PI3K/Akt/mTOR.

PI3K/Akt/mTOR

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.

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.

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.

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.

ROLES OF AKT IN DETERMINING THE HALLMARKS OF CANCER
Self-sufficiency in growth signals and insensitivity to anti-growth signals: Akt overexpression can mediate an increase in cellular response to growth factors in the extra-cellular space Akt promotes cytoplasmic localization of CKIs, such as p21 and p27, thereby inhibiting their function Akt stabilizes cyclin D1 e D3 levels Akt facilitates MDM2 nuclear localization and its inhibitory action on p53

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

ROLES OF AKT IN DETERMINING THE HALLMARKS OF CANCER
Inhibition of apoptosis Akt inactivates the proapoptic factors Bad and (pro)caspase-9 Akt activates IKK enhancing NFκB transcriptional activity on antiapoptotic genes Akt inactivates Forkhead transcription factors, inhibiting FasL synthesis

I SEGNI CARDINALI DEL CANCRO E I MOLTEPLICI RUOLI DI AKT
Potenziale replicativo illimitato: Akt aumenta l’attività telomerasica fosforilando hTERT

Angiogenesi: Akt attiva la nitrossido sintetasi endoteliale (eNOS), promuovendo il processo angiogenico

Invasività e metastasi : Akt contribuisce al potenziale invasivo stimolando la produzione di metalloproteinasi della matrice (MMPs)

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.

mTOR Inhibitors: Exploiting New Targets in Cancer
Cancer Cell Endothelial Cell Growth Factors Nutrients VEGFR PDGFR-b PI3K mTOR Akt Protein Synthesis mTOR Tumor Cell Growth & Proliferation Bioenergetics Vascular Cell Growth Angiogenic Factors Vascular Pericyte

mTOR Coordinates Growth and Nutrient Signaling
Blood Vessel Nutrient Availability Growth Factors Increased Nutrient Uptake Nutrients mTOR Secretion of Angiogenic Growth Factors M G1 G2 Cell Cycle Activation S

mTOR is a Central Regulator of Growth and Metabolism
Nutrients Growth Factors mTOR is an intracellular serine/threonine kinase mTOR is a central regulator that senses changes in Availability of growth factors1,2 Availability of nutrients1,2 Availability of fuel/energy3 mTOR regulation can affect Angiogenesis4 Cell growth3 Nutrient uptake, utilization5 Metabolism3 mTOR mTOR is a central controller of cell growth and proliferation in normal cells mTOR integrates signals from a variety of upstream sources, including nutrients and growth factors In 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) References Harris 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 Synthesis Cell Growth & Proliferation Bioenergetics Angiogenesis Normal Cell

mTOR Integrates Growth Factor Signaling
TSC2 ↑Glucose TSC1 AMPK Amino Acids ↑ATP ↓Glucose ↓ATP mTOR pathway, PI3K-AKT-mTOR, is a downstream component of several growth factor signaling pathways1 mTOR activation turns on the synthesis of proteins involved in cell growth2 mTOR is a critical integrator of signaling that coordinates cell growth control3 PI3K Growth Signaling Akt mTOR Growth factors are important determinants for biological processes for cell growth, proliferation, metabolism, and survival Growth factors, such as IGF, EGF, PDGF, and VEGF, bind to and activate receptors located on the cell surface Receptors activate intracellular signaling cascades that regulate cell growth, angiogenesis, and nutrient uptake Regulation of growth factor-stimulated signaling is important for ensuring normal cell growth mTOR is a key integration point for information received from upstream receptors References Shaw and Cantley. Nature. 2006;441(7092): Wang X. Physiology (Bethesda). 2006;21: Sarbassov et al. Curr Opin Cell Biol. 2005;17(6): Protein Synthesis Cell Growth & Proliferation Bioenergetics Angiogenesis

