PAK1 Levels Research Articles

Revealing the Molecular Signatures of miR-185-5p on Breast Cancer Cells Using Proteomic Analysis.

Breast cancer is a heterogeneous type of disease in which genetic and environmental factors play a crucial role. There are several types of treatment for breast cancer (BC) patients. However, the biggest problem in the treatment of breast cancer is the resistance that occurs during the treatment with chemotherapeutic agents. Usnic acid, a secondary metabolite of lichen, has been identified as a drug candidate molecule in cancer treatment. The determination of miRNA target proteins is essential for the understanding of molecular mechanisms of miRNA-related tumorigenesis. We determined that mir-185-5p has therapeutic potential at the miRNA level by applying usnic acid to BT-474 breast cancer cells in a previous study. Herein, we aimed to investigate the molecular mechanisms of miR-185-5p on BT-474 breast cancer cells using a proteomics approach. We explored the changes in the protein expression level of BT-474 breast cancer cells in response to the up-regulation of miR-185-5p after applying usnic acid as a novel candidate anti-- cancer drug molecule. We performed quantitative proteome analysis based on an LC-MS/MS assay, which was validated by western blotting. The differentially expressed proteins were analyzed using the latest data available in bioinformatics tools. The up-regulated expression of YWHAE, Cathepsin D, and the down-regulated levels of PAK-1 were demonstrated by western blot assay. According to the results, 86 proteins showing >2-fold change were identified as differentially expressed between breast cancer and normal breast epithelial cells. The apoptosis pathway was the main clade containing most of the proteins regulated by miR-185-5p. The results indicate that miR-185-5p modulates apoptosis signaling pathways in BT-474 breast cancer cells. Breast cancer inhibition due to increased expression of YWHAE, Cathepsin D, and decreased expression of PAK-1 is likely to be mediated by inducing miR-185-5p mediated apoptosis. In this study, the identification of miR-185-5p protein targets demonstrated the potential for the development of targeted therapy and the development of miRNA-based therapeutics and presented it as a biomarker for breast cancer diagnosis, prognosis, and treatment response. In this regard, proteome analyses provided an understanding of the molecular mechanism underlying the effect of miR-185-5p on breast cancer.

Diallyl disulfide downregulating RhoGDI2 induces differentiation and inhibit invasion via the Rac1/Pak1/LIMK1 pathway in human leukemia HL-60 cells.

Leukemia is a type of disease in which hematopoietic stem cells proliferate clonally at the genetic level. We discovered previously by high-resolution mass spectrometry that diallyl disulfide (DADS), which is one of the effective ingredients of garlic, reduces the performance of RhoGDI2 from APL HL-60 cells. Although RhoGDI2 is oversubscribed in several cancer categories, the effect of RhoGDI2 in HL-60 cells has remained unexplained. We aimed to investigate the influence of RhoGDI2 on DADS-induced differentiation of HL-60 cells to elucidate the association among the effect of inhibition or over-expression of RhoGDI2 with HL-60 cell polarization, migration and invasion, which is important for establishing a novel generation of inducers to elicit leukemia cell polarization. Co-transfection with RhoGDI2-targeted miRNAs apparently decreases the malignant biological behavior of cells and upregulates cytopenias in DADS-treated HL-60 cell lines, which increases CD11b and decreases CD33 and mRNA levels of Rac1, PAK1 and LIMK1. Meanwhile, we generated HL-60 cell lines with high-expressing RhoGDI2. The proliferation, migration and invasion capacity of such cells were significantly increased by the treated with DADS, while the reduction capacity of the cells was decreased. There was a reduction in CD11b and an increase in CD33 production, as well as an increase in the mRNA levels of Rac1, PAK1 and LIMK1. It also confirmed that inhibition of RhoGDI2 attenuates the EMT cascade via the Rac1/Pak1/LIMK1 pathway, thereby inhibiting the malignant biological behavior of HL-60 cells. Thus, we considered that inhibition of RhoGDI2 expression might be a new therapeutic direction for the treatment of human promyelocytic leukemia. The anti-cancer property of DADS against HL-60 leukemia cells might be regulated by RhoGDI2 through the Rac1-Pak1-LIMK1 pathway, which provides new evidence for DADS as a clinical anti-cancer medicine.

Mechanism by Which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency.

