Molecular Biology of Osteosarcoma and Genetic Factors that ... · - spontaneous Osteosarcoma approx. 8000 times higher risk (radiation risk unknown) Li-Fraumeni Syndrom (dominant,

M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis

Molecular Biology of Osteosarcoma and

Genetic Factors that influence Risk

following Radiation Exposure

M.Rosemann

National Center for Radiation Sciences

Helmholtz-Center for Health and Environment


The Radium-Dial Painters (US Radium Corporation)

painting of aircraft-instruments

with radioactive-fluorescence

ink

accidental oral uptake of Ra226

(10-1000µCi/year, T1/2=1600 y)

accumulation in all skeletal

regions (earth alkaline metal

incorporated into bone similar

to calcium)

http://www.lastfm.de/music/Kraftwerk/_/Radioaktivit%C3%A4t

http://www.lastfm.de/music/Kraftwerk/_/Radioaktivit%C3%A4t


Bone seeking alpha-emitters are potent inducer of bone-cancer in man

(Rowland et al 1978, Fry 1978, Mays et al 1978, Koshurnikova et al 2000)

Bone sarcomas – the first human tumor with

a clear association to radiation exposure


Common sources of Radium

Radium soap from the 1920s,

can still be found on selected

Munich flea-markets

Uranium-ore from a mine in Bulgaria

(Probojniza, Stara Planina mountains)

Ra226 produced as a daughter product


Ra224 (T1/2=3.5 days) was used till in the 40s and 50s for

the treatment off Ankylosis spondylitis and Bone tuberculosis

(up to 1000MBq per patient)

more than 56 cases of Osteosarcoma (expected 0.002)

Ra224 was used in Germany till 2007

for the treatment of Ankylosis

spondylitis (10 x 1MBq injections)

Therapeutic applications of Ra-compounds

Th227 and Ra223 again in clinical

trials in Norway and US (Algeta Inc)

for the treatment of breast and

prostate cancer and metastasis


Karyogram of a radiation-induced Osteosarcoma

• Aneuploidie

• many numerical chromosomal aberrations

• several alterations resulting in marker chromosomes

• several deletions


Karyotypes of three radiation-induced Osteosarcoma

case 1

case 2

case 3

• chromosomal aberrations differ between tumors (genetic heterogeneity)

and between cells of a single tumor (intra-tumor heterogeneity)

• Absence of recurrend chromosomal alterations make cytogenetic studies

in Osteosarcoma virtually impossible.

• Complexity of cytogentic alterations might shed light on defects in chromosomal

maintenance mechanism in Osteosarcoma


Molecular Karyotypes of human Osteosarcoma

• Osteosarcoma following radiotherapy exhibit

a significant higher degree of genome wide

allelic losses than spontaneous osteosarcoma

post RTx

osteosarcoma

spontaneous

osteosarcoma

Genome wide Loss-of-Heterozygosity (LOH)

post RTx OS


Th227 induced Osteosarcomas in mice:

radiological and histological appearance

Histology using

EvG staining

Murine radiogenic osteosarcomas resemble human spontaneous bone tumours

(Gössner et al 1976).


Radiation-induced Osteosarcoma and Retroviruses

224 Ra

Primary Osteosarcoma

Tumor

cells

cell -free

extract multiple Osteoma


Osteoma contain viral

particles

RFB virus was isolated

from cell free extract

active oncogene: v-Fos

(Reilly & Finkel 1971)

The v-Fos Oncogene alone can only induce

benign Osteoma.


The v-Fos Oncogene is the viral homologue of the

cellular c-Fos Gene.

c-Fos build a complex with c-Jun, forming the AP1

Transcription factor.

AP1 regulates differentiation, proliferation and apoptosis

following cellular stress. It is involved in tissue regeneration and

Wound healing.

In bone: Involved in the normal regulation of anabolism

(osteoblasts) and catabolism (osteoclasts).

Unregulated overexpression of c-Fos leads to impairment of bone homeostasis

(abnormal proliferation of osteoblasts)

C-Jun

C-Fos


Osteoma

cells

15 Gy

gamma

Injection into

isogenic mice Osteosarcoma !!

Irradiation of Osteoma cells containing activated RFB retrovirus

can lead to full malignancy (Osteosarcoma).


Question : What happend with the RFB-retrovirus

following irradiation ?

