PAK1 Expression Research Articles

Fisetin Modulates Human Oral Squamous Cell Carcinoma Proliferation by Blocking PAK4 Signaling Pathways.

ObjectiveHuman oral squamous cell carcinoma (OSCC) is a major cause of mortality and morbidity worldwide. There is an urgent need to identify bioactive molecules and potential target genes that could inhibit carcinogenesis for OSCC therapy. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a naturally occurring flavonoid, has been previously shown to have anti-proliferative activities in OSCC; however, its molecular mechanism is unknown.MethodsColony formation, cell viability, Boyden chamber, wound healing, and tumor xenograft assays were used to detect the impact of fisetin on OSCC cells in vitro and in vivo. Western blot analysis was used to examine the corresponding protein expression.ResultsFisetin treatment significantly inhibited proliferation and promoted apoptosis by repressing PAK4 expression. Moreover, fisetin treatment attenuated cell migration by blocking PAK4 signaling pathways. In addition, the tumor xenograft showed anti-tumor growth effects of fisetin exposure in vivo.ConclusionFisetin may represent a potential therapeutic strategy for human OSCC by targeting PAK4 signaling pathways.

Systemic analysis of the expression and prognostic significance of PAKs in breast cancer

PAKs (p21-activated kinases) are reported to play crucial roles in a variety of cellular processes and participate in the progression of human cancers. However, the expression and prognostic values of PAKs remain poorly explored in breast cancers. In our study, we examined the mRNA and protein expression levels of PAKs and the prognostic value. We also analyzed the interaction network, genetic alteration, and functional enrichment of PAKs. The results showed that the mRNA levels of PAK1, PAK2, PAK4 and PAK6 were significantly up-regulated in breast cancer compared with normal tissues, while the reverse trend for PAK3 and PAK5 was found, furthermore, the proteins expression of PAK1, PAK2 and PAK4 in breast cancer tissues were higher than that in normal breast tissues. Survival analysis revealed breast cancer patients with low mRNA expression of PAK3 and PAK5 showed worse RFS, conversely, elevated PAK4 levels predicted worse RFS. In addition, the breast cancer patients with PAKs genetic alterations correlated with worse OS. These results indicated that PAKs might be promising potential biomarkers for breast cancer.

Mononuclear phagocyte system blockade improves therapeutic exosome delivery to the myocardium.

Rationale: Exosomes are emerging as a promising drug delivery carrier. However, rapid uptake of exosomes by the mononuclear phagocyte system (MPS) remains an obstacle for drug delivery into other targeted organs, including the heart. We hypothesized that prior blocking of uptake of exosomes by the MPS would improve their delivery to the targeted organs.Methods: Exosomes were isolated from the cell culture medium. Fluorescence-labeled exosomes were tracked in vitro and in vivo by fluorescence imaging. The expression of clathrin heavy chain (Cltc), cavolin1, Pak1 and Rhoa, known genes for endocytosis, were profiled in various cell lines and organs by qPCR. The knockdown efficiency of siRNA against Cltc was analyzed by Western blotting. Exosomecontrol and exosomeblocking were constructed by encapsulating isolated exosomes with siControl or siClathrin via electroporation, while exosometherapeutic was constructed by encapsulating isolated exosomes with miR-21a. Doxorubicin-induced cardiotoxicity model was used to verify the therapeutic efficiency of the exosome-based miR-21a delivery by echocardiography.Results: Exosomes were preferentially accumulated in the liver and spleen, mainly due to the presence of abundant macrophages. Besides the well-known phagocytic effect, efficient endocytosis also contributes to the uptake of exosomes by macrophages. Cltc was found to be highly expressed in the macrophages compared with other endocytosis-associated genes. Accordingly, knockdown of Cltc significantly decreased the uptake of exosomes by macrophages in vitro and in vivo. Moreover, prior injection of exosomeblocking strikingly improved the delivery efficiency of exosomes to organs other than spleen and liver. Consistently, compared with the direct injection of exosometherapeutic, prior injection of exosomeblocking produced a much better therapeutic effect on cardiac function in the doxorubicin-induced cardiotoxicity mouse model.Conclusions: Prior blocking of endocytosis of exosomes by macrophages with exosomeblocking successfully and efficiently improves the distribution of following exosometherapeutic in targeted organs, like the heart. The established two-step exosome delivery strategy (blocking the uptake of exosomes first followed by delivery of therapeutic exosomes) would be a promising method for gene therapy.

