Download Origin of oncogenes?

Oncogenes and Proto-oncogenes
Jekyll and Hyde
A double edged sword
Origin of oncogenes?
Oncogene hypothesis
Retroviral oncogenes and cell proto-oncogenes
(v-onc)
(c-onc)
The role of c-onc in cancer
How many c-onc in human genome?
Tumor viruses and cancer
The viral origin of the majority of all
malignant tumors…..have now been
documented beyond any reasonable doubt.
It…..would be rather difficult to assume a
fundamentally different etiology for human
cancer.
Ludwik Gross 1970
Endogenous retroviruses
and
cancer
Epidemiological observation of cancer is
inconsistent with viral etiology for human cancer
Two schools of thought from virologists firmly believed in viral
etiology:
•Can reactivation of endogenous viruses cause cancer?
Ans: Could be in some cases in animals, but no evidence in
human
•Can reactivation of integrated viral oncogene(s) cause cancer?
Ans: No such evidence in human cancer
Murine versus Human endogenous viruses
Murine: 4-5% of the genome
*Some endogenous viruses are infectious.
*Continue to be infected in recent evolution
Human: 1% of the genome
•No infectious endogenous viruses have been
found so far.
•Pattern of human endogenous retroviral
sequences are fixed, and no evidence of recent
infection in evolution.
Origin of
Endogenous viruses
Murine
Human
Figure 4.1 The Biology of Cancer (© Garland Science 2007)
Figure 4.1a (part 1 of 2) The Biology of Cancer (© Garland Science 2007)
Figure 4.1a (part 2 of 2) The Biology of Cancer (© Garland Science 2007)
Figure 4.1b The Biology of Cancer (© Garland Science 2007)
Figure 4.1c The Biology of Cancer (© Garland Science 2007)
Gene transfer technology
and
cancer
1972 Calcium phosphate DNA transfection method
Cooper and Temin
RSV transformed cells ---> DNA ---> NIH 3T3 cells --> transformation
1978-1979
3-MC (carcinogen) ---> C3H 10T1/2 cells
transformation---> isolate DNA
---> transfect NIH 3T3 cells ---> transformation
DNA transfection using chemically transformed cells and
Subsequent tumor formation in mouse using the cells
Figure 4.2 The Biology of Cancer (© Garland Science 2007)
Figure 4.2 (part 1 of 2) The Biology of Cancer (© Garland Science 2007)
Figure 4.2 (part 2 of 2) The Biology of Cancer (© Garland Science 2007)
T24 bladder carcinoma cells DNA transfected NIH 3T3
A transformed focus
Magnification of the focus
Figure 4.3 The Biology of Cancer (© Garland Science 2007)
Surrounding
Normal cells
How many oncogenes are needed to
transform cells?
One hit, two hits or multiple hits phenomenon?
* Only 0.1% of a genome set was transfected to
induce transformation.
If one hit: 0.001 probability : yes
If two hits: 0.000001 probability : no
•Any other way to show the hitness?
(Ans: serial dilution kinetics)
Human cancer genes = viral oncogenes?
DNA blotting and hybridization
Mid 1970’s: Southern blot
Figure 4.4 The Biology of Cancer (© Garland Science 2007)
Figure 4.4 (part 1 of 4) The Biology of Cancer (© Garland Science 2007)
Figure 4.4 (part 2 of 4) The Biology of Cancer (© Garland Science 2007)
Figure 4.4 (part 3 of 4) The Biology of Cancer (© Garland Science 2007)
Figure 4.4 (part 4 of 4) The Biology of Cancer (© Garland Science 2007)
Southern blot: DNA
Northern blot: RNA
Western blot: protein
Eastern blot: ? (carbohydrates)
T24 DNA transformed NIH 3T3 lines
Southern blot
H-ras probe
Figure 4.5 The Biology of Cancer (© Garland Science 2007)
Amplification of proto-oncogenes in
cancer
Myc: promyelocytic leukemia line HL60
N-Myc: neuroblastoma
ErbB (EGFR): stomach, lung, brain, and breast
cancers
ErbB2 (HER2): 25 to 30% breast cancer
Table 4.1 The Biology of Cancer (© Garland Science 2007)
Human breast carcinoma with amplified ErbB2/Neu
Southern blot using ErbB2 probe
Figure 4.6a The Biology of Cancer (© Garland Science 2007)
Kaplan-Meier plot
Figure 4.6b The Biology of Cancer (© Garland Science 2007)
Amplified ErbB2 gene
Elevated ErbB2
transcription
Elevated ErbB2
translation
Figure 4.6c The Biology of Cancer (© Garland Science 2007)
Gene cluster analysis: 160 genes on 17q
25%
Figure 4.7 The Biology of Cancer (© Garland Science 2007)
Activation of proto-oncogenes by
point mutation(s)
Early 1980’s: Cloning and sequence
analysis of bladder and colon cancer
genes:
H-Ras; K-Ras
Codon 12, 61 and 13 (less frequent)
Sequential transfection with human cancer cells DNA
Figure 4.8 The Biology of Cancer (© Garland Science 2007)
Localization of an activating oncogene in human cancer DNA
Figure 4.9 The Biology of Cancer (© Garland Science 2007)
H-ras oncogene activation: codon 12 Gly Val
Figure 4.10 The Biology of Cancer (© Garland Science 2007)
Table 4.2 The Biology of Cancer (© Garland Science 2007)
Homogeneously Staining regions (HSRs)
and
Double minutes (DMs)
HSRs: tandem gene amplification in the chromosome
DMs: amplified genes broken off from chromosome
N-myc in
Neuroblastoma
FISH analysis
HSRs
DMs
Figure 4.11a The Biology of Cancer (© Garland Science 2007)
Kaplan-Meier plot
Figure 4.11b The Biology of Cancer (© Garland Science 2007)
Table 4.3 The Biology of Cancer (© Garland Science 2007)
Gene translocation
C-myc translocation: Burkitt’s lymphoma
Mimicking promoter insertion activation of
c-myc transcription in chicken leukemia
Figure 4.12 The Biology of Cancer (© Garland Science 2007)
Figure 4.13a The Biology of Cancer (© Garland Science 2007)
Chromosomal translocation
Figure 4.13b The Biology of Cancer (© Garland Science 2007)
C-Myc translocation: transcriptional
activation
Ig heavy chain : 75%
Ig lambda chain: 16%
Ig kapa chain: 9%
What is the role of EBV in Burkitt’s
lymphoma?