LTR LTR Gag Pol Env

(Schmidt, Strauss, Luz, Erfle, GSF

1980 – 1990)

Virus itself was not changed,

neither in its structure nor in it

sequence.

FBR Retrovirus

P 53 gene

c - myc

FBR LTR

But the virus was found to be

amplified and reintegrated

into new chromosomal loci of

the osteosarcoma cells

destruction of p53 by intragenic

insertion

or

transactivation of c-myc by the

viral LTR promoter


The lesson from radiation and RFB-virus :

A single activated Oncogene (v-Fos) is an early event in

Osteosarcoma development.

It is part of a transcription-factor complex and transactivates

genes for cell-cycle progression.

Activated v-Fos alone produces only benign

Osteoma.

If Tumor-Suppressor Genes (like p53) are destroyed by the

radiation+retroviral rearrangements, benign Osteoma transform

into fully malignant Osteosarcoma.


Promotion

Mutation in Tumor-

Suppressor-Genes

Multistep-Model of Carcinogenesis

Initiation

Activation of

Oncogenes

Osteoma

Progression

Activation of genes that promote

metastasis, invasion, angiogenesis

immunological tolerance etc.

Germline Mutation

in Susceptibility Genes

cell with

de-novo

mutation

early

Osteosarcoma

aggressive

metastasing

Osteosarcoma


Familial Bilateral Retinoblastom (dominant, mutant Rb1)

- approximately 1000 fold increased risk for ionizing

radiation induced Osteosarcoma

Werner Syndrome (recessive, mutant Wrn-Helicase)

- spontaneous Osteosarcoma approx. 5000 times higher risk

(radiation risk unknown)

Rothmund-Thomson Syndrome (recessive, mutant RecQ4 Helicase)

- spontaneous Osteosarcoma approx. 8000 times higher risk

(radiation risk unknown)

Li-Fraumeni Syndrom (dominant, mutant P53)

- approximately 1000 fold increased risk for spontaneous Osteosarcoma

- additionally increased incidence of OS after irradiation in mouse

knockout model.

Inherited Predisposition for Osteosarcoma

through Germline-Mutations


A large portion of tumours still considered as sporadic (90%

of all) might in fact be associated with a multi-gene

mechanism of tumour predisposition

low high

Tumour - Susceptibility

1

2 3

4


Strategies to identify single susceptibility

genes in a multi-genetic disease:

in humans: Genome wide association studies (GWAS)

in groups of affected and unaffected person

search for Gene variants (SNP) that show different frequency

in healthy controls vs. patients

(requires several 1000 cases and controls

impractical for radiation-induced cancer)

mouse genetics: - dedicated breeding strategies (mapping gene loci)

- manipulation of selected genes

- controlled radiation-exposure


Mouse model for radiation induced Osteosarcoma

Histology using

EvG staining


0

0,1

0,2

0,3

0,4

0,5

0,6

300 400 500 600 700 800 900

Age / days

OS Inc

Balb/c

CBA/CA

Genetic background governs Osteosarcoma

Susceptibility following 227Th incorporation

Inbred mouse strains differ in latency and incidence

of

osteosarcoma following Ra224, Th227 or Pu239 exposure

(Finkel et al 1961, Luz et al 1982, Ellenders 2001)


Objective:

• Are the observed strain differences genetically

determined ?

• Can we map susceptibility loci in the mouse genome ?

• To which extend can genetic factors influence

osteosarcoma susceptibility ?


0

0.1

0.2

0.3

0.4

0.5

0.6

300 400 500 600 700 800 900

Age / days

OS Incid

Balb/c

CBA

F2

0

0.1

0.2

0.3

0.4

0.5

0.6

300 400 500 600 700 800 900

Age [days]

OS Incid.

Balb/c

CBA

F1


Balb/c CBA

B x C Balb/c

Bx(BxC)

OS free OS

D14Mit166

B C

Each of the backcrossed

mice is characterised by

an individual haplotype

pattern of homozygote and

heterozygote regions

Balb/c B x C

Bx(BxC)

Genotyping backcrossed Balb x (Balb x CBA) mice


Linkage analysis for a

multicomponent airplane

Which component came

from the same supplier in

all crashed airplanes?


