Transcript
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CANCER GENETICS 2
Two classes of genes in which mutations cause transformation-
Oncogenes were initially identified as genes carried by viruses that cause transformation of theirtarget cells. A major class of the viral oncogenes have cellular counterparts that are involved in normal cell
functions. The cellular genes are called proto-oncogenes, and in certain cases their mutation or aberrantactivation in the cell to form an oncogene is associated with tumor formation. The generation of an oncogene
represents a gain-of-function in which a cellular proto-oncogene is inappropriately activated. This can
involve a mutational change in the protein, or constitutive activation, overexpression, or failure to turn
off expression at the appropriate time.
Dominant Oncogenes- These are genes whose normal activity promotes cell Proliferation. Gain of function
mutations in tumour cells create forms that are excessively or inappropriately active. A single mutant allele may
affect the phenotype of the cell. The non-mutant versions are properly called proto-oncogenes.
Tumor suppressors are detected by deletions (or other inactivating mutations) that aretumorigenic. The mutations representloss-of-function in genes that usually impose some constraint on the cell
cycle or cell growth; the release of the constraint is tumorigenic. It is necessary for both copies of the gene to be
inactivated.
Transforming viruses carry oncogenes
Transformation may occur spontaneously, may be caused by certain chemical agents, and, most notably, may
result from infection with tumor viruses. There are many classes of tumor viruses, including both DNA and
RNA viruses, and they occur widely in the avian and animal kingdoms.
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The oncogenes carried by the DNA viruses specify proteins that inactivate tumor suppressors, so their action in
part mimics loss-of-function of the tumor suppressors. The oncogenes carried by retroviruses are derived from
cellular genes and therefore may mimic the behaviour of gain-of-function mutations in animal protooncogenes.
DNA Tumor Viruses May Kill or Transform Cells
The response of a cell to infection depends on its species
and phenotype and falls into one of two classes-
Permissive cells are productively infected. The
virus proceeds through a lytic cycle that is divided
into the usual early and late stages. The cycle ends with
release of progeny viruses and (ultimately) cell
death.
Nonpermissive cells cannot be productively
infected, and viral replicationis abortive. Some of the
infected cells are transformed; in this case, the
phenotype of the individual cell changes and the
culture is perpetuated in an unrestrained manner.
A common mechanism underlies transformation by
DNA tumor viruses. Oncogenic potential resides in
a single function or group of related functions that
are active early in the viral lytic cycle. When
transformation occurs, the relevant gene(s) are
integrated into the genomes of transformed cells
and expressed constitutively.
Cells transformed by polyomaviruses contain
integrated copies of part or all of the viral genome.
The integrated sequences always include the
early region. So, here the early region is very
essential for viral replication. The T antigens have
transforming activity, which rests upon their abilityto interact with cellular proteins. This is
independent of their ability to interact directly with
the viral genome.
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Retroviruses activate or incorporate cellular genes
The retroviral genome is flanked by two long terminal repeat (LTR) sequences at both the 5- and 3- ends.
In the integrated virus (provirus) each LTR consists of three regions: 1) the R sequence, 2) the U3 region, and
3) the U5 region. LTR is mainly the regulatory element of viral replication and transcription. Original
retroviral sequences usually organized into the genes gag-pol-env, coding for coat proteins, reverse
transcriptase, and other enzyme activities.
In 1911, Peyton Rous discovered that cancer could be induced in healthy chickens by injecting them with a cell-
free extract of the tumor of a sick chicken. This was the first demonstration of an oncogenic virus that is, a
virus capable of causing cancer. The tumor was a sarcoma, a tumo r ofconnective tissue. The virus was named
the Rous sarcoma virus (RSV).
The Rous sarcoma virus has only 4 genes (bottom panel):
gag, which encodes the capsid protein
pol, which encodes the reverse transcriptase
env, which encodes the envelope protein
src, which encodes a tyrosine kinase, an enzyme that attaches phosphate groups to Tyr residues on a
variety of host cell proteins.
Cellular Origin ofv-onc
1. RSV mRNAs (gag-pol mRNA, env
mRNA, src mRNA) were converted to
cDNA (gag-pol cDNA, env cDNA, src
cDNA).
2. All cDNAs were annealed to excess
mRNA (gag-pol mRNA, env mRNA)
from src deleted RSV genome (no src
mRNA).
3. Except src cDNA all other cDNA will be
hybridized with mRNA.
