Oncogenes

1
Cellular Oncogenes
Earl Ruley Professor of Microbiology and
Immunology AA4210 MCN 3-1379
2
Oncogenes

• Oncogenes function in signaling pathways that
  regulate cell proliferation, differentiation and
  survival
• In cancer cells, proto-oncogenes are converted
  into oncogenes by mutations (activating
  mutations) that deregulate their function
• Increased expression
• Deregulated proteins

3
Cellular proto-oncogene
Activated Oncogene
4
The three types of somatic mutations that convert
cellular proto-oncogenes into activated
oncogenes.
Alberts, B., Bray, D., Lewis, J., Raff, M.,
Roberts, K., and Watson, J. D. (2000) Molecular
Biology of the Cell, Third Ed., Garland Science
Publishing, New York
5
Discovery of Oncogenes in Human Tumors

• Genes capable of transforming cultured cells to a
  malignant State
• Genes associated with tumor specific chromosome
  abnormalities
• Chromosome translocations
• Double minute chromosomes and heterogeneous
  staining regions (HSRs)
• Many human oncogenes were previously
  characterized as genes associated with oncogenic
  retroviruses

6
Isolation of EJ Ras Oncogene by DNA-Mediated Gene
Transfer
Watson, J. D., Hopkins, N. H., Roberts, J. W.,
Steitz, J. A., and Weiner, A. M. (1987) Molecular
Biology of the Gene, Fourth Ed., 2. 2 vols., The
Benjamin/Cummings Publishing Co., Inc., Menlo Park
7
How is EJ ras activated?
Transforming Actitivity
Gene
WT
EJ
Codon 12
Gly
Val
8
cMyc Translocated in Plasmacytomas
Southern
Northern
Shen-Ong, G. L., Keath, E. J., Piccoli, S. P.,
and Cole, M. D. (1982) Cell 31, 443-452
9
Abl Gene Translocation in Chronic Myelogenous
Leukemia
9
Abl
Bcr
22
Ph
9q
Bcr-Abl Fusion Protein
10
MYCN and chromosome 17 FISH. (a) Cell line Kelly
showing MYCN hsr (red) inserted into 17q (green)
(two copies). (b), (c) No apparent association
between MYCN hsrs (red) and chromosome 17
material (green) in cell lines LS and PER107. (d)
Primary tumor Case 1 MYCN hsr (red) inserted
into one arm of an isochromosome 17q (green). (e)
G-banded image of 16p13 hsr in a primary
neuroblastoma tumor. (f) Chromosome 17 wcp probe
(red) hybridizing to both ends of the 16p hsr.
O'Neill, S., Ekstrom, L., Lastowska, M.,
Roberts, P., Brodeur, G. M., Kees, U. R., Schwab,
M., and Bown, N. (2001) Genes Chromosomes Cancer
30, 87-90
11

• A Giemsa stained metaphase showing double minutes
  at the time of the diagnosis of MDS.
• Metaphase and interphase cells showing the c-MYC
  amplification by fluorescence in situ
  hybridization. Mathew, S., Lorsbach, R. B.,
  Shearer, P., Sandlund, J. T., and Raimondi, S. C.
  (2000) Leukemia 14, 1314-1315

12
Double minute chromosomes (DMs) and HSRs result
from gene amplification. The regions of amplified
DNA are larger than genes (e.g. several hundred
Kb). DMs and HSRs are frequently observed in
cells selected to express more of a specific
protein e.g. increased expression of
dihydrofolate reductase in cells resistant to
methotrexate. Since DMs lack centromeres they
are unstable and are quickly lost from cells in
the absence of selection. This implies oncogene
amplification confers a selective advantage to
cells.
13
The seven types of proteins that participate in
controlling cell growth. Cancer can result from
expression of mutant forms of these proteins
growth factors (I), growthfactor receptors (II),
signal-transduction proteins (III), transcription
factors (IV), pro- or anti-apoptotic proteins
(V), cellcycle control proteins (VI), and
DNArepair proteins (VII). Mutations changing the
structure or expression of proteins in classes I
IV generally give rise to dominantly active
oncogenes. The class VI proteins mainly act as
tumor suppressors mutations in the genes
encoding these proteins act recessively to
release cells from control and surveillance,
greatly increasing the probability that the
mutant cells will become tumor cells. Class VII
mutations greatly increase the probability of
mutations in the other classes. Virus-encoded
proteins that activate growth-factor receptors
(Ia) also can induce cancer.
Lodish, H., Berk, A., Zipursky, L. S.,
Matsudaria, P., Baltimore, D., and Darnell, J.
(2000) Molecular Cell Biology, W.H. Freeman and
Company, New York
14
Activation
Tumor
Category
Growth Factors
PDGF B chain Fibroblast GFs
gt Expression gt Expression
Osteosarcoma Carcinomas
Growth Factor Receptors
Carcinomas MEN
gt Expression Mutant Protein
Epidermal GF Receptors CSF-1 Receptor
Signal Transducers
Many CML
Mutant Protein Mutant Protein
GTP binding proteins (Ras) Tyrosine kinases (Abl)
Transcription Factors
Lymphoma Osteosarcoma
gt Expression gt Expression
Myc Fos
Anti-Apoptosis
Leukemia
Bcl2
gt Expression
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Elucidation of Oncogene Functions

