Nutrient and Growth Factor Control of Cell Cycle Progression and Survival in Cancer Cells  Bio 714 Cell Biology Feb, 2014

Hurdles in Progression to Cancer I Constitutive Growth Factor Signaling avoid quiescence - cell cycle exit into G0 II Suppression of “Gatekeeper Functions” to get past cell cycle checkpoints III Suppression of Apoptosis avoid default cell death programs IV Acquire Immortality - telomerase expression overcome cell senescence - cancer stem cells? V Stimulate Angiogenesis provide nutrition VI Acquire the ability to migrate and invade gain access to circulation (metastasis) VII Breakdown Caretaker Function genomic instability needed for many mutations

Cell Cycle Control and Cancer I Constitutive Growth Factor Signaling avoid quiescence - cell cycle exit into G0 II Suppression of “Gatekeeper Functions” to get past cell cycle checkpoints III Suppression of Apoptosis avoid default cell death programs IV Acquire Immortality - telomerase expression overcome cell senescence - cancer stem cells? V Stimulate Angiogenesis provide nutrition VI Acquire the ability to migrate and invade gain access to circulation (metastasis) VII Breakdown Caretaker Function genomic instability needed for many mutations

Regulation of Cell Cycle Progression (Quiescence) Growth Factor Signals Tyrosine kinases Ras/Raf/MEK/MAPK Restriction Point G1-pm G1-ps S G2 M Gatekeepers Myc SV40 Early Region (Suppression of p53, Rb and PP2A) Cell Growth Checkpoint (mTOR)

Cooperating Oncogenes and Survival Signals in Tumorigenesis Weinberg and colleagues demonstrated that signaling oncogenes (Ras) cooperate with gatekeeper override oncogenes (Myc) to transform primary rodent cells - and with SV40 early region genes to transform human cells Activated Ras by itself - induces apoptosis Thus, Myc suppresses the apoptosis induced by activated Ras Signals that lead to elevated Myc provide a “Survival Signal”

Foster DA, Yellen P, Xu L, Saqcena M. Genes & Cancer (2011)

Conventional View of Cell Cycle Points: Figure 8.8 The Biology of Cancer (© Garland Science 2007) Conventional View of Cell Cycle Points: The Restriction Point, originally characterized by Arthur Pardee, is a point in G1 where cells no longer require growth factors and commit to completing the cell cycle In the absence of growth factors, cells exit the cell cycle into quiescence or G0 Zetterberg and colleagues have mapped the Restriction Point to a site ~ 3.5 hr after mitosis - where cyclin D is elevated Leland Hartwell described a site in the Yeast cell cycle called START that is late in G1 - where cells evaluate whether there is sufficient nutrition to complete cell division From: Weinberg, The Biology of Cancer, 2007 In some texts, the Restricition Point is referred to as the mammalian equivalent of START - and located near the site where cyclin E is activated Rapamycin treatment results in the activation of TGF- signaling and arrest at the cyclin E site - that can be clearly distinguished both temporally and genetically from the growth factor-dependent Restriction Point

Protozoans & Metazoans are… different! Vander Heiden MG, Cantley LC, Thompson CB. Science (2009)

G1 S Restriction Point (R) - GF Restriction Point: Point in G1 after which cells no longer need GF permissive signals to divide.

Mammalian Restriction Point is Analogous to START in Yeast Cell Cycle: START vs. R Yeast Mammalian Restriction Point Pyronnet S, Sonenberg N Curr. Opin. Genetics Dev. (2001) Cooper GM. The Cell: A Molecular Approach (2000) Mammalian Restriction Point is Analogous to START in Yeast Lodish H, et al. Molecular Cell Biology (2008)

What is a Restriction Point ? 3.5 hr + GF R - GF G0 M G1 S Competence Factor (PDGF) Progression Factor (IGF-1) Based on Zetterberg and Larsson, PNAS (1985) Based on Pledger and Stiles, PNAS (1979)

Where is the Restriction Point ? Dowdy SF, Curr. Opin. Gen Dev (2002) Weinberg RA, Biology of Cancer (2006) Lodish H, Molecular Cell Biology (2008) Boonstra J, Adv Enzyme Regul (2007) Zetterberg A, Exp Cell Res (2005) http://medicinembbs.blogspot.com/ 2011_10_01_archive.html … and where is the Nutrient Sensing ?

