P21-activated Protein Kinase Research Articles

MiR-216a-5p act as a tumor suppressor, regulating the cell proliferation and metastasis by targeting PAK2 in breast cancer.

Our study aimed to investigate the expression of microRNA-216a-5p (miR-216a-5p) in breast cancer (BC) and its effect on the proliferation and metastasis of BC cells by regulating the expression of p21-activated protein kinase 2 (PAK2) gene. A total of 50 cases of cancer tissue specimens and corresponding para-carcinoma normal tissue specimens were collected from the breast surgery department of our hospital from July 2016 to December 2017. BC MCF-7 cell line and normal breast epithelial MCF-10A cells were cultured. MiR-NC (negative control), LV-p21-activated protein kinase 2 (PAK2) and/or miR-216a-5p mimics were synthesized and transfected. The protein and mRNA expression level in BC tissues and cells were detected by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay, respectively. Additionally, the Luciferase Reporter Assays, cell proliferation detection, clone formation assays and transwell migration and invasion assay were performed to determine the functional alteration of BC cells, respectively. The results of qRT-PCR demonstrated that miR-216a-5p was decreased in both BC tissues and cells compared with that in normal controls. Online target gene prediction software and Dual-Luciferase reporter assay were used for target identification, and PAK2 was identified as a functional target of miR-216a-5p in BC cells. The results were further clarified with the Western blot (WB) experiment. In vitro, cell functions were detected by Cell Counting Kit-8 (CCK-8), crystal violet staining and transwell experiment, respectively. The results indicated that decreased expression of PAK2 resulting from the up-regulation of miR-216a-5p could restrain the proliferation, clone formation, invasion and migration abilities of BC cells. We showed that miR-216a-5p played a role as antioncogene in BC, which provides a new therapeutic target for the treatment of BC.

Porcine Hemagglutinating Encephalomyelitis Virus Activation of the Integrin α5β1-FAK-Cofilin Pathway Causes Cytoskeletal Rearrangement To Promote Its Invasion of N2a Cells.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic virus that causes diffuse neuronal infection with neurological damage and high mortality. Virus-induced cytoskeletal dynamics are thought to be closely related to this type of nerve damage. Currently, the regulation pattern of the actin cytoskeleton and its molecular mechanism remain unclear when PHEV enters the host cells. Here, we demonstrate that entry of PHEV into N2a cells induces a biphasic remodeling of the actin cytoskeleton and a dynamic change in cofilin activity. Viral entry is affected by the disruption of actin kinetics or alteration of cofilin activity. PHEV binds to integrin α5β1 and then initiates the integrin α5β1-FAK signaling pathway, leading to virus-induced early cofilin phosphorylation and F-actin polymerization. Additionally, Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division cycle 42 (Cdc42), and downstream regulatory gene p21-activated protein kinases (PAKs) are recruited as downstream mediators of PHEV-induced dynamic changes of the cofilin activity pathway. In conclusion, we demonstrate that PHEV utilizes the integrin α5β1-FAK-Rac1/Cdc42-PAK-LIMK-cofilin pathway to cause an actin cytoskeletal rearrangement to promote its own invasion, providing theoretical support for the development of PHEV pathogenic mechanisms and new antiviral targets.IMPORTANCE PHEV, a member of the Coronaviridae family, is a typical neurotropic virus that primarily affects the nervous system of piglets to produce typical neurological symptoms. However, the mechanism of nerve damage caused by the virus has not been fully elucidated. Actin is an important component of the cytoskeleton of eukaryotic cells and serves as the first obstacle to the entry of pathogens into host cells. Additionally, the morphological structure and function of nerve cells depend on the dynamic regulation of the actin skeleton. Therefore, exploring the mechanism of neuronal injury induced by PHEV from the perspective of the actin cytoskeleton not only helps elucidate the pathogenesis of PHEV but also provides a theoretical basis for the search for new antiviral targets. This is the first report to define a mechanistic link between alterations in signaling from cytoskeleton pathways and the mechanism of PHEV invading nerve cells.

