Phosphorylation of p70S6 kinase predicts overall survival in patients with clear margin‐resected hepatocellular carcinoma

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitors play a key role in regulating signal transduction by blocking the mTOR pathway and combining anticancer and immunosuppressive properties. This study was undertaken to determine the prevalence and clinicopathological relevance of phospho‐p70S6 (p‐p70S6) kinase in hepatocellular carcinoma (HCC) and to investigate the effects of rapamycin on HCC in vitro. Methods: A total of 196 patients with HCCs were treated either with surgical resection (n=106) or liver transplantation (n=90). Tumour tissue was investigated for p‐p70S6, phospho‐AKT, Ki‐67, Cyclin‐D1 and apoptosis, and staining results were correlated with clinicopathologically relevant parameters. Results: Overall, p‐p70S6 was detected in 24.5% (48/196) of HCCs. In the resection group, 26.4% (28/106) of HCC were positive and 22.2% (20/90) in the transplant group. p‐p70S6 was significantly associated with elevated Cyclin‐D1 immunoexpression and was correlated with decreased overall survival (P=0.011) in patients resected with a clear margin. In multivariate COX regression analysis, p‐p70S6 was identified as an independent prognostic parameter in patients resected with a clear margin. Rapamycin induced apoptosis and growth inhibition by G0/G1 cell cycle arrest in vitro. However, in HCC patients p‐p70S6 kinase was not associated with proliferation or apoptosis. Conclusions: Activation of p70S6 kinase indicates aggressive tumour behaviour in patients with clear margin‐resected HCC. Identification of p‐p70S6 kinase in HCC selects high‐risk patients who may benefit from drugs targeting the mTOR pathway.     

Chronic Treatment with FK506 Increases p70 S6 Kinase Activity Associated with Reduced Nitric Oxide Synthase Activity in Rabbit Hearts

FK506, an immunosuppressant, modulates phosphorylation of nitric oxide (NO) synthase, and induces cardiac hypertrophy in clinical settings. Having recently reported that chronic treatment with an inhibitor of NO synthase induces cardiac hypertrophy associated with the activation of 70-kD S6 kinase (p70^S6K), which plays an important role in cardiac hypertrophy by regulating protein synthesis, we investigated the effects of chronic administration of FK506 on NO synthase and p70^S6K activities in hearts. Twenty rabbits were divided into four groups: untreated rabbits, those treated with low-dose FK506 (0.10 mg/kg), those treated with medium-dose FK506 (0.20 mg/kg), and those treated with high-dose FK506 (0.40 mg/kg). FK506 was administered intravenously twice a day. After 4 weeks of treatment with FK506, calcium-dependent NO synthase activity in myocardium in the high-dose FK506 group was lower (P < 0.05) than in the untreated group. p70^S6K activity in myocardium in the high-dose group was higher (P < 0.05) than in the untreated group. There was a significant (P < 0.05) inverse correlation between NO synthase and p70^S6K activities in myocardium. However, the endothelial-dependent vasodilation of aortic rings or plasma levels of NO metabolites during experimental protocols did not differ among the groups studied. These findings suggest that chronic treatment of FK506 activates p70^S6K and reduces NO synthase activity in rabbit hearts. Reduced NO synthase and/or activated p70^S6K activities in hearts might contribute to the cardiac hypertrophy observed in some patients receiving FK506.     

胰岛素对血管平滑肌细胞4E结合蛋白1和核糖体蛋白S6激酶磷酸化的刺激作用

目的研究胰岛素对血管平滑肌细胞(VSMC)蛋白质合成翻译过程的两个调节子,4E-结合蛋白1(4E-BP1)和核糖体蛋白S6激酶,磷酸化的调节作用及其生物学意义。方法培养大鼠胸主动脉VSMC。用3H-亮氨酸和3H-TdR掺入法分别测定蛋白质合成和DNA合成;免疫印迹法检测4E-BP1和核糖体蛋白S6激酶的磷酸化。结果与对照相比,100nmol/L胰岛素显著增加了VSMC的3H-亮氨酸和3H-TdR掺入。同样浓度的胰岛素作用于VSMC,可以诱导4E-BP1和核糖体蛋白S6激酶发生磷酸化。二者的磷酸化分别于胰岛素刺激后,30min和10min达高峰。结论胰岛素可以刺激VSMC的4E-BP1和核糖体蛋白S6激酶发生磷酸化,这可能是胰岛素发挥促进VSMC生长作用的机制。     

NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase

The A₁/A₂ adenosine agonist 5′-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H₂O₂ (200 μM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0 ± 3.2 min in the NECA group versus 29.5 ± 2.2 min in untreated cells, P < 0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3 ± 3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H₂O₂-induced loss of potential. The direct GSK-3β inhibitor SB216763 (3 μM) also prolonged the time to fluorescence loss (49.2 ± 2.1 min, P < 0.001 versus control), and its protection could not be blocked by rapamycin (42.2 ± 2.3 min, P < 0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0 ± 3.8% of the risk zone in control hearts to 11.8 ± 2.0% (P < 0.001), and rapamycin blocked this NECA-induced protection (38.3 ± 3.7%). A comparable protective effect was seen for SB216763 (1 μM) with infarct size reduction to 13.5 ± 2.3% (P < 0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA’s cardioprotection at reperfusion. Returned for 1st revision: 3 November 2005 1st revision received: 3 February 2006 Returned for 2nd revision: 23 February 2006 2nd revision received: 1 March 2006     

