Identification of RSK substrates using an analog-sensitive kinase approach.

The p90 ribosomal S6 kinases (RSK) family of serine/threonine kinases comprises four isoforms (RSK1-4) that lie downstream of the ERK1/2 mitogen-activated protein kinase pathway. RSKs are implicated in fine tuning of cellular processes such as translation, transcription, proliferation, and motility. Previous work showed that pathogens such as Cardioviruses could hijack any of the four RSK isoforms to inhibit PKR activation or to disrupt cellular nucleocytoplasmic trafficking. In contrast, some reports suggest nonredundant functions for distinct RSK isoforms, whereas Coffin-Lowry syndrome has only been associated with mutations in the gene encoding RSK2. In this work, we used the analog-sensitive kinase strategy to ask whether the cellular substrates of distinct RSK isoforms differ. We compared the substrates of two of the most distant RSK isoforms: RSK1 and RSK4. We identified a series of potential substrates for both RSKs in cells and validated RanBP3, PDCD4, IRS2, and ZC3H11A as substrates of both RSK1 and RSK4, and SORBS2 as an RSK1 substrate. In addition, using mutagenesis and inhibitors, we confirmed analog-sensitive kinase data showing that endogenous RSKs phosphorylate TRIM33 at S1119. Our data thus identify a series of potential RSK substrates and suggest that the substrates of RSK1 and RSK4 largely overlap and that the specificity of the various RSK isoforms likely depends on their cell- or tissue-specific expression pattern.

Marine-Based Candidates as Potential RSK1 Inhibitors: A Computational Study.

Manzamines are chemically related compounds extracted from the methanolic extract of Acanthostrongylophora ingens species. Seven compounds were identified by our research group and are being characterized. As their biological target is unknown, this work is based on previous screening work performed by Mayer et al., who revealed that manzamine A could be an inhibitor of RSK1 kinase. Within this work, the RSK1 N-terminal kinase domain is exploited as a target for our work and the seven compounds are docked using Autodock Vina software. The results show that one of the most active compounds, Manzamine A N-oxide (5), with an IC50 = 3.1 µM, displayed the highest docking score. In addition, the compounds with docking scores lower than the co-crystalized ligand AMP-PCP (-7.5 and -8.0 kcal/mol) for ircinial E (1) and nakadomarin A (7) were found to be inferior in activity in the biological assay. The docking results successfully managed to predict the activities of four compounds, and their in silico results were in concordance with their biological data. The ß-carboline ring showed noticeable receptor binding, which could explain its reported biological activities, while the lipophilic side of the compound was found to fit well inside the hydrophobic active site.

RSK1 vs. RSK2 Inhibitory Activity of the Marine ß-Carboline Alkaloid Manzamine A: A Biochemical, Cervical Cancer Protein Expression, and Computational Study.

Manzamines are complex polycyclic marine-derived ß-carboline alkaloids with reported anticancer, immunostimulatory, anti-inflammatory, antibacterial, antiviral, antimalarial, neuritogenic, hyperlipidemia, and atherosclerosis suppression bioactivities, putatively associated with inhibition of glycogen synthase kinase-3, cyclin-dependent kinase 5, SIX1, and vacuolar ATPases. We hypothesized that additional, yet undiscovered molecular targets might be associated with Manzamine A's (MZA) reported pharmacological properties. We report here, for the first time, that MZA selectively inhibited a 90 kDa ribosomal protein kinase S6 (RSK1) when screened against a panel of 30 protein kinases, while in vitro RSK kinase assays demonstrated a 10-fold selectivity in the potency of MZA against RSK1 versus RSK2. The effect of MZA on inhibiting cellular RSK1 and RSK2 protein expression was validated in SiHa and CaSki human cervical carcinoma cell lines. MZA's differential binding and selectivity toward the two isoforms was also supported by computational docking experiments. Specifically, the RSK1-MZA (N- and C-termini) complexes appear to have stronger interactions and preferable energetics contrary to the RSK2-MZA ones. In addition, our computational strategy suggests that MZA binds to the N-terminal kinase domain of RSK1 rather than the C-terminal domain. RSK is a vertebrate family of cytosolic serine-threonine kinases that act downstream of the ras-ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes, including growth, survival, and proliferation. Consequently, our findings have led us to hypothesize that MZA and the currently known manzamine-type alkaloids isolated from several sponge genera may have novel pharmacological properties with unique molecular targets, and MZA provides a new tool for chemical-biology studies involving RSK1.

