Role Of Pak1 Research Articles

Pak1 kinase controls cell shape through ribonucleoprotein granules.

Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.

Pak1 maintains epidermal stem cells by regulating Langerhans cells and is required for skin carcinogenesis.

Pak1 (serine/threonine p21-activated kinases) was previously reported to have oncogenic activity in several cancers. However, its roles in the cancer microenvironment are poorly understood. We demonstrated that Pak1 expression in Langerhans cells (LCs) is essential for the maintenance of epidermal stem cells and skin tumor development. We found that PAK1 is localized in LCs by immunohistochemistry. Furthermore, the number of LCs significantly decreased in MSM/Ms Pak1 homozygous knockout mice (MSM/Ms-Pak1-/-). F1 hybrid (FVB/N×MSM/Ms) Pak1 heterozygous knockout mice (F1-Pak1+/-) had increased numbers of Th17 cells in the skin. Therefore, Pak1 knockdown cells were prepared using LC-derived XS52 cells (XS52-Pak1KD) and co-cultured with keratinocyte-derived C5N cells. As a result, XS52-Pak1KD cell supernatants promoted C5N cell proliferation. We then carried out DMBA/TPA skin carcinogenesis experiments using F1-Pak1+/- mice. Of note, F1-Pak1+/- mice exhibited stronger resistance to skin tumors than control mice. F1-Pak1+/- mice had fewer epidermal stem cells in the skin bulge. Our study suggested that Pak1 regulates the epidermal stem cell number by changing the properties of LCs and functions in skin carcinogenesis. We clarified a novel role of Pak1 in regulating LCs as a potential therapeutic target in skin immune disease and carcinogenesis.

Potential target identification for breast cancer and screening of small molecule inhibitors: A bioinformatics approach

In the current study, we investigated the role of PAK1 (P21 (RAC1) Activated Kinase 1) gene in breast cancer and to this end, we performed differential gene expression analysis of PAK1 in breast cancer tissues compared to the normal adjacent tissue. We also studied its significance in protein-protein interaction (PPI) network, and analysed biological pathways, cellular processes, and role of PAK1 in different diseases. We found PAK1 to have significant role in breast cancer pathways such as integrin signaling, axonal guidance signaling, signaling by Rho family GTPases, ERK5 signaling. Additionally, it has been found as hub gene in PPI network, suggesting its possible regulatory role in breast carcinogenesis. Moreover, PAK1 had role in progression of various diseases as neoplasia, tumorigenesis, lymphatic neoplasia. Thereby, PAK1 can be used as a therapeutic target in breast cancer. Further, we put our efforts in identification of potential small molecules inhibitors against PAK1 by developing a composite virtual screening protocol involving molecular dynamics (MD) and molecular docking. The chemical library of compounds from NCI diversity sets, Pubchem and eMolecules were screened against PAK1 protein and hits which showed good binding affinity were considered for MD simulation study. Moreover, to assess binding of selected hits, MMGBSA (Molecular Mechanics-Generalized Born Surface Area) analysis was performed using AMBER (Assisted Model Building with Energy Refinement) package. MMGBSA calculations exhibited that the identified ligands showed good binding affinity with PAK1. Highlights The PAK1 has been found to be upregulated in breast cancer samples and is a potential oncogene playing role in different cellular functions and processes. The molecular docking studies revealed ligands showed good binding affinity towards PAK1 protein. The residues Glu345, Leu347, Thr406, Asp299, Asp393 and Gly350 were found to make H-bond interactions with small molecule inhibitors. The residues Ile276, Val284, Ala297, Tyr346, Leu396 and Asp407 were found to make hydrophobic interactions. The RMSD analysis confirmed stability of complexes throughout 40 ns production period. The MD simulations studies revealed the binding site flexibility, binding free energy of complexes and per-residue contribution in ligand binding. Communicated by Ramaswamy H. Sarma

PAK1 promotes proliferation, migration and invasion of hepatocellular carcinoma by facilitating EMT via directly up-regulating Snail

