2 Model of PI3K activation Model of PI3K activation. Autophosphorylation of ligand-activated receptor tyrosine kinases (RTKs) causes recruitment of inactive heterodimeric class IA phosphatidylinositol 3-kinases (PI3Ks) through the interaction of phosphotyrosine residues on the receptor and SRC-homology 2 (SH2) domains on the PI3K p85 regulatory subunit, or the adaptor proteins IRS1 and IRS2. IRS1 and IRS2 are phosphorylated by the activated receptor, generating docking sites for the SH2 domains of p85 and inducing proper assembly of the signalling complex. These SH2–phosphotyrosine interactions bring PI3K in close proximity to its substrate at the plasma membrane and relieve the inhibitory action of p85 on the p110 catalytic subunit, which is then free to convert PtdIns(4,5)P2 (PIP2) into PtdIns(3,4,5)P3 (PIP3). Alternatively, binding of PI3K to activated RAS can also stabilize its membrane localization and activate the catalytic domain. This occurs by recruitment of the adaptor proteins SHC, GRB2 and GAB2 to activated RTKs. C2, C2 domain; CD, catalytic domain; p85 BD, p85-binding domain; RBD, RAS-binding domain.
3 Minding your Ps: the PtdIns(4,5)P2–PtdIns(3,4,5)P3 cycle Minding your Ps: the PtdIns(4,5)P2–PtdIns(3,4,5)P3 cycle. Phosphatidylinositol phosphates are composed of a membrane-associated phosphatidic acid group and a glycerol moiety that is linked to a cytosolic phosphorylated inositol head group. Phosphatidylinositol 3-kinase (PI3K) can phosphorylate PtdIns(4,5)P2 (PIP2) at the D3 position to form the second messenger PtdIns(3,4,5)P3 (PIP3). Phosphorylation at the D3 position is necessary for binding to the pleckstrin-homology domain of AKT (not shown). Dephosphorylation of PIP3 to regenerate PIP2 is accomplished by the 3-phosphatase PTEN. Additionally, PIP3 can be dephosphorylated at the D5 position by SHIP1 or SHIP2 to generate PtdIns(3,4)P2, another potential second messenger.
4 Regulation of AKT activity Regulation of AKT activity. Activation of AKT is initiated by membrane translocation, which occurs after cell stimulation and PtdIns(3,4,5)P3 (PIP3) production. Localization of AKT to the plasma membrane is accomplished by an interaction between its pleckstrin-homology (PH) domain and PIP3. At the membrane, association with carboxy-terminal modulator protein (CTMP) prevents AKT from becoming phosphorylated and fully active. Phosphorylation of CTMP by an as yet unidentified kinase releases CTMP from AKT and allows AKT to be phosphorylated by PDK1 and PDK2 at Thr308 and Ser473, respectively. Phosphorylation at these two sites causes full activation of AKT. C2, C2 domain; CD, catalytic domain; p85 BD, p85-binding domain.Please close this window to return to the main article.
5 PI3K signalling: the big picture PI3K signalling: the big picture. Activation of class IA phosphatidylinositol 3-kinases (PI3Ks) occurs through stimulation of receptor tyrosine kinases (RTKs) and the concomitant assembly of receptor–PI3K complexes. These complexes localize at the membrane where the p110 subunit of PI3K catalyses the conversion of PtdIns(4,5)P2 (PIP2) to PtdIns(3,4,5)P3 (PIP3). PIP3 serves as a second messenger that helps to activate AKT. Through phosphorylation, activated AKT mediates the activation and inhibition of several targets, resulting in cellular growth, survival and proliferation through various mechanisms. Additionally, PI3K has been shown to regulate the activity of other cellular targets, such as the serum and glucocorticoid-inducible kinase (SGK), the small GTP-binding proteins RAC1 and CDC42, and protein kinase C (PKC), in an AKT-independent manner through poorly characterized mechanisms. The activity of these targets leads to survival, cytoskeletal rearrangement and transformation. GSK3 , glycogen synthase kinase-3 ; NF- B, nuclear factor of B; PDK1/2, 3-phosphoinositide-dependent protein kinase 1/2.
6 I SEGNI CARDINALI DEL CANCRO E I MOLTEPLICI RUOLI DI AKT Autosufficienza rispetto ai segnali di crescita e insensibilità ai segnali antiproliferativi :La sovraespressione di Akt può mediare un aumento della risposta cellulare ai livelli di fattori di crescita presenti nell’ambiente extracellulareAkt promuove la localizzazione nucleare di Mdm2, favorendone l’azione di inibizione su p53Akt promuove la localizzazione citoplasmatica di CKI quali p21 e p27, inibendone la funzioneAkt stabilizza i livelli di cicline D1 e D3
7 I SEGNI CARDINALI DEL CANCRO E I MOLTEPLICI RUOLI DI AKT Inibizione del processo apoptotico:Akt inattiva i fattori proapoptotici Bad e (pro)caspasi-9Akt attiva IKK promuovendo la trascrizione di geni antiapoptotici da parte di NFκBAkt inattiva i fattori di trascrizione Forkhead, inibendo la sintesi di FasL
8 Phosphoinositide 3-kinase (PtdIns 3-kinase)–Akt signaling suppresses and PTEN promotes p53 function. (a) Phosphorylation of Mdm2 by Akt induces translocation of Mdm2 into the nucleus where it binds to p53. Mdm2 ligates ubiquitin to p53, which targets it for degradation by the proteasome. (b) The PTEN tumor suppressor protein inhibits PtdIns 3-kinase signaling, including activation of Akt. Blockade of the PtdIns 3-kinase–Akt signaling pathway by PTEN restricts Mdm2 to the cytoplasm, where the Mdm2 is degraded. Thus, PTEN protects p53 from Mdm2 and allows cells to respond to damage or mutation with an apoptotic response
9 I SEGNI CARDINALI DEL CANCRO E I MOLTEPLICI RUOLI DI AKT Potenziale replicativo illimitato:Akt aumenta l’attività telomerasica fosforilando hTERT
10 I SEGNI CARDINALI DEL CANCRO E I MOLTEPLICI RUOLI DI AKT Angiogenesi:Akt attiva la nitrossido sintetasi endoteliale (eNOS), promuovendo il processo angiogenico
11 I SEGNI CARDINALI DEL CANCRO E I MOLTEPLICI RUOLI DI AKT Invasività e metastasi :Akt contribuisce al potenziale invasivo stimolando la produzione di metalloproteinasi della matrice (MMPs)
12 Substrates for Akt affect cell survival Substrates for Akt affect cell survival. Activation of phosphoinositide 3-kinase (PtdIns 3-kinase)–Akt signaling promotes the phosphorylation and loss of function of pro-apoptotic targets and augments the function of anti-apoptotic targets. Akt suppresses the activity of targets that promote apoptosis; among these are Bad, forkhead and human caspase 9. Whether caspase 9 is a bona fide substrate for Akt is uncertain, however, as the Akt phosphorylation motif in human caspase 9 is not conserved in murine caspase 9. Akt inhibits cell cycle arrest by phosphorylating cdk inhibitors p21 and p27 and promotes the activity of NF- B and the expression of proteins that promote cell survival by direct phosphorylation of IKK and as a component of a cascade that promotes phosphorylation of the p65 Rel subunit of NF- B.