The "block and lock" strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The "block" refers to a compound's ability to inhibit latent HIV-1 proviral transcription, while the "lock" refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.

PAK1 overexpression promotes myxofibrosarcoma angiogenesis through STAT5B-mediated CSF2 transactivation: clinical and therapeutic relevance of amplification and nuclear entry.

Myxofibrosarcoma is genetically complex without established nonsurgical therapies. In public datasets, PAK1 was recurrently gained with mRNA upregulation. Using myxofibrosarcoma cells, we explored the oncogenic underpinning of PAK1 with genetic manipulation and a pan-PAK inhibitor (PF3758309). Myxofibrosarcoma specimens were analyzed for the levels of PAK1, phospho-PAKT423, CSF2 and microvascular density (MVD) and those of PAK1 gene and mRNA. PAK1-expressing xenografts were assessed for the effects of PF3758309 and CSF2 silencing. Besides pro-proliferative and pro-migrator/pro-invasive attributes, PAK1 strongly enhanced angiogenesis in vitro, which, not phenocopied by PAK2-4, was identified as CSF2-mediated using antibody arrays. PAK1 underwent phosphorylation at tyrosines153,201,285 and threonine423 to facilitate nuclear entry, whereby nuclear PAK1 bound STAT5B to co-transactivate the CSF2 promoter, increasing CSF2 secretion needed for angiogenesis. Angiogenesis driven by PAK1-upregulated CSF2 was negated by CSF2 silencing, anti-CSF2, and PF3758309. Clinically, overexpressed whole-cell phospho-PAKT423, related to PAK1 amplification, was associated with increased grades, stages, and PAK1 mRNA, higher MVD, and CSF2 overexpression. Overexpressed whole-cell phospho-PAKT423 and CSF2 independently portended shorter metastasis-free survival and disease-specific survival, respectively. In vivo, both CSF2 silencing and PF3758309 suppressed PAK1-driven tumor proliferation and angiogenesis. Conclusively, the nuclear entry of overexpressed/activated PAK1 endows myxofibrosarcomas with pro-angiogenic function, highlighting the vulnerable PAK1/STAT5B/CSF2 regulatory axis.

The role of circRNA polyribonucleotide nucleoside transferase 1 on Gestational Diabetes Mellitus.

This study aimed to focus on the mechanism of circRNA polyribonucleotide nucleoside transferase 1 (circ-PNPT1)-mediated miR-889-3p/PAK1 on gestational diabetes mellitus (GDM). Placental tissues from normal pregnancy and GDM patients were collected to detect the levels of circ-PNPT1, miR-889-3p, and PAK1. The high glucose-induced human trophoblast cells HTR-8/SVneo were adopted to stimulate the GDM model in vitro (HG group) and were transfected with lentivirus to silence circ-PNPT1 (si-circ-PNPT1 group) and mimic to overexpress miR-889-3p (miR-889-3p group). Cell proliferation, apoptosis, migration, and invasion were detected by CKK-8, flow cytometry, Transwell, and scratch assay, respectively. The results showed that the expressions of circ-PNPT1 and PAK1 in the GDM patients were up-regulated, and miR-889-3p was down-regulated (P< 0.05). Compared with cells in the control group, the circ-PNPT1 and PAK1 in the HG group were up-regulated, and miR-889-3p was down-regulated (P< 0.05). The cell proliferation, migration, and invasion abilities were weakened, and the apoptosis rate increased (P< 0.05). E-cadherin protein was elevated, and the N-cadherin and Vimentin decreased (P< 0.05). Compared with the HG group, the expressions of circ-PNPT1 and PAK1 in the other two groups decreased, and miR-889-3p increased (P< 0.05). The cell proliferation, migration, and invasion were enhanced, and the apoptosis rate decreased (P< 0.05). E-cadherin, N-cadherin, and Vimentin decreased (P< 0.05). There were targeted binding sites for miR-889-3p with circ-PNPT1 and PAK1, indicating circ-PNPT1 promoted HG-induced trophoblast dysfunction through the miR-889-3p/PAK1 axis.

LINC01232 Promotes Metastasis and EMT by Regulating miR-506-5p/PAK1 Axis in Gastric Cancer.

