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Jonathan Chernoff - One of the best experts on this subject based on the ideXlab platform.
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targeting group i p21 activated kinases to control malignant peripheral nerve sheath tumor growth and metastasis
Oncogene, 2017Co-Authors: Galina Semenova, Dina Stepanova, Cara Dubyk, E Handorf, S M Deyev, Alexander J Lazar, Jonathan ChernoffAbstract:Malignant peripheral nerve sheath tumors (MPNSTs) are devastating sarcomas for which no effective medical therapies are available. Over 50% of MPSNTs are associated with mutations in NF1 tumor suppressor gene, resulting in activation of Ras and its effectors, including the Raf/Mek/Erk and PI3K/Akt/mTORC1 signaling cascades, and also the WNT/β-catenin pathway. As Group I p21-activated kinases (Group I Paks, PAK1/2/3) have been shown to modulate Ras-driven oncogenesis, we asked if these enzymes might regulate signaling in MPNSTs. In this study we found a strong positive correlation between the activity of PAK1/2/3 and the stage of human MPNSTs. We determined that reducing Group I Pak activity diminished MPNST cell proliferation and motility, and that these effects were not accompanied by significant blockade of the Raf/Mek/Erk pathway, but rather by reductions in Akt and β-catenin activity. Using the small molecule PAK1/2/3 inhibitor Frax1036 and the MEK1/2 inhibitor PD0325901, we showed that the combination of these two agents synergistically inhibited MPNST cell growth in vitro and dramatically decreased local and metastatic MPNST growth in animal models. Taken together, these data provide new insights into MPNST signaling deregulation and suggest that co-targeting of PAK1/2/3 and MEK1/2 may be effective in the treatment of patients with MPNSTs.
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kinase activity of pak2 an effector of rac cdc42 and its interaction with β pix is required for murine hematopoietic stem cell shape f actin formation directional migration in vitro and for hspc homing to bone marrow in vivo
Blood, 2013Co-Authors: Pavankumar N G Reddy, Jonathan Chernoff, Rachelle Kosoff, Maria Radu, Jenna Wood, Chad E Harris, Meaghan Mcguinness, David A WilliamsAbstract:Hematopoietic stem and progenitor cell (HSPC) migration, marrow homing and engraftment are key physiological processes regulating hematopoiesis post transplantation. These processes are the result of the orchestrated actions of multiple extracellular stimuli, which regulate actin remodeling, cell polarity, chemotaxis and cell-cell interactions. In HSPC, the Rho GTPases Rac and CDC42 act as molecular switches that integrate extracellular stimuli in a spatially regulated manner to control cell migration and mediate homing to marrow and mobilization as well as cell survival/ proliferation pathways to mediate engraftment (Gu et al., Science 2003; Cancelas et al., Nature Medicine 2005; Wang et al., Blood 2006) . Using an inhibitory peptide against Group A p21 activated kinases (PAK1-3), key effectors of Rac/ CDC42 and individual PAK1 & 2 genetic knock-out mice, we recently demonstrated that Pak kinases, specifically Pak2, are important for HSPC homing and engraftment ( Dorrance et al., Blood 2013 ). Pak2 is a multi-domain protein that contains a C-terminal kinase domain and multiple N-terminal protein-interaction domains. Among these is a non-classical SH3-binding site for the guanine-nucleotide-exchange factor β-PIX, which was shown to be critical for both activation of Rac1 and its localization to and induction of membrane ruffles ( Klooster et al., Journal of Cell Biology 2006 ). In this study we further explored the role of these domains of Pak2 in key HSPC functions, including homing to bone marrow in vivo . We employed a multi-cistronic retrovirus vector that simultaneously deleted floxed endogenous Pak2 gene sequences and rescued with either wild type (WT), a kinase dead (KD) mutant (K278A, defective in auto/ trans phosphorylation) or a Δβ-PIX mutant, (P185/R186A, that cannot bind to β-PIX). As previously demonstrated deletion of Pak2 (Pak2 Δ/Δ) was associated with abnormal SDF-1 stimulated cell protrusions containing F-actin (as demonstrated by confocal and electron microscopy) and these HSPC displayed decreased directional migration (Euclidean distance in Pak2Δ/Δ vs. Pak2WT/WT: 39.6µm ±9.6 vs. 96.6µm ±21.6; P<0.05). This phenotype of abnormal cell protrusions and decreased directional migration was rescued by expressing Pak2-WT (Pak2WT/WT vs. Pak2-WT: 96.6µm ±21.6 vs. 74.0µm ±18.7; P: not significant) but not by expressing Pak2-KD (Pak2WT/WT vs. Pak2-KD: 96.6µm ±21.6 vs. 33.6µm ±6.3; P<0.05) demonstrating the requirement