P21 Protein-activated Kinase Research Articles

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).

Effect of empagliflozin on cytoskeletal repair in the hippocampus of obese mice.

We aimed to investigate the effect of empagliflozin on hippocampal phosphorylated protein levels in obese mice. Sixteen obese mice successfully modeled on high-fat diet were randomly divided into high-fat feeding group (group H) and empagliflozin group (group H + empagliflozin, group E), eight mice in each group, and eight C57BL/6J male normal mice were selected as the control group (normal control, group C). Group E was treated with empagliflozin 10 mg/kg/d for 12 weeks, while mice in groups H and C were treated with equal amounts of saline. The spatial learning memory ability of the mice was determined by the Morris water maze experiment. Further, their body weights and serological indices were measured. Finally, total proteins were extracted from hippocampal tissues for functional analysis by the phosphorylated proteomics method. The results showed that escape latency was prolonged, retention time in the target quadrant was shortened, and the number of loop penetrations was reduced in the obese mice induced by a high-calorie diet compared with normal controls, whereas escape latency was shortened, retention time in the target quadrant was increased, and the number of loop penetrations was increased after empagliflozin treatment. Phosphoproteomics in the high-fat/control (H/C), empagliflozin/high-fat (E/H), and E/C groups showed 844, 1,552, and 1,512 differentially significant phosphorylation sites, respectively. The proteins corresponding to these differentially phosphorylated sites were mainly involved in neurodegenerative pathways and actin cytoskeleton regulation. Notably, myosin heavy chain 10 (MYH10), p21 protein-activated kinase 4 (PAK4), phosphatidylinositol 3 -phosphate 5-kinase (PIKfyve), and other differentially phosphorylated proteins were involved in actin cytoskeleton regulation. We concluded that empagliflozin protects cognitive functions by inducing serine phosphorylation in MYH10, PAK4, and PIKfyve in the hippocampal tissue of obese mice.

Integrative Proteomic and Phosphoproteomic Analyses of Hypoxia-Treated Pulmonary Artery Smooth Muscle Cells.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the main causes of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Hypoxia is an important factor related to PAH and can induce the excessive proliferation of PASMCs and inhibit apoptosis. To explore the possible mechanism of hypoxia-related PAH, human PASMCs are exposed to hypoxia for 24 h and tandem mass tag (TMT)-based quantitative proteomic and phosphoproteomic analyses are performed. Proteomic analysis revealed 134 proteins are significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]), of which 48 proteins are upregulated and 86 are downregulated. Some of the changed proteins are verified by using qRT-PCR and Western blotting. Phosphoproteomic analysis identified 404 significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]) phosphopeptides. Among them, 146 peptides are upregulated while 258 ones are downregulated. The kinase-substrate enrichment analysis revealed kinases such as P21 protein-activated kinase 1/2/4 (PAK1/2/4), protein-kinase cGMP-dependent 1 and 2 (PRKG1/2), and mitogen-activated protein-kinase 4/6/7 (MAP2K4/6/7) are significantly enriched and activated. For all the significantly changed proteins or phosphoproteins, a comprehensive pathway analysis is performed. In general, this study furthers our understanding of the mechanism of hypoxia-induced PAH.

Gut Bacterial Metabolite Urolithin A Decreases Actin Polymerization and Migration in Cancer Cells.

Urolithin A (UA) is a gut-derived bacterial metabolite from ellagic acid found in pomegranates, berries, and nuts can downregulate cell proliferation and migration. Cell proliferation and cell motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explores whether UA can modify actin cytoskeleton in cancer cells. The effect of UA on globular over filamentous actin ratio is determined utilizing Western blotting, immunofluorescence, and flow cytometry. Rac1 and PAK1 levels are measured by quantitative RT-PCR and immunoblotting. As a result, a 24 h treatment with UA (20µm) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin and wound healing. The effect of UA on actin polymerization is mimicked by pharmacological inhibition of Rac1 and PAK1. The effect is also mirrored by knock down using siRNA. UA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization and migration. Thus, use of dietary UA in cancer prevention or as adjuvant therapy is promising.

P21 protein-activated kinase 1 is associated with severe regressive autism, and epilepsy.

