World Cancer Online presentation by Warren Ladiges 1

Exercise delays age-related cancer driven by mitochondrial ROS and tissue hypoxia.

Warren Ladiges

Seattle, Washington

2

Acknowledgements

Linda Enns, PhDJorming Goh, PhD

Supported by the National Cancer Institute and the National Institute for Aging

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Mitochondrial Free Radical Theory of Aging

• Mitochondria are the principal source of endogenous ROS. • Mitochondrial genome has no protective histones, limited

repair capacity, and close proximity to the electron transport chain and codes essential subunits of the electron transport system.

• The mutation rate of mtDNA is reported to be as much as two orders of magnitude greater than that of nuclear DNA.

• Complexes I, II, III and aconitase contain redox sensitive Fe-S centers.

• Loss of membrane potential and induction of permeability transition can lead to apoptosis/cell loss.

• Mutation of mtDNA encoded proteins can lead to increased ROS from defective respiratory proteins.

Mitochondrial ROS as signaling molecules

• Collective data suggest that release of mROS has evolved as a method of communication between mitochondria and cellular processes to maintain homeostasis and promote adaptation to stress.

• mROS regulate physiology but specific molecular targets are not well known.

• mH2O2 can reversibly change protein function by modifying a thiol group to a disulphide group on a cysteine residue.

5

Interest in the catalase antioxidant enzyme

• Human catalase gene• Target specificity

– Peroxisomal– Nuclear– Mitochondrial

• We generated transgenic mouse lines for all three

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Mitochondrial localization of the human catalase transgene in mCAT mice.

• MCAT (C) and WT (D) mouse embryonic fibroblast cultures stained for human catalase (green) and the mitochondrial marker prohibitin (red) with a sytox nuclear counterstain (blue). Scale bars indicate 20 µm.

Embryonic fibroblasts from mCATtransgenic mice show site specific

localization for mitochondria

100

80

60

40

20

0 10 20 30 4015 255 35

MCATWL4033 -

MCATWL4403 +

MCATWL4033 +

MCATWL4403 -

45

%S u

r viv

ing

AGE (months)

Founder 1WT

WT

Founder 1

Founder 2Founder 2

26.1 mo 30.6 mo (+18%)P < 0.0002

100x in heart

7x in heart

Transgenic mice overexpressing human catalase targeted to mitochondria (mCAT)

Schriner et al., Science 308:1909-11, 2005

.-.-

O 2

O 2

H 2O 2H 2O + O 2OH.

Free radical injury

Fe 2+ Catalase

mitochondria

GPx

SOD

.-.-

O 2

O 2

H 2O 2H 2 2OH.

Free radical injury

Fe 2+ Catalase

mitochondria

GPx, Prx

SOD

O + O

.-.-

O 2

O 2

H 2O 2H 2O + O 2OH.

Free radical injury

Fe 2+ Catalase

mitochondria

GPx

SOD

.-.-

O 2

O 2

H 2O 2H 2 2OH.

Free radical injury

Fe 2+ Catalase

mitochondria

GPx, Prx

SOD

O + O

No significant lifespan extension by

pCAT or nCAT

mCAT Phenotypes

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• Reduced non-hematopoietic neoplasms (Trueting, Ladiges, 2008, J Gerontology)

• Reduced invasive cancer in a mouse model (Goh, Ladiges, 2011, BMC Cancer)

• Reduced cardiac pathology and dysfunction (Dai, Rabinovitch, 2012, Circulation Research).

• Preserved insulin sensitivity in mice fed a high-fat diet (D. Neufer, JCI 2009 Feb 2. pii: 37048)

Prevents age-associated reductions in mitochondrial function and insulin resistance (G Shulman Cell Metab. 2010 Dec 1;12(6):668-74)

• Reduced cone cell death in retinitis pigmentosa (P. Campochiaro, Molecular Therapy 2009 17(5):778-86)

• Enhanced exercise performance (D. Duan, PLoS One 2009 4(8):e6673) • Reduced age-dependent hearing loss (T. Prolla, PNAS, 106:19432-7, 2009 )• Reduced MPTP-induced Parkinson’s disease (M. Vila, Ann Neurol.

68:184-92, 2010.• Reduced a-beta induced Alzheimers Disease (H. Reddy, Hum Mol Genet.

2012 Apr 5 )

Age-associated cancers are driven by increasing levels of mitochondrial ROS. This is based on our published studies of decreased cancer in mice expressing mitochondrial targeted catalase (mCAT).

