Chronic mTOR Inhibition Research Articles

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4+ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA–treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4+ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.

MTOR inhibition improves mitochondria function/biogenesis and delays cardiovascular aging in kidney transplant recipients with chronic graft dysfunction.

CVD remains the major cause of mortality with graft functioning in Kidney transplant recipients (KTRs), with an estimated risk of CV events about 50-fold higher than in the general population. Many strategies have been considered to reduce the CV risk such as the use of mTOR inhibitors. We evaluate whether chronic mTOR inhibition might influence CV aging in KTRs studying the molecular mechanisms involved in this effect. We retrospectively analyzed 210 KTRs with stable graft function on therapy with CNI and mycophenolic acid (Group A, 105 pts.), or with CNI and mTORi (Everolimus, Group B, 105 pts.). The presence of mTOR inhibitor in immunosuppressive therapy was associated to increase serum levels of Klotho with concomitant reduction in FGF-23, with a significant decrease in left ventricular mass. In addition, KTRs with mTORi improved mitochondrial function/biogenesis in PBMC with more efficient oxidative phosphorylation, antioxidant capacity and glutathione peroxidase activity. Finally, group B KTRs presented reduced levels of inflammaging markers such as reduced serum pentraxin-3 and p21ink expression in PBMC. In conclusion, we demonstrated that mTOR inhibition in immunosuppressive protocols prevents the occurrence and signs of CV aging in KTRs.

Sex-dependent lifespan extension of Apc Min/+ FAP mice by chronic mTOR inhibition.

Apc Min/+ mice model familial adenomatous polyposis (FAP), a disease that causes numerous colon polyps leading to colorectal cancer. We previously showed that chronic treatment of Apc Min/+ females with the anti-aging drug, rapamycin, restored a normal lifespan through reduced polyposis and anemia prevention. Lifespan extension by chronic rapamycin in wildtype UM-HET3 mice is sex-dependent with females gaining the most benefit. Whether Apc Min/+ mice have a similar sex-dependent response to chronic mTOR inhibition is not known. To address this knowledge gap and gain deeper insight into how chronic mTOR inhibition prevents intestinal polyposis, we compared male and female Apc Min/+ mice responses to chronic treatment with a rapamycin-containing diet. Animals were fed a diet containing either 42 ppm microencapsulate rapamycin or empty capsules, one group was used to determine lifespan and a second group with similar treatment was harvested at 16 weeks of age for cross-sectional studies. We found that the survival of males is greater than females in this setting (P < 0.0197). To explore the potential basis for this difference we analyzed factors affected by chronic rapamycin. Immunoblot assays showed that males and females exhibited approximately the same level of mTORC1 inhibition using phosphorylation of ribosomal protein S6 (rpS6) as an indirect measure. Immunohistochemistry assays of rpS6 phosphorylation showed that rapamycin reduction of mTORC1 activity was on the same level, with the most prominent difference being in intestinal crypt Paneth cells in both sexes. Chronic rapamycin also reduced crypt depths in both male and female Apc Min/+ mice (P < 0.0001), consistent with reduced crypt epithelial cell proliferation. Finally, chronic rapamycin prevented anemia equally in males and females. In males and females, these findings link rapamycin-mediated intestinal polyposis prevention with mTORC1 inhibition in Paneth cells and concomitant reduced epithelial cell proliferation.

