TAM biomarker CD163 was conducted to evaluate TAM infiltration in CRC. Transwell assays and an ectopic liver metastasis model were established to evaluate the metastatic ability of tumour cells. RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS) were applied to identify the differentially expressed genes and proteins in CRC cells and in TAM-derived extracellular vesicles (EVs). Cholesterol content measurement, a membrane fluidity assay and filipin staining were performed to evaluate cholesterol efflux in CRC cells. RESULTS: Our results showed that TAM infiltration is positively correlated with CRC metastasis. TAMs can facilitate the migration and invasion of MC-38 and CT-26 cells via EVs. According to the RNA-seq data, TAM-EVs increase cholesterol efflux and enhance membrane fluidity in CRC cells by regulating ABCA1 expression, thus affecting the motility of CRC cells. Mechanistically, DOCK7 packaged in TAM-EVs can activate RAC1 in CRC cells and subsequently upregulate ABCA1 expression by phosphorylating AKT and FOXO1. Moreover, IHC analysis of ABCA1 in patients with liver-metastatic CRC indicated that ABCA1 expression is significantly greater in metastatic liver nodules than in primary CRC tumours. CONCLUSIONS: Overall, our findings suggest that DOCK7 delivered via TAM-EVs could regulate cholesterol metabolism in CRC cells and CRC cell metastasis through the RAC1/AKT/FOXO1/ABCA1 axis. DOCK7 could thus be a new therapeutic target for controlling CRC metastasis.

HMGB1 promotes mitochondrial transfer between hepatocellular carcinoma cells through RHOT1 and RAC1 under hypoxia.

Mitochondrial transfer plays an important role in various diseases, and many mitochondrial biological functions can be regulated by HMGB1. To explore the role of mitochondrial transfer in hepatocellular carcinoma (HCC) and its relationship with HMGB1, field emission scanning electron microscopy, immunofluorescence, and flow cytometry were used to detect the mitochondrial transfer between HCC cells. We found that mitochondrial transfer between HCC cells was confirmed using tunnel nanotubes (TNTs). The transfer of mitochondria from the highly invasive HCC cells to the less invasive HCC cells could enhance the migration and invasion ability of the latter. The hypoxic conditions increased the mitochondrial transfer between HCC cells. Then the mechanism was identified using co-immunoprecipitation, luciferase reporter assay, and chromatin immunoprecipitation. We found that RHOT1, a mitochondrial transport protein, promoted mitochondrial transfer and the migration and metastasis of HCC cells during this process. Under hypoxia, HMGB1 further regulated RHOT1 expression by increasing the expression of NFYA and NFYC subunits of the NF-Y complex. RAC1, a protein associated with TNTs formation, promoted mitochondrial transfer and HCC development. Besides, HMGB1 regulated RAC1 aggregation to the cell membrane under hypoxia. Finally, the changes and significance of related molecules in clinical samples of HCC were analyzed using bioinformatics and tissue microarray analyses. We found that HCC patients with high HMGB1, RHOT1, or RAC1 expression exhibited a relatively shorter overall survival period. In conclusion, under hypoxic conditions, HMGB1 promoted mitochondrial transfer and migration and invasion of HCC cells by increasing the expression of mitochondrial transport protein RHOT1 and TNTs formation-related protein RAC1.

Tight junction protein cingulin variant is associated with cancer susceptibility by overexpressed IQGAP1 and Rac1-dependent epithelial-mesenchymal transition.