Insulin-stimulated Rac1-GTP binding is not impaired by palmitate treatment in L6 myotubes.

Ras‐related C3 botulinum toxin substrate 1 (Rac1) is required for normal insulin‐stimulated glucose transport in skeletal muscle and evidence indicates Rac1 may be negatively regulated by lipids. We investigated if insulin‐stimulated activation of Rac1 (i.e., Rac1‐GTP binding) is impaired by accumulation of diacylglycerols (DAG) and ceramides in cultured muscle cells. Treating L6 myotubes with 100 nmol/L insulin resulted in increased Rac1‐GTP binding that was rapid (occurring within 2 min), relatively modest (+38 ± 19% vs. basal, P < 0.001), and short‐lived, returning to near‐basal levels within 15 min of continuous treatment. Incubating L6 myotubes overnight in 500 μmol/L palmitate increased the accumulation of DAG and ceramides (P < 0.05 vs. no fatty acid control). Despite significant accumulation of lipids, insulin‐stimulated Rac1‐GTP binding was not impaired during palmitate treatment (P = 0.39 vs. no fatty acid control). Nevertheless, phosphorylation of Rac1 effector protein p21‐activated kinase (PAK) was attenuated in response to palmitate treatment (P = 0.02 vs. no fatty acid control). Palmitate treatment also increased inhibitory phosphorylation of insulin receptor substrate‐1 and attenuated insulin‐stimulated phosphorylation of Akt at both Thr308 and Ser473 (all P < 0.05 vs. no fatty acid control). Such signaling impairments resulted in near complete inhibition of insulin‐stimulated translocation of glucose transporter protein 4 (GLUT4; P = 0.10 vs. basal during palmitate treatment). In summary, our finding suggests that Rac1 may not undergo negative regulation by DAG or ceramides. We instead provide evidence that attenuated PAK phosphorylation and impaired GLUT4 translocation during palmitate‐induced insulin resistance can occur independent of defects in insulin‐stimulated Rac1‐GTP binding.

Abstract 3345: MAZ is a downstream target of CCN1 and promotes aggressive behavior of pancreatic cancer cells via CRAF-ERK signaling pathway

Abstract MAZ (Myc-associated zinc-finger protein) is a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression has been shown to associate with the progression of pancreatic ductal adenocarcinoma cancer (PDAC). However, underlying mechanisms of action of MAZ in PDAC progression are largely unknown. Here we present evidence that MAZ mRNA expression and protein level were increased in human PDAC cell lines, tissue samples, subcutaneous tumor xenograft model in a nude mouse, and spontaneous cancer in the genetically engineered PDAC mouse model. We found that MAZ is predominantly expressed in pancreatic cancer stem cells. Functional analysis demonstrates that the depletion of MAZ in PDAC cells results in inhibition of invasive phenotypes such as phenotypic shift, migration, invasion, and the sphere-forming ability of PDAC cells. Mechanistically, we found no direct effect of MAZ on the expression of K-Ras mutants, but evidently MAZ increases the activity of CRAF-ERK-signaling, which is a downstream signaling target of K-Ras. Activation of CRAF-ERK-signaling by MAZ is mediated via p21-activated protein kinases (PAK) and protein kinase B (AKT/PKB) signaling cascades and promotes invasive phenotypes of PDAC cells. Moreover, a matricellular oncoprotein CCN1 regulates MAZ expression in PDAC cells. Thereby, we proposed that CCN1-induced expression of MAZ promotes invasive phenotypes of PDAC cells is not through direct K-Ras activation as presently thought but instead through the activation of CRAF-ERK signals. Collectively, these studies highlight key players that could help in clinical management, prognosis, and therapeutic strategies. This project is funded by VA Merit Award grants (SB &amp; SKB). Citation Format: Gargi Maity, Inamul Haque, Arnab Ghosh, Gopal Dhar, Vijayalaxmi Gupta, Sandipto Sarkar, Imaan Azeem, Douglas McGregor, Abhishek Choudhary, Donald R Campbell, Sushanta K. Banerjee, Snigdha Banerjee. MAZ is a downstream target of CCN1 and promotes aggressive behavior of pancreatic cancer cells via CRAF-ERK signaling pathway [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 3345.

