Transcript Cancer

Cancer
What is Cancer?
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uncontrolled cell growth (as
opposed to steady-state
replacement of cells)
usually accompanied by dedifferentiation of cells
cancerous mass = tumor or
neoplasm
Natural selection: cells which grow
faster than others will take up
more and more space.
– Our cells have multiple
defenses against cells
overgrowing their allotted
locations.
– Cancer occurs when those
defenses have been removed.
– starts with one transformed
cell
Basic types of cancer
• Sarcoma is derived from
mesodermal tissue such
as bone, muscle.
• Carcinoma is derived
from ectoderm or
endoderm such as skin,
nerves.
• Leukemia is derived from
white blood cells (WBC)
from bone marrow.
• Lymphoma is derived
from WBCs from lymph
glands.
Genetic Phenomenon
• Cancer involves changes in DNA sequence i.e. it
is genetic
• Cancer is not epigenetic i.e. changes in patterns
of gene expression without DNA changes.
• If cancer were epigenetic, it might be easier to
reverse.
• Epigenetic changes, such as DNA methylation
and histone modification, do occur in cancer, but
they are rarely or never the underlying cause.
Why we think cancer is genetic:
1. Most mutagens are
carcinogens:
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Many carcinogens are
activated in the liver:
need to treat with liver
extract before doing
Ames test.
Ames test: hisSalmonella are reverted
to his+ by mutagens.
They will then grow on
medium lacking histidine.
Why we think this (cont)
2. Tumors are clonal:
– X-inactivation.
– 2 alleles of Glucose 6-phosphate
dehydrogenase, located on X.
– Only 1 is expressed in any given tumor.
Why we think this (cont)
3. Cancer runs in families:
• Survey of 4000 visitors to a
general clinic,
– 50% had little or no cancer in
their families,
– 7% had 3 or more close
relatives with it.
• Of the 7% of clinic visitors with
3 or more close relatives with
cancer:
– Pedigrees show that there are
many types of cancer in these
families
– Implying a general propensity
to cancer rather than a
specific type
Types of Cancer in Families
– Li-Fraumeni syndrome is a rare hereditary disorder
that increases greatly the susceptibility to cancer. The
syndrome is a mutation in the tumor suppressor gene,
which normally helps control cell growth.
– Xeroderma pigmentosum--lack of DNA repair in skin
leads to skin cancers, esp. with sunlight exposure.
– Also several others. --specific chromosome breakage
syndromes e.g. ataxia-telangiectasia. Many breaks,
tendency to breast cancer.
Cancer is a progressive disease
• Needs 5-6 mutations for full-blown cancer.
• Involves natural selection--in a slowgrowing tumor, a faster growing mutant
will take over.
Stages of Cancer
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Initiation: A mutation that transforms
the cell, leaving it capable of
unrestrained growth.
Promotion: No growth unless cell
enters S phase (many cells are
arrested and need a promoter, a
mitogen, to get them started)
Progression:
A. Angiogenesis--invasion of tumor
by blood vessels
B. Invasiveness--ability to
penetrate basal membranes.
Tumors that can't do this are
benign, those that can are
malignant
C. Metastasis--ability to go through
the blood and colonize other
tissues
Cervical Cancer example
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Cervix is a multi-layered tissue with a basal
membrane.
Normally, all cell division occurs only in layer
next to basal membrane.
Dysplasia - patches of cells multiply above
the basal membrane.
– Seen in Pap smear.
– Often remains harmless and sometime
reverts to normal.
– With carcinoma in situ, (badly named-it is not full-blown cancer), all layers in
an area are de-differentiated and
dividing.
– Easy to cure at this point with surgery.
Malignant carcinoma - crosses basal
membrane and invades underlying
connective tissue.
Tumor Inducing Viruses
• Key to our current understanding of
cancer.
