Activation Of P70 S6 Kinase Research Articles

Pharmacodynamic Monitoring of NFAT- Regulated Gene Expression and P70S6 Kinase Activity in Kidney Transplant Patients With BKV Infection.

Purpose: Measuring drug levels has been unhelpful in guiding management of patients with BKV infection. We measured NFAT-regulated gene expression and p70S6k activity in kidney transplant recipients (KTR) with BKV and correlated the results with clinical outcome. Methods: KTR with BK viruria > 1 million copies/ml or any detectable viremia on tacrolimus (TAC), mycophenolic acid (MPA) and prednisone were randomized to MPA reduction or conversion to everolimus (EVR). The primary endpoint (PE) was clearance of viremia or halving of viruria at 3 months (M). As a sub-study, peripheral blood was collected pre- (T0) and 1.5 hours post- (T1.5) TAC dose at enrollment and 3 M after randomization. Cells were stimulated with PMA/ionomycin. RT- PCR quantified the expression of NFAT-regulated genes IL-2, IFN-γ, and GM-CSF. Residual expression of each gene was calculated as T1.5/T0 x 100 and the average value expressed as mean residual expression (MRE). P70S6K activity was detected by flow cytometry and expressed as mean fluorescence intensity (MFI). The Mann-Whitney U test for comparison of median values was used. Results: 14 KTR with BKV enrolled. At enrollment, KTR with BKV viremia had significantly (SS) lower MRE than those with viruria alone (17.1% CI 14.3-36.4 vs. 43.3% CI 27.7-74.3 P= 0.04) despite similar TAC troughs (8.4, CI 7.0-10.4 vs 9.4, CI 8.6-10, P= 0.35) and P70S6K activity (5264 MFI CI 3379-8659 vs. 8128 MFI CI 2830-11593, P= 0.5). 12/14 KTR have data at 3 M of which 3 reached the PE. Median MRE at enrollment was SS higher in KTR who reached the PE (70.9% CI 27.5-98.1 vs. 17.1% CI 14.4-35, P =0.01). There was no SS difference between those that did and did not reach the PE in TAC levels at enrollment (9.7 CI 6.7-12.7 vs. 8.4 CI 7.4-10.4, P=0.35); at 3 M (5.7 CI 3.7-8.8 vs. 7.6 CI 5.6-8.8, P= 0.35) or P70S6K at enrollment (6765 CI-2904-15434 vs. 4151 CI 2390-7008, P=0.48); at 3 M (2959 CI -5777-14215 vs. 6508 CI 3109-7626, P=1.0). MRE at 3 M did not correlate with outcome (64.01 CI -7.2-128 vs. 53.1 CI 32.3-65.8, P=0.37). Conclusion: MRE of NFAT regulated genes at the time of BKV diagnosis, but not TAC levels or P70S6K activity, correlated with severity of BKV infection and outcome. Pharmacodynamic monitoring of NFAT regulated gene expression may play an important role in prognosis of BKV infection and may aid providers in the management of BKV associated disease. DISCLOSURES:Webber, A.: Grant/Research Support, Novartis, Astellas. Wojciechowski, D.: Grant/Research Support, Novartis, Bristol-Myers Squibb. Vincenti, F.: Grant/Research Support, Pfizer, Alexion, Novartis, Astellas, Bristol-Myers Squibb, Genetech.

FINAL RESULTS OF A PROSPECTIVE MULTI-INSTITUTIONAL PHASE II STUDY OF EVEROLIMUS (RAD001), AN MTOR INHIBITOR, IN PEDIATRIC PATIENTS WITH RECURRENT OR PROGRESSIVE LOW-GRADE GLIOMA. A POETIC CONSORTIUM TRIAL