PAK1 expression determines poor prognosis and immune evasion in metastatic renal cell carcinoma patients

BackgroundPrevious studies have shown the prognostic value of PAK1 expression in different tumor patients, including nonmetastatic renal cell carcinoma. In this study, we explored the prognostic and drug predictive value of PAK1 expression in metastatic renal cell carcinoma (mRCC) patients treated with tyrosine kinase inhibitors (TKIs). Materials and MethodsWe retrospectively enrolled 138 mRCC patients treated with TKIs from a single institution from 2005 to 2014. Analyses were based on 111 patients who met our inclusion criteria. The validation set enrolled 538 RCC patients from The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma cohort (TCGA KIRC) between 1998 and 2013 in North America. PAK1 expression was assessed by immunohistochemistry (IHC) on tissue microarrays. ResultsHigh PAK1 expression was associated with short overall survival (OS) (P < 0.001) and progression-free survival (PFS) (P = 0.008). Multivariate analyses further indicated that PAK1 expression was an independent prognostic factor for OS (hazard ratio 3.301 [95% confidence interval 2.579–10.899], P < 0.001) and PFS (hazard ratio 3.108 [95% confidence interval 1.795–5.381], P < 0.001). Subgroup analyses suggested that PAK1 was more significant in patients with the intermediate risk group of Heng risk criteria (OS, P = 0.004). Of note, patients treated with Sunitinib showed improved outcome in the low PAK1 subgroup (OS, P = 0.002; PFS, P = 0.013). Finally, relationship was found between PAK1 expression and natural killer cell-mediated cytotoxicity according to gene profile investigation. ConclusionsHigh PAK1 expression predicted dismal prognosis in mRCC patients treated with TKIs. Besides, PAK1 was a potential predictor for TKIs treatments.

Gestational diabetes and maternal obesity are associated with sex-specific changes in miRNA and target gene expression in the fetus.

Background/ObjectivePregnancies complicated by gestational diabetes (GDM) or maternal obesity have been linked to the development of diabetes, obesity and fatty liver disease later in life with sex-specific manifestations. Alterations in miRNA expression in offspring exposed to GDM and maternal obesity and effects on hepatic development are unknown. Here we describe how exposure to maternal obesity in utero leads to sex-specific changes in miRNA and target gene expression in human fetal liver.MethodsCandidate miRNA expression was measured in 2nd trimester amniotic fluid (AF) from women with GDM. Targets of differentially expressed miRNAs were determined and pathway enrichment of target genes was performed. MiRNA and target gene expression were measured in a separate cohort of 2nd trimester primary human fetal hepatocytes (PHFH) exposed to maternal obesity via QPCR and western blot. All studies were IRB approved.ResultsGDM exposed AF had significant increases in miRNAs 199a-3p, 503-5p, and 1268a (fold change (FC) ≥1.5, p<0.05). Female offspring specific analysis showed enrichment in miRNAs 378a-3p, 885-5p, and 7-1-3p (p<0.05). MiRNA gene targets were enriched in hepatic pathways. Key genes regulating de novo lipogenesis were upregulated in obesity exposed PHFH, especially in males. Significantly altered miRNAs in GDM AF were measured in obese exposed PHFH, with consistent increases in miRNAs 885-5p, 199-3p, 503-5p, 1268a and 7-1-3p (FC ≥1.5, p<0.05). Female PHFH exposed to maternal obesity had increased expression of miR-885-5p, miR-199-3p, miR-503-5p, miR-1268s and miR-7-1-3p, (p<0.05), corresponding to decreased target genes expression for ABCA1, PAK4 and INSR. In male PHFHs, no miRNA changes were measured but there was increased expression of ABCA1, PAK4, and INSR (p<0.05).ConclusionOur data suggest sex-specific changes in miRNA and gene expression in PHFH may be one mechanism contributing to the sexual dimorphism of metabolic disease in offspring exposed to GDM and maternal obesity in utero.