of Pak2 kinase activity in SDF1-induced cell polarization and directed cell migration. Interestingly, we found abnormal F-actin clustering associated with defective polarization (by confocal microscopy) and decreased velocity of cell migration in time-lapsed video microscopy when Pak2-deletion was rescued with Pak2-Δβ-PIX (velocity of migration Pak2WT/WT vs. Pak2-Δβ-PIX, 0.32µm/minute ±0.02 vs. 0.13µm/minute ±0.02; P<0.001), indicating the requirement of β-PIX exchange factor interaction with Pak2 in directed migration. To test whether these in vitro phenotypes were associated with changes in homing efficiency to bone marrow, we performed in vivo homing assays of rescued HSPC. Transduced, GFP-sorted Lin-Sca1+Kit+ cells of each genotype were injected into lethally-irradiated C57BL/6 recipient mice (N= 12-29 /genotype). Twelve hours post-transplantation the number of EGFP+ cells in the bone marrow was determined and percent homing is calculated. Compared to Pak2 WT/WT, Pak2Δ/Δ HSPC displayed reduced homing (99.26%± 4.9 vs. 53.4% ± 4.2; P< 0.0001). The homing defect was rescued by Pak2-WT (Pak2WT/WT vs. Pak2-WT rescue: 99.26%± 4.9 vs. 86% ± 8.5; P: not significant). However neither Pak2-KD nor Pak2-Δβ-PIX rescued in vivo homing: 99.26% ±4.9 vs. 38.9% ±3.7 vs. 33.0%± 6.0; P< 0.0001 each mutant vs.Pak2WT/WT) proving the necessity of kinase activity and interaction with β-PIX for bone marrow homing. Taken together we show that both Pak2-kinase activity and its interaction with β-PIX exchange factor are required for coordinated HSPC F-actin formation and cell polarization, directed cell migration in vitro and homing to bone marrow in vivo . These data directly link the in vitro effects of Pak2 kinase with in vivo bone marrow homing. Note All p values are calculated by Mann Whitney test. Disclosures: No relevant conflicts of interest to declare.
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Role of p21-activated kinases in cardiovascular development and function
Cellular and Molecular Life Sciences, 2013Co-Authors: Mollie L. Kelly, Artyom Astsaturov, Jonathan ChernoffAbstract:p21-activated kinases (Paks) are a group of six serine/threonine kinases (PAK1-6) that are involved in a variety of biological processes. Recently, Paks, more specifically PAK1, -2, and -4, have been shown to play important roles in cardiovascular development and function in a range of model organisms including zebrafish and mice. These functions include proper morphogenesis and conductance of the heart, cardiac contractility, and development and integrity of the vasculature. The mechanisms underlying these effects are not fully known, but they likely differ among the various Pak isoforms and include both kinase-dependent and -independent functions. In this review, we discuss aspects of Pak function relevant to cardiovascular biology as well as potential therapeutic implications of small-molecule Pak inhibitors in cardiovascular disease.
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frax597 a small molecule inhibitor of the p21 activated kinases inhibits tumorigenesis of neurofibromatosis type 2 nf2 associated schwannomas
Journal of Biological Chemistry, 2013Co-Authors: Silvia Licciulli, Jonathan Chernoff, Jasna Maksimoska, Chun Zhou, Scott Troutman, Smitha Kota, Qin Liu, Sergio G Duron, David A Campbell, Jeffrey FieldAbstract:The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.
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PAK1 kinase links erbb2 to β catenin in transformation of breast epithelial cells
Cancer Research, 2013Co-Authors: Luis E Ariasromero, Olga Villamarcruz, Min Huang, Klaus P Hoeflich, Jonathan ChernoffAbstract:p21-Activated kinase-1 (PAK1) is frequently upregulated in human breast cancer and is required for transformation of mammary epithelial cells by ErbB2. Here, we show that loss of PAK1, but not the closely related Pak2, leads to diminished expression of β-catenin and its target genes. In MMTV-ErbB2 transgenic mice, loss of PAK1 prolonged survival, and mammary tissues of such mice showed loss of β-catenin. Expression of a β-catenin mutant bearing a phospho-mimetic mutation at Ser 675, a specific PAK1 phosphorylation site, restored transformation to ErbB2-positive, PAK1-deficient mammary epithelial cells. Mice bearing xenografts of ErbB2-positive breast cancer cells showed tumor regression when treated with small-molecule inhibitors of Pak or β-catenin, and combined inhibition by both agents was synergistic. These data delineate a signaling pathway from ErbB2 to Pak to β-catenin that is required for efficient transformation of mammary epithelial cells, and suggest new therapeutic strategies in ErbB2-positive breast cancer.