BackgroundLong non-coding RNA LINC01232 plays an important role in the progression of metastasis in several cancers. However, the function of LINC01232 in gastric cancer is limited. Authors aimed to investigate the role and mechanism of LINC01232 in the metastasis of gastric cancer.MethodsThe expression levels and correlation of LINC01232, miR-506-5p, and PAK1 were analyzed by GEPIA or ENCORI, and the abundance of LINC01232 and miR-506-5p was measured in tissues and cells via qRT-PCR, the location of LINC01232 in gastric cells was analyzed by nuclear and cytoplasmic fractionation, while the protein levels of PAK1, E-cadherin and vimentin were additionally quantified by Western blotting. Interactions between LINC01232, miR-506-5p, and PAK1 were detected through luciferase reporter assays, qRT-PCR and Western blotting. Cellular viability was evaluated through CCK8 assays, migration ability was measured by transwell assays, invasion ability was tested by wound healing experiment.ResultsLINC01232 was overexpressed in gastric cancer tissues and cells, and mainly located in nucleus. The inhibition of LINC01232 could suppress migration, invasion and EMT of gastric cancer cells. MiR-506-5p was downregulated in gastric cancer tissues and cells. LINC01232 sponged miR-506-5p to accelerate migration and EMT. PAK1 was certified to be a target of miR-506-5p, inhibition of PAK1 could interrupt LINC01232 overexpression-induced migration of gastric cancer cells.ConclusionThe LINC01232/miR-506-5p/PAK1 axis promotes metastasis of gastric cancer cells.

Helicobacter pylori regulates ILK to influence autophagy through Rac1 and RhoA signaling pathways in gastric epithelial cells

The ability of Helicobacter pylori to manipulate host autophagy is an important pathogenic mechanism. We found an inverse correlation between the expression of ILK and the autophagy marker protein LC3B in H. pylori-positive human samples, H. pylori-infected mice models and H. pylori-infected GES-1 cell lines. When the ILK-knockdown GES-1 cells were infected by H. pylori, CagA were significantly degraded, autophagosomes accumulation and autolysosomes formation were significantly increased, and LC3B protein levels and ratio of LC3BII to LC3BI were also remarkably upregulated. And chloroquine treatment increased LC3B levels in ILK-knockdown GES-1 cells. The expression levels of both Rac1 and RhoA were downregulated in GES-1 cells after H. pylori infection and were decreased in ILK-knockdown GES-1 cells. The mRNA and protein levels of PAK1, MLC, and LIMK were significantly decreased and cofilin mRNA and protein levels were significantly increased in GES-1 cells treated with the Rac1 inhibitor NSC 23766. The mRNA and protein levels of ROCK1, ROCK2, MLC, and LIMK1 were significantly reduced and cofilin mRNA and protein levels were significantly increased in GES-1 cells treated with the RhoA inhibitor CCG-1423. F-actin was significantly reduced in Rac1- or RhoA-inhibited GES-1 cells. F-actin depolymerization induced autophagosomes accumulation, autolysosomes formation, and the increase of LC3B levels in GES-1 cells. Therefore, these findings revealed that ILK could serve as a novel regulator to affect Rac1/PAK1 and RhoA/ROCKs signaling pathways, thereby influencing H. pylori-induced autophagy.

P21-Activated Kinase 1 Is Permissive for the Skeletal Muscle Hypertrophy Induced by Myostatin Inhibition.

Skeletal muscle, the most abundant tissue in the body, plays vital roles in locomotion and metabolism. Understanding the cellular processes that govern regulation of muscle mass and function represents an essential step in the development of therapeutic strategies for muscular disorders. Myostatin, a member of the TGF-β family, has been identified as a negative regulator of muscle development. Indeed, its inhibition induces an extensive skeletal muscle hypertrophy requiring the activation of Smad 1/5/8 and the Insulin/IGF-I signaling pathway, but whether other molecular mechanisms are involved in this process remains to be determined. Using transcriptomic data from various Myostatin inhibition models, we identified Pak1 as a potential mediator of Myostatin action on skeletal muscle mass. Our results show that muscle PAK1 levels are systematically increased in response to Myostatin inhibition, parallel to skeletal muscle mass, regardless of the Myostatin inhibition model. Using Pak1 knockout mice, we investigated the role of Pak1 in the skeletal muscle hypertrophy induced by different approaches of Myostatin inhibition. Our findings show that Pak1 deletion does not impede the skeletal muscle hypertrophy magnitude in response to Myostatin inhibition. Therefore, Pak1 is permissive for the skeletal muscle mass increase caused by Myostatin inhibition.

