Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity.

Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin

UNLABELLED The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

The role of phospholipase D in modulating the MTOR signaling pathway in polycystic kidney disease

The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD.

Role of somatostatins in gastroenteropancreatic neuroendocrine tumor development and therapy

The incidence and prevalence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have increased in the past 20 years. GEP-NETs are heterogeneous tumors, in terms of clinical and biological features, that originate from the pancreas or the intestinal tract. Some GEP-NETs grow very slowly, some grow rapidly and do not cause symptoms, and others cause hormone hypersecretion and associated symptoms. Most GEP-NETs overexpress receptors for somatostatins. Somatostatins inhibit the release of many hormones and other secretory proteins; their effects are mediated by G protein-coupled receptors that are expressed in a tissue-specific manner. Most GEP-NETs overexpress the somatostatin receptor SSTR2; somatostatin analogues are the best therapeutic option for functional neuroendocrine tumors because they reduce hormone-related symptoms and also have antitumor effects. Long-acting formulations of somatostatin analogues stabilize tumor growth over long periods. The development of radioactive analogues for imaging and peptide receptor radiotherapy has improved the management of GEP-NETs. Peptide receptor radiotherapy has significant antitumor effects, increasing overall survival times of patients with tumors that express a high density of SSTRs, particularly SSTR2 and SSTR5. The multi-receptor somatostatin analogue SOM230 (pasireotide) and chimeric molecules that bind SSTR2 and the dopamine receptor D2 are also being developed to treat patients with GEP-NETs. Combinations of radioactive labeled and unlabeled somatostatin analogues and therapeutics that inhibit other signaling pathways, such as mammalian target of rapamycin (mTOR) and vascular endothelial growth factor, might be the most effective therapeutics for GEP-NETs.

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis

BACKGROUND/AIMS: Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. METHODS: Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. RESULTS: Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. CONCLUSIONS: Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.

Pulse mTOR inhibitor treatment effectively controls cyst growth but leads to severe parenchymal and glomerular hypertrophy in rat polycystic kidney disease

The efficacy of mammalian target of rapamycin (mTOR) inhibitors is currently tested in patients affected by autosomal dominant polycystic kidney disease. Treatment with mTOR inhibitors has been associated with numerous side effects. However, the renal-specific effect of mTOR inhibitor treatment cessation in polycystic kidney disease is currently unknown. Therefore, we compared pulse and continuous everolimus treatment in Han:SPRD rats. Four-week-old male heterozygous polycystic and wild-type rats were administered everolimus or vehicle by gavage feeding for 5 wk, followed by 7 wk without treatment, or continuously for 12 wk. Cessation of everolimus did not result in the appearance of renal cysts up to 7 wk postwithdrawal despite the reemergence of S6 kinase activity coupled with an overall increase in cell proliferation. Pulse everolimus treatment resulted in striking noncystic renal parenchymal enlargement and glomerular hypertrophy that was not associated with compromised kidney function. Both treatment regimens ameliorated kidney function, preserved the glomerular-tubular connection, and reduced proteinuria. Pulse treatment at an early age delays cyst development but leads to striking glomerular and parenchymal hypertrophy. Our data might have an impact when long-term treatment using mTOR inhibitors in patients with autosomal dominant polycystic kidney disease is being considered.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Neuroprotection through excitability and mTOR required in ALS motoneurons to delay disease and extend survival.

Delaying clinical disease onset would greatly reduce neurodegenerative disease burden, but the mechanisms influencing early preclinical progression are poorly understood. Here, we show that in mouse models of familial motoneuron (MN) disease, SOD1 mutants specifically render vulnerable MNs dependent on endogenous neuroprotection signaling involving excitability and mammalian target of rapamycin (mTOR). The most vulnerable low-excitability FF MNs already exhibited evidence of pathology and endogenous neuroprotection recruitment early postnatally. Enhancing MN excitability promoted MN neuroprotection and reversed misfolded SOD1 (misfSOD1) accumulation and MN pathology, whereas reducing MN excitability augmented misfSOD1 accumulation and accelerated disease. Inhibiting metabotropic cholinergic signaling onto MNs reduced ER stress, but enhanced misfSOD1 accumulation and prevented mTOR activation in alpha-MNs. Modulating excitability and/or alpha-MN mTOR activity had comparable effects on the progression rates of motor dysfunction, denervation, and death. Therefore, excitability and mTOR are key endogenous neuroprotection mechanisms in motoneurons to counteract clinically important disease progression in ALS.