BACKGROUND: Purpose: The ras/raf signaling pathway is crucial in the development of pediatric low-grade gliomas (LGGs). Aberrant ras/raf signaling is involved in tumorigenesis through promotion of cell proliferation, survival, and differentiation in sporadic LGG. Everolimus (RAD001) is a potent and selective inhibitor of mTOR, a downstream element of the ras/raf pathway. The activity, safety and pharmacokinetics of everolimus in pediatric patients with radiographic recurrent/progressive LGG are presented. METHODS: Pediatric patients with radiographic progressive or recurrent LGGs without neurofibromatosis type I were treated with oral everolimus 5mg/m2/dose once daily. Therapy was provided for 28 days (one cycle) and could be repeated for a total of 12 cycles. Response, as determined by standard 2-D MRI criteria, was assessed for all patients. Pharmacokinetics, pharmacogenetics, pharmacodynamic parameters including inhibition of p70s6 kinase activity, 4E-BP1 phosphorylation inhibition and suppression of cMyc expression, as well as the toxicity profile of everolimus were evaluated. RESULTS: Twenty-three patients with a median age of 9 years (range, 3–17 years) were enrolled, all of whom had received prior chemotherapy (average # regimens = 2.7) including progression after a carboplatin-containing regimen. Median number of cycles of therapy was 10 (range, 1-12). Responses were determined by blinded central review and included 4 patients with PR (>50% decrease) and 13 with stable disease. Six patients had progressive disease by one year. Overall therapy was well tolerated; two patients discontinued therapy due to mouth sores (n = 1) and withdrawal of consent (n = 1). Everolimus PK parameters were similar to those previously reported in both adult and pediatric patients and drug trough levels were maintained above 5ng/ml. Pharmacodynamic analysis demonstrated inhibition of downstream targets of mTOR including phospho-S6 kinase, 4E-BP1 phosphorylation and cMyc expression within 14 days of initiation of everolimus therapy and this inhibition was maintained throughout the treatment period. CONCLUSIONS: Everolimus was well tolerated in pediatric patients with progressive or recurrent LGGs and demonstrated activity in this multiply recurrent/progressive patient population. Incorporation of everolimus into frontline LGG therapy is being proposed. SECONDARY CATEGORY: Clinical Neuro-Oncology.

SGK3 is an androgen-inducible kinase promoting prostate cancer cell proliferation through activation of p70 S6 kinase and up-regulation of cyclin D1.

Both androgen and phosphatidylinositol 3-kinase (PI3K) signaling are critical for cell proliferation of androgen receptor (AR)-positive prostate cancer cells, but the underlying mechanisms are still not fully understood. Here we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a Ser/Thr kinase functioning downstream of PI3K, is an AR transcriptional target and promotes prostate cancer cell proliferation. SGK3 expression is up-regulated by androgen DHT via AR. We identified an AR-binding region at the sgk3 locus, which confers androgen responsiveness of sgk3 promoters. Interestingly, we found that androgen/AR-dependent SGK3 expression requires estrogen receptor (ER) (including both isoforms, ERα and ERβ). Depletion of ER blocked DHT-induced SGK3 expression. Functionally, knockdown of SGK3 expression significantly decreased LNCaP prostate cancer cell proliferation by inhibiting G1 to S phase cell cycle progression. We further provided evidence that SGK3 promotes p70 S6 kinase (p70S6K) activation and increases cyclin D1 levels. In summary, our study identifies SGK3 as an AR target and provides a novel androgen-induced cell proliferation mechanism mediated by the AR-SGK3-p70S6K-cyclin D1 pathway in prostate cancer cells.

Transforming Growth Factor-β Signaling Participates in the Maintenance of the Primordial Follicle Pool in the Mouse Ovary

Physiologically, only a few primordial follicles are activated to enter the growing follicle pool each wave. Recent studies in knock-out mice show that early follicular activation depends on signaling from the tuberous sclerosis complex, the mammalian target of rapamycin complex 1 (mTORC1), phosphatase and tensin homolog deleted on chromosome 10, and phosphatidylinositol 3-kinase (PI3K) pathways. However, the manner in which these pathways are normally regulated, and whether or not TGF-β acts on them are poorly understood. So, this study aims to identify whether or not TGF-β acts on the process. Ovary organ culture experiments showed that the culture of 18.5 days post-coitus (dpc) ovaries with TGF-β1 reduced the total population of oocytes and activated follicles, accelerated oocyte growth was observed in ovaries treated with TGF-βR1 inhibitor 2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine (SD208) compared with control ovaries, the down-regulation of TGF-βR1 gene expression also activated early primordial follicle oocyte growth. We further showed that there was dramatically more proliferation of granulosa cells in SD208-treated ovaries and less proliferation in TGF-β1-treated ovaries. Western blot and morphological analyses indicated that TGF-β signaling manipulated primordial follicle growth through tuberous sclerosis complex/mTORC1 signaling in oocytes, and the mTORC1-specific inhibitor rapamycin could partially reverse the stimulated effect of SD208 on the oocyte growth and decreased the numbers of growing follicles. In conclusion, our results suggest that TGF-β signaling plays an important physiological role in the maintenance of the dormant pool of primordial follicles, which functions through activation of p70 S6 kinase 1 (S6K1)/ribosomal protein S6 (rpS6) signaling in mouse ovaries.

