Polymeric nanofiber leveraged co-delivery of anti-stromal PAK1 inhibitor and paclitaxel enhances therapeutic effects in stroma-rich 3D spheroid models.

The role of tumor stroma in solid tumors has been widely recognized in cancer progression, metastasis and chemoresistance. Cancer-associated fibroblasts (CAFs) play a crucial role in matrix remodeling and promoting cancer cell stemness and resistance via reciprocal crosstalk. Residual tumor tissue after surgical removal as well as unresectable tumors face therapeutic challenges to achieve curable outcome. In this study, we propose to develop a dual delivery approach by combining p21-activated kinase 1 (PAK1) inhibitor (FRAX597) to inhibit tumor stroma and chemotherapeutic agent paclitaxel (PTX) to kill cancer cells using electrospun nanofibers. First, the role of the PAK1 pathway was established in CAF differentiation, migration and contraction using relevant in vitro models. Second, polycaprolactone polymer-based nanofibers were fabricated using a uniaxial electrospinning technique to incorporate FRAX597 and/or PTX, which showed a uniform texture and a prolonged release of both drugs for 16 days. To test nanofibers, stroma-rich 3D heterospheroid models were set up which showed high resistance to PTX nanofibers compared to stroma-free homospheroids. Interestingly, nanofibers containing PTX and FRAX597 showed strong anti-tumor effects on heterospheroids by reducing the growth and viability by > 90 % compared to either of single drug-loaded nanofibers. These effects were reflected by reduced intra-spheroidal expression levels of collagen 1 and α-smooth muscle actin (α-SMA). Overall, this study provides a new therapeutic strategy to inhibit the tumor stroma using PAK1 inhibitor and thereby enhance the efficacy of chemotherapy using nanofibers as a local delivery system for unresectable or residual tumor. Use of 3D models to evaluate nanofibers highlights these models as advanced in vitro tools to study the effect of controlled release local drug delivery systems before animal studies.

[Computer-aided prediction and molecular mechanism investigation of active components in compound Kushen injection inhibiting p21-activated kinase 1].

Targeting p21-activated kinase 1 (PAK1) is a novel strategy for pancreatic cancer treatment. Compound Kushen injection contains many anti-pancreatic cancer components, but the specific targets are unknown. In this study, 14α-hydroxymatrine, an active component of Kushen injection, was found to possess high binding free energy with the allosteric site of PAK1 by molecular docking based virtual screening. Molecular dynamics simulations suggested that 14α-hydroxymatrine caused the α1 and α2 helices of the allosteric site of PAK1 to extend outward to form a deep allosteric regulatory pocket. Meanwhile, 14α-hydroxymatrine induced the ß-folding region at the adenosine triphosphate (ATP)-binding pocket of PAK1 to close inward, resulting in the ATP-binding pocket in a "semi-closed" state which caused the inactivation of PAK1. After removal of 14α-hydroxymatrine, PAK1 showed a tendency to change from the inactive conformation to the active conformation. We supposed that 14α-hydroxymatrine of compound Kushen injection might be a reversible allosteric inhibitor of PAK1. This study used modern technologies and methods to study the active components of traditional Chinese medicine, which laid a foundation for the development and utilization of natural products and the search for new treatments for pancreatic cancer.

Dual-inhibition of NAMPT and PAK4 induces anti-tumor effects in 3D-spheroids model of platinum-resistant ovarian cancer.