mTOR Integrates Nutrient Signaling
↓Glucose ↓ATP Growth Signaling PI3K Akt ↑Glucose mTOR senses availability of amino acids, metabolic fuel, and energy1 Nutrients and energy stores are essential for protein synthesis, cell growth, proliferation, and survival1,2,3 mTOR activation supports growth and survival by increasing cell access to nutrients and metabolic fuels4 AMPK TSC1 ↑ATP TSC2 Amino Acids mTOR mTOR 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 angiogenesis Nutrients (amino acids, glucose, cholesterol, iron, zinc): mTOR is a sensor of nutrient availability Amino 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 nutrients Depending 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 survival Energy 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. References Marshall 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 Synthesis Cell Growth & Proliferation Bioenergetics Angiogenesis

mTOR Pathway Regulates Bioenergetics
Bioenergetics refers to nutrient utilization and metabolism mTOR senses nutrient and energy availability in a cell mTOR pathway activation controls bioenergetics by increasing nutrient transporter expression and production of angiogenic growth factors mTOR pathway activation controls bioenergetics by enabling the influx of glucose, amino acids, and other important molecules that are metabolic fuels used to generate ATP Targeting the mTOR pathway can impact the bioenergetics of the cell Nutrients (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 metabolism Increased transporter expression and/or angiogenesis are ways in which a cell can gain access to additional nutrients for essential cell functions mTOR is activated by nutrients mTOR is inhibited by low levels of nutrients and energy References Dann 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.

mTOR Pathway is Deregulated by Mutations in Cancer
Akt PI3K ER Abl Ras EGF IGF Nutrients VEGF Growth Signaling Normal cell growth, proliferation, and metabolism are maintained by a number of mTOR regulators1,2 Regulators of mTOR activity mTOR activating mTOR deactivating Deregulation of mTOR can result in loss of growth control and metabolism1,3 Mutations in the mTOR pathway have been linked to specific cancers4 PTEN TSC2 TSC1 The importance of mTOR in regulating normal cell growth, cell division and angiogenesis is highlighted by the number of proteins involved in its activation or inhibition mTOR is deregulated in cancer by increased upstream signaling, loss-of-function mutations in upstream inhibitors, and activating mutations in mTOR activators Increased 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). References Averous 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): mTOR Protein Synthesis Cell Growth & Proliferation Bioenergetics Angiogenesis Cancer Cell

mTOR Pathway is Deregulated in Many Cancers
Brain Thyroid Oral SCC Breast Lung Blood Pancreas Kidney Ovary Colon Uterus Prostate Deregulation 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 as Inappropriate signaling through members of the human epidermal growth factor receptor (HER/EGFR) family in lung, colon, and breast cancers1-3 Activation of the estrogen receptor (ER) through ligand-independent pathways linked to mTOR in breast cancer4 High levels of IGF-1 or expression of IGF-1 receptor (IGF-1R) in breast, prostate, lung, thyroid, and kidney cancers, melanoma, and sarcoma5-11 Increased 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 melanoma12-22 Formation of the Bcr-Abl fusion gene, which causes Ph+ chronic myelogenous leukemia (CML)23 Deregulated signaling or cross-talk through mTOR-linked pathways can increase mTOR activity; mTOR inhibition could counteract this deregulated signaling Combining 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) Skin Sarcoma

mTOR Pathway is Deregulated in Select Cancers
p-Akt, 42%16 PI3K, 18%–26%27,28 PTEN, 15%–41%25 HER2, 30%–36%26,27 p-Akt, 23%–50%18 PTEN, 24%22 Ras, 30%12 EGFR, 32%–60%1 Breast Lung TSC1/TSC231,32 IGF-1/IGF-1R33 VHL34 NET TSC1/TSC240 p-Akt, 38%38 PTEN, 31%39 TGFa/TGFb1, %–100%35 VHL, 30%–50%36,37 IGF-1/IGF-IR, %-69%9 p-Akt, 46%15 PI3K, 20%–32%13,41 PTEN, 35%41 Ras, 50%12 EGFR, 70%42 Kidney In 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