CXCL4L1 and CXCL4 signaling in human lymphatic and microvascular endothelial cells and activated lymphocytes: involvement of mitogen-activated protein (MAP) kinases, Src and p70S6 kinase

CXC chemokines influence a variety of biological processes, such as angiogenesis, both in a physiological and pathological context. Platelet factor-4 (PF-4)/CXCL4 and its variant PF-4var/CXCL4L1 are known to favor angiostasis by inhibiting endothelial cell proliferation and chemotaxis. CXCL4L1 in particular is a potent inhibitor of angiogenesis with anti-tumoral characteristics, both through regulation of neovascularization and through attraction of activated lymphocytes. However, its underlying signaling pathways remain to be elucidated. Here, we have identified various intracellular pathways activated by CXCL4L1 in comparison with other CXCR3 ligands, including CXCL4 and interferon-γ-induced protein 10/CXCL10. Signaling experiments show involvement of the mitogen-activated protein kinase (MAPK) family in CXCR3A-transfected cells, activated lymphocytes and human microvascular endothelial cells (HMVEC). In CXCR3A transfectants, CXCL4 and CXCL4L1 activated p38 MAPK, as well as Src kinase within 30 and 5 min, respectively. Extracellular signal-regulated kinase (ERK) phosphorylation occurred in activated lymphocytes, yet was inhibited in microvascular and lymphatic endothelial cells. CXCL4L1 and CXCL4 counterbalanced the angiogenic chemokine stromal cell-derived factor-1/CXCL12 in both endothelial cell types. Notably, inhibition of ERK signaling by CXCL4L1 and CXCL4 in lymphatic endothelial cells implies that these chemokines might also regulate lymphangiogenesis. Furthermore, CXCL4, CXCL4L1 and CXCL10 slightly enhanced forskolin-stimulated cAMP production in HMVEC. Finally, CXCL4, but not CXCL4L1, induced activation of p70S6 kinase within 5 min in HMVEC. Our findings confirm that the angiostatic chemokines CXCL4L1 and CXCL4 activate both CXCR3A and CXCR3B and bring new insights into the complexity of their signaling cascades.

Resveratrol inhibits BMP-4-stimulated VEGF synthesis in osteoblasts: Suppression of S6 kinase

Resveratrol is well known as a natural polyphenol abundantly found in red wine. We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates vascular endothelial growth factor (VEGF) synthesis via p70 S6 kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of resveratrol on the BMP-4-stimulated VEGF synthesis in MC3T3-E1 cells. Resveratrol significantly suppressed BMP-4-stimulated release and expression levels of VEGF mRNA. SRT1720, an activator of SIRT1 with potencies greater than resveratrol, also reduced VEGF release and the mRNA levels. Both resveratrol and SRT1720 markedly attenuated the BMP-4-induced phosphorylation of p70 S6 kinase without affecting the BMP-4-induced phosphorylation of Smad1/5/8. These findings strongly suggest that resveratrol attenuates BMP-4-stimulated VEGF synthesis through suppression of the activation of p70 S6 kinase in osteoblasts, and that the inhibitory effect is mediated at least in part by SIRT1 activation.

δ-Tocotrienol oxazine derivative antagonizes mammary tumor cell compensatory response to CoCl2-induced hypoxia.

In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival. Studies were conducted to determine the role of δ-tocotrienol and a semisynthetic δ-tocotrienol oxazine derivative, compound 44, on +SA mammary tumor cell hypoxic response. Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response. CoCl2-induced hypoxia was also associated with a large increase in Akt/mTOR signaling, activation of downstream targets p70S6K and eIF-4E1, and a significant increase in VEGF production, and combined treatment with compound 44 blocked this response. Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6kinase and eIF-4E1. These findings demonstrate that treatment with the δ-tocotrienol oxazine derivative, compound 44, significantly attenuates +SA mammary tumor cell compensatory responses to hypoxia and suggests that this compound may provide benefit in the treatment of rapidly growing solid breast tumors.