Ovarian cancer follows a characteristic progression pattern, forming multiple tumor masses enriched with cancer stem cells (CSCs) within the abdomen. Most patients develop resistance to standard platinum-based drugs, necessitating better treatment approaches. Targeting CSCs by inhibiting NAD+ synthesis has been previously explored. Nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway for NAD+ synthesis is an attractive drug target in this pathway. KPT-9274 is an innovative drug targeting both NAMPT and p21 activated kinase 4 (PAK4). However, its effectiveness against ovarian cancer has not been validated. Here, we show the efficacy and mechanisms of KPT-9274 in treating 3D-cultured spheroids that are resistant to platinum-based drugs. In these spheroids, KPT-9274 not only inhibited NAD+ production in NAMPT-dependent cell lines, but also suppressed NADPH and ATP production, indicating reduced mitochondrial function. It also downregulated of inflammation and DNA repair-related genes. Moreover, the compound reduced PAK4 activity by altering its mostly cytoplasmic localization, leading to NAD+-dependent decreases in phosphorylation of S6 Ribosomal protein, AKT, and ß-Catenin in the cytoplasm. These findings suggest that KPT-9274 could be a promising treatment for ovarian cancer patients who are resistant to platinum drugs, emphasizing the need for precision medicine to identify the specific NAD+ producing pathway that a tumor relies upon before treatment.

[Blocking PAK1 kinase activity promotes the differentiation of acute megakaryocytic leukemia cells and induces their apoptosis].

Objective: To investigate the effect of blocking P21 activated kinase 1 (PAK1) activity on the proliferation, differentiation, and apoptosis of acute megakaryocytic leukemia (AMKL) cell lines (CHRF and CMK) . Methods: Cell counts were used to detect the effects of PAK1 inhibitors (IPA-3 and G5555) on AMKL cell proliferation inhibition and colony formation, and flow cytometry was used to detect its effects on AMKL cell cycle. The effect of PAK1 inhibitor on the expression of cyclin D1 and apoptosis-related protein Cleaved caspase 3 was detected using Western blot, while interference with the protein expression level of PAK1 in AMKL cells was assessed using lentivirus-mediated shRNA transfection technology. Flow cytometry was used to detect the effects of knockdown of PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in AMKL cells. Results: PAK1 inhibitors inhibited the proliferation of AMKL cells in a dose-dependent manner and reduced the ability of cell colony formation, and the difference was statistically significant when compared with the control group (P<0.05) . Moreover, they also reduced the percentage of AMKL cells in S phase, and Western blot detection showed that the expression levels of phosphorylated PAK1 and cyclin D1 decreased significantly. Finally, PAK1 inhibitors induced AMKL cell apoptosis by up-regulating Cleaved caspase 3 and showed different abilities to increase the content of polyploid DNA in megakaryocytes. Only high concentrations of IPA-3 and low doses of G5555 increased the number of polyploid megakaryocytes, while knockdown of PAK1 kinase activity promoted AMKL cell differentiation and increased the apoptosis rate. Conclusion: PAK1 inhibitor significantly arrests AMKL cell growth and promotes cell apoptosis. Knocking down the expression of PAK1 promotes the formation of polyploid DNA and induces AMKL cell apoptosis. The above findings indicate that inhibiting the activity of PAK1 may control AMKL effectively.

p21-Activated kinases as promising therapeutic targets in hematological malignancies.

The p21-Activated Kinases (PAKs) are a family of six serine/threonine kinases that were originally identified as downstream effectors of the Rho GTPases Cdc42 and Rac. Since the first PAK was discovered in 1994, studies have revealed their fundamental and biological importance in the development of physiological systems. Within the cell, PAKs also play significant roles in regulating essential cellular processes such as cytoskeletal dynamics, gene expression, cell survival, and cell cycle progression. These processes are often deregulated in numerous cancers when different PAKs are overexpressed or amplified at the chromosomal level. Furthermore, PAKs modulate multiple oncogenic signaling pathways which facilitate apoptosis escape, uncontrolled proliferation, and drug resistance. There is growing insight into the critical roles of PAKs in regulating steady-state hematopoiesis, including the properties of hematopoietic stem cells (HSC), and the initiation and progression of hematological malignancies. This review will focus on the most recent studies that provide experimental evidence showing how specific PAKs regulate the properties of leukemic stem cells (LSCs) and drug-resistant cells to initiate and maintain hematological malignancies. The current understanding of the molecular and cellular mechanisms by which the PAKs operate in specific human leukemia or lymphomas will be discussed. From a translational point of view, PAKs have been suggested to be critical therapeutic targets and potential prognosis markers; thus, this review will also discuss current therapeutic strategies against hematological malignancies using existing small-molecule PAK inhibitors, as well as promising combination treatments, to sensitize drug-resistant cells to conventional therapies. The challenges of toxicity and non-specific targeting associated with some PAK inhibitors, as well as how future approaches for PAK inhibition to overcome these limitations, will also be addressed.