The Preeclamptic Environment Promotes the Activation of Transcription Factor Kappa B by P53/RSK1 Complex in a HTR8/SVneo Trophoblastic Cell Line.

Preeclampsia is a pregnancy disorder associated with shallow placentation, forcing placental cells to live in hypoxic conditions. This activates the transcription factor kappa B (NFκB) in maternal and placental cells. Although the role of NFκB in preeclampsia is well documented, its mechanism of activation in trophoblastic cells has been never studied. This study investigates the mechanism of NFκB activation in a first trimester trophoblastic cell line (HTR8/SVneo) stimulated by a medium containing serum from preeclamptic (PE) or normotensive (C) women in hypoxic (2% O2) or normoxic (8% O2) conditions. The results indicate that in HTR8/SVneo cells, the most widely studied NFκB pathways, i.e., canonical, non-canonical and atypical, are downregulated in environment PE 2% O2 in comparison to C 8% O2. Therefore, other pathways may be responsible for NFκB activation. One such pathway depends on the activation of NFκB by the p53/RSK1 complex through its phosphorylation at Serine 536 (pNFκB Ser536). The data generated by our study show that inhibition of the p53/RSK1 pathway by p53-targeted siRNA results in a depletion of pNFκB Ser536 in the nucleus, but only in cells incubated with PE serum at 2% O2. Thus, the p53/RSK1 complex might play a critical role in the activation of NFκB in trophoblastic cells and preeclamptic placentas.

Effect of Chronic Alcohol Abuse on Anabolic and Catabolic Signaling Pathways in Human Skeletal Muscle.

BACKGROUND: Animal studies showed that alcoholic myopathy is characterized by the reduction in myofiber cross-sectional area (CSA) and by impaired anabolic signaling. The goal of this study was to compare changes in CSA and fiber type composition with modifications in anabolic and catabolic signaling pathways at the early stages of alcohol misuse in humans. METHODS: Skeletal muscle samples from 7 male patients with chronic alcohol abuse (AL; 47.7 ± 2.0 years old; alcohol misuse duration 7.7 ± 0.6 years) were compared with muscle from a control group of 7 healthy men (C; 39.7 ± 5.0 years old). Biopsies from vastus lateralis muscles were taken and analyzed for the changes in fiber type composition, fiber CSA, and for the alterations in anabolic and catabolic signaling pathways. RESULTS: AL patients did not have detectable clinical myopathy symptoms or muscle fiber atrophy, but the relative proportion of fast fibers was increased. There was a significant decrease in IGF-1 in plasma and IRS-1 protein content in muscle of AL group. Levels of total and phosphorylated p70S6K1, GSK3ß, and p90RSK1 were not different between AL and C groups. Muscle of AL patients had increased mRNA expression of HSP70 and HSP90. A marker of anabolic pathway p-4E-BP1 was decreased, while catabolic markers (MuRF-1, MAFbx, ubiquitinated proteins) were increased in AL patients when compared with C group. CONCLUSIONS: At the early stages of alcohol misuse in humans, changes in the regulation of anabolic and catabolic signaling pathways precede the development of skeletal muscle atrophy and manifestation of clinical symptoms of alcoholic myopathy.

Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex.

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.

Study of Drug Targets Associated With Oncogenesis and Cancer Cell Survival and the Therapeutic Activity of Engineered Ashwagandha Extract Having Differential Withanolide Constitutions.