Jeffrey Field - One of the best experts on this subject based on the ideXlab platform.
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frax597 a small molecule inhibitor of the p21 activated kinases inhibits tumorigenesis of neurofibromatosis type 2 nf2 associated schwannomas
Journal of Biological Chemistry, 2013Co-Authors: Silvia Licciulli, Jonathan Chernoff, Jasna Maksimoska, Chun Zhou, Scott Troutman, Smitha Kota, Qin Liu, Sergio G Duron, David A Campbell, Jeffrey FieldAbstract:The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.
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p21 activated kinase 1 PAK1 dependent phosphorylation of raf 1 regulates its mitochondrial localization phosphorylation of bad and bcl 2 association
Journal of Biological Chemistry, 2005Co-Authors: Shenghao Jin, Ya Zhuo, Weining Guo, Jeffrey FieldAbstract:Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which PAK1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by PAK1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for PAK1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a PAK1-Raf-1-BAD pathway that is involved in cell survival signaling.
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Akt Phosphorylation of Serine 21 on PAK1 Modulates Nck Binding and Cell Migration
Molecular and cellular biology, 2003Co-Authors: Guo-lei Zhou, Gary M Bokoch, Ya Zhuo, Charles C. King, Benjamin H. Fryer, Jeffrey FieldAbstract:The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated PAK1 in vitro. The major site phosphorylated by Akt on PAK1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on PAK1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not PAK1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length PAK1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of PAK1 in vitro with Akt or coexpression of constitutively active Akt with PAK1 reduced Nck binding to PAK1. In HeLa cells, green fluorescent protein-tagged PAK1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of PAK1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.
Rakesh Kumar - One of the best experts on this subject based on the ideXlab platform.
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Potential role of p21 Activated Kinase 1 (PAK1) in the invasion and motility of oral cancer cells
BMC Cancer, 2016Co-Authors: Muraleedharan Parvathy, Rakesh Kumar, Sreeharshan Sreeja, Madhavan Radhakrishna PillaiAbstract:Background Oral cancer malignancy consists of uncontrolled division of cells primarily in and around the floor of the oral cavity, gingiva, oropharynx, lower lip and base of the tongue. According to GLOBOCAN 2012 report, oral cancer is one of the most common cancers among males and females in India. Even though significant advancements have been made in the field of oral cancer treatment modalities, the overall prognosis for the patients has not improved in the past few decades and hence, this demands a new thrust for the identification of novel therapeutic targets in oral cancer. p21 Activated Kinases (PAKs) are potential therapeutic targets that are involved in numerous physiological functions. PAKs are serine-threonine kinases and they serve as important regulators of cytoskeletal dynamics and cell motility, transcription through MAP kinase cascades, death and survival signalling, and cell-cycle progression. Although PAKs are known to play crucial roles in cancer progression, the role and clinical significance of PAKs in oral cancer remains poorly understood. Results Our results suggest that PAK1 is over-expressed in oral cancer cell lines. Stimulation of Oral Squamous Cell Carcinoma (OSCC) cells with serum growth factors leads to PAK1 re-localization and might cause a profound cytoskeletal remodelling. PAK1 was also found to be involved in the invasion, migration and cytoskeletal remodelling of OSCC cells. Conclusions Our study revealed that PAK1 may play a crucial role in the progression of OSCC. Studying the role of PAK1 and its substrates is likely to enhance our understanding of oral carcinogenesis and potential therapeutic value of PAKs in oral cancer.
