AMPK – Akt – mTOR AXIS IS A METABOLIC CHECKPOINT OF THE CELL in normal and malignant cells AMPK – Akt – mTOR AXIS IS A METABOLIC CHECKPOINT OF THE CELL Dr. Tomas Koltai

HISTORY STARTS IN RAPA NUI (isla de Pascua) (Easter Island) Where Streptomyces hygroscopicus, a bacteria, was found in the soil. It elaborated a product that showed strong antifungal activity, besides immunosuppression and inhibition of cell proliferation. This product was named RAPAMYCIN

Looking for the molecular site of action of RAPAMYCIN A protein was identified in the cell which was called TARGET OF RAPAMYCIN (or TOR). Nowadays it is mTOR (mammalian target of rapamycin) Heitman J, Mowa NR, Hall MN, (1991) Science 253:905-909

Rapamycin is a powerful inhibitor of mTOR But it does not act alone. It forms a complex with other protein: FKBP12. This complex of rapamycin and FKPB12 binds to mTOR and inhibits it. mTOR FKBP12 RAPAMYCIN INACTIVATION OF mTOR

...is precisely the inhibition of the phosphokinase mTOR The mechanism that allows rapamycin to produce immunosuppressive, antiproliferative and antifungal activity… ...is precisely the inhibition of the phosphokinase mTOR Rapamycin

mTOR proteins Are big size proteins (280 Kd). Present in all eucariotic cells. Have phosphokinase activity. There are two different mTORs in mammals: mTOR1 and mTOR2 forming two different associations with other proteins: mTOR1 + RAPTOR = mTORC1 mTOR2 + RICTOR = mTORC2

mTor and its inhibition by the FKBP12-rapamycin complex

mTORC1 is inhibited by rapamycin mTORC2 shows little or no response to rapamycin inhibition. But if it is administered for a long time, some inhibition may appear.

All previous steps are absolutely necessary for cell proliferation. Actions of mTORC1 Has a role in protein synthesis. Induces ribosomal biogenesis. Stimulates RNAm translation. Checks nutrient and energy availability All previous steps are absolutely necessary for cell proliferation.

Cell may undergo autophagy through inhibition of mTOR. If the requiered nutrients or energy are not available… Cell may undergo autophagy through inhibition of mTOR. GENOMIC STRESS AMP ATP AMPKinase mTOR P53 mTOR is an inhibitor of autophagia AUTOPHAGIA

mTOR is a metabolic regulator of the cell Regulators of mTOR activities Growth factors Nutrients Energy Stress Hypoxia

GROWTH FACTORS REGULATING mTOR IRS PI3K AKT TSC2 TSC1 Rheb mTORC1 IGF Extracellular Cell Membrane IGFR IRS PI3K Intracellular AKT GROWTH FACTORS REGULATING mTOR TSC2 TSC1 Rheb RICTOR RAPTOR mTORC1 TRANSCRIPTION mTORC2 AUTOPHAGIA S6K E4 BP ACTINE ORGANIZATION PROTEIN SYNTHESIS RIBOSOMAL GENESIS

HYPOXIA REGULATING mTOR HIPOXIA TSC2 HIF TSC1 Rheb mTORC1 mTORC2 Cell Membrane Extracellular cytoplasm HYPOXIA REGULATING mTOR REDD1 Y REDD2 HIPOXIA TSC2 HIF TSC1 Rheb RICTOR RAPTOR mTORC1 mTORC2

STRESS REGULATING mTOR STRESS: Due to DNA damage P53 activation TSC2 AMPK activation TSC1 Rheb RICTOR RAPTOR mTORC1 TRANSCRIPCION mTORC2

Nutrient availability REGULATING mTOR NUTRIENTS Nutrient availability REGULATING mTOR Cell Membrane Rheb mTORC1 RICTOR RAPTOR TRANSCRIPTION mTORC2 AUTOPHAGIA S6K E4 BP ACTIN ORGANIZATION RIBOSOMAL BIOGENESIS PROTEIN SYNTHESIS

ENERGY REGULATING mTOR INCREASED AMP/ATP RATIO LOW ENERGY AVAILABLE TSC2 AMPK TSC1 LKB1 Rheb RICTOR RAPTOR mTORC1 mTORC2

MAPKINASES ACTIVATION REGULATING mTOR Membrane ACTIVATION OF RAS-MAPK PATHWAY MAPKINASES ACTIVATION REGULATING mTOR TSC2 TSC1 Rheb RICTOR RAPTOR mTORC1 mTORC2

AMPK – Akt – mTOR pathway is a metabolic check point

Mechanism of the metabolic check point mTORC1 is activated when AMPK activity is decreased. AMPK is activated when the AMP/ATP ratio increases (which means low energy). Activated AMPK decreases all those activities that are energy demanding through inhibition of mTORC1 by two pathways.

Mechanism of check point in simple words AMPK “informs” mTOR if there is enough energy. If there is enough energy, mTOR stimulates protein synthesis and proliferative activity. If tehre is energy shortage, mTOR is deactivated and there is no synthesis nor proliferation. In extreme cases, autophagia is started.

What is AMPK? It is a serine/treonine kinasa that senses available celular energy. It is activated by metabolic stress like physical exercise, hypoxia, glucose decrease, etc. It is regulated by AMP/ATP ratio and by kinases LKB1 and CaMKKbeta (calmodulin dependent protein kinase kinase beta). The activaction of AMPK suppresses metabolic activities that requiere ATP and increases those that generate ATP.