Answer:

right engine


Balb/c Allele

confers susceptibility

CBA Allele


Linkage of osteosarcoma susceptibility

to multiple loci in the mouse genome

cM 0

10

20

30

40

50

60

70

80

90

100

11 12 13 14 15 16 17 18 19 X

1 2 3 4 5 6 7 8 9 10

106

115

178

54

13

9

247

167

411

444

369

185 182

254 167

403

46

172

166 24

223

25

223

148

105

64

246

218

35

157

125

165 119

83

79 147

155

133 83

173

68

217*

229

262

176

205

292

158

80

305

234

225 91

0.003

0.002

0.001 0.0001 0.005

Susceptibility loci

in C3H x 102

(Rosemann et al 2002)


Linkage analysis shows:

• genetic loci exhibit significant association

with susceptibility of mice for Th227 induced

osteosarcoma

• the strain differences between Balb/c and CBA

are determined by 5 loci

• the alleles derived from the

more sensitive Balb/c strain are

associated with susceptibility

at 3 of the loci, whereas at

2 loci the allele from the

relative resistant CBA strain


11 12 13 14 15 16 17 18 19

X

1 2 3 4 5 6 7 8 9 10

106

115

178

54

13

9

247

167

411

444

369

185 182

254 167

403

46

172

166 24

223

25

223

148

105

64

246

218

35

157

125

165 119

83

79 147

155

133 8

3

173

68

217*

229

262

176

205

292

158

80

305

234

225 91

0.003

0.0

02

0.001 0.000

1

0.005


0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 200 400 600 800

LT / days

OS Incid

CBA

Balb

Osteosarcoma incidence in the two

parental inbred strains

Balb/c

CBA/CA

Balb/c

CBA/CA


Inheritance of multiple susceptibility loci –

Incidences as compared to parental strains

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 200 400 600 800

LT / days

OS Incid

5 x S

CBA

Balb

5 x R

BBBCC

CCCBB


low high

Tumor - Susceptibility

1

2

3

4

Congenital tumor predisposition by multigenetic

inheritance of low-penetrance alleles

Random segregation of multiple low penetrance gene variants could cause congenital

cancer predisposition in the progeny of „normal“ parents.

Balmain & Nagase, Nat Genet 1998


Summary

• mice with susceptibility alleles at 5 loci develop

100% osteosarcoma within 550 days after exposure

• mice harbouring resistance alleles at 5 loci did not

developed any tumor up to 700 days p.i.

Only 1 of 11 mice developed a tumor with 720days

latency.

• by random segregation of maternal and paternal

alleles some of the progenitor mice inherit only

resistance or only susceptibility genotypes.

These mice show much more pronounced phenotypes

than either of the parental inbred strains.


Somatic changes follow germline predisposition

-1 0 1 2 3 4

Cen

Tel

D14Mit60

D14Mit234

D14Mit115

D14Mit225

D14Mit92

24cM

31cM

47cM

50cM 51cM 52cM 53cM

62cM

D14Mit219

D14Mit166

D14Mit125

LOD

BALB x CBA

C3H x 102

D14Mit 99

D14Mit 207

D14Mit 34

D14Mit 90

D14Mit 225

D14Mit 125

D14Mit 234

D14Mit 113

D14Mit 97

D14Mit 133

D14Mit 37

1st hit germline inherited-susceptibility

Rb1

P16

2nd hit somatic radiation-induced allelic loss

Rosemann et al, IJC 2006

Somatic losses at the Rb1 locus are fully complemen-tory with LOH at the P16 locus on chromosome 4


nt 3090 nt 3090 nt 3090

BALB/c F1 hybrid CBA

F1 normal tissue

CBA Allele lost

F1 normal tissue

F1 tumor

F1 tumor

BALB/c Allele lost

Rb1 gene - Loss-of-heterozygosity in F1 osteosarcoma


Somatic changes follow germline predisposition

-1 0 1 2 3 4

Cen

Tel

D14Mit60

D14Mit234

D14Mit115

D14Mit225

D14Mit92

24cM

31cM

47cM

50cM 51cM 52cM 53cM

62cM

D14Mit219

D14Mit166

D14Mit125

LOD

BALB x CBA

C3H x 102

D14Mit 99

D14Mit 207

D14Mit 34

D14Mit 90

D14Mit 225

D14Mit 125

D14Mit 234

D14Mit 113

D14Mit 97

D14Mit 133

D14Mit 37

1st hit germline inherited-susceptibility

Rb1

2nd hit somatic radiation-induced allelic loss

Rosemann et al, IJC 2006


Rb1 promoter variant defines the high-risk allele

The Rb1 promoter

region ....