Abstract PR10: Multimodality imaging of human lung squamous cell carcinoma reveals unique metabolic dependencies that are effectively targeted with metabolic-based therapies

Abstract Altered metabolism is known to commonly contribute to cancer growth, forming the conceptual basis for the development of metabolic therapies as cancer treatments. However, the specific metabolic characteristics of individual cancer types in vivo are still largely unknown, limiting the translatability of metabolic therapies in the clinic. In this study we used a multimodality imaging approach to identify metabolic dependencies in lung squamous cell carcinomas (SCC) in order to guide precise treatments with metabolic therapies. We performed in vivo metabolic profiling and molecular analysis of lung SCC using positron emission tomography (PET) imaging, liquid chromatography mass spectrometry (LC-MS), and immunohistochemistry (IHC). PET imaging lung SCC tumors with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-Glutamine PET tracers identifed a conserved metabolic reliance on both glucose and glutamine that we confirmed with LC-MS based metabolomics and quantitative IHC. The mTOR kinase is a central regulator of growth and metabolism and is readily inhibited using allosteric and catalytic kinase inhibitors. Using the mTOR catalytic kinase inhibitor MLN128, we successfully inhibited glycolysis in lung SCC. However, lung SCC tumors were able to metabolically adapt to chronic mTOR inhibition through upregulation of glutamine metabolism. We discovered that chronic mTOR inhibition induced phosphorylation and inactivation of GSK3a/b resulting in upregulation and activation of cMYC and cJUN—both central regulators of the glutaminase (GLS) enzyme and glutaminolysis. Importantly, phospho-GSK3α/β and phospho-cJUN proteins serve as functional biomarkers that predict MLN128 resistance and upregulation of glutaminolysis. To overcome MLN128 resistance we treated lung SCC tumors with the glutaminase inhibitor CB-839. The combination of MLN128 and CB-839 effectively overcame therapy resistance in genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) of lung SCC. Lastly, data mining from The Lung Cancer Genome Atlas coupled to our PET imaging and IHC biomarkers analysis allowed us to construct a metabolic signature that is conserved in human lung SCC as well as a broad spectrum of hypermetabolic human tumors. These tumors include lung SCC, head and neck squamous cell carcinoma (HNSCC), osteosarcomas (OS), and triple-negative breast cancer (TNBC). Importantly, this metabolic signature is predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase. We therefore propose a clinically translatable treatment strategy for lung SCC patients that is driven by PET imaging and IHC to first stratify patients by their metabolic signature, which is then used to guide the delivery of precise metabolic-based therapies that target key metabolic nodes such as mTOR and GLS. This abstract is also being presented as Poster B11. Citation Format: Milica Momcilovic, David B. Shackelford. Multimodality imaging of human lung squamous cell carcinoma reveals unique metabolic dependencies that are effectively targeted with metabolic-based therapies [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR10.

The GSK3 Signaling Axis Regulates Adaptive Glutamine Metabolism in Lung Squamous Cell Carcinoma

Altered metabolism is a hallmark of cancer growth, forming the conceptual basis for development of metabolic therapies as cancer treatments. We performed invivo metabolic profiling and molecular analysis of lung squamous cell carcinoma (SCC) to identify metabolic nodes for therapeutic targeting. Lung SCCs adapt to chronic mTOR inhibition and suppression of glycolysis through the GSK3α/β signaling pathway, which upregulates glutaminolysis. Phospho-GSK3α/β protein levels are predictive of response to single-therapy mTOR inhibition while combinatorial treatment with the glutaminase inhibitor CB-839 effectively overcomes therapy resistance. In addition, we identified a conserved metabolic signature in a broad spectrum of hypermetabolic human tumors that may be predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase.

A molecular cascade modulates MAP1B and confers resistance to mTOR inhibition in human glioblastoma.

Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy. To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures. An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization. These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

Age-Related Neurodegeneration Prevention Through mTOR Inhibition: Potential Mechanisms and Remaining Questions.