BackgroundHepatocellular carcinoma (HCC) is a primary cause of cancer mortality. PAK1 plays key roles in many types of cancers. However, the role of PAK1 in HCC is not clear. MethodsqRT-PCR and Western blotting were used to determine expressions of PAK1, Snail and epithelial mesenchymal transition (EMT)-related proteins. Luciferase reporter assay was used to measure the interaction between PAK1 and Snail. Wound healing, transwell, colony formation assays and flow cytometry were used to assess cell migration, invasion, proliferation and apoptosis. Mouse tumor xenograft model was used to determine the effect of PAK1 on tumor growth in vivo. ResultsPAK1 and Snail were up-regulated in HCC cells. PAK1 knockdown suppressed cell proliferation, migration and invasion, and increased apoptosis of HCC cells. PAK1 knockdown also inhibited tumor growth in vivo. Mechanistically, PAK1 promoted EMT by targeting Snail. Knockdown of PAK1 could up-regulate pro-apoptotic proteins but down-regulate proliferation-related proteins via suppressing β-catenin signaling pathway. ConclusionPAK1 promotes EMT process by increasing Snail, and facilitates progression of HCC by activating β-catenin pathway.

Late-onset megaconial myopathy in mice lacking group I Paks

BackgroundGroup I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria.MethodsWe analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation.ResultsdKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis.ConclusionsConditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

A Stemness Screen Reveals C3ORF54/INKA1 As a Gate-Keeper of Human Stem Cell Latency

The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC.Our previous work on LSC subsets from >80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen.Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP.Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation.Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency.In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac.In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. DisclosuresTakayanagi:Kyowa Hakko Kirin Co., Ltd.: Employment.

Abstract 4755: Role of PAK4 in cancer immune cell exclusion

Abstract Lack of T-cell infiltration is the main mechanism of primary resistance to checkpoint blockade therapies. Here, we performed a transcriptomic analysis of metastatic melanoma biopsies taken from patients treated with anti-PD1 (n=23) with biopsies pre- (n=17) and during treatment (n=22), and investigated cancer cell intrinsic mechanisms of immune evasion. We classified our samples based on their T-cell infiltration status using a validated set of immune genes for T-cell infiltration (Spranger et al., 2015), normalized within gene and scored the samples from 0 (least infiltrated) to 1 (most infiltrated). In order to identify genes that anti-correlate with infiltration, we performed differential gene expression analysis between non-infiltrated (n= 18) and infiltrated tumors (n=21) regardless clinical response and condition. The analysis resulted in total of 1904 genes enriched in the non-infiltrated group (log2foldchange > 1, q-value < 0.05 and infiltration threshold = 0.5). Since Wnt signaling pathway has been previously suggested as a potential mechanism of immune exclusion, we aimed to select targetable proteins implicated in β-catenin signaling. Interestingly, p21 (RAC1) Activated Kinase 4 (PAK4) was significantly over-expressed in the non-infiltrated group (log2folchange = 1.04, q-value = 1.08e-05). PAK4 is a kinase involved in cell migration, proliferation and apoptosis and it is known that it can directly phosphorylate β-catenin to promote Wnt signaling. In addition, PAK4 was found to be also significantly enriched in on-treatment samples of non-responders (n=7) compared to responders (n=7) (mean of non-responder = 3.68, mean of responders = 2.69, p-value = 0.007). We next aimed to determine genes negatively associated with infiltration using Pearson correlation coefficient. PAK4 was on the top 5 of genes negatively associated with CD8A (pcc = -0.56, p-value 1.8e-04), HLA-DMB (pcc = -0.64, p-value 8.4e-06), TNF (pcc = -0.66, p-value 4.8e-06) among other immune genes. Altogether, this data suggests that PAK4 have an active role in T-cell exclusion. In order to validate our finding, we analyzed 472 melanoma samples from the TCGA. We first performed RNA-seq deconvolution to estimate the infiltration status of each sample and then determined genes that anti-correlated with the different immune cell subtypes. In line with the hypothesis that PAK4 was causatively excluding T-cell from tumors, we found that PAK4 performed the strongest anti-correlation with dendritic cells (pcc= -0.45, p-value = 2.2e-16) and CD8+ T-cells (pcc = -0.22, p-value = 1.12e-06). PAK4 expression was enriched in TCGA non-infiltrated samples (p-value = 4.92e-09, infiltration threshold = 0.5). In summary, this data suggests that PAK4 might be driving T-cell exclusion and that inhibition of PAK4 kinase activity with small molecules could facilitate T-cell infiltration and synergize with current checkpoint blockade therapies. Citation Format: Gabriel Abril-Rodriguez, Catherine S. Grasso, Jesse M. Zaretsky, Beata Berent-Maoz, Siwen Hu-Lieskovan, Antoni Ribas. Role of PAK4 in cancer immune cell exclusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4755.