mTOR Activation Supports Cancer Cell Growth
Nutrients Cancer cells have deregulated growth Key proteins are regulated by mTOR activation: Cell cycle regulators1 Proangiogenic factors2 Amino acid and glucose transporters3,4 mTOR activation supports cancer cell growth by stimulating the synthesis of proteins important for cell growth, angiogenesis, nutrient uptake, and metabolism Growth Signaling mTOR S6K1 4E-BP1 elF-4E S6 Protein Synthesis Activation 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-BP1 mTOR pathway activation stimulates cell growth through cyclin D1, an important component of a cell cycle checkpoint for DNA replication mTOR increases production of the HIF-1 protein, a transcriptional regulator of angiogenic growth factors, such as VEGF and PDGF mTOR 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. References Hidalgo 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 1 LAT1 Cyclin D HIF-1a Cell Growth Angio- genesis Nutrient Uptake & Metabolism

mTOR Activates Cell Cycle Progression
Protein Synthesis G1 Cyclin D1 In 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 phase After 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 divide mTOR inhibition delays cell cycle progression by decreased translation of cyclin D1 and blockage at the restriction point mTOR 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 lymphoma References de Boer et al. Blood. 1995;86: Nelsen et al. J Biol Chem. 2003;278: S Restriction point Israels and Israels. Oncologist. 2000;5: , with permission.

mTOR Pathway Activation Promotes Angiogenesis Secretion of Angiogenic Growth Factors
Angiogenesis enables cancer cells access to growth factors, nutrient and energy resources1 mTOR activation elevates protein synthesis of HIF-1a and HIF-2a2 HIF turns on several hypoxic stress genes including VEGF and PDGF-b3 Cancer cells secrete the proangiogenic factors that promote the formation of new vessels1,4,5 mTOR Protein Synthesis VHL HIF-1/2 mTOR 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 metabolism In 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 promote Cell survival under anaerobic conditions Angiogenesis Metastasis HIF-2 is controlled by mTOR in a similar manner, and in some tissues HIF-2 is an important factor in angiogenesis and tumor progression Secretion 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 production References Gupta 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 Genes Angiogenic Factors Secretion

mTOR Pathway Activation Promotes Angiogenesis Growth Control of Vascular Cells
VEGF PDGF Endothelial Cell Cancer Cell VEGFR PDGFR-b PI3K mTOR Akt Protein Synthesis mTOR In the cancer cell, hypoxia activation of mTOR leads to protein synthesis of hypoxic stress genes, including angiogenic growth factors, such as VEGF and PDGF When 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 cells Growth and proliferation of both endothelial cells and pericytes are essential for angiogenesis VHL HIF-1/2 Tumor Vascular Cell Growth Tumor Hypoxic Stress Genes Angiogenesis Angiogenic Growth Factors Vascular Pericyte

mTOR Activation Increases Nutrient Uptake
Amino Acids Glucose Nutrients Cancer cells have increased nutrient and metabolic needs Adequate amino acids, glucose, and ATP are required to sustain cancer cell growth Nutrients and metabolic fuel are taken up via nutrient transporters mTOR activation can increase the expression of nutrient transporters Cancer cell access to nutrients and metabolic fuel support unregulated cell growth GLUT 1 LAT mTOR Cancer 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 cells Amino 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 surface Additional nutrient transporters at the cell surface allow the uptake of extracellular amino acids and glucose Amino acid and glucose transporters are upregulated in several cancers, highlighting the cancer cell requirement for additional nutrient and energy sources mTOR inhibition suppresses access of cancer cells to necessary metabolic fuel and energy stores References Cooper 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 Synthesis Amino Acid and Glucose Transporters