4. Purify src cDNA and probe chicken genomic DNA by southern blot.
Positive hybridization indicates cellular origin of v-src called c-src.
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Retrovirus Transduces Cell
Retrovirus oncogenes d
lar Genes
The virus gains a copy of a pr
genome. Sometimes the copy
sequence typically because it
cases, the difference is suffi
oncogene into an oncogene. In
in the viral sequence that
oncogene.
The viral oncogenes and the
described by using prefixes v
So the oncogene carried by R
v-src, and the proto-oncoge
genomes is called c-src.
rived from normal cellular g
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oto-oncogene from a cellular
is different from the cellular
has been truncated. In some
cient to convert the proto-
other cases, mutations occur
converts the copy into an
ir cellular counterparts are
for viral and c for cellular.
us sarcoma virus is called
e related to it in cellular
nes
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DNA from Tu
Some oncogenes can be detected by
are transfected with DNA obtained fro
Oncogenes derived from the c-ras fa
several active genes in both man an
individual genes, N-ras, H-ras, and K-
collectively as p21ras.
Functions of cellular pro
or Cell can Transform No
using a direct assay for transformation in
animal tumors.
mily are often detected in the transfection
rat, dispersed in the genome. (There are a
as, are closely related, and code for protein
to-oncogenes
The functions of the cellular prot
transforming retroviruses all have to
growth. As shown in the figure, the g
classes: those encoding secreted gr
receptors, cytoplasmic signal transd
transcription factors. Overexpressi
any of these proteins would be exp
regulate cell proliferation. The specif
proto-oncogenes are shown in the nex
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mal Cell
hich "normal" recipient cells
ssay. The family consists of
lso some pseudogenes.) The
roducts ~21 kD and known
o-oncogenes picked up by
do with the regulation of cell
enes fall into four functional
wth factors, growth factor
ction proteins, and nuclear
n or constitutive activity of
ected to activate genes that
ic functions of some of these
t slide.
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Functions of selected proto-oncogenes-
Sustaining Proliferative Signaling
Cancer cell can produce growth factor themselves that functions in a autocrine manner. Cancer cells may
send signals to stimulate normal cells within the supporting tumor-associated stroma, which reciprocate
by supplying the cancer cells with various growth factors.
Receptor signaling can also be deregulated by elevating the levels of receptor proteins rendering such
cells hyper-responsive to otherwise-limiting amounts of growth factor ligand. e.g EGFR
The same outcome can result from structural alterations in the receptor molecules that facilitate ligand-
independent firing. e.g. EGFR
Somatic mutations activate additional downstream pathways e.g. Ras, Raf, PI3-kinase
Disruptions of negative-feedback mechanisms that attenuate proliferative signaling e.g. Ras, PTEN,
mTOR
Growth factor receptor kinases can be mutated to oncogenes
The protein tyrosine kinases constitute a major
class of oncoproteins, and fall into two general
groups: transmembrane receptors for growth
factors; and cytoplasmic enzymes.
A (generalized) relationship between a growth
factor receptor and an oncogenic variantis very
crucial. The wild-type receptor is regulated by
ligand binding. In the absence of ligand, the
monomers do not interact. Growth factor
binding triggers an interaction, allowing the
receptor to form dimers. This in turn activates the
receptor, and triggers signal transduction. By
contrast, the oncogenic variant spontaneously
forms dimers that are constitutively active.
Different types of events may be responsible for
the constitutive dimerization and activation in
different growth factor receptors.
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Mutational Pattern of EGFR
The protein tyrosine kinases constitute a major class of oncoproteins, and fall
into two general groups: transmembrane receptors for growth factors and
cytoplasmic enzymes.
The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is
the cell-surface receptor for members of the epidermal growth factor family
(EGF-family) of extracellular protein ligands.
1. Gene amplification
2. Partial gene deletion
3. Activating Missense mutation
Extracellular domains of EGFR are responsible for ligand binding and
dimerization. Deletion in domain I and II [2-7 (VIII) EGFR] makes EGFR
constitutively active even in the absence of EGF. Missense mutation makes the
EGFR constitutively active.
Ligand binds to the extracellular domain of the receptor and results in
receptor dimerization and phosphorylation of the intracellular domains.