DOUBLE MECHANISM of mTOR INHIBITION BY AMPK: THROUGH STIMULATING TUBEROUS SCLEROSIS COMPLEX DIRECT INHIBITION OF mTORC1 LKB1 OTHER ACTIVATORS OF AMPK: resveratrol, quercetina,berberine. INCREASED AMP/ATP RATIO TSC2 AMPK TSC1 METFORMIN Rheb RAPTOR mTORC1 Gwinn DM et al. Mol Cell 2008 30(2):214-226

Akt DOUBLE MECHANISM OF SELENIUM ON THE METABOLIC CHECKPOINT STIMULATION/ACTIVATION OF AMPK DIRECT INHIBITION OF Akt LKB1 TSC2 AMPK SELENIO TSC1 Rheb RAPTOR mTORC1 Akt Lee YK et al. Carcinogenesis 2010; 31(6):1092-1099

Incresead AMP/ATP ratio LKB1 CaMKK Calcium Abundance Incresead AMP/ATP ratio LKB1 CaMKK Other kinases Decreased fatty acid synthesis EF2K Decreased protein synthesis AMPK ACC1/FAS TSC2 ACC2 HMG-Co A reductase p53 Increased fatty Acid oxidation Decreased colesterol synthesis mTOR Cell cycle arrest Fay JR et al. Cancer Prev Res 2009;2(4):301-309 (ACC: acetilCoa carboxilasa/ HMG-Co A: hidroximetilglutaril-Co a reductasa)

ANTAGONISTIC ACTIVITIES BETWEEN Akt and AMPK: central axis of energetic control FOXO PI3K AMPK Akt TSC2 TSC1 RHEB-GTP RAPTOR PRAS40 mTOR mTORC1 GBL

Deactivators of mTORC1 Lack of environmental amino acids (particularly leucine) Decreased energy (via AMPK) Hypoxia (via REDD1 y REDD2 and HIF) Stress (hypoxia, DNA damage through P53) Drugs: rapamycin, metformin, AICAR

The circuit 1) Negative biofeedback from TSC-mTORC1-S6K1 on the pathway IRS-PI3K-Akt This biofeedback is important in diabetes and obesity. It also explains the failure in cancer treatment of certain tumors with rapamycin and rapalogs. 2) mTORC2 activates Akt Akt activation is an important step in cancer.

Diferences and similarities between rapamycin and metformin actions on this check point Inhibits mTOR Increases Akt activity by the negative feedback circuit Decreases negative feedback on IRS1 Acts directly on Raptor METFORMIN Inhibits mTOR Does not increase Akt activity Does not Acts indirectly through AMPK Zakikhani M et al. 2010. Breast Cancer Res Treat. 5 february 2010

AMPK and Raptor: Matching Cell Growth to Energy Supply Interaction Raptor-AMPK Growth is dependent on cellular energy Hardy DG. Molecular Cell 30, May 9, 2008;263-265

LKB1-AMPK-mTORC1 and metformin LKB1: liver kinase B 1 AMPK: adenosine monophosphate activated protein kinase mTORC1: mammalian target of rapamicine complex 1 AMPK is activated by phosphorylation dependent on LKB1 If LKB1 is inhibited AMPK remains inactive. LKB1 inhibition produces hyperglycemia with increased gluconeogenesis yand lipogenesis. LKB1’s activity is necessary for AMPK activation under energy stress (decreased ATP and increased AMP). Metformin requieres a functioning LKB1 for its glucose lowering activity. If LKB1 is absent, metformin lacks glucose lowering activity. According to Rocha metformin may activate AMPK even when LKB1 is absent. Shaw RJ et al. Science 2005; 310(5754):1642-1646 Rocha GZ. Clini Cancer Res 2011;17:3993-4005

AMPK and cancer: 2 contradictions Under isquemia, AMPK activates PFK-2(phosphofructokinase) La PFK-2 protects cells against isquemia. Malignant cells have high levels of PFK-2. CONCLUSION AMPK may protect tumor cells against isquemia. On the other side, it decreases protien synthesis through inhibition of mTORC1.

AMPK activators in cancer may produce Growth arrest of malignant cells with increased expression of P53 and CDK inhibitors like P21 y P27. 1) Rattan et al. J Biol Chem 2005;280:39582-93// 2) Imamura et al. Biochem Biophys Res Commun 2001;287:562-7// 3) Xiang X et al Biochem Biophys Res Commun 2004;321:161-7 Inhibition of breast cancer cells growth with decreased protein synthesis and inhibition of mTOR (Zakikhani et al. Cancer Res 2006;66:10269-73) Inhibition of growth of p53 deficient tumor cells. Buzzai et al. Cancer Res 2007;67:6745-52 Delayed tumor growth and smaller size. Anisimov et al Exp Gerontol 2005;40:685-93 Preventive action in the development of experimental tumors. Schneider et al. Gastroenterology 2001;120:1263-70 Retarded tumor growth. Huang et al Biochem J, 2008;412:221-221

Akt’s activity in cell metabolism control

Akt Stimulates glycolysis Induces resistance to apoptosis Activates hexokinase Through GSK3 (glycogen synthase kinase 3) produces translocation of hexokinase to mitochondrial membrane where it binds to the voltage dependent annion channel (VDAC) suppressing apoptosis.