Regulation Of mTOR Signaling Research Articles

Abstract 2789: Iron chelation inhibits mTOR activity in cancer cells

Abstract Mammalian target of rapamycin (mTOR) plays a critical role in regulating cell proliferation and survival through sensing environmental cues. mTOR functions as at least two complexes (mTORC1/2). In cancer cells, mTOR activity is often deregulated. Intracellular iron levels are higher in cancer cells than in normal cells mainly due to the increased requirement of iron for proliferation. Thus, cancer cells are more sensitive to iron chelation than normal cells. Iron chelators have been used as traditional treatment for iron overload diseases. Recent studies have shown that iron chelators possess potent anticancer activity. However, the underlying mechanism is not well understood. In this study, we found that ciclopirox olamine (CPX), an iron chelator and off-patent antifungal drug, inhibited cell proliferation and induced cell death in human head and neck cancer cells (PCI-13 and FaDu), breast adenocarcinoma (MDA-MB-231), human rhabdomyosarcoma (Rh30), and skin squamous cell carcinoma (SRB-M7) cells. Similar results were observed using Dp44mT, another iron chelator. Concurrently, CPX and Dp44mT inhibited mTORC1 signaling in the cells. This is evidenced by the findings that treatment with the iron chelators inhibited phosphorylation of 4E-BP1 and S6K1, two best known effector molecules of mTORC1, which could be blocked by addition of ferrous sulfate, but not cuprous sulfate. At the same time, the addition of ferrous sulfate prevented cancer cell death. The findings suggest that the inhibitory effect of CPX or Dp44mT on mTORC1 function was attributed to selective chelation of iron in the cells. Mechanistically, chelation of iron did not activate protein phosphatase 2A (PP2A), but induced raptor (S792) phosphorylation and disrupted mTORC1 integrity. Of note, the effect of iron chelation on mTORC2 was cell line dependent. Further research may enhance our understanding the role of iron in the regulation of mTOR signaling. Citation Format: Chaowei Shang, Hongyu Zhou, Shile Huang. Iron chelation inhibits mTOR activity in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2789. doi:10.1158/1538-7445.AM2014-2789

Abstract 4046: N-myc interactor impacts autophagy via modulation of GSK3β/mTOR signaling

Abstract Autophagy is a cellular mechanism for removal of dysfunctional cellular organelles, large protein complexes. It drives a specialized form of program cell death termed as programmed cell death II. Recent studies have demonstrated a prominent role of the process of autophagy in cancer progression and drug sensitivity. Under conditions of stress, cancer cells try to utilize autophagy to leverage a survival advantage; however, excessive autophagy renders cells vulnerable. Our previous work has demonstrated a pivotal role of N-myc interactor(NMI) in impeding breast cancer progression and metastasis. Loss of NMI is one of the key events that precedes and contributes to invasive progression of breast cancer. Here we provide evidence that NMI plays a crucial role in regulation of mTOR signaling ultimately impacting the autophagic response in breast cancer cells. Our initial observations showed that breast cancer cells restored for NMI expression contained more acidic vacuoles as determined by staining with Lysotracker. Further indication of autophagy was evidenced by the formation of distinct punctae in NMI cells upon transfection with GFP-LC3 which was ablated upon treatment with an autophagy inhibitor. Cells restored for expression of NMI showed decreased levels of p62 along with an increased LC3II processing. The mTOR signaling pathway plays a pivotal role in driving the autophagy. We found that p70S6K phosphorylation dramatically decreases in NMI expressing cells indicating an inhibition of mTOR signaling in these cells. Our observations show that active GSK3β, a negative modulator of mTOR, is responsible for mediating the effects of NMI. Conversely, NMI silenced cells showed indication of inactive GSK3β concomitant with activated mTOR signaling. Functionally, NMI mediated sensitization of cells to autophagy rendered breast cancer cells incapable of sustaining stress of cisplatin treatment. The sensitivity of these cells to cisplatin was notably reduced upon inhibition of the autophagic process. This suggests a possible role for autophagy in NMI induced drug sensitivity. Collectively, our results suggest that NMI plays an important role in modulation of autophagy via activation of GSK3β and consequent inhibition of mTOR signaling. Citation Format: Brandon J. Metge, Aparna Mitra, Lalita A. Shevde, Rajeev S. Samant. N-myc interactor impacts autophagy via modulation of GSK3β/mTOR signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4046. doi:10.1158/1538-7445.AM2014-4046

Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway.