The MAZ transcription factor is a downstream target of the oncoprotein Cyr61/CCN1 and promotes pancreatic cancer cell invasion via CRAF–ERK signaling

Myc-associated zinc-finger protein (MAZ) is a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression is associated with the progression of pancreatic ductal adenocarcinoma (PDAC). However, the mechanism of action of MAZ in PDAC progression is largely unknown. Here, we present evidence that MAZ mRNA expression and protein levels are increased in human PDAC cell lines, tissue samples, a subcutaneous tumor xenograft in a nude mouse model, and spontaneous cancer in the genetically engineered PDAC mouse model. We also found that MAZ is predominantly expressed in pancreatic cancer stem cells. Functional analysis indicated that MAZ depletion in PDAC cells inhibits invasive phenotypes such as the epithelial-to-mesenchymal transition, migration, invasion, and the sphere-forming ability of PDAC cells. Mechanistically, we detected no direct effects of MAZ on the expression of K-Ras mutants, but MAZ increased the activity of CRAF-ERK signaling, a downstream signaling target of K-Ras. The MAZ-induced activation of CRAF-ERK signaling was mediated via p21-activated protein kinase (PAK) and protein kinase B (AKT/PKB) signaling cascades and promoted PDAC cell invasiveness. Moreover, we found that the matricellular oncoprotein cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) regulates MAZ expression via Notch-1-sonic hedgehog signaling in PDAC cells. We propose that Cyr61/CCN1-induced expression of MAZ promotes invasive phenotypes of PDAC cells not through direct K-Ras activation but instead through the activation of CRAF-ERK signaling. Collectively, these results highlight key molecular players in PDAC invasiveness and may help inform therapeutic strategies to improve clinical management and outcomes of PDAC.

Development of anti-fungal pesticides from protein kinase inhibitor-based anticancer agents

Repurposing the novel p21-activated protein kinase inhibitor compound 15 identified its antifungal activity against five selected species of phytopathogenic fungi. Lead optimization based on its structure gave rise to a focused library of 20 derivatives, among which compound 3c demonstrated increased activity over compound 15 and even comparable to that of some commercialized fungicides in the market including carbendazim, tebuconazole, and pyraclostrobin. This study showed that p21-activated protein kinase inhibitor compound 15 was able to serve as a molecular platform to develop effective fungicides against fungal phytopathogens and indicate that screening existing protein kinase inhibitors might be an effective way to identify lead compounds for antifungal pesticides development.

Staphylococcus aureus α-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells.

Exposure of cultured human airway epithelial model cells (16HBE14o-, S9) to Staphylococcus aureus α-toxin (hemolysin A, Hla) induces changes in cell morphology and cell layer integrity that are due to the inability of the cells to maintain stable cell-cell or focal contacts and to properly organize their actin cytoskeletons. The aim of this study was to identify Hla-activated signaling pathways involved in regulating the phosphorylation level of the actin-depolymerizing factor cofilin. We used recombinant wild-type hemolysin A (rHla) and a variant of Hla (rHla-H35L) that is unable to form functional transmembrane pores to treat immortalized human airway epithelial cells (16HBE14o-, S9) as well as freshly isolated human nasal tissue. Our results indicate that rHla-mediated changes in cofilin phosphorylation require the formation of functional Hla pores in the host cell membrane. Formation of functional transmembrane pores induced hypophosphorylation of cofilin at Ser3, which was mediated by rHla-induced attenuation of p21-activated protein kinase and LIM kinase activities. Because dephosphorylation of pSer3-cofilin results in activation of this actin-depolymerizing factor, treatment of cells with rHla resulted in loss of actin stress fibers from the cells and destabilization of cell shape followed by the appearance of paracellular gaps in the cell layers. Activation of protein kinase A or activation of small GTPases (Rho, Rac, Cdc42) do not seem to be involved in this response.