Activated EGFR leads to activation of the oncogene KRAS, which in turn
activates the oncogene BRAF, mitogen-activated protein kinase kinase (MEK),
and mitogen-activated protein kinase (MAPK), and leads to expression of
growth-promoting genes. In addition to activation ofKRAS, EGFR activates the
oncogene PIK3CA which phosphorylates phosphatidylinositol-2-phosphate
(PIP2) to phosphatidylinositol-3-phosphate (PIP3), which in turn activates
AKTand several downstream effectors, leading to protein synthesis, cell
growth and survival, proliferation, migration, and angiogenesis.
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Intracellular Signaling Networks Activated by EGFR- (A) A subset of intracellular signaling
components influenced by epidermal growth factor receptor (EGFR) activation are intertwined in a complex
network. Through a combination ofstimulatory (black arrows) or inhibitory (red lines) signals, several key
positive feedback loops (blue circular arrows) and negative feedback loops (red circular arrows) emerge
in the network and exert significant influence on its behaviour. For example, inhibition of Ras by Ras-GAP or
EGFR by protein kinase C (PKC) serves a negative feedback function. On the other hand, H2O2 inhibits
protein tyrosine phosphatases (PTPs) and thus prolongs or increases activity of EGFR by a positive
feedback mechanism.
(B) A conceptual representation of a bow tie or
hourglass network, as described by Kitano
(2004). A wide input layer (green) includes
multiple RTKs that all influence a relatively small
number of core processes (magenta), including
phosphoinositide 3-kinase (PI-3K) signaling, MAPK
signaling, and Ca2+ signaling. Feedback processes
within the core define specific emergent properties
of the system. The behavior of the core processes is
read out by a wide output layer (orange) that
consists of diverse transcriptional responses and
cytoskeletal changes. Extensive negative and
positive feedback loops exist between the core
processes and the input layer. Similar feedback
exists between the output layer and the core
processes, in addition to feedforward regulation
by core processes (e.g., MAPK signaling) of
immediate early gene products described by
Murphy and Blenis (2006). An additional layer of
system control also occurs between the input and
output layers.
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Ras proto-oncogene is activated by point mutation
1. The c-ras family contains three genes: H-ras, K-ras, and N-
ras. v-ras genes are derived by point mutations ofc-ras
gene. Each of the c-ras proto-oncogenes can give rise to a
transforming oncogene by a single base mutation. The
mutations in several independent human tumors cause
substitution of a single amino acid, most commonly at
position 12 or 61, in one of the Ras proteins. Almost anymutation at either gly 12 or gln 61converts c-ras proto-
oncogene into oncogenes.
All three c-ras genes have glycine at position 12. If it is
replaced in vitro by any other of the 19 amino acids exceptproline, the mutated c-ras gene can transform cultured
cells. The particular substitution influences the strength ofthe transforming ability.
Position 61 is occupied by glutamine in wild-type c-ras
genes. Its change to another amino acid usually creates a
gene with transforming potential. Some substitutions areless effective than others; proline and glutamic acid are the
only substitutions that have no effect.
2. The Ras proteins encoded by these genes are small G-
proteins.
3. The proteins transmit growth signals from cell surface
receptors.
4. The Ras proteins are activated by binding GTP.
5. The proteins are inactivated by GTP to GDP hydrolysis.The effect of the mutations is to increase Ras activity by
inhibiting the hydrolysis of bound GTP to GDP.
6. Mutations in the c-ras genes inactivate the Ras GTPase
7. Mutated Ras proteins are constitutively active
8. Constitutively active Ras proteins result in
uncontrolled cell growth.
Amino acid substitutions in Ras family proteins
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Ras proliferation signaling pathway
Binding of growth factors to receptor tyrosine kinases stimulates the autophosphorylation of specifictyrosines on the receptors. The phosphorylated receptor then binds to an adaptor protein called GRB2 which,
in turn, recruitsSOS (son of sevenless) to the plasma membrane. SOS is a guanine nucleotide exchange factor
(GEF) which displaces GDP from Ras, subsequently allowing the binding of GTP that recruits and activates Raf.
Raf initates a cascade of protein phosphorylation by firstphosphorylating MEK (Mitogen-activated protein
kinases). Phosphorylated MEK in turn phosphorylates ERK (Extracellular signal-regulated kinases).
Phosphorylated ERK moves from the cytoplasm into the nucleus where it subsequently phosphorylates a
number of transcription factors, including the specific transcription factor called Elk-1 (ETS domain-
containing protein). Phosphorylated transcription factors turn on transcription (gene expression) of specific
sets of target genes. The activity of Ras is limited by the hydrolysis of GTP back to GDP by GTPase activating
proteins (GAP).