Ex1 Ex2 - E27 SP2 ATF E2

(82% CG)

1 821 1221 1372

4.5kb mRNA

6bp Insertion

CBA

Balb

contains a 6bp insertion

in the Balb/c strain ....

that generates a novel Wt1 transcription factor binding site

and an additional Sp1 module (according to promotor structure

modelling).


PCR amplification of the Rb1 promotor to

distinguish between Balb/c and CBA allele

B C 0


age / days

Tu

mo

r fr

ee s

urv

ival

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

200 300 400 500 600 700

Rb1 B/C

Rb1 B/B

MIXED

Mice with the Rb1 promotor variant show significantly

reduced osteosarcoma incidence following Th227 injection


Reduced gene-expression from the CBA-allele

of the Rb1-Promotor

after transient transfection of ROS17/2.8 cells with Rb1 Promotor / CAT - constructs. CAT activity was measured by incubating cell extracts with Promega FastCAT substrate in the presence of Acetyl-CoA

+ B B B B B B C C C C C C inverse

Acetyl-CA

CA

CAT-Reporter

SP2 ATF E2

CAT-Reporter

6bp Insertion

SP2 ATF E2

CAT-Reporter

E2 ATF SP2


0.000

0.100

0.200

0.300

0.400

0.500

0.600

rel. CAT-Act. [AU]

* after transient transfection of ROS17/2.8 cells with Rb1 Promotor / CAT - constructs. CAT activity was

determined as the ratio between acetyl-Chloramphenicol and Chloramphenicol. Normalisation for equal

transfection efficiency using luciferase activity from cotransfected RFB_pGL3 luciferase expression vectors.

Balb/

c 36

CBA

5

neg.C

ontr

inv.

orient

Rb1-Promotor Reporter Assay


Rb1-Expression in Balb/c vs. CBA/CA mouse embryos

RNA was extracted at day 16 p.c. from 6 Balb/c and 6 CBA/CA embryos (derived from 3 pregnant females of each strain). Rb1 mRNA was determined by Northern Blotting using a 32P-labelled mouse specific Rb1 probe

B B C C B B C C B B C C

Rb1

28S

18S


functional !! molecular !!

Rb1

genetic !!

linkage with OS

predisposition

(LOD=4.5)

6bp insertion

in core promotor

region

differential

expression

of the Rb1

mRNA

Indications, that Rb1 promoter variants could

modify osteosarcoma predisposition following

radiation exposure


Cell – Cycle regulation by Rb

• unphosphorylated Rb has tumour-suppressor properties by blocking DNA-synthesis

• S-phase is initiated by Rb-phosphorylation (temporal loss of activity)

• deleted/mutated Rb behaves like constituitive phosphorylated Rb (cells cannot stop

DNA synthesis)

Cyclin D1

CDK4/6

Rb

Rb P

P

G1

G2

G0

p16

S M

E2F


Conditional bone specific Rb1- and P16

mutant mice

bGal-Cre reporter

(ROSA26)

knee femur osteocytes


In-vitro Rb1+/- osteoblast model

Bone explant osteoblast cell line AP expression

Rb1 flox WT

Rb1


Osteosarcoma induction in k.o. mice following Th-

227 incorporation

Cre + : 11/24 OS

Cre - : 2/18 OS

p=0.004 Cox‘s F-Test

P16 ko + wt: 24/70 OS

wt: 22/76 OS

p=0.036 Gehans Wilcoxon-Test

Rb1 +/- P16 +/-


Conclusion

- Common Gene-Variants (polymorphic alles) can cause

inherited predisposition for radiation-induced cancer

- Allelic variants with reduced gene-expression

can modify cancer-risk in a quantitative manner

(low-penetrance modifier genes).

- Multiple of low-penetrance modifier genes in the

genome can interact to confer a more sever increase

in cancer-risk in individual cases.

- Cell-cycle regulation of Rb1 and other proteins

frequently altered in Osteosarcoma

Documents

Molecular Biology of Osteosarcoma and Genetic Factors that ... · - spontaneous Osteosarcoma approx. 8000 times higher risk (radiation risk unknown) Li-Fraumeni Syndrom (dominant,