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Phosphorylated PAK1 level in the cytoplasm correlates with shorter survival time in patients with glioblastoma.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2007Co-Authors: Hiroshi Aoki, Tomohisa Yokoyama, Keishi Fujiwara, Ana M Tari, Raymond Sawaya, Dima Suki, Kenneth R Hess, Kenneth D Aldape, Seiji Kondo, Rakesh KumarAbstract:Glioblastoma is the most common primary malignant tumor in the brain. It aggressively invades the surrounding parenchyma, often allowing the tumor to progress after surgery. Accumulating evidence has shown that phosphorylated p21-activated kinase 1 (PAK1), a mediator of small guanosine triphosphatases, plays a role in the proliferation, survival, and invasiveness of cancer cells. Thus, we examined patterns of PAK1 expression in glioblastoma and sought to determine whether the level of phosphorylated PAK1 in glioblastoma cells is associated with patient survival time. We carried out immunohistochemical staining for phosphorylated PAK1 in tumor specimens from 136 patients with glioblastoma; the tumors were classified according to PAK1 protein levels in the cytoplasm and nucleus. We compared the patients' overall survival times using Kaplan-Meier analysis and estimated the effects of levels of cytoplasmic or nuclear phosphorylated PAK1. We then down-regulated PAK1 by using small interfering RNA to knock down PAK1 in two glioblastoma cell lines to determine whether PAK1 contributed to cell viability and invasion. Median overall survival was significantly shorter in patients with tumors showing a moderate or high level of cytoplasmic phosphorylated PAK1 than in patients with tumors showing no cytoplasmic phosphorylated PAK1. The level of nuclear phosphorylated PAK1 was not related to survival time. Knockdown of PAK1 suppressed the invasion, but not the viability, of U87-MG and U373-MG cells. The presence of phosphorylated PAK1 in the cytoplasm of glioblastoma cells is associated with shorter survival, and PAK1 plays a role in the invasiveness of glioblastoma. These data suggest that PAK1 might be a potential target for the management of glioblastoma.
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association between PAK1 expression and subcellular localization and tamoxifen resistance in breast cancer patients
Journal of the National Cancer Institute, 2006Co-Authors: Caroline Holm, Rakesh Kumar, Suresh K Rayala, Karin Jirstrom, Olle Stal, Goran LandbergAbstract:Background: p21-activated kinase 1 (PAK1) phosphorylates many proteins in both normal and transformed cells. Its ability to phosphorylate and thereby activate the estrogen receptor alpha (ER alpha) potentially limits the effectiveness of antiestrogen treatment in breast cancer. Here we studied associations between PAK1 expression and subcellular localization in tumor cells and tamoxifen resistance. Methods: PAK1 protein expression was evaluated in 403 primary breast tumors from premenopausal patients who had been randomly assigned to 2 years of adjuvant tamoxifen or no treatment. Tamoxifen response was evaluated by comparing recurrence-free survival in relation to PAK1 and ER alpha expression in untreated versus tamoxifen-treated patients. Tamoxifen responsiveness of human MCF-7 breast cancer cells that inducibly expressed constitutively active PAK1 or that transiently overexpressed wild-type PAK1 (Wt-PAK1) or PAK1 that lacked functional nuclear localization signals (PAK1 Delta NLS) was evaluated by analyzing cyclin D1 promoter activation and protein levels as markers for ER alpha activation. The response to tamoxifen in relation to PAK1 expression was analyzed in naturally tamoxifen-resistant Ishikawa human endometrial cancer cells. All statistical tests were two-sided. Results: Among patients who had ER alpha-positive tumors with low PAK1 expression, those treated with tamoxifen had better recurrence-free survival than those who received no treatment (hazard ratio [HR] = 0.502, 95% confidence interval [CI] = 0.331 to 0.762; P = .001) whereas there was no difference in recurrence-free survival between treatment groups for patients whose tumors had high cytoplasmic (HR = 0.893, 95% CI = 0.420 to 1.901; P = .769) or any nuclear PAK1 expression (HR = 0.955, 95% CI = 0.405 to 2.250; P = .916). In MCF-7 cells, overexpression of Wt-PAK1, but not of PAK1 Delta NLS, compromised tamoxifen response by stimulating cyclin D1 expression. Treatment of Ishikawa cells with tamoxifen led to an increase in the amount of nuclear PAK1 and PAK1 kinase activity, suggesting that tamoxifen, to some extent, regulates PAK1 expression. Conclusions: Our data support a role for PAK1, particular PAK1 localized to the nucleus, in ER alpha signaling and in tamoxifen resistance. (Less)
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Association between PAK1 expression and subcellular localization and tamoxifen resistance in breast cancer patients.