With the global aging population, Alzheimer's disease, Parkinson's disease and mild cognition impairment are increasing in prevalence. The success of rapamycin as an agent to extend lifespan in various organisms, including mice, brings hope that chronic mTOR inhibition could also refrain age-related neurodegeneration. Here we review the evidence suggesting that mTOR inhibition - mainly with rapamycin - is a valid intervention to delay age-related neurodegeneration. We discuss the potential mechanisms by which rapamycin may facilitate neurodegeneration prevention or restoration of cognitive function. We also discuss the known side effects of rapamycin and provide evidence to alleviate exaggerated concerns regarding its wider clinical use. We explore the small molecule alternatives to rapamycin and propose future directions for their development, mainly by exploring the possibility of targeting the downstream effectors of mTOR: S6K1 and especially S6K2. Finally, we discuss the strengths and weaknesses of the models used to determine intervention efficacy for neurodegeneration. We address the difficulties of interpreting data using the common way of investigating the efficacy of interventions to delay/prevent neurodegeneration by observing animal behavior while these animals are under treatment. We propose an experimental design that should isolate the variable of aging in the experimental design and resolve the ambiguity present in recent literature.

PPARγ activation attenuates glucose intolerance induced by mTOR inhibition with rapamycin in rats

mTOR inhibition with rapamycin induces a diabetes-like syndrome characterized by severe glucose intolerance, hyperinsulinemia, and hypertriglyceridemia, which is due to increased hepatic glucose production as well as reduced skeletal muscle glucose uptake and adipose tissue PPARγ activity. Herein, we tested the hypothesis that pharmacological PPARγ activation attenuates the diabetes-like syndrome associated with chronic mTOR inhibition. Rats treated with the mTOR inhibitor rapamycin (2 mg·kg(-1)·day(-1)) in combination or not with the PPARγ ligand rosiglitazone (15 mg·kg(-1)·day(-1)) for 15 days were evaluated for insulin secretion, glucose, insulin, and pyruvate tolerance, skeletal muscle and adipose tissue glucose uptake, and insulin signaling. Rosiglitazone corrected fasting hyperglycemia, attenuated the glucose and insulin intolerances, and abolished the increase in fasting plasma insulin and C-peptide levels induced by rapamycin. Surprisingly, rosiglitazone markedly increased the plasma insulin and C-peptide responses to refeeding in rapamycin-treated rats. Furthermore, rosiglitazone partially attenuated rapamycin-induced gluconeogenesis, as evidenced by the improved pyruvate tolerance and reduced mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Rosiglitazone also restored insulin's ability to stimulate glucose uptake and its incorporation into glycogen in skeletal muscle of rapamycin-treated rats, which was associated with normalization of Akt Ser(473) phosphorylation. However, the rapamycin-mediated impairments of adipose tissue glucose uptake and incorporation into triacylglycerol were unaffected by rosiglitazone. Our findings indicate that PPARγ activation ameliorates some of the disturbances in glucose homeostasis and insulin action associated with chronic rapamycin treatment by reducing gluconeogenesis and insulin secretion and restoring muscle insulin signaling and glucose uptake.

Adaptation to chronic mTOR inhibition in cancer and in aging

The mTOR [mammalian (or mechanistic) target of rapamycin] protein kinase co-ordinates catabolic and anabolic processes in response to growth factors and nutrients and is a validated anticancer drug target. Rapamycin and related allosteric inhibitors of mTORC1 (mTOR complex 1) have had some success in specific tumour types, but have not exhibited broad anticancer activity, prompting the development of new ATP-competitive mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2. In common with other targeted kinase inhibitors, tumours are likely to adapt and acquire resistance to mTOR inhibitors. In the present article, we review studies that describe how tumour cells adapt to become resistant to mTOR inhibitors. mTOR is a central signalling hub which responds to an array of signalling inputs and activates a range of downstream effector pathways. Understanding how this signalling network is remodelled and which pathways are invoked to sustain survival and proliferation in the presence of mTOR inhibitors can provide new insights into the importance of the various mTOR effector pathways and may suggest targets for intervention to combine with mTOR inhibitors. Finally, since chronic mTOR inhibition by rapamycin can increase lifespan and healthspan in nematodes, fruitflies and mice, we contrast these studies with tumour cell responses to mTOR inhibition.

