oncogenesis

Introduction

Oncogenesis

Early Det.

Assays

Conclusion

Molecular, Genetics and

Epigenetics of oncogenesis

oncogenesis

Introduction

Herry HERMAN1,2
of Orthopaedic Surgery and Traumatology, School of
Medicine Padjadaran Univ.-Hasan Sadikin General Hospital,
Bandung, Indonesia
2Institute of Molecular Genetics Montpellier, France

Introduction

Oncogenesis

Early Det

Assays

Conclusion

Proposed stages of
oncogenesis

Early Det.

Assays

Conclusion

Demography and
Significance

400 new cases every year (US 200 millions

population)

Account for 60% of the primary bone

malignancy in adolescent

20% of cases present with metastases

4 out 5 resected cases develop metastases

Need identification at early stages

1Dept

oncogenesis

Oncogenesis

oncogenesis

Introduction

Oncogenesis

Early Det.

Assays

Conclusion

Oncogenesis
Two Steps (Hits) Oncogenesis
Statistic based prediction
Retinoblastoma is genotypically recessive, only
manifest when both Rb gene copies are mutated ie
genotype is rb/rb, carrier (i.g. RB/rb) are normal
Descendents from a carrier and a normal parent
theoritically should be 0% for Retinoblastoma
Observation
50% of the children develop Retinoblastoma

Lisa L Wang Journal of Cancer 2005 Vol; Issue:294

oncogenesis

Introduction

Oncogenesis

Early Det.

Assays

Conclusion

Oncogenesis
Two Steps (Hits) Oncogenesis
Genotype

Clinical

Germline

RB/rb

RB/RB

Children

RB/rb

RB/RB

0%RB?

Somatic
Tumor

rb/rb

50%RB!

2 Hits : Germline inactivating mutation in previous

generation + somatic inactivating mutation in tumor

oncogenesis

Introduction

Oncogenesis

Early Det.

Assays

Conclusion

Oncogenesis
Two Steps (Hits) Oncogenesis

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det.

Assays

Conclusion

Pathways to
malignancy

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy
Current multi steps oncogenesis

Molecular view of oncogenesis

Cancer is the result of imbalance between genetic
events; promoting and preventing cancer
Promotion of cancer is mediated by
Oncogenes
Prevention of cancer is mediated by Tumor
Suppressor Genes (TSG)

b
e
n
i
g
n

Stimulate own
proliferation
Ignore order to stop
proliferation
Old cells live on

m
a
l
i
g
n

limitless proliferation
Paving in roads
Founding of new colonies

Hanahan and Weinberg, Cell 2000

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy

Pathways to malignancy

Genes implicated in oncogenesis

Cell Cycle / Rb Pathways

where first two steps of
oncogenesis applies
E2F activate DNA
synthesis genes
Rb inactivate E2F and
prevent cell division
CDKs and Cyclins
release E2F by
phosphorylating Rb

Lisa L Wang Journal of Cancer 2005;294

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy

Pathways to malignancy

Programmed Cell Death (Apoptosis) Evasion

How apoptosis evasion promotes oncogenesis

cell division or G1S is

inhibited by p53 through
p21
cell death or apoptosis is
mediated by P53 and
BAX and is positively
modulated by p14ARF
cell survival is mediated
by BCL2 and MYC and
modulated by MDM2

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy

Pathways to malignancy

Genomic Instability Pathway`

Genomic Instability Pathway

For familial cancer, the second mutation may suffice to give rise to
cancer i.e. Retinoblastoma, since the first mutation had already
occurred in the previous. generation
For sporadic cancer, no mutations was inherited from the parents,
mutations on both gene copies need to occur somatically in the
individual.
A non stable genome, where chromosomes are reshuffled randomly,
increase the random chances of having both gene copies to be
inactivated.

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy

Barriers are the two

hits
TSG Recql4,
BLM, WRN
OG : any genes that
promote genomic
instability

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy

The Telomeric Pathway

The Telomeric Pathway

Telomerase system :

Chromosomes have telomere,

acting as bumper, protecting it

Telomerase / Reverse
transcriptase

However, telomere is
progressively shortened with
each cell division. Loss of
telomere jeopardize
chromosome integrity, halts cell
proliferation and induces death

RNA template

Cancer keeps telomere length

with telomerases system or the
alternative pathway (ALT)
cancer utilizes almost exclusively
the ALT pathway

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy

Pathways to malignancy

The Telomeric Pathway

can perturbation of a single pathway cause cancer?