Mutations in the Effector Domain of RhoV GTPase Impair Its Binding to Pak1 Protein Kinase

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.

Abstract 2419: Genome-wide profiling of PAK4 DNA-binding sites and transcriptome reveals its potential transcriptional control on DNA repair-related genes in ovarian cancer cells

Abstract Oncogenic role of p21-activated protein 4 (PAK4) in relation to cell growth and migration through various signalling cascades has been well studied in multiple cancer types. Such role might be contributed largely by cytoplasmic PAK4 as most of the known PAK4 signalling events occur in the cytoplasm. However, recent studies have demonstrated that PAK4 is capable of shuttling between nucleus and cytoplasm. The precise function of nuclear PAK4 (nPAK4) remains substantially unexplored. We have previously shown that PAK4 can act as a transcription factor. Herein, we aim to identify nPAK4-regulated genes by delineating the PAK4-binding landscape in ovarian cancer genome and mapping the PAK4 transcriptome in ovarian cancer cells. PAK4-enriched genomic regions were identified by chromatin immunoprecipitation with a PAK4 antibody followed by next-generation sequencing (ChIP-seq) in OVCAR3 cells. PAK4 transcriptome data was generated using RNA extracted from SKOV3 cells stably expressing PAK4 in the Affymetrix GeneChIP® Human Transcriptome Array 2.0 system. Gene lists generated from both ChIP-seq and microarray dataset were subjected to pathway enrichment analysis by Partek® PathwayTM. Among the 20 most significantly enriched pathways from microarray studies, several pathways in which PAK4 has no known reported function by far have been identified. Notably, DNA repair pathways that are known to influence drug chemosensitivity in ovarian cancer, such as the nucleotide excision repair (p=0.008), homologous recombination (p=0.012), mismatch repair (p=0.018) and DNA replication (p=0.018) pathways were significantly enriched. In concordance with the findings, the nucleotide excision repair pathway was also significantly enriched (p<0.006) in the gene list derived from promoter-restricted PAK4 binding sites in the ovarian cancer genome. Among the many candidates that were deregulated by PAK4, cyclin-dependent kinase 7 (CDK7), a core component in the CAK subcomplex crucial to nucleotide excision repair pathway, was further validated. The transcription start site (TSS) of cyclin-dependent kinase 7 (CDK7) was bound by PAK4. CDK7 mRNA expression was upregulated upon PAK4 overexpression. Quantitative-PCR showed downregulated CDK7 mRNA expression upon siRNA-mediated knockdown of PAK4 in OVCAR3 and TUOS3. This study points to the possible role of PAK4 in regulating gene transcription as PAK4 is mapped to several core promoter/TSS regions in the ovarian cancer genome. Our findings also shed light into the potential role of PAK4 in regulating drug chemosensitivity via its transcriptional control on genes implicated in DNA repair pathways. We postulate CDK7 as a direct transcriptional target of PAK4 and thereby contributes to chemoresistance in ovarian cancer cells. Citation Format: Ivy Tsz-Lo Wong, Oscar Gee-Wan Wong, Yiming Qin, Junwen Wang, Annie Nga-Yin Cheung. Genome-wide profiling of PAK4 DNA-binding sites and transcriptome reveals its potential transcriptional control on DNA repair-related genes in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2419. doi:10.1158/1538-7445.AM2017-2419

