M.Rosemann, Institute for Radiation Biology Helmholtz ......EYF YVDFREYEYD LK 31-1 66 Liquid REYEYD LKWEFPRENL EFG consensus Structure of Flt3 ITDs in spontaneous and ... following

Multistep Carcinogenesis 2015 DoReMi Mol Mech Rad Carc

Multistep Carcinogenesis

M.Rosemann, Institute for Radiation Biology

Helmholtz Center Munich,

Research Centre for Health and Environment


The Hallmarks of Cancer

D.Hanahan, Cell 2011


The Hallmarks of Cancer


Impairments of the normal cellular homeostasis

in a multicellular organisms


Cancer Formation:

Several cellular defence mechanisms have to be

bypassed in one and the same cell

These cellular alterations have to be persistent

(i.e. stable inheritable from one cell to ist progeny



Chemical carcinogenesis in a mouse skin tumor model (Benz-Pyrene + TPA)

B(a)P

B(a)P

B(a)P

B(a)P

TPA

TPA

TPA

TPA: 12-O-Tetradodecanoyl-Phorbol-13-Acetat (Phorbolester)

B(a)P: Benz(a)Pyren

Hyperplasia,

no malignant carcinoma

Adenoma, Carcinoma

Hyperplasia,

no malignant carcinoma

Adenoma, Carcinoma


Promotion

Mutation in Tumor-

Suppressor-Genes

Multistep-Model of Carcinogenesis

Initiation

Activation of

Oncogenes

Hyperplasia

malignant

Tumor

Progression

Activation of genes that promote

metastasis, invasion, angiogenesis

immunological tolerance etc.

Carcinoma in situ


Colorectal Cancer

• 11% of cancer-

related deaths

• Tumor

progression may

take 10-35 years

• Adenomatous

polyp develops

into carcinoma


Stepwise

Transformation of

normal epithelial

tissue giving rise to

malignant carcinoma


Adenoma-Carcinoma-Sequence (Colon carcinoma) transition from normal epithelium to adenoma and to carcinoma

is associated with aquired molecular events, including:

stepwise pattern of mutational activation of oncogenes and inactivation of TS genes,

resulting in cancer (Vogelstein et al. 1988)

deletion activation

deletion

mutation

APC=brake on Wnt-signaling,

transcriptional activation

of ß-catenin

activation of oncogenes

G-protein,

signal transduction

p53 mutation,

no: DNA-repair,

apoptosis, cell cycle

arrest etc.


Multistep Colorectal Carcinoma formation and

the associated genetic alterations

Loss or

Mutation

Of APC Gene

DCC Loss P53 Mutation K-Ras Mut.

Activation

Fearon and Vogelstein, Cell 1990


metastasis processes

metastases = new malignant tumor growing at different places/organs

of the body;

only malignant tumors build metastases

steps in metastasis processes:

cells have to penetrate several times extracellular matrix and basal layer,

migrate through the body, attach to the new localisation, penetrate again the

wall of vessels and basal membrans


Individual cancer risk is determined by :

- NATURE : inherited, genetic factors

(cancer predisposition)

- NORTURE : exposure to carcinogenic noxae

(dose, dose-rate, type of radiation)

- CHANCE : random fluctuations in biochemical processes

(errors in DNA replication/DNA repair)


The energy of ionizing radiation is deposited in living matter

in the form of discret ionisations.

Their spatial pattern match the structure of the DNA double-helix.

Ionizing radiation thus has a high efficiancy to produce DNA breaks.

DNA is a single-copy molecule

Chromosomal DNA with a Mr > 6x104 kDa is several hundred times

larger than the largest protein and has thus the biggest target size.

DNA – the primary target of

radiation damage

A - dose of about 2 Gy deposits in the entire exposed

human body about 150 Joule energy

(less than the thermal energy of a cup of hot coffee).

Why than has ionizing radiation such a sever biological

effect ?


Initial radiation effect causes DNA fragmentation by double-strand breaks)

Living cells can repair most of these damages

Dose [Gy] 0 0.2 0.5 1 2 5 10 20

Repair 0 20’ 40’ 60’ 90’ 2h 3h 5h

following 20Gy

intact, chromo-

somale DNA

fragmented

DNA


V79 cells (Chin. Hamsters ovary cells)

2 Gy gamma-Irradiation, 6 h

ROS17/2.8 cells (Osteoblasten, Ratte)

4 Gy gamma-irradiation, 12 h

Incomplete or miss-repair can result in fixed genetic alterations such as

Chromosomal translocation or deletions/losses of DNA (micronuclei)


Chromatid type

Aberration

S-Phase Mitosis

Loss of

acentric fragments

If an essential gene

is lost, cell dies.

If no essential gene is lost,

cell might survive

(but might have lost

Tumor-Suppressor-Genes)

Trans-

location

If a stable translocation

is generated, cell survives

(but might acquire an

activated Oncogens )

Chromosomal – Aberrations might contribute to tumor-development

Chromosome type

Aberration

Deletion

Deletion


Ionizing Radiation causes stochastic damage

to the genome …..


Ionizing Radiation causes stochastic damage

to the genome …..

…. Is the process of malignant transformation

after irradiation a manifestation of genetic disorder ?