The p21-activated kinase (PAK) family of proteins function as key effectors of RHO family GTPases in mammalian cells to regulate many pathways including Ras/Raf/MEK/ERK and Wnt/β-catenin, amongst others. Here we report an individual with a novel autosomal dominant disorder characterized by severe regressive autism, intellectual disability, and epilepsy. Exome sequencing of the proband and her parents revealed a de novo variant in the PAK1 gene ([NM_001128620] c.362C>T/p.Pro121Leu). Studies in patient cells showed a clear effect on PAK1 protein function, including altered phosphorylation of targets (JNK and ERK), decreased abundance of β-catenin, and concomitant altered expression downstream of these key regulators. Our findings add PAK1 to the list of PAK proteins and kinases which when mutated cause rare genetic diseases.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Pseudo-allergic reaction caused by Qingkailing injection partially via the PI3K-Rac1 signaling pathway in RBL-2H3 cells.

Qingkailing injection (QKLI) is a kind of multi-component traditional Chinese medicine injection. It has been widely used in clinical practice, but in recent years, it has caused more and more adverse reactions, mainly manifested as pseudo-allergic symptoms. To explore the potential mechanism of the pseudo-allergic reaction by QKLI, basophilic leukemia cell line 2H3 (RBL-2H3) was chosen. The results showed that QKLI at doses of 5, 10 and 20 mL L-1 activated phosphoinositide 3-kinase (PI3K) activity and also increased the levels of Ras-related C3 botulinum toxin substrate 1 (Rac1), p21 protein-activated kinase 1 (Pak1), LIM kinase (Limk1) and cofilin (an actin polymerization regulator) proteins. What's more, QKLI aggravated the depolymerization of F-actin. NSC23766, a Rac1 inhibitor, reversed the previous results in QKLI-treated RBL-2H3 cells. In addition, when the Rac1 gene was knocked down using lentiviral vector-loaded shRNA in RBL-2H3 cells, the PI3K activity and depolymerization of F-actin were downregulated, hinting that the pseudo-allergic reaction was significantly reduced. In general, the pseudo-allergic reaction induced by QKLI was likely to be based on PI3K-Rac1 signaling pathways partially.

Estrogen-regulated feedback loop limits the efficacy of estrogen receptor–targeted breast cancer therapy

Endocrine therapy resistance invariably develops in advanced estrogen receptor-positive (ER+) breast cancer, but the underlying mechanisms are largely unknown. We have identified C-terminal SRC kinase (CSK) as a critical node in a previously unappreciated negative feedback loop that limits the efficacy of current ER-targeted therapies. Estrogen directly drives CSK expression in ER+ breast cancer. At low CSK levels, as is the case in patients with ER+ breast cancer resistant to endocrine therapy and with the poorest outcomes, the p21 protein-activated kinase 2 (PAK2) becomes activated and drives estrogen-independent growth. PAK2 overexpression is also associated with endocrine therapy resistance and worse clinical outcome, and the combination of a PAK2 inhibitor with an ER antagonist synergistically suppressed breast tumor growth. Clinical approaches to endocrine therapy-resistant breast cancer must overcome the loss of this estrogen-induced negative feedback loop that normally constrains the growth of ER+ tumors.

Epigallocatechin-3-gallate reduces the proliferation of benign prostatic hyperplasia cells via regulation of focal adhesions

AimsBenign prostatic hyperplasia (BPH) is the most common urological disease that is characterized by the excessive growth of prostatic epithelial and stromal cells. Pharmacological therapy for BPH has limited use due to the many side effects so there is a need for new agents including natural compounds such as epigallocatechin-3-gallate (EGCG). This study was undertaken to assess the role of EGCG, suppressing the formation of BPH by reducing inflammation and oxidative stress, in cytoskeleton organization and ECM interactions via focal adhesions. Main methodsWe performed MTT assay to investigate cell viability of BPH-1 cells, wound healing assay to examine cell migration, immunofluorescence assay for F-actin organization and paxillin distribution and finally immunoblotting to investigate focal adhesion protein levels in the presence and absence of EGCG. Key findingsWe found that EGCG inhibits cell proliferation at the concentration of 89.12μM, 21.2μM and 2.39μM for 24, 48 and 72h, respectively as well as inhibitory effects of EGCG on BPH-1 cell migration were observed in a wound healing assay. Furthermore, it was determined by immunofluorescence labeling that EGCG disrupts F-actin organization and reduces paxillin distribution. Additionally, EGCG decreases the activation of FAK (Focal Adhesion Kinase) and the levels of paxillin, RhoA (Ras homolog gene family, member A), Cdc42 (cell division cycle 42) and PAK1 (p21 protein-activated kinase 1) in a dose-dependent manner. SignificanceFor the first time, by this study, we found evidence that BPH-1 cell proliferation could be inhibited with EGCG through the disruption of cytoskeleton organization and ECM interactions. Consequently, EGCG might be useful in the prevention and treatment of diseases characterized by excessive cell proliferation such as BPH.