Striatal-enriched Tyrosine Protein Phosphatase (STEP) in the Mechanisms of Depressive Disorders.

Striatal-enriched tyrosine protein phosphatase (STEP) is expressed mainly in the brain. Its dysregulation is associated with Alzheimer's and Huntington's diseases, schizophrenia, fragile X syndrome, drug abuse and stroke/ischemia. However, an association between STEP and depressive disorders is still obscure. The review discusses the theoretical foundations and experimental facts concerning possible relationship between STEP dysregulation and depression risk. STEP dephosphorylates and inactivates several key neuronal signaling proteins such as extracellular signal-regulating kinase 1 and 2 (ERK1/2), stress activated protein kinases p38, the Src family tyrosine kinases Fyn, Pyk2, NMDA and AMPA glutamate receptors. The inactivation of these proteins decreases the expression of brain derived neurotrophic factor (BDNF) necessary for neurogenesis and neuronal survival. The deficit of BDNF results in progressive degeneration of neurons in the hippocampus and cortex and increases depression risk. At the same time, a STEP inhibitor, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153), increases BDNF expression in the hippocampus and attenuated the depressivelike behavior in mice. Thus, STEP is involved in the mechanism of depressive disorders and it is a promising molecular target for atypical antidepressant drugs of new generation.

Pak1 mediates the stimulatory effect of insulin and curcumin on hepatic ChREBP expression.

Insulin can stimulate hepatic expression of carbohydrate-responsive element-binding protein (ChREBP). As recent studies revealed potential metabolic beneficial effects of ChREBP, we asked whether its expression can also be regulated by the dietary polyphenol curcumin. We also aimed to determine mechanisms underlying ChREBP stimulation by insulin and curcumin. The effect of insulin on ChREBP expression was assessed in mouse hepatocytes, while the effect of curcumin was assessed in mouse hepatocytes and with curcumin gavage in mice. Chemical inhibitors for insulin signaling molecules were utilized to identify involved signaling molecules, and the involvement of p21-activated protein kinase 1 (Pak1) was determined with its chemical inhibitor and Pak1-/- hepatocytes. We found that both insulin and curcumin-stimulated ChREBP expression in Akt-independent but MEK/ERK-dependent manner, involving the inactivation of the transcriptional repressor Oct-1. Aged Pak1-/- mice showed reduced body fat volume. Pak1 inhibition or its genetic deletion attenuated the stimulatory effect of insulin or curcumin on ChREBP expression. Our study hence suggests the existence of a novel signaling cascade Pak1/MEK/ERK/Oct-1 for both insulin and curcumin in exerting their glucose-lowering effect via promoting hepatic ChREBP production, supports the recognition of beneficial functions of ChREBP, and brings us a new overview on dietary polyphenols.

Salinity stress, enhancing basal and induced immune responses in striped catfish Pangasianodon hypophthalmus (Sauvage)

In the Mekong Delta, striped catfish are faced with chronic salinity stress related to saltwater intrusion induced by global climatic changes. In this study, striped catfish juveniles were submitted to a prolonged salinity stress (up to 10ppt) over three weeks followed by infection with a virulent bacterial strain, Edwardsiella ictaluri. Osmoregulatory parameters were investigated. In addition, a label free quantitative proteomics workflow was performed on kidneys. The workflow consisted of an initial global profiling of relative peptide abundances (by LC/MS, peak area quantification based on extracted ion currents), followed by identification (by MS/MS). The aim of the study was to highlight specific functional pathways modified during realistic salinity stress, particularly those involved in immunity. In kidney proteome, 2483 proteins were identified, of which 400 proteins were differentially expressed between the freshwater and the saline water conditions. Several pathways and functional categories were highlighted, mostly related to energy metabolism, protein metabolism, actin cytoskeleton, signaling, immunity, and detoxification. In particular, the responsiveness of proteins involved in small GTPases and Mitogen Activated Protein Kinase p38 signaling, phagolysosome maturation, and T-cells regulation is discussed. Statement of significanceIn the Mekong River Delta (Vietnam), striped catfish production is threatened by extensive sea water intrusion exacerbated by sea level rise. In fish, the effect of chronic exposure to salinity stress on immune capacities and response to disease has been poorly investigated. This study aims to highlight the main molecular changes occurring in the kidney during acclimation to salinity stress, particularly those involved in the immune defences of fish.