Journal of the National Cancer Institute, 2006Co-Authors: Caroline Holm, Rakesh Kumar, Suresh K Rayala, Karin Jirstrom, Olle Stal, Goran LandbergAbstract:p21-activated kinase 1 (PAK1) phosphorylates many proteins in both normal and transformed cells. Its ability to phosphorylate and thereby activate the estrogen receptor alpha (ERalpha) potentially limits the effectiveness of antiestrogen treatment in breast cancer. Here we studied associations between PAK1 expression and subcellular localization in tumor cells and tamoxifen resistance. PAK1 protein expression was evaluated in 403 primary breast tumors from premenopausal patients who had been randomly assigned to 2 years of adjuvant tamoxifen or no treatment. Tamoxifen response was evaluated by comparing recurrence-free survival in relation to PAK1 and ERalpha expression in untreated versus tamoxifen-treated patients. Tamoxifen responsiveness of human MCF-7 breast cancer cells that inducibly expressed constitutively active PAK1 or that transiently overexpressed wild-type PAK1 (Wt-PAK1) or PAK1 that lacked functional nuclear localization signals (PAK1DeltaNLS) was evaluated by analyzing cyclin D1 promoter activation and protein levels as markers for ERalpha activation. The response to tamoxifen in relation to PAK1 expression was analyzed in naturally tamoxifen-resistant Ishikawa human endometrial cancer cells. All statistical tests were two-sided. Among patients who had ERalpha-positive tumors with low PAK1 expression, those treated with tamoxifen had better recurrence-free survival than those who received no treatment (hazard ratio [HR] = 0.502, 95% confidence interval [CI] = 0.331 to 0.762; P = .001) whereas there was no difference in recurrence-free survival between treatment groups for patients whose tumors had high cytoplasmic (HR = 0.893, 95% CI = 0.420 to 1.901; P = .769) or any nuclear PAK1 expression (HR = 0.955, 95% CI = 0.405 to 2.250; P = .916). In MCF-7 cells, overexpression of Wt-PAK1, but not of PAK1DeltaNLS, compromised tamoxifen response by stimulating cyclin D1 expression. Treatment of Ishikawa cells with tamoxifen led to an increase in the amount of nuclear PAK1 and PAK1 kinase activity, suggesting that tamoxifen, to some extent, regulates PAK1 expression. Our data support a role for PAK1, particular PAK1 localized to the nucleus, in ERalpha signaling and in tamoxifen resistance.
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CRIPak, a novel endogenous PAK1 inhibitor
Oncogene, 2005Co-Authors: Amjad H. Talukder, Qing-chang Meng, Rakesh KumarAbstract:p21-activated protein kinase 1 (PAK1) plays an important role in several cellular processes, including cytoskeleton reorganization, promotion of the cell survival, and the estrogen receptor (ER) signaling. PAK1 expression and activity is deregulated in a number of cancers. PAK1 is activated by a variety of physiological signals; however, less is known about the negative regulators of PAK1. Here, we report a negative regulator of PAK1. By performing a yeast two-hybrid screen of a mammary gland library, we identified cysteine-rich inhibitor of PAK1 (CRIPak) as a novel PAK1-interacting protein. We found that CRIPak is an intronless gene that localized to chromosome 4p16.3. It contains 13 zinc-finger domains and has three trypsin inhibitor-like, cysteine-rich domains and is widely expressed in a number of human cells and tissues. We further found that CRIPak interacted with PAK1 through the N-terminal regulatory domain and inhibited PAK1 kinase in both in vitro and in vivo assays. CRIPak inhibited PAK1-mediated LIM kinase activation and enhancement of ER transactivation. Conversely, selective inhibition of the endogenous CRIPak resulted in an increased PAK1 activity, and consequently, increased cytoskeleton remodeling and PAK1-mediated ER transactivation activity. The hormonal stimulation of cells enhanced CRIPak expression and promoted its colocalization with ER in the nuclear compartment. Our findings suggest that CRIPak is a novel negative regulator of the PAK1 and has a role in the modulation of PAK1-mediated ER transactivation in breast cancer cells.
Hiroshi Maruta - One of the best experts on this subject based on the ideXlab platform.
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The serum/PDGF-dependent "melanogenic" role of the minute level of the oncogenic kinase PAK1 in melanoma cells proven by the highly sensitive kinase assay.