Abstract PR06: CRISPR screens identified drivers of endocrine resistance and synthetic lethal vulnerabilities in breast cancer

Abstract Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapies, although in advanced disease therapeutic resistance almost invariably develops. Using genome-wide CRISPR screens we identified genes essential for the growth of ER+ breast cancers. More importantly, we also identified genes whose loss confers endocrine resistance. Notably the expression of c-src tyrosine kinase (CSK) is driven by estrogen yet loss of CSK promoted estrogen independent growth. A synthetic lethality screen for CSK loss identified the p21 protein-activated kinase (PAK2) which becomes activated by SRC-family kinases (SFK) in the absence of CSK. CSK loss or PAK2 over-expression predict worse clinical outcome in breast cancer cohorts. Inhibitors of PAK2 or SFK block estrogen-independent breast tumor growth. These findings reveal an estrogen-induced negative feedback loop that constrains the growth of ER+ tumors thereby limiting the efficacy of therapies that inhibit ER and suggest a previously unappreciated therapeutic route to overcoming endocrine resistance. Citation Format: Tengfei Xiao, Wei Li, Xiaoqing Wang, Han Xu, Qiu Wu, Peng Jiang, Jixin Yang, Teng Fei, Chongzhi Zang, Qi Liao, Jonathan Rennhack, Eran Andrechek, Nanlin Li, Rinath Jeselsohn, Shirley X. Liu, Myles Brown. CRISPR screens identified drivers of endocrine resistance and synthetic lethal vulnerabilities in breast cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR06.

LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells.

LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage.

Activation of the SDF1/CXCR4 pathway retards muscle atrophy during cancer cachexia.

Cancer cachexia is a life-threatening syndrome that affects most patients with advanced cancers and causes severe body weight loss, with rapid depletion of skeletal muscle. No treatment is available. We analyzed microarray data sets to identify a subset of genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing rodents but not in those with diabetes, disuse, uremia or fasting. Ingenuity Pathways Analysis indicated that three genes belonging to the C-X-C motif chemokine receptor 4 (CXCR4) pathway were downregulated only in muscles atrophying because of cancer: stromal cell-derived factor 1 (SDF1), adenylate cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). Notably, we found that, in the Rectus Abdominis muscle of cancer patients, the expression of SDF1 and CXCR4 was inversely correlated with that of two ubiquitin ligases induced in muscle wasting, atrogin-1 and MuRF1, suggesting a possible clinical relevance of this pathway. The expression of all main SDF1 isoforms (α, β, γ) also declined in Tibialis Anterior muscle from cachectic mice bearing murine colon adenocarcinoma or human renal cancer and drugs with anticachexia properties restored their expression. Overexpressing genes of this pathway (that is, SDF1 or CXCR4) in cachectic muscles increased the fiber area by 20%, protecting them from wasting. Similarly, atrophying myotubes treated with either SDF1α or SDF1β had greater total protein content, resulting from reduced degradation of overall long-lived proteins. However, inhibiting CXCR4 signaling with the antagonist AMD3100 did not affect protein homeostasis in atrophying myotubes, whereas normal myotubes treated with AMD3100 showed time- and dose-dependent reductions in diameter, until a plateau, and lower total protein content. This further confirms the involvement of a saturable pathway (that is, CXCR4). Overall, these findings support the idea that activating the CXCR4 pathway in muscle suppresses the deleterious wasting associated with cancer.

Transcriptome Analysis of Peripheral Blood in Chronic Inflammatory Demyelinating Polyradiculoneuropathy Patients Identifies TNFR1 and TLR Pathways in the IVIg Response.