mTOR Coordinates Cancer Cell Growth
Blood Vessel Nutrient Availability Production of Transporters Increased Nutrient Uptake mTOR coordinates cancer cell growth mTOR activation promotes entry into the cell cycle1, thus committing the cell to divide Cells 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 angiogenesis3 mTOR activation leads to increased expression of transporters at the cell membrane and increases the secretion of proangiogenic molecules, such as VEGF4,5,6 Secretion of VEGF induces capillary outgrowth from nearby blood vessels7 Capillary outgrowth toward the tumor cells provides new sources of vital oxygen, glucose, and amino acids Additional nutrient availability enables tumor growth and survival8,9 References Nelsen 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 Factors Glucose Transporter mTOR Mutations in Cancer Amino Acid Transporter M G1 G2 Cancer Cell Growth Cancer Cell S

mTOR Inhibition May Disrupt Cancer Cell Growth by Various Ways
Blood Vessel Nutrient Availability DECREASED mTOR inhibition may disrupt cancer cell growth by various ways mTOR inhibitors will slow/stop entry into the cell cycle by decreasing cyclin D1 mRNA translation In vitro, mTOR inhibitors can suppress translation/secretion of proangiogenic factors, such as VEGF and PDGF Decreased VEGF secretion should result in decreased blood supply to the tumor cells Decreased 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 survival Secretion of Angiogenic Growth Factors mTOR Glucose Transporter DECREASED Amino Acid Transporter M G1 G2 Cancer Cell Growth Cancer Cell S

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.

Target of rapamycin is a central regulator of cell growth and proliferation in response to environmental and nutritional conditions. Target of rapamycin (TOR) signalling is regulated by growth factors, amino acids, ATP and O2 levels; second messengers (for example, phosphatidic acid); and, possibly, mitochondrial stress. Signalling through TOR seems to regulate several downstream pathways that impinge on cell-cycle progression, translation initiation, transcriptional stress responses, protein stability and survival. Dashed lines indicate pathways that are best described in yeast. ATP, adenosine triphosphate; ASK1, apoptosis-signal-regulating kinase 1; S6K1, ribosomal p70 S6 kinase 1; eIF4E, eukaryotic translation initiation factor 4E; 4E-BP1, eIF4E-binding protein 1.

a | A simplified overview of the PI3K/AKT/mTOR pathway
a | A simplified overview of the PI3K/AKT/mTOR pathway. Ligand binding to various receptor tyrosine kinases initiates a cascade that leads to the activation of mTOR, which has a key role in cell growth and proliferation and regulation of apoptosis. Temsirolimus binds to FK506-binding protein 12 (FKBP12), and the resultant protein–drug complex inhibits the kinase activity of mTOR6, 9. b | Temsirolimus

Chemical structure of rapamycin and its analogues currently in clinical trials as anticancer chemotherapeutic agents. Bars indicate the chemical modifications to rapamycin.