• Growth Factors
• Growth Factor Receptors
• Signal Transducers
• Nuclear Receptors
• Transcription Factors
• Anti-Apoptosis

16
Growth Factors PDGF
17
(No Transcript)
18
Growth Factor Receptors EGF, CSF-1
19
(No Transcript)
20
c Fms has Properties of a CSF-1 (M-CSF) Receptor
CSF-1 stimulates c Fms Kinase Activity
c FMS binds 125I CSF-1
Sherr, C. J., Rettenmier, C. W., Sacca, R.,
Roussel, M. F., Look, A. T., and Stanley, E. R.
(1985) Cell 41, 665-676
21
Architecture and domain organization for a
variety of RTKs. The extracellular portion of the
receptors is on top and the cytoplasmic portion
is on bottom. Legend for the domain types is on
the right side. The TK domain of the PDGF
receptor contains a large insertion, represented
as a break in the TK domain symbol. The lengths
of the receptors are only approximately to scale.
Hubbard, S. R. (1999) Prog Biophys Mol Biol 71,
343-358
22
Regulated intramembrane proteolysis of a tyrosine
kinase receptor. Binding of a ligand to its
tyrosine kinase receptor induces activation and
autophosphorylation of the receptor and the
creation of docking sites for signaling proteins
containing SH2 domains. In this way, different
signaling pathways, such as that containing
mitogen-activated protein (MAP) kinase, are
activated. In a separate signaling pathway,
PLC-gamma activates PKC, which then activates
the metalloprotease TACE. This enzyme cleaves off
the ectodomain of the receptor and allows
intramembrane cleavage of the remaining part by
gamma-secretase. The cleaved cytoplasmic region
of the receptor then moves to the nucleus, where
it may affect the transcription of target genes.
From   Heldin Science, Volume
294(5549).December 7, 2001.2111, 2113
23
Signal Transducers Ras
24
ras Mutations in Human Tumors
Cancer or site or tumor Mutation
frequency Predominant ras