0%10%20%30%40%50%60%70%80%90%

100%

HP Neo Non HP neo

Prevalence (%)

WTMCAT

p=0.224

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

HP Neo Non HP neo

Seve

rity in af

fected

mice

WTMCAT

p=0.39

p=0.001*

0.00

0.50

1.00

1.50

2.00

2.50

HP Neo Non HP neo

Lesion burden

WTMCAT

p=0.008

p=0.67

*

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Benign Non-HP Malignant Non-HP

Lesion burden

WTMCAT

*p=0.51

p=0.007

A B

C D

0%10%20%30%40%50%60%70%80%90%

100%

HP Neo Non HP neo

Prevalence (%)

WTMCAT

p=0.224

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

HP Neo Non HP neo

Seve

rity in af

fected

mice

WTMCAT

p=0.39

p=0.001*

0.00

0.50

1.00

1.50

2.00

2.50

HP Neo Non HP neo

Lesion burden

WTMCAT

p=0.008

p=0.67

*

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Benign Non-HP Malignant Non-HP

Lesion burden

WTMCAT

*p=0.51

p=0.007

A B

C D

Treuting et al., 2008 Goh et al., 2011 mCAT positive mCAT negative

Primary mouse fibroblasts expressing mCAT have lower levels of ROS compared to fibroblasts from wild type littermates (A) and are more resistant to H2O2 challenge (B).A B

Microenvironment: fibroblasts

Cancer is driven by increasing levels of tumor cell-generated mitochondrial ROS, which activates tissue fibroblasts to release pro-tumor factors.

H2O2

Breast cancer cells

mCAT

mCAT

Pro-tumor factors

Mouse breast cancer model. The 4T1 mouse breast carcinoma mimics triple negative breast cancer in women. It is highly metastatic to lungs, lymph nodes, liver and bone within several weeks after implantation in the mammary fat pad of immune competent Balb/c mice.

Ten thousand 4T1 tumor cells are injected into the left fourth mammary fat pad of 16 to 18 month old female Balb/c mice. Tumors are palpable within 10 days and metastasis to lymph node and lungs can occur within 30 days.

134th mammary fat pad

Primary tumor

Metastatic lymph node

Pulmonary metastasis

Mice with higher VO2 rates had decreased tumor mass. N = 8, at 19 months of age.

4000 5000 60000

100

200

300

400

500

600

700Tumor mass (mg)

Tumor mass (mg)*

**** *

*

*

*

*

*

R2 = 0.85P = 0.003

VO2 (ml/kg/hr)

Protein expression levels in 4T1 breast tumors from 19 month BALB/c mice varied according to tumor volume (weight).

Decreasing tumor volume

HIF1 alpha

Hypoxic microenvironment: fibroblasts

The data suggest that age-associated cancer is driven by tissue hypoxia.

VEGFOthers

Breast cancer cells

Tissue hypoxia is a characteristic of old age. Old mice with decreased metabolic rate have increased tumor mass and a hypoxic tissue environment to maintain HIF1a in an active state.

HIF1a is a transcription factor responsive to low tissue oxygen levels, and activation of angiogenesis, energy metabolism, nutrient transport, cell cycle and cell migration.

HIF1 alpha

Hypoxic microenvironment fibroblasts

Working hypothesis: a 2-hit attack on stromal fibroblasts drives age-related cancer.

VEGF

H2O2

Breast cancer cells

1 21. Tumor cell mitochondria generate H2O2 diffuses into the micro-environment, activating HIF1a in stromal fibroblasts.

2. A hypoxic microenvironment triggers fibroblasts to activate HIF1a, which transcribes the generation of tumor growth factors.

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Mouse tumor cells grow better when co-cultured with wt fibroblasts compared to mCAT fibroblasts. *p≤0.05 for average of three different tumor cell lines.

No fibroblasts wt fibroblasts added mCAT fibroblasts added

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

p≤0.05

Tum

or c

ell c

ount

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Conditioned medium (CM) from fibroblasts from old mCAT mice prevents increased tumor cell proliferation, *p≤0.05 for average of three different tumor cell lines.

Old WT CM Old mCAT CM0

5

10

15

20

25

30

35

Tumor cell proliferation

*

Per

cen

t inc

reas

e in

pro

lifer

atio

n

Can the two-hit attack be targeted?

• Our observations provide the rationale for combining an anti-hypoxic approach with mitochondrial antioxidants to decrease cancer risk.