Ashwagandha (Withania somnifera) has gained worldwide popularity for a multitude of health benefits inclusive of cancer-preventive and curative effects. Despite numerous research data supporting the benefits of this wonder herb, the actual use of ashwagandha for cancer treatment in clinics is limited. The primary reason for this is the inconsistent therapeutic outcome due to highly variable composition and constitution of active ingredients in the plant extract impacting ashwagandha's pharmacology. We investigate here an engineered yield: an ashwagandha extract (Oncowithanib) that has a unique and fixed portion of active ingredients to achieve consistent and effective therapeutic activity. Using the MCF7 cell line, Oncowithanib was studied for its anti-neoplastic efficacy and drug targets associated with cell cycle regulation, translation machinery, and cell survival and apoptosis. Results demonstrate a dose-dependent decline in Oncowithanib-treated MCF7 cell viability and reduced colony-forming ability. Treated cells showed increased cell death as evidenced by enhancement of Caspase 3 enzyme activity and decreased expressions of cell proliferation markers such as Ki67 and Aurora Kinase A. Oncowithanib treatment was also found to be associated with expressional suppression of key cellular kinases such as RSK1, Akt1, and mTOR in MCF7 cells. Our findings indicate that Oncowithanib decreases MCF7 cell survival and propagation, and sheds light on common drug targets that might be good candidates for the development of cancer therapeutics. Further in-depth investigations are required to fully explore the potency and pharmacology of this novel extract. This study also highlights the importance of the standardization of herbal extracts to get consistent therapeutic activity for the disease indication.

The potential of some functional group compounds substituted 8-Manzamine A as RSK1 inhibitors: molecular docking and molecular dynamics simulations.

Cancer, an incurable global disease, demands urgent anti-cancer drug development. Marine alkaloids like Manzamine and its derivatives show promise as RSK inhibitors against cancer cell invasion. Replacing the hydrogen at the 8-position of Manzamine A with a hydroxyl group has been shown to significantly enhance its biological activity. In this article, we designed various functional group compounds (A1-A21) substituted 8-Manzamine A by docking, MM-GBSA, molecular dynamics (MD) simulation, and well-tempered metadynamics (WT-MetaD) simulations to evaluate their potential as RSK1 inhibitors. Ligands A1-A21 were docked in the RSK1 N-terminal kinase domain (PDB ID: 2Z7Q) using the Glide module. The calculation of binding energy was performed using Prime MM-GB/SA, while MD simulations were conducted with the Desmond module of Schrodinger suite 2023. Compound A5 exhibits the highest G-score (-7.01) compared to 8-Hydroxymanzamine A (-6.08). Additionally, compounds A6, A10, A12, A17, A11, A4, and A13 demonstrate increased activity against RSK1 when compared to both 8-Hydroxymanzamine A and Manzamine A. Residues LEU68, VAL76, LEU141, PHE143, LEU144, PHE150, ASP148, GLU191, and LEU194 of RSK1 protein play a key role in binding with ligands. An MD simulation of Compound A5 was carried out to explore the dynamic interactions within the protein-ligand complex. Furthermore, WT-MetaD simulations validated the docking study results and identified the most energetically favored conformations for the A5/RSK1 complex. Ligands A5, A6, A10, A12, A17, A11, A4, and A13, featuring diverse functional groups and good Glide scores, may have the potential for significant RSK1 activity and merit further development.Communicated by Ramaswamy H. Sarma.

Functional group modifications at the 8-position of Manzamine A enhance its inhibitory activity against RSK1.Amine and carbonyl groups at position 8 of Manzamine A enhance its inhibitory activity, especially when accompanied by an aryl group.This research lays a solid groundwork for future drug development targeting RSK1, offering promising candidates for further investigation.

Alternative glucose uptake mediated by ß-catenin/RSK1 axis under stress stimuli in mammalian cells.