Isoform Non-small-cell lung cancer
33 K Colorectal 44 K Pancreas 90 K
Thyroid     Follicular 53 H, K,
N     Undifferentiated papillary 60 H, K,
N     Papillary 0 Seminoma 43 K,
N Melanoma 13 N Bladder 10 H Liver
30 N Kidney 10 H Myelodysplastic
syndrome 40 N, K Acute myelogenous
leukemia 30 N K Kirsten H Harvey N
neuroblastoma.
Adjei, A. A. (2001) J Natl Cancer Inst 93,
1062-1074
25
Activation of Ras following binding of a hormone
(e.g., EGF) to an RTK. The adapter protein GRB2
binds to a specific phosphotyrosine on the
activated RTK and to Sos, which in turn interacts
with the inactive Ras GDP. The guanine
nucleotide exchange factor (GEF) activity of
Sos then promotes formation of the active Ras
GTP. Note that Ras is tethered to the membrane by
a farnesyl anchor (see Figure 3-36b). See L.
Buday and J. Downward, 1993, Cell 73611 J. P.
Olivier et al., 1993, Cell 73179 S. E. Egan et
al., 1993, Nature 36345 E. J. Lowenstein et
al., 1992, Cell 70431 M. A. Simon et al., 1993,
Cell 73169.
Lodish, H., Berk, A., Zipursky, L. S.,
Matsudaria, P., Baltimore, D., and Darnell, J.
(2000) Molecular Cell Biology, W.H. Freeman and
Company, New York
26
Intrinsic Ras GTPase (in vitro)
Discovery of Ras GAP (GTPase Activating Protein)
Gibbs, J. B., Sigal, I. S., Poe, M., and
Scolnick, E. M. (1984) Proc Natl Acad Sci U S A
81, 5704-5708 Trahey, M., and McCormick, F.
(1987) Science 238, 542-545
Ras-Induced Germinal VesicleBreakdown and Ras
GTPase in Xenopus Ooctyes
GVBD
27
Inactive
Active
Activation cycle of Ras proteins. GNEFS guanine
nucleotide exchange factors, also known as
guanine nucleotide dissociation stimulators
(GDS) GAP guanine triphosphate-activating
protein GAPS guanine triphosphatase-activating
proteins GDP guanosine diphosphate GTP
guanosine triphosphate SOS son of sevenless
CDC25 a guanine nucleotide exchange factor Pi
inorganic phosphate C3G a guanine nucleotide
exchange factor. Adjei, A. A. (2001) J Natl
Cancer Inst 93, 1062-1074
28
Simplified drawing of ras signaling and its
effector pathways. P13K phosphoinositide
3'-kinase PLC phospholipase C PKC protein
kinase C MEK mitogen-activated protein kinase
kinase JNK Jun amino-terminal kinase SAPK
stress-activated protein kinase. The
well-characterized Ras/Raf/mitogen-activated
protein (MAP) kinase pathway illustrates a
typical MAP kinase-signaling module. Raf is an
MAPKKK (MEKK). MEK is an MAPKK. Activated MAPK
(ERK, i.e., extracellular signal-regulated
kinase) phosphorylates and activates various
transcription factors in the nucleus, which
control cellular responses. Although simplified,
note the cross-talk and redundancy of the
signaling pathways. BAD pro-apoptotic protein
of the Bcl-2 family DAG diacyl glycerol SEK
stress-activated protein (SAP)/Erk-Kinase Eg
for example PDGFR platelet-derived growth
factor receptor IGF-IR insulin-like growth
factor receptor type 1 MET/HGF-R hepatocyte
growth factor receptor (a product of the c-met
proto-oncogene). Adjei, A. A. (2001) J Natl
Cancer Inst 93, 1062-1074
29
Ras as a Target of Chemical Carcinogens
Zarbl, H., Sukumar, S., Arthur, A. V.,
Martin-Zanca, D., and Barbacid, M. (1985) Nature
315, 382-385
30
Transcription Factors Fos, Jun, Myc
31
c-Fos Transcripts are induced by Growth Factors
Cochran, B. H., Zullo, J., Verma, I. M., and
Stiles, C. D. (1984) Science 226, 1080-1082
32
c Jun is a Component of Activator Protein 1
(AP-1)
Purification of AP1 Protein
AP-1 Sites are TPA Inducible Elements
Footprint
Similarity to vJun and cJun
Angel, P., Imagawa, M., Chiu, R., Stein, B.,
Imbra, R. J., Rahmsdorf, H. J., Jonat, C.,
Herrlich, P., and Karin, M. (1987) Cell 49,
729 Bohmann, D., Bos, T. J., Admon, A.,
Nishimura, T., Vogt, P. K., and Tjian, R. (1987)
Science 238, 1386-1392
33
Myc as a Transcription Factor
Amati, B., Frank, S. R., Donjerkovic, D., and
Taubert, S. (2001) Biochim Biophys Acta 1471,
M135-145
34
Anti-Apoptosis Factors Bcl2
35
Cory, S., and Adams, J. M. (2002) Nat Rev Cancer
2, 647-656
Three subfamilies of Bcl2-related proteins.  
Known -helical regions are indicated, as are the
four regions (BH14) that are most highly
conserved among family members. Most members have
a carboxy-terminal hydrophobic domain that aids
association with intracellular membranes, the
exceptions being A1 and many of the BH3-only
proteins (Bad, Bid, Noxa, Bmf and Puma). Several
other multidomain homologues (for example,
Boo/Diva, Bcl-Rambo, Bcl-G, Bcl-B) have been
described, but their function is not yet clear.
TM, transmembrane domain.
36
Inactivation of the retinoblastoma (Rb) pathway
for example, by loss of cell-cycle inhibitor
Ink4a, which can prevent cyclin-DCdk4 from
phosphorylating Rb unleashes the transcription
factor E2f1, which increases expression of Arf, a
protein that is encoded by the same locus as
Ink4a (Ref. 136). Arf, which is also a
transcriptional target of Myc, sequesters Mdm2, a
negative regulator of p53. Raised p53 levels can
either impose growth arrest, typically by
inducing the Waf1 cell-cycle inhibitor, or
promote apoptosis through targets such as Bax,
Puma and Noxa. The apoptotic targets seem to also
require the p53 relative p63 or p73 (Ref. 152).
Circles/ovals denote oncogene products
rectangles denote known or likely tumour
suppressors. For more detail, see Refs 46,136.
ATM, ataxia telengiectasia mutated Chk2,
checkpoint 2 NF-B, nuclear factor-B.
Cory, S., and Adams, J. M. (2002) Nat Rev Cancer
2, 647-656
37
The molecular circuitry of cancer
Hahn, W. C., and Weinberg, R. A. (2002) Nat Rev
Cancer 2, 331-341
38
Model for Colorectal Carcinoma
Benign Adenoma
Cancerous Adenoma
Invasive Colon Cancer
39
Epigenetic differentiation of genetically
abnormal myeloid leukemic cells to non-malignant
mature macrophages and granulocytes by IL-6 (A)
leukemic cell (B) macrophage (C) colony with
macrophages (DG) stages in differentiation to
granulocytes.
J Lotem and L Sachs Seminars in CANCER BIOLOGY,
Vol. 12, 2002 pp. 339346