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

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

Abstract 14365: Identification of MicroRNAs and Their Target Genes in Microvascular Disease in Diabetes

Cardiovascular disease is the leading cause of death in patients with diabetes. Among coronary vascular defects, obstructive coronary artery disease (CAD) is the primary cause of cardiac mortality. However, there is growing evidence that patients with coronary microvascular disease (CMD, also known as non-obstructive coronary disease) also experience chest pain and occasional heart attack. Furthermore, recent studies show that diabetes is the risk factor of CMD. However, the molecular mechanisms by which diabetes develops CMD is not fully understood. MicroRNAs (miRNAs) are a non-coding small RNA that regulates gene expression at the post-transcriptional level, and the change of miRNA profile is implicated in many diseases. In this study, we identified the core miRNAs which are involved in the development of coronary endothelial dysfunction in diabetes. We used an inducible type 2 diabetic (T2D) mouse model generated by high-fat diet and a single injection of low-dose streptozotocin. Our T2D mice did not show detectable atherosclerotic plaque, but developed CMD evidenced by reduced coronary flow velocity reserve. We conducted a miRNA array using mouse coronary endothelial cells (MCECs) isolated from diabetic and control mice, and selected seven miRNAs based on the result. Next, real-time PCR assays were carried out with seven miRNAs and found that miR210 and miR342-5p were downregulated, and miR378 was upregulated in MCECs of diabetic mice compared to the control. To identify the target genes of miR342-5p and miR378, we inhibited miR342-5p or overexpressed miR378 in MCECs. After testing 93 genes that are involved in endothelial functions, we found that inhibition of miR342-5p decreased the levels of Atp2a3 , Opa1 , Sod3 , and Vegfb , whereas miR378 overexpression significantly decreased Aggf1, HK1, Mapk3, Pak1, Panx1, Stim1, Stim2, Vcam1, and Vegfb and increased Casp2 and Gja1 . Western blotting data revealed that the expression levels of Affg1, Opa1, and Pak1 were significantly decreased in the MCECs from diabetic mice compared to the control. In summary, these data suggest that miR342-5p and miR378 could be a potential therapeutic target for CMD in diabetic patients, and Affg1 Opa1 and Pak1 are the downstream mRNAs of miR342-5p or miR378.

A requirement for PAK1 to support mitochondrial function and maintain cellular redox balance via electron transport chain proteins to prevent β-cell apoptosis

Objectivep21 (Cdc42/Rac1) activated Kinase 1 (PAK1) is a candidate susceptibility factor for type 2 diabetes (T2D). PAK1 is depleted in the islets from T2D donors, compared to control individuals. In addition, whole-body PAK1 knock out (PAK1-KO) in mice worsens the T2D-like effects of high-fat diet. The current study tested the effects of modulating PAK1 levels only in β-cells. Materials/methodsβ-cell-specific inducible PAK1 KO (βPAK1-iKO) mice were generated and used with human β-cells and T2D islets to evaluate β-cell function. ResultsβPAK1-iKO mice exhibited glucose intolerance and elevated β-cell apoptosis, but without peripheral insulin resistance. β-cells from βPAK-iKO mice also contained fewer mitochondria per cell. At the cellular level, human PAK1-deficient β-cells showed blunted glucose-stimulated insulin secretion and reduced mitochondrial function. Mitochondria from human PAK1-deficient β-cells were deficient in the electron transport chain (ETC) subunits CI, CIII, and CIV; NDUFA12, a CI complex protein, was identified as a novel PAK1 binding partner, and was significantly reduced with PAK1 knockdown. PAK1 knockdown disrupted the NAD+/NADH and NADP+/NADPH ratios, and elevated ROS. An imbalance of the redox state due to mitochondrial dysfunction leads to ER stress in β-cells. PAK1 replenishment in the β-cells of T2D human islets ameliorated levels of ER stress markers. ConclusionsThese findings support a protective function for PAK1 in β-cells. The results support a new model whereby the PAK1 in the β-cell plays a required role upstream of mitochondrial function, via maintaining ETC protein levels and averting stress-induced β-cell apoptosis to retain healthy functional β-cell mass.