Genetic requirements for the transformation of human cells (I) (Hahn et al., Nature 400:464, 1999; MCB 22;2111, 2002) Genetic effect Molecular Target Cell cycle target Ras Growth factor signals Restriction point SV40 Large T p53 G1/S checkpoint Rb All G1 checkpoints SV40 small t PP2A Cell Growth checkpoint (?) Genetic requirements for the transformation of human cells (II) (Boehm et al., MCB 25:6464, 2005) Genetic effect Molecular Target Cell cycle target Ras Growth factor signals Restriction point p53 KO p53 G1/S checkpoint Rb KO Rb All G1 checkpoints Myc Gene expression Cell Growth checkpoint (?) PTEN KO mTORC1 Cell Growth checkpoint (?)

Foster DA, Yellen P, Xu L, Saqcena M. Genes & Cancer (2011)

GF / nutrient deprivation mediate cell cycle arrest Immortalized primary cell line (BJ hTERT)

GF / nutrient deprivation mediate cell cycle arrest

Sequential Blocking Experiments: 1st Block = No thymidine incorporation 2nd Block + 3H-Thymidine 1st Block = Thymidine incorporation 2nd Block + 3H-Thymidine

R & Nutrient Sensing Checkpoints are distinguishable First Block: -GF First Block: -EAA A temporal relationship can be established whereby the GF-dependent R is upstream from sites that are sensitive to EAA, Q, and mTOR suppression 3H-TdR Inc. (%Ctrl) First Block: -Q First Block: +Rapa. Second Block Conditions

Temporal Mapping of Checkpoints from G0 Indicates that the mTOR control point is at least two hr downstream from the amino acid checkpoints – likely very close to the G1/S boundry

GF / Amino acid deprivation and mTOR inhibition impact differentially on PI3K/mTOR signaling

GF / AA deprivation and mTOR inhibition: Cell cycle regulators

Summary Data support a model where there is GF-dependent R where multi-cellular organisms determine whether it is appropriate for a cell to divide During G1-ps, cells that have been given the green light to divide, determine whether they have the means/raw materials to double the mass of a cell, Replicate its genome, and divide into two daughter cells The late G1 “Metabolic Checkpoints” in late G1 collectively represent a “Cell Growth” checkpoint that responds to nutrients that is evolutionarily equivalent to START in the yeast cell cycle – or mSTART TOR/mTOR is likely the ultimate arbiter for determining nutrient sufficiency

Metabolic Checkpoints are Dysregulated in Cancer Cells Ctrl -GF -EAA -Q +Rapa. % Cells MCF 7 Ctrl -GF -EAA -Q +Rapa. # Cells B Ctrl -GF -EAA -Q +Rapa. % Cells MDA MB 231 Ctrl -GF -EAA -Q +Rapa. C Ctrl -GF -EAA -Q +Rapa. % Cells Panc-1 Ctrl -GF -EAA -Q +Rapa.