13 mTOR Inhibitors: Exploiting New Targets in Cancer Cancer CellEndothelial CellGrowth FactorsNutrientsVEGFRPDGFR-bPI3KmTORAktProtein SynthesismTORTumorCell Growth & ProliferationBioenergeticsVascular Cell GrowthAngiogenic FactorsVascular Pericyte
14 mTOR Coordinates Growth and Nutrient Signaling Blood VesselNutrient AvailabilityGrowth FactorsIncreased Nutrient UptakeNutrientsmTORSecretion of Angiogenic Growth FactorsMG1G2Cell Cycle ActivationS
15 mTOR is a Central Regulator of Growth and Metabolism NutrientsGrowth FactorsmTOR is an intracellular serine/threonine kinasemTOR is a central regulator that senses changes inAvailability of growth factors1,2Availability of nutrients1,2Availability of fuel/energy3mTOR regulation can affectAngiogenesis4Cell growth3Nutrient uptake, utilization5Metabolism3mTORmTOR is a central controller of cell growth and proliferation in normal cellsmTOR integrates signals from a variety of upstream sources, including nutrients and growth factorsIn a nutrient-rich environment, mTOR acts to induce protein synthesis of molecules necessary for angiogenesis, cell growth, and nutrient uptake. Nutrient availability is an important determinant of cell metabolism and the pathways used to generate energy (ATP)ReferencesHarris and Lawrence. Sci STKE. 2003;(212):re15.Huang et al. Cancer Biol Ther. 2003;2(3):Wullschleger et al. Cell. 2006;124(3):Humar et al. FASEB J. 2002;16(8):Edinger and Thompson. Mol Biol Cell. 2002;13(7):Protein SynthesisCell Growth & ProliferationBioenergeticsAngiogenesisNormal Cell
16 mTOR Integrates Growth Factor Signaling TSC2↑GlucoseTSC1AMPKAminoAcids↑ATP↓Glucose↓ATPmTOR pathway, PI3K-AKT-mTOR, is a downstream component of several growth factor signaling pathways1mTOR activation turns on the synthesis of proteins involved in cell growth2mTOR is a critical integrator of signaling that coordinates cell growth control3PI3KGrowthSignalingAktmTORGrowth factors are important determinants for biological processes for cell growth, proliferation, metabolism, and survivalGrowth factors, such as IGF, EGF, PDGF, and VEGF, bind to and activate receptors located on the cell surfaceReceptors activate intracellular signaling cascades that regulate cell growth, angiogenesis, and nutrient uptakeRegulation of growth factor-stimulated signaling is important for ensuring normal cell growthmTOR is a key integration point for information received from upstream receptorsReferencesShaw and Cantley. Nature. 2006;441(7092):Wang X. Physiology (Bethesda). 2006;21:Sarbassov et al. Curr Opin Cell Biol. 2005;17(6):Protein SynthesisCell Growth & ProliferationBioenergeticsAngiogenesis
17 mTOR Integrates Nutrient Signaling ↓Glucose↓ATPGrowthSignalingPI3KAkt↑GlucosemTOR senses availability of amino acids, metabolic fuel, and energy1Nutrients and energy stores are essential for protein synthesis, cell growth, proliferation, and survival1,2,3mTOR activation supports growth and survival by increasing cell access to nutrients and metabolic fuels4AMPKTSC1↑ATPTSC2AminoAcidsmTORmTOR is a sensor that acts as a biochemical switch, ensuring that adequate supplies of energy and nutrients are available to support cell growth, cell metabolism, and angiogenesisNutrients (amino acids, glucose, cholesterol, iron, zinc): mTOR is a sensor of nutrient availabilityAmino acids are taken into the cell via transporters located at the surface of the cell. When cell signaling and/or metabolic activity in the cell are increased, there is an increased need for essential amino acids. Cells meet this need by synthesizing more transporters thus allowing uptake of more nutrientsDepending on the tissue type, glucose can be used by the cell as a carbon source to build fatty acids, amino acids, or other macromolecules. Additionally, glucose can be used as a metabolic fuel to obtain the energy required for cell survivalEnergy for cell processes is provided by ATP, an energy-storing molecule found in cells. mTOR senses ATP availability in the cell through its regulation by AMPK.When resources are low (low ATP, low oxygen, low amino acids, and/or low glucose), cells experience nutrient deprivation and a slowed metabolism. In nutrient- and energy-poor environments, there is a relative increase in the activity of AMP kinase (AMPK). The increased activity of AMPK inhibits the mTOR pathway by phosphorylating and activating TSC2, thus ensuring biological processes do not occur in the absence of adequate nutrient and energy resources.ReferencesMarshall S. Sci STKE. 2006;(346):re7.Herman and Kahn. J Clin Invest. 2006;116:Motoshima et al. J Physiol. 2006;574:63-71.Edinger and Thompson. Mol Biol Cell. 2002;13:Protein SynthesisCell Growth & ProliferationBioenergeticsAngiogenesis
18 mTOR Pathway Regulates Bioenergetics Bioenergetics refers to nutrient utilization and metabolismmTOR senses nutrient and energy availability in a cellmTOR pathway activation controls bioenergetics by increasing nutrient transporter expression and production of angiogenic growth factorsmTOR pathway activation controls bioenergetics by enabling the influx of glucose, amino acids, and other important molecules that are metabolic fuels used to generate ATPTargeting the mTOR pathway can impact the bioenergetics of the cellNutrients (amino acids, glucose, cholesterol, fatty acids) are used to build proteins, lipids or other macromolecules used for cell growth, proliferation, and survival. In most tissues, they are also consumed to generate energy for cell metabolismIncreased transporter expression and/or angiogenesis are ways in which a cell can gain access to additional nutrients for essential cell functionsmTOR is activated by nutrientsmTOR is inhibited by low levels of nutrients and energyReferencesDann and Thomas. FEBS Lett. 2006;580:Edinger and Thompson. Mol Biol Cell. 2002;13:Edinger and Thompson. Oncogene. 2004;23:Herman and Kahn. J Clin Invest. 2006;116:Jequier. Ann N Y Acad Sci. 2002;967:Lynch et al. Am J Physiol Endocrinol Metab. 2001;281:E25-E34.Marshall. Sci STKE. 2006;346:re7.Motoshima et al. J Physiol. 2006;574:63-71.