BACKGROUND: Cingulin (CGN) is a pivotal cytoskeletal adaptor protein located at tight junctions. This study investigates the link between CGN mutation and increased cancer susceptibility through genetic and mechanistic analyses and proposes a potential targeted therapeutic approach. METHODS: In a high-cancer-density family without known pathogenic variants, we performed tumor-targeted and germline whole-genome sequencing to identify novel cancer-associated variants. Subsequently, these variants were validated in a 222 cancer patient cohort, and CGN c.3560C > T was identified as a potential cancer-risk allele. Both wild-type (WT) (c.3560C > C) and variant (c.3560C > T) were transfected into cancer cell lines and incorporated into orthotopic xenograft mice model for evaluating their effects on cancer progression. Western blot, immunofluorescence analysis, migration and invasion assays, two-dimensional gel electrophoresis with mass spectrometry, immunoprecipitation assays, and siRNA applications were used to explore the biological consequence of CGN c.3560C > T. RESULTS: In cancer cell lines and orthotopic animal models, CGN c.3560C > T enhanced tumor progression with reduced sensitivity to oxaliplatin compared to the CGN WT. The variant induced downregulation of epithelial marker, upregulation of mesenchymal marker and transcription factor, which converged to initiate epithelial-mesenchymal transition (EMT). Proteomic analysis was conducted to investigate the elements driving EMT in CGN c.3560C > T. This exploration unveiled overexpression of IQGAP1 induced by the variant, contrasting the levels observed in CGN WT. Immunoprecipitation assay confirmed a direct interaction between CGN and IQGAP1. IQGAP1 functions as a regulator of multiple GTPases, particularly the Rho family. This overexpressed IQGAP1 was consistently associated with the activation of Rac1, as evidenced by the analysis of the cancer cell line and clinical sample harboring CGN c.3560C > T. Notably, activated Rac1 was suppressed following the downregulation of IQGAP1 by siRNA. Treatment with NSC23766, a selective inhibitor for Rac1-GEF interaction, resulted in the inactivation of Rac1. This intervention mitigated the EMT program in cancer cells carrying CGN c.3560C > T. Consistently, xenograft tumors with WT CGN showed no sensitivity to NSC23766 treatment, but NSC23766 demonstrated the capacity to attenuate tumor growth harboring c.3560C > T. CONCLUSIONS: CGN c.3560C > T leads to IQGAP1 overexpression, subsequently triggering Rac1-dependent EMT. Targeting activated Rac1 is a strategy to impede the advancement of cancers carrying this specific variant.

Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints.

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

Tigecycline causes loss of cell viability mediated by mitochondrial OXPHOS and RAC1 in hepatocellular carcinoma cells.

BACKGROUND: Despite recent advances in locoregional, systemic, and novel checkpoint inhibitor treatment, hepatocellular carcinoma (HCC) is still associated with poor prognosis. The feasibility of potentially curative liver resection (LR) and transplantation (LT) is limited by the underlying liver disease and a shortage of organ donors. Especially after LR, high recurrence rates present a problem and circulating tumor cells are a major cause of extrahepatic recurrence. Tigecycline, a commonly used glycylcycline antibiotic, has been shown to have antitumorigenic effects and could be used as a perioperative and adjuvant therapeutic strategy to target circulating tumor cells. We aimed to investigate the effect of tigecycline on HCC cell lines and its mechanisms of action. METHODS: Huh7, HepG2, Hep3B, and immortalized hepatocytes underwent incubation with clinically relevant tigecycline concentrations, and the influence on proliferation, migration, and invasion was assessed in two- and three-dimensional in vitro assays, respectively. Bioinformatic analysis was used to identify specific targets of tigecycline. The expression of RAC1 was detected using western blot, RT-PCR and RNA sequencing. ELISA and flow cytometry were utilized to measure reactive oxygen species (ROS) generation upon tigecycline treatment and flow cytometry to detect alterations in cell cycle. Changes in mitochondrial function were detected via seahorse analysis. RNA sequencing was performed to examine involved pathways. RESULTS: Tigecycline treatment resulted in a significant reduction of mitochondrial function with concomitantly preserved mitochondrial size, which preceded the observed decrease in HCC cell viability. The sensitivity of HCC cells to tigecycline treatment was higher than that of immortalized non-cancerous THLE-2 hepatocytes. Tigecycline inhibited both migratory and invasive properties. Tigecycline application led to an increase of detected ROS and an S-phase cell cycle arrest. Bioinformatic analysis identified RAC1 as a likely target for tigecycline and the expression of this molecule was increased in HCC cells as a result of tigecycline treatment. CONCLUSION: Our study provides evidence for the antiproliferative effect of tigecycline in HCC. We show for the first time that this effect, likely to be mediated by reduced mitochondrial function, is associated with increased expression of RAC1. The reported effects of tigecycline with clinically relevant and achievable doses on HCC cells lay the groundwork for a conceivable use of this agent in cancer treatment.