The Use of Nanomedicine to Target Signaling by the PAK Kinases for Disease Treatment.

P21-activated kinases (PAKs) are serine/threonine kinases involved in the regulation of cell survival, proliferation, inhibition of apoptosis, and the regulation of cell morphology. Some members of the PAK family are highly expressed in several types of cancer, and they have also been implicated in several other medical disorders. They are thus considered to be good targets for treatment of cancer and other diseases. Although there are several inhibitors of the PAKs, the utility of some of these inhibitors is reduced for several reasons, including limited metabolic stability. One way to overcome this problem is the use of nanoparticles, which have the potential to increase drug delivery. The overall goals of this review are to describe the roles for PAK kinases in cell signaling and disease, and to describe how the use of nanomedicine is a promising new method for administering PAK inhibitors for the purpose of disease treatment and research. We discuss some of the basic mechanisms behind nanomedicine technology, and we then describe how these techniques are being used to package and deliver PAK inhibitors.

PAK1 Silencing Attenuated Proinflammatory Macrophage Activation and Foam Cell Formation by Increasing PPARγ Expression.

Macrophage polarization in response to environmental cues has emerged as an important event in the development of atherosclerosis. Compelling evidences suggest that P21-activated kinases 1 (PAK1) is involved in a wide variety of diseases. However, the potential role and mechanism of PAK1 in regulation of macrophage polarization remains to be elucidated. Here, we observed that PAK1 showed a dramatically increased expression in M1 macrophages but decreased expression in M2 macrophages by using a well-established in vitro model to study heterogeneity of macrophage polarization. Adenovirus-mediated loss-of-function approach demonstrated that PAK1 silencing induced an M2 macrophage phenotype-associated gene profiles but repressed the phenotypic markers related to M1 macrophage polarization. Additionally, dramatically decreased foam cell formation was found in PAK1 silencing-induced M2 macrophage activation which was accompanied with alternation of marker account for cholesterol efflux or influx from macrophage foam cells. Moderate results in lipid metabolism and foam cell formation were found in M1 macrophage activation mediated by AdshPAK1. Importantly, we presented mechanistic evidence that PAK1 knockdown promoted the expression of PPARγ, and the effect of macrophage activation regulated by PAK1 silencing was largely reversed when a PPARγ antagonist was utilized. Collectively, these findings reveal that PAK1 is an independent effector of macrophage polarization at least partially attributed to regulation of PPARγ expression, which suggested PAK1-PPARγ axis as a novel therapeutic strategy in atherosclerosis management.

Drug discovery targeting p21-activated kinase 4 (PAK4): a patent review.

Introduction: The Ser/Thr protein kinase PAK4 is a downstream regulator of Cdc42, mediating cytoskeleton remodeling, and cell motility, and inhibiting apoptosis and transcriptional regulation. Nowadays, efforts in PAK4 inhibitor development are focusing on improving inhibitory selectivity, cellular potency, and in vivo pharmacokinetic properties, and identifying the feasibility of immunotherapy combination in oncology therapy.Areas covered: This review summarized the development of PAK4 inhibitors that reported on patents in the past two decades. According to their binding features, these inhibitors were classified into type I, type I 1/2, and PAMs. Their designing ideas and SAR were elucidated in this review. Moreover, synergistic therapy of PAK4 inhibitors with PD-1/PD-L1 or CAR-T were also summarized .Expert opinion: In the past years, preclinical and clinical studies of PAK4 inhibitors ended in failure due to poor selectivity, cellular activity, or pharmacokinetic issues. There are researchers questioning the reliability of PAK4 as a drug target, particularly PAK4-related therapy is concerned with the distinguishment of the non-kinase functions and catalytic functions triggered by PAK4 phosphorylation. Meanwhile, synergistic effects of PAK4 inhibitors with PD-1/PD-L1 and CAR-T immunotherapy shed light for the development of PAK4 inhibitors.