19 mTOR Pathway is Deregulated by Mutations in Cancer AktPI3KERAblRasEGFIGFNutrientsVEGFGrowth SignalingNormal cell growth, proliferation, and metabolism are maintained by a number of mTOR regulators1,2Regulators of mTOR activitymTOR activatingmTOR deactivatingDeregulation of mTOR can result in loss of growth control and metabolism1,3Mutations in the mTOR pathway have been linked to specific cancers4PTENTSC2TSC1The importance of mTOR in regulating normal cell growth, cell division and angiogenesis is highlighted by the number of proteins involved in its activation or inhibitionmTOR is deregulated in cancer by increased upstream signaling, loss-of-function mutations in upstream inhibitors, and activating mutations in mTOR activatorsIncreased mTOR activity results in the increased protein synthesis of more than 100 genes and proteins involved in cellular responses. Many of the proteins that are regulated by mTOR support the growth, metabolic requirements, and survival of cancer cells.Deregulation of the mTOR-linked pathways increase the risk of developing cancer or have been identified in many cancers (details on specific mutation rates in cancer are listed on page 11).ReferencesAverous and Proud. Oncogene Oct 16;25(48):Mamane et al. Oncogene. 2006;25(48):Ellisen. Cell Cycle. 2005;4(11):Kaper et al. Cancer Res. 2006;66(3):mTORProtein SynthesisCell Growth & ProliferationBioenergeticsAngiogenesisCancer Cell
20 mTOR Pathway is Deregulated in Many Cancers BrainThyroidOral SCCBreastLungBloodPancreasKidneyOvaryColonUterusProstateDeregulation of the pathway can include overexpression of growth factors, overexpression or mutations of growth factor receptors, loss of tumor suppressor genes, and gain-of-function mutations in mTOR-linked pathways, such asInappropriate signaling through members of the human epidermal growth factor receptor (HER/EGFR) family in lung, colon, and breast cancers1-3Activation of the estrogen receptor (ER) through ligand-independent pathways linked to mTOR in breast cancer4High levels of IGF-1 or expression of IGF-1 receptor (IGF-1R) in breast, prostate, lung, thyroid, and kidney cancers, melanoma, and sarcoma5-11Increased Ras or PI3K signaling through activating mutations or loss-of-function mutations in tumor suppressor genes in pancreas, colon, thyroid, lung, leukemia, brain, gastric, breast, ovarian, prostate, endometrial, and oral squamous cell cancers and melanoma12-22Formation of the Bcr-Abl fusion gene, which causes Ph+ chronic myelogenous leukemia (CML)23Deregulated signaling or cross-talk through mTOR-linked pathways can increase mTOR activity; mTOR inhibition could counteract this deregulated signalingCombining an agent that directly targets mTOR with an agent that targets a deregulation in an mTOR-linked pathway could produce more profound anticancer activity than either agent alone, particularly in tumors that have lost function of the tumor suppressor gene, PTEN24(References on page 10)SkinSarcoma
21 mTOR Pathway is Deregulated in Select Cancers p-Akt, 42%16PI3K, 18%–26%27,28PTEN, 15%–41%25HER2, 30%–36%26,27p-Akt, 23%–50%18PTEN, 24%22Ras, 30%12EGFR, 32%–60%1BreastLungTSC1/TSC231,32IGF-1/IGF-1R33VHL34NETTSC1/TSC240p-Akt, 38%38PTEN, 31%39TGFa/TGFb1, %–100%35VHL, 30%–50%36,37IGF-1/IGF-IR, %-69%9p-Akt, 46%15PI3K, 20%–32%13,41PTEN, 35%41Ras, 50%12EGFR, 70%42KidneyIn the cancers shown, signaling through mTOR is stimulated by defects in one or more of the several pathway components upstream of mTOR (growth factor receptors, PI3-K, Akt, PTEN, TSC1/TSC2) or by stimulation of PI3K by mutant Ras/Raf/MAPK pathway components. In certain types of renal cell cancer and some neuroendocrine tumors, loss of function of VHL eliminates the mechanism for clearance of hypoxia-inducible factor 1 (HIF-1), resulting in the transcription of numerous “hypoxia-associated” proteins, which drive angiogenesis and other cellular functions. HIF-1 translation is controlled by mTOR; inhibiting mTOR may be one approach to overcoming the effects of VHL loss.Cappuzzo et al. J Natl Cancer Inst. 2005;97:Cunningham et al. N Engl J Med. 2004;351:Slamon et al. N Engl J Med. 2001;344:Ali and Coombes. Nat Rev Cancer. 