Cells adapt to stress conditions by increasing glucose uptake as cytoprotective strategy. The efficiency of glucose uptake is determined by the translocation of glucose transporters (GLUTs) from cytosolic vesicles to cellular membranes in many tissues and cells. GLUT translocation is tightly controlled by the activation of Tre-2/BUB2/CDC16 1 domain family 4 (TBC1D4) via its phosphorylation. The mechanisms of glucose uptake under stress conditions remain to be clarified. In this study, we surprisingly found that glucose uptake is apparently increased for the early response to three stress stimuli, glucose starvation and the exposure to lipopolysaccharide (LPS) or deoxynivalenol (DON). The stress-induced glucose uptake was mainly controlled by the increment of ß-catenin level and the activation of RSK1. Mechanistically, ß-catenin directly interacted with RSK1 and TBC1D4, acting as the scaffold protein to recruit activated RSK1 to promote the phosphorylation of TBC1D4. In addition, ß-catenin was further stabilized due to the inhibition of GSK3ß kinase activity which is caused by activated RSK1 phosphorylating GSK3ß at Ser9. In general, this triple protein complex consisting of ß-catenin, phosphorylated RSK1, and TBC1D4 were increased in the early response to these stress signals, and consequently, further promoted the phosphorylation of TBC1D4 to facilitate the translocation of GLUT4 to the cell membrane. Our study revealed that the ß-catenin/RSK1 axis contributed to the increment of glucose uptake for cellular adaption to these stress conditions, shedding new insights into cellular energy utilization under stress.

Discovery of potential RSK1 inhibitors for cancer therapy using virtual screening, molecular docking, molecular dynamics simulation, and MM/GBSA calculations.

The p90 ribosomal protein S6 Kinase (RSK) family belongs to Ser/Thr protein kinases that includes four isoform RSK1-4 in mammals. The ribosomal protein S6 Kinase 1 (RSK1) is also known as ribosomal protein S6 kinase alpha-1 (RPS6KA1) is a special protein due to their two catalytic regions that is associated with abundantly various cancers and it is proposed as a drug target. Several RSK1 isoform inhibitors have been reported but none of them are used in clinical studies. Thus, we aimed to perform ligand pharmacophore mapping with the known inhibitor and structure-based virtual screening studies to determine potential candidates against RSK1-terminal kinase domains CTKD and NTKD. The studied compounds from the databases (ApexBio, ChEMBL, ChemDiv). The molecular docking study was performed with the resulted candidates by using CDOCKER and Glide/SP methods. The four candidates with the highest docking scores were used for further 100-ns molecular dynamics (MD) simulations and Molecular Mechanics Generalised Born and Surface Area (MM/GBSA) calculations. The root mean square deviation (RMSD) for protein complexes were found between 2 Å and 4 Å. Solvent accessible surface area (SASA), radius of gyration (Rg), and polar surface area (PSA) values were calculated for compounds. The binding free energies were calculated between -72.22 kcal/mol and -82.44 kcal/mol. The interaction diagrams showed that hydrogen bond, alkyl, and π-alkyl interactions were observed with specific residues such as Leu144, Lys94, Asp142 for RSK1-NTKD, and Cys532, Cys556, Lys447, Asn540 for RSK1-CTKD. The identified compounds may be potential inhibitor candidates of RSK1 following the preclinical studies.Communicated by Ramaswamy H. Sarma.

Plakophilin 3 facilitates G1/S phase transition and enhances proliferation by capturing RB protein in the cytoplasm and promoting EGFR signaling.

Plakophilin 3 (PKP3) is a component of desmosomes and is frequently overexpressed in cancer. Using keratinocytes either lacking or overexpressing PKP3, we identify a signaling axis from ERK to the retinoblastoma (RB) protein and the E2F1 transcription factor that is controlled by PKP3. RB and E2F1 are key components controlling G1/S transition in the cell cycle. We show that PKP3 stimulates the activity of ERK and its target RSK1. This inhibits expression of the transcription factor RUNX3, a positive regulator of the CDK inhibitor CDKN1A/p21, which is also downregulated by PKP3. Elevated CDKN1A prevents RB phosphorylation and E2F1 target gene expression, leading to delayed S phase entry and reduced proliferation in PKP3-depleted cells. Elevated PKP3 expression not only increases ERK activity but also captures phosphorylated RB (phospho-RB) in the cytoplasm to promote E2F1 activity and cell-cycle progression. These data identify a mechanism by which PKP3 promotes proliferation and acts as an oncogene.