We have studied the response to intravenous immunoglobulins (IVIg) by a transcriptomic approach in 11 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients (CIDP duration = 6 [0.83–6.5] years). RNA was extracted from cells in whole blood collected before and 3 weeks after IVIg treatment, and hybridized on Illumina chips. After RNA quality controls, gene expression was analyzed using statistical tests fitted for microarrays (R software, limma package), and a pathway analysis was performed using DAVID software. We identified 52 genes with expression that varied significantly after IVIg (fold change [FC] > 1.2, P < 0.001, false discovery rate [FDR] <0.05). Among these 52 genes, 7 were related to immunity, 3 were related to the tumor necrosis factor (TNF)-α receptor 1 (TNFR1) pathway (inhibitor of caspase-activated DNase (ICAD): FC = 1.8, P = 1.7E-7, FDR = 0.004; p21 protein-activated kinase 2 [PAK2]: FC = 1.66, P = 2.6E-5, FDR = 0.03; TNF-α-induced protein 8-like protein 1 [TNFAIP8L1]: P = 1.00E-05, FDR = 0.026), and 2 were related to Toll-like receptors (TLRs), especially TLRs 7 and 9, and were implicated in autoimmunity. These genes were UNC93B1 (FC = 1.6, P = 2E-5, FDR = 0.03), which transports TLRs 7 and 9 to the endolysosomes, and RNF216 (FC = 1.5, P = 1E-05, FDR = 0.03), which promotes TLR 9 degradation. Pathway analysis showed that the TNFR1 pathway was significantly lessened by IVIg (enrichment score = 24, Fischer exact test = 0.003). TNF-α gene expression was higher in responder patients than in nonresponders; however, it decreased after IVIg in responders (P = 0.04), but remained stable in nonresponders. Our data suggest the actions of IVIg on the TNFR1 pathway and an original mechanism involving innate immunity through TLRs in CIDP pathophysiology and the response to IVIg. We conclude that responder patients have stronger inflammatory activity that is lessened by IVIg.

1α,25(OH)2D3 Induces Actin Depolymerization in Endometrial Carcinoma Cells by Targeting RAC1 and PAK1

Background: Cell proliferation and motility require actin reorganization, which is under control of various signalling pathways including ras-related C3 botulinum toxin substrate 1 (RAC1), p21 protein-activated kinase 1 (PAK1) and actin related protein 2 (ARP2). Tumour cell proliferation is modified by 1α,25-Dihydroxy-Vitamin D3 (1α,25(OH)₂D₃), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. The present study explored whether 1α,25(OH)₂D₃ modifies actin cytoskeleton in Ishikawa cells, a well differentiated endometrial carcinoma cell line. Methods: To this end, actin cytoskeleton was visualized by confocal microscopy. Globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry, transcript levels by qRT-PCR and protein abundance by immunoblotting. Results: A 24 hour treatment with 1α,25(OH)₂D₃ (100 nM) significantly decreased RAC1 and PAK1 transcript levels and activity, decreased ARP2 protein levels and depolymerized actin. The effect of 1α,25(OH)₂D₃ on actin polymerization was mimicked by pharmacological inhibition of RAC1 and PAK1. Conclusions: 1α,25(OH)₂D₃ leads to disruption of RAC1 and PAK1 activity with subsequent actin depolymerization of endometrial carcinoma cells.

Macrocyclic Lactones Block Melanoma Growth, Metastases Development and Potentiate Activity of Anti–BRAF V600 Inhibitors