2002;2:Hankinson et al. Lancet. 1998;351:Chan et al. Science. 1998;279:Minuto et al. Cancer Res. 1986;46:Belfiore et al. Biochimie. 1999;81:Schips et al. Am J Clin Pathol. 2004;122:All-Ericsson et al. Invest Ophthalmol Vis Sci. 2002;43:1-8.Burrow et al. J Surg Oncol. 1998;69:21-27.Bos. Cancer Res. 1989;49:Samuels et al. Science. 2004;304:554.Levine et al. Clin Cancer Res. 2005;11:Itoh et al. Cancer. 2002;94:Zhou et al. Clin Cancer Res. 2004;10:Mandal et al. Br J Cancer. 2005;92:David et al. Clin Cancer Res. 2004;10:Dai et al. J Clin Oncol. 2005;23:Lim et al. J Clin Pathol. 2005;58:Sansal and Sellers. J Clin Oncol. 2004;22:Soria et al. Clin Cancer Res. 2002;8:Kantarjian et al. N Engl J Med. 2002;346:Hynes and Lane. Nat Rev Cancer. 2005;5:Li et al. Science. 1997;275:Yu and Huang. Oncogene. 2000;19:Saal et al. Cancer Res. 2005;65:Hawthorne and Yu. Cancer Biol Ther. 2004;3:Bhargava et al. Mod Pathol. 2005;18:Stephens et al. Nature. 2004;431:Verhoef et al. Eur J Pediatr. 1999;158:Francalanci et al. Am J Surg Path. 2003;27:Van Gompel and Chen. Surgery. 2004;136:1297.Hammel et al. Gastroenterology. 2000;119:Gomella et al. Cancer Res. 1989;49:Herman et al. Proc Natl Acad Sci. 1994;91:Gnarra et al. Nature Gen. 1994;7:85-90.Horiguchi et al. J Urol. 2003;169:Shin Lee et al. J Surg Oncol. 2003;84:Bjornsson et al. Am J Pathol. 1996;149:Frattini et al. Cancer Res. 2005;65:11227.Ooi et al. Mod Pathol. 2004;17:Colon% Incidence of mutation in select cancer
22 mTOR Activation Supports Cancer Cell Growth NutrientsCancer cells have deregulated growthKey proteins are regulated by mTOR activation:Cell cycle regulators1Proangiogenic factors2Amino acid and glucose transporters3,4mTOR activation supports cancer cell growth by stimulating the synthesis of proteins important for cell growth, angiogenesis, nutrient uptake, and metabolismGrowth SignalingmTORS6K14E-BP1elF-4ES6Protein SynthesisActivation of mTOR pathway is linked to increased protein synthesis by modulating elements that are important in a number of cellular processes, including growth, proliferation, angiogenesis, and nutrient uptake.mTOR stimulates and regulates the synthesis of several proteins at the translation level through its phosphorylation of S6K1 and 4E-BP1mTOR pathway activation stimulates cell growth through cyclin D1, an important component of a cell cycle checkpoint for DNA replicationmTOR increases production of the HIF-1 protein, a transcriptional regulator of angiogenic growth factors, such as VEGF and PDGFmTOR activation stimulates increased expression of glucose and amino acid transporters. Increased transporter expression allows the cell to take up additional metabolic fuel and extracellular nutrients.ReferencesHidalgo and Rowinsky. Oncogene. 2000;19(56):Slomiany and Rosenzweig. J Pharmacol Exp Ther. 2006;318(2):Dann and Thomas. FEBS Lett. 2006;580:Wieman et al. Mol Biol Cell Feb 14 (Ahead of print).Glut 1LAT1Cyclin DHIF-1aCellGrowthAngio-genesisNutrientUptake &Metabolism
23 mTOR Activates Cell Cycle Progression Protein SynthesisG1Cyclin D1In the cell cycle, the G1 and G2 phases define gap periods between DNA synthesis (S) and mitosis (M)In response to growth factor stimuli, cyclin/cyclin-dependent kinase (CDK) combinations promote passage through the cycle—cyclin D/CDK4/6 controls the G1 phase; cyclin E/CDK2 controls the late G1 phaseAfter cells pass through a critical point in late G1, termed the restriction point, they no longer require growth factor stimulation to complete passage through the cell cycle and are committed to dividemTOR inhibition delays cell cycle progression by decreased translation of cyclin D1 and blockage at the restriction pointmTOR inhibition, resulting in decreased cyclin D1 mRNA translation, may suppress the growth of malignant cells characterized by overexpression of cyclin D1, such as mantle cell lymphomaReferencesde Boer et al. Blood. 1995;86:Nelsen et al. J Biol Chem. 2003;278:SRestriction pointIsraels and Israels. Oncologist. 2000;5: , with permission.