Tiam1 methylation by NSD2 promotes Rac1 signaling activation and colon cancer metastasis.

Metastasis is a major cause of cancer therapy failure and mortality. However, targeting metastatic seeding and colonization remains a significant challenge. In this study, we identified NSD2, a histone methyltransferase responsible for dimethylating histone 3 at lysine 36, as being overexpressed in metastatic tumors. Our findings suggest that NSD2 overexpression enhances tumor metastasis both in vitro and in vivo. Further analysis revealed that NSD2 promotes tumor metastasis by activating Rac1 signaling. Mechanistically, NSD2 combines with and activates Tiam1 (T lymphoma invasion and metastasis 1) and promotes Rac1 signaling by methylating Tiam1 at K724. In vivo and in vitro studies revealed that Tiam1 K724 methylation could be a predictive factor for cancer prognosis and a potential target for metastasis inhibition. Furthermore, we have developed inhibitory peptide which was proved to inhibit tumor metastasis through blocking the interaction between NSD2 and Tiam1. Our results demonstrate that NSD2-methylated Tiam1 promotes Rac1 signaling and cancer metastasis. These results provide insights into the inhibition of tumor metastasis.

Microglial Rac1 is essential for experience-dependent brain plasticity and cognitive performance.

Microglia, the largest population of brain immune cells, continuously interact with synapses to maintain brain homeostasis. In this study, we use conditional cell-specific gene targeting in mice with multi-omics approaches and demonstrate that the RhoGTPase Rac1 is an essential requirement for microglia to sense and interpret the brain microenvironment. This is crucial for microglia-synapse crosstalk that drives experience-dependent plasticity, a fundamental brain property impaired in several neuropsychiatric disorders. Phosphoproteomics profiling detects a large modulation of RhoGTPase signaling, predominantly of Rac1, in microglia of mice exposed to an environmental enrichment protocol known to induce experience-dependent brain plasticity and cognitive performance. Ablation of microglial Rac1 affects pathways involved in microglia-synapse communication, disrupts experience-dependent synaptic remodeling, and blocks the gains in learning, memory, and sociability induced by environmental enrichment. Our results reveal microglial Rac1 as a central regulator of pathways involved in the microglia-synapse crosstalk required for experience-dependent synaptic plasticity and cognitive performance.

Integrin ß4 promotes DNA damage-related drug resistance in triple-negative breast cancer via TNFAIP2/IQGAP1/RAC1.

Anti-tumor drug resistance is a challenge for human triple-negative breast cancer (TNBC) treatment. Our previous work demonstrated that TNFAIP2 activates RAC1 to promote TNBC cell proliferation and migration. However, the mechanism by which TNFAIP2 activates RAC1 is unknown. In this study, we found that TNFAIP2 interacts with IQGAP1 and Integrin ß4. Integrin ß4 activates RAC1 through TNFAIP2 and IQGAP1 and confers DNA damage-related drug resistance in TNBC. These results indicate that the Integrin ß4/TNFAIP2/IQGAP1/RAC1 axis provides potential therapeutic targets to overcome DNA damage-related drug resistance in TNBC.

Cell aggregation activates small GTPase Rac1 and induces CD44 cleavage by maintaining lipid raft integrity.

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.

Circular RNA CSPP1 motivates renal cell carcinoma carcinogenesis and the Warburg effect by targeting RAC1 through microRNA-493-5p.