• DNA viruses--complicated systems:
– Epstein-Barr virus --Burkitt's lymphoma
– hepatitis B--Liver cancer
– papilloma virus (genital warts)--cervical
cancer
– HTLV I and II --leukemia
DNA viruses
• DNA viruses:
normally replicate in
the cell as plasmids,
but occasionally
integrate into the
genome, where viral
promoters can by
chance activate
oncogenes.
• Very complicated
genomes
RNA viruses
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RNA viruses: retroviruses.
First one Rous sarcoma virus
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structure: LTR--gag--pol--env--LTR
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Discovered by Peyton Rous in 1917
Chickens could get cancer from a
filterable extract of tumors.
LTR--long terminal repeat. For
integration and transcription (promoter)
gag—inner core RNA-binding proteins
pol--reverse transcriptase and other
enzymes
env--envelope glycoproteins (virus has
membrane)
expression--interesting splicing and
cleavage of RNA and polyproteins
Oncogenes
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Two very similar retroviruses: RSV and
ALV
Rous Sarcoma Virus is 10 kb and
acutely transforming (tumors in
weeks).
Avian Leukosis Virus is 8.5 kb and
weakly transforming: latent period of
months or more, and often has no
effect.
temperature sensitive mutants of RSV:
replicate at all temperatures, but
transform at 35o but not 41o. That is, a
genetic separation of transformation
from replication--different genes
involved. Temp sensitive mutants due
to mutant protein that is unstable at
high temps.
Transforming gene at 3' end of RSV.
Called src.
Transforming Retroviruses
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Most transforming retroviruses are replicationdefective.
– They only replicate in mixed infection with
normal (non-oncogenic) retrovirus. e.g.
Abelson MuLV has gag deleted at 3' end
fused to abl oncogene, generating a
fusion protein, and nothing else in
genome.
source of viral oncogenes
– Varmus + Bishop 1976 used a src probe
against chicken genomic DNA in a
Southern blot
– got a band i.e. src is a normal cellular
gene as well as a viral gene.
True of all viral oncogenes:
– v-onc = viral version. No introns
– c-onc = cellular version has introns
– v-oncs are also often mutated, partially
deleted, or fused to other sequences as
compared to c-oncs.
– presumably got into viruses by viral
genome integrating next to c-onc and a
mistake occurring allowing c-onc to be
transcribed along with viral genome.
– A certain amount of mutation at this point
would give a v-onc surrounded by LTRs.
In the cell, oncogenes do not normally cause
cancer, only their mutated versions do. The
normal, non-carcingeneic versions are often
called “proto-oncogenes”.
Gene Transfer Experiments
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Gene Transfer experiments are another
approach to identifying oncogenes
Showed that oncogenes in human tumors are
the same as oncogenes identified from
retroviruses.
Normal fibroblasts will multiply in Petri dishes,
but they have 2 specific properties of interest:
1. contact inhibition: they stop growing when they
touch, leading to a monolayer.
2. finite number (50-60) of cell divisions before death
Partially Transformed Cells
• When transformed, cells lose
contact inhibition (they pile up)
and become immortal.
• NIH 3T3 mouse cells are
partially transformed: immortal
but still contact inhibited. That
is, they grow in a monolayer
• However, mutagens, etc. will
create foci (plural of focus) of
piled up cells starting with a
single transformed cell.
• Basis for oncogene assay.
The Experiment
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--extract DNA from bladder carcinoma EJ.
Use to transfect 3T3 cells (CaPO4
precipitation induces pinocytosis). See foci
of transformed cells, meaning that some of
the human DNA can transform.
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now, extract DNA from foci: contains mostly
mouse DNA with just a bit of human
containing the oncogene.
Re-transfect fresh 3T3 cells, get new foci.
This removes more of the extraneous
human DNA.
Clone DNA from the foci, screen with Alu
sequence
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Alu sequences are very common throughout
the human genome and thus are linked to
most human genes. Not found in mouse DNA
at all.
Then sequence the resulting clones.