The Role of PAK1 in a Gut Manipulation Model of Ileus

Feeding intolerance is prevalent in surgical, critically ill, and trauma patients leading to prolonged hospital and intensive care unit stays, increased infectious and thromboembolic complications, and increased patient care costs. Ileus is a slowing of intestinal motility which contributes to feeding intolerance. At present there are no effective pharmacological treatments for preventing or treating feeding ileus. In previous studies, we have shown that upregulation of PAK1 activity inhibited intestinal contractility in an intestinal edema model of ileus. The purpose of this study was to determine if PAK1 also contributed to decreased intestinal contractility in a gut manipulation model of ileus. We hypothesized that, after gut manipulation, increased inflammatory mediators cause PAK1 activation leading to decreased intestinal contractility. We utilized both mouse and rat models. Ileus was induced by gently rolling the entire length of the small intestine between two cotton tipped applicators three times. Contractile activity was measured ex vivo in an organ bath system. PAK1 levels and Raf phosphorylation were measured by western blotting. In both mouse and rat models, we found that PAK1 expression was increased after gut manipulation. Inhibition of PAK1, as well as PAK1 genetic ablation, attenuated the decreased contractility induced by gut manipulation. PAK1 inhibition also improved response to carbachol in mice after gut manipulation. Pretreatment with a Rho kinase inhibitor blocked the effects of PAK1 inhibitors on contractile activity. CXCL1 and LIX, both CXCR2 ligands, were significantly upregulated within 1 hour after gut manipulation in intestinal smooth muscle. We found that CXCL1 can increase Raf1 phosphorylation, a known PAK1 target, and decrease MLC phosphorylation in primary human intestinal smooth muscle cells. CXCL1 also reduced the response to carbachol in rats. Taken together, these data suggest that PAK1 mediates the inhibitory effects of several inflammatory cytokines on intestinal contractility after gut manipulation. PAK1 is a potential drug target for the treatment of ileus.

Abstract 1024: Targeting PAK1 activity in breast cancer: Inhibition of cell growth, survival, motility, and signaling

Abstract Breast cancer (BrCa) is one of the most common cancers diagnosed and is the second leading cause of cancer-related deaths of women in the United States. BrCa is divided into four major molecular subtypes; these categories are based upon the expression of hormone receptors (i.e., estrogen receptor (ER) and progesterone receptor (PR)), HER2/neu receptor, and proliferation rate. Of these subtypes triple negative (ER-, PR-, HER2-) BrCa are among the most lethal, are highly aggressive, have poor prognosis, limited treatment options, and lack effective targeted therapies. Our lab has shown p-21 activated kinase 1 (PAK1) protein expression increases with BrCa progression in human tumor samples. PAK1 is highly expressed in invasive tumor tissues in comparison to normal tissues. The role of PAK1 in BrCa development and progression is through its activity as a serine/threonine kinase involved in the regulation of several key oncogenic pathways (i.e., WNT/β-catenin and MAPK/ERK/JNK). Although PAK1 expression is increased in invasive tumor tissues, our studies have found that in human BrCa cell cultures PAK1 expression is independent of hormonal receptor status. Our hypothesis for this study is that triple negative BrCa cell lines are more reliant upon PAK1's activity for cell growth, survival, and motility. To test our hypothesis two triple negative BrCa cell lines, MDA-MB-231 and MDA-MB-468, and two ER+ BrCa cell lines, T47D and ZR75-1, were used. Our results demonstrate there are significantly higher levels of PAK1 protein in T47D and MDA-MB-468, compared to ZR75-1 and MDA-MB-231. To determine if the cells were reliant on the activity of PAK1 for cell viability, a trypan blue exclusion assay was performed. The PAK1 inhibitor 1,1′-Dithiodi-2-napthol (IPA-3) significantly inhibited all four BrCa cell lines, cell growth and viability in a dose-dependent manner. IPA-3 further inhibited cell proliferation of MDA-MB-468 (IC50 = 11.5μM), MDA-MB-231 (IC50 = 14.5μM), ZR75-1 (IC50 = 13.3μM), and T47D (IC50 = 17.1μM). IPA-3 also inhibited the phosphorylation and nuclear translocation of PAK1 in a dose-dependent manner in MDA-MB-231 cells. The inhibition of PAK1 by IPA-3 also significantly inhibited the migration of MDA-MB-231 cells in a time- and dose-dependent manner. The regulation of PAK1 activation by the WNT/β-catenin pathway was further explored in ER+ and triple negative BrCa cell lines. Additional studies are currently underway investigating the role of PAK1 activation on the MAPK/ERK/JNK pathway and its effects on cell migration and invasion. These studies explore the role of PAK-1 in triple negative BrCa cell growth, survival, motility, and oncogenic signaling. Further investigation of PAK1's targets within these signaling pathways will provide opportunities for the development of novel prevention and/or therapeutic strategies. Citation Format: Alexandra M. Fajardo, Tristan Browne, Hannah Graff, Kelly Kleier, Kyle Neltner, Courtney McCall, Brad Meyer, Larry Douglass, Julia Carter. Targeting PAK1 activity in breast cancer: Inhibition of cell growth, survival, motility, and signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1024. doi:10.1158/1538-7445.AM2015-1024