24 mTOR Pathway Activation Promotes Angiogenesis Secretion of Angiogenic Growth Factors Angiogenesis enables cancer cells access to growth factors, nutrient and energy resources1mTOR activation elevates protein synthesis of HIF-1a and HIF-2a2HIF turns on several hypoxic stress genes including VEGF and PDGF-b3Cancer cells secrete the proangiogenic factors that promote the formation of new vessels1,4,5mTORProtein SynthesisVHLHIF-1/2mTOR activation stimulates the translation of HIF-1, which ultimately increases production of proangiogenic factors, such as VEGF-A and PDGF-Overexpression of HIF-1 has been associated with aggressive disease and poor prognosis in cancers of the breast, ovary, cervix, esophagus, brain, and head and neck; loss of HIF-1 activity decreases tumor growth, vascularization, and energy metabolismIn hypoxic cells, such as those found in tumors, HIF-1 translocates to the nucleus and combines with HIF-1, ultimately initiating the transcription of hypoxia-regulated genes, such as those for VEGF-A and inducible nitric oxide synthetase (iNOS), which promoteCell survival under anaerobic conditionsAngiogenesisMetastasisHIF-2 is controlled by mTOR in a similar manner, and in some tissues HIF-2 is an important factor in angiogenesis and tumor progressionSecretion of proangiogenic factors from the cancer cell promotes the proliferation of endothelial cells and recruitment of vascular pericytes, cell types that are necessary for new vessel growth.mTOR inhibition can have a compound effect by decreasing HIF-1 levels and inhibiting VEGF productionReferencesGupta and Qin. World J Gastroenterol. 2003;9(6):Majumder et al. Nat Med Jun;10(6):Stoeltzing et al. J Natl Cancer Inst ;96:Yoshimura et al. Clin Cancer Res. 2004;10:Yu et al. World J Gastroenterol ;12:Hypoxic Stress GenesAngiogenic FactorsSecretion
25 mTOR Pathway Activation Promotes Angiogenesis Growth Control of Vascular Cells VEGFPDGFEndothelial CellCancer CellVEGFRPDGFR-bPI3KmTORAktProtein SynthesismTORIn the cancer cell, hypoxia activation of mTOR leads to protein synthesis of hypoxic stress genes, including angiogenic growth factors, such as VEGF and PDGFWhen these angiogenic factors are secreted from the cancer cell, they bind to receptors located on the surface of vascular cells (endothelial cells and pericytes)Activated receptors initiate growth signaling within the vascular cellsGrowth and proliferation of both endothelial cells and pericytes are essential for angiogenesisVHLHIF-1/2TumorVascular Cell GrowthTumorHypoxic Stress GenesAngiogenesisAngiogenicGrowth FactorsVascular Pericyte
26 mTOR Activation Increases Nutrient Uptake Amino AcidsGlucoseNutrientsCancer cells have increased nutrient and metabolic needsAdequate amino acids, glucose, and ATP are required to sustain cancer cell growthNutrients and metabolic fuel are taken up via nutrient transportersmTOR activation can increase the expression of nutrient transportersCancer cell access to nutrients and metabolic fuel support unregulated cell growthGLUT 1LATmTORCancer cells rely on glycolysis to provide the energy (ATP) for cellular processes. In fact, generation of energy (ATP) via glycolysis is a hallmark of transformed cancer cellsAmino acids are used as building blocks for the synthesis of proteins and other macromolecules in cells. Because of a high metabolic requirement in cancer cells, additional nutrients are required to maintain cell processes. Cancer cells meet this need, in part, by increasing the number of transporters at the cell surfaceAdditional nutrient transporters at the cell surface allow the uptake of extracellular amino acids and glucoseAmino acid and glucose transporters are upregulated in several cancers, highlighting the cancer cell requirement for additional nutrient and energy sourcesmTOR inhibition suppresses access of cancer cells to necessary metabolic fuel and energy storesReferencesCooper et al. Br J Cancer 2003;89:Fuchs and Bode. Semin Cancer Biol. 2005;15:Kobayashi et al. J Surg Oncol. 2005;90:Oliver et al. Eur J Cancer 2004;40:Pelicano et al. Oncogene 2006;25:Xu et al. Cancer Res. 2005;65:Protein SynthesisAmino Acid andGlucose Transporters
27 mTOR Coordinates Cancer Cell Growth Blood VesselNutrient AvailabilityProduction of TransportersIncreased Nutrient UptakemTOR coordinates cancer cell growthmTOR activation promotes entry into the cell cycle1, thus committing the cell to divideCells require a lot of nutrients and energy resources to maintain the metabolic requirements found in cancer2. Cancer cells have to find additional sources of nutrients and energy once the cellular supplies are low. Cancer cells gain access to additional resources through increased availability to extracellular nutrients and through angiogenesis3mTOR activation leads to increased expression of transporters at the cell membrane and increases the secretion of proangiogenic molecules, such as VEGF4,5,6Secretion of VEGF induces capillary outgrowth from nearby blood vessels7Capillary outgrowth toward the tumor cells provides new sources of vital oxygen, glucose, and amino acidsAdditional nutrient availability enables tumor growth and survival8,9ReferencesNelsen et al. J Biol Chem. 2003;278:Shaw. Curr Opin Cell Biol. 2006;18(6):Hickey and Simon. Curr Top Dev Biol. 2006;76:Wieman et al. Mol Biol Cell Feb 14 (ahead of print).Fuchs and Bode. Semin Cancer Biol. 2005;15(4):Edinger and Thompson. Mol Biol Cell. 2002;13(7):Bernanke and Velkey. Anat Rec. 2002;269(4):Nishida et al. Vasc Health Risk Manag. 2006;2(3):Brahimi-Horn and Pouyssegur. Int Rev Cytol. 2005;242:Secretion of Angiogenic Growth FactorsGlucose TransportermTORMutations in CancerAmino Acid TransporterMG1G2Cancer Cell GrowthCancer CellS
28 mTOR Inhibition May Disrupt Cancer Cell Growth by Various Ways Blood VesselNutrient AvailabilityDECREASEDmTOR inhibition may disrupt cancer cell growth by various waysmTOR inhibitors will slow/stop entry into the cell cycle by decreasing cyclin D1 mRNA translationIn vitro, mTOR inhibitors can suppress translation/secretion of proangiogenic factors, such as VEGF and PDGFDecreased VEGF secretion should result in decreased blood supply to the tumor cellsDecreased blood supply should result in decreased delivery of nutrient resources (amino acids, oxygen, and glucose)Decreased nutrient supply in combination with direct mTOR inhibition should present a significant barrier to tumor cell growth and survivalSecretion of Angiogenic Growth FactorsmTORGlucose TransporterDECREASEDAmino Acid TransporterMG1G2Cancer Cell GrowthCancer CellS
29 The target of rapamycin signalling pathway The target of rapamycin signalling pathway. Growth factors such as insulin-like growth factor (IGF) activate receptors on the cell surface. These signal to phosphatidylinositol 3-kinase (PI3K), which can be inhibited by PTEN, through insulin-receptor substrates (IRS). PI3K, in turn, activates phosphoinositide-dependent protein kinase 1 (PDK1) and AKT. AKT phosphorylates the TSC1–TSC2 complex, which is thought to act as a negative regulator of the small GTP-binding protein RHEB that might be the direct activator of TOR. TOR binds to raptor and either directly or indirectly controls the activation of ribosomal p70 S6 kinase 1 (S6k1) to regulate ribosomal protein translation and ribosome biogenesis. TOR directly phosphorylates and inactivates eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), the suppressor of eIF4E. Release of eIF4E from phosphorylated 4E-BP1 enables the formation of the eIF4F complex, which is required for cap-dependent translation of mRNAs such as cyclin D1 and c-MYC, which have extensive 5'-untranslated-region secondary structure. PABP, poly-A-binding protein.