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.

CXCR4 and CXCR7 transduce through mTOR in human renal cancer cells.

Treatment of metastatic renal cell carcinoma (mRCC) has improved significantly with the advent of agents targeting the mTOR pathway, such as temsirolimus and everolimus. However, their efficacy is thought to be limited by feedback loops and crosstalk with other pathways leading to the development of drug resistance. As CXCR4–CXCL12–CXCR7 axis has been described to have a crucial role in renal cancer; the crosstalk between the mTOR pathway and the CXCR4–CXCL12–CXCR7 chemokine receptor axis has been investigated in human renal cancer cells. In SN12C and A498, the common CXCR4–CXCR7 ligand, CXCL12, and the exclusive CXCR7 ligand, CXCL11, activated mTOR through P70S6K and 4EBP1 targets. The mTOR activation was specifically inhibited by CXCR4 antagonists (AMD3100, anti-CXCR4-12G5 and Peptide R, a newly developed CXCR4 antagonist) and CXCR7 antagonists (anti-CXCR7-12G8 and CCX771, CXCR7 inhibitor). To investigate the functional role of CXCR4, CXCR7 and mTOR in human renal cancer cells, both migration and wound healing were evaluated. SN12C and A498 cells migrated toward CXCL12 and CXCL11; CXCR4 and CXCR7 inhibitors impaired migration and treatment with mTOR inhibitor, RAD001, further inhibited it. Moreover, CXCL12 and CXCL11 induced wound healing while was impaired by AMD3100, the anti CXCR7 and RAD001. In SN12C and A498 cells, CXCL12 and CXCL11 promoted actin reorganization characterized by thin spikes at the cell periphery, whereas AMD3100 and anti-CXCR7 impaired CXCL12/CXCL11-induced actin polymerization, and RAD001 treatment further reduced it. In addition, when cell growth was evaluated in the presence of CXCL12, CXCL11 and mTOR inhibitors, an additive effect was demonstrated with the CXCR4, CXCR7 antagonists and RAD001. RAD001-resistant SN12C and A498 cells recovered RAD001 sensitivity in the presence of CXCR4 and CXCR7 antagonists. In conclusion, the entire axis CXCR4–CXCL12–CXCR7 regulates mTOR signaling in renal cancer cells offering new therapeutic opportunities and targets to overcome resistance to mTOR inhibitors.

Gβγ interacts with mTOR and promotes its activation

Diverse G protein-coupled receptors depend on Gβγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gβγ. Thus, we tested the hypothesis that Gβγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gβγ. Cell stimulation with serum modulates Gβγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gβγ heterodimers containing different Gβ subunits, except Gβ4. Both, mTORC1 and mTORC2 complexes interact with Gβ1γ2 which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gβγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gβγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gβγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gβγ signaling interfaces.

Rapamycin improves sociability in the BTBR T+Itpr3/J mouse model of autism spectrum disorders