Drug discoveries & therapeutics, 2016Co-Authors: Binh Cao Quan Nguyen, Shinkichi Tawata, Cheong-yong Yun, Eung Gook Kim, Hiroshi MarutaAbstract:We previously demonstrated that the oncogenic kinase PAK4, which both melanomas and normal melanocytes express at a very high level, is essential for their melanogenesis. In the present study, using the highly sensitive "Macaroni-Western" (IP-ATP-Glo) kinase assay, we investigated the melanogenic potential of another oncogenic kinase PAK1, which melanoma (B16F10) cells express only at a very minute level. After transfecting melanoma cells with PAK1-shRNA for silencing PAK1 gene, melanin content, tyrosinase activity, and kinase activity of PAK1 were compared between the wild-type and transfectants. We found that (i) PAK1 is significantly activated by melanogenic hormones such as IBMX (3-isobutyl-1-methyl xanthine) and α-MSH (melanocyte-stimulating hormone), (ii) silencing the endogenous PAK1 gene in melanoma cells through PAK1-specific shRNA reduces both melanin content and tyrosinase activity in the presence of both serum and melanogenic hormones to the basal level, (iii) the exogenously added wild-type PAK1 in the melanoma cells boosts the α-MSH-inducible melanin level by several folds without affecting the basal, and (iv) α-MSH/IBMX-induced melanogenesis hardly takes place in the absence of either serum or PAK1, clearly indicating that PAK1 is essential mainly for serum- and α-MSH/IBMX-dependent melanogenesis, but not the basal, in melanoma cells. The outcome of this study might provide the first scientific basis for explaining why a wide variety of herbal PAK1-blockers such as CAPE (caffeic acid phenethyl ester), curcumin and shikonin in cosmetics are useful for skin-whitening.
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Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture.
Drug discoveries & therapeutics, 2015Co-Authors: Binh Cao Quan Nguyen, Shinkichi Tawata, Hiroshi MarutaAbstract:Cucurbitacin I (CBI) is a triterpene from a bitter melon called Goya grown in Okinawa, Japan, and directly inhibits both the Tyr-kinase JAK2 and the G protein RAC, leading to the inactivation of PAK1 (RAC/CDC42-activated kinase 1). Bio 30, a propolis produced in New Zealand, contains CAPE (caffeic acid phenethyl ester) as the major anti-cancer ingredient which directly down-regulates RAC, leading to the inactivation of PAK1. Since PAK1 is essential for the growth of RAS cancer cells such as A549 cell line which carry an oncogenic K-RAS mutant, and the melanogenesis in skin cells, here using these PAK1-blockers as model compounds, we introduce a new approach to the quick assessment of PAK1-blockers in cell culture. First, combining the immuno-precipitation (IP) of PAK1 from cell lysate and the in vitro ATP_Glo kinase assay kit (called "Macaroni-Western" assay), we confirmed that both CBI and Bio 30 inactivate PAK1 in A549 lung cancer cells in 24 h, and inhibit their PAK1-dependent growth in 72 h. Furthermore, we verified that CBI inhibits the PAK1/PAK4-dependent melanogenesis in melanoma cells by far more than 50%, while Bio 30 inhibits the melanogenesis only by 50%, with only a merginal effect on their growth per se. Since the "Macaroni-Western" kinase assay and melanogenesis are both rather simple and quick, the combination of these two cell culture assays would be highly useful for selecting both "potent" (highly cell-permeable) and "safe" (non-toxic) natural or synthetic PAK1-blockers.
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herbal therapeutics that block the oncogenic kinase PAK1 a practical approach towards PAK1 dependent diseases and longevity
Phytotherapy Research, 2014Co-Authors: Hiroshi MarutaAbstract:Over 35 years research on PAKs, RAC/CDC42(p21)-activated kinases, comes of age, and in particular PAK1 has been well known to be responsible for a variety of diseases such as cancer (mainly solid tumors), Alzheimer's disease, acquired immune deficiency syndrome and other viral/bacterial infections, inflammatory diseases (asthma and arthritis), diabetes (type 2), neurofibromatosis, tuberous sclerosis, epilepsy, depression, schizophrenia, learning disability, autism, etc. Although several distinct synthetic PAK1-blockers have been recently developed, no FDA-approved PAK1 blockers are available on the market as yet. Thus, patients suffering from these PAK1-dependent diseases have to rely on solely a variety of herbal therapeutics such as propolis and curcumin that block PAK1 without affecting normal cell growth. Furthermore, several recent studies revealed that some of these herbal therapeutics significantly extend the lifespan of nematodes (C. elegans) and fruit flies (Drosophila), and PAK1-deficient worm lives longer than the wild type. Here, I outline mainly pathological phenotypes of hyper-activated PAK1 and a list of herbal therapeutics that block PAK1, but cause no side (harmful) effect on healthy people or animals. Copyright © 2013 John Wiley & Sons, Ltd.
