Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis.

The manifestations of tuberous sclerosis complex (TSC) in humans include epilepsy, autism spectrum disorders (ASD) and intellectual disability. Previous studies suggested the linkage of TSC to altered cerebral blood flow and metabolic dysfunction. We previously reported a significant elevation in cerebral blood flow in an animal model of TSC and autism of young Eker rats. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin could restore normal oxygen consumption and cerebral blood flow. In this study, we investigated whether inhibiting a component of the mTOR signaling pathway, p70 ribosomal S6 kinase (S6K1), would yield comparable effects. Control Long Evans and Eker rats were divided into vehicle and PF-4708671 (S6K1 inhibitor, 75 mg/kg for 1 h) treated groups. Cerebral regional blood flow (14C-iodoantipyrine) was determined in isoflurane anesthetized rats. We found significantly increased basal cortical (+ 32%) and hippocampal (+ 15%) blood flow in the Eker rats. PF-4708671 significantly lowered regional blood flow in the cortex and hippocampus of the Eker rats. PF-4708671 did not significantly lower blood flow in these regions in the control Long Evans rats. Phosphorylation of S6-Ser240/244 and Akt-Ser473 was moderately decreased in Eker rats but only the latter reached statistical significance upon PF-4708671 treatment. Our findings suggest that moderate inhibition of S6K1 with PF-4708671 helps to restore normal cortical blood flow in Eker rats and that this information might have therapeutic potential in tuberous sclerosis complex and autism.

An inulin-type fructan CP-A from Codonopsis pilosula alleviates TNBS-induced ulcerative colitis based on serum-untargeted metabolomics.

Ulcerative colitis (UC) is an inflammatory disease with abdominal pain, diarrhea, and bloody stool as the main symptoms. Several studies have confirmed that polysaccharides are effective against UC. It is commonly accepted that the traditional benefits of Radix Codonopsis can be attributed to its polysaccharide contents, and inulin-type fructan CP-A is the main active monomer in the polysaccharide components. Herein, we established a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced UC rat model and lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) to investigate the effect of CP-A on UC. Untargeted metabolomics studies were conducted to identify differential metabolites using ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) and enrich metabolic pathways in rat serum. The in vivo assays demonstrated that CP-A reduces colonic macroscopic injury, disease activity index (DAI), histopathological score, interleukin (IL)-8, and tumor necrosis factor-α (TNF-α) levels, as well as the expression of intercellular adhesion molecules. On the other hand, CP-A increases IL-10 and transforming growth factor-ß (TGF-ß) levels. The in vitro experiments indicated that CP-A treatment could reduce nitric oxide (NO) and IL-1ß after LPS stimulation. The metabolomics results suggested that CP-A therapy for UC may be related to the mammalian target of rapamycin (mTOR) signaling pathway. The in vitro and in vivo validation of the pathway showed similar results, indicating that CP-A alleviates UC by preventing the activation of mTOR/p70S6K signaling pathway. These findings offer a fresh approach to treating UC and a theoretical foundation for the future advancement of CP-A.NEW & NOTEWORTHY We report that an inulin-type fructan from Codonopsis pilosula CP-A exhibits a therapeutic effect on experimental colitis. Its mechanism may be to alleviate intestinal inflammation by preventing the activation of mammalian target of rapamycin (mTOR)/p70S6K signaling pathway. These findings offer a fresh approach to treating ulcerative colitis (UC) and a theoretical foundation for the future advancement of CP-A.

CDK7 and CDK9 inhibition interferes with transcription, translation, and stemness, and induces cytotoxicity in GBM irrespective of temozolomide sensitivity.

