Nutrient and Growth Factor Control of Cell Cycle Progression and Survival in Cancer Cells 

Bio 714Cell BiologyFeb, 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 Apoptosisavoid default cell death programs

IV Acquire Immortality - telomerase expressionovercome cell senescence - cancer stem cells?

V Stimulate Angiogenesisprovide 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 Apoptosisavoid default cell death programs

IV Acquire Immortality - telomerase expressionovercome cell senescence - cancer stem cells?

V Stimulate Angiogenesisprovide nutrition

VI Acquire the ability to migrate and invade gain access to circulation (metastasis)

VII Breakdown Caretaker Function genomic instability needed for many mutations

(Quiescence)

Gatekeepers Myc SV40 Early Region (Suppression of p53, Rb and PP2A)

Restriction Point

Growth Factor Signals Tyrosine kinases Ras/Raf/MEK/MAPK

G0

G1-pm SG1-ps G2 M

Regulation of Cell Cycle Progression

Cell GrowthCheckpoint(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)

Figure 8.8 The Biology of Cancer (© Garland Science 2007)

Conventional View of Cell Cycle

Zetterberg and colleagues have mapped the Restriction Point to a site ~ 3.5 hr after mitosis - where cyclin D is elevated

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

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

In some texts, the Restricition Point is referred to as the mammalian equivalent of START - and located near the site where cyclin E is activatedRapamycin 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

From: Weinberg, The Biology of Cancer, 2007

Protozoans & Metazoans are… different!

Vander Heiden MG, Cantley LC, Thompson CB. Science (2009)

Restriction Point: Point in G1 after which cells no longer need GF permissive signals to divide.

Restriction Point (R)

G0

G1 S

G0

R - GF

Mammalian Restriction Point is Analogous to START in Yeast

• Lodish H, et al. Molecular Cell Biology (2008)

Yeast Mammalian

Cooper GM. The Cell: A Molecular Approach (2000)

RestrictionPoint

Pyronnet S, Sonenberg NCurr. Opin. Genetics Dev. (2001)

Cell Cycle: START vs. R

Based on Zetterberg and Larsson, PNAS (1985)

Progression Factor (IGF-

1)

Competence Factor

(PDGF)

Based on Pledger and Stiles, PNAS (1979)

What is a Restriction Point ?

G0

G1 S

R

- GF

M

3.5 hr + GF

Where is the Restriction Point ?

… and where is the Nutrient Sensing ?

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

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 checkpointRb 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 checkpointRb 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)

Immortalized primary cell line (BJ hTERT)

GF / nutrient deprivation mediate cell cycle arrest

GF / nutrient deprivation mediate cell cycle arrest

1st Block

2nd Block

+ 3H-Thymidine= No thymidine incorporation

1st Block

2nd Block + 3H-Thymidine

= Thymidine incorporation

Sequential Blocking Experiments:

R & Nutrient Sensing Checkpoints are distinguishable

3 H-T

dR

Inc.

(%

Ctr

l)

First Block: -GF

First Block: -Q

First Block: -EAA

First Block: +Rapa.

Second Block Conditions

A temporal relationship can be established whereby the GF-dependent R is upstream from sites that are sensitive to EAA, Q, and mTOR suppression

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, determinewhether 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 evolutionarilyequivalent 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

A

Ctrl -GF -EAA -Q +Rapa.

% C

ells

MCF 7

Ctrl -GF -EAA -Q +Rapa.

# C

ell

s

MCF 7

B

Ctrl -GF -EAA -Q +Rapa.

% C

ells

MDA MB 231

Ctrl -GF -EAA -Q +Rapa.

MDA MB 231

C

Ctrl -GF -EAA -Q +Rapa.

% C

ells

Panc-1

Ctrl -GF -EAA -Q +Rapa.

Panc-1

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

Metabolic Dereguation in Cancer: The Warburg Effect

Lactate

~90%

Glutamine

Glutamate

NADPH

NAD

Glutamine supports anaplerosis

Glucose

Pyruvate

Lipid Synthesis

Based on: DeBerardinis RJ, et al. PNAS (2011)

Gao P, et al. Nature (2009)

Glutamine is “conditionally essential”

• 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)

Glutamine in cell culture

A

B

Genetic mutations determine differential glutamine sensitivity displayed by cancer

cells

Mu

tan

t K

-Ras

WT

K-R

asGlutamine deprivation causes loss of cell

proliferation

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

arrest

Ctrl Cape. Pacli. C+P Ctrl Cape. Pacli. C+P

% N

on

-via

ble

cel

ls

Ctrl Cape. Pacli. C+P Ctrl Cape. Pacli. C+P

% N

on

-via

ble

cel

ls

Glutamine deprivation creates synthetic lethality for cytotoxic drugs – Capecitabine and Paclitaxel

AOA and EGCG inhibit glutamine utilization

Gln

Glu

GDH

Glu +

α-KG + Asp

GOT

% C

ells

Aminotransferase inhibitor Amino-oxyacetate (AOA)

Glutamate dehydrogenase inhibitorEpigallocatechin gallate (EGCG)

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

cancer cells

AOA

• 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).

Conclusions

Ras

Raf

Mek

MAPK

Cyclin D

Restriction Point Cell Growth CheckpointGrowth Factor Signals

PTEN

mTORC1

Rheb

TSC1/2

Akt

PDK1mTORC2

Ser473 T308

S6K

FKBP38

Myc

PLD1

Insulin/IGF1

TGF-

Cyclin E

mTOR Signals

PIP2

PI3K

AMPK

LKB1

Energystatus

Aminoacids

PIP3

AMP

Complementing oncogenic alterations dysregulate Restriction Pointand Cell Growth checkpoints

Conclusions

The GF-dependent R can be distinguished from late G1 metaboliccheckpoints 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

Restriction Point

G1-pm G1-ps S

Growth FactorSignals

NutritionalSufficiencyAmino acidsFatty acidsEnergyATPO2

Cyclin D-CDK4/6

Cyclin E-CDK2

Cyclin A-CDK2

Cell GrowthCheckpoint

(START)

mTOR

TGF-

NutritionalSufficiency

Cell GrowthCommitmentCellSize

PLD

Rheb

G0

RalA

Vps34