ALT system :

Yes but mostly No

Recombination
mediated

In an invitro system, disruption of Rb and P53 does allow increase

proliferation, however this soon will be followed by apoptosis
induced by the telomere shortening

no telomerases
no RNA template

Acquisition of telomere maintenance permit cell to grow indefinitely

however does not cause cancer.
Activation of late stage oncogenes, such as RAS, c-fos and c-myc,
initiate oncogenesis

oncogenesis

Introduction

Oncogenesis
Oncogenesis

Early Det

Assays

Conclusion

Pathways to malignancy
Summary

- The Cell Cycle/ Rb patways

- The Apoptosis / p53 pathways
- The Telomere Pathways
- The Genomic Instability Pathways
- The Metastasis Pathways

Introduction

Oncogenesis

Early
EarlyDetect.
Det

Assays

Introduction

Biology
Oncogenesis

Early Det

Assays

Conclusion

Cell Cycles\1Cell undergoing cycling.swf

Cell Cycles\3cell_cycle Checkpoints.swf
Cell Cycles\4Spindle Check points.swf`

Cell Cycles\2factors of cell cycles+G1SCP.swf

apoptosis\p53 pada G1S checkpoint.swf
apoptosis\apoptosis general2.swf
apoptosis\p53pathway general.swf
apoptosis\apoptosis_07 well animated.swf
apoptosis\necrosis vs apoptosis macros.swf
apoptosis\Indyction_Of_Apoptosis EARLY PHASE
(GOOD).swf
apoptosis\procaspase 8 activation.swf
apoptosis\ntlup good static diagram for whole pathway.swf
apoptosis\complete pathway simpler animation.swf
apoptosis\apop-fas complete pathway.swf
apoptosis\Microscopic view of apoptotic hepatocytes.swf

Multiple pathways dysregulation mediate oncogenesis

oncogenesis

oncogenesis

Conclusion

Oncogenesis in the
clinical setting

oncogenesis

Introduction

Oncogenesis

Early
EarlyDetect.
Det

Assays

Conclusion

What about sporadic

cancer?

Remember that predisposing factors may manifest as familial

genetic syndromes or as acquired germ line mutations.
Know that many familial syndromes were attributed to the mutation
of genes involved in oncogenesis

Unfortunately most sporadic cancer diagnosed

in the clinic are already at the advance stage,
with microscopic metastasis. They have
already deregulated all the critical pathways.

P53 is already mutated in patients with Li-Fraumeni syndromes. Rb

is mutated in patients with Retinoblastoma, Recq1l4 is mutated in
patients with Werner and Bloom syndromes.

Screening general population or even

orthopaedic patients for cancer markers may
not be feasible.

In the absence of familial syndromes, remember that germline

mutation manifest as disease or cancer shared by brother/sisters
not shared by previous generation

oncogenesis

Introduction

Oncogenesis

Early
EarlyDetect.
Det

Assays

Conclusion

oncogenesis

What to do in the presence of

predisposing factors?
Regular screening for progression genetic abnormalities by
cytogenetics.
Molecular genetics work up for critical genes in the absence of
cytogenetic evidence
- Southern Blot
- SSCP
Epigenetics work up in the absence of cytogenetic and genetics
evidence.
Regular Radiologic or bone scan work up

Introduction

Oncogenesis

Early Det

Cytogenetics
The Principles
Make a metaphase or
interphase spread
Identify and line up
each chromosome
pairs side by side
Look for structure
abnormalities

Assays
Assays

Conclusion

oncogenesis

Introduction

Oncogenesis

Early Det

Assays
Assays

Conclusion

oncogenesis

Introduction

Oncogenesis

Early Det

Assays
Assays

Cytogenetics

Cytogenetics

How to Identify each Chromosome?

Examples of chromosomal aberration

oncogenesis

Introduction

Oncogenesis

Early Det

Assays
Assays

Conclusion

oncogenesis

Introduction

Oncogenesis

Early Det

Assays
Assays

Cytogenetics

Southern Blotting

How does chromosomal aberration bring about gene dysregulation

The Principles

Conclusion

Conclusion

Restrict the
genome with
Cutter
Query gene of
interest with
specific probe
Compare banding
pattern to that of
normal samples

oncogenesis

Introduction

Oncogenesis

Early Det

Assays
Assays

Conclusion

oncogenesis

Introduction

Oncogenesis

Early Det

Assays

Conclusion

Transcript Detection
The Principles

Concluding remarks

Isolate RNA from Cell

Prime RNA with
appropriate primers

If nothing remembered from this

presentation, at least know that
more than 1 pathways are
required for oncogenesis

Reverse transcribed
RNA into cDNA
with Reverse
Transcriptase

Effort of early detection should be

directed toward patients with
predisposing factors

PCR and run on gel,

only active gene
will give out bands

oncogenesis

Introduction

Oncogenesis

Early Det

Assays

Conclusion

Acknowledgments
Dept. Orthopaedic Surgery
and Traumatology
European Molecular
Biology Organization
Robert Feils Lab