Turned out to by H-ras, already known from
Harvey rat sacroma virus
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Non-tumor DNA does not transform.
had one mutation: Gly converted to Val
other human tumors processed this way
yield other oncogenes.
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but not all work.
Activating Oncogenes
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Normally, cellular oncogenes are proto-oncogenes: they have
a regular cellular function and aren’t involved with cancer.
Two basic ways of converting proto-oncogenes into
oncogenes:
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mutate the protein
make lots of the normal protein
There are a variety of ways to accomplish these events.
Types of Mutation that Create
Oncogenes
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Base changes and other simple
alterations in protein structure. e.g. H-ras
described above as the cause of bladder
carcinoma in the transfection
experiments.
Creation of a fusion protein:
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Starts out a normal (often highly
expressed) cellular protein
Ends up with oncogene sequences,
creating a N-terminal deleted oncogene
with other sequences added.
This can have abnormal function.
Created by translocation (e.g. Philadelphia
chromosome (short 22--actually a
t(9;22))that joins c-abl with bcr (breakpoint
cluster region) gene.
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Creates an unusually active Abl protein that
causes chronic myeloid leukemia.
Types of Mutation that Create Oncogenes (cont.)
Types of mutation to create active oncogenes from
proto-oncogenes:
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Replacing normal weak promoter of proto-oncogene
with strong promoter, leading to over-expression.
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Insertional mutagenesis: the LTR is often a strong
promoter for sequences 3' to it.
The cause of oncogenesis in weakly oncogenic viruses
like ALV.
ALV often inserts in c-myc gene just before proteincoding sequences, giving lots of normal Myc protein.
Gene amplification.
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Tumors often become insensitive to chemotherapeutic
agents due to gene amplification: many duplications of
target gene.
Classic case is methotrexate resistance due to
amplification of dihydrofolate reductase gene.
Gives homogeneously staining regions of chromosome
due to many tandem copies and/or double minute
chromosomes, containing copies of the gene but no
centromere.
Many tumors have this:
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myc in lung cancer
erb -B2 in breast and ovarian tumors
Get hundreds of copies of the gene
Comparative genome hybridization: label DNA
from normal cells and from tumor cells with red
and green fluorescent tags. Hybridize to normal
chromosomes and scan to see red/green ratio.
Deviations show regions of amplification or loss in
the tumor line.
Tumor Suppressor genes
• A distinction:
– Oncogenes act in a dominant fashion: one mutant copy plus one
normal copy gives a tumor.
– Tumor suppressor genes are recessive: one mutant and one
normal is still wild type--need both copies mutant to give a tumor.
• Wild-type oncogenes (proto-oncogenes) promote cell
proliferation; mutant versions enhance this property.
• On the other hand, tumor suppressors regulate and
inhibit cell proliferation; mutant versions remove controls
on proliferation.
• Tumor suppressor genes mostly found by cloning familial
cancer genes and chromosome regions commonly
deleted in tumor cells.
Retinoblastoma
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Retinoblasts are retinal precursor cells
that are only found in young children.
Inherited and spontaneous forms of
retinoblastoma.
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Theory of Knudson (1971): both
copies of the Rb gene need to be
inactivated by mutation to give a
tumor.
Inherited form:
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Inherited form is autosomal dominant
and usually both eyes are affected.
Spontaneous is almost always one eye
only.
People start with one copy knocked
out: RB+ RBThey thus need only a single mutation
to get a tumor.
Mutation rate is around 1 in 106;
8-10 million cells per retina means that
at least one cell in both retinas usually
gets hit and forms a tumor.
Spontaneous:
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Need 2 hits,
Each with a 10-6 rate, or 1 in 1012--quite
rare.
How to Lose a Gene
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Many events leading from an RB
heterozygote to cancer are not
classical mutations, but rather
"loss of heterozygoisty" (LOH),
where one copy of the gene is
deleted or inactivated, or
converted to the same allele as
the other copy.
Various causes: spontaneous
mutation or deletion, nondisjunction, mitotic recombination.