• Attenuation of mitochondrial H2O2– Prevention of HIF1a activation

• Transition from a hypoxic microenvironment to a normoxic microenvironment– Deactivation of HIF1a

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Exercise increases metabolic rate

• Regular physical activity is associated with an increased life expectancy and decreased mortality.

• It reduces the risk of age-related disease.

• Epidemiological studies show a correlation between exercise and decreased cancer risk but lack underlying causal mechanisms.

• In general, larger training volumes are associated with increased health benefits.

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Types of exercise training

• Forced exercise– Tread mill– Swimming

• Voluntary wheel running– All mice run, but distance is strain and age-

dependent• Advantages and disadvantages

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Voluntary running wheel setup. A. An individual mouse is given access to a running wheel that either freely rotates (runner) or is locked (non-runner). Rotation of the running wheel transmits an electronic signal wirelessly to a hub and the number of revolutions is recorded on the Wheel Manager software. B. Each colored line reflects distance ran by a different mouse. Daily running activity is nocturnal.A B

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Experimental design• Female Balb/c mice were randomized into

runner and non-runner groups at 16 months of age. Runners were allowed free access to an in-cage running wheel, while the in-cage wheel was locked for non-runners.

• 4T1 breast tumor cells were implanted at 18 months of age after two months of voluntary wheel running. Mice were allowed to continue running for another month.

• Weekly palpation and tumor measurement.• Mice were terminated at 19 months of age, for a

total running time of three months. 24

Results. Running distance was inversely correlated with tumor mass in old female mice, which had been running for two months and then implanted with 4T1 breast cancer cells and allowed to run an additional month.

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Reddish brown stain shows greatly reduced CD34 positive vascular endothelial cells in tumor tissue from a long distance runner, but an extensive increase in these cells in tumor tissue from a short distance runner.

A. Long distance runner B. Short distance runner

There was no difference in tumor volume between runners and non-runners at the

end of the study.• This observation suggests that running causes

only modest suppression of tumor growth and reinforces the rationale for adding other anti-cancer modalities.

• We hypothesized that the exercise induced stromal cell resistance generated before tumor onset was not strong enough to completely prevent tumor growth so tumor cells were still able to grow and generate H2O2.

• mCAT is a logical complementary approach.27

Transgenic expression of mitochondrial catalase enhances the anti-tumor effect of voluntary wheel running in a model of mouse breast cancer. N = 3-5 per cohort, *p ≤ 0.05.

Nonrunners Runners0

100

200

300

400

500

600

700

800

WTmCAT*

28

*

Tum

or v

olum

e (g

m)

HIF1 alpha

Exercise

Mitochondrial antioxidants

Anti-angiogenic compounds?

Hypoxic microenvironment fibroblasts

Hypothesis: Targeting the pro-tumor two-hit attack with mitochondrial antioxidants and exercise will prevent cancer progression.

Normoxic microenvironment

VEGF

H2O2

Breast cancer cells

1 2

Summary and conclusions

• Tumors develop a two-hit attack to survive and proliferate in a submissive microenvironment.

• Tumor cells take advantage of a hypoxic microenvironment by the activation of the oxygen sensitive transcription gene HIF1 alpha in stromal fibroblasts, which triggers a pathway of pro-tumor signaling.

• Tumor cell mitochondria generate H2O2, which diffuses into the microenvironment further activating HIF1 alpha.

• The two-hit attack can be targeted with exercise and mitochondrial anti-oxidants. Future studies are needed to validate this concept.

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Mitochondrial Free Radical Theory of Aging

• Mitochondria are the principal source of endogenous ROS • Mitochondrial genome has no protective histones, limited

repair capacity, and close proximity to the electron transport chain and codes essential subunits of the electron transport system

• The mutation rate of mtDNA is reported to be as much as two orders of magnitude greater than that of nuclear DNA

• Complexes I, II, III and aconitase contain redox sensitive Fe-S centers

• Loss of membrane potential and induction of permeability transition can lead to apoptosis/cell loss

• Mutation of mtDNA encoded proteins can lead to increased ROS from defective respiratory proteins and further mutation, damage and apoptosis: mitochondrial ROS “vicious cycle”.

AcknowledgementsSeattle, Washington

Jorming Goh, PhD; Christina Pettan-Brewer, DVM, MSc; Ruby Magalindan, DVM; Linda Enns, PhD; Sy Fatemie, DVM, PhD; Shu Xian Lee; Emma Endicott; Heather Hopkins; Supported in part by NCI and NIA, National Institutes of Health.

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