[Other abbreviations are: MEK = MAPK/ERK kinase, ERK = extracellular receptor-stimulated kinase, MAPK =
mitogen-activated protein kinase. Kinases are enzymes that add phosphates to molecules using ATP. Mitogens
are factors (such as growth factors) that stimulate cell division.]
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Proto-oncogenes can be activated by translocationTranslocation to a new chromosomal location is another of the mechanisms by which oncogenes are activated. A
reciprocal translocation occurs when an illegitimate recombination occurs between two chromosomes.
When c-mycis translocated to the Ig locus, its level of expression is usually increased (range from 2-10X).
Why does translocation activate the c-mycgene? The event has two consequences: c-mycisbrought into a
new region, one in which an Ig or TCR gene was actively expressed ; and the structure of the c-mycgene
may itself be changed. c-myc exhibits three means of oncogene activation: retroviral insertion,chromosomaltranslocation, and gene amplification.
Mice carrying a c-myc gene linked to a B lymphocyte-specific enhancer (the IgH enhancer) develop lymphomas.
The tumors represent both immature and mature B lymphocytes, suggesting that overexpression of c-myc is
tumorigenic throughout the B cell lineage.
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The Philadelphia translocation generates a new oncogene
Its observed in Philadelphia (PH1) chromosome present in patients with chronic myelogenous leukemia
(CML). This reciprocal translocation is too small to be visible in the karyotype, but links a 5000 kb region from
the end of chromosome 9 carrying c-ablto the bcr gene of chromosome 22. The bcr(breakpoint cluster
region) was originally named to describe a region of ~5.8 kb within which breakpoints occur on chromosome
22.
The bcrregion lies within a large (>90 kb) gene, which is now known as the bcrgene. The breakpoints in CMLusually occur within one of two introns in the middle of the gene . The same gene is also involved in
translocations that generate another disease, ALL (acute lymphoblastic leukemia); in this case, the breakpoint in
the bcrgene occurs in the first intron.
The c-ablgene is expressed by alternative splicing that uses either of the first two exons. The breakpoints in both
CML and ALL occur in the intron that precedes the first common exon. Although the exact breakpoints on both
chromosomes 9 and 22 vary in individual cases, the common outcome is the production of a transcript
coding for a Bcr-Abl fusion protein, in which N-terminal sequences derived from bcrare linked to c-abl
sequences. In ALL, the fusion protein has ~45 kD of the Bcr protein; in CML the fusion protein has ~70 kD of
the Bcr protein.
In each case, the fusion protein
contains -140 kD of the usual -145
kD c-Abl protein, that is, it has lost
just a few N-terminal amino acids of
the c-abl sequence. Changes at the
N-terminus are involved in
activating the oncogenic activity of
v-abl, a transforming version of the
gene carried in a retrovirus. The c-
ablgene codes for a tyrosine kinase
activity; this activity is essential for
transforming potential in oncogenic
variants. Deletion (or replacement)
of the N-terminal region activates
the kinase activity and transforming
capacity. So the N-terminus
provides a domain that usually
regulates kinase activity; its loss
may cause inappropriate activation.
Why is the fusion protein oncogenic?
The Bcr-Abl protein activates the Ras pathway for transformation. It may have multiple ways of doing so,
including activation of the adaptors Grb2 and Shc. Both the Bcr and Abl regions of the joint protein may be
important in transforming activity.
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Nondefective retroviruses activate proto-oncogenes
Some proto-oncogenes are activated by events that change their expression. The ability of a retrovirus to
transform without expressing a v-onc sequence was first noted during analysis of the bursal lymphomas
caused by the transformation of B lymphocytes with avian leukemia virus (ALV). In each case, the
transforming potential of the retrovirus is due to the ability of its LTR.
In many independent tumors, the virus has integrated into the cellular genome within or close to the c-myc gene.
The gene consists of three exons; the first represents a long nontranslated leader, and the second two code for
the c-Myc protein. The simplest insertions to explain are those that occur within the first intron. The LTR
provides a promoter, and transcription reads through the two coding exons. Transcription of c-myc under
viral control differs from its usual control: the level of expression is increased (because the LTR provides an
efficient promoter).