Amyloid-ß induced membrane damage instigates tunneling nanotube-like conduits by p21-activated kinase dependent actin remodulation.

Alzheimer's disease (AD) pathology progresses gradually via anatomically connected brain regions. Direct transfer of amyloid-ß1-42 oligomers (oAß) between connected neurons has been shown, however, the mechanism is not fully revealed. We observed formation of oAß induced tunneling nanotubes (TNTs)-like nanoscaled f-actin containing membrane conduits, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that oAß propagate from one cell to another via TNT-like structures. Preceding the formation of TNT-like conduits, we detected oAß-induced plasma membrane (PM) damage and calcium-dependent repair through lysosomal-exocytosis, followed by massive endocytosis to re-establish the PM. Massive endocytosis was monitored by an influx of the membrane-staining dye TMA-DPH and PM damage was quantified by propidium iodide influx in the absence of Ca2+. The massive endocytosis eventually caused accumulation of internalized oAß in Lamp1 positive multivesicular bodies/lysosomes via the actin cytoskeleton remodulating p21-activated kinase1 (PAK1) dependent endocytic pathway. Three-dimensional quantitative confocal imaging, structured illumination superresolution microscopy, and flowcytometry quantifications revealed that oAß induces activation of phospho-PAK1, which modulates the formation of long stretched f-actin extensions between cells. Moreover, the formation of TNT-like conduits was inhibited by preventing PAK1-dependent internalization of oAß using the small-molecule inhibitor IPA-3, a highly selective cell-permeable auto-regulatory inhibitor of PAK1. The present study reveals that the TNT-like conduits are probably instigated as a consequence of oAß induced PM damage and repair process, followed by PAK1 dependent endocytosis and actin remodeling, probably to maintain cell surface expansion and/or membrane tension in equilibrium.

PAK4-NAMPT Dual Inhibition Sensitizes Pancreatic Neuroendocrine Tumors to Everolimus.

Metastatic pancreatic neuroendocrine tumors (PNET) remain an unmet clinical problem. Chronologic treatment in PNETs includes observation (watchful protocol), surgery, targeted therapy, and chemotherapy. However, increasing evidence illustrates that the outcomes of targeted therapeutic options for the treatment of advanced PNETs show minimal response. The FDA-approved mTOR inhibitor everolimus does not shrink these tumors. It only delays disease progression in a subset of patients, while a significant fraction acquires resistance and shows disease progression. Thus, there is a need for more effective targeted approaches to sensitize PNETs to everolimus for better treatment outcomes. Previously, we showed that mTOR regulator p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyl transferase (NAMPT) were aberrantly expressed in PNET tissue and promoted everolimus resistance. In this report, we demonstrate that PAK4-NAMPT dual inhibitor KPT-9274 can synergize with everolimus (growth inhibition, colony suppression, and glucose uptake assays). KPT-9274-everolimus disrupted spheroid formation in multiple PNET models. Molecular analysis showed alteration of mTORC2 through downregulation of RICTOR as a mechanism supporting synergy with everolimus in vitro KPT-9274 suppressed ß-catenin activity via inhibition of PAK4, highlighting the cross-talk between Rho GTPases and Wnt signaling in PNETs. KPT-9274, given at 150 mg/kg in combination with sub-MTD everolimus (2.5 mg/kg), significantly suppressed two PNET-derived xenografts. These studies bring forward a well-grounded strategy for advanced PNETs that fail to respond to single-agent everolimus.

Ganoderic Acid A Promotes Amyloid-ß Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer's Disease (Mouse Model) through Autophagy Induced by Activating Axl.

