S6 Kinase 1 Inhibition Research Articles

Structure-based identification of potential inhibitors of ribosomal protein S6 kinase 1, targeting cancer therapy: a combined docking and molecular dynamics simulations approach

Ribosomal protein S6 kinase 1 (S6K1), commonly known as P70-S6 kinase 1 (p70S6), is a key protein kinase involved in cellular signaling pathways that regulate cell growth, proliferation, and metabolism. Its significant role is reported in the PIK3/mTOR signaling pathway and is associated with various complex diseases, including diabetes, obesity, and different types of cancer. Due to its involvement in various physiological and pathological conditions, S6K1 is considered as an attractive target for drug design and discovery. One way to target S6K1 is by developing small molecule inhibitors that specifically bind to its ATP-binding site, preventing its activation and thus inhibiting downstream signaling pathways necessary for cell growth and survival. In this study, we have conducted a multitier virtual screening of a pool of natural compounds to identify potential S6K1 inhibitors. We performed molecular docking on IMPPAT 2.0 library and selected top hits based on their binding affinity, ligand efficiency, and specificity towards S6K1. The selected hits were further assessed based on different filters of drug-likeliness where two compounds (Hecogenin and Glabrene) were identified as potential leads for S6K1 inhibition. Both compounds showed appreciable affinity, ligand efficiency and specificity towards S6K1 binding pocket, drug-like properties, and stable protein-ligand complexes in molecular dynamics (MD) simulations. Finally, our study has suggested that Hecogenin and Glabrene can be potential S6K1 inhibitors which are presumably implicated in the therapeutic management of associated diseases such as diabetes, obesity, and varying types of cancer. Communicated by Ramaswamy H. Sarma

Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPKT172 and ULK1S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1G93A protein aggregates with autophagosomes and accelerated SOD1G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.

Pharmacological inhibition of S6K1 facilitates platelet activation by enhancing Akt phosphorylation

Platelet activation and thrombus formation is a delicate process involving a series of crosstalk between different pathways. P70 ribosomal S6 kinase1 (S6K1) is a member of serine/threonine kinases and can be phosphorylated by 3-phosphoinositide-dependent protein kinase 1 (PDK1). S6K1 is widely reported to play important roles in cancers and metabolic diseases, but the role of S6K1 and the importance of phosphorylation on Thr229 in platelet activation have not been defined. PF-4708671 is a recently synthesized highly specific inhibitor of S6K1. In this study, we tested PF-4708671 to assess the role of S6K1 in platelet. PF-4708671 facilitated mouse and human platelet aggregation and ATP secretion induced by collagen, thrombin, and adenosine diphosphate through enhanced Akt and Gsk3β phosphorylation. PF-4708671 also accelerated integrin αIIbβ3-mediated clot retraction and spreading. Intravenously given PF-4708671 shortened the occlusion time in arterial thrombosis model. Further results demonstrated that S6K1 was phosphorylated by PDK1 on Thr229 in the resting platelets and dephosphorylated in response to agonist stimulation. PDK1-deficient mice showed higher aggregation when PI3K–Akt–Gsk3β signaling was blocked by the Gsk3β-inhibitor SB216763. Thus, S6K1 Thr229 phosphorylation might function as a regulator that prevents platelets from activation. S6K1 inhibition may yield potential pro-thrombotic effects and should be used cautiously when considered as a therapy.

P53 modulates the effect of ribosomal protein S6 kinase1 (S6K1) on cisplatin toxicity in chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is characterized by the expression of the oncoprotein, BCR-ABL. BCR-ABL inhibitors revolutionized CML chemotherapy while blast crisis (BC) CML patients are less responsive. Since suppression of ribosomal protein S6 kinase1 (S6K1) phosphorylation reverses the resistance to BCR-ABL inhibitor in CML cells and S6K1 inhibitors augment cisplatin toxicity in lung cancer cells, we speculated that combination of S6K1 inhibitor and cisplatin may be beneficial for eliminating BC CML cells. To our surprise, S6K1 inhibition decreased cisplatin-induced DNA damage and cell death only in p53-/− BC CML cells but not in p53+/+ BC CML cells. During the progression of CML, p53 expression either decreases or mutates. Moreover, the expression of p53 affects drug response of CML cells. Our results confirmed that S6K1 inhibition reversed cisplatin toxicity is dependent on p53 expression in CML cells. Moreover, p53 attenuated the phosphorylation and localization of S6K1 via attenuating 3-phosphoinositide dependent protein kinase-1 (PDK1) phosphorylation. Furthermore, S6K1 acts via DNA-PKcs to regulate H2AX phosphorylation and PARP cleavage, respectively. Taken together, our results suggest that p53/PDK1/S6K1 is a novel pathway regulating cisplatin toxicity in BC CML cells.