Inactivation by methylation of the
non-mutant copy also occurs.
Sometimes can see deletion or
loss of marker loci in region of the
RB gene (13q14) in tumor cells.
Why is RB a dominant trait?
• Semantics.
• At the whole body level, it is dominant
because you need to inherit it from only 1
parent.
• At the cellular level, it is recessive
because both copies need to be knocked
out to get the transformed cell.
• A Rb homozygote would probably be
dead.
Other Cases
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Wilm's tumor.
– Part of WAGR syndrome (Wilm's, aniridia, genital abnormalities, retardation)
found in heterozygous deletion of 11p.
– Homozygous deletion is dead as are virtually all homozygous deletions of more
than 1 or 2 genes.
– Apparently 4 separate genes involved in syndrome.
– WT is childhood kidney tumor same deal as RB
• spontaneous is one side only
• inherited is usually bilateral
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Li-Fraumeni syndrome: loss of p53, a 53 kdal protein that is the “central
guardian of the genome”, preventing replication or killing cells that have
DNA damage.
– Loss of 17p region where p53 is seen in many tumors: colon, lung breast etc.
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Ataxia telangiectasia. The disease has many symptoms:
neurodegeneration, immunodeficiency, premature aging, radiation
sensitivity
– The ATM protein is part of the pathway that signals DNA damage
– cloned by using cosmids with normal DNA to rescue an AT cell line from its
radiation-sensitive phenotype
What are oncogenes and tumor
suppresors?
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A lot of fundamental cell
processes have been
investigated as part of
understanding oncogenes.
Several basic types:
1. growth factors
2. growth factor receptors at the
cell surface
3. signal transduction proteins
4. transcription factors
5. cell cycle regulatory proteins
6. DNA damage detection and
repair proteins
Growth Factors / Analogs
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Some oncogenes are growth factors or
analogs. e.g. sis is very similar to PDGF
(platelet-derived growth factor)
– If turned on in a cell carrying PDGF
receptors, it will self-stimulate to
unrestrained growth.
– Normally PDGF is made in one cell and
the receptors are on other cells
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Opposite side of this: some oncogenes
are growth factor receptor analogs:
– e.g. erbB is a mutated version of
epidermal growth factor receptor (EGF).
– Normally EGF receptor is a
transmembrane protein that binds EGF on
outside and a kinase on the inside that is
activated only when EGF is bound outside
membrane.
– erbB oncogene deletes most of the
outside part, leading to a kinase that is
always on.
– Just like continuous application of EGF-continuous growth and division.
Intracellular Transducers and
Transcription Factors
• Some oncogenes are
intracellular transducers
– Lots of targets: lots of
pathways and interactions
– e.g. src, ras, abl
• Some oncogenes are
transcription factors:
– cause sets of genes to be
activated or inactivated
inappropriately.
– e.g. fos/jun combine to
form transcription factor
AP1, which binds to a
specific DNA sequence for
transcription.
Cell Cycle Control
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Complex and not fully understood yet. Many
overlapping control systems
In general a cell can: stay in interphase, divide, or
undergo programmed cell death (apoptosis).
Checkpoints: the cell cannot proceed past them until
certain conditions are met.
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G1 -> S
G2 -> mitosis
metaphase spindle attachment
G1-S checkpoint. The main control point for
cells
with damaged DNA
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the main control protein is pRb (product of RB1, the
retinoblastoma gene).
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It has 2 states: phosphorylated (inactive) and
dephosphorylated (active).
pRb acts on the transcription factor E2F1. If E2F1 is active,
the cell can move from G1 phase to S phase, the first step
in getting the cell to divide.
The role of pRb is to inhibit E2F1: when pRb is active
(dephosphorylated), E2F1 is inactivated, so the cell stays in
G1.
If pRb is phosphorylated (by a cascade of kinases), it
becomes inactive, and E2F1 becomes active; the cell then
enters S phase.