30 Figure 1. The mTOR Network (A) The mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. (Left) In addition to mTOR, mTORC1 contains RAPTOR, mLST8, and PRAS40. mTORC1 drives cellular growth by controlling numerous processes that regulate protein synthesis and degradation. Diverse positive and negative growth signals influence the activity of mTORC1, many of which converge upon the TSC1/2 complex.
31 Figure 1. The mTOR Network (B) Model of mTORC1 coregulation by RHEB and PRAS40. (Left) When AKT is inactive, TSC1/2 inhibits RHEB while PRAS40 inhibits mTORC1. (Middle) Upon activation, AKT promotes mTORC1 activity by phosphorylating both TSC1/2 and PRAS40. This results in GTP-loading of RHEB, which directly activates mTORC1 and release of mTORC1 from PRAS40 repression. (Right) In tsc2 null cells, RHEB strongly activates mTORC1. This in turn inhibits AKT by way of the negative feedback loop (described in the text). Even though PRAS40 is dephosphorylated in this state, its ability to repress mTORC1 is overrun by the greatly elevated Rheb activity.
32 Figure 1. The mTOR Network (A) The mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. (Right) mTORC2 also contains mLST8, but instead of RAPTOR and PRAS40, mTORC2 contains the RICTOR, mSIN1, and PROTOR proteins. Currently, the only characterized substrate of mTORC2 is the AKT kinase, which suggests mTORC2 functions downstream in the PI3K pathway to regulate cell growth, proliferation, and survival. mTORC2 also regulates PKCα phosphorylation, but it is not known if this is direct, or if mTORC2 can regulate other AGC-family kinases. Activation and inhibition induced by direct phosphorylation is indicated by a phosphate (P).(
33 Figure 1. The mTOR Network (A) The mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. (Left) In addition to mTOR, mTORC1 contains RAPTOR, mLST8, and PRAS40. mTORC1 drives cellular growth by controlling numerous processes that regulate protein synthesis and degradation. Diverse positive and negative growth signals influence the activity of mTORC1, many of which converge upon the TSC1/2 complex. (Right) mTORC2 also contains mLST8, but instead of RAPTOR and PRAS40, mTORC2 contains the RICTOR, mSIN1, and PROTOR proteins. Currently, the only characterized substrate of mTORC2 is the AKT kinase, which suggests mTORC2 functions downstream in the PI3K pathway to regulate cell growth, proliferation, and survival. mTORC2 also regulates PKCα phosphorylation, but it is not known if this is direct, or if mTORC2 can regulate other AGC-family kinases. Activation and inhibition induced by direct phosphorylation is indicated by a phosphate (P).(B) Model of mTORC1 coregulation by RHEB and PRAS40. (Left) When AKT is inactive, TSC1/2 inhibits RHEB while PRAS40 inhibits mTORC1. (Middle) Upon activation, AKT promotes mTORC1 activity by phosphorylating both TSC1/2 and PRAS40. This results in GTP-loading of RHEB, which directly activates mTORC1 and release of mTORC1 from PRAS40 repression. (Right) In tsc2 null cells, RHEB strongly activates mTORC1. This in turn inhibits AKT by way of the negative feedback loop (described in the text). Even though PRAS40 is dephosphorylated in this state, its ability to repress mTORC1 is overrun by the greatly elevated Rheb activity.
34 Figure 2. mTOR May Function Both Upstream and Downstream of AKT (A) Full activation of AKT requires phosphorylation on two sites, T308 by PDK1 and S473 by mTORC2. Activated AKT phosphorylates many substrates including TSC2 and the FOXOs. Akt phosphorylates mTORC1 directly, but this connection is just beginning to be understood.(B) Eliminating mTORC2 ablates AKT S473 phosphorylation in mouse embryo fibroblasts. This does not affect the phosphorylation of TSC2. In contrast, one AKT phosphorylation site in the FOXO1 and FOXO3 transcription factors is reduced, while phosphorylation at another AKT site is unaffected. One possible model to explain this finding is that S473-unphosphorylated AKT retains enough catalytic activity to phosphorylate some of its targets but not others.(C) Alternatively, a substitute kinase, which is likely to be regulated by PDK1, can compensate for S473-deficient AKT. In the case of FOXO, it is known that SGK can phosphorylate some of the same sites as AKT
35 Figure 4. Linking Cell Autonomous and Systemic Nutrient Sensing by mTOR mTORC1 is an ancient regulator of cell growth that is activated by intracellular nutrients. The ancient function of mTORC2 is unclear, but it may have evolved to indirectly sense nutrients by way of insulin signaling. Circulating glucose triggers the release of insulin into the bloodstream. In peripheral tissues harboring growth factor responsive cells, insulin activates the PI3K-mTORC2-AKT pathway. In individual cells, activation of AKT promotes survival, nutrient influx, and energy (ATP) generation. Signals from intracellular nutrients, energy, and from AKT itself subsequently activate mTORC1, which drives protein synthesis and promotes cell growth. Negative feedback mechanisms modulate PI3K-AKT activity, which may serve to balance nutrient intake with expenditure. Since all cells are not equally responsive to insulin or nutrients, cells originating from diverse tissues may have differential requirements for each mTOR complex.