Rapamycin-Induced Feedback Activation of eIF4E-EIF4A Dependent mRNA Translation in Pancreatic Cancer.

Pancreatic cancer cells adapt molecular mechanisms to activate the protein synthesis to support tumor growth. This study reports the mTOR inhibitor rapamycin's specific and genome-wide effect on mRNA translation. Using ribosome footprinting in pancreatic cancer cells that lack the expression of 4EBP1, we establish the effect of mTOR-S6-dependent mRNAs translation. Rapamycin inhibits the translation of a subset of mRNAs including p70-S6K and proteins involved in the cell cycle and cancer cell growth. In addition, we identify translation programs that are activated following mTOR inhibition. Interestingly, rapamycin treatment results in the translational activation of kinases that are involved in mTOR signaling such as p90-RSK1. We further show that phospho-AKT1 and phospho-eIF4E are upregulated following mTOR inhibition suggesting a feedback activation of translation by rapamycin. Next, targeting eIF4E and eIF4A-dependent translation by using specific eIF4A inhibitors in combination with rapamycin shows significant growth inhibition in pancreatic cancer cells. In short, we establish the specific effect of mTOR-S6 on translation in cells lacking 4EBP1 and show that mTOR inhibition leads to feedback activation of translation via AKT-RSK1-eIF4E signals. Therefore, targeting translation downstream of mTOR presents a more efficient therapeutic strategy in pancreatic cancer.

ZCL-082, a boron-containing compound, induces apoptosis of non-Hodgkin's lymphoma via targeting p90 ribosomal S6 kinase 1/NF-κB signaling pathway.

INTRODUCTION: Despite the rapid progress in the diagnosis and treatment, the prognosis of some types of non-Hodgkin's lymphoma (NHL), especially those with double-hit or double-expressor genotypes, remains poor. Novel targets and compounds are needed to improve the prognosis of NHL. METHODS: We investigated the effect of ZCL-082, a novel boron-containing compound with anti-proliferating activity against ovarian cancer cells, on NHL cells and human peripheral blood mononuclear cells by CCK-8 assay, Annexin V/PI double staining assay, RH123/PI double staining, Western blot, and immunohistochemistry. NF-κB pathway activity was analyzed using luciferase reporter gene assay and RT-PCR. The location of p65 was detected by immunofluorescence and nuclear/cytoplasmic fractionation assay. Immunoprecipitation and chromatin immunoprecipitation assays were used to detect the binding between p65 and p300. CETSA and molecular docking assay were carried out to test the interaction between ZCL-082 and p90 ribosomal S6 kinase 1 (RSK1). Kinase reaction was conducted to examine the inhibition of RSK1 kinase activity by ZCL-082. RESULTS: We found that ZCL-082 can induce the apoptosis of various NHL cell lines in vitro and in vivo. ZCL-082 significantly inhibits TNFα- or LPS-induced NF-κB activation without disturbing TNFα-induced IκBα degradation or the nuclear translocation and DNA-binding ability of p65. However, ZCL-082 markedly suppresses the phosphorylation of p65 on Ser536 and the interaction between p65 and p300. The overexpression of the phosphomimetic mutant of p65 at Ser536 partially abrogates ZCL-082-induced cell death. We further found that ZCL-082 directly binds to and inhibits the activity of RSK1. RSK1 can phosphorylate RelA/p65 on Ser536 and its overexpression is associated with the poor prognosis of lymphoma. The overexpression of RSK1 partially rescues ZCL-082-induced cell death. Molecular docking studies show that ZCL-082 fits well with the N-terminal kinase domain of RSK1. Furthermore, the combination of ZCL-082 and BCL-2 inhibitor ABT-199 has a synergistic apoptosis-inducing effect against double-hit lymphoma cell line OCI-Ly10. DISCUSSION: We found that ZCL-082 is a highly promising anti-lymphoma compound that targets RSK1 and interferes with the RSK1/NF-κB signaling pathway. The combination of ZCL-082 with BCL-2 inhibitor may represent a novel strategy to improve the outcome of double-hit or double-expressor lymphoma.