Sabatini Nature Reviews Cancer advance online publication;
The main points of contact between the mTORC1 (mammalian target of rapamycin complex 1) and PI3K (phosphatidylinositol 3-kinase)–Akt (also known as protein kinase B) pathways are emphasized. A main function of the mTORC1 pathway is to regulate the accumulation of cell mass by activating mRNA translation and ribosome biogenesis and by inhibiting autophagy. mTORC1 directly phosphorylates and activates S6K1 (ribosomal S6 kinase 1), which is an important regulator of cell size. Phosphorylation of S6K1 by PDPK1 (3-phosphoinositide-dependent protein kinase 1) is also important for its activation, but for clarity this connection is not shown. S6K1 inhibits IRS1 (insulin receptor substrate 1) by directly phosphorylating it, a connection that in the mTOR field is frequently called 'the feedback loop' and is responsible for the inhibition of Akt that is caused by high mTORC1 activity. By phosphorylating the 4EBP (eukaryotic translation initiation factor 4E binding protein) family of proteins mTORC1 represses their capacity to inhibit the mRNA cap-binding protein eIF4E (eukaryotic initiation factor 4E). Less is known about how mTORC1 activates S6K2 and CLIP-170 (cytoplasmic linker protein 170, also known as restin) and it is likely that many direct substrates of mTORC1 remain to be discovered. The TSC1 (tuberous sclerosis 1)–TSC2 heterodimer is a key negative regulator of mTORC1 that functions by suppressing Rheb (Ras homologue enriched in brain), a small GTP-binding protein that activates mTORC1. Mammals contain two Rhebs, RHEB1 and RHEB2, which can both activate mTORC1 signalling. Insulin and other growth factors, energy status and DNA damage signal to TSC1–TSC2 by regulating kinases that directly phosphorylate TSC2. Hypoxia induces the expression of REDD1 (regulated in development and DNA-damage responses 1) and REDD2, which activate TSC1–TSC2 through an unknown mechanism. It is unknown how osmotic and heat-shock stress, as well as amino acids, signal to mTORC1 and it might be that mechanisms apart from the Redds are involved in the regulation of mTORC1 by hypoxia. mTORC2 directly phosphorylates Akt on the hydrophobic site in the C-terminal tail, which together with the PDK1-mediated phosphorylation of the activation loop is necessary for full Akt activation. How mTORC2 is regulated is unknown but its activity does respond to growth factors; this is mediated through tyrosine kinase (TK) receptors. mTORC2 can be considered upstream of mTORC1 because by activating Akt it leads to the inhibition of TSC1–TSC2, which causes the activation of Rheb and mTORC1. AMPK, AMP-activated protein kinase; ERK, extracellular signal-regulated kinase; FKBP12, intracellular receptor for rapamycin; Foxo, Forkhead box; GSK3, glycogen synthase kinase 3;HIF1 , hypoxia-induced factor 1 ; LKB1, serine–threonine kinase 11; MDM2, mouse double minute 2; NF1, neurofibromatosis 1; PKC , protein kinase C ; PTEN, phosphatase and tensin homologue; RAC1, Ras-related C3 Botulinum toxin substrate 1; RSK, ribosomal protein S6 kinase. Sabatini Nature Reviews Cancer advance online publication; published online 17 August 2006 | doi: /nrc1974

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.

(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.

(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). (

(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.

mTOR Inhibitors Suppress Nutrient Availability
VEGF PDGF Nutrients Growth Factors VEGFR PDGFR-b PI3K mTOR Akt Protein Synthesis mTOR mTOR inhibition suppresses nutrient availability by two mechanisms In the cancer cell mTOR inhibition suppresses nutrient availability by decreasing the synthesis of new transporters, which are essential to take up nutrients from the extracellular environment1-4 mTOR inhibition should decrease the production and secretion of angiogenic factors from the cancer cell5 In the vascular cells (endothelial cells and vascular pericytes) Because of decreased synthesis and secretion of angiogenic growth factors from the cancer cells (described above), there will be less VEGF and PDGF to bind receptors on the vascular cells Decreased growth signaling leads to a decrease in growth and proliferation of vascular cells6 mTOR inhibition within the vascular cells may further suppress vascular cell growth7 The net result of decreased endothelial and pericyte growth is decreased angiogenesis8 Decreased angiogenesis results in a decreased availability of new sources of nutrients References 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): Edinger. Biochem Soc Trans. 2005;33(Pt 1): Del Bufalo et al. Cancer Res. 2006;66(11): Bergers et al. J Clin Invest. 2003;111(9): Guba et al. Blood. 2005;105(11): Armulik et al. Circ Res. 2005;97(6): Angiogenic Factors Nutrient Transporters Tumor Vascular Cell Growth Secretion VEGF, PDGF Nutrient Uptake Angiogenesis