Estradiol enhances CIP2A expression by the activation of p70 S6 kinase

Cancerous inhibitor of PP2A (CIP2A) stimulates the proliferation of various cancer cells, and 17β-estradiol (E₂) enhances the proliferation of breast cancer cells. E₂ activates epidermal growth factor receptor (EGFR), stimulating the MEK1/2 and PI3K pathways, and CIP2A expression is increased by the MEK1/2-induced transcription factor ETS1. It is possible for E₂ to increase CIP2A expression. This study examined whether E₂ could increase CIP2A expression and whether CIP2A is highly expressed in estrogen receptor (ER)-positive breast cancer tissues. E₂ increased CIP2A expression at the translational level in a c-MYC-independent manner in MCF-7 cells. E₂-enhanced proliferation was impaired without CIP2A expression. E₂-stimulated EGFR activated the MAPK and PI3K pathways, which converged to activate p70 S6 kinase (S6K). Phosphorylation at all the three phosphorylation sites (S424/T421, T229, and T389) on S6K was required for the phosphorylation of eukaryotic initiation factor 4B (eIF4B), which was responsible for the increase in CIP2A translation. Furthermore, CIP2A expression was higher in ER-positive tissues than in ER-negative tissues. This is the first study, to our knowledge, to demonstrate that CIP2A is a key factor in E₂-enhanced proliferation and that estrogen regulates CIP2A expression by non-genomic action through EGFR.

Vascular tumors have increased p70 S6-kinase activation and are inhibited by topical rapamycin

Vascular tumors are endothelial cell neoplasms whose cellular and molecular mechanisms, leading to tumor formation, are poorly understood, and current therapies have limited efficacy with significant side effects. We have investigated mechanistic (mammalian) target of rapamycin (mTOR) signaling in benign and malignant vascular tumors, and the effects of mTOR kinase inhibitor as a potential therapy for these lesions. Human vascular tumors (infantile hemangioma and angiosarcoma) were analyzed by immunohistochemical stains and western blot for the phosphorylation of p70 S6-kinase (S6K) and S6 ribosomal protein (S6), which are activated downstream of mTOR complex-1 (mTORC1). To assess the function of S6K, tumor cells with genetic knockdown of S6K were analyzed for cell proliferation and migration. The effects of topical rapamycin, an mTOR inhibitor, on mTORC1 and mTOR complex-2 (mTORC2) activities, as well as on tumor growth and migration, were determined. Vascular tumors showed increased activation of S6K and S6. Genetic knockdown of S6K resulted in reduced tumor cell proliferation and migration. Rapamycin fully inhibited mTORC1 and partially inhibited mTORC2 activities, including the phosphorylation of Akt (serine 473) and PKCα, in vascular tumor cells. Rapamycin significantly reduced vascular tumor growth in vitro and in vivo. As a potential localized therapy for cutaneous vascular tumors, topically applied rapamycin effectively reduced tumor growth with limited systemic drug absorption. These findings reveal the importance of mTOR signaling pathways in benign and malignant vascular tumors. The mTOR pathway is an important therapeutic target in vascular tumors, and topical mTOR inhibitors may provide an alternative and well-tolerated therapy for the treatment of cutaneous vascular lesions.

Inhibition of ERK1/2 Worsens Intestinal Ischemia/Reperfusion Injury

BackgroundThe role of extracellular signal-regulated protein kinase (ERK) in intestinal ischemia/reperfusion (I/R) injury has not been well investigated. The aim of the current study was to examine the effect of inhibition of the ERK pathway in an in vitro and in vivo model of intestinal I/R injury.MethodsERK1/2 activity was inhibited using the specific inhibitor, U0126, in intestinal epithelial cells under hypoxia/reoxygenation conditions and in mice subjected to 1 hour of intestinal ischemia followed by 6 hours reperfusion. In vitro, cell proliferation was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, apoptosis by DNA fragmentation, and migration using an in vitro model of intestinal wound healing. Cells were also transfected with a p70S6K plasmid and the effects of overexpression similarly analyzed. In vivo, the effects of U0126 on intestinal cell proliferation and apoptosis, intestinal permeability, lung and intestinal neutrophil infiltration and injury, and plasma cytokine levels were measured. Survival was also assessed after U0126. Activity of p70S6 kinase (p70S6K) was measured by Western blot.ResultsIn vitro, inhibition of ERK1/2 by U0126 significantly decreased cell proliferation and migration but enhanced cell apoptosis. Overexpression of p70S6K promoted cell proliferation and decreased cell apoptosis. In vivo, U0126 significantly increased cell apoptosis and decreased cell proliferation in the intestine, increased intestinal permeability, intestinal and lung neutrophil infiltration, and injury, as well as systemic pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Mortality was also significantly increased by U0126. Inhibition of ERK1/2 by U0126 also abolished activity of p70S6K both in vitro and in vivo models.ConclusionPharmacologic inhibition of ERK1/2 by U0126 worsens intestinal IR injury. The detrimental effects are mediated, at least in part, by inhibition of p70S6K, the major effector of mammalian target of rapamycin pathway.