Association of ribosomal protein S6 kinase 1 with cellular radiosensitivity of non-small lung cancer

Purpose: Ribosomal S6 kinase 1 (S6K1) plays an important role in cell proliferation, protein translation and cell survival. This study investigated the possibility of using S6K1 as a new target in the radiotherapy of non-small cell lung cancer (NSCLC) and its potential mechanism.Materials and methods: shRNA interference technology was applied to silence the expression of S6K1 in A549 and H460, and clonogenic assay was performed to measure the radiosensitizing effects. DNA repair was monitored by γH2AX foci formation. Cell cycle and apoptosis were measured by flow cytometry assays. Protein expression was analyzed by Western blotting. S6K1 and lentivirus containing programmed cell death 4 (PDCD4), a substrate of S6K1, were co-transfected into cells and radiosensitivity was also detected.Results: S6K1 knockdown significantly enhanced the radiosensitivity of NSCLC cells. The notable findings in response to this combined treatment were prolonged delay in radiation-induced DNA DSB repair, robust G2/M checkpoint arrest, increased apoptosis and upregulation of PDCD4. Moreover, PDCD4 knockdown reversed the radiosensitizing effects of S6K1 inhibition in NSCLC.Conclusions: S6K1 is a promising tumor-specific target for the enhancement of NSCLC radiosensitivity and its effects may be mediated by increased expression of PDCD4.

S6K1 inhibition enhances the apoptotic cell death of breast cancer cells in response to Bcl-2/Bcl-xL inhibition by the downregulation of survivin.

Breast cancer cells possess a deregulated apoptotic pathway with increased expression levels of anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins and ribosomal S6 kinase 1 (S6K1) protein activity. Therefore, combined interference of anti-apoptotic Bcl-2 family and S6K1 protein expression may be a reasonable therapeutic strategy for the treatment of patients with breast cancer. In the present study, it was identified that the administration of a combination of ABT263 [navitoclax; a Bcl-2/Bcl-extra large (Bcl-xL) inhibitor] and PF4708671 (an S6K1 inhibitor) markedly increased apoptotic cell death in the BT474 breast cancer cells compared with the administration of either agent alone. Furthermore, the downregulation of Bcl-2/Bcl-xL and S6K1 with small interfering RNA induced a significant increase in cell death compared with RNA interference of either agent alone. Notably, combination treatment with ABT263 and PF4708671 decreased the expression level of survivin protein, with this ectopic expression of survivin attenuating cell death. Thus, the present study determined that the combined inhibition of Bcl-2/Bcl-xL and S6K1 may be a good strategy for treating patients with breast cancer.

MTORC1 drives HIF-1α and VEGF-A signalling via multiple mechanisms involving 4E-BP1, S6K1 and STAT3.

Recent clinical trials using rapalogues in tuberous sclerosis complex (TSC) show regression in volume of typically vascularised tumours including angiomyolipomas (AMLs) and sub-ependymal giant cell astrocytomas (SEGAs). By blocking mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signalling, rapalogue efficacy is likely to occur in part through suppression of hypoxia inducible factors (HIFs) and vascular endothelial growth factors (VEGFs). We show that rapamycin reduces HIF-1α protein levels, and to a lesser extent VEGF-A levels, in renal cystadenoma cells in a Tsc2+/− mouse model. We establish that mTORC1 drives HIF-1α protein accumulation through enhanced transcription of HIF-1α mRNA, a process that is blocked by either inhibition or knockdown of signal transducer and activation of transcription 3 (STAT3). Furthermore, we demonstrate that STAT3 is directly phosphorylated by mTORC1 on Ser727 during hypoxia, promoting HIF-1α mRNA transcription. mTORC1 also regulates HIF-1α synthesis on a translational level via co-operative regulation of both initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase-1 (S6K1), whilst HIF-1α degradation remains unaffected. We therefore propose that mTORC1 drives HIF-1α synthesis in a multi-faceted manner through 4E-BP1/eIF4E, S6K1 and STAT3. Interestingly, we observe a disconnect between HIF-1α protein levels and VEGF-A expression. While both S6K1 and 4E-BP1 regulate HIF-1α translation, VEGF-A is primarily under the control of 4E-BP1/eIF4E. S6K1 inhibition reduces HIF-1α but not VEGF-A expression, suggesting that mTORC1 mediates VEGF-A expression via both HIF-1α-dependent and -independent mechanisms. Our work has important implications for the treatment of vascularised tumours, where mTORC1 acts as a central mediator of STAT3, HIF-1α, VEGF-A and angiogenesis via multiple signalling mechanisms.