Phosphorylation of pRb is done by cyclins (proteins
whose concentration varies with the cell’s position in the
cell cycle) and CDKs (cyclin-dependent kinases).
G2-M checkpoint. Cells must have completed DNA
repair to pass this point
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Mitosis is initiated by the MPF (maturation promoting factor) protein
complex, composed of cyclins and CDKs which have built up over the
course of the cell cycle.
Genome Integrity
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Mutation is a constant problem. Many
mechanisms prevent cells with seriously mutated
DNA from dividing.
Malignant cells usually undergo chromosomal
rearrangements, leading to new fused genes and
loss of heterozygosity.
Spindle checkpoint. During mitosis, cells can only
proceed into anaphase when all of the
chromosomes are properly attached to the
spindle. A protein complex on the kinetochore is
displaced when the kinetochore is attached to the
spindle.
Telomerase. This enzyme prevents the loss of
DNA at the ends of chromosomes, an inevitable
consequence of replication. It is inactive in most
cells, which results in them dying after 60 or so
cell divisions. However, it is re-activated in 85%
of successful tumor cells, resulting in cellular
immortality. This is one of the most common
markers of cancer.
DNA Damage Detection
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Several DNA repair systems are tumor
suppressor genes.
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BRCA1 and BRCA2: genes implicated in
breast cancer. Also ATM, the ataxia
telangiectasia protein. Part of a multiprotein BASC complex that scans the DNA
for damage
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Xeroderma pigmentosum: DNA damage
caused by sunlight isn't repaired, leading to
skin tumors.
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Several forms, involving nucleotide excision DNA repair
enzymes.
Hereditary non-polyposis colon cancer
(doesn't form polyps).
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Autosomal dominant
When the genomes of HNPCC patients were scanned
for LOH, many micro-satellite loci had changes in repeat
number, all over the genome.
Related to E. coli mutator system MutHLS,
• Mutations in these genes increase mutation rate in
E. coli up to 1000 x.
• Mismatch repair system: removes mismatched DNA
bases on newly synthesized strand and resynthesizes that stretch of DNA.
Human analogue of MutS gene mapped to region of
HNPCC gene, and turned out to be mutant in HNPCC
patients.
One human homologue of MutL is also responsible for
much of HNPCC
p53 and Apoptosis
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p53 is the central regulator of genome integrity, “the
guardian of the genome”.
p53 is a transcription factor, the protein product of
the TP53 gene.
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In normal cells, the p53 level is low because p53 is rapidly
degraded by being ubiquitinated and processed by the
proteosomes.
DNA damage and other stresses cause p53 to become
phosphorylated, which stabilizes it.
This leads to a buildup of p53, which causes transcription of
genes that inhibit the cell cycle and also initiate apoptosis.
Mutations in TP53 are very common, especially in
late stage tumors.
It is the gene mutated in Li-Fraumeni syndrome,
which causes a general increase in all kinds of
tumors.
Also mutated in cigarette smoke-induced tumors,
sunlight-induced tumors, aflatoxin-induced tumors,
cervical cancer (papilloma virus-induced).
Apoptosis is programmed cell death: a signal
(internal or external) activates a mechanism that
kills the cell.
Very important is embryonic development
Cells with DNA damage undergo apoptosis
Cancer often develops due to a failure of apoptosis.
The apoptosis process is carried out by a set of
proteases called caspases.
Cancer as Multi-step Process
Progression of colorectal cancer
(familial adenomatous cancer)
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Adenomas are polyps seen in
the colon. They start as
abnormal crypt cells, progress to
benign polyps, and finally
become cancerous.
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The following shows one way an
FAP case may develop:
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Normal colon epithelium has
mutation in APC tumor
suppressor gene causing rapidly
proliferating epithelium.
Activation by mutation of KRAS
(ras-K) leads to polyp formation.
Loss of heterozygosity tumor
suppressor gene in 18q (exact
gene not clear) leads to late
stage polyp.
Mutation in p53 leads to
carcinoma.