IntroductionMelanoma is the most aggressive form of skin cancer. Targeted antimelanoma therapies such as BRAF V600 inhibitors have shown spectacular results. However, these therapies are restricted to BRAF V600 mutated melanoma and display both adverse effects and resistance outbreaks. MethodsA high-content screening campaign identified compounds that displayed antimelanoma activities that can potentiate anti–BRAF V600 inhibitors. We identified ivermectin, a US Food and Drug Administration (FDA)-approved macrocyclic lactone widely used as an anthelmintic and insecticidal agent. ResultsMacrocyclic lactones possess in vitro cytotoxicity against either BRAF wild type (wt) or BRAF V600 mutated melanoma cells lines. Daily intraperitoneal injections of ivermectin strongly reduce pulmonary metastasis implantation in vivo in murine and melanoma models. Interestingly, these macrocyclic lactones are also able to trigger vemurafenib-dependent cytotoxicity in vitro in BRAF-wt melanoma cells, although vemurafenib action was thought to be restricted to melanoma bearing the BRAF V600 mutation. These macrocyclic lactones are also able to increase the in vitro antimelanoma activity of another BRAF V600 clinical inhibitor, dabrafenib. Serine/threonine p21-activated protein kinase 1 (PAK1) is the key target of ivermectin responsible for its antimelanoma activity. ConclusionIvermectin is a promising agent that could strongly increase melanoma therapy efficiency because it is cytotoxic for BRAF-wt and BRAF mutated melanomas and because it increases the efficiency of antimutated BRAF inhibitors independent of the BRAF status of tumors. Because ivermectin is FDA approved and a study of ivermectin would be able to include all patients in the same protocol, this combination treatment should be next investigated in clinical studies.

Cx43 increases serum induced filopodia formation via activation of p21-activated protein kinase 1

In a previous study we could show that connexin 43 (Cx43) expression increased the migration of cells in a channel-independent manner involving the MAPK p38. We analyzed here the mechanism by which Cx43 enhanced p38 activation and migration related changes of the actin cytoskeleton. HeLa cells were used as a model system for the controlled expression of Cx43 and truncated Cx43 proteins. The expression of Cx43 altered the actin cytoskeleton organization in response to serum stimulation. Cx43 expressing HeLa cells had significantly more filopodial protrusions per cell than empty-vector transfected control cells. The expression of the channel incompetent carboxyl tail of Cx43 was sufficient to enhance the filopodia formation whereas the N-terminal, channel-building part, had no such effect. The enhanced filopodia formation was p38 dependent since the p38 blocker SB203580 significantly diminished it. Immunoprecipitation revealed an interaction of the upstream regulator of p38, p21-activated protein kinase 1 (PAK1), with Cx43 resulting in an enhanced phosphorylation of PAK1. Moreover, p38 activation, filopodia formation and cell migration were significantly reduced by blocking the PAK1 activity with its pharmacological inhibitor, IPA-3. The p38 target Hsp27, which favors the actin polymerization in its phosphorylated form, was significantly more phosphorylated characterizing it as a potential candidate molecule to enhance the serum-induced actin polymerization in Cx43 expressing cells. Our results provide a novel mechanism by which Cx43 can modify actin cytoskeletal dynamics and may thereby enhance cell migration.

Abstract 124: P21-activated protein kinase 1 high expression is a positive prognostic factor in human primary and metastatic pancreatic cancer

Abstract Background: P21-activated protein kinase 1 (PAK1), a main downstream effector of small Rho GTPases, is overexpressed in many malignancies. Its overexpression was associated with poor prognosis in some tumor types including breast cancer, gastric cancer, colorectal cancer and so on. However, the expression and clinical relevance of PAK1 expression in human pancreatic cancer remain unknown. Methods: The present study was designed to investigate the clinical and prognostic significance of PAK1 expression in pancreatic carcinoma. We examined the expression of PAK1 by immunohistochemistry in 72 primary pancreatic carcinoma samples and 20 liver metastatic samples. The relationships between PAK1 and clinicopathological parameters and prognosis in primary and metastatic pancreatic cancer were analyzed. Results: Among the total 92 cases, primary pancreatic cancer samples had significantly higher rate (38/72, 52.8%) of high PAK1 expression than liver metastatic samples (5/20, 25.0%) (p = 0.028). In 72 primary pancreatic cancer patients, high PAK1 expression was associated with younger age (p = 0.038) and moderately or well differentiated tumor (p = 0.007). Moreover, a positive relationship was found between high PAK1 expression and overall survival (OS) (p&amp;lt;0.005). Patients with high PAK1 expression had a better OS than those with low PAK1 expression. Univariate and multivariate analysis by Cox regression including PAK1 and other prognostic pathological markers demonstrated high PAK1 immunostaining as an independent prognostic factor for survival in pancreatic cancer patients (P&amp;lt;0.005). Conclusions: For the first time, we report that PAK1 is a novel prognostic marker for pathologically confirmed human pancreatic cancer. Reduced expression of p21-activated protein kinase 1 correlates with poor histological differentiation in pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Shuqiang Yuan, Feng Wang, Han Juan, Hengying Pu, Zhiwei Zhou, Rui-hua Xu. P21-activated protein kinase 1 high expression is a positive prognostic factor in human primary and metastatic pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 124. doi:10.1158/1538-7445.AM2015-124