Inhibition of PAK1 suppresses pancreatic cancer by stimulation of anti-tumour immunity through down-regulation of PD-L1.

Immunotherapies have not yielded significant clinical benefits for pancreatic ductal adenocarcinoma (PDA) because of the existence of an immunosuppressive tumour microenvironment (TME) characterized by a desmoplastic stroma containing infiltrated immune cells and activated pancreatic stellate cells (PSCs). This study aims to investigate the involvement of PAK1 in anti-tumour immunity. In PDA patients, low PAK1 expression, low activation of PSC and high CD8+ T cell/PAK1 ratios correlated with longer overall survival. In a murine PDA model, PAK1 knockout increased intra-tumoral CD4+ and CD8+ T cells, inhibited PSCs activation and extended survival. Inhibition of PAK1 reduced PSC-stimulated PDA cell proliferation and migration, blocked PSC-mediated protection of PDA cells from killing by cytotoxic lymphocytes and decreased intrinsic and PSC-stimulated PD-L1 expression in PDA cells, which further sensitized PDA cells to cytotoxic lymphocytes. Inhibition of PAK1 stimulates anti-tumour immunity by increasing intra-tumoral CD4+ and CD8+ T cells, and by sensitizing PDA cells to killing by cytotoxic lymphocytes via down-regulation of intrinsic and PSC-stimulated PD-L1 expression. PAK1 inhibitors, especially in combination with immune checkpoint inhibitors may result in improved efficacy of immunotherapy of PDA.

PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells

Summaryp21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to podosomes: transient adhesive structures that degrade the extracellular matrix to facilitate rapid myeloid cell migration. We find that treatment of transforming growth factor β (TGF-β)-differentiated monocytic (THP-1) cells with a PAK4-targeted inhibitor significantly reduces podosome formation and induces the formation of focal adhesions. This switch in adhesions confers a diminution of matrix degradation and reduced cell migration. Furthermore, reduced PAK4 expression causes a significant reduction in podosome number that cannot be rescued by kinase-dead PAK4, supporting a kinase-dependent role. Concomitant with PAK4 depletion, phosphorylation of Akt is perturbed, whereas a specific phospho-Akt signal is detected within the podosomes. Using superresolution analysis, we find that PAK4 specifically localizes in the podosome ring, nearer to the actin core than other ring proteins. We propose PAK4 kinase activity intersects with the Akt pathway at the podosome ring:core interface to drive regulation of macrophage podosome turnover.

Circ-TFCP2L1 Promotes the Proliferation and Migration of Triple Negative Breast Cancer through Sponging miR-7 by Inhibiting PAK1.

CircRNAs are essential factors that have been verified to regulate various forms of carcinogenesis. However, the role of circRNAs in triple negative breast cancer (TNBC) tumourigenesis is not well clarified. In this study, we explored the circRNA expression profiles and possible modulation mechanism of circRNAs on triple negative breast cancer tumourigenesis. We used three pairs of triple negative breast cancer tissues and adjacent noncancerous tissues to perform a human circRNA microarray for screening of circRNA expression patterns in TNBC. The results showed that circ-TFCP2L1 was significantly up-regulated in TNBC tissues and cells, tending to have a shorter disease-free survival of TNBC patients. In vitro loss-of-function experiments showed that knockdown of circ-TFCP2L1 significantly suppressed the proliferation and migration of TNBC cells. Moreover, the results showed that the proliferation and migration capabilities and PAK1 expression in TNBC cells treated with si-circ-TFCP2L1 + miR-7 mimics were significantly suppressed compared with the normal group. Therefore, circ-TFCP2L1 was identified as a sponge of miR-7 functionally targeting PAK1 and further promoting the proliferation and migration of TNBC cells. Taken together, the results from our study reveal a novel regulatory mechanism and offer novel insight into the role of circ-TFCP2L1 in progression of triple negative breast cancer.