The Expression of Paks and Its Clinical Significance in T-Cell Lymphoblastic Lymphoma

Background: T-cell lymphoblastic lymphoma (T-LBL) is a highly aggressive non-Hodgkin's lymphoma with poor prognosis and lacks of standard treatment approaches. PAK2, a member of the p21 activated kinase (PAKs) family, is a component of the gene-expression-based classifier which made great contributions to prognostic prediction of T-LBL(Cai et al. Leukemia 2020). This study aims to analyze the expression of PAKs in human T-LBL cell lines and tissues to clarify its clinical significance and evaluate the therapeutic activity of PAKs inhibitor in T-LBL. Methods: The mRNA and protein expression levels of PAKs in human T-lymphoid cell lines (H9) and T-LBL cell lines (Jurkat, SUP-T1 and CCRF-CEM) were detected by quantitative real-time PCR and western blot, respectively. We retrospectively collected 69 Formalin-fixed, Paraffin-embedded (FFPE) samples and corresponding clinical information from T -LBL patients between September 2000 and May 2015 at Sun Yat-sen University Cancer Center (SYSUCC). Affymetrix Human Gene 2.0 ST microarray (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the expression of PAKs. Mann-Whitney U test was used for comparing the expression differences between relapsed and non-relapsed patients. Cumulative relapse-free survival (RFS) time was calculated using the Kaplan-Meier method (Expression level higher than the upper quartile (P75) was defined as PAK1 or PAK 2 high expression. High expression (median as cutoff point) of both PAK1 and PAK2 was defined as PAK1/2 high expression). Pearson's chi-square test and Fisher's exact test were used to compare the distribution of clinical variables. Correlations between PAK1, PAK2 and NOTCH1 were evaluated using Spearman's correlation coefficient. Two PAK inhibitors, PF3758309 (PF) and FRAX597, were used to block PAK kinase activity pharmacologically. Cell viability was determined using the viability assay kit CCK-8. Cell cycle and cell apoptosis were analyzed by flow cytometry. All statistical analyses were performed using SPSS 24.0 software (SPSS, Armonk, NY, USA) or GraphPad Prism 8.0 (GraphPad, La Jolla, CA, USA). A P value &amp;lt; 0.05 was considered significant. The study protocol was approved by the Institutional Review Board of SYSUCC. Results: The mRNA and protein expression levels of PAK1 in T-LBL cell lines of Jurkat, SUP-T1 and CCRF-CEM cell lines were significantly higher than those in T-lymphoid cell lines H9 cell line (P&amp;lt;0.05) (Figure 1). Of the 69 T-LBL patients, 44 (63.8%) were male and the median age at diagnosis was 30 years (range: 16-44). The majority (72.5%) of the patients received acute lymphoblastic leukemia (ALL)-type chemotherapy regimens. The PAK2 mRNA expression level of 32 patients with relapsed disease was significantly higher than that of 37 cases without relapse (P=0.012), and no difference was found in mRNA expression of PAK1, 3, 4, 7(Figure 2). Patients with high PAK1 and PAK2 expression had significantly shorter median RFS than those with low PAK1 and PAK2 expression (PAK1 mRFS 31.5 months vs not reached(NR), HR=3.001, P=0.028; PAK2 mRFS 25.7 months vs NR, HR=3.981, P=0.027)(Figure 3). Patients with high PAK1/2 mRNA expression experienced earlier relapse than patients with low PAK1/2 mRNA expression (mRFS 26.0 months vs NR, HR=2.721, P=0.032)(Figure 3). PAK1/2 mRNA expression was found to be associated with hemoglobin concentration, Ki-67 expression, pleural and pericardia effusion, bone marrow involvement and chemotherapy response (P&amp;lt;0.05)(Table 1). Further, the PAK1 mRNA was correlated with the expression of NOTCH1, which is frequently mutated in T-LBL (r=0.5716, P&amp;lt;0.0001)(Figure 4). The PAK inhibitors PF and FRAX597 demonstrated strong anti-tumor activity in vitro (Table 2). Both inhibitors suppressed cell proliferation in a time- and dose-dependent manner (Figure 5). Both inhibitors induced cell cycle arrest in the G1/0 phase, accompanied by corresponding S phase reductions in all tested cells(Figure 6). The synergistic effect between PAK inhibitor PF and doxorubicin was also observed (Figure 7). Besides, PF could inhibit the phosphorylation of PAK1/2, Cyclin D1 and NF-κB in Jurkat cell line(Figure 8). Conclusions: PAK1 and PAK2 play certain roles in the occurrence and recurrence of T-LBL, and their potential as novel biomarkers deserves further exploring. Our results underscore the potential of PAK inhibitor as effective target therapy for T-LBL. Disclosures No relevant conflicts of interest to declare.

