Accelerated Aging and Cancer in ERCC1-XPF-Deficient Mouse Models

Accelerated aging and cancer in ERCC1-XPF-deficient mouse models

Laura Niedernhofer, M.D., Ph.D. Department of Molecular Genetics and Biochemistry University of Pittsburgh Cancer Institute Outline

• Deficiency of ERCC1-XPF DNA repair endonuclease causes accelerated aging.

• New mouse models of ERCC1-XPF deficiency.

• Strategies to identify types of DNA damage that promote cancer and aging, and their sources. Nucleotide Excision Repair

xeroderma pigmentosum • photosensitivity • pigmentation abnormalities • atrophic skin • skin cancer (>2000x↑) • neurodegeneration • 7 complementation groups XPA - XPG NER: Damage recognition

Rad XPE 23B DDB1 XPC CSB RNA polII CSA

Global: Transcription-coupled: genome wide transcribed strand NER: Open complex formation

Rad 23B XPC XPD XPB TFIIH

Rad 23B XPC

XPB XPD TFIIH NER: Damage excision

XPF ERCC1 XPG

X PA

RPA RPA NER: Gap filling DNA synthesis

RFC LIG I PCNA pol δ, ε

LIG III XRCC1 pol κ ERCC1 ?

XPF ERCC1 XPG

X PA

RPA RPA Ercc1-/- phenotype

Ercc1-/- wild type

wild type Xpa-/-

ERCC1 has function(s) distinct from NER Progeroid syndrome due to a mutation in XPF

3 yrs old 16 yrs old

Nature (2006) 444:1038-43

ERCC1 and XPF function exclusively as a heterodimer Mutations in XPF lead to two diseases Genome-wide expression profiling

1) Ercc1-/- mice vs. wild type littermates

2) Old wild type mice vs. young wild type mice

Liver: 1) life limiting 2) age-associated changes 3) p53 stabilization Pathways significantly altered in Ercc1-/- mice

DNA repair ↑ apoptosis ↑ GH / IGF1 hormonal axis ↓ oxidative metabolism ↓ glycogen synthesis ↑ fatty acid synthesis ↑ anti-oxidant defenses ↑ Confirmation by qRT-PCR

800800 Liver 15 days 600 600 21 days 400400

200200 wild type relativewild-type to 0

% of mRNA expression expression mRNA of % 0

1 3 f1 f 3 p R lR r R 1 1 1 11 11 t12 21 1 gIg pb rl r h h io io p px xx xt 5t 5 I fb f P P G D fb fb o oo os ds g g G Dg m h hG Ga d I I I IgH mp p R a HE E R Gene expression changes in Ercc1-/- mice are progressive and systemic Confirmation of biological endpoints

Ercc1-/- wild-type Liver

Serum IGF1(ng/L)

TUNEL assay to measure apoptosis Confirmation of biological endpoints

800

600

400 Serum IGF1(ng/L) * 200 serum IGF-1 (ng/ml)

0 control Ercc1-/- Similarity between progeria due to ERCC1-deficiency and aging: all genes: 32% (p<0.05) all pathways: 86% growth hormone axis: >95% Histologic comparison of Ercc1-/- mice and aged mice Ercc1 -/- wt littermate wt 24 mths

15 days

-BrdU

21 days

TUNEL

IGFBP1

5 weks

anti-BrdU to identify proliferating nuclei Histologic comparison of Ercc1-/- mice and aged mice

Ercc1 -/ - wt littermate wt 24 mths

IGFBP-1 expression Histologic comparison of Ercc1-/- mice and aged mice

Ercc1 -/ - wt littermate wt 24 months

Oil Red O stain for liver triglycerides Does genotoxic stress induce a similar response in a normal host?

0.1 mg/kg MMC (100X below LD) intraperitoneal weekly x 5 wks

Serum IGF1(ng/L) 500 Liver * * * 400 *

300 *

200

relative to wild-type 100 * untreated % of mRNA expression mRNA of % * *** 0

3 l R 1 1 1 1 1 1 f1 p rlR h 1 o p 1 x 1 x 2st2 5 % mRNA relative to untreated % mRNA relative f Ig Pr o i o o tt 51d Ig fb P GhrG i D fb oxm h s G a Ig D fbp1Ig p G R Igfbp3 Ig HmH EphxE Rad

600

550 500 *

(ng/ml) 450 Serum IGF1 400 untreated MMC A common response to stress mediated by the somatotroph axis

Biological process: Ercc1 -/- old age Ghr-/-; Igf1+/- CR GH / IGF1 hormonal axis ↓ ↓ ↓ ↓ oxidative metabolism ↓ ↓ ↓ ↓ glycogen synthesis ↑ ↑ ↑ ↑ fatty acid synthesis ↑ ↑ ↑ ↑ peroxisome biogenesis ↑ ↑ ↑ ↑ anti-oxidant defenses ↑ - ↑ ↑

A. Bartke R. Miller Model:

DNA lesions that are cytotoxic Implications:

1. Prevention of DNA damage (or improving DNA repair) may delay aging.

2. Cancer therapy with genotoxins may cause accelerated aging in cancer survivors.

3. Progeria caused by defects in the DNA damage response is accelerated aging.

4. Mouse models of human progerias are a valid and rapid system for studying aging. Acknowledgments

Dr. Anwaar Ahmad Andria Robinson Nikhil Bhagwat Brooke McClendon Hillary Shane

Jan Hoeijmakers: George Garinis Ellen van Drunen

University of Pittsburgh Cancer Institute: Rick Wood, Rob Sobol, Chris Bakkenist, Yong Wan, Vesna Rapic-Otrin