Cardiotoxic effects of [3-[2-(diethylamino)ethyl]-1H-indol-4-yl] acetate and 3-[2-[ethyl(methyl)amino]ethyl]-1H-indol-4-ol

Two synthetic tryptamines, namely [3-[2-(diethylamino)ethyl]-1H-indol-4-yl] acetate (4-AcO-DET) and 3-[2-[ethyl(methyl)amino]ethyl]-1H-indol-4-ol (4-HO-MET), are abused by individuals seeking recreational hallucinogens. These new psychoactive substances (NPSs) can cause serious health problems because their adverse effects are mostly unknown. In the present study, we evaluated the cardiotoxicity of 4-AcO-DET and 4-HO-MET using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, electrocardiography (ECG), and the human ether-a-go-go-related gene (hERG) assay. In addition, we analyzed the expression level of p21 (CDC42/RAC)-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, 4-AcO-DET- and 4-HO-MET-treated H9c2 cells proliferated in a concentration-dependent manner. Moreover, both substances increased QT intervals (as determined using ECG) in Sprague–Dawley rats and inhibited potassium channels (as verified by the hERG assay) in Chinese hamster ovary cells. However, there was no change in PAK1 expression. Collectively, the results indicated that 4-AcO-DET and 4-HO-MET might cause adverse effects on the cardiovascular system. Further studies are required to confirm the relationship between PAK1 expression and cardiotoxicity. The findings of the present study would provide science-based evidence for scheduling the two NPSs.

Melittin inhibits proliferation, migration and invasion of bladder cancer cells by regulating key genes based on bioinformatics and experimental assays.

The antitumour effect of melittin (MEL) has recently attracted considerable attention. Nonetheless, information regarding the functional role of MEL in bladder cancer (BC) is currently limited. Herein, we investigated the effect of MEL on critical module genes identified in BC. In total, 2015 and 4679 differentially expressed genes (DEGs) associated with BC were identified from the GSE31189 set and The Cancer Genome Atlas database, respectively. GSE‐identified DEGs were mapped and analysed using Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes analyses to determine BC‐involved crucial genes and signal pathways. Coupled with protein–protein interaction network and Molecular Complex Detection analyses, Modules 2 and 4 were highlighted in the progression of BC. In in‐vitro experiments, MEL inhibited the proliferation, migration, and invasion of UM‐UC‐3 and 5637 cells. The expression of NRAS, PAK2, EGFR and PAK1 in Module 4—enriched in the MAPK signalling pathway—was significantly reduced after treatment with MEL at concentrations of 4 or 6 μg/mL. Finally, quantitative reverse transcription‐polymerase chain reaction and Western blotting analyses revealed MEL inhibited the expression of genes at the mRNA (ERK1/2, ERK5, JNK and MEK5), protein (ERK5, MEK5, JNK and ERK1/2) and phosphorylation (p‐ERK1/2, p‐JNK, and p‐38) levels. This novel evidence indicates MEL exerts effects on the ERK5‐MAK pathway—a branch of MAPK signalling pathway. Collectively, these findings provide a theoretical basis for MEL application in BC treatment.