mTOR Inhibition is a Novel Approach for Blocking Angiogenesis
VEGF mAb PDGF VEGF TKI VEGFR PDGFR-b mTOR PI3K Protein Synthesis Akt Angiogenesis inhibitors are currently approved therapies for the treatment of cancer Inhibitors of mTOR have dual mechanisms for inhibiting cancer cells mTOR inhibitors directly suppress the growth of cancer cells and vascular cells by decreasing protein synthesis of important growth-related proteins. mTOR inhibitors act indirectly on vascular cells by blocking synthesis and secretion of proangiogenic factors, such as VEGF and PDGF mTOR inhibitors may have enhanced activity in combination with VEGF monoclonal antibodies (mAb) or tyrosine kinase inhibitors (TKI) Preclinical and clinical studies indicate a synergistic response in patients with combination therapy involving approved angiogenesis inhibitors Targeting angiogenesis downstream of the receptor may provide therapeutic benefit to patients in whom approved treatments have failed mTOR inhibitors have an additional benefit of inhibiting growth factors by blocking synthesis and secretion of proangiogenic factors, such as VEGF and PDGF References Lane and Lebwohl. Semin Oncol. 2006;33:S18-S25. Maxwell et al. Nature. 1999;399: Wan et al. Neoplasia. 2006;8: Yoshimura et al. Clin Cancer Res. 2004;10: Yu et al. World J Gastroenterol. 2006;12: VHL HIF1/2 Tumor mTOR Hypoxic Stress Genes Angiogenic Growth Factors Vascular Cell Growth Angiogenesis

Cell Growth & Proliferation
mTOR Inhibition May Enhance the Antitumor Effects of Targeted Therapies Growth Factor mAb Growth factor inhibitors target either the growth factor or the receptor on the cell surface mTOR inhibitors target cancer cell growth downstream of growth factor receptors Combining an mTOR inhibitor with a growth factor receptor inhibitor may be a more effective strategy for cancer treatment mTOR inhibitors may be effective in patients that are refractory to growth factor inhibition TKI PI3K Akt mTOR Protein Synthesis Cell Growth & Proliferation Bioenergetics Angiogenesis

mTOR Inhibition May Enhance the Antitumor Effects of Other Therapies
Chemotherapy Radiation mTOR Inhibition Cancer 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 studies Growth Factor Signaling Inhibitors Antiestrogens Antiangiogenics

Combination Therapy Rationale mTOR Inhibition May Enhance the Antitumor Effects of Other Therapies
Agent Rationale EGFR inhibitors Defects in the mTOR signaling pathway may counter the effects of EGFR inhibitors on cell growth and proliferation. Combined treatment has been beneficial in preclinical studies1 Cytotoxic chemotherapy Cytotoxic drugs such as the platinum derivatives, taxanes, anthracyclines, and gemcitabine have shown improved antitumor effects in preclinical models when used in combination with mTOR inhibitors2-4 Antiangiogenic agents mTOR inhibition affects angiogenesis through mechanisms that enhance and complement those of anti-VEGF/anti-VEGFR signaling inhibitors5 Antiestrogens Defects in the mTOR signaling pathway may render estrogen-dependent tumor cells resistant to antiestrogens and aromatase inhibitors. Combinations effective preclinically6-8 Radiation In preclinical studies, mTOR inhibition enhances cell killing induced by radiation, possibly by interfering with repair of damage to DNA9 References Goudar 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.

Summary Rationale for Targeting mTOR
Targeting deregulated pathways has been a successful clinical strategy mTOR is a central regulator of cancer cell growth and metabolism Deregulation of components of the mTOR pathway occurs in many types of hematologic and solid tumors Targeting the mTOR pathway can impact the bioenergetics of the cell, a new approach in the treatment of cancer mTOR is a unique target in cancer that may provide therapeutic benefit to patients with disease refractory to currently approved therapies Therapeutic strategies combining mTOR inhibitors with other targeted therapies or cytotoxic agents may provide enhanced anticancer activity

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