Circular RNAs (circRNAs) take on regulatory roles in renal cell carcinoma (RCC). The research's goal was to figure out circ-CSPP1's role and molecular mechanism in RCC. The results clarified that circ-CSPP1 expression was enhanced in RCC. Down-regulating circ-CSPP1 refrained the proliferation, migration, invasion, and Warburg effect (aerobic glycolysis), but accelerated apoptosis of RCC cells. The luciferase activity assay exhibited that circ-CSPP1 could perform as an endogenous sponge for miR-493-5p. Elevating miR-493-5p repressed RCC progression. The bioinformatics website starBase confirmed that ras-related C3 botulinum toxin substrate 1 (RAC1) was a target gene of miR-493-5p. Circ-CSPP1 up-regulated RAC1 by sponging miR-493-5p, and elevating RAC1 could turn around the effect of down-regulating circ-CSPP1 on RCC cells. Taken together, circ-CSPP1 is identified as a novel RCC-promoting RNA that could serve as a latent therapeutic target for RCC therapy.

MiR-22-3p suppresses NSCLC cell migration and EMT via targeting RAC1 expression.

Previous studies have demonstrated the tumor-suppressive function of microRNA-22-3p (miR-22-3p) in several cancers, whereas the significance of miR-22-3p in non-small cell lung cancer (NSCLC) remains unclear. In this study, we explored the biological function and molecular mechanism of miR-22-3p in NSCLC cells. First, we assessed the expression of miR-22-3p in NSCLC tissues and cells based on RT-qPCR and TCGA database. Compared with normal lung tissues and cells, miR-22-3p expression was dramatically decreased in lung cancer tissues and cells. miR-22-3p expression was also correlated with lymph node metastasis and tumor size, but not TNM stages. We further explored the in vitro function of miR-22-3p on the migration and epithelial-mesenchymal transition (EMT) of NSCLC cells. The results showed that overexpression of miR-22-3p suppressed the migration and EMT of NSCLC cells, whereas silencing miR-22-3p showed the opposite effect. Luciferase assay demonstrated that RAS-related C3 botulinum toxin substrate 1 (RAC1) was the target gene for miR-22-3p. Mechanistically, we demonstrated that miR-22-3p suppressed the cell migration and EMT via downregulation of RAC1 because the inhibitory effect of miR-22-3p on cell migration and EMT of NSCLC cells was reversed by RAC1 overexpression. Based on these novel data, the miR-22-3p/RAC1 axis may be an alternative target in the therapeutic intervention of NSCLC.

The Evaluation of Rac1 Signaling as a Potential Therapeutic Target of Alzheimer's Disease.

The Small GTPase Rac1 is critical for various fundamental cellular processes, including cognitive functions. The cyclical activation and inactivation of Rac1, mediated by Rac guanine nucleotide exchange factors (RacGEFs) and Rac GTPase-activating proteins (RacGAPs), respectively, are essential for activating intracellular signaling pathways and controlling cellular processes. We have recently shown that the Alzheimer's disease (AD) therapeutic drug donepezil activates the Rac1-PAK pathway in the nucleus accumbens (NAc) for enhanced aversive learning. Also, PAK activation itself in the NAc enhances aversive learning. As aversive learning allows short-term preliminary AD drug screening, here we tested whether sustained Rac1 activation by RacGAP inhibition can be used as an AD therapeutic strategy for improving AD-learning deficits based on aversive learning. We found that the RacGAP domain of breakpoint cluster region protein (Bcr) (Bcr-GAP) efficiently inhibited Rac1 activity in a membrane ruffling assay. We also found that, in striatal/accumbal primary neurons, Bcr knockdown by microRNA mimic-expressing adeno-associated virus (AAV-miRNA mimic) activated Rac1-PAK signaling, while Bcr-GAP-expressing AAV inactivated it. Furthermore, conditional knockdown of Bcr in the NAc of wild-type adult mice enhanced aversive learning, while Bcr-GAP expression in the NAc inhibited it. The findings indicate that Rac1 activation by RacGAP inhibition enhances aversive learning, implying the AD therapeutic potential of Rac1 signaling.

Rac1 activation in oral squamous cell carcinoma as a predictive factor associated with lymph node metastasis.