Knockdown of RhoGDI2 represses human gastric cancer cell proliferation, invasion and drug resistance via the Rac1/Pak1/LIMK1 pathway.

Gastric cancer (GC) is the fifth most common primary malignancy in humans. Rho GDP dissociation inhibitor 2 (RhoGDI2) is overexpressed in multiple cancer types, but the role of RhoGDI2 in GC has not been elucidated. This study aims to determine the level of RhoGDI2 in GC and to confirm the effect of its inhibition or overexpression on GC cell migration, invasion and chemosensitivity. RhoGDI2 level is significantly enhanced in human GC tissue samples in comparison with normal gastric epithelium and corresponding para-cancerous samples. The expression of RhoGDI2 is correlated with clinicopathological parameters and prognosis. Transfection in combination with miRNA targeting of RhoGDI2 in GC cell lines remarkably downregulates GC cell migration and invasion and reduces the mRNA levels of Rac1, Pak1 and LIMK1. The inhibition of RhoGDI2 downregulates GC cell migration and invasion by attenuating the EMT cascade via the Rac1/Pak1/LIMK1 pathway. Knockdown of RhoGDI2 is a potential therapeutic strategy for GC.

Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

ABSTRACT Although the treatment of brain tumors by targeting kinase-regulated macroautophagy/autophagy, is under investigation, the precise mechanism underlying autophagy initiation and its significance in glioblastoma (GBM) remains to be defined. Here, we report that PAK1 (p21 [RAC1] activated kinase 1) is significantly upregulated and promotes GBM development. The Cancer Genome Atlas analysis suggests that the oncogenic role of PAK1 in GBM is mainly associated with autophagy. Subsequent experiments demonstrate that PAK1 indeed serves as a positive modulator for hypoxia-induced autophagy in GBM. Mechanistically, hypoxia induces ELP3-mediated PAK1 acetylation at K420, which suppresses the dimerization of PAK1 and enhances its activity, thereby leading to subsequent PAK1-mediated ATG5 (autophagy related 5) phosphorylation at the T101 residue. This event not only protects ATG5 from ubiquitination-dependent degradation but also increases the affinity between the ATG12–ATG5 complex and ATG16L1 (autophagy related 16 like 1). Consequently, ELP3-dependent PAK1 (K420) acetylation and PAK1-mediated ATG5 (T101) phosphorylation are required for hypoxia-induced autophagy and brain tumorigenesis by promoting autophagosome formation. Silencing PAK1 with shRNA or small molecule inhibitor FRAX597 potentially blocks autophagy and GBM growth. Furthermore, SIRT1-mediated PAK1-deacetylation at K420 hinders autophagy and GBM growth. Clinically, the levels of PAK1 (K420) acetylation significantly correlate with the expression of ATG5 (T101) phosphorylation in GBM patients. Together, this report uncovers that the acetylation modification and kinase activity of PAK1 plays an instrumental role in hypoxia-induced autophagy initiation and maintaining GBM growth. Therefore, PAK1 and its regulator in the autophagy pathway might represent potential therapeutic targets for GBM treatment. Abbreviations: 3-MA: 3-methyladenine; Ac-CoA: acetyl coenzyme A; ATG5: autophagy related 5; ATG16L1, autophagy related 16 like 1; BafA1: bafilomycin A1; CDC42: cell division cycle 42; CGGA: Chinese Glioma Genome Atlas; CHX, cycloheximide; ELP3: elongator acetyltransferase complex subunit 3; GBM, glioblastoma; HBSS: Hanks balanced salts solution; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K1: mitogen-activated protein kinase kinase 1; MAPK14, mitogen-activated protein kinase 14; PAK1: p21 (RAC1) activated kinase 1; PDK1: pyruvate dehydrogenase kinase 1; PGK1, phosphoglycerate kinase 1; PTMs: post-translational modifications; RAC1: Rac family small GTPase 1; SQSTM1: sequestosome 1; TCGA, The Cancer Genome Atlas.