MicroRNA-455-3p promotes TGF-\u03b2 signaling and inhibits osteoarthritis development by directly targeting PAK2

MicroRNAs (miRNAs, miR) play a key role in the pathogenesis of osteoarthritis (OA). Few studies have examined the regulatory role of P21-activated kinases (PAKs), a family of serine/threonine kinases, in OA. The aim of this study was to determine whether miR-455-3p can regulate cartilage degeneration in OA by targeting PAK2. MiR-455-3p knockout mice showed significant degeneration of the knee cartilage. MiR-455-3p expression increased and PAK2 expression decreased in the late stage of human adipose-derived stem cell (hADSC) chondrogenesis and in chondrocytes affected by OA. Furthermore, in both miR-455-3p-overexpressing chondrocytes and PAK2-suppressing chondrocytes, cartilage-specific genes were upregulated, and hypertrophy-related genes were downregulated. A luciferase reporter assay confirmed that miR-455-3p regulates PAK2 expression by directly targeting the 3′-untranslated regions (3′UTRs) of PAK2 mRNA. IPA-3, a PAK inhibitor, inhibited cartilage degeneration due to OA. Moreover, suppressing PAK2 promoted R-Smad activation in the TGF/Smad signaling pathway in chondrocytes. Altogether, our results suggest that miR-455-3p promotes TGF-β/Smad signaling in chondrocytes and inhibits cartilage degeneration by directly suppressing PAK2. These results thus indicate that miR-455-3p and PAK2 are novel potential therapeutic agents and targets, respectively, for the treatment of OA.

Mechanisms involved in follistatin-induced hypertrophy and increased insulin action in skeletal muscle.

BackgroundSkeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor β (TGF‐β) superfamily, including activin A, which causes atrophy. TGF‐β superfamily ligands also negatively regulate insulin‐sensitive proteins, but whether this pathway contributes to insulin action remains to be determined.MethodsTo elucidate if TGF‐β superfamily ligands regulate insulin action, we used an adeno‐associated virus gene editing approach to overexpress an activin A inhibitor, follistatin (Fst288), in mouse muscle of lean and diet‐induced obese mice. We determined basal and insulin‐stimulated 2‐deoxy‐glucose uptake using isotopic tracers in vivo. Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans, we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux‐en‐Y gastric bypass (RYGB).ResultsFst288 muscle overexpression markedly increased in vivo insulin‐stimulated (but not basal) glucose uptake (+75%, P < 0.05) and increased protein expression and intracellular insulin signalling of AKT, TBC1D4, PAK1, pyruvate dehydrogenase‐E1α, and p70S6K, while decreasing TBC1D1 signaling (P < 0.05). Fst288 increased both basal and insulin‐stimulated protein synthesis, but no correlation was observed between the Fst288‐driven hypertrophy and the increase in insulin‐stimulated glucose uptake. Importantly, Fst288 completely normalized muscle glucose uptake in insulin‐resistant diet‐induced obese mice. RYGB surgery doubled circulating Fst and reduced activin A (−24%, P < 0.05) concentration 1 week after surgery before any significant weight loss in morbidly obese normoglycemic patients, while major weight loss after 1 year did not further change the concentrations.ConclusionsWe here present evidence that Fst is a potent regulator of insulin action in muscle, and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4, pyruvate dehydrogenase‐E1α, and PAK1 as Fst targets. Circulating Fst more than doubled post‐RYGB surgery, a treatment that markedly improved insulin sensitivity, suggesting a role for Fst in regulating glycaemic control. These findings demonstrate the therapeutic potential of inhibiting TGF‐β superfamily ligands to improve insulin action and Fst's relevance to muscle wasting‐associated insulin‐resistant conditions in mice and humans.

Knockdown of PAK1 Inhibits the Proliferation and Invasion of Non-Small Cell Lung Cancer Cells Through the ERK Pathway.

The p21-activated kinase (PAK) family of serine/threonine kinases plays a pivotal role in various human tumors, as supported by our previous report on the overexpressed PAK isoforms in non-small cell lung cancer (NSCLC). To better understand the role of PAKs in tumorigenesis, the authors examined PAK1 expression patterns and its significance in NSCLC. It was demonstrated by immunohistochemical staining that PAK1 was increased and localized in the cytoplasm in 151 of 207 cases. High levels of PAK1 expression correlated with a histologic type of tumor (squamous cell carcinoma), tumor node metastasis stage, and lymph nodal status. We also examined the biological role of PAK1 in lung cancer cell lines transfected with PAK1-small interfering RNA. Decreased expression of PAK1 inhibited lung cancer cell proliferation and invasion, which is the major cause of lung cancer malignancy. Downregulated expression of PAK1 hampered rapidly accelerated fibrosarcoma/mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase pathway activity but did not affect Wnt/β-catenin signaling. Our findings suggest that PAK1 is an important oncogene in NSCLC, as decreased expression of PAK1 inhibited the proliferation and invasion of NSCLC cells by blocking the ERK pathway. These results provide evidence for using PAK1 inhibition as potential anticancer therapy.