Overactivation of the mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of syndromic forms of autism spectrum disorders (ASDs), such as tuberous sclerosis complex, neurofibromatosis 1, and fragile X syndrome. Administration of mTORC1 (mTOR complex 1) inhibitors (e.g. rapamycin) in syndromic mouse models of ASDs improved behavior, cognition, and neuropathology. However, since only a minority of ASDs are due to the effects of single genes (∼10%), there is a need to explore inhibition of mTOR activity in mouse models that may be more relevant to the majority of nonsyndromic presentations, such as the genetically inbred BTBR T(+)Itpr3(tf)/J (BTBR) mouse model of ASDs. BTBR mice have social impairment and exhibit increased stereotypic behavior. In prior work, d-cycloserine, a partial glycineB site agonist that targets the N-methyl-d-aspartate (NMDA) receptor, was shown to improve sociability in both Balb/c and BTBR mouse models of ASDs. Importantly, NMDA receptor activation regulates mTOR signaling activity. The current study investigated the ability of rapamycin (10mg/kg, i.p.×four days), an mTORC1 inhibitor, to improve sociability and stereotypic behavior in BTBR mice. Using a standard paradigm to assess mouse social behavior, rapamycin improved several measures of sociability in the BTBR mouse, suggesting that mTOR overactivation represents a therapeutic target that mediates or contributes to impaired sociability in the BTBR mouse model of ASDs. Interestingly, there was no effect of rapamycin on stereotypic behaviors in this mouse model.

MTOR signaling in autophagy regulation in the kidney.

Cells possess adaptive biosynthetic systems to maintain cellular energy levels for survival under adverse environmental conditions. Autophagy is an evolutionarily conserved cellular catabolic process that breaks down and recycles cytosolic material including macromolecules and organelles through lysosomal degradation. This catabolic process, represented by macroautophagy, is induced by a variety of cellular stresses such as nutrient starvation, which causes a shortage of cellular energy for cells to maintain cellular homeostasis and essential biological activities. In contrast, upon nutrient availability, cells stimulate anabolic processes. The mechanistic/mammalian target of rapamycin, a serine/threonine protein kinase, is a key player in stimulating cellular anabolism in response to nutrients and growth factors, and plays a crucial role in suppressing autophagy activity. Growing evidence has suggested that autophagy activity is required for the maintenance and physiological functions of renal cells including proximal tubular cells and podocytes. In this article, we discuss recent progress in the regulation of autophagy by mechanistic/mammalian target of rapamycin signaling.

Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling

Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

The mechanical activation of mTOR signaling: an emerging role for late endosome/lysosomal targeting

It is well recognized that mechanical signals play a critical role in the regulation of skeletal muscle mass, and the maintenance of muscle mass is essential for mobility, disease prevention and quality of life. Furthermore, over the last 15 years it has become established that signaling through a protein kinase called the mammalian (or mechanistic) target of rapamycin (mTOR) is essential for mechanically-induced changes in protein synthesis and muscle mass, however, the mechanism(s) via which mechanical stimuli regulate mTOR signaling have not been defined. Nonetheless, advancements are being made, and an emerging body of evidence suggests that the late endosome/lysosomal (LEL) system might play a key role in this process. Therefore, the purpose of this review is to summarize this body of evidence. Specifically, we will first explain why the Ras homologue enriched in brain (Rheb) and phosphatidic acid (PA) are considered to be direct activators of mTOR signaling. We will then describe the process of endocytosis and its involvement in the formation of LEL structures, as well as the evidence which indicates that mTOR and its direct activators (Rheb and PA) are all enriched at the LEL. Finally, we will summarize the evidence that has implicated the LEL in the regulation of mTOR by various growth regulatory inputs such as amino acids, growth factors and mechanical stimuli.

Abstract 151: A Novel Role for DDiT4L in Regulation of mTOR and Autophagy in the Heart