Studying the Structures of Relaxed and Fuzzy Interactions: The Diverse World of S100 Complexes.

S100 proteins are small, dimeric, Ca2+-binding proteins of considerable interest due to their associations with cancer and rheumatic and neurodegenerative diseases. They control the functions of numerous proteins by forming protein-protein complexes with them. Several of these complexes were found to display "fuzzy" properties. Examining these highly flexible interactions, however, is a difficult task, especially from a structural biology point of view. Here, we summarize the available in vitro techniques that can be deployed to obtain structural information about these dynamic complexes. We also review the current state of knowledge about the structures of S100 complexes, focusing on their often-asymmetric nature.

Dimethyl Fumarate Targets MSK1, RSK1, 2 and IKKα/ß Kinases and Regulates NF-κB /p65 Activation in Psoriasis: A Demonstration of the Effect on Peripheral Blood Mononuclear Cells, Drawn from Two Patients with Severe Psoriasis Before and After Treatment with Dimethyl Fumarate.

BACKGROUND: Dimethyl fumarate (DMF) has an inhibitory effect on the production of pro-inflammatory proteins from different cells which participate in the immune reaction in psoriatic skin. Most recently it was shown that DMF is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases (RSK1, 2), determined by X-ray crystallography. DMF binds to a specific cysteine residue in RSK2 and in the closely related mitogen and stress-activated kinases 1 (MSK1) which inhibits further downstream activation. OBJECTIVES: The aim of this study was to review the literature on the effects of DMF on activation of MSK1, RSK1, 2 kinases, and downstream transcription factors NF-κB/p65 and IκBα in cells contributing to the pathogenesis of psoriasis. We also hypothesized and studied if treatment with DMF would inhibit the activation of MSK1, RSK1, 2 kinases in peripheral blood mononuclear cells (PBMCs) in psoriatic patients. METHODS: PBMCs were purified from patients with severe psoriasis before and after 90 days of treatment with DMF. Cells were stimulated with anisomycin, IL-1ß or EGF for 10 and 20 minutes. The levels of phosphorylation of MSK1, RSK1, 2 or NF-κB/p65, IκBα were analyzed by Western blotting. RESULTS: Our case study showed that treatment with DMF inhibited the activation of MSK1 and RSK1, 2 kinases in PBMCs in patients. This supports that DMF is the active metabolite in vivo in psoriatic patients during DMF treatment. CONCLUSION: Pro-inflammatory proteins are induced through activation of MSK1 and NF-κB/p65 at (S276). The extracellular signal-regulated kinases (ERK1/2) control cell survival by activating both MSK1 and RSK1, 2 kinases. P-RSK1, 2 activates P-κBα and NF-κB/p65 at (S536). The phosphorylation of NF-κB/p65 at (S276) and (S536) controls different T cell and dendritic cell functions. DMF´s inhibitory effect on MSK1 and RSK1, 2 kinase activations reduces multiple immune reactions in psoriatic patients.

MAP Kinase-Mediated Activation of RSK1 and MK2 Substrate Kinases.

Mitogen-activated protein kinases (MAPKs) control essential eukaryotic signaling pathways. While much has been learned about MAPK activation, much less is known about substrate recruitment and specificity. MAPK substrates may be other kinases that are crucial to promote a further diversification of the signaling outcomes. Here, we used a variety of molecular and cellular tools to investigate the recruitment of two substrate kinases, RSK1 and MK2, to three MAPKs (ERK2, p38α, and ERK5). Unexpectedly, we identified that kinase heterodimers form structurally and functionally distinct complexes depending on the activation state of the MAPK. These may be incompatible with downstream signaling, but naturally they may also form structures that are compatible with the phosphorylation of the downstream kinase at the activation loop, or alternatively at other allosteric sites. Furthermore, we show that small-molecule inhibitors may affect the quaternary arrangement of kinase heterodimers and thus influence downstream signaling in a specific manner.