Evidence that chronic inhibition of the mammalian Target of Rapamycin (mTOR) reduces skin blood flow in humans

Chronic mTOR inhibition causes vascular dysfunction in rodents. We hypothesized that a similar effect would be observed in humans. 6 middle‐aged persons participated. mTOR was antagonized by topical application of 8% rapamycin (RAPA) ointment to skin. RAPA was applied twice daily for 7 days to an 8×8 cm area of skin on the abdomen while a second site was similarly treated with ointment vehicle only. We examined the effect of these treatments on skin blood flow (SkBF) as monitored by laser‐Doppler flowmetry (LDF). Local skin temperature (Tloc) was controlled with combined LDF/local heating probe holders. Tloc was maintained initially at 34°C and then was raised to 43°C to cause maximal local vasodilation (maxLDF).ResultsAt Tloc=34°C, SkBF was 72±4.3% maxLDF at RAPA treated sites and was 80±4.9% maxLDF at sites that received vehicle only (p&lt;0.03 between sites). maxLDF (in mV) when Tloc was 43°C did not differ between RAPA and vehicle treated sites (p&gt;;0.05). We conclude that chronic RAPA treatment reduces SkBF levels at normothermic skin temperatures in middle‐aged persons without an effect on maximal vasodilation. (supported by NIH Grant AG041365)

Major involvement of mTOR in the PPARγ-induced stimulation of adipose tissue lipid uptake and fat accretion

Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)γ agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPARγ agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPARγ activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPARγ activation.

Acetylation of histone H3 prevents resistance development caused by chronic mTOR inhibition in renal cell carcinoma cells

Chronic mTOR inhibition may induce resistance development in renal cell carcinoma (RCC). We analyzed whether long-term exposure of RCC cells to the mTOR-inhibitor RAD001 evokes resistance and whether additional targeting histone deacetylases (HDAC) by valproic acid (VPA) overcomes RAD001 resistance. It is demonstrated that responsiveness to either drug alone is lost over time, evidenced by increased cell growth, proliferation and de-differentiation. However, drug sensitivity was conserved when RAD001 and VPA were applied in concert. Molecular analysis particularly revealed strong re-activation of Akt under chronic RAD001 or diminished histone H3 acetylation under chronic VPA single drug exposure. Combined drug application did not inactivate Akt but rather resulted in H3 acetylation remaining high while RCC cell growth was still reduced. siRNA experiments revealed that histone H3 acetylation is responsible for preserving drug sensitivity in RCCs.

321 HDAC BLOCKADE COUNTERACTS RESISTANCE DEVELOPMENT CAUSED BY CHRONIC MTOR INHIBITION

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2012321 HDAC BLOCKADE COUNTERACTS RESISTANCE DEVELOPMENT CAUSED BY CHRONIC MTOR INHIBITION Jasmina Makarevic, Igor Tsaur, Eva Weich, Axel Haferkamp, and Roman Blaheta Jasmina MakarevicJasmina Makarevic Frankfurt, Germany More articles by this author , Igor TsaurIgor Tsaur Frankfurt, Germany More articles by this author , Eva WeichEva Weich Frankfurt, Germany More articles by this author , Axel HaferkampAxel Haferkamp Frankfurt, Germany More articles by this author , and Roman BlahetaRoman Blaheta Frankfurt, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.381AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES mTOR-targeting is an attractive approach to prostate cancer therapy. However, the benefit of long-term application has not been evaluated in detail. Growth and invasive behavior of prostate cancer cells under chronic exposure to the mTOR-inhibitor temsirolimus was, therefore, investigated. Further investigation was designed to determine whether additionally targeting histone deacetylase (HDAC) by an HDAC-inhibitor (valproic acid, VPA) might counteract undesired modification of the tumor caused by chronic use of temsirolimus. METHODS Prostate cancer cells were either treated with temsirolimus over 12 months, starting at 1 nM and increasing stepwise to 5 μM (PC3TEM), or only treated with medium (PC3). 3 days before experiment begin, temsirolimus containing culture medium was replaced by a temsirolimus free medium and tumor cells analyzed thereafter, or PC3TEM (versus PC3) then re-incubated with low-dosed (10 nM) temsirolimus to evaluate drug responsiveness. Growth, proliferation, adhesion to matrix proteins or endothelial cells and invasion of PC3 versus PC3TEM were investigated. Expression of cell cycle regulating proteins (cdk1, cdk2, cdk4, cyclin A,B,D3,E, p21, p27), mTOR related intracellular signaling (Akt, mTOR, rictor, raptor, p70S6k) and the expression profile of alpha and beta integrin subtypes were also evaluated. Additionally, HDAC was blocked in PC3TEM cells and the consequences on invasive growth investigated. RESULTS PC3 TEM accumulated in the G2/M-phase, accompanied by cdk1, cdk2, cyclin B elevation and reduction of p21 and p27. Application of 10 nM temsirolimus to PC3TEM did not diminish cell growth and proliferation, whereas proliferative activity in PC3 was significantly diminished by 10 nM temsirolimus. PC3TEM cell adhesion and invasion was enhanced by 10 nM temsirolimus, compared to temsirolimus treated PC3 control cells. The target proteins Akt, mTOR, rictor (but not raptor) and p70S6k were all elevated (total and activated) in PC3TEM compared to PC3 cells. Distinct modifications were also seen with respect to alpha and beta integrin expression. Additional application of VPA blocked growth and adhesion of PC3TEM cells, reverted integrin modulation and also deactivated proteins of the mTOR signaling pathway. CONCLUSIONS Chronic use of the mTOR inhibitor temsirolimus causes prostate cancer cell resistance. HDAC-inhibition counteracts this process, possibly by cross-communicating with the mTOR signaling axis. Specific targeting of HDAC may, therefore, enhance the benefit of an mTOR-inhibitor based regimen. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e130 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jasmina Makarevic Frankfurt, Germany More articles by this author Igor Tsaur Frankfurt, Germany More articles by this author Eva Weich Frankfurt, Germany More articles by this author Axel Haferkamp Frankfurt, Germany More articles by this author Roman Blaheta Frankfurt, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

mTOR inhibitors are currently used as immunosuppressants in transplanted patients and as promising anti-cancer agents. However, new-onset diabetes is a frequent complication occurring in patients treated with mTOR inhibitors such as rapamycin (Sirolimus). Here, we investigated the mechanisms associated with the diabetogenic effects of chronic Sirolimus administration in rats and in in vitro cell cultures. Sirolimus was administered to rats fed either a standard or high-fat diet for 21 days. Metabolic parameters were measured in vivo and in ex vivo tissues. Insulin sensitivity was assessed by glucose tolerance tests and euglycaemic hyperinsulinaemic clamps. Rapamycin effects on glucose metabolism and insulin signalling were further evaluated in cultured myotubes. Sirolimus induced a decrease in food intake and concomitant weight loss. It also induced specific fat mass loss that was independent of changes in food intake. Despite these beneficial effects, Sirolimus-treated rats were glucose intolerant, hyperinsulinaemic and hyperglycaemic, but not hyperlipidaemic. The euglycaemic hyperinsulinaemic clamp measurements showed skeletal muscle is a major site of Sirolimus-induced insulin resistance. At the molecular level, long-term Sirolimus administration attenuated glucose uptake and metabolism in skeletal muscle by preventing full insulin-induced Akt activation and altering the expression and translocation of glucose transporters to the plasma membrane. In rats fed a high-fat diet, these metabolic defects were exacerbated, although Sirolimus-treated animals were protected from diet-induced obesity. Taken together, our data demonstrate that the diabetogenic effect of chronic rapamycin administration is due to an impaired insulin action on glucose metabolism in skeletal muscles.