Long non-coding RNA LINC01296 promotes progression of oral squamous cell carcinoma through activating the MAPK/ERK signaling pathway via the miR-485-5p/PAK4 axis.

IntroductionLong intergenic non-protein-coding RNA 1296 (LINC01296), a newly identified lncRNA, can function as an oncogenic driver to promote the development of multiple carcinomas. However, the effect of LINC01296 on oral squamous cell carcinoma (OSCC) is still unclear.Material and methodsWe determined the expression and role of LINC01296 in OSCC tissues and cell lines. The cell viability, migration and invasion were determined by MTT, wound healing assay and transwell assay, respectively. Flow cytometry was used for detecting cell cycle and apoptosis. The interaction and association between LINC01296, microRNA-485-5p (miR-485-5p) and p21 (RAC1) activated kinase 4 (PAK4) were analyzed by RNA immunoprecipitation (RIP) and luciferase reporter assays. The xenograft mouse model was established to detect the effect of LINC01296 on OSCC tumor growth.ResultsOur study showed that LINC01296 was over-expressed in OSCC tissues and cell lines. The level of LINC01296 was positively correlated with the patient’s tumor node metastasis (TNM) stage and nodal invasion. Knockdown of LINC01296 effectively inhibits cell viability, migration and invasion but promotes cell apoptosis in vitro. The in vivo experiment showed that LINC01296 knockdown inhibited OSCC tumor growth. The following analysis indicated that LINC01296 acted as a ceRNA for miR-485-5p, and PAK4 was identified as a direct target of miR-485-5p. Furthermore, we found that the effects of LINC01296 on OSCC progression were through regulating the expression of PAK4/p-MEK/p-ERK via sponging miR-485-5p.ConclusionsLINC01296 promote the cell cycle, proliferation, migration and invasion, and inhibit apoptosis of OSCC cells through activating the MAPK/ERK signaling pathway via sponging miR-485-5p to regulate PAK4 expression. These results suggested that the LINC01296/miR-485-5p/PAK4 axis was closely associated with OSCC progression. Our study provides a new insight into the molecular pathogenesis of OSCC, and may supply novel biomarkers for diagnosis and therapy of OSCC.

Melatonin-Mediated Pak2 Activation Reduces Cardiomyocyte Death Through Suppressing Hypoxia Reoxygenation Injury-Induced Endoplasmic Reticulum Stress.

Cardiac reperfusion injury has been found to be associated with endoplasmic reticulum (ER) stress. Recently, p21-activated kinase 2 (Pak2) has been identified as a primary mediator of ER stress in chronic myocardial injury. Melatonin, a biological clock-related hormone, has been demonstrated to attenuate heart reperfusion burden by modulating ER stress and mitochondrial function. The aim of our study was to explore whether reperfusion-induced ER stress is modulated by melatonin through Pak2. Hypoxia reoxygenation (HR) was used in vitro to mimic reperfusion injury in cardiomyocytes. ER stress, oxidative stress, calcium overload, and cell death were measured through Western blotting, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and immunofluorescence with the assistance of siRNA transfection and pathway blocker treatment. The results of our study demonstrated that HR decreased the levels of Pak2 in cardiomyocytes in vitro, and inactivation of Pak2 was associated with ER stress, oxidative stress, calcium overload, caspase-12 activation, and cardiomyocytes apoptosis in vitro. Interestingly, melatonin treatment attenuated HR-mediated ER stress, redox imbalance, calcium overload, and caspase-12-related cardiomyocytes apoptosis, and these protective effects were dependent on Pak2 upregulation. Knockdown of Pak2 abolished the beneficial actions exerted by melatonin on HR-treated cardiomyocytes in vitro. Finally, we found that melatonin reversed Pak2 expression by activating the AMPK pathway and blockade of the AMPK pathway suppressed Pak2 upregulation and cardiomyocytes survival induced by melatonin in the presence of HR stress. Overall, our study reports that the AMPK-Pak2 axis, a novel signaling pathway modulated by melatonin, sends prosurvival signals for cardiomyocytes reperfusion injury through attenuation of ER stress in vitro.