Alzheimer's disease (AD) is thought to be caused by amyloid-ß (Aß) accumulation in the central nervous system due to deficient clearance. The aim of the present study was to investigate the effect of ganoderic acid A (GAA) on Aß clearance in microglia and its anti-AD activity. Aß degradation in BV2 microglial cells was determined using an intracellular Aß clearance assay. GAA stimulated autophagosome formation via the Axl receptor tyrosine kinase (Axl)/RAC/CDC42-activated kinase 1 (Pak1) pathway was determined by Western blot analyses, and fluorescence-labeled Aß42 was localized in lysosomes in confocal laser microscopy images. The in vivo anti-AD activity of GAA was evaluated by object recognition and Morris water maze (MWM) tests in an AD mouse model following intracerebroventricular injection of aggregated Aß42. The autophagy level in the hippocampus was assayed by immunohistochemical assessment against microtubule-associated proteins 1A/1B light-chain 3B (LC3B). Intracellular Aß42 levels were significantly reduced by GAA treatment in microglial cells. Additionally, GAA activated autophagy according to increased LC3B-II levels, with this increased autophagy stimulated by upregulating Axl and Pak1 phosphorylation. The effect of eliminating Aß by GAA through autophagy was reversed by R428, an Axl inhibitor, or IPA-3, a Pak1 inhibitor. Consistent with the cell-based assay, GAA ameliorated cognitive deficiency and reduced Aß42 levels in an AD mouse model. Furthermore, LC3B expression in the hippocampus was up-regulated by GAA treatment, with these GAA-specific effects abolished by R428. GAA promoted Aß clearance by enhancing autophagy via the Axl/Pak1 signaling pathway in microglial cells and ameliorated cognitive deficiency in an AD mouse model.

Inhibiting p21-activated kinase (PAK7) enhances radiosensitivity in hepatocellular carcinoma.

OBJECTIVE: In light of the upregulation of p21-activated kinase (PAK7) in a variety of cancers, including hepatocellular carcinoma (HCC), we aimed to investigate the effect of PAK7 on the sensitivity of HCC cells to radiotherapy. METHODS: PAK7 expression was determined in normal adult liver epithelial THLE-2 and human HCC cell lines. The effect of ionizing radiation (IR) on the HCC cell viability was evaluated by Sulforhodamine B (SRB) assay. HCC cell lines Mahlavu and Huh7 were chosen to assess the effect of PAK7 shRNAs on the viability, clone formation, apoptosis, cycle distribution and γ-H2AX expression after exposure to IR. RESULTS: As compared to THLE-2 cells, PAK7 was upregulated in poorly differentiated Mahlavu and SK-Hep-1 cells, but moderately or lowly expressed in well-differentiated Huh7 and HepG2 cells. HCC cells with moderate or low expression of PAK7 presented a decreased viability at 2 Gy IR, which had no significant effect on PAK7high HCC cells. Mahlavu and Huh7 cells transfected with PAK7 shRNAs showed increased inhibitory effect of IR on viability. In addition, PAK7 shRNAs reduced clone formation, facilitated the cell apoptosis, arrested cells at G2/M phase, and increased γ-H2AX expression. Moreover, changes above were more evident in the HCC cells co-treated with IR and PAK7 shRNAs. CONCLUSION: PAK7 downregulation could inhibit the viability, promote the apoptosis, arrest cells in G2/M phase, and induce the DNA damage in HCC cells, thereby enhancing the radiosensitivity in HCC.

CDK4/6 inhibition synergizes with inhibition of P21-Activated Kinases (PAKs) in lung cancer cell lines.