Carbon Monoxide–Releasing Molecule-401 Suppresses Polymorphonuclear Leukocyte Migratory Potential by Modulating F-Actin Dynamics

PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMNwith CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P<0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distributionand fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P<0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophilmigratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics.

Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK.

Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK.

P21-activated protein kinase 1 induces the invasion of gastric cancer cells through c-Jun NH2-terminal kinase-mediated activation of matrix metalloproteinase-2.

Gastric cancer(GC) is one of the most common malignancies worldwide. The prognosis of GC is poor, mostly due to widespread metastasis. p21-activated kinase1 (Pak1), the best characterized member of an evolutionarily conserved family of serine/threonine kinases, plays an important role in the regulation of cell morphogenesis, motility, mitosis and angiogenesis. By qRT-PCR and Gelatin zymograph assay, we demonstrated in the present study that stable overexpression of Pak1 induced matrix metalloproteinase (MMP)-2 mRNA expression and activity in the human MKN45 GC cell line. Conversely, knockdown of endogenous Pak1 expression by small interfering RNA (siRNA) decreased MMP-2 mRNA expression and activity in the MKN45 GC cells. Activation of c-Jun N-terminal kinase (JNK) was required for Pak1-induced upregulation of MMP-2 mRNA level and activity. Moreover, upregulation of MMP-2 by Pak1 via the JNK pathway notably promoted the invasion of MKN45 GC cells. Overexpression of MMP-2 mRNA was once again confirmed to be associated with GC metastasis. In conclusion, our results demonstrated for the first time that Pak1 stimulated MMP-2 mRNA expression and activity in MKN45 GC cells. The JNK signaling pathway was involved in Pak1 modulation of MMP-2, which was important for MKN45 GC cell invasiveness.

A fucose specific lectin from Aspergillus flavus induced interleukin-8 expression is mediated by mitogen activated protein kinase p38.

Aspergillus flavus is an ubiquitous, opportunistic fungus responsible to cause invasive fungal allergic diseases, including bronchopulmonary invasive aspergillosis in persons with altered immune function. Lectins have been implicated as interaction mediators between the pathogenic fungi and human host. We isolated L-fucose specific lectin from A. flavus (FFL) and purified it to homogeneity with a combination of ion exchange and hydrophobic interaction chromatography methods. Its hemagglutination activity was significantly inhibited by 125 μM L-fucose as compared to other sugars and sugar derivatives. We, then used human cell line L-132, and U937 cell line to explore the possible cytotoxicity and proinflammatory effect of this fucose-specific lectin. The lectin induced the expression of proinflammatory cytokine interleukin-8 (IL-8) in a dose-dependent manner, and it was found to be associated with the p38 mitogen activated protein kinase (MAPK). The p38MAPK signalling pathway regulates the transcription factor activator protein-1 (AP-1) activity, which is the integration point of many signals that can differentially affect the expression and transcriptional activity of a cell. We observed activation of c-Jun, a critical component of the AP-1 complex, mediated by p38MAPK upon the FFL treatment in L-132 cells. Finally, inhibition of p38MAPK by a specific inhibitor attenuates the c-Jun, suggesting the p38MAPK involvement in the c-Jun activation, which in turn transcriptionally activates the induction of IL-8 in response to the lectin. Thus, this study showed a potential lectin-mediated mechanism to modulate the immune response during host-fungus interactions.

Rac1/p21-activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes.