Differential p70S6k and 4E-BP1 regulation by insulin and amino acids in vascular endothelial and smooth muscle cells

Abstract Differential stimulation of vascular endothelial and smooth muscle cells proliferation is responsible for atherosclerotic lesions. Amino acids and insulin modulate p70S6k and 4E-BP1 activity, regulating cell growth and proliferation. We hypothesised that nutritional (amino acids) and hormonal (insulin) signals differently modulate protein anabolism in human vascular endothelial (HUVEC) and smooth muscle (HVSMC) cells. We evaluated p70S6kinase and 4E-BP1 phosphorylation in the two cell types, grown in amino acid-free medium with or without insulin (INS, 100 nM) or/and amino acids mixture (AA, 3 mM) and with the selective addition or deprivation of branched chain amino acids (BCAA, 0.5 mM). INS stimulated p70S6k and 4E-BP1 phosphorylation transiently in HUVEC and persistently in HVSMC. AA and INS+AA stimulated p70S6k and 4E-BP1 phosphorylation persistently in HUVEC and HVSMC. AA, but not BCAA alone or BCAA-deprived AA, induced p70S6k phosphorylation in HUVEC. BCAA deprivation decreased the p70S6k phosphorylation induced by AA with or without insulin in HVSMC. These results show that anabolic stimuli modulate p70S6k and 4E-BP1 activity differently in the two vascular cell types, suggesting that insulin stimulates protein synthesis for a longer time in HUSMC than in HUVEC. We speculate that hyperinsulinaemia frequently associated with atherosclerosis could induce a selective HVSMC proliferation.     

Involvement of phosphoinositide-3-kinase and p70 S6 kinase in regulation of proliferation of rat lactotrophs in culture

Phosphoinositide-3-kinase (PI-3K) and p70 S6 kinase (p70^56k) are suggested as important molecules for mediating mitogenic actions of growth factors and cytokines in variety of cell types. The purpose of the present study was to investigate whether these kinases were involved in mediation of the mitogenic actions of not only the growth factor insulin but also cyclic adenosine monophosphate (cAMP) and estrogen on rat cultured lactotrophs. Treatment with wortmannin or LY294002, a PI-3K inhibtor, or rapamycin, a p70^S6k inhibitor, decreased basal levels of 5-bromo-2-deoxyuridine (BrdU)-labeling indices of lactotrophs in a dose-dependent manner. These inhibitors were effective in blocking an increase in BrdU-labeling indices induced by insulin. LY294002 and rapamycin also suppressed an increase in BrdU-labeling indices induced by forskolin, an adenylate cyclase activator, or dibutyryl cAMP, a membrane-permeable cAMP analog, as well as that induced by estradiol, a physiologic extracellular activator of lactotroph proliferation. However, the dibutyryl cAMP-, but not insulin-induced proliferation, acquired a resistance to LY294002 and rapamycin by pretreatment with bromocriptine, a dopaminergic agonist that is able to suppress lactotroph proliferation. These results suggest that the mitogenic actions of cAMP and estradiol on rat lactotrophs are mediated by PI-3K and p70^S6k, and that dopaminergic inhibition modifies the PI-3K and p70^S6k dependence of the regulation of lactotroph proliferation.     

Preclinical validation of a novel compound targeting p70S6 kinase in breast cancer

Breast cancer is a frequent and treatable disease. However, when recurrent, breast cancer often becomes refractory to therapy and progresses into metastatic forms that are typically incurable. Thus, understanding and targeting the critical pathways underlying breast cancer recurrence is urgently needed to eradicate primary disease and achieve better prognosis. Recently, we have demonstrated that the ribosomal protein p70S6K is activated in residual breast cancer cells as a result of post-surgical inflammation and that interfering with its activity in the peri-operative setting strongly suppresses recurrence in a mouse model. In order to develop clinically-exploitable treatments targeting p70S6K, we have tested a newly generated compound, called FS-115. FS-115 potently inhibited p70S6K1 (IC₅₀ 35nM) with high selectivity over other AGC kinases or PI3K pathway kinases. In vitro, treatment with FS-115 efficiently blocked p70S6K activity in breast cancer cell lines and impaired colony formation and anchorage independent growth. Pharmacokinetic profiling showed that FS-115 exhibited high oral bioavailability, optimal plasma distribution and high brain penetrance. In nude mice, FS-115 strongly suppressed tumor take-rate and primary tumor growth. Oral dosing with FS-115 in a peri-operative schedule was effective in decreasing local recurrence of breast cancer and a long-term treatment schedule was well tolerated and efficiently suppressed distant metastasis formation. Altogether, we propose that FS-115 might be a good candidate for the treatment of breast cancer patients at high risk to relapse.