What is new in the pathology of pancreatic neuroendocrine tumors?

The diagnostics of pancreatic neuroendocrine tumors (PanNEN) have changed in recent years especially concerning the World Health Organization (WHO) classification, TNM staging and grading. Furthermore, some new prognostic and predictive immunohistochemical markers have been introduced. Most progress, however, has been made in the molecular pathogenesis of these neoplasms. Using next generation sequencing techniques, the mammalian target of rapamycin (mTOR) pathway, hypoxia and epigenetic changes were identified as key players in tumorigenesis. In this article the most important developments of morphological as well as immunohistochemical diagnostics together with the molecular background of PanNEN are summarized.

Rapamycin impairs endothelial cell function in human internal thoracic arteries.

BACKGROUND Definitive fate of the coronary endothelium after implantation of a drug-eluting stent remains unclear, but evidence has accumulated that treatment with rapamycin-eluting stents impairs endothelial function in human coronary arteries. The aim of our study was to demonstrate this phenomenon on functional, morphological and biochemical level in human internal thoracic arteries (ITA) serving as coronary artery model. METHODS After exposure to rapamycin for 20 h, functional activity of ITA rings was investigated using the organ bath technique. Morphological analysis was performed by scanning electron microscopy and evaluated by two independent observers in blinded fashion. For measurement of endothelial nitric oxide synthase (eNOS) release, mammalian target of rapamycin (mTOR) and protein kinase B (PKB) (Akt) activation, Western blotting on human mammary epithelial cells-1 and on ITA homogenates was performed. RESULTS Comparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 μM rapamycin. Electron microscopic evaluation of the endothelial layer showed no differences between controls and samples exposed to 10 μM rapamycin. Western blots after 20-h incubation with rapamycin (10 nM-1 μM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1. Furthermore, 1 μM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged. CONCLUSIONS The present data suggests that 20-h exposure of ITA rings to rapamycin reduces endothelium-mediated relaxation through down-regulation of Akt-phosphorylation via the mTOR signalling axis within the ITA tissue without injuring the endothelial cell layer.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Targeting class IA PI3K isoforms selectively impairs cell growth, survival, and migration in glioblastoma.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

Nutrient-responsive mTOR signalling grows on Sterile ground

The control of cell growth, that is cell size, is largely controlled by mTOR (the mammalian target of rapamycin), a large serine/threonine protein kinase that regulates ribosome biogenesis and protein translation. mTOR activity is regulated both by the availability of growth factors, such as insulin/IGF-1 (insulin-like growth factor 1), and by nutrients, notably the supply of certain key amino acids. The last few years have seen a remarkable increase in our understanding of the canonical, growth factor-regulated pathway for mTOR activation, which is mediated by the class I PI3Ks (phosphoinositide 3-kinases), PKB (protein kinase B), TSC1/2 (the tuberous sclerosis complex) and the small GTPase, Rheb. However, the nutrient-responsive input into mTOR is important in its own right and is also required for maximal activation of mTOR signalling by growth factors. Despite this, the details of the nutrient-responsive signalling pathway(s) controlling mTOR have remained elusive, although recent studies have suggested a role for the class III PI3K hVps34. In this issue of the Biochemical Journal, Findlay et al. demonstrate that the protein kinase MAP4K3 [mitogen-activated protein kinase kinase kinase kinase-3, a Ste20 family protein kinase also known as GLK (germinal centre-like kinase)] is a new component of the nutrient-responsive pathway. MAP4K3 activity is stimulated by administration of amino acids, but not growth factors, and this is insensitive to rapamycin, most likely placing MAP4K3 upstream of mTOR. Indeed, MAP4K3 is required for phosphorylation of known mTOR targets such as S6K1 (S6 kinase 1), and overexpression of MAP4K3 promotes the rapamycin-sensitive phosphorylation of these same targets. Finally, knockdown of MAP4K3 levels causes a decrease in cell size. The results suggest that MAP4K3 is a new component in the nutrient-responsive pathway for mTOR activation and reveal a completely new function for MAP4K3 in promoting cell growth. Given that mTOR activity is frequently deregulated in cancer, there is much interest in new strategies for inhibition of this pathway. In this context, MAP4K3 looks like an attractive drug target since inhibitors of this enzyme should switch off mTOR, thereby inhibiting cell growth and proliferation, and promoting apoptosis.