BACKGROUND: Glioblastoma (GBM) is refractory to current treatment modalities while side effects of treatments result in neurotoxicity and cognitive impairment. Here we test the hypothesis that inhibiting CDK7 or CDK9 would effectively combat GBM with reduced neurotoxicity. METHODS: We examined the effect of a CDK7 inhibitor, THZ1, and multiple CDK9 inhibitors (SNS032, AZD4573, NVP2, and JSH150) on GBM cell lines, patient-derived temozolomide (TMZ)-resistant and responsive primary tumor cells and glioma stem cells (GSCs). Biochemical changes were assessed by western blotting, immunofluorescence, multispectral imaging, and RT-PCR. In vivo, efficacy was assessed in orthotopic and subcutaneous xenograft models. RESULTS: CDK7 and CDK9 inhibitors suppressed the viability of TMZ-responsive and resistant GBM cells and GSCs at low nanomolar concentrations, with limited cytotoxic effects in vivo. The inhibitors abrogated RNA Pol II and p70S6K phosphorylation and nascent protein synthesis. Furthermore, the self-renewal of GSCs was significantly reduced with a corresponding reduction in Sox2 and Sox9 levels. Analysis of TCGA data showed increased expression of CDK7, CDK9, SOX2, SOX9, and RPS6KB1 in GBM; supporting this, multispectral imaging of a TMA revealed increased levels of CDK9, Sox2, Sox9, phospho-S6, and phospho-p70S6K in GBM compared to normal brains. RNA-Seq results suggested that inhibitors suppressed tumor-promoting genes while inducing tumor-suppressive genes. Furthermore, the studies conducted on subcutaneous and orthotopic GBM tumor xenograft models showed that administration of CDK9 inhibitors markedly suppressed tumor growth in vivo. CONCLUSIONS: Our results suggest that CDK7 and CDK9 targeted therapies may be effective against TMZ-sensitive and resistant GBM.

ABBV-744 induces autophagy in gastric cancer cells by regulating PI3K/AKT/mTOR/p70S6k and MAPK signaling pathways.

The mortality rates of gastric cancer remain high due to limited therapeutic strategies. As a highly selective inhibitor of the BD2 domain of BET family proteins, ABBV-744 has potent chemotherapeutic activity against various human solid tumors. However, whether ABBV-744 has potential anti-tumor effects in gastric cancer remain largely unknown. In this study, we evaluated the effect of ABBV-744 on gastric cancer cells and explored the possible underlying mechanisms. We found that ABBV-744 inhibited the growth of gastric cancer cells and patient-derived tumor organoids in a dose-dependent manner. Cellular experiments revealed that ABBV-744 induced mitochondria damage, reactive oxygen species accumulation, cell cycle arrest and apoptotic cell death in gastric cancer cells. Transcriptomic analysis using RNA-sequencing data identified autophagy as a crucial pathway involved in the cell death caused by ABBV-744. Mechanically, further studies showed that ABBV-744 induced autophagy flux in gastric cancer cells by inactivating PI3K/AKT/mTOR/p70S6k and activating the MAPK signaling pathways. In vivo mouse xenograft studies demonstrated that ABBV-744 significantly suppressed the growth of gastric cancer cells via inducing autophagy. Taken together, our results suggest that ABBV-744 is a novel drug candidate for gastric cancer.

Diosmin nanocrystal gel alleviates imiquimod-induced psoriasis in rats via modulating TLR7,8/NF-κB/micro RNA-31, AKT/mTOR/P70S6K milieu, and Tregs/Th17 balance.

Diosmin is a flavonoid with promising anti-inflammatory and antioxidant properties. However, it has difficult physicochemical characteristics since its solubility demands a pH level of 12, which has an impact on the drug's bioavailability. The aim of this work is the development and characterization of diosmin nanocrystals using anti-solvent precipitation technique to be used for topical treatment of psoriasis. Results revealed that diosmin nanocrystals stabilized with hydroxypropyl methylcellulose (HPMC E15) in ratio (diosmin:polymer; 1:1) reached the desired particle size (276.9 ± 16.49 nm); provided promising colloidal properties and possessed high drug release profile. Additionally, in-vivo assessment was carried out to evaluate and compare the activities of diosmin nanocrystal gel using three different doses and diosmin powder gel in alleviating imiquimod-induced psoriasis in rats and investigating their possible anti-inflammatory mechanisms. Herein, 125 mg of 5% imiquimod cream (IMQ) was applied topically for 5 consecutive days on the shaved backs of rats to induce psoriasis. Diosmin nanocrystal gel especially in the highest dose used offered the best anti-inflammatory effect. This was confirmed by causing the most statistically significant reduction in the psoriasis area severity index (PASI) score and the serum inflammatory cytokines levels. Furthermore, it was capable of maintaining the balance between T helper (Th17) and T regulatory (Treg) cells. Moreover, it tackled TLR7/8/NF-κB, miRNA-31, AKT/mTOR/P70S6K and elevated the TNFAIP3/A20 (a negative regulator of NF-κB) expression in psoriatic skin tissues. This highlights the role of diosmin nanocrystal gel in tackling imiquimod-induced psoriasis in rats, and thus it could be a novel promising therapy for psoriasis.