Role of PAK4 in pancreas development and pancreatic cancer progression

Role of PAK4 in pancreas development and pancreatic cancer progression

Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

BackgroundKey effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).MethodsWe used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.ResultsImmunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.ConclusionsOur data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1360-4) contains supplementary material, which is available to authorized users.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

IntroductionBreast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.MethodsPAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.ResultsWe demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 × 10−4 and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.ConclusionsTaken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0564-5) contains supplementary material, which is available to authorized users.

Three‐dimensional Collagen IV and Hormone Prolactin Regulate Breast Cancer Cell Invasion: Role of PAK1 and Matrix Metalloproteinases

Three‐dimensional (3D) matrix is a critical component of mammary tissue development not only under physiological but also in pathophysiological conditions. The hormone/cytokine prolactin (PRL) also contributes to the pathogenesis of breast cancer via activation of JAK2/ STAT‐ and MAPK‐dependent pathways. Serine‐threonine kinase PAK1 is one of the targets of PRL‐activated JAK2. We showed previously that tyrosine kinase JAK2 phosphorylates PAK1 in response to PRL on three tyrosines (Tyr 153, 201 and 285). We show herein that the PAK1 phosphorylation is required for maximal PRL‐dependent invasion of human breast cancer cells through Matrigel and that 3D collagen IV acts synergistically with PRL‐activated PAK1 to induce the expression and secretion of MMP‐1 and MMP‐3. In contrast, PRL‐activated PAK1 decreases the 3D collagen IV induction of MMP‐2. MMP‐9 expression and secretion are stimulated by 3D collagen I, not collagen IV, and are not affected by PRL but are down‐regulated by PAK1. Using shRNA or pharmacological inhibitors, we show that MMP‐1 and ‐3 are required and MMP‐2 contributes to PRL‐ dependent invasion. Erk 1/2 signaling appears to be required for the enhanced expression and secretion of MMP‐1, and ‐3 and enhanced PRL‐dependent invasion. p38 MAPK and JNK 1/2 pathways participate in production of MMP‐1 and ‐3 as well as in PRL/PAK1‐dpendent cell invasion. The NFkB pathway is required for MMP‐3 and ‐9 secretion. Together, these data illustrate complex interaction between cellular substrate and PRL regulation of breast cancer cell progression, implicating a pivotal role for PAK1 tyrosyl phosphorylation in MMP regulation.This work was supported by National Institutes of Health Grant R01 DK88127 (to MD).

Novel insights into mechanisms for Pak1-mediated regulation of cardiac Ca(2+) homeostasis.