Theoretically, small molecule CDK4/6 inhibitors (CDK4/6is) represent a logical therapeutic option in non-small cell lung cancers since most of these malignancies have wildtype RB, the key target of CDKs and master regulator of the cell cycle. Unfortunately, CDK4/6is are found to have limited clinical activity as single agents in non-small cell lung cancer. To address this problem and to identify effective CDK4/6i combinations, we screened a library of targeted agents for efficacy in four non-small cell lung cancer lines treated with CDK4/6 inhibitors Palbociclib or Abemaciclib. The pan-PAK (p21-activated kinase) inhibitor PF03758309 emerged as a promising candidate with viability ratios indicating synergy in all 4 cell lines and for both CDK4/6is. It is noteworthy that the PAKs are downstream effectors of small GTPases Rac1 and Cdc42 and are overexpressed in a wide variety of cancers. Individually the compounds primarily induced cell cycle arrest; however, the synergistic combination induced apoptosis, accounting for the synergy. Surprisingly, while the pan-PAK inhibitor PF03758309 synergizes with CDK4/6is, no synergy occurs with group I PAK inhibitors FRAX486 or FRAX597. Cell lines treated only with Ribociclib, FRAX486 or FRAX597 underwent G1/G0 arrest, whereas combination treatment with these compounds predominantly resulted in autophagy. Combining high concentrations of FRAX486, which weakly inhibits PAK4, and Ribociclib, mimics the autophagy and apoptotic effect of PF03758309 combined with Ribociclib. FRAX597, a PAKi that does not inhibit PAK4 did not reduce autophagy in combination with Ribociclib. Our results suggest that a unique combination of PAKs plays a crucial role in the synergy of PAK inhibitors with CDK4/6i. Targeting this unique PAK combination, could greatly improve the efficacy of CDK4/6i and broaden the spectrum of cancer treatment.

Kaempferol ameliorates the regulatory effects of PVT1/miR-214 on epithelial-mesenchymal transition through the PAK4/ß-catenin axis in SRA01/04 cells.

Aim: To investigate whether kaempferol exhibits a protective effect on high glucose-induced epithelial-mesenchymal transition (EMT) by mediating the PVT1/miR-214 and PAK4/ß-catenin pathways in SRA01/04 cells. Methods & methods: qRT-PCR and western blot assays were used for gene and protein determination, and migration and invasion assays were conducted. A coimmunoprecipitation assay was used for determining protein interactions. Results: High glucose effectively upregulated PVT1 expression, downregulated miR-214 expression and promoted cell migration and invasion. Kaempferol attenuated high glucose-induced EMT by increasing PVT1 expression and decreasing miR-214 expression. PAK4 was identified as a direct target of miR-214. PAK4 overexpression could rescue the effects of PVT1 deficiency on SRA01/04 cells. Conclusion: Kaempferol ameliorated the regulatory effects of PVT1/miR-214 on high glucose-induced EMT through PAK4/ß-catenin in SRA01/04 cells.

PAK4 suppresses motor neuron degeneration in hSOD1G93A -linked amyotrophic lateral sclerosis cell and rat models.

OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MN). CREB pathway-mediated inhibition of apoptosis contributes to neuron protection, and PAK4 activates CREB signalling in diverse cell types. This study aimed to investigate PAK4's effect and mechanism of action in ALS. METHODS: We analysed RNA levels by qRT-PCR, protein levels by immunofluorescence and Western blotting, and apoptosis by flow cytometry and TUNEL staining. Cell transfection was performed for in vitro experiment. Mice were injected intraspinally to evaluate PAK4 function in vivo experiment. Rotarod test was performed to measure motor function. RESULTS: The expression and activation of PAK4 significantly decreased in the cell and mouse models of ALS as the disease progressed, which was caused by the negative regulation of miR-9-5p. Silencing of PAK4 increased the apoptosis of MN by inhibiting CREB-mediated neuroprotection, whereas overexpression of PAK4 protected MN from hSOD1G93A -induced degeneration by activating CREB signalling. The neuroprotective effect of PAK4 was markedly inhibited by CREB inhibitor. In ALS models, the PAK4/CREB pathway was inhibited, and cell apoptosis increased. In vivo experiments revealed that PAK4 overexpression in the spinal neurons of hSOD1G93A mice suppressed MN degeneration, prolonged survival and promoted the CREB pathway. CONCLUSIONS: PAK4 protects MN from degeneration by activating the anti-apoptotic effects of CREB signalling, suggesting it may be a therapeutic target in ALS.

