BACKGROUND： Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy， the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study， we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy. METHODS AND RESULTS： Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart， we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload， Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls， and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover， we discovered that the Pak1 activator FTY720， a sphingosine-like analog， was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile， FTY720 failed to exert such an effect on Pak1(cko) mice， suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation. CONCLUSIONS： These results， for the first time， establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.
p21-activated kinases (PAKs) are serine/threonine protein kinases that serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAK1 has been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity， invasion， and actin cytoskeleton organization. To better understand the role of PAK1 in tumorigenesis， PAK1 genomic copy number and expression were determined for a large panel of breast， lung， and head and neck tumors. PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer， and PAK1 protein expression was associated with lymph node metastasis. Breast cancer cells with PAK1 genomic amplification rapidly underwent apoptosis after inhibition of this kinase. Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous nonsmall cell lung carcinomas (NSCLCs)， and selective PAK1 inhibition was associated with delayed cell-cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small-molecule inhibitors， and several synergistic combination therapies， including combination with antagonists of inhibitor of apoptosis proteins， were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked inhibitor of apoptosis efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together， our results provide evidence for dysregulation of PAK1 in breast and squamous NSCLCs and a role for PAK1 in cellular survival and proliferation in these indications.
Abstract 912: P21-activated kinase 4 (PAK4) as a predictive marker for gemcitabine in pancreatic cancer cell line
PURPOSE： -activated kinases () are involved in cytoskeletal reorganization， gene transcription， and survival， and oncogenic transformation. Therefore， we hypothesized that expression levels could predict the sensitivity of to treatment， and could be therapeutic targets. MATERIALS AND METHODS： viability inhibition by was evaluated in lines (Capan-1， Capan-2， MIA PaCa-2， -1， Aspc-1， SNU-213， and SNU-410). expression and mRNA of molecules was detected by immunoblot analysis and polymerase chain reaction. To define the function of ， was controlled using siRNA.： Capan-2， -1， and SNU-410 were resistant to treatment. Immunoblot analysis of molecules reported to indicate sensitivity showed higher expression of and lower expression of (hENT1)， a well-known predictive marker for activity， in the resistant lines. Knockdown of using siRNA induced the upregulation of hENT1. In resistant lines (Capan-2， -1， and SNU-410)， knockdown of by siRNA resulted in restoration of sensitivity to . CONCLUSION： could be a predictive marker of sensitivity and a potential therapeutic target to increase sensitivity in .
Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486.
Fragile X syndrome (FXS) is the most common inherited form of autism and intellectual disability and is caused by the silencing of a single gene， fragile X mental retardation 1 (Fmr1). The Fmr1 KO mouse displays phenotypes similar to symptoms in the human condition--including hyperactivity， repetitive behaviors， and seizures--as well as analogous abnormalities in the density of dendritic spines. Here we take a hypothesis-driven， mechanism-based approach to the search for an effective therapy for FXS. We hypothesize that a treatment that rescues the dendritic spine defect in Fmr1 KO mice may also ameliorate autism-like behavioral symptoms. Thus， we targeted a protein that regulates spines through modulation of actin cytoskeleton dynamics： p21-activated kinase (PAK). Our results demonstrate that a potent small molecule inhibitor of group I PAKs reverses dendritic spine phenotypes in Fmr1 KO mice. Moreover， this PAK inhibitor--which we call FRAX486--also rescues seizures and behavioral abnormalities such as hyperactivity and repetitive movements， thereby supporting the hypothesis that a drug treatment that reverses the spine abnormalities can also treat neurological and behavioral symptoms. Finally， a single administration of FRAX486 is sufficient to rescue all of these phenotypes in adult Fmr1 KO mice， demonstrating the potential for rapid， postdiagnostic therapy in adults with FXS.
Although remarkable clinical response rates in melanoma have been observed using vemurafenib or dabrafenib in patients with tumors carrying oncogenic mutations in BRAF， a substantial unmet medical need remains for the subset of patients with wild-type BRAF tumors.To investigate the role of p21-activated kinases (PAKs) in melanoma， we determined PAK1 genomic copy number and protein expression for a panel of human melanoma tissues. PAK1 was inhibited in vitro and in vivo using RNA interference or PF-3758309 inhibitor treatment in a panel of melanoma cell lines with known BRAF and RAS (rat sarcoma) genotype to better understand its role in melanoma cell proliferation and migration. Tumorigenesis was assessed in vivo in female NCR nude mice and analyzed with cubic spline regression and area under the curve analyses. All statistical tests were two-sided.Strong cytoplasmic PAK1 protein expression was prevalent in melanomas (27%) and negatively associated with activating mutation of the BRAF oncogene (P < .001). Focal copy number gain of PAK1 at 11q13 was also observed in 9% of melanomas (n = 87; copy number ≥ 2.5) and was mutually exclusive with BRAF mutation (P < .005). Selective PAK1 inhibition attenuated signaling through mitogen-activated protein kinase (MAPK) as well as cytoskeleton-regulating pathways to modulate the proliferation and migration of BRAF wild-type melanoma cells. Treatment of BRAF wild-type melanomas with PF-3758309 PAK inhibitor decreased tumor growth for SK-MEL23 and 537MEL xenografts (91% and 63% inhibition， respectively; P < .001) and MAPK pathway activation in vivo.Taken together， our results provide evidence for a functional role of PAK1 in BRAF wild-type melanoma and therapeutic use of PAK inhibitors in this indication.
miRNA-7 attenuation in Schwannoma tumors stimulates growth by upregulating three oncogenic signaling pathways.