Small GTPases of the Ras superfamily are capable of activating E2F-dependent transcription leading to cell proliferation, but the molecular mechanisms are poorly understood. In this study, using immortalized chicken DT40 B cell lines to investigate the role of the Vav/Rac signalling cascade on B cell proliferation, it is shown that the proliferative response triggered by B cell receptor activation is dramatically reduced in the absence of Vav3 expression. Analysis of this proliferative defect shows that in the absence of Vav3 expression, retinoblastoma protein (RB) phosphorylation and the subsequent E2F activation do not take place. By combining pharmacological and genetic approaches, phosphatidylinositol-3-kinase and phospholipase Cγ2 (PLCγ2) were identified as the key regulatory signalling molecules upstream of the Vav3/Rac pathway leading to RB phosphorylation and E2F transcription factor activation. Additionally, vav3(-/-) and plcγ2(-/-) DT40 B cells were not able to activate the RB-E2F complex wild-type phenotype when these genetically modified cells were transfected with constitutively active forms of RhoA or Cdc42. However, when these knockout cells were transfected with different constitutively active versions of PLCγ, Vav or Rac1, not only activation of the RB-E2F complex wild-type phenotype was recovered but also the cellular proliferation. Furthermore, by evaluating the effect of two known effector mutants of Rac1 (Rac1(Q61L/F37A) and Rac1(Q61L/Y40C) ), the RB-E2F complex activation dependency on p21-activated kinase (PAK) and protein kinase Cε (PKCε) activities was established, being independent of both actin cytoskeleton reorganization and Ras activity. These results suggest that PAK1 and PKCε may be potential therapeutic targets to stop uncontrolled B cell proliferation mediated by the Vav/Rac pathway.

Macrocyclic Lactones Block Melanoma Growth, Metastases Development and Potentiate Activity of Anti–BRAF V600 Inhibitors

IntroductionMelanoma is the most aggressive form of skin cancer. Targeted antimelanoma therapies such as BRAF V600 inhibitors have shown spectacular results. However, these therapies are restricted to BRAF V600 mutated melanoma and display both adverse effects and resistance outbreaks. MethodsA high-content screening campaign identified compounds that displayed antimelanoma activities that can potentiate anti–BRAF V600 inhibitors. We identified ivermectin, a US Food and Drug Administration (FDA)-approved macrocyclic lactone widely used as an anthelmintic and insecticidal agent. ResultsMacrocyclic lactones possess in vitro cytotoxicity against either BRAF wild type (wt) or BRAF V600 mutated melanoma cells lines. Daily intraperitoneal injections of ivermectin strongly reduce pulmonary metastasis implantation in vivo in murine and melanoma models. Interestingly, these macrocyclic lactones are also able to trigger vemurafenib-dependent cytotoxicity in vitro in BRAF-wt melanoma cells, although vemurafenib action was thought to be restricted to melanoma bearing the BRAF V600 mutation. These macrocyclic lactones are also able to increase the in vitro antimelanoma activity of another BRAF V600 clinical inhibitor, dabrafenib. Serine/threonine p21-activated protein kinase 1 (PAK1) is the key target of ivermectin responsible for its antimelanoma activity. ConclusionIvermectin is a promising agent that could strongly increase melanoma therapy efficiency because it is cytotoxic for BRAF-wt and BRAF mutated melanomas and because it increases the efficiency of antimutated BRAF inhibitors independent of the BRAF status of tumors. Because ivermectin is FDA approved and a study of ivermectin would be able to include all patients in the same protocol, this combination treatment should be next investigated in clinical studies.

Infectious Entry Pathway of Enterovirus B Species.

Enterovirus B species (EV-B) are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The biogenesis of neutral multivesicular bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered by the virus binding to their receptors on the plasma membrane, and they are not efficiently recycled like other cellular pathways used by circulating receptors. Therefore, the best “markers” of these pathways may be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of enterovirus uncoating and genome release from the endosomes to start efficient replication.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression and metastasis.

F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.

Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development.