Activation of c-myc in the other two classes of
insertions reflects different mechanisms. The
retroviral genome may be inserted within or
upstream of the first intron, but in reverse
orientation, so that its promoter points in the
wrong direction. The retroviral genome also may be
inserted downstream of the c-myc gene. In these
cases, the enhancer in the viral LTR may be
responsible for activating transcription of c-
Myc, either from its normal promoter or from a
fortuitous promoter. In all of these cases, the
coding sequence o/c-myc is unchanged, so
oncogenicity is attributed to the loss of normal
control and increased expression of the gene.
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Oncoproteins may regulate gene expression
1. The oncogene v-rel was identified as the transforming function of the avian (turkey)
reticuloendotheliosis virus and is a truncated version of c-rel, (lacking the ~100 C-terminal amino
acids, and has a small number of point mutations in the remaining sequence) and belongs to the
transcription factor NF-KB dimer of two subunits, p65 and p50. The two subunits of NF-KB have
related sequences, and c-relhas 60% similarity with p50. When I-KB is phosphorylated, it is degraded
and therefore releases NF-KB, which enters the nucleus and activates transcription of target genes. When
v-Rel forms dimers with cellular family members, it may influence their activities either
negatively or positively, thus changing the pattern of gene expression.
2. The activator protein 1 (AP-1) is a transcription factor which is a heterodimeric protein composed ofproteins belonging to the c-Fos, c-Jun. Mutations ofv-jun or v-fos thatabolish the ability to bind DNA
or that damage the trans-activation function also render the product non-transforming, providing a
direct proof that ability to activate transcription is required for transforming activity.
3. The cellular gene c-erbA codes for a thyroid hormone receptor v-erbA are truncated at both ends
and have a small number of substitutions relative to c-erbA. Hormone binding is altered; the c-erbA
product binds triiodothyronine (T3) with high affinity, but the v-erbA product has little or no affinity for
the ligand in mammalian cells. This suggests thatloss of the ligand-binding capacity (perhaps together
with other changes) may create a protein whose function has become independent of the hormone.
The consequence of losing the response to ligand is that the factor can no longer be stimulated to activate
transcription. These results place v-erbA as a dominant negative oncogene.
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Upstream and downstream of mTOR
The mammalian target of rapamycin (mTOR) also known as mechanistic target of rapamycin is
a protein that in humans is encoded by the FRAP1 gene. mTOR belongs to the phosphatidylinositol 3-kinase-
related kinase protein family. mTOR is a serine/threonine protein kinase that regulates cell growth, cell
proliferation, cell motility, cell survival, protein synthesis, and transcription.
The regulation mTOR activity by growth factors is mediated by the PI3K/Akt signaling pathway leading to
phosphorylation and inhibition of TSC2 by Akt and to the subsequent activation of Rheb, which activates mTOR
by an as yet unknown mechanism.
[AktThe serine/threonine protein kinase Akt a downstream effector of PI3K, has emerged as a critical mediator
of mTOR activity. The rate-limiting step in Akt activation is the binding of PIP3 to the pleckstrin homology (PH)
domain of Akt and the subsequent translocation of Akt to the plasma membrane Akt is then phosphorylated by 3-
phosphoinositide- dependent kinase-1 (PDK1) and by another as yet unknown PI3K-dependent kinase. Both
phosphorylation events are required for full activation of Akt. Overexpression of an activated form of Akt in HEK
293 cells promotes 4E-BP1 phosphorylation in the absence of growth factors and in a wortmannin-resistant and
rapamycin-sensitive manner.]
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Prospects for phosphoinositide 3-kinase inhibition as a cancer treatment
The phosphoinositide 3-kinases (PI3-kinases) are a family of lipid kinases that have a key role in the regulation of
many cellular processes including proliferation, survival, carbohydrate metabolism, and motility.
Many additional downstream targets of class I PI3-kinases have been identified; those shown here have
particularly well-defined roles and probably represent the major functional pathways for transmission of PI3-
kinase signals. Enzymes marked with a star have been identified as oncoproteins; underlining indicates known
tumour suppressor function. MEK, mitogen-activated protein kinase kinase; ERK, extracellular regulated kinase;
PDK1, phosphoinositide-dependent kinase 1; PKB, PKC, protein kinases B and C; Casp9, caspase 9; BAD, bcl2
antagonist of cell death; FKHLR1, forkhead transcription factor; IKK, IB kinase; GSK3, glycogen synthase kinase
3; PLC, phospholipase C-; Btk/Tec, Brutons (and related) tyrosine kinase.
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