Tumor P70S6K hyperactivation is inversely associated with tumor-infiltrating lymphocytes in triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is characterized by large heterogeneity and relative lack of available targeted therapies. To find therapeutic strategies for distinct patients with TNBC, several approaches have been used for TNBC clustering, including recently immune and phosphoproteomic patterns. Based on 70-kDa ribosomal protein S6 kinase (P70S6K)-TNBC clustering, the current study explores the immune profiling in TNBC tumors. METHODS: Stromal tumor-infiltrating lymphocytes (sTILs) were evaluated in human TNBC tumor samples. Furthermore, immunohistochemistry staining for CD8, CD4, Foxp3, and CD20 was performed in tissue microarrays (TMA) sections. RESULTS: Histological analysis showed decreased sTILs, CD20+ cells, and CD8+/CD4+ ratio in high phosphorylated P70S6K (p-P70S6K) tumors. Moreover, p-P70S6K score was directly correlated with CD4+ and Foxp3+ T cells, while it was inversely correlated with CD8+/CD4+ and CD8+/Foxp3+ ratios. CONCLUSION: sTIL infiltration and lymphocyte profiling vary in the context of hyperactivation of P70S6K in TNBC tumors.

Brucein D imparts a growth inhibitory effect in multiple myeloma cells by abrogating the Akt-driven signaling pathway.

The Akt signaling pathway is an oncogenic cascade activated in the bone marrow microenvironment of multiple myeloma (MM) cells and contributes to their uncontrolled proliferation. Abrogation of Akt signaling has been presented as one of the prime therapeutic targets in the treatment of MM. In the present report, we have investigated the effect of Brucein D (BD) on Akt-driven signaling events in MM cells. BD (300 nM) substantially inhibited cell viability and imparted growth-inhibitory effects in U266 cells as evidenced by cell viability assays and flow cytometric analysis. Effect of BD on cell viability was evaluated by MTT assay. Apoptotic cells and cell cycle arrest by BD were analyzed by flow cytometer. The results of the TUNEL assay and western blotting showed that BD induces apoptosis of MM cells by activating caspase-8 and 9 with subsequent reduction in the expression of antiapoptotic proteins (Bcl-2, Bcl-xl, survivin, cyclin D1, COX-2, VEGF, MMP-9). Analysis of activated kinases by Phospho-Kinase Array Kit revealed that Akt, p70S6K, HSP60, p53, and WNK1 were strongly expressed in untreated cells and BD treatment reversed this effect. Using transfection experiments, AKT depletion led to a decrease in phosphorylation of Akt, mTOR, p70S6K, and WNK. However, Akt overexpression led to increase in phosphorylation of these proteins. Depletion of Akt potentiated the apoptosis-inducing effect of BD whereas overexpression displayed resistance to BD-induced apoptosis suggesting the role of Akt in chemoresistance. Taken together, BD mitigates Akt-dependent signaling pathways in MM cells to impart its anticancer activity.

Understanding the Polyamine and mTOR Pathway Interaction in Breast Cancer Cell Growth.