13 15 20 O M

30 31 O M O M

Flt3 genomic PCR Exon13/Intron13/Exon14

internal tandem duplications: 15, 31 (liquidator AML)

13 (victim AML)

20,30 (unexposed AML)

Flt3 internal tandem duplications

in Radiation-induced Acute Myeloid Leukaemia

Flt3 ITD in about 25% of all AML

(spontaneous + radiation-induced)

ITD

FL

wt Flt3 Flt3 ITD


AMLcase / Duplication Exposure duplicated AA

Allele [bp] Group

573 613

13-1 129 Victim SQLQMVQ VTGSSDNEYF YVDFREYEYD LKWEFPRENL EFG

13-2 48 Victim YF YVDFREYEYD LKWE

15-1 ~ 24 Liquid. n.d.

15-2 114 Liquid. VTGSSDNEYF YVDFREYEYD LKWEFPRENL EF

15-3 48 Liquid. GSSDNEYF YVDFREYE

20-1 21 Contr. VDFREYE

30-1 45 Contr. EYF YVDFREYEYD LK

31-1 66 Liquid REYEYD LKWEFPRENL EFG

consensus

Structure of Flt3 ITDs in spontaneous and

radiation-associated Acute Myeloid Leukaemia

All Flt3 ITDs are in-frame and affect the juxtamembrane domain

of the protein


Bone Tumors induced in mice following α-particle Th227 incorporation

227Th


1 2 3 4 5

Transak- spez. DNA Bindung Protein-WW

tivierung

E5 E6 E7 E8 E9

spont (2)

viral (7) ACC-->ACT

(18)

6 Fälle: keine p53 cDNA amplifizierbar

P53 Mutations radiation-induced osteosarcoma in mice

C.Dalke, PhD 2000


Stochastics and Specifity

Ionizing Radiation causes a genome-wide stochastic

damage to the DNA.

During the carcinogenic process, out of this pool of random

genetic changes specific gene-alterations are selected that

confer a growth advantage to the tumor cells.

Persistent genetic alterations in Radiation Carcinogenesis

can be activated Oncogenes or inactivated Tumor-

Suppressor Genes


The selection of a few specific gene-alterations out of a

large pool of random genetic damages is a very inefficient

mechanism.


The selection of a few specific gene-alterations out of a

large pool of random genetic damages is a very inefficient

mechanism.

Therefore, you need

• several billions irradiated cells in a person,

• several thousands of exposed person

• and several years or decades after exposure

to find a single radiation-associated tumor.


In the context of cancer as a multifactorial disease

- NATURE : inherited, genetic factors

(cancer predisposition)

- NORTURE : exposure to carcinogenic noxae

(dose, dose-rate, type of radiation)

- CHANCE : random fluctuations in biochemical processes

(errors in DNA replication/DNA repair)


Rb1 : familial bilateral Retinoblastom

patients develop Bone sarcoma following RT

Ercc1 – Ercc5: Xeroderma Pigmentosum

Basalioma and squamous cell carcinoma of skin

following UV-B

Patched: Gorlin-Syndrome (NBCC)

Basalioma following RT

P53: Li-Fraumeni-Syndrome

Lymphoma, Soft-tissue sarcoma following X-ray

(P53+/- mouse)

ATM: Ataxia Telangiectasia

(Lymphoma, Breast carcinoma ..., severe acute radiation effects following RT)

Inherited cancer syndroms

(showing increased cancer risk after radiation exposure):


NBCCS (Gorlin-Syndrome)

Ataxia Telangiectasia


Occurence of secondary tumors (mainly Osteosarcoma)

following RT in Retinoblastoma-Patients

Rb families

congenital predisposition

sporadic cases

Rb familie patients carry a heterozygote germline defect

in a tumor-suppressor gene.

Tumor formation following RT in these patients require

inactivation of the wt allele (second hit)


Promotion

Mutation in Tumor-

Suppressor-Genes

Multistep-Model of Carcinogenesis

Initiation

Activation of

Oncogenes

Hyperplasia

malignant

Tumor

Progression

Activation of genes that promote

metastasis, invasion, angiogenesis

immunological tolerance etc.

Germline Mutation

in Repair-Genes

Adenoma


Accelerated

Initiation (2nd Hit)

Accelerated

Promotion Progression

(Transformation)

Multistep Carcinogenesis with induced or inherited

genomic instability

Germline

Predispo-

sition (Mutation

in a repair-

Gene)


- The malignant transformation of a cell and the formation of a tumor

requires several cellular and molecular functions to be altered.

- These alterations have to be irreversible.

- Depending on the degree of these alterations, tumors with different

stage of malignancy can be found.

- The process of malignant transformation is a time-dependent process.

- Ionizing radiation can contribute to this process, but not in a

deterministic manner.

- The stepwise carcinogenic process can be dramatically accelerated in

patients carrying germline mutations in cancer-predisposing genes.

S U M M A R Y

Documents

M.Rosemann, Institute for Radiation Biology Helmholtz ......EYF YVDFREYEYD LK 31-1 66 Liquid REYEYD LKWEFPRENL EFG consensus Structure of Flt3 ITDs in spontaneous and ... following