A role for PAK1 mediated phosphorylation of ß-catenin Ser552 in the regulation of insulin secretion.

The presence of adherens junctions and the associated protein ß-catenin are requirements for the development of glucose-stimulated insulin secretion (GSIS) in ß-cells. Evidence indicates that modulation of ß-catenin function in response to changes in glucose levels can modulate the levels of insulin secretion from ß-cells but the role of ß-catenin phosphorylation in this process has not been established. We find that a Ser552Ala version of ß-catenin attenuates glucose-stimulated insulin secretion indicating a functional role for Ser552 phosphorylation of ß-catenin in insulin secretion. This is associated with alterations F/G actin ratio but not the transcriptional activity of ß-catenin. Both glucose and GLP-1 stimulated phosphorylation of the serine 552 residue on ß-catenin. We investigated the possibility that an EPAC-PAK1 pathway might be involved in this phosphorylation event. We find that reduction in PAK1 levels using siRNA attenuates both glucose and GLP-1 stimulated phosphorylation of ß-catenin Ser552 and the effects of these on insulin secretion in ß-cell models. Furthermore, both the EPAC inhibitor ESI-09 and the PAK1 inhibitor IPA3 do the same in both ß-cell models and mouse islets. Together this identifies phosphorylation of ß-catenin at Ser552 as part of a cell signalling mechanism linking nutrient and hormonal regulation of ß-catenin to modulation of insulin secretory capacity of ß-cells and indicates this phosphorylation event is regulated downstream of EPAC and PAK1 in ß-cells.

p21-activated kinases as viable therapeutic targets for the treatment of high-risk Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults. Unfortunately, there have been minimal recent advancements in improving patient outcomes, especially in metastatic and recurrent diseases. In this study, we investigated the biological role of p21-activated kinases (PAKs) in ES, and the ability to therapeutically target them in high-risk disease. Via informatics analysis, we established the inverse association of PAK1 and PAK4 expression with clinical stage and outcome in ES patients. Through expression knockdown and small-molecule inhibition of PAKs, utilizing FRAX-597, KPT-9274, and PF-3758309 in multiple ES cell lines and patient-derived xenograft models, we further explored the role of PAKs in ES tumor growth and metastatic capabilities. In vitro studies in several ES cell lines indicated that diminishing PAK1 and PAK4 expression reduces tumor cell viability, migratory, and invasive properties. In vivo studies using PAK4 inhibitors, KPT-9274 and PF-3758309 demonstrated significant inhibition of primary and metastatic tumor formation, while transcriptomic analysis of PAK4-inhibitor-treated tumors identified concomitant suppression of Notch, ß-catenin, and hypoxia-mediated signatures. In addition, the analysis showed enrichment of anti-tumor immune regulatory mechanisms, including interferon (IFN)-É£ and IFN-α responses. Altogether, our molecular and pre-clinical studies are the first to establish a critical role for PAKs in ES development and progression, and consequently as viable therapeutic targets for the treatment of high-risk ES in the near future.

Chemical composition and pharmacological properties of Macaranga-type Pacific propolis: A review.

Propolis, a resinous substance, is collected from plants and processed by honeybees to seal holes and cracks in beehives, protecting them from microbial infection. Based on the plant source and geographical location, propolis is categorized into seven groups. Of these, Pacific propolis, found in the Pacific islands, originates from Macaranga spp. and is, therefore, known as Macaranga-type Pacific propolis. Okinawa propolis and Taiwanese propolis, which are both Macaranga-type propolis, are rich in prenylated flavonoids from the same botanical source, Macaranga tanarius, and are used locally as traditional remedies. They are reported to have a wide range of pharmacological benefits, including antioxidant, anti-inflammation, antimicrobial, anticancer, antidiabetic, anti-Alzheimer's, anti-melanogenic, and longevity-extending effects. However, not much is known about their mode of action, and recently, the extract of Okinawa propolis and its major prenylated flavonoids were found to selectively inhibit the oncogenic kinase, p21-activated kinase 1 (PAK1). PAK1 enables cross-talking among several signaling pathways, causing many diseases/disorders. The existing results reviewed here support the use of Macaranga-type Pacific propolis for the effective development of safe herbal drugs and functional foods. Furthermore, its mode of action by modulating PAK1 can be explored, and the geographical and seasonal effects on its chemistry and biology, and its pharmacokinetics and toxicology should be studied as well.