Polarization and sprouting of endothelial cells by angiopoietin-1 require PAK2 and paxillin-dependent Cdc42 activation.

Binding of angiopoietin-1 (Ang-1) to its receptor Tie2 on endothelial cells (ECs) promotes vessel barrier integrity and angiogenesis. Here, we identify PAK2 and paxillin as critical targets of Ang-1 responsible for EC migration, polarization, and sprouting. We found that Ang-1 increases PAK2-dependent paxillin phosphorylation and remodeling of focal adhesions and that PAK2 and paxillin are required for EC polarization, migration, and angiogenic sprouting in response to Ang-1. Our findings show that Ang-1 triggers Cdc42 activation at the leading edges of migrating ECs, which is dependent on PAK2 and paxillin expression. We also established that the polarity protein Par3 interacts with Cdc42 in response to Ang-1 in a PAK2- and paxillin-dependent manner. Par3 is recruited at the leading edges of migrating cells and in focal adhesion, where it forms a signaling complex with PAK2 and paxillin in response to Ang-1. These results show that Ang-1 triggers EC polarization and angiogenic sprouting through PAK2-dependent paxillin activation and remodeling of focal adhesions, which are necessary for local activation of Cdc42 and the associated polarity complex. We have shown that PAK2 controls a signaling pathway important for angiogenic sprouting that links focal adhesions to polarity signaling in ECs.

Melatonin ameliorates endoplasmic reticulum stress in N2a neuroblastoma cell hypoxia-reoxygenation injury by activating the AMPK-Pak2 pathway.

Endoplasmic reticulum (ER) stress has been identified as a primary factor involved in brain ischemia-reperfusion injury progression. p21-activated kinase 2 (Pak2) is a novel ER function regulator. The aim of our study is to explore the influence of Pak2 on ER stress and determine whether melatonin attenuates ER stress-mediated cell death by modulating Pak2 expression in vitro using N2a cells. The results of our study demonstrated that hypoxia-reoxygenation (HR) injury repressed the levels of Pak2, an effect that was accompanied by activation of ER stress. In addition, decreased Pak2 was associated with oxidative stress, calcium overload, and caspase-12-mediated apoptosis activation in HR-treated N2a cells. Interestingly, melatonin treatment reversed the decreased Pak2 expression under HR stress. Knockdown of Pak2 abolished the protective effects of melatonin on ER stress, oxidative stress, and caspase-12-related N2a cells death. Additionally, we found that Pak2 was regulated by melatonin via the AMPK pathway; inhibition of AMPK prevented melatonin-mediated Pak2 upregulation, a result that was accompanied by an increase in N2a cell death. Altogether, these results identify the AMPK-Pak2 axis as a new signaling pathway responsible for ER stress and N2a cell viability under HR injury. Modulation of the AMPK-Pak2 cascade via supplementation of melatonin might be considered an effective approach to attenuate reperfusion-mediated N2a cell damage via repression of ER stress.