36 Autophagy is regulated by mammalian target of rapamycin (mTOR) through the action of phosphotidylinositol (PtdIns)-3-kinases. mTOR is a serine/threonine protein kinase that acts as a central regulator of cell growth and survival. The class I PtdIns-3-kinase product PtdIns(3)-phosphate indirectly activates Akt1 (also known as protein kinase B) and stimulates mTOR. Phosphatase and tensin homologue (PTEN) is a PtdIns(3,4,5)P3 3-phosphatase, which lowers the activity of Akt1 and mTOR. Akt1 is another serine/threonine protein kinase that, in addition to activating mTOR, negatively regulates the activity of various downstream effectors including glycogen synthase kinase 3 (GSK3). GSK3 in turn plays a role in the regulation of several physiological processes and mediates apoptosis. Forkhead box O (FOXO) is a transcription factor that is phosphorylated by Akt1, which causes it to be retained as an inactive form in the cytosol. When FOXO enters the nucleus it blocks cell-cycle progression and cell growth. The mTOR complexes (TORC) control cell growth and cell-cycle progression in response to nutrient availability and certain growth factors; TOR acts in part as a global regulator of protein translation. TORC1 (also known as CRTC1) blocks autophagy and is sensitive to rapamycin, whereas TORC2 (also known as CRTC2) regulates Akt1 activation and is insensitive to rapamcyin. However, exposure of certain cell types to rapamycin for 24–48 hours can destabilize TORC2 and inhibit Akt1 activation. The class III PtdIns-3-kinase generates PtdIns(3)-phosphate and activates autophagy. Therefore, use of a pan-specific PtdIns-3-kinase inhibitor or a class III selective inhibitor would block autophagy, whereas a class-I-selective inhibitor would activate autophagy. mLST8, mammalian orthologue of mTOR-interacting protein from Saccharomyces cerevisiae Lst8; PRAS40, proline-rich AKT-substrate, 40 kDa; RAPTOR, regulatory-associated protein of mTOR; RICTOR, rapamycin-insensitive companion of mTOR; SIN1, stress-activated protein-kinase-interacting protein.
37 The cellular process of autophagy The cellular process of autophagy. Conditions such as nutrient starvation, pathogen infection and other environmental stressors, can induce autophagy. Autophagy begins with the isolation of double-membrane-bound structures inside an intact cell. Previously, these structures were believed to be derived from the ribosome-free region of the rough endoplasmic reticulum, but recent studies indicate that they might originate from a pre-existing membrane structure called a phagophore, or could be formed de novo. These membrane structures elongate and mature, and microtubule-associated protein 1 light chain 3 (LC3) is recruited to the membrane. The elongated double membranes form autophagosomes, which sequester cytoplasmic proteins and organelles such as mitochondria. The formation of the pre-autophagosomal structure can be inhibited by the phosphatidylinositol 3-phosphate kinase (PI3K) inhibitor 3-methyladenine (3-MA). Sequestration requires ATP and is regulated mainly by class III PI3K. The autophagosomes mature with acidification by the H+-ATPase and fuse with lysosomes to become autolysosomes (also known as the degradative autophagic vacuoles). Microtubules are important mediators of this fusion process. This process is inhibited by the H+-ATPase inhibitor bafilomycin A1, or by microtubule inhibitors such as vinblastine and nocodazole. Eventually, the sequestered contents are degraded by lysosomal hydrolases for recycling. One assay for autophagic cells is to detect the presence of membrane-bound LC3, which accumulates on autophagosomes.
38 The molecular regulation of autophagy The molecular regulation of autophagy. In the presence of growth factors, growth factor receptor signalling activates class I phosphatidylinositol 3-phosphate kinase (PI3K) at the plasma membrane to keep cells from undergoing autophagy. PI3K activates the downstream target AKT, leading to activation of mammalian target of rapamycin (mTOR), which results in inhibition of autophagy. p70S6 kinase (p70S6K) might be a good candidate for the control of autophagy downstream of mTOR. Overexpression of the phosphatase and tensin homologue (PTEN) gene, by an inducible promoter, antagonizes class I PI3K to induce autophagy. RAS has a dual effect on autophagy — when it activates class I PI3K, autophagy is inhibited, but when it selectively activates the RAF1–mitogen-activated protein kinase kinase (MEK)–extracellular signal-regulated kinase (ERK) cascade, autophagy is stimulated. Rapamycin, an inhibitor of mTOR, induces autophagy. A complex of class III PI3K and beclin 1 (BECN1) at the trans-Golgi network acts to induce autophagy. This pathway is inhibited by 3-methyladenine (3-MA). Downregulation of BCL2, or upregulation of BCL2–adenovirus E1B 19-kD-interacting protein 3 (BNIP3) or HSPIN1 at the mitochondria, also induces autophagy, indicating that BCL2 protects against autophagy, BNIP3 and HSPIN1 trigger autophagy. Autophagy is also induced by the cell death-associated protein kinase (DAPK) and the death-associated related protein kinase 1 (DRP1).
