molecular biology of osteosarcoma and genetic factors that ... · - spontaneous osteosarcoma...
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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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Karyogram of a radiation-induced Osteosarcoma
• Aneuploidie
• many numerical chromosomal aberrations
• several alterations resulting in marker chromosomes
• several deletions
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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).
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Radiation-induced Osteosarcoma and Retroviruses
224 Ra
Primary Osteosarcoma
Tumor
cells
cell -free
extract multiple Osteoma
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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.
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Osteoma
cells
15 Gy
gamma
Injection into
isogenic mice Osteosarcoma !!
Irradiation of Osteoma cells containing activated RFB retrovirus
can lead to full malignancy (Osteosarcoma).
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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.
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Mouse model for radiation induced Osteosarcoma
Histology using
EvG staining
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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)
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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 ?
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Linkage analysis for a
multicomponent airplane
Which component came
from the same supplier in
all crashed airplanes?
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Answer:
right engine
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Balb/c Allele
confers susceptibility
CBA Allele
confers susceptibility
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)
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
confers susceptibility
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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.
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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).
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
PCR amplification of the Rb1 promotor to
distinguish between Balb/c and CBA allele
B C 0
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
Conditional bone specific Rb1- and P16
mutant mice
bGal-Cre reporter
(ROSA26)
knee femur osteocytes
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
In-vitro Rb1+/- osteoblast model
Bone explant osteoblast cell line AP expression
Rb1 flox WT
Rb1
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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 +/-
M.Rosemann, Genetics of Radiation induced Bone Tumorigenesis
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