Hypotheses:
•Promoted immune response of B-cell gene
rearrangement myc translocation
•Chronic infection viral reactivation chromosomal instability
What is the role of malaria in Burkitt’s
lymphoma?
Hypothesis:
Malaria CIDR-1 EBV reactivation
C-myc in cancer
•Promoter insertion
•Gene amplification
•Chromosomal translocation
Table 4.4 The Biology of Cancer (© Garland Science 2007)
Why gene translocation often
seen in leukemia?
CML
Philadelphia
chromosome
Figure 4.15a The Biology of Cancer (© Garland Science 2007)
Chromos 9
Chromos 22
Figure 4.15b The Biology of Cancer (© Garland Science 2007)
Table 4.5 The Biology of Cancer (© Garland Science 2007)
Activation of cellular tyrosine kinases
Non-receptor tyrosine kinases: ex Src
Receptor tyrosine kinases: ex EGFR
Src Protein
unique
SH3
SH2
PTK/SH1
N
membrane
binding
myristoylation
site
K295
ATP
binding
Y416
(+)
regulatory
C
Y527
(−)
Autoinhibition of Cytoplasmic PTK and PTP
Figure 4.14 The Biology of Cancer (© Garland Science 2007)
PTK Receptors & Oncogenes
Kinase
1
src
fes
fgr
fyn
lyn
lck
.
etc
2
fps
yes
abl
syn
hck
tkl
3
EGFR
neu
v-erbB
sea
eph
ltk
.
etc
4
InsR
IGF-1R
met
ros
trk
5
PDGFRs
v-ros
CSF1R
kit
ret
flt
FGFR (3 loops)
flg (
“ )
bek (
“ )
6
7
v-fms
Flk
Flt
Tie1
Tie2
Location and Function of Retroviral
Oncogene Products
PDGF
sis
Extracellular
Intracellular
GTP binding:
K-ras, H-ras
Kinase
Kinase
Receptor PTKs:
erbB2, fms, (ros), (kit)
Non-receptor PTKs:
src, fps/fes, yes, fgr, abl
Serine/Threonine Kinases:
mos, rel, mil/raf
DNA binding;
Transcription Factors:
myc, fos, rel, ski, myb, jun, ets
Nucleus
Products and Functions of Various Oncogenes
Growth factors: ex.sis of Simian sarcoma virus (SSV)
Growth factor receptors: receptor protein tyrosine kinases (RPTKs); ex. verbB of Avian erythroblostosis virus (AEV); fms of SM-FeSV; ros of
ASV UR2
Cytoplasmic PTKs: ex.src of RSV; fps/fes of FSV & FeSV
GTP-binding protein: ex.ras of Ki-MuSV and Ha-MuSV
Serine/threonine Protein Kinases: ex.mos of Mo-MuSV; raf of MSV;
mhl/mil of MH2
Nuclear proteins (transcription factors): ex.myc of MC29; myb of AMV;
rel of REV-T; jun of ASV17.
Signaling Adaptors: crk of ATV110
Lipid and protein kinase (PI3 Kinase) : Avian sarcoma virus
Activation of Proto-oncogenes
A) Point mutations
single mutation:ex. c-ras codons 12 or 61 mutation resulting in enhanced binding to GTP and
persisted activation of ras;
ex. c-neu conversion is due to oncogenic neu conversion is due to a single
mutation in the transmembrane domain;
ex. c-src can be activated by a single point mutation.
multiple point mutations: most of the oncogenes contain several point mutations.
B) Deletions
Almost invariably retroviral oncogenes suffer 5' and 3' truncations comparing to their proto-oncogenes.
The 5' truncated gene is fused in-frame to one of the viral gene, most often the 5' gag gene, resulting in the
generation of a fusion protein. The 3' region of the proto-oncogene is also frequently fused to the viral
sequence. Those mutations result in constitutive activation of the proto-oncogenes. Aside from the terminal
truncations, internal deletions can also activate a proto-oncogene in a fashion equivalent to those of point
mutations.
C) Amplification
Several types of human tumors have been found to contain amplified proto-oncogenes. Examples
include myc amplification in carcinomas of lung, breast, cervix and erbB/erb2 amplification in squamous cell
carcinoma and astrocytoma. Although it is not clear whether the amplified genes also harbor mutations and
either or both is responsible for the oncogenesis, it has been demonstrated experimentally for several protooncogenes that mere overexpression of them can lead to cell transformation.
D) Gene translocation and rearrangement
This type of gene aberration not only can lead to structural mutation, but could also result in deregulated
expression. The best known examples are the c-abl translocation leading to formation of Philadelphia
chromosome in chronic myelogenous leukemia (CML) and the c-myc translocation in Burkitt's and certain Tand B-cell lymphomas. The resulting rearranged genes could still code for the original proteins, or in other
cases, code for novel fusion proteins.
Table 4.6 The Biology of Cancer (© Garland Science 2007)