Cancer Genetics 2

7/30/2019 Cancer Genetics 2

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Cancer genetics-Aayudh Das Page 1

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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Cancer genetics-Aayudh Das

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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Cancer genetics-Aayudh Das

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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Cancer Genetics 2