OBJECTIVES: Secondary lymph node metastasis (SLNM) indicates a poor prognosis, and limiting it can improve the survival rate in early-stage tongue squamous cell carcinoma (TSCC). Many factors have been identified as predictors of SLNM; however, there is no unified view. Ras-related C3 botulinum toxin substrate 1 (Rac1) was found to be a promoter of the epithelial-mesenchymal transition (EMT) and is also attracting attention as a new therapeutic target. This study aims to investigate the role of Rac1 in metastasis and its relationship with pathological findings in early-stage TSCC. MATERIALS AND METHODS: Rac1 expression levels of 69 cases of stage I/II TSCC specimens and their association with clinicopathological characteristics were evaluated by immunohistochemical staining. The role of Rac1 in oral squamous cell carcinoma (OSCC) was examined after Rac1 in OSCC cell lines was silenced in vitro. RESULTS: High Rac1 expression was significantly associated with the depth of invasion (DOI), tumor budding (TB), vascular invasion, and SLNM (p < 0.05). Univariate analyses revealed that Rac1 expression, DOI, and TB were factors significantly associated with SLNM (p < 0.05). Moreover, our multivariate analysis suggested that Rac1 expression was the only independent determinant of SLNM. An in vitro study revealed that Rac1 downregulation tended to decrease cell migration and proliferation. CONCLUSION: Rac1 was suggested to be an important factor in the metastasis of OSCC, and it could be useful as a predictor of SLNM.

Impaired Insulin Signaling Mediated by the Small GTPase Rac1 in Skeletal Muscle of the Leptin-Deficient Obese Mouse.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lepob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lepob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lepob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lepob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lepob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.

A missense variant at the RAC1-PAK1 binding site of RAC1 inactivates downstream signaling in VACTERL association.

RAC1 at 7p22.1 encodes a RAC family small GTPase that regulates actin cytoskeleton organization and intracellular signaling pathways. Pathogenic RAC1 variants result in developmental delay and multiple anomalies. Here, exome sequencing identified a rare de novo RAC1 variant [NM_018890.4:c.118T > C p.(Tyr40His)] in a male patient. Fetal ultrasonography indicated the patient to have multiple anomalies, including persistent left superior vena cava, total anomalous pulmonary venous return, esophageal atresia, scoliosis, and right-hand polydactyly. After birth, craniofacial dysmorphism and esophagobronchial fistula were confirmed and VACTERL association was suspected. One day after birth, the patient died of respiratory failure caused by tracheal aplasia type III. The molecular mechanisms of pathogenic RAC1 variants remain largely unclear; therefore, we biochemically examined the pathophysiological significance of RAC1-p.Tyr40His by focusing on the best characterized downstream effector of RAC1, PAK1, which activates Hedgehog signaling. RAC1-p.Tyr40His interacted minimally with PAK1, and did not enable PAK1 activation. Variants in the RAC1 Switch II region consistently activate downstream signals, whereas the p.Tyr40His variant at the RAC1-PAK1 binding site and adjacent to the Switch I region may deactivate the signals. It is important to accumulate data from individuals with different RAC1 variants to gain a full understanding of their varied clinical presentations.

RUNX1/ETO regulates reactive oxygen species (ROS) levels in t(8,21) acute myeloid leukaemia via FLT3 and RAC1.

Reactive oxygen species (ROS) homeostasis is crucial for leukaemogenesisand deregulation would hamper leukaemic progression. Although the regulatory effects of RUNX1/ETO has been extensively studied, its underlying molecular mechanims in ROS production in t(8,21) AML is yet to be fully elucidated. Here, we report that RUNX1/ETO could directly control FLT3 by occupying several DNA elements on FLT3 locus. The possible hijacking mechanism by RUNX1/ETO over FLT3 mediated ROS modulation in AML t(8;21) was made apparent when suppression of RUNX1/ETO led to decrement in ROS levels and the direct oxidative marker FOXO3 but not in FLT3 and RAC1 suppressed t(8,21) AML cell line Furthermore, nuclear import of RUNX1/ETO was aberrated following RUNX1/ETO and RAC1 suppression suggesting association in ROS control. A different picture was depicted in non t(8;21) cells where suppression of RAC1 and FLT3 led to decreased levels of FOXO3a and ROS. Results alltogether indicate a possible dysregulation of ROS levels by RUNX1/ETO in t(8,21) AML.