Aegeline from Aegle marmelos stimulates glucose transport via Akt and Rac1 signaling, and contributes to a cytoskeletal rearrangement through PI3K/Rac1

Aegeline is an alkaloidal-amide, isolated from the leaves of Aegle marmelos and have shown antihyperglycemic as well as antidyslipidemic activities in the validated animal models of type 2 diabetes mellitus. Here we delineate, aegeline enhanced GLUT4 translocation mediated 2-deoxy-glucose uptake in both time and concentration-dependent manner. 2-deoxy-glucose uptake was completely stymied by the transport inhibitors (wortmannin and genistein) in C2C12 myotubes. Pharmacological inhibition of Akt (also known as protein kinase B) and Ras-related C3 botulinum toxin substrate 1 (Rac1) suggest that both Akt and Rac1 operate aegeline-stimulated glucose transport via distinct parallel pathways. Moreover, aegeline activates p21 protein-activated kinase 1 (PAK1) and cofilin (an actin polymerization regulator). Rac1 inhibitor (Rac1 inhib II) and PAK1 inhibitor (IPA-3) completely blocked aegeline-induced phosphorylation of cofilin and p21 protein-activated kinase 1 (PAK1). In summary, these findings suggest that aegeline stimulates the glucose transport through Akt and Rac1 dependent distinct parallel pathways and have cytoskeletal roles via stimulation of the PI3-kinase-Rac1-PAK1-cofilin pathway in the skeletal muscle cells. Therefore, multiple targets of aegeline in the improvement of insulin sensitivity of the skeletal muscle cells may be suggested.

Clinicopathological and cellular signature of PAK1 in human bladder cancer.

Bladder cancer (BC) is the ninth most common cancer and the 13th most common cause of cancer death. Although p21 protein-activated kinase (PAK) regulates cell growth, motility, and morphology, the expression and function of PAK1 associated with the clinicopathological and cellular signature of human BC are not clear. This study was to examine the expression of PAK1 in human BC, the association of PAK1 with clinicopathological features, and the effect of PAK1 on cell proliferation, migration, and invasion in BC cells. A total of 54 BC and 12 normal bladder tissue specimens were retrieved. Among 54 BC patients, 39 cases were superficial BC and 15 cases were invasive BC. Histological examination revealed 29 patients with low-grade and 25 patients with high-grade papillary urothelial carcinomas. Immunohistochemical staining showed that PAK1 was overexpressed in BC tissue compared with normal bladder tissue. The overexpression of PAK1 was significantly associated with tumor size, histological grade, and lymph node metastasis, but not with gender, age, clinical stage, tumor number, and recurrence. Furthermore, the cytoplasmic distribution of PAK1 was observed in BC cells. Knocking down of PAK1 using lentiviral transduction decreased BC cell proliferation, migration, and invasion. In conclusion, we demonstrated that the overexpression of PAK1 is closely associated with the clinicopathological features of BC, suggesting that PAK1 may play an important role in the development and progression of BC and may be a potential therapeutic target for the treatment of BC.

Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase.

Children with Neurofibromatosis type 1 (NF1) are increasingly recognized to have high prevalence of social difficulties and autism spectrum disorders (ASD). We demonstrated selective social learning deficit in mice with deletion of a single Nf1 gene (Nf1+/−), along with greater activation of mitogen activated protein kinase pathway in neurons from amygdala and frontal cortex, structures relevant to social behaviors. The Nf1+/− mice showed aberrant amygdala glutamate/GABA neurotransmission; deficits in long-term potentiation; and specific disruptions in expression of two proteins associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (ADAM22) and heat shock protein 70 (HSP70), respectively. All of these amygdala disruptions were normalized by co-deletion of p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1+/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide novel insights and therapeutic targets for NF1 and ASD patients.