Effect of IRS4 levels on PI 3-kinase signalling.

Insulin receptor substrate 1 (IRS1) and IRS2 are well-characterized adapter proteins that relay signals from receptor tyrosine kinases to downstream components of signalling pathways. In contrast, the function of IRS4 is not well understood. IRS4 overexpression has been associated with acute lymphoblastic leukaemia and subungual exostosis, while point mutations of IRS4 have been found in melanomas. Here, we show that while IRS4 expression is low in most cancer cell lines, IRS4 mRNA and protein levels are markedly elevated in certain cells including the NCI-H720, DMS114, HEK293T and HEK293AAV lines. Surprisingly, IRS4 expression was also strongly induced when HEK293 cells were infected with retroviral particles and selected under puromycin, making IRS4 expression a potential off-target effect of retroviral expression vectors. Cells with high expression of IRS4 displayed high phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, as well as elevated Akt and p70 S6 kinase activities, even in the absence of growth factors. PI 3-kinase (PI3K) signalling in these cells depends on IRS4, even though these cells also express IRS1/2. Knockdown of IRS4 also inhibited cell proliferation in cells with high levels of IRS4. Together, these findings suggest IRS4 as a potential therapeutic target for cancers with high expression of this protein.

Inhibition of breast cancer local relapse by targeting p70S6 kinase activity

Inhibition of breast cancer local relapse by targeting p70S6 kinase activity

Rosiglitazone inhibits insulin-like growth factor-1-induced polycystic kidney disease cell growth and p70S6 kinase activation

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic kidney disorders. Thiazolidinediones (TZDs) are anti-diabetic drugs that have been shown to suppress polycystic kidney diseases (PKD) development. However, their underlying mechanism of action remains largely unknown. Insulin-like growth factor-1 (IGF-1) expression increases with the progression of cystic lesions in ADPKD and murine PKD, thus the increased expression of IGF-1 may contribute to the progression of cystic lesions. p70S6 kinase (p70S6K) is an important downstream signaling molecule of IGF-1 and is implicated in the regulation of cell cycle progression and cell proliferation. In the present study, we found that IGF-1 increased the growth of cyst-lining epithelial cells by 15-20% in a dose-dependent manner, while no effect on the proliferation of normal renal cortical tubular epithelial cells (RCTEC) was observed. Rosiglitazone, a TZD, was found to inhibit the IGF-1-induced growth of cyst-lining epithelial cells when applied at a dose of 50-200 µM. However, the IGF-1-induced growth of immortalized epithelial cells from >30 individual renal cysts obtained from 11 ADPKD patients (WT9-12 cells) was inhibited with a 12.5-µM dose of rosiglitazone. Moreover, rosiglitazone (at the same concentration) was shown to inhibit the IGF-1-induced activation of p70S6K. TZDs are known to exert antitumor properties via peroxisome proliferator-activated receptor (PPAR)γ-dependent and -independent mechanisms. The present study showed that PPARγ small interfering RNA (siRNA) did not block the effect of rosiglitazone in inhibiting the IGF-1-induced phosphorylation of p70S6K. In conclusion, cyst-lining epithelial cells were found to be more sensitive to IGF-1 compared with normal cells. Rosiglitazone inhibited the proliferation of cyst-lining epithelial cells; more specifically, it inhibited the proliferation-promoting activity of IGF-1 in these cells. This effect of rosiglitazone was demonstrated to be partially due to the inhibition of IGF-1-induced activation of p70S6K. Increased IGF-1 expression was identified in early-stage PKD, indicating that rosiglitazone is more suitable for the treatment of early-stage PKD.

Abstract 34: A phase I study of a p70S6 kinase inhibitor (LY2584702) in patients (pts) with solid tumors.