The polyamines putrescine, spermidine and spermine are nutrient-like polycationic molecules involved in metabolic processes and signaling pathways linked to cell growth and cancer. One important pathway is the PI3K/Akt pathway where studies have shown that polyamines mediate downstream growth effects. Downstream of PI3K/Akt is the mTOR signaling pathway, a nutrient-sensing pathway that regulate translation initiation through 4EBP1 and p70S6K phosphorylation and, along with the PI3K/Akt, is frequently dysregulated in breast cancer. In this study, we investigated the effect of intracellular polyamine modulation on mTORC1 downstream protein and general translation state in two breast cancer cell lines, MCF-7 and MDA-MB-231. The effect of mTORC1 pathway inhibition on the growth and intracellular polyamines was also measured. Results showed that polyamine modulation alters 4EBP1 and p70S6K phosphorylation and translation initiation in the breast cancer cells. mTOR siRNA gene knockdown also inhibited cell growth and decreased putrescine and spermidine content. Co-treatment of inhibitors of polyamine biosynthesis and mTORC1 pathway induced greater cytotoxicity and translation inhibition in the breast cancer cells. Taken together, these data suggest that polyamines promote cell growth in part through interaction with mTOR pathway. Similarly intracellular polyamine content appears to be linked to mTOR pathway regulation. Finally, dual inhibition of polyamine and mTOR pathways may provide therapeutic benefits in some breast cancers.

Therapeutic efficacy of the novel SHP2 degrader SHP2-D26, alone or in combination, against lung cancer is associated with modulation of p70S6K/S6, Bim and Mcl-1.

SHP2, a protein tyrosine phosphatase, plays a critical role in fully activating oncogenic signaling pathways such as Ras/MAPK downstream of cell surface tyrosine receptors (e.g., EGFR), which are often activated in human cancers, and thus has emerged as an attractive cancer therapeutic target. This study focused on evaluating the therapeutic potential of the novel SHP2 degrader, SHP2-D26 (D26), either alone or in combination, against non-small cell lung cancer (NSCLC) cells. While all tested NSCLC cell lines responded to D26 with IC50s of < 8 µM, a few cell lines (4/14) were much more sensitive than others with IC50s of ≤ 4 µM. There was no clear association between basal levels of SHP2 and cell sensitivities to D26. Moreover, D26 rapidly and potently decreased SHP2 levels in different NSCLC cell lines in a sustained way regardless of cell sensitivities to D26, suggesting that additional factors may impact cell response to D26. We noted that suppression of p70S6K/S6, but not ERK1/2, was associated with cell responses to D26. In the sensitive cell lines, D26 effectively increased Bim levels while decreasing Mcl-1 levels accompanied with the induction of apoptosis. When combined with the third generation EGFR inhibitor, osimertinib (AZD9291), synergistic effects on decreasing the survival of different osimertinib-resistant cell lines were observed with enhanced induction of apoptosis. Although D26 alone exerted moderate inhibition of the growth of NSCLC xenografts, the combination of osimertinib and D26 effectively inhibited the growth of osimertinib-resistant xenografts, suggesting promising efficacy in overcoming acquired resistance to osimertinib.

Erythropoietin and Bacillus Calmette-Guérin Vaccination Mitigate 3-Nitropropionic Acid-Induced Huntington-like Disease in Rats by Modulating the PI3K/Akt/mTOR/P70S6K Pathway and Enhancing the Autophagy.

Oxidative stress and mitochondrial dysfunction are among the mechanisms expected to explain the pathogenesis of Huntington's disease. Erythropoietin (EPO) and the Bacillus Calmette-Guérin (BCG) vaccine have neuroprotective effects against neurodegenerative diseases; however, the full mechanisms of their action are currently unclear. Here, for the first time, we investigated the neuroprotective effect of BCG vaccination in Huntington-like disease induced by 3-nitropropionic acid (3-NP) and its combination with EPO. Male Wistar rats were randomized into five groups: saline-treated control; 3-NP group (20 mg/kg/day, i.p.) for 7 days; EPO-treated group (5000 IU/kg/day, i.p.) for 14 days after 3-NP administration; live BCG vaccine prophylactic group (5000 cfu/g, i.p.) 10 days prior to 3-NP administration; and live BCG vaccine (5000 cfu/g, i.p.) 10 days before 3-NP administration, followed by EPO treatment (5000 IU/kg/day, i.p.) for 14 days. In a histopathological examination, striatum neurodegeneration was evidenced in the 3-NP injected rats. Administration of 3-NP elevated the levels of p-PI3K, p-Akt, p-mTOR, p-P70S6K, BAX, malondialdehyde, nitric oxide, and cytochrome oxidase while reduced the levels of BCL-2, superoxide dismutase, reduced glutathione, and the autophagy marker microtubule-associated protein light chain 3 in the striatum. EPO and BCG ameliorated the biochemical, histopathological, and behavioral derangements induced by 3-NP, with prominent neuroprotection observed in rats administered the BCG prophylactic combined with EPO treatment. These results highlight the role played by EPO and BCG in the management of 3-NP-induced Huntington-like disease by inhibiting the PI3K/Akt/mTOR/P70S6K pathway and enhancing the autophagy.

p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice.