Summary Statement

Our results confirm that inhibition of p70S6K represents a valuable opportunity for restraining loco-regional relapse and metastasis in breast cancer and identify in FS-115 a promising candidate-inhibitor to move from preclinical to clinical treatments.     

Second window of protection following myocardial preconditioning: an essential role for PI3 kinase and p70S6 kinase

Ischaemic preconditioning (IPC) protects the heart against myocardial infarction acutely as well as several hours later (e.g. 24-48 h). The mechanism of the profound cardioprotection is not completely explored. We hypothesized that PI3K/PDK1/Akt/mTOR/p70S6K-mediated pro-survival pathway is involved in delayed cardioprotection induced by IPC. Under Hypnorm-Diazepam anaesthesia, male New Zealand White rabbits were either sham-operated (SC) or preconditioned by four cycles of 5-min ischaemia and 10-min reperfusion on day 1. Twenty-four hours after recovery, the animals were anaesthetized with sodium pentobarbitone and subjected to 30-min ischaemia followed by 180-min reperfusion. Wortmannin (0.6 mg/kg, i.v.), an irreversible PI3 kinase (PI3K) inhibitor, rapamycin (0.25 mg/kg, i.v.), which prevents the phosphorylation of p70S6 kinase (p70S6K), or DMSO (control vehicle) was given 15 min prior to IPC. IPC significantly reduced infarct size compared to the control group (SC) (31.9 +/- 5.8% (n = 7) vs. 54.9 +/- 2.9% (n = 6), P < 0.05). Wortmannin and rapamycin alone had no effect on infarct size (56.3 +/- 1.6% (n = 6) and 54.7 +/- 3.8% (n = 6), respectively). However, when wortmannin or rapamycin were given prior to IPC the protection was completely abolished (49.9 +/- 2.8% (n = 6), 45.1 +/- 4.6% (n = 7), P < 0.05 vs. IPC). Western blot analysis showed that wortmannin, at a dose of 0.6 mg/kg, and rapamycin, at a dose of 0.25 mg/kg, were sufficient to prevent phosphorylation of Akt and p70S6K, respectively, when the inhibitors were given prior to IPC. We conclude that PI3K/PDK1/Akt/mTOR/p70S6K-signalling pathway plays an essential role in the development of the cardioprotection against infarction in rabbits.     

Lesion-targeted thrombopoietin potentiates vasculogenesis by enhancing motility and enlivenment of transplanted endothelial progenitor cells via activation of Akt/mTOR/p70S6kinase signaling pathway

Thrombopoietin (TPO), a physiological regulator of megakaryocyte and platelet development, is a multifunctional positive regulator in early hematopoiesis by hematopoietic stem cells. In this study, we investigated the effect of TPO on endothelial progenitor cells (EPCs) for therapeutic vasculogenesis in vitro and in vivo, and the intracellular signaling mechanism exerting the activity of EPCs. 7-day culture-expanded EPCs derived from human peripheral blood mononuclear cells were applied to each assay. Flow cytometry demonstrated the expression of c-Mpl, the receptor of TPO, in cultured EPCs. In vitro experiments revealed enhanced migration and survival of cultured EPCs by TPO. In vivo, TPO was intramuscularly administered into the foci of ischemic hindlimbs in athymic nude mice, immediately followed by intravenous injection of cultured EPCs, to assess the booster effect of TPO on vascular regeneration. At day 4 post-transplantation, transplanted EPCs were 1.7-fold higher in TPO-treated animals compared to control. At day 28, blood perfusion was recovered in the TPO-treated group, accompanied by an increase in microvascular density. The signaling transduction pathway underlying TPO-mediated activities of cultured EPCs was assessed by Western blotting. TPO induced sequential phosphorylations of Akt to p70S6kinase through mTOR. Inhibition of the PI3-kinase/Akt/mTOR/p70S6kinase signaling pathway negated the biological functions of cultured EPCs, either migration (by LY294002 for PI3-kinase and Rapamycin for mTOR) or survival and tubulogenesis (by Rapamycin). These findings provide evidence that TPO possesses booster potential for therapeutic vasculogenesis, by activating the PI3-kinase/Akt/mTOR/p70S6kinase pathway crucial to the biological activities of EPCs.     

Deleted in liver cancer 2 suppresses cell growth via the regulation of the Raf‐1–ERK1/2–p70S6K signalling pathway

Background: Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase‐activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation. Aims: In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain. Methods and results: On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ‐stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ‐stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector‐stable clone, suggesting that downregulation of RhoA activity in DLC2γ‐stable clones inhibited cell cycle progression. In the DLC2γ‐stable clone, the levels of Raf‐1 and extracellular signal‐regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ–GAP defective mutant‐stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2‐stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho‐p70S6K and phospho‐ERK1/2 were upregulated. Conclusion: Our data show that DLC2 inhibits the activity of Raf‐1–ERK1/2–p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.