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Herbal Therapeutics that Block the Oncogenic Kinase PAK1: A Practical Approach towards PAK1‐dependent Diseases and Longevity
Phytotherapy research : PTR, 2013Co-Authors: Hiroshi MarutaAbstract:Over 35 years research on PAKs, RAC/CDC42(p21)-activated kinases, comes of age, and in particular PAK1 has been well known to be responsible for a variety of diseases such as cancer (mainly solid tumors), Alzheimer's disease, acquired immune deficiency syndrome and other viral/bacterial infections, inflammatory diseases (asthma and arthritis), diabetes (type 2), neurofibromatosis, tuberous sclerosis, epilepsy, depression, schizophrenia, learning disability, autism, etc. Although several distinct synthetic PAK1-blockers have been recently developed, no FDA-approved PAK1 blockers are available on the market as yet. Thus, patients suffering from these PAK1-dependent diseases have to rely on solely a variety of herbal therapeutics such as propolis and curcumin that block PAK1 without affecting normal cell growth. Furthermore, several recent studies revealed that some of these herbal therapeutics significantly extend the lifespan of nematodes (C. elegans) and fruit flies (Drosophila), and PAK1-deficient worm lives longer than the wild type. Here, I outline mainly pathological phenotypes of hyper-activated PAK1 and a list of herbal therapeutics that block PAK1, but cause no side (harmful) effect on healthy people or animals. Copyright © 2013 John Wiley & Sons, Ltd.
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a clue to the therapy of neurofibromatosis type 2 nf2 merlin is a PAK1 inhibitor
Cancer Journal, 2004Co-Authors: Yumiko Hirokawa, Anjali Tikoo, John Huynh, Tamara Utermark, Oliver C Hanemann, Marco Giovannini, Guanghui Xiao, Joseph R Testa, John L Wood, Hiroshi MarutaAbstract:BACKGROUND Neurofibromatosis type 2 is a group of tumors caused by loss-of-function mutations of a tumor suppressor gene encoding NF2/ merlin. Development of chemotherapeutics for this disease, which often threatens the life of young children, has been hampered by a limited information on the signaling function of NF2. NF2 can inhibit Ras-induced malignant transformation. However, the primary (signaling) target of NF2 in the oncogenic pathway has not been previously identified. RESULTS Here, using a series of NF2 constructs, we show that NF2 inhibits directly the Rac/CDC42-dependent Ser/Thr kinase PAK1, which is essential for both Ras transformation and neurofibromatosis type 1 (NF1), through two separate domains. A mutant of NF2, that lacks the PAK1-inhibiting domain of 78 amino acids (NF78C, residues 447-524), fails to suppress Ras transformation. Furthermore, PAK1-specific inhibitors CEP-1347 and WR-PAK18 selectively inhibit the growth of NF2-deficient cancer cells, but not NF2-positive cells. CONCLUSIONS These results suggest that PAK1 is essential for the malignant growth of NF2-deficient cells, and that PAK1-blocking drugs could be potentially useful for the treatment of neurofibromatosis types 2, in addition to Ras-induced cancers and neurofibromatosis type 1.
Audrey Minden - One of the best experts on this subject based on the ideXlab platform.
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Pdx1-Cre-driven conditional gene depletion suggests PAK4 as dispensable for mouse pancreas development.
Scientific reports, 2017Co-Authors: Miao Zhao, Parisa Rabieifar, Tânia D. F. Costa, Ting Zhuang, Audrey Minden, Matthias Löhr, Rainer Heuchel, Staffan StrömbladAbstract:Constitutive depletion of p21-activated kinase 4 (PAK4) in the mouse causes embryonic lethality associated with heart and brain defects. Given that conventional gene depletion of PAK1 or PAK3 caused functional deficits in the mouse pancreas, while gene depletion of PAK5 or PAK6 did not, we asked if PAK4 might have a functional role in pancreas development. We therefore introduced conditional, Pdx1-Cre-mediated, pancreatic PAK4 gene depletion in the mouse, verified by loss of PAK4 protein expression in the pancreas. PAK4 knock-out (KO) mice were born at Mendelian ratios in both genders. Further, morphological and immunohistochemical examinations and quantifications indicated that exocrine, endocrine and ductal compartments retained the normal proportions and distributions upon PAK4 gene depletion. In addition, body weight records and a glucose tolerance test revealed no differences between WT and PAK4 KO mice. Together, this suggests that PAK4 is dispensable for mouse pancreas development. This will facilitate future use of our Pdx1-Cre-driven conditional PAK4 KO mouse model for testing in vivo potential functions of PAK4 in pancreatic disease models such as for pancreatitis and different pancreatic cancer forms.