Metabolic Dereguation in Cancer: The Warburg Effect Preferential utilization of glucose through aerobic glycolysis by cancer cells leading to lactate production, independent of the oxygen availability. Only 2 ATPs are formed from glucose to lactate as opposed to 36 ATPs realized by engaging TCA cycle and oxidative phosphorylation. http://cronachedal900.blogspot.com/2012/12/otto-warburg-cura-cancro.html

Glutamine supports anaplerosis Glucose ~90% Pyruvate Lipid Synthesis NAD Lactate NADPH Glutamate Glutamine Based on: DeBerardinis RJ, et al. PNAS (2011)

Glutamine is “conditionally essential” Gao P, et al. Nature (2009)

Glutamine in cell culture Cancer cell propagation in mouse ascites. In 1950s, Harry Eagle* formulated a media which can support cell culture in vitro (DMEM). Key ingredient: Glutamine! “Added glutamine since serum had lots of glutaminase in it…” –Jim Darnell, Jr. Started adding excess glutamine (over 10-fold greater than any other amino acids), so that it is not a “growth-limiting” component of the medium. *Eagle, H. Nutrition needs of mammalian cells in tissue culture. Science (1955)

Genetic mutations determine differential glutamine sensitivity displayed by cancer cells

Glutamine deprivation causes loss of cell proliferation Mutant K-Ras WT K-Ras

Dual inhibition of K-Ras/MAPK and PI3K/mTOR pathway restores glutamine-mediated G1 cell cycle arrest

Glutamine deprivation creates synthetic lethality for cytotoxic drugs – Capecitabine and Paclitaxel Ctrl Cape. Pacli. C+P % Non-viable cells Ctrl Cape. Pacli. C+P Ctrl Cape. Pacli. C+P % Non-viable cells

AOA and EGCG inhibit glutamine utilization Aminotransferase inhibitor Amino-oxyacetate (AOA) Glutamate dehydrogenase inhibitor Epigallocatechin gallate (EGCG) Glu + α-KG + Asp GOT % Cells Gln Glu GDH

Blocking glutamine utilization creates synthetic lethality to cell cycle phase-specific cytotoxic drugs in K-Ras mutant cancer cells AOA

Conclusions Amino acids and mTOR mediate distinct late-G1 metabolic checkpoints. Cancer cells with K-Ras mutation override the amino acid-mediated G1 cell cycle checkpoints and these cells arrest in S and G2/M. Thus, the aberrant response to amino acid deprivation could prove to be an Achilles’ heel in K-Ras mutant cancer cells – by sensitizing cancer cells to agents that kill cells in S-phase. Ras is mutated in ~30% of all cancers, occurs in over 90% of pancreatic cancers , which has 5-year survival rate of less than 5%. However, it is considered “therapeutically undruggable” owing in part to its extremely high affinity with GTP (in the picomolar range).

Complementing oncogenic alterations dysregulate Restriction Point and Cell Growth checkpoints Restriction Point Cell Growth Checkpoint Growth Factor Signals PTEN mTORC1 Rheb TSC1/2 Akt PDK1 mTORC2 Ser473 T308 S6K FKBP38 Myc PLD1 Insulin/IGF1 TGF- Cyclin E mTOR Signals PIP2 PI3K AMPK LKB1 Energy status Amino acids PIP3 AMP Ras Raf Mek MAPK Cyclin D

Conclusions The GF-dependent R can be distinguished from late G1 metabolic checkpoints and mTOR The G1 metabolic checkpoints – like R – are dysregulated in human cancer cells Cooperating genetic alterations in cancer cells disable both R and the late metabolic checkpoints that collectively may represent a “Cell Growth” checkpoint with mTOR as the final arbitor Surprisingly, mTOR, which is widely known to be regulated by amino acids, blocked cell cycle progression well downstream of the amino acid sites It is hypothesized that other nutrient inputs – such as glucose and phosphatidic acid (lipids) may be required for complete activation of mTOR and progression into S-phase

G1-pm G1-ps S Cell Size G0 PLD mTOR Growth Factor Signals Nutritional Sufficiency Cell Growth Checkpoint (START) Restriction Point G1-pm G1-ps S Nutritional Sufficiency Amino acids Fatty acids Energy ATP O2 Cyclin D-CDK4/6 Cyclin E-CDK2 G0 Cyclin A-CDK2 Rheb RalA PLD mTOR Vps34 TGF- Cell Size Commitment Cell Growth