Introduction: DDiT4L is a known negative regulator of mTOR signaling in skeletal muscle; however its role in the heart is unknown. We have recently showed increased DDiT4L mRNA in a murine transgenic model of pathological but not physiological hypertrophy. Here we test the hypothesis that DDiT4L is a regulator of mTOR signaling in the heart and may play a role in pathological hypertrophy and heart failure. Methods: We investigated the regulation of DDiT4L in murine models of hypertrophy and in cultured neonatal rat ventricular cardiomyocytes (NRVMs). Loss and gain of function of DDiT4L in mTOR regulation and autophagy was investigated using confocal imaging, immunoblotting, and qRT-PCR in NRVMs. Results: DDiT4L gene and protein expression was increased four-fold in pressure overload hypertrophy (n = 4-6, p<0.001), but not in a swim model of physiological hypertrophy. DDiT4L gene expression also significantly increased in a genetic model of dilated cardiomyopathy model (n = 4, p<0.001). In NRVMs, DDiT4L was induced by cardiac stressors such as pathological stretch, hypoxia, and glucose deprivation (n = 3-5 in duplicate, p<0.05-0.01). Increased DDiT4L expression correlated with inhibition of mTOR signaling, and an increase in autophagy markers. siRNA ablation of DDiT4L revealed that inhibition of mTOR signaling by DDiT4L was necessary for glucose deprivation induced autophagy, as determined by imaging of GFP-LC3 autophagosomes (n = 3 in duplicate, p<0.01), and immunoblotting of autophagy markers. Conversely, adenoviral-driven overexpression of DDiT4L inhibited mTOR signaling and significantly increased basal autophagy (n = 3 in duplicate, p<0.05). In TAC mice, the increase in DDiT4L protein expression correlated to inhibition of mTOR signaling, increases in autophagy markers (p<0.01), and preceded the transition to LV dilation and HF. Conclusion: Our data suggests that DDiT4L expression is altered in diverse models of pathological hypertrophy and precedes the development of LV dilatation and overt heart failure. DDiT4L inhibition of mTOR and modulation of autophagy may play a role in the progression to heart failure. DDiT4L may represent a novel therapeutic target to prevent this transition.

Targeting mTOR: Evaluating the Therapeutic Potential of Resveratrol for Cancer Treatment

Resveratrol (3,4',5-trihydroxystilbene; RSV), a natural polyphenol found in a variety of daily food including grapes and red wine, has long been suspected to have multifaceted health beneficial properties, including anti-inflammation, anti-oxidant, and anticancer activities. Over the past few years, numerous studies have suggested that suppressing the activity of mammalian target of rapamycin (mTOR), a critical regulator of cell metabolism, growth, and proliferation, may provide a key mechanism underlying the anticarcinogenic properties of resveratrol. It has been found that resveratrol targets multiple components of the phosphatidylinositol 3- kinase(PI3K)/Akt and mTOR signaling pathways, including PI3K, Akt, PTEN, and DEPTOR, suggesting that this natural compound and its derivatives may offer a promising new cancer treatment. In the current review, we discuss recent findings on the molecular mechanisms regulating mTOR signaling and the therapeutic potential of resveratrol for cancer treatment by targeting mTOR.

52. Roles of the mTOR signaling pathway in hibernating ground squirrels, a differential suppression of active protein synthesis

For many small mammals, survival during the winter months is a serious challenge because of cold environmental temperatures and limited food availability. The solution can be hibernation, an altered physiological state characterized by seasonal heterothermy and entry into long periods of torpor that are interspersed with short arousals back to euthermia. During torpor, metabolic rate is strongly depressed, often to just 1–5% of resting metabolic rate in euthermia. The thirteen-lined ground squirrel, Spermophilus tridecemlineatus , is a primary model species for hibernation research. A critical part of torpor is the coordinated strong suppression of nonessential ATP-expensive cell functions such as protein synthesis to achieve major energy savings. The mTOR signalling pathway is a critical component of the insulin receptor network and the primary cellular process that is responsible for regulating protein synthesis, through its direct interaction with P70S6K and 4E-BP, two regulators of ribosome assembly. Activity of the mTOR signalling pathway in skeletal and cardiac muscle was assessed at six different time points over the torpor-arousal cycle: active cold room (ACR), entrance of torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IBA). Western blotting showed that phosphorylation of mTOR at the Ser2448 residue was strongly reduced in skeletal muscle (87%) during LT indicating reduced mTOR activity; however, phosphorylation of this residue in cardiac muscle was enhanced during EN and EA stages (by 2.9 and 3.2-fold, respectively), while remaining relatively constant during torpor. Evaluation of TSC2, an upstream inhibitor of the mTOR complex in its inactive form, showed reduced phosphorylation of TSC2 Thr 1462 in skeletal muscle (66%) during LT. Suppression of mTOR activity also lead to inactivation of selected downstream substrates; e.g. phospho-4EBPThr46 and phospho-70S6K Thr 389 contents decreased in skeletal muscle by 74% and 45% respectively during LT. Meanwhile, levels of phospho-S6 protein Ser 235 , a component of the 40S ribosome in its active form, were also selectively reduced skeletal muscle (62%) during LT. Differential regulation of mTOR signalling during torpor suggests its alternative roles in tissue dependent responses. Funded by NSERC Canada. For more information go to: www.carleton.ca/∼kbstorey .

Abstract 4180: miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration.

Abstract Background: Glioblastoma is an aggressive tumor which originates from the glial cells and accounts for nearly 50% of adult brain tumors. These tumors are characterized by rapid proliferation, aggressive metastatic potential, and resistance to radio- and chemotherapy. MicroRNAs (miRNAs) are 22-nucleotides long RNAs that regulate gene expression post-transcriptionally. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that is used for the treatment of cardiovascular disease and various metabolic disorders. In addition, treatment with Fenofibrate results in the proliferation of peroxisomes and a marked reduction in the growth of Glioblastoma cells. Methods: To evaluate the effects of treatment on miRNA expression, we used TaqMan quantitative real-time PCR assays from Life Technologies (Grand Island, NY). miRNAs were cloned into BLOCK-iT Pol II miR RNAi Expression Vectors (Life Technologies). Cell growth was evaluated using Guava ViaCount Reagent (Guava Technologies, Hayward, CA). Migration of miRNA-expressing cells was assessed 24h after plating on 8 μm membranes (BD Biosciences, San Jose, CA). Results: In this study, we utilized the LN229 Glioblastoma cell line and found that among the miRNAs up-regulated by Fenofibrate treatment, miR-634 and miR-136 targeted CYR61, a protein involved in migration and proliferation of cancer cells. We additionally found that miR-136 was up-regulated by Fenofibrate treatment while miR-155, another miRNA previously shown to target CYR61, was down-regulated by Fenofibrate. All three miRNAs were predicted and validated to directly target the 3’-UTR of Cyr61. Over-expression of miR-136 and miR-634 miRNAs negatively affected proliferation, but not migration, of LN229 cells. Forced expression of miR-155 reduced migration and addition of recombinant Cyr61 reversed this impairment in migration. Investigation of the signaling pathways regulated by miR-634, revealed an increased phosphorylation of p70 S6 kinase (Thr389), suggesting an induction of the mammalian target of rapamycin (mTOR) complex 1 pathway. Induction of the mTOR pathway was dependent on Akt and Erk activation. In miR-634 overexpressing cells, TSC2, a negative regulator of mTOR signaling, was found to be decreased. Further studies on the differential roles of miRs-136, -155, and -634 in controlling cell growth, cell size, and migration will provide important information on the molecular mechanisms involved in the anticancer properties of Fenofibrate. Citation Format: Duane Jeansonne, Marco Pacifici, Adam Lassak, Krzysztof Reiss, Francesca Peruzzi. miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2013-4180

Abstract 2322: The KIAA1549:BRAF fusion gene regulates mTOR signaling and gliomagenesis in a cell type- and brain region-specific manner.

Abstract The two most common genetic alterations in low-grade pediatric brain tumors (glioma) are NF1 gene inactivation and tandem duplications involving the BRAF kinase gene. In this study, we sought to define the role of KIAA1549:BRAF fusion (fusion BRAF; f-BRAF) gene in glioma development. We demonstrate that f-BRAF regulates cerebellar NSC proliferation by activating the mTOR pathway downstream of MAPK. Also, f-BRAF expression in cerebellar neuroglial progenitors is sufficient to induce glioma-like lesions in mice. Interestingly, f-BRAF expression does not increase the proliferation of NSCs derived from other brain regions, nor does it increase primary astrocyte proliferation. These brain region- and cell type-specific growth promoting effects of f-BRAF are mediated by tuberin/Rheb-dependent modulation of mTOR, leading to S6-kinase activation and p27 degradation. Finally, we demonstrate that mTOR activation is also observed in fusion BRAF-associated human sporadic pilocytic astrocytomas. Our findings highlight the importance of spatial, cellular and molecular heterogeneity in brain tumor development, and provide compelling preclinical evidence for the use of mTOR pathway inhibitors for both sporadic and familial low-grade gliomas. Citation Format: Aparna Kaul, Yi-Hsien Chen, Ryan J. Emnett, Sonika Dahiya, David Gutmann. The KIAA1549:BRAF fusion gene regulates mTOR signaling and gliomagenesis in a cell type- and brain region-specific manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2013-2322

TCTP as a Regulator of mTOR Signaling

TCTP (Translationally Controlled Tumor Protein) is a highly conserved protein that has been suggested as a tumor associated antigen as shown by its deregulation in many different types of cancers. TCTP is thought to be a guanine nucleotide exchange factor (GEF) for Rheb, a key player of the mTOR signal transduction cascade. In this study, the contribution of TCTP toward the mTOR pathway was investigated by over‐expression, gene silencing, and mutational analysis of TCTP in HEK293 cells. Specifically, the overall effects of treatment were studied by performing cell proliferation assays and mTOR signaling was monitored via the phosphorylation status of the protein S6, the downstream effector which gets phosphorylated upon mTOR activation. Overexpression was achieved using a recombinant human TCTP construct and silencing was achieved using specific siRNAs. We constructed site‐directed TCTP mutants at three serine residues at positions 46, 53 and 64 based on their ability to be phosphorylated to determine if these serines have an effect on TCTPs activity. Our results from over‐expression, silencing and mutational analysis of TCTP indicate that silencing of TCTP influences cell viability and mTOR signaling in a manner contrary to what has been reported previously using over‐expression studies and provide interesting insights into TCTP's role in regulating the mTOR pathway.

SYK regulates mTOR signaling in AML

Spleen Tyrosine Kinase (SYK) was recently identified as a new target in acute myeloid leukemia (AML); however, its mechanistic role in this disease is poorly understood. Based on the known interaction between SYK and mTOR signaling in lymphoma, we hypothesized that SYK may regulate mTOR signaling in AML. Both small-molecule inhibition of SYK and SYK-directed shRNA suppressed mTOR and its downstream signaling effectors, as well as its upstream activator, AKT. Moreover, the inhibition of multiple nodes of the PI3K signaling pathway enhanced the effects of SYK suppression on AML cell viability and differentiation. Evaluation of the collateral MAPK pathway revealed a heterogeneous response to SYK inhibition in AML with down-regulation of MEK and ERK phosphorylation in some AML cell lines but a paradoxical increase in MEK/ERK phosphorylation in RAS-mutated AML. These studies reveal SYK as a regulator of mTOR and MAPK signaling in AML and demonstrate that inhibition of PI3K pathway activity enhances the effects of SYK inhibition on AML cell viability and differentiation.

Enchondroma resulting from loss of a Stk11-dependent switch of proliferative chondrocytes to a postmitotic fate

Stk11 (also known as liver kinase b1 (Lkb1)) is a serine-threonine protein kinase that acts upstream of the AMP-activated protein kinase (AMPK) family in coupling energy homeostasis to cell growth, proliferation and survival. Through chondrocyte-specific removal of Lkb1 activity, we showed that Stk11 is required for the normal switch of mitotic chondrocytes to a postmitotic hypertrophic chondrocyte fate. Consequently, it led to a dramatic overgrowth of the growth plate in the Stk11-mutant postnatal skeletal elements. To determine the molecular mechanisms underlying Stk11 action, we examined the mTOR pathway, which is inhibited through AMPK in growth regulation. Strikingly, rapamycin treatment of the pregnant mouse was able to rescue the delay in chondrocyte hypertrophy in Stk11-mutant embryos, suggesting that Stk11 inhibition of mTOR signaling is critical for the switch in chondrocyte fate. Since the dramatic overgrowth of the growth plate is characteristic of enchondroma, we also examined the tumorigenicity of the mutant chondrocytes both in vitro and in vivo . In contrast to wild-type chondrocytes isolated from the postnatal day-30 growth plate, Stk11-mutant chondrocytes proliferated and formed colonies in monolayer and anchorage-independent agar cultures, indicative of a neoplastic transformation in vitro . Similarly, allotransplantation of mutant chondrocytes into immune-deficient NOG mice also resulted in tumor formation in vivo . Gene Ontology analysis of gene expression profiles indicated an augmented activity of cell proliferation and cell cycle regulators within the enchondroma-chondrocyte population compared to chondrocytes in the normal growth plate. Taken together, these data demonstrated an unexpected role of Stk11 in balancing proliferative and nonproliferative hypertrophic states of chondrocyte development through the regulation of mTOR signaling. Stk11 is a known tumor suppressor; our findings raise the possibility that loss of Stk11 may play a role in human enchondroma, a possibility that we are investigating.

Fluorinated N,N′-diarylureas as AMPK activators

Adenosine monophosphate-activated kinase (AMPK) plays a central role in regulating energy homeostasis in eukaryotic cells. AMPK also regulates lipid synthesis by inhibiting acetyl-CoA carboxylase (ACC) and regulates mTOR signaling by activating TSC2. Due to its important roles in cell metabolism, AMPK is an attractive target for metabolic diseases, such as type II diabetes and obesity. AMPK activators, such as metformin, that are used for diabetes treatment are also effective anticancer agents. However, the efficacies of many known AMPK activators are relatively low. For example, metformin activates AMPK at millimolar levels. In this study, we identified a novel family of AMPK activators, namely fluorinated N,N′-diarylureas, that activate AMPK at 1–3μM concentrations. These novel agents strongly inhibit the proliferation of colon cancer cells. We studied the potential mechanisms of these agents, performed a structure–activity relationship (SAR) study and identified several fluorinated N,N′-diarylureas as potent AMPK activators.

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTORSer2448 and a reduced expression of active phospho-p70S6KThr389 and phospho-4E-BP1Thr37/46, two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTORSer2448, phospho-p70S6KThr389 or phospho-4E-BP1Thr37/46. XLID CUL4B mutants did not affect protein level of active phospho-mTORSer2448, phospho-p70S6KThr389 or phospho-4E-BP1Thr37/46. Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.

Negative regulation of mTOR activity by LKB1-AMPK signaling in non-small cell lung cancer cells

To investigate the role of LKB1 in regulation of mTOR signaling in non-small cell lung cancer (NSCLC) cells. LKB1 protein expression and phosphorylation of AMPK, 4E-BP1 and S6K in the cells were assessed using Western blotting in various NSCLC cell lines (A549, H460, H1792, Calu-1 and H1299). Energy stress was mimicked by treating the cells with 2-deoxyglucose (2-DG). Compound C was used to inhibit AMPK activity. Cell growth was measured using the MTS assay. LKB1 protein was expressed in LKB1 wild-type Calu-1, H1299 and H1792 cells, but it was undetected in LKB1 mutant A549 and H460 cells. Treatment of the LKB1 wild-type cells with 2-DG (5, 10 and 25 mmol/L) augmented the phosphorylation of AMPK in dose- and time-dependent manners. In the LKB1 wild-type cells, 2-DG dramatically suppressed the phosphorylation of two mTOR targets, 4E-BP1 and S6K, whereas the LKB1 mutant A549 and H460 cells were highly resistant to 2-DG-induced inhibition on mTOR activity. In addition, stable knockdown of LKB1 in H1299 cells impaired 2-DG-induced inhibition on mTOR activity. Pretreatment of H1299 and H1792 cells with the AMPK inhibitor compound C (10 μmol/L) blocked 2-DG-induced inhibition on mTOR activity. 2-DG inhibited the growth of H1299 cells more effectively than that of H460 cells; stable knockdown of LKB1 in H1299 cells attenuated the growth inhibition caused by 2-DG. In non-small cell lung cancer cells, LKB1/AMPK signaling negatively regulates mTOR activity and contributes to cell growth inhibition in response to energy stress.