Increased mTOR Activity Research Articles

Loss of LKB1 and PTEN tumor suppressor genes in the ovarian surface epithelium induces papillary serous ovarian cancer

Epithelial ovarian cancer presents mostly with serous, endometrioid or mucinous histology but is treated as a single disease. The development of histotype-specific therapy has been challenging because of the relative lack of studies attributing disrupted pathways to a distinct histotype differentiation. mTOR activation is frequently associated with poor prognosis in serous ovarian cancer, which is the most common and most deadly histotype. However, the mechanisms dysregulating mTOR in the pathogenesis of ovarian cancer are unknown. We detected copy number loss and correlated lower expression levels of LKB1, TSC1, TSC2 and PTEN tumor suppressor genes for upstream regulators of mTOR activity in up to 80% in primary ovarian serous tumor databases, with LKB1 allelic loss-predominant. Reduced LKB1 protein was usually associated with increased mTOR activity in both serous ovarian cancer cell lines and primary tumors. Conditional deletion of Lkb1 in murine ovarian surface epithelial (OSE) cells caused papillary hyperplasia and shedding but not tumors. Simultaneous deletion of Lkb1 and Pten, however, led to development of high-grade ovarian serous histotype tumors with 100% penetrance that expressed WT1, ERα, PAX8, TP53 and cytokeratin 8, typical markers used in the differential diagnosis of serous ovarian cancer. Neither hysterectomy nor salpingectomy interfered with progression of ovarian tumorigenesis, suggesting that neither uterine nor Fallopian tube epithelial cells were contributing to tumorigenesis. These results implicate LKB1 loss in the OSE in the pathogenesis of serous ovarian cancer and provide a compelling rationale for investigating the therapeutic potential of targeting LKB1 signaling in patients with this deadly disease.

Mammalian Target of Rapamycin (mTOR) Activity Dependent Phospho-Protein Expression in Childhood Acute Lymphoblastic Leukemia (ALL)

Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25–30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments.

1134 GRADE ASSOCIATED AUTOPHAGY REGULATION VIA AMPK AND MTOR CONTRIBUTES TO UROTHELIAL CANCER CELL SURVIVAL

You have accessJournal of UrologyBladder Cancer: Basic Research (III)1 Apr 20131134 GRADE ASSOCIATED AUTOPHAGY REGULATION VIA AMPK AND MTOR CONTRIBUTES TO UROTHELIAL CANCER CELL SURVIVAL Rani U Ojha, Shrawan K Singh, Shalmoli Bhattacharyya, Rakesh Singh, Aruna Rakha, Kusum Joshi, Arup K Mandal, and Vivekanand Jha Rani U OjhaRani U Ojha Chandigarh, India More articles by this author , Shrawan K SinghShrawan K Singh Chandigarh, India More articles by this author , Shalmoli BhattacharyyaShalmoli Bhattacharyya Chandigarh, India More articles by this author , Rakesh SinghRakesh Singh Chandigarh, India More articles by this author , Aruna RakhaAruna Rakha Chandigarh, India More articles by this author , Kusum JoshiKusum Joshi Chandigarh, India More articles by this author , Arup K MandalArup K Mandal Chandigarh, India More articles by this author , and Vivekanand JhaVivekanand Jha Chandigarh, India More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.749AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelial cell carcinoma (UCC) of urinary bladder has high propensity for recurrence and progression. Effective treatment strategies are needed in this direction. Evidences suggest that targeting autophagy may be an useful approach to treat various cancers, however the status and role of autophagy in UCC is largely unknown. To evaluate autophagy in different grades of UCC and autophagy modulators as potential adjunctive therapeutic agent in the management of UCC the study was conducted. METHODS Material consisted of UCC tissue samples from patients with low and high grade tumor, normal urothelial tissues and T24 cell line. The autophagic vesicles (AVs) and expression of various autophagy proteins in tissue samples were studied by transmission electron microscopy and western blotting respectively. Cell viability, mitochondrial membrane potential, ATP, AMPK and mTOR were measured after inducing autophagy by starvation. The therapeutic implication of autophagy was studied using cisplatin alone or in combination with various autophagy inhibitors (AI). RESULTS High grade UCC had more number of AVs and significantly higher expression of autophagic proteins. On starvation primary culture cells derived from high and low grade UCC showed significant induction of autophagy, activation of AMPK and inhibition of mTOR. Increased mTOR activation and decreased autophagic response were observed after AMPK inhibition. Autophagy inhibition by wortmannin, 3-MA and chloroquine led to ATP depletion, mitochondrial membrane hypo-polarization and caspase 3 and 9 dependent cell death. Treatment with cisplatin significantly increased autophagy in UCC. Treatment with combination of cisplatin and AI led to significantly more cell death in UCC as compared to cisplatin or AI alone. All the above findings were related to grade of UCC and were also observed in T24 cells. CONCLUSIONS This novel study shows that autophagy is related to the grade of Urothelial cell carcinoma of bladder and regulated via AMPK - mTOR pathway for tumor cell survival. Autophagy inhibition causes urothelial cancer cell death through intrinsic apoptotic pathway. The potential application of autophagy inhibitors as an adjunct to chemotherapy in UCC needs further exploration. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e463 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rani U Ojha Chandigarh, India More articles by this author Shrawan K Singh Chandigarh, India More articles by this author Shalmoli Bhattacharyya Chandigarh, India More articles by this author Rakesh Singh Chandigarh, India More articles by this author Aruna Rakha Chandigarh, India More articles by this author Kusum Joshi Chandigarh, India More articles by this author Arup K Mandal Chandigarh, India More articles by this author Vivekanand Jha Chandigarh, India More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Rheb Activation in Subventricular Zone Progenitors Leads to Heterotopia, Ectopic Neuronal Differentiation, and Rapamycin-Sensitive Olfactory Micronodules and Dendrite Hypertrophy of Newborn Neurons

Mammalian target of rapamycin (mTOR) hyperactivity in perinatal neural progenitor cells (NPCs) of tuberous sclerosis complex 1 (Tsc1) heterozygote mice leads to heterotopia and abnormal neuronal morphogenesis as seen in patients with tuberous sclerosis. Considering that pathological hyperactive mTOR also occurs in individuals carrying no genetic mutations, we examined whether increasing mTOR activity in neonatal NPCs of wild-type mice would recapitulate the above phenotypes. Electroporation of a plasmid encoding constitutively active Ras-homolog enriched in brain (Rheb(CA)) into subventricular zone NPCs increased mTOR activity in newborn cells. At 19 d post-electroporation (dpe), heterotopia and ectopic cells with a neuronal morphology were observed along the migratory path [rostral migratory stream (RMS)] and in the olfactory bulb (OB). These ectopic cells displayed action potentials and received synaptic inputs identifying them as synaptically integrated neurons. RMS heterotopias contained astrocytes, neurons, and entrapped neuroblasts. Immunostaining at 3 dpe revealed the presence of Mash1(+) Olig2(-) cells in the migratory route accompanied by ectopic neuronal differentiation and altered direction and speed of neuroblast migration at 7 dpe, suggesting a non-cell-autonomous disruption of migration. At >19 dpe, newborn Rheb(CA)-expressing neurons displayed altered distribution and formed micronodules in the OB. In addition, they displayed increased dendritic complexity along with altered membrane biophysics and increased frequency of GABAergic synaptic inputs. OB heterotopia, micronodules, and dendrite hypertrophy were notably prevented by rapamycin treatment, suggesting their mTOR dependence. Collectively, these data show that increasing mTOR activity in neonatal NPCs of wild-type mice recapitulate the pathologies observed in Tsc1 mutant mice. In addition, increased mTOR activity in individuals without known mutations could significantly impact neurogenesis and circuit formation.

Geropotency: Increased malignant potential of aging neural progenitors

Geropotency: Increased malignant potential of aging neural progenitors

Deregulation of miR-100, miR-99a and miR-199b in tissues and plasma coexists with increased expression of mTOR kinase in endometrioid endometrial carcinoma

BackgroundAlterations of mTOR gene expression have been implicated in the pathogenesis of endometrioid endometrial cancer however only few studies explored the cause of increased mTOR activation in this malignancy. miRNAs are small, noncoding RNAs, which were proven to regulated gene expression at the posttranscriptional level. The study aimed to explore deregulation of miRNAs targeting mTOR kinase (miR-99a, miR-100 and miR-199b) as a possible cause of its altered expression in EEC tissues. In addition expression of the three miRNAs was investigated in plasma of EEC patients and was assessed in terms of diagnostic and prognostic utility.MethodsWe investigated expression of mTOR kinase transcripts in 46 fresh tissue samples. Expression of miR-99a, miR-100 and miR-199b was investigated in the same group of fresh samples, and in additional 58 FFPE sections as well as in 48 plasma samples using qPCR. Relative quantification was performed using experimentally validated endogenous controls.ResultsmTOR kinase expression was increased in EEC tissues and was accompanied by decreased expression of all three miRNAs. Down-regulation of the investigated miRNAs was discovered in plasma of EEC patients and miRNA signatures classified EEC tissues (miR-99a/miR-100/miR-199b) and plasma (miR-99a/miR-199b) samples with higher accuracy in comparison to single miRNAs. We also revealed that miR-100 was an independent prognostic marker of overall survival.ConclusionsWe conclude that increased expression of mTOR kinase coexists with down-regulation of its targeting miRNAs, which could suggest a new mechanism of mTOR pathway alterations in EEC. In addition, our findings implicate that miRNA signatures can be considered promising biomarkers for early detection and prognosis of endometrioid endometrial carcinoma.

Abstract 73: Aberrant Activation of γ2-AMPK Increases Cardiac Growth Through Cellular Hypertrophy and Hyperplasia

AMP-activated protein kinase (AMPK) is an energy sensor and a key regulator of cell metabolism, hence a promising drug target. Point mutations in the regulatory γ2-subunit (encoded by PRKAG2 gene) have been shown to cause a unique form of cardiomyopathy in humans characterized by cardiac growth, arrhythmias and glycogen storage. In previous studies, we demonstrated that the mutation of prkag2 (N488I) caused aberrant activation of AMPK leading to glycogen storage. However, elimination of glycogen storage by inhibiting glycogen synthase activity failed to normalize heart weight (HW) of the mutant mice. Here, we aimed to determine whether cardiac growth in PRKAG2 ardiomyopathy was dueto cellular hypertrophy or hyperplasia. We used transgenic mice expressing a mutant PRKAG2 (N488I) in the heart (TGγ2 N488I ) that faithfully recapitulated PRKAG2 cardiomyopathy. We determined HW and cardiac myocyte size in adult (2 months) and postnatal (2 weeks) hearts in WT and TGγ2 N488I . At 2 months, TGγ2 N488I hearts show a 2.4-fold increase in HW/BW (body weight) (10.3 ± 1.44 vs. 4.3± 0.17 mg/g) as well as cross-sectional cell surface area compared WT hearts (325 ± 13 vs. 155 ± 5.4 μm 2 ,p<0.01), suggesting cellular hypertrophy in adult TGγ2 N488I heart. Furthermore, we observed increased mTOR activity evidenced by enhanced phosphorylation of mTOR (Ser2448) as well as its downstream targets S6 and 4E-BP. The HW of TGγ2 N488I was partially inhibited by treatment with rapamycin, an inhibitor of mTOR. Interestingly, the length and width of isolated cardiomyocytes from 2 weeks old mice were not different between the WT and TGγ2 N488I heart in spite of a 50% increase of HW of TGγ2 N488I mice. We observed a 2- fold increase in the expression of a proliferation marker, proliferating cell nuclear antigen (PCNA) during postnatal cardiac growth. Expression of Cyclin genes including cyclin D1, D2 and E1 was greatly increased in TGγ2 N488I hearts (2.4 - 4 fold, p<0.01 vs. WT). Taken together, these data indicate that aberrant γ2-AMPK activation stimulates cardiac growth through increased cell number during postnatal growth period and increased cell size at adulthood. These results suggest a novel role of γ2-AMPK in the growth of cardiac myocytes.

RESTORATION OF RETINAL GANGLION CELL DENDRITIC ARBORS AFTER AXONAL INJURY IN VIVO.

PurposeDendrites are major determinants of how retinal neurons integrate and process incoming information. We showed that retinal ganglion cells (RGCs) undergo shrinkage of dendritic arbors soon after axotomy, but the molecular mechanisms that underlie injury‐induced dendritic remodeling are poorly understood. Here, we investigated the role of the mTOR (mammalian target of rapamycin) pathway in RGC dendritic structure and synaptic input after acute optic nerve lesion.MethodsAdult transgenic mice carrying the yellow fluorescent protein (YFP) gene were subjected to optic nerve axotomy. Retinal mTOR activity was manipulated by intraocular injection of siRNA against the mTOR repressor REDD2 or by intraperitoneal administration of the mTOR inhibitor rapamycin. Retinal whole‐mounts were prepared and RGC dendritic trees were 3‐D reconstructed.ResultsOptic nerve axotomy leads to marked downregulation of mTOR activity in RGCs correlating with dendritic shrinkage prior to RGC death onset. siREDD2 stimulated mTOR activity in injured RGCs leading to increased dendritic length, surface and complexity compared to control retinas. Importantly, increased mTOR activity in RGCs restored glutamatergic bipolar cell inputs onto RGC dendritic shafts.ConclusionWe report a novel role for the mTOR pathway in the restoration of RGC dendritic arbors and excitatory synaptic input after acute optic nerve lesion.

New therapies for tuber-less sclerosis

Tuberous sclerosis complex (TSC) is well known to neurologists because of the high prevalence of epilepsy, autism spectrum disorders, and intellectual disabilities seen in individuals with this genetic disease.1 TSC has been documented in the medical literature for more than 100 years, with clear descriptions of cortical malformations (tubers), skin lesions, epilepsy, and developmental delay. Our understanding of the genetics, cellular signaling pathways, and pathogenesis of TSC has increased dramatically over the past decade. A key discovery was that the proteins encoded by the TSC1 and TSC2 genes normally function to inhibit the mammalian target of rapamycin (mTOR) serine/threonine kinase.2 This was a seminal breakthrough because mTOR kinase activity normally controls cell size, cell proliferation, and differentiation. Because all of these processes are abnormal in TSC, increased mTOR activity was implicated in the pathogenesis of TSC. The mTOR …

Abstract P198: Aberrant Activation of γ2-AMPK Causes Cardiac Hypertrophy Independent of Glycogen Storage

AMP-activated protein kinase (AMPK) is an energy sensor and a key regulator of cell metabolism, hence a promising drug target. The cardiac functions of AMPK have not been understood. Point mutations in the regulatory γ2-subunit (encoded by PRKAG2 gene) have been shown to cause a unique form of cardiomyopathy in humans characterized by cardiac hypertrophy, arrhythmias and glycogen storage. We have previously shown that PRKAG2 mutation caused aberrant activation of AMPK in the absence of energy deficit and subsequently triggered re-routing of excessive glucose into glycogen pool. In this study, we addressed two questions: 1) whether cardiac hypertrophy in PRKAG2 cardiomyopathy was secondary of glycogen storage; 2) which hypertrophic signaling pathways are involved. We sought to reduce glycogen storage in transgenic mice expressing a mutant PRKAG2 (N488I) in the heart (TGγ2 N488I ) by crossing them to knock-in mice harboring a mutation in the muscle form of glycogen synthase (GYS1 KI ) that greatly reduced GYS activity in response to glucose-6-phosphate. Compared to TGγ2 N488I , TGγ2 N488I -GYS1-KI (double mutant) hearts showed lower GYS activity (0.7 ± 0.07 vs. 6.9 ± 0.49 nmol/min/mg, p<0.0001) and reduced glycogen content (35 ± 4.5 vs. 169 ± 40 umol/g, p<0.0001). Nonetheless, cardiac hypertrophy remained in the double mutant. The heart weight to body weight ratios were 6.8 ± 0.7 mg/g for TGγ2 N488I , 6.7 ± 0.5 mg/g for the double mutant compared to 4.0 ± 0.2 mg/g in the wild type. Furthermore, we have observed significant changes in FOXO (forkhead-O transcription factor) and mTOR (mammalian target of rapamycin) pathways in the TGγ2 N488I hearts. Increased phosphorylation of FOXO3a (Ser321, Ser253) and FoxO1a (Ser256) led to nuclear exclusion and degradation of FOXO proteins. Increased mTOR activity was evidenced by enhanced phosphorylation of Ser2448 as well as its downstream targets S6 and 4E-BP. Taken together these data indicate that aberrant γ2-AMPK activation causes cardiac hypertrophy independent of excessive glycogen accumulation. We found that increased mTOR activity and decreased FOXO signaling contributes to cardiac hypertrophy in TGγ2 N488I mice, suggesting novel mechanisms underlying cardiac hypertrophy caused by abnormal γ2-AMPK activity.

Mtor Complex 1 Plays Critical Roles in Hematopoiesis and Pten-Loss-Evoked Leukemogenesis

Abstract 391The mammalian Target of Rapamycin (mTor) is an evolutionary-conserved serine/threonine protein kinase that plays critical roles in energy homeostasis, and cell-decision processes, such as cell growth and proliferation. Genetic alterations that lead to increased mTor activity in humans and mouse models have suggested a critical role for this kinase and its signal-transduction pathway in hematopoietic-stem cell (HSC) biology as well leukemogenesis, and has garnered much attention as a therapeutic target in human hematopoietic diseases. Further confirmation for a critical function of mTor in hematopoiesis has been suggested through pharmacological inhibition with rapamycin. However, recent evidence has shown that rapamycin is an incomplete inhibitor of mTor and its two associated complexes (mTorc1 and mTorc2). We thus sought to definitively discern the role of mTorc1 in hematopoiesis and leukemogenesis by utilizing mice containing inducible “knock-out” alleles of the essential mTorc1 component Raptor.To conditionally ablate the Raptor gene, mice with flanking loxP sites (floxed) of exon 6 of raptor were crossed with mice expressing Cre under the interferon-inducible Mx promoter (MxCre), and deletion induced in 4–6 week old offspring with injections of polyIpolyC. Induced MxCre:raptor homozoygous mice (KO) and controls (WT) were then analyzed for hematopoietic pertubations. Deletion of raptor leads to a complete ablation of mTorc1 biochemical activity in HSC and progenitors (HSPC) as assessed by phosphoflow analysis of key mTorc1 substrates in response to stem-cell factor. Furthermore, cell-type specific pertubations of a major negative feedback loop from mTor to the Akt pathway were uncovered by this analysis, with increased pAkt levels apparent only in early monocytic cells. Loss of mTorc1 activity produced a non-lethal hematopoietic disorder characterized by leucopenia, transient hypochromic anemia, and thrombocytopenia. Bone marrow (BM) of KO mice was characterized by an apparent lack of normal hematopoiesis and a striking expansion of monoyctic cells at the expense of neutrophils and B lymphocytes. These data suggest a critical role for mTorc1 in the execution of myelomonocytic development. Splenomegaly was prevalent in KO animals, reflecting extramedullary hematopoiesis. As many of these hematopoietic alterations could result from defective HSPC function, we examined the HSPC compartment in more detail. Raptor loss leads to an accumulation of mostly G0 immunophenotypically-defined HSC and multi-potent progenitors in the BM. Metabolic and transcriptional profiling of HSC/MPP indicated pertubations in major metabolic pathways in the KO cells, including decreases in cholesterol and steroid biosynthesis, and alterations in transamination reactions, among others. Strikingly, loss of mTorc1 in HSC/MPP leads to alterations in expression of genes that play critical roles in HSC. To examine the relevancy of these findings to HSC function we performed a series of competitive transplantation experiments. Results from these experiments indicate that mTorc1 is required in a cell-autonomous manner for HSC fitness and proper myelomonocytic differentiation, likely through the control of key metabolic and gene-expression programs.While mTorc1/Raptor loss resulted in profound hematopoietic dysfunction, KO mice survived for greater than 1 year after induced deletion of the raptor gene and maintained hematopoiesis in the absence of Raptor. We next asked whether loss of mTorc1 could be a reasonable therapeutic target in leukemia by generating mice with compound floxed alleles of both Raptor and the Pten gene (deleted again with Cre under the Mx promoter). While homozygous Pten deletion results in a rapidly-fatal myeloproliferative disease in both WT and induced Raptor heterozygous mice, homozygous deletion of Raptor leads to a significantly prolonged survival. Furthermore, through transplantation experiments, we show that mTorc1 is required in a cell-autonomous manner for Pten-loss-evoked leukemogenesis. These studies have demonstrated critical functions of mTorc1 in benign hematopoiesis, suggest rapamycin-independent functions of mTorc1 in this system, and imply that mTorc1 ablation can have beneficial effects in mouse models of leukemogenesis. Disclosures:Scadden:Fate Therapeutics: Equity Ownership. Armstrong:Epizyme: Consultancy.

GLUT1 enhances mTOR activity independently of TSC2 and AMPK.

Enhanced GLUT1 expression in mesangial cells plays an important role in the development of diabetic nephropathy by stimulating signaling through several pathways resulting in increased glomerular matrix accumulation. Similarly, enhanced mammalian target of rapamycin (mTOR) activation has been implicated in mesangial matrix expansion and glomerular hypertrophy in diabetes. We sought to examine whether enhanced GLUT1 expression increased mTOR activity and, if so, to identify the mechanism. We found that levels of GLUT1 expression and mTOR activation, as evidenced by S6 kinase (S6K) and 4E-BP-1 phosphorylation, changed in tandem in cell lines exposed to elevated levels of extracellular glucose. We then showed that increased GLUT1 expression enhanced S6K phosphorylation by 1.7- to 2.9-fold in cultured mesangial cells and in glomeruli from GLUT1 transgenic mice. Treatment with the mTOR inhibitor, rapamycin, eliminated the GLUT1 effect on S6K phosphorylation. In cells lacking functional tuberous sclerosis complex (TSC) 2, GLUT1 effects on mTOR activity persisted, indicating that GLUT1 effects were not mediated by TSC. Similarly, AMP kinase activity was not altered by enhanced GLUT1 expression. Conversely, enhanced GLUT1 expression led to a 2.4-fold increase in binding of mTOR to its activator, Rheb, and a commensurate 2.1-fold decrease in binding of Rheb to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) consistent with mediation of GLUT1 effects by a metabolic effect on GAPDH. Thus, GLUT1 expression appears to augment mesangial cell growth and matrix protein accumulation via effects on glycolysis and decreased GAPDH interaction with Rheb.

Role of mTOR in podocyte function and diabetic nephropathy in humans and mice.

Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly controlled mTOR activity was crucial to maintaining glomerular podocyte function, while dysregulation of mTOR facilitated glomerular diseases. Genetic deletion of mTOR complex 1 (mTORC1) in mouse podocytes induced proteinuria and progressive glomerulosclerosis. Furthermore, simultaneous deletion of both mTORC1 and mTORC2 from mouse podocytes aggravated the glomerular lesions, revealing the importance of both mTOR complexes for podocyte homeostasis. In contrast, increased mTOR activity accompanied human diabetic nephropathy, characterized by early glomerular hypertrophy and hyperfiltration. Curtailing mTORC1 signaling in mice by genetically reducing mTORC1 copy number in podocytes prevented glomerulosclerosis and significantly ameliorated the progression of glomerular disease in diabetic nephropathy. These results demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis and suggest that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy.

Resistin Promotes Cardiac Hypertrophy via the AMP-activated Protein Kinase/Mammalian Target of Rapamycin (AMPK/mTOR) and c-Jun N-terminal Kinase/Insulin Receptor Substrate 1 (JNK/IRS1) Pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Abstract 4005: The leukemia-associated fusion protein DEK-NUP214 induces proliferation through an mTOR-dependent mechanism

Abstract Acute myeloid leukemia (AML) is characterized by the dysregulated proliferation and impaired differentiation of myeloid precursor cells. The majority of these leukemias harbor genetic translocations, which determine both the molecular mechanistics and the prognosis of the disease. The t(6;9)(p22;q34) chromosomal translocation is found in 1% of AML, where it is associated with young age and poor prognosis. The translocation occurs between specific introns in the gene DEK on chromosome 6 and the gene NUP214 on chromosome 9, creating the fusion gene DEK-NUP214. The role of DEK-NUP214 in leukemogenesis is still largely uncharacterized. To obtain an experimental model of the disease, we expressed the fusion gene in the myeloid cell line U937 and studied the phenotype of the stable clones. We show that cells expressing DEK-NUP214 proliferate faster and also sustain their proliferative capacity longer in culture than their normal counterparts. Cellular proliferation is regulated by a wide range of signaling pathways, but many converge on the activating phosphorylation of the mechanistic target of rapamycin (mTOR) at Ser2448. Western blot analysis of the clones revealed that cells expressing DEK-NUP214 have higher levels of both phosphorylated and total mTOR protein. To determine the effect on downstream cellular functions, we proceeded to study mTOR-dependent translation and metabolism. We performed a global translation assay where the incorporation of radioactively labeled amino acids into newly synthesized proteins reflects the rate of translation. The results show that cells expressing DEK-NUP214 have a markedly increased translation rate. Cellular metabolism was studied by measuring the consumption of glucose and the production of lactate in cell supernatant. We demonstrate that cells expressing DEK-NUP214 produce less lactate, despite equal glucose consumption and increased proliferation. In concordance with increased mTOR activity, our results suggest that cells expressing DEK-NUP214 shift their metabolism from glycolysis to oxidative phosphorylation. The increased activity of mTOR thus leads to translational and metabolic changes that could play a role in the leukemogenic effect of DEK-NUP214. Interestingly, proliferation induced by DEK-NUP214 is highly dependent on mTOR. Treatment with the mTOR inhibitor everolimus (RAD001) reduces the proliferation of the DEK-NUP214 cells to the level of the control cells, without affecting the control cells. This pivotal role of mTOR suggests that leukemias harboring the t(6;9)(p22;q34) translocation may be susceptible to treatment with either rapamycin or the novel mTOR inhibitors that are approaching the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4005. doi:10.1158/1538-7445.AM2011-4005

Abstract 2836: Rapamycin prolongs overall survival and progression-free survival in a mouse model of lung cancer in never smokers resistant to erlotinib

Abstract Lung cancer in never smokers accounts for approximately 25,000 cases/yr in the US, and is characterized by distinct molecular alterations such as mutations in EGFR. Despite initial responses to EGFR inhibitors such as erlotinib, tumors invariably recur with selection of tumor cells that have mutations in EGFR that confer resistance. Previously we showed that rapamycin, an inhibitor of mTOR, could inhibit tobacco carcinogen-induced lung tumorigenesis. Here, we used an inducible, genetically engineered mouse model of lung cancer in never smokers resistant to erlotinib (CC10-rtTA, EGFRL858R/T790M) to test whether rapamycin could prevent lung tumorigenesis. Prior to detection of tumors, mTOR was activated in lung tissues by the mutant EGFR transgene. When given to achieve trough drug levels achievable in humans, rapamycin decreased tumor incidence by 52% compared to vehicle at 8 wk, as assessed by microCT. Rapamycin increased overall survival by more than 3 fold (HR=0.075, p<0.0001), which was dependent upon continued rapamycin treatment. To extend these studies, we treated mice with a new irreversible EGFR TKI, WZ4002, and randomised them to consolidation treatment with rapamycin or vehicle. Mice treated with rapamycin had increased progression-free survival and overall survival (p = 0.014 and 0.0007, respectively). Tumors from mice that progressed on rapamycin continued to show inhibition of mTOR, with no evidence of feedback activation of Akt. In a series of 7 resected human NSCLC specimens with T790M mutations, 7/7 displayed increased mTOR activation, highlighting the potential clinical application of rapamycin as a chemopreventive agent in EGFR-driven lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2836. doi:10.1158/1538-7445.AM2011-2836

Akt/mammalian target of rapamycin signaling pathway regulates neurite outgrowth in cerebellar granule neurons stimulated by methylcobalamin

Methylcobalamin (MeCbl), a vitamin B12 analog, promotes neurite outgrowth by activating Akt in neurons. However, Akt is involved in many cellular functions, and the downstream signal of Akt that promotes neurite outgrowth in neurons in the presence of MeCbl remains obscure. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple cellular functions including neurite outgrowth. mTOR is regarded as important for the regeneration of injured nerves. In this study, we examined the relationship between MeCbl and mTOR activity and found that MeCbl increases mTOR activity via the activation of Akt and promotes neurite outgrowth in cerebellar granule neurons via the activation of mTOR.

Single-cell Tsc1 knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in mice

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by mutations in Tsc1 or Tsc2 that lead to mammalian target of rapamycin (mTOR) hyperactivity. Patients with TSC suffer from intractable seizures resulting from cortical malformations known as tubers, but research into how these tubers form has been limited because of the lack of an animal model. To address this limitation, we used in utero electroporation to knock out Tsc1 in selected neuronal populations in mice heterozygous for a mutant Tsc1 allele that eliminates the Tsc1 gene product at a precise developmental time point. Knockout of Tsc1 in single cells led to increased mTOR activity and soma size in the affected neurons. The mice exhibited white matter heterotopic nodules and discrete cortical tuber-like lesions containing cytomegalic and multinucleated neurons with abnormal dendritic trees resembling giant cells. Cortical tubers in the mutant mice did not exhibit signs of gliosis. Furthermore, phospho-S6 immunoreactivity was not upregulated in Tsc1-null astrocytes despite a lower seizure threshold. Collectively, these data suggest that a double-hit strategy to eliminate Tsc1 in discrete neuronal populations generates TSC-associated cortical lesions, providing a model to uncover the mechanisms of lesion formation and cortical hyperexcitability. In addition, the absence of glial reactivity argues against a contribution of astrocytes to lesion-associated hyperexcitability.

Hydrogen peroxide induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells

A novel mechanism for H2O2-induced autophagic cell death in GSH-depleted RAW 264.7 cells, a murine macrophage cell line, is proposed. Under GSH-depleted conditions, H2O2-induced autophagic cell, characterized by an increased LC3-II/I ratio, a decreased level of p62 and the formation of autophagic vacuoles, was inhibited by bafilomycin A1 and by Atg5 siRNA transfection, whereas the cell death was not inhibited by zVAD-fmk, by PI3K inhibitors or by Beclin 1 siRNA transfection. In addition, H2O2 treatment reduced the activity of mTOR and promoted the ubiquitination and degradation of Rheb, a key upstream activator of mTOR. Furthermore, proteasome inhibition with MG132 restored the expression of Rheb and increased mTOR activity, resulting in an increased viability of H2O2-treated cells. Collectively, these findings demonstrate that H2O2 induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells.

Termination of autophagy and reformation of lysosomes regulated by mTOR

Autophagy is an evolutionarily conserved process to catabolize cytoplasmic proteins and organelles1, 2. During starvation, the target of rapamycin (TOR), a nutrient-responsive kinase, is inhibited, thereby inducing autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of autophagosome cargo in autolysosomes3-5, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly defined. Here we show that mTOR signaling is inhibited during autophagy initiation, but reactivated with prolonged starvation. mTOR reactivation is autophagy-dependent, and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell – a process we identify in multiple animal species. Thus, an evolutionarily-conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.