Abstract Introduction: LY2584702 is an inhibitor of p70S6 kinase, a downstream effector of the AKT pathway which is activated in multiple tumor types. Suppressing p70S6 kinase activity is expected to inhibit cell growth, cell proliferation, and possibly cell death. A phase 1 study was conducted to evaluate the safety and tolerability of LY2584702 in Japanese pts with solid tumors. Methods: Patients received a single oral dose of LY2584702 on Day1, followed by the same doses given twice daily (BID) from Day3 to Day30 (Cycle 1), and were evaluated for occurrence of dose limiting toxicity (DLT). Other measurements included safety, pharmacokinetics, pharmacodynamics (PD biomarkers) and efficacy. The first 3 pts were assigned to 50 mg BID LY2584702, and the following 6 pts assigned to 75 mg BID. Study treatment was repeated until progressive disease or intolerable toxicity occurred. Results: Cancer types included colon, gastric and esophagus cancers. All pts in the trial completed Cycle 1 without showing DLT. There were no serious adverse events or discontinuations due to adverse events during the study. Adverse events of Grade 3 were observed in 2 pts receiving 50 mg BID LY2584702: one with increased alkaline phosphatase, and another with increased bilirubin. Of these 9 pts, 5 had aPTT elongation and 4 had PT/INR elongation. After a single dose, the PK parameters showed relatively high variability; tmax, t1/2, and apparent clearance ranged from 5 to 8 h, 14.7 to 28.1 h, and 5.18 to 7.79 L/h, respectively. Accumulation ratio of exposure ranged from 1.41 to 2.07. While PD biomarkers (pS6RP, pAKT, and pPRAS40) showed no meaningful findings, a pattern in the kinetics of these biomarkers was observed after LY2584702 exposure; there was a reduction in the level of expression of pS6 positive cells from the entire epidermis, and 3 PD biomarkers in PBMCs showed the same kinetic pattern with consistent down-regulation. No complete or partial responses were observed, but 2 pts (colon and esophagus cancers) exhibited long-term stable disease (one for 183 days; the other 137 days). Conclusion: No remarkable safety concern was revealed during the phase 1 study of LY2584702 in Japanese solid tumor pts. Despite a limited number of pts and high variability, Cmax and AUC in this study were generally consistent with those in a previous US phase 1 study. Coagulation response was down-regulated after LY2584702 exposure, which would have opposite effects of everolimus. In addition, since a pattern of kinetics of PD biomarkers was observed in both PBMCs and skin biopsies after LY2584702 exposure, our PD marker analysis may encourage their future potential as biomarker candidates for PI3K-Akt pathway. Citation Format: Toshihiko Doi, Naoto Yoshizuka, Risa Sekiguchi, Risa Nasu, Akihiko Kido, Leslie H. Brail, Paul Matthew Westwood, Atsushi Ohtsu. A phase I study of a p70S6 kinase inhibitor (LY2584702) in patients (pts) with solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 34. doi:10.1158/1538-7445.AM2013-34

Inhibition of S6 kinase suppresses the apoptotic effect of eIF4E ablation by inducing TGF-β-dependent G1 cell cycle arrest

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cap-dependent translation through its direct activation of ribosomal protein p70 S6 kinase (S6 kinase) and indirect activation of eukaryotic initiation factor 4E (eIF4E). We recently reported that inhibition of eIF4E expression caused apoptosis in cancer cells in the absence of serum. This was indicated by treatment with the mTORC1 inhibitor rapamycin, which suppressed both S6 kinase and 4E-BP1 phosphorylation (dephosphorylated 4E-BP1 binds and inactivates eIF4E), or by knockdown of eIF4E. We report here that knockdown of eIF4E also causes apoptosis in the presence of serum. This was unexpected because rapamycin induces G1 cell cycle arrest in the presence of serum. Upon investigation, we have found that inactivated S6 kinase prevents the apoptotic effect observed by singular knockdown of eIF4E and results in G1 cell cycle arrest. This effect is dependent on TGF-β (transforming growth factor-β) signaling which contributes to G1 cell cycle arrest. Suppression of S6 kinase phosphorylation alone is insufficient to mediate cell cycle arrest, indicating that complete G1 cell cycle arrest is due to suppression of both S6 kinase and eIF4E. These data indicate that the cytostatic effect of rapamycin is suppression of both S6 kinase and eIF4E, while the cytotoxic effects are due suppression of eIF4E in the absence of S6 kinase-dependent activation of TGF-β signals. Our findings place an importance on the evaluating the activity/expression level of S6 kinase and eIF4E as readouts for rapamycin/rapalog efficacy.