Micro RNAs (miRNA) negatively regulate protein-coding genes at the posttranscriptional level and are critical in tumorigenesis. Schwannomas develop from proliferation of dedifferentiated Schwann cells， which normally wrap nerve fibers to help support and insulate nerves. In this study， we carried out high-throughput miRNA expression profiling of human vestibular schwannomas by using an array representing 407 known miRNAs to explore the role of miRNAs in tumor growth. Twelve miRNAs were found to be significantly deregulated in tumor samples as compared with control nerve tissue， defining a schwannoma-typical signature. Among these miRNAs， we focused on miR-7， which was one of the most downregulated in these tumors and has several known oncogene targets， including mRNAs for epidermal growth factor receptor (EGFR) and p21-activated kinase 1 (Pak1). We found that overexpression of miR-7 inhibited schwannoma cell growth both in culture and in xenograft tumor models in vivo， which correlated with downregulation of these signaling pathways. Furthermore， we identified a novel direct target of miR-7， the mRNA for associated cdc42 kinase 1 (Ack1)， with the expression levels of miR-7 and Ack1 being inversely correlated in human schwannoma samples. These results represent the first miRNA profiling of schwannomas and the first report of a tumor suppressor function for miR-7 in these tumors that is mediated by targeting the EGFR， Pak1， and Ack1 oncogenes. Our findings suggest miR-7 as a potential therapeutic molecule for schwannoma treatment， and they prompt clinical evaluation of drugs that can inhibit the EGFR， Pak1， and Ack1 signaling pathways to treat this tumor type.
目的 研究二烯丙基二硫(diallyl disulfide，DADS)对人胃癌MGC803细胞Rac1-Pak1/Rock1通路分子的影响.方法 30 mg·L-1 DADS 分别处理MGC803细胞不同时间后，采用RT-PCR、Western blot分别检测Rac1、Pak1、Rock1、cofilin1和destrin的mRNA和蛋白水平.结果 DADS可下调MGC803细胞Rac1、Pak1和Rock1 mRNA和蛋白水平(P<0.05)，对cofilin1和destrin的表达无影响，但可抑制cofilin1磷酸化(P<0.01). 结论 DADS可下调Rac1、Pak1和Rock1表达和磷酸化cofilin1;DADS抑制 Rac1-Pak1/Rock1通路可能与DADS抗人胃癌MGC803细胞迁移侵袭作用机制有关.
Gestational trophoblastic disease (GTD) includes frankly malignant choriocarcinoma (CCA) and placental site trophoblastic tumor and potentially malignant hydatidiform mole. p21-Activated kinase (PAK) 4 promotes cell motility. This study investigated the role of PAK4 in the pathogenesis of GTD. PAK4 messenger RNA and protein expressions in clinical samples and cell lines of normal placentas and GTD were determined by quantitative real-time polymerase chain reaction and western blot， respectively. The effects of human chorionic gonadotropin (hCG) and phosphoinositide 3 kinase (PI3K) on the expression and activation of PAK4 were investigated by treating CCA JEG3 and JAR cells with anti-hCG antibody and PI3K inhibitor， respectively. The effects of PAK4 on CCA cell proliferation， migration and invasion were assessed by corresponding functional assays. We demonstrated overexpression of PAK4 in GTD and CCA cell lines at both RNA and protein level. hCG is one of the upstream regulators of PAK4 expression， whereas activation of PAK4 is PI3K/PKB dependent in JEG3 and JAR cells. Significant correlation was found between PAK4 expression and proliferation index minichromosome maintenance complex component 7 (P = 0.007). In JEG3 and JAR cells， stably transfected PAK4 increased proliferation， migration and invasion， whereas small interfering RNA knockdown of PAK4 decreased proliferation， migration and invasion along with downregulated CDK6 and membrane-type 1 matrix metalloproteinase (MT1-MMP) and upregulated p16. We further found PAK4-mediated transcription of MT1-MMP in CCA cells by luciferase reporter assay. Our results demonstrated for the first time that overexpressed PAK4 was involved in the pathogenesis of GTD， promoting proliferation and enhancing cell migration and invasion in CCA cells.
Oncogenic activation of Pak1-dependent pathway of macropinocytosis determines BCG entry into bladder cancer cells
Bacille Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is used widely as a vaccine for tuberculosis and is used as an effective treatment for superficial bladder carcinoma. Despite being the most successful cancer biotherapy， its mechanism of action and response determinants remain obscure. Here， we establish a model system to analyze BCG interaction with bladder cancer cells， using it to show that these cells vary dramatically in their susceptibility to BCG infection. Unexpectedly， the uptake of BCG by bladder cancer cells occurs by macropinocytosis rather than phagocytosis. BCG entry into bladder cancer cells relied upon Rac1， Cdc42， and their effector kinase Pak1. The difference in susceptibility between BCG-permissive and -resistant bladder cancer cells was due to oncogenic activation of signaling pathways that activate macropinocytosis， with phosphoinositide 3-kinase inhibitor activation stimulating BCG uptake independently of Akt. Similarly， activated Ras strongly activated Pak1-dependent uptake of BCG. These results reveal that oncogenic activation of macropinocytosis determines BCG uptake by bladder cancer cells， implying that tumor responsiveness to BCG may be governed by the specific mutations present in the treated cancer cell.
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.