S6K1 inhibition enhances tamoxifen-induced cell death in MCF-7 cells through translational inhibition of Mcl-1 and survivin

S6 kinase 1 (S6K1) was suggested to be a marker for endocrine therapy resistance in breast cancer. We examined whether tamoxifen's effect can be modulated by S6K1 inhibition. S6K1 inhibition by PF4708671, a selective inhibitor of S6K1, acts synergistically with tamoxifen in S6K1-high MCF-7 cells. Similarly, the knockdown of S6K1 with small interfering RNA (siRNA) significantly sensitized MCF-7 cells to tamoxifen. Inhibition of S6K1 by PF4708671 led to a marked decrease in the expression levels of the anti-apoptotic proteins Mcl-1 and survivin, which was not related to mRNA levels. In addition, suppression of Mcl-1 or survivin, using specific siRNA, further enhanced cell sensitivity to tamoxifen. These results showed that inhibition of S6K1 acts synergistically with tamoxifen, via translational modulation of Mcl-1 and survivin. Based on these findings, we propose that targeting S6K1 may be an effective strategy to overcome tamoxifen resistance in breast cancer.

Genistein sensitizes hepatic insulin signaling and modulates lipid regulatory genes through p70 ribosomal S6 kinase-1 inhibition in high-fat–high-fructose diet-fed mice

Context: Genistein reduces high-calorie diet-induced insulin resistance and fat accumulation in animals, but the mechanism is unresolved.Objective: This study explores whether action of genistein is associated with p70 ribosomal S6 kinase-1 (S6K1) inhibition.Materials and methods: Adult male mice were fed either normal diet or high-fat–high-fructose diet (HFFD) for 15 days, after which animals in each dietary group were divided into two groups and administered either genistein (1 mg kg−1 day−1, p.o.) in 0.5 ml of 30% dimethylsulfoxide (DMSO) or 30% DMSO (0.5 ml) for the next 45 days. At the end of the study, their liver was analyzed for lipid content. Semi-quantitative RT-PCR and western blotting methods were used to analyze lipid regulatory genes and insulin signaling proteins, respectively.Results: Genistein significantly (p < 0.05) lowered HFFD-induced body and liver weight gain and plasma and hepatic lipid levels. Histology showed a 2.5-fold increase of lipid in HFFD compared to control. Genistein treatment to HFFD-fed animals significantly decreased lipid accumulation (by 40%) compared to HFFD. Insulin-stimulated tyrosine phosphorylation of insulin receptor-β and insulin receptor substrates-1 (IRS-1), IRS-1 associated phospatidylinositol-3kinase (PI3K) and Akt Ser473 phosphorylation were improved while IRS-1 serine phosphorylation was significantly (p < 0.05) decreased by genistein in HFFD. Significant (p < 0.05) increase in adenosine monophosphate-activated protein kinase (AMPK) Thr172 phosphorylation and decrease in S6K1 Thr389 phosphorylation were observed in HFFD-plus genistein compared to HFFD. Genistein downregulated lipogenic genes and upregulated fatty acid oxidative genes in HFFD-fed mice.Conclusion: Genistein improves insulin signaling and attenuates fat accumulation in liver through S6K1 inhibition.

Antisense Inhibition of S6 Kinase 1 Produces Improved Glucose Tolerance and Is Well Tolerated for 4 Weeks of Treatment in Rats

p70 ribosomal S6 kinase 1 (S6K1) is implicated in the pathogenesis of type 2 diabetes as knockout mice are hypoinsulinemic, hypersensitive to insulin treatment and are less susceptible to obesity-induced insulin resistance. Although S6K1 knockout mice provide important information on the biology of this target, the therapeutic relevance of S6K1 inhibition in adult animals is unknown. Thus, this research evaluated the potential safety and efficacy of S6K1 inhibition using antisense oligonucleotides (ASO) in mature Sprague-Dawley rats. Male rats treated with S6K1 ASO (25 or 50 mg/kg, 2×/week × 4 weeks) had a marked reduction (>90%) of S6K1 mRNA in the liver and epididymal fat and no effect on hepatic S6K2 expression. The decrease in S6K1 mRNA translated to decreased (>80%) S6K1 protein and kinase activity in the liver at the 50-mg/kg dose. The animals tolerated the S6K1 treatment well with no signs of clinical toxicity. A reduction in body weight gain was observed within 2 weeks of S6K1 ASO treatment. At 4 weeks, body weight gain was reduced by up to 25% in the 50 mg/kg group with a commensurate decrease (14%) in food consumption. A decrease in heart weight in the 50 mg/kg group was observed and not associated with cardiac injury or dysfunction. In an oral glucose tolerance test, S6K1-ASO-treated animals demonstrated a dose-dependent improvement in systemic glucose utilization and had reduced fasting insulin levels. Hepatic gene microarray analysis identified dose-dependent elevations in igfbp1, acss2 and acat2 gene expression in S6K1-ASO-treated animals. These results suggest that inhibition of S6K1 for up to 4 weeks may be therapeutically relevant to induce insulin sensitization and attenuate weight gain with low risk for serious toxicity.