Targeting effector pathways in RAC1P29S-driven malignant melanoma.

Malignant melanoma is characterized by mutations in a number of driver genes, most notably BRAF and NRAS. Recent genomic analyses revealed that 4-9% of sun-exposed melanomas bear activating mutations in RAC1, which encodes a small GTPase that is known to play key roles in cell proliferation, survival, and migration. The RAC1 protein activates several effector pathways, including Group A p21-activated kinases (PAKs), phosphoinositol-3-kinases (PI3Ks), in particular the beta isoform, and the serum-response factor/myocardin-related transcription factor (SRF/MRTF). Having previously shown that inhibition of Group A PAKs impedes oncogenic signalling from RAC1P29S, we here extend this analysis to examine the roles of PI3Ks and SRF/MRTF in melanocytes and/or in a zebrafish model. We demonstrate that a selective Group A PAK inhibitor (Frax-1036), a pan-PI3K (BKM120), and two PI3Kß inhibitors (TGX221, GSK2636771) impede the growth of melanoma cells driven by mutant RAC1 but not by mutant BRAF, while other PI3K selective inhibitors, including PI3Kα, δ and γ, are less effective. Using these compounds as well as an SRF/MRTF inhibitor (CCG-203,971), we observed similar results in vivo, using embryonic zebrafish development as a readout. These results suggest that targeting Group A PAKs, PI3Kß, and/or SRF/MRTF represent a promising approach to suppress RAC1 signalling in malignant melanoma.

Targeting PAK4 Inhibits Ras-Mediated Signaling and Multiple Oncogenic Pathways in High-Risk Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most prevalent pediatric soft-tissue sarcoma. Multimodal treatment, including surgery and traditional chemotherapy with radiotherapy, has contributed to improvements in overall survival rates. However, patients with recurrent or metastatic disease have 5-year survival rates of less than 30%. One reason for the lack of therapeutic advancement is identification and targeting of critical signaling nodes. p21-activated kinases (PAK) are a family of serine/threonine kinases downstream of multiple critical tumorigenic receptor tyrosine kinase receptors and oncogenic regulators, including IGFR and RAS signaling, that significantly contribute to aggressive malignant phenotypes. Here, we report that RMS cell lines and tumors exhibit enhanced PAK4 expression levels and activity, which are further activated by growth factors involved in RMS development. Molecular perturbation of PAK4 in multiple RMS models in vitro and in vivo resulted in inhibition of RMS development and progression. Fusion-positive and -negative RMS models were sensitive to two PAK4 small-molecule inhibitors, PF-3758309 and KPT-9274, which elicited significant antitumor and antimetastatic potential in several primary and metastatic in vivo models, including a relapsed RMS patient-derived xenograft model. Transcriptomic analysis of PAK4-targeted tumors revealed inhibition of the RAS-GTPase, Hedgehog, and Notch pathways, along with evidence of activation of antitumor immune response signatures. This PAK4-targeting gene signature showed prognostic significance for patients with sarcoma. Overall, our results show for the first time that PAK4 is a novel and viable therapeutic target for the treatment of high-risk RMS. SIGNIFICANCE: These data demonstrate a novel oncogenic role for PAK4 in rhabdomyosarcoma and show that targeting PAK4 activity is a promising viable therapeutic option for advanced rhabdomyosarcoma.