Targeting p21-activated kinase 1 inhibits growth and metastasis via Raf1/MEK1/ERK signaling in esophageal squamous cell carcinoma cells

Backgroundp21-activated kinase 1 (PAK1) plays a fundamental role in promoting the development and progression of several cancers and is a potential therapeutic target. However, the biological function and underlying mechanism of PAK1 in esophageal squamous cell carcinoma (ESCC) remain unclear.MethodsThe expression of PAK1 was detected in both ESCC cell lines and clinical samples. Cell growth was measured by MTT, focus formation and soft agar assays. Cell migration and invasion were detected by wound healing and transwell assays. Animal models of subcutaneous tumourigenicity and tail vein metastasis were performed to determine the inhibitory effect of pharmacological inhibitor IPA-3 on tumor growth and metastasis of ESCC cells.ResultsWe found that PAK1 was frequently overexpressed in ESCC. Ectopic expression of PAK1 promoted cellular growth, colony formation and anchorage-independent growth. Overexpressing PAK1 also enhanced migration, invasion and the expression of MMP-2 and MMP-9 in ESCC cells. In contrast, silencing PAK1 by lentiviral knockdown or a specific inhibitor IPA-3 resulted in a contrary effect. Subsequent investigations revealed that Raf1/MEK1/ERK signaling pathway was involved in PAK1-mediated effect. Enhanced expression of Raf1 attenuated the inhibitory functions of PAK1 shRNA. Whereas blocking of Raf1 by shRNA or specific inhibition of MEK1 by U0126 antagonized the oncogenetic effect of PAK1 on ESCC cells. More importantly, Pharmacological inhibition of PAK1 by IPA-3 significantly suppressed tumor growth and lung metastasis of ESCC cells in vivo.ConclusionsThese data support that PAK1 is an ideal target for the development of potential therapeutic drugs for ESCC patients even with metastasis.

Numb inhibits cell proliferation, invasion, and epithelial-mesenchymal transition through PAK1/β-catenin signaling pathway in ovarian cancer.

ObjectiveThe present study aimed to investigate the expression of Numb in ovarian cancer tissues and to assess the effect of Numb on cell proliferation, invasion, and EMT in ovarian cancer.MethodsReal-time PCR and Western blotting were used to detect the mRNA and protein expression of Numb, PAK1, β-catenin, and epithelial–mesenchymal transition (EMT)-related proteins. MTT was employed to check the effect of Numb on proliferation of ovarian cancer cells. Transwell assay was performed to examine the functions of Numb and PAK1 on migration and invasion of ovarian cancer cells.ResultsThe Numb expression was significantly downregulated while PAK1 and β-catenin were significantly upregulated in both ovarian cancer tissues and cell lines. Silencing of Numb promoted cell proliferation, migration, invasion, and EMT in ovarian cancer cell lines while overexpressed Numb reversed the above effects. Moreover, the EMT process induced by the inhibition of Numb was regulated through Numb-mediated PAK1/β-catenin signaling pathway.ConclusionNumb was downregulated and associated with cell proliferation, invasion, and EMT in ovarian cancer through regulating PAK1/β-catenin signaling, providing a novel potential biomarker and potential therapeutic target for ovarian cancer.

Prognostic significance of PAK family kinases in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a clonal and heterogeneous disease characterized by a myriad of genetic defects. Genetic abnormalities are powerful prognostic factors. P21-activated kinases (PAKs) are a kind of serine/threonine protein kinases, which is regulator of plenty of oncogenic signaling pathways. The clinical and prognostic value of PAKs in AML is unclear. A total of 155 AML patients with PAK expression data from The Cancer Genome Atlas database were enrolled in this study. Eighty-four patients underwent chemotherapy only, 71 also underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the chemotherapy-only group, high PAK3 and PAK7 expression were both bound up with poor EFS and OS (all P < 0.05). However, high PAK2 expressers had better EFS and OS (all P < 0.05). Multivariate analysis demonstrated that high PAK7 expression was an adverse independent prognostic factor in patients who received chemotherapy only. PAKs have no influence in EFS and OS in patients who underwent allo-HSCT. In conclusion, high PAK2 expression is a favorable prognostic factor, as to the high expression of PAK3 and PAK7, they are poor prognostic factors, and PAK7 has better prognostic value, but their prognostic effects can be offset by allo-HSCT.