Identifying Optimal Biologic Doses of Everolimus (RAD001) in Patients With Cancer Based on the Modeling of Preclinical and Clinical Pharmacokinetic and Pharmacodynamic Data

To use preclinical and clinical pharmacokinetic (PK)/pharmacodynamic (PD) modeling to predict optimal clinical regimens of everolimus, a novel oral mammalian target of rapamycin (mTOR) inhibitor, to carry forward to expanded phase I with tumor biopsy studies in cancer patients. Inhibition of S6 kinase 1 (S6K1), a molecular marker of mTOR signaling, was selected for PD analysis in peripheral blood mononuclear cells (PBMCs) in a phase I clinical trial. PK and PD were measured up to 11 days after the fourth weekly dose. A PK/PD model was used to describe the relationship between everolimus concentrations and S6K1 inhibition in PBMCs of cancer patients and in PBMCs and tumors of everolimus-treated CA20948 pancreatic tumor-bearing rats. Time- and dose-dependent S6K1 inhibition was demonstrated in human PBMCs. In the rat model, a relationship was shown between S6K1 inhibition in tumors or PBMCs and antitumor effect. This allowed development of a direct-link PK/PD model that predicted PBMC S6K1 inhibition-time profiles in patients. Comparison of rat and human profiles simulated by the model suggested that a weekly 20- to 30-mg dose of everolimus would be associated with an antitumor effect in an everolimus-sensitive tumor and that daily administration would exert a greater effect than weekly administration at higher doses. A direct-link PK/PD model predicting the time course of S6K1 inhibition during weekly and daily everolimus administration allowed extrapolation from preclinical studies and first clinical results to select optimal doses and regimens of everolimus to explore in future clinical trials.

Regulation of S6 Kinase 1 Activation by Phosphorylation at Ser-411

Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G(1) phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1.

Peroxisome Proliferator-Activated Receptor-γ and Retinoic Acid X Receptor α Represses theTGFβ1Gene via PTEN-Mediated p70 Ribosomal S6 Kinase-1 Inhibition: Role for Zf9 Dephosphorylation

Peroxisome proliferator-activated receptor (PPAR)-gamma and retinoic acid X receptor (RXR) heterodimer regulates cell growth and differentiation. Zinc finger transcription factor-9 (Zf9), whose phosphorylation promotes target genes, is a transcription factor essential for transactivation of the transforming growth factor (TGF)-beta1 gene. This study investigated whether activation of PPARgamma-RXR heterodimer inhibits TGFbeta1 gene transcription and Zf9 phosphorylation and, if so, what signaling pathway regulates it. Either 15-deoxy-delta(12,14)-prostaglandin J2 (PGJ2) or 9-cis-retinoic acid (RA) treatment decreased the TGFbeta1 mRNA level in L929 fibroblasts. PGJ2 + RA, compared with individual treatment alone, synergistically inhibited the TGFbeta1 gene expression, which was abrogated by PPARgamma antagonists. Likewise, PGJ2 + RA decreased luciferase expression from the TGFbeta1 gene promoter. Promoter deletion analysis of the TGFbeta1 gene revealed that pGL3-323 making up to -323-base pair region, but lacking PPAR-responsive elements, responded to PGJ2 + RA. PGJ2 + RA treatment inhibited the activity of p70 ribosomal S6 kinase-1 (S6K1), abolishing Zf9 phosphorylation at serine as did rapamycin [a mammalian target of rapamycin (mTOR) inhibitor]. Zf9 dephosphorylation by PGJ2 + RA was reversed by transfection of cells with the plasmid encoding constitutively active S6K1 (CA-S6K1). Transfection with dominant negative S6K1 inhibited the TGFbeta1 gene. TGFbeta1 gene repression by PGJ2 + RA was consistently antagonized by CA-S6K1. Ectopic expression of PPARgamma1 and RXRalpha repressed pGL3-323 transactivation with S6K1 inhibition, which was abrogated by CA-S6K1 transfection. PGJ2 + RA induced phosphatase and tensin homolog deleted on chromosome 10 (PTEN), whose overexpression repressed the TGFbeta1 gene through S6K1 inhibition, decreasing extracellular signal-regulated kinase 1/2-90-kDa ribosomal S6 kinase 1 and Akt-mTOR phosphorylations. Data indicate that activation of PPARgamma-RXR heterodimer represses the TGFbeta1 gene and induces Zf9 dephosphorylation via PTEN-mediated S6K1 inhibition, providing insight into pharmacological manipulation of the TGFbeta1 gene regulation.