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abstract 1864 in vivoefficacy of the pak4 allosteric modulator kpt 9274 against a triple negative breast cancer model
Cancer Research, 2016Co-Authors: Chetan K. Rane, William Senapedis, Erkan Baloglu, Sharon Shacham, Audrey MindenAbstract:The p21-activated kinases (PAK) belong to a family of serine threonine kinases that promote cell survival and play an important role in cell proliferation, cell cycle regulation and cell shape determination. There are six mammalian PAK proteins which can be subdivided into two groups by sequence homology and mode of activation- Group A PAKs consisting of PAK 1, 2 and 3 and Group B PAKs consisting of PAK 4, 5 and 6. We have found that PAK4 protein levels are elevated in breast cancer, including Her2 positive and triple negative breast cancers, while it is expressed at low levels in normal mammary tissue, making it an attractive drug target. PAK inhibitors are being tested for effectiveness against solid tumors, but generation of highly specific PAK4 inhibitors has been a challenge. Furthermore, PAK4 has been reported to have kinase-independent functions. Therefore inhibiting its kinase activity alone might not be sufficient in blocking its tumorigenic potential. Our lab has previously reported the effectiveness of PAK4 allosteric modulators (PAM; KPT-8752 and KPT-9274) against multiple breast cancer cell lines. These novel PAK4 inhibitors reduce steady state protein levels and were able to block cell growth, cell migration and induce apoptosis in breast cancer cell lines, without affecting the control cells. Here, we tested the efficacy of the orally bioavailable PAM, KPT-9274 against tumors formed by the triple negative breast cancer cell line, MDA-MB-231. Following six weeks of treatment with orally administered KPT-9274 (150mg/kg bidx4), there was almost a five-fold reduction in tumor volume and tumor weight in the treatment group as compared to the control group. The treatment did not significantly affect mice body weight. After six weeks of treatment, the tumors were excised and analyzed for PAK4 levels. We observed a significant decrease in PAK4 levels in excised tumors from the treatment group as compared to those from the control group. PAK1 levels were monitored to see any off-target effects, but their levels were unchanged. Our results indicate that PAK4 plays a key functional role in triple negative breast cancer and treatment with an orally administered KPT-9274 was capable of specifically binding and inhibiting PAK4, and consequently reducing tumor growth. Future studies analyzing the effects of KPT-9274 in blocking PAK4 mediated functions that promote tumorigenesis are ongoing. Additional studies of the effectiveness of KPT-9274 on mammary fat pad tumors formed by MDA-MB-231 and the ER positive cell line, MCF7 are under investigation. Citation Format: Chetan Rane, William Senapedis, Erkan Baloglu, Sharon Shacham, Audrey G. Minden. In vivo efficacy of the PAK4 allosteric modulator KPT-9274 against a triple-negative breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1864.
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The pak4 protein kinase in breast cancer.
ISRN oncology, 2012Co-Authors: Audrey MindenAbstract:Paks4, along with Paks5, and 6 are members of the group B family of p21-activated kinases (Paks). The Paks play multiple different roles in controlling cell morphology, cell growth, proliferation, and signaling. Pak4 has essential roles in embryonic development (Qu et al., 2003), but in adults high levels of Pak4 are frequently associated with cancer. Pak4 has been implicated in several types of cancer (Wells and Jones, 2010; Eswaran et al., 2009; Liu et al., 2008; and Liu et al., 2010) and it is strongly linked to breast cancer (Liu et al., 2008; Liu et al. 2010; Yu et al., 2009; Rafn et al., 2012; and So et al., 2012). Breast tumors and breast cancer cell lines frequently have high levels of Pak4 (Liu et al., 2008), and overexpression of Pak4 in mammary epithelial cells leads to tumorigenesis in mice (Liu et al., 2010). This paper summarizes the current work on the role of Pak4 in breast cancer.
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pak4 6 in cancer and neuronal development
Cellular logistics, 2012Co-Authors: Audrey MindenAbstract:PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.
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PAK4–6 in cancer and neuronal development
Cellular logistics, 2012Co-Authors: Audrey MindenAbstract:PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.