A series of recent studies report novel roles for Pak1, a key member of the highly conserved family of serine-threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1, in cardiac physiology and cardioprotection. Previous studies had identified Pak1 in the regulation of hypertrophic remodeling that could potentially lead to heart failure. This article provides a review of more recent findings on the roles of Pak1 in cardiac Ca2+ homeostasis. These findings identified crucial roles for Pak1 in cardiomyocyte Ca2+ handling and demonstrated that it functions through unique mechanisms involving regulation of the post-transcriptional activity of key Ca2+-handling proteins, including the expression of Ca2+-ATPase SERCA2a, along with the speculative possibility of an involvement in the maintenance of transverse (T)-tubular structure. They highlight important regulatory functions of Pak1 in Ca2+ homeostasis in cardiac cells, and identify novel potential therapeutic strategies directed at manipulation of Pak1 signaling for the management of cardiac disease, particularly heart failure.

Abstract A36: Interruption of K-Ras/PAK1/Crk pathway has a dramatic anti-tumor effect in NSCLC

Abstract Purposes of the study: The key downstream effector(s) of mutant K-Ras involved in lung cancer metastasis are not known. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic K-Ras signal in non-small cell lung cancer (NSCLC). Experimental procedures: We have carried out three types of experiments. Exp.1: NSCLC clinical specimens were used to examine correlation between K-Ras mutation, activation of PAK1/Crk axis and expression of adhesion molecules. K-Ras genotype was determined by sequencing K-Ras on exon 2 and the activation of PAK1/Crk axis was determined by quantifying PAK1 phosphorylation (Thr 423) and Crk phosphorylation (Ser 41) by immunohistochemical staining of the surgically resected, paraffin embedded NSCLC specimens. Exp.2: In order to assess the role of proto-oncogene c-Crk as a downstream effector of K-Ras, we inhibited K-Ras in NSCLC cells and examined Crk phosphorylation. Specifically, we inhibited K-Ras in NSCLC cells by exposing cells to a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and examined p-Crk-II (Ser41) by western blotting. Exp.3: We examined the functional role of K-Ras/PAK1/Crk axis in controlling malignant behavior of NSCLC cells. For this purpose, we interrupted the K-Ras/PAK1/Crk axis in NSCLC cells by combination of K-Ras inhibition (FTI + GGTI) and PAK1 inhibition and examined the motility and morphology of the cells in a wound healing assay. Findings: Exp.1: Immunohistochemical analysis of the specimens demonstrated a complete and inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. PAK1/Crk phosphorylation was also associated with disease stage at presentation (Pearson's R = 0.57). Furthermore, we observed a positive correlation between K-Ras mutation on codons 12 or 13 and PAK1/Crk phosphorylation (Pearson's R = 0.67) even in a small number of analyzed specimens. Exp.2: K-Ras inhibition by combination of FTI and GGTI completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change in cells exposed to individual inhibitors or inhibitor diluents. Exp.3: Combination of K-Ras inhibition (FTI + GGTI) and PAK1 inhibition resulted in a dramatic morphological change in H157 cells. H157 cells which typically harbor a mesenchymal morphology, obtained a tightly conjoined epithelial phenotype with a smooth border when exposed to K-Ras and PAK1 inhibitors. Cells exposed to K-Ras inhibitors and PAK1 inhibitor almost completely lost their motility as well. These dramatic effects were not seen in cells exposed to either K-Ras inhibitor combination, PAK1 inhibitor or inhibitor diluents. Conclusion: Our data provide strong evidence that proto-oncogene c-Crk is operative downstream of K-Ras in NSCLC. Considering that Crk as an oncogene has cell transforming ability, it seems that the oncogenic K-Ras signal is transduced through Crk. Our previous work demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of K-Ras signaling, bring forward the important role of K-Ras/PAK1/Crk axis as a key pathway in transduction of the oncogenic K-Ras signal in lung cancer metastasis. The very significant anti-tumor effects that we observed as a result of K-Ras/PAK1/Crk pathway inhibition, is further supportive of the prominent role of this pathway in transduction of the oncogenic K-Ras signal in NSCLC. Citation Format: Fariborz (Fred) Mortazavi, Jie Lu, Aljilani Amir, Ryan Phan, Fuyuhiko Tamanoi. Interruption of K-Ras/PAK1/Crk pathway has a dramatic anti-tumor effect in NSCLC. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A36. doi: 10.1158/1557-3125.RASONC14-A36

Potential compensation among group I PAK members in hindlimb ischemia and wound healing.

PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on in vitro studies and its role in angiogenesis in vivo has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6–8 week old PAK1−/− and PAK1+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1−/− mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1−/− mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1−/− but not PAK1+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1−/− mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1−/− and PAK1+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.

Abstract A067: Targeting p21-activated kinases in breast cancer

Abstract Luminal breast cancer is the most commonly diagnosed cancer worldwide and mechanisms of innate and acquired resistance are not yet well understood. P21-activated kinases (PAKs) are members of the STE20 family of serine/threonine kinases that lie downstream of receptor tyrosine kinases and Rho family GTPases. PAKs regulate many cellular processes that are commonly perturbed in cancer, including migration, polarization and proliferation, and are therapeutic targets of interest. PAK1 is also overexpressed in several cancers and we recently showed that focal genomic amplification of PAK1 occurs in ~8% of luminal breast cancer (Proc Natl Acad Sci 2011; 108:7177). PAK1 amplification has also been associated with poor patient outcome and PAK1 is a putative mediator of resistance to anti-estrogen therapy (Nature 2012; 486:346). The aim of this study was to further characterize PAK family kinases in breast adenocarcinoma. Luminal breast cancer cells with PAK1 focal amplification rapidly underwent apoptosis following inhibition of this kinase. To identify potential mechanisms of acquired resistance we have derived PAK1-resistant cell lines and begun characterizing via genomic and proteomics approaches. Furthermore, in contrast to the role of PAK1 in luminal breast cancer, elevated expression of PAK4 was associated with poor outcome in basal-like breast cancer. We developed a novel small molecule inhibitor with high potency and selectivity of group II PAKs to delineate the role of PAK4 signaling in basal breast cancer. Together, our results provide evidence for dysregulation of PAK1 and PAK4 in differing subtypes of breast cancer and roles in for these kinases in cellular survival, proliferation and migration in this tumor indication. Citation Format: Klaus Hoeflich, Christy Ong, Karen Lyle, Adrian Jubb, Wei Zhou, Adrian Harris, Marcia Belvin, Lori Friedman, Hartmut Koeppen, Joachim Rudolph, Steven Staben. Targeting p21-activated kinases in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A067.

Abstract B31: Pak1 and β-catenin inhibition blocks tumor progression in ErbB2-driven breast cancer models

Abstract p21-activated kinase-1 (Pak1) is frequently upregulated in human breast cancer and is required for transformation of mammary epithelial cells by ErbB2. However, its role in tumorigenesis in vivo, and the particular signaling pathways affected, are not defined. In this work, we examined the distinct roles of Pak1 and Pak2 in cellular and animal models of ErbB2-driven breast cancer. We found that inhibition of Pak1, but not Pak2, impedes transformation by ErbB2 in a 3D cell culture system, but that loss of either Pak1 or Pak2 causes loss of both Erk and Akt activation. A phospho-proteomic screen revealed that Pak1-deficient, but not Pak2-deficient, ErbB2 cells showed almost total loss of β-catenin expression that is accompanied by a significant reduction in almost all known β-catenin 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 the ability of ErbB2 to transform Pak1-deficient mammary epithelial cells. Lastly, we showed that small molecule inhibitors of Pak or β-catenin blocked transformation by ErbB2 in 3D culture and tumorigenesis by ErbB2 in mouse xenografts, and combined inhibition by both agents was synergistic. These findings establish Pak1 as a new target in ErbB2-driven breast cancer and define a new mechanism of action primarily through the β-catenin, but not the Erk or Akt, signaling pathways. Citation Format: Luis E. Arias-Romero, Olga Villamar-Cruz, Jonathan Chernoff. Pak1 and β-catenin inhibition blocks tumor progression in ErbB2-driven breast cancer models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B31.