Novel withanolides target medullary thyroid cancer through inhibition of both RET phosphorylation and the mammalian target of rapamycin pathway

Despite development of current targeted therapies for medullary thyroid cancer (MTC), long-term survival remains unchanged. Recently isolated novel withanolide compounds from Solanaceae physalis are highly potent against MTCs. We hypothesize that these withanolides uniquely inhibit RET phosphorylation and the mammalian target of rapamycin (mTOR) pathway in MTC cells as a mechanism of antiproliferation and apoptosis. MTC cells were treated with novel withanolides and MTC-targeted drugs. In vitro studies assessed cell viability and proliferation (MTS; trypan blue assays), apoptosis (flow cytometry with Annexin V/PI staining; confirmed by Western blot analysis), long-term cytotoxic effects (clonogenic assay), and suppression of key regulatory proteins such as RET, Akt, and mTOR (by Western blot analysis). The novel withanolides potently reduced MTC cell viability (half maximal inhibitory concentration [IC(50)], 270-2,850 nmol/L; 250-1,380 nmol/L for vandetanib; 360-1,640 nmol/L for cabozantinib) with induction of apoptosis at <1,000 nmol/L of drug. Unique from other targeted therapies, withanolides suppressed RET and Akt phosphorylation and protein expression (in a concentration- and time-dependent manner) as well as mTOR activity and translational activity of 4E-BP1 and protein synthesis mediated by p70S6kinase activation at IC(50) concentrations. Novel withanolides from Physalis selectively and potently inhibit MTC cells in vitro. Unlike other MTC-targeted therapies, these compounds uniquely inhibit both RET kinase activity and the Akt/mTOR prosurvival pathway. Further translational studies are warranted to evaluate their clinical potential.

Abstract B48: Anticancer effect of guava leaves (Psidium guajava L.) in prostate cancer cells

Abstract This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer. Citation Format: Woong Mo Yang, Mi Hye Kim, Sang-Yoon Park, Kwang Seok Ahn, Seok-Geun Lee. Anticancer effect of guava leaves (Psidium guajava L.) in prostate cancer cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B48.

Effects of AMPK activation and combined treatment with AMPK activators and somatostatin on hormone secretion and cell growth in cultured GH-secreting pituitary tumor cells

We investigated the effects of the AMPK activator AICAR as compared to somatostatin-14 on cell viability and GH secretion in human GH-secreting pituitary adenomas in vitro and in rat GH3 cells.Overnight treatment with AICAR increased phospho-(threonine-172) AMPK levels (activated AMPK) in cultured human adenomas.As to the effects on cell viability, four adenomas out of 15 were responsive to AICAR (0.4mM) and five adenomas were responsive to SS-14 (100nM). One adenoma was responsive to both somatostatin and AICAR. The effects of cotreatment with SS-14 and AICAR were investigated in eight adenomas. In two adenomas, the effects of AICAR+SS-14 did not exceed the effect of AICAR. In two adenomas which were not responsive to either AICAR or SS-14, the cotreatment was able to reduce cell viability versus control. Two adenomas were not responsive to any treatment.As to the effects on GH secretion, nine adenomas out of 15 were responsive to AICAR. Twelve adenomas were responsive to SS-14. Eight adenomas were responsive to both AICAR and SS-14. Cotreatment exceeded the effect of single treatments in 4 out of 10 adenomas.In GH3 cells, AICAR reduced the activity of p70S6 kinase, which plays an important role in cell growth. SS-14 did not affect significantly AMPK phosphorylation and p70S6K activity but it was able to enhance the inhibitory effect of AICAR on phospho-S6 levels. Moreover, AICAR and SS-14 reduced ERK phosphorylation with a different time course. The combined treatment reduced phospho-ERK levels at any time point.

Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling

Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing and microarray analysis, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR-binding regions in or near these genes, and gene expression is regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 pathways. We found that overexpression of p85α, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85α expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of insulin receptor substrate 1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85α expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a glucocorticoid response element was identified in the p85α GR-binding regions. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85α plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes.