Akt-mTOR Axis Research Articles

Taurine activates the AKT-mTOR axis to restore muscle mass and contractile strength in human 3D in vitro models of steroid myopathy.

Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin-proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.

First person – Sheeza Mughal

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Sheeza Mughal is first author on ‘ Taurine activates the AKT–mTOR axis to restore muscle mass and contractile strength in human 3D in vitro models of steroid myopathy’, published in DMM. Sheeza is a PhD Student in the lab of Javier Ramón-Azcón at the Institute for Bioengineering of Catalonia (IBEC) in Barcelona, Spain, and is interested in developing disease models to simplify understanding of otherwise complex diseases.

Ferroptosis-related gene glutathione peroxidase 4 promotes reprogramming of glucose metabolism via Akt-mTOR axis in intrahepatic cholangiocarcinoma.

The role of the ferroptosis-related gene glutathione peroxidase 4 (GPX4) in oncology has been extensively investigated. However, the clinical implications of GPX4 in patients with intrahepatic cholangiocarcinoma (ICC) remain unknown. This study aimed to evaluate the prognostic impact of GPX4 and its underlying molecular mechanisms in patients with ICC. Fifty-seven patients who underwent surgical resection for ICC between 2010 and 2017 were retrospectively analyzed. Based on the immunohistochemistry, patients were divided into GPX4 high (n = 15) and low (n = 42) groups, and clinical outcomes were assessed. Furthermore, the roles of GPX4 in cell proliferation, migration and gene expression were analyzed in ICC cell lines in vitro and in vivo. The results from clinical study showed that GPX4 high group showed significant associations with high SUVmax on 18F-fluorodeoxyglucose-positron emission tomography (≥8.0, P = 0.017), multiple tumors (P = 0.004), and showed glucose transporter 1 (GLUT1) high expression with a trend toward significance (P = 0.053). Overall and recurrence-free survival in the GPX4 high expression group were significantly worse than those in the GPX4 low expression group (P = 0.038 and P < 0.001, respectively). In the experimental study, inhibition of GPX4 attenuated cell proliferation and migration in ICC cell lines. Inhibition of GPX4 also decreased the expression of glucose metabolism-related genes, such as GLUT1 or HIF1α. Mechanistically, these molecular changes are regulated in Akt-mechanistic targets of rapamycin axis. In conclusion, this study suggested the pivotal value of GPX4 serving as a prognostic marker for patients with ICC. Furthermore, GPX4 can mediate glucose metabolism of ICC.

Isovitexin alleviates hepatic fibrosis by regulating miR-21-mediated PI3K/Akt signaling and glutathione metabolic pathway: based on transcriptomics and metabolomics

BackgroundEffective drugs for the treatment of hepatic fibrosis have not yet been identified. Isovitexin (IVT) is a promising hepatoprotective agent owing to its efficacy against acute liver injury. However, the role of IVT in liver fibrosis has not been reported. PurposeTo explore the effect of IVT on liver fibrosis both in vitro and in vivo. Study design and methodsA mouse model of liver fibrosis induced by carbon tetrachloride (CCl4) and two types of hepatic stellate cell models induced by platelet-derived growth factor-BB (PDGF-BB) were established to evaluate the effect of IVT on hepatic fibrosis. Transcriptomics and metabolomics were used to predict the underlying targets of IVT and were validated by a combination of in vitro and in vivo experiments. Exploration of miRNA and N6-methyladenosine (m6A) modifications was also carried out to detect the key upstream targets of the above targets. ResultsIVT reduced collagen deposition and hepatic stellate cell activation to alleviate liver fibrosis. The transcriptomics and metabolomics analyses showed that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling and the glutathione (GSH) metabolic pathway may be the main regulatory processes of IVT in hepatic fibrosis. Both the in vitro and in vivo experiments confirmed the inhibitory effect of IVT on the PTEN–PI3K–Akt–mTOR axis and activation of the GSH metabolic pathway. A miR-21 mimic inhibited the effects of IVT on these two pathways, suggesting that miR-21 is the hub for IVT regulation of PI3K–Akt signaling and the GSH metabolic pathway. IVT also increased pri-miR-21 level and reduced the m6A enrichment of pri-miR-21, demonstrating that IVT may regulate pri-miR-21 through m6A modification, thereby affecting the maturation of miR-21. ConclusionThis study is the first to propose a protective effect of IVT against liver fibrosis. The mechanism of IVT against hepatic fibrosis is based on the regulation of miR-21, targeting PTEN–Akt signaling and the GSH metabolic pathway, which is also a novel discovery.

Stonin 2 activates lysosomal-mTOR axis for cell survival in oral cancer

Aberrant expression of various genes is associated with the progression of oral squamous cell carcinoma. Stonin 2, an endocytic protein, has a prominent role in clathrin-associated endocytosis. Its position in oral cancer is still unknown. Here, we report that STON2 expression increases with an increase in the grade of the oral cancer tissue. Further, STON2 overexpressed cells possess a higher rate of proliferation and migraton in oral cancer cells. STON2 helps maintain lysosomal functions by preserving the lysosomal membrane integrity. It activates the Akt-mTOR axis and retains the mTOR on the membrane of the lysosomes. Further, we have identified an inhibitor of STON2, i.e., Trifluoperazine dihydrochloride (TFP), which targets the lysosomal axis by disrupting the Akt-mTOR pathway and causes lysosomal membrane permeabilization. Intererstingly, TFP shows a decrease in cell vaibility on the oral cancer cells and it was observed that cell viability is restored in TFP-treated STON2 overexpressed cells. Moreover, the lysosomal activity and the Akt-mTOR expression are restored in STON2 overexpressed cells co-treated with TFP, establishing TFP targets STON2 to showcase its anti-cancer effects in oral cancer. In conclusion, STON2 might serve as a potential biomarker in oral cancer, and its inhibition could functions as a novel anti-cancer mechanims against oral cancer.

The cannabinoid receptor 1 antagonist AM6545 stimulates the Akt-mTOR axis and in vivo muscle protein synthesis in a dexamethasone-induced muscle atrophy model

Cannabinoid receptor 1 (CB1) antagonists were shown to stimulate in vitro muscle protein synthesis, but this has never been confirmed in vivo. Therefore, this study investigated whether treatment with the CB1 antagonist AM6545 upregulates in vivo muscle anabolism. Chronic AM6545 treatment stimulated the Akt-mTOR axis and protein synthesis (+22%; p = 0.002) in the Tibialis Anterior, which protected mice from dexamethasone-induced muscle loss (−1% vs. −6% compared to healthy controls; p = 0.02). Accordingly, acute AM6545 treatment stimulated protein synthesis (+44%; p = 0.04) in the Tibialis Anterior but not Soleus. The anabolic upregulation was accompanied by ERK1/2 activation, whereas protein kinase A signaling remained unaffected, suggesting a CB1-independent mechanism. The present study for the first time shows that the CB1 antagonist AM6545 can upregulate the Akt-mTOR axis and in vivo muscle protein synthesis. However, future work applying genetic approaches should further uncover the signaling pathways via which AM6545 enhances muscle anabolism.

A first-in-human phase I/Ib dose-escalation clinical trial of the autophagy inducer ABTL0812 in patients with advanced solid tumours

BackgroundABTL0812 is an autophagy inducer that promotes cancer cell death by activation of cytotoxic autophagy selectively in tumour cells. ABTL0812 induces endoplasmic reticulum stress and blocks the Akt-mTOR axis; both actions converge to activate a robust and sustained autophagy leading to cancer cell death. Preclinical data supported the initiation of clinical trials in patients with cancer. Patients and methodsThis first-in-human trial consisted of an escalation phase (3 + 3 design), followed by an expansion phase, to assess safety and tolerability of ABTL0812. Secondary objectives were determining the recommended phase II dose (RP2D), clinical antitumour activity, pharmacokinetics (PK) and pharmacodynamics (PD). ResultsA total of 29 patients were enrolled and treated; fifteen patients were treated in four escalation dosing cohorts (ranging from 500 mg once a day to 2000 mg twice a day) and fourteen in the expansion phase (dosed with 1300 mg three times a day). No maximum tolerated dose was attained, and RP2D was determined by PK/PD modelling. Most drug-related adverse events were gastrointestinal grade I–II. Correlation between drug levels and pAkt/Akt ratio was found. Two cases of long-term (>1 year) stable disease were observed. ConclusionsABTL0812 is safe and has an acceptable tolerability profile, allowing a long-term oral dosing. RP2D of 1300 mg three times a day was determined according to PK/PD modelling, and preliminary antitumour efficacy was observed. Clinical trial registration numberNCT02201823.

MTOR Pathway in Gastroenteropancreatic Neuroendocrine Tumor (GEP-NETs).

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) originate from neuroendocrine cells in the gastrointestinal tract. They are heterogeneous, and though initially considered rare tumors, the incidence of GEP-NENs has increased in the last few decades. Therapeutic approaches for the metastatic disease include surgery, radiological intervention by chemoembolisation, radiofrequency ablation, biological therapy in addition to somatostatin analogs, and PRRT therapy (177Lu-DOTATATE). The PI3K-AKT-mTOR pathway is essential in the regulation of protein translation, cell growth, and metabolism. Evidence suggests that the mTOR pathway is involved in malignant progression and resistance to treatment through over-activation of several mechanisms. PI3K, one of the main downstream of the Akt-mTOR axis, is mainly involved in the neoplastic process. This pathway is frequently deregulated in human tumors, making it a central target in the development of new anti-cancer treatments. Recent molecular studies identify potential targets within the PI3K/Akt/mTOR pathway in GEP-NENs. However, the use of target therapy has been known to lead to resistance due to several mechanisms such as feedback activation of alternative pathways, inactivation of protein kinases, and deregulation of the downstream mTOR components. Therefore, the specific role of targeted drugs for the management of GEP-NENs is yet to be well-defined. The variable clinical presentation of advanced neuroendocrine tumors is a significant challenge for designing studies. This review aims to highlight the role of the PI3K/Akt/mTOR pathway in the development of neuroendocrine tumors and further specify its potential as a therapeutic target in advanced stages.

Abstract 2914: Hexokinase 2-mediated metabolic reprogramming and apoptosis inhibition supports hepatocellular carcinoma radiation resistance

Abstract Purpose: Hepatocellular carcinoma (HCC) cells are metabolically distinct from normal hepatocytes by selectively upregulating hexokinase 2 (HK2), a key enzyme that catalyzes the first committed step of aerobic glycolysis, which provides genetic proof of concept that HK2 can be inhibited to treat HCC without adverse physiological consequences. The present study was designed to investigate the role of HK2-mediated metabolic reprogramming and apoptosis inhibition in radiation resistance of HCC cells. Experimental design: Acquired radiation-resistant cells were generated by sequential irradiation and recovery, and proteomics approach was used to illustrate the biological changes and molecular mechanisms involved. qRT-PCR was performed to evaluate the expression of glycolysis genes including HK2. TCGA and GEO analysis were used to investigate the prognostic value of HK2. Intrinsic radiation responsive and resistant HCC cells were also used to further identify the resistant role of HK2. Either lentivirus-mediated HK2 knockdown or pharmacological blockade by 3-bromopyruvate (BrPA) was used to inhibit HK2. Radiation resistance was evaluated by DNA damage response, in vitro cytotoxicity and colony formation assay. Apoptosis and ROS levels were measured with Annexin V and DC-FDA. Cytochrome c release and HK2 mitochondrial translocation were detected by immunofluorescence and western blots. 2-NBDG uptake, HK2 activity, lactate production, and 13C isotopomer tracing experiment were conducted to illustrate the glycolytic activities. HK2 translation efficiency was measured by polyribosome analysis. The upstream AKT-mTOR signaling were detected by western blots and rescued by pharmacological inhibitors. Xenografts were treated with radiation, BrPA, or a combination of radiation and BrPA. Results: Aerobic glycolysis was significantly activated and was indispensable for cell survival in both acquired and intrinsic radiation-resistant HCC cells. Mechanically, both up-regulation of mRNA expression of hexokinase (HK2) after sequential irradiation and increased translation of HK2 mRNA after one-fractional irradiation were mediated by the activation of AKT-mTOR axis. In addition, high HK2 expression was significantly associated with poor overall survival of HCC patients. Inhibition of HK2 expression or its mitochondrial translocation re-sensitized HCC cells to radiation therapy by promoting mitochondrial cytochrome c release and inhibiting of glycolysis both in vitro and in vivo. Conclusion: AKT-mTOR-HK2 axis plays crucial roles in both acquired and intrinsic radiation resistance of HCC cells by facilitating aerobic glycolysis and inhibiting cytochrome c release. Targeting HK2 could be an attractive and beneficial approach to improve the therapeutic effects of radiotherapy in HCC patients. Citation Format: Yuan Fang, Yizhi Zhan, Yiyi Li, Wei Wang, Dehua Wu, Yi Ding. Hexokinase 2-mediated metabolic reprogramming and apoptosis inhibition supports hepatocellular carcinoma radiation resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2914.

Mechanisms behind Temsirolimus Resistance Causing Reactivated Growth and Invasive Behavior of Bladder Cancer Cells In Vitro.

Background: Although mechanistic target of rapamycin (mTOR) inhibitors, such as temsirolimus, show promise in treating bladder cancer, acquired resistance often hampers efficacy. This study evaluates mechanisms leading to resistance. Methods: Cell growth, proliferation, cell cycle phases, and cell cycle regulating proteins were compared in temsirolimus resistant (res) and sensitive (parental—par) RT112 and UMUC3 bladder cancer cells. To evaluate invasive behavior, adhesion to vascular endothelium or to immobilized extracellular matrix proteins and chemotactic activity were examined. Integrin α and β subtypes were analyzed and blocking was done to evaluate physiologic integrin relevance. Results: Growth of RT112res could no longer be restrained by temsirolimus and was even enhanced in UMUC3res, accompanied by accumulation in the S- and G2/M-phase. Proteins of the cdk-cyclin and Akt-mTOR axis increased, whereas p19, p27, p53, and p73 decreased in resistant cells treated with low-dosed temsirolimus. Chemotactic activity of RT112res/UMUC3res was elevated following temsirolimus re-exposure, along with significant integrin α2, α3, and β1 alterations. Blocking revealed a functional switch of the integrins, driving the resistant cells from being adhesive to being highly motile. Conclusion: Temsirolimus resistance is associated with reactivation of bladder cancer growth and invasive behavior. The α2, α3, and β1 integrins could be attractive treatment targets to hinder temsirolimus resistance.

The immunosuppressant drug azathioprine restrains adipogenesis of muscle Fibro/Adipogenic Progenitors from dystrophic mice by affecting AKT signaling

Fibro/Adipogenic Progenitors (FAPs) define a stem cell population playing a pro-regenerative role after muscle damage. When removed from their natural niche, FAPs readily differentiate into adipocytes or fibroblasts. This digressive differentiation potential, which is kept under tight control in the healthy muscle niche, contributes to fat and scar infiltrations in degenerative myopathies, such as in Duchenne Muscular Dystrophy (DMD). Controlling FAP differentiation by means of small molecules may contribute to delay the adverse consequences of the progressive pathological degeneration while offering, at the same time, a wider temporal window for gene therapy and cell-based strategies. In a high content phenotypic screening, we identified the immunosuppressant, azathioprine (AZA) as a negative modulator of FAP adipogenesis. We show here that AZA negatively affects the adipogenic propensity of FAPs purified from wild type and mdx mice by impairing the expression of the master adipogenic regulator, peroxisome proliferator-activated receptor γ (PPARγ). We show that this inhibition correlates with a decline in the activation of the AKT-mTOR axis, the main pathway that transduces the pro-adipogenic stimulus triggered by insulin. In addition, AZA exerts a cytostatic effect that has a negative impact on the mitotic clonal process that is required for the terminal differentiation of the preadipocyte-committed cells.

Neuregulin-1β attenuates sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway.

The aim of this study was to investigate the protective effects of neuregulin-1β (NRG-1β) on sepsis-induced diaphragm atrophy and the possible underlying mechanisms. Sprague-Dawley rats were randomly divided into sham, sepsis and NRG groups. Sepsis was induced by cecal ligation and puncture (CLP). In the NRG group, rats received tail vein injections of NRG-1β (10μg/kg) every 12h for 72h after CLP. At 3days after surgery, diaphragm contractile forces were measured by determining the force-frequency curve and muscle fiber areas by hematoxylin-eosin staining. Moreover, the NRG-1 expression level in the diaphragm was detected by Western blotting. Furthermore, the proteins in the PI3K/Akt signaling pathway and its downstream Akt-mTOR and Akt-FOXO axes were detected by Western blotting analysis. In L6 myotubes treated with lipopolysaccharide (LPS) and NRG-1β, PI3K/Akt signaling pathway-related protein expression was further determined using the PI3K inhibitor LY294002. Exogenous NRG-1β could compensate for sepsis-induced diminished NRG-1 in the diaphragm and attenuate the reduction in diaphragm contractile forces and muscle fiber areas during sepsis. Moreover, NRG-1β treatment could activate the PI3K/Akt signaling pathway in the diaphragm during sepsis. The inhibition of p70S6K and 4E-BP1 on the Akt-mTOR axis and the increased expression of Murf1 on the Akt-FOXO axis were reversed after NRG-1 treatment. In addition, NRG-1β could activate the PI3K/Akt signaling pathway in L6 myotubes treated with LPS, while the PI3K inhibitor LY294002 blocked the effects of NRG-1β. NRG-1 expression in the diaphragm was reduced during sepsis, and exogenously administered recombinant human NRG-1β could attenuate sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway.

Hexanoic, Octanoic and Decanoic Acids Promote Basal and Insulin-Induced Phosphorylation of the Akt-mTOR Axis and a Balanced Lipid Metabolism in the HepG2 Hepatoma Cell Line.

Metabolic illnesses such as non-alcoholic fatty liver disease (NAFLD) are in constant increase worldwide. Highly consumed long chain fatty acids (LCFA) are among the most obesogenic and steatogenic nutrients. Hepatic steatosis is associated with several complications such as insulin resistance. Growing evidence points to medium chain fatty acids (MCFA), more efficiently oxidized than LCFA, as a promising dietary alternative against NAFLD. However, reports on the hepatic effects of MCFA are sometimes conflicting. In this study we exposed HepG2 cells, a human hepatocellular model, to 0.25 mM of hexanoic (C6), or octanoic (C8), and decanoic (C10) acids separately or in a C8 + C10 equimolar mix reflecting commercially available MCFA-rich oils. We found that C6, a poorly studied MCFA, as well as C8 and C10 did not provoke the deleterious lipid anabolism runaway typically induced by LCFA palmitate. MCFA tended, instead, to promote a balanced metabolic profile and were generally non-cytotoxic. Accordingly, mitochondrial integrity was mostly preserved following MCFA treatment. However, treatments with C8 induced a mitochondrial membrane potential decrease, suggesting prolonged exposure to this lipid could be problematic. Finally, MCFA treatments maintained optimal insulin sensitivity and even fostered basal and insulin-dependent phosphorylation of the Akt-mTOR pathway. Overall, MCFA could constitute an effective nutritional tool to manage liver steatosis and hepatic insulin resistance.

RAPTOR up-regulation contributes to resistance of renal cancer cells to PI3K-mTOR inhibition.

The outlook for patients with advanced renal cell cancer (RCC) has been improved by targeted agents including inhibitors of the PI3 kinase (PI3K)-AKT-mTOR axis, although treatment resistance is a major problem. Here, we aimed to understand how RCC cells acquire resistance to PI3K-mTOR inhibition. We used the RCC4 cell line to generate a model of in vitro resistance by continuous culture in PI3K-mTOR kinase inhibitor NVP-BEZ235 (BEZ235, Dactolisib). Resistant cells were cross-resistant to mTOR inhibitor AZD2014. Sensitivity was regained after 4 months drug withdrawal, and resistance was partially suppressed by HDAC inhibition, supporting an epigenetic mechanism. BEZ235-resistant cells up-regulated and/or activated numerous proteins including MET, ABL, Notch, IGF-1R, INSR and MEK/ERK. However, resistance was not reversed by inhibiting or depleting these pathways, suggesting that many induced changes were passengers not drivers of resistance. BEZ235 blocked phosphorylation of mTOR targets S6 and 4E-BP1 in parental cells, but 4E-BP1 remained phosphorylated in resistant cells, suggesting BEZ235-refractory mTORC1 activity. Consistent with this, resistant cells over-expressed mTORC1 component RAPTOR at the mRNA and protein level. Furthermore, BEZ235 resistance was suppressed by RAPTOR depletion, or allosteric mTORC1 inhibitor rapamycin. These data reveal that RAPTOR up-regulation contributes to PI3K-mTOR inhibitor resistance, and suggest that RAPTOR expression should be included in the pharmacodynamic assessment of mTOR kinase inhibitor trials.

Activation of AKT-mTOR Signaling Directs Tenogenesis of Mesenchymal Stem Cells.

Tendon repair is a clinical challenge because of the limited understanding on tenogenesis. The synthesis of type I collagen (Collagen I) and other extracellular matrix are essential for tendon differentiation and homeostasis. Current studies on tenogenesis focused mostly on the tenogenic transcriptional factors while the signaling controlling tenogenesis on translational level remains largely unknown. Here, we showed that mechanistic target of rapamycin (mTOR) signaling was activated by protenogenic growth factor, transforming growth factors beta1, and insulin-like growth factor-I. The expression of mTOR was upregulated during tenogenesis of mesenchymal stem cells (MSCs). Moreover, mTOR was downregulated in human tendinopathy tissues and was inactivated upon statin treatment. Both inhibition and depletion of AKT or mTOR significantly reduced type I collagen production and impaired tenogenesis of MSCs. Tendon specific-ablation of mTOR resulted in tendon defect and reduction of Collagen I. However, there is no evident downregulation of tendon associated collagens at the transcription level. Our study demonstrated that AKT-mTOR axis is a key mediator of tendon differentiation and provided a novel therapeutic target for tendinopathy and tendon injuries. Stem Cells 2018;36:527-539.

Inhibiting 4EBP1 in Glioblastoma.

Glioblastoma is the most common and aggressive adult brain cancer. Tumors show frequent dysregulation of the PI3K-mTOR pathway. Although a number of small molecules target the PI3K-AKT-mTOR axis, their preclinical and clinical efficacy has been limited. Reasons for treatment failure include poor penetration of agents into the brain and observations that blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma. Clinical trials using allosteric mTOR inhibitors (rapamycin and rapalogs) to treat patients with glioblastoma have also been unsuccessful or uncertain, in part, because rapamycin inefficiently blocks the mTORC1 target 4EBP1 and feeds back to activate PI3K-AKT signaling. Inhibitors of the mTOR kinase (TORKi) such as TAK-228/MLN0128 interact orthosterically with the ATP- and substrate-binding pocket of mTOR kinase, efficiently block 4EBP1 in vitro, and are currently being investigated in the clinical trials. Preclinical studies suggest that TORKi have poor residence times of mTOR kinase, and our data suggest that this poor pharmacology translates into disappointing efficacy in glioblastoma xenografts. RapaLink-1, a TORKi linked to rapamycin, represents a drug with improved pharmacology against 4EBP1. In this review, we clarify the importance of 4EBP1 as a biomarker for the efficacy of PI3K-AKT-mTOR inhibitors in glioblastoma. We also review mechanistic data by which RapaLink-1 blocks p-4EBP1 and discuss future clinical strategies for 4EBP1 inhibition in glioblastoma. Clin Cancer Res; 24(1); 14-21. ©2017 AACR.

Enrichment of PI3K-AKT-mTOR Pathway Activation in Hepatic Metastases from Breast Cancer.

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K-AKT-mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing-based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K-AKT-mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K-AKT-mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results:PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K-AKT-mTOR signaling network. Clin Cancer Res; 23(16); 4919-28. ©2017 AACR.

Kinase-driven metabolic signalling as a predictor of response to carboplatin\u2013paclitaxel adjuvant treatment in advanced ovarian cancers

Background:The biological mechanisms underlying early- and advanced-stage epithelial ovarian cancers (EOCs) are still poorly understood. This study explored kinase-driven metabolic signalling in early and advanced EOCs, and its role in tumour progression and response to carboplatin–paclitaxel treatment.Methods:Tumour epithelia were isolated from two independent sets of primary EOC (n=72 and 30 for the discovery and the validation sets, respectively) via laser capture microdissection. Reverse phase protein microarrays were used to broadly profile the kinase-driven metabolic signalling of EOC with particular emphasis on the LBK1–AMPK and AKT–mTOR axes. Signalling activation was compared between early and advanced lesions, and carboplatin–paclitaxel-sensitive and -resistant tumours.Results:Advanced EOCs were characterised by a heterogeneous kinase-driven metabolic signature and decreased phosphorylation of the AMPK–AKT–mTOR axis compared to early EOC (P<0.05 for AMPKα T172, AMPKα1 S485, AMPKβ1 S108, AKT S473 and T308, mTOR S2448, p70S6 S371, 4EBP1 S65, GSK-3 α/β S21/9, FOXO1 T24/FOXO3 T32, and FOXO1 S256). Advanced tumours with low relative activation of the metabolic signature and increased FOXO1 T24/FOXO3 T32 phosphorylation (P=0.041) were associated with carboplatin–paclitaxel resistance.Conclusions:If validated in a larger cohort of patients, the decreased AMPK–AKT–mTOR activation and phosphorylation of FOXO1 T24/FOXO3 T32 may help identify carboplatin–paclitaxel-resistant EOC patients.

Synthetic sulfoglycolipids targeting the serine–threonine protein kinase Akt

The serine–threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)–Akt–mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

Fingolimod ameliorates the development of experimental autoimmune encephalomyelitis by inhibiting Akt–mTOR axis in mice

Fingolimod is a new immunosuppressive agent approved by Food and Drug Administration (FDA) for treating multiple sclerosis (MS). It acts as a functional antagonist to downregulate the S1P1 receptor, which is known to signal through the Akt–mTOR pathway. We investigated the mechanism of fingolimod action in the classical animal model of MS: experimental autoimmune encephalomyelitis (EAE). Fingolimod treatment significantly reduced clinical scores and histopathology in this model, even when treatment was begun after the onset of pathology. The Akt–mTOR signaling pathway was shown to be activated in the EAE model, by measuring the abundance of downstream activation markers, pAkt and ps6k. And this pathway was inhibited when EAE mice were treated with fingolimod. Mice with EAE exhibited an increased frequency of Th1 cells in the spleen, with concomitant increases in the mRNA levels of Tbet and Ifng and increased IFN-γ production by activated splenocytes; the frequency of Treg cells, as well as mRNA levels of Foxp3 and Tgfb, was reduced, as was TGF-β production by activated splenocytes. After treatment with fingolimod, these parameters were reversed, suggesting that fingolimod treatment inhibits the Akt–mTOR axis in EAE, which affects the differentiation and function of Th1 and Treg cells. These results provide an insight into the mechanism of action of fingolimod treatment and may provide new ideas for treating EAE and MS.