BACKGROUND & AIMS: Progression of chronic liver disease (CLD) to liver cirrhosis and liver cancer is a major global cause of morbidity and mortality. Treatment options capable of inhibiting progression of liver fibrosis when etiological treatment of CLD is not available or fails have yet to be established. We investigated the role of serine/threonine kinase p70 ribosomal protein S6 kinase (p70S6K) as checkpoint of fibrogenesis in hepatic stellate cells (HSCs) and as target for the treatment of liver fibrosis. APPROACH & RESULTS: Immunohistochemistry was used to assess p70S6K expression in liver resection specimen. Primary human or murine HSCs from wild-type or p70S6K-/- mice as well as LX-2 cells were used for in vitro experiments. Specific small interfering RNA or CEP-1347 were used to silence or inhibit p70S6K and assess its functional relevance in viability, contraction and migration assays, fluorescence-activated cell sorting, and Western blot. These results were validated in vivo by a chemical model of fibrogenesis using wild-type and p70S6K-/- mice. Expression of p70S6K was significantly increased in human cirrhotic vs noncirrhotic liver-tissue and progressively increased in vitro through activation of primary human HSCs. Conversely, p70S6K induced fibrogenic activation of HSCs in different models, including the small interfering RNA-based silencing of p70S6K in HSC lines, experiments with p70S6K-/- cells, and the pharmacological inhibition of p70S6K by CEP-1347. These findings were validated in vivo as p70S6K-/- mice developed significantly less fibrosis upon exposure to CCl4. CONCLUSIONS: We establish p70S6K as a checkpoint of fibrogenesis in vitro and in vivo and CEP-1347 as potential treatment option that can safely be used for long-term 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 Gsk3ß phosphorylation. PF-4708671 also accelerated integrin αIIbß3-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-Gsk3ß signaling was blocked by the Gsk3ß-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.

The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth.

BACKGROUND: As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC. METHODS: Three NSCLC cell lines (A549, SK-MES-1, and NCI-H460) were treated with PF-4708671 at five different concentrations, including 0.1µM, 0.3µM, 1µM, 3µM and 10µM, and protein levels were determined by Western-blot. Then, PF-4708671's effects were assessed both in vitro (cell proliferation, apoptosis, cell cycle distribution, and invasion) and in vivo. RESULTS: The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo. CONCLUSION: These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.

Everolimus.

Everolimus is an immunosuppressant that blocks growth factor-mediated proliferation of haematopoietic and nonhaematopoietic cells. Oral everolimus 0.75 or 1.5mg twice daily significantly reduced the incidence of the primary composite endpoint, efficacy failure 6 months after transplantation, compared with azathioprine 1-3 mg/kg/day, in adult cardiac transplant recipients. All patients also received baseline immunosuppression with cyclosporin and corticosteroids. The incidence of efficacy failure remained significantly lower in everolimus recipients than in those receiving azathioprine 1 and 2 years after cardiac transplantation. However, graft and patient survival rates at 1 year were similar in patients receiving everolimus or azathioprine. The incidence of graft vasculopathy 2 years after transplantation was significantly lower in cardiac transplant recipients receiving everolimus 0.75 mg twice daily than in those receiving azathioprine. The combined incidence of biopsy-confirmed acute rejection, graft loss, death, or loss to follow-up was similar in adult patients receiving everolimus 1.5 or 3 mg/day or mycophenolate mofetil (MMF) 2 g/day 1 or 3 years after renal transplantation. Patients also received baseline immunosuppression with cyclosporin and corticosteroids. Compared with azathioprine and MMF, everolimus is associated with a lower incidence of cytomegalovirus infection in cardiac and renal transplant recipients. Everolimus has been associated with thrombocytopenia, leucopenia and elevated serum lipids and creatinine.