39 The role of programmed cell death in tumor development Figure 1. The role of programmed cell death in tumor development. (Top row) In normal cells, apoptosis eliminates damaged or displaced cells to ensure proper tissue homeostasis. Other growth-limiting stimuli, such as nutrient or essential factor limitation, may trigger autophagy to allow transient survival, but cells will ultimately die by either apoptosis or type II programmed cell death if starvation is prolonged.(Second row) Autophagic-defective cells may have decreased survival in response to nutrient or essential factor limitation,but might also exhibit less efficient cell death in response to normal growth-restricting stimuli, or organelle damage. This could result in excessive cell growth or propagation of damaged cells, which could contribute to tumor formation or progression.(Third row) Apoptotic-resistant cells that fail to die in response to damage or improper environmental survival signals allowfor the propagation of damaged or displaced cells, ultimately promoting tumor formation and progression. Such apoptotic lesions can simultaneously impair therapeutic treatments that work by inducing apoptosis (see text).(Bottom row) Tumor cells that depend on aerobic glycolysis to meet energy demands have now been shown to be sensitive to necrotic cell death in response to DNA-alkylating agents, independent of their capacity for apoptosis (see text and accompanying article by Zong et al. 2004). These results provide important insights into an effective class of chemotherapeutic agents, thus facilitating tumor-specific treatment design.Deirdre A. Nelson et al. Genes Dev. 2004; 18:
40 hypoxicHypothetical model for the double role of macroautophagy in cancer. Depending on their maturation stage, a switch from the inhibition of macroautophagy to maximum activation, could be beneficial for some types of cancer, especially those forming solid tumors. (a) In the early stages of the tumor development, a decrease in protein degradation by autophagy would shift the balance between protein synthesis and degradation towards synthesis, increasing intracellular protein content and thus favoring cellular growth. Also, the low rates of autophagy might render these cells resistant to death after some courses of treatment because autophagic programmed cell death (PCD) could not be activated. (b) In advanced stages of cancer, activation of autophagy might allow the survival of the cells located in central areas of the tumor (blue), which show low vascularization. To compensate for the low supply of nutrients coming to these cells from the blood stream, they could activate autophagy to degrade dispensable intracellular macromolecules, thereby obtaining the building blocks required for the synthesis of the more essential components. Some of these tumoral cells could also activate autophagy when exposed to different cancer treatments as a defensive mechanism. By eliminating intracellular damaged structures before they accumulate inside, these cells prevent the activation of PCD.autophagy
41 Differenze nella risposta di cellule normali e tumorali allo stress metabolico Jin, S. et al. J Cell Sci 2007;120:Fig. 2. Differential response of normal and tumor cells to metabolic stress. Normal cells regulate cell growth in response to nutrient availability by modulating the activity of the PI 3-kinase pathway, which through mTOR promotes cell growth and downregulates the catabolic process of autophagy. In periods of starvation, normal cells downregulate mTOR, which slows cell growth, while upregulating autophagy to allow adaptation to metabolic stress. In contrast, tumor cells frequently acquire mutations that constitutively activate the PI 3-kinase pathway that efficiently promotes cell growth in the presence of nutrients. In starvation conditions, however, tumor cells inefficiently adapt to metabolic stress through the failure to downregulate cell growth and upregulate autophagy, which can result in apoptotic or necrotic cell death though metabolic catastrophe.
42 In che modo è possibile manipolare il metabolismo delle cellule tumorali in modo da indurre la morte cellulare attraverso una catastrofe metabolica?Fig. 3. How manipulation of tumor cell metabolism can be used to induce cell death by metabolic catastrophe. The difference between normal and tumor cell metabolism can be exploited for cancer therapy by promoting metabolic catastrophe. mTOR inhibitors, such as rapamycin and its analogues in this case, are an effective means to limit tumor cell growth (Faivre et al., 2006 ). Alternatively, metabolic catastrophe can be achieved by therapeutic starvation, by restricting nutrient availability, uptake and utilization, by enhancing consumption or by preventing catabolism through autophagy
45 Chemical structure of rapamycin and its analogues currently in clinical trials as anticancer chemotherapeutic agents. Bars indicate the chemical modifications to rapamycin.
46 Regulation of target of rapamycin signalling by raptor Regulation of target of rapamycin signalling by raptor. a | Under amino-acid restriction, raptor binds with higher affinity to target of rapamycin (TOR), inactivating the kinase and preventing the phosphorylation of substrates, such as 4E-BP1, which are bound to raptor through TOR signalling (TOS) motifs. b | Under nutrient-replete conditions, raptor binds less tightly to TOR, presumably as a consequence of allosteric changes in TOR structure, which activates the kinase activity of TOR. This facilitates phosphorylation of 4E-BP1 and assembly of the eIF4F complex, shown in Fig. 4. The question remains whether the binding of the rapamycin–FKBP12 complex to TOR directly inhibits kinase activity and therefore mimics nutrient deprivation, as depicted in c, or induces conformational changes in TOR that displace substrates from the catalytic domain, as depicted in d.
47 mTOR Inhibition May Enhance the Antitumor Effects of Other Therapies ChemotherapyRadiationmTOR InhibitionCancer therapy with mTOR inhibition may potentially be used with other approaches to cancer monotherapy or multimodality therapy. Several multiagent combinations are being investigated in clinical trials. The figure shows combinations for which a rationale has been developed in preclinical studiesGrowth Factor Signaling InhibitorsAntiestrogensAntiangiogenics
48 Combination Therapy Rationale mTOR Inhibition May Enhance the Antitumor Effects of Other Therapies AgentRationaleEGFR inhibitorsDefects in the mTOR signaling pathway may counter the effects of EGFR inhibitors on cell growth and proliferation. Combined treatment has been beneficial in preclinical studies1Cytotoxic chemotherapyCytotoxic drugs such as the platinum derivatives, taxanes, anthracyclines, and gemcitabine have shown improved antitumor effects in preclinical models when used in combination with mTOR inhibitors2-4Antiangiogenic agentsmTOR inhibition affects angiogenesis through mechanisms that enhance and complement those of anti-VEGF/anti-VEGFR signaling inhibitors5AntiestrogensDefects in the mTOR signaling pathway may render estrogen-dependent tumor cells resistant to antiestrogens and aromatase inhibitors. Combinations effective preclinically6-8RadiationIn preclinical studies, mTOR inhibition enhances cell killing induced by radiation, possibly by interfering with repair of damage to DNA9ReferencesGoudar et al. Mol Cancer Ther. 2005;4:Mondesire et al. Clin Cancer Res. 2004;10:Bruns et al. Clin Cancer Res. 2004;10:Grünwald et al. Cancer Res. 2002;62:O’Reilly et al. Proc Am Assoc Cancer Res. 2005;46:715. Abstract 3038.de Graffenried et al. Clin Cancer Res. 2004;10:Zhang et al. Proc Am Assoc Cancer Res. 2003;44(2nd ed):739. Abstract 3715.Boulay et al. Clin Cancer Res. 2005;11:Manegold et al. Proc Am Assoc Cancer Res. 2006;47:1032. Abstract 4397.