RSK1 promotes murine breast cancer growth and metastasis.

INTRODUCTION: Triple-negative breast cancer (TNBC), representing over 15% of all breast cancers, has a poorer prognosis than other subtypes. There is no effective targeted treatment available for the TNBC sufferers. Ribosomal S6 kinases (RSKs) have been previously proposed as drug targets for TNBC based on observations that 85% of these tumors express activated RSKs. MATERIALS AND METHODS: Herein we examined an involvement of RSK1 (p90 ribosomal S6 kinase 1) in a regulation of TNBC growth and metastatic spread in an animal model, which closely imitates human disease. Mice were inoculated into mammary fat pad with 4T1 cells or their RSK1-depleted variant. We examined tumor growth and formation of pulmonary metastasis. Boyden chamber, wound healing and soft agarose assays were performed to evaluate cells invasion, migration and anchorage-independent growth. RESULTS: We found that RSK1 promoted tumor growth and metastasis in vivo. After 35 days all animals inoculated with control cells developed tumors while in the group injected with RSK1-negative cells, there were 75% tumor-bearing mice. Average tumor mass was estimated as 1.16 g and 0.37 g for RSK1-positive vs. -negative samples, respectively (p < 0.0001). Quantification of the macroscopic pulmonary metastases indicated that mice with RSK1-negative tumors developed approximately 85% less metastatic foci on the lung surface (p < 0.001). This has been supported by in vitro data presenting that RSK1 promoted anchorage-independent cell growth and migration. Moreover, RSK1 knock-down corresponded with decreased expression of cell cycle regulating proteins, i.e. cyclin D3, CDK6 and CDK4. CONCLUSIONS: We provide evidence that RSK1 supports tumor growth and metastatic spread in vivo as well as in vitro migration and survival in non-adherent conditions. Further studies of RSK1 involvement in TNBC progression may substantiate our findings, laying the foundations for development of anti-RSK1-based therapeutic strategies in the management of patients with TNBC.

Hypoxia-induced ADAM 17 expression is mediated by RSK1-dependent C/EBPß activation in human lung fibroblasts.

Hypoxia was identified as a mediator of lung fibrosis in patients with chronic obstructive asthma (COA). Overexpression of a disintegrin and metalloproteinase 17 (ADAM 17) and connective tissue growth factor (CTGF) leads to development of tissue fibrosis. However, the signaling pathway in hypoxia-induced ADAM 17 expression remains poorly defined. In this study, we investigated the roles that ribosomal S-6 kinase 1 (RSK1)/CCAAT/enhancer-binding protein ß (C/EBPß)-dependent ADAM 17 expression plays in hypoxia-induced CTGF expression in human lung fibroblasts. We observed that hypoxia caused increases in ADAM 17 expression and ADAM 17-luciferase activity in WI-38 cells. Hypoxia-induced CTGF-luciferase activity and CTGF expression were reduced in cells transfected with small interfering (si)RNA of ADAM 17 in WI-38 cells. Moreover, hypoxia-induced ADAM 17 expression was reduced by RSK1 siRNA and C/EBPß siRNA. Hypoxia caused time-dependent increases in RSK1 phosphorylation at Thr359/Ser363. Exposure of cells to hypoxia resulted in increased C/EBPß phosphorylation at Thr266 and C/EBPß-luciferase activity in time-dependent manners, and these effects were suppressed by RSK1 siRNA. Hypoxia induced recruitment of C/EBPß to the ADAM 17 promoter. Furthermore, CTGF-luciferase activity induced by hypoxia was attenuated by RSK1 siRNA and C/EBPß siRNA. These results suggest that hypoxia instigates the RSK1-dependent C/EBPß signaling pathway, which in turn initiates binding of C/EBPß to the ADAM 17 promoter and ultimately induces ADAM 17 expression in human lung fibroblasts. Moreover, RSK1/C/EBPß-dependent ADAM 17 expression is involved in hypoxia-induced CTGF expression. Our results suggest possible therapeutic approaches for treating hypoxia-mediated lung fibrosis in COA.

Involvement of Polo-like kinase 1 (Plk1) in quiescence regulation of cancer stem-like cells of the gastric cancer cell lines.

Cancer stem cells (CSCs) have been hypothesized to initiate tumor growth and be resistant to chemoradiotherapy, and these processes appear to be closely related to CSC quiescence. Here, a CSC-like cell population with a high level of CD44 expression was obtained from the human gastric cancer cell lines MKN45 and MKN74. Using a PKH26-labeling retention assay, quiescent CSC-like cells with low levels of Ki67 and PCNA expression were found in spheres formed in serum-free medium, and exhibited resistance to drug and radiation treatments. Polo-like kinase 1 (Plk1) and ribosomal S6 kinase 1 (RSK1) were silenced in the quiescent CSC-like cells. The Plk1-specific inhibitors inhibited the activation of RSK1 and induced quiescence in the CSC-like cells, but increased RSK1 activity and resulted in apoptosis in non-CSCs. Furthermore, RSK1 silencing by inhibitors activated Plk1 and had no effect on the growth of spheres in the CSC-like cells, but did not affect phosphorylation of Plk1 and led to decreased proliferation in non-CSCs. Our results showed that Plk1 and RSK1 play important roles in the conversion of CSCs between active and quiescent states.

Protein phosphatase 2Cδ/Wip1 regulates phospho-p90RSK2 activity in lesional psoriatic skin.

OBJECTIVES: P90 ribosomal S6 kinase (RSK) 1 and 2 are serine/threonine protein kinases believed to mediate proliferation and apoptosis via the extracellular signal-regulated kinases (ERK1/2) signaling pathway. Macrophage migration inhibitory factor (MIF) and epidermal growth factor (EGF) are activators of this pathway and are elevated in the serum of patients with psoriasis compared with healthy controls. Studies on COS-7 cell cultures have shown that protein phosphatase 2Cδ (PP2Cδ) decreases the activity of RSK2 following EGF stimulation. We therefore hypothesize that PP2Cδ regulates RSK2 activity in psoriasis. METHODS: In paired biopsies from nonlesional (NL) and lesional (L) skins, we analyzed the level of RSK1, 2 phosphorylation and the expression of PP2Cδ isoforms, integrin-linked kinase-associated serine/threonine phosphatase (ILKAP) and wild-type p53-induced phosphatase 1 (Wip1) by Western blotting, immunofluorescence and coimmunoprecipitation with monoclonal antibody for RSK2. The induction of Wip1 by MIF or EGF was studied in cultured normal human keratinocytes. RESULTS: The protein level of RSK1, 2 phosphorylated at T573/T577 was significantly increased in L compared with NL psoriatic skin, while phosphorylation at S380/S386 was reduced in L compared with NL psoriatic skin when assayed by Western blotting and immunofluorescence microscopy. ILKAP expression was significantly higher in L than in NL skin, whereas Wip1 was expressed in similar amounts but showed increased coimmunoprecipitation with RSK2 in L compared with NL psoriatic skin. In cultured normal human keratinocytes stimulated with MIF, Wip1 phosphorylation and Wip1 expression were increased after 24 hours, but not when costimulated with dimethyl fumarate (DMF). The increased coimmunoprecipitation of Wip1 with RSK2 was significantly induced by EGF or MIF activation at 24 hours and could be significantly inhibited by DMF or the ERK1/2 inhibitor PD98059. CONCLUSION: The complex formation of Wip1 with RSK2 indicates a direct interaction reducing P-RSK2 (S386) activation in L skin and indicates that Wip1 has a role in the pathogenesis of psoriasis.