Systemic analysis of the expression and prognostic significance of PAKs in breast cancer

PAKs (p21-activated kinases) are reported to play crucial roles in a variety of cellular processes and participate in the progression of human cancers. However, the expression and prognostic values of PAKs remain poorly explored in breast cancers. In our study, we examined the mRNA and protein expression levels of PAKs and the prognostic value. We also analyzed the interaction network, genetic alteration, and functional enrichment of PAKs. The results showed that the mRNA levels of PAK1, PAK2, PAK4 and PAK6 were significantly up-regulated in breast cancer compared with normal tissues, while the reverse trend for PAK3 and PAK5 was found, furthermore, the proteins expression of PAK1, PAK2 and PAK4 in breast cancer tissues were higher than that in normal breast tissues. Survival analysis revealed breast cancer patients with low mRNA expression of PAK3 and PAK5 showed worse RFS, conversely, elevated PAK4 levels predicted worse RFS. In addition, the breast cancer patients with PAKs genetic alterations correlated with worse OS. These results indicated that PAKs might be promising potential biomarkers for breast cancer.

The lncRNA FAL1 protects against hypoxia-reoxygenation- induced brain endothelial damages through regulating PAK1.

Dysregulation of cerebral microvascular endothelial cells plays an important role in the pathogenesis of stroke. However, the underlying mechanisms still need to be elucidated. In the current study, we found that the long non-coding RNA (lncRNA) FAL1 was significantly reduced in response to oxygen-glucose deprivation and reoxygenation (OGD/R) stimulation in human primary brain microvascular endothelial cells (HBMVECs). Interestingly, overexpression of FAL1 ameliorated OGD/R-induced oxidative stress by reducing the production of reactive oxygen species (ROS) and increasing the level of reduced glutathione (GSH). Also, overexpression of FAL1 suppressed OGD/R-induced secretions of interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and high mobility group box-1 (HMGB-1). We then found that OGD/R-induced reduction of cell viability and release of lactate dehydrogenase (LDH) were prevented by overexpression of FAL1. Additionally, exposure to OGD/R significantly reduced the phosphorylated levels of PAK1 and AKT as well as the total level of proliferating cell nuclear antigen (PCNA), which was restored by overexpression of FAL1. Importantly, overexpression of FAL1 restored OGD/R-induced reduction in the expression of endothelial nitric oxide synthase (eNOS) and the subsequent release of nitric oxide (NO). Our results implicate that FAL1 might be involved in the process of brain endothelial cell damage.

LINC00460 promotes hepatocellular carcinoma development through sponging miR-485-5p to up-regulate PAK1

LncRNAs can function as significant regulators of tumor development. However, their roles in hepatocellular carcinoma (HCC) remain poorly investigated. LINC00460 has been identified in several cancers, which can act as an oncogene. In this study, we observed that LINC00460 was significantly up-regulated in HCC cells, which implied that LINC00460 was involved in HCC development. Then, LINC00460 was silenced in Hep3B and Huh-7 cells and we found that knockdown of LINC00460 greatly inhibited HCC cell proliferation. In addition, HCC cell apoptosis was induced and meanwhile, cell cycle progression was blocked by down-regulation of LINC00460 in vitro. Furthermore, we proved that Hep3B and Huh-7 cell migration and invasion capacity was repressed by decrease of LINC00460. Recently, a growing number of studies have indicated the correlation between lncRNAs and microRNAs. Currently, we displayed that miR-485-5p was greatly decreased in HCC cells and LINC00460 could sponge miR-485-5p to regulate HCC progression. The binding association between LINC00460 and miR-485-5p was confirmed using dual-luciferase reporter assay, RNA pulled down and RIP assay in our research. Subsequently, PAK1 was predicted as a downstream target of miR-485-5p and we demonstrated that miR-485-5p suppressed PAK1 levels in vitro. Finally, in vivo experiments were conducted to validate that knockdown of LINC00460 repressed HCC development through modulating miR-485-5p to increase PAK1. Taken these together, we indicated that LINC00460 promoted HCC progression through sponging miR-485-5p and up-regulating PAK1.

MiR-29a inhibits cell proliferation and migration by targeting the CDC42/PAK1 signaling pathway in cervical cancer

Cervical cancer is the second most common gynecological malignancy worldwide and the tumorigenesis mechanisms of cervical cancer are still unclear. This study aimed to reveal the role of miR-29a in cervical cancer. The expression level of miR-29a and CDC42 was measured using qRT-PCR. Cell proliferation, apoptosis, migration, and invasion were detected using colony formation, flow cytometry analysis, wound-healing assay, and Transwell assay, respectively. Luciferase reporter assay was used to determine the binding of miR-29a with CDC42. CDC42/p21-activated kinase 1 (PAK1) pathway-related proteins were measured by western blotting. MiR-29a was downregulated and CDC42 was upregulated in cervical cancer cells. Luciferase reporter assay showed that miR-29a negatively regulated the expression of CDC42 by directly targeting 3'-UTR of CDC42. Cell proliferation, migration, and invasion were markedly inhibited, whereas cell apoptosis was significantly increased in Hela and CaSki cells transfected with miR-29a mimics. These effects were partly recovered by CDC42 overexpression. Protein levels of PAK1, p-PAK1, p-LIMK, and p-cofilin were significantly downregulated by miR-29a mimics, which was reversed by CDC42 overexpression and was increased by the miR-29a inhibitor. MiR-29a inhibited cell proliferation, migration, and invasion, as well as promoted cell apoptosis through repressing the PAK1/LIMK signaling pathway by targeting CDC42 in cervical cancer.

Transcriptional regulation of ataxia-telangiectasia and Rad3-related protein by activated p21-activated kinase-1 protects keratinocytes in UV-B-induced premalignant skin lesions.

Sun-induced skin lesions, in particular actinic keratosis, are generally considered as premalignant skin lesions that can progress into squamous cell carcinoma (SCC) and invasive SCC if left untreated. Therefore, understanding the molecular mechanisms by which the ultraviolet-B (UV-B)-exposed cells are being protected and the signaling pathways that promote the progression of certain premalignant skin lesions to malignant lesions will permit us to prevent or cure skin cancers. In the current study, we found that phospho-p21-activated kinase-1 (Pak1) and Pak1 expression was high in clinical samples of sunlight-induced premalignant skin lesions assessed by immunohistochemistry. Further, we observed that phospho-Pak1 and Pak1 levels are high in UV-B-exposed hairless SKH mouse model skin samples as compared with unexposed skin tissue. Our results from cell line and animal models showed that Pak1 is activated in response to UV-B radiation, and this activated Pak1 translocates from the cytoplasm to the nucleus. Inside the nucleus, Pak1 via C-Fos binds to a specific promoter region of DNA repair kinase ATR (ataxia-telangiectasia and Rad3-related protein) and acts as a transcriptional regulator of ATR. Results from our analysis showed that Pak1 overexpression, knockdown and Pak1 knockout cell line models showed that Pak1 confers protection to keratinocytes from UV-B-induced apoptosis and DNA damage via ATR. To our knowledge, this is the first study that evaluates the functional and clinical significance of a signaling molecule, Pak1, in sun-induced premalignant skin lesions and indicates that increased Pak1 activation and expression could serve as an early warning sign of progression toward non-melanoma skin cancer, if ignored.

P21-activated kinase 1 regulates resistance to BRAF inhibition in human cancer cells.

BRAF is a commonly mutated oncogene in various human malignancies and a target of a new class of anti-cancer agents, BRAF-inhibitors (BRAFi). The initial enthusiasm for these agents, based on the early successes in the management of metastatic melanoma, is now challenged by the mounting evidence of intrinsic BRAFi-insensitivity in many BRAF-mutated tumors, by the scarcity of complete responses, and by the inevitable emergence of drug resistance in initially responsive cases. These setbacks put an emphasis on discovering the means to increase the efficacy of BRAFi and to prevent or overcome BRAFi-resistance. We explored the role of p21-activated kinases (PAKs), in particular PAK1, in BRAFi response. BRAFi lowered the levels of active PAK1 in treated cells. An activated form of PAK1 conferred BRAFi-resistance on otherwise sensitive cells, while genetic or pharmacologic suppression of PAK1 had a sensitizing effect. While activation of AKT1 and RAC1 proto-oncogenes increased BRAFi-tolerance, the protective effect was negated in the presence of PAK inhibitors. Furthermore, combining otherwise ineffective doses of PAK- and BRAF-inhibitors synergistically affected intrinsically BRAFi-resistant cells. Considering the high incidence of PAK1 activation in cancers, our findings suggests PAK inhibition as a strategy